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1
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Babawale PI, Guerrero-Plata A. Differential Responses of Pediatric and Adult Primary Epithelial Cells to Human Metapneumovirus and Respiratory Syncytial Virus Infection. Viruses 2025; 17:380. [PMID: 40143308 PMCID: PMC11946536 DOI: 10.3390/v17030380] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2024] [Revised: 03/02/2025] [Accepted: 03/04/2025] [Indexed: 03/28/2025] Open
Abstract
Human metapneumovirus (HMPV) and respiratory syncytial virus (RSV) are pneumoviruses causing lower respiratory tract infections, primarily in infants and children rather than in healthy adults. Human bronchial epithelial cells serve as a viral replication target and source of the innate immune response to these viruses. To better understand the immune responses induced by RSV and HMPV in the pediatric airway epithelium, we comparatively studied pediatric and adult epithelial responses. We used normal human bronchial epithelial (NHBE) cells cultured in an air-liquid interface culture system (ALI), which helps to mimic the architecture of the human lower respiratory tract epithelium. Our results demonstrate differential viral replication patterns and reduced interferons; and inflammatory cytokines' expression in pediatric cells compared to adult cells. However, pediatric epithelial cells expressed an increased mucus response and induced a stronger pro-inflammatory response in monocyte-derived dendritic cells. These findings reveal age-dependent immune epithelial responses that may contribute to more severe infections by HMPV and RSV.
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Affiliation(s)
| | - Antonieta Guerrero-Plata
- Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA;
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2
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Mei W, Faraj Tabrizi S, Godina C, Lovisa AF, Isaksson K, Jernström H, Tavazoie SF. A commonly inherited human PCSK9 germline variant drives breast cancer metastasis via LRP1 receptor. Cell 2025; 188:371-389.e28. [PMID: 39657676 PMCID: PMC11770377 DOI: 10.1016/j.cell.2024.11.009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2024] [Revised: 08/12/2024] [Accepted: 11/10/2024] [Indexed: 12/12/2024]
Abstract
Identifying patients at risk for metastatic relapse is a critical medical need. We identified a common missense germline variant in proprotein convertase subtilisin/kexin type 9 (PCSK9) (rs562556, V474I) that is associated with reduced survival in multiple breast cancer patient cohorts. Genetic modeling of this gain-of-function single-nucleotide variant in mice revealed that it causally promotes breast cancer metastasis. Conversely, host PCSK9 deletion reduced metastatic colonization in multiple breast cancer models. Host PCSK9 promoted metastatic initiation events in lung and enhanced metastatic proliferative competence by targeting tumoral low-density lipoprotein receptor related protein 1 (LRP1) receptors, which repressed metastasis-promoting genes XAF1 and USP18. Antibody-mediated therapeutic inhibition of PCSK9 suppressed breast cancer metastasis in multiple models. In a large Swedish early-stage breast cancer cohort, rs562556 homozygotes had a 22% risk of distant metastatic relapse at 15 years, whereas non-homozygotes had a 2% risk. Our findings reveal that a commonly inherited genetic alteration governs breast cancer metastasis and predicts survival-uncovering a hereditary basis underlying breast cancer metastasis.
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Affiliation(s)
- Wenbin Mei
- Laboratory of Systems Cancer Biology, The Rockefeller University, New York, NY, USA
| | | | - Christopher Godina
- Division of Oncology, Department of Clinical Sciences in Lund, Lund University Cancer Center/Kamprad, Lund, Sweden
| | - Anthea F Lovisa
- Laboratory of Systems Cancer Biology, The Rockefeller University, New York, NY, USA
| | - Karolin Isaksson
- Division of Surgery, Department of Clinical Sciences in Lund, Lund University and Department of Surgery Kristianstad Hospital, Lund, Sweden
| | - Helena Jernström
- Division of Oncology, Department of Clinical Sciences in Lund, Lund University Cancer Center/Kamprad, Lund, Sweden
| | - Sohail F Tavazoie
- Laboratory of Systems Cancer Biology, The Rockefeller University, New York, NY, USA.
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3
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Li S, Gao Y, Zhai H, Guan X, Yang X, Hou Q, Zhang X, Li LF, Wang X, Huang S, Qiu HJ, Li Y. Immune responses induced by a recombinant C-strain of classical swine fever virus expressing the F317L protein of African swine fever virus. Vet Microbiol 2024; 298:110239. [PMID: 39243670 DOI: 10.1016/j.vetmic.2024.110239] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2024] [Revised: 08/15/2024] [Accepted: 08/28/2024] [Indexed: 09/09/2024]
Abstract
African swine fever (ASF), a highly infectious and devastating disease affecting both domestic pigs and wild boars, owes its etiology to African swine fever virus (ASFV). ASFV encodes more than 165 proteins. However, novel immunogenic proteins remain unknown. This study aimed to determine the antigenicity of the F317L protein (pF317L) of ASFV. The results revealed that pF317L was able to react with convalescent pig sera, indicating that pF317L could be a candidate antigen. The antigenic potential of pF317L expressed by rHCLV-F317L, a recombinant virus in the backbone of C-strain (a lapinized live attenuated classical swine fever virus) was further investigated in rabbits and pigs. The results revealed that antibodies and cell-mediated immune responses against pF317L were induced in either rabbits or pigs inoculated with rHCLV-F317L. Importantly, anti-pF317L antibodies from rabbits or pigs immunized with rHCLV-F317L significantly inhibited ASFV replication in vitro. In conclusion, pF317L demonstrates favorable immunogenic properties, positioning it as a promising candidate for the development of protective antigens in the ongoing endeavor to formulate efficacious ASF vaccine strategies.
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Affiliation(s)
- Shuwen Li
- State Key Laboratory for Animal Disease Control and Prevention Harbin Veterinary Research Institute, CAAS, 678 Haping Road, Harbin, Heilongjiang 150069, China; College of Life Science and Engineering, Foshan University, 33 Guangyun Road, Foshan, Guangdong 528231, China
| | - Yuxuan Gao
- State Key Laboratory for Animal Disease Control and Prevention Harbin Veterinary Research Institute, CAAS, 678 Haping Road, Harbin, Heilongjiang 150069, China
| | - Huanjie Zhai
- State Key Laboratory for Animal Disease Control and Prevention Harbin Veterinary Research Institute, CAAS, 678 Haping Road, Harbin, Heilongjiang 150069, China
| | - Xiangyu Guan
- State Key Laboratory for Animal Disease Control and Prevention Harbin Veterinary Research Institute, CAAS, 678 Haping Road, Harbin, Heilongjiang 150069, China
| | - Xiaoke Yang
- State Key Laboratory for Animal Disease Control and Prevention Harbin Veterinary Research Institute, CAAS, 678 Haping Road, Harbin, Heilongjiang 150069, China
| | - Qinghe Hou
- State Key Laboratory for Animal Disease Control and Prevention Harbin Veterinary Research Institute, CAAS, 678 Haping Road, Harbin, Heilongjiang 150069, China
| | - Xinyu Zhang
- State Key Laboratory for Animal Disease Control and Prevention Harbin Veterinary Research Institute, CAAS, 678 Haping Road, Harbin, Heilongjiang 150069, China; College of Life Science and Engineering, Foshan University, 33 Guangyun Road, Foshan, Guangdong 528231, China
| | - Lian-Feng Li
- State Key Laboratory for Animal Disease Control and Prevention Harbin Veterinary Research Institute, CAAS, 678 Haping Road, Harbin, Heilongjiang 150069, China
| | - Xiao Wang
- Department of Pathogenic Biology, School of Basic Medical Sciences, Binzhou Medical University, 346 Guanhai Road, Yantai, Shandong 264000, China.
| | - Shujian Huang
- College of Life Science and Engineering, Foshan University, 33 Guangyun Road, Foshan, Guangdong 528231, China.
| | - Hua-Ji Qiu
- State Key Laboratory for Animal Disease Control and Prevention Harbin Veterinary Research Institute, CAAS, 678 Haping Road, Harbin, Heilongjiang 150069, China; College of Life Science and Engineering, Foshan University, 33 Guangyun Road, Foshan, Guangdong 528231, China.
| | - Yongfeng Li
- State Key Laboratory for Animal Disease Control and Prevention Harbin Veterinary Research Institute, CAAS, 678 Haping Road, Harbin, Heilongjiang 150069, China.
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4
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Loiola RA, Hachani J, Duban-Deweer S, Sevin E, Bugno P, Kowalska A, Rizzi E, Shimizu F, Kanda T, Mysiorek C, Mazurek M, Gosselet F. Secretome of brain microvascular endothelial cells promotes endothelial barrier tightness and protects against hypoxia-induced vascular leakage. Mol Med 2024; 30:132. [PMID: 39187765 PMCID: PMC11348522 DOI: 10.1186/s10020-024-00897-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2024] [Accepted: 08/12/2024] [Indexed: 08/28/2024] Open
Abstract
Cell-based therapeutic strategies have been proposed as an alternative for brain and blood vessels repair after stroke, but their clinical application is hampered by potential adverse effects. We therefore tested the hypothesis that secretome of these cells might be used instead to still focus on cell-based therapeutic strategies. We therefore characterized the composition and the effect of the secretome of brain microvascular endothelial cells (BMECs) on primary in vitro human models of angiogenesis and vascular barrier. Two different secretome batches produced in high scale (scHSP) were analysed by mass spectrometry. Human primary CD34+-derived endothelial cells (CD34+-ECs) were used as well as in vitro models of EC monolayer (CMECs) and blood-brain barrier (BBB). Cells were also exposed to oxygen-glucose deprivation (OGD) conditions and treated with scHSP during reoxygenation. Protein yield and composition of scHSP batches showed good reproducibility. scHSP increased CD34+-EC proliferation, tubulogenesis, and migration. Proteomic analysis of scHSP revealed the presence of growth factors and proteins modulating cell metabolism and inflammatory pathways. scHSP improved the integrity of CMECs, and upregulated the expression of junctional proteins. Such effects were mediated through the activation of the interferon pathway and downregulation of Wnt signalling. Furthermore, OGD altered the permeability of both CMECs and BBB, while scHSP prevented the OGD-induced vascular leakage in both models. These effects were mediated through upregulation of junctional proteins and regulation of MAPK/VEGFR2. Finally, our results highlight the possibility of using secretome from BMECs as a therapeutic alternative to promote brain angiogenesis and to protect from ischemia-induced vascular leakage.
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Affiliation(s)
- Rodrigo Azevedo Loiola
- UR 2465, Laboratory of the Blood-Brain Barrier (LBHE), Sciences Faculty Jean Perrin, Artois University, 62300, Lens, France
| | - Johan Hachani
- UR 2465, Laboratory of the Blood-Brain Barrier (LBHE), Sciences Faculty Jean Perrin, Artois University, 62300, Lens, France
| | - Sophie Duban-Deweer
- UR 2465, Laboratory of the Blood-Brain Barrier (LBHE), Sciences Faculty Jean Perrin, Artois University, 62300, Lens, France
| | - Emmanuel Sevin
- UR 2465, Laboratory of the Blood-Brain Barrier (LBHE), Sciences Faculty Jean Perrin, Artois University, 62300, Lens, France
| | - Paulina Bugno
- Pure Biologics S.A., Duńska 11, 54-427, Wroclaw, Poland
| | | | - Eleonora Rizzi
- UR 2465, Laboratory of the Blood-Brain Barrier (LBHE), Sciences Faculty Jean Perrin, Artois University, 62300, Lens, France
| | - Fumitaka Shimizu
- Department of Neurology and Clinical Neuroscience, Graduate School of Medicine, Yamaguchi University, Ube, Japan
| | - Takashi Kanda
- Department of Neurology and Clinical Neuroscience, Graduate School of Medicine, Yamaguchi University, Ube, Japan
| | - Caroline Mysiorek
- UR 2465, Laboratory of the Blood-Brain Barrier (LBHE), Sciences Faculty Jean Perrin, Artois University, 62300, Lens, France
| | | | - Fabien Gosselet
- UR 2465, Laboratory of the Blood-Brain Barrier (LBHE), Sciences Faculty Jean Perrin, Artois University, 62300, Lens, France.
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5
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Bathula NV, Friesen JJ, Casmil IC, Wayne CJ, Liao S, Soriano SKV, Ho CH, Strumpel A, Blakney AK. Delivery vehicle and route of administration influences self-amplifying RNA biodistribution, expression kinetics, and reactogenicity. J Control Release 2024; 374:28-38. [PMID: 39097193 DOI: 10.1016/j.jconrel.2024.07.078] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2024] [Revised: 07/05/2024] [Accepted: 07/30/2024] [Indexed: 08/05/2024]
Abstract
Self-amplifying RNA (saRNA) is a next-generation RNA platform derived from an alphavirus that enables replication in host cytosol, offering a promising shift from traditional messenger RNA (mRNA) therapies by enabling sustained protein production from minimal dosages. The approval of saRNA-based vaccines, such as the ARCT-154 for COVID-19 in Japan, underscores its potential for diverse therapeutic applications, including vaccine development, cancer immunotherapy, and gene therapy. This study investigates the role of delivery vehicle and administration route on saRNA expression kinetics and reactogenicity. Employing ionizable lipid-based nanoparticles (LNPs) and polymeric nanoparticles, we administered saRNA encoding firefly luciferase to BALB/c mice through six routes (intramuscular (IM), intradermal (ID), intraperitoneal (IP), intranasal (IN), intravenous (IV), and subcutaneous (SC)), and observed persistent saRNA expression over a month. Our findings reveal that while LNPs enable broad route applicability and stability, pABOL (poly (cystamine bisacrylamide-co-4-amino-1-butanol)) formulations significantly amplify protein expression via intramuscular delivery. Notably, the disparity between RNA biodistribution and protein expression highlight the nuanced interplay between administration routes, delivery vehicles, and therapeutic outcomes. Additionally, our research unveiled distinct biodistribution profiles and inflammatory responses contingent upon the chosen delivery formulation and route. This research illuminates the intricate dynamics governing saRNA delivery, biodistribution and reactogenicity, offering essential insights for optimizing therapeutic strategies and advancing the clinical and commercial viability of saRNA technologies.
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Affiliation(s)
- Nuthan Vikas Bathula
- Michael Smith Laboratories, University of British Columbia, Vancouver V6T 1Z4, Canada; School of Biomedical Engineering, University of British Columbia, Vancouver V6T 1Z3, Canada
| | - Josh J Friesen
- Michael Smith Laboratories, University of British Columbia, Vancouver V6T 1Z4, Canada; School of Biomedical Engineering, University of British Columbia, Vancouver V6T 1Z3, Canada
| | - Irafasha C Casmil
- Michael Smith Laboratories, University of British Columbia, Vancouver V6T 1Z4, Canada
| | - Christopher J Wayne
- Michael Smith Laboratories, University of British Columbia, Vancouver V6T 1Z4, Canada
| | - Suiyang Liao
- Michael Smith Laboratories, University of British Columbia, Vancouver V6T 1Z4, Canada; Biochemistry and Molecular Biology, University of British Columbia, Vancouver V6T 2A1, Canada
| | - Shekinah K V Soriano
- Michael Smith Laboratories, University of British Columbia, Vancouver V6T 1Z4, Canada; School of Biomedical Engineering, University of British Columbia, Vancouver V6T 1Z3, Canada
| | - Chia Hao Ho
- Michael Smith Laboratories, University of British Columbia, Vancouver V6T 1Z4, Canada
| | - Anneke Strumpel
- Michael Smith Laboratories, University of British Columbia, Vancouver V6T 1Z4, Canada; School of Biomedical Engineering, University of British Columbia, Vancouver V6T 1Z3, Canada; RWTH Aachen University, Templergraben 55, Aachen 52062, Germany
| | - Anna K Blakney
- Michael Smith Laboratories, University of British Columbia, Vancouver V6T 1Z4, Canada; School of Biomedical Engineering, University of British Columbia, Vancouver V6T 1Z3, Canada.
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6
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Rowe T, Davis W, Wentworth DE, Ross T. Differential interferon responses to influenza A and B viruses in primary ferret respiratory epithelial cells. J Virol 2024; 98:e0149423. [PMID: 38294251 PMCID: PMC10878268 DOI: 10.1128/jvi.01494-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2023] [Accepted: 12/02/2023] [Indexed: 02/01/2024] Open
Abstract
Influenza B viruses (IBV) cocirculate with influenza A viruses (IAV) and cause periodic epidemics of disease, yet antibody and cellular responses following IBV infection are less well understood. Using the ferret model for antisera generation for influenza surveillance purposes, IAV resulted in robust antibody responses following infection, whereas IBV required an additional booster dose, over 85% of the time, to generate equivalent antibody titers. In this study, we utilized primary differentiated ferret nasal epithelial cells (FNECs) which were inoculated with IAV and IBV to study differences in innate immune responses which may result in differences in adaptive immune responses in the host. FNECs were inoculated with IAV (H1N1pdm09 and H3N2 subtypes) or IBV (B/Victoria and B/Yamagata lineages) and assessed for 72 h. Cells were analyzed for gene expression by quantitative real-time PCR, and apical and basolateral supernatants were assessed for virus kinetics and interferon (IFN), respectively. Similar virus kinetics were observed with IAV and IBV in FNECs. A comparison of gene expression and protein secretion profiles demonstrated that IBV-inoculated FNEC expressed delayed type-I/II IFN responses and reduced type-III IFN secretion compared to IAV-inoculated cells. Concurrently, gene expression of Thymic Stromal Lymphopoietin (TSLP), a type-III IFN-induced gene that enhances adaptive immune responses, was significantly downregulated in IBV-inoculated FNECs. Significant differences in other proinflammatory and adaptive genes were suppressed and delayed following IBV inoculation. Following IBV infection, ex vivo cell cultures derived from the ferret upper respiratory tract exhibited reduced and delayed innate responses which may contribute to reduced antibody responses in vivo.IMPORTANCEInfluenza B viruses (IBV) represent nearly one-quarter of all human influenza cases and are responsible for significant clinical and socioeconomic impacts but do not pose the same pandemic risks as influenza A viruses (IAV) and have thus received much less attention. IBV accounts for greater severity and deaths in children, and vaccine efficacy remains low. The ferret can be readily infected with human clinical isolates and demonstrates a similar course of disease and immune responses. IBV, however, generates lower antibodies in ferrets than IAV following the challenge. To determine whether differences in initial innate responses following infection may affect the development of robust adaptive immune responses, ferret respiratory tract cells were isolated, infected with IAV/IBV, and compared. Understanding the differences in the initial innate immune responses to IAV and IBV may be important in the development of more effective vaccines and interventions to generate more robust protective immune responses.
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Affiliation(s)
- Thomas Rowe
- Centers for Disease Control and Prevention, Influenza Division, Atlanta, Georgia, USA
- Center for Vaccines and Immunology, University of Georgia, Athens, Georgia, USA
| | - William Davis
- Centers for Disease Control and Prevention, Influenza Division, Atlanta, Georgia, USA
| | - David E. Wentworth
- Centers for Disease Control and Prevention, Influenza Division, Atlanta, Georgia, USA
| | - Ted Ross
- Center for Vaccines and Immunology, University of Georgia, Athens, Georgia, USA
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Sarkar R, Patra U, Mukherjee A, Mitra S, Komoto S, Chawla-Sarkar M. Rotavirus circumvents the antiviral effects of protein ISGylation via proteasomal degradation of Ube1L. Cell Signal 2023; 112:110891. [PMID: 37722521 DOI: 10.1016/j.cellsig.2023.110891] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2023] [Revised: 08/10/2023] [Accepted: 09/15/2023] [Indexed: 09/20/2023]
Abstract
Among the ramified cellular responses elicited in response to pathogenic stimuli, upregulation and covalent conjugation of an Ubiquitin-like modifier ISG15 to lysine residues of target proteins (ISGylation) through sequential action of three enzymes E1 (Ube1L), E2 (Ube2L6) and E3 (Herc5) have emerged as an important regulatory facet governing innate immunity against numerous viral infections. In the present study, we investigated the interplay between host ISGylation system and Rotavirus (RV). We observed that RV infection upregulates the expression of free ISG15 but prevents protein ISGylation. Analysing the expression of ISGylation machinery components revealed that RV infection results in steady depletion of Ube1L protein with the progression of infection. Indeed, restoration of Ube1L expression caused induction in protein ISGylation during RV infection. Subsequent investigation revealed that ectopic expression of RV non-structural protein 5 (NSP5) fosters proteolytic ubiquitylation of Ube1L, thereby depleting it in an ubiquitin-proteasome-dependent manner. Moreover, pan-Cullin inhibition also abrogates proteolytic ubiquitylation and rescued depleted Ube1L in RV-NSP5 expressing cells, suggesting the involvement of host cellular Cullin RING Ligases (CRLs) in proteasomal degradation of Ube1L during RV-SA11 infection. Reciprocal co-immunoprecipitation analyses substantiated a molecular association between Ube1L and RV-NSP5 during infection scenario and also under ectopically overexpressed condition independent of intermediate RNA scaffold and RV-NSP5 hyperphosphorylation. Interestingly, clonal overexpression of Ube1L reduced expression of RV proteins and RV infectivity, which are restored in ISG15 silenced cells, suggesting that Ube1L is a crucial anti-viral host cellular determinant that inhibits RV infection by promoting the formation of ISG15 conjugates.
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Affiliation(s)
- Rakesh Sarkar
- Division of Virology, ICMR-National Institute of Cholera and Enteric Diseases, Kolkata, West Bengal, India
| | - Upayan Patra
- Division of Virology, ICMR-National Institute of Cholera and Enteric Diseases, Kolkata, West Bengal, India
| | - Arpita Mukherjee
- Division of Virology, ICMR-National Institute of Cholera and Enteric Diseases, Kolkata, West Bengal, India
| | - Suvrotoa Mitra
- Division of Virology, ICMR-National Institute of Cholera and Enteric Diseases, Kolkata, West Bengal, India
| | - Satoshi Komoto
- Department of Virology and Parasitology, School of Medicine, Fujita Health University, Aichi, Japan
| | - Mamta Chawla-Sarkar
- Division of Virology, ICMR-National Institute of Cholera and Enteric Diseases, Kolkata, West Bengal, India.
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Xu T, Zhu C, Chen J, Song F, Ren X, Wang S, Yi X, Zhang Y, Zhang W, Hu Q, Qin H, Liu Y, Zhang S, Tan Z, Pan Z, Huang P, Ge M. ISG15 and ISGylation modulates cancer stem cell-like characteristics in promoting tumor growth of anaplastic thyroid carcinoma. J Exp Clin Cancer Res 2023; 42:182. [PMID: 37501099 PMCID: PMC10373324 DOI: 10.1186/s13046-023-02751-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2023] [Accepted: 07/06/2023] [Indexed: 07/29/2023] Open
Abstract
BACKGROUND Anaplastic thyroid carcinoma (ATC) was a rare and extremely malignant endocrine cancer with the distinct hallmark of high proportion of cancer stem cell-like characteristics. Therapies aiming to cancer stem-like cells (CSCs) were emerging as a new direction in cancer treatment, but targeting ATC CSCs remained challenging, mainly due to incomplete insights of the regulatory mechanism of CSCs. Here, we unveiled a novel role of ISG15 in the modulation of ATC CSCs. METHODS The expression of ubiquitin-like proteins were detected by bioinformatics and immunohistochemistry. The correlation between ISG15 expression and tumor stem cells and malignant progression of ATC was analyzed by single-cell RNA sequence from the Gene Expression Omnibus. Flow cytometry combined with immunofluorescence were used to verify the enrichment of ISG15 and ISGyaltion in cancer stem cells. The effect and mechanism of ISG15 and KPNA2 on cancer stem cell-like characteristics of ATC cells were determined by molecular biology experiments. Mass spectrometry combined with immunoprecipitation to screen the substrates of ISG15 and validate its ISGylation modification. Nude mice and zebrafish xenograft models were utilized to demonstrate that ISG15 regulates stem cell characteristics and promotes malignant progression of ATC. RESULTS We found that among several ubiquitin proteins, only ISG15 was aberrantly expressed in ATC and enriched in CSCs. Single-cell sequencing analysis revealed that abnormal expression of ISG15 were intensely associated with stemness and malignant cells in ATC. Inhibition of ISG15 expression dramatically attenuated clone and sphere formation of ATC cells, and facilitated its sensitivity to doxorubicin. Notably, overexpression of ISGylation, but not the non-ISGylation mutant, effectively reinforced cancer stem cell-like characteristics. Mechanistically, ISG15 mediated the ISGylation of KPNA2 and impeded its ubiquitination to promote stability, further maintaining cancer stem cell-like characteristics. Finally, depletion of ISG15 inhibited ATC growth and metastasis in xenografted mouse and zebrafish models. CONCLUSION Our studies not only provided new insights into potential intervention strategies targeting ATC CSCs, but also uncovered the novel biological functions and mechanisms of ISG15 and ISGylation for maintaining ATC cancer stem cell-like characteristics.
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Affiliation(s)
- Tong Xu
- Center for Clinical Pharmacy, Cancer Center, Department of Pharmacy, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China
| | - Chaozhuang Zhu
- Zhejiang University of Technology, Hangzhou, Zhejiang, China
| | - Jinming Chen
- Hangzhou Medical College, Hangzhou, Zhejiang, China
| | - Feifeng Song
- Center for Clinical Pharmacy, Cancer Center, Department of Pharmacy, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China
| | - Xinxin Ren
- Center for Clinical Pharmacy, Cancer Center, Department of Pharmacy, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China
| | - Shanshan Wang
- Center for Clinical Pharmacy, Cancer Center, Department of Pharmacy, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China
| | - Xiaofen Yi
- Center for Clinical Pharmacy, Cancer Center, Department of Pharmacy, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China
| | - Yiwen Zhang
- Center for Clinical Pharmacy, Cancer Center, Department of Pharmacy, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China
| | - Wanli Zhang
- Zhejiang University of Technology, Hangzhou, Zhejiang, China
| | - Qing Hu
- Center for Clinical Pharmacy, Cancer Center, Department of Pharmacy, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China
| | - Hui Qin
- Center for Clinical Pharmacy, Cancer Center, Department of Pharmacy, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China
| | - Yujia Liu
- Center for Clinical Pharmacy, Cancer Center, Department of Pharmacy, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China
| | - Song Zhang
- Center for Clinical Pharmacy, Cancer Center, Department of Pharmacy, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China
| | - Zhuo Tan
- Key Laboratory of Endocrine Gland Diseases of Zhejiang Province, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China
- Otolaryngology & Head and Neck Center, Cancer Center, Department of Head and Neck Surgery, Affiliated People's Hospital, Zhejiang Provincial People's Hospital, Hangzhou Medical College, Zhejiang, Hangzhou, China
| | - Zongfu Pan
- Center for Clinical Pharmacy, Cancer Center, Department of Pharmacy, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China.
- Key Laboratory of Endocrine Gland Diseases of Zhejiang Province, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China.
| | - Ping Huang
- Center for Clinical Pharmacy, Cancer Center, Department of Pharmacy, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China.
- Key Laboratory of Endocrine Gland Diseases of Zhejiang Province, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China.
| | - Minghua Ge
- Key Laboratory of Endocrine Gland Diseases of Zhejiang Province, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China.
- Otolaryngology & Head and Neck Center, Cancer Center, Department of Head and Neck Surgery, Affiliated People's Hospital, Zhejiang Provincial People's Hospital, Hangzhou Medical College, Zhejiang, Hangzhou, China.
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9
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Bermúdez-Méndez E, Angelino P, van Keulen L, van de Water S, Rockx B, Pijlman GP, Ciuffi A, Kortekaas J, Wichgers Schreur PJ. Transcriptomic Profiling Reveals Intense Host-Pathogen Dispute Compromising Homeostasis during Acute Rift Valley Fever Virus Infection. J Virol 2023; 97:e0041523. [PMID: 37306574 PMCID: PMC10308945 DOI: 10.1128/jvi.00415-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2023] [Accepted: 05/13/2023] [Indexed: 06/13/2023] Open
Abstract
Rift Valley fever virus (RVFV) (family Phenuiviridae) can cause severe disease, and outbreaks of this mosquito-borne pathogen pose a significant threat to public and animal health. Yet many molecular aspects of RVFV pathogenesis remain incompletely understood. Natural RVFV infections are acute, characterized by a rapid onset of peak viremia during the first days post-infection, followed by a rapid decline. Although in vitro studies identified a major role of interferon (IFN) responses in counteracting the infection, a comprehensive overview of the specific host factors that play a role in RVFV pathogenesis in vivo is still lacking. Here, the host in vivo transcriptional profiles in the liver and spleen tissues of lambs exposed to RVFV are studied using RNA sequencing (RNA-seq) technology. We validate that IFN-mediated pathways are robustly activated in response to infection. We also link the observed hepatocellular necrosis with severely compromised organ function, which is reflected as a marked downregulation of multiple metabolic enzymes essential for homeostasis. Furthermore, we associate the elevated basal expression of LRP1 in the liver with RVFV tissue tropism. Collectively, the results of this study deepen the knowledge of the in vivo host response during RVFV infection and reveal new insights into the gene regulation networks underlying pathogenesis in a natural host. IMPORTANCE Rift Valley fever virus (RVFV) is a mosquito-transmitted pathogen capable of causing severe disease in animals and humans. Outbreaks of RVFV pose a significant threat to public health and can result in substantial economic losses. Little is known about the molecular basis of RVFV pathogenesis in vivo, particularly in its natural hosts. We employed RNA-seq technology to investigate genome-wide host responses in the liver and spleen of lambs during acute RVFV infection. We show that RVFV infection drastically decreases the expression of metabolic enzymes, which impairs normal liver function. Moreover, we highlight that basal expression levels of the host factor LRP1 may be a determinant of RVFV tissue tropism. This study links the typical pathological phenotype induced by RVFV infection with tissue-specific gene expression profiles, thereby improving our understanding of RVFV pathogenesis.
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Affiliation(s)
- Erick Bermúdez-Méndez
- Department of Virology and Molecular Biology, Wageningen Bioveterinary Research, Lelystad, The Netherlands
- Laboratory of Virology, Wageningen University & Research, Wageningen, The Netherlands
- Institute of Microbiology, Lausanne University Hospital, Lausanne, Switzerland
- Institute of Microbiology, University of Lausanne, Lausanne, Switzerland
| | - Paolo Angelino
- Institute of Microbiology, Lausanne University Hospital, Lausanne, Switzerland
- Institute of Microbiology, University of Lausanne, Lausanne, Switzerland
- Bioinformatics Core Facility, Swiss Institute of Bioinformatics, Lausanne, Switzerland
| | - Lucien van Keulen
- Department of Bacteriology, Host-Pathogen Interaction and Diagnostics Development, Wageningen Bioveterinary Research, Lelystad, The Netherlands
| | - Sandra van de Water
- Department of Virology and Molecular Biology, Wageningen Bioveterinary Research, Lelystad, The Netherlands
| | - Barry Rockx
- Department of Virology and Molecular Biology, Wageningen Bioveterinary Research, Lelystad, The Netherlands
| | - Gorben P. Pijlman
- Laboratory of Virology, Wageningen University & Research, Wageningen, The Netherlands
| | - Angela Ciuffi
- Institute of Microbiology, Lausanne University Hospital, Lausanne, Switzerland
- Institute of Microbiology, University of Lausanne, Lausanne, Switzerland
| | - Jeroen Kortekaas
- Department of Virology and Molecular Biology, Wageningen Bioveterinary Research, Lelystad, The Netherlands
- Laboratory of Virology, Wageningen University & Research, Wageningen, The Netherlands
| | - Paul J. Wichgers Schreur
- Department of Virology and Molecular Biology, Wageningen Bioveterinary Research, Lelystad, The Netherlands
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10
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Mertowska P, Smolak K, Mertowski S, Grywalska E. Immunomodulatory Role of Interferons in Viral and Bacterial Infections. Int J Mol Sci 2023; 24:10115. [PMID: 37373262 DOI: 10.3390/ijms241210115] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2023] [Revised: 06/02/2023] [Accepted: 06/09/2023] [Indexed: 06/29/2023] Open
Abstract
Interferons are a group of immunomodulatory substances produced by the human immune system in response to the presence of pathogens, especially during viral and bacterial infections. Their remarkably diverse mechanisms of action help the immune system fight infections by activating hundreds of genes involved in signal transduction pathways. In this review, we focus on discussing the interplay between the IFN system and seven medically important and challenging viruses (herpes simplex virus (HSV), influenza, hepatitis C virus (HCV), lymphocytic choriomeningitis virus (LCMV), human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), and SARS-CoV coronavirus) to highlight the diversity of viral strategies. In addition, the available data also suggest that IFNs play an important role in the course of bacterial infections. Research is currently underway to identify and elucidate the exact role of specific genes and effector pathways in generating the antimicrobial response mediated by IFNs. Despite the numerous studies on the role of interferons in antimicrobial responses, many interdisciplinary studies are still needed to understand and optimize their use in personalized therapeutics.
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Affiliation(s)
- Paulina Mertowska
- Department of Experimental Immunology, Medical University of Lublin, 20-093 Lublin, Poland
| | - Konrad Smolak
- Department of Experimental Immunology, Medical University of Lublin, 20-093 Lublin, Poland
| | - Sebastian Mertowski
- Department of Experimental Immunology, Medical University of Lublin, 20-093 Lublin, Poland
| | - Ewelina Grywalska
- Department of Experimental Immunology, Medical University of Lublin, 20-093 Lublin, Poland
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11
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Ren J, Antony F, Rouse BT, Suryawanshi A. Role of Innate Interferon Responses at the Ocular Surface in Herpes Simplex Virus-1-Induced Herpetic Stromal Keratitis. Pathogens 2023; 12:437. [PMID: 36986359 PMCID: PMC10058014 DOI: 10.3390/pathogens12030437] [Citation(s) in RCA: 15] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2023] [Revised: 03/06/2023] [Accepted: 03/08/2023] [Indexed: 03/14/2023] Open
Abstract
Herpes simplex virus type 1 (HSV-1) is a highly successful pathogen that primarily infects epithelial cells of the orofacial mucosa. After initial lytic replication, HSV-1 enters sensory neurons and undergoes lifelong latency in the trigeminal ganglion (TG). Reactivation from latency occurs throughout the host's life and is more common in people with a compromised immune system. HSV-1 causes various diseases depending on the site of lytic HSV-1 replication. These include herpes labialis, herpetic stromal keratitis (HSK), meningitis, and herpes simplex encephalitis (HSE). HSK is an immunopathological condition and is usually the consequence of HSV-1 reactivation, anterograde transport to the corneal surface, lytic replication in the epithelial cells, and activation of the host's innate and adaptive immune responses in the cornea. HSV-1 is recognized by cell surface, endosomal, and cytoplasmic pattern recognition receptors (PRRs) and activates innate immune responses that include interferons (IFNs), chemokine and cytokine production, as well as the recruitment of inflammatory cells to the site of replication. In the cornea, HSV-1 replication promotes type I (IFN-α/β) and type III (IFN-λ) IFN production. This review summarizes our current understanding of HSV-1 recognition by PRRs and innate IFN-mediated antiviral immunity during HSV-1 infection of the cornea. We also discuss the immunopathogenesis of HSK, current HSK therapeutics and challenges, proposed experimental approaches, and benefits of promoting local IFN-λ responses.
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Affiliation(s)
- Jiayi Ren
- Department of Pathobiology, College of Veterinary Medicine, Auburn University, 240B Greene Hall, Auburn, AL 36849, USA
| | - Ferrin Antony
- Department of Pathobiology, College of Veterinary Medicine, Auburn University, 240B Greene Hall, Auburn, AL 36849, USA
| | - Barry T. Rouse
- College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA
| | - Amol Suryawanshi
- Department of Pathobiology, College of Veterinary Medicine, Auburn University, 240B Greene Hall, Auburn, AL 36849, USA
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12
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Teimoori A, Mirshahabi H, Khansarinejad B, Soleimanjahi H, Karimi H, Rasti M, Shatizadeh Malekshahi S. Significant alteration of IFN stimulated genes expression in MA104 cells infected with bovine rotavirus RF strain. J Immunoassay Immunochem 2023; 44:56-65. [PMID: 36052996 DOI: 10.1080/15321819.2022.2118061] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
The pattern recognition receptors (PRRs) trigger signaling cascades, such as nuclear factor kappa B (NF-κB) and interferon regulatory factors (IRFs). Rotavirus (RV) countermeasures against innate responses and understanding of these processes will improve our knowledge regarding immunopathogenesis of RV infection. In this study, we investigated the effect of RV RF strain on the important ISG candidate genes engaging in virus infections for which little information is known in RV RF strain. To this end, MA104 cells were mock/infected with RF followed by incubation in the presence or absence of IFN-α and the expression of MX1, OAS1, STAT1, ISG15, and ISG56 mRNA was analyzed by real-time PCR. All of ISGs' mRNAs showed higher expression levels in IFN I treated cells compared to virus-infected cells except for ISG56. Infecting the cells with RV and treatment with IFN type I led to overexpression of ISG56 compared to cells were either infected with the virus or only treated with IFN I. In conclusion, we showed that the RV RF strain efficiently blocks type I IFN-induced gene expression particularly ISG15, MX1, STAT, and OSA1 as antiviral proteins. Furthermore, viruses may use some ISGs such as ISG 56 to regulate IFN I signaling pathway, negatively.
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Affiliation(s)
- Ali Teimoori
- Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.,Department of Microbiology, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran
| | - Hessam Mirshahabi
- Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.,Department of Microbiology and Virology, Zanjan University of Medical Sciences, Zanjan, Iran
| | - Behzad Khansarinejad
- Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.,Department of Microbiology and Immunology, Arak University of Medical Sciences, Arak, Iran
| | - Hoorieh Soleimanjahi
- Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
| | - Hesam Karimi
- Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
| | - Mojtaba Rasti
- Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
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13
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Munnur D, Banducci-Karp A, Sanyal S. ISG15 driven cellular responses to virus infection. Biochem Soc Trans 2022; 50:1837-1846. [PMID: 36416643 DOI: 10.1042/bst20220839] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2022] [Revised: 11/01/2022] [Accepted: 11/04/2022] [Indexed: 11/24/2022]
Abstract
One of the hallmarks of antiviral responses to infection is the production of interferons and subsequently of interferon stimulated genes. Interferon stimulated gene 15 (ISG15) is among the earliest and most abundant proteins induced upon interferon signalling, encompassing versatile functions in host immunity. ISG15 is a ubiquitin like modifier that can be conjugated to substrates in a process analogous to ubiquitylation and referred to as ISGylation. The free unconjugated form can either exist intracellularly or be secreted to function as a cytokine. Interestingly, ISG15 has been reported to be both advantageous and detrimental to the development of immunopathology during infection. This review describes recent findings on the role of ISG15 in antiviral responses in human infection models, with a particular emphasis on autophagy, inflammatory responses and cellular metabolism combined with viral strategies of counteracting them. The field of ISGylation has steadily gained momentum; however much of the previous studies of virus infections conducted in mouse models are in sharp contrast with recent findings in human cells, underscoring the need to summarise our current understanding of its potential antiviral function in humans and identify knowledge gaps which need to be addressed in future studies.
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Affiliation(s)
- Deeksha Munnur
- Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, U.K
| | - Adrianna Banducci-Karp
- Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, U.K
| | - Sumana Sanyal
- Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, U.K
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14
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Toro A, Lage-Vickers S, Bizzotto J, Vilicich F, Sabater A, Pascual G, Ledesma-Bazan S, Sanchis P, Ruiz MS, Arevalo AP, Porfido JL, Abbate M, Seniuk R, Labanca E, Anselmino N, Navone NM, Alonso DF, Vazquez E, Crispo M, Cotignola J, Gueron G. Pin-Pointing the Key Hubs in the IFN-γ Pathway Responding to SARS-CoV-2 Infection. Viruses 2022; 14:2180. [PMID: 36298734 PMCID: PMC9610092 DOI: 10.3390/v14102180] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2022] [Revised: 08/30/2022] [Accepted: 09/28/2022] [Indexed: 11/06/2022] Open
Abstract
Interferon gamma (IFN-γ) may be potential adjuvant immunotherapy for COVID-19 patients. In this work, we assessed gene expression profiles associated with the IFN-γ pathway in response to SARS-CoV-2 infection. Employing a case-control study from SARS-CoV-2-positive and -negative patients, we identified IFN-γ-associated pathways to be enriched in positive patients. Bioinformatics analyses showed upregulation of MAP2K6, CBL, RUNX3, STAT1, and JAK2 in COVID-19-positive vs. -negative patients. A positive correlation was observed between STAT1/JAK2, which varied alongside the patient's viral load. Expression of MX1, MX2, ISG15, and OAS1 (four well-known IFN-stimulated genes (ISGs)) displayed upregulation in COVID-19-positive vs. -negative patients. Integrative analyses showcased higher levels of ISGs, which were associated with increased viral load and STAT1/JAK2 expression. Confirmation of ISGs up-regulation was performed in vitro using the A549 lung cell line treated with Poly (I:C), a synthetic analog of viral double-stranded RNA; and in different pulmonary human cell lines and ferret tracheal biopsies infected with SARS-CoV-2. A pre-clinical murine model of Coronavirus infection confirmed findings displaying increased ISGs in the liver and lungs from infected mice. Altogether, these results demonstrate the role of IFN-γ and ISGs in response to SARS-CoV-2 infection, highlighting alternative druggable targets that can boost the host response.
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Affiliation(s)
- Ayelen Toro
- Laboratorio de Inflamación y Cáncer, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
- Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), CONICET—Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
| | - Sofia Lage-Vickers
- Laboratorio de Inflamación y Cáncer, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
- Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), CONICET—Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
| | - Juan Bizzotto
- Laboratorio de Inflamación y Cáncer, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
- Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), CONICET—Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
- Instituto de Tecnología (INTEC), Universidad Argentina de la Empresa (UADE), Buenos Aires C1073AAO, Argentina
| | - Felipe Vilicich
- Laboratorio de Inflamación y Cáncer, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
| | - Agustina Sabater
- Laboratorio de Inflamación y Cáncer, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
- Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), CONICET—Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
- Instituto de Tecnología (INTEC), Universidad Argentina de la Empresa (UADE), Buenos Aires C1073AAO, Argentina
| | - Gaston Pascual
- Laboratorio de Inflamación y Cáncer, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
- Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), CONICET—Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
| | - Sabrina Ledesma-Bazan
- Laboratorio de Inflamación y Cáncer, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
- Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), CONICET—Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
| | - Pablo Sanchis
- Laboratorio de Inflamación y Cáncer, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
- Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), CONICET—Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
| | - Maria Sol Ruiz
- Laboratorio de Inflamación y Cáncer, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
- Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), CONICET—Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
| | - Ana Paula Arevalo
- Laboratory Animals Biotechnology Unit, Institut Pasteur de Montevideo, Montevideo 11400, Uruguay
| | - Jorge L. Porfido
- Laboratory Animals Biotechnology Unit, Institut Pasteur de Montevideo, Montevideo 11400, Uruguay
| | - Mercedes Abbate
- Laboratorio de Inflamación y Cáncer, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
- Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), CONICET—Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
| | - Rocio Seniuk
- Laboratorio de Inflamación y Cáncer, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
- Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), CONICET—Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
| | - Estefania Labanca
- Department of Genitourinary Medical Oncology and The David H. Koch Center for Applied Research of Genitourinary Cancers, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Nicolas Anselmino
- Department of Genitourinary Medical Oncology and The David H. Koch Center for Applied Research of Genitourinary Cancers, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Nora M. Navone
- Department of Genitourinary Medical Oncology and The David H. Koch Center for Applied Research of Genitourinary Cancers, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Daniel F. Alonso
- Centro de Oncología Molecular y Traslacional y Plataforma de Servicios Biotecnológicos, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Bernal B1876BXD, Argentina
| | - Elba Vazquez
- Laboratorio de Inflamación y Cáncer, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
- Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), CONICET—Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
| | - Martina Crispo
- Laboratory Animals Biotechnology Unit, Institut Pasteur de Montevideo, Montevideo 11400, Uruguay
| | - Javier Cotignola
- Laboratorio de Inflamación y Cáncer, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
- Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), CONICET—Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
| | - Geraldine Gueron
- Laboratorio de Inflamación y Cáncer, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
- Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), CONICET—Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina
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15
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Interferon-Stimulated Gene 15 Knockout in Mice Impairs IFNα-Mediated Antiviral Activity. Viruses 2022; 14:v14091862. [PMID: 36146669 PMCID: PMC9502845 DOI: 10.3390/v14091862] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2022] [Revised: 08/19/2022] [Accepted: 08/19/2022] [Indexed: 11/25/2022] Open
Abstract
Type I interferon (IFN) plays an important role in the host defense against viral infection by inducing expression of interferon-stimulated genes (ISGs). In a previous study, we found that porcine interferon-stimulated gene 15 (ISG15) exhibited antiviral activity against PRV in vitro. To further investigate the antiviral function of ISG15 in vivo, we utilized ISG15 knockout (ISG15-/-) mice in this study. Here, we demonstrate that ISG15-/- mice were highly susceptible to PRV infection in vivo, as evidenced by a considerably reduced survival rate, enhanced viral replication and severe pathological lesions. However, we observed no significant difference between female and male infected WT and ISG15-/- mice. Moreover, ISG15-/- mice displayed attenuated antiviral protection as a result of considerably reduced expression of IFNβ and relevant ISGs during PRV replication. Furthermore, excessive production of proinflammatory cytokines may be closely related to encephalitis and pneumonia. In further studies, we found that the enhanced sensitivity to PRV infection in ISG15-/- mice might be caused by reduced phosphorylation of STAT1 and STAT2, thereby inhibiting type I IFN-mediated antiviral activity. Based on these findings, we conclude that ISG15 is essential for host type I IFN-mediated antiviral response.
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16
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Al-Hourani K, Ramamurthy N, Marchi E, Eichinger R, Li L, Fabris P, Drakesmith AH, Klenerman P. Innate triggering and antiviral effector functions of Activin A. Wellcome Open Res 2022. [DOI: 10.12688/wellcomeopenres.17237.1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Background: First-line defence against viral infection is contingent upon rapid detection of conserved viral structural and genomic motifs by pattern recognition receptors, followed by activation of the type I IFN response and establishment of an antiviral state. Novel antiviral functions of bone morphogenetic protein and related activin cytokines, acting in conjunction with, and independently of, type I IFN, have recently been described. How these antiviral effects are mediated and triggered by viral infection has not been defined. Methods: Microarray and RNAseq data from hepatoma-derived cell lines stimulated with Activin A in vitro were interrogated both by pathway analysis and for evidence of IFN-stimulated gene induction. Liver tissue obtained from patients with chronic HCV were examined by real-time quantitative polymerase chain reaction (RT-qPCR) for evidence of Activin A induction. Activin expression by peripheral blood mononuclear cells exposed to nucleic acid analogues was quantified by RT-qCR, whereas induction dynamics in acute infection was investigated in in vitro Sendai virus infection and a murine influenza A. Results: Transcriptomic analyses delineated strikingly congruent patterns of gene regulation in hepatocytes stimulated with recombinant Activin A and IFNα in vitro. Activin A mRNA, encoded by INHBA, is induced upon activation of RIG-I, MDA5 and TLR7/8 viral nucleic acid sensors in vitro, across multiple cell lines and in human peripheral blood mononuclear cells. In vivo, imurine influenza A also upregulated Inhba mRNA in the lung; this local upregulation of Inhba is retained in MAVS knockout mice, indicating roles for non-RIG-I-like receptors in its induction. Activin induction and signalling were also detectable in patients with chronic viral hepatitis. Conclusions: These data suggest Activin A is triggered in parallel with type I IFN responses and can trigger related antiviral effector functions, with implications for the development of targeted antiviral therapies and revealing novel facets of Activin biology.
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Zhao X, Wang J, Wang Y, Zhang M, Zhao W, Zhang H, Zhao L. Interferon‑stimulated gene 15 promotes progression of endometrial carcinoma and weakens antitumor immune response. Oncol Rep 2022; 47:110. [PMID: 35445736 PMCID: PMC9073416 DOI: 10.3892/or.2022.8321] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2021] [Accepted: 12/14/2021] [Indexed: 11/24/2022] Open
Abstract
Endometrial carcinoma (EC) is one of the most common gynecological cancers with a poor prognosis. Therefore, clarifying the details of the molecular mechanisms is of great importance for EC diagnosis and clinical management. Interferon-stimulated gene 15 (ISG15) plays an important role in the development of various cancers. However, its role in EC remains unclear. High ISG15 expression was observed in EC, which was associated with poor clinical outcomes and pathological stage of patients with EC, thus representing a promising marker for EC progression. Further exploratory analysis revealed that the elevated ISG15 levels in EC were driven by aberrant DNA methylation, independent of copy number variation and specific transcription factor aberrations. Accordingly, knockdown of ISG15 by small interfering RNA attenuated the malignant cellular phenotype of EC cell lines, including proliferation and colony formation in vitro. Finally, investigation of the molecular mechanisms indicated that ISG15 promoted the cell cycle G1/S transition in EC. Furthermore, ISG15 promoted EC progression by activating the MYC proto-oncogene protein signaling pathway. Moreover, ECs with high levels of ISG15 harbored a more vital immune escape ability, evidenced not only by significantly less invasive CD8+ T cells, but also higher expression of T cell inhibitory factors, such as programmed death-ligand 1. These results suggest a tumor-promoting role of ISG15 in EC, which may be a promising marker for diagnosis, prognosis and therapeutic immunity.
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Affiliation(s)
- Xiwa Zhao
- Department of Obstetrics and Gynecology, The Fourth Hospital, Hebei Medical University, Shijiazhuang, Hebei 050011, P.R. China
| | - Jingjing Wang
- The Research Center, The Fourth Hospital, Hebei Medical University, Shijiazhuang, Hebei 050011, P.R. China
| | - Yaojie Wang
- The Research Center, The Fourth Hospital, Hebei Medical University, Shijiazhuang, Hebei 050011, P.R. China
| | - Mengmeng Zhang
- Department of Obstetrics and Gynecology, The Fourth Hospital, Hebei Medical University, Shijiazhuang, Hebei 050011, P.R. China
| | - Wei Zhao
- Department of Obstetrics and Gynecology, The Fourth Hospital, Hebei Medical University, Shijiazhuang, Hebei 050011, P.R. China
| | - Hui Zhang
- Department of Obstetrics and Gynecology, The Fourth Hospital, Hebei Medical University, Shijiazhuang, Hebei 050011, P.R. China
| | - Lianmei Zhao
- The Research Center, The Fourth Hospital, Hebei Medical University, Shijiazhuang, Hebei 050011, P.R. China
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18
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Xu X, Liu L, Feng J, Li X, Zhang J. Comparative transcriptome analysis reveals potential anti-viral immune pathways of turbot (Scophthalmus maximus) subverted by megalocytivirus RBIV-C1 for immune evasion. FISH & SHELLFISH IMMUNOLOGY 2022; 122:153-161. [PMID: 35150827 DOI: 10.1016/j.fsi.2022.02.005] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/24/2021] [Revised: 01/15/2022] [Accepted: 02/02/2022] [Indexed: 06/14/2023]
Abstract
Successful viral infection and multiplication chiefly rely on virus subversion mechanisms against host anti-viral immune responses. In this study, in order to reveal the anti-viral immune-related pathways suppressed by megalocytivirus infection, transcriptome analysis was performed on the head-kidney of turbot (Scophthalmus maximus) infected with lethal dose of RBIV-C1 at 3, 6 and 9 days post challenge (dpc). The results showed that, compared to unchallenged groups, 190, 1220, and 3963 DEGs were detected in RBIV-C1 infected groups at 3, 6 and 9 dpc, respectively, of which, DEGs of complement components and pattern recognition proteins were up-regulated at 3 dpc and down-regulated at 6 and 9 dpc, DEGs of cytokines were up-regulated at 6 dpc and down-regulated at 9 dpc. Expression trend analysis revealed that DEGs of profiles 9 and 13 featured decreased expression patterns and were significantly enriched into 10 immune-related pathways, i.e., complement and coagulation cascades, cytokine-cytokine receptor interaction, chemokine signaling pathway, B/T cell receptor signaling pathway, antigen processing and presentation, and so on. Further co-expression network analysis (WGCNA) revealed positive correlated innate immune related pathways at 3 and 6 dpc, and negative correlated innate and adaptive immune related pathways at 9 dpc. This study revealed a set of anti-viral immune genes/pathways that would also be potential targets subverted by RBIV-C1 for immune evasion, which can serve as a valuable resource for future studies on the molecular mechanisms of anti-viral immune defense of turbot and immune escape of megalocytivirus.
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Affiliation(s)
- Xiudan Xu
- School of Ocean, Yantai University, 30 Qingquan Road, Yantai, 264005, China
| | - Ling Liu
- School of Ocean, Yantai University, 30 Qingquan Road, Yantai, 264005, China
| | - Jixing Feng
- School of Ocean, Yantai University, 30 Qingquan Road, Yantai, 264005, China
| | - Xuepeng Li
- School of Ocean, Yantai University, 30 Qingquan Road, Yantai, 264005, China
| | - Jian Zhang
- School of Ocean, Yantai University, 30 Qingquan Road, Yantai, 264005, China.
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19
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Dai X, Zhang T, Hua D. Ubiquitination and SUMOylation: protein homeostasis control over cancer. Epigenomics 2021; 14:43-58. [PMID: 34875856 DOI: 10.2217/epi-2021-0371] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Ubiquitination and SUMOylation are two essential components of the ubiquitination proteasome system playing fundamental roles in protein homeostasis maintenance and signal transduction, perturbation of which is associated with tumorigenesis. By comparing the mechanisms of ubiquitination and SUMOylation, assessing their crosstalk, reviewing their differential associations with cancer and identifying unaddressed yet important questions that may lead the field trend, this review sheds light on the similarities and differences of ubiquitination and SUMOylation toward the improved harnessing of both post-translational modification machineries, as well as forecasts novel onco-therapeutic opportunities through cell homeostasis control.
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Affiliation(s)
- Xiaofeng Dai
- Wuxi School of Medicine, Jiangnan University, Wuxi, 214122,China
| | - Tongxin Zhang
- Wuxi School of Medicine, Jiangnan University, Wuxi, 214122,China
| | - Dong Hua
- Wuxi School of Medicine, Jiangnan University, Wuxi, 214122,China.,Wuxi People's Hospital, Wuxi, 214023, China.,Affiliated Hospital of Jiangnan University, Wuxi, 214122, China
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20
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Hsieh YY, Tung SY, Pan HY, Chang TS, Wei KL, Chen WM, Deng YF, Lu CK, Lai YH, Wu CS, Li C. Fusobacterium nucleatum colonization is associated with decreased survival of helicobacter pylori-positive gastric cancer patients. World J Gastroenterol 2021; 27:7311-7323. [PMID: 34876791 PMCID: PMC8611209 DOI: 10.3748/wjg.v27.i42.7311] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/11/2021] [Revised: 06/10/2021] [Accepted: 09/16/2021] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND An increased amount of Fusobacterium nucleatum (F. nucleatum) is frequently detected in the gastric cancer-associated microbiota of the Taiwanese population. F. nucleatum is known to exert cytotoxic effects and play a role in the progression of colorectal cancer, though the impact of F. nucleatum colonization on gastric cancer cells and patient prognosis has not yet been examined.
AIM To identify F. nucleatum-dependent molecular pathways in gastric cancer cells and to determine the impact of F. nucleatum on survival in gastric cancer.
METHODS Coculture of F. nucleatum with a gastric cancer cell line was performed, and changes in gene expression were investigated. Genes with significant changes in expression were identified by RNA sequencing. Pathway analysis was carried out to determine deregulated cellular functions. A cohort of gastric cancer patients undergoing gastrectomy was recruited, and nested polymerase chain reaction was performed to detect the presence of F. nucleatum in resected cancer tissues. Statistical analysis was performed to determine whether F. nucleatum colonization affects patient survival.
RESULTS RNA sequencing and subsequent pathway analysis revealed a drastic interferon response induced by a high colonization load. This response peaked within 24 h and subsided after 72 h of incubation. In contrast, deregulation of actin and its regulators was observed during prolonged incubation under a low colonization load, likely altering the mobility of gastric cancer cells. According to the clinical specimen analysis, approximately one-third of the gastric cancer patients were positive for F. nucleatum, and statistical analysis indicated that the risk for colonization increases in late-stage cancer patients. Survival analysis demonstrated that F. nucleatum colonization was associated with poorer outcomes among patients also positive for Helicobacter pylori (H. pylori).
CONCLUSION F. nucleatum colonization leads to deregulation of actin dynamics and likely changes cancer cell mobility. Cohort analysis demonstrated that F. nucleatum colonization leads to poorer prognosis in H. pylori-positive patients with late-stage gastric cancer. Hence, combined colonization of F. nucleatum and H. pylori is a predictive biomarker for poorer survival in late-stage gastric cancer patients treated with gastrectomy.
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Affiliation(s)
- Yung-Yu Hsieh
- Department of Gastroenterology and Hepatology, Chiayi Chang Gung Memorial Hospital, Chiayi 61301, Taiwan
- College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
| | - Shui-Yi Tung
- Department of Gastroenterology and Hepatology, Chiayi Chang Gung Memorial Hospital, Chiayi 61301, Taiwan
- College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
| | - Hung-Yu Pan
- Department of Applied Mathematics, National Chiayi University, Chiayi 60035, Taiwan
| | - Te-Sheng Chang
- Department of Gastroenterology and Hepatology, Chiayi Chang Gung Memorial Hospital, Chiayi 61301, Taiwan
- College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
| | - Kuo-Liang Wei
- Department of Gastroenterology and Hepatology, Chiayi Chang Gung Memorial Hospital, Chiayi 61301, Taiwan
- College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
| | - Wei-Ming Chen
- Department of Gastroenterology and Hepatology, Chiayi Chang Gung Memorial Hospital, Chiayi 61301, Taiwan
- College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
| | - Yi-Fang Deng
- Department of Gastroenterology and Hepatology, Chiayi Chang Gung Memorial Hospital, Chiayi 61301, Taiwan
| | - Chung-Kuang Lu
- Department of Gastroenterology and Hepatology, Chiayi Chang Gung Memorial Hospital, Chiayi 61301, Taiwan
| | - Yu-Hsuan Lai
- Department of Gastroenterology and Hepatology, Chiayi Chang Gung Memorial Hospital, Chiayi 61301, Taiwan
| | - Cheng-Shyong Wu
- Department of Gastroenterology and Hepatology, Chiayi Chang Gung Memorial Hospital, Chiayi 61301, Taiwan
- College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
| | - Chin Li
- Department of Biomedical Sciences, National Chung Cheng University, Chiayi 62130, Taiwan
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21
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Loiola RA, García-Gabilondo M, Grayston A, Bugno P, Kowalska A, Duban-Deweer S, Rizzi E, Hachani J, Sano Y, Shimizu F, Kanda T, Mysiorek C, Mazurek MP, Rosell A, Gosselet F. Secretome of endothelial progenitor cells from stroke patients promotes endothelial barrier tightness and protects against hypoxia-induced vascular leakage. Stem Cell Res Ther 2021; 12:552. [PMID: 34702368 PMCID: PMC8549346 DOI: 10.1186/s13287-021-02608-y] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2021] [Accepted: 09/25/2021] [Indexed: 01/04/2023] Open
Abstract
BACKGROUND Cell-based therapeutic strategies have been proposed as an alternative for brain repair after stroke, but their clinical application has been hampered by potential adverse effects in the long term. The present study was designed to test the effect of the secretome of endothelial progenitor cells (EPCs) from stroke patients (scCM) on in vitro human models of angiogenesis and vascular barrier. METHODS Two different scCM batches were analysed by mass spectrometry and a proteome profiler. Human primary CD34+-derived endothelial cells (CD34+-ECs) were used for designing angiogenesis studies (proliferation, migration, and tubulogenesis) or in vitro models of EC monolayer (confluent monolayer ECs-CMECs) and blood-brain barrier (BBB; brain-like ECs-BLECs). Cells were treated with scCM (5 μg/mL) or protein-free endothelial basal medium (scEBM-control). CMECs or BLECs were exposed (6 h) to oxygen-glucose deprivation (OGD) conditions (1% oxygen and glucose-free medium) or normoxia (control-5% oxygen, 1 g/L of glucose) and treated with scCM or scEBM during reoxygenation (24 h). RESULTS The analysis of different scCM batches showed a good reproducibility in terms of protein yield and composition. scCM increased CD34+-EC proliferation, tubulogenesis, and migration compared to the control (scEBM). The proteomic analysis of scCM revealed the presence of growth factors and molecules modulating cell metabolism and inflammatory pathways. Further, scCM decreased the permeability of CMECs and upregulated the expression of the junctional proteins such as occludin, VE-cadherin, and ZO-1. Such effects were possibly mediated through the activation of the interferon pathway and a moderate downregulation of Wnt signalling. Furthermore, OGD increased the permeability of both CMECs and BLECs, while scCM prevented the OGD-induced vascular leakage in both models. These effects were possibly mediated through the upregulation of junctional proteins and the regulation of MAPK/VEGFR2 activity. CONCLUSION Our results suggest that scCM promotes angiogenesis and the maturation of newly formed vessels while restoring the BBB function in ischemic conditions. In conclusion, our results highlight the possibility of using EPC-secretome as a therapeutic alternative to promote brain angiogenesis and protect from ischemia-induced vascular leakage.
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Affiliation(s)
| | - Miguel García-Gabilondo
- Neurovascular Research Laboratory, Vall d'Hebron Institut de Recerca, Universitat Autònoma de Barcelona, 08035, Barcelona, Catalonia, Spain
| | - Alba Grayston
- Neurovascular Research Laboratory, Vall d'Hebron Institut de Recerca, Universitat Autònoma de Barcelona, 08035, Barcelona, Catalonia, Spain
| | - Paulina Bugno
- Pure Biologics S.A., Duńska 11, 54-427, Wroclaw, Poland
| | | | - Sophie Duban-Deweer
- UR 2465, Blood-Brain Barrier Laboratory (LBHE), Univ. Artois, 62300, Lens, France
| | - Eleonora Rizzi
- UR 2465, Blood-Brain Barrier Laboratory (LBHE), Univ. Artois, 62300, Lens, France
| | - Johan Hachani
- UR 2465, Blood-Brain Barrier Laboratory (LBHE), Univ. Artois, 62300, Lens, France
| | - Yasuteru Sano
- Department of Neurology and Clinical Neuroscience, Graduate School of Medicine, Yamaguchi University, Ube, Japan
| | - Fumitaka Shimizu
- Department of Neurology and Clinical Neuroscience, Graduate School of Medicine, Yamaguchi University, Ube, Japan
| | - Takashi Kanda
- Department of Neurology and Clinical Neuroscience, Graduate School of Medicine, Yamaguchi University, Ube, Japan
| | - Caroline Mysiorek
- UR 2465, Blood-Brain Barrier Laboratory (LBHE), Univ. Artois, 62300, Lens, France
| | | | - Anna Rosell
- Neurovascular Research Laboratory, Vall d'Hebron Institut de Recerca, Universitat Autònoma de Barcelona, 08035, Barcelona, Catalonia, Spain
| | - Fabien Gosselet
- UR 2465, Blood-Brain Barrier Laboratory (LBHE), Univ. Artois, 62300, Lens, France.
- Laboratory of the Blood-Brain Barrier, Sciences Faculty Jean Perrin, Artois University, Lens, France.
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22
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Wani SA, Sahu AR, Khan RIN, Praharaj MR, Saxena S, Rajak KK, Muthuchelvan D, Sahoo A, Mishra B, Singh RK, Mishra BP, Gandham RK. Proteome Modulation in Peripheral Blood Mononuclear Cells of Peste des Petits Ruminants Vaccinated Goats and Sheep. Front Vet Sci 2021; 8:670968. [PMID: 34631844 PMCID: PMC8493254 DOI: 10.3389/fvets.2021.670968] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2021] [Accepted: 05/31/2021] [Indexed: 12/03/2022] Open
Abstract
In the present study, healthy goats and sheep (n = 5) that were confirmed negative for peste des petits ruminants virus (PPRV) antibodies by monoclonal antibody-based competitive ELISA and by serum neutralization test and for PPRV antigen by s-ELISA were vaccinated with Sungri/96. A quantitative study was carried out to compare the proteome of peripheral blood mononuclear cells (PBMCs) of vaccinated goat and sheep [5 days post-vaccination (dpv) and 14 dpv] vs. unvaccinated (0 day) to divulge the alteration in protein expression following vaccination. A total of 232 and 915 proteins were differentially expressed at 5 and 14 dpv, respectively, in goats. Similarly, 167 and 207 proteins were differentially expressed at 5 and 14 dpv, respectively, in sheep. Network generated by Ingenuity Pathway Analysis was “infectious diseases, antimicrobial response, and inflammatory response,” which includes the highest number of focus molecules. The bio functions, cell-mediated immune response, and humoral immune response were highly enriched in goats at 5 dpv and at 14 dpv. At the molecular level, the immune response produced by the PPRV vaccine virus in goats is effectively coordinated and stronger than that in sheep, though the vaccine provides protection from virulent virus challenge in both. The altered expression of certain PBMC proteins especially ISG15 and IRF7 induces marked changes in cellular signaling pathways to coordinate host immune responses.
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Affiliation(s)
- Sajad Ahmad Wani
- Division of Veterinary Biotechnology, Indian Council of Agricultural Research - Indian Veterinary Research Institute, Bareilly, India.,College of Pharmacy, Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University, Columbus, OH, United States
| | - Amit Ranjan Sahu
- Division of Veterinary Biotechnology, Indian Council of Agricultural Research - Indian Veterinary Research Institute, Bareilly, India
| | - Raja Ishaq Nabi Khan
- Division of Veterinary Biotechnology, Indian Council of Agricultural Research - Indian Veterinary Research Institute, Bareilly, India
| | - Manas Ranjan Praharaj
- Systems Biology Lab, Department of Biotechnology -National Institute of Animal Biotechnology, Hyderabad, India
| | - Shikha Saxena
- Division of Veterinary Biotechnology, Indian Council of Agricultural Research - Indian Veterinary Research Institute, Bareilly, India
| | - Kaushal Kishor Rajak
- Division of Biological Products, Indian Council of Agricultural Research - Indian Veterinary Research Institute, Bareilly, India
| | - Dhanavelu Muthuchelvan
- Division of Virology, Indian Council of Agricultural Research - Indian Veterinary Research Institute, Mukteswar, India
| | - Aditya Sahoo
- Division of Veterinary Biotechnology, Indian Council of Agricultural Research - Indian Veterinary Research Institute, Bareilly, India
| | - Bina Mishra
- Division of Biological Products, Indian Council of Agricultural Research - Indian Veterinary Research Institute, Bareilly, India
| | - R K Singh
- Division of Veterinary Biotechnology, Indian Council of Agricultural Research - Indian Veterinary Research Institute, Bareilly, India
| | - Bishnu Prasad Mishra
- Division of Veterinary Biotechnology, Indian Council of Agricultural Research - Indian Veterinary Research Institute, Bareilly, India
| | - Ravi Kumar Gandham
- Division of Veterinary Biotechnology, Indian Council of Agricultural Research - Indian Veterinary Research Institute, Bareilly, India.,Systems Biology Lab, Department of Biotechnology -National Institute of Animal Biotechnology, Hyderabad, India
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23
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He W, Li C, Dong L, Yang G, Liu H. Tandem Mass Tag-Based Quantitative Proteomic Analysis of ISG15 Knockout PK15 Cells in Pseudorabies Virus Infection. Genes (Basel) 2021; 12:genes12101557. [PMID: 34680952 PMCID: PMC8535405 DOI: 10.3390/genes12101557] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2021] [Revised: 09/14/2021] [Accepted: 09/28/2021] [Indexed: 12/24/2022] Open
Abstract
Pseudorabies virus (PRV) is recognized as one of the most important pathogens of swine and poses a serious threat to the swine industry worldwide. Available commercial vaccines fail to protect against the emergence of new PRV strains. Therefore, the new protein targets against PRV highlight the urgent need for uncovering the molecular determinants of host cellular proteins following PRV infection. Interferon-stimulated gene 15 (ISG15) demonstrates an outstanding antiviral response. However, the molecular mechanism of ISG15 that affects PRV replication is incompletely known. Here, we performed a tandem mass tag (TMT)-based approach to quantitatively identify protein expression changes in PRV-infected ISG15 knockout PK15 (ISG15−/−-PK15) cells. In total, 4958 proteins were identified by using TMT coupled with LC-MS/MS in this study. In the PRV- and mock-infected groups, 241 differentially expressed proteins (DEPs) were identified, 162 upregulated and 79 downregulated proteins at 24 h post-infection (hpi), among which AFP, Vtn, Hsp40, Herc5, and Mccc1 may play important roles in PRV propagation. To ensure the validity and reliability of the proteomics data, the randomly selected DEPs were verified by RT-qPCR and Western blot analysis, and the results were consistent with the TMT results. Bioinformatics analyses further demonstrated that the DEPs are mainly involved in various biological processes and signaling pathways, such as signal transduction, the digestive system, and the PI3K-AKT pathway. These findings may provide new insight into molecular mechanisms for PRV infection, which is helpful for identifying potential protein targets for antiviral agents.
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24
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Mathieu NA, Paparisto E, Barr SD, Spratt DE. HERC5 and the ISGylation Pathway: Critical Modulators of the Antiviral Immune Response. Viruses 2021; 13:1102. [PMID: 34207696 PMCID: PMC8228270 DOI: 10.3390/v13061102] [Citation(s) in RCA: 39] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2021] [Revised: 06/02/2021] [Accepted: 06/04/2021] [Indexed: 01/10/2023] Open
Abstract
Mammalian cells have developed an elaborate network of immunoproteins that serve to identify and combat viral pathogens. Interferon-stimulated gene 15 (ISG15) is a 15.2 kDa tandem ubiquitin-like protein (UBL) that is used by specific E1-E2-E3 ubiquitin cascade enzymes to interfere with the activity of viral proteins. Recent biochemical studies have demonstrated how the E3 ligase HECT and RCC1-containing protein 5 (HERC5) regulates ISG15 signaling in response to hepatitis C (HCV), influenza-A (IAV), human immunodeficiency virus (HIV), SARS-CoV-2 and other viral infections. Taken together, the potent antiviral activity displayed by HERC5 and ISG15 make them promising drug targets for the development of novel antiviral therapeutics that can augment the host antiviral response. In this review, we examine the emerging role of ISG15 in antiviral immunity with a particular focus on how HERC5 orchestrates the specific and timely ISGylation of viral proteins in response to infection.
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Affiliation(s)
- Nicholas A. Mathieu
- Gustaf H. Carlson School of Chemistry and Biochemistry, Clark University, 950 Main St., Worcester, MA 01610, USA;
| | - Ermela Paparisto
- Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, Western University, 1151 Richmond St., London, ON N6A 5C1, Canada; (E.P.); (S.D.B.)
| | - Stephen D. Barr
- Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, Western University, 1151 Richmond St., London, ON N6A 5C1, Canada; (E.P.); (S.D.B.)
| | - Donald E. Spratt
- Gustaf H. Carlson School of Chemistry and Biochemistry, Clark University, 950 Main St., Worcester, MA 01610, USA;
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25
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Occhigrossi L, Rossin F, D'Eletto M, Farrace MG, Ciccosanti F, Petrone L, Sacchi A, Nardacci R, Falasca L, Del Nonno F, Palucci I, Smirnov E, Barlev N, Agrati C, Goletti D, Delogu G, Fimia GM, Piacentini M. Transglutaminase 2 Regulates Innate Immunity by Modulating the STING/TBK1/IRF3 Axis. THE JOURNAL OF IMMUNOLOGY 2021; 206:2420-2429. [PMID: 33941660 DOI: 10.4049/jimmunol.2001122] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/02/2020] [Accepted: 03/16/2021] [Indexed: 12/17/2022]
Abstract
We have recently shown that type 2 transglutaminase (TG2) plays a key role in the host's inflammatory response during bacterial infections. In this study, we investigated whether the enzyme is involved in the regulation of the STING pathway, which is the main signaling activated in the presence of both self- and pathogen DNA in the cytoplasm, leading to type I IFN (IFN I) production. In this study, we demonstrated that TG2 negatively regulates STING signaling by impairing IRF3 phosphorylation in bone marrow-derived macrophages, isolated from wild-type and TG2 knockout mice. In the absence of TG2, we found an increase in the IFN-β production and in the downstream JAK/STAT pathway activation. Interestingly, proteomic analysis revealed that TG2 interacts with TBK1, affecting its interactome composition. Indeed, TG2 ablation facilitates the TBK1-IRF3 interaction, thus indicating that the enzyme plays a negative regulatory effect on IRF3 recruitment in the STING/TBK1 complex. In keeping with these findings, we observed an increase in the IFNβ production in bronchoalveolar lavage fluids from COVID-19-positive dead patients paralleled by a dramatic decrease of the TG2 expression in the lung pneumocytes. Taken together, these results suggest that TG2 plays a negative regulation on the IFN-β production associated with the innate immunity response to the cytosolic presence of both self- and pathogen DNA.
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Affiliation(s)
- Luca Occhigrossi
- Department of Biology, University of Rome "Tor Vergata," Rome, Italy
| | - Federica Rossin
- Department of Biology, University of Rome "Tor Vergata," Rome, Italy
| | - Manuela D'Eletto
- Department of Biology, University of Rome "Tor Vergata," Rome, Italy
| | | | - Fabiola Ciccosanti
- National Institute for Infectious Diseases IRCCS "L. Spallanzani," Rome, Italy
| | - Linda Petrone
- National Institute for Infectious Diseases IRCCS "L. Spallanzani," Rome, Italy
| | - Alessandra Sacchi
- National Institute for Infectious Diseases IRCCS "L. Spallanzani," Rome, Italy
| | - Roberta Nardacci
- National Institute for Infectious Diseases IRCCS "L. Spallanzani," Rome, Italy
| | - Laura Falasca
- National Institute for Infectious Diseases IRCCS "L. Spallanzani," Rome, Italy
| | - Franca Del Nonno
- National Institute for Infectious Diseases IRCCS "L. Spallanzani," Rome, Italy
| | - Ivana Palucci
- Institute of Microbiology, Università Cattolica del Sacro Cuore - Fondazione Policlinico Gemelli, Rome, Italy
| | | | - Nick Barlev
- Institute of Cytology, Saint Petersburg, Russia
| | - Chiara Agrati
- National Institute for Infectious Diseases IRCCS "L. Spallanzani," Rome, Italy
| | - Delia Goletti
- National Institute for Infectious Diseases IRCCS "L. Spallanzani," Rome, Italy
| | - Giovanni Delogu
- Institute of Microbiology, Università Cattolica del Sacro Cuore - Fondazione Policlinico Gemelli, Rome, Italy
| | - Gian Maria Fimia
- National Institute for Infectious Diseases IRCCS "L. Spallanzani," Rome, Italy.,Department of Molecular Medicine, University of Rome "La Sapienza," Rome, Italy
| | - Mauro Piacentini
- National Institute for Infectious Diseases IRCCS "L. Spallanzani," Rome, Italy .,Institute of Cytology, Saint Petersburg, Russia
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26
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Investigating the effects of IDO1, PTGS2, and TGF-β1 overexpression on immunomodulatory properties of hTERT-MSCs and their extracellular vesicles. Sci Rep 2021; 11:7825. [PMID: 33837229 PMCID: PMC8035148 DOI: 10.1038/s41598-021-87153-7] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2020] [Accepted: 03/25/2021] [Indexed: 02/07/2023] Open
Abstract
The therapeutic potential of mesenchymal stem cells (MSCs) is out of the question. Yet, recent drawbacks have resulted in a strategic shift towards the application of MSC-derived cell-free products such as extracellular vesicles (EVs). Recent reports revealed that functional properties of MSCs, including EV secretion patterns, correlate with microenvironmental cues. These findings highlight the urgent need for defining the optimal circumstances for EV preparation. Considering the limitations of primary cells, we employed immortalized cells as an alternative source to prepare therapeutically sufficient EV numbers. Herein, the effects of different conditional environments are explored on human TERT-immortalized MSCs (hTERT-MSCs). The latter were transduced to overexpress IDO1, PTGS2, and TGF-β1 transgenes either alone or in combination, and their immunomodulatory properties were analyzed thereafter. Likewise, EVs derived from these various MSCs were extensively characterized. hTERT-MSCs-IDO1 exerted superior inhibitory effects on lymphocytes, significantly more than hTERT-MSCs-IFN-γ. As such, IDO1 overexpression promoted the immunomodulatory properties of such enriched EVs. Considering the limitations of cell therapy like tumor formation and possible immune responses in the host, the results presented herein might be considered as a feasible model for the induction of immunomodulation in off-the-shelf and cell-free therapeutics, especially for autoimmune diseases.
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27
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Abstract
Immune response is a highly coordinated cascade involving all the subsets of peripheral blood mononuclear cells (PBMCs). In this study, RNA sequencing (RNA-Seq) analysis of PBMC subsets was done to delineate the systems biology behind immune protection of the vaccine in sheep and goats. The PBMC subsets studied were CD4+, CD8+, CD14+, CD21+, and CD335+ cells from day 0 and day 5 of sheep and goats vaccinated with Sungri/96 peste des petits ruminants virus. Assessment of the immune response processes enriched by the differentially expressed genes (DEGs) in all the subsets suggested a strong dysregulation toward the development of early inflammatory microenvironment, which is very much required for differentiation of monocytes to macrophages, and activation as well as the migration of dendritic cells into the draining lymph nodes. The protein-protein interaction networks among the antiviral molecules (IFIT3, ISG15, MX1, MX2, RSAD2, ISG20, IFIT5, and IFIT1) and common DEGs across PBMC subsets in both species identified ISG15 to be a ubiquitous hub that helps in orchestrating antiviral host response against peste des petits ruminants virus (PPRV). IRF7 was found to be the key master regulator activated in most of the subsets in sheep and goats. Most of the pathways were found to be inactivated in B lymphocytes of both the species, indicating that 5 days postvaccination (dpv) is too early a time point for the B lymphocytes to react. The cell-mediated immune response and humoral immune response pathways were found more enriched in goats than in sheep. Although animals from both species survived the challenge, a contrast in pathway activation was observed in CD335+ cells. IMPORTANCE Peste des petits ruminants (PPR) by PPR virus (PPRV) is an World Organisation for Animal Health (OIE)-listed acute, contagious transboundary viral disease of small ruminants. The attenuated Sungri/96 PPRV vaccine used all over India against this PPR provides long-lasting robust innate and adaptive immune response. The early antiviral response was found mediated through type I interferon-independent interferon-stimulated gene (ISG) expression. However, systems biology behind this immune response is unknown. In this study, in vivo transcriptome profiling of PBMC subsets (CD4+, CD8+, CD14+, CD21+, and CD335+) in vaccinated goats and sheep (at 5 days postvaccination) was done to understand this systems biology. Though there are a few differences in the systems biology across cells (specially the NK cells) between sheep and goats, the coordinated response that is inclusive of all the cell subsets was found to be toward the induction of a strong innate immune response, which is needed for an appropriate adaptive immune response.
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Bussienne C, Marquet R, Paillart JC, Bernacchi S. Post-Translational Modifications of Retroviral HIV-1 Gag Precursors: An Overview of Their Biological Role. Int J Mol Sci 2021; 22:ijms22062871. [PMID: 33799890 PMCID: PMC8000049 DOI: 10.3390/ijms22062871] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2021] [Revised: 03/06/2021] [Accepted: 03/09/2021] [Indexed: 11/24/2022] Open
Abstract
Protein post-translational modifications (PTMs) play key roles in eukaryotes since they finely regulate numerous mechanisms used to diversify the protein functions and to modulate their signaling networks. Besides, these chemical modifications also take part in the viral hijacking of the host, and also contribute to the cellular response to viral infections. All domains of the human immunodeficiency virus type 1 (HIV-1) Gag precursor of 55-kDa (Pr55Gag), which is the central actor for viral RNA specific recruitment and genome packaging, are post-translationally modified. In this review, we summarize the current knowledge about HIV-1 Pr55Gag PTMs such as myristoylation, phosphorylation, ubiquitination, sumoylation, methylation, and ISGylation in order to figure out how these modifications affect the precursor functions and viral replication. Indeed, in HIV-1, PTMs regulate the precursor trafficking between cell compartments and its anchoring at the plasma membrane, where viral assembly occurs. Interestingly, PTMs also allow Pr55Gag to hijack the cell machinery to achieve viral budding as they drive recognition between viral proteins or cellular components such as the ESCRT machinery. Finally, we will describe and compare PTMs of several other retroviral Gag proteins to give a global overview of their role in the retroviral life cycle.
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Abstract
Post-translational modifications of cellular substrates with ubiquitin and ubiquitin-like proteins (UBLs), including ubiquitin, SUMOs, and neural precursor cell-expressed developmentally downregulated protein 8, play a central role in regulating many aspects of cell biology. The UBL conjugation cascade is initiated by a family of ATP-dependent enzymes termed E1 activating enzymes and executed by the downstream E2-conjugating enzymes and E3 ligases. Despite their druggability and their key position at the apex of the cascade, pharmacologic modulation of E1s with potent and selective drugs has remained elusive until 2009. Among the eight E1 enzymes identified so far, those initiating ubiquitylation (UBA1), SUMOylation (SAE), and neddylation (NAE) are the most characterized and are implicated in various aspects of cancer biology. To date, over 40 inhibitors have been reported to target UBA1, SAE, and NAE, including the NAE inhibitor pevonedistat, evaluated in more than 30 clinical trials. In this Review, we discuss E1 enzymes, the rationale for their therapeutic targeting in cancer, and their different inhibitors, with emphasis on the pharmacologic properties of adenosine sulfamates and their unique mechanism of action, termed substrate-assisted inhibition. Moreover, we highlight other less-characterized E1s-UBA6, UBA7, UBA4, UBA5, and autophagy-related protein 7-and the opportunities for targeting these enzymes in cancer. SIGNIFICANCE STATEMENT: The clinical successes of proteasome inhibitors in cancer therapy and the emerging resistance to these agents have prompted the exploration of other signaling nodes in the ubiquitin-proteasome system including E1 enzymes. Therefore, it is crucial to understand the biology of different E1 enzymes, their roles in cancer, and how to translate this knowledge into novel therapeutic strategies with potential implications in cancer treatment.
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Affiliation(s)
- Samir H Barghout
- Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada (S.H.B., A.D.S.); Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada (S.H.B., A.D.S.); and Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tanta University, Tanta, Egypt (S.H.B.)
| | - Aaron D Schimmer
- Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada (S.H.B., A.D.S.); Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada (S.H.B., A.D.S.); and Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tanta University, Tanta, Egypt (S.H.B.)
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Narovlyansky AN, Poloskov VV, Ivanova AM, Kravchenko SK, Babayeva FE, Sychevskaya KA, Mezentseva MV, Suetina IA, Russu LI, Izmest'eva AV, Ospelnikova TP, Sarymsakov AA, Ershov FI. [Interferon-regulating activity of the celagrip antiviral drug and its influence on formation of reactive oxygen species and expression of innate immunity genes in the follicular lymphoma patients]. Vopr Virusol 2020; 65:284-293. [PMID: 33533212 DOI: 10.36233/0507-4088-2020-65-5-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2020] [Accepted: 11/15/2020] [Indexed: 06/12/2023]
Abstract
INTRODUCTION Medicines from the group of interferon inducers (IFNs) "swith on" the synthesis of type 1 interferons (IFN-I) and induce the expression of IFN-stimulated genes (ISGs) that regulate innate immunity reactions and protect the host from infectious agents and the tumour pathology.The purpose of the study was to determine the role of the drug celagrip (CA) in the activation of innate immunity genes and the effect on the production of reactive oxygen species (ROS) in patients with follicular lymphoma (FL). OBJECTIVES to study the intensity of ROS production and the level of expression of the IFN-α2, IFN-λ1, ISG15, BCL2, P53(TP53) and USP18 genes in response to the treatment of blood cells of patients with FL with the preparation of CA. MATERIAL AND METHODS The study involved primary cancer patients diagnosed with follicular lymphoma (FL) and healthy volunteers. A kinetic analysis of the dynamics of production of reactive oxygen species (ROS) was performed in whose blood cells, and the expression of the group of genes was determined by real-time PCR in response to CA processing. RESULTS AND DISCUSSION ROS production by blood cells of patients with FL and volunteers in the presence of CA significantly decreased (P < 0.05). The level of gene expression of ISG15, P53(TR53) and USP 18 in the group of patients with FL was significantly higher than that in the group of volunteers. When treating blood cells with CA, it becomes possible to divide patients with FL into groups with a positive and negative response in accordance with the level of expression of the USP18 gene. We divided FL patients into groups with a positive and negative response in accordance with the level of USP18 gene expression after treatment of blood cells with CA. CONCLUSIONS The CA drug reduces the production of ROS and simultaneously stimulates the activity of the innate immunity genes ISG15, P53(TP53) and USP18 in the blood cells of patients with FL.
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Affiliation(s)
- A N Narovlyansky
- National Research Centre for Epidemiology and Microbiology named after the honorary academician N.F. Gamaleya
| | - V V Poloskov
- National Research Centre for Epidemiology and Microbiology named after the honorary academician N.F. Gamaleya
| | - A M Ivanova
- National Research Centre for Epidemiology and Microbiology named after the honorary academician N.F. Gamaleya
| | | | | | | | - M V Mezentseva
- National Research Centre for Epidemiology and Microbiology named after the honorary academician N.F. Gamaleya
| | - I A Suetina
- National Research Centre for Epidemiology and Microbiology named after the honorary academician N.F. Gamaleya
| | - L I Russu
- National Research Centre for Epidemiology and Microbiology named after the honorary academician N.F. Gamaleya
| | - A V Izmest'eva
- National Research Centre for Epidemiology and Microbiology named after the honorary academician N.F. Gamaleya
| | - T P Ospelnikova
- National Research Centre for Epidemiology and Microbiology named after the honorary academician N.F. Gamaleya
| | | | - F I Ershov
- National Research Centre for Epidemiology and Microbiology named after the honorary academician N.F. Gamaleya
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Zhao J, Zhu L, Xu L, Huang J, Sun X, Xu Z. Porcine interferon lambda 3 (IFN-λ3) shows potent anti-PRRSV activity in primary porcine alveolar macrophages (PAMs). BMC Vet Res 2020; 16:408. [PMID: 33115475 PMCID: PMC7594293 DOI: 10.1186/s12917-020-02627-6] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2019] [Accepted: 10/19/2020] [Indexed: 02/07/2023] Open
Abstract
Background Porcine reproductive and respiratory syndrome virus (PRRSV) is a serious viral disease of swine. At present, there are vaccines for the control of PRRSV infection, but the effect is not satisfactory. The recombination of attenuated vaccines causes significant difficulties with the prevention and control of PRRSV. Type III interferons (IFNs), also called IFN-λs, were newly identified and showed potent antiviral activity within the mucosal surface and immune organs. Results Therefore, primary porcine alveolar macrophages (PAMs) were used for this investigation. To this end, we found that the replication of PRRSV in PAMs was significantly reduced after pre-treatment with IFN-λ3, and such inhibition was dose- and time-dependent. The plaque formation of PRRSV abrogated entirely, and virus yields were reduced by four orders of magnitude when the primary PAMs were treated with IFN-λ3 at 1000 ng/ml. In addition, IFN-λ3 in our study was able to induce the expression of interferon-stimulated genes 15 (ISG15), 2′-5′-oligoadenylate synthase 1 (OAS1), IFN-inducible transmembrane 3 (IFITM3), and myxoma resistance protein 1(Mx1) in primary PAMs. Conclusions IFN-λ3 had antiviral activity against PRRSV and can stimulate the expression of pivotal interferon-stimulated genes (ISGs), i.e., ISG15, Mx1, OAS1, and IFITM3. So, IFN-λ3 may serve as a useful antiviral agent. Supplementary Information The online version contains supplementary material available at 10.1186/s12917-020-02627-6.
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Affiliation(s)
- Jun Zhao
- College of Veterinary Medicine, Sichuan Agricultural University, Cheng Du, Sichuan Province, China
| | - Ling Zhu
- College of Veterinary Medicine, Sichuan Agricultural University, Cheng Du, Sichuan Province, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Cheng Du, Sichuan Province, China
| | - Lei Xu
- College of Veterinary Medicine, Sichuan Agricultural University, Cheng Du, Sichuan Province, China
| | - Jianbo Huang
- College of Veterinary Medicine, Sichuan Agricultural University, Cheng Du, Sichuan Province, China
| | - Xiangang Sun
- College of Veterinary Medicine, Sichuan Agricultural University, Cheng Du, Sichuan Province, China
| | - Zhiwen Xu
- College of Veterinary Medicine, Sichuan Agricultural University, Cheng Du, Sichuan Province, China. .,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Cheng Du, Sichuan Province, China.
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Abstract
The intracellular protozoan parasite Toxoplasma gondii is capable of infecting most nucleated cells, where it survives in a specially modified compartment called the parasitophorous vacuole (PV). Interferon gamma (IFN-γ) is the major cytokine involved in activating cell-autonomous immune responses to inhibit parasite growth within this intracellular niche. In HeLa cells, IFN-γ treatment leads to ubiquitination of susceptible parasite strains, recruitment of the adaptors p62 and NDP52, and engulfment in microtubule-associated protein 1 light chain 3 (LC3)-positive membranes that restrict parasite growth. IFN-γ-mediated growth restriction depends on core members of the autophagy (ATG) pathway but not the initiation or degradative steps in the process. To explore the connection between these different pathways, we used permissive biotin ligation to identify proteins that interact with ATG5 in an IFN-γ-dependent fashion. Network analysis of the ATG5 interactome identified interferon-stimulated gene 15 (ISG15), which is highly upregulated by IFN treatment, as a hub connecting the ATG complex with other IFN-γ-induced genes, suggesting that it forms a functional link between the pathways. Deletion of ISG15 resulted in impaired recruitment of p62, NDP52, and LC3 to the PV and loss of IFN-γ-restricted parasite growth. The function of ISG15 required conjugation, and a number of ISGylated targets overlapped with the IFN-γ-dependent ATG5 interactome, including the adapter p62. Collectively, our findings establish a role for ISG15 in connecting the ATG pathway with IFN-γ-dependent restriction of T. gondii in human cells.IMPORTANCE Interferon(s) provide the primary defense against intracellular pathogens, a property ascribed to their ability to upregulate interferon-stimulated genes. Due to the sequestered niche occupied by Toxoplasma gondii, the host has elaborated intricate ways to target the parasite within its vacuole. One such mechanism is the recognition by a noncanonical autophagy pathway that envelops the parasite-containing vacuole and stunts growth in human cells. Remarkably, autophagy-dependent growth restriction requires interferon-γ, yet none of the classical components of autophagy are induced by interferon. Our studies draw a connection between these pathways by demonstrating that the antiviral protein ISG15, which is normally upregulated by interferons, links the autophagy-mediated control to ubiquitination of the vacuole. These findings suggest a similar link between interferon-γ signaling and autophagy that may underlie defense against other intracellular pathogens.
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Fieulaine S, Witte MD, Theile CS, Ayach M, Ploegh HL, Jupin I, Bressanelli S. Turnip yellow mosaic virus protease binds ubiquitin suboptimally to fine-tune its deubiquitinase activity. J Biol Chem 2020; 295:13769-13783. [PMID: 32732284 PMCID: PMC7535911 DOI: 10.1074/jbc.ra120.014628] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2020] [Revised: 07/27/2020] [Indexed: 11/21/2022] Open
Abstract
Single-stranded, positive-sense RNA viruses assemble their replication complexes in infected cells from a multidomain replication polyprotein. This polyprotein usually contains at least one protease, the primary function of which is to process the polyprotein into mature proteins. Such proteases also may have other functions in the replication cycle. For instance, cysteine proteases (PRO) frequently double up as ubiquitin hydrolases (DUB), thus interfering with cellular processes critical for virus replication. We previously reported the crystal structures of such a PRO/DUB from Turnip yellow mosaic virus (TYMV) and of its complex with one of its PRO substrates. Here we report the crystal structure of TYMV PRO/DUB in complex with ubiquitin. We find that PRO/DUB recognizes ubiquitin in an unorthodox way: It interacts with the body of ubiquitin through a split recognition motif engaging both the major and the secondary recognition patches of ubiquitin (Ile44 patch and Ile36 patch, respectively, including Leu8, which is part of the two patches). However, the contacts are suboptimal on both sides. Introducing a single-point mutation in TYMV PRO/DUB aimed at improving ubiquitin-binding led to a much more active DUB. Comparison with other PRO/DUBs from other viral families, particularly coronaviruses, suggests that low DUB activities of viral PRO/DUBs may generally be fine-tuned features of interaction with host factors.
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Affiliation(s)
- Sonia Fieulaine
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France.
| | - Martin D Witte
- Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts, USA
| | - Christopher S Theile
- Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts, USA
| | - Maya Ayach
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France
| | - Hidde L Ploegh
- Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts, USA
| | - Isabelle Jupin
- Laboratory of Molecular Virology, Jacques Monod Institute, CNRS, UMR, Université de Paris, Paris, France
| | - Stéphane Bressanelli
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France.
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Alcalá S, Sancho P, Martinelli P, Navarro D, Pedrero C, Martín-Hijano L, Valle S, Earl J, Rodríguez-Serrano M, Ruiz-Cañas L, Rojas K, Carrato A, García-Bermejo L, Fernández-Moreno MÁ, Hermann PC, Sainz B. ISG15 and ISGylation is required for pancreatic cancer stem cell mitophagy and metabolic plasticity. Nat Commun 2020; 11:2682. [PMID: 32472071 PMCID: PMC7260233 DOI: 10.1038/s41467-020-16395-2] [Citation(s) in RCA: 78] [Impact Index Per Article: 15.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2019] [Accepted: 05/01/2020] [Indexed: 02/06/2023] Open
Abstract
Pancreatic cancer stem cells (PaCSCs) drive pancreatic cancer tumorigenesis, chemoresistance and metastasis. While eliminating this subpopulation of cells would theoretically result in tumor eradication, PaCSCs are extremely plastic and can successfully adapt to targeted therapies. In this study, we demonstrate that PaCSCs increase expression of interferon-stimulated gene 15 (ISG15) and protein ISGylation, which are essential for maintaining their metabolic plasticity. CRISPR-mediated ISG15 genomic editing reduces overall ISGylation, impairing PaCSCs self-renewal and their in vivo tumorigenic capacity. At the molecular level, ISG15 loss results in decreased mitochondrial ISGylation concomitant with increased accumulation of dysfunctional mitochondria, reduced oxidative phosphorylation (OXPHOS) and impaired mitophagy. Importantly, disruption in mitochondrial metabolism affects PaCSC metabolic plasticity, making them susceptible to prolonged inhibition with metformin in vivo. Thus, ISGylation is critical for optimal and efficient OXPHOS by ensuring the recycling of dysfunctional mitochondria, and when absent, a dysregulation in mitophagy occurs that negatively impacts PaCSC stemness. The ubiquitin-like modifier ISG15 exerts post-translational protein regulation through ISGylation. Here, the authors show that ISGylation is necessary for pancreatic cancer stem cell self-renewal and tumourigenesis by supporting the recycling of non-functional mitochondria.
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Affiliation(s)
- Sonia Alcalá
- Department of Biochemistry, Universidad Autónoma de Madrid (UAM) and Instituto de Investigaciones Biomédicas "Alberto Sols" (IIBM), CSIC-UAM, Madrid, Spain. .,Chronic Diseases and Cancer Area 3-Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain.
| | - Patricia Sancho
- IIS Aragón, Hospital Universitario Miguel Servet, Zaragoza, Spain
| | - Paola Martinelli
- Institute for Cancer Research, Comprehensive Cancer Center, Medical University Vienna, Vienna, Austria
| | - Diego Navarro
- Department of Biochemistry, Universidad Autónoma de Madrid (UAM) and Instituto de Investigaciones Biomédicas "Alberto Sols" (IIBM), CSIC-UAM, Madrid, Spain.,Chronic Diseases and Cancer Area 3-Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain
| | - Coral Pedrero
- Department of Biochemistry, Universidad Autónoma de Madrid (UAM) and Instituto de Investigaciones Biomédicas "Alberto Sols" (IIBM), CSIC-UAM, Madrid, Spain.,Chronic Diseases and Cancer Area 3-Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain
| | - Laura Martín-Hijano
- Department of Biochemistry, Universidad Autónoma de Madrid (UAM) and Instituto de Investigaciones Biomédicas "Alberto Sols" (IIBM), CSIC-UAM, Madrid, Spain.,Chronic Diseases and Cancer Area 3-Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain
| | - Sandra Valle
- Department of Biochemistry, Universidad Autónoma de Madrid (UAM) and Instituto de Investigaciones Biomédicas "Alberto Sols" (IIBM), CSIC-UAM, Madrid, Spain.,Chronic Diseases and Cancer Area 3-Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain
| | - Julie Earl
- Chronic Diseases and Cancer Area 3-Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain.,Medical Oncology Department, Ramón y Cajal University Hospital, Alcala University, Madrid, Spain.,Biomedical Research Network in Cancer (CIBERONC, CB16/12/00446), Madrid, Spain
| | | | - Laura Ruiz-Cañas
- Department of Biochemistry, Universidad Autónoma de Madrid (UAM) and Instituto de Investigaciones Biomédicas "Alberto Sols" (IIBM), CSIC-UAM, Madrid, Spain.,Chronic Diseases and Cancer Area 3-Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain
| | - Katerin Rojas
- Department of Biochemistry, Universidad Autónoma de Madrid (UAM) and Instituto de Investigaciones Biomédicas "Alberto Sols" (IIBM), CSIC-UAM, Madrid, Spain
| | - Alfredo Carrato
- Chronic Diseases and Cancer Area 3-Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain.,Medical Oncology Department, Ramón y Cajal University Hospital, Alcala University, Madrid, Spain.,Biomedical Research Network in Cancer (CIBERONC, CB16/12/00446), Madrid, Spain
| | | | - Miguel Ángel Fernández-Moreno
- Department of Biochemistry, Universidad Autónoma de Madrid (UAM) and Instituto de Investigaciones Biomédicas "Alberto Sols" (IIBM), CSIC-UAM, Madrid, Spain.,Centro de Investigación Biomédica en Red en Enfermedades Raras (CIBERER), Madrid, Spain.,Instituto de Investigación Sanitaria Hospital 12 de Octubre (imas12), Madrid, Spain
| | | | - Bruno Sainz
- Department of Biochemistry, Universidad Autónoma de Madrid (UAM) and Instituto de Investigaciones Biomédicas "Alberto Sols" (IIBM), CSIC-UAM, Madrid, Spain. .,Chronic Diseases and Cancer Area 3-Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain.
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Drug repurposing of pyrimidine analogs as potent antiviral compounds against human enterovirus A71 infection with potential clinical applications. Sci Rep 2020; 10:8159. [PMID: 32424333 PMCID: PMC7235037 DOI: 10.1038/s41598-020-65152-4] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2020] [Accepted: 04/28/2020] [Indexed: 01/15/2023] Open
Abstract
Enterovirus A71 (EV-A71) is one of the aetiological agents for the hand, foot and mouth disease (HFMD) in young children and a potential cause of neurological complications in afflicted patients. Since its discovery in 1969, there remains no approved antiviral for EV-A71 and other HFMD-causing enteroviruses. We set out to address the lack of therapeutics against EV-A71 by screening an FDA-approved drug library and found an enrichment of hits including pyrimidine antimetabolite, gemcitabine which showed 90.2% of inhibition on EV-A71 infection. Gemcitabine and other nucleoside analogs, LY2334737 and sofosbuvir inhibition of EV-A71 infection were disclosed using molecular and proteomic quantification, and in vitro and in vivo efficacy evaluation. Gemcitabine displayed a significant reduction of infectious EV-A71 titres by 2.5 logs PFU/mL and was shown to target the early stage of EV-A71 viral RNA and viral protein synthesis process especially via inhibition of the RNA dependent RNA polymerase. In addition, the drug combination study of gemcitabine's synergistic effects with interferon-β at 1:1 and 1:2 ratio enhanced inhibition against EV-A71 replication. Since gemcitabine is known to metabolize rapidly in vivo, other nucleoside analogs, LY2334737 and sofosbuvir conferred protection in mice against lethal EV-A71 challenge by potentially reducing the death rate, viral titers as well on virus-induced pathology in the limb muscle tissue of mice. Additionally, we found that gemcitabine is competent to inhibit other positive-sense RNA viruses of the Flaviviridae and Togaviridae family. Overall, these drugs provide new insights into targeting viral factors as a broad-spectrum antiviral strategy with potential therapeutic value for future development and are worthy of potential clinical application.
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Clasman JR, Everett RK, Srinivasan K, Mesecar AD. Decoupling deISGylating and deubiquitinating activities of the MERS virus papain-like protease. Antiviral Res 2020; 174:104661. [PMID: 31765674 PMCID: PMC7114298 DOI: 10.1016/j.antiviral.2019.104661] [Citation(s) in RCA: 38] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2019] [Revised: 11/12/2019] [Accepted: 11/18/2019] [Indexed: 11/17/2022]
Abstract
Coronavirus papain-like proteases (PLPs or PLpro), such as the one encoded in the genome of the infectious Middle East Respiratory Syndrome (MERS) virus, have multiple enzymatic activities that promote viral infection. PLpro acts as a protease and processes the large coronavirus polyprotein for virus replication. PLpro also functions as both a deubiquitinating (DUB) and deISGylating (deISG) enzyme and removes ubiquitin (Ub) and interferon-stimulated gene 15 (ISG15) from cellular proteins. Both DUB and deISG activities are implicated in suppressing innate immune responses; however, the precise role of each activity in this process is still unclear due in part to the difficulties in separating each activity. In this study, we determine the first structure of MERS PLpro in complex with the full-length human ISG15 to a resolution of 2.3 Å. This structure and available structures of MERS PLpro-Ub complexes were used as molecular guides to design PLpro mutants that lack either or both DUB/deISG activities. We tested 13 different PLpro mutants for protease, DUB, and deISG activitites using fluorescence-based assays. Results show that we can selectively modulate DUB activity at amino acid positions 1649 and 1653 while mutation of Val1691 or His1652 of PLpro to a positive charged residue completely impairs both DUB/deISG activities. These mutant enzymes will provide new functional tools for delineating the importance of DUB versus deISG activity in virus-infected cells and may serve as potential candidates for attenuating the MERS virus in vivo for modified vaccine design efforts.
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Affiliation(s)
- Jozlyn R Clasman
- Department of Biological Sciences, Purdue University, West Lafayette, IN, USA
| | - Renata K Everett
- Department of Biological Sciences, Purdue University, West Lafayette, IN, USA
| | - Karthik Srinivasan
- Department of Biological Sciences, Purdue University, West Lafayette, IN, USA
| | - Andrew D Mesecar
- Department of Biological Sciences, Purdue University, West Lafayette, IN, USA; Department of Biochemistry, Purdue University, West Lafayette, IN, USA; Center for Cancer Research, Purdue University, West Lafayette, IN, USA.
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Interferon-stimulated genes inhibit caprine parainfluenza virus type 3 replication in Madin-Darby bovine kidney cells. Vet Microbiol 2020; 241:108573. [DOI: 10.1016/j.vetmic.2019.108573] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2019] [Revised: 12/19/2019] [Accepted: 12/30/2019] [Indexed: 12/21/2022]
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Wang TY, Wang YF, Zhang Y, Shen JJ, Guo M, Yang J, Lau YL, Yang W. Identification of Regulatory Modules That Stratify Lupus Disease Mechanism through Integrating Multi-Omics Data. MOLECULAR THERAPY. NUCLEIC ACIDS 2019; 19:318-329. [PMID: 31877408 PMCID: PMC6938958 DOI: 10.1016/j.omtn.2019.11.019] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/21/2018] [Revised: 11/10/2019] [Accepted: 11/11/2019] [Indexed: 11/05/2022]
Abstract
Although recent advances in genetic studies have shed light on systemic lupus erythematosus (SLE), its detailed mechanisms remain elusive. In this study, using datasets on SLE transcriptomic profiles, we identified 750 differentially expressed genes (DEGs) in T and B lymphocytes and peripheral blood cells. Using transcription factor (TF) binding data derived from chromatin immunoprecipitation sequencing (ChIP-seq) experiments from the Encyclopedia of DNA Elements (ENCODE) project, we inferred networks of co-regulated genes (NcRGs) based on binding profiles of the upregulated DEGs by significantly enriched TFs. Modularization analysis of NcRGs identified co-regulatory modules among the DEGs and master TFs vital for each module. Remarkably, the co-regulatory modules stratified the common SLE interferon (IFN) signature and revealed SLE pathogenesis pathways, including the complement cascade, cell cycle regulation, NETosis, and epigenetic regulation. By integrative analyses of disease-associated genes (DAGs), DEGs, and enriched TFs, as well as proteins interacting with them, we identified a hierarchical regulatory cascade with TFs regulated by DAGs, which in turn regulates gene expression. Integrative analysis of multi-omics data provided valuable molecular insights into the molecular mechanisms of SLE.
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Affiliation(s)
- Ting-You Wang
- Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong
| | - Yong-Fei Wang
- Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong
| | - Yan Zhang
- Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong
| | - Jiangshan Jane Shen
- Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong; Collaborative Innovation Center for Birth Defect Research and Transformation of Shandong Province, Jining Medical University, Jining, China; Lupus Research Institute, Affiliated Hospital of Jining Medical University, Jining, China
| | - Mengbiao Guo
- Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong
| | - Jing Yang
- Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong
| | - Yu Lung Lau
- Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong.
| | - Wanling Yang
- Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong.
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Carriere J, Rao Y, Liu Q, Lin X, Zhao J, Feng P. Post-translational Control of Innate Immune Signaling Pathways by Herpesviruses. Front Microbiol 2019; 10:2647. [PMID: 31798565 PMCID: PMC6868034 DOI: 10.3389/fmicb.2019.02647] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2019] [Accepted: 10/30/2019] [Indexed: 12/21/2022] Open
Abstract
Herpesviruses constitute a large family of disease-causing DNA viruses. Each herpesvirus strain is capable of infecting particular organisms with a specific cell tropism. Upon infection, pattern recognition receptors (PRRs) recognize conserved viral features to trigger signaling cascades that culminate in the production of interferons and pro-inflammatory cytokines. To invoke a proper immune response while avoiding collateral tissue damage, signaling proteins involved in these cascades are tightly regulated by post-translational modifications (PTMs). Herpesviruses have developed strategies to subvert innate immune signaling pathways in order to ensure efficient viral replication and achieve persistent infection. The ability of these viruses to control the proteins involved in these signaling cascades post-translationally, either directly via virus-encoded enzymes or indirectly through the deregulation of cellular enzymes, has been widely reported. This ability provides herpesviruses with a powerful tool to shut off or restrict host antiviral and inflammatory responses. In this review, we highlight recent findings on the herpesvirus-mediated post-translational control along PRR-mediated signaling pathways.
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Affiliation(s)
| | | | | | | | | | - Pinghui Feng
- Section of Infection and Immunity, Ostrow School of Dentistry, University of Southern California, Los Angeles, CA, United States
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U L26 Attenuates IKKβ-Mediated Induction of Interferon-Stimulated Gene (ISG) Expression and Enhanced Protein ISGylation during Human Cytomegalovirus Infection. J Virol 2019; 93:JVI.01052-19. [PMID: 31534044 DOI: 10.1128/jvi.01052-19] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2019] [Accepted: 09/11/2019] [Indexed: 11/20/2022] Open
Abstract
Viruses must negotiate cellular antiviral responses in order to replicate. Human cytomegalovirus (HCMV) is a prevalent betaherpesvirus that encodes a number of viral gene products that modulate cellular antiviral signaling. The HCMV UL26 gene has previously been found to attenuate cytokine-activated NF-κB signaling, yet the role that UL26 plays in modulating the host cell's global transcriptional response to infection is not clear. Here, we find that infection with a UL26 deletion virus (ΔUL26) induces a proinflammatory transcriptional environment that includes substantial increases in the expression of cytokine signaling genes relative to wild-type HCMV. These increases include NF-κB-regulated genes as well as interferon-stimulated genes (ISGs), such as ISG15 and bone marrow stromal cell antigen 2 (BST2). The ΔUL26 mutant-mediated induction of ISG15 expression was found to drive increases in global protein ISGylation during ΔUL26 mutant infection. However, short hairpin RNA (shRNA) and CRISPR-mediated targeting of ISG15 indicated that its induction does not restrict HCMV infection. In contrast, shRNA-mediated targeting of BST2 demonstrated that BST2 restricts HCMV cell-to-cell spread. In addition, the increased expression of both of these ISGs and the global enhancement in protein ISGylation were found to be dependent on the activity of the canonical inhibitor of NF-κB kinase beta (IKKβ). Both CRISPR-based and pharmacologically mediated inhibition of IKKβ blocked the induction of ISG15 and BST2. These results suggest significant cross-talk between the NF-κB and interferon signaling pathways and highlight the importance of IKK signaling and the HCMV UL26 protein in shaping the antiviral response to HCMV.IMPORTANCE Modulation of cellular antiviral signaling is a key determinant of viral pathogenesis. Human cytomegalovirus (HCMV) is a significant source of morbidity in neonates and the immunosuppressed that contains many genes that modulate antiviral signaling, yet how these genes contribute to shaping the host cell's transcriptional response to infection is largely unclear. Our results indicate that the HCMV UL26 protein is critical in preventing the establishment of a broad cellular proinflammatory transcriptional environment. Further, we find that the host gene IKKβ is an essential determinant governing the host cell's antiviral transcriptional response. Given their importance to viral pathogenesis, continuing to elucidate the functional interactions between viruses and the cellular innate immune response could enable the development of therapeutic strategies to limit viral infection.
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Contact-dependent delivery of IL-2 by dendritic cells to CD4 T cells in the contraction phase promotes their long-term survival. Protein Cell 2019; 11:108-123. [PMID: 31691194 PMCID: PMC6954898 DOI: 10.1007/s13238-019-00662-0] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2019] [Accepted: 09/18/2019] [Indexed: 01/17/2023] Open
Abstract
Common γ chain cytokines are important for immune memory formation. Among them, the role of IL-2 remains to be fully explored. It has been suggested that this cytokine is critically needed in the late phase of primary CD4 T cell activation. Lack of IL-2 at this stage sets for a diminished recall response in subsequent challenges. However, as IL-2 peak production is over at this point, the source and the exact mechanism that promotes its production remain elusive. We report here that resting, previously antigen-stimulated CD4 T cells maintain a minimalist response to dendritic cells after their peak activation in vitro. This subtle activation event may be induced by DCs without overt presence of antigen and appears to be stronger if IL-2 comes from the same dendritic cells. This encounter reactivates a miniature IL-2 production and leads a gene expression profile change in these previously activated CD4 T cells. The CD4 T cells so experienced show enhanced reactivation intensity upon secondary challenges later on. Although mostly relying on in vitro evidence, our work may implicate a subtle programing for CD4 T cell survival after primary activation in vivo.
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Gulati NM, Gallagher JR, McCraw DM, Harris AK. Probing the Structural Organization of Virions and Genomic Ribonucleoprotein Complexes from Type B Influenza Virus by Cryo-electron Microscopy. MICROSCOPY AND MICROANALYSIS : THE OFFICIAL JOURNAL OF MICROSCOPY SOCIETY OF AMERICA, MICROBEAM ANALYSIS SOCIETY, MICROSCOPICAL SOCIETY OF CANADA 2019; 25:1302-1303. [PMID: 31413663 PMCID: PMC6693676 DOI: 10.1017/s1431927619007244] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/10/2023]
Affiliation(s)
- Neetu M. Gulati
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - John R. Gallagher
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Dustin M. McCraw
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Audray K. Harris
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
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McCarthy RR, Everett HE, Graham SP, Steinbach F, Crooke HR. Head Start Immunity: Characterizing the Early Protection of C Strain Vaccine Against Subsequent Classical Swine Fever Virus Infection. Front Immunol 2019; 10:1584. [PMID: 31396205 PMCID: PMC6663987 DOI: 10.3389/fimmu.2019.01584] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2019] [Accepted: 06/25/2019] [Indexed: 01/18/2023] Open
Abstract
Classical Swine Fever Virus (CSFV) is an ongoing threat to the pig industry due to the high transmission and mortality rates associated with infection. Live attenuated vaccines such as the CSFV C strain vaccine are capable of protecting against infection within 5 days of vaccination, but the molecular mechanisms through which this early protection is mediated have yet to be established. In this study, we compared the response of pigs vaccinated with the C strain to non-vaccinated pigs both challenged with a pathogenic strain of CSFV. Analysis of transcriptomic data from the tonsils of these animals during the early stages after vaccination and challenge reveals a set of regulated genes that appear throughout the analysis. Many of these are linked to the ISG15 antiviral pathway suggesting it may play a role in the rapid and early protection conferred by C strain vaccination.
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Affiliation(s)
- Ronan R McCarthy
- Virology Department, Animal and Plant Health Agency, Addlestone, United Kingdom
| | - Helen E Everett
- Virology Department, Animal and Plant Health Agency, Addlestone, United Kingdom
| | - Simon P Graham
- Virology Department, Animal and Plant Health Agency, Addlestone, United Kingdom.,School of Veterinary Medicine, University of Surrey, Guildford, United Kingdom.,The Pirbright Institute, Pirbright, United Kingdom
| | - Falko Steinbach
- Virology Department, Animal and Plant Health Agency, Addlestone, United Kingdom.,School of Veterinary Medicine, University of Surrey, Guildford, United Kingdom
| | - Helen R Crooke
- Virology Department, Animal and Plant Health Agency, Addlestone, United Kingdom
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44
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Wani SA, Sahu AR, Khan RIN, Pandey A, Saxena S, Hosamani N, Malla WA, Chaudhary D, Kanchan S, Sah V, Rajak KK, Muthuchelvan D, Mishra B, Tiwari AK, Sahoo AP, Sajjanar B, Singh YP, Gandham RK, Mishra BP, Singh RK. Contrasting Gene Expression Profiles of Monocytes and Lymphocytes From Peste-Des-Petits-Ruminants Virus Infected Goats. Front Immunol 2019; 10:1463. [PMID: 31333643 PMCID: PMC6624447 DOI: 10.3389/fimmu.2019.01463] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2019] [Accepted: 06/10/2019] [Indexed: 01/06/2023] Open
Abstract
In this study, transcriptome analysis of PPRV infected PBMC subsets-T helper cells, T cytotoxic cells, monocytes, and B lymphocytes was done to delineate their role in host response. PPRV was found to infect lymphocytes and not monocytes. The established receptor for PPRV-SLAM was found downregulated in lymphocytes and non-differentially expressed in monocytes. A profound deviation in the global gene expression profile with a large number of unique upregulated genes (851) and downregulated genes (605) was observed in monocytes in comparison to lymphocytes. ISGs-ISG15, Mx1, Mx2, RSAD2, IFIT3, and IFIT5 that play a role in antiviral response and the genes for viral sensors-MDA5, LGP2, and RIG1, were found to be upregulated in lymphocytes and downregulated in monocytes. The transcription factors-IRF-7 and STAT-1 that regulate expression of most of the ISGs were found activated in lymphocytes and not in monocytes. Interferon signaling pathway and RIG1 like receptor signaling pathway were found activated in lymphocytes and not in monocytes. This contrast in gene expression profiles and signaling pathways indicated the predominant role of lymphocytes in generating the antiviral response against PPRV in goats, thus, giving us new insights into host response to PPRV.
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Affiliation(s)
- Sajad Ahmad Wani
- Division of Veterinary Biotechnology, ICAR-Indian Veterinary Research Institute (IVRI), Bareilly, India.,Division of Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University, Columbus, OH, United States
| | - Amit Ranjan Sahu
- Division of Veterinary Biotechnology, ICAR-Indian Veterinary Research Institute (IVRI), Bareilly, India.,Genomics and Computational Biology, DBT-National Institute of Animal Biotechnology, Hyderabad, India
| | - Raja Ishaq Nabi Khan
- Division of Veterinary Biotechnology, ICAR-Indian Veterinary Research Institute (IVRI), Bareilly, India
| | - Aruna Pandey
- Division of Veterinary Biotechnology, ICAR-Indian Veterinary Research Institute (IVRI), Bareilly, India
| | - Shikha Saxena
- Division of Veterinary Biotechnology, ICAR-Indian Veterinary Research Institute (IVRI), Bareilly, India
| | - Neelima Hosamani
- Genomics and Computational Biology, DBT-National Institute of Animal Biotechnology, Hyderabad, India
| | - Waseem Akram Malla
- Division of Veterinary Biotechnology, ICAR-Indian Veterinary Research Institute (IVRI), Bareilly, India
| | - Dheeraj Chaudhary
- Division of Virology, ICAR-Indian Veterinary Research Institute (IVRI), Mukteswar, India
| | - Sonam Kanchan
- Division of Veterinary Biotechnology, ICAR-Indian Veterinary Research Institute (IVRI), Bareilly, India
| | - Vaishali Sah
- Division of Animal Genetics and Breeding, ICAR-Indian Veterinary Research Institute (IVRI), Bareilly, India
| | - Kaushal Kishor Rajak
- Division of Biological Products, ICAR-Indian Veterinary Research Institute, Bareilly, India
| | - D Muthuchelvan
- Division of Virology, ICAR-Indian Veterinary Research Institute (IVRI), Mukteswar, India
| | - Bina Mishra
- Division of Biological Products, ICAR-Indian Veterinary Research Institute, Bareilly, India
| | - Ashok Kumar Tiwari
- Division of Biological Standardization, ICAR-Indian Veterinary Research Institute, Bareilly, India
| | - Aditya P Sahoo
- ICAR- Directorate of Foot and Mouth Disease, Mukteswar, India
| | - Basavaraj Sajjanar
- Division of Veterinary Biotechnology, ICAR-Indian Veterinary Research Institute (IVRI), Bareilly, India
| | - Yash Pal Singh
- ARIS Cell, ICAR-Indian Veterinary Research Institute (IVRI), Bareilly, India
| | - Ravi Kumar Gandham
- Division of Veterinary Biotechnology, ICAR-Indian Veterinary Research Institute (IVRI), Bareilly, India.,Genomics and Computational Biology, DBT-National Institute of Animal Biotechnology, Hyderabad, India
| | - Bishnu Prasad Mishra
- Division of Veterinary Biotechnology, ICAR-Indian Veterinary Research Institute (IVRI), Bareilly, India
| | - Raj Kumar Singh
- Division of Veterinary Biotechnology, ICAR-Indian Veterinary Research Institute (IVRI), Bareilly, India
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Liu W, Xiang Y, Zhang W, Jia P, Yi M, Jia K. Expression pattern, antiviral role and regulation analysis of interferon-stimulated gene 15 in black seabream, Acanthopagrus schlegelii. FISH & SHELLFISH IMMUNOLOGY 2018; 82:60-67. [PMID: 30041052 DOI: 10.1016/j.fsi.2018.07.041] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/28/2018] [Revised: 07/17/2018] [Accepted: 07/19/2018] [Indexed: 05/04/2023]
Abstract
Interferon stimulated gene 15 (ISG15) is an IFN inducible ubiquitin-like protein and plays a critical role in immune response against viral infection. In this study, an ISG15 gene (AsISG15) was cloned and characterized from the marine fish black seabream, Acanthopagrus schlegelii. The full-length cDNA of AsISG15 was 1302 bp and encoded 155 amino acids containing two ubiquitin-like motifs and a LRGG conjugation domain. Multiple alignment and phylogenetic tree showed that AsISG15 shared 31-70% amino acid identity with other known ISG15s and had a closer evolutionary relationship with teleost ISG15s. In vitro, AsISG15 expression was inducible by poly I:C, LPS and red spotted nervous necrosis virus (RGNNV) in cultured black seabream brain cells. In vivo, AsISG15 was ubiquitously expressed in all examined tissues with higher expression levels in eye and gill, and the expression was significantly up-regulated in most tissues post RGNNV infection, especially in liver, spleen and kidney. The testing of antiviral activity showed that silencing AsISG15 significantly increased RGNNV replication in RGNNV infected AsS cells, and the LRGG domain was crucial for the anti-RGNNV activity of AsISG15. By promoter-driven luciferase reporter assay, we demonstrated that two IFN-stimulated response elements within the promoter region of AsISG15 and the promoter-proximal intron were essential for AsISG15 expression. Furthermore, our results showed that the gamma-IFN activation sequence located in the intron was required for the intron mediated enhancement for AsISG15 expression. Our results would provide insights for understanding the underlying regulation mechanism of ISG15 in teleost.
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Affiliation(s)
- Wei Liu
- Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, Guangdong, China; School of Marine Sciences, Sun Yat-sen University, Guangdong, China.
| | - Yangxi Xiang
- Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, Guangdong, China; School of Marine Sciences, Sun Yat-sen University, Guangdong, China.
| | - Wanwan Zhang
- Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, Guangdong, China; School of Marine Sciences, Sun Yat-sen University, Guangdong, China.
| | - Peng Jia
- Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, Guangdong, China; School of Marine Sciences, Sun Yat-sen University, Guangdong, China.
| | - Meisheng Yi
- Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, Guangdong, China; School of Marine Sciences, Sun Yat-sen University, Guangdong, China.
| | - Kuntong Jia
- Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, Guangdong, China; School of Marine Sciences, Sun Yat-sen University, Guangdong, China.
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Suzuki H, Tsuji R, Sugamata M, Yamamoto N, Yamamoto N, Kanauchi O. Administration of plasmacytoid dendritic cell-stimulative lactic acid bacteria is effective against dengue virus infection in mice. Int J Mol Med 2018; 43:426-434. [PMID: 30365042 DOI: 10.3892/ijmm.2018.3955] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2018] [Accepted: 10/17/2018] [Indexed: 11/06/2022] Open
Abstract
Dengue virus (DENV), a mosquito‑borne flavivirus, causes an acute febrile illness that is a major public health problem in the tropics and subtropics globally. However, methods to prevent or treat DENV infection have not been well established. It was previously demonstrated that Lactococcus lactis strain plasma (LC‑plasma) has the ability to stimulate plasmacytoid dendritic cells (pDCs). As pDCs are key immune cells that control viral infection by producing large amounts of type I interferons (IFN), the present study evaluated the effect of LC‑plasma on DENV infection using a mouse infectious DENV strain. Mice were divided into two groups and the test group was orally administered LC‑plasma for two weeks. Two weeks following administration, the mice were infected with DENV and the relative viral titers and the expression of the inflammatory genes in DENV‑infected tissue were measured using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). The relative viral titers were notably lower in the DENV‑infected tissues compared with the control group when LC‑plasma was orally administered prior to DENV infection. Furthermore, the expression of the inflammatory genes associated with DENV infection was also reduced by LC‑plasma administration. To investigate how LC‑plasma administration controls DENV infection, the present study examined anti‑viral gene expression, which is critical for the viral clearance induced by type I IFN. Two weeks subsequent to the administration of LC‑plasma, the expression of anti‑viral gene was measured using RT‑qPCR. Oral intake of LC‑plasma enhanced anti‑viral gene expression in DENV‑infected spleen tissue. To clarify the detailed mechanism, in vitro co‑culture studies using bone‑marrow derived DC (BMDC) were performed. BMDC were stimulated with LC‑plasma in combination with anti‑IFN‑α/β antibody and the expression of anti‑viral genes was measured. In vitro studies revealed that the effect of LC‑plasma on anti‑viral genes was dependent on type I IFN. Based on these results, LC‑plasma may be effective against DENV infection by stimulating pDCs, which results in the increased production of anti‑viral factors.
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Affiliation(s)
- Hiroaki Suzuki
- Research Laboratories for Health Science and Food Technologies, Kirin Company, Ltd., Yokohama‑shi, Kanagawa 236‑0004, Japan
| | - Ryohei Tsuji
- Research Laboratories for Health Science and Food Technologies, Kirin Company, Ltd., Yokohama‑shi, Kanagawa 236‑0004, Japan
| | - Miho Sugamata
- Research Laboratories for Health Science and Food Technologies, Kirin Company, Ltd., Yokohama‑shi, Kanagawa 236‑0004, Japan
| | - Naoki Yamamoto
- National Institute of Infectious Diseases, Tokyo 162‑8640, Japan
| | - Norio Yamamoto
- Department of Infection Control Science, Graduate School of Medicine, Juntendo University, Tokyo 113‑8421, Japan
| | - Osamu Kanauchi
- Research Laboratories for Health Science and Food Technologies, Kirin Company, Ltd., Yokohama‑shi, Kanagawa 236‑0004, Japan
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Strand-Specific Dual RNA Sequencing of Bronchial Epithelial Cells Infected with Influenza A/H3N2 Viruses Reveals Splicing of Gene Segment 6 and Novel Host-Virus Interactions. J Virol 2018; 92:JVI.00518-18. [PMID: 29976658 DOI: 10.1128/jvi.00518-18] [Citation(s) in RCA: 36] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2018] [Accepted: 05/24/2018] [Indexed: 02/08/2023] Open
Abstract
Host-influenza virus interplay at the transcript level has been extensively characterized in epithelial cells. Yet, there are no studies that simultaneously characterize human host and influenza A virus (IAV) genomes. We infected human bronchial epithelial BEAS-2B cells with two seasonal IAV/H3N2 strains, Brisbane/10/07 and Perth/16/09 (reference strains for past vaccine seasons) and the well-characterized laboratory strain Udorn/307/72. Strand-specific RNA sequencing (RNA-seq) of the infected BEAS-2B cells allowed for simultaneous analysis of host and viral transcriptomes, in addition to pathogen genomes, to reveal changes in mRNA expression and alternative splicing (AS). In general, patterns of global and immune gene expression induced by the three IAVs were mostly shared. However, AS of host transcripts and small nuclear RNAs differed between the seasonal and laboratory strains. Analysis of viral transcriptomes showed deletions of the polymerase components (defective interfering-like RNAs) within the genome. Surprisingly, we found that the neuraminidase gene undergoes AS and that the splicing event differs between seasonal and laboratory strains. Our findings reveal novel elements of the host-virus interaction and highlight the importance of RNA-seq in identifying molecular changes at the genome level that may contribute to shaping RNA-based innate immunity.IMPORTANCE The use of massively parallel RNA sequencing (RNA-seq) has revealed insights into human and pathogen genomes and their evolution. Dual RNA-seq allows simultaneous dissection of host and pathogen genomes and strand-specific RNA-seq provides information about the polarity of the RNA. This is important in the case of negative-strand RNA viruses like influenza virus, which generate positive (complementary and mRNA) and negative-strand RNAs (genome) that differ in their potential to trigger innate immunity. Here, we characterize interactions between human bronchial epithelial cells and three influenza A/H3N2 strains using strand-specific dual RNA-seq. We focused on this subtype because of its epidemiological importance in causing significant morbidity and mortality during influenza epidemics. We report novel elements that differ between seasonal and laboratory strains highlighting the complexity of the host-virus interplay at the RNA level.
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48
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Wang M, Huang Y, He M, Peng WJ, Tian DY. Effects of hepatitis E virus infection on interferon production via ISG15. World J Gastroenterol 2018; 24:2173-2180. [PMID: 29853735 PMCID: PMC5974579 DOI: 10.3748/wjg.v24.i20.2173] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/29/2018] [Revised: 04/19/2018] [Accepted: 04/23/2018] [Indexed: 02/06/2023] Open
Abstract
AIM To assess the effects of hepatitis E virus (HEV) on the production of type I interferons (IFNs) and determine the underlying mechanisms.
METHODS We measured the production of interferon (IFN)-alpha and -beta (-α/β) in genotype 3 HEV-infected C3A cells at different time points (0, 8, 12, 24, 48, 72 and 120 h) by enzyme-linked immunosorbent assay (ELISA). The expression levels of IFN-stimulated gene (ISG)15 in HEV-infected C3A cells at different time points were tested by western blotting. The plasmid-expressing open reading frame 3 (ORF3) or control plasmids (green fluorescent protein-expressing) were transfected into C3A cells, and the levels of IFN-α/β and ISG15 were evaluated, respectively. Furthermore, the plasmid-expressing ISG15 or small interfering RNA-inhibiting ISG15 was transfected into infected C3A cells. Then, the production of IFN-α/β was also measured by ELISA.
RESULTS We showed that genotype 3 HEV could enhance the production of IFN-α/β and induce elevation of ISG15 in C3A cells. HEV ORF3 protein could enhance the production of IFN-α/β and the expression of ISG15. Additionally, ISG15 silencing enhanced the production of IFN-α/β. Overexpression of ISG15 resulted in the reduction of IFN-α/β.
CONCLUSION HEV may promote production of IFN-α/β and expression of ISG15 via ORF3 in the early stages, and increased ISG15 subsequently inhibited the production of IFN-α/β.
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Affiliation(s)
- Min Wang
- Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
| | - Ying Huang
- The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510700, Guangdong Province, China
| | - Man He
- Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
| | - Wen-Ju Peng
- Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
| | - De-Ying Tian
- Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
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Gargan S, Ahmed S, Mahony R, Bannan C, Napoletano S, O'Farrelly C, Borrow P, Bergin C, Stevenson NJ. HIV-1 Promotes the Degradation of Components of the Type 1 IFN JAK/STAT Pathway and Blocks Anti-viral ISG Induction. EBioMedicine 2018; 30:203-216. [PMID: 29580840 PMCID: PMC5952252 DOI: 10.1016/j.ebiom.2018.03.006] [Citation(s) in RCA: 39] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2017] [Revised: 02/24/2018] [Accepted: 03/07/2018] [Indexed: 01/12/2023] Open
Abstract
Anti-retroviral therapy successfully suppresses HIV-1 infection, but fails to provide a cure. During infection Type 1 IFNs normally play an essential role in viral clearance, but in vivo IFN-α only has a modest impact on HIV-1 infection, suggesting its possible targeting by HIV. Here, we report that the HIV protein, Vif, inhibits effective IFN-α signalling via degradation of essential JAK/STAT pathway components. We found that STAT1 and STAT3 are specifically reduced in HEK293T cells expressing Vif and that full length, infectious HIV-1 IIIB strain promotes their degradation in a Vif-dependent manner. HIV-1 IIIB infection of myeloid ThP-1 cells also reduced the IFN-α-mediated induction of the anti-viral gene, ISG15, but not MxA, revealing a functional consequence of this HIV-1-mediated immune evasion strategy. Interestingly, while total STAT levels were not reduced upon in vitro IIIB infection of primary human PBMCs, IFN-α-mediated phosphorylation of STAT1 and STAT3 and ISG induction were starkly reduced, with removal of Vif (IIIBΔVif), partially restoring pSTATs, ISG15 and MxB induction. Similarly, pSTAT1 and pSTAT3 expression and IFN-α-induced ISG15 were reduced in PBMCs from HIV-infected patients, compared to healthy controls. Furthermore, IFN-α pre-treatment of a CEM T lymphoblast cells significantly inhibited HIV infection/replication (measured by cellular p24), only in the absence of Vif (IIIBΔVif), but was unable to suppress full length IIIB infection. When analysing the mechanism by which Vif might target the JAK/STAT pathway, we found Vif interacts with both STAT1 and STAT3, (but not STAT2), and its expression promotes ubiquitination and MG132-sensitive, proteosomal degradation of both proteins. Vif's Elongin-Cullin-SOCS-box binding motif enables the formation of an active E3 ligase complex, which we found to be required for Vif's degradation of STAT1 and STAT3. In fact, the E3 ligase scaffold proteins, Cul5 and Rbx2, were also found to be essential for Vif-mediated proteasomal degradation of STAT1 and STAT3. These results reveal a target for HIV-1-Vif and demonstrate how HIV-1 impairs the anti-viral activity of Type 1 IFNs, possibly explaining why both endogenous and therapeutic IFN-α fail to activate more effective control over HIV infection.
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Affiliation(s)
- Siobhan Gargan
- Intracellular Immunology Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland
| | - Suaad Ahmed
- Intracellular Immunology Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland
| | - Rebecca Mahony
- Intracellular Immunology Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland
| | - Ciaran Bannan
- Intracellular Immunology Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland; School of Medicine, Trinity College Dublin, Ireland; Department of GU Medicine and Infectious Diseases, St. James's Hospital, Dublin, Ireland
| | - Silvia Napoletano
- Intracellular Immunology Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland
| | - Cliona O'Farrelly
- Intracellular Immunology Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland; School of Medicine, Trinity College Dublin, Ireland
| | - Persephone Borrow
- Nuffield Department of Clinical Medicine, University of Oxford, United Kingdom
| | - Colm Bergin
- School of Medicine, Trinity College Dublin, Ireland; Department of GU Medicine and Infectious Diseases, St. James's Hospital, Dublin, Ireland
| | - Nigel J Stevenson
- Intracellular Immunology Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland.
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Irreversible inactivation of ISG15 by a viral leader protease enables alternative infection detection strategies. Proc Natl Acad Sci U S A 2018; 115:2371-2376. [PMID: 29463763 PMCID: PMC5877979 DOI: 10.1073/pnas.1710617115] [Citation(s) in RCA: 57] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
In response to viral infection, cells mount a potent inflammatory response that relies on ISG15 and ubiquitin posttranslational modifications. Many viruses use deubiquitinases and deISGylases that reverse these modifications and antagonize host signaling processes. We here reveal that the leader protease, Lbpro, from foot-and-mouth disease virus (FMDV) targets ISG15 and to a lesser extent, ubiquitin in an unprecedented manner. Unlike canonical deISGylases that hydrolyze the isopeptide linkage after the C-terminal GlyGly motif, Lbpro cleaves the peptide bond preceding the GlyGly motif. Consequently, the GlyGly dipeptide remains attached to the substrate Lys, and cleaved ISG15 is rendered incompetent for reconjugation. A crystal structure of Lbpro bound to an engineered ISG15 suicide probe revealed the molecular basis for ISG15 proteolysis. Importantly, anti-GlyGly antibodies, developed for ubiquitin proteomics, are able to detect Lbpro cleavage products during viral infection. This opens avenues for infection detection of FMDV based on an immutable, host-derived epitope.
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