• P. gingivalis
• gingipains
• neutrophils granules
• periodontal pathogens
• serine proteases

• Neutrophils/immunology
• Neutrophils/microbiology
• Neutrophils/metabolism
• Porphyromonas gingivalis/enzymology
• Porphyromonas gingivalis/immunology
• Humans
• Proteolysis
• Gingipain Cysteine Endopeptidases
• Adhesins, Bacterial/metabolism
• Cysteine Endopeptidases/metabolism
• Cytoplasmic Granules/metabolism
• Apoptosis

• R01 DE028296 NIDCR NIH HHS
• F31 DE031948 NIDCR NIH HHS
• National Institute of Dental and Craniofacial Research

• Clostripain
• Cryo-EM
• Cysteine protease
• MicroED
• Microcrystal electron diffraction

• P. gingivalis
• apoptosis
• efferocytosis
• necroptosis
• periodontitis
• pyroptosis

• Humans
• Phagocytosis
• Apoptosis
• Cell Death
• Immunity, Innate
• Animals
• Host-Pathogen Interactions
• Adaptive Immunity
• Cytokines/metabolism
• Antigen-Presenting Cells/immunology
• Immunity, Cellular
• Virulence
• Lysosomes/metabolism

• R01 DE030939 NIDCR NIH HHS
• DE022597 NIDCR NIH HHS
• R01 DE028296 NIDCR NIH HHS
• F31 DE031948 NIDCR NIH HHS
• DE031756 NIDCR NIH HHS
• R01 DE031756 NIDCR NIH HHS
• DE030939 NIDCR NIH HHS
• DE028296 NIDCR NIH HHS
• R01 DE022597 NIDCR NIH HHS

• Porphyromonas gingivalis
• cytokines
• gingipains
• innate immunity
• neutrophils
• phagocytosis
• superoxides

• airway macrophages
• children
• cystic fibrosis
• inflammation
• lung disease
• neutrophils
• resolution
• scavenger receptor

• National Institutes of Health
• Cystic Fibrosis Foundation
• Nederlandse Cystic Fibrosis Stichting

• Animals
• Porphyromonas gingivalis
• Periodontitis/microbiology
• Inflammation
• Atherosclerosis
• Lipids

• National Natural Science Foundation of China

• Bacterial proteases
• Host immunity
• bacterial pathogenesis
• polymicrobialinteractions
• therapeutics

Neutrophil-derived networks of DNA-composed extracellular fibers covered with antimicrobial molecules, referred to as neutrophil extracellular traps (NETs), are recognized as a physiological microbicidal mechanism of innate immunity. The formation of NETs is also classified as a model of a cell death called NETosis. Despite intensive research on the NETs formation in response to pathogens, the role of specific bacteria-derived virulence factors in this process, although postulated, is still poorly understood. The aim of our study was to determine the role of gingipains, cysteine proteases responsible for the virulence of P. gingivalis, on the NETosis process induced by this major periodontopathogen. We showed that NETosis triggered by P. gingivalis is gingipain dependent since in the stark contrast to the wild-type strain (W83) the gingipain-null mutant strain only slightly induced the NETs formation. Furthermore, the direct effect of proteases on NETosis was documented using purified gingipains. Notably, the induction of NETosis was dependent on the catalytic activity of gingipains, since proteolytically inactive forms of enzymes showed reduced ability to trigger the NETs formation. Mechanistically, gingipain-induced NETosis was dependent on proteolytic activation of protease-activated receptor-2 (PAR-2). Intriguingly, both P. gingivalis and purified Arg-specific gingipains (Rgp) induced NETs that not only lacked bactericidal activity but instead stimulated the growth of bacteria species otherwise susceptible to killing in NETs. This protection was executed by proteolysis of bactericidal components of NETs. Taken together, gingipains play a dual role in NETosis: they are the potent direct inducers of NETs formation but in the same time, their activity prevents P. gingivalis entrapment and subsequent killing. This may explain a paradox that despite the massive accumulation of neutrophils and NETs formation in periodontal pockets periodontal pathogens and associated pathobionts thrive in this environment.

Periodontitis, or gum disease, is characterized by chronic inflammation and erosion of the tooth-supporting tissues. The condition is fuelled by bacterial accumulation on the tooth surface below the gum line that resists the host innate immune response, including massive accumulation of neutrophils. Despite possessing a formidable array of bactericidal machineries, including neutrophil extracellular traps (NETs) formation whereby neutrophils release DNA-composed fibers decorated with bactericidal proteins and peptides to efficiently trap and kill bacteria. Nevertheless, neutrophils in periodontitis are unable to clear the infection due to the presence of key periodontal pathogens, including Porphyromonas gingivalis. This bacterium secretes a variety of virulence factors, including proteases (gingipains) that allow the organism to manipulate the host immune response to benefit the entire dysbiotic microbial community. Here, we describe a unique strategy whereby P. gingivalis trigger NET formation through gingipain-dependent cleavage of Protease Activated Receptor (PAR)-2 on the neutrophil surface. Importantly, NETs formed in this way are deficient in antibacterial activity but instead, supports bacterial growth due to degradation of bactericidal components by gingipains. This finding may explain a paradox that dysbiotic bacteria flourished in periodontal pockets in spite of massive accumulation of neutrophils and abundant NETs formation.

• Adhesins, Bacterial/immunology
• Adhesins, Bacterial/metabolism
• Animals
• Bacteroidaceae Infections/immunology
• Bacteroidaceae Infections/metabolism
• Bacteroidaceae Infections/microbiology
• Bacteroidaceae Infections/pathology
• Cells, Cultured
• Cysteine Endopeptidases/immunology
• Cysteine Endopeptidases/metabolism
• Extracellular Traps/immunology
• Extracellular Traps/microbiology
• Female
• Gingipain Cysteine Endopeptidases
• Humans
• Mice
• Mice, Inbred C57BL
• Neutrophils/immunology
• Neutrophils/microbiology
• Neutrophils/pathology
• Peritonitis/immunology
• Peritonitis/metabolism
• Peritonitis/microbiology
• Porphyromonas gingivalis/immunology
• Porphyromonas gingivalis/pathogenicity
• Receptor, PAR-2/immunology
• Receptor, PAR-2/metabolism
• Signal Transduction

• R01 DE022597 NIDCR NIH HHS
• R21 DE026280 NIDCR NIH HHS
• The National Science Center, Poland
• National Institutes of Health (US)
• National Institutes of Health
• the Deutsche Forschungsgemeinschaft

• M6P/IGF2R
• efferocytosis
• macrophages
• plasminogen
• tissue homeostasis

• Bacterial Proteins/chemistry
• Bacterial Proteins/metabolism
• Crystallography, X-Ray
• Cysteine Endopeptidases
• Cysteine Proteases/chemistry
• Cysteine Proteases/metabolism
• Enterobacteriaceae/enzymology
• Epithelial Cells/metabolism
• Humans
• Molecular Structure
• Substrate Specificity
• Symbiosis

• R21 CA181027 NCI NIH HHS
• U54 GM094586 NIGMS NIH HHS
• R21 CA186077 NCI NIH HHS
• T32 AI007244 NIAID NIH HHS
• P41 CA196276 NCI NIH HHS

Gingipain cysteine proteases are considered key virulence factors of Porphyromonas gingivalis. They significantly influence antibacterial and homeostatic functions of macrophages, neutrophils, the complement system, and cytokine networks. Recent data indicate the role of P. gingivalis in T cell differentiation; however, the involvement of gingipains in this process remains elusive. Therefore, the aim of this study was to investigate the contribution of danger signals triggered by the gingipains on the generation of Th17 cells, which play a key role in protection against bacterial diseases but may cause chronic inflammation and bone resorption. To this end we compared the effects of the wild-type strain of P. gingivalis (W83) with its isogenic mutant devoid of gingipain activity (ΔKΔRAB), and bacterial cells pretreated with a highly-specific inhibitor of gingipains activity (KYTs). Antigen presenting cells (APCs), both professional (dendritic cells), and non-professional (gingival keratinocytes), exposed to viable bacteria expressed high amounts of cytokines (IL-6, IL-21, IL-23). These cytokines are reported to either stimulate or balance the Th17-dependent immune response. Surprisingly, cells infected with P. gingivalis devoid of gingipain activity showed increased levels of all tested cytokines compared to bacteria with fully active enzymes. The effect was dependent on both the reduction of cytokine proteolysis and the lack of cross-talk with other bacterial virulence factors, including LPS and fimbriae that induce de novo synthesis of cytokines. The profile of lymphocyte T differentiation from naive T cells showed enhanced generation of Th17 in response to bacteria with inactive gingipains. Moreover, we found that gingipain-dependent induction of Th17 cells was highly specific, since other T cell-subsets remained unchanged. Finally, inhibition of IL-6 signaling in dendritic cells led to a significant depletion of the Th17 population. Cumulatively, this study revealed a previously undisclosed role of gingipain activity in the process of Th17 differentiation reliant on blocking signaling through IL-6. Since inactivation of gingipains accelerates the skewing of T cells toward Th17 cells, which are detrimental in periodontitis, IL-6 signaling may serve as an attractive target for treatment of the disease.

• IL-6
• P. gingivalis
• Th17
• dendritic cells
• gingipains

Periodontal disease is a prevalent chronic inflammatory condition characterised by an aberrant host response to a pathogenic plaque biofilm resulting in local tissue damage and frustrated healing that can result in tooth loss. Cysteine proteases (gingipains) from the key periodontal pathogen Porphyromonas gingivalis have been implicated in periodontal disease pathogenesis by inhibiting inflammation resolution and are linked with systemic chronic inflammatory conditions such as rheumatoid arthritis. Efficient clearance of apoptotic cells is essential for the resolution of inflammation and tissue restoration. Here we sought to characterise the innate immune clearance of apoptotic cells and its modulation by gingipains. We examined the capacity of gingipain-treated macrophages to migrate towards and phagocytose apoptotic cells. Lysine gingipain treatment of macrophages impaired macrophage migration towards apoptotic neutrophils. Furthermore, lysine gingipain treatment reduced surface expression levels of CD14, a key macrophage receptor for apoptotic cells, which resulted in reduced macrophage interactions with apoptotic cells. Additionally, while apoptotic cells and their derived secretome were shown to inhibit TNF-α-induced expression by P. gingivalis lipopolysaccharide, we demonstrated that gingipain preparations induced a rapid inflammatory response in macrophages that was resistant to the anti-inflammatory effects of apoptotic cells or their secretome. Taken together, these data indicate that P. gingivalis may promote the chronic inflammation seen in periodontal disease patients by multiple mechanisms, including rapid, potent gingipain-mediated inflammation, coupled with receptor cleavage leading to defective clearance of apoptotic cells and reduced anti-inflammatory responses. Thus, gingipains represent a potential therapeutic target for intervention in the management of chronic periodontal disease.

• CD44
• Endocytosis
• Hyaluronate
• Osteoarthritis
• Proteoglycan

Excessive ethanol consumption affects virtually any organ, both by indirect and direct mechanisms. Considerable research in the last two decades has widened the knowledge about the paramount importance of proinflammatory cytokines and oxidative damage in the pathogenesis of many of the systemic manifestations of alcoholism. These cytokines derive primarily from activated Kupffer cells exposed to Gram-negative intestinal bacteria, which reach the liver in supra-physiological amounts due to ethanol-mediated increased gut permeability. Reactive oxygen species (ROS) that enhance the inflammatory response are generated both by activation of Kupffer cells and by the direct metabolic effects of ethanol. The effects of this increased cytokine secretion and ROS generation lie far beyond liver damage. In addition to the classic consequences of endotoxemia associated with liver cirrhosis that were described several decades ago, important research in the last ten years has shown that cytokines may also induce damage in remote organs such as brain, bone, muscle, heart, lung, gonads, peripheral nerve, and pancreas. These effects are even seen in alcoholics without significant liver disease. Therefore, alcoholism can be viewed as an inflammatory condition, a concept which opens the possibility of using new therapeutic weapons to treat some of the complications of this devastating and frequent disease. In this review we examine some of the most outstanding consequences of the altered cytokine regulation that occurs in alcoholics in organs other than the liver.

• Alcoholism
• Cytokines
• Brain
• Bone
• Muscle
• Oxidative damage
• Atherosclerosis
• Sepsis
• Lung
• Chronic pancreatitis
• Alcoholic liver disease

• Alcohol Drinking/adverse effects
• Alcohol Drinking/metabolism
• Alcohol Drinking/mortality
• Alcoholism/diagnosis
• Alcoholism/etiology
• Alcoholism/metabolism
• Alcoholism/mortality
• Animals
• Cytokines/metabolism
• Ethanol/adverse effects
• Humans
• Inflammation/diagnosis
• Inflammation/etiology
• Inflammation/metabolism
• Inflammation/mortality
• Inflammation Mediators/metabolism
• Liver/drug effects
• Liver/metabolism
• Liver/pathology
• Liver Diseases, Alcoholic/diagnosis
• Liver Diseases, Alcoholic/etiology
• Liver Diseases, Alcoholic/metabolism
• Liver Diseases, Alcoholic/mortality
• Oxidative Stress/drug effects
• Prognosis
• Reactive Oxygen Species/metabolism
• Risk Assessment
• Risk Factors

• Ablation
• Hepatocellular carcinoma
• Nanosecond pulsed electric field

The Triggering Receptor Expressed on Myeloid cells 1 (TREM-1) is a cell surface receptor of the immunoglobulin superfamily, with the capacity to amplify pro-inflammatory cytokine production and regulate apoptosis. Polymorphonuclear neutrophils (PMNs) are the first line of defence against infection, and a major source of TREM-1. Porphyromonas gingivalis is a Gram-negative anaerobe highly implicated in the inflammatory processes governing periodontal disease, which is characterized by the destruction of the tooth-supporting tissues. It expresses a number of virulence factors, including the cysteine proteinases (or gingipains). The aim of this in vitro study was to investigate the effect of P. gingivalis on TREM-1 expression and production by primary human PMNs, and to evaluate the role of its gingipains in this process. After 4 h of challenge, P. gingivalis enhanced TREM-1 expression as identified by quantitative real-time PCR. This was followed by an increase in soluble (s)TREM-1 secretion over a period of 18 h, as determined by ELISA. At this time-point, the P. gingivalis-challenged PMNs exhibited diminished TREM-1 cell-membrane staining, as identified by flow cytometry and confocal laser scanning microscopy. Furthermore engagement of TREM-1, by means of anti-TREM-1 antibodies, enhanced the capacity of P. gingivalis to stimulate interleukin (IL)-8 production. Conversely, antagonism of TREM-1 using a synthetic peptide resulted in reduction of IL-8 secretion. Using isogenic P. gingivalis mutant strains, we identified the Arg-gingipain to be responsible for shedding of sTREM-1 from the PMN surface, whereas the Lys-gingipain had the capacity to degrade TREM-1. In conclusion, the differential regulation of TREM-1 by the P. gingivalis gingipains may present a novel mechanism by which P. gingivalis manipulates the host innate immune response helping to establish chronic periodontal inflammation.

• Porphyromonas gingivalis
• gingipain adhesins
• oral infection
• protein structure
• virulence factor

• Apo E
• atherosclerosis
• cathepsin G
• lesion progression

• P. gingivalis
• biofilm
• immunoediting hypothesis
• pathobiont
• tumor

The neutrophil-specific protease membrane-type 6 matrix metalloproteinase (MT6-MMP)/MMP-25/leukolysin is implicated in multiple sclerosis and cancer yet remains poorly characterized. To characterize the biological roles of MT6-MMP, it is critical to identify its substrates for which only seven are currently known. Here, we biochemically characterized MT6-MMP, profiled its tissue inhibitor of metalloproteinase inhibitory spectrum, performed degradomics analyses, and screened 26 chemokines for cleavage using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. MT6-MMP processes seven each of the CXC and CC chemokine subfamilies. Notably, cleavage of the neutrophil chemoattractant CXCL5 activates the chemokine, thereby increasing its agonist activity, indicating a feed-forward mechanism for neutrophil recruitment. Likewise, cleavage also activated CCL15 and CCL23 to increase monocyte recruitment. Utilizing the proteomics approach proteomic identification of cleavage site specificity (PICS), we identified 286 peptidic cleavage sites spanning from P6 to P6′ from which an unusual glutamate preference in P1 was identified. The degradomics screen terminal amine isotopic labeling of substrates (TAILS), which enriches for neo-N-terminal peptides of cleaved substrates, was used to identify 58 new native substrates in fibroblast secretomes after incubation with MT6-MMP. Vimentin, cystatin C, galectin-1, IGFBP-7, and secreted protein, acidic and rich in cysteine (SPARC) were among those substrates we biochemically confirmed. An extracellular “moonlighting” form of vimentin is a chemoattractant for THP-1 cells, but MT6-MMP cleavage abolished monocyte recruitment. Unexpectedly, the MT6-MMP-cleaved vimentin potently stimulated phagocytosis, which was not a property of the full-length protein. Hence, MT6-MMP regulates neutrophil and monocyte chemotaxis and by generating “eat-me” signals upon vimentin cleavage potentially increases phagocytic removal of neutrophils to resolve inflammation.

Accumulation of neutrophils in the site of inflammation is a typical mechanism of innate immunity. The accumulated neutrophils are exposed to stressogenic factors usually associated with inflammation. Here, we studied response of human peripheral blood neutrophils subjected to short, febrile-range heat stress. We show that 90 min heat stress slowed down the spontaneous apoptosis of neutrophils. In the absence of typical markers of apoptosis the heat-shocked neutrophils induced antiinflammatory effect in human monocyte-derived macrophages (hMDMs), yet without being engulfed. Importantly, the expression of FcγRIII (CD16) was sharply reduced. Surprisingly, concentration of the soluble CD16 did not change in heat-shocked neutrophil supernates indicating that the reduction of the cell surface CD16 was achieved mainly by inhibition of fresh CD16 delivery. Inhibitors of 90 kDa heat shock protein (HSP90), a molecular chaperone found in membrane platforms together with CD16 and CD11b, significantly increased the observed effects caused by heat shock. The presented data suggest a novel systemic aspect of increased temperature which relies on immediate modification by heat of a neutrophil molecular pattern. This effect precedes cell death and may be beneficial in the initial phase of inflammation providing a nonphlogistic signal to macrophages before it comes from apoptotic cells.

Pulmonary accumulation of neutrophils is typical for active smokers who are also predisposed to multiple inflammatory and infectious lung diseases. We show that human neutrophil exposure to cigarette smoke extract (CSE) leads to an atypical cell death sharing features of apoptosis, autophagy and necrosis. Accumulation of tar-like substances in autophagosomes is also apparent. Before detection of established cell death markers, CSE-treated neutrophils are effectively recognized and non-phlogistically phagocytosed by monocyte-derived macrophages. Blockade of LOX-1 and scavenger receptor A, but not MARCO or CD36, as well as pre-incubation with oxLDL, inhibited phagocytosis, suggesting that oxLDL-like structures are major phagocytosis signals. Specific lipid (β-carotene and quercetin), but not aqueous, antioxidants increased the pro-phagocytic effects of CSE. In contrast to non-phlogistic phagocytosis, degranulation of secondary granules, as monitored by lactoferrin release, was apparent on CSE exposure, which is likely to promote pulmonary inflammation and tissue degradation. Furthermore, CSE-exposed neutrophils exhibited a compromised ability to ingest the respiratory pathogen, Staphylococcus aureus, which likely contributes to bacterial persistence in the lungs of smokers and is likely to promote further pulmonary recruitment of neutrophils. These data provide mechanistic insight into the lack of accumulation of apoptotic neutrophil populations in the lungs of smokers and their increased susceptibility to degradative pulmonary diseases and bacterial infections.

• Adhesins, Bacterial/metabolism
• Animals
• Antimicrobial Cationic Peptides/metabolism
• Bacterial Adhesion/physiology
• Chronic Periodontitis/immunology
• Chronic Periodontitis/microbiology
• Complement Activation
• Cysteine Endopeptidases/metabolism
• Gingipain Cysteine Endopeptidases
• Gingiva/microbiology
• Heme/metabolism
• Humans
• Immune Evasion/physiology
• Immunity, Innate/physiology
• Inflammation/microbiology
• Porphyromonas gingivalis/enzymology
• Protein Denaturation
• Receptors, Proteinase-Activated/metabolism
• Signal Transduction
• Substrate Specificity
• Virulence Factors

• R01 DE009761 NIDCR NIH HHS
• DE 09761 NIDCR NIH HHS

• Antimicrobial peptides
• Complement
• Inflammation
• Phagocytosis
• Protease
• Proteolysis
• Virulence

• Animals
• Antimetabolites
• Bromodeoxyuridine
• Cell Proliferation
• Data Interpretation, Statistical
• Dextrans
• Ferrosoferric Oxide
• Fluorescent Antibody Technique
• Infarction, Middle Cerebral Artery/pathology
• Iron
• Ischemic Attack, Transient/pathology
• Macrophages/physiology
• Magnetic Resonance Imaging
• Magnetite Nanoparticles
• Male
• Mice
• Mice, Inbred C57BL
• Microglia/pathology
• Monocytes/physiology
• Neutrophil Infiltration
• Oxides
• Phagocytes/physiology

• G9219675 Medical Research Council

• ATP-Binding Cassette Transporters/metabolism
• Amino Acid Sequence
• Animals
• Antigens, Surface/metabolism
• Apoptosis
• Caenorhabditis elegans/cytology
• Caenorhabditis elegans/embryology
• Caenorhabditis elegans/metabolism
• Caenorhabditis elegans Proteins/metabolism
• Embryonic Development
• Germ Cells/cytology
• Humans
• Ligands
• Membrane Proteins/metabolism
• Mice
• Milk Proteins/metabolism
• Models, Biological
• Molecular Sequence Data
• Mutation/genetics
• Phagocytosis
• Phosphatidylserines/metabolism
• Phospholipid Transfer Proteins/chemistry
• Phospholipid Transfer Proteins/metabolism
• Recombinant Fusion Proteins/metabolism
• Sequence Homology, Amino Acid
• Signal Transduction

• R01 GM067848 NIGMS NIH HHS
• R25 GM056929 NIGMS NIH HHS
• GM067848 NIGMS NIH HHS
• GM56929 NIGMS NIH HHS