Cladosporium cladosporioides ameliorates visceral hypersensitivity in irritable bowel syndrome-like mice by inhibiting dectin-1 signaling and mast cell activation

Abstract

BACKGROUND

Gut mycobiota dysbiosis contributes to visceral hypersensitivity (VH) in irritable bowel syndrome (IBS); however, the roles of colony-specific fungi in IBS-VH development remain unclear.

AIM

To investigate the effects of mucosal mycobiota in IBS-VH pathogenesis in vitro and in vivo along with the underlying mechanisms.

METHODS

In this study, 36 patients with diarrhea-predominant IBS and 29 healthy controls were enrolled for screening of biomarker fungi using internal transcribed spacer region sequencing. The relationship between fungal abundance and VH in patients was assessed. A trinitrobenzene sulfonic acid (TNBS)-induced IBS mouse model was used to explore the effect of a specific fungal species on IBS-VH. Colonic inflammatory cytokine levels, activation of mast cells, and dectin-1 signaling were assayed. A human mast cell line (HMC-1) was used to investigate the underlying mechanisms in vitro.

RESULTS

The results showed that Cladosporium was a characteristic fungal genus in the colonic mucosa of patients with diarrhea-predominant IBS. Oral gavage of Cladosporium cladosporioides (C. cladosporioides) improved IBS-VH in mice. In terms of the mechanisms, application of C. cladosporioides reduced colonic mucosal inflammatory cytokine levels, inhibited the activation of mast cells, and downregulated dectin-1/nuclear factor kappa B (NF-κB) signaling in the TNBS-induced IBS mouse model. Studies in HMC-1 cells indicated that cotreatment with C. cladosporioides inhibited the dectin-1 agonist curdlan-induced activation of NF-κB signaling and upregulation of inflammatory cytokines.

CONCLUSION

Cladosporium, a characteristic colony-specific fungal genus enriched in IBS patients, improved TNBS-induced IBS-VH in mice by inhibiting dectin-1/NF-κB signaling and mast cell activation. C. cladosporioides is a promising fungal species that protects against the IBS-VH.

Key Words: Cladosporium; Mycobiota; Mast cell; Dectin-1; Visceral hypersensitivity

Core Tip: Gut mycobiota are linked to visceral hypersensitivity (VH) in irritable bowel syndrome (IBS); however, the specific role of fungi in IBS remains unexplored. This study explored the role of the gut mycobiota in VH associated with diarrhea-predominant IBS. We identified Cladosporium as a key fungal genus enriched in diarrhea-predominant IBS patients and demonstrated that Cladosporium cladosporioides (C. cladosporioides) alleviates IBS-VH in mice. Mechanistically, C. cladosporioides reduces inflammatory cytokine levels, inhibits mast cell activation, and downregulates dectin-1/nuclear factor kappa B signaling. These findings suggest C. cladosporioides as a potential therapeutic probiotic for IBS-VH, highlighting the role of this gut fungus in modulating IBS pathogenesis.

INTRODUCTION

Irritable bowel syndrome (IBS) is a gastrointestinal disease with a prevalence of 7%-21% worldwide[1]. Due to recurrent abdominal pain and altered bowel habits, IBS has a substantial impact on an individual’s life quality and social function[2]. However, the pathophysiological mechanisms of IBS are not fully understood. Multiple mechanisms, including dysfunction of the gut-brain axis, visceral hypersensitivity (VH), gut microbiota dysbiosis, impaired gut barrier function, and altered gut motility, contribute to the development of IBS. Among these factors, VH is a central mechanism and, as such, serves as a key biomarker in IBS[3]. More than 60% of patients with IBS are diagnosed with VH, which is also considered to underlie the suboptimal therapeutic response observed in IBS patients[4].

As a disorder of gut-brain interactions, microbiota play a critical role in the cross talk between the gut and brain. The involvement of bacterial microbiota in IBS has been widely studied. Previous studies have shown that Lactiplantibacillus plantarum D266 regulates gut physiology and enteric neurons through tryptophan metabolism and that Bifidobacterium longum NCC3001 reduces anxiety scores, thus improving quality of life in patients with IBS[5,6]. However, we have noted that fungal dysbiosis is also associated with VH, both in patients with IBS and in rat models[7]. Although the compositional changes of mycobiota in IBS have been revealed by the internal transcribed spacer (ITS) region sequencing, the variations within fungal species should not be overlooked. It has been demonstrated that Candida albicans has genetic and phenotypic diversities, which were linked to the differential levels of visceral sensitivity[8]. Identification of the specific fungal strains contributing to the development or amelioration of IBS is of significance not only to confirm the involvement of mycobiota in IBS but also to build the cause-and-effect relationship between mycodysbiosis and IBS as well as to develop new treatments for IBS.

C-type lectin receptors, including dectin-1, dectin-2, and mincle, and toll-like receptors mediate the cellular response to fungi. Our previous work revealed the involvement of dectin-1 in primary sensory neurons in VH in a mouse model of IBS[9]. Mast cells, which have been shown to play a critical pathophysiological role in the development of VH in IBS[10-13], respond to fungal stimulation via dectin-1 signaling[14,15]. Therefore, the roles of mast cells in mycobiota alterations associated with VH in IBS deserve further investigation.

Gut mycobiota include both the luminal and mucosa-associated mycobiota. Mucosal resident fungi are in close proximity to host cells, enabling their direct interactions. In the current study, we compared the colonic mucosal mycobiomes of patients with IBS and healthy volunteers as well as identified the characteristic mucosal fungi in IBS patients. Furthermore, we evaluated the effects of an IBS-specific fungus, Cladosporium cladosporioides (C. cladosporioides), on VH using a trinitrobenzene sulfonic acid (TNBS)-induced IBS-like mouse model and explored the potential mechanisms.

MATERIALS AND METHODS

Patient characteristics and sample collection

Thirty-six patients with diarrhea-predominant IBS (IBS-D), meeting the Rome IV diagnostic criteria, and 29 contemporaneously age- and gender-matched healthy controls were enrolled in this study between June 2019 and December 2019 at the Department of Gastroenterology of Peking University First Hospital (Beijing, China). The IBS severity scoring system (IBS-SSS) was employed, since previous studies have demonstrated that it can be used to evaluate the VH of IBS patients[16,17]. The flow chart of this study is shown in Supplementary Figure 1. Colonic mucosal tissues were collected from the cecum during colonoscopy, immediately snap-frozen with liquid nitrogen, and stored at -80 °C. All patients with IBS-D and healthy volunteers provided written informed consent. This study was approved by the Ethics Committee of Peking University First Hospital (No. 2019-074), registered on Chinese Clinical Trials Registry (ChiCTR2400079961, Registered January 17, 2024). All authors had access to the study data and reviewed and approved the final manuscript.

Fungal ITS region amplicon sequencing

The gut mycobiota profiles were evaluated based on the fungal ITS amplicon sequences. ITS region sequencing was performed by CapitalBio Technology using the Illumina NovaSeq platform. Barcoded fungal ITS-1-1F amplicons were generated using Phusion High-Fidelity PCR Master Mix (New England Biolabs, MA, United States) with the following primer pair: Forward 5’-TTGGTCATTTAGAGGAAGTAA-3’ and reverse 5’-GCTGCGTTCTTCATCGATGC-3’. Sequencing libraries were generated using the TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, CA, United States). After data filtration using QIIME and chimeric read removal using the UCHIME algorithm, sequences with ≥ 97% similarity were assigned to the same operational taxonomic units (OTUs) and then annotated against the UNITE database. Alpha and beta diversity were analyzed to evaluate community diversity. Partial least squares discriminant analysis, principal coordinate analysis (PCoA), and constrained PCoA as well as heatmap creation were performed using R software. Linear discriminant analysis effect size analysis was conducted using the online tool http://huttenhower.sph.harvard.edu/galaxy. The default filtering value for the linear discriminant analysis score was set to 4.

Mouse model with VH establishment

The IBS-like VH mouse model was established by colonic TNBS stimulation as previously described[18]. After an overnight fast, the TNBS-treated plus vehicle group (TNBS + vehicle group) and TNBS-treated plus C. cladosporioides gavage group (TNBS + C. cladosporioides group) were placed under anesthesia with 1% pentobarbital (50 mg/kg, intraperitoneal) and perfused intrarectally with 0.1 mL of 130 μg/mL TNBS (P2297, Sigma-Aldrich, Shanghai, China) dissolved in 30% ethanol using a flexible catheter (outer diameter = 2 mm) inserted into the descending colon to a depth of 2 cm from the anus. Meanwhile, the saline plus vehicle group (saline + vehicle group) and saline plus C. cladosporioides (6721, American Type Culture Collection, Manassas, VA, United States) gavage group (saline + C. cladosporioides group) received an enema containing 0.1 mL of 0.9% NaCl. Mice were maintained in the tail-up position for 2 minutes to prevent drug leakage and then allowed to recover for 28 days, after which VH was confirmed using the abdominal withdrawal reflex response to graded colorectal distention.

Animal experimental design and colony-specific fungi, Cladosporium, gavage treatment protocol

Nine-week-old male C57BL/6J mice (20 ± 0.3 g) were purchased from SpePharm Biotechnology (Beijing, China) and housed (5 mice/cage) in a specific pathogen-free room at a constant temperature and humidity (22 ± 1 °C, 55% ± 10%) with a 12-hour light/dark cycle. Water and food were provided ad libitum. The experimental design was approved by the Ethics Committee of Peking University First Hospital and followed the institutional guidelines for the care and use of laboratory animals (Ethics No. LA2021501).

After 1 week of acclimation, the mice were randomly divided into four groups (n = 7 mice/group): Saline + vehicle group, saline + C. cladosporioides group, TNBS + vehicle group, and TNBS + C. cladosporioides group. From day 1, the saline + C. cladosporioides and TNBS + C. cladosporioides groups were treated with a 200-μL C. cladosporioides spore suspension (5 × 10⁶ spores/mL) by gavage three times a week. The saline + vehicle and TNBS + vehicle groups were administered 200 μL of sterile water by gavage following the same schedule. The flow chart of model establishment and fungal gavage is shown in Figure 1A. The fungal spore suspension was prepared as follows. Lyophilized C. cladosporioides was suspended in sterile distilled water and incubated on potato dextrose agar at 28 °C. After reaching the logarithmic growth phase, spores were collected and suspended in sterile distilled water, filtered to remove hyphae, centrifuged at 1400 × g for 5 minutes, and finally resuspended at 5 × 10⁶ spores/mL.

Figure 1 Administration of Cladosporium cladosporioides attenuated visceral hypersensitivity and gut motility in trinitrobenzene sulfonic acid-treated mice. A: Schematic diagram showing the timeline of model establishment, Cladosporium cladosporioides gavage, sample collection and behavioral observation; B: Visceral hypersensitivity in mice evaluated by CRD-AWR at 20, 40, 60, and 80 mmHg. Tow-way ANOVA; C: Fecal weight, frequency, and water content; D: Representative images of hematoxylin and eosin-stained tissues (n = 7). Scale bar = 100 μm. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. TNBS: Trinitrobenzene sulfonic acid.

Evaluation of visceral sensitivity

VH was evaluated by measuring the intensity of the abdominal withdrawal reflex after colorectal distention as previously reported[19]. On day 28, mice were fasted for 12 hours and anesthetized by 2% isoflurane inhalation for 10 seconds. Subsequently, a catheter (2-mm diameter) with a balloon (1-mL maximum volume) lubricated with glycerol was inserted into the rectum until the balloon was 1-cm deep from the anal margin. The mice were fixed in a fitted box, and their tails were fastened. After recovery from anesthesia, the balloon was inflated to pressures of 20, 40, 60, and 80 mmHg for 20 seconds each. The mean abdominal withdrawal reflex score was calculated from three repeated distention tests to represent visceral sensitivity.

The abdominal withdrawal reflex score was evaluated according to the A1-Chaer standard[19] by two researchers blinded to the grouping information. Briefly, a score of 0 indicated no behavioral response to graded colorectal distention; 1, brief head movement followed by immobility; 2, contraction of abdominal muscles; 3, lifting of the abdomen; 4, body arching and lifting of the pelvis.

Sacrifice and sample collection

On day 26, we measured stool frequency for 2 hours and calculated the water content of the mouse feces. On day 27, the colon fecal contents were collected, snap-frozen with liquid nitrogen, and stored at -80 °C. On day 28 after VH evaluation, blood was collected by eyeball enucleation after anesthesia, and the mice were subsequently euthanized by cervical dislocation. The colorectal tissues were removed and stored at -80 °C in a 40 g/L formaldehyde solution. Plasma was obtained after blood centrifugation at 3000 rpm and 4 °C for 10 minutes.

Hematoxylin and eosin staining

Fresh colon samples were immediately fixed in 4% paraformaldehyde solution (DF0135, Leagen, Beijing, China) for 24 hours, embedded in paraffin, cut into 4-μm-thick sections, and stained with hematoxylin and eosin. Stained areas were viewed under a light microscope (Olympus, Tokyo, Japan), and the adipocyte size was measured using ImageJ software 1.53t (National Institutes of Health, Bethesda, MD, United States) at 40 × magnification.

Enzyme-linked immunosorbent assay

We analyzed the concentrations of interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and monocyte chemoattractant protein-1 (MCP-1) in colonic tissue and homogenates of a human mast cell line (HMC-1) using an enzyme-linked immunosorbent assay (ELISA) kit, according to the manufacturer’s instructions (Mouse IL-1β, KE10003-96T, Proteintech, Wuhan, Hubei Province, China; Mouse TNF-α, ELM-TNFa-1, RayBiotech, Guangzhou, Guangdong Province, China; Mouse MCP-1, SEA087Mu, Cloud-Clone, Wuhan, Hubei Province, China; Human IL-1β, KE00021, Proteintech, Wuhan, Hubei Province, China; Human TNF-α, KE00154, Proteintech, Wuhan, Hubei Province, China). The plasma tryptase levels were determined using an ELISA kit (SEB070Mu, Cloud-Clone, Wuhan, Hubei Province, China), according to the manufacturer’s instructions.

Reverse transcription-quantitative polymerase chain reaction

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the dectin-1/spleen tyrosine kinase (syk)/caspase recruitment domain family member 9 (card9) pathway as well as tryptase, IL-1β, TNF-α, and MCP-1 mRNA expression. Total RNA was isolated from colon samples using TRIzol reagent (P108, GenStar, Beijing, China), according to the manufacturer’s protocol. First-strand cDNA was synthesized from total RNA using a reverse transcription cDNA synthesis kit (A231, GenStar, Beijing, China). The cDNA was analyzed by qPCR with a SYBR Green PCR kit (A311, GenStar, Beijing, China) on a 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, United States), following the manufacturer’s instructions. Relative gene expression was calculated using the 2^-ΔΔCt method, with GAPDH used as an internal control. The primer sequences for the genes are listed in Supplementary Tables 1 and 2.

Western blot analysis

Western blotting was used to detect dectin-1/nuclear factor kappa B (NF-κB) pathway protein expression in colon tissue and HMC-1 cells. Colonic biopsy samples or cells were homogenized in ice-cold radioimmunoprecipitation assay lysis buffer (P0013B, Beyotime, Shanghai, China) supplemented with protease inhibitors (P1050, Beyotime, Shanghai, China). The homogenate was centrifuged at 12000 rpm for 15 minutes, and the protein concentrations were determined using a bicinchoninic acid assay. Protein samples were electrophoresed on 12% Tris sodium dodecyl sulfate-polyacrylamide gel for 30 minutes at 80 V and then 90 minutes at 120 V. The separated proteins were transferred onto polyvinylidene difluoride membranes (IPVH00010, MilliporeSigma, Germany) for 1 hour at 300 mA. The membranes were blocked with tris-buffered saline containing Tween 20 supplemented with 0.5% skim milk (232100, Becton Dickinson, Franklin Lakes, NJ, United States) for 1 hour at 24 °C, probed with primary antibodies against dectin-1 (ab140039, 1:1000, Abcam, United Kingdom), p-syk (2715, 1:1000, CST, Beverly, MA, United States), syk (13198T, 1:1000, CST, Beverly, MA, United States), p-p65 (8242T, 1:1000, CST, Beverly, MA, United States), p65 (3033, 1:1000, CST, Beverly, MA, United States), tryptase (ab151757, 1:1000, Abcam, United Kingdom), and β-actin (AC026, 1:400000, ABclonal, Wuhan, Hubei Province, China) at 4 °C overnight, and then washed in tris-buffered saline containing Tween 20 for 40 minutes. The membranes were probed with the corresponding horseradish peroxidase-conjugated secondary antibodies (1:16000, ZSGB-Bio, Beijing, China) for 1 hour at room temperature. Signals were quantified using ImageJ 1.53t (National Institutes of Health) and normalized to the controls.

Cell culture

HMC-1 cells (DSMZ, Braunschweig, Germany) were grown in RPMI 1640 medium (C11875500BT, Thermo, MA, United States) supplemented with 10% fetal bovine serum (F8687, Sigma, St. Louis, MO, United States) and 1% penicillin-streptomycin (15140163, Thermo, MA, United States) at 37 °C and 50 mL/L CO₂. Cell viability was determined with cell counting kit 8 (CCK8) assays. Briefly, 5000 cells/well were seeded into 96-well plates and were incubated with different concentrations of C. cladosporioides at multiplicities of infection (MOIs) of 0.25:1, 0.5:1, 1:1, 2:1, 5:1, 10:1, and 15:1. After 48 hours, 10 μL of CCK8 solution (CA1210, Solarbio, Beijing, China) was added to each well and the cells were incubated for 8 hours before the absorbance was measured at 450 nm.

Detection of mast cell activation

The early-phase degranulation of mast cells was detected using a β-hexosaminidase release assay. HMC-1 cells (2 × 10⁶/mL) were stimulated with or without spores (MOIs of 0.25:1, 0.5:1, 1:1, 2:1, 5:1, and 10:1) of C. cladosporioides or with 10 μM inomycin in supplemented RPMI 1640 medium for 2 hours at 37 °C. After centrifugation, the supernatants were collected and the remaining cells were lysed with 0.1% Triton X-100 (P1080, Solarbio, Beijing, China). Then, 50 μL of each sample was mixed with 50 μL of 7.5 mmol/L p-nitrophenyl-N-acetyl-β-D-glucosaminide (487052, Sigma-Aldrich, Shanghai, China) dissolved in 0.1 M citric acid buffer (pH = 4.5) in 96-well plates, mixed gently, and then incubated at 37 °C for 120 minutes. The reaction was stopped with 100 μL of 0.2 M glycine-NaOH (pH = 10.7). The absorbance at 405 nm was measured using a microplate reader (Thermo Fisher Scientific, Waltham, MA, United States). The percentage of β-hexosaminidase released was calculated as follows: Optical density (OD) in supernatant/(OD in supernatant + OD in pellet) × 100%. The late-phase detection of cytokine release of mast cells is detailed in the ELISA section.

Curdlan-stimulated activation of dectin-1

HMC-1 cells were incubated in 48-well plates with different concentrations of C. cladosporioides at MOIs of 0.25:1, 0.5:1, 1:1, 2:1, 5:1, and 10:1, with or without the dectin-1 agonist curdlan (tlrl-curd, InvivoGen, Toulouse, France) at 350 μg/mL for 24 hours. Cells were harvested, and the inflammatory cytokines IL-1 and TNF-α, along with the dectin-1/NF-κB signaling proteins were extracted for subsequent detection, as described for ELISA and western blotting.

Statistical analysis

Data analysis was performed using the Student’s t-test for pairwise comparisons between groups, while two-way analysis of variance (ANOVA) or one-way ANOVA followed by Tukey’s post hoc test was used for multiple group comparisons (GraphPad Prism 10, GraphPad Software, San Diego, CA, United States); P < 0.05 was considered significant. Nonparametric tests were used to assess non-normally distributed data. Spearman’s correlation analysis was used to analyze the relationship between Cladosporium abundance and visceral sensitivity. Repeated-measurement data are presented as the mean ± SD, enumeration data are presented as a n (%), and the abdominal withdrawal reflex scores are presented as (P₂₅, P₇₅).

RESULTS

Screening of the characteristic fungi in the intestinal mucosa of patients with IBS

A total of 36 IBS-D patients and 29 healthy volunteers were recruited in this study. Their colonic mucosal tissues were collected from the cecum during colonoscopy. Ultimately, DNA libraries were successfully constructed for 14 samples from the patients and 12 samples from the control group. The characteristics of the participants are shown in Table 1. The demographic parameters, diet, anxiety scores, and depression scores did not differ between the two groups. ITS sequencing indicated that the Simpson index of mycobiota diversity was higher in patients with IBS than in healthy volunteers (Figure 2A, P < 0.05). The composition of fungal genera in the mucosal tissues varied between the IBS patients and the healthy controls (Figure 2B). PCoA and partial least squares discriminant analysis indicated that the β-diversity of the mucosal mycobiota community differed between the patients and healthy controls (P < 0.05, Adonis test; Figure 2C and D) at the OTU level. The variable importance in the projection scores revealed that OTU22 (Cladosporium spp.) was the top contributor to differences between the groups (Figure 2E). Linear discriminant analysis effect size analysis indicated that Cladosporium and Fomitopsis were biomarkers for patients with IBS (Figure 2F and G). Cladosporium and Cryptococcus showed the most significant differences between the two groups (fold change > 2, false discovery rate < 0.1; P < 0.05, Wilcoxon test; Figure 2H). Cladosporium was much more abundant in patients with IBS (P < 0.01, P-adj. = 0.079), while Cryptococcus was more abundant in healthy controls (P < 0.01, P-adj. = 0.079). The other 20 predominant mucosal genera are shown in Figure 2I. Spearman correlation analysis showed that the abundance of Cladosporium was negatively associated with the IBS-SSS score (r = -0.76, P < 0.01, Figure 2J). These results suggest that the intestinal mucosal fungal composition differs between the IBS and control groups and that Cladosporium is an IBS-specific fungus showing a negative correlation with VH.

Figure 2 Colonic mucosal mycobiota differ between patients with diarrhea-predominant irritable bowel syndrome and healthy controls. A: Simpson index of α-diversity; B: Venn plot of gut mucosal mycobiota; C: Principal coordinate analysis of β-diversity based on unweighted UniFrac distances; D: Partial least squares discriminant analysis of β-diversity; E: VIP scores of the top 15 operational taxonomic units; F: Biomarker fungus for patients with diarrhea-predominant irritable bowel syndrome (IBS-D) revealed by linear discriminant analysis effect size LDA; G: Linear discriminant analysis effect size cladogram of biomarker fungus; H: Mucosal fungi with differential abundances identified based on fold change and Wilcoxon analysis (a, refers to IBS-D. b refers to healthy controls); I: Bar plot of 20 most abundant mucosal genera in patients with IBS-D and healthy controls; J: Spearman correlation between Cladosporium abundance and irritable bowel syndrome severity scoring system score (r = -0.73, P < 0.01). IBS: Irritable bowel syndrome; PCoA: Principal coordinate analysis; OUT: Operational taxonomic unit; IBS-SSS: Irritable bowel syndrome severity scoring system.

Table 1 Characteristics of study participants, n (%)/mean ± SD.

	IBS (n = 14)	Healthy controls (n = 12)
Age	49.0 ± 12.0	49.9 ± 8.4
Sex (% females)	7 (50.0)	7 (58.3)
BMI	22.4 ± 3.5	22.2 ± 2.9
Alcohol use	2 (14.3)	1 (8.3)
Current smoker	1 (7.1)	1 (8.3)
HADS depression score	3.9 ± 2.1	3.5 ± 1.9
HADS anxiety score	4.6 ± 2.6	3.3 ± 2.1
Medication use (%)
PPI	1 (7.1)	-
Probiotics	1 (7.1)	-
SSRI	0	-
Spasmolytics	1 (7.1)	-
Antibiotics	0	-
Diet model (% vegetarian)	8 (57.1)	5 (41.7)
IBS-SSS score	233.7 ± 131.1	-
Abdominal pain	54.6 ± 15.5
Bloating	54.9 ± 15.8
Abdominal pain frequency	55 ± 26
Bowel dissatisfaction	60.0 ± 19.3
Quality of life	59.2 ± 18.3
Diarrhea frequency > 4 times/week (%)	11 (78.6)	-

IBS: Irritable bowel syndrome; BMI: Body mass index; HADS: Hospital Anxiety and Depression Scale; PPI: Protein-protein interaction; SSRI: Selective serotonin reuptake inhibitor; IBS-SSS: Irritable bowel syndrome severity scoring system.

Protective role of C. cladosporioides in a TNBS-induced IBS-like VH mouse model

The negative correlation between Cladosporium abundance and the VH level attracted us to explore the role of Cladosporium in a mouse model of TNBS-induced VH. BLAST analysis of the OTU22 sequence with the National Center for Biotechnology Information and UNITE databases indicated a 100% match with the species C. cladosporioides. Hence, the potential effect of C. cladosporioides, a typical species of Cladosporium, on VH in IBS was examined. Intrarectal administration of TNBS was used to induce an IBS model in mice, and sterile water was used as a vehicle control. C. cladosporioides was administered to mice via oral gavage three times a week from day 1 of modeling. The results of ITS sequencing at 27 days post oral gavage showed that C. cladosporioides effectively colonized the gut. Notably, the bacterial and fungal microecology in IBS mice shifted toward that in the control group (Supplementary Figures 2 and 3).

Compared to the saline plus vehicle group, the TNBS-treated plus vehicle group showed greater visceral sensitivity at 20 (TNBS + vehicle vs saline + vehicle, P < 0.05, Figure 1B), 40 (TNBS + vehicle vs saline + vehicle, P < 0.05, Figure 1B), 60 (TNBS + vehicle vs saline + vehicle, P < 0.05, Figure 1B) and 80 mmHg (TNBS + vehicle vs saline + vehicle, P < 0.05, Figure 1B). Moreover, the TNBS-treated plus vehicle group showed enhanced intestinal motility and watery stools, which was consistent with the IBS-D subtype. After 4 weeks of gavage with C. cladosporioides, visceral sensitivity at 60 mmHg in the TNBS-treated plus C. cladosporioides gavage group was significantly lower than that treated with TNBS alone [(1.6, 2.3) vs (2.3, 3.1), P < 0.05; Figure 1B]. Gavage with C. cladosporioides did not alter the extent of visceral sensitivity in the vehicle-treated mice. The TNBS-treated plus vehicle group showed a higher fecal weight, frequency, and water content than the saline plus vehicle group, which was reversed by gavage with C. cladosporioides (P < 0.05; Figure 1C). Gavage with C. cladosporioides did not change the fecal parameters in the saline group. In addition, hematoxylin and eosin staining of the intestinal tissues showed an intact epithelial structure with no obvious infiltration of inflammatory cells or tissue edema in any of the four groups (Figure 1D).

C. cladosporioides downregulated dectin-1/NF-κB signaling

Next, we explored the potential mechanisms mediating the attenuating effects of C. cladosporioides on IBS-like symptoms. The expression of the dectin-1 and its associated signaling molecules in colonic tissue was detected. The mRNA expression of dectin-1/syk/card9 signaling pathway members in the TNBS-treated plus vehicle group was significantly higher than that in the saline plus vehicle group (Figure 3A). Importantly, the elevated mRNA levels of the dectin-1/syk/card9 signaling pathway members were suppressed by C. cladosporioides gavage (P < 0.05, Figure 3A). Furthermore, western blot analysis confirmed that gavage with C. cladosporioides inhibited the upregulation of dectin-1 (P < 0.0001) and phosphorylation of the downstream syk and NF-κB p65 proteins in the TNBS-treated mice (Figure 3B and C). These findings suggest that application of C. cladosporioides may suppress dectin-1/NF-κB signaling via unknown mechanisms.

Figure 3 Gavage with Cladosporium cladosporioides downregulated dectin-1/nuclear factor kappa B signaling. A: Reverse transcription-quantitative polymerase chain reaction of dectin-1/spleen tyrosine kinase/caspase recruitment domain family member 9 mRNA expression among groups. One outlier identified in saline + vehicle group in the mRNA expression analysis of caspase recruitment domain family member 9 and removed with Turkey outlier analysis; B: Western blot analysis of dectin-1/p65 protein expression; C: Quantification of dectin-1 protein levels normalized to β-actin levels. Data are presented as the mean ± SD. Two-way ANOVA (n = 7). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. TNBS: Trinitrobenzene sulfonic acid; Syk: Spleen tyrosine kinase; Card9: Caspase recruitment domain family member 9; NF-κB: Nuclear factor kappa B.

Mast cell activation and mucosal microinflammation are suppressed by C. cladosporioides in the VH mouse model

Activation of mast cells and the associated microinflammation is a key mechanism of VH in IBS; hence, we determined whether the expression of mast cell-associated tryptase and cytokines in colonic mucosa were influenced by C. cladosporioides treatment. The protein (P < 0.05, Figure 4A and B) and mRNA (Figure 4C) levels of tryptase and its expression in serum increased in the TNBS-treated plus vehicle group, all of which were rescued by gavage with C. cladosporioides (P < 0.05). Gavage with C. cladosporioides in TNBS-treated mice also decreased the mRNA expression of Il-1β, Tnf-α, and Mcp1 in the colonic tissue (P < 0.05, Figure 4D). ELISA of colonic tissue also confirmed that the increased protein level of IL-1β was reversed by C. cladosporioides gavage (P < 0.05, Figure 4E and F). Spearman correlation between the Cladosporium genus, colonic IL-1β, and serum mast cell tryptase is shown in Supplementary Figure 4. These results indicate that C. cladosporioides suppressed mast cell activation and inhibited microinflammation in the colonic mucosa of the VH mouse model.

Figure 4 Gavage with Cladosporium cladosporioides inhibited colonic inflammation and mast cell activation. A: Mast cell tryptase expression based on western blot analysis of colonic mucosa; B: Quantification of tryptase protein levels normalized to β-actin levels; C: Mast cell tryptase mRNA expression based on reverse transcription-quantitative polymerase chain reaction of colonic mucosa. One outlier identified and removed with Turkey outlier analysis in saline + Cladosporium cladosporioides group; D: Cytokine mRNA expression based on reverse transcription-quantitative polymerase chain reaction of colonic mucosa; E: Serum mast cell tryptase detected using enzyme-linked immunosorbent assay; F: Cytokine expression in colonic mucosa detected using enzyme-linked immunosorbent assay. Data are presented as the mean ± SD. Two-way ANOVA (n = 7). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. TNBS: Trinitrobenzene sulfonic acid; MCP: Monocyte chemoattractant protein; TNF: Tumor necrosis factor; IL: Interleukin.

C. cladosporioides suppresses mast cell activation by downregulating dectin-1/NF-κB signaling

To further explore the mechanisms leading to suppression of mast cell activation, we assessed whether C. cladosporioides influenced the dectin-1 signaling pathway in mast cells. We used HMC-1 cells, a human mast cell line, to perform the in-vitro studies. The potential effect of C. cladosporioides on cell survival was determined using a CCK8 assay. The cell viability was not reduced at a MOI of 0.25-10 for C. cladosporioides, but it also increased at a MOI of 0.25-5, although it was reduced at a MOI of 15 (Figure 5A). A β-hexosaminidase release assay showed no obvious degranulation in HMC-1 cells treated with C. cladosporioides at a MOI of 0.25-10 (Figure 5B). However, C. cladosporioides at a MOI of 5-10 significantly suppressed the mRNA expression of IL-1β and TNF-α in HMC-1 cells (P < 0.05, Figure 5C). Analysis of the cell homogenates confirmed the reduced levels of inflammatory cytokines at a MOI of 5-10 (P < 0.05, Figure 5D). As a positive control, application of curdlan (350 μg/mL), a dectin-1 agonist, significantly upregulated the expression of dectin-1/NF-κB pathway molecules and the expression of IL-1β and TNF-α. However, all of these changes were significantly inhibited by combined treatment with C. cladosporioides at a MOI of 0.25-10 (Figure 5E and F) (P < 0.05, Figure 5G), especially at higher MOIs. These findings indicate that C. cladosporioides can suppress the release of inflammatory factors from mast cells by downregulating the dectin-1/NF-κB pathway.

Figure 5 Cladosporium cladosporioides suppresses mast cell activation by inhibiting dectin-1/nuclear factor kappa B signaling. A: Cell counting kit 8 assay of mast cell viability following coculture with Cladosporium cladosporioides (C. cladosporioides) at different multiplicities of infection (MOIs) for 48 hours. n = 6, one outlier identified and removed with Turkey outlier analysis in MOI 5; B: Mast cell degranulation detected using β-hexosaminidase releasing assay after treatment at different MOIs for 2 hours; C: Interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) mRNA expression in mast cells detected using reverse transcription-quantitative polymerase chain reaction after treatment at different MOIs for 12 hours; D: IL-1β and TNF-α expression in mast cells detected using enzyme-linked immunosorbent assay after treatment at different MOIs for 24 hours; E: Dectin-1/nuclear factor kappa B expression in mast cells stimulated with curdlan (350 μg/mL) and cocultured with C. cladosporioides at different MOIs for 24 hours; F: Quantification of dectin-1 protein levels normalized to β-actin levels, and p-p65 levels normalized to p65 levels; G: IL-1β and TNF-α expression detected using enzyme-linked immunosorbent assay after treatment with curdlan and C. cladosporioides at different MOIs for 24 hours. Data are presented as the mean ± SD. One-way ANOVA followed by Tukey’s test and Student’s t-test (n = 3). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 compared to the vehicle group; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001 compared to the curdlan 350 μg/mL group. MOI: Multiplicity of infection; Iono: Ionomycin; Cur 350: Curdlan 350 μg/mL; TNF: Tumor necrosis factor; IL: Interleukin.

DISCUSSION

The management of abdominal pain patients with VH as a biological marker is a significant challenge in the clinical treatment of IBS. Recent studies have shown that the gut-microbiota-brain axis plays a crucial role in the development of VH. While extensive research has focused on intestinal bacteria, emerging evidence suggests that intestinal fungi are also implicated in VH development[7]. However, the influence of intestinal fungi on host homeostasis remains largely unclear. One of the critical reasons can be attributed to the low abundance of intestinal fungi, making them difficult to be isolated and characterized. The few existing studies primarily focus on fecal fungi, whose correlation with the local microenvironment in the gut is weaker than that of mucosal fungi. Hence, we collected the colonic mucosal samples in the current studies. However, a challenge in creating a library of mucosal fungi lies in the low yield of mucosal fungal DNA extraction. It is notable that libraries were successfully constructed only in one-third of samples in this study. The results of ITS sequencing highlighted a characteristic fungal genus of the intestinal mucosal mycobiome, Cladosporium, in the IBS-D patients. A previous study showed that Cladosporium exacerbated airway hyper-responsiveness[20]. However, our study indicated that the severity of abdominal pain in IBS patients seems to have a negative correlation with the abundance of Cladosporium (r = -0.76, P < 0.01, Figure 2J), suggesting that Cladosporium may play a potential protective role against IBS-VH.

We further investigated the role of Cladosporium in an IBS-like VH model in mice. Our studies indicated that C. cladosporioides gavage not only ameliorated VH but also improved gut motility and fecal parameters. Therefore, C. cladosporioides shows the ability to improve the overall intestinal function. Notably, C. cladosporioides gavage shifted the bacterial and fungal microecology in IBS mice toward that of control mice. Based on these findings, we propose that the increased Cladosporium abundance in patients with IBS is likely a compensatory response to elevated VH over a long time. Under this hypothesis, the host may selectively maintain or recruit Cladosporium as an adaptive mechanism to alleviate chronic mucosal stress, although this endogenous compensation might not be sufficient to fully reverse the disease phenotype in all patients. Unlike the previous findings where fungal dysbiosis was predominantly characterized by the overgrowth of pro-inflammatory taxa like Candida albicans[8], our study identified C. cladosporioides as a potential protective symbiont in the IBS-D mucosa. The significant negative correlation between its abundance and IBS-SSS scores suggests a potential probiotic effect in the host. In recent years, emerging evidence has shown the probiotic effects of gut fungi. The probiotic fungi Saccharomyces boulardii and Saccharomyces cerevisiae have been shown to relieve abdominal discomfort[21], regulate gastrointestinal motility, and improve the abnormal intestinal changes in IBS[22]. Like these two probiotic fungi, C. cladosporioides represents a promising candidate to act as an autochthonous probiotic that may contribute to intestinal homeostasis.

Next, we sought to investigate the mechanisms underlying the potential protective action of Cladosporium in IBS. The pattern recognition receptor dectin-1 is a major mediator of fungal recognition, which further activates host immunity[23]. Previous studies have indicated that dectin-1 signaling in primary sensory neurons induces sensitization of the transient receptor potential vanilloid 1 and participates in TNBS-induced IBS-VH in mice[9,24,25]. The current work further confirmed that the intestinal dectin-1/NF-κB pathway was significantly activated in the IBS-VH mouse model, consistent with previous findings[7,26]. Of note, this upregulation of the dectin-1/NF-κB pathway was significantly inhibited by gavage with C. cladosporioides, suggesting that C. cladosporioides may abrogate IBS-VH by inhibiting dectin-1/NF-κB signaling. However, the role of dectin-1 in fungal recognition and visceral sensation is more complex. A previous study showed that although viable spores of C. cladosporioides possess the intrinsic potential to trigger dectin-1 as fungal entities, they do not elicit functional activation of the dectin-1 receptor[20]. This failure to activate dectin-1 - and the subsequent suppression of the NF-κB pathway - likely underlies the fungus’ ability to stabilize mast cells and mitigate VH.

Mast cells participate in the immune recognition of fungi[15], which are also crucial effector cells in IBS-VH[27]. Multiple studies have shown that the number of mast cells was significantly increased in the jejunum, cecum, ascending colon, descending colon, and rectum of patients with IBS than that in healthy individuals[28-30]. In addition to the increased number, the tryptase and histamine activities associated with the mast cells were also elevated in the colonic mucosa[31], which can activate the enteric neurons and visceral afferents, leading to neuronal hyperexcitability[32-34]. In this study, we further confirmed that mast cells were significantly activated in the IBS-like VH mouse model. Importantly, gavage with C. cladosporioides significantly decreased mucosal mast cell activation, which might be attributed to the inhibition of the dectin-1 signaling pathway. Our study provides a mechanistic bridge by showing that inhibition of the dectin-1/NF-κB pathway by C. cladosporioides leads to a significant reduction in mast cell degranulation and the release of inflammatory cytokines like IL-1β and TNF-α. This reduction in the inflammatory milieu surrounding enteric nerves may account for the improved abdominal withdrawal reflex scores observed in our TNBS-induced mouse model.

In-vitro experiments with HMC-1 cells showed that treatment with C. cladosporioides reduced both the basal release and the dectin-1 agonist-induced release of inflammatory cytokines. In terms of the mechanisms, dectin-1/NF-κB signaling was suppressed along with the treatment of C. cladosporioides. Therefore, inhibition of mast cell activation could partially account for the potential protective effect of C. cladosporioides against IBS-VH. While our data demonstrate the suppression of the dectin-1/NF-κB signaling axis by C. cladosporioides, the upstream molecular triggers remain to be identified. This inhibitory effect may result from several distinct mechanisms. Metabolites produced by Cladosporium spp., including cladosporone A and cladosporol D, have been reported to inhibit cyclooxygenase-2 activity; calphostin C suppresses protein kinase C activity; and ergosterol peroxide inhibits the NF-κB and mitogen-activated protein kinases signaling pathways[35-37]. Therefore, C. cladosporioides may attenuate the dectin-1/NF-κB signaling pathway and mast cell inflammatory mediator expression through its bioactive products. Alternatively, β-glucan hiding - a strategy where fungi mask their stimulatory cell-wall components - could allow C. cladosporioides to bypass dectin-1 recognition, thereby reducing the overall inflammatory tone of the mucosa[20]. Lastly, the induction of immune tolerance in mucosal mast cells through chronic interaction with C. cladosporioides might recalibrate their threshold for degranulation. The exact molecular mechanisms involved in these effects need further investigation.

This study has some limitations. First, due to the low abundance of mucosal fungi, the number of successfully constructed libraries was limited. Further studies with larger sample sizes or the development of novel detection methods are required to investigate the composition and diversity of the mucosal mycobiota in the samples from IBS-D patients. Second, we used ITS sequencing to screen for the characteristic fungi at the genus level, consequently identifying Cladosporium. But it remains challenging to identify the specific Cladosporium species responsible for this finding. Although C. cladosporioides is a representative member of the genus Cladosporium[38], isolation and culture of C. cladosporioides from the patients’ samples will give more direct evidence for its distinct role in IBS-VH. Third, although our gavage experiments in mice demonstrated functional effects, the direct causal link in humans remains to be established. Future research employing fecal fungal transfer or patient-derived strain interventions is necessary to fully distinguish between a causal role and a compensatory association in the context of IBS-VH. Last, future research will focus on a multi-omics approach. Specifically, coupling untargeted metabolomics of C. cladosporioides with receptor-blocking assays will be essential to identify the active anti-inflammatory mediators and their downstream signaling pathways in mast cells.

CONCLUSION

In this study, we found that Cladosporium is a characteristic fungal genus of the intestinal mucosa in IBS-D patients and that its representative member, C. cladosporioides, was shown to attenuate VH in an IBS mouse model. The effect is at least partly due to the suppression of mast cell activation through inhibiting dectin-1/NF-κB signaling. Our findings emphasize the important role of fungi in IBS-VH pathogenesis and shed light on the development of new therapeutic approaches targeting gut mycobiota. Overall, our study elucidates the intricate fungi-host interactions in IBS and reveals Cladosporium as a potential probiotic fungus associated with promoting intestinal homeostasis.

ACKNOWLEDGEMENTS

The authors thank Professor Zhe Wan of Research Center for Medical Mycology of Peking University for her kind support in fungi-related experimental operations.