Crohn’s disease proteolytic microbiota enhances inflammation through PAR2 pathway in gnotobiotic mice

ABSTRACTEmerging evidence implicates microbial proteolytic activity in ulcerative colitis (UC), but whether it also plays a role in Crohn’s disease (CD) remains unclear. We investigated the effects of colonizing adult and neonatal germ-free C57BL/6 mice with CD microbiota, selected based on high (CD-HPA) or low fecal proteolytic activity (CD-LPA), or microbiota from healthy controls with LPA (HC-LPA) or HPA (HC-HPA). We then investigated colitogenic mechanisms in gnotobiotic C57BL/6, and in mice with impaired Nucleotide-binding Oligomerization Domain-2 (NOD2) and Protease-Activated Receptor 2 (PAR2) cleavage resistant mice (Nod2−/−; R38E-PAR2 respectively). At sacrifice, total fecal proteolytic, elastolytic, and mucolytic activity were analyzed. Microbial community and predicted function were assessed by 16S rRNA gene sequencing and PICRUSt2. Immune function and colonic injury were investigated by inflammatory gene expression (NanoString) and histology. Colonization with HC-LPA or CD-LPA lowered baseline fecal proteolytic activity in germ-free mice, which was paralleled by lower acute inflammatory cell infiltrate. CD-HPA further increased proteolytic activity compared with germ-free mice. CD-HPA mice had lower alpha diversity, distinct microbial profiles and higher fecal proteolytic activity compared with CD-LPA. C57BL/6 and Nod2−/− mice, but not R38E-PAR2, colonized with CD-HPA had higher colitis severity than those colonized with CD-LPA. Our results indicate that CD proteolytic microbiota is proinflammatory, increasing colitis severity through a PAR2 pathway

Proteolytic bacteria expansion during colitis amplifies inflammation through cleavage of the external domain of PAR2

International audienceImbalances in proteolytic activity have been linked to the development of inflammatory bowel diseases (IBD) and experimental colitis. Proteases in the intestine play important roles in maintaining homeostasis, but exposure of mucosal tissues to excess proteolytic activity can promote pathology through protease-activated receptors (PARs). Previous research implicates microbial proteases in IBD, but the underlying pathways and specific interactions between microbes and PARs remain unclear. In this study, we investigated the role of microbial proteolytic activation of the external domain of PAR2 in intestinal injury using mice expressing PAR2 with a mutated N-terminal external domain that is resistant to canonical activation by proteolytic cleavage. Our findings demonstrate the key role of proteolytic cleavage of the PAR2 external domain in promoting intestinal permeability and inflammation during colitis. In wild-type mice expressing protease-sensitive PAR2, excessive inflammation leads to the expansion of bacterial taxa that cleave the external domain of PAR2, exacerbating colitis severity. In contrast, mice expressing mutated protease-resistant PAR2 exhibit attenuated colitis severity and do not experience the same proteolytic bacterial expansion. Colonization of wild-type mice with proteolytic PAR2-activating Enterococcus and Staphylococcus worsens colitis severity. Our study identifies a previously unknown interaction between proteolytic bacterial communities, which are shaped by inflammation, and the external domain of PAR2 in colitis. The findings should encourage new therapeutic developments for IBD by targeting excessive PAR2 cleavage by bacterial proteases

Active thrombin produced by the intestinal epithelium controls mucosal biofilms

International audienceProteolytic homeostasis is important at mucosal surfaces, but its actors and their precise role in physiology are poorly understood. Here we report that healthy human and mouse colon epithelia are a major source of active thrombin. We show that mucosal thrombin is directly regulated by the presence of commensal microbiota. Specific inhibition of luminal thrombin activity causes macroscopic and microscopic damage as well as transcriptomic alterations of genes involved in host-microbiota interactions. Further, luminal thrombin inhibition impairs the spatial segregation of microbiota biofilms, allowing bacteria to invade the mucus layer and to translocate across the epithelium. Thrombin cleaves the biofilm matrix of reconstituted mucosa-associated human microbiota. Our results indicate that thrombin constrains biofilms at the intestinal mucosa. Further work is needed to test whether thrombin plays similar roles in other mucosal surfaces, given that lung, bladder and skin epithelia also express thrombin

BL-7010 Demonstrates Specific Binding to Gliadin and Reduces Gluten-Associated Pathology in a Chronic Mouse Model of Gliadin Sensitivity

<div><p>Celiac disease (CD) is an autoimmune disorder in individuals that carry DQ2 or DQ8 MHC class II haplotypes, triggered by the ingestion of gluten. There is no current treatment other than a gluten-free diet (GFD). We have previously shown that the BL-7010 copolymer poly(hydroxyethyl methacrylate-<i>co</i>-styrene sulfonate) (P(HEMA-<i>co</i>-SS)) binds with higher efficiency to gliadin than to other proteins present in the small intestine, ameliorating gliadin-induced pathology in the HLA-HCD4/DQ8 model of gluten sensitivity. The aim of this study was to investigate the efficiency of two batches of BL-7010 to interact with gliadin, essential vitamins and digestive enzymes not previously tested, and to assess the ability of the copolymer to reduce gluten-associated pathology using the NOD-DQ8 mouse model, which exhibits more significant small intestinal damage when challenged with gluten than HCD4/DQ8 mice. In addition, the safety and systemic exposure of BL-7010 was evaluated <i>in vivo</i> (in rats) and <i>in vitro</i> (genetic toxicity studies). <i>In vitro</i> binding data showed that BL-7010 interacted with high affinity with gliadin and that BL-7010 had no interaction with the tested vitamins and digestive enzymes. BL-7010 was effective at preventing gluten-induced decreases in villus-to-crypt ratios, intraepithelial lymphocytosis and alterations in paracellular permeability and putative anion transporter-1 mRNA expression in the small intestine. In rats, BL-7010 was well-tolerated and safe following 14 days of daily repeated administration of 3000 mg/kg. BL-7010 did not exhibit any mutagenic effect in the genetic toxicity studies. Using complementary animal models and chronic gluten exposure the results demonstrate that administration of BL-7010 is effective and safe and that it is able to decrease pathology associated with gliadin sensitization warranting the progression to Phase I trials in humans.</p></div

Administration of BL-7010 (Polymers A and B) decreased paracellular permeability and PAT-1 mRNA levels in gliadin-sensitized mice.

<p>(A) Gliadin-sensitized mice had higher ⁵¹Cr-EDTA flux in comparison to non-sensitized control mice, and administration of Polymers A or B decreased it to the same level as the non-sensitized control mice. (B) Gliadin-sensitized mice had higher mRNA levels of PAT-1 in comparison to non-sensitized control mice, and administration of Polymers A or B to sensitized mice decreased expression to the same level as non-sensitized control mice. Bars represent the mean +/− SEM, statistical analysis was completed via one-way ANOVA and Bonferroni's post-hoc test (**p<0.01, ***p<0.001, ****p<0.0001) (n = 12–13).</p

An overlay of sensograms showing Polymer A interaction with gliadin at a series of concentrations of 10-640 ng/mL.

<p>KD was determined to be 12.1 nM, with kon of 2.39×105 1/M·s and koff of 2.89×10-3 1/s. B. An overlay of sensograms showing Polymer B interacting with gliadin, with KD of 2.44 nM, kon of 6.11×105 1/M·s and koff of 1.49×10-3 1/s using Biacore Evaluation Software (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109972#s2" target="_blank">Methods</a>). For both graphs, colored lines indicate experimental sensograms at various concentrations, while corresponding black lines denote fitting of experimental data using 1∶1 binding model with global Rmax.</p

Administration of BL-7010 (Polymers A and B) decreased CD3⁺ intraepithelial cells in villi tips of gliadin-sensitized mice.

<p>Small intestinal CD3⁺ stained small intestinal tissues from (A) non-sensitized mice (control) (B) gliadin-sensitized mice (gluten) (C) gliadin-sensitized mice, receiving Polymer A (D) gliadin-sensitized mice, receiving Polymer B. (E) Quantification of CD3⁺ cells/100 enterocytes in villi tips. Stained sections were viewed via optical microscopy (20X magnification). Bars represent the mean +/− SEM, statistical analysis was completed via one-way ANOVA and Bonferroni's post-hoc test (***p<0.001, ****p<0.0001) (n = 11–13).</p

Interaction of BL-7010 (Polymer A) with gliadin, BSA, proteolytic enzymes and vitamins.

1<p>“No Interaction” means that no interaction signal (no increase in resonance units) was detected while increasing the concentration of the tested material and therefore no kinetics parameters could be calculated.</p><p>Interaction of BL-7010 (Polymer A) with gliadin, BSA, proteolytic enzymes and vitamins.</p