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

ABSTRACT Emerging 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.

[1]  A. Getgood,et al.  Human osteoarthritis knee joint synovial fluids cleave and activate Proteinase-Activated Receptor (PAR) mediated signaling , 2023, Scientific Reports.

[2]  Asha A. Nair,et al.  Gut Microbial β-Glucuronidases Regulate Host Luminal Proteases and are depleted in Irritable Bowel Syndrome , 2022, Nature Microbiology.

[3]  Kelly C. Weldon,et al.  Multi-omics analyses of the Ulcerative Colitis gut microbiome link Bacteroides vulgatus proteases with disease severity , 2021, Nature Microbiology.

[4]  A. Lesner,et al.  Gut Serpinome: Emerging Evidence in IBD , 2021, International journal of molecular sciences.

[5]  P. Moayyedi,et al.  Novel fecal biomarkers that precede clinical diagnosis of ulcerative colitis. , 2020, Gastroenterology.

[6]  E. Chang,et al.  Inflammatory Bowel Diseases (IBD) (Inflammatory Bowel Diseases and the Microbiome: Searching the Crime Scene for Clues). , 2020, Gastroenterology.

[7]  Hongbo Hu,et al.  Targeting NF-κB pathway for the therapy of diseases: mechanism and clinical study , 2020, Signal Transduction and Targeted Therapy.

[8]  Gavin M Douglas,et al.  PICRUSt2 for prediction of metagenome functions , 2020, Nature Biotechnology.

[9]  Yutaka Suzuki,et al.  Association of colitis with gut-microbiota dysbiosis in clathrin adapter AP-1B knockout mice , 2020, PloS one.

[10]  T. Yi,et al.  The role of IL-22 in intestinal health and disease , 2020, The Journal of experimental medicine.

[11]  A. Gargouri,et al.  Fecal Serine Protease Profiling in Inflammatory Bowel Diseases , 2020, Frontiers in Cellular and Infection Microbiology.

[12]  C. Dinarello,et al.  IL-1R3 blockade broadly attenuates the functions of six members of the IL-1 family, revealing their contribution to models of disease , 2019, Nature Immunology.

[13]  D. Schuppan,et al.  Lactobacilli Degrade Wheat Amylase Trypsin Inhibitors to Reduce Intestinal Dysfunction Induced by Immunogenic Wheat Proteins. , 2019, Gastroenterology.

[14]  Kevin S. Bonham,et al.  Multi-omics of the gut microbial ecosystem in inflammatory bowel diseases , 2019, Nature.

[15]  C. Southward,et al.  Duodenal bacterial proteolytic activity determines sensitivity to dietary antigen through protease-activated receptor-2 , 2019, Nature Communications.

[16]  U. Ijaz,et al.  Differential ratio amplicons (R amp) for the evaluation of RNA integrity extracted from complex environmental samples , 2019, Environmental microbiology.

[17]  M. Jordana,et al.  Microbial Regulation of Enteric Eosinophils and Its Impact on Tissue Remodeling and Th2 Immunity , 2019, bioRxiv.

[18]  Morris A. Swertz,et al.  Gut microbiota composition and functional changes in inflammatory bowel disease and irritable bowel syndrome , 2018, Science Translational Medicine.

[19]  Eric A. Franzosa,et al.  Gut microbiome structure and metabolic activity in inflammatory bowel disease , 2018, Nature Microbiology.

[20]  M. Surette,et al.  Commensal microbiota induces colonic barrier structure and functions that contribute to homeostasis , 2018, Scientific Reports.

[21]  W. Turpin,et al.  Determinants of IBD Heritability: Genes, Bugs, and More. , 2018, Inflammatory bowel diseases.

[22]  Xinrui Li,et al.  Toll-like Receptors and Inflammatory Bowel Disease , 2018, Front. Immunol..

[23]  Nima Hamidi,et al.  Worldwide incidence and prevalence of inflammatory bowel disease in the 21st century: a systematic review of population-based studies , 2017, The Lancet.

[24]  Jimmy D Bell,et al.  NOX1 loss-of-function genetic variants in patients with inflammatory bowel disease , 2017, Mucosal Immunology.

[25]  A. Lambeir,et al.  Regulation of intestinal permeability: The role of proteases , 2017, World journal of gastroenterology.

[26]  M. Surette,et al.  Transplantation of fecal microbiota from patients with irritable bowel syndrome alters gut function and behavior in recipient mice , 2017, Science Translational Medicine.

[27]  Harry Sokol,et al.  A microbial signature for Crohn's disease , 2017, Gut.

[28]  J. Carrero,et al.  Germ-free and Antibiotic-treated Mice are Highly Susceptible to Epithelial Injury in DSS Colitis. , 2016, Journal of Crohn's & colitis.

[29]  M. Hollenberg,et al.  Thrombin-Mediated Direct Activation of Proteinase-Activated Receptor-2: Another Target for Thrombin Signaling , 2016, Molecular Pharmacology.

[30]  Paul J. McMurdie,et al.  DADA2: High resolution sample inference from Illumina amplicon data , 2016, Nature Methods.

[31]  N. Vergnolle.  Protease inhibition as new therapeutic strategy for GI diseases , 2016, Gut.

[32]  G. Kaplan,et al.  The global burden of IBD: from 2015 to 2025 , 2015, Nature Reviews Gastroenterology &Hepatology.

[33]  M. Surette,et al.  Ecobiotherapy Rich in Firmicutes Decreases Susceptibility to Colitis in a Humanized Gnotobiotic Mouse Model , 2015, Inflammatory bowel diseases.

[34]  J. Griffin,et al.  EPCR-dependent PAR2 activation by the blood coagulation initiation complex regulates LPS-triggered interferon responses in mice. , 2015, Blood.

[35]  Ashwin N. Ananthakrishnan,et al.  Epidemiology and risk factors for IBD , 2015, Nature Reviews Gastroenterology &Hepatology.

[36]  M. A. Rosillo,et al.  Dietary squalene supplementation improves DSS-induced acute colitis by downregulating p38 MAPK and NFkB signaling pathways. , 2015, Molecular nutrition & food research.

[37]  Carla J. Davidson,et al.  Culture and molecular-based profiles show shifts in bacterial communities of the upper respiratory tract that occur with age , 2015, The ISME Journal.

[38]  I. Carroll,et al.  Enteric bacterial proteases in inflammatory bowel disease- pathophysiology and clinical implications. , 2013, World journal of gastroenterology.

[39]  Michael C. Wu,et al.  Fecal Protease Activity Is Associated with Compositional Alterations in the Intestinal Microbiota , 2013, PloS one.

[40]  Jesse R. Zaneveld,et al.  Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences , 2013, Nature Biotechnology.

[41]  Susan Holmes,et al.  phyloseq: An R Package for Reproducible Interactive Analysis and Graphics of Microbiome Census Data , 2013, PloS one.

[42]  F. Guarner,et al.  Phylogenetic Analysis of Dysbiosis in Ulcerative Colitis During Remission , 2013, Inflammatory bowel diseases.

[43]  Gilles Dietrich,et al.  Food-Grade Bacteria Expressing Elafin Protect Against Inflammation and Restore Colon Homeostasis , 2012, Science Translational Medicine.

[44]  K. Orth,et al.  Manipulation of kinase signaling by bacterial pathogens , 2011, The Journal of cell biology.

[45]  Dirk Haller,et al.  Bacterial proteases in IBD and IBS , 2011, Gut.

[46]  M. Hollenberg,et al.  Neutrophil Elastase Acts as a Biased Agonist for Proteinase-activated Receptor-2 (PAR2)* , 2011, The Journal of Biological Chemistry.

[47]  Marcel Martin.  Cutadapt removes adapter sequences from high-throughput sequencing reads , 2011 .

[48]  Andrea K. Bartram,et al.  Generation of Multimillion-Sequence 16S rRNA Gene Libraries from Complex Microbial Communities by Assembling Paired-End Illumina Reads , 2011, Applied and Environmental Microbiology.

[49]  J. Sallenave,et al.  Overexpression of Elastin Affects the Protease to Anti-Protease Balance and Protects Mice from Colitis , 2019 .

[50]  G. Schneider,et al.  Helicobacter pylori HtrA is a new secreted virulence factor that cleaves E‐cadherin to disrupt intercellular adhesion , 2010, EMBO reports.

[51]  Paramvir S. Dehal,et al.  FastTree 2 – Approximately Maximum-Likelihood Trees for Large Alignments , 2010, PloS one.

[52]  Thomas F. Tedder,et al.  Innate and Adaptive Immunity Cooperate Flexibly to Maintain Host-Microbiota Mutualism , 2009, Science.

[53]  A. Ashkar,et al.  Interleukin-15 and NK1.1+ Cells Provide Innate Protection against Acute Salmonella enterica Serovar Typhimurium Infection in the Gut and in Systemic Tissues , 2008, Infection and Immunity.

[54]  S. Brand,et al.  Linking genetic susceptibility to Crohn's disease with Th17 cell function: IL‐22 serum levels are increased in Crohn's disease and correlate with disease activity and IL23R genotype status , 2008, Inflammatory bowel diseases.

[55]  F. Hoentjen,et al.  Pathophysiology of Inflammatory Bowel Diseases , 2008 .

[56]  F. Hirayama,et al.  Helicobacter pylori Activates Gastric Epithelial Cells to Produce Interleukin-8 via Protease-Activated Receptor 2 , 2008, Digestion.

[57]  F. Nagy,et al.  A pilot study of fecal serine-protease activity: a pathophysiologic factor in diarrhea-predominant irritable bowel syndrome. , 2007, Clinical gastroenterology and hepatology : the official clinical practice journal of the American Gastroenterological Association.

[58]  Y. Naito,et al.  Interleukin-8 production via protease-activated receptor 2 in human esophageal epithelial cells. , 2007, International Journal of Molecular Medicine.

[59]  Manuela M. Santos,et al.  Contribution of Hfe expression in macrophages to the regulation of hepatic hepcidin levels and iron loading. , 2005, Blood.

[60]  S. Aspengren,et al.  PAR-2 activation in intestinal epithelial cells potentiates interleukin-1beta-induced chemokine secretion via MAP kinase signaling pathways. , 2005, Cytokine.

[61]  A. Takayanagi,et al.  Interleukin-22, a member of the IL-10 subfamily, induces inflammatory responses in colonic subepithelial myofibroblasts. , 2005, Gastroenterology.

[62]  N. Vergnolle,et al.  CLINICAL RELEVANCE OF PROTEINASE ACTIVATED RECEPTORS (PARS) IN THE GUT , 2005, Gut.

[63]  G. Núñez,et al.  Cell death and immunity: NODs: intracellular proteins involved in inflammation and apoptosis , 2003, Nature Reviews Immunology.

[64]  P. Rutgeerts,et al.  A review of activity indices and efficacy endpoints for clinical trials of medical therapy in adults with Crohn's disease. , 2002, Gastroenterology.

[65]  Kevin F. Jones,et al.  Conserved DegP Protease in Gram-Positive Bacteria Is Essential for Thermal and Oxidative Tolerance and Full Virulence inStreptococcus pyogenes , 2001, Infection and Immunity.

[66]  Mourad Sahbatou,et al.  Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease , 2001, Nature.

[67]  M. Karin,et al.  Mammalian MAP kinase signalling cascades , 2001, Nature.

[68]  J. Gordon,et al.  Molecular analysis of commensal host-microbial relationships in the intestine. , 2001, Science.

[69]  D. Kell,et al.  The Kyoto Encyclopedia of Genes and Genomes—KEGG , 2000, Yeast.

[70]  N. Vergnolle,et al.  Review article: proteinase‐activated receptors — novel signals for gastrointestinal pathophysiology , 2000, Alimentary pharmacology & therapeutics.

[71]  김명욱.  Pathophysiology , 1990, Definitions.

[72]  R. F. Harvey,et al.  A SIMPLE INDEX OF CROHN'S-DISEASE ACTIVITY , 1980, The Lancet.

[73]  K. Orth,et al.  Host – pathogen interactions Manipulation of kinase signaling by bacterial pathogens , 2011 .

[74]  梶川 洋和.  Helicobacter pylori activates gastric epithelial cells to produce interleukin-8 via protease-activated receptor 2 , 2008 .