Fungal-Associated Invasive and Inflammatory Diseases LIRIC-INSERM U995-Equipe2 Lille, France

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1 Polymorphisms in the Mannose Binding Lectin gene are associated with the defect of the mannose binding lectin functional activity in Crohn s disease patients Laura Choteau; Francis Vasseur; Frederic Lepretre; Martin Figeac; Corine Gower-Rousseau; Laurent Dubuquoy; Daniel Poulain; Jean-Frederic Colombel; Boualem Sendid; Samir Jawhara Fungal-Associated Invasive and Inflammatory Diseases LIRIC-INSERM U995-Equipe2 Lille, France

2 Inflammatory Bowel Disease Ulcerative colitis Crohn s disease Baumgart et Sandborn (2012) Lancet.

3 IBD: multifactorial disorders Etiologic Hypotheses Persistent colonization/infection: Mycobacteria Helicobacter sp. Listeria Toxigenic E. coli C. albicans IBD Genetics: NOD2, STAT1, 2 or 4 IL12B IL23R IL10 MBL2 Dysbiosis: Protective bacteria Aggressive commensals Dysregulated immune response and defective mucosal integrity: Loss of tolerance Aggressive cellular activation Altered mucus/increased permeability Desreumaux and Colombel (2002) Ann Pharm Fr

4 NOD2 plays a key role in intestinal homeostasis Philpott et al. (2014) Nature Review

5 Link between inflammatory bowel disease and Candida albicans Candida albicans? Anti-Saccharomyces cerevisiae antibodies (ASCA) IBD Colonized with C. albicans Gut lumen Neutrophil Bloodstream Lymphocyte T Macrophage Sendid et al. (1998) Am J Gasterenterol Standaert-Vitse et al. (2009) Am J Gasterenterol Poulain et al. (2009) Digest. Dis.

6 Candida albicans Opportunistic pathogen yeast causes the candidiasis ranked the 4 th most frequent nosocomial infection 30 70% mortality rates for patients with systemic candidiasis commensal yeast resides in digestive tract and vagina polymorphic fungus Blastospores filamentous form

7 Cell wall structure of C. albicans Mannans (PPM) Glycolipids (PLM) Mannoproteins β-glucans Chitin

8 C. albicans colonization and ASCA in familial Crohn's disease C. albicans was isolated frequently from stool samples from CD patients Regradeless of ASCA levels in IC, ASCA status is stable during the time course of CD Sendid et al. (1998) Am J Gasterenterol Standaert-Vitse et al. (2009) Am J Gastroenterol

9 Role of MBL in Crohn s diseases Gut lumen MBL Neutrophil Macrophage Monocyte Lymphocyte T MBL levels are inversely correlated with ASCA levels in CD patients MBL deficiency was found to be related to CD in a pediatric cohort Seibold et al, Gut, 2006 Bak-Romaniszyn et al. Scand J Gastroenterol, 2011

10 Mannose Binding Lectin Activation of complement lectin patways Opsonization of microorganisms Coagulation cascades MBL Yeasts Yeasts MBL MASP C 4 C 2 MBL Collectin receptor MASP Prothrombin Thrombin C 3 C3 receptor /Mac1 Platelet activation Garred et al, Genes Immunity, 2006 Van Asbeck et al, BMC microbiology, 2008 Héja et al, J Bio Chem, 2012 Garred et al, Genes Immunity, 2006 Van Asbeck et al, BMC microbiology, 2008 Héja et al, J Bio Chem, 2012

11 The question is whether MBL is also produced and released locally in the gut? MBL2 MBL? Gut lumen MBL Bloodstream

12 Detection of MBL-A and MBL-C mrna and proteins in the gastrointestinal tract of wild-type mice Detection of MBL-A and MBL-C mrna in the gastrointestinal tract of mice MBL-A MBL-C Liver Stomach Caecum Colon Liver Stomach Caecum Colon Expression of MBL proteins in the gastrointestinal tract of mice Liver colon stomach caecum 35Kd WT stomach MBL-KO stomach Choteau et al. Mucosol. Immunol. 2015

13 C. albicans colonization increases the expression of MBL-A and MBL-C in the gastrointestinal tract of mice Single oral gavage with C. albicans 10 7 End of experiment WT mice D0 D14 Expression of MBL proteins in the gastrointestinal tract of mice colonized or not with C. albicans MBL-A MBL-C Choteau et al. Mucosol. Immunol. 2015

14 Mouse intestinal explant culture produced MBL after C. albicans sensing alone or with pioglitazone treatment The level of MBL-A from colons of wild-type mice increased in the supernatant at 3hrs after C. albicans sensing combined or not with pioglitazone. Mouse colon MBL detection in the supernatant C. albicans/ Pioglitazone 37 C 3H

15 MBL deficiency exacerbates intestinal inflammation and C. albicans colonization Single oral gavage with C. albicans 10 7 End of experiment MBL-KO mice D0 Colitis was induced by administering mouse 1.5% DSS D14 Histological analysis of colon sections MBL deficiency promoted C. albicans colonization in mice WT CaD CFU of C. albicans /mg of stomach WT P<0.001 Stomach MBL-KO CFU of C. albicans /mg of caecum P< Caecum WT MBL-KO MBL-KO CaD CFU of C. albicans /mg of colon 80 P< MBL-KO Stomach WT MBL-KO CFU of C. albicans /mg of feces WT P< MBL-KO

16 The aims of the study were to assess in 70 CD patients and 30 age- and sex-matched healthy control subjects the relationship between : Clinical CD phenotypes MBL serum concentrations MBL functional activity MBL2, and NOD2 polymorphisms ASCA levels

17 Clinical characteristics of the Crohn s disease patients Crohn s disease patients (n=70) Mean age of onset (years) 23 Female/male 42/27 Montreal classification Age at diagnosis A1: 16-years 17 (24.6%) A2: years 48 (69.6%) A3: >40-years 3 (4.3%) Behavior B1: Non-stricturing/non-penetrating 37 (53.6%) B2: Stricturing 16 (23.2%) B3: Penetrating 14 (20.3%) Location L1: Terminal ileum 15 (21.7%) L2: Colon 14 (20.3%) L3: Ileocolon 34 (49.3%)

18 Development of assay for MBL-MASP functional activity Fluorogenic thrombin substrate 1 MASP MBL Mannans 2 3 Cleavage of the fluorogenic thrombin substrate by the MBL-MASP complex Time (min) Time (min)

19 Polymorphisms in the MBL gene are associated with the defect of the MBL activity in Crohn s disease patients MBL levels (ng/ml) MBL concentration p=0.80 Healthy subjects CD patients MBL-MASP activity (% FE / ng of MBL) The functional activity of MBL-MASP Healthy subjects B1 B2 B3 CD patients MBL-MASP activity (%FE) P< (r = 0.8) MBL level (ng/ml) in healthy subjects MBL-MASP activity (%FE) P< (r = 0.75) MBL level (ng/ml) in CD patients Choteau et al. Sci. Rep. 2016

20 Polymorphisms in the MBL gene are associated with the defect of the MBL activity in Crohn s disease patients p= ASCA levels (AU) p< ASCA levels (AU) p< Healthy subjects 200 CD patients 0 p<0.015 (r = -0.72) B1 B2 B3 CD patients ASCA Levels MBL level (ng/ml) in CD patients with B3 Choteau et al. Sci. Rep. 2016

21 MBL level (ng/ml) Polymorphisms in the MBL gene are associated with the defect of the MBL activity in Crohn s disease patients MBL level vs. MBL2 genotyping p< G_G G_A MBL2 rs MBL-MASP activity vs. MBL2 genotyping MBL-MASP activity (%FE/ng of MBL) G_G p<0.05 G_A MBL2 rs ASCA levels (AU) ASCA level vs. MBL2 genotyping 250 p< G_G G_A MBL2 rs MBL-MASP activity vs. NOD2 genotyping MBL-MASP activity (%FE/ng of MBL) C_C p<0.05 C_T NOD2 rs

22 Role of MBL2 and NOD2 in Crohn s diseases Dysbiosis and intestinal cell lesions The MBL2 variant rs was associated with a low level of MBL in CD patients. The MBL2 variant rs was associated with an impaired MBL-MASP functional activity in CD patients. The MBL2 variant rs was associated with a higher level of ASCA. The NOD2 variant rs was significantly correlated with the impairment in MBL-MASP functional activity. MBL/MASP? NOD2 MBL ASCA

23 Conclusion Our findings provide evidence that CD patients with severe clinical phenotypes have an impairment of MBL-MASP functional activity, and that this defect is associated with MBL2 and NOD2 variants. In the clinical study: Perspectives Further analysis of the cells and molecular pathways that regulate innate immune responses in the intestine is ongoing (NOD2 and MBL2) Modification of the gut microbiota and their impact on MBL levels

24 Inserm U995/Team2, Lille France Acknowledgments Dubuquoy s team (Inserm U995/Team1) Guerardel s team (CNRS, UMR 8576) Plateforme d Interaction Moléculaire (IMPRT-IFR114) Bayry s team (Inserm U1138) Kuchler s team (Medical University of Vienna) Plow s team (Cleveland Clinic, Cleveland ) Roger s team (University of Lausanne, Switzerland)

25 Principe de mesure de l activation plaquettaire par le complexe MBL-MASP

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27 Short-chain fatty acids (acetate or butyrate)

28 Short-chain fatty acids (acetate or butyrate)

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