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DOP086 Macrophages from Crohn's disease patients showed a defect to control adherent-invasive Escherichia coli replication influenced by genetic host factors

Buisson A.*1,2, Vazeille E.1,2, Ouchchane L.3, Goutte M.1,2, Hugot J.-P.4, Dubois A.2, Minet Quinard R.5, Bouvier D.5, Bommelaer G.1,2, Barnich N.2

1University Hospital Estaing, Gastroenterology Department, Clermont-Ferrand, France 2UMR 1071 Inserm/Université d'Auvergne; USC-INRA 2018, Microbes, Intestine, Inflammation and Susceptibility of the host, Clermont-Ferrand, France 3University Hospital G Montpied, Biostatistics unit, clermont-Ferrand, France 4Assistance Publique Hôpitaux de Paris et Université, Paris Diderot, Paris, UMR843, Inserm, Paris, France 5University Hospital G. Montpied, Biochemistry laboratory, Clermont-Ferrand, France

Background

Adherent-invasive E. coli (AIEC) abnormally colonize ileal mucosa from one third of Crohn's disease (CD) patients. AIEC are able to survive within macrophages, which are pivotal cells in eliminating intracellular bacteria. We recently suggested that macrophages from CD patients could have an impaired ability to restrict intracellular AIEC replication.

We aimed to confirm that macrophages from CD patients could have an impaired ability to restrict intracellular AIEC replication compared to ulcerative colitis (UC) patients or healthy volunteers (HV). We also investigated the factors associated with this deficiency and analyzed macrophages behaviour in response to AIEC infection in CD patients.

Methods

Peripheral blood monocyte-derived macrophages (MDM) were obtained from 95 CD patients, 30 UC patients and 15 HV, genotyped for CD-associated polymorphisms implicated in autophagy (ULK, LRRK2, NOD2, IRGM and ATG16L1), ER stress (XBP1), or ubiquitin-proteasome system (CYLD, USP40). The numbers of intracellular bacteria were determined at 1h and 10h post-infection using gentamicin assay. Cytokine secretion was quantified by ELISA, LDH by colorimetry assay and CD163 by western blot.

Results

The AIEC uptake (1h post-infection) within MDM did not differ according to MDM origin. The AIEC survival (10h post-infection) within MDM from CD patients compared to UC patients or HV (p=0.0019). In multivariate analysis, AIEC survival within MDM from CD patients was positively correlated with IL1-β secretion (p<0.0001) and was decreased in the presence of ULK1 (p=0.046), XBP1 (p=0.014) and CYLD (p=0.0008) mutations. AIEC were able to replicate within MDM from CD patients but not within MDM from UC patients or HV (p<0.001). In multivariate analysis, AIEC intracellular replication was increased in CD patients with IRGM mutation (p=0.045). AIEC infection leads to specific inflammatory response (increased secretion of TNF-α, IL-1β and IL-8 compared to UC patients and HV, p<0.001 for both) which does not depend on MDM origin but is associated with intestinal inflammation exclusively in CD patients. MDM from CD patients exhibit a specific phenotype at baseline (low LDH secretion, p<0.001) compared to UC patients. AIEC infection modified MDM behaviour from CD patients in weakening MDM (increase of LDH secretion, p<0.05) and decreasing macrophages activation (decrease of CD163, p<0.05).

Conclusion

We confirmed that MDM from CD patients are deficient to control AIEC replication compared to UC or healthy controls and identified the key role of autophagy (especially IRGM), ER stress and ubiquitin-proteasome system genes. We also reported a specific inflammatory response of AIEC-infected macrophages depending on the intestinal inflammation.