TLR4, IL10RA, and NOD2 mutation in paediatric Crohn’s disease patients: an association with Mycobacterium avium subspecies paratuberculosis and TLR4 and IL10RA expression

Abstract

Mycobacterium avium subspecies paratuberculosis (MAP) has been implicated in the pathogenesis of Crohn's disease (CD). The role of CD susceptibility genes in association with these microbes is not known. Sixty-two early onset paediatric CD patients and 46 controls with known MAP status were analysed for an association with 34 single nucleotide polymorphisms (SNPs) from 18 CD susceptibility genes. Functional studies on peripheral blood mononuclear cells (PBMCs) were conducted on 17 CD patients with known CD mutations to assess IL-6, IL-10, and TNF-α expression upon stimulation with MAP precipitated protein derivative (PPD) and lipopolysaccharide (LPS). In addition, surface expression of IL10R and TLR4 on resting B cells, NK cells, T cells, and monocytes was assessed. A mutation in TLR4 (rs4986790) and IL10RA (rs22291130) was significantly associated with MAP-positive CD patients compared to MAP-negative CD patients (27.6 vs. 6.1%, p=0.021, and 62.1 vs. 33.3%, p=0.024, respectively). PPD and LPS significantly increased IL-6, IL-10, and TNF-α production in PBMCs. IL-10 and TNF-α production were significantly lower in a subgroup of CD patients (5/12) with a known NOD2 mutation. Receptor for IL-10 was significantly higher expressed on NK cells (CD56low) and on NK T cells harbouring a NOD2 mutations compared to wildtype cells (p=0.031 and 0.005, respectively). TLR4 was significantly higher expressed on NK cells (CD56high) harbouring a NOD2 mutations compared to wildtype cells (p=0.038).