P-191 Loss of TLR2 Worsens Spontaneous Colitis in MDR1A Deficiency Through Commensal-Induced Pyroptosis

Background

Variants of the multidrug resistance gene (MDR1/ABCB1) have been associated with increased susceptibility to severe Ulcerative Colitis (UC). However, the innate immune mechanisms modulating colonic inflammation in MDR1A deficiency have not been established yet. The aim of this study was to investigate the role of TLR/IL-1R signaling pathways including the common adaptor MyD88 in the pathogenesis of chronic colitis in mice lacking MDR1A.

Methods

Mice (SPF) double or triple knockout in TLR2, MD-2, MyD88 and MDR1A were generated (F7; FVB/N). Male mice (n>6) were examined at 5 or 10 weeks of age. Colitis activity was assessed by standard clinical parameters and histopathology. Real-time qRT-PCR and protein arrays were employed to determine cyto-/chemokine profiles in colonic tissues and 16S rRNA analysis to cover the dominant bacterial populations. CD11b+- myeloid cells were cultured in the presence of non-pathogenic E.coli-eGFP and cell death was examined by TUNEL (-/+ caspase-1 inhibitor) and caspase-1 activity assays.

Results

Genetic deletion of TLR2 in MDR1A deficiency resulted in fulminant pancolitis with early expansion of CD11b+/Ly6C+-myeloid cells producing IL-1ß in the lamina propria. Blockade of IL-1ß activity by treatment with IL-1 receptor antagonist inhibited colitis acceleration in TLR2/MDR1A deficiency. Colitis exacerbation via IL-1ß in TLR2/MDR1A double-knockout mice required the commensal microbiota and depended on the lipopolysaccharide (LPS) co-receptor MD-2 and the adaptor protein MyD88. Of note, loss of TLR2 in the context of MDR1A deficiency neither affected Paneth cell gene expression nor provoked major shifts in commensal composition. However, in contrast to TLR2-expressing WT or MDR1A, TLR2/MDR1A double-deficient CD11b+-myeloid cells hyperresponded to nonpathogenic E. coli or LPS with increased IL-1ß secretion and cell death via caspase-1, demonstrating “balloon-shaped” vesicles around the nucleus with absence of nuclear fragmentation; these features are consistent with pyroptosis.

Conclusion(s)

Our findings imply pyroptosis as a maladaptive host defense strategy for clearance of commensal gut bacteria in the context of combined deficiencies in MDR1A and TLR2, leading to spontaneous and uncontrolled colitis progression through aberrant MD-2 and IL-1R signaling via MyD88.