TLR2 and TLR4 regulate B cell responses in human inflammatory bowel disease

INTRODUCTION: The pathogenesis of inflammatory bowel disease (IBD) is believed to be mediated primarily by T cells through an aberrant immunologic response to commensal bacteria. The innate immune response also plays an important role as evidenced by the elevation of Toll-like receptor (TLR) expression and IL-8 production, which promote a chronic, inflammatory milieu in IBD. Remarkably, the role of the most abundant cells in the gastrointestinal tract, the B cell, is largely undefined in this disease which is characterized by mucosal inflammation. We have found that surface levels of TLR2 are increased on human B cells in other mucosal inflammatory diseases, but the role of B cell-expressed TLRs in the gastrointestinal immune response and their effect in IBD is unclear. Thus, we sought to study human B cell function in patients with Crohn's disease and ulcerative colitis in order to better explain their role in this chronic inflammatory disease. METHODS: Blood and fresh ileocolonic tissue samples were analyzed from patients with IBD and healthy volunteers. Surface levels of TLR2 and TLR4 were assessed on B cells in whole blood and tissues by flow cytometry. B cells were purified from blood and stimulated with the TLR2 ligand Pam3CSK4 or TLR4 ligands E. coli LPS, R. sphaeoroides LPS or LPS-free ES-62. Cell-free supernatants were collected and assayed for cytokines by ELISA. The Inflammatory Bowel Disease Questionnaire (IBDQ) and Crohn's Disease Activity Index (CDAI) were collected at the time of the blood draw as a measure of disease severity and these levels were plotted against TLR2, TLR4, and cytokine levels. RESULTS: Our data shows that B cells from ill Crohn's disease (CD) patients constitutively secrete IL-8 as compared to healthy controls and patients in remission. The TLR2 ligand Pam3CSK4 further induced IL-8 secretion by B cells, with IL-8 levels correlating to disease activity, but this effect was not seen when stimulated by TLR4 ligands. Whereas E. Coli LPS had no measurable effect on TLR4 B cells, alternative TLR4 ligands, including ES-62 from parasitic helminths, blocked TLR2-mediated activation of B cells. Our data also revealed that an increased percentage of circulating B cells seen in whole blood samples from IBD patients are surface TLR2- and TLR4-positive with a quantitative increase in levels according to the patient's disease severity. This data was duplicated on mucosal B cells from ileocecal resections which demonstrated high levels of TLR2 positive B cells in patients with active CD. CONCLUSIONS: Inflammatory bowel disease activity correlated with B cells TLR2 expression and CD activity correlated with constitutive IL-8 production, suggesting that IL-8 from TLR2B cells contributes to active inflammation, likely through alteration of neutrophil mobilization and timely neutrophil-mediated repair of mucosal injury. We conclude that TLR2 and TLR4 on B cells play an important role in sensing specific microbial antigens and modulating mucosal inflammation by novel mechanisms.