Human cathelicidins improve colonic epithelial defenses against Salmonella typhimurium modulating cellular permeability, TLR4 and pro-inflammatory cytokines

MARIN M; BURUCÚA M; HOLANI R; BLYTH G; DROUIN D; ODEÓN A; COBO E

Mar del Plata

Otro; REUNIÓN CONJUNTA SAIC SAI SAIF 2018; 2018

SAIC, SAI, SAIF

The intestinal mucosa contributes to frontline gut defenses by forming a barrier and preventing the entry of pathogenic microbes. One remarkable innate role of the colonic epithelium is to secrete cathelicidin, a peptide with broad antimicrobial and immunomodulatory functions. In this study, the effect of cathelicidin in the maintenance of epithelial integrity, Toll-like receptor recognition, and initiation of inflammatory response against Salmonella typhimurium were investigated. Tight junction gene levels and ZO-1 immunolocalization were studied in T84 cells infected with a virulent and drug-resistant S. typhimurium definitive type 104 strain (MOI 5, 4-16h) ± synthetic LL37 (0-40 μg/mL). Normal (ntLL37) and LL37 know-down (shLL37) HT29 colonic cells were pre-stimulated with LL37 (0-20 μg/mL) and infected with S. typhimurium (MOI 1-5, 4-24h). Antimicrobial activity in cell lysates was achieved by bacterial counting and TLR4, TLR9, IL1β and IL18 expression by RT-qPCR. For comparisons a non-paired, two-tailed Student?s t-test was used (P < 0.05). Exogenous human cathelicidin restored the epithelial integrity in S. typhimurium-infected colonic epithelial by mostly post-translational effects associated with the reorganization of ZO-1 tight junction proteins. No changes were observed in occludin and claudin gene expression. Endogenous cathelicidin showed to contribute to preventing S. typhimurium internalization as studied in intestinal epithelial cells genetically deficient in the only human cathelicidin LL37. Moreover, supplementation of shLL37 cells (i.e., lacking endogenous cathelicidins) with synthetic LL37 (restorative effect) reduced the grade of S. typhimurium internalization in a dose-dependent manner (~50-90% inhibition). Mechanistically, shLL37 cells had significant lower gene expression of TLR4 and IL1β than normal cells in response to S. typhimurium (0.5-1.5 folds). Thus, cathelicidins aided in the early epithelial response against enteric S. typhimurium controlling the invasion of and maintaining the barrier integrity. Endogenous synthesis of cathelicidins occurred key in these functions and principally in the production of sensing TLR4 and pro-inflammatory cytokines.