Multiple TLRs Are Expressed in Human Cholangiocytes and Mediate Host Epithelial Defense Responses to Cryptosporidium parvum via Activation of NF-κB

Abstract

Infection of epithelial cells by Cryptosporidium parvum triggers a variety of host-cell innate and adaptive immune responses including release of cytokines/chemokines and up-regulation of antimicrobial peptides. The mechanisms that trigger these host-cell responses are unclear. Thus, we evaluated the role of TLRs in host-cell responses during C. parvum infection of cultured human biliary epithelia (i.e., cholangiocytes). We found that normal human cholangiocytes express all known TLRs. C. parvum infection of cultured cholangiocytes induces the selective recruitment of TLR2 and TLR4 to the infection sites. Activation of several downstream effectors of TLRs including IL-1R-associated kinase, p-38, and NF-κB was detected in infected cells. Transfection of cholangiocytes with dominant-negative mutants of TLR2 and TLR4, as well as the adaptor molecule myeloid differentiation protein 88 (MyD88), inhibited C. parvum-induced activation of IL-1R-associated kinase, p-38, and NF-κB. Short-interfering RNA to TLR2, TLR4, and MyD88 also blocked C. parvum-induced NF-κB activation. Moreover, C. parvum selectively up-regulated human β-defensin-2 in directly infected cells, and inhibition of TLR2 and TLR4 signals or NF-κB activation were each associated with a reduction of C. parvum-induced human β-defensin-2 expression. A significantly higher number of parasites were detected in cells transfected with a MyD88 dominant-negative mutant than in the control cells at 48–96 h after initial exposure to parasites, suggesting MyD88-deficient cells were more susceptible to infection. These findings demonstrate that cholangiocytes express a variety of TLRs, and suggest that TLR2 and TLR4 mediate cholangiocyte defense responses to C. parvum via activation of NF-κB.

Innate immune response to double-stranded RNA in biliary epithelial cells is associated with the pathogenesis of biliary atresia

“…14,17,18,29,30 In the TLR family, TLR3 recognizes dsRNA and is located in the endosomal membranes with the ligand-binding domain facing the lumen of the endosomes and the signaling domain positioned on the cytoplasmic side. Therefore, TLR3 allows cells to detect dsRNA that is phagocytosed from the extracellular space, where it is released by infected cells that are undergoing lysis or necrosis.…”

A Cellular Micro-RNA, let-7i, Regulates Toll-like Receptor 4 Expression and Contributes to Cholangiocyte Immune Responses against Cryptosporidium parvum Infection

“…We also previously demonstrated that TLR2 and TLR4 signals mediate cholangiocyte responses including production of human ␤-defensin 2 against C. parvum via TLR-associated activation of NF-B (11). Dominant negative TLR/MyD88 expressing cholangiocytes have diminished defenses against C. parvum infection in vitro (11). Thus, TLRs and the mechanisms involved in their regulation are key elements for cholangiocyte defense against C. parvum infection.…”

“…We and others previously reported that human cholangiocytes express all 10 known TLRs and produce a variety of inflammatory cytokines/chemokines and antimicrobial peptides in response to microbial infection, suggesting a key but poorly understood role for cholangiocytes in epithelial defense (9 -14). We also previously demonstrated that TLR2 and TLR4 signals mediate cholangiocyte responses including production of human ␤-defensin 2 against C. parvum via TLR-associated activation of NF-B (11). Dominant negative TLR/MyD88 expressing cholangiocytes have diminished defenses against C. parvum infection in vitro (11).…”

Immune response of newborn BALB/c mice to Cryptosporidium infection

“…Non-coding RNAs are critical regulators of mucosal immunity to infection, while release of exosomes from epithelial cells may be a relatively unexplored, important part of mucosal anti-parasite defense. Conversely, it appears that Cryptosporidium has also developed strategies of immune evasion to escape host immunity, at least at the early stage of infection (Chen et al 2005).…”