NF-kappaB p65-Dependent Transactivation of miRNA Genes following Cryptosporidium parvum Infection Stimulates Epithelial Cell Immune Responses

Abstract

Cryptosporidium parvum is a protozoan parasite that infects the gastrointestinal epithelium and causes diarrheal disease worldwide. Innate epithelial immune responses are key mediators of the host's defense to C. parvum. MicroRNAs (miRNAs) regulate gene expression at the posttranscriptional level and are involved in regulation of both innate and adaptive immune responses. Using an in vitro model of human cryptosporidiosis, we analyzed C. parvum-induced miRNA expression in biliary epithelial cells (i.e., cholangiocytes). Our results demonstrated differential alterations in the mature miRNA expression profile in cholangiocytes following C. parvum infection or lipopolysaccharide stimulation. Database analysis of C. parvum-upregulated miRNAs revealed potential NF-κB binding sites in the promoter elements of a subset of miRNA genes. We demonstrated that mir-125b-1, mir-21, mir-30b, and mir-23b-27b-24-1 cluster genes were transactivated through promoter binding of the NF-κB p65 subunit following C. parvum infection. In contrast, C. parvum transactivated mir-30c and mir-16 genes in cholangiocytes in a p65-independent manner. Importantly, functional inhibition of selected p65-dependent miRNAs in cholangiocytes increased C. parvum burden. Thus, we have identified a panel of miRNAs regulated through promoter binding of the NF-κB p65 subunit in human cholangiocytes in response to C. parvum infection, a process that may be relevant to the regulation of epithelial anti-microbial defense in general.

Author Summary

MicroRNAs (miRNAs) are newly identified small non-coding RNAs that regulate gene expression at the posttranscriptional level. While much of our understanding of the cellular processes modulated by miRNAs has come from studies on development and tumorigenesis, the role of miRNAs in immune responses is now being gradually uncovered. Nevertheless, whether miRNA-mediated posttranscriptional gene regulation is involved in the fine-tuning of epithelial cell immune responses against pathogen infection remains undefined. Cryptosporidium parvum is a protozoan parasite that infects gastrointestinal epithelium. TLR/NF-κB-mediated innate immune responses by epithelial cells are critical to the host's defense to infection. Using an in vitro model of human cryptosporidiosis, we show here differential alterations in the miRNA expression profile in biliary epithelial cells following C. parvum infection. Promoter binding of NF-κB p65 subunit accounts for the upregulation of a panel of miRNA genes in cells infected by C. parvum. Importantly, functional inhibition of several NF-κB p65-dependent miRNAs in epithelial cells increases C. parvum infection burden. Our findings suggest that host epithelial cells activate NF-κB signaling to regulate miRNA expression in response to C. parvum infection. Moreover, NF-κB-mediated miRNA expression is involved in epithelial anti-microbial defense. Our study provides new insights into epithelial cell immunoregulation.