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-kappaB 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-kappaB 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-kappaB 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.

Figure 1. Expression profiling of mature miRNAs in cholangiocytes following C. parvum infection and LPS stimulation.

(A) miRNA expression profile in H69 cells following C. parvum infection. The left panel shows a heat-map of selected miRNAs that showed changes in expression in H69 cells following C. parvum infection. The horizontal axis indicates samples of non-infected cells (n = 3; Control-1, -2, and -3) and cells after exposure to live C. parvum for 12 h (n = 3, C. parvum-1, -2, and -3). The right panel shows expression of miRNAs in H69 cells following C. parvum infection. Cellular levels of miRNAs were presented as the log₂ (Hy5/Hy3) ratios which passed the filtering criteria variation across the samples. p values are from the t' test. hsa = Homo sapiens. (B) Comparison of miRNA expression patterns in H69 cells following C. parvum infection for 12 h and LPS stimulation for 8 h. Graphics indicate those miRNAs showing an increased or decreased expression (including those significant change when p< = 0.05 and those with a tendency to change when 0.05<p< = 0.20) in cells after treatment with LPS (n = 3) or exposure to C. parvum (n = 3). A complete description of miRNA expression profiles in cells was listed in Table S1.

Figure 2. Altered expression of selected miRNAs confirmed by real-time PCR, Northern blot and Luminex bead analyses.

(A) Alterations of selected miRNA expression in cells after exposure to C. parvum for various periods of time as assessed by real-time PCR. The amount of mature miRNAs was obtained by normalizing to the level of snRNA RNU6B in the samples. Data are expressed as the amount of mature miRNAs in the infected samples relative to the control uninfected samples and representative of three independent experiments. (B) Alterations of selected miRNA expression in cells after exposure to C. parvum for 12 h as determined by Northern blot. snRNA RNU6B was used as a control to ensure equal loading. Representative Northern blots (C. parvum infected cells vs non-infected control) from three independent experiments are shown. (C) Total RNA isolated from C. parvum oocysts was also blotted to demonstrate the specificity of the probes. (D) Alterations of selected miRNA expression in cells after exposure to C. parvum for 12 h as assessed by bead-based miRNA Luminex analysis. The amount of mature miRNAs was obtained by normalizing the samples to the positive control beads provided by the company (Luminex). Data are representative of three independent experiments. *, p<0.05 vs. the non-infected control.

Figure 3. Differential expression of primary transcripts of C. parvum-upregulated mature miRNAs in H69 cells.

H69 cells were exposed to C. parvum for 2 h to 24 h and primary transcripts (pri-miRNAs) of select miRNAs were quantified by real-time PCR. The amount of pri-miRNAs was obtained by normalizing to the level of GAPDH in the samples. Data are expressed as the amount of pri-miRNAs in the infected samples relative to the control uninfected samples and representative of three independent experiments. *, p<0.05 vs. the non-infected control.

Figure 4. Promoter binding of p65 transactivates miR-125b-1 gene to increase miR-125b expression following C. parvum infection.

(A) p65-dependent upregulation of pri-miR-125b-1 in cholangiocytes following C. parvum infection. Data are presented as the relative expression level of pri-miR-125b-1 in H69 cells following C. parvum infection in the presence or absence of SC-514 as assessed by real-time PCR. (B) C. parvum increases promoter element binding of p65 to mir-125b-1 gene. The schematic diagram shows two potential binding sites in the putative promoter element of mir-125b-1 gene. ChIP analysis revealed increased binding of p65 to the binding site at −1059, but not at −2455, of mir-125b-1 promoter element in cells following infection. Representative ChIP gels are shown in the upper panel and densitometry analysis of the gels in the lower panel. (C) H69 cells were transfected with various luciferase reporter constructs spanning the potential p65 binding sites of the mir-125b-1 promoter. The transfected cells were exposed to C. parvum in the presence or absence of SC-514. Luciferase activity was measured and presented as the ratio of the activity of the test construct with the control luciferase reporter construct. Six reporter constructs containing the mutants of the two potential NF-κB binding sites were also utilized for the analysis as indicated. (D) H69 cells were co-transfected with the pCMV-p65 to overexpress p65 and the luciferase reporter construct containing the mir-125b-1 promoter for 24 h followed by measurement of luciferase activity. *, p<0.05 vs. the non-infected control (in A and C) or empty pCMV vector control (in D); ^#, p<0.05 vs. C. parvum infected cells (in A and C).

Figure 5. Functional inhibition of selected p65-dependent miRNAs in cholangiocytes increases C. parvum infection burden in vitro.

(A) A similar number of parasites was detected in cells transfected with Drosha siRNA or treated with SC-514 after initial exposure to C. parvum for 2 h as quantified by real-time PCR. (B) Transfection of cells with Drosha siRNA or treated with SC-514 increased C. parvum infection burden in cholangiocytes in vitro 24 h after initial exposure to the parasite. (C) Effects of anti-miRs on C. parvum after initial exposure to C. parvum for 2 h. (D) Transfection of cells with anti-miRs on C. parvum infection burden in cholangiocytes 24 h after initial exposure to the parasite. *, p<0.05 vs. non-treated cells or cells transfected with a control siRNA (in B) or non-specific control anti-miR (in D). (E) Effects of anti-miRs or SC-514 on C. parvum burden in cholangiocytes in vitro 24 h after initial exposure to the parasite as assessed by immunofluorescent microscopy. C. parvum parasites were stained in green and nuclei in blue. Bars = 5 µm.