Cryptosporidial Infection Suppresses Intestinal Epithelial Cell MAPK Signaling Impairing Host Anti-Parasitic Defense

Abstract

Cryptosporidium is a genus of protozoan parasites that infect the gastrointestinal epithelium of a variety of vertebrate hosts. Intestinal epithelial cells are the first line of defense and play a critical role in orchestrating host immunity against Cryptosporidium infection. To counteract host defense response, Cryptosporidium has developed strategies of immune evasion to promote parasitic replication and survival within epithelial cells, but the underlying mechanisms are largely unclear. Using various models of intestinal cryptosporidiosis, we found that Cryptosporidium infection caused suppression of mitogen-activated protein kinase (MAPK) signaling in infected murine intestinal epithelial cells. Whereas expression levels of most genes encoding the key components of the MAPK signaling pathway were not changed in infected intestinal epithelial cells, we detected a significant downregulation of p38/Mapk, MAP kinase-activated protein kinase 2 (Mk2), and Mk3 genes in infected host cells. Suppression of MAPK signaling was associated with an impaired intestinal epithelial defense against C. parvum infection. Our data suggest that cryptosporidial infection may suppress intestinal epithelial cell MAPK signaling associated with the evasion of host antimicrobial defense.

Keywords: Cryptosporidium; MAPK; cryptosporidiosis; defense; intestinal epithelium; p38/MAPK.

Figure 1

Suppression of MAPK signaling in intestinal epithelium following C. parvum infection. (A) Heatmap of expression levels of MAPK-controlled genes in host cells following C. parvum infection. IEC4.1 cells were exposed to C. parvum infection for 24 h. Total RNA was isolated for genome-wide transcriptome analysis via microarray. Expression levels of MAPK-controlled genes and selected inflammatory genes not directly related to the MAPK signaling are presented as the log2 (Hy5/Hy3), which passed the filtering criteria variation across the samples (n = 3). ^a p < 0.05, vs., control; ^b p < 0.01, vs. control. (B) Suppression of Il-6 and Tnf-α expression and inhibition of phosphorylation of p38/Mapk in C. parvum-infected intestinal epithelial cells in response to MAPK activator stimulation. IEC4.1 cells were exposed to C. parvum infection for 24 h and then treated with the MAPK activator anisomycin for up to 4 h. Anisomycin-mediated expression levels of IL-6 and TNF-α were measured. Phosphorylation of p38/Mapk was assessed using Western blot. Gapdh was also blotted for control. “+” and “−” represent cells treated with and without the according reagents, respectively. Representative gel images were shown. Data represent three independent experiments.

Figure 2

Expression profile of genes key to the MAPK signaling pathway in intestinal epithelial cells following C. parvum infection. Heatmap of expression levels of genes key to the MAPK signal pathway in host cells following C. parvum infection, presented as the log2 (Hy5/Hy3) ratios, which passed the filtering criteria variation across the samples (n = 3). IEC4.1 cells were exposed to C. parvum infection for 24 h and RNA was isolated for genome-wide transcriptome analysis via microarray. Expression levels of genes key to the MAPK signal pathway are shown. ^a p < 0.05, vs. control; ^b p < 0.01, vs. control.

Figure 3

Downregulation of p38/Mapk, Mk2 and Mk3 genes in intestinal epithelial cells following C. parvum infection. (A) RNA levels of a panel of inflammatory genes in IEC4.1 cells following C. parvum infection. Cells were exposed to C. parvum infection for 8 and 24 h. RNA levels of these genes were measured by using real-time quantitative PCR. (B) RNA levels of p38/Mapk, Mk2 and Mk3 genes in IEC4.1 cells following C. parvum infection. Cells were exposed to C. parvum infection for 8 and 24 h. RNA levels of p38/Mapk, Mk2 and Mk3 genes were measured. (C) Protein level of p38/Mapk in IEC4.1 cells following C. parvum infection. Cells were exposed to C. parvum infection for 24 h and 48 h. Protein level of p38/Mapk was assessed by using Western blot. Gapdh was also blotted for control. Data represent three independent experiments.

Figure 4

Downregulation of p38/Mapk, Mk2 and Mk3 genes in intestinal epithelium of neonatal mice following C. parvum infection in vivo. (A) Immunofluorescent staining of ileum from neonatal mice with and with C. parvum infection. Mice at the age of 5 days after birth received C. parvum oocysts by oral gavage (10⁵ oocysts each mouse). Mice which received phosphate buffered saline by oral gavage were used as control. Tissue sections were triple stained with anti-C. parvum (showing in red), anti-PCNA (showing proliferating cells in green) and DAPI (showing nuclei in blue). (B) RNA levels of p38/Mapk, Mk2 and Mk3 genes in isolated intestinal epithelium from infected neonatal animals. RNA levels of p38/Mapk, Mk2 and Mk3 genes were measured by using real-time PCR. (C) Protein level of p38/Mapk in isolated intestinal epithelium from infected neonatal animals. Protein level of p38/Mapk was assessed by using Western blot. Gapdh was also blotted for control. DAPI = 4′,6-diamidino-2-phenylindole; PCNA = Proliferating cell nuclear antigen.

Figure 5

Downregulation of p38/Mapk, Mk2 and Mk3 genes in mulINTEP1 cells following C. parvum infection. RNA levels of p38/Mapk, Mk2 and Mk3 genes in mulINTEP1 cells following C. parvum infection. Cells were exposed to C. parvum infection for 48 and 72 h. RNA levels of p38/Mapk, Mk2 and Mk3 genes were measured by using real-time PCR. Data represent three independent experiments.

Figure 6

Suppression of MAPK signaling impairs intestinal epithelial defense against C. parvum infection. (A) Activation of MAPK signaling decreased the infection burden of C. parvum in host cells. IEC4.1 cells were exposed to C. parvum infection for 24 h in the presence or absence of the MAPK activator anisomycin or inhibitor SP600125. Infection burden of C. parvum was quantified. (B) Suppression of MAPK signaling in intestinal epithelial cells on the attachment and invasion of C. parvum to host cells. Cells were exposed to C. parvum infection for 2 h (for attachment and invasion) in the presence or absence of the MAPK activator anisomycin or inhibitor SP600125. Infection burden of C. parvum was quantified. The expression levels of IL-6 and TNF- were measured in cells treated with anisomycin or SP600125 to confirm their effects on MAPK signaling. “+” and “−” represent cells treated with and without the according reagents, respectively. Data represent three independent experiments.