Fig. 6: Impaired cellular response to IFN-γ stimulation in infected epithelial cells and its association with C. parvum-induced type I IFN signaling activation.
From: Cryptosporidium uses CSpV1 to activate host type I interferon and attenuate antiparasitic defenses
a Pre-treatment of IEC4.1 cells with IFN-γ decreased C. parvum infection burden. Cells were first treated with IFN-γ (10 ng/ml) for 8 h, exposed to C. parvum for 24 h, and cp18S was quantified by RT-qPCR. b Impaired cellular response to IFN-γ stimulation in infected IEC4.1 cells associated with type I IFN signaling. IEC4.1 and Ifnar1−/− IEC4.1 cells were first exposed to C. parvum infection for 24 h followed by IFN-γ stimulation for additional 2 h. Cellular response to IFN-γ stimulation was reflected by the expression level of the Ido1 gene. c Knockdown of CSpV1-dsRNA delivery in infected IEC4.1 cells through transfection of the specific siRNA combinations (siPOOLs) partially restored the induction of Ido1 gene expression in response to IFN-γ stimulation. A non-specific control siRNA (siControl) was used for control. Data (in a–c) are from three biological replicates and presented as mean values ± SD. p values were determined by two-way ANOVA followed by Tukey’s HSD test (in a–c). d Heatmaps representing altered expression levels of selected genes for the key components associated with IFN-γ signaling in infected IEC4.1 cells or neonatal intestinal epithelium. IEC4.1 cells (24 h p.i.) or intestinal epithelium from Ifnar1fl/fl and Villin.Ifnar1−/− neonates (5 days old) after infection (48 h and 72 h p.i.) were collected, followed by RNA-Seq. Expression levels of genes for key components associated with IFN-γ signaling are shown. Data are from three RNA-Seq biological replicates for each group. e Protein content of the Ifngr1 subunit of IFN-γ receptor in IEC4.1 and Ifnar1−/− IEC4.1 cells, intestinal epithelium from Ifnar1fl/fl and Villin.Ifnar1−/− neonates as assessed by Western. Gapdh was used as a loading control. Representative gels from three independent experiments are shown. f Intestinal epithelium of Ifnar1fl/fl and Villin.Ifnar1−/− neonatal mice (either uninfected or 72h-infected animals) showed a similar staining for Ifngr1 protein by immunofluorescence. Representative images from 4 independent experiments are shown. Blue: DAPI (DNA), green: Ifngr1, red: C. parvum (arrows). Bars = 50 µm. Source data are provided as a Source Data file.
