Cryptosporidium parvum induces B7-H1 expression in cholangiocytes by down-regulating microRNA-513

Abstract

Expression of B7 costimulatory molecules represents an important compartment of immune response of epithelial cells after microbial infection. We report here that the protozoan parasite Cryptosporidium parvum induced B7-H1 expression in cultured human cholangiocytes. Induced expression of B7-H1 was identified in cells after exposure to infective C. parvum parasite or parasite lysate. Interestingly, the level of microRNA-513 (miR-513) was reduced in cells after exposure to C. parvum, which resulted in a relief of 3' untranslated region-mediated translational suppression of B7-H1. Overexpression of miR-513 through transfection of miR-513 precursor inhibited C. parvum-induced B7-H1 protein expression. Moreover, enhanced apoptotic cell death was identified in activated human T cells after coculture with C. parvum-infected cholangiocytes. The apoptosis of activated T cells was partially blocked by a neutralizing antibody to B7-H1 or transfection of cholangiocytes with miR-513 precursor. These data suggest a role of miR-513 in regulating B7-H1 expression by cholangiocytes in response to C. parvum infection.

FIGURE 1

C. parvum infection induces B7-H1 expression in cultured cholangiocytes. (A and B) Time-dependent expression of B7-H1 at the message (A) and protein (B) levels in H69 cells following C. parvum infection. H69 cells were exposed to culture medium with viable C. parvum oocysts (oocysts with host cells in a 10:1 ratio) or C. parvum lysate from the same amount of oocysts for up to 24 h followed by real-time PCR and Western blot analyses. PCR reactions were performed in triplicate. A representative Western blot from three independent experiments is shown in B. Actin was blotted as a loading control. (C and D) Expression of B7-H1 expression in H69 cells induced by C. parvum as assessed by immunofluoscent staining. Cells were exposed to viable C. parvum oocysts for 24 h followed by immunoconfocal microscopy for B7-H1. Arrows indicate parasites stained in red. *p < 0.05 vs. the non-infected control; ^#, p < 0.05, vs. cells exposed to C. parvum oocysts.

FIGURE 2

C. parvum infection does not induce TNF-α production in cholangiocytes in vitro. H69 cells were exposed to viable C. parvum oocysts followed by analysis for TNF-α at message level by real-time PCR and at protein level by bead-based multiplex sandwich immunoassay. No increase of TNF-α expression either at the message level (A) or at protein level (B) was detected in H69 cells after exposure to viable C. parvum oocysts or lysate for 8 h or 24 h, respectively. As the positive control, a significant increase of TNF-α mRNAs (A), but not at the protein level (B), was detected in H69 cells after exposure to LPS. * p < 0.05, vs. the non-stimulated control.

FIGURE 3

C. parvum infection decreases expression of miR-513 in cholangiocytes. Cells were exposed to C. parvum for 12 h followed by real-time PCR (A) and Luminex FlexmiR Select (B) analyses for miR-513. Experiments were performed in triplicate. *, p < 0.05 ANOVA versus the control.

FIGURE 4

Relief of miR-513-mediated B7-H1 translational suppression occurs in cholangiocytes following C. parvum infection. (A) The schematic of B7-H1 mRNA shows a potential binding site in the B7-H1 3′-UTR for miR-513. (B) The complementary miR-513-binding site in the B7-H1 3′-UTR was inserted downstream of a luciferase reporter on the pMIR-Report plasmid. A control plasmid with the mutant 3′-UTR sequence was also generated. (C) H69 cells were transfected with the reporter constructs and were harvested 24 h after transfection. Luciferase activities were measured and normalized to the control β-gal level. A scrambled antisense oligonucleotide and a non-specific precursor were used as controls. These data are representative of three independent experiments. *, p < 0.05, vs. the empty vector; ^#, p < 0.05, vs. the B7-H1 3′-UTR reporter construct.

FIGURE 5

Decreased miR-513 expression impacts C. parvum-induced B7-H1 protein expression in cholangiocytes. (A) Transfection of miR-513 precursor reduces C. parvum-induced B7-H1 protein expression. H69 cells were transfected with miR-513 precursor or a control non-specific precursor for 48 h, exposed to C. parvum oocysts for 24 h followed and then assayed for B7-H1 by Western blot. A representative Western blot from three independent experiments and densitometric levels of B7-H1 signals are also shown. (B) MicroRNA-513 precursor transfection does not affect C. parvum-induced B7-H1 mRNA expression. H69 cells were transfected with miR-513 precursor or a control non-specific precursor for 48 h and then exposed to viable C. parvum oocysts for 24 h followed by real-time PCR analysis for B7-H1 mRNA. *, p < 0.05, vs. non-infected control cells.

FIGURE 6

MicroRNA-513 influences B7-H1-associated apoptotic cell death in co-cultured Jurkat cells. H69 cells were first exposed to C. parvum oocysts for 24 h and then co-cultured with Jurkat cells in the presence of PMA with or without neutralizing antibodies to B7-H1 or Fas for 24 h. Jurkat cells were then harvested for apoptosis assay by DAPI staining. These data are representative of three independent experiments. Ab = antibody; *, p < 0.05, vs. Jurkat cells co-cultured with non-C. parvum infected H69 cells as the control; #, p < 0.05, vs. Jurkat cells co-cultured with C. parvum oocysts-treated H69 cells without antibody or precursor treatment.