Differential regulation of beta-defensin gene expression during Cryptosporidium parvum infection

Abstract

Invasion of enterocytes by pathogenic microbes evokes both innate and adaptive immune responses, and microbial pathogens have developed strategies to overcome the initial host immune defense. beta-Defensins are potentially important endogenous antibiotic-like effectors of innate immunity expressed by intestinal epithelia. In this study, the interplay between the enteric protozoan parasite Cryptosporidium parvum and host epithelial beta-defensin expression was investigated. Using human and murine models of infection, we demonstrated that C. parvum infection differentially regulates beta-defensin gene expression. Downregulation of murine beta-defensin-1 mRNA and protein was observed in both in vitro and in vivo models of infection. Infection of the human colonic HT29 cell line with the parasite resulted in differential effects on various members of the defensin gene family. Partial reduction in human beta-defensin-1 (hBD-1), induction of hBD-2, and no effect on hBD-3 gene expression was observed. Recombinant hBD-1 and hBD-2 peptides exhibited significant antimicrobial activity against C. parvum sporozoites in vitro. These findings demonstrate that C. parvum infection of enterocytes may affect the expression of various defensins in different ways and suggest that the overall outcome of the effect of antimicrobial peptides on early survival of the parasite may be complex.

FIG. 1.

Effect of C. parvum infection on mBD-1 and mBD-3 expression in CMT-93 cells. (a) RT-PCR demonstrated that C. parvum downregulated mBD-1 mRNA expression in CMT-93 cells at 24 and 48 h postinfection, with a greater effect observed at 24 h. In contrast, the parasite had no effect on the expression of mBD-3 in CMT-93 cells. (b) Western blots showing downregulation of mBD-1 peptide expression in CMT-93 cells 24 and 48 h after infection with C. parvum.

FIG. 2.

Effect of C. parvum infection on mBD-1 expression in the intestine of mice. (a) Measurement of C. parvum oocyst production in infected neonatal BALB/c mice. Oocyst excretion first became patent on day 4, the peak of infection occurred around day 7, and recovery was achieved by day 21. (b) Results of RT-PCR showing dynamic changes in colonic mBD-1 mRNA expression in infected BALB/c mice. There was marked downregulation on day 4, complete inhibition on day 7, and expression recovery in some mice (two of four) on day 14 postinfection. (c) Densitometric analysis of RT-PCR data showing significant downregulation of mBD-1 mRNA expression on days 4 and 14 (P < 0.02), and no defensin expression was observed on day 7.

FIG. 3.

Role of IFN-γ in downregulation of mBD-1 expression by C. parvum infection. (a) Oocyst production by C. parvum-infected neonatal BALB/c IFN-γ KO mice. The mice developed an acute infection that peaked around day 7, followed by a low-level chronic infection lasting until at least day 42. (b) RT-PCR analysis showed downregulation of mBD-1 mRNA at the peak of infection (day 7), but expression had recovered by day 14. (c) Densitometry analysis of data showing significant inhibition of mBD-1 at day 7 (P < 0.03) but not at day 14.

FIG. 4.

Regulation of human β-defensin gene expression in HT29 cells during C. parvum infection. (a) RT-PCR analysis showing that C. parvum infection downregulated hBD-1 mRNA 24 h after infection. (b) Infection upregulated hBD-2 mRNA expression in HT29 cells 24 h after infection. (c) Infection did not induce hBD-3 mRNA expression in HT29 cells, although cells stimulated with IFN-γ (40 ng/ml) did express hBD-3.

FIG. 5.

Effect of proinflammatory cytokines on human β-defensin gene expression in HT29 cells. Cells were incubated with recombinant human interleukin-1β (IL-1β) (20 ng/ml), IFN-γ (40 ng/ml), or interleukin-6 (IL-6) (40 ng/ml) for 24 h. RT-PCR was performed on RNA extracted from control and stimulated cells. There was constitutive expression of hBD-1 mRNA in unstimulated cells, and this was not affected by stimulation with proinflammatory cytokines. In contrast, hBD-2 mRNA expression was upregulated after stimulation with interleukin-1β but not with IFN-γ or interleukin-6. hBD-3 mRNA was not detected in unstimulated cells but was upregulated in response to all three cytokines.

FIG. 6.

Effect of treatment of C. parvum sporozoites with recombinant human β-defensins on in vitro intracellular development. Purified C. parvum sporozoites (4 × 10⁵) were exposed to 10⁻⁵ M rhBD-1 or hBD-2 or medium only for 1 h and then added to CMT-93 cell monolayers in a 24-well plate. After 24 h, the cells were fixed and stained with Giemsa stain, and the number of intracellular parasites was determined by microscopic examination. Treatment with either defensin reduced the viability of sporozoites compared with that of sporozoites incubated in medium only (P < 0.05).

FIG. 7.

Flow cytometry measurement of in vitro killing of C. parvum sporozoites by recombinant human β-defensins. Purified C. parvum sporozoites were exposed to 10⁻⁵ M rhBD-1 or rhBD-2 or incubation medium only for 1 h and then stained with CFSE. The representative scatter plot analyses were obtained after the following treatments: the upper plot is an untreated sample, with the right gate representing viable sporozoites as determined by size and granularity and the left gate representing dead parasites; the middle plot shows sporozoites treated with rhBD-1; the bottom plot shows sporozoites treated with rhBD-2. Analysis of flow cytometry data indicated that exposure to rhBD-1 or rhBD-2 reduced the percentages of viable parasites by 20 and 40%, respectively.