Lymphocytes and not IFN-gamma mediate expression of iNOS by intestinal epithelium in murine cryptosporidiosis

Abstract

We hypothesized that unrecognized differences in epithelial expression of inducible nitric oxide synthase (iNOS), resulting from engineered immunodeficiency, could explain the contradictory findings of prior studies regarding the importance of nitric oxide (NO) in murine models of Cryptosporidium parvum infection. Severe combined immunodeficient mice (SCID) failed to constitutively or inducibly express epithelial iNOS or increase NO synthesis in response to C. parvum infection. In contrast, mice lacking IFN-gamma alone induced both epithelial iNOS expression and NO synthesis in response to infection. Accordingly, lymphocytes mediate epithelial expression of iNOS and NO synthesis independent of IFN-gamma in response to C. parvum infection. These findings in large part explain the contradictory conclusions of prior studies regarding the role of iNOS in C. parvum infection.

Fig. 1

Constitutive (a, b) and induced NO responses to C. parvum infection (c, d, e, f) are absent in mice lacking lymphocytes but not IFNγ. Immunohistochemistry for iNOS expression by ileal epithelium and plasma NO concentrations were measured in uninfected mice and at peak infection in wild-type BALB/c, SCID, IFNγ deficient (GKO), and SCID mice treated with anti-IFNγ antibodies (SCID + anti- IFNγ). Numbers of mice are indicated at the base of each bar. Differences in iNOS expression were quantified by densitometric analysis of Western blots from 3 mice of each genotype (e, f). *+p<0.05, ** p<0.01 one-way ANOVA versus wild-type BALB/c within the corresponding time period.

Fig. 2

Number of oocysts excreted (a) and histological severity of C. parvum infection (b) is greatest in mice lacking IFNγ. Infection severity scores (scale 1-4) were assigned to the ileum, cecum and colon of wild-type BALB/c, SCID, IFNγ deficient (GKO), and SCID mice treated with anti-IFNγ antibodies (SCID + anti- IFNγ) at the time of peak infection (2 −vs- 4-weeks). Numbers of mice are indicated at the base of each bar. *p<0.05, ** p<0.01, *** p<0.001 one-way ANOVA versus wild-type BALB/c within the corresponding time period. ***p<0.001 Mann-Whitney Rank Sum between scores obtained from the ileum and cecum versus wild type BALB/c mice.