Local peroxynitrite formation contributes to early control of Cryptosporidium parvum infection

Abstract

In intestinal inflammation, mucosal injury is often exacerbated by the reaction of NO with neutrophil-derived superoxide to form the potent oxidant peroxynitrite. Peroxynitrite also has antimicrobial properties that aid in the killing mechanism of macrophages and neutrophils. Cryptosporidium parvum parasitizes intestinal epithelium, resulting in loss of epithelial cells and mucosal inflammation. Synthesis of NO is significantly increased and arises from the induced expression of inducible nitric oxide synthase (iNOS) by the infected epithelium. Inhibition of iNOS results in intensified epithelial parasitism and oocyst excretion. We hypothesized that formation of peroxynitrite is restricted to sites of iNOS expression by the epithelium and contributes to host defense in C. parvum infection. Accordingly, the location and biological effects of peroxynitrite formation were examined in neonatal piglets infected with C. parvum. Infected piglets were treated daily with a selective iNOS inhibitor [L-N6-(1-iminoethyl)-lysine] or one of two peroxynitrite scavengers [5,10,15,20-tetrakis(N-methyl-4'-pyridyl)porphyrinato iron(III) or uric acid] or received vehicle. At peak infection, peroxynitrite formation was restricted to sites of iNOS expression by parasitized epithelium and lamina propria of the apical villi. Peroxynitrite formation was dependent on iNOS activity and was inhibited by treatment with peroxynitrite scavengers. Scavengers increased the number of intracellular parasites and the number of infected epithelial cells present per villus and significantly exacerbated oocyst excretion. Recovery from infection was not delayed by ongoing treatment with scavenger. The present results are the first to demonstrate an in vivo role for peroxynitrite formation in acute mucosal defense against a noninvasive intestinal epithelial pathogen.

FIG. 1.

Immunohistochemistry for NT and iNOS expression in ileal mucosa from uninfected and C. parvum-infected piglets on days 1, 3, 5, 8, and 11 of experimental infection. Immunohistochemistry for control piglets is shown for day 5 only and was similar at all time points. H+E, hematoxylin and eosin.

FIG. 2.

Immunoblot results for iNOS (130 kDa) and immunoreactive bands of NT (a stable “footprint” of peroxynitrite action) performed using ileal mucosal protein obtained from piglets at the onset (day 1), peak (days 3 to 5), and recovery (days 8 to 11) stages of infection with C. parvum. Equal protein loading was demonstrated by immunoblotting for actin (40 kDa). Tyrosine-nitrated proteins were immunoprecipitated from equal starting concentrations of total mucosal protein (500 μg). +, nitrotyrosine-labeled bovine serum albumin. M, molecular mass marker (marker shown for iNOS = 132 kDa; marker for actin = 43 kDa).

FIG. 3.

Immunohistochemistry and immunoblots for detection of immunoreactive bands of tyrosine-nitrated proteins in ileal mucosa from piglets infected with C. parvum. Immunoblots are shown from piglets treated daily with phosphate-buffered saline (PBS; 2 ml i.p.; n = 4) or the selective iNOS inhibitor L-NIL (3 mg/kg i.p.; n = 3) (day 4 postinfection) and piglets treated daily with PBS (5 ml i.v.; n = 3) or the peroxynitrite scavenger uric acid (10 mg/kg i.v.; n = 3) (day 11 postinfection). Two piglets treated with the peroxynitrite scavenger FeTMPyP (5 mg/kg i.v.) (day 4 postinfection) are shown paired with their PBS-treated littermate (1 ml i.v.). Tyrosine-nitrated proteins were immunoprecipitated from equal starting concentrations of total mucosal protein (500 μg). +, nitrotyrosine-labeled bovine serum albumin. Negative (Neg.) control, primary antibody omitted.

FIG. 4.

Numbers of fecal oocysts excreted by C. parvum-infected piglets. Piglets were treated daily for 11 days with phosphate-buffered saline (5 ml i.v.) or the peroxynitrite scavenger uric acid (10 mg/kg i.v.). In the lower graph, piglets were treated daily with phosphate-buffered saline (1 ml i.v.) or the peroxynitrite scavenger FeTMPyP (5 mg/kg i.v.) and euthanized on day 4 for histologic studies (shown in Table 1). n, number of piglets. The data are mean ± SE. *, significantly different (P < 0.05) from the value for saline-treated piglets as calculated by Student's t test.