Coinfection with the intestinal nematode Heligmosomoides polygyrus markedly reduces hepatic egg-induced immunopathology and proinflammatory cytokines in mouse models of severe schistosomiasis

Abstract

Infection with the trematode helminth Schistosoma mansoni results in a parasite egg-induced, CD4 T-cell-mediated, hepatointestinal granulomatous and fibrosing inflammation that varies greatly in severity, with a higher frequency of milder forms typically occurring in regions where the disease is endemic. One possible explanation for this is that in these regions the degree of inflammation is lessened by widespread concurrent infection with gastrointestinal nematodes. We tested this hypothesis by establishing a murine coinfection model in which mice were infected with the intestinal nematode parasite Heligmosomoides polygyrus prior to infection with S. mansoni. In CBA mice that naturally display a severe form of schistosomiasis, preinfection with H. polygyrus resulted in a marked reduction in schistosome egg-induced hepatic immunopathology, which was associated with significant decreases in the levels of interleukin-17 (IL-17), gamma interferon, tumor necrosis factor alpha, IL-23, IL-6, and IL-1beta and with increases in the levels of IL-4, IL-5, IL-10, and transforming growth factor beta in mesenteric lymph node cells, purified CD4 T cells, and isolated liver granuloma cells. There also were increases in liver Ym1 and forkhead box P3 transcription factor expression. In another model of high-pathology schistosomiasis induced in C57BL/6 mice by immunization with schistosome egg antigens in complete Freund's adjuvant, coinfection with the nematodes also resulted in a marked inhibition of hepatic immunopathology accompanied by similar shifts in cytokine production. These findings demonstrate that intestinal nematodes prevent Th1- and Th17-cell-mediated inflammation by promoting a strong Th2-polarized environment associated with increases in the levels of alternatively activated macrophages and T regulatory cells, which result in significant amelioration of schistosome-induced immunopathology.

FIG. 1.

Hepatic immunopathology, worm burdens, and egg counts in CBA mice coinfected with H. polygyrus and S. mansoni. (A) Hepatic granulomatous inflammation, measured as described in Materials and Methods, was significantly decreased in CBA mice coinfected with H. polygyrus and S. mansoni compared with control CBA mice infected with S. mansoni alone. (B and C) There were no significant differences in the numbers of (B) S. mansoni worms or (C) S. mansoni eggs between CBA mice infected with schistosomes alone and CBA mice coinfected with H. polygyrus. Thirteen to 23 1-mm² fields were counted for each liver section, and 10 livers were counted for each mouse group. The immunopathology, worm, and egg data are representative of the results of two or three independent experiments. Sm, S. mansoni; Hp, H. polygyrus; ns, not significant.

FIG. 2.

Cytokine production by SEA-stimulated bulk MLNC, CD4 T cells, and GC in CBA mice coinfected with H. polygyrus and S. mansoni. Cytokine levels in 48-h supernatants from SEA-stimulated bulk MLNC, CD4 T cells plus APC, and GC were measured by ELISA. (A) The levels of IL-17, IFN-γ, and TNF-α produced by bulk MLNC, CD4 T cells, and GC were significantly lower in CBA mice coinfected with H. polygyrus and S. mansoni than in CBA mice infected with S. mansoni alone. (B) The levels of IL-4 and TGF-β, but not the level of IL-5 or IL-10, produced by bulk MLNC were significantly higher in CBA mice coinfected with H. polygyrus and S. mansoni than in CBA mice infected with S. mansoni alone. IL-4, IL-5, and IL-10 production, but not TGF-β production, was significantly increased in CD4 T cells, and production of all four cytokines was significantly increased in GC. The bars indicate the means of triplicate determinations, and the error bars indicate the standard deviations; background cytokine levels from unstimulated cells were subtracted. The results shown are from one experiment that was representative of three experiments in which similar results were obtained. Sm, S. mansoni; Hp, H. polygyrus; ns, not significant.

FIG. 3.

mRNA transcripts from livers of CBA mice coinfected with H. polygyrus and S. mansoni. mRNA levels were measured by real-time quantitative RT-PCR as described in Materials and Methods. (A) The mRNA transcript levels of IL-17, IFN-γ, TNF-α, IL-23p19, IL-6, and IL-1β, as well as the chemokines CXCL1 and CXCL2, were decreased and (B) the levels of IL-4 and IL-10, as well as the chemokine CCL11, were increased in CBA mice coinfected with H. polygyrus and S. mansoni compared with CBA mice infected with S. mansoni alone. The bars indicate the means of two independent experiments using mRNA from three livers in each experiment, and the error bars indicate the standard deviations. All P values are at least <0.05. Sm, S. mansoni; Hp, H. polygyrus.

FIG. 4.

Ym1 and Foxp3 expression in livers from CBA mice coinfected with H. polygyrus and S. mansoni. mRNA levels were measured by real-time quantitative RT-PCR as described in Materials and Methods. The mRNA transcript levels of Ym1 and Foxp3 were significantly greater in CBA mice coinfected with H. polygyrus and S. mansoni than in CBA mice infected with S. mansoni alone. The bars indicate the means of two independent experiments using mRNA from three livers in each experiment, and the error bars indicate the standard deviations. Sm, S. mansoni; Hp, H. polygyrus.

FIG. 5.

Hepatic immunopathology in SEA/CFA-immunized BL/6 mice coinfected with H. polygyrus and S. mansoni. Hepatic granulomatous inflammation, measured as described in Materials and Methods, was significantly increased in SEA/CFA-immunized mice compared with unimmunized BL/6 mice (P < 0.001) but was significantly decreased in SEA/CFA-immunized BL/6 mice coinfected with H. polygyrus compared with SEA/CFA-immunized BL/6 mice infected with schistosomes alone. The results shown are from one experiment representative of three in which similar results were obtained. Sm, S. mansoni; Hp, H. polygyrus; Imm, SEA/CFA immunization.

FIG. 6.

Cytokine production by SEA-stimulated bulk MLNC, CD4 T cells, and GC in SEA/CFA-immunized BL/6 mice coinfected with H. polygyrus and S. mansoni. Cytokine levels in 48-h supernatants from SEA-stimulated bulk MLNC, CD4 T cells plus APC, and GC were measured by ELISA. (A) The levels of IL-17, IFN-γ, and TNF-α produced by bulk MLNC, CD4 T cells, and GC were significantly lower in SEA/CFA-immunized BL/6 mice coinfected with H. polygyrus than in SEA/CFA-immunized BL/6 mice infected with S. mansoni alone. (B) The levels of IL-4, IL-5, and IL-10 produced by bulk MLNC were not significantly different in SEA/CFA-immunized mice coinfected with H. polygyrus and SEA/CFA-immunized mice infected with S. mansoni alone, but the level of TGF-β was higher in SEA/CFA-immunized mice coinfected with H. polygyrus than in SEA/CFA-immunized mice infected with S. mansoni alone. IL-4, IL-5, and IL-10 production, but not TGF-β production, by CD4 T cells was significantly greater in SEA/CFA-immunized BL/6 mice coinfected with H. polygyrus than in SEA/CFA-immunized BL/6 mice infected with S. mansoni alone, and the levels of all four cytokines were significantly greater in GC from SEA/CFA-immunized BL/6 mice coinfected with H. polygyrus than in GC from SEA/CFA-immunized BL/6 mice infected with S. mansoni alone. The bars indicate the means of triplicate determinations, and the error bars indicate standard deviations; background cytokine levels from unstimulated cells were subtracted. The results shown are from one experiment that was representative of three experiments in which similar results were obtained. Sm, S. mansoni; Hp, H. polygyrus; Imm, SEA/CFA immunization; ns, not significant.