Involvement of IL-18 in the expansion of unique hepatic T cells with unconventional cytokine profiles during Schistosoma mansoni infection

Abstract

Infection with schistosomes invokes severe fibrotic granulomatous responses in the liver of the host. Schistosoma mansoni infection induces dramatic fluctuations in Th1 or Th2 cytokine responses systemically; Th1 reactions are provoked in the early phase, whilst Th2 responses become dominant after oviposition begins. In the liver, various unique immune cells distinct from those of conventional immune competent organs or tissues exist, resulting in a unique immunological environment. Recently, we demonstrated that S. mansoni infection induces unique CD4+ T cell populations exhibiting unconventional cytokine profiles in the liver of mice during the period between Th1- and Th2-phases, which we term the transition phase. They produce both IFN-γ and IL-4 or both IFN-γ and IL-13 simultaneously. Moreover, T cells secreting triple cytokines IFN-γ, IL-13 and IL-4 were also induced. We term these cells Multiple Cytokine Producing Hepatic T cells (MCPHT cells). During the transition phase, when MCPHT cells increase, IL-18 secretion was up-regulated in the liver and sera. In S. mansoni-infected IL-18-deficient mice, expansion of MCPHT cells was curtailed. Thus our data suggest that IL-18 produced during S. mansoni infection play a role in the expansion of MCPHT cells.

Figure 1. Serum levels of IL-18 are up-regulated during the transition phase of S. mansoni infection.

(A, B) Hepatic lymphocytes were isolated from S. mansoni-infected mice at 6 weeks PI. Cytokine production was analyzed by ICS after TCR stimulation. (C) Sera were obtained at indicted time points, and serum levels of IL-18 were determined by ELISA. Data represent the mean values+SD of three or four mice in each experimental time point. ND, not detected. (A, B) Similar results were obtained in three independent experiments.

Figure 2. S. mansoni infection triggers the increase of IL-18 within a liver.

The concentrations of IL-18 in the liver of S. mansoni-infected mice were measured as described in the Materials and Methods. Data are expressed as mean values+SD of three or four mice in each experimental time point. *0.02<P<0.05 (Mann-Whitney U test). Data shown are a representative of three independent experiments.

Figure 3. IL-18 plays a role in the expansion of MCPHT cells during S. mansoni infection.

(A–D) Hepatic lymphocytes were isolated from wild type (WT) or IL-18-deficient (IL-18KO) mice at indicated time points of S. mansoni infection, and the proportions and absolute numbers of γ4, γ13 (A and C), or triple positive (B and D) cells were analyzed by ICS upon TCR ligation. (A and B) One example using hepatic lymphocytes prepared at 6 weeks PI was exhibited. (C and D) Upper graphs; the percentages express the proportions in CD4-positive (γ4 or γ13 cells, C) or in lymphocyte (triple positive cells, D) population. Lower graphs; the absolute numbers of γ4, γ13 (C), or triple positive (D) cells were demonstrated. Data are expressed as mean values+SD of three or four mice in each experimental time point. Data shown are a representative of three independent experiments. (C) *0.02<P<0.05 (Mann-Whitney U test). (E) EPG of WT and IL-18 KO mice were analyzed at 4 and 6 weeks PI. Data represent the mean values+SD of four mice in each experimental time point. This is one representative of three independent experiments. (C–E) Open bars represent WT mice, and filled bars do IL-18KO mice.

Figure 4. IL-18 receptor is expressed upon some MCPHT cells.

(A) Hepatic lymphocytes were isolated from S. mansoni-infected mice at 6 weeks PI and ICS was conducted after TCR ligation. The expression levels of IL-18 receptor α (IL-18Rα) upon γ4 (upper panels) or γ13 (lower panels) cells were analyzed. Cells were stained with anti-IL-18Rα (red line) or its respective isotype control (black line). One representative result is shown. (B) The proportions of IL-18Rα-positive (filled bars) or -negative (open bars) population in γ4 (upper graph) or γ13 (lower graph) cells are shown. Data shown are a representative of four independent experiments.

Figure 5. iNKT cells are not the major population of MCPHT cells induced during the transition phase.

Hepatic lymphocytes were isolated from S. mansoni- infected or non-infected, naïve mice and the expressions of iNKT cell-specific TCR were analyzed with αGalCer/CD1d tetramer after TCR stimulation at 6 weeks PI. αGalCer-unloaded tetramer was used for the negative controls. The numbers in the insets represent percentages of tetramer-positive populations in CD4⁺ T cells sampled from naïve mice, γ4, γ13, or IFN-γ⁻ IL-4⁻ (γ⁻4⁻) cells from the infected mice. Similar results were obtained in two independent experiments.

Figure 6. Infection with increased numbers of cercariae augments the induction of MCPHT cells.

(A–D) Hepatic lymphocytes were prepared from S. mansoni-infected mice at indicated time points, and the proportions and absolute numbers of γ4, γ13 (A and C), or triple positive (B and D) cells were investigated by ICS. (A and B) One example using hepatic lymphocytes prepared at 6 weeks PI was displayed. (C and D) Upper graphs; the proportions in CD4-positive (γ4 or γ13 cells, C) or in lymphocyte (triple positive cells, D) population are shown. Lower graphs; the absolute numbers of γ4, γ13 (C), or triple positive (D) cells are shown. Data are expressed as mean values+SD of three or four mice in each experimental time point. Data shown are a representative of three independent experiments. (C–D) Open bars represent the mice infected with 25 cercariae, and filled bars do those with 250 cercariae. *0.02<P<0.05 (Mann-Whitney U test).