IL-22 Protects Against Liver Pathology and Lethality of an Experimental Blood-Stage Malaria Infection - PubMed (original) (raw)
IL-22 Protects Against Liver Pathology and Lethality of an Experimental Blood-Stage Malaria Infection
Béatris Mastelic et al. Front Immunol. 2012.
Abstract
The host response following malaria infection depends on a fine balance between levels of pro-inflammatory and anti-inflammatory mediators resulting in the resolution of the infection or immune-mediated pathology. Whilst other components of the innate immune system contribute to the pro-inflammatory milieu, T cells play a major role. For blood-stage malaria, CD4(+) and γδ T cells are major producers of the IFN-γ that controls parasitemia, however, a role for TH17 cells secreting IL-17A and other cytokines, including IL-17F and IL-22 has not yet been investigated in malaria. TH17 cells have been shown to play a role in some protozoan infections, but they also are a source of pro-inflammatory cytokines known to be involved in protection or pathogenicity of infections. In the present study, we have investigated whether IL-17A and IL-22 are induced during a Plasmodium chabaudi infection in mice, and whether these cytokines contribute to either protection or to pathology induced during the infection. Although small numbers of IL-17- and IL-22-producing CD4 T cells are induced in the spleens of infected mice, a more pronounced induction is observed in the liver, where increases in mRNA for IL-17A and, to a lesser extent, IL-22 were observed and CD8(+) T cells, rather than CD4 T cells, are a major source of these cytokines in this organ. Although the lack of IL-17 did not affect the outcome of infection or pathology, lack of IL-22 resulted in 50% mortality within 12 days after infection with significantly greater weight loss at the peak of infection and significant increase in alanine transaminase in the plasma in the acute infection. As parasitemias and temperature were similar in IL-22 KO and wild-type control mice, our observations support the idea that IL-22 but not IL-17 provides protection from the potentially lethal effects of liver damage during a primary P. chabaudi infection.
Keywords: IL-17; IL-22; Plasmodium chabaudi; TH17; liver damage; malaria.
Figures
Figure 1
TH17 cells signature cytokines, IL-17A and IL-17F, mRNA transcripts are increased in the liver during a primary P. chabaudi infection. C57BL/6 mice were infected with 105 P. chabaudi and sacrificed post-infection at the time points indicated. Spleen and liver mononuclear cells were harvested from naïve and infected mice and analyzed by real-time PCR. (A) Representative course of infection in C57BL/6 mice. IFN-γ, IL-17A, IL-17F, and IL-22 mRNA levels were determined in naïve and infected splenocytes (B) and liver mononuclear cells (C). Data are normalized to Ubiquitin expression. Test samples were expressed as the fold increase of gene expression compared with expression in normal uninfected mice (mean of expression from three uninfected mice were set at one) versus infected mice samples. Negative values represent levels of gene expression lower than those measured in naïve mice. Data are representative of two experiments and are obtained in groups of five mice per time points (mean ± SEM).
Figure 2
Gating strategies for IL-17 producing cells. IL-17-fate reporter (Il17aCreR26ReYFP) and C57BL/6 mice were infected with 105 P. chabaudi and sacrificed at days 3, 5, and 7 post-infection. (A) Representative flow cytometry plots of live cells gating in spleen and liver mononuclear cells. (B) Gating strategy for CD3+, CD4+, CD8+, NK+, and γδ+ T cells, and CD3−NK+ cells. (C) Total cell numbers of the several T cells populations in C57BL/6 splenocytes and liver mononuclear cells during the first 7 days of P. chabaudi infection. (D) Representative flow cytometry plots of IL-17EYFP or IL-17A+ cells in CD4+ and CD8+ T cells of infected splenocytes and liver mononuclear cells of reporter and C57BL/6 mice, respectively.
Figure 3
In vivo kinetics of IL-17A in spleen and liver of mice infected with P. chabaudi. IL-17-fate reporter (Il17aCreR26ReYFP) and C57BL/6 mice were infected with 105_P. chabaudi_ and sacrificed at days 3, 5, and 7 post-infection. (A) Total numbers of IL-17AeYFP- and IL-17+-cells (detected by intracellular staining) in splenocytes and hepatocytes from naïve and infected IL-17-fate reporter mice (left panel) and C57BL/6 mice (right panel), respectively. The numbers represent the median and range of three mice. The fraction of CD4+, CD8+, γδ+, NK T cells, and NK cells contributing to IL-17A+ production in spleen and liver at day 7 and 5, respectively. (B) Representative flow cytometry plots of IL-17AeYFP versus IFN-γ expression in CD4+ and CD8+ T cells, in liver cells, and splenocytes of naïve and day 5 and 7 infected mice. Numbers in quadrants refer to the percentages of cells in each quadrant. Data are representative of two experiments and are obtained in groups of three mice per time points (mean ± SEM).
Figure 4
In vivo kinetics of IL-22 in spleen and liver of mice infected with P. chabaudi. C57BL/6 mice were infected with 105_P. chabaudi_ and sacrificed at days 3, 5, and 7 post-infection. (A) Total numbers of IL-22+ cells in splenocytes and hepatocytes from naïve and infected C57BL/6 mice. The fraction of CD4+, CD8+, γδ+, NK T cells, and NK cells contributing to IL-22 production in infected splenocytes and mononuclear liver cells on days 5 and 7 post-infection. The numbers represent the median and range of four mice. Representative flow cytometry plots of IL-22 versus IL-17A (left panels) and IL-22 versus IFN-γ (right panels) and expression in CD4+, and CD8+ T in naïve, day 5 and 7 infected liver mononuclear cells (B) and splenocytes (C). Numbers in quadrants refer to the percentages of cells in each quadrant. Data are representative of two experiments and are from groups of four mice per time point.
Figure 5
Parasitemia and pathology in IL-17-fate reporter and IL-17 KO mice. (A) Il17aCre+/+R26ReYFP and conventional (B) _Il17A_−/− mice were infected with 105 P. chabaudi, five mice per group. The heterozygous reporter mice in (A) also act as a control for the homozygous mice as the IL-17 gene is not deleted in these mice. WT C57BL/6 mice were used as controls in (A,B). Weight loss and hypothermia (pathology) were monitored daily. Parasitemias are presented as percentage of parasitized RBC, and the error bars represents the SEMs. Tail blood was collected to measure ALT variation during the infection, using a colorimetric end-point method (adapted from Reitman and Frankel, 1957). Briefly, 20 μl of test serum was added to 100 μl of the ALT substrate and incubated for 30 min at 37°C. Hundred microliters of 2,4-dinitrophenylhydrazine was then added and incubated a further 20 min at RT. One milliliter of 0.4 M NaOH was added to stop the reaction and the OD determined at 490 nm. Water was used as blank. OD values were then converted into the equivalent enzyme units (U/ml) using a standard curve derived from known concentrations of a pyruvate standard. The standard curve for ALT was performed using pyruvate standards. Data are means and SEM of five mice and representative of two experiments. Significant differences are shown using a Mann–Whitney _U_-test of five mice [*significant _P_-value (0.01–0.05)].
Figure 6
Lack of IL-22 but not IL-17A results in increased mortality and liver damage during a P. chabaudi infection. C57BL/6 mice were treated with either 0.5 mg of anti-IL-22 antibody or an isotype control, every other day, from day 0 to 14 post-infection. IL-22 KO mice and anti-IL-22 treated C57BL/6 mice were infected at day 0 with 105 P. chabaudi, five mice per group. Weight loss and hypothermia were monitored daily. Parasitemias are presented as percentage of parasitized RBC, and the error bars represents the SEMs. Tail blood was collected to measure ALT variation during the infection, using a Cobas C111 chemistry analyzer. Data are means and SEM of five mice and representative of two experiments. Significant differences are shown using a Mann–Whitney _U_-test of five mice [*significant _P_-value (0.01–0.05), **very significant _P_-value (0.001–0.01)]. (A) Survival, parasitemia, pathology, and liver damage in IL-22 KO and WT mice. (B) Parasitemia and ALT levels in anti-IL-22 treated or control mice. (C) Plasma IL-1β, TNF-α, and IFN-γ levels were determined by ELISA in naïve and day 7 and 9 infected IL-22 KO mice and WT mice. The numbers and the median are represented of four mice.
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