IL-22 Protects Against Liver Pathology and Lethality of an Experimental Blood-Stage Malaria Infection

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

Regulatory B cells shape the development of Th2 immune responses in BALB/c mice infected with Leishmania major through IL-10 production.

Recent evidence indicates that B cells are required for susceptibility to infection with Leishmania major in BALB/c mice. In this study, we analyzed the role of the IL-10 produced by B cells in this process. We showed that B cells purified from the spleen of BALB/c mice produced IL-10 in response to stimulation with L. major in vitro. In vivo, early IL-10 mRNA expression is detected after L. major infection in B cells from draining lymph nodes of susceptible BALB/c, but not of resistant C57BL/6 mice. Although adoptive transfer of naive wild-type B cells prior to infection in B cell-deficient BALB/c mice restored Th2 cell development and susceptibility to infection with L. major of these otherwise resistant mice, adoptive transfer of IL-10(-/-) B cells mice did not. B cells stimulated by L. major, following in vitro or in vivo encounter, express the CD1d and CD5 molecules and the IL-10 produced by these cells downregulate IL-12 production by L. major-stimulated dendritic cells. These observations indicate that IL-10 secreting B cells are phenotypically and functionally regulatory B cells. Altogether these results demonstrate that the IL-10 produced by regulatory CD1d+ CD5+ B cells in response to L. major is critical for Th2 cell development in BALB/c mice

Environmental and T cell-intrinsic factors limit the expansion of neonatal follicular T helper cells but may be circumvented by specific adjuvants.

Follicular Th (T(FH)) cells have emerged as a new Th subset providing help to B cells and supporting their differentiation into long-lived plasma cells or memory B cells. Their differentiation had not yet been investigated following neonatal immunization, which elicits delayed and limited germinal center (GC) responses. We demonstrate that neonatal immunization induces CXCR5(high)PD-1(high) CD4(+) T(FH) cells that exhibit T(FH) features (including Batf, Bcl6, c-Maf, ICOS, and IL-21 expression) and are able to migrate into the GCs. However, neonatal T(FH) cells fail to expand and to acquire a full-blown GC T(FH) phenotype, as reflected by a higher ratio of GC T(FH)/non-GC CD4(+) T cells in immunized adults than neonates (3.8 × 10(-3) versus 2.2 × 10(-3), p = 0.01). Following the adoptive transfer of naive adult OT-II CD4(+) T cells, OT-II T(FH) cells expand in the vaccine-draining lymph nodes of immunized adult but not infant recipients, whereas naive 2-wk-old CD4(+) OT-II cells failed to expand in adult hosts, reflecting the influence of both environmental and T cell-intrinsic factors. Postponing immunization to later in life increases the number of T(FH) cells in a stepwise manner, in direct correlation with the numbers of GC B cells and plasma cells elicited. Remarkably, adjuvantation with CpG oligonucleotides markedly increased GC T(FH) and GC B cell neonatal responses, up to adult levels. To our knowledge, this is the first demonstration that the T(FH) cell development limits early life GC responses and that adjuvants/delivery systems supporting T(FH) differentiation may restore adultlike early life GC B cell responses

<i>P</i>. <i>chabaudi</i>-specific IgG B-cell responses are abrogated in the absence of IL-21 signaling.

<p>(A) IgG, (B) IgG subtypes (day 32 post-infection) and (C) IgM antibodies specific for a lysate of <i>P</i>. <i>chabaudi</i>-infected rbc determined by ELISA. Antibody units (AU) were calculated based on the <i>P</i>. <i>chabaudi</i>-specific antibody levels of a hyper-immune standard plasma defined as 1000 U. In the cases where levels of antibodies were below background, arbitrary values of 2 log lower than the mean value observed in WT C57BL/6 mice were set to be able to perform the statistical test. (D) MSP1₂₁-specific IgG-producing ASC in BM obtained from one femur and one tibia, and (E) MBC per spleen, determined by ELISPOT 32 days post-infection. Statistical significance was obtained using the Kruskal-Wallis test comparing each time point with its respective basal level (day 0 post-infection) (*, P<0.05; **, P<0.01), or comparing with the data obtained from the WT C57BL/6 group (# #, P<0.01). The Mann Whitney U test was used in the case of IgG subtypes, comparing <i>Il21</i>^<i>-/-</i> vs WT C57BL/6 mice (#, P<0.05). Bars represent median values. Data are representative of at least two independent experiments and were obtained in groups of 3–8 mice per time point.</p

IL-21 is produced during <i>P</i>. <i>chabaudi</i> infection and required to control chronic infection.

<p>(A) IL-21 mRNA in spleen cells of <i>P</i>. <i>chabaudi</i>-infected mice measured by real-time quantitative RT-PCR. Parasitemia (B) and total rbc counts (C) were determined in WT C57BL/6 (closed circles), <i>Il21</i>^<i>-/-</i> (open circles) and <i>Il21r</i>^<i>-/-</i> (open squares) mice. (D) Individual examples of spleens from <i>Il21r</i>^<i>-/-</i> (a) <i>Il21</i>^<i>-/-</i> (b) and WT C57BL/6 (c) mice at day 120 post-infection, and a spleen from an age-matched WT C57BL/6 naïve mouse (d). Bar, 1 cm. (E) Total number of nucleated live splenocytes were determined with a hemocytometer in WT C57BL/6 (black bars), <i>Il21</i>^<i>-/-</i> (open bars) and <i>Il21r</i>^<i>-/-</i> (stripped bars) mice. (F) Numbers of Ter119⁺ and Ter119^– cells in the spleen of WT C57BL/6 (black bars) and <i>Il21r</i>^<i>-/-</i> (striped bars) at day 32 post-infection. Data are representative of two or more independent experiments and are obtained in groups of 5–10 mice per time point. Statistical significance was obtained using Mann Whitney U test or Kruskal-Wallis test. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. Error bars correspond to mean ± SEM.</p

<i>P</i>. <i>chabaudi</i>-specific IgG B-cell responses are abrogated in the absence of IL-21 signaling.

<p>(A) IgG, (B) IgG subtypes (day 32 post-infection) and (C) IgM antibodies specific for a lysate of <i>P</i>. <i>chabaudi</i>-infected rbc determined by ELISA. Antibody units (AU) were calculated based on the <i>P</i>. <i>chabaudi</i>-specific antibody levels of a hyper-immune standard plasma defined as 1000 U. In the cases where levels of antibodies were below background, arbitrary values of 2 log lower than the mean value observed in WT C57BL/6 mice were set to be able to perform the statistical test. (D) MSP1₂₁-specific IgG-producing ASC in BM obtained from one femur and one tibia, and (E) MBC per spleen, determined by ELISPOT 32 days post-infection. Statistical significance was obtained using the Kruskal-Wallis test comparing each time point with its respective basal level (day 0 post-infection) (*, P<0.05; **, P<0.01), or comparing with the data obtained from the WT C57BL/6 group (# #, P<0.01). The Mann Whitney U test was used in the case of IgG subtypes, comparing <i>Il21</i>^<i>-/-</i> vs WT C57BL/6 mice (#, P<0.05). Bars represent median values. Data are representative of at least two independent experiments and were obtained in groups of 3–8 mice per time point.</p

Disruption of IL-21 Signaling Affects T Cell-B Cell Interactions and Abrogates Protective Humoral Immunity to Malaria

<div><p>Interleukin-21 signaling is important for germinal center B-cell responses, isotype switching and generation of memory B cells. However, a role for IL-21 in antibody-mediated protection against pathogens has not been demonstrated. Here we show that IL-21 is produced by T follicular helper cells and co-expressed with IFN-γ during an erythrocytic-stage malaria infection of <i>Plasmodium chabaudi</i> in mice. Mice deficient either in IL-21 or the IL-21 receptor fail to resolve the chronic phase of <i>P</i>. <i>chabaudi</i> infection and <i>P</i>. <i>yoelii</i> infection resulting in sustained high parasitemias, and are not immune to re-infection. This is associated with abrogated <i>P</i>. <i>chabaudi</i>-specific IgG responses, including memory B cells. Mixed bone marrow chimeric mice, with T cells carrying a targeted disruption of the <i>Il21</i> gene, or B cells with a targeted disruption of the <i>Il21r</i> gene, demonstrate that IL-21 from T cells signaling through the IL-21 receptor on B cells is necessary to control chronic <i>P</i>. <i>chabaudi</i> infection. Our data uncover a mechanism by which CD4+ T cells and B cells control parasitemia during chronic erythrocytic-stage malaria through a single gene, <i>Il21</i>, and demonstrate the importance of this cytokine in the control of pathogens by humoral immune responses. These data are highly pertinent for designing malaria vaccines requiring long-lasting protective B-cell responses.</p></div

IL-21 is co-expressed with IFN-γ and IL-10 during <i>P</i>. <i>chabaudi</i> infection.

<p>(A-C) Flow cytometry plots showing individual examples for days 8 and 15 post-infection of different cytokine combinations studied in CD3⁺CD4⁺ T cells from the spleen of WT C57BL/6 mice. (D) IL-21-producing CD4⁺ T cells (red) overlaid on the plots corresponding to IFN-γ vs IL-10 on gated CD3⁺CD4⁺ T cells. Cumulative data showing the percentage (E) and total numbers (F) of IL-21-producing CD4⁺ T cells co-expressing IFN-γ, IL-4 and IL-10 in the spleen of WT C57BL/6 mice. The differential combination of expression (+) or absence of expression (–) of each cytokine (indicated in the bottom left) is shown for each subset at different days post-infection. Data are representative of at least two independent experiments and were obtained in groups of 4–6 mice per time point. Statistical significance was obtained using the Kruskal-Wallis test comparing each time point, corresponding to each cytokine combination with its respective basal level (day 0 post-infection). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. Bars represent median values.</p

Mice deficient in IL-21 signaling fail to develop immunity to a secondary <i>P</i>. <i>chabaudi</i> infection.

<p>(A) Scheme describing the experimental approach. CQ = chloroquine. (B and C) <i>P</i>. <i>chabaudi</i>-infected mice were treated with chloroquine to eliminate parasitemia as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004715#sec009" target="_blank">materials and methods</a>, and re-infected with 10⁵<i>P</i>. <i>chabaudi-</i>infected rbc (day 0 post-secondary infection). The graphs show the course of secondary <i>P</i>. <i>chabaudi</i> infection in WT C57BL/6 (black circles), <i>Il21</i>^<i>-/-</i> (red circles) and <i>Il21r</i>^<i>-/-</i> (brown circles) mice; course of primary infection in <i>Il21</i>^<i>-/-</i> (gray circles) and <i>Il21r</i>^<i>-/-</i> (gray squares) are overlaid. (D and E) Number of Tfh cells per spleen post-primary and secondary infection, respectively. (F and G) Number of IFN-γ⁺CD4⁺ T cells per spleen post-primary and secondary infection, respectively. Data are representative of two independent experiments and are obtained in groups of 3–10 mice per time point. Statistical significance was obtained using Mann Whitney U test (**, P<0.01) or Kruskal-Wallis test (#, P<0.05). Error bars correspond to mean ± SEM.</p