IL-27 receptor signalling restricts the formation of pathogenic, terminally differentiated Th1 cells during malaria infection by repressing IL-12 dependent signals

Abstract

The IL-27R, WSX-1, is required to limit IFN-γ production by effector CD4⁺ T cells in a number of different inflammatory conditions but the molecular basis of WSX-1-mediated regulation of Th1 responses in vivo during infection has not been investigated in detail. In this study we demonstrate that WSX-1 signalling suppresses the development of pathogenic, terminally differentiated (KLRG-1⁺) Th1 cells during malaria infection and establishes a restrictive threshold to constrain the emergent Th1 response. Importantly, we show that WSX-1 regulates cell-intrinsic responsiveness to IL-12 and IL-2, but the fate of the effector CD4⁺ T cell pool during malaria infection is controlled primarily through IL-12 dependent signals. Finally, we show that WSX-1 regulates Th1 cell terminal differentiation during malaria infection through IL-10 and Foxp3 independent mechanisms; the kinetics and magnitude of the Th1 response, and the degree of Th1 cell terminal differentiation, were comparable in WT, IL-10R1⁻/⁻ and IL-10⁻/⁻ mice and the numbers and phenotype of Foxp3⁺ cells were largely unaltered in WSX-1⁻/⁻ mice during infection. As expected, depletion of Foxp3⁺ cells did not enhance Th1 cell polarisation or terminal differentiation during malaria infection. Our results significantly expand our understanding of how IL-27 regulates Th1 responses in vivo during inflammatory conditions and establishes WSX-1 as a critical and non-redundant regulator of the emergent Th1 effector response during malaria infection.

Figure 1. WSX-1 signalling restrains the Th1 response during malaria infection.

WT and WSX-1^−/− mice were infected i.v. with 10⁴ with P. berghei NK65 pRBC. (A) Representative plots showing T-bet expression by splenic CD4⁺ effector (CD44⁺ CD62L⁻) T cells from naïve and infected WT and WSX-1^−/− mice. (B, C) The (B) frequencies and (C) total numbers of splenic CD4⁺ effector T-bet⁺ T cells in WT and WSX-1^−/− mice. (D) Representative plots showing IFN-γ and TNF expression by splenic Th1 effector CD4⁺ T cells from naïve and infected WT and WSX-1^−/− mice following in vitro PMA and ionomycin stimulation. (E–F) The Mean fluorescence intensity of (E) TNF and (F) IFN-γ expression by CD4⁺ effector Th1 cells from naïve and infected WT and WSX-1^−/− mice. (G) The frequencies of polyfunctional CD4⁺ effector Th1 cells expressing IFN-γ and TNF within the spleen of naïve and infected WT and WSX-1^−/− mice. The results are the mean +/− SEM of the group with 3–5 mice per group. The results are representative of 5 independent experiments. * P<0.05 between WT and WSX-1^−/− mice.

Figure 2. WSX-1 signalling inhibits the development of terminally differentiated KLRG-1⁺ Th1 cells during malaria infection.

WT and WSX-1^−/− mice were infected i.v. with 10⁴ P. berghei NK65 pRBC. (A) Representative plots showing KLRG-1 expression by splenic Th1 effector CD4⁺ T cells from naïve and infected WT and WSX-1^−/− mice. (B, C) The (B) frequencies and (C) total numbers of splenic Th1 effector CD4⁺ T cells from naïve and infected WT and WSX-1^−/− mice expressing KLRG-1. (D) Representative histograms showing the expression of KLRG-1 by CD4⁺ effector T-bet⁺ and T-bet⁻ T cells derived from naïve (dashed) and D14 infected (solid line) WT (black) and WSX-1^−/− (grey) mice. The results are the mean +/− SEM of the group with 3–5 mice per group. The results are representative of 7 independent experiments. * P<0.05 between WT and WSX-1^−/− mice.

Figure 3. Phenotypic profiling of T-bet⁺ cells in WT and WSX-1^−/− mice reveals dysregulated expression of multiple disparate pathways.

WT and WSX-1^−/− mice were infected i.v. with 10⁴ P. berghei NK65 pRBC. (A–D) Expression of cytokine receptors and regulatory receptors by splenic Th1 effector CD4⁺ T cells from naïve and infected WSX-1^−/− mice. (A,C) Representative histograms showing expression of each receptor by splenic Th1 effector CD4+ T cells from WT (red lines) and WSX-1^−/− mice (blue lines) on days 9 and 14 of infection. (B,D) The mean fluorescence intensity of receptor expression by splenic Th1 effector CD4⁺ T cells from naïve and infected WT and WSX-1^−/− mice. The results are the mean +/− SEM of the group with 3–5 mice per group. The results are representative of 4 independent experiments. * P<0.05 between WT and WSX-1^−/− mice.

Figure 4. Splenic Th1 cells from infected WSX-1^−/− mice are hyper-responsive to IL-12 and IL-2.

(A–F) Splenocytes derived from naïve and P. berghei NK65 infected (day 9 and 14) WT and WSX-1^−/− mice were treated with (A, C, E) rIL-12p70 or (B, D, F) rIL-2 for 10 minutes and the responsiveness of effector CD4⁺ T cell populations was determined by the levels of pSTAT4 and pSTAT5 expression respectively. (A, B) Representative histograms demonstrating the level of (A) pSTAT4 and (B) pSTAT5 expression in Th1 effector CD4⁺ T cells from WT and WSX-1^−/− mice following rIL-12p70 and rIL-2 stimulation respectively. (C, D) Representative histograms demonstrating the level of (C) pSTAT4 and (D) pSTAT5 in T-bet⁺ and T-bet⁻ effector CD4⁺ T cells from infected WSX-1^−/− mice following rIL-12p70 and rIL-2 stimulation respectively. (E, F) Representative histograms demonstrating the level of (E) pSTAT4 and (F) pSTAT5 in T-bet⁺KLRG-1⁺ and T-bet⁺KLRG-1⁻ effector CD4⁺ T cells from infected WSX-1^−/− mice following rIL-12p70 and rIL-2 stimulation respectively. (G) The plasma levels of IL-12p70 and IL-2 in naïve and infected (D14) WT and WSX-1^−/− mice, as measured by cytokine bead array. (H) Splenic CD4⁺ T cells purified from WT and WSX-1^−/− mice on day 14 of infection were restimulated in vitro with anti-CD3 and the frequencies of CD4⁺ effector Th1 cells expressing Il-2 was determined after 4 days by intracellular staining. (G) The results are the mean +/− SEM of the group with 3–5 mice per group. * P<0.05 between infected WT and WSX-1^−/− mice. (A–F) The results are representative of 4 independent experiments.

Figure 5. Neutralisation of IL-12p40, but not IL-2, attenuates the Th1 response in WSX-1^−/− mice during malaria infection.

WT and WSX-1^−/− mice were infected i.v. with 10⁴ P. berghei NK65 pRBC. (A–F) Groups of WSX-1^−/− mice were injected with 250 µg of anti-IL-12 or anti-IL-2 on days 7, 9, 11 and 13 of infection, or isotype Abs in control groups. (A, D) Representative plots from day 14 of infection showing (A) T-bet expression by splenic CD4⁺ effector T cells and (D) KLRG-1 expression by Th1 effector CD4⁺ T cells from WT, WSX-1^−/− and treated WSX-1^−/− mice. The frequencies (B, E) and total numbers (C, F) of splenic effector and effector Th1 CD4⁺ T cells expressing (B, C) T-bet and (E, F) KLRG-1, respectively. (G) The peripheral parasite burdens in WT, WSX-1^−/− and ani-IL-12p40 treated WSX-1^−/− mice were assessed on thin smears by microscopy. (H) The level of hepatic pathology in WT, WSX-1^−/− mice and WSX-1^−/− mice treated with ani-IL-2 or anti-IL-12p40 mAbs was examined on day 13 of infection by histological examination of H & E stained tissue sections (20x magnification). n = areas of necrosis. The results are the mean +/− SEM of the group with 3–5 mice per group. The results are representative of 4 independent experiments. * P<0.05 between WT and WSX-1^−/− mice; † P<0.05 between WT and ani-IL-2mAb treated WSX-1^−/− mice; ‡ P<0.05 between WT and anti-IL-12p40 mAb treated WSX-1^−/− mice; ∼P<0.05 between ani-IL-2mAb treated WSX-1^−/− mice and anti-IL-12p40 mAb treated WSX-1^−/− mice.

Figure 6. IL-10 does not control the magnitude or terminal differentiation of the Th1 response during malaria infection.

WT, WSX-1^−/−, IL-10^−/− and IL-10R1^−/− mice were infected i.v. with 10⁴ P. berghei NK65 pRBC. (A) Representative plots showing T-bet expression by splenic CD4⁺ effector T cells from each strain of mice on day 14 of infection. (B–C) The (B) frequencies and (C) total numbers of splenic effector CD4⁺ T cells from each strain of mice expressing T-bet on day 14 of infection. (D) Representative plots showing KLRG-1 expression by splenic Th1 effector CD4⁺ T cells from each strain of mice on day 14 of infection. (E, F) The (E) frequencies and (F) total numbers of effector T-bet⁺ CD4⁺ T cells from each strain of mice expressing KLRG-1 on day 14 of infection. The results are the mean +/− SEM of the group with 3–5 mice per group. The results are representative of 3 independent experiments. * P<0.05 between WT and WSX-1^−/− mice; † P<0.05 between WSX-1^−/− and IL-10^−/− mice; ∼P<0.05 between WSX-1^−/− and IL-10R1^−/− mice; +P<0.05 between WT and IL-10^−/− mice.

Figure 7. The Foxp3⁺ Treg response is unaltered in WSX-1^−/− mice during malaria infection.

WT and WSX-1^−/− mice were infected with P. berghei NK65. (A) Representative plots showing the expression of Foxp3 vs Tbet by splenic CD4⁺ T cells from naive and infected WT and WSX-1^−/− mice. (B–E) The (B, D) frequencies and (C, E) total numbers of splenic CD4⁺ T cells from naive and infected WT and WSX-1^−/− mice expressing FoxP3 and splenic CD4⁺FoxP3⁺ T cells from naive and infected WT and WSX-1^−/− mice expressing T-bet, respectively. (F) Representative plots showing the expression of Foxp3 vs IFN-γ by splenic CD4⁺ T cells from naive and infected WT and WSX-1^−/− mice following in vitro stimulation with PMA and ionomycin. (G–H) The (G) frequencies and (H) total numbers of splenic CD4⁺FoxP3⁺ T cells from naive and infected WT and WSX-1^−/− mice expressing IFN-γ. (I–J) The (I) frequencies and (J) total numbers of splenic CD4⁺FoxP3⁺ T cells from naive and infected (D14) WT and WSX-1^−/− mice expressing CXCR3. The results are the mean +/− SEM of the group with 3–5 mice per group. The results are representative of 3 independent experiments. * P<0.05 between WT and WSX-1^−/− mice.