Endogenous IL-27 during toxoplasmosis limits early monocyte responses and their inflammatory activation by pathological T cells

Abstract

Mice that lack the genes for IL-27, or the IL-27 receptor, and infected with Toxoplasma gondii develop T cell-mediated pathology. Here, studies were performed to determine the impact of endogenous IL-27 on the immune response to T. gondii in wild-type (WT) mice. Analysis of infected mice revealed the early production of IL-27p28 by a subset of Ly6C^hi, inflammatory monocytes, and sustained IL-27p28 production at sites of acute and chronic infection. Administration of anti-IL-27p28 prior to infection resulted in an early (day 5) increase in levels of macrophage and granulocyte activation, as well as enhanced effector T cell responses, as measured by both cellularity, cytokine production, and transcriptional profiling. This enhanced acute response led to immune pathology, while blockade during the chronic phase of infection resulted in enhanced T cell responses but no systemic pathology. In the absence of IL-27, the enhanced monocyte responses observed at day 10 were a secondary consequence of activated CD4⁺ T cells. Thus, in WT mice, IL-27 has distinct suppressive effects that impact innate and adaptive immunity during different phases of this infection.

Importance: The molecule IL-27 is critical in limiting the immune response to the parasite Toxoplasma gondii. In the absence of IL-27, a lethal, overactive immune response develops during infection. However, when exactly in the course of infection this molecule is needed was unclear. By selectively inhibiting IL-27 during this parasitic infection, we discovered that IL-27 was only needed during, but not prior to, infection. Additionally, IL-27 is only needed in the active areas in which the parasite is replicating. Finally, our work found that a previously unstudied cell type, monocytes, was regulated by IL-27, which contributes further to our understanding of the regulatory networks established by this molecule.

Keywords: IL-27p28; immunity; inflammation; neutralizing antibody; toxoplasma.

Fig 1

IL-27p28 expression in T. gondii infection. (A) Kinetics of circulating IL-27p28 during toxoplasmosis. (B and C) Expression of IL-27p28 was determined among CD64⁺ monocytes and macrophages at indicated dates of post infection. (D) Cell type expression of IL-27p28 in the spleen of infected mice at 5 dpi was determined by flow cytometry. (E and F) 10,000 monocyte events were pooled from 3 infected reporter mice, for 30,000 total events. Unsupervised UMAP analysis was performed based on the expression of 15 surface markers (see Materials and Methods), excluding IL-27p28 GFP. Supervised gating of IL-27p28-GFP⁺ and GFP^– cells was then overlayed over the UMAP output (E). (F) Expression of six surface markers most highly associated with differences between GFP⁺ and GFP^– clusters are shown.

Fig 2

IL-27p28 neutralization results in pathology similar to p28 deficiency in acute toxoplasmosis. (A) Survival of IgG isotype controls (n = 10) and anti-IL-27p28 treated mice (n = 10) with T. gondii infection. (B) Serum levels of IL-27p28 were measured in naïve (left two bars) and infected (right two bars) with isotype or blockade treatment. Liver histology (C) and serum ALT (D) in indicated groups at 10 days post infection. (E) IFN-γ level in the serum (left) or from splenocytes incubated with T.gondii antigen (right) at 10 dpi were determined by ELISA (n = 5–8, from 2 to 3 experiments, mean ± SEM). * and ** indicate P ≤ 0.05 and 0.01, respectively.

Fig 3

IL-27p28 blockade results in differential transcript and cytokine expression profiles at 5 dpi. (A) A schematic of IL-27 blockade is shown (left). Gene expression between isotype and αIL-27p28 treated CD45⁺ cells was then compared, with the top 35 differentially expressed genes shown. (B) CIBERSORT (23), a bioinformatics technique that identifies cell types based on gene expression profiles, assigned immune cell types to the profile identified in (A). Warmer colors indicate greater association with the profile, while cooler colors indicate less association. (C) Serum from naive, isotype, or anti-IL-27p28 treated mice was collected at 5 dpi and analyzed by multiplexed cytokine analysis. Unbiased hierarchical clustering was then performed, and three dominant clusters are shown.

Fig 4

Inflammatory monocyte responses are enhanced in the absence of IL-27 and impacted by CD4⁺ T cell responses. (A) Splenocytes from WT and IL-27R^−/− mice at 10 dpi were isolated, incubated with BFA and GolgiStop for 4 h before analyzing monocyte expression of TNFα by flow cytometry. Representative plots are shown (left) and quantified (right). Statistical analysis was performed using Welch’s t-test. ** indicates P ≤ 0.01. (B) CD4⁺ T cells were depleted from WT and IL-27R KO mice at 7 dpi. Splenocytes were then isolated at 11 dpi, and monocyte TNFα expression was analyzed as above. Representative flow plots are shown (left) and quantified (right). Statistical analysis was performed using one-way ANOVA, followed by Tukey’s multiple comparison test. *, **, and **** indicate P ≤ 0.05, 0.01, and 0.0001, respectively.

Fig 5

T cell responses to anti-IL-27p28 treatment in acute toxoplasmosis. (A) T. gondii-specific CD4⁺ (top) and CD8⁺ (bottom) T cells from the spleens of mice treated with either isotype (left) or anti-IL-27p28 (right) antibodies were analyzed by flow cytometry at 10 dpi for their expression of IFNγ following stimulations with PMA + ionomycin treatment. Representative flow plots are shown (left) and cell numbers quantified (right). (B) IFNγ-Thy1.1 reporters were treated with isotype (left) or anti-p28 antibodies (right), infected as above and CD4⁺ (top) and CD8⁺ (bottom) T cells were measured for reporter expression from splenocytes at 10 dpi. Representative flow plots of CD11a^hiThy1.1⁺ cells are shown (left) and the percentage and numbers of tetramer⁺ CD11a^hiThy1.1⁺ cells quantified (right). (C) T. gondii-specific and (D) polyclonal, Foxp3⁻CD25⁺ CD4⁺ (top) and CD8⁺ (bottom) T cells in the spleens of isotype IgG and anti-IL-27p28 antibody mice at 10 dpi were analyzed. (E) Comparison of T cell subset by CXCR3 and KLRG1 expression among T.gondii experienced CD4⁺ and CD8⁺ T cells is provided for the indicated groups. (F) Microarray analysis of CD4⁺, T. gondii-specific KLRG1^lo and KLRG1^hi cells from splenocytes of mice treated with anti-IL-27p28 or isotype control antibodies was performed. Volcano plots showcasing alterations in gene expression during blockade are shown, with Venn diagrams of the numbers upregulated and downregulated genes in each populations shown below. Representative and combined data collected (mean ± SEM, n = 5–9) from 2 to 3 independent experiments. *, **, and **** indicate P ≤ 0.05, 0.01, and 0.0001, respectively.