Macrophage migration inhibitory factor: a downregulator of early T cell-dependent IFN-gamma responses in Plasmodium chabaudi adami (556 KA)-infected mice

Abstract

Neutralization of macrophage migration inhibitory factor (MIF) increases anti-tumor cytotoxic T cell responses in vivo and IFN-γ responses in vitro, suggesting a plausible regulatory role for MIF in T cell activation. Considering that IFN-γ production by CD4(+) T cells is pivotal to resolve murine malaria and that secretion of MIF is induced by Plasmodium chabaudi adami parasites, we investigated the effect of MIF deficiency on the infection with this pathogen. Infections with P. c. adami 556 KA parasites were more efficiently controlled in MIF-neutralized and MIF-deficient (knockout [KO]) BALB/c mice. The reduction in parasitemia was associated with reduced production of IL-4 by non-T/non-B cells throughout patent infection. At day 4 postinfection, higher numbers of activated CD4(+) cells were measured in MIF KO mice, which secreted more IFN-γ, less IL-4, and less IL-10 than did CD4(+) T cells from wild-type mice. Enhanced IFN-γ and decreased IL-4 responses also were measured in MIF KO CD4(+) T cells stimulated with or without IL-12 and anti-IL-4 blocking Ab to induce Th1 polarization. However, MIF KO CD4(+) T cells efficiently acquired a Th2 phenotype when stimulated in the presence of IL-4 and anti-IL-12 Ab, indicating normal responsiveness to IL-4/STAT6 signaling. These results suggest that by promoting IL-4 responses in cells other than T/B cells during early P. c. adami infection, MIF decreases IFN-γ secretion in CD4(+) T cells and, additionally, has the intrinsic ability to render CD4(+) T cells less capable of acquiring a robust Th1 phenotype when stimulated in the presence of IL-12.

Figure 1

A. Western Blot analysis of equal amounts of undiluted sera from naive (1, 2) and infected (3, 4) mice (peak infection) indicate systemic release of MIF during P. c. adami DK infection. Kinetics of P. c. adami DK infection (B), peak (C) and cumulative parasitemia (D, E) in wild type and MIF KO BALB/c mice. Data represent the compilation of two independent experiments (n= 7, 8 mice per group).Values were compared using a non parametric Student T Test. ***P<0.001;**P<0.01.

Figure 2

A. Kinetics of P. c. adami DK infection (A), peak (B) and cumulative parasitemia (C, D) in control mice and in mice treated with MIF neutralizing Ab. Data represent the compilation of two independent experiments (n= 7, 8 mice per group).Values were compared using a non parametric Student T Test. ***P<<0.001;**P<0.01.

Figure 3

Cytokine responses in total (SPC), CD4⁺ T and CD90⁻ splenic cell cultures from wild type (WT) and MIF KO P.c. adami infected mice at day 4 (A–C) and day 8 (D–E) post-infection. Concentrations of IFN-γ (A, D), TNF-α (B, E) and IL-10 (C, F) were determined in 48 h culture supernatants by ELISA; 8 or more mice per group were assessed, expect for data from CD90⁻ cells, for which 3–4 mice were considered. Values were compared between WT and MIF KO cells using a non parametric Student T Test. **P<0.01;*P<0.05.

Figure 4

IL-4 responses in total (SPC), CD4⁺ T and CD90⁻ splenic cells from wild type (WT) and MIF KO P.c. adami infected mice at day 4 (A), day 8 (B) and day 12 post-infection (C). IL-4 concentrations were determined in 48 h culture supernatants by ELISA. Eight mice per group were assessed. Values were compared between WT and MIF KO cells for each cell type using a non-parametric Student T Test. ***P<0.001;**P<0.01.

Figure 5

Percentages and absolute numbers of total and activated CD4⁺ T cells in P.c.adami infected mice (day 4 post-infection). Spleen cell suspensions from wild type (WT) and MIF KO mice were stained with anti-CD69 and anti-CD4 monoclonal Ab for analysis by flow cytometry. The percentages of CD4⁺ CD69⁺ cells (A) and their absolute numbers (B), as well as the absolute numbers of splenic CD4⁺ T cells (C) were determined in 8–10 mice per group (naive mice are represented with dashed bars in C). Values were compared using a non-parametric Student T Test. **P<0.01, *P<0.05.

Figure 6

Determination of T-bet and Gata3 gene expression levels in CD4 ⁺ T cells purified from naive and P.c. adami DK-infected mice at day 4 post-infection infection by RT-PCR. Relative expression levels of T-bet (A) and Gata3 (B) were normalized to GADPH by densitometry, and T-bet/Gata3 ratios were estimated (C). Naive mice are represented with dashed bars; data from 4 mice per group is represented. Values were compared between WT and MIF KO cells using a non parametric Student T Test. *P<0.05.

Figure 7

In vitro proliferation of purified CD4⁺ T cells from naive wild type (WT) and MIF KO mice in response to stimulation with anti-CD3 and anti-CD28 Ab. In three independent experiments, CD4⁺ T cells were labelled with CFSE (A,B) or remained unlabelled prior to stimulation for 48 h. Mitotic profiles from WT (A) and MIF KO (B) CD4⁺ T cells were analysed by flow cytometry (MODFIT program) (A,B) and the stimulation indexes were accordingly determined (C). Alternatively, in experiments using unlabelled CD4⁺ T cells, the cellular metabolic activity was determined with the Cell Titer 96 aqueous non-radioactive cell proliferation assay (Promega, USA) and represented as the absorbance at 490nm (D). Values were compared using a non-parametric Student T Test. **P<0.01.

Figure 8

Cytokine levels in culture supernatants from WT and MIF KO CD4⁺ T cells stimulated for 48 h with anti-CD3 and anti-CD28 Abs. IFN-γ (A) and IL-4 (B) levels were assessed by ELISA. Values were compared using a non-parametric Student T Test. ***P<0.001; **P<0.01. Intracellular protein expression levels of T-bet (C) and Gata3 (D) were assessed by flow cytometry in resting (open bars) and stimulated (dashed bars) CD4⁺ T cells (four mice per group) for 48 h (C).

Figure 9

Cytokine responses in purified CD4⁺ T cells from naive wild type (WT) and MIF KO mice differentiated under Th1 or Th2 polarizing conditions. In 4 independent experiments, purified CD4⁺ T cells were stimulated for 3 days with plate bound anti-CD3 and soluble anti-CD28 Ab in the presence of IL-12p70 and anti-IL-4 Ab (Th1 differentiation) or in the presence of IL-4, with anti-IFN-γ and anti-IL-12 Ab (Th2 differentiation). The cells were split and further cultured in the presence of IL-2 for 3 additional days, after which they were plated for 24 h and stimulated with anti-CD3 Ab. Supernatants were analysed for IFN-γ (A), IL-4 (B) and IL-13 (C) content by ELISA. Cytokine values were compared using a non-parametric Student T Test. ***P<0.001; *P<0.05.

Figure 10

Effect of MIF neutralizing antibody on IFN-γ (A) and IL-4 (B) responses in WT CD4⁺ T cells cultured under Th1 or Th2 polarizing conditions. In 4 independent experiments, purified CD4⁺ T cells were stimulated for 3 days with plate bound anti-CD3 and soluble anti-CD28 Ab in the presence of IL-12p70 and anti-IL-4 Ab (Th1 differentiation) or in the presence of IL-4, with anti-IFN-γ and anti-IL-12 Ab (Th2 differentiation). Anti-MIF antibody was added at a final concentration of 100 µg/ml during the first three days of differentiation. The cells were split and further cultured in the presence of IL-2 for 3 additional days, after which they were plated for 24 h and stimulated with anti-CD3 Ab. Supernatants were analysed for IFN-γ (A) and IL-4 (B) content by ELISA. Values were compared between untreated and anti-MIF-treated cells using a non-parametric Student T Test. **P<0.01.