Interferon regulatory factor modulation underlies the bystander suppression of malaria antigen-driven IL-12 and IFN-γ in filaria-malaria co-infection

Abstract

In areas where polyparasitism is highly prevalent, the impact of multiple parasites on the host response is underestimated. In particular, the presence of helminth infection coincident with malaria profoundly alters the production of malaria-specific IFN-γ, IL-12p70, CXCL9, CXCL10 and CXCL11, cytokines/chemokines known to be critical in mediating malaria-specific immunity. In order to elucidate the mechanisms underlying the suppression of malaria-specific cytokines/chemokines, we assessed the expression of malaria-specific IL-12Rβ1, IL-12Rβ2 and interferon regulatory factor (IRF)-1 in blood obtained from 18 filaria-infected (Fil(+)) and 17 filaria-uninfected (Fil(-)) individuals in a filaria-malaria co-endemic region of Mali. We found that Fil(+) individuals had significantly lower RNA expression of IRF-1 but not IL-12Rβ1 or IL-12Rβ2 in response to malaria antigen stimulation. We also measured the frequency of IL-12-producing DCs from these subjects and found that Fil(+) subjects had lower frequencies of IL-12(+) mDCs after malaria antigen stimulation than did the Fil(-) subjects. Modeling these data in vitro, we found that mDCs pre-exposed to live microfilariae not only produced significantly lower levels of CXCL-9, CXCL-10, IL-12p35, IL-12p40, IL-12p19 and CXCL-11 following stimulation with malaria antigen but also markedly downregulated the expression of IRF-1, IRF-2 and IRF-3 compared with microfilaria-unexposed mDCs. siRNA-inhibition of irf-1 in mDCs downregulated the production of IL-12p70 through repression of IL-12p35. Our data demonstrate that the modulation of IRFs seen in filarial (and presumably other tissue-invasive helminths) infection underlies the suppression of malaria-specific cytokines/chemokines that play a crucial role in immunity to malaria.

Figure 1

Patent filarial infection is associated with lower expression of IRF-1 in response to malaria antigen stimulation that can be reversed with anti-IL10 antibodies. Whole blood cells from filaria-infected and-uninfected subjects were stimulated with MalAg or left unstimulated for 24 h, and RNA expression was assessed by reverse transcriptase real-time PCR (RT-PCR). Expression levels of il12rb1, il12rb2 and irf-1 (A) at baseline, (B) in response to MalAg alone, or (C) in response to MalAg in the presence of neutralizing antibody to IL-10 are shown. The graph represents all the date obtained from the study subjects (n = 38). (A and B) Each symbol represents a subject and the bar represents the geometric mean. (C) Six Fil+subjects were assessed in the presence of neutralizing antibodies to IL-10 (square) or of the isotype control antibody (circle). Statistical significance was determined using Mann-Whitney (B) or Wilcoxon signed-rank test (C).

Figure 2

Patent filarial infection is associated not only with lower frequency of IL-12p70/40-producing mDCs but also with the per-cell cytokine production following malaria antigen stimulation. Whole blood cells were cultured as in Fig. 1, with Brefeldin A added in the last 12 h of incubation. The cells were then stained with antibodies to mDC and pDC surface markers (Alexa fluor 700-mouse anti-human CD3, CD19, CD19, PE-Cy7-mouse anti-human CD123 and PE-Cy5-mouse anti-human CD11c), and antibodies specific to IL-12p70/40, IFN-β and IL-10. The (A, C) net frequency and (B, D) iGMFI of cytokine-producing (A, B) mDCs and (C, D) pDCs were determined. The graph represents all the date obtained from the study subjects (n = 38). Each symbol represents the frequency or the iGMFI of mDCs or pDCs from one subject and the bars represent the geometric mean. Statistical significance was determined using the Mann–Whitney test.

Figure 3

Pre-exposure of monocyte-derived mDCs and pDCs to Brugia malayi mf suppresses the production of Th1-associated proinflammatory cytokines in response to malaria antigen stimulation in mDCs but not pDCs. mDCs and pDCs were differentiated in vitro from monocytes for 7 days using a cocktail of GM-CSF/IL-4 and IFN-β/IL-3 respectively, exposed to mf for 48 h, then harvested, counted and stimulated with MalAg for 24 h. The supernatants were screened for the indicated cytokines and the cells were used for RNA purification and RT-PCR. (A, C, D and F) represent the net concentration of cytokines measured in culture supernatants, and (B and E) represent the fold change of mRNA expression of the listed cytokines in MalAg-stimulated over the unstimulated DCs. The figure represents data from 32 individual experiments; each symbol represents a monocyte donor, and the bars join the data point from an individual sample exposed or unexposed to mf. Statistical significance was determined comparing cytokine values from the DC-exposed or -unexposed to mf using the Wilcoxon signed-rank test. The p-values were corrected for multiple comparisons using Holm's correction.

Figure 4

Pre-exposure of monocyte-derived DCs induces a down-regulation of interferon regulatory factors 1, 2 and 3 in mDCs but not pDCs. In vitro generated (A) mDCs and (B) pDCs were exposed to mf for 48 h, stimulated with MalAg for 24 h and the cells were used for RNA purification and RT-PCR. The fold change of RNA expression over unstimulated DCs or mf-pre-exposed DCs from 22 experiments is shown. Each symbol represents a value from one monocyte donor, and the lines join the values from mDCs and mf pre-exposed DCs from the same volunteer. Statistical significance was determined using Wilcoxon signed-rank test.

Figure 5

Pre-exposure of blood derived mDCs and pDCs with B. malayi mf induce a downregulation of Th1-associated proinflammatory cytokines and suppression of IRF-1 and 2 in mDCs but not pDCs. mDCs and pDCs were purified from blood using mDCs and BDCA-4/Neuropilin-1 MicroBead Kit, pre-exposed to mf and stimulated with MalAg for 24 h. (A, B) The supernatants were collected and screened for cytokines and (C, D) the cells were used for RNA purification and RT-PCR. The figure represents data from 15 individual experiments/donors. Each symbol represents cells from one volunteer and the line joins the cytokine values from the same donor. Comparisons were done using the Wilcoxon signed-rank tests and the p-values were corrected for multiple comparisons using Holm's correction.

Figure 6

Inhibition of IRF-1 down-regulate the production of IL-12p70 in mDCs. Monocyte-derived mDCs were transfected with either IRF-1 siRNA or control siRNA and stimulated with (A, B) Poly (I:C) or (C) MalAg for 24 h. (A, C) The cells were used for RNA extraction and RT-PCR and (B) the supernatants were collected and screened for cytokine production. The figure represents data from 18 experiments/donors; each symbol represents data from one volunteer with the lines joining data points from the same cells treated with either control siRNA or IRF-1 siRNA constructs. The bars represent the geometric mean and the error bars the 95% confidence interval. Statistical significance was estimated using the Wilcoxon signed-rank test.