Interleukin-6 (IL-6) and IL-17 synergistically promote viral persistence by inhibiting cellular apoptosis and cytotoxic T cell function

Abstract

Interleukin-6 (IL-6) plays an important role in the development and progression of inflammatory responses, autoimmune diseases, and cancers. Many viral infections, including Theiler's murine encephalomyelitis virus (TMEV), result in the vigorous production of IL-6. However, the role of IL-6 in the development of virus-induced inflammatory responses is unclear. The infection of susceptible mice with TMEV induces the development of chronic demyelinating disease, which is considered a relevant infectious model for multiple sclerosis. In this study, we demonstrate that resistant C57BL/6 mice carrying an IL-6 transgene (IL-6 Tg) develop a TMEV-induced demyelinating disease accompanied by an increase in viral persistence and an elevated Th17 cell response in the central nervous system. Either IL-6 or IL-17 induced the expression of Bcl-2 and Bcl-xL at a high concentration. The upregulated expression of prosurvival molecules in turn inhibited target cell destruction by virus-specific CD8(+) T cells. More interestingly, IL-6 and IL-17 synergistically promoted the expression of these prosurvival molecules, preventing cellular apoptosis at a much lower (<5-fold) concentration. The signals involved in the synergy appear to include the activation of both STAT3 and NF-κB via distinct cytokine-dependent pathways. Thus, the excessive IL-6 promotes the generation of Th17 cells, and the resulting IL-6 and IL-17 synergistically promote viral persistence by protecting virus-infected cells from apoptosis and CD8(+) T cell-mediated target destruction. These results suggest that blocking both IL-6 and IL-17 functions are important considerations for therapies of chronic viral diseases, autoimmune diseases, and cancers.

Importance: This study indicates that an excessive level of IL-6 cytokine produced following viral infection promotes the development of IL-17-producing pathogenic helper T cells. We demonstrate here for the first time that IL-6 together with IL-17 synergistically enhances the expression of survival molecules to hinder critical host defense mechanisms removing virus-infected cells. This finding has an important implication in controlling not only chronic viral infections but also autoimmune diseases and cancers, which are associated with prolonged cell survival.

FIG 1

IL-6 KO mice infected with TMEV exhibit reduced Th17 response and disease development. IL-6 KO and control B6 mice (n = 3) were treated with PBS or LPS at days 0 and 5 of TMEV infection. (A) Levels of IL-17 and IFN-γ produced by CD4⁺ T cells from the CNS of mice at 8 days after viral infection were assessed using ELISA after restimulation with UV-TMEV for 3 days. **, P < 0.01 for WT versus IL-6 KO mice. Data are representative of three independent experiments. (B) Levels of IL-6 and KC produced in the CNS of virus-infected WT and IL-6 KO mice treated with either PBS or LPS were determined at 8 days postinfection using specific ELISA. (C) The incidence and severity of demyelinating disease in LPS-treated WT B6 (n = 7) and IL-6 KO (n = 7) mice infected with TMEV was monitored weekly.

FIG 2

B6 IL-6 Tg mice infected with TMEV develop demyelinating disease and exhibit elevated viral load. (A) Development of demyelinating disease in TMEV-infected WT (n = 10) and IL-6 Tg (n = 10) mice was monitored. (B) Viral loads in the CNS (n = 3) of infected mice were determined using plaque assays at 8 and 21 days postinfection. (C) Flow cytometry of CNS-infiltrating cells bearing CD45 and CD11b in WT (n = 3) and IL-6 Tg mice (n = 3) at 8 days after virus infection. The bar graph shows the number of CD45^int CD11b⁺ microglia, CD45^hi CD11b⁺ macrophages, and CD45^hi CD11⁻ lymphocytes in the CNS of infected mice. (D) Levels of IL-6 and KC in the CNS of virus-infected WT B6 and IL-6 Tg mice were assessed using specific ELISA at 8 days postinfection. Sp Cord, spinal cord.

FIG 3

B6 IL-6 Tg mice infected with TMEV exhibit elevated Th17 response. (A) Flow-cytometric analysis of intracellular IL-17 and IFN-γ production by CNS-infiltrating CD4⁺ cells from WT and IL-6 Tg mice (n = 3 each) after restimulation with viral epitope peptides at 8 days postinfection. The bar graphs depict the quantity of cytokine-secreting cells among the CNS-infiltrating CD4⁺ cells. (B) ELISAs for IL-17 and IFN-γ levels produced by CNS-infiltrating cells at 8 days postinfection after restimulation with mixed CD4 epitope peptides. (C) Flow-cytometric analysis of intracellular production of IL-17 and IFN-γ by CNS-infiltrating CD8⁺ cells in WT and IL-6 Tg mice (n = 3) after restimulation with viral epitope peptides at 8 days postinfection. The presented data are representative of three independent experiments.

FIG 4

IL-6 promotes the expression of prosurvival proteins independently from IL-17. (A) Intracellular Bcl-2 and Bcl-xL expression in BM cells from naive WT and IL-6 KO mice was assessed after 24 h of stimulation with PBS, 100 ng/ml IL-6, or 100 ng/ml IL-17. (B) Comparison of Bcl-2 and Bcl-xL levels expressed by CNS-resident microglia and CNS-infiltrating macrophages in WT (n = 3) and IL-6 Tg mice (n = 3) at 8 days after TMEV infection. (C) Bcl-2 and Bcl-xL expression levels in microglia and macrophages from IL-6 Tg mice treated with either control antibody (n = 3) or anti-IL-17 antibody (n = 3) at 8 days postinfection. In each panel, the numbers in histograms represent relative median fluorescence intensity differences between the cells stained with isotype antibody (filled histogram) and anti-Bcl antibodies (open histogram). The data are representative of three independent experiments.

FIG 5

IL-6 and IL-17 synergistically promote the expression of prosurvival proteins to prevent virus infection-induced cellular apoptosis. (A) Flow-cytometric analysis of Bcl-2 and Bcl-xL expression in BM cells from IL-6 KO mice after 24 h of stimulation with IL-6, IL-17, or IL-6/IL-17 combination. (B) Flow-cytometric assessment for the effects of IL-6, IL-17, and IL-6/IL-17 combination on viral infection-induced cell apoptosis by staining with propidium iodide and allophycocyanin-conjugated annexin V. BM cells from WT or IL-6 KO mice were infected with TMEV in vitro at an MOI of 10 for 24 h in the presence or absence of the cytokines before flow cytometry analysis. The data are representative of two to three independent experiments.

FIG 6

IL-6 and IL-17 are synergistic with respect to the inhibition of antiviral cytotoxic T cell activity against target cells. (A) Spleen cells from naive IL-6 KO mice were pulsed with VP2_121-130 or OVA_323-339 peptides and labeled with a lower or higher concentration of CFSE as target cells, respectively. The labeled target cells and effector spleen cells isolated at 8 days from TMEV-infected IL-6 KO mice were cocultured for 60 h in the presence of different doses of cytokines and their combination. The numbers in each histogram show the percentages of the lower (VP2_121-130) and higher (OVA_323-339) concentrations of the overall CFSE-labeled cell population. (B) Levels of infectious virus in the CNS of TMEV-infected WT (littermate [LM]) mice and IL-6 Tg mice left untreated or treated with anti-IL-17 antibody (n = 3/group) were quantified using plaque assays at 8 days postinfection. (C) Levels of infectious virus in the CNS of WT and IL-6 KO mice infected with TMEV in PBS or 100 ng IL-17 (n = 3/group) were quantified using plaque assays at 7 days postinfection. **, P < 0.01; ***, P < 0.001.

FIG 7

Common (STAT3 and NF-κB) and distinct (TRAF6) signaling pathways by IL-6 and IL-17 are involved in the upregulation of Bcl molecules. (A) Flow cytometry of intracellular phosphorylated STAT3 in IL-6 KO BM cells pretreated with the control (DMSO) or an inhibitor of STAT3, S31-201 (100 μM), for 10 min prior to stimulation with IL-6, IL-17, or IL-6/IL-17 for 15 min. (B) Flow cytometry of intracellular Bcl-2 and Bcl-xL in IL-6 KO BM cells pretreated with DMSO or S31-201 for 10 min prior to stimulation with the cytokines listed for panel A for 24 h. (C) Expression of Bcl-2 and Bcl-xL in IL-6 KO BM cells was determined after pretreatment with 100 μM control peptide or 100 μM TRAF6 inhibitory peptide prior to stimulation with the cytokines for 24 h. (D) Expression of Bcl-2 and Bcl-xL was determined as described for panel C after pretreatment with 10 μM SB202190, 10 μM U0126, 100 μM PDTC, or 10 μM MG-132 for 2 h and then stimulated with the cytokines for 24 h. The presented data are representatives of at least two separate experiments.