Hyun Seok Kang

Th17 cells enhance viral persistence and inhibit T cell cytotoxicity in a model of chronic virus infection

Persistent viral infection and its associated chronic diseases are a global health concern. Interleukin (IL) 17–producing Th17 cells have been implicated in the pathogenesis of various autoimmune diseases, and in protection from bacterial or fungal infection. However, the role of Th17 cells in persistent viral infection remains unknown. We report that Th17 cells preferentially develop in vitro and in vivo in an IL-6–dependent manner after Theiler’s murine encephalomyelitis virus infection. Th17 cells promote persistent viral infection and induce the pathogenesis of chronic demyelinating disease. IL-17 up-regulates antiapoptotic molecules and, consequently, increases persistent infection by enhancing the survival of virus-infected cells and blocking target cell destruction by cytotoxic T cells. Neutralization of IL-17 augments virus clearance by eliminating virus-infected cells and boosting lytic function by cytotoxic T cells, leading to the prevention of disease development. Thus, these results indicate a novel pathogenic role of Th17 cells via IL-17 in persistent viral infection and its associated chronic inflammatory diseases.

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 Theilers 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.

Distinct subregions of Swi1 manifest striking differences in prion transmission and SWI/SNF function.

ABSTRACT We have recently reported that the yeast chromatin-remodeling factor Swi1 can exist as a prion, [SWI+], demonstrating a link between prionogenesis and global transcriptional regulation. To shed light on how the Swi1 conformational switch influences Swi1 function and to define the sequence and structural requirements for [SWI+] formation and propagation, we functionally dissected the Swi1 molecule. We show here that the [SWI+] prion features are solely attributable to the first 327 amino acid residues (N), a region that is asparagine rich. N was aggregated in [SWI+] cells but diffuse in [swi−] cells; chromosomal deletion of the N-coding region resulted in [SWI+] loss, and recombinant N peptide was able to form infectious amyloid fibers in vitro, enabling [SWI+] de novo formation through a simple transformation. Although the glutamine-rich middle region (Q) was not sufficient to aggregate in [SWI+] cells or essential for SWI/SNF function, it significantly modified the Swi1 aggregation pattern and Swi1 function. We also show that excessive Swi1 incurred Li+/Na+ sensitivity and that the N/Q regions are important for this gain of sensitivity. Taken together, our results provide the final proof of “protein-only” transmission of [SWI+] and demonstrate that the widely distributed “dispensable” glutamine/asparagine-rich regions/motifs might have important and divergent biological functions.

IL-1 signal affects both protection and pathogenesis of virus-induced chronic CNS demyelinating disease.

BackgroundTheiler’s virus infection induces chronic demyelinating disease in mice and has been investigated as an infectious model for multiple sclerosis (MS). IL-1 plays an important role in the pathogenesis of both the autoimmune disease model (EAE) and this viral model for MS. However, IL-1 is known to play an important protective role against certain viral infections. Therefore, it is unclear whether IL-1-mediated signaling plays a protective or pathogenic role in the development of TMEV-induced demyelinating disease.MethodsFemale C57BL/6 mice and B6.129S7-Il1r1tm1Imx/J mice (IL-1R KO) were infected with Theiler’s murine encephalomyelitis virus (1 x 106 PFU). Differences in the development of demyelinating disease and changes in the histopathology were compared. Viral persistence, cytokine production, and immune responses in the CNS of infected mice were analyzed using quantitative PCR, ELISA, and flow cytometry.ResultsAdministration of IL-1β, thereby rending resistant B6 mice susceptible to TMEV-induced demyelinating disease, induced a high level of Th17 response. Interestingly, infection of TMEV into IL-1R-deficient resistant C57BL/6 (B6) mice also induced TMEV-induced demyelinating disease. High viral persistence was found in the late stage of viral infection in IL-1R-deficient mice, although there were few differences in the initial anti-viral immune responses and viral persistent levels between the WT B6 and IL-1R-deficiecent mice. The initial type I IFN responses and the expression of PDL-1 and Tim-3 were higher in the CNS of TMEV-infected IL-1R-deficient mice, leading to deficiencies in T cell function that permit viral persistence.ConclusionsThese results suggest that the presence of high IL-1 level exerts the pathogenic role by elevating pathogenic Th17 responses, whereas the lack of IL-1 signals promotes viral persistence in the spinal cord due to insufficient T cell activation by elevating the production of inhibitory cytokines and regulatory molecules. Therefore, the balance of IL-1 signaling appears to be extremely important for the protection from TMEV-induced demyelinating disease, and either too much or too little signaling promotes the development of disease.

Anticapsid Immunity Level, Not Viral Persistence Level, Correlates with the Progression of Theiler's Virus-Induced Demyelinating Disease in Viral P1-Transgenic Mice

ABSTRACT Intracranial infection of Theilers murine encephalomyelitis virus (TMEV) induces demyelination and a neurological disease in susceptible SJL/J (SJL) mice that resembles multiple sclerosis. While the virus is cleared from the central nervous system (CNS) of resistant C57BL/6 (B6) mice, it persists in SJL mice. To investigate the role of viral persistence and its accompanying immune responses in the development of demyelinating disease, transgenic mice expressing the P1 region of the TMEV genome (P1-Tg) were employed. Interestingly, P1-Tg mice with the B6 background showed severe reductions in both CD4+ and CD8+ T-cell responses to capsid epitopes, while P1-Tg mice with the SJL background displayed transient reductions following viral infection. Reduced antiviral immune responses in P1-Tg mice led to >100- to 1,000-fold increases in viral persistence at 120 days postinfection in the CNS of mice with both backgrounds. Despite the increased CNS TMEV levels in these P1-Tg mice, B6 P1-Tg mice developed neither neuropathological symptoms nor demyelinating lesions, and SJL P1-Tg mice developed significantly less severe TMEV-induced demyelinating disease. These results strongly suggest that viral persistence alone is not sufficient to induce disease and that the level of T-cell immunity to viral capsid epitopes is critical for the development of demyelinating disease in SJL mice.

TLR3 signaling is either protective or pathogenic for the development of Theiler's virus-induced demyelinating disease depending on the time of viral infection

BackgroundWe have previously shown that toll-like receptor 3 (TLR3)-mediated signaling plays an important role in the induction of innate cytokine responses to Theilers murine encephalomyelitis virus (TMEV) infection. In addition, cytokine levels produced after TMEV infection are significantly higher in the glial cells of susceptible SJL mice compared to those of resistant C57BL/6 mice. However, it is not known whether TLR3-mediated signaling plays a protective or pathogenic role in the development of demyelinating disease.MethodsSJL/J and B6;129S-Tlr3tm1Flv /J (TLR3KO-B6) mice, and TLR3KO-SJL mice that TLR3KO-B6 mice were backcrossed to SJL/J mice for 6 generations were infected with Theilers murine encephalomyelitis virus (2 × 105 PFU) with or without treatment with 50 μg of poly IC. Cytokine production and immune responses in the CNS and periphery of infected mice were analyzed.ResultsWe investigated the role of TLR3-mediated signaling in the protection and pathogenesis of TMEV-induced demyelinating disease. TLR3KO-B6 mice did not develop demyelinating disease although they displayed elevated viral loads in the CNS. However, TLR3KO-SJL mice displayed increased viral loads and cellular infiltration in the CNS, accompanied by exacerbated development of demyelinating disease, compared to the normal littermate mice. Late, but not early, anti-viral CD4+ and CD8+ T cell responses in the CNS were compromised in TLR3KO-SJL mice. However, activation of TLR3 with poly IC prior to viral infection also exacerbated disease development, whereas such activation after viral infection restrained disease development. Activation of TLR3 signaling prior to viral infection hindered the induction of protective IFN-γ-producing CD4+ and CD8+ T cell populations. In contrast, activation of these signals after viral infection improved the induction of IFN-γ-producing CD4+ and CD8+ T cells. In addition, poly IC-pretreated mice displayed elevated PDL-1 and regulatory FoxP3+ CD4+ T cells in the CNS, while poly IC-post-treated mice expressed reduced levels of PDL-1 and FoxP3+ CD4+ T cells.ConclusionsThese results suggest that TLR3-mediated signaling during viral infection protects against demyelinating disease by reducing the viral load and modulating immune responses. In contrast, premature activation of TLR3 signal transduction prior to viral infection leads to pathogenesis via over-activation of the pathogenic immune response.

The Role of Interleukin-6 in the Expression of PD-1 and PDL-1 on Central Nervous System Cells following Infection with Theiler's Murine Encephalomyelitis Virus

ABSTRACT Infection with Theilers murine encephalomyelitis virus (TMEV) in the central nervous system (CNS) of susceptible mice results in an immune-mediated demyelinating disease which is considered a relevant viral model of human multiple sclerosis. We previously demonstrated that the expression of positive costimulatory molecules (CD40, CD80, and CD86) is higher on the microglia of TMEV-resistant C57BL/6 (B6) mice than the microglia of TMEV-susceptible SJL/J (SJL) mice. In this study, we analyzed the expression levels of the negative costimulatory molecules PD-1 and PDL-1 in the CNS of TMEV-infected SJL mice and B6 mice. Our results indicated that TMEV infection induces the expression of both PD-1 and PDL-1 on microglia and macrophages in the CNS but not in the periphery. The expression of PD-1 only on CNS-infiltrating macrophages and not on resident microglia was considerably higher (>4-fold) in TMEV-infected SJL mice than TMEV-infected B6 mice. We further demonstrated that interleukn-6 (IL-6) is necessary to induce the maximal expression of PDL-1 but not PD-1 after TMEV infection using IL-6-deficient mice and IL-6-transgenic mice in conjunction with recombinant IL-6. In addition, cells from type I interferon (IFN) receptor knockout mice failed to upregulate PD-1 and PDL-1 expression after TMEV infection in vitro, indicating that type I IFN signaling is associated with the upregulation. However, other IFN signaling may also participate in the upregulation. Taken together, these results strongly suggest that the expression of PD-1 and PDL-1 in the CNS is primarily upregulated following TMEV infection via type I IFN signaling and the maximal expression of PDL-1 additionally requires IL-6 signaling.

Preferential Induction of Protective T Cell Responses to Theiler's Virus in Resistant (C57BL/6 X SJL)F1 Mice

ABSTRACT Infection of the central nervous system (CNS) with Theilers murine encephalomyelitis virus (TMEV) induces an immune-mediated demyelinating disease in susceptible mouse strains such as SJL/J (H-2s) but not in strains such as C57BL/6 (H-2b). In addition, it has been shown that (C57BL/6 × SJL/J)F1 mice (F1 mice), which carry both resistant and susceptible MHC haplotypes (H-2b/s), are resistant to both viral persistence and TMEV-induced demyelinating disease. In this study, we further analyzed the immune responses underlying the resistance of F1 mice. Our study shows that the resistance of F1 mice is associated with a higher level of the initial virus-specific H-2b-restricted CD8+ T cell responses than of the H-2s-restricted CD8+ T cell responses. In contrast, pathogenic Th17 responses to viral epitopes are lower in F1 mice than in susceptible SJL/J mice. Dominant effects of resistant genes expressed in antigen-presenting cells of F1 mice on regulation of viral replication and induction of protective T cell responses appear to play a crucial role in disease resistance. Although the F1 mice are resistant to disease, the level of viral RNA in the CNS was intermediate between those of SJL/J and C57BL/6 mice, indicating the presence of a threshold of viral expression for pathogenesis.

Epitope-Specific CD8+ T Cells Play a Differential Pathogenic Role in the Development of a Viral Disease Model for Multiple Sclerosis

ABSTRACT Theilers virus-induced demyelinating disease has been extensively investigated as a model for persistent viral infection and multiple sclerosis (MS). However, the role of CD8+ T cells in the development of disease remains unclear. To assess the role of virus-specific CD8+ T cells in the pathogenesis of demyelinating disease, a single amino acid substitution was introduced into the predominant viral epitope (VP3 from residues 159 to 166 [VP3159-166]) and/or a subdominant viral epitope (VP3173-181) of susceptible SJL/J mice by site-directed mutagenesis. The resulting variant viruses (N160V, P179A, and N160V/P179A) failed to induce CD8+ T cell responses to the respective epitopes. Surprisingly, mice infected with N160V or N160V/P179A virus, which lacks CD8+ T cells against VP3159-166, did not develop demyelinating disease, in contrast to wild-type virus or P179A virus lacking VP3173-181-specific CD8+ T cells. Our findings clearly show that the presence of VP3159-166-specific CD8+ T cells, rather than viral persistence itself, is strongly correlated with disease development. VP3173-181-specific CD8+ T cells in the central nervous system (CNS) of these virus-infected mice expressed higher levels of transforming growth factor β, forkhead box P3, interleukin-22 (IL-22), and IL-17 mRNA but caused minimal cytotoxicity compared to that caused by VP3159-166-specific CD8+ T cells. VP3159-166-specific CD8+ T cells exhibited high functional avidity for gamma interferon production, whereas VP3173-181-specific CD8+ T cells showed low avidity. To our knowledge, this is the first report indicating that the induction of the IL-17-producing CD8+ T cell type is largely epitope specific and that this specificity apparently plays a differential role in the pathogenicity of virus-induced demyelinating disease. These results strongly advocate for the careful consideration of CD8+ T cell-mediated intervention of virus-induced inflammatory diseases.

Predominant Clonal Accumulation of CD8+ T Cells with Moderate Avidity in the Central Nervous Systems of Theiler's Virus-Infected C57BL/6 Mice

ABSTRACT Induction of antigen-specific CD8+ T cells bearing a high-avidity T-cell receptor (TCR) is thought to be an important factor in antiviral and antitumor immune responses. However, the relationship between TCR diversity and functional avidity of epitope-specific CD8+ T cells accumulating in the central nervous system (CNS) during viral infection is unknown. Hence, analysis of T-cell diversity at the clonal level is important to understand the fate and function of virus-specific CD8+ T cells. In this study, we examined the Vβ diversity and avidity of CD8+ T cells specific to the predominant epitope (VP2121-130) of Theilers murine encephalomyelitis virus. We found that Vβ6+ CD8+ T cells, associated with epitope specificity, predominantly expanded in the CNS during viral infection. Further investigations of antigen-specific Vβ6+ CD8+ T cells by CDR3 spectratyping and sequencing indicated that distinct T-cell clonotypes are preferentially increased in the CNS compared to the periphery. Among the epitope-specific Vβ6+ CD8+ T cells, MGX-Jβ1.1 motif-bearing cells, which could be found at a high precursor frequency in naïve mice, were expanded in the CNS and tightly associated with gamma interferon production. These T cells displayed moderate avidity for the cognate epitope rather than the high avidity normally observed in memory/effector T cells. Therefore, our findings provide new insights into the CD8+ T-cell repertoire during immune responses to viral infection in the CNS.