BackgroundCCR5 is a CC chemokine receptor involved in the migration of effector leukocytes including macrophages, NK, and T cells into inflamed tissues. Also, the role of CCR5 in CD4+Foxp3+ regulatory T cell (Treg) homing has recently begun to grab attention. Japanese encephalitis (JE) is defined as severe neuroinflammation of the central nervous system (CNS) following infection with mosquito-borne flavivirus JE virus. However, the potential contribution of CCR5 to JE progression via mediating CD4+Foxp3+ Treg homing has not been investigated.MethodsInfected wild-type (Ccr5+/+) and CCR5-deficient (Ccr5−/−) mice were examined daily for mortality and clinical signs, and neuroinflammation in the CNS was evaluated by infiltration of inflammatory leukocytes and cytokine expression. In addition, viral burden, NK- and JEV-specific T cell responses were analyzed. Adoptive transfer of CCR5+CD4+Foxp3+ Tregs was used to evaluate the role of Tregs in JE progression.ResultsCCR5 ablation exacerbated JE without altering viral burden in the extraneural and CNS tissues, as manifested by increased CNS infiltration of Ly-6Chi monocytes and Ly-6Ghi granulocytes. Compared to Ccr5+/+ mice, Ccr5−/− mice unexpectedly showed increased responses of IFN-γ+NK and CD8+ T cells in the spleen, but not CD4+ T cells. More interestingly, CCR5-ablation resulted in a skewed response to IL-17+CD4+ Th17 cells and correspondingly reduced CD4+Foxp3+ Tregs in the spleen and brain, which was closely associated with exacerbated JE. Our results also revealed that adoptive transfer of sorted CCR5+CD4+Foxp3+ Tregs into Ccr5−/− mice could ameliorate JE progression without apparently altering the viral burden and CNS infiltration of IL-17+CD4+ Th17 cells, myeloid-derived Ly-6Chi monocytes and Ly-6Ghi granulocytes. Instead, adoptive transfer of CCR5+CD4+Foxp3+ Tregs into Ccr5−/− mice resulted in increased expression of anti-inflammatory cytokines (IL-10 and TGF-β) in the spleen and brain, and transferred CCR5+ Tregs were found to produce IL-10.ConclusionsCCR5 regulates JE progression via governing timely and appropriate CNS infiltration of CD4+Foxp3+ Tregs, thereby facilitating host survival. Therefore, this critical and extended role of CCR5 in JE raises possible safety concerns regarding the use of CCR5 antagonists in human immunodeficiency virus (HIV)-infected individuals who inhabit regions in which both HIV and flaviviruses, such as JEV and West Nile virus, are endemic.

The importance of TLR2 and TLR9 in the recognition of infection with herpes simplex virus (HSV) and HSV-caused diseases has been described, but some discrepancies remain concerning the benefits of these responses. Moreover, the impact of TLR2/9 on innate and adaptive immune responses within relevant mucosal tissues has not been elucidated using natural mucosal infection model of HSV. Here, we demonstrate that dual TLR2/9 recognition is essential to provide resistance against mucosal infection with HSV via an intravaginal route. Dual TLR2/9 ablation resulted in the highly enhanced mortality with exacerbated symptoms of encephalitis compared with TLR2 or TLR9 deficiency alone, coinciding with highly increased viral load in central nervous system tissues. TLR2 appeared to play a minor role in providing resistance against mucosal infection with HSV, since TLR2-ablated mice showed higher survival rate compared with TLR9-ablated mice. Also, the high mortality in dual TLR2/9-ablated mice was closely associated with the reduction in early monocyte and NK cell infiltration in the vaginal tract (VT), which was likely to correlate with low expression of cytokines and CCR2 ligands (CCL2 and CCL7). More interestingly, our data revealed that dual TLR2/9 recognition of HSV infection plays an important role in the functional maturation of TNF-α and iNOS-producing dendritic cells (Tip-DCs) from monocytes as well as NK cell activation in VT. TLR2/9-dependent maturation of Tip-DCs from monocytes appeared to specifically present cognate Ag, which effectively provided functional effector CD4+ and CD8+ T cells specific for HSV Ag in VT and its draining lymph nodes. TLR2/9 expressed in monocytes was likely to directly facilitate Tip-DC-like features after HSV infection. Also, dual TLR2/9 recognition of HSV infection directly activated NK cells without the aid of dendritic cells through activation of p38 MAPK pathway. Taken together, these results indicate that dual TLR2/9 recognition plays a critical role in providing resistance against mucosal infection with HSV, which may involve a direct regulation of Tip-DCs and NK cells in VT. Therefore, our data provide a more detailed understanding of TLR2/9 role in conferring antiviral immunity within relevant mucosal tissues after mucosal infection with HSV.

BackgroundJapanese encephalitis (JE), a leading cause of viral encephalitis, is characterized by extensive neuroinflammation following infection with neurotropic JE virus (JEV). Indoleamine 2,3-dioxygenase (IDO) has been identified as an enzyme associated with immunoregulatory function. Although the regulatory role of IDO in viral replication has been postulated, the in vivo role of IDO activity has not been fully addressed in neurotropic virus-caused encephalitis.MethodsMice in which IDO activity was inhibited by genetic ablation or using a specific inhibitor were examined for mortality and clinical signs after infection. Neuroinflammation was evaluated by central nervous system (CNS) infiltration of leukocytes and cytokine expression. IDO expression, viral burden, JEV-specific T-cell, and type I/II interferon (IFN-I/II) innate responses were also analyzed.ResultsElevated expression of IDO activity in myeloid and neuron cells of the lymphoid and CNS tissues was closely associated with clinical signs of JE. Furthermore, inhibition of IDO activity enhanced resistance to JE, reduced the viral burden in lymphoid and CNS tissues, and resulted in early and increased CNS infiltration by Ly-6Chi monocytes, NK, CD4+, and CD8+ T-cells. JE amelioration in IDO-ablated mice was also associated with enhanced NK and JEV-specific T-cell responses. More interestingly, IDO ablation induced rapid enhancement of type I IFN (IFN-I) innate responses in CD11c+ dendritic cells (DCs), including conventional and plasmacytoid DCs, following JEV infection. This enhanced IFN-I innate response in IDO-ablated CD11c+ DCs was coupled with strong induction of PRRs (RIG-I, MDA5), transcription factors (IRF7, STAT1), and antiviral ISG genes (Mx1, Mx2, ISG49, ISG54, ISG56). IDO ablation also enhanced the IFN-I innate response in neuron cells, which may delay the spread of virus in the CNS. Finally, we identified that IDO ablation in myeloid cells derived from hematopoietic stem cells (HSCs) dominantly contributed to JE amelioration and that HSC-derived leukocytes played a key role in the enhanced IFN-I innate responses in the IDO-ablated environment.ConclusionsInhibition of IDO activity ameliorated JE via enhancement of antiviral IFN-I/II innate and adaptive T-cell responses and increased CNS infiltration of peripheral leukocytes. Therefore, our data provide valuable insight into the use of IDO inhibition by specific inhibitors as a promising tool for therapeutic and prophylactic strategies against viral encephalitis caused by neurotropic viruses.

Japanese encephalitis virus (JEV) is a re-emerging zoonotic flavivirus that poses an increasing threat to global health and welfare due to rapid changes in climate and demography. Although the CCR2-CCL2 axis plays an important role in trafficking CD11b(+) Ly-6C(hi) monocytes to regulate immunopathological diseases, little is known about their role in monocyte trafficking during viral encephalitis caused by JEV infection. Here, we explored the role of CCR2 and its ligand CCL2 in JE caused by JEV infection using CCR2- and CCL2-ablated murine models. Somewhat surprisingly, the ablation of CCR2 and CCL2 resulted in starkly contrasting susceptibility to JE. CCR2 ablation induced enhanced resistance to JE, whereas CCL2 ablation highly increased susceptibility to JE. This contrasting regulation of JE progression by CCR2 and CCL2 was coupled to central nervous system (CNS) infiltration of Ly-6C(hi) monocytes and Ly-6G(hi) granulocytes. There was also enhanced expression of CC and CXC chemokines in the CNS of CCL2-ablated mice, which appeared to induce CNS infiltration of these cell populations. However, our data revealed that contrasting regulation of JE in CCR2- and CCL2-ablated mice was unlikely to be mediated by innate natural killer and adaptive T-cell responses. Furthermore, CCL2 produced by haematopoietic stem cell-derived leucocytes played a dominant role in CNS accumulation of Ly-6C(hi) monocytes in infected bone marrow chimeric models, thereby exacerbating JE progression. Collectively, our data indicate that CCL2 plays an essential role in conferring protection against JE caused by JEV infection. In addition, blockage of CCR2, but not CCL2, will aid in the development of strategies for prophylactics and therapeutics of JE.