Ajit Mahadev Patil

Impaired cross-presentation of CD8α+ CD11c+ dendritic cells by Japanese encephalitis virus in a TLR2/MyD88 signal pathway-dependent manner.

Cross‐presentation is the pathway by which exogenous antigens are routed for presentation by MHC class I molecules leading to activation of antiviral CD8+ T‐cell responses. However, there is little information describing the modulation of cross‐presentation and the impact of pathogen‐derived signals associated with Japanese encephalitis virus (JEV), which is one of the most common causes of encephalitis in humans. In this study, we demonstrate that JEV infection could suppress in vivo cross‐presentation of soluble and cell‐associated antigens, thereby generating weak CD8+ T‐cell responses to exogenous antigens, as evaluated by CFSE dilution of adoptively transferred CD8+ T cells and in vivo CTL killing activity. Furthermore, CD8α+CD11c+ dendritic cells (DCs), which are known to be far more efficient at cross‐presenting soluble antigens, played a specific role in contributing to JEV‐mediated inhibition of the cross‐presentation of exogenous antigens through interference with effective antigen uptake. Finally, this study provides evidence that TLR2‐MyD88 and p38 MAPK signal pathway might be involved in JEV‐mediated inhibition of cross‐presentation of soluble and cell‐associated antigens. These observations suggest that the modulation of cross‐presentation of exogenous antigens through TLR signaling has important implications for antiviral immune responses against JEV infection and the development of effective vaccination strategies.

CCR5 ameliorates Japanese encephalitis via dictating the equilibrium of regulatory CD4 + Foxp3 + T and IL-17 + CD4 + Th17 cells

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.

Distinct Upstream Role of Type I IFN Signaling in Hematopoietic Stem Cell-Derived and Epithelial Resident Cells for Concerted Recruitment of Ly-6Chi Monocytes and NK Cells via CCL2-CCL3 Cascade.

Type I interferon (IFN-I)-dependent orchestrated mobilization of innate cells in inflamed tissues is believed to play a critical role in controlling replication and CNS-invasion of herpes simplex virus (HSV). However, the crucial regulators and cell populations that are affected by IFN-I to establish the early environment of innate cells in HSV-infected mucosal tissues are largely unknown. Here, we found that IFN-I signaling promoted the differentiation of CCL2-producing Ly-6Chi monocytes and IFN-γ/granzyme B-producing NK cells, whereas deficiency of IFN-I signaling induced Ly-6Clo monocytes producing CXCL1 and CXCL2. More interestingly, recruitment of Ly-6Chi monocytes preceded that of NK cells with the levels peaked at 24 h post-infection in IFN-I–dependent manner, which was kinetically associated with the CCL2-CCL3 cascade response. Early Ly-6Chi monocyte recruitment was governed by CCL2 produced from hematopoietic stem cell (HSC)-derived leukocytes, whereas NK cell recruitment predominantly depended on CC chemokines produced by resident epithelial cells. Also, IFN-I signaling in HSC-derived leukocytes appeared to suppress Ly-6Ghi neutrophil recruitment to ameliorate immunopathology. Finally, tissue resident CD11bhiF4/80hi macrophages and CD11chiEpCAM+ dendritic cells appeared to produce initial CCL2 for migration-based self-amplification of early infiltrated Ly-6Chi monocytes upon stimulation by IFN-I produced from infected epithelial cells. Ultimately, these results decipher a detailed IFN-I–dependent pathway that establishes orchestrated mobilization of Ly-6Chi monocytes and NK cells through CCL2-CCL3 cascade response of HSC-derived leukocytes and epithelium-resident cells. Therefore, this cascade response of resident–to-hematopoietic–to-resident cells that drives cytokine–to-chemokine–to-cytokine production to recruit orchestrated innate cells is critical for attenuation of HSV replication in inflamed tissues.

Prophylactic and therapeutic modulation of innate and adaptive immunity against mucosal infection of herpes simplex virus.

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are the most common cause of genital ulceration in humans worldwide. Typically, HSV-1 and 2 infections via mucosal route result in a lifelong latent infection after peripheral replication in mucosal tissues, thereby providing potential transmission to neighbor hosts in response to reactivation. To break the transmission cycle, immunoprophylactics and therapeutic strategies must be focused on prevention of infection or reduction of infectivity at mucosal sites. Currently, our understanding of the immune responses against mucosal infection of HSV remains intricate and involves a balance between innate signaling pathways and the adaptive immune responses. Numerous studies have demonstrated that HSV mucosal infection induces type I interferons (IFN) via recognition of Toll-like receptors (TLRs) and activates multiple immune cell populations, including NK cells, conventional dendritic cells (DCs), and plasmacytoid DCs. This innate immune response is required not only for the early control of viral replication at mucosal sites, but also for establishing adaptive immune responses against HSV antigens. Although the contribution of humoral immune response is controversial, CD4+ Th1 T cells producing IFN-γ are believed to play an important role in eradicating virus from the hosts. In addition, the recent experimental successes of immunoprophylactic and therapeutic compounds that enhance resistance and/or reduce viral burden at mucosal sites have accumulated. This review focuses on attempts to modulate innate and adaptive immunity against HSV mucosal infection for the development of prophylactic and therapeutic strategies. Notably, cells involved in innate immune regulations appear to shape adaptive immune responses. Thus, we summarized the current evidence of various immune mediators in response to mucosal HSV infection, focusing on the importance of innate immune responses.

CD11c hi Dendritic Cells Regulate Ly-6C hi Monocyte Differentiation to Preserve Immune-privileged CNS in Lethal Neuroinflammation

Although the roles of dendritic cells (DCs) in adaptive defense have been defined well, the contribution of DCs to T cell-independent innate defense and subsequent neuroimmunopathology in immune-privileged CNS upon infection with neurotropic viruses has not been completely defined. Notably, DC roles in regulating innate CD11b+Ly-6Chi monocyte functions during neuroinflammation have not yet been addressed. Using selective ablation of CD11chiPDCA-1int/lo DCs without alteration in CD11cintPDCA-1hi plasmacytoid DC number, we found that CD11chi DCs are essential to control neuroinflammation caused by infection with neurotropic Japanese encephalitis virus, through early and increased infiltration of CD11b+Ly-6Chi monocytes and higher expression of CC chemokines. More interestingly, selective CD11chi DC ablation provided altered differentiation and function of infiltrated CD11b+Ly-6Chi monocytes in the CNS through Flt3-L and GM-CSF, which was closely associated with severely enhanced neuroinflammation. Furthermore, CD11b+Ly-6Chi monocytes generated in CD11chi DC-ablated environment had a deleterious rather than protective role during neuroinflammation, and were more quickly recruited into inflamed CNS, depending on CCR2, thereby exacerbating neuroinflammation via enhanced supply of virus from the periphery. Therefore, our data demonstrate that CD11chi DCs provide a critical and unexpected role to preserve the immune-privileged CNS in lethal neuroinflammation via regulating the differentiation, function, and trafficking of CD11b+Ly-6Chi monocytes.

Functional restoration of exhausted CD4(+) and CD8(+) T cells in chronic viral infection by vinegar-processed flos of Daphne genkwa.

T-cell exhaustion has become an important issue in chronic infection because exhausted antigen-specific T cells show impaired abilities to eradicate persistently infected pathogens and produce effector cytokines, such as IFN-γ and TNF-α. Thus, strategies to either restore endogenous exhausted T cell responses or provide functional T cells are needed for therapeutics of chronic infection. Despite promising developments using antibodies and cell immunotherapy, there have been no reported attempts to restore exhausted T cells using treatment with materials derived from natural resources. Here, using a mouse model of chronic infection with lymphocytic choriomeningitis virus (LCMV), we found that vinegar-processed flowers (flos) of Daphne genkwa (vp-genkwa), which was composed mainly of four index components, restored exhausted CD4(+) and CD8(+) T cells significantly, as corroborated by evidence that vp-genkwa treatment enhanced functional LCMV-specific CD4(+) and CD8(+) T cells, both quantitatively and qualitatively. Furthermore, pretreatment with vp-genkwa prevented the generation of exhausted LCMV-specific CD8(+) T cells. Such restorations of exhausted LCMV-specific CD4(+) and CD8(+) T cells by vp-genkwa were closely associated with reduced viral burden in sera and tissues. More interestingly, vp-genkwa treatment induced down-regulation of negative molecules, such as PD-1 and Tim-3, in exhausted CD4(+) and CD8(+) T cells with more apparent down-regulation of Tim-3, suggesting that Tim-3 molecule may be a major target in restoring exhausted T cell responses. Collectively, these results provide valuable new insights into the use of vp-genkwa to develop a therapeutic strategy for chronic human diseases, such as hepatitis B and C virus, human immunodeficiency virus, and cancers.

Amelioration of Japanese encephalitis by blockage of 4-1BB signaling is coupled to divergent enhancement of type I/II IFN responses and Ly-6C hi monocyte differentiation

BackgroundJapanese encephalitis (JE), a neuroinflammation caused by zoonotic JE virus, is the major cause of viral encephalitis worldwide and poses an increasing threat to global health and welfare. To date, however, there has been no report describing the regulation of JE progression using immunomodulatory tools for developing therapeutic strategies. We tested whether blocking the 4-1BB signaling pathway would regulate JE progression using murine JE model.MethodsInfected wild-type and 4-1BB-knockout (KO) 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, JEV-specific T cell, and type I/II IFN (IFN-I/II) innate responses were analyzed.ResultsBlocking the 4-1BB signaling pathway significantly increased resistance to JE and reduced viral burden in extraneural tissues and the CNS, rather than causing a detrimental effect. In addition, treatment with 4-1BB agonistic antibody exacerbated JE. Furthermore, JE amelioration and reduction of viral burden by blocking the 4-1BB signaling pathway were associated with an increased frequency of IFN-II-producing NK and CD4+ Th1 cells as well as increased infiltration of mature Ly-6Chi monocytes in the inflamed CNS. More interestingly, DCs and macrophages derived from 4-1BB KO mice showed potent and rapid IFN-I innate immune responses upon JEV infection, which was coupled to strong induction of PRRs (RIG-I, MDA5), transcription factors (IRF7), and antiviral ISG genes (ISG49, ISG54, ISG56). Further, the ablation of 4-1BB signaling enhanced IFN-I innate responses in neuron cells, which likely regulated viral spread in the CNS. Finally, we confirmed that blocking the 4-1BB signaling pathway in myeloid cells derived from hematopoietic stem cells (HSCs) played a dominant role in ameliorating JE. In support of this finding, HSC-derived leukocytes played a dominant role in generating the IFN-I innate responses in the host.ConclusionsBlocking the 4-1BB signaling pathway ameliorates JE via divergent enhancement of IFN-II-producing NK and CD4+ Th1 cells and mature Ly-6Chi monocyte infiltration, as well as an IFN-I innate response of myeloid-derived cells. Therefore, regulation of the 4-1BB signaling pathway with antibodies or inhibitors could be a valuable therapeutic strategy for the treatment of JE.

Blockage of indoleamine 2,3-dioxygenase regulates Japanese encephalitis via enhancement of type I/II IFN innate and adaptive T-cell responses.

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.

CCL2, but not its receptor, is essential to restrict immune privileged central nervous system‐invasion of Japanese encephalitis virus via regulating accumulation of CD11b+ Ly‐6Chi monocytes

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‐6Chi 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‐6Chi monocytes and Ly‐6Ghi 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‐6Chi 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.

Ablation of CD11c(hi) dendritic cells exacerbates Japanese encephalitis by regulating blood-brain barrier permeability and altering tight junction/adhesion molecules.

Japanese encephalitis (JE), characterized by extensive neuroinflammation following infection with neurotropic JE virus (JEV), is becoming a leading cause of viral encephalitis due to rapid changes in climate and demography. The blood-brain barrier (BBB) plays an important role in restricting neuroinvasion of peripheral leukocytes and virus, thereby regulating the progression of viral encephalitis. In this study, we explored the role of CD11c(hi) dendritic cells (DCs) in regulating BBB integrity and JE progression using a conditional depletion model of CD11c(hi) DCs. Transient ablation of CD11c(hi) DCs resulted in markedly increased susceptibility to JE progression along with highly increased neuro-invasion of JEV. In addition, exacerbated JE progression in CD11c(hi) DC-ablated hosts was closely associated with increased expression of proinflammatory cytokines (IFN-β, IL-6, and TNF-α) and CC chemokines (CCL2, CCL3, CXCL2) in the brain. Moreover, our results revealed that the exacerbation of JE progression in CD11c(hi) DC-ablated hosts was correlated with enhanced BBB permeability and reduced expression of tight junction and adhesion molecules (claudin-5, ZO-1, occluding, JAMs). Ultimately, our data conclude that the ablation of CD11c(hi) DCs provided a subsidiary impact on BBB integrity and the expression of tight junction/adhesion molecules, thereby leading to exacerbated JE progression. These findings provide insight into the secondary role of CD11c(hi) DCs in JE progression through regulation of BBB integrity and the expression of tight junction/adhesion molecules.