Japanese encephalitis (JE) is major emerging neurologic disease caused by JE virus. To date, the impact of TLR molecules on JE progression has not been addressed. Here, we determined whether each TLR modulates JE, using several TLR-deficient mouse strains (TLR2, TLR3, TLR4, TLR7, TLR9). Surprisingly, among the tested TLR-deficient mice there were contrasting results in TLR3−/− and TLR4−/− mice, i.e. TLR3−/− mice were highly susceptible to JE, whereas TLR4−/− mice showed enhanced resistance to JE. TLR3 ablation induced severe CNS inflammation characterized by early infiltration of inflammatory CD11b+Ly-6Chigh monocytes along with profoundly increased viral burden, proinflammatory cytokine/chemokine expression as well as BBB permeability. In contrast, TLR4−/− mice showed mild CNS inflammation manifested by reduced viral burden, leukocyte infiltration and proinflammatory cytokine expression. Interestingly, TLR4 ablation provided potent in vivo systemic type I IFN innate response, as well as ex vivo type I IFN production associated with strong induction of antiviral PRRs (RIG-I, MDA5), transcription factors (IRF-3, IRF-7), and IFN-dependent (PKR, Oas1, Mx) and independent ISGs (ISG49, ISG54, ISG56) by alternative activation of IRF3 and NF-κB in myeloid-derived DCs and macrophages, as compared to TLR3−/− myeloid-derived cells which were more permissive to viral replication through impaired type I IFN innate response. TLR4 ablation also appeared to mount an enhanced type I IFN innate and humoral, CD4+ and CD8+ T cell responses, which were mediated by altered immune cell populations (increased number of plasmacytoid DCs and NK cells, reduced CD11b+Ly-6Chigh monocytes) and CD4+Foxp3+ Treg number in lymphoid tissue. Thus, potent type I IFN innate and adaptive immune responses in the absence of TLR4 were closely coupled with reduced JE lethality. Collectively, these results suggest that a balanced triggering of TLR signal array by viral components during JE progression could be responsible for determining disease outcome through regulating negative and positive factors.

In this study, we examined the effects of murine chemokine DNA, as genetic adjuvants given mucosally, on the systemic and distal mucosal immune responses to plasmid DNA encoding gB of herpes simplex virus (HSV) by using the mouse model. The CC chemokines macrophage inflammatory protein 1␤ (MIP-1␤) and monocyte chemotactic protein 1 (MCP-1) biased the immunity to the Th2-type pattern as judged by the ratio of immunoglobulin isotypes and interleukin-4 cytokine levels produced by CD4 ؉ T cells. The CXC chemokine MIP-2 and the CC chemokine MIP-1␣, however, mounted immune responses of the Th1-type pattern, and such a response rendered recipients more resistant to HSV vaginal infection. In addition, MIP-1␣ appeared to act via the upregulation of antigen-presenting cell (APC) function and the expression of costimulatory molecules (B7-1 and B7-2), whereas MIP-2 enhanced Th1-type CD4 ؉ T-cell-mediated adaptive immunity by increasing gamma interferon secretion from activated NK cells. Our results emphasize the value of using the mucosal route to administer DNA modulators such as chemokines that function as adjuvants by regulating the activity of innate immunity. Our findings provide new insight into the value of CXC and CC chemokines, which act on different innate cellular components as the linkage signals between innate and adaptive immunity in mucosal DNA vaccination.Mucosal surfaces represent the primary site for the transmission of several viruses including human immunodeficiency virus and herpes simplex virus (HSV). In consequence, immunity at mucosal sites represents an important issue in vaccine development. Mucosae have numerous innate defenses, some of which serve to alert and direct the nature of subsequent acquired immune events (1, 10). Interest has recently focused on cytokines and chemokines and the role they appear to play as modulators of the adaptive immune responses (12, 31). Accordingly, members of both types of molecules induced nonspecifically upon infection are involved in regulating the inflammatory reaction and the subsequent adaptive Th1 or Th2 type of T-cell reaction that occurs in the draining lymphoid tissue. Consequently, manipulating the expression of cytokines and chemokines during exposure to infectious agents or vaccine represents a valuable approach to achieve optimal protection.Most information on immunomodulatory effects of immune mediators has emphasized cytokines (34, 40). However, various chemokines may represent even more useful innate modulators since these molecules are involved in the recruitment and activation of cells related to innate immunity (35, 39). Currently, little is known about the nonspecific adjuvant effect of chemokines, and the two previous studies which used chemokine DNA given systemically with antigen (Ag) provided conflicting data (13,18). In the present study, we investigated whether genetic cotransfer of certain chemokines along with plasmid DNA encoding viral Ag to a mucosal site can affect the efficiency of subsequent acquired mucosal and systemic immune re...

The main targets for the immunosuppressive calcineurin inhibitors, cyclosporin A (CsA) and tacrolimus, have been considered to be activated T cells, but not antigen-presenting cells. Here we demonstrate that CsA and tacrolimus, but not rapamycin, inhibit major histocompatibility complex (MHC)-restricted antigen presentation in dendritic cells (DCs). Microencapsulated ovalbumin (OVA) was efficiently captured, processed, and presented on both class I MHC molecules IntroductionDendritic cells (DCs) play a key role in the initiation of primary immune responses. 1 DCs can acquire and process antigens in the periphery, and migrate to secondary lymphoid tissues where they prime primary T-cell responses. Two distinct pathways have been known for the presentation of antigenic peptides on major histocompatibility complex (MHC) molecules. 2 Exogenous antigens internalized by phagocytosis or endocytosis are normally processed and loaded on class II MHC molecules in a post-Golgi compartment. In contrast, endogenous antigens are partially digested by proteosome, transferred into endoplasmic reticulum (ER) by a transporter associated with antigen presentation (TAP), and then loaded on class I MHC molecules. It is also known that DCs can uptake antigens from cells undergoing apoptosis or necrosis for presentation to class I MHC-restricted cytotoxic T lymphocytes (CTLs). This form of antigen presentation has been termed "cross-presentation." Crosspresentation has been demonstrated to occur for many viral antigens and also for tumor antigens. 3,4 Cross-presentation may be a mechanism by which naive T cells can be primed to antigens that are present in nonprofessional antigen-presenting cells (APCs). In the absence of such a mechanism, viral or tumor antigens expressed in nonprofessional APCs could escape immunosurveillance because CTL responses can only be induced efficiently for the antigens presented via class I MHC molecules on professional APCs. [5][6][7] Thus far, the main cellular targets for CsA and tacrolimus have been considered to be activated T cells, but not APCs. 8 Even though chemically unrelated, CsA and tacrolimus bind to and inhibit the same protein, calcineurin, in a calcium-dependent signaling pathway after formation of a complex with cyclophilin A and FK-506 binding proteins (FKBPs), respectively, resulting in inhibition of the transcription of interleukin 2 (IL-2) and other lymphokines. 9,10 Rapamycin has structural similarity with tacrolimus and even forms a complex with the same FKBPs that form a complex with tacrolimus. 11 However, the FKBP-rapamycin complex interacts with a protein distinct from calcineurin, termed the mammalian target of rapamycin, mTOR. 12 Rapamycin suppresses T-cell activation at a different level, mainly through inhibition of cell-cycle progression via suppression of p70 S6 kinase activation induced by growthpromoting lymphokines. 8 Because T-cell responses can only be initiated on recognition of peptide-MHC complexes presented by professional APCs, inhibitors of antigen processing p...

Dendritic cells (DCs) are potent initiators of T cell-mediated immunity that undergo maturation during viral infections. However, few reports describing the interactions of DCs with Japanese encephalitis virus (JEV), which remains the most frequent cause of acute and epidemic viral encephalitis, are available. In this study, we investigated the interaction of JEV with DCs and macrophages. JEV replicated its viral RNA in both cells with different efficiency, and JEV infection of macrophages followed the classical activation pathway of up-regulation of tested costimulatory molecules and proinflammatory cytokine production (IL-6, TNF-α, and IL-12). On the contrary, JEV-infected DCs failed to up-regulate costimulatory molecules such as CD40 and MHC class II. Of more interest, along with production of proinflammatory cytokines, DCs infected by JEV released antiinflammatory cytokine IL-10, which was not detected in macrophages. Moreover, signaling through MyD88 molecule, a pan-adaptor molecule of TLRs, and p38 MAPK in JEV-infected DCs was found to play a role in the production of cytokines and subversion of primary CD4+ and CD8+ T cell responses. We also found that IL-10 released from JEV-infected DCs led to a reduction in the priming of CD8+ T cells, but not CD4+ T cells. Taken together, our data suggest that JEV induces functional impairment of DCs through MyD88-dependent and -independent pathways, which subsequently leads to poor CD4+ and CD8+ T cell responses, resulting in boosting viral survival and dissemination in the body.