IPS-1 Is Essential for the Control of West Nile Virus Infection and Immunity

The innate immune response is essential for controlling West Nile virus (WNV) infection but how this response is propagated and regulates adaptive immunity in vivo are not defined. Herein, we show that IPS-1, the central adaptor protein to RIG-I-like receptor (RLR) signaling, is essential for triggering of innate immunity and for effective development and regulation of adaptive immunity against pathogenic WNV. IPS-1^−/− mice exhibited increased susceptibility to WNV infection marked by enhanced viral replication and dissemination with early viral entry into the CNS. Infection of cultured bone-marrow (BM) derived dendritic cells (DCs), macrophages (Macs), and primary cortical neurons showed that the IPS-1-dependent RLR signaling was essential for triggering IFN defenses and controlling virus replication in these key target cells of infection. Intriguingly, infected IPS-1^−/− mice displayed uncontrolled inflammation that included elevated systemic type I IFN, proinflammatory cytokine and chemokine responses, increased numbers of inflammatory DCs, enhanced humoral responses marked by complete loss of virus neutralization activity, and increased numbers of virus-specific CD8+ T cells and non-specific immune cell proliferation in the periphery and in the CNS. This uncontrolled inflammatory response was associated with a lack of regulatory T cell expansion that normally occurs during acute WNV infection. Thus, the enhanced inflammatory response in the absence of IPS-1 was coupled with a failure to protect against WNV infection. Our data define an innate/adaptive immune interface mediated through IPS-1-dependent RLR signaling that regulates the quantity, quality, and balance of the immune response to WNV infection.     

The Essential,Nonredundant Roles of RIG-I and MDA5 in Detecting and Controlling West Nile Virus Infection

Virus recognition and response by the innate immune system are critical components of host defense against infection. Activation of cell-intrinsic immunity and optimal priming of adaptive immunity against West Nile virus (WNV), an emerging vector-borne virus, depend on recognition by RIG-I and MDA5, two cytosolic pattern recognition receptors (PRRs) of the RIG-I-like receptor (RLR) protein family that recognize viral RNA and activate defense programs that suppress infection. We evaluated the individual functions of RIG-I and MDA5 both in vitro and in vivo in pathogen recognition and control of WNV. Lack of RIG-I or MDA5 alone results in decreased innate immune signaling and virus control in primary cells in vitro and increased mortality in mice. We also generated RIG-I^−/− × MDA5^−/− double-knockout mice and found that a lack of both RLRs results in a complete absence of innate immune gene induction in target cells of WNV infection and a severe pathogenesis during infection in vivo, similar to findings for animals lacking MAVS, the central adaptor molecule for RLR signaling. We also found that RNA products from WNV-infected cells but not incoming virion RNA display at least two distinct pathogen-associated molecular patterns (PAMPs) containing 5′ triphosphate and double-stranded RNA that are temporally distributed and sensed by RIG-I and MDA5 during infection. Thus, RIG-I and MDA5 are essential PRRs that recognize distinct PAMPs that accumulate during WNV replication. Collectively, these experiments highlight the necessity and function of multiple related, cytoplasmic host sensors in orchestrating an effective immune response against an acute viral infection.     

CD22 Is Required for Protection against West Nile Virus Infection

West Nile virus (WNV) is a RNA virus of the family Flaviviridae and the leading cause of mosquito-borne encephalitis in the United States. Humoral immunity is essential for protection against WNV infection; however, the requirements for initiating effective antibody responses against WNV infection are still unclear. CD22 (Siglec-2) is expressed on B cells and regulates B cell receptor signaling, cell survival, proliferation, and antibody production. In this study, we investigated how CD22 contributes to protection against WNV infection and found that CD22 knockout (Cd22^−/−) mice were highly susceptible to WNV infection and had increased viral loads in the serum and central nervous system (CNS) compared to wild-type (WT) mice. This was not due to a defect in humoral immunity, as Cd22^−/− mice had normal WNV-specific antibody responses. However, Cd22^−/− mice had decreased WNV-specific CD8⁺ T cell responses compared to those of WT mice. These defects were not simply due to reduced cytotoxic activity or increased cell death but, rather, were associated with decreased lymphocyte migration into the draining lymph nodes (dLNs) of infected Cd22^−/− mice. Cd22^−/− mice had reduced production of the chemokine CCL3 in the dLNs after infection, suggesting that CD22 affects chemotaxis via controlling chemokine production. CD22 was not restricted to B cells but was also expressed on a subset of splenic DCIR2⁺ dendritic cells that rapidly expand early after WNV infection. Thus, CD22 plays an essential role in controlling WNV infection by governing cell migration and CD8⁺ T cell responses.     

A Role for Ifit2 in Restricting West Nile Virus Infection in the Brain

Previous studies have demonstrated that type I interferon (IFN-I) restricts West Nile virus (WNV) replication and pathogenesis in peripheral and central nervous system (CNS) tissues. However, the in vivo role of specific antiviral genes that are induced by IFN-I against WNV infection remains less well characterized. Here, using Ifit2^−/− mice, we defined the antiviral function of the interferon-stimulated gene (ISG) Ifit2 in limiting infection and disease in vivo by a virulent North American strain of WNV. Compared to congenic wild-type controls, Ifit2^−/− mice showed enhanced WNV infection in a tissue-restricted manner, with preferential replication in the CNS of animals lacking Ifit2. Virological analysis of cultured macrophages, dendritic cells, fibroblasts, cerebellar granule cell neurons, and cortical neurons revealed cell type-specific antiviral functions of Ifit2 against WNV. In comparison, small effects of Ifit2 were observed on the induction or magnitude of innate or adaptive immune responses. Our results suggest that Ifit2 restricts WNV infection and pathogenesis in different tissues in a cell type-specific manner.     

IL-10 Signaling Blockade Controls Murine West Nile Virus Infection

West Nile virus (WNV), a mosquito-borne single-stranded RNA flavivirus, can cause significant human morbidity and mortality. Our data show that interleukin-10 (IL-10) is dramatically elevated both in vitro and in vivo following WNV infection. Consistent with an etiologic role of IL-10 in WNV pathogenesis, we find that WNV infection is markedly diminished in IL-10 deficient (IL-10^−/−) mice, and pharmacologic blockade of IL-10 signaling by IL-10 neutralizing antibody increases survival of WNV-infected mice. Increased production of antiviral cytokines in IL-10^−/− mice is associated with more efficient control of WNV infection. Moreover, CD4⁺ T cells produce copious amounts of IL-10, and may be an important cellular source of IL-10 during WNV infection in vivo. In conclusion, IL-10 signaling plays a negative role in immunity against WNV infection, and blockade of IL-10 signaling by genetic or pharmacologic means helps to control viral infection, suggesting a novel anti-WNV therapeutic strategy.     

Deficient IFN Signaling by Myeloid Cells Leads to MAVS-Dependent Virus-Induced Sepsis

The type I interferon (IFN) signaling response limits infection of many RNA and DNA viruses. To define key cell types that require type I IFN signaling to orchestrate immunity against West Nile virus (WNV), we infected mice with conditional deletions of the type I IFN receptor (IFNAR) gene. Deletion of the Ifnar gene in subsets of myeloid cells resulted in uncontrolled WNV replication, vasoactive cytokine production, sepsis, organ damage, and death that were remarkably similar to infection of Ifnar ^−/− mice completely lacking type I IFN signaling. In Mavs^−/−×Ifnar^−/− myeloid cells and mice lacking both Ifnar and the RIG-I-like receptor adaptor gene Mavs, cytokine production was muted despite high levels of WNV infection. Thus, in myeloid cells, viral infection triggers signaling through MAVS to induce proinflammatory cytokines that can result in sepsis and organ damage. Viral pathogenesis was caused in part by massive complement activation, as liver damage was minimized in animals lacking complement components C3 or factor B or treated with neutralizing anti-C5 antibodies. Disease in Ifnar ^−/− and CD11c Cre⁺ Ifnar ^f/f mice also was facilitated by the proinflammatory cytokine TNF-α, as blocking antibodies diminished complement activation and prolonged survival without altering viral burden. Collectively, our findings establish the dominant role of type I IFN signaling in myeloid cells in restricting virus infection and controlling pathological inflammation and tissue injury.     

Regulatory role of CD1d in neurotropic virus infection

The GDVII strain of Theiler's murine encephalomyelitis virus (TMEV) causes an acute fatal polioencephalomyelitis in mice. Infection of susceptible mice with the DA strain of TMEV results in an acute polioencephalomyelitis followed by chronic immune-mediated demyelination with virus persistence in the central nervous system (CNS); DA virus infection is used as an animal model for multiple sclerosis. CD1d-restricted natural killer T (NKT) cells can contribute to viral clearance and regulation of autoimmune responses. To investigate the role of CD1d in TMEV infection, we first infected CD1d-deficient mice (CD1^−/−) and wild-type BALB/c mice with GDVII virus. Wild-type mice were more resistant to virus than CD1^−/− mice (50% lethal dose titers: wild-type mice, 10 PFU; CD1^−/− mice, 1.6 PFU). Wild-type mice had fewer viral antigen-positive cells with greater inflammation in the CNS than CD1^−/− mice. Second, an analysis of DA virus infection in CD1^−/− mice was conducted. Although both wild-type and CD1^−/− mice had similar clinical signs during the first 2 weeks after infection, CD1^−/− mice had an increase in neurological deficits over those observed in wild-type mice at 3 to 5 weeks after infection. Although wild-type mice had no demyelination, 20 and 60% of CD1^−/− mice developed demyelination at 3 and 5 weeks after infection, respectively. TMEV-specific lymphoproliferative responses, interleukin-4 (IL-4) production, and IL-4/gamma interferon ratios were higher in CD1^−/− mice than in wild-type mice. Thus, CD1d-restricted NKT cells may play a protective role in TMEV-induced neurological disease by alteration of the cytokine profile and virus-specific immune responses.     

Targeted Deletion of FGL2 Leads to Increased Early Viral Replication and Enhanced Adaptive Immunity in a Murine Model of Acute Viral Hepatitis Caused by LCMV WE

Mounting effective innate and adaptive immune responses are critical for viral clearance and the generation of long lasting immunity. It is known that production of inhibitory factors may result in the inability of the host to clear viruses, resulting in chronic viral persistence. Fibrinogen-like protein 2 (FGL2) has been identified as a novel effector molecule of CD4⁺CD25⁺ Foxp3⁺ regulatory T (Treg) cells that inhibits immune activity by binding to FCγRIIB expressed primarily on antigen presenting cells (APC). In this study, we show that infection of mice with Lymphocytic Choriomeningitis Virus WE (LCMV WE) leads to increased plasma levels of FGL2, which were detected as early as 2 days post-infection (pi) and persisted until day 50 pi. Mice deficient in FGL2 (fgl2^−/−) had increased viral titers of LCMV WE in the liver early p.i but cleared the virus by day 12 similar to wild type mice. Dendritic cells (DC) isolated from the spleens of LCMV WE infected fgl2^−/− had increased expression of the DC maturation markers CD80 and MHC Class II compared to wild type (fgl2^+/+). Frequencies of CD8⁺ and CD4⁺ T cells producing IFNγ in response to ex vivo peptide re-stimulation isolated from the spleen and lymph nodes were also increased in LCMV WE infected fgl2 ^−/− mice. Increased frequencies of CD8⁺ T cells specific for LCMV tetramers GP₃₃ and NP₃₉₆ were detected within the liver of fgl2^−/− mice. Plasma from fgl2^−/− mice contained higher titers of total and neutralizing anti-LCMV antibody. Enhanced anti-viral immunity in fgl2^−/− mice was associated with increased levels of serum alanine transaminase (ALT), hepatic necrosis and inflammation following LCMV WE infection. These data demonstrate that targeting FGL2 leads to early increased viral replication but enhanced anti-viral adaptive T & B cell responses. Targeting FGL2 may enhance the efficacy of current anti-viral therapies for hepatotropic viruses.     

Decreased antiviral immune response within the central nervous system of aged mice is associated with increased lethality of West Nile virus encephalitis

West Nile virus (WNV) is an emerging pathogen that causes disease syndromes ranging from a mild flu‐like illness to encephalitis. While the incidence of WNV infection is fairly uniform across age groups, the risk of lethal encephalitis increases with advanced age. Prior studies have demonstrated age‐related, functional immune deficits that limit systemic antiviral immunity and increase mortality; however, the effect of age on antiviral immune responses specifically within the central nervous system (CNS) is unknown. Here, we show that aged mice exhibit increased peripheral organ and CNS tissue viral burden, the latter of which is associated with alterations in activation of both myeloid and lymphoid cells compared with similarly infected younger animals. Aged mice exhibit lower MHCII expression by microglia, and higher levels of PD1 and lower levels of IFNγ expression by WNV‐specific CD8⁺ T cells in the CNS and CD8⁺CD45⁺ cells. These data indicate that the aged CNS exhibits limited local reactivation of T cells during viral encephalitis, which may lead to reduced virologic control at this site.     

Human Herpesvirus 6A Infection and Immunopathogenesis in Humanized Rag2?/?γc?/? Mice

Although serious human diseases have been correlated with human herpesvirus 6A (HHV-6A) and HHV-6B, the lack of animal models has prevented studies which would more definitively link these viral infections to disease. HHV-6A and HHV-6B have recently been classified as two distinct viruses, and in this study we focused specifically on developing an in vivo model for HHV-6A. Here we show that Rag2^−/−γc^−/− mice humanized with cord blood-derived human hematopoietic stem cells produce human T cells that express the major HHV-6A receptor, CD46. Both cell-associated and cell-free viral transmission of HHV-6A into the peritoneal cavity resulted in detectable viral DNA in at least one of the samples (blood, bone marrow, etc.) analyzed from nearly all engrafted mice. Organs and cells positive for HHV-6A DNA were the plasma and cellular blood fractions, bone marrow, lymph node, and thymic samples; control mice had undetectable viral DNA. We also noted viral pathogenic effects on certain T cell populations. Specific thymocyte populations, including CD3⁻ CD4⁺ CD8⁻ and CD3⁺ CD4⁻ cells, were significantly modified in humanized mice infected by cell-associated transmission. In addition, we detected significantly increased proportions of CD4⁺ CD8⁺ cells in the blood of animals infected by cell-free transmission. These findings provide additional evidence that HHV-6A may play a role in human immunodeficiencies. These results indicate that humanized mice can be used to study HHV-6A in vivo infection and replication as well as aspects of viral pathogenesis.     

2��-O Methylation of the Viral mRNA Cap by West Nile Virus Evades Ifit1-Dependent and -Independent Mechanisms of Host Restriction In Vivo

Prior studies have shown that 2′-O methyltransferase activity of flaviviruses, coronaviruses, and poxviruses promotes viral evasion of Ifit1, an interferon-stimulated innate immune effector protein. Viruses lacking 2′-O methyltransferase activity exhibited attenuation in primary macrophages that was rescued in cells lacking Ifit1 gene expression. Here, we examined the role of Ifit1 in restricting pathogenesis in vivo of wild type WNV (WNV-WT) and a mutant in the NS5 gene (WNV-E218A) lacking 2′-O methylation of the 5′ viral RNA cap. While deletion of Ifit1 had marginal effects on WNV-WT pathogenesis, WNV-E218A showed increased replication in peripheral tissues of Ifit1 ^−/− mice after subcutaneous infection, yet this failed to correlate with enhanced infection in the brain or lethality. In comparison, WNV-E218A was virulent after intracranial infection as judged by increased infection in different regions of the central nervous system (CNS) and a greater than 16,000-fold decrease in LD₅₀ values in Ifit1 ^−/− compared to wild type mice. Ex vivo infection experiments revealed cell-type specific differences in the ability of an Ifit1 deficiency to complement the replication defect of WNV-E218A. In particular, WNV-E218A infection was impaired in both wild type and Ifit1 ^−/− brain microvascular endothelial cells, which are believed to participate in blood-brain barrier (BBB) regulation of virus entry into the CNS. A deficiency of Ifit1 also was associated with increased neuronal death in vivo, which was both cell-intrinsic and mediated by immunopathogenic CD8⁺ T cells. Our results suggest that virulent strains of WNV have largely evaded the antiviral effects of Ifit1, and viral mutants lacking 2′-O methylation are controlled in vivo by Ifit1-dependent and -independent mechanisms in different cell types.     

Melanoma Differentiation-Associated Gene 5 (MDA5) Is Involved in the Innate Immune Response to Paramyxoviridae Infection In Vivo

The early host response to pathogens is mediated by several distinct pattern recognition receptors. Cytoplasmic RNA helicases including RIG-I and MDA5 have been shown to respond to viral RNA by inducing interferon (IFN) production. Previous in vitro studies have demonstrated a direct role for MDA5 in the response to members of the Picornaviridae, Flaviviridae and Caliciviridae virus families ((+) ssRNA viruses) but not to Paramyxoviridae or Orthomyxoviridae ((−) ssRNA viruses). Contrary to these findings, we now show that MDA5 responds critically to infections caused by Paramyxoviridae in vivo. Using an established model of natural Sendai virus (SeV) infection, we demonstrate that MDA5^−/− mice exhibit increased morbidity and mortality as well as severe histopathological changes in the lower airways in response to SeV. Moreover, analysis of viral propagation in the lungs of MDA5^−/− mice reveals enhanced replication and a distinct distribution involving the interstitium. Though the levels of antiviral cytokines were comparable early during SeV infection, type I, II, and III IFN mRNA expression profiles were significantly decreased in MDA5^−/− mice by day 5 post infection. Taken together, these findings indicate that MDA5 is indispensable for sustained expression of IFN in response to paramyxovirus infection and provide the first evidence of MDA5-dependent containment of in vivo infections caused by (−) sense RNA viruses.     

IL-1β Signaling Promotes CNS-Intrinsic Immune Control of West Nile Virus Infection

West Nile virus (WNV) is an emerging flavivirus capable of infecting the central nervous system (CNS) and mediating neuronal cell death and tissue destruction. The processes that promote inflammation and encephalitis within the CNS are important for control of WNV disease but, how inflammatory signaling pathways operate to control CNS infection is not defined. Here, we identify IL-1β signaling and the NLRP3 inflammasome as key host restriction factors involved in viral control and CNS disease associated with WNV infection. Individuals presenting with acute WNV infection displayed elevated levels of IL-1β in their plasma over the course of infection, suggesting a role for IL-1β in WNV immunity. Indeed, we found that in a mouse model of infection, WNV induced the acute production of IL-1β in vivo, and that animals lacking the IL-1 receptor or components involved in inflammasome signaling complex exhibited increased susceptibility to WNV pathogenesis. This outcome associated with increased accumulation of virus within the CNS but not peripheral tissues and was further associated with altered kinetics and magnitude of inflammation, reduced quality of the effector CD8⁺ T cell response and reduced anti-viral activity within the CNS. Importantly, we found that WNV infection triggers production of IL-1β from cortical neurons. Furthermore, we found that IL-1β signaling synergizes with type I IFN to suppress WNV replication in neurons, thus implicating antiviral activity of IL-1β within neurons and control of virus replication within the CNS. Our studies thus define the NLRP3 inflammasome pathway and IL-1β signaling as key features controlling WNV infection and immunity in the CNS, and reveal a novel role for IL-1β in antiviral action that restricts virus replication in neurons.     

Interferon Regulatory Factor-1 Protects from Fatal Neurotropic Infection with Vesicular Stomatitis Virus by Specific Inhibition of Viral Replication in Neurons

The innate immune system protects cells against invading viral pathogens by the auto- and paracrine action of type I interferon (IFN). In addition, the interferon regulatory factor (IRF)-1 can induce alternative intrinsic antiviral responses. Although both, type I IFN and IRF-1 mediate their antiviral action by inducing overlapping subsets of IFN stimulated genes, the functional role of this alternative antiviral action of IRF-1 in context of viral infections in vivo remains unknown. Here, we report that IRF-1 is essential to counteract the neuropathology of vesicular stomatitis virus (VSV). IFN- and IRF-1-dependent antiviral responses act sequentially to create a layered antiviral protection program against VSV infections. Upon intranasal infection, VSV is cleared in the presence or absence of IRF-1 in peripheral organs, but IRF-1^−/− mice continue to propagate the virus in the brain and succumb. Although rapid IFN induction leads to a decline in VSV titers early on, viral replication is re-enforced in the brains of IRF-1^−/− mice. While IFN provides short-term protection, IRF-1 is induced with delayed kinetics and controls viral replication at later stages of infection. IRF-1 has no influence on viral entry but inhibits viral replication in neurons and viral spread through the CNS, which leads to fatal inflammatory responses in the CNS. These data support a temporal, non-redundant antiviral function of type I IFN and IRF-1, the latter playing a crucial role in late time points of VSV infection in the brain.     

Activated mouse CD4+Foxp3? T cells facilitate melanoma metastasis via Qa-1-dependent suppression of NK-cell cytotoxicity

The regulatory activities of mouse CD4⁺Foxp3⁺ T cells on various immune cells, including NK cells, have been well documented. Under some conditions, conventional CD4⁺Foxp3⁻ T cells in the periphery are able to acquire inhibitory function on other T cells, but their roles in controlling innate immune cells are poorly defined. As a potential cellular therapy for cancer, ex vivo activated CD4⁺Foxp3⁻ effector T cells are often infused back in vivo to suppress tumor growth and metastasis. Whether such activated T cells could affect NK-cell control of tumorigenesis is unclear. In the present study, we found that mitogen-activated CD4⁺Foxp3⁻ T cells exhibited potent suppressor function on NK-cell proliferation and cytotoxicity in vitro, and notably facilitated B16 melanoma metastasis in vivo. Suppression of NK cells by activated CD4⁺Foxp3⁻ T cells is cell-cell contact dependent and is mediated by Qa-1:NKG2A interaction, as administration of antibodies blocking either Qa-1 or NKG2A could completely reverse this suppression, and significantly inhibited otherwise facilitated melanoma metastasis. Moreover, activated CD4⁺Foxp3⁻ cells from Qa-1 knockout mice completely lost the suppressor activity on NK cells, and failed to facilitate melanoma metastasis when transferred in vivo. Taken together, our findings indicate that innate anti-tumor response is counter regulated by the activation of adaptive immunity, a phenomenon we term as “activation-induced inhibition”. Thus, the regulatory role of activated CD4⁺Foxp3⁻ T cells in NK-cell activity must be taken into consideration in the future design of cancer therapies.     

Inducible Deletion of CD28 Prior to Secondary Nippostrongylus brasiliensis Infection Impairs Worm Expulsion and Recall of Protective Memory CD4+ T Cell Responses

IL-13 driven Th2 immunity is indispensable for host protection against infection with the gastrointestinal nematode Nippostronglus brasiliensis. Disruption of CD28 mediated costimulation impairs development of adequate Th2 immunity, showing an importance for CD28 during the initiation of an immune response against this pathogen. In this study, we used global CD28^−/− mice and a recently established mouse model that allows for inducible deletion of the cd28 gene by oral administration of tamoxifen (CD28^−/loxCre^+/−+TM) to resolve the controversy surrounding the requirement of CD28 costimulation for recall of protective memory responses against pathogenic infections. Following primary infection with N. brasiliensis, CD28^−/− mice had delayed expulsion of adult worms in the small intestine compared to wild-type C57BL/6 mice that cleared the infection by day 9 post-infection. Delayed expulsion was associated with reduced production of IL-13 and reduced serum levels of antigen specific IgG1 and total IgE. Interestingly, abrogation of CD28 costimulation in CD28^−/loxCre^+/− mice by oral administration of tamoxifen prior to secondary infection with N. brasiliensis resulted in impaired worm expulsion, similarly to infected CD28^−/− mice. This was associated with reduced production of the Th2 cytokines IL-13 and IL-4, diminished serum titres of antigen specific IgG1 and total IgE and a reduced CXCR5⁺ T_FH cell population. Furthermore, total number of CD4⁺ T cells and B220⁺ B cells secreting Th1 and Th2 cytokines were significantly reduced in CD28^−/− mice and tamoxifen treated CD28^−/loxCre^+/− mice compared to C57BL/6 mice. Importantly, interfering with CD28 costimulatory signalling before re-infection impaired the recruitment and/or expansion of central and effector memory CD4⁺ T cells and follicular B cells to the draining lymph node of tamoxifen treated CD28^−/loxCre^+/− mice. Therefore, it can be concluded that CD28 costimulation is essential for conferring host protection during secondary N. brasiliensis infection.     

TLR2 Directing PD-L2 Expression Inhibit T Cells Response in Schistosoma japonicum Infection

Toll-like receptor 2 (TLR2) was shown to be an important immune receptor involved in the recognition of schistosome antigens, especially soluble egg antigen (SEA). In mice models with Schistosoma japonicum acute infection, we observed enhanced T cell-mediated immune responses in TLR2 knock out (TLR2^−/−) mice compared with B6 mice. In Schistosoma japonicum chronic infection models, programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2) expression as well as TLR2 expression gradually increased in B6 mice, while only PD-L2 expression significantly decreased in TLR2^−/− mice. Meanwhile, Programmed Death 1(PD-1) expression on CD4⁺T cells was down-regulated in TLR2^−/− mice after a large number of egg appeared. We also found that stimulation with schistosome antigens, especially SEA, could up-regulate PD-L2 expression on BMDCs in a TLR2-dependent manner in vitro. Schistosome antigens primed-BMDCs with impaired expression of TLR2 or PD-L2 could induce CD4⁺T cells to produce low level of IL-10 or high level of IFN-γ. Our results indicated that TLR2 signaling can direct PD-L2 expression on DCs, which binds to PD-1 mainly on CD4⁺T cells, to help inhibit T cells response in Schistosoma japonicum infection.     

PI3Kγ Drives Priming and Survival of Autoreactive CD4+ T Cells during Experimental Autoimmune Encephalomyelitis

The class IB phosphoinositide 3-kinase gamma enzyme complex (PI3Kγ) functions in multiple signaling pathways involved in leukocyte activation and migration, making it an attractive target in complex human inflammatory diseases including MS. Here, using pik3cg ^−/− mice and a selective PI3Kγ inhibitor, we show that PI3Kγ promotes development of experimental autoimmune encephalomyelitis (EAE). In pik3cg^−/− mice, EAE is markedly suppressed and fewer leukocytes including CD4⁺ and CD8⁺ T cells, granulocytes and mononuclear phagocytes infiltrate the CNS. CD4⁺ T cell priming in secondary lymphoid organs is reduced in pik3cg^−/− mice following immunisation. This is attributable to defects in DC migration concomitant with a failure of full T cell activation following TCR ligation in the absence of p110γ. Together, this results in suppressed autoreactive T cell responses in pik3cg^−/− mice, with more CD4⁺ T cells undergoing apoptosis and fewer cytokine-producing Th1 and Th17 cells in lymphoid organs and the CNS. When administered from onset of EAE, the orally active PI3Kγ inhibitor AS605240 caused inhibition and reversal of clinical disease, and demyelination and cellular pathology in the CNS was reduced. These results strongly suggest that inhibitors of PI3Kγ may be useful therapeutics for MS.