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Resolution of viral infections of the central nervous system (CNS) is contingent on eliciting protective immunity with minimal pathology. C57BL/6 (B6) mice elucidate efficient innate and anti-viral T cell responses which render them resistant to infection with a virulent HSV 17+ virus strain. Virus replication is limited to ganglia and brainstem. However, susceptible mice such as 129S6 and BALB/c mice harbor increased virus in the CNS. These mice show increased myeloid derived suppressor cells (MDSC) and Tregs along with associated cytokines such as G-CSF, GM-CSF, IL-10 and IL-33 resulting in reduced numbers of virus specific CD8 T cells and effector T cells with increased expression of suppressive markers such as PD-1 and LAG-3. This impaired immune response resulted in disease and mortality. Neutralization of checkpoint inhibitors or immunosuppressive cytokines and depletion of myeloid cells or Tregs did not alter susceptibility rates. However, reducing expression of IL-33 in the CNS had a beneficial effect on T cell activation and the overall outcome of disease. Intriguingly, immunizing susceptible mice with a HSV thymidine kinase mutant virus incapable of infecting CNS induced memory T cells that suppressed MDSCs and protected from a subsequent challenge with HSV 17+. Interestingly IFNg, B cells or antibody were not essential but instead revealed the importance of the T cell myeloid interaction in determining disease outcomes. IL-33 plays critical roles as an immune-modulatory Th2 cytokine regulating immune-mediated diseases and tissue function especially in the CNS and as an alarmin in response to pathogens. This study shows that tempering IL-33 responses is essential to predicting improved outcomes to CNS viral infections.

A major impact of commensal microbiota on many aspects of host physiology including regulation of host immunity extends beyond the gut to remote sites, including the brain. Studies in mouse models have shown that the commensal Bacteroides fragilis and its capsular PSA can prevent various inflammatory disorders including colitis, experimental autoimmune encephalomyelitis and asthma. However, little is known about the interaction of gut bacteria and their products with the immune system during viral infection. We assessed the immunomodulatory potential of PSA in a murine model of herpes simplex virus (HSV) encephalitis (HSE). Fatal HSE results from inflammation of the brain in susceptible 129 strain of mice due to uncontrolled invasion by Ly6Chigh inflammatory monocytes (IM) and neutrophils. Naïve 129 mice pretreated with PSA or PBS were then given a 1-week course of the anti-viral drug acyclovir (ACV) from day 4-post HSV infection to simulate a clinical setting. PSA, but not PBS-treated mice survived with dramatically reduced brain inflammation including fewer degranulating IM and altered cytokine and chemokine profiles. Mesenteric lymph node cellularity was increased and IL-10 secreting ICOS+ CD73+ CD4+ T cells and CD73+ CD8+ T cells were induced by PSA treatment. Importantly, IL-10 deficient and Rag knockout mice were not protected, suggesting T/B cells derived IL-10 as the protective mechanism. Interestingly, B cells were required for PSA mediated protection. We infer that the microbiota augment immunity to HSV using pathways distinct from those required for protection in chronic autoimmunity models, and that probiotics in combination with antivirals might act synergistically to limit HSV induced inflammatory diseases.

The neurotropic coronavirus JHM strain of mouse hepatitis virus persists in oligodendroglia despite the presence of virus-specific CD8 T cells. Expression of programmed death 1 (PD-1) and B7-H1 were studied during acute and persistent infection to examine whether this negative regulatory mechanism contributes to CNS viral persistence. The majority of CNS-infiltrating CD8 T cells expressed PD-1, with the highest levels on virus-specific CD8 T cells. Moreover, despite control of infectious virus, CD8 T cells within the CNS of persistently infected mice maintained high PD-1 expression. Analysis of virus-susceptible target cells in vivo revealed that B7-H1 expression was regulated in a cell type-dependent manner. Oligodendroglia and microglia up-regulated B7-H1 following infection; however, although B7-H1 expression on oligodendroglia was prominent and sustained, it was significantly reduced and transient on microglia. Infection of mice deficient in the IFN-gamma or IFN-alpha/beta receptor demonstrated that B7-H1 expression on oligodendroglia is predominantly regulated by IFN-gamma. Ab blockade of B7-H1 on oligodendroglia in vitro enhanced IFN-gamma secretion by virus-specific CD8 T cells. More efficient virus control within the CNS of B7-H1-deficient mice confirmed inhibition of CD8 T cell function in vivo. Nevertheless, the absence of B7-H1 significantly increased morbidity without altering demyelination. These data are the first to demonstrate glia cell type-dependent B7-H1 regulation in vivo, resulting in adverse effects on antiviral CD8 T cell function. However, the beneficial role of PD-1:B7-H1 interactions in limiting morbidity highlights the need to evaluate tissue-specific intervention strategies.

MicroRNAs (miRNAs) are a class of powerful post-transcriptional regulators implicated in the control of diverse biological processes, including regulation of hematopoiesis and the immune response. To define the biological functions of miR-142, which is specifically and abundantly expressed in immune cells, we created a mouse line with targeted deletion of this gene. Our analysis of miR-142-/- mice revealed a critical role for this miRNA in the development and homeostasis of lymphocytes. Marginal zone B cells expand in the knockout spleen, while the number of T and B1 B cells in the periphery is reduced. Abnormal development of hematopoietic lineages in miR-142-/- animals is accompanied by a profound immunodeficiency, manifested by hypoimmunoglobulinemia and failure to mount a productive immune response to soluble antigens and virus. miR-142-/- B cells express elevated levels of BAFF receptor (BAFF-R) and as a result proliferate more robustly in response to BAFF stimulation. Lowering the BAFF-R gene dose in miR-142-/- mice rescued the B cell expansion defect, suggesting that BAFF-R is a bona fide miR-142 target through which it controls B cell homeostasis. Collectively, our results uncover miR-142 as an essential regulator of lymphocyte ontogenesis and suggest that lesions in this miRNA gene may lead to primary immunodeficiency.

Inflammation of the brainstem (BS) of wildtype (wt) 129 mice due to uncontrolled invasion by Ly6Chigh inflammatory monocytes (IM) and neutrophils is causally implicated in fatal HSV induced encephalitis (HSE). Anti-viral treatment with acyclovir following virus entry into BS cannot protect mice, as inflammation continues unabated. We showed previously that IL-10 regulates IM infiltration into BS. We determined the role of IFNγ, a key pleiotropic cytokine that directs the host immune response by inducing potent inflammatory or regulatory activities, in HSE. IFNγ knockout (GKO) mice developed HSE much quicker than wt or Rag mice. Surprisingly, mortality did not correlate with the ability to control virus replication but rather with massive expansion and infiltration of neutrophils into the BS. Neutrophil expansion in the absence of IFNγ was not due to increased IL-17 but rather to augmented secretion of G-CSF especially in CNS. Depletion of G-CSF by administration of anti G-CSF Ab or 25 mg IVIG (high dose), which contains anti G-CSF Ab, but not 4 mg (low dose) inhibited progenitor proliferation and neutrophil output from bone marrow and protected GKO mice. Importantly, reduction of serum G-CSF levels resulted in renewed IL-10 secretion by ICOS+ CD4 T cells and Tregs, which limited IM expansion. This study reveals that the complex interplay of IFNγ with G-CSF and IL-10 determines the ability to skew neutrophil or monocyte output from the bone marrow in response to infection.

The current dogma is that episodes of transient immunodeficiency triggered by various stressors reactivate herpesviruses that are ubiquitous in the human population. We established a novel model of Herpes simplex virus 1 (HSV) latency in immunodeficient B6.Rag mice inoculated with either a low (LD) or high dose (HD) inoculum by using IVIG and acyclovir to suppress virus replication and promote latency. Remarkably, heat stress induced in vivo reactivation (IVR) of latent HD-Rag mice resulted in uncontrolled HSV replication and fatal encephalitis (HSE) in all mice compared to transient reactivation in LD-Rag mice that all survived. The major difference between the two groups was the massive invasion of inflammatory Ly6Chigh monocytes (IM) into the brainstem (BS) of latent HD-Rag but not LD-Rag mice. T cell transfer prevented IVR induced fatal HSE in HD-Rag recipients by reducing inflammation, especially IM invasion of the BS. Surprisingly, IVR of lethally irradiated latent HD-Rag and wildtype B6 mice induced only transient reactivation resulting in 100% survival; notably, IM were absent in the BS of these mice. Thus, counter intuitively, our data show that IM resident at sites of latency promote rather than suppress HSV reactivation and by modulating IM invasion into the BS, T cells control virus reactivation. This is a novel finding with important clinical implications, especially for treatment of neonates and immunosuppressed cancer patients undergoing transplant therapy.

CD8+ T cells infiltrating the CNS control infection by the neurotropic JHM strain of mouse hepatitis virus. Differential susceptibility of infected cell types to clearance by perforin or IFN-γ uncovered distinct, nonredundant roles for these antiviral mechanisms. To separately evaluate each effector function specifically in the context of CD8+ T cells, pathogenesis was analyzed in mice deficient in both perforin and IFN-γ (PKO/GKO) or selectively reconstituted for each function by transfer of CD8+ T cells. Untreated PKO/GKO mice were unable to control the infection and died of lethal encephalomyelitis within 16 days, despite substantially higher CD8+ T cell accumulation in the CNS compared with controls. Uncontrolled infection was associated with limited MHC class I up-regulation and an absence of class II expression on microglia, coinciding with decreased CD4+ T cells in CNS infiltrates. CD8+ T cells from perforin-deficient and wild-type donors reduced virus replication in PKO/GKO recipients. By contrast, IFN-γ-deficient donor CD8+ T cells did not affect virus replication. The inability of perforin-mediated mechanisms to control virus in the absence of IFN-γ coincided with reduced class I expression. These data not only confirm direct antiviral activity of IFN-γ within the CNS but also demonstrate IFN-γ-dependent MHC surface expression to guarantee local T cell effector function in tissues inherently low in MHC expression. The data further imply that IFN-γ plays a crucial role in pathogenesis by regulating the balance between virus replication in oligodendrocytes, CD8+ T cell effector function, and demyelination.

DC-SIGN is a critical determinant of susceptibility to flaviviral infections. To study its role in immunity to West Nile Virus (WNV), we used mice that lacked the murine homolog SIGNR1. Following infection with a lethal dose of WNV, knockout (KO) but not wildtype (WT) mice were resistant to WNV; all WT mice succumbed by day 9 pi. CNS inflammation in WT and KO mice was similar. CD45high infiltrates in brains were similar at day 6-8 pi; macrophages and DCs were predominant in both KO and WT mice at day 6 pi but Ly6Chigh inflammatory macrophages was significantly increased in WT compared to KO brains (45% Vs 23%). T cell infiltrates were increased in WT compared to KO brains at day 8 pi, yet they did not protect. Analysis of T cell activation revealed increased CD62Llow CD44high effector CD4+ (40% Vs 10%) and CD8+T cells (35% Vs 5%) in the spleens of KO compared to WT mice. Enhanced T cell activation was supported by an increase in IFN-γ secreting T cells in the KO spleen. Higher levels of PD-1+ CD4+ and CD8+ T cells observed in WT compared to KO spleens possibly contributed to the reduced activation. Treatment of WT mice with a blocking mAb 22D1 and an RNA aptamer selected for binding to SIGNR1 that transiently reduces surface SIGNR1 expression significantly protected mice from mortality. These data suggest that SIGN-R1 plays an inhibitory role in activating WNV specific T cells and is a potential target for immunotherapeutic approaches for WNV and other flaviviral infections.

Susceptible mouse strains like 129 mice develop HSV encephalitis (HSE) characterized by large focal inflammatory CNS lesions associated with massive accumulation of monocytes and neutrophils, resulting in rapid onset of death. In contrast, regulated CNS inflammatory responses protect resistant C57BL/6 mice against HSE. Disease progression was not affected by early inhibition of HSV replication consistent with immune mediated pathology and not virus cytopathology, causing death. Intravenous immunoglobulin (IVIG) possesses potent anti-inflammatory activities and is highly effective in treating various inflammatory diseases. We report here that IVIG protects against fatal HSE by inhibiting CNS inflammation through a mechanism(s) independent of HSV antibody. Both sialylated and non-sialylated but not deglycosylated IgGs mediated protection. IVIG treatment profoundly altered host immune responses, promoting lymphocytic rather than monocytic infiltration of the CNS accompanied by phenotypic alteration of macrophages and a dramatic expansion of CD4+ Tregs that accumulated in peripheral lymphoid organs. However the Tregs were not solely responsible for inhibiting inflammation. IL-10 secreted by APCs was primarily responsible for suppressing CNS inflammation. Despite controlling virus replication IVIG treated IL-10 deficient mice were unable to control CNS inflammation and succumbed eventually. In addition, FcγR2b was critical for IVIG’s immunomodulatory activity.