Herpes simplex virus infection, Acyclovir and IVIG treatment all independently cause gut dysbiosis.

Herpes simplex virus 1 (HSV) is a ubiquitous human virus resident in a majority of the global population as a latent infection. Acyclovir (ACV), is the standard of care drug used to treat primary and recurrent infections, supplemented in some patients with intravenous immunoglobulin (IVIG) treatment to suppress infection and deleterious inflammatory responses. As many diverse medications have recently been shown to change composition of the gut microbiome, we used Illumina 16S rRNA gene sequencing to determine the effects of ACV and IVIG on the gut bacterial community. We found that HSV, ACV and IVIG can all independently disrupt the gut bacterial community in a sex biased manner when given to uninfected C57BL/6 mice. Treatment of HSV infected mice with ACV or IVIG alone or together revealed complex interactions between these drugs and infection that caused pronounced sex biased dysbiosis. ACV reduced Bacteroidetes levels in male but not female mice, while levels of the Anti-inflammatory Clostridia (AIC) were reduced in female but not male mice, which is significant as these taxa are associated with protection against the development of graft versus host disease (GVHD) in hematopoietic stem cell transplant (HSCT) patients. Gut barrier dysfunction is associated with GVHD in HSCT patients and ACV also decreased Akkermansia muciniphila, which is important for maintaining gut barrier functionality. Cumulatively, our data suggest that long-term prophylactic ACV treatment of HSCT patients may contribute to GVHD and also potentially impact immune reconstitution. These data have important implications for other clinical settings, including HSV eye disease and genital infections, where ACV is given long-term.

Bacteroides fragilis polysaccharide A induces IL-10 secreting B and T cells that prevent viral encephalitis.

The gut commensal Bacteroides fragilis or its capsular polysaccharide A (PSA) can prevent various peripheral and CNS sterile inflammatory disorders. Fatal herpes simplex encephalitis (HSE) results from immune pathology caused by uncontrolled invasion of the brainstem by inflammatory monocytes and neutrophils. Here we assess the immunomodulatory potential of PSA in HSE by infecting PSA or PBS treated 129S6 mice with HSV1, followed by delayed Acyclovir (ACV) treatment as often occurs in the clinical setting. Only PSA-treated mice survived, with dramatically reduced brainstem inflammation and altered cytokine and chemokine profiles. Importantly, PSA binding by B cells is essential for induction of regulatory CD4+ and CD8+ T cells secreting IL-10 to control innate inflammatory responses, consistent with the lack of PSA mediated protection in Rag-/-, B cell- and IL-10-deficient mice. Our data reveal the translational potential of PSA as an immunomodulatory symbiosis factor to orchestrate robust protective anti-inflammatory responses during viral infections.

The Ca2+ sensor STIM1 regulates the type I interferon response by retaining the signaling adaptor STING at the endoplasmic reticulum.

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER) signaling adaptor that is essential for the type I interferon response to DNA pathogens. Aberrant activation of STING is linked to the pathology of autoimmune and autoinflammatory diseases. The rate-limiting step for the activation of STING is its translocation from the ER to the ER-Golgi intermediate compartment. Here, we found that deficiency in the Ca2+ sensor stromal interaction molecule 1 (STIM1) caused spontaneous activation of STING and enhanced expression of type I interferons under resting conditions in mice and a patient with combined immunodeficiency. Mechanistically, STIM1 associated with STING to retain it in the ER membrane, and coexpression of full-length STIM1 or a STING-interacting fragment of STIM1 suppressed the function of dominant STING mutants that cause autoinflammatory diseases. Furthermore, deficiency in STIM1 strongly enhanced the expression of type I interferons after viral infection and prevented the lethality of infection with a DNA virus in vivo. This work delineates a STIM1-STING circuit that maintains the resting state of the STING pathway.

Dominant Role of the Gut Microbiota in Chemotherapy Induced Neuropathic Pain.

Chemotherapy induced peripheral neuropathy (CIPN), a toxic side effect of some cancer treatments, negatively impacts patient outcomes and drastically reduces survivor's quality of life (QOL). Uncovering the mechanisms driving chemotherapy-induced CIPN is urgently needed to facilitate the development of effective treatments, as currently there are none. Observing that C57BL/6 (B6) and 129SvEv (129) mice are respectively sensitive and resistant to Paclitaxel-induced pain, we investigated the involvement of the gut microbiota in this extreme phenotypic response. Reciprocal gut microbiota transfers between B6 and 129 mice as well as antibiotic depletion causally linked gut microbes to Paclitaxel-induced pain sensitivity and resistance. Microglia proliferated in the spinal cords of Paclitaxel treated mice harboring the pain-sensitive B6 microbiota but not the pain-resistant 129 microbiota, which exhibited a notable absence of infiltrating immune cells. Paclitaxel decreased the abundance of Akkermansia muciniphila, which could compromise barrier integrity resulting in systemic exposure to bacterial metabolites and products - that acting via the gut-immune-brain axis - could result in altered brain function. Other bacterial taxa that consistently associated with both bacteria and pain as well as microglia and pain were identified, lending support to our hypothesis that microglia are causally involved in CIPN, and that gut bacteria are drivers of this phenotype.

IFNγ inhibits G-CSF induced neutrophil expansion and invasion of the CNS to prevent viral encephalitis.

Emergency hematopoiesis facilitates the rapid expansion of inflammatory immune cells in response to infections by pathogens, a process that must be carefully regulated to prevent potentially life threatening inflammatory responses. Here, we describe a novel regulatory role for the cytokine IFNγ that is critical for preventing fatal encephalitis after viral infection. HSV1 encephalitis (HSE) is triggered by the invasion of the brainstem by inflammatory monocytes and neutrophils. In mice lacking IFNγ (GKO), we observed unrestrained increases in G-CSF levels but not in GM-CSF or IL-17. This resulted in uncontrolled expansion and infiltration of apoptosis-resistant, degranulating neutrophils into the brainstem, causing fatal HSE in GKO but not WT mice. Excessive G-CSF in GKO mice also induced granulocyte derived suppressor cells, which inhibited T-cell proliferation and function, including production of the anti-inflammatory cytokine IL-10. Unexpectedly, we found that IFNγ suppressed G-CSF signaling by increasing SOCS3 expression in neutrophils, resulting in apoptosis. Depletion of G-CSF, but not GM-CSF, in GKO mice induced neutrophil apoptosis and reinstated IL-10 secretion by T cells, which restored their ability to limit innate inflammatory responses resulting in protection from HSE. Our studies reveals a novel, complex interplay among IFNγ, G-CSF and IL-10, which highlights the opposing roles of G-CSF and IFNγ in regulation of innate inflammatory responses in a murine viral encephalitis model and reveals G-CSF as a potential therapeutic target. Thus, the antagonistic G-CSF-IFNγ interactions emerge as a key regulatory node in control of CNS inflammatory responses to virus infection.

Effects of Acyclovir and IVIG on Behavioral Outcomes after HSV1 CNS Infection.

Herpes simplex virus 1 (HSV) encephalitis (HSE) has serious neurological complications, involving behavioral and cognitive impairments that cause significant morbidity and a reduced quality of life. We showed that HSE results from dysregulated central nervous system (CNS) inflammatory responses. We hypothesized that CNS inflammation is casually involved in behavioral abnormalities after HSE and that treatment with ACV and pooled human immunoglobulin (IVIG), an immunomodulatory drug, would improve outcomes compared to mice treated with phosphate buffered saline (PBS) or ACV alone. Anxiety levels were high in HSV-infected PBS and ACV-treated mice compared to mice treated with ACV + IVIG, consistent with reports implicating inflammation in anxiety induced by lipopolysaccharide (LPS) or stress. Female, but not male, PBS-treated mice were cognitively impaired, and unexpectedly, ACV was protective, while the inclusion of IVIG surprisingly antagonized ACV's beneficial effects. Distinct serum proteomic profiles were observed for male and female mice, and the antagonistic effects of ACV and IVIG on behavior were paralleled by similar changes in the serum proteome of ACV- and ACV + IVIG-treated mice. We conclude that inflammation and other factors mediate HSV-induced behavioral impairments and that the effects of ACV and IVIG on behavior involve novel mechanisms.

Altered lymphopoiesis and immunodeficiency in miR-142 null mice.

MicroRNAs (miRNAs) are a class of powerful posttranscriptional 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 preferentially and abundantly expressed in immune cells, we created a mouse line with a 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, whereas 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 B-cell-activating factor (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 rescues 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 lymphopoiesis, and suggest that lesions in this miRNA gene may lead to primary immunodeficiency.

Establishment of HSV1 latency in immunodeficient mice facilitates efficient in vivo reactivation.

The establishment of latent infections in sensory neurons is a remarkably effective immune evasion strategy that accounts for the widespread dissemination of life long Herpes Simplex Virus type 1 (HSV1) infections in humans. Periodic reactivation of latent virus results in asymptomatic shedding and transmission of HSV1 or recurrent disease that is usually mild but can be severe. An in-depth understanding of the mechanisms regulating the maintenance of latency and reactivation are essential for developing new approaches to block reactivation. However, the lack of a reliable mouse model that supports efficient in vivo reactivation (IVR) resulting in production of infectious HSV1 and/or disease has hampered progress. Since HSV1 reactivation is enhanced in immunosuppressed hosts, we exploited the antiviral and immunomodulatory activities of IVIG (intravenous immunoglobulins) to promote survival of latently infected immunodeficient Rag mice. Latently infected Rag mice derived by high dose (HD), but not low dose (LD), HSV1 inoculation exhibited spontaneous reactivation. Following hyperthermia stress (HS), the majority of HD inoculated mice developed HSV1 encephalitis (HSE) rapidly and synchronously, whereas for LD inoculated mice reactivated HSV1 persisted only transiently in trigeminal ganglia (Tg). T cells, but not B cells, were required to suppress spontaneous reactivation in HD inoculated latently infected mice. Transfer of HSV1 memory but not OVA specific or naïve T cells prior to HS blocked IVR, revealing the utility of this powerful Rag latency model for studying immune mechanisms involved in control of reactivation. Crossing Rag mice to various knockout strains and infecting them with wild type or mutant HSV1 strains is expected to provide novel insights into the role of specific cellular and viral genes in reactivation, thereby facilitating identification of new targets with the potential to block reactivation.

Anti-inflammatory activity of intravenous immunoglobulins protects against West Nile virus encephalitis.

West Nile virus (WNV), an important global human pathogen, targets neurons to cause lethal encephalitis, primarily in elderly and immunocompromised patients. Currently, there are no approved therapeutic agents or vaccines to treat WNV encephalitis. Recent studies have suggested that inflammation is a major contributor to WNV encephalitis morbidity. In this study we evaluated the use of IVIG (intravenous immunoglobulins - a clinical product comprising pooled human IgG) as an anti-inflammatory treatment in a model of lethal WNV infection. We report here that IVIG and pooled human WNV convalescent sera (WNV-IVIG) inhibited development of lethal WNV encephalitis by suppressing central nervous system (CNS) infiltration by CD45(high) leukocytes. Pathogenic Ly6C(high) CD11b(+) monocytes were the major infiltrating subset in the CNS of infected control mice, whereas IVIG profoundly reduced infiltration of these pathogenic Ly6C(high) monocytes into the CNS of infected mice. Interestingly, WNV-IVIG was more efficacious than IVIG in controlling CNS inflammation when mice were challenged with a high-dose inoculum (10(5) versus 10(4) p.f.u.) of WNV. Importantly, adsorption of WNV E-glycoprotein neutralizing antibodies did not abrogate IVIG protection, consistent with virus neutralization not being essential for IVIG protection. These findings confirmed the potent immunomodulatory activity of generic IVIG, and emphasized its potential as an effective immunotherapeutic drug for encephalitis and other virus induced inflammatory diseases.

The case for immunomodulatory approaches in treating HSV encephalitis.

HSV encephalitis (HSE) is the most prevalent sporadic viral encephalitis. Although safe and effective antiviral therapies and greatly improved noninvasive diagnostic procedures have significantly improved outcomes, mortality (~20%) and debilitating neurological sequelae in survivors remain unacceptably high. An encouraging new development is that the focus is now shifting away from the virus exclusively, to include consideration of the host immune response to infection in the pathology underlying development of HSE. In this article, the authors discuss results from recent studies in experimental mouse models, as well as clinical reports that demonstrate a role for exaggerated host inflammatory responses in the brain in the development of HSE that is motivating researchers and clinicians to consider new therapeutic approaches for treating HSE. The authors also discuss results from a few studies that have shown that immunomodulatory drugs can be highly protective against HSE, which supports a role for deleterious host inflammatory responses in HSE. The impressive outcomes of some immunomodulatory approaches in mouse models of HSE emphasize the urgent need for clinical trials to rigorously evaluate combination antiviral and immunomodulatory therapy in comparison with standard antiviral therapy for treatment of HSE, and support for such an initiative is gaining momentum.

Passively administered pooled human immunoglobulins exert IL-10 dependent anti-inflammatory effects that protect against fatal HSV encephalitis.

HSV-1 is the leading cause of sporadic encephalitis in humans. HSV infection of susceptible 129S6 mice results in fatal encephalitis (HSE) caused by massive inflammatory brainstem lesions comprising monocytes and neutrophils. During infection with pathogenic microorganisms or autoimmune disease, IgGs induce proinflammatory responses and recruit innate effector cells. In contrast, high dose intravenous immunoglobulins (IVIG) are an effective treatment for various autoimmune and inflammatory diseases because of potent anti-inflammatory effects stemming in part from sialylated IgGs (sIgG) present at 1-3% in IVIG. We investigated the ability of IVIG to prevent fatal HSE when given 24 h post infection. We discovered a novel anti-inflammatory pathway mediated by low-dose IVIG that protected 129S6 mice from fatal HSE by modulating CNS inflammation independently of HSV specific antibodies or sIgG. IVIG suppressed CNS infiltration by pathogenic CD11b(+) Ly6C(high) monocytes and inhibited their spontaneous degranulation in vitro. FcγRIIb expression was required for IVIG mediated suppression of CNS infiltration by CD45(+) Ly6C(low) monocytes but not for inhibiting development of Ly6C(high) monocytes. IVIG increased accumulation of T cells in the CNS, and the non-sIgG fraction induced a dramatic expansion of FoxP3(+) CD4(+) T regulatory cells (Tregs) and FoxP3(-) ICOS(+) CD4(+) T cells in peripheral lymphoid organs. Tregs purified from HSV infected IVIG treated, but not control, mice protected adoptively transferred mice from fatal HSE. IL-10, produced by the ICOS(+) CD4(+) T cells that accumulated in the CNS of IVIG treated, but not control mice, was essential for induction of protective anti-inflammatory responses. Our results significantly enhance understanding of IVIG's anti-inflammatory and immunomodulatory capabilities by revealing a novel sIgG independent anti-inflammatory pathway responsible for induction of regulatory T cells that secrete the immunosuppressive cytokine IL-10 and further reveal the therapeutic potential of IVIG for treating viral induced inflammatory diseases.

Target-dependent B7-H1 regulation contributes to clearance of central nervous system infection and dampens morbidity.

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.

Interleukin-12 (IL-12), but not IL-23, deficiency ameliorates viral encephalitis without affecting viral control.

The relative contributions of interleukin-12 (IL-12) and IL-23 to viral pathogenesis have not been extensively studied. IL-12p40 mRNA rapidly increases after neurotropic coronavirus infection. Infection of mice defective in both IL-12 and IL-23 (p40(-/-)), in IL-12 alone (p35(-/-)), and in IL-23 alone (p19(-/-)) revealed that the symptoms of coronavirus-induced encephalitis are regulated by IL-12. IL-17-producing cells never exceeded background levels, supporting a redundant role of IL-23 in pathogenesis. Viral control, tropism, and demyelination were all similar in p35(-/-), p19(-/-), and wild-type mice. Reduced morbidity in infected IL-12 deficient mice was also not associated with altered recruitment or composition of inflammatory cells. However, gamma interferon (IFN-gamma) levels and virus-specific IFN-gamma-secreting CD4 and CD8 T cells were all reduced in the central nervous systems (CNS) of infected p35(-/-) mice. Transcription of the proinflammatory cytokines IL-1beta and IL-6, but not tumor necrosis factor, were initially reduced in infected p35(-/-) mice but increased to wild-type levels during peak inflammation. Furthermore, although transforming growth factor beta mRNA was not affected, IL-10 was increased in the CNS in the absence of IL-12. These data suggest that IL-12 does not contribute to antiviral function within the CNS but enhances morbidity associated with viral encephalitis by increasing the ratio of IFN-gamma to protective IL-10.

The immune response to herpes simplex virus type 1 infection in susceptible mice is a major cause of central nervous system pathology resulting in fatal encephalitis.

This study was undertaken to investigate possible immune mechanisms in fatal herpes simplex virus type 1 (HSV-1) encephalitis (HSE) after HSV-1 corneal inoculation. Susceptible 129S6 (129) but not resistant C57BL/6 (B6) mice developed intense focal inflammatory brain stem lesions of primarily F4/80(+) macrophages and Gr-1(+) neutrophils detectable by magnetic resonance imaging as early as day 6 postinfection (p.i.). Depletion of macrophages and neutrophils significantly enhanced the survival of infected 129 mice. Immunodeficient B6 (IL-7R(-/-) Kit(w41/w41)) mice lacking adaptive cells (B6-E mice) and transplanted with 129 bone marrow showed significantly accelerated fatal HSE compared to B6-E mice transplanted with B6 marrow or control nontransplanted B6-E mice. In contrast, there was no difference in ocular viral shedding in B6-E mice transplanted with 129 or B6 bone marrow. Acyclovir treatment of 129 mice beginning on day 4 p.i. (24 h after HSV-1 first reaches the brain stem) reduced nervous system viral titers to undetectable levels but did not alter brain stem inflammation or mortality. We conclude that fatal HSE in 129 mice results from widespread damage in the brain stem caused by destructive inflammatory responses initiated early in infection by massive infiltration of innate cells.

Induction of class I antigen processing components in oligodendroglia and microglia during viral encephalomyelitis.

Glia exhibit differential susceptibility to CD8 T cell mediated effector mechanisms during neurotropic coronavirus infection. In contrast to microglia, oligodendroglia are resistant to CD8 T cell perforin-mediated viral control in the absence of IFN gamma. Kinetic induction of MHC Class I expression by microglia and oligodendroglia in vivo was thus analyzed to assess responses to distinct inflammatory signals. Flow cytometry demonstrated delayed Class I surface expression by oligodendroglia compared with microglia. Distinct kinetics of Class I protein upregulation correlated with cell type specific transcription patterns of genes encoding Class I heavy chains and antigen processing components. Microglia isolated from naïve mice expressed high levels of these mRNAs, whereas they were near detection limits in oligodendroglia; nevertheless, Class I protein was undetectable on both cell types. Infection induced modest mRNA increases in microglia, but dramatic transcriptional upregulation in oligodendroglia coincident with IFN alpha or IFN gamma mRNA increases in infected tissue. Ultimately mRNAs reached similar levels in both cell types at their respective time points of maximal Class I expression. Expression of Class I on microglia, but not oligodendroglia, in infected IFN gamma deficient mice supported distinct IFN requirements for Class I presentation. These data suggest an innate immune preparedness of microglia to present antigen and engage CD8 T cells early following infection. The delayed, yet robust, IFN gamma dependent capacity of oligodendroglia to express Class I suggests strict control of immune interactions to avoid CD8 T cell recognition and potential presentation of autoantigen to preserve myelin maintenance.

Inhibition of interferon-gamma signaling in oligodendroglia delays coronavirus clearance without altering demyelination.

Infection of the central nervous system (CNS) by the neurotropic JHM strain of mouse hepatitis virus (JHMV) induces an acute encephalomyelitis associated with demyelination. To examine the anti-viral and/or regulatory role of interferon-gamma (IFN-gamma) signaling in the cell that synthesizes and maintains the myelin sheath, we analyzed JHMV pathogenesis in transgenic mice expressing a dominant-negative IFN-gamma receptor on oligodendroglia. Defective IFN-gamma signaling was associated with enhanced oligodendroglial tropism and delayed virus clearance. However, the CNS inflammatory cell composition and CD8(+) T-cell effector functions were similar between transgenic and wild-type mice, supporting unimpaired peripheral and CNS immune responses in transgenic mice. Surprisingly, increased viral load in oligodendroglia did not affect the extent of myelin loss, the frequency of oligodendroglial apoptosis, or CNS recruitment of macrophages. These data demonstrate that IFN-gamma receptor signaling is critical for the control of JHMV replication in oligodendroglia. In addition, the absence of a correlation between increased oligodendroglial infection and the extent of demyelination suggests a complex pathobiology of myelin loss in which infection of oligodendroglia is required but not sufficient.

Vaccine-induced memory CD8+ T cells cannot prevent central nervous system virus reactivation.

Noncytopathic viruses use multiple strategies to evade immune detection, challenging a role for vaccine induced CTL in preventing microbial persistence. Recrudescence of neurotropic coronavirus due to loss of T cell-mediated immune control provided an experimental model to test T cell vaccination efficacy in the absence of Ab. Challenge virus was rapidly controlled in vaccinated Ab-deficient mice coincident with accelerated recruitment of memory CD8+ T cells and enhanced effector function compared with primary CD8+ T cell responses. In contrast to primary effectors, reactivated memory cells persisted in the CNS at higher frequencies and retained ex vivo cytolytic activity. Nevertheless, despite earlier and prolonged T cell-mediated control in the CNS of vaccinated mice, virus ultimately reactivated. Apparent loss of memory CD8+ effector function in vivo was supported by a prominent decline in MHC expression on CNS resident target cells, presumably reflecting diminished IFN-gamma. Severely reduced MHC expression on glial cells at the time of recrudescence suggested that memory T cells, although fully armed to exert antiviral activity upon Ag recognition in vitro, are not responsive in an environment presenting few if any target MHC molecules. Paradoxically, effective clearance of viral Ag thus affords persisting virus a window of opportunity to escape from immune surveillance. These studies demonstrate that vaccine-induced T cell memory alone is unable to control persisting virus in a tissue with strict IFN-dependent MHC regulation, as evident in immune privileged sites.