Manohar B. Mutnal

Murine Cytomegalovirus Infection of Neural Stem Cells Alters Neurogenesis in the Developing Brain

Background Congenital cytomegalovirus (CMV) brain infection causes serious neuro-developmental sequelae including: mental retardation, cerebral palsy, and sensorineural hearing loss. But, the mechanisms of injury and pathogenesis to the fetal brain are not completely understood. The present study addresses potential pathogenic mechanisms by which this virus injures the CNS using a neonatal mouse model that mirrors congenital brain infection. This investigation focused on, analysis of cell types infected with mouse cytomegalovirus (MCMV) and the pattern of injury to the developing brain. Methodology/Principal Findings We used our MCMV infection model and a multi-color flow cytometry approach to quantify the effect of viral infection on the developing brain, identifying specific target cells and the consequent effect on neurogenesis. In this study, we show that neural stem cells (NSCs) and neuronal precursor cells are the principal target cells for MCMV in the developing brain. In addition, viral infection was demonstrated to cause a loss of NSCs expressing CD133 and nestin. We also showed that infection of neonates leads to subsequent abnormal brain development as indicated by loss of CD24(hi) cells that incorporated BrdU. This neonatal brain infection was also associated with altered expression of Oct4, a multipotency marker; as well as down regulation of the neurotrophins BDNF and NT3, which are essential to regulate the birth and differentiation of neurons during normal brain development. Finally, we report decreased expression of doublecortin, a marker to identify young neurons, following viral brain infection. Conclusions MCMV brain infection of newborn mice causes significant loss of NSCs, decreased proliferation of neuronal precursor cells, and marked loss of young neurons.

Glial cells suppress postencephalitic CD8+ T lymphocytes through PD-L1

Engagement of the programmed death (PD)−1 receptor on activated cells by its ligand (PD‐L1) is a mechanism for suppression of activated T‐lymphocytes. Microglia, the resident inflammatory cells of the brain, are important for pathogen detection and initiation of innate immunity, however, a novel role for these cells as immune regulators has also emerged. PD‐L1 on microglia has been shown to negatively regulate T‐cell activation in models of multiple sclerosis and acute viral encephalitis. In this study, we investigated the role of glial cell PD‐L1 in controlling encephalitogenic CD8+ T‐lymphocytes, which infiltrate the brain to manage viral infection, but remain to produce chronic neuroinflammation. Using a model of chronic neuroinflammation following murine cytomegalovirus (MCMV)‐induced encephalitis, we found that CD8+ T‐cells persisting within the brain expressed PD‐1. Conversely, activated microglia expressed PD‐L1. In vitro, primary murine microglia, which express low basal levels of PD‐L1, upregulated the co‐inhibitory ligand on IFN‐γ‐treatment. Blockade of the PD‐1: PD‐L1 pathway in microglial: CD8+ T‐cell co‐cultures increased T‐cell IFN‐γ and interleukin (IL)−2 production. We observed a similar phenomenon following blockade of this co‐inhibitory pathway in astrocyte: CD8+ T‐cell co‐cultures. Using ex vivo cultures of brain leukocytes, including microglia and CD8+ T‐cells, obtained from mice with MCMV‐induced chronic neuroinflammation, we found that neutralization of either PD‐1 or PD‐L1 increased IFN‐γ production from virus‐specific CD8+ T‐cells stimulated with MCMV IE1168–176 peptide. These data demonstrate that microglia and astrocytes control antiviral T‐cell responses and suggest a therapeutic potential of PD1: PD‐L1 modulation to manage the deleterious consequences of uncontrolled neuroinflammation. GLIA 2014;62:1582–1594

Infiltrating Regulatory B Cells Control Neuroinflammation following Viral Brain Infection

Previous studies have demonstrated the existence of a subset of B lymphocytes, regulatory B cells (Bregs), which modulate immune function. In this study, in vivo and in vitro experiments were undertaken to elucidate the role of these Bregs in controlling neuroinflammation following viral brain infection. We used multicolor flow cytometry to phenotype lymphocyte subpopulations infiltrating the brain, along with in vitro cocultures to assess their anti-inflammatory and immunoregulatory roles. This distinctive subset of CD19+CD1dhiCD5+ B cells was found to infiltrate the brains of chronically infected animals, reaching highest levels at the latest time point tested (30 d postinfection). B cell–deficient Jh−/− mice were found to develop exacerbated neuroimmune responses as measured by enhanced accumulation and/or retention of CD8+ T cells within the brain, as well as increased levels of microglial activation (MHC class II). Conversely, levels of Foxp3+ regulatory T cells were found to be significantly lower in Jh−/− mice when compared with wild-type (Wt) animals. Further experiments showed that in vitro–generated IL-10–secreting Bregs (B10) were able to inhibit cytokine responses from microglia following stimulation with viral Ags. These in vitro–generated B10 cells were also found to promote proliferation of regulatory T cells in coculture studies. Finally, gain-of-function experiments demonstrated that reconstitution of Wt B cells into Jh−/− mice restored neuroimmune responses to levels exhibited by infected Wt mice. Taken together, these results demonstrate that Bregs modulate T lymphocyte as well as microglial cell responses within the infected brain and promote CD4+Foxp3+ T cell proliferation in vitro.

Excess neutrophil infiltration during cytomegalovirus brain infection of interleukin-10-deficient mice

Wild-type mice control murine cytomegalovirus (MCMV) brain infection, but identical infection is lethal to animals deficient in interleukin (IL)-10. Here, we report that MCMV-infected IL-10 knockout (KO) mice displayed a marked increase in neutrophil infiltration into the infected, IL-10-deficient brain when compared to wild-type animals. Enhanced microglial cell activation, determined by MHC class II up-regulation, overexpression of CXCL2, and elevated P-selectin mRNA levels were observed. In vivo blocking of CXCL2 attenuated neutrophil infiltration and significantly improved the outcome of infection. Collectively, these data indicate that the absence of IL-10 results in pathologic neutrophil infiltration into MCMV-infected brains.

Persistent Humoral Immune Responses in the CNS Limit Recovery of Reactivated Murine Cytomegalovirus

Background Experimental infection of the mouse brain with murine CMV (MCMV) elicits neuroimmune responses that terminate acute infection while simultaneously preventing extensive bystander damage. Previous studies have determined that CD8+ T lymphocytes are required to restrict acute, productive MCMV infection within the central nervous system (CNS). In this study, we investigated the contribution of humoral immune responses in control of MCMV brain infection. Methodology/Principal Findings Utilizing our MCMV brain infection model, we investigated B-lymphocyte-lineage cells and assessed their role in controlling the recovery of reactivated virus from latently infected brain tissue. Brain infiltrating leukocytes were first phenotyped using markers indicative of B-lymphocytes and plasma cells. Results obtained during these studies showed a steady increase in the recruitment of B-lymphocyte-lineage cells into the brain throughout the time-course of viral infection. Further, MCMV-specific antibody secreting cells (ASC) were detected within the infiltrating leukocyte population using an ELISPOT assay. Immunohistochemical studies of brain sections revealed co-localization of CD138+ cells with either IgG or IgM. Additional immunohistochemical staining for MCMV early antigen 1 (E1, m112–113), a reported marker of viral latency in neurons, confirmed its expression in the brain during latent infection. Finally, using B-cell deficient (Jh−/−) mice we demonstrated that B-lymphocytes control recovery of reactivated virus from latently-infected brain tissue. A significantly higher rate of reactivated virus was recovered from the brains of Jh−/− mice when compared to Wt animals. Conclusion Taken together, these results demonstrate that MCMV infection triggers accumulation and persistence of B-lymphocyte-lineage cells within the brain, which produce antibodies and play a significant role in controlling reactivated virus.

T-cell reconstitution during murine acquired immunodeficiency syndrome (MAIDS) produces neuroinflammation and mortality in animals harboring opportunistic viral brain infection

BackgroundHighly active antiretroviral therapy (HAART) restores inflammatory immune responses in AIDS patients which may unmask previous subclinical infections or paradoxically exacerbate symptoms of opportunistic infections. In resource-poor settings, 25% of patients receiving HAART may develop CNS-related immune reconstitution inflammatory syndrome (IRIS). Here we describe a reliable mouse model to study underlying immunopathological mechanisms of CNS-IRIS.MethodsUtilizing our HSV brain infection model and mice with MAIDS, we investigated the effect of immune reconstitution on MAIDS mice harboring opportunistic viral brain infection. Using multi-color flow cytometry, we quantitatively measured the cellular infiltrate and microglial activation.ResultsInfection with the LP-BM5 retroviral mixture was found to confer susceptibility to herpes simplex virus (HSV)-1 brain infection to normally-resistant C57BL/6 mice. Increased susceptibility to brain infection was due to severe immunodeficiency at 8 wks p.i. and a marked increase in programmed death-1 (PD-1) expression on CD4+ and CD8+ T-cells. Both T-cell loss and opportunistic brain infection were associated with high level PD-1 expression because PD-1-knockout mice infected with LP-BM5 did not exhibit lymphopenia and retained resistance to HSV-1. In addition, HSV-infection of MAIDS mice stimulated peripheral immune cell infiltration into the brain and its ensuing microglial activation. Interestingly, while opportunistic herpes virus brain infection of C57BL/6 MAIDS mice was not itself lethal, when T-cell immunity was reconstituted through adoptive transfer of virus-specific CD3+ T-cells, it resulted in significant mortality among recipients. This immune reconstitution-induced mortality was associated with exacerbated neuroinflammation, as determined by MHC class II expression on resident microglia and elevated levels of Th1 cytokines in the brain.ConclusionsTaken together, these results indicate development of an immune reconstitution disease within the central nervous system (CNS-IRD). Experimental immune reconstitution disease of the CNS using T-cell repopulation of lymphopenic murine hosts harboring opportunistic brain infections may help elucidate neuroimmunoregulatory networks that produce CNS-IRIS in patients initiating HAART.

Reduced lymphocyte infiltration during cytomegalovirus brain infection of interleukin-10-deficient mice

Interleukin (IL)-10 deficiency results in highly elevated levels of interferon (IFN)-γ, as well as the IFN-γ-inducible chemokines CXCL9 and CXCL10 within murine cytomegalovirus (MCMV)-infected brains. To test the hypothesis that these elevated chemokine levels would result in enhanced brain infiltration, we compared immune cell infiltration in response to MCMV brain infection between wild-type and IL-10 knockout (KO) mice. Longitudinal analysis following adoptive transfer of cells from β-actin-luciferase transgenic wild-type mice showed maximal brain infiltration by peripheral immune cells occurred at 5 days post infection. Although the overall percentage of CD45(hi) cells infiltrating the brain was not altered by IL-10 deficiency, paradoxically, despite elevated chemokine levels, reduced T lymphocyte (CD8+) and natural killer (NK) (CD49b+) cell infiltration into the brain was observed in IL-10-deficient animals. This decreased lymphocyte infiltration was associated with elevated levels of the lymph node homing receptor L-selectin/CD62L on CD8+ T cells. Lymph node cells obtained from MCMV-infected mice deficient in IL-10 also displayed reduced migration towards CXCL10 when compared to wild-type animals. Taken together, these data show that despite elevated chemokine levels, absence of IL-10 results in reduced lymphocyte infiltration into MCMV-infected brains.

Chronic reactive gliosis following regulatory T cell depletion during acute MCMV encephalitis

Long‐term, persistent central nervous system inflammation is commonly seen following brain infection. Using a murine model of viral encephalitis (murine cytomegalovirus, MCMV) we have previously shown that post‐encephalitic brains are maintained in an inflammatory state consisting of glial cell reactivity, retention of brain‐infiltrating tissue‐resident memory CD8+ T‐cells, and long‐term persistence of antibody‐producing cells of the B‐lineage. Here, we report that this neuroinflammation occurs concomitantly with accumulation and retention of immunosuppressive regulatory T‐cells (Tregs), and is exacerbated following their ablation. However, the extent to which these Tregs function to control neuroimmune activation following MCMV encephalitis is unknown. In this study, we used Foxp3‐diphtheria toxin receptor‐GFP (Foxp3‐DTR‐GFP) transgenic mice, which upon administration of low‐dose diphtheria toxin (DTx) results in the specific depletion of Tregs, to investigate their function. We found treatment with DTx during the acute phase of viral brain infection (0–4 dpi) resulted in depletion of Tregs from the brain, exacerbation of encephalitis (i.e., increased presence of CD4+ and CD8+ T‐cells), and chronic reactive phenotypes of resident glial cells (i.e., elevated MHC Class II as well as PD‐L1 levels, sustained microgliosis, and increased glial fibrillary acidic protein (GFAP) expression on astrocytes) versus untreated, infected animals. This chronic proinflammatory environment was associated with reduced cognitive performance in spatial learning and memory tasks (Barnes Maze) by convalescent animals. These data demonstrate that chronic glial cell activation, unremitting post‐encephalitic neuroinflammation, and its associated long‐term neurological sequelae in response to viral brain infection are modulated by the immunoregulatory properties of Tregs. GLIA 2015;63:1982–1996

Pathogenic and immunogenic responses in turkeys following in ovo exposure to avian metapneumovirus subtype C.

Commercial turkey eggs, free of antibodies to avian metapneumovirus subtype C (aMPV/C), were inoculated with aMPV/C at embryonation day (ED) 24. There was no detectable effect of virus inoculation on the hatchability of eggs. At 4 days post inoculation (DPI) (the day of hatch (ED 28)) and 9 DPI (5 days after hatch), virus replication was detected by quantitative RT-PCR in the turbinate, trachea and lung but not in the thymus or spleen. Mild histological lesions characterized by lymphoid cell infiltration were evident in the turbinate mucosa. Virus exposure inhibited the mitogenic response of splenocytes and thymocytes and upregulated gene expression of IFN-γ and IL-10 in the turbinate tissue. Turkeys hatching from virus-exposed eggs had aMPV/C-specific IgG in the serum and the lachrymal fluid. At 3 week of age, in ovo immunized turkeys were protected against a challenge with pathogenic aMPV/C.