Shuxian Hu

Regulatory T-cells (Tregs) play pivotal roles during infection, cancer, and autoimmunity. In our previous study, we demonstrated a role for the PD-1:PD-L1 pathway in controlling cytolytic responses of CD8⁺ T lymphocytes against microglial cells presenting viral peptides. In this study, we investigated the role of Tregs in suppressing CD8⁺ T-cell-mediated cytotoxicity against primary microglial cells. Using in vitro cytotoxicity assays and flow cytometry, we demonstrated a role for Tregs in suppressing antigen-specific cytotoxic T-lymphocyte (CTL) responses against microglia loaded with a model peptide (SIINFEKL). We went on to show a significant decrease in the frequency of IFN-γ- and TNF-producing CD8⁺ T-cells when cultured with Tregs. Interestingly, a significant increase in the frequency of granzyme B- and Ki67-producing CTLs was observed. We also observed a significant decrease in the production of interleukin (IL)-6 by microglia. On further investigation, we found that Tregs significantly reduced MHC class 1 (MHC-1) expression on IFN-γ-treated microglial cells. Taken together, these studies demonstrate an immunosuppressive role for Tregs on CTL responses generated against primary microglia. Hence, modulation of Treg cell activity in combination with negative immune checkpoint blockade may stimulate anti-viral T-cell responses to more efficiently clear viral infection from microglial cell reservoirs.

The role of select pro- and anti-inflammatory mediators in driving microglial cell polarization into classically (M1), or alternatively, (M2) activated states, as well as the subsequent differential responses of these induced phenotypes, was examined. Expression of PD-L1, MHC-II, MHC-I, arginase 1 (Arg-1), and inducible nitric oxide synthase (iNOS) was assessed using multi-color flow cytometry. We observed that both pro- and anti-inflammatory mediators induced PD-L1 expression on non-polarized microglia. Moreover, IFN-γ stimulated significant MHC class I and II expression on these cells. Interestingly, we observed that only IL-4 treatment induced Arg-1 expression, indicating M2 polarization. These M2 cells were refractory to subsequent depolarization and maintained their alternatively activated state. Furthermore, PD-L1 expression was significantly induced on these M2-polarized microglia after treatment with pro-inflammatory mediators, but not anti-inflammatory cytokines. In addition, we observed that only LPS induced iNOS expression in microglial cells, indicating M1 polarization. Furthermore, IFN-γ significantly increased the percentage of M1-polarized microglia expressing iNOS. Surprisingly, when these M1-polarized microglia were treated with either IL-6 or other anti-inflammatory cytokines, they returned to their non-polarized state, as demonstrated by significantly reduced expression of iNOS. Taken together, these results demonstrate differential responses of microglial cells to mediators present in dissimilar microenvironments.

Peripheral neuropathy is currently the most common neurological complication in HIV-infected individuals, occurring in 35–50% of patients undergoing combination anti-retroviral therapy. Data have shown that distal symmetric polyneuropathy develops in mice by 6 weeks following infection with the LP-BM5 retrovirus mixture. Previous work from our laboratory has demonstrated that glial cells modulate antiviral T-cell effector responses through the programmed death (PD)-1: PD-L1 pathway, thereby limiting the deleterious consequences of unrestrained neuroinflammation. Using the MouseMet electronic von Frey system, we assessed hind-paw mechanical hypersensitivity in LP-BM5-infected wild-type (WT) and PD-1 KO animals. Using multi-color flow cytometry, we quantitatively assessed cellular infiltration and microglial activation. Using real-time RT-PCR, we assessed viral load, expression of IFN-γ, iNOS, and MHC class II. Using western blotting, we measured protein nitrosylation within the lumbar spinal cord (LSC) and dorsal root ganglion (DRG). Histochemical staining was performed to analyze the presence of CD3, ionized calcium binding adaptor molecule (Iba)-1, MHCII, nitrotyrosine, isolectin B4 (IB4) binding, and neurofilament 200 (NF200). Statistical analyses were carried out using graphpad prism. Hind-paw mechanical hypersensitivity observed in LP-BM5-infected animals was associated with significantly increased lymphocyte infiltration into the spinal cord and DRG. We also observed elevated expression of IFN-γ (in LSC and DRG) and MHC II (on resident microglia in LSC). We detected elevated levels of 3-nitrotyrosine within the LSC and DRG of LP-BM5-infected animals, an indicator of nitric oxide (NO)-induced protein damage. Moreover, we observed 3-nitrotyrosine in both small (IB4+) and large (NF200+) DRG sensory neurons. Additionally, infected PD-1 KO animals displayed significantly greater mechanical hypersensitivity than WT or uninfected mice at 4 weeks post-infection (p.i.). Accelerated onset of hind-paw hypersensitivity in PD-1 KO animals was associated with significantly increased infiltration of CD4+ and CD8+ T lymphocytes, macrophages, and microglial activation at early time points. Importantly, we also observed elevated levels of 3-nitrotyrosine and iNOS in infected PD-1 KO animals when compared with WT animals. Results reported here connect peripheral immune cell infiltration and reactive gliosis with nitrosative damage. These data may help elucidate how retroviral infection-induced neuroinflammatory networks contribute to nerve damage and neuropathic pain.

Previous work from our laboratory has demonstrated that during acute viral brain infection, glial cells modulate antiviral T cell effector responses through the PD-1: PD-L1 pathway, thereby limiting the deleterious consequences of unrestrained neuroinflammation. Here, we evaluated the PD-1: PD-L1 pathway in development of brain-resident memory T cells (bTRM) following murine cytomegalovirus (MCMV) infection. Flow cytometric analysis of immune cells was performed at 7, 14, and 30 days post-infection (dpi) to assess the shift of brain-infiltrating CD8+ T cell populations from short-lived effector cells (SLEC) to memory precursor effector cells (MPEC), as well as generation of bTRMs. In wild-type (WT) animals, we observed a switch in the phenotype of brain-infiltrating CD8+ T cell populations from KLRG1+ CD127− (SLEC) to KLRG1− CD127+ (MPEC) during transition from acute through chronic phases of infection. At 14 and 30 dpi, the majority of CD8+ T cells expressed CD127, a marker of memory cells. In contrast, fewer CD8+ T cells expressed CD127 within brains of infected, PD-L1 knockout (KO) animals. Notably, in WT mice, a large population of CD8+ T cells was phenotyped as CD103+ CD69+, markers of bTRM, and differences were observed in the numbers of these cells when compared to PD-L1 KOs. Immunohistochemical studies revealed that brain-resident CD103+ bTRM cells were localized to the parenchyma. Higher frequencies of CXCR3 were also observed among WT animals in contrast to PD-L1 KOs. Taken together, our results indicate that bTRMs are present within the CNS following viral infection and the PD-1: PD-L1 pathway plays a role in the generation of this brain-resident population.

Highly 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.

Background: Production of reactive oxygen species (ROS) and proinflammatory cytokines by microglial cells in response to viral brain infection contributes to both pathogen clearance and neuronal damage. In the present study, we examined the effect of herpes simplex virus (HSV)-1-induced, NADPH oxidase-derived ROS in activating mitogen-activated protein kinases (MAPKs) as well as driving cytokine and chemokine expression in primary murine microglia. Methods: Oxidation of 2', 7'-dichlorodihydrofluorescin diacetate (H₂DCFDA) was used to measure production of intracellular ROS in microglial cell cultures following viral infection. Virus-induced cytokine and chemokine mRNA and protein levels were assessed using real-time RT-PCR and ELISA, respectively. Virus-induced phosphorylation of microglial p38 and p44/42 (ERK1/2) MAPKs was visualized using Western Blot, and levels of phospho-p38 were quantified using Fast Activated Cell-based ELISA (FACE assay). Diphenyleneiodonium (DPI) and apocynin (APO), inhibitors of NADPH oxidases, were used to investigate the role of virus-induced ROS in MAPK activation and cytokine, as well as chemokine, production. Results: Levels of intracellular ROS were found to be highly elevated in primary murine microglial cells following infection with HSV and the majority of this virus-induced ROS was blocked following DPI and APO treatment. Correspondingly, inhibition of NADPH oxidase also decreased virus-induced proinflammatory cytokine and chemokine production. In addition, microglial p38 and p44/42 MAPKs were found to be phosphorylated in response to viral infection and this activation was also blocked by inhibitors of NADPH oxidase. Finally, inhibition of either of these ROS-induced signaling pathways suppressed cytokine (TNF-α and IL-1β) production, while chemokine (CCL2 and CXCL10) induction pathways were sensitive to inhibition of p38, but not ERK1/2 MAPK. Conclusions: Data presented herein demonstrate that HSV infection induces proinflammatory responses in microglia through NADPH oxidase-dependent ROS and the activation of MAPKs.

Heme oxygenase (HO)-1 has been shown to attenuate oxidative injury and reduce apoptosis. HO-1 can be induced by various stimuli released during cellular injury, such as heme. Deleterious free heme is degraded by HO-1 to carbon monoxide, iron and biliverdin, which have potent anti-oxidant and anti-inflammatory properties. In this study, we tested the hypothesis that upregulation of HO-1 would inhibit production of the free radical (NO) by interlukin (IL)-1β-activated human astrocytes.

Substances of abuse, such as opiates, have a variety of immunomodulatory properties that may influence both neuroinflammatory and neurodegenerative disease processes. The chemokine CCL2, which plays a pivotal role in the recruitment of inflammatory cells in the nervous system, is one of only a few chemokines produced by neurons. We hypothesized that morphine may alter expression of CCL2 by human neurons. Primary neuronal cell cultures and highly purified astrocyte and microglial cell cultures were prepared from human fetal brain tissue. Cell cultures were treated with morphine, and cells were examined by RNase protection assay for mRNA. Culture supernatants were assayed by ELISA for CCL2 protein. β-funaltrexamine (β-FNA) was used to block μ-opioid receptor (MOR)s. Morphine upregulated CCL2 mRNA and protein in neuronal cultures in a concentration- and time-dependent fashion, but had no effect on CCL2 production in astrocyte or microglial cell cultures. Immunocytochemical analysis also demonstrated CCL2 production in morphine-stimulated neuronal cultures. The stimulatory effect of morphine was abrogated by β-FNA, indicating an MOR-mediated mechanism. Morphine stimulates CCL2 production by human neurons via a MOR-related mechanism. This finding suggests another mechanism whereby opiates could affect neuroinflammatory responses.