Cutting Edge: IFN-? Produced by Brain-Resident Cells Is Crucial To Control Cerebral Infection with Toxoplasma gondii.
ABSTRACT: In vitro studies demonstrated that microglia and astrocytes produce IFN-? in response to various stimulations, including LPS. However, the physiological role of IFN-? production by brain-resident cells, including glial cells, in resistance against cerebral infections remains unknown. We analyzed the role of IFN-? production by brain-resident cells in resistance to reactivation of cerebral infection with Toxoplasma gondii using a murine model. Our study using bone marrow chimeric mice revealed that IFN-? production by brain-resident cells is essential for upregulating IFN-?-mediated protective innate immune responses to restrict cerebral T. gondii growth. Studies using a transgenic strain that expresses IFN-? only in CD11b(+) cells suggested that IFN-? production by microglia, which is the only CD11b(+) cell population among brain-resident cells, is able to suppress the parasite growth. Furthermore, IFN-? produced by brain-resident cells is pivotal for recruiting T cells into the brain to control the infection. These results indicate that IFN-? produced by brain-resident cells is crucial for facilitating both the protective innate and T cell-mediated immune responses to control cerebral infection with T. gondii.
Project description:Toxoplasma gondii induces a potent IL-12 response early in infection that results in IFN-?-dependent control of parasite growth. It was previously shown that T. gondii soluble tachyzoite antigen (STAg) injected 48 hr before intraperitoneal infection reduces lipoxin A4 and 5-lipoxygenase (5-LO)-dependent systemic IL-12 and IFN-? production as well as hepatic immunopathology. This study investigated the ability of STAg-pretreatment to control the fatal intestinal pathology that develops in C57BL/6 mice orally infected with 100 T. gondii cysts. STAg-pretreatment prolonged the animals' survival by decreasing tissue parasitism and pathology, mainly in the ilea. Protection was associated with decreases in the systemic IFN-? levels and IFN-? and TNF message levels in the ilea and with increased TGF-? production in this tissue, but protection was independent of 5-LO and IL-4. STAg-pretreatment decreased CD4(+) T cell, NK cell, CD11b(+) monocyte and CD11b(+)CD11c(+) dendritic cell numbers in the lamina propria and increased CD8(+) T cells in the intestinal epithelial compartment. In parallel, decreases were observed in iNOS and IL-17 expression in this organ. These results demonstrate that pretreatment with STAg can induce the recruitment of protective CD8(+) T cells to the intraepithelial compartment and decrease proinflammatory immune mechanisms that promote intestinal pathology in T. gondii infection.
Project description:Experimental murine herpes simplex virus (HSV)-1 brain infection stimulates microglial cell-driven proinflammatory chemokine production which precedes the presence of brain-infiltrating systemic immune cells. In the present study, we investigated the phenotypes and infiltration kinetics of leukocyte trafficking into HSV-infected murine brains. Using real-time bioluminescence imaging, the infiltration of luciferase-positive splenocytes, transferred via tail vein injection into the brains of HSV-infected animals, was followed over an 18-day time course. Flow cytometric analysis of brain-infiltrating leukocytes at 5, 8, 14, and 30 days postinfection (d.p.i.), was performed to assess their phenotype. A predominantly macrophage (CD45(high)CD11b(+)Ly6C(high)) and neutrophil (CD45(high)CD11b(+)Ly6G(+)) infiltration was seen early during infection, with elevated levels of TNF-alpha mRNA expression. By 14 d.p.i., the phenotypic profile shifted to a predominantly lymphocytic (CD45(high)CD3(+)) infiltrate. This lymphocyte infiltrate was detected until 30 d.p.i., when infectious virus could not be recovered, with CD8(+) and CD4(+) T cells present at a 3:1 ratio, respectively. This T lymphocyte infiltration paralleled increased IFN-gamma mRNA expression in the brain. Activation of resident microglia (CD45(int)CD11b(+)) was also detected until 30 d.p.i., as assessed by MHC class II expression. Activated microglial cells were further identified as the predominant source of IL-1beta. In addition, infected mice given primed immunocytes at 4 d.p.i. showed a significant increase in mortality. Taken together, these results demonstrate that intranasal infection results in early macrophage and neutrophil infiltration into the brain followed by prolonged microglial activation and T lymphocyte retention. Similar prolonged neuroimmune activation may contribute to the neuropathological sequelae observed in herpes encephalitis patients.
Project description:<h4>Background</h4>It has become increasingly evident that the immune and nervous systems are closely intertwined, relying on one another during regular homeostatic conditions. Prolonged states of imbalance between neural and immune homeostasis, such as chronic neuroinflammation, are associated with a higher risk for neural damage. Toxoplasma gondii is a highly successful neurotropic parasite causing persistent subclinical neuroinflammation, which is associated with psychiatric and neurodegenerative disorders. Little is known, however, by what means neuroinflammation and the associated neural impairment can be modulated by peripheral inflammatory processes.<h4>Methods</h4>Expression of immune and synapse-associated genes was assessed via quantitative real-time PCR to investigate how T. gondii infection-induced chronic neuroinflammation and associated neuronal alterations can be reshaped by a subsequent acute intestinal nematode co-infection. Immune cell subsets were characterized via flow cytometry in the brain of infected mice. Sulfadiazine and interferon-?-neutralizing antibody were applied to subdue neuroinflammation.<h4>Results</h4>Neuroinflammation induced by T. gondii infection of mice was associated with increased microglia activation, recruitment of immune cells into the brain exhibiting Th1 effector functions, and enhanced production of Th1 and pro-inflammatory molecules (IFN-?, iNOS, IL-12, TNF, IL-6, and IL-1?) following co-infection with Heligmosomoides polygyrus. The accelerated cerebral Th1 immune response resulted in enhanced T. gondii removal but exacerbated the inflammation-related decrease of synapse-associated gene expression. Synaptic proteins EAAT2 and GABA<sub>A</sub>?1, which are involved in the excitation/inhibition balance in the CNS, were affected in particular. These synaptic alterations were partially recovered by reducing neuroinflammation indirectly via antiparasitic treatment and especially by application of IFN-?-neutralizing antibody. Impaired iNOS expression following IFN-? neutralization directly affected EAAT2 and GABA<sub>A</sub>?1 signaling, thus contributing to the microglial regulation of neurons. Besides, reduced CD36, TREM2, and C1qa gene expression points toward inflammation induced synaptic pruning as a fundamental mechanism.<h4>Conclusion</h4>Our results suggest that neuroimmune responses following chronic T. gondii infection can be modulated by acute enteric nematode co-infection. While consecutive co-infection promotes parasite elimination in the CNS, it also adversely affects gene expression of synaptic proteins, via an IFN-?-dependent manner.
Project description:Neurotropic recombinant strain of Mouse Hepatitis Virus, RSA59, induces meningo-encephalitis, myelitis and demyelination following intracranial inoculation. RSA59 induced neuropathology is partially caused by activation of CNS resident microglia, as demonstrated by changes in cellular morphology and increased expression of a microglia/macrophage specific calcium ion binding factor, Iba1. Affymetrix Microarray analysis for mRNA expression data reveals expression of inflammatory mediators that are known to be released by activated microglia. Microglia-specific cell surface molecules, including CD11b, CD74, CD52 and CD68, are significantly upregulated in contrast to CD4, CD8 and CD19. Protein analysis of spinal cord extracts taken from mice 6 days post-inoculation, the time of peak inflammation, reveals robust expression of IFN-?, IL-12 and mKC. Data suggest that activated microglia and inflammatory mediators contribute to a local CNS microenvironment that regulates viral replication and IFN-? production during the acute phase of infection, which in turn can cause phagolysosome maturation and phagocytosis of the myelin sheath, leading to demyelination.
Project description:Murine cytomegalovirus (MCMV) brain infection stimulates microglial cell-driven proinflammatory chemokine production which precedes the presence of brain-infiltrating systemic immune cells. Here, we show that in response to MCMV brain infection, antigen-specific CD8(+) T cells migrated into the brain and persisted as long-lived memory cells. The role of these persistent T cells in the brain is unclear because most of our understanding of antimicrobial T cell responses comes from analyses of lymphoid tissue. Strikingly, memory T cells isolated from the brain exhibited an effector phenotype and produced IFN-? upon restimulation with viral peptide. Furthermore, we observed time-dependent and long-term activation of resident microglia, indicated by chronic MHC class II up-regulation and TNF-? production. The immune response in this immunologically restricted site persisted in the absence of active viral replication. Lymphocyte infiltrates were detected until 30 days post-infection (p.i.), with CD8(+) and CD4(+) T cells present at a 3:1 ratio, respectively. We then investigated the role of IFN-? in chronic microglial activation by using IFN-?-knockout (GKO) mice. At 30 days p.i., GKO mice demonstrated a similar phenotypic brain infiltrate when compared to wild-type mice (Wt), however, MHC class II expression on microglia isolated from these GKO mice was significantly lower compared to Wt animals. When IFN-? producing CD8(+) T cells were reconstituted in GKO mice, MHC class II up-regulation on microglial cells was restored. Taken together, these results suggest that MCMV brain infection results in long-term persistence of antigen-specific CD8(+) T cells which produce IFN-? and drive chronic microglial cell activation. This response was found to be dependent on IFN-? production by viral Ag-specific T cells during the chronic phase of disease.
Project description:<b>Background:</b>The ability to distinguish resident microglia from infiltrating myeloid cells by flow cytometry-based surface phenotyping is an important technique for examining age-related neuroinflammation. The most commonly used surface markers for the identification of microglia include CD45 (low-intermediate expression), CD11b, Tmem119, and P2RY12.<br><br><b>Methods:</b>In this study, we examined changes in expression levels of these putative microglia markers in in vivo animal models of stroke, cerebral amyloid angiopathy (CAA), and aging as well as in an ex vivo LPS-induced inflammation model.<br><br><b>Results:</b>We demonstrate that Tmem119 and P2RY12 expression is evident within both CD45<sup>int</sup> and CD45<sup>high</sup> myeloid populations in models of stroke, CAA, and aging. Interestingly, LPS stimulation of FACS-sorted adult microglia suggested that these brain-resident myeloid cells can upregulate CD45 and downregulate Tmem119 and P2RY12, making them indistinguishable from peripherally derived myeloid populations. Importantly, our findings show that these changes in the molecular signatures of microglia can occur without a contribution from the other brain-resident or peripherally sourced immune cells.<br><br><b>Conclusion:</b>We recommend future studies approach microglia identification by flow cytometry with caution, particularly in the absence of the use of a combination of markers validated for the specific neuroinflammation model of interest. The subpopulation of resident microglia residing within the "infiltrating myeloid" population, albeit small, may be functionally important in maintaining immune vigilance in the brain thus should not be overlooked in neuroimmunological studies.
Project description:Fetal placental macrophages and microglia (resident brain macrophages) have a common origin in the fetal yolk sac. Yolk-sac-derived macrophages comprise the permanent pool of brain microglia throughout an individual's lifetime. Inappropriate fetal microglial priming may therefore have lifelong neurodevelopmental consequences, but direct evaluation of microglial function in a living fetus or neonate is impossible. We sought to test the hypothesis that maternal obesity would prime both placental macrophages and fetal brain microglia to overrespond to an immune challenge, thus providing a window into microglial function using placental cells. Obesity was induced in C57BL/6?J mice using a 60% high-fat diet. On embryonic day 17.5, fetal brain microglia and corresponding CD11b?+?placental cells were isolated from fresh tissue. Cells were treated with media or lipopolysaccharide (LPS). Tumor necrosis factor-alpha (TNF-?) production by stimulated and unstimulated cells was quantified via ELISA. We demonstrate for the first time that the proinflammatory cytokine production of CD11b?+?placental cells is strongly correlated with that of brain microglia (Spearman's ??=?0.73, p?=?0.002) in the setting of maternal obesity. Maternal obesity-exposed CD11b?+?cells had an exaggerated response to LPS compared to controls, with a 5.1-fold increase in TNF-? production in placentas (p?=?0.003) and 3.8-fold increase in TNF-? production in brains (p?=?0.002). In sex-stratified analyses, only male obesity-exposed brains and placentas had significant increase in TNF-? production in response to LPS. Taken together, these data suggest that maternal obesity primes both placental macrophages and fetal brain microglia to overproduce a proinflammatory cytokine in response to immune challenge. Male brain and placental immune response is more marked than female in this setting. Given that fetal microglial priming may impact neuroimmune function throughout the lifespan, these data could provide insight into the male predominance of certain neurodevelopmental morbidities linked to maternal obesity, including cognitive dysfunction, autism spectrum disorder, and ADHD. Placental CD11b+ macrophages may have the potential to serve as an accessible biomarker of aberrant fetal brain immune activation in maternal obesity. This finding may have broader implications for assaying the impact of other maternal exposures on fetal brain development.
Project description:Inflammation is implicated in ischemic stroke and is involved in abnormal homeostasis. Activation of the immune system leads to breakdown of the blood-brain barrier and, thereby, infiltration of immune cells into the brain. Upon cerebral ischemia, infiltrated macrophages and microglia (resident CNS immune cell) are activated, change their phenotype to M1 or M2 based on the microenvironment, migrate toward damaged tissue, and are involved in repair or damage. Those of M1 phenotype release pro-inflammatory mediators, which are associated with tissue damage, while those of M2 phenotype release anti-inflammatory mediators, which are related to tissue recovery. Moreover, late inflammation continually stimulates immune cell infiltration and leads to brain infarction. Therefore, regulation of M1/M2 phenotypes under persistent inflammatory conditions after cerebral ischemia is important for brain repair. Herein, we focus on apoptosis signal-regulating kinase 1 (ASK1), which is involved in apoptotic cell death, brain infarction, and production of inflammatory mediators after cerebral ischemia. We hypothesized that ASK1 is involved in the polarization of M1/M2 phenotype and the function of microglia and macrophage during the late stage of ischemia/hypoxia. We investigated the effects of ASK1 in mice subjected to middle cerebral artery occlusion and on BV2 microglia and RAW264.7 macrophage cell lines subjected to oxygen-glucose deprivation. Our results showed that ASK1 silencing effectively reduced Iba-1 or CD11b-positive cells in ischemic areas, suppressed pro-inflammatory cytokines, and increased anti-inflammatory mediator levels at 7 days after cerebral ischemia. In cultured microglia and macrophages, ASK1 inhibition, induced by NQDI-1 drug, decreased the expression and release of M1-associated factors and increased those of M2-associated factors after hypoxia/reperfusion (H/R). At the gene level, ASK1 inhibition suppressed M1-associated genes and augmented M2-associated genes. In gap closure assay, ASK1 inhibition reduced the migration rate of microglia and macrophages after H/R. Taken together, our results provide new information that suggests ASK1 controls the polarization of M1/M2 and the function of microglia and macrophage under sustained-inflammatory conditions. Regulation of persistent inflammation via M1/M2 polarization by ASK1 is a novel strategy for repair after ischemic stroke.
Project description:Microglia, as well as other tissue-resident macrophages, arise from yolk sac progenitors. Thus, it is likely that the central nervous system environment is critical for the acquisition of a distinct microglial phenotype. Several microRNAs that are enriched in the brain play crucial roles in brain development and may also play a role in the differentiation of microglia.To track the differentiation of hematopoietic cells into microglia, lineage-negative bone marrow cells were co-cultured with astrocytes in the absence or presence of microRNAs or their inhibitors. Microglia-like cells were identified as small, round cells that were immunopositive for CD11b, Iba1, CX3CR1, and triggering receptor expressed on myeloid cells (TREM)-2.Five microRNAs (miR-101a, miR-139-3p, miR-214*, miR-218, and miR-1186) were identified as modifiers of the differentiation of bone marrow-derived microglia-like cells. Among them, miR-101a facilitated the differentiation of bone marrow cells into microglia-like cells most potently. Small, round cells expressing CD11b, Iba1, CX3CR1, and TREM-2 were predominant in cells treated by miR-101a. miR-101a was abundantly expressed in non-microglial brain cells. Transfection of miR-101a into microglia significantly increased the production of IL-6 in response to LPS. Finally, miR-101a downregulated the expression of MAPK phosphatase-1.miR-101a, which is enriched in the brain, promotes the differentiation of bone marrow cells into microglia-like cells.
Project description: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-?-treatment. Blockade of the PD-1: PD-L1 pathway in microglial: CD8(+) T-cell co-cultures increased T-cell IFN-? 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-? 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.