Project description:The chemokine receptor CCR5 is a major co-receptor for human immunodeficiency virus 1 (HIV-1) mediating infection of target cells1,2. Beyond its function as co-receptor, CCR5 also influences the course of HIV disease and progression to AIDS3. However, it is unclear how CCR5 affects HIV-associated damage to the central nervous system (CNS) and development of HIV-associated neurocognitive disorders (HAND) independently of its co-receptor function. Here we show in a transgenic model of brain damage induced by HIV envelope protein gp1204 that genetic ablation of CCR5 prevents neuronal injury and loss and limits microglial activation, but fails to abrogate astrocytosis. CCR5 deficiency also protected gp120-transgenic mice against impairment of spatial learning and memory. Thus, CCR5-deficiency revealed that astrocytosis, a prominent pathological feature of AIDS brains5, can occur independently from neuronal demise and behavioral impairment. Since the viral envelope protein expressed in the transgenic mouse model originated from HIV-1 LAV4, a CXCR4-preferring virus that can infect macrophages6, CCR5 appeared to exert its control of neuronal injury predominantly in an indirect fashion and independently from its function as a co-receptor. Using analysis of genome-wide CNS gene expression we identified a subset of 734 genes that were differentially regulated in association with neuronal injury in the presence of CCR5. This subset of genes indicated that neuronal injury was associated with activation of macrophages and microglia. A set of 1305 genes was only differentially regulated in association with gp120 in the absence of CCR5 and indicated changes to leukocyte function and the anti-viral immune response besides a down-regulation of components in the GABAergic neurotransmission system. Interestingly, the most significantly differentially expressed genes were observed in a separate subset of 461 genes that was regulated in association with gp120 but independently of the CCR5 genotype. The finding that differential regulation of the 461 and 1305 gene sets was not necessarily linked to neuronal damage and loss suggested that altered expression of at least some of these factors may represent a protective adaptive response of the brain to the presence of viral gp120. Additional experiments using quantitative RT-PCR, protein assays and flow cytometry further supported the notion that CCR5 deficiency blunted microglial activation, a hallmark of HIV-associated brain injury7-9 without significantly affecting the expression of viral gp120. These results provide in vivo evidence for a significant role of CCR5 in HIV-associated CNS injury and behavioral impairment that is independent of the molecules’ function as HIV co-receptor. To characterize the neuroprotective effect of CCR5 ablation in the presence of HIV gp120, we next performed a genome-wide gene expression analysis using whole brain RNA preparations of mice from lines 1 and 2. We decided to collect samples from two time points before and after the ages of when we analyzed histopathology (6 months) and behavior (8 to 9 months) in order to identify genes for which differential regulation may be affected by an interaction of HIVgp120 expression and age. Accordingly, we collected brain tissue of gp120tg, CCR5KO x gp120tg, WT and CCR5KO mice at five different ages: 1.5, 3, 6, 12, and 20 (line 1) or 16 (line 2) months.

Project description:The chemokine receptor CCR5 is a major co-receptor for human immunodeficiency virus 1 (HIV-1) mediating infection of target cells1,2. Beyond its function as co-receptor, CCR5 also influences the course of HIV disease and progression to AIDS3. However, it is unclear how CCR5 affects HIV-associated damage to the central nervous system (CNS) and development of HIV-associated neurocognitive disorders (HAND) independently of its co-receptor function. Here we show in a transgenic model of brain damage induced by HIV envelope protein gp1204 that genetic ablation of CCR5 prevents neuronal injury and loss and limits microglial activation, but fails to abrogate astrocytosis. CCR5 deficiency also protected gp120-transgenic mice against impairment of spatial learning and memory. Thus, CCR5-deficiency revealed that astrocytosis, a prominent pathological feature of AIDS brains5, can occur independently from neuronal demise and behavioral impairment. Since the viral envelope protein expressed in the transgenic mouse model originated from HIV-1 LAV4, a CXCR4-preferring virus that can infect macrophages6, CCR5 appeared to exert its control of neuronal injury predominantly in an indirect fashion and independently from its function as a co-receptor. Using analysis of genome-wide CNS gene expression we identified a subset of 734 genes that were differentially regulated in association with neuronal injury in the presence of CCR5. This subset of genes indicated that neuronal injury was associated with activation of macrophages and microglia. A set of 1305 genes was only differentially regulated in association with gp120 in the absence of CCR5 and indicated changes to leukocyte function and the anti-viral immune response besides a down-regulation of components in the GABAergic neurotransmission system. Interestingly, the most significantly differentially expressed genes were observed in a separate subset of 461 genes that was regulated in association with gp120 but independently of the CCR5 genotype. The finding that differential regulation of the 461 and 1305 gene sets was not necessarily linked to neuronal damage and loss suggested that altered expression of at least some of these factors may represent a protective adaptive response of the brain to the presence of viral gp120. Additional experiments using quantitative RT-PCR, protein assays and flow cytometry further supported the notion that CCR5 deficiency blunted microglial activation, a hallmark of HIV-associated brain injury7-9 without significantly affecting the expression of viral gp120. These results provide in vivo evidence for a significant role of CCR5 in HIV-associated CNS injury and behavioral impairment that is independent of the molecules’ function as HIV co-receptor.

Project description:During normal neurodevelopment, microglia perform a range of different functions that are crucial for the proper development, circuit integration, and function of neurons. These functions include certain types of synaptic pruning during critical stages of neurodevelopment (Schafer et al., 2012), and the production of cytokines and chemokines during normal physiological tasks, proteins which are important for homeostasis in addition to immune defense (Avital et al., 2003; Goshen et al., 2007). Microglia and the inflammatory cytokines they produce (e.g., in response to injury or infection) are also increasingly implicated in neural dysfunction during development and the risk of neuropsychiatric disorders. Increasing evidence suggests many neurological disorders emerge because of disrupted neuronal developmental trajectory (Marin, 2016). Considering the critical role of microglia for integration of neurons into their proper circuitry as well in proper synapse formation, there is a pressing need to understand the mechanisms underlying normal and abnormal microglia development, their interactions with specific neuronal subsets, and to understand the utility of these same analyses for human tissue. Moreover, as there are pronounced sex differences in the presentation of many neurological disorders, it is critical to understand these mechanisms in both males and females. Here we study developmental gene expression changes in male and female mice from E18 to P60. We isolated microglia from male and female hippocampi of mice from different developmental ages ranging from embryonic day 18 (E18) to postnatal day 60 (P60). Hippocampus was selected based on its importance to mental health, its marked vulnerability relative to other brain regions in response to diverse threats to homeostasis (epilepsy, stroke, and cardiac arrest (Fujioka et al., 2000; Petito, Feldmann, Pulsinelli, & Plum, 1987; Salmenpera, Kalviainen, Partanen, & Pitkanen, 1998; Sapolsky, Uno, Rebert, & Finch, 1990)), and due to evidence of morphological sex differences in microglial development in this region (Schwarz, Sholar, & Bilbo, 2012). It was found that developmental gene expression changes were more advanced in females at P60 and that treatment with LPS, a potent immune activator, could normalize developmental gene expression differences in males.

Project description:Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system, representing the leading cause of non-traumatic neurologic disease in young adults. This disease is three times more common in women, yet more severe in men, but the mechanisms underlying these sex differences remain largely unknown. MS is initiated by autoreactive T helper cells, but CNS-resident and CNS-infiltrating myeloid cells are the key proximal effector cells regulating disease pathology. We have previously shown that genetic ablation of p38α MAP kinase broadly in the myeloid lineage is protective in the autoimmune model of MS, experimental autoimmune encephalomyelitis (EAE), but only in females, and not males. To precisely define the mechanisms responsible, we used multiple genetic approaches and bone marrow chimeras to ablate p38α in microglial cells, peripheral myeloid cells, or both. Deletion of p38α in both cell types recapitulated the previous sex difference, with reduced EAE severity in females. Unexpectedly, deletion of p38α in the periphery was protective in both sexes. In contrast, deletion of p38α in microglia exacerbated EAE in males only, revealing opposing roles of p38α in microglia vs. periphery. Bulk transcriptional profiling revealed that p38α regulated the expression of distinct gene modules in male vs. female microglia. Single-cell transcriptional analysis of WT and p38α-deficient microglia isolated from the inflamed CNS revealed a diversity of complex microglial states, connected by distinct convergent transcriptional trajectories. Microglial p38α deficiency in males resulted in enhanced transition from homeostatic to disease-associated microglial states, with the downregulation of regulatory genes such as Atf3, Rgs1, Socs3, and Btg2, and increased expression of inflammatory genes such as Cd74, Trem2, and MHC class I and II genes. The effect of p38α deficiency was divergent in female microglia, with an upregulation of a small subset of inflammatory genes that overlapped with males, but mostly a unique transcriptional profile that also included an upregulation of potentially tissue reparative genes. Taken together, these results reveal the presence of a p38α-dependent sex-specific molecular pathway in microglia that is protective in CNS autoimmunity in males, suggesting that autoimmunity in males and females is driven by distinct cellular and molecular pathways, thus informing the design of novel future sex-specific therapeutic approaches.

Project description:Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system, representing the leading cause of non-traumatic neurologic disease in young adults. This disease is three times more common in women, yet more severe in men, but the mechanisms underlying these sex differences remain largely unknown. MS is initiated by autoreactive T helper cells, but CNS-resident and CNS-infiltrating myeloid cells are the key proximal effector cells regulating disease pathology. We have previously shown that genetic ablation of p38α MAP kinase broadly in the myeloid lineage is protective in the autoimmune model of MS, experimental autoimmune encephalomyelitis (EAE), but only in females, and not males. To precisely define the mechanisms responsible, we used multiple genetic approaches and bone marrow chimeras to ablate p38α in microglial cells, peripheral myeloid cells, or both. Deletion of p38α in both cell types recapitulated the previous sex difference, with reduced EAE severity in females. Unexpectedly, deletion of p38α in the periphery was protective in both sexes. In contrast, deletion of p38α in microglia exacerbated EAE in males only, revealing opposing roles of p38α in microglia vs. periphery. Bulk transcriptional profiling revealed that p38α regulated the expression of distinct gene modules in male vs. female microglia. Single-cell transcriptional analysis of WT and p38α-deficient microglia isolated from the inflamed CNS revealed a diversity of complex microglial states, connected by distinct convergent transcriptional trajectories. Microglial p38α deficiency in males resulted in enhanced transition from homeostatic to disease-associated microglial states, with the downregulation of regulatory genes such as Atf3, Rgs1, Socs3, and Btg2, and increased expression of inflammatory genes such as Cd74, Trem2, and MHC class I and II genes. Taken together, these results reveal the presence of a p38α-dependent male-specific molecular pathway in microglia that is protective in CNS autoimmunity, suggesting that autoimmunity in males and females is driven by distinct cellular and molecular pathways, thus informing the design of novel future sex-specific therapeutic approaches.

Dataset Information

RNA-seq analysis of HIV-1 gp120 stimulated, p38α knockout and wild-type microglia isolated from mouse brain.

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