Browsing by Subject "Microglia"

Synapse formation and elimination are two developmental processes that concurrently take place in the neonatal brain. Dysregulation of these two processes have been implicated in the etiology and progression of neurodevelopmental and neurodegenerative diseases. Previous work has found that in mice, the first three postnatal weeks are highly active periods of synapse remodeling throughout the entire brain. Glial cells called astrocytes are highly complex neural derived cells that are born and mature during this period. As they mature, astrocytes instruct the formation of synapses through contact with synaptic components and through the secretion of various synaptogenic factors. Microglia by contrast are the tissue resident macrophages of the central nervous system (CNS). During the first three postnatal weeks, microglia sculpt developing synaptic circuits by engulfment of synaptic components through various phagocytic mechanisms. While the field has steadily grown our understanding of the importance of these two cell types in synapse formation and elimination separately, few studies have addressed the possibility of communication between these two cell types to regulate their respective functions at synapses. Here I used the developing visual thalamocortical circuit as a model system to investigate the molecular cross talk between astrocytes and microglia. To address the impact of this communication on synapse development and function, I focused on one factor called Hevin/Sparcl1 which has previously been shown to be necessary and sufficient for thalamocortical synapse formation and plasticity. Previous studies have shown that Hevin induces thalamocortical synapse formation during the second postnatal week in mouse visual cortex. Hevin orchestrates this process by bridging pre-and post-synaptic cell adhesion molecules, Nrxn1α and Nlgn1B. Curiously, I found that despite high levels of Hevin in the maturing primary visual cortex, thalamocortical synapse numbers decrease even during the time when Hevin expression is at its peak. This refinement process, I determined, was dependent on microglia. Using super resolution microscopy, I found that only a subset thalamocortical synapses have Hevin at their cleft and that loss of Hevin aberrantly enhances microglia phagocytic activity. These initial findings suggested that Hevin likely functioned to spare only specific synapses from microglia mediated elimination. To interrogate this possibility, I used an in vitro microglia culture system to assess the transcriptional responses of microglia to Hevin treatment. Surprisingly, this treatment led to robust transcriptional changes in microglia that were distinct from well described immunological stimulation. This screen implicated Toll-like receptors (TLR) 2 and 4 in this transcriptional response. Further studies using our in vitro culture system showed that proteolytic cleavage of Hevin was required to upregulate TLR2 expression in microglia and that its C-terminus alone was sufficient to upregulate TLR2. Moving in vivo, I found that TLR2 expression is strongly developmentally regulated and highly heterogeneously expressed by microglia in the mouse primary visual cortex. Using overexpression studies in vivo, I also found that microglia strongly upregulate TLR2 in response to Hevin or Hevin’s C-terminus and that these TLR2 high microglia have enhanced phagocytic activity both in normal development and after Hevin/Hevin C-terminal overexpression. These findings indicate that Hevin function is regulated by proteolytic cleavage and suggest that Hevin is a dual signal in synaptic development: both to stimulate synapse formation by neurons and enhance synapse elimination by microglia. I next sought to test the functional relevance of the microglia specific response to Hevin. To do this, I used co-immunoprecipitation studies to identify candidate receptors for Hevin on microglia. I found that Hevin and its C-terminus interacted with both TLR2 and TLR4 but seemed to have a stronger affinity for TLR4. Therefore, I used TLR4 KO mice to test if microglia could still be stimulated by Hevin in vivo. I found that TLR4 KO microglia were no longer responsive to Hevin overexpression and had reduced phagocytic capacity compared to WT microglia. Ultimately, I found that TLR4 KO mice had impaired thalamocortical synapse refinement and impaired circuit plasticity. Taken together, my results identify astrocyte-derived Hevin as a synaptogenic molecule that links thalamocortical synapse formation with synaptic refinement mediated by microglia.

Naturally-occurring cell death is a fundamental developmental mechanism for regulating cell numbers and sculpting developing organs. This is particularly true in the central nervous system, where large numbers of neurons and oligodendrocytes are eliminated via apoptosis during normal development. Given the profound impact of death upon these two major cell populations, it is surprising that developmental death of another major cell type – the astrocyte – has rarely been studied. It is presently unclear whether astrocytes are subject to significant amounts of developmental death, or how it occurs. Here we address these questions using mouse retinal astrocytes as our model system. We show that the total number of retinal astrocytes declines by over 3-fold during a death period spanning postnatal days 5-14. Surprisingly, these astrocytes do not die by apoptosis, the canonical mechanism underlying the vast majority of developmental cell death. Instead, we find that microglia kill and engulf astrocytes to mediate their developmental removal. Genetic ablation of microglia inhibits astrocyte death, leading to a larger astrocyte population size at the end of the death period. However, astrocyte death is not completely blocked in the absence of microglia, apparently due to the ability of astrocytes to engulf each other. Nevertheless, mice lacking microglia showed significant anatomical changes to the retinal astrocyte network, with functional consequences for the astrocyte-associated vasculature leading to retinal hemorrhage. These results establish a novel modality for naturally-occurring cell death, and demonstrate its importance for formation and integrity of the retinal gliovascular network.

The field of "perinatal programming" has increasingly implicated an adverse early-life environment in the etiology of many chronic health problems and mental disorders. The following dissertation research is based on the hypothesis that the programming of brain and behavior by an altered early-life environment is propagated by inflammatory mechanisms in the placenta and developing brain. Offspring outcomes of two different maternal environmental exposures--air pollution and a "Western diet" (both highly relevant for the modern world)--were assessed in a mouse model in order to identify mechanisms common to developmental programming more generally.

The first set of experiments characterized the long-term behavioral and metabolic consequences of prenatal air pollution exposure in adult offspring. The male offspring of diesel exhaust particle (DEP)-exposed dams were predisposed to obesity, insulin resistance, and increased anxiety following placement on a high-fat diet (HFD) in adulthood. Furthermore, DEP/HFD male offspring exhibited evidence of macrophage priming, both in microglia and peripheral macrophages. The next experiment examined whether prenatal air pollution exposure could also synergize with a simultaneous "second hit" (i.e., maternal stress) during gestation. The offspring of mothers exposed to both air pollution and stress during gestation were more anxious as adults, but only the male offspring of this group also exhibited impaired cognition, in conjunction with neuroinflammatory changes. A further experiment revealed that prenatal air pollution exposure altered microglial maturation in a TLR4- and sex-dependent manner, consistent with the previous results. However, we found limited evidence of a placental immune response to DEP, potentially due to analysis too late in gestation.

The second set of experiments characterized the enduring behavioral and metabolic consequences of maternal consumption of a "Western diet" (HFD in combination with BCAA supplementation) prior to and during gestation and lactation. The adult offspring of HFD-fed dams were more anxious in adulthood, despite being placed on a low-fat diet at weaning. Male HFD offspring were also hyperactive, whereas female HFD offspring exhibited more severe metabolic disturbances. Furthermore, there was evidence of microglial priming and peripheral macrophage priming in male HFD offspring, similar to the prenatal air pollution model. The next experiment also found evidence of altered microglial development due to maternal HFD, in conjunction with widespread, sex-specific immune gene regulation in the placenta in response to maternal diet. Moreover, maternal HFD decreased placental serotonin production, and also programmed long-term alterations in serotonergic function in the prefrontal cortex of adult HFD offspring. Taken together, these experiments define sexually dimorphic innate immune mechanisms in the placenta and developing brain that may underlie the long-term metabolic and behavioral consequences of maternal environmental exposures.

Huntington’s Disease (HD) is an inherited, progressive, fatal, and late-onset neurodegenerative disorder. Its neuropathogenesis is characterized by selective loss of medium spiny neurons (MSNs) in the striatum and cortical neurons in the cortex. It is caused by an expansion of CAG repeats in the exon-1 of IT-15 gene, which results in a mutated polyglutamine domain in the huntingtin protein (HTT) (Collaborative, 1993). The mutation that causes HD was identified almost thirty years ago, yet the ultimate cause of neuron death is still uncertain. This dissertation is based on the hypothesis that monocytes infiltrate the brain of late stage HD patients and drive neuropathogenesis. To test this hypothesis, I developed a novel hybrid brain-slice experimental model in which human monocytes (MO) isolated from HD and non-disease control patients (CTRL) are engrafted into living postnatal striatal brain tissues prepared from neonatal rats.

In the first set of experiments, the inflammatory response of stimulated HD and CTRL MO were tested. HD MO exhibited an increased inflammatory response demonstrated by increased production of cytokine IL-10. Furthermore, a late-stage HD patient exhibited an increased production of multiple cytokines, including IL-10.

The second set of experiments utilized a brain-tissue based model to measure the impact of engrafting HD MO on MSN health. In brain slices engraftment with HD MO, there was a decrease in the number of healthy MSNs. A further experiment revealed that stimulating MO prior to their engraftment differentially influenced the survival of MSNs depending on whether the MO came from HD or CTRL patients. Measurement of cytokine production from these unstimulated/stimulated engraftment experiments revealed a trend for increased cytokine production in samples taken from a moderate stage HD patient.

The third set of experiments assessed the ability of HD MO to influence the endogenous macrophages within the brain slice tissue, microglia. MO engraftment, independent of its genotype, altered microglia morphology within brain slices. Further results from these experiments demonstrated that the presence of HD MO increased microglial density and upregulated their phagocytosis of MSNs. While the mechanisms underlying these multicellular interactions remain to be determined, it is possible that such an increase in phagocytosis could lead to the observation of fewer healthy MSNs as quantified in the second set of experiments.

Together, these three sets of experiments support the application of a novel model to study neuropathogenesis and highlight that the genotype of infiltrating MO has the potential to quantifiably alter intercellular interactions and neuronal health in the context of HD.

The pathogenesis of Alzheimer’s disease (AD) is a critical unsolved question, and while recent studies have demonstrated a strong association between altered brain immune responses and disease progression, the mechanistic cause of neuronal dysfunction and death is unknown. We have previously described the unique CVN-AD mouse model of AD, in which immune-mediated nitric oxide is lowered to mimic human levels, resulting in a mouse model that demonstrates the cardinal features of AD, including amyloid deposition, hyperphosphorylated and aggregated tau, behavioral changes and age-dependent hippocampal neuronal loss. Using this mouse model, we studied longitudinal changes in brain immunity in relation to neuronal loss and, contrary to the predominant view that AD pathology is driven by pro-inflammatory factors, we find that the pathology in CVN-AD mice is driven by local immune suppression. Areas of hippocampal neuronal death are associated with the presence of immunosuppressive CD11c+ microglia and extracellular arginase, resulting in arginine catabolism and reduced levels of total brain arginine. Pharmacologic disruption of the arginine utilization pathway by an inhibitor of arginase and ornithine decarboxylase protected the mice from AD-like pathology and significantly decreased CD11c expression. Our findings strongly implicate local immune-mediated amino acid catabolism as a novel and potentially critical mechanism mediating the age-dependent and regional loss of neurons in humans with AD.

There is a large interest in identifying, lineage tracing, and determining the physiologic roles of monophagocytes in Alzheimer’s disease. While Cx3cr1 knock-in fluorescent reporting and Cre expressing mice have been critical for studying neuroimmunology, mice that are homozygous null or hemizygous for CX3CR1 have perturbed neural development and immune responses. There is, therefore, a need for similar tools in which mice are CX3CR1+/+. Here, we describe a mouse where Cre is driven by the Cx3cr1 promoter on a bacterial artificial chromosome (BAC) transgene (Cx3cr1-CreBT) and the Cx3cr1 locus is unperturbed. Similarly to Cx3cr1-Cre knock-in mice, these mice express Cre in Ly6C-, but not Ly6C+, monocytes and tissue macrophages, including microglia. These mice represent a novel tool that maintains the Cx3cr1 locus while allowing for selective gene targeting in monocytes and tissue macrophages.

The study of immunity in Alzheimer’s requires the ability to identify and quantify specific immune cell subsets by flow cytometry. While it is possible to identify lymphocyte subsets based on cell lineage-specific markers, the lack of such markers in brain myeloid cell subsets has prevented the study of monocytes, macrophages and dendritic cells. By improving on tissue homogenization, we present a comprehensive protocol for flow cytometric analysis, that allows for the identification of several cell types that have not been previously identified by flow cytometry. These cell types include F4/80hi macrophages, which may be meningeal macrophages, IA/IE+ macrophages, which may represent perivascular macrophages, and dendritic cells. The identification of these cell types now allows for their study by flow cytometry in homeostasis and disease.

Stress exposure is the most cited factor in the development of both anxiety and depressive disorders. Both anxiety and depressive disorders have an increased prevalence in females; however, capturing this using pre-clinical models of stress has proven to be challenging. Therefore, we employed High Mobility Group Box 1 (HMGB1), an established pharmacological model of stress. Using this model, we sought to investigate the mechanisms which underlie HMGB1 associated behavioral changes in both male and female mice. Previous work demonstrated that circulating HMGB1 throughout the brain via the third ventricle causes depression like behavioral changes decreased sucrose preference, decreased sucrose consumption, decreased social preference, and increased immobility in the tail suspension test. Due to the known role of the cortico-limbic circuit in depression, we hypothesized that infusion of HMGB1 directly to the medial Prefrontal Cortex (mPFC) would also cause a depressive-like phenotype previously reported. Alternatively, we observed changes strongly associated with an anxiety-like phenotype. Previous work has implicated HMGB1 in chronic and acute stress responses; however, a majority of CNS cell types express receptors for HMGB1. Extracellular HMGB1 signals like a cytokine binding to many receptors that participate in pro-inflammatory signaling; yet a cell specific role for microglia the resident immune cell of the brain has not been clearly delineated. HMGB1’s effect on behavior has been causally linked to the activation of both RAGE and TLR4 pathways. Both pathways rely on concurrent activation of Myeloid differentiation primary response 88 (MyD88), though MyD88 independent activation is possible. We hypothesize that microglia reactivity is a necessary mechanism by which HMGB1 alters behavior and in this context increases anxiety. Moreover, we posit that MyD88 may be critical to increased microglial reactivity in response to HMGB1. To test this hypothesis, we utilized a pharmacological model of stress whereby dsHMGB1 was administered locally to the mPFC via cannula. Both male and female mice, aged 12-20 weeks, underwent stereotaxic surgery for the implantation of an intracerebral ventricular (ICV) guide cannula into the left medial prefrontal cortex. After a recovery period, mice were infused with artificial cerebrospinal fluid (aCSF) or recombinant HMGB1 (dsHMGB1) for five consecutive days. Following dosing, behaviors related to anxiety, despair and sociability were assayed. Various behavioral tests were conducted, including the Open Field Test (OFT), Elevated Plus Maze (EPM), Social Preference Assay, Novelty Suppressed Feeding (NSF), Home cage Feeding Assay, Light Dark Box (LDB) test, and Tail Suspension Test (TST). Baseline weights were established, and daily weight measurements were taken before and during dosing periods. Weight changes were calculated from baseline. To determine if female behavioral changes were also correlated with estrus vaginal cytology was performed to determine the stage of the estrous cycle. Smears were obtained and stained using the hematoxylin-eosin method, with cell types counted to define each stage of the cycle. We found that both male and female mice showed increased anxiety-like behavior in the EPM and Home cage Feeding Assay. For females specifically, we found that dsHMGB1 treatment caused significant changes in weight and that neither the behavioral phenotypes nor changes in weight were correlated with estrus cycle changes. Understanding the importance of microglial reactivity in stress response, we sought to evaluate microglial functional alterations in response to HMGB1. 16 hours after the final dose of dsHMGB1 or aCSF, mice were perfused and brain sections containing the medial prefrontal cortex were processed for immunohistochemical staining. Fluorescent images were captured for analysis of microglial activation. Microglia were reconstructed individually in IMARIS software and measurements for volume and branching were taken. We found that male and female demonstrated similar alterations in morphology wherein dsHMGB1 reduced the average number of Sholl intersections in more distal parts of the microglia. Though we again observed female specific effects relative to changes in Iba1 volume, ratio of TMEM119:Iba1 volume, and phagocytic index. Overall dsHMGB1 alters morphology of microglia indiscriminately of sex but additional changes related to reactivity were only observed in female mice. HMGB1 is recognized by multiple receptors including RAGE and TLR4 and activation of these receptors is often dependent on the concurrent activation of other adaptor proteins like MyD88 and TRAF6. Microglial RAGE, microglial TLR4, and MyD88 have been previously shown to be necessary mediators of neuroinflammatory stress responses. In this model, we determined the manner in which HMGB1 alters transcriptional activity of these key signaling molecules. Mice underwent the previously outlined surgery, recovery, and dosing timeline. 16 hours after the final dose of either aCSF or dsHMGB1 was administered, mice were sacrificed, and frontal cortex was removed using microdissection techniques. We utilized a cell specific isolation protocol to separate microglia and non-microglia cells by their expression of CD11b. RNA was extracted from these two populations of cells, and cDNA synthesis was performed. Quantitative real-time PCR (qPCR) was conducted to assess gene expression related to HMGB1-dependent signaling pathways. We found that female mice showed a robust pro-inflammatory phenotype defined by increased expression of both RAGE and MyD88 in microglia as well as increased MyD88 in non-microglial cells. For males, no significant changes in transcriptional activity were observed; however, it is of note that the directionality of male expression in the HMGB1 treated group is opposite of that seen in females. Together the data thus far established that HMGB1 exerts functional alterations in microglia in both sex specific and sex indiscriminate ways. We hypothesized that MyD88 specifically in microglia could be a key factor in conferring stress induced behavioral changes. To test this, we employed a transgenic mouse line with conditional deletion of MyD88 (cKO mice) in microglia. We utilized the surgery, recovery, and dosing timeline previously outlined to investigate anxiety-related behaviors. We found the female cKO mice did not show increased anxiety-like behavior following HMGB1 infusion and that male cKO mice did show increased anxiety-like behavior in response to HMGB1. This demonstrates that microglial MyD88 is necessary for HMGB1 induced anxiety in adult female mice. Taken together, the primary aim of this work is to examine the mechanisms of HMGB1 induced behavioral changes in a cell specific manner in both sexes. We found that HMGB1 acts on microglial cells in the context of anxiety as measured by changes in transcriptional changes, morphology, and phagocytic activity. Surprisingly, female but not male mice demonstrate concurrent changes in microglial reactivity and pro-inflammatory associated transcriptional changes. Specifically, female microglia show increased phagocytic capacity and decreased TMEM119 volume relative to Iba1, evidence of dysregulated homeostasis. Moreover, both RAGE, a putative HMBG1 receptor, and the adaptor molecule MyD88 were increased following dsHMGB1 in the microglia isolated from mPFC. Finally, we determined if microglial MyD88 conditional knockout (cKO) mice demonstrate a unique behavioral phenotype following dsHMGB1 infusion to the mPFC. These data appear to demonstrate that female mice rely on microglial mediated activation of MyD88 to respond to increased HMGB1 in the mPFC, whereas males do not. Without microglial MyD88, females do not demonstrate an anxiety-like response following dsHMGB1 administration whereas cKO males continue to show HMGB1-induced behavioral changes. Taken together these data elucidate a role for microglia in HMGB1 mediated behavioral responses. Furthermore, we have identified a potential sex-specific microglial mechanism of action underlying the impact of HMGB1 on behavior.

Developmental cell death is essential for nervous system development, sculpting the developing tissue by controlling cell numbers. While developmental neuron death has been studied extensively, the most abundant cell type of the nervous system – the astrocyte – has often been overlooked. Our lab recently showed that astrocytes in the developing retina undergo an unusual non-apoptotic form of death that eliminates a vast proportion of the original population. Further, we found that microglia are the major effectors of astrocyte death. However, the mechanisms that induce microglia to kill astrocytes remain mysterious. It is important to understand these astrocyte death mechanisms because astrocytes play a crucial role in patterning the retinal blood vessel network. Developmental perturbations to astrocyte number have large effects on their patterning, and in turn cause severe vascular patterning defects – some of which resemble vasculopathies typical of human blinding disorders. Because death has such a major impact on astrocyte number, it presumably has an outsized impact on this critical patterning process. We therefore sought to identify the non-apoptotic mechanisms that drive astrocyte death. Previously, we showed that astrocyte numbers modulate microglial phagocytic activity – increasing this activity as astrocyte numbers rise and decreasing it as astrocyte numbers decline. This observation suggested that astrocytes themselves are the source of cues that drive their own death via recruitment of phagocytic microglia. Here we identify the membrane lipid phosphatidylserine (PtdSer) as one such astrocyte-derived “eat-me” cue. PtdSer is best known as an “eat-me” signal expressed on the surface of apoptotic cells. We show that PtdSer is also externalized on the cell surface of apparently normal astrocytes during the developmental death period. Moreover, using a genetic approach to increase cell-surface PtdSer, we show that it is sufficient to drive astrocyte death. For these studies, we used an astrocyte-specific mouse knockout of Tmem30a, an obligate subunit of the flippase enzymes that normally remove PtdSer from the cell surface. In these knockout animals, microglia are recruited to Tmem30a mutant astrocytes, engulf them, and cause a significant acceleration of cell number decline. This excess astrocyte loss has functional consequences for the development of the vasculature: The astrocytic template for angiogenesis is overly sparse, which leads to vascular patterning defects and delayed angiogenesis. Interestingly, these defects can be rescued by blocking the function of a phagocytic signaling pathway that can recognize PtdSer exposure, suggesting that the excess PtdSer exposure in the Tmem30a knockout animals is responsible for the increase in astrocyte death. Altogether our findings highlight the broad impact of dysregulated astrocyte death. Understanding how astrocyte population size is controlled will provide new insights into death mechanisms that are crucial for development not only in the retina but may also sculpt glial populations elsewhere in the central nervous system.

Opioid drug abuse represents a serious public health concern with few effective therapeutic strategies. A primary goal for researchers modeling substance abuse disorders has been the delineation of the biological and environmental factors that shape an individual’s susceptibility or resistance to the reinforcing properties of abused substances. Early-life environmental conditions are frequently implicated as critical mediators for later-life health outcomes, although the cellular and molecular mechanisms that underlie these effects have historically been challenging to identify. Previous work has shown that a neonatal handling procedure in rats (which promotes enriched maternal care) attenuates morphine conditioning, reduces morphine-induced glial activation in the nucleus accumbens (NAc), and increases microglial expression of the anti-inflammatory cytokine interleukin-10 (IL-10). The experiments described in this dissertation were thus designed to address if inflammatory signaling in the NAc may underlie the effects of early-life experience on later-life opioid drug-taking. The results demonstrate that neonatal handling attenuates intravenous self-administration of the opioid remifentanil in a drug concentration-dependent manner. Transcriptional profiling of the NAc reveals a suppression of pro-inflammatory cytokine and chemokine signaling molecules and an increase in anti-inflammatory IL-10 in handled rats following repeated exposure to remifentanil. To directly test the hypothesis that anti-inflammatory signaling can alter drug-taking behavior, bilateral intracranial injections of plasmid DNA encoding IL-10 (pDNA-IL-10) or control pDNA were delivered into the NAc of naïve rats. pDNA-IL-10 treatment reduces remifentanil self-administration in a drug concentration-dependent manner, similar to the previous observations in handled rats. Additional experiments confirmed that neither handling nor pDNA-IL-10 treatment alters operant responding for food or sucrose rewards. These results help define the conditions under which ventral striatal neuroimmune signaling may influence motivated behaviors for highly reinforcing opioid drugs.

The interactions between the brain and the immune system are extensive and each has a profound influence on the other. The hippocampus is a brain region that is strongly impacted by the immune system, especially considering its large population of microglia, the resident immune cells of the brain. Cytokines and chemokines, the signaling molecules from immune cells, signal within the central nervous system (CNS) as well, and they are critical in hippocampal function. The relationship between the immune system and the hippocampus may underlie its particular vulnerability to diseases and disorders of the nervous system and the periphery. Conversely, immune signaling within the hippocampus is affected by alterations in hippocampal resilience and flexibility, such that increased hippocampal plasticity reduces vulnerability to immune challenges. The balance between risk and resilience in the hippocampus is modulated by immune signaling, especially by microglia.

The hippocampus is vulnerable to immune challenges, disease and injury, but it is simultaneously a region capable of profound plasticity and flexibility. The following dissertation experiments were designed to assess the roles of microglia and their signaling molecules, cytokines and chemokines, during normal hippocampal processes, such as learning and memory and response to immune challenge. The first set of experiments examined the effects of a neonatal bacterial infection in rats on hippocampal-dependent learning and memory as well as neuronal and microglial signaling in adulthood. In the first experiment, neonatally infected rats have impaired memory during fear conditioning following an immune challenge in adulthood. The impairment is caused by the exaggerated expression of the pro-inflammatory cytokine, interleukin (IL)-1β, within the hippocampus during learning. Hippocampal microglia are the primary source of IL-1β and the microglia in neonatally infected rats are "primed" by the infection into adulthood. In the second experiment, neonatally infected rats are more accurate on a Morris Water maze task following minimal training in adulthood, but have significantly impaired memory for a reversal platform location. In addition to improved accuracy, they have lower neural activation as measured by Arc protein expression within the dentate gyrus (DG) of the hippocampus. The next set of experiments assessed the effects of increasing hippocampal plasticity on immune signaling within the hippocampus. Following 7 weeks of environmental enrichment (EE), enriched rats had an attenuated pro-inflammatory response within the hippocampus in response to an in vivo peripheral immune challenge. The reduced immune response was specific to a subset of cytokines and chemokines and occurred only within the hippocampus and not adjacent cortical regions. Enrichment increased glial antigen expression within the DG as well. In another group of enriched rats, an ex vivo stimulation of isolated hippocampal microglia from EE rats demonstrated that the reduced microglial reactivity observed in vivo requires influence of other neural cell types on microglia phenotype, such that microglia within the DG of EE rats are smaller than controls. Taken together, these experiments define cellular and molecular mechanisms of hippocampal vulnerability and resilience as a function of interactions between the brain and the immune system.

Malignant glioma formation involves an abundant inflammatory infiltrate dominated by glioma-associated macrophages and microglia (GAMM). GAMM constitutes a large portion of the glioma mass and tumor microenvironment. They are actively involved in tissue repair and immune surveillance, however in the tumor microenvironment (TME), they are subverted to promote tumor progression. The human poliovirus receptor, hCD155h, is constitutively expressed in members of the mononuclear phagocytic system and is upregulated ectopically in the neoplastic compartment of malignant gliomas (and solid cancers in general). Intratumor treatment with the highly attenuated rhino:poliovirus chimera, PVSRIPO, has a dual effect of releasing neoantigens by oncolysis and activating the GAMM component via sublethal infection, leading to a substantial but transient immune therapy effect. In a phase I clinical trial, PVSRIPO treatment resulted in 21% long-term survival with durable radiographic responses in patients with recurrent glioblastoma (Desjardins et al. New England Journal of Medicine, 2018). Therefore, studying the mechanisms of PVSRIPO immunotherapy in mouse brain tumor models to decipher contributions of viral infection to GAMM vs. malignant cells is critical to improving the therapeutic efficacy in ongoing clinical trials. We recapitulated the clinical trial scenario in an immunocompetent intracerebral mouse tumor model (CT2A-CD155) and obtained baseline and post treatment brain in a time series. Histopathology studies, combined with detailed multiplex IHC/IF and RNAseq were performed on tumor bearing brains. We found the PVSRIPO therapy induced intense engagement of the GAMM infiltrate accompanied by substantial, but transient tumor regression. There were extensive microglia activation and proliferation in adjacent brain parenchyma and even part of the contralateral cortex. This occurred against a backdrop of sustained innate antiviral inflammation and is associated with an induction of the PD-L1 immune checkpoint on GAMM. In contrast to transient antitumor effects observed after PVSRIPO monotherapy, combining PVSRIPO with PD1/PD-L1 blockade led to durable remission. Our work implicates GAMM as active drivers of inflammation and reveals broad neuroinflammatory activation of the CNS-resident myeloid compartment upon polio virotherapy of malignant glioma.