Microglial MyD88 Dependent Pathways are Regulated in a Sex Specific Manner in the context of HMGB1 induced anxiety

Abstract

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.