Impaired cortical neuronal homeostasis and cognition after diffuse traumatic brain injury are dependent on microglia and type I interferon responses.

Neuropsychiatric complications including depression and cognitive decline develop in the years after traumatic brain injury (TBI), negatively affecting quality of life. Microglial and type 1 interferon (IFN-I) responses are associated with the transition from acute to chronic neuroinflammation after diffuse TBI in mice. Thus, the purpose of this study was to determine if impaired neuronal homeostasis and increased IFN-I responses intersected after TBI to cause cognitive impairment. Here, the RNA profile of neurons and microglia after TBI (single nucleus RNA-sequencing) with or without microglia depletion (CSF1R antagonist) was assessed 7 dpi. There was a TBI-dependent suppression of cortical neuronal homeostasis with reductions in CREB signaling, synaptogenesis, and synaptic migration and increases in RhoGDI and PTEN signaling (Ingenuity Pathway Analysis). Microglial depletion reversed 50% of TBI-induced gene changes in cortical neurons depending on subtype. Moreover, the microglial RNA signature 7 dpi was associated with increased stimulator of interferon genes (STING) activation and IFN-I responses. Therefore, we sought to reduce IFN-I signaling after TBI using STING knockout mice and a STING antagonist, chloroquine (CQ). TBI-associated cognitive deficits in novel object location and recognition (NOL/NOR) tasks at 7 and 30 dpi were STING dependent. In addition, TBI-induced STING expression, microglial morphological restructuring, inflammatory (Tnf, Cd68, Ccl2) and IFN-related (Irf3, Irf7, Ifi27) gene expression in the cortex were attenuated in STINGKO mice. CQ also reversed TBI-induced cognitive deficits and reduced TBI-induced inflammatory (Tnf, Cd68, Ccl2) and IFN (Irf7, Sting) cortical gene expression. Collectively, reducing IFN-I signaling after TBI with STING-dependent interventions attenuated the prolonged microglial activation and cognitive impairment.

Amplified Gliosis and Interferon-Associated Inflammation in the Aging Brain following Diffuse Traumatic Brain Injury.

Traumatic brain injury (TBI) is associated with chronic psychiatric complications and increased risk for development of neurodegenerative pathology. Aged individuals account for most TBI-related hospitalizations and deaths. Nonetheless, neurobiological mechanisms that underlie worsened functional outcomes after TBI in the elderly remain unclear. Therefore, this study aimed to identify pathways that govern differential responses to TBI with age. Here, adult (2 months of age) and aged (16-18 months of age) male C57BL/6 mice were subjected to diffuse brain injury (midline fluid percussion), and cognition, gliosis, and neuroinflammation were determined 7 or 30 d postinjury (dpi). Cognitive impairment was evident 7 dpi, independent of age. There was enhanced morphologic restructuring of microglia and astrocytes 7 dpi in the cortex and hippocampus of aged mice compared with adults. Transcriptional analysis revealed robust age-dependent amplification of cytokine/chemokine, complement, innate immune, and interferon-associated inflammatory gene expression in the cortex 7 dpi. Ingenuity pathway analysis of the transcriptional data showed that type I interferon (IFN) signaling was significantly enhanced in the aged brain after TBI compared with adults. Age prolonged inflammatory signaling and microgliosis 30 dpi with an increased presence of rod microglia. Based on these results, a STING (stimulator of interferon genes) agonist, DMXAA, was used to determine whether augmenting IFN signaling worsened cortical inflammation and gliosis after TBI. DMXAA-treated Adult-TBI mice showed comparable expression of myriad genes that were overexpressed in the cortex of Aged-TBI mice, including Irf7, Clec7a, Cxcl10, and Ccl5 Overall, diffuse TBI promoted amplified IFN signaling in aged mice, resulting in extended inflammation and gliosis.SIGNIFICANCE STATEMENT Elderly individuals are at higher risk of complications following traumatic brain injury (TBI). Individuals >70 years old have the highest rates of TBI-related hospitalization, neurodegenerative pathology, and death. Although inflammation has been linked with poor outcomes in aging, the specific biological pathways driving worsened outcomes after TBI in aging remain undefined. In this study, we identify amplified interferon-associated inflammation and gliosis in aged mice following TBI that was associated with persistent inflammatory gene expression and microglial morphologic diversity 30 dpi. STING (stimulator of interferon genes) agonist DMXAA was used to demonstrate a causal link between augmented interferon signaling and worsened neuroinflammation after TBI. Therefore, interferon signaling may represent a therapeutic target to reduce inflammation-associated complications following TBI.

Chronic Cortical Inflammation, Cognitive Impairment, and Immune Reactivity Associated with Diffuse Brain Injury Are Ameliorated by Forced Turnover of Microglia.

Traumatic brain injury (TBI) is associated with an increased risk of cognitive, psychiatric, and neurodegenerative complications that may develop after injury. Increased microglial reactivity following TBI may underlie chronic neuroinflammation, neuropathology, and exaggerated responses to immune challenges. Therefore, the goal of this study was to force turnover of trauma-associated microglia that develop after diffuse TBI and determine whether this alleviated chronic inflammation, improved functional recovery and attenuated reduced immune reactivity to lipopolysaccharide (LPS) challenge. Male mice received a midline fluid percussion injury (mFPI) and 7 d later were subjected to a forced microglia turnover paradigm using CSF1R antagonism (PLX5622). At 30 d postinjury (dpi), cortical gene expression, dendritic complexity, myelin content, neuronal connectivity, cognition, and immune reactivity were assessed. Myriad neuropathology-related genes were increased 30 dpi in the cortex, and 90% of these gene changes were reversed by microglial turnover. Reduced neuronal connectivity was evident 30 dpi and these deficits were attenuated by microglial turnover. TBI-associated dendritic remodeling and myelin alterations, however, remained 30 dpi independent of microglial turnover. In assessments of functional recovery, increased depressive-like behavior, and cognitive impairment 30 dpi were ameliorated by microglia turnover. To investigate microglial priming and reactivity 30 dpi, mice were injected intraperitoneally with LPS. This immune challenge caused prolonged lethargy, sickness behavior, and microglial reactivity in the TBI mice. These extended complications with LPS in TBI mice were prevented by microglia turnover. Collectively, microglial turnover 7 dpi alleviated behavioral and cognitive impairments associated with microglial priming and immune reactivity 30 dpi.SIGNIFICANCE STATEMENT A striking feature of traumatic brain injury (TBI), even mild injuries, is that over 70% of individuals have long-term neuropsychiatric complications. Chronic inflammatory processes are implicated in the pathology of these complications and these issues can be exaggerated by immune challenge. Therefore, our goal was to force the turnover of microglia 7 d after TBI. This subacute 7 d postinjury (dpi) time point is a critical transitional period in the shift toward chronic inflammatory processes and microglia priming. This forced microglia turnover intervention in mice attenuated the deficits in behavior and cognition 30 dpi. Moreover, microglia priming and immune reactivity after TBI were also reduced with microglia turnover. Therefore, microglia represent therapeutic targets after TBI to reduce persistent neuroinflammation and improve recovery.

Traumatic Brain Injury Causes Chronic Cortical Inflammation and Neuronal Dysfunction Mediated by Microglia.

Traumatic brain injury (TBI) can lead to significant neuropsychiatric problems and neurodegenerative pathologies, which develop and persist years after injury. Neuroinflammatory processes evolve over this same period. Therefore, we aimed to determine the contribution of microglia to neuropathology at acute [1 d postinjury (dpi)], subacute (7 dpi), and chronic (30 dpi) time points. Microglia were depleted with PLX5622, a CSF1R antagonist, before midline fluid percussion injury (FPI) in male mice and cortical neuropathology/inflammation was assessed using a neuropathology mRNA panel. Gene expression associated with inflammation and neuropathology were robustly increased acutely after injury (1 dpi) and the majority of this expression was microglia independent. At 7 and 30 dpi, however, microglial depletion reversed TBI-related expression of genes associated with inflammation, interferon signaling, and neuropathology. Myriad suppressed genes at subacute and chronic endpoints were attributed to neurons. To understand the relationship between microglia, neurons, and other glia, single-cell RNA sequencing was completed 7 dpi, a critical time point in the evolution from acute to chronic pathogenesis. Cortical microglia exhibited distinct TBI-associated clustering with increased type-1 interferon and neurodegenerative/damage-related genes. In cortical neurons, genes associated with dopamine signaling, long-term potentiation, calcium signaling, and synaptogenesis were suppressed. Microglial depletion reversed the majority of these neuronal alterations. Furthermore, there was reduced cortical dendritic complexity 7 dpi, reduced neuronal connectively 30 dpi, and cognitive impairment 30 dpi. All of these TBI-associated functional and behavioral impairments were prevented by microglial depletion. Collectively, these studies indicate that microglia promote persistent neuropathology and long-term functional impairments in neuronal homeostasis after TBI.SIGNIFICANCE STATEMENT Millions of traumatic brain injuries (TBIs) occur in the United States alone each year. Survivors face elevated rates of cognitive and psychiatric complications long after the inciting injury. Recent studies of human brain injury link chronic neuroinflammation to adverse neurologic outcomes, suggesting that evolving inflammatory processes may be an opportunity for intervention. Here, we eliminate microglia to compare the effects of diffuse TBI on neurons in the presence and absence of microglia and microglia-mediated inflammation. In the absence of microglia, neurons do not undergo TBI-induced changes in gene transcription or structure. Microglial elimination prevented TBI-induced cognitive changes 30 d postinjury (dpi). Therefore, microglia have a critical role in disrupting neuronal homeostasis after TBI, particularly at subacute and chronic timepoints.

Interleukin-1 receptor on hippocampal neurons drives social withdrawal and cognitive deficits after chronic social stress.

Chronic stress contributes to the development of psychiatric disorders including anxiety and depression. Several inflammatory-related effects of stress are associated with increased interleukin-1 (IL-1) signaling within the central nervous system and are mediated by IL-1 receptor 1 (IL-1R1) on several distinct cell types. Neuronal IL-1R1 is prominently expressed on the neurons of the dentate gyrus, but its role in mediating behavioral responses to stress is unknown. We hypothesize that IL-1 acts on this subset of hippocampal neurons to influence cognitive and mood alterations with stress. Here, mice subjected to psychosocial stress showed reduced social interaction and impaired working memory, and these deficits were prevented by global IL-1R1 knockout. Stress-induced monocyte trafficking to the brain was also blocked by IL-1R1 knockout. Selective deletion of IL-1R1 in glutamatergic neurons (nIL-1R1-/-) abrogated the stress-induced deficits in social interaction and working memory. In addition, viral-mediated selective IL-1R1 deletion in hippocampal neurons confirmed that IL-1 receptor in the hippocampus was critical for stress-induced behavioral deficits. Furthermore, selective restoration of IL-1R1 on glutamatergic neurons was sufficient to reestablish the impairments of social interaction and working memory after stress. RNA-sequencing of the hippocampus revealed that stress increased several canonical pathways (TREM1, NF-κB, complement, IL-6 signaling) and upstream regulators (INFγ, IL-1ß, NF-κB, MYD88) associated with inflammation. The inductions of TREM1 signaling, complement, and leukocyte extravasation with stress were reversed by nIL-1R1-/-. Collectively, stress-dependent IL-1R1 signaling in hippocampal neurons represents a novel mechanism by which inflammation is perpetuated and social interactivity and working memory are modulated.

Comparison between midline and lateral fluid percussion injury in mice reveals prolonged but divergent cortical neuroinflammation.

Animal models are critical for determining the mechanisms mediating traumatic brain injury-induced (TBI) neuropathology. Fluid percussion injury (FPI) is a widely used model of brain injury typically applied either midline or parasagittally (lateral). Midline FPI induces a diffuse TBI, while lateral FPI induces both focal cortical injury (ipsilateral hemisphere) and diffuse injury (contralateral hemisphere). Nonetheless, discrete differences in neuroinflammation and neuropathology between these two versions of FPI remain unclear. The purpose of this study was to compare acute (4-72 h) and subacute (7 days) neuroinflammatory responses between midline and lateral FPI. Midline FPI resulted in longer righting reflex times than lateral FPI. At acute time points, the inflammatory responses to the two different injuries were similar. For instance, there was evidence of monocytes and cytokine mRNA expression in the brain with both injuries acutely. Midline FPI had the highest proportion of brain monocytes and highest IL-1ß/TNFα mRNA expression 24 h later. NanoString nCounter analysis 7 days post-injury revealed robust and prolonged expression of inflammatory-related genes in the cortex after midline FPI compared to lateral FPI; however, Iba-1 cortical immunoreactivity was increased with lateral FPI. Thus, midline and lateral FPI caused similar cortical neuroinflammatory responses acutely and mRNA expression of inflammatory genes was detectable in the brain 7 days later. The primary divergence was that inflammatory gene expression was greater and more diverse subacutely after midline FPI. These results provide novel insight to variations between midline and lateral FPI, which may recapitulate unique temporal pathogenesis.

Sleep Disruption Exacerbates and Prolongs the Inflammatory Response to Traumatic Brain Injury.

Traumatic brain injury (TBI) alters stress responses, which may influence neuroinflammation and behavioral outcome. Sleep disruption (SD) is an understudied post-injury environmental stressor that directly engages stress-immune pathways. Thus, we predicted that maladaptive changes in the hypothalamic-pituitary-adrenal (HPA) axis after TBI compromise the neuroendocrine response to SD and exacerbate neuroinflammation. To test this, we induced lateral fluid percussion TBI or sham injury in female and male C57BL/6 mice aged 8-10 weeks that were then left undisturbed or exposed to 3 days of transient SD. At 3 days post-injury (DPI) plasma corticosterone (CORT) was reduced in TBI compared with sham mice, indicating altered HPA-mediated stress response to SD. This response was associated with approach-avoid conflict behavior and exaggerated cortical neuroinflammation. Post-injury SD specifically enhanced neutrophil trafficking to the injured brain in conjunction with dysregulated aquaporin-4 (AQP4) polarization. Delayed and persistent effects of post-injury SD were determined 4 days after SD concluded at 7 DPI. SD prolonged anxiety-like behavior regardless of injury and was associated with increased cortical Iba1 labeling in both sham and TBI mice. Strikingly, TBI SD mice displayed an increased number of CD45+ cells near the site of injury, enhanced cortical glial fibrillary acidic protein (GFAP) immunolabeling, and persistent expression of Trem2 and Tlr4 7 DPI compared with TBI mice. These results support the hypothesis that post-injury SD alters stress-immune pathways and inflammatory outcomes after TBI. These data provide new insight to the dynamic interplay between TBI, stress, and inflammation.

Traumatic brain injury-induced neuronal damage in the somatosensory cortex causes formation of rod-shaped microglia that promote astrogliosis and persistent neuroinflammation.

Microglia undergo dynamic structural and transcriptional changes during the immune response to traumatic brain injury (TBI). For example, TBI causes microglia to form rod-shaped trains in the cerebral cortex, but their contribution to inflammation and pathophysiology is unclear. The purpose of this study was to determine the origin and alignment of rod microglia and to determine the role of microglia in propagating persistent cortical inflammation. Here, diffuse TBI in mice was modeled by midline fluid percussion injury (FPI). Bone marrow chimerism and BrdU pulse-chase experiments revealed that rod microglia derived from resident microglia with limited proliferation. Novel data also show that TBI-induced rod microglia were proximal to axotomized neurons, spatially overlapped with dense astrogliosis, and aligned with apical pyramidal dendrites. Furthermore, rod microglia formed adjacent to hypertrophied microglia, which clustered among layer V pyramidal neurons. To better understand the contribution of microglia to cortical inflammation and injury, microglia were eliminated prior to TBI by CSF1R antagonism (PLX5622). Microglial elimination did not affect cortical neuron axotomy induced by TBI, but attenuated rod microglial formation and astrogliosis. Analysis of 262 immune genes revealed that TBI caused profound cortical inflammation acutely (8 hr) that progressed in nature and complexity by 7 dpi. For instance, gene expression related to complement, phagocytosis, toll-like receptor signaling, and interferon response were increased 7 dpi. Critically, these acute and chronic inflammatory responses were prevented by microglial elimination. Taken together, TBI-induced neuronal injury causes microglia to structurally associate with neurons, augment astrogliosis, and propagate diverse and persistent inflammatory/immune signaling pathways.