Use of adeno-associated viruses for transgenic modulation of microglia structure and function: A review of technical considerations and challenges.

Microglia play a central role in the etiology of many neuropathologies. Transgenic tools are a powerful experiment approach to gain reliable and specific control over microglia function. Adeno-associated virus (AAVs) vectors are already an indispensable tool in neuroscience research. Despite ubiquitous use of AAVs and substantial interest in the role of microglia in the study of central nervous system (CNS) function and disease, transduction of microglia using AAVs is seldom reported. This review explores the challenges and advancements made in using AAVs for expressing transgenes in microglia. First, we will examine the functional anatomy of the AAV capsid, which will serve as a basis for subsequent discussions of studies exploring the relationship between capsid mutations and microglia transduction efficacy. After outlining the functional anatomy of AAVs, we will consider the experimental evidence demonstrating AAV-mediated transduction of microglia and microglia-like cell lines followed by an examination of the most promising experimental approaches identified in the literature. Finally, technical limitations will be considered in future applications of AAV experimental approaches.

SARS-CoV-2 S1 subunit produces a protracted priming of the neuroinflammatory, physiological, and behavioral responses to a remote immune challenge: A role for corticosteroids.

Long COVID is a major public health consequence of COVID-19 and is characterized by multiple neurological and neuropsychatric symptoms. SARS-CoV-2 antigens (e.g., spike S1 subunit) are found in the circulation of Long COVID patients, have been detected in post-mortem brain of COVID patients, and exhibit neuroinflammatory properties. Considering recent observations of chronic neuroinflammation in Long COVID patients, the present study explores the idea that antigens derived from SARS-CoV-2 might produce a long-term priming or sensitization of neuroinflammatory processes, thereby potentiating the magnitude and/or duration of the neuroinflammatory response to future inflammatory insults. Rats were administered S1 or vehicle intra-cisterna magna and 7d later challenged with vehicle or LPS. The neuroinflammatory, physiological, and behavioral responses to LPS were measured at various time points post-LPS. We found that prior S1 treatment potentiated many of these responses to LPS suggesting that S1 produces a protracted priming of these processes. Further, S1 produced a protracted reduction in basal brain corticosteroids. Considering the anti-inflammatory properties of corticosteroids, these findings suggest that S1 might disinhibit innate immune processes in brain by reducing anti-inflammatory drive, thereby priming neuroinflammatory processes. Given that hypocortisolism is observed in Long COVID, we propose that similar S1-induced innate immune priming processes might play role in the pathophysiology of Long COVID.

Interleukin-1beta and inflammasome expression in spinal cord following chronic constriction injury in male and female rats.

Females represent a majority of chronic pain patients and show greater inflammatory immune responses in human chronic pain patient populations as well as in animal models of neuropathic pain. Recent discoveries in chronic pain research have revealed sex differences in inflammatory signaling, a key component of sensory pathology in chronic neuropathic pain, inviting more research into the nuances of these sex differences. Here we use the chronic constriction injury (CCI) model to explore similarities and differences in expression and production of Inflammatory cytokine IL-1beta in the lumbar spinal cord, as well as its role in chronic pain. We have discovered that intrathecal IL-1 receptor antagonist reverses established pain in both sexes, and increased gene expression of inflammasome NLRP3 is specific to microglia and astrocytes rather than neurons, while IL-1beta is specific to microglia in both sexes. We report several sex differences in the expression level of the genes coding for IL-1beta, as well as the four inflammasomes responsible for IL-1beta release: NLRP3, AIM2, NLRP1, and NLRC4 in the spinal cord. Total mRNA, but not protein expression of IL-1beta is greater in females than males after CCI. Also, while CCI increases all four inflammasomes in both sexes, there are sex differences in relative levels of inflammasome expression. NLRP3 and AIM2 are more highly expressed in females, whereas NLRP1 expression is greater in males.

Exploring the immunogenic properties of SARS-CoV-2 structural proteins: PAMP:TLR signaling in the mediation of the neuroinflammatory and neurologic sequelae of COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) produces an array of neurologic and neuropsychiatric symptoms in the acute and post-acute phase of infection (PASC; post-acute sequelae of SARS-CoV-2 infection). Neuroinflammatory processes are considered key factors in the etiology of these symptoms. Several mechanisms underpinning the development of inflammatory events in the brain have been proposed including SARS-CoV-2 neurotropism and peripheral inflammatory responses (i.e., cytokine storm) to infection, which might produce neuroinflammation via immune-to-brain signaling pathways. In this review, we explore evidence in support of an alternate mechanism whereby structural proteins (e.g., spike and spike S1 subunit) derived from SARS-CoV-2 virions function as pathogen-associated molecular patterns (PAMPs) to elicit proinflammatory immune responses in the periphery and/or brain via classical Toll-Like Receptor (TLR) inflammatory pathways. We propose that SARS-CoV-2 structural proteins might directly produce inflammatory processes in brain independent of and/or in addition to peripheral proinflammatory effects, which might converge to play a causal role in the development of neurologic/neuropsychiatric symptoms in COVID-19.

Immunization with a heat-killed preparation of Mycobacterium vaccae NCTC 11659 enhances auditory-cued fear extinction in a stress-dependent manner.

Stress-related psychiatric disorders including anxiety disorders, mood disorders, and trauma and stressor-related disorders, such as posttraumatic stress disorder (PTSD), affect millions of people world-wide each year. Individuals with stress-related psychiatric disorders have been found to have poor immunoregulation, increased proinflammatory markers, and dysregulation of fear memory. The "Old Friends" hypothesis proposes that a lack of immunoregulatory inputs has led to a higher prevalence of inflammatory disorders and stress-related psychiatric disorders, in which inappropriate inflammation is thought to be a risk factor. Immunization with a soil-derived saprophytic bacterium with anti-inflammatory and immunoregulatory properties, Mycobacterium vaccae NCTC 11659, can lower proinflammatory biomarkers, increase stress resilience, and, when given prior to or after fear conditioning in a rat model of fear-potentiated startle, enhance fear extinction. In this study, we investigated whether immunization with heat-killed M. vaccae NCTC 11659 would enhance fear extinction in contextual or auditory-cued fear conditioning paradigms and whether M. vaccae NCTC 11659 would prevent stress-induced exaggeration of fear expression or stress-induced resistance to extinction learning. Adult male Sprague Dawley rats were immunized with M. vaccae NCTC 11659 (subcutaneous injections once a week for three weeks), and underwent either: Experiment 1) one-trial contextual fear conditioning; Experiment 2) two-trial contextual fear conditioning; Experiment 3) stress-induced enhancement of contextual fear conditioning; Experiment 4) stress-induced enhancement of auditory-cued fear conditioning; or Experiment 5) stress-induced enhancement of auditory-cued fear conditioning exploring short-term memory. Immunizations with M. vaccae NCTC 11659 had no effect on one- or two-trial contextual fear conditioning or contextual fear extinction, with or without exposure to inescapable stress. However, inescapable stress increased resistance to auditory-cued fear extinction. Immunization with M. vaccae NCTC 11659 prevented the stress-induced increase in resistance to auditory-cued fear extinction learning. Finally, in an auditory-cued fear conditioning paradigm exploring short-term memory and fear acquisition, immunization with M. vaccae did not prevent fear acquisition, either with or without exposure to inescapable stress, consistent with the hypothesis that M. vaccae NCTC 11659 has no effect on fear acquisition but enhances fear extinction. These data are consistent with the hypothesis that increased immunoregulation following immunization with M. vaccae NCTC 11659 promotes stress resilience, in particular by preventing stress-induced resistance to fear extinction, and may be a potential therapeutic intervention for trauma- and stressor-related disorders such as PTSD.

Mycobacterium vaccae NCTC 11659, a Soil-Derived Bacterium with Stress Resilience Properties, Modulates the Proinflammatory Effects of LPS in Macrophages.

Inflammatory conditions, including allergic asthma and conditions in which chronic low-grade inflammation is a risk factor, such as stress-related psychiatric disorders, are prevalent and are a significant cause of disability worldwide. Novel approaches for the prevention and treatment of these disorders are needed. One approach is the use of immunoregulatory microorganisms, such as Mycobacterium vaccae NCTC 11659, which have anti-inflammatory, immunoregulatory, and stress-resilience properties. However, little is known about how M. vaccae NCTC 11659 affects specific immune cell targets, including monocytes, which can traffic to peripheral organs and the central nervous system and differentiate into monocyte-derived macrophages that, in turn, can drive inflammation and neuroinflammation. In this study, we investigated the effects of M. vaccae NCTC 11659 and subsequent lipopolysaccharide (LPS) challenge on gene expression in human monocyte-derived macrophages. THP-1 monocytes were differentiated into macrophages, exposed to M. vaccae NCTC 11659 (0, 10, 30, 100, 300 µg/mL), then, 24 h later, challenged with LPS (0, 0.5, 2.5, 250 ng/mL), and assessed for gene expression 24 h following challenge with LPS. Exposure to M. vaccae NCTC 11659 prior to challenge with higher concentrations of LPS (250 ng/mL) polarized human monocyte-derived macrophages with decreased IL12A, IL12B, and IL23A expression relative to IL10 and TGFB1 mRNA expression. These data identify human monocyte-derived macrophages as a direct target of M. vaccae NCTC 11659 and support the development of M. vaccae NCTC 11659 as a potential intervention to prevent stress-induced inflammation and neuroinflammation implicated in the etiology and pathophysiology of inflammatory conditions and stress-related psychiatric disorders.

Effects of Mycobacterium vaccae NCTC 11659 and Lipopolysaccharide Challenge on Polarization of Murine BV-2 Microglial Cells.

Previous studies have shown that the in vivo administration of soil-derived bacteria with anti-inflammatory and immunoregulatory properties, such as Mycobacterium vaccae NCTC 11659, can prevent a stress-induced shift toward an inflammatory M1 microglial immunophenotype and microglial priming in the central nervous system (CNS). It remains unclear whether M. vaccae NCTC 11659 can act directly on microglia to mediate these effects. This study was designed to determine the effects of M. vaccae NCTC 11659 on the polarization of naïve BV-2 cells, a murine microglial cell line, and BV-2 cells subsequently challenged with lipopolysaccharide (LPS). Briefly, murine BV-2 cells were exposed to 100 µg/mL whole-cell, heat-killed M. vaccae NCTC 11659 or sterile borate-buffered saline (BBS) vehicle, followed, 24 h later, by exposure to 0.250 µg/mL LPS (Escherichia coli 0111: B4; n = 3) in cell culture media vehicle (CMV) or a CMV control condition. Twenty-four hours after the LPS or CMV challenge, cells were harvested to isolate total RNA. An analysis using the NanoString platform revealed that, by itself, M. vaccae NCTC 11659 had an "adjuvant-like" effect, while exposure to LPS increased the expression of mRNAs encoding proinflammatory cytokines, chemokine ligands, the C3 component of complement, and components of inflammasome signaling such as Nlrp3. Among LPS-challenged cells, M. vaccae NCTC 11659 had limited effects on differential gene expression using a threshold of 1.5-fold change. A subset of genes was assessed using real-time reverse transcription polymerase chain reaction (real-time RT-PCR), including Arg1, Ccl2, Il1b, Il6, Nlrp3, and Tnf. Based on the analysis using real-time RT-PCR, M. vaccae NCTC 11659 by itself again induced "adjuvant-like" effects, increasing the expression of Il1b, Il6, and Tnf while decreasing the expression of Arg1. LPS by itself increased the expression of Ccl2, Il1b, Il6, Nlrp3, and Tnf while decreasing the expression of Arg1. Among LPS-challenged cells, M. vaccae NCTC 11659 enhanced LPS-induced increases in the expression of Nlrp3 and Tnf, consistent with microglial priming. In contrast, among LPS-challenged cells, although M. vaccae NCTC 11659 did not fully prevent the effects of LPS relative to vehicle-treated control conditions, it increased Arg1 mRNA expression, suggesting that M. vaccae NCTC 11659 induces an atypical microglial phenotype. Thus, M. vaccae NCTC 11659 acutely (within 48 h) induced immune-activating and microglial-priming effects when applied directly to murine BV-2 microglial cells, in contrast to its long-term anti-inflammatory and immunoregulatory effects observed on the CNS when whole-cell, heat-killed preparations of M. vaccae NCTC 11659 were given peripherally in vivo.

SARS-CoV-2 spike S1 subunit induces neuroinflammatory, microglial and behavioral sickness responses: Evidence of PAMP-like properties.

SARS-CoV-2 infection produces neuroinflammation as well as neurological, cognitive (i.e., brain fog), and neuropsychiatric symptoms (e.g., depression, anxiety), which can persist for an extended period (6 months) after resolution of the infection. The neuroimmune mechanism(s) that produces SARS-CoV-2-induced neuroinflammation has not been characterized. Proposed mechanisms include peripheral cytokine signaling to the brain and/or direct viral infection of the CNS. Here, we explore the novel hypothesis that a structural protein (S1) derived from SARS-CoV-2 functions as a pathogen-associated molecular pattern (PAMP) to induce neuroinflammatory processes independent of viral infection. Prior evidence suggests that the S1 subunit of the SARS-CoV-2 spike protein is inflammatory in vitro and signals through the pattern recognition receptor TLR4. Therefore, we examined whether the S1 subunit is sufficient to drive 1) a behavioral sickness response, 2) a neuroinflammatory response, 3) direct activation of microglia in vitro, and 4) activation of transgenic human TLR2 and TLR4 HEK293 cells. Adult male Sprague-Dawley rats were injected intra-cisterna magna (ICM) with vehicle or S1. In-cage behavioral monitoring (8 h post-ICM) demonstrated that S1 reduced several behaviors, including total activity, self-grooming, and wall-rearing. S1 also increased social avoidance in the juvenile social exploration test (24 h post-ICM). S1 increased and/or modulated neuroimmune gene expression (Iba1, Cd11b, MhcIIα, Cd200r1, Gfap, Tlr2, Tlr4, Nlrp3, Il1b, Hmgb1) and protein levels (IFNγ, IL-1ß, TNF, CXCL1, IL-2, IL-10), which varied across brain regions (hypothalamus, hippocampus, and frontal cortex) and time (24 h and 7d) post-S1 treatment. Direct exposure of microglia to S1 resulted in increased gene expression (Il1b, Il6, Tnf, Nlrp3) and protein levels (IL-1ß, IL-6, TNF, CXCL1, IL-10). S1 also activated TLR2 and TLR4 receptor signaling in HEK293 transgenic cells. Taken together, these findings suggest that structural proteins derived from SARS-CoV-2 might function independently as PAMPs to induce neuroinflammatory processes via pattern recognition receptor engagement.

Microglia: Neuroimmune-sensors of stress.

Exposure to stressors disrupts homeostasis and results in the release of stress hormones including glucocorticoids, epinepherine and norepinepherine. Interestingly, stress also has profound affects on microglia, which are tissue-resident macrophages in the brain parenchyma. Microglia express a diverse array of receptors, which also allows them to respond to stress hormones derived from peripheral as well as central sources. Here, we review studies of how exposure to acute and chronic stressors alters the immunophenotype and function of microglia. Further, we examine a causal for stress hormones in these effects of stress on microglia. We propose that microglia serve as immunosensors of the stress response, which puts them in the unique position to sense and respond rapidly to alterations in homeostasis and integrate the neural response to threats.

Comparing the effects of two different strains of mycobacteria, Mycobacterium vaccae NCTC 11659 and M. vaccae ATCC 15483, on stress-resilient behaviors and lipid-immune signaling in rats.

Stress-related disorders, such as posttraumatic stress disorder (PTSD), are highly prevalent and often difficult to treat. In rodents, stress-related, anxiety-like defensive behavioral responses may be characterized by social avoidance, exacerbated inflammation, and altered metabolic states. We have previously shown that, in rodents, subcutaneous injections of a heat-killed preparation of the soil-derived bacterium Mycobacterium vaccae NCTC 11659 promotes stress resilience effects that are associated with immunoregulatory signaling in the periphery and the brain. In the current study, we sought to determine whether treatment with a heat-killed preparation of the closely related M. vaccae type strain, M. vaccae ATCC 15483, would also promote stress-resilience in adult male rats, likely due to biologically similar characteristics of the two strains. Here we show that immunization with either M. vaccae NCTC 11659 or M. vaccae ATCC 15483 prevents stress-induced increases in hippocampal interleukin 6 mRNA expression, consistent with previous studies showing that M. vaccae NCTC 11659 prevents stress-induced increases in peripheral IL-6 secretion, and prevents exaggeration of anxiety-like defensive behavioral responses assessed 24 h after exposure to inescapable tail shock stress (IS) in adult male rats. Analysis of mRNA expression, protein abundance, and flow cytometry data demonstrate overlapping but also unique effects of treatment with the two M. vaccae strains on immunological and metabolic signaling in the host. These data support the hypothesis that treatment with different M. vaccae strains may immunize the host against stress-induced dysregulation of physiology and behavior.