Impact of age and sex on neuroinflammation following SARS-CoV-2 infection in a murine model.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, is known to infect people of all ages and both sexes. Senior populations have the greatest risk of severe COVID-19, and sexual dimorphism in clinical outcomes has been reported. Neurological symptoms are widely observed in COVID-19 patients, with many survivors exhibiting persistent neurological and cognitive impairment. The present study aims to investigate the impact of age and sex on the neuroinflammatory response to SARS-CoV-2 infection using a mouse model. Wild-type C57BL/6J mice were intranasally inoculated with SARS-CoV-2 lineage B.1.351, a variant known to infect mice. Older male mice exhibited a significantly greater weight loss and higher viral loads in the lung at 3 days post infection. Notably, no viral RNA was detected in the brains of infected mice. Nevertheless, expression of IL-6, TNF-α, and CCL-2 in the lung and brain increased with viral infection. RNA-seq transcriptomic analysis of brains showed that SARS-CoV-2 infection caused significant changes in gene expression profiles, implicating innate immunity, defense response to virus, and cerebrovascular and neuronal functions. These findings demonstrate that SARS-CoV-2 infection triggers a neuroinflammatory response, despite the lack of detectable virus in the brain. Aberrant activation of innate immune response, disruption of blood-brain barrier and endothelial cell integrity, and suppression of neuronal activity and axonogenesis underlie the impact of SARS-CoV-2 infection on the brain. Understanding the role of these affected pathways in SARS-CoV-2 pathogenesis helps identify appropriate points of therapeutic interventions to alleviate neurological dysfunction observed during COVID-19.

Impact of age and sex on neuroinflammation following SARS-CoV-2 infection in a murine model.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent for the worldwide COVID-19 pandemic, is known to infect people of all ages and both sexes. Senior populations have the greatest risk of severe disease, and sexual dimorphism in clinical outcomes has been reported in COVID-19. SARS-CoV-2 infection in humans can cause damage to multiple organ systems, including the brain. Neurological symptoms are widely observed in patients with COVID-19, with many survivors suffering from persistent neurological and cognitive impairment, potentially accelerating Alzheimer's disease. The present study aims to investigate the impact of age and sex on the neuroinflammatory response to SARS-CoV-2 infection using a mouse model. Wild-type C57BL/6 mice were inoculated, by intranasal route, with SARS-CoV-2 lineage B.1.351 variant known to infect mice. Older animals and in particular males exhibited a significantly greater weight loss starting at 4 dpi. In addition, male animals exhibited higher viral RNA loads and higher titers of infectious virus in the lung, which was particularly evident in males at 16 months of age. Notably, no viral RNA was detected in the brains of infected mice, regardless of age or sex. Nevertheless, expression of IL-6, TNF-α, and CCL-2 in the lung and brain was increased with viral infection. An unbiased brain RNA-seq/transcriptomic analysis showed that SARS-CoV-2 infection caused significant changes in gene expression profiles in the brain, with innate immunity, defense response to virus, cerebravascular and neuronal functions, as the major molecular networks affected. The data presented in this study show that SARS-CoV-2 infection triggers a neuroinflammatory response despite the lack of detectable virus in the brain. Age and sex have a modifying effect on this pathogenic process. Aberrant activation of innate immune response, disruption of blood-brain barrier and endothelial cell integrity, and supression of neuronal activity and axonogenesis underlie the impact of SARS-CoV-2 infection on the brain. Understanding the role of these affected pathways in SARS-CoV-2 pathogenesis helps identify appropriate points of therapeutic interventions to alleviate neurological dysfunction observed during COVID-19.

Transplanting Microglia for Treating CNS Injuries and Neurological Diseases and Disorders, and Prospects for Generating Exogenic Microglia.

Microglia are associated with a wide range of both neuroprotective and neuroinflammatory functions in the central nervous system (CNS) during development and throughout lifespan. Chronically activated and dysfunctional microglia are found in many diseases and disorders, such as Alzheimer's disease, Parkinson's disease, and CNS-related injuries, and can accelerate or worsen the condition. Transplantation studies designed to replace and supplement dysfunctional microglia with healthy microglia offer a promising strategy for addressing microglia-mediated neuroinflammation and pathologies. This review will cover microglial involvement in neurological diseases and disorders and CNS-related injuries, current microglial transplantation strategies, and different approaches and considerations for generating exogenic microglia.

Cell Reprogramming for Regeneration and Repair of the Nervous System.

A persistent barrier to the cure and treatment of neurological diseases is the limited ability of the central and peripheral nervous systems to undergo neuroregeneration and repair. Recent efforts have turned to regeneration of various cell types through cellular reprogramming of native cells as a promising therapy to replenish lost or diminished cell populations in various neurological diseases. This review provides an in-depth analysis of the current viral vectors, genes of interest, and target cellular populations that have been studied, as well as the challenges and future directions of these novel therapies. Furthermore, the mechanisms by which cellular reprogramming could be optimized as treatment in neurological diseases and a review of the most recent cellular reprogramming in vitro and in vivo studies will also be discussed.

Microglia and Macrophages in Neuroprotection, Neurogenesis, and Emerging Therapies for Stroke.

Stroke remains the number one cause of morbidity in the United States. Within weeks to months after an ischemic event, there is a resolution of inflammation and evidence of neurogenesis; however, years following a stroke, there is evidence of chronic inflammation in the central nervous system, possibly by the persistence of an autoimmune response to brain antigens as a result of ischemia. The mechanisms underlying the involvement of macrophage and microglial activation after stroke are widely acknowledged as having a role in ischemic stroke pathology; thus, modulating inflammation and neurological recovery is a hopeful strategy for treating the long-term outcomes after ischemic injury. Current treatments fail to provide neuroprotective or neurorestorative benefits after stroke; therefore, to ameliorate brain injury-induced deficits, therapies must alter both the initial response to injury and the subsequent inflammatory process. This review will address differences in macrophage and microglia nomenclature and summarize recent work in elucidating the mechanisms of macrophage and microglial participation in antigen presentation, neuroprotection, angiogenesis, neurogenesis, synaptic remodeling, and immune modulating strategies for treating the long-term outcomes after ischemic injury.

Role of Macrophages and Microglia in Zebrafish Regeneration.

Currently, there is no treatment for recovery of human nerve function after damage to the central nervous system (CNS), and there are limited regenerative capabilities in the peripheral nervous system. Since fish are known for their regenerative abilities, understanding how these species modulate inflammatory processes following injury has potential translational importance for recovery from damage and disease. Many diseases and injuries involve the activation of innate immune cells to clear damaged cells. The resident immune cells of the CNS are microglia, the primary cells that respond to infection and injury, and their peripheral counterparts, macrophages. These cells serve as key modulators of development and plasticity and have been shown to be important in the repair and regeneration of structure and function after injury. Zebrafish are an emerging model for studying macrophages in regeneration after injury and microglia in neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. These fish possess a high degree of neuroanatomical, neurochemical, and emotional/social behavioral resemblance with humans, serving as an ideal simulator for many pathologies. This review explores literature on macrophage and microglial involvement in facilitating regeneration. Understanding innate immune cell behavior following damage may help to develop novel methods for treating toxic and chronic inflammatory processes that are seen in trauma and disease.

Microglial response patterns following damage to the zebrafish olfactory bulb.

The inherent plasticity of the zebrafish olfactory system serves as a useful model for examining immune cell responses after injury. Microglia are the resident immune cells of the CNS that respond to damage by migrating to the site of injury and phagocytizing neuronal debris. While the olfactory system is renowned for its ability to recover from damage, the specific mechanisms of microglial involvement in olfactory system plasticity are unknown. To approach the potentially time-dependent effects of microglial activation after injury, we performed a time course analysis of microglial response profiles and patterns following different forms of damage: deafferentation by cautery ablation of the olfactory organ, deafferentation by chemical ablation of the olfactory epithelium, and direct lesioning of the olfactory bulb. Our aim was to demonstrate that immunocytochemistry and microscopy methods in zebrafish can be used to determine the timing of distinct microglial response patterns following various forms of injury. We found that permanent and temporary forms of damage to the olfactory bulb resulted in different microglial response profiles from 1 to 72 h after injury, suggesting that there may be critical timepoints in which microglia are activated that contribute to tissue and neuronal repair with a regenerative outcome versus a degenerative outcome. These distinctions between the different forms of damage suggest temporal changes relative to the potential for regeneration, since cautery deafferentation is permanent and unrecoverable while chemical ablation deafferentation and direct lesioning is reversible and can be used to observe the microglial relationship in neural regeneration and functional recovery in future studies.

Increased cell-free mitochondrial DNA is a marker of ongoing inflammation and better neurocognitive function in virologically suppressed HIV-infected individuals.

Cell-free mitochondrial DNA (mtDNA) is a highly immunogenic molecule that is associated with several inflammatory conditions and with neurocognitive impairment during untreated HIV infection. Here, we investigate how cell-free mtDNA in cerebrospinal fluid (CSF) is associated with inflammation, neuronal damage, and neurocognitive functioning in the context of long-term suppressive antiretroviral therapy (ART). We quantified the levels of cell-free mtDNA in the CSF from 41 HIV-infected individuals with completely suppressed HIV RNA levels in blood plasma (<50 copies/mL) by droplet digital PCR. We measured soluble CD14, soluble CD163, interferon γ-induced protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor-α (TNF-α), neopterin, and neurofilament light chain (NFL) by immunoassays in CSF supernatant or blood plasma. Higher levels of mtDNA in CSF were associated with higher levels of MCP-1 (r = 0.56, p < 0.01) in CSF and TNF-α (r = 0.43, p < 0.01) and IL-8 (r = 0.44, p < 0.01) in blood plasma. Subjects with a previous diagnosis of AIDS showed significantly higher levels of mtDNA (p < 0.01) than subjects without AIDS. The associations between mtDNA and MCP-1 in CSF and TNF-α in blood remained significant after adjusting for previous diagnosis of AIDS (p < 0.01). Additionally, higher levels of mtDNA were associated with a lower CD4 nadir (r = -0.41, p < 0.01) and lower current CD4% (r = -0.34, p = 0.03). Paradoxically, higher levels of mtDNA in CSF were significantly associated with better neurocognitive performance (r = 0.43, p = 0.02) and with less neuronal damage (i.e. lower NFL). Higher cell-free mtDNA is associated with inflammation during treated HIV infection, but the impact on neurocognitive functioning and neuronal damage remains unclear and may differ in the setting of suppressive ART.

Replication of Human Herpesviruses Is Associated with Higher HIV DNA Levels during Antiretroviral Therapy Started at Early Phases of HIV Infection.

UNLABELLED: Asymptomatic replication of human herpesviruses (HHV) is frequent in HIV-infected men and is associated with increased T-cell activation and HIV disease progression. We hypothesized that the presence of replication of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) (the most frequently detected HHV) might influence HIV DNA decay during antiretroviral therapy (ART). We investigated 607 peripheral blood mononuclear cell (PBMC) samples from 107 CMV-seropositive, HIV-infected men who have sex with men, who started ART within a median of 3 months from their estimated date of infection (EDI) and were monitored for a median of 19 months thereafter. Levels of HIV, CMV, and EBV DNA and cellular HIV RNA were measured by droplet digital PCR (ddPCR) for each time point. Using a general linear mixed-effect regression model, we evaluated associations between the presence of detectable CMV DNA and EBV DNA levels and HIV DNA decay and cellular HIV RNA levels, while adjusting for peak HIV RNA, nadir CD4(+)count, CD4/CD8 ratio, CMV IgG levels, time from EDI to ART initiation, time from ART initiation to virologic suppression, detectable CMV DNA pre-ART, and age. The presence of intermittent CMV DNA in PBMC during ART was significantly associated with slower decay of HIV DNA (P= 0.011) but not with increased cellular HIV RNA transcription or more detectable 2-long terminal repeat circles. Higher levels of EBV DNA were also associated with higher levels of HIV DNA (P< 0.001) and increased unspliced cellular HIV RNA transcription (P= 0.010). These observations suggest that replication of HHV may help maintain a larger HIV DNA reservoir, but the underlying mechanisms remain unclear. IMPORTANCE: Over three-fourths of HIV-infected men have at least one actively replicating human herpesvirus (HHV) in their mucosal secretions at any one time. Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) are the most common, and although it is often asymptomatic, such CMV and EBV replication is associated with higher levels of immune activation and HIV disease progression. We hypothesized that HHV-associated activation of HIV-infected CD4(+)T cells might lead to increased HIV DNA. This study found that detectable CMV in blood cells of HIV-infected men was associated with slower decay of HIV DNA even during antiretroviral therapy (ART) that was started during early HIV infection. Similarly, levels of EBV DNA were associated with higher levels of HIV DNA during ART. If this observation points to a causal pathway, interventions that control CMV and EBV replication may be able to reduce the HIV reservoir, which might be relevant to current HIV cure efforts.

Mitochondrial injury and cognitive function in HIV infection and methamphetamine use.

OBJECTIVE: In this work, we evaluated the association of human immunodeficiency virus (HIV) infection and methamphetamine (METH) use with mitochondrial injury in the brain and its implication on neurocognitive impairment. DESIGN: Mitochondria carry their genome (mtDNA) and play a critical role in cellular processes in the central nervous system. METH is commonly used in HIV-infected populations. HIV infection and METH use can cause damage to mtDNA and lead to neurocognitive morbidity. We evaluated HIV infection and METH use with mitochondrial injury in the brain. METHODS: We obtained white and gray matter from Brodmann areas 7, 8, 9, 46 of the following: HIV-infected individuals with history of past METH use (HIV+METH+, n = 16), HIV-infected individuals with no history of past METH use (HIV+METH-, n = 11), and HIV-negative controls (HIV-METH-, n = 30). We used the 'common deletion', a 4977 bp mutation, as a measurement of mitochondrial injury, and quantified levels of mtDNA and 'common deletion' by droplet digital PCR, and evaluated in relation to neurocognitive functioning [Global Deficit Score (GDS)]. RESULTS: Levels of mtDNA and mitochondrial injury were highest in white matter of Brodmann area 46. A higher relative proportion of mtDNA carrying the 'common deletion' was associated with lower GDS (P < 0.01) in HIV+METH+ but higher GDS (P < 0.01) in HIV+METH-. CONCLUSIONS: Increased mitochondrial injury was associated with worse neurocognitive function in HIV+METH- individuals. Among HIV+METH+ individuals, an opposite effect was seen.

Cell-free mitochondrial DNA in CSF is associated with early viral rebound, inflammation, and severity of neurocognitive deficits in HIV infection.

Cell-free mitochondiral DNA (mtDNA) is an immunogenic molecule associated with many inflammatory conditions. We evaluated the relationship between cell-free mtDNA in cerebrospinal fluid (CSF) and neurocognitive performance and inflammation during HIV infection. In a cross-sectional analysis, we evaluated the association of mtDNA levels with clinical assessments, inflammatory markers, and neurocognitive performance in 28 HIV-infected individuals. In CSF, we measured mtDNA levels by droplet digital PCR, and soluble CD14 and CD163, neurofilament light, and neopterin by ELISA. In blood and CSF, we measured soluble IP-10, MCP-1, TNF-α, and IL-6 by ELISA, and intracellular expression of IL-2, IFN-γ, and TNF-α in CD4(+) and CD8(+) T cells by flow cytometry. We also evaluated the relationship between CSF pleocytosis and mtDNA longitudinally in another set of five individuals participating in an antiretroviral treatment (ART) interruption study. Cell-free CSF mtDNA levels strongly correlated with neurocognitive performance among individuals with neurocognitive impairment (NCI) (r = 0.77, p = 0.001). CSF mtDNA also correlated with levels of IP-10 in CSF (r = 0.70, p = 0.007) and MCP-1 in blood plasma (r = 0.66, p = 0.01) in individuals with NCI. There were no significant associations between inflammatory markers and mtDNA in subjects without NCI, and levels of mtDNA did not differ between subjects with and without NCI. MtDNA levels preceded pleocytosis and HIV RNA following ART interruption. Cell-free mtDNA in CSF was strongly associated with the severity of neurocognitive dysfunction and inflammation only in individuals with NCI. Our findings suggest that within a subset of subjects cell-free CSF mtDNA is associated with inflammation and degree of NCI.

Gut Lactobacillales are associated with higher CD4 and less microbial translocation during HIV infection.

OBJECTIVE: Early HIV infection is characterized by a dramatic depletion of CD4 T cells in the gastrointestinal tract and translocation of bacterial products from the gut into the blood. In this study, we evaluated if gut bacterial profiles were associated with immune status before and after starting antiretroviral therapy (ART). DESIGN: We evaluated the gut microbiota of men recently infected with HIV (n = 13) who were participating in a randomized, double-blind controlled trial of combination ART and maraviroc versus placebo and who were followed for 48 weeks. METHODS: To evaluate the gut microbiota of participants, we pyrosequenced the bacterial populations from anal swabs collected before and longitudinally after the initiation of ART. Associations of the gut flora with clinical variables (lymphocyte profiles and viral loads), activation and proliferation markers in peripheral blood mononuclear cells and gut biopsies (measured by flow cytometry) and markers of microbial translocation (lipopolysaccharide and soluble CD14) were performed by regression analyses using R statistical software. RESULTS: Using pyrosequencing, we identified that higher proportions of Lactobacillales in the distal gut of recently HIV-infected individuals were associated with lower markers of microbial translocation, higher CD4% and lower viral loads before ART was started. Similarly, during ART, higher proportions of gut Lactobacillales were associated with higher CD4%, less microbial translocation, less systemic immune activation, less gut T lymphocyte proliferation, and higher CD4% in the gut. CONCLUSION: Shaping the gut microbiome, especially proportions of Lactobacillales, could help to preserve immune function during HIV infection.