Journal of Neuroinflammation

Background and ObjectivesHerpes simplex virus type 1 (HSV-1) is a neurotropic pathogen capable of invading the central nervous system (CNS) and increasingly associated with chronic neuroinflammation, cognitive impairment, and neurodegenerative disease. While microglia orchestrate the initial immune response to HSV-1, the molecular mechanisms that regulate their sustained neuroinflammatory activity in vivo remain poorly understood. MethodsTo define the transcriptional and epigenetic mechanisms that shape microglial responses during acute HSV-1 infection in vivo, we have, for the first time, integrated single-nucleus RNA sequencing, chromatin accessibility profiling, and spatial transcriptomics in a physiologically relevant intranasal HSV-1 infection model. ResultsSingle-cell multiome analysis of CD11b nuclei identified transcriptionally and epigenetically distinct microglial and macrophage populations. HSV-1 infection redistributed monocyte-lineage states, with a marked overrepresentation of interferon (IFN)-responsive microglia and macrophage-associated populations. These states exhibited differential amplification of STAT1/2-, IRF1-, and CEBPB-centered regulons, distinguishing IFN-responsive microglia from macrophage-enriched populations rather than reflecting uniform activation. Homeostatic microglial gene signatures (e.g., ApoE, Cst3) were reduced in response to HSV-1 infection. Spatial transcriptomics localized HSV-1 antigen to discrete brainstem regions, which were enriched for predicted STAT-, IRF-, and CEBPB-regulated targets identified through single-nuclei analysis. DiscussionUsing a multiomic framework, we demonstrate that HSV-1 infection drives transcriptional and epigenetic remodeling of microglial populations, characterized by a dominance of IFN-responsive states and a loss of homeostatic signatures. These findings provide mechanistic insight into how localized viral infection can reprogram microglial regulatory landscapes to maintain persistent HSV-1-associated neuroinflammation, contributing to long-term neurological vulnerability and neurodegenerative disease risk.

Early infection in life has been implicated in increasing the risk for neurological disorders. Here we performed single-cell sequencing of microglia and monocytes from 6-month-old WT and 5xFAD mice subjected to one dose of LPS (1mg/kg) at postnatal day 9. We successfully mapped disease-associated microglia (DAM) and perivascular macrophages in our data and demonstrated a subpopulation of microglia that adopted a monocyte-like profile, marked by Lyz2, Tmsb10, Lgals1and Lgals3. This unique subset appeared in response to early systemic LPS challenge and AD pathology but diminished in the presence of double stimulus. Different cytokines were altered in the brain and periphery as seen using mesoscale plates. GM-CSF and MIP-1 levels were altered in an amyloid-{beta}(A{beta})-dependent manner in hippocampus. MIP-1{beta} and IFN-{gamma} were altered upon early LPS stimulation. In the periphery, we found MMP-9 was significantly increased in serum samples from 5xFAD mice. Interestingly, early LPS stimulation significantly elevated TNF- in serum from WT and 5xFAD mice, but was reduced in the hippocampus due to A{beta} pathology. The LPS treatment in 5xFAD mice had a tendency to improve the short-term memory deficit. Taken together, we observed long-lasting effects from early life stress, including activation of inflammation in the periphery and brain through modulation of different signaling cascades.

The novel coronavirus SARS-CoV-2 has caused significant global morbidity and mortality and continues to burden patients with persisting neurological dysfunction. COVID-19 survivors develop debilitating symptoms to include neuro-psychological dysfunction, termed "Long COVID", which can cause significant reduction of quality of life. Despite vigorous model development, the possible cause of these symptoms and the underlying pathophysiology of this devastating disease remains elusive. Mouse adapted (MA10) SARS-CoV-2 is a novel mouse-based model of COVID-19 which simulates the clinical symptoms of respiratory distress associated with SARS-CoV-2 infection in mice. In this study, we evaluated the long-term effects of MA10 infection on brain pathology and neuroinflammation. 10-week and 1-year old female BALB/cAnNHsd mice were infected intranasally with 104 plaque-forming units (PFU) and 103 PFU of SARS-CoV-2 MA10, respectively, and the brain was examined 60 days post-infection (dpi). Immunohistochemical analysis showed a decrease in the neuronal nuclear protein NeuN and an increase in Iba-1 positive amoeboid microglia in the hippocampus after MA10 infection, indicating long-term neurological changes in a brain area which is critical for long-term memory consolidation and processing. Importantly, these changes were seen in 40-50% of infected mice, which correlates to prevalence of LC seen clinically. Our data shows for the first time that MA10 infection induces neuropathological outcomes several weeks after infection at similar rates of observed clinical prevalence of "Long COVID". These observations strengthen the MA10 model as a viable model for study of the long-term effects of SARS-CoV-2 in humans. Establishing the viability of this model is a key step towards the rapid development of novel therapeutic strategies to ameliorate neuroinflammation and restore brain function in those suffering from the persistent cognitive dysfunction of "Long-COVID".

Survivors of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection frequently experience lingering neurological symptoms, including impairment in attention, concentration, speed of information processing and memory. This long-COVID cognitive syndrome shares many features with the syndrome of cancer therapy-related cognitive impairment (CRCI). Neuroinflammation, particularly microglial reactivity and consequent dysregulation of hippocampal neurogenesis and oligodendrocyte lineage cells, is central to CRCI. We hypothesized that similar cellular mechanisms may contribute to the persistent neurological symptoms associated with even mild SARS-CoV-2 respiratory infection. Here, we explored neuroinflammation caused by mild respiratory SARS-CoV-2 infection - without neuroinvasion - and effects on hippocampal neurogenesis and the oligodendroglial lineage. Using a mouse model of mild respiratory SARS-CoV-2 infection induced by intranasal SARS-CoV-2 delivery, we found white matter-selective microglial reactivity, a pattern observed in CRCI. Human brain tissue from 9 individuals with COVID-19 or SARS-CoV-2 infection exhibits the same pattern of prominent white matter-selective microglial reactivity. In mice, pro-inflammatory CSF cytokines/chemokines were elevated for at least 7-weeks post-infection; among the chemokines demonstrating persistent elevation is CCL11, which is associated with impairments in neurogenesis and cognitive function. Humans experiencing long-COVID with cognitive symptoms (48 subjects) similarly demonstrate elevated CCL11 levels compared to those with long-COVID who lack cognitive symptoms (15 subjects). Impaired hippocampal neurogenesis, decreased oligodendrocytes and myelin loss in subcortical white matter were evident at 1 week, and persisted until at least 7 weeks, following mild respiratory SARS-CoV-2 infection in mice. Taken together, the findings presented here illustrate striking similarities between neuropathophysiology after cancer therapy and after SARS-CoV-2 infection, and elucidate cellular deficits that may contribute to lasting neurological symptoms following even mild SARS-CoV-2 infection.

Metabolic syndrome (MetS) puts patients more at risk for neurodegenerative diseases such as Alzheimers disease (AD). Microglia are implicated as causal factors in AD, however, the effect of MetS on microglia has not been characterized. To address this, we contrasted New Zealand Obese (NZO) with C57BL/6J (B6J) mice in combination with a high fat/high sugar diet (HFD). Irrespective of diet, NZO mice displayed a broader array of MetS-relevant phenotypes compared to B6J mice fed a HFD. Single cell RNA-sequencing of microglia predicted transcriptional shifts indicative of reduced responsiveness and increased vascular interactions in NZO, but not B6J HFD mice. Significant cerebrovascular fibrin deposition and increased perivascular accumulation of microglia were observed in NZO relative to B6J HFD mice. Further, compared to the widely used B6J.APP/PS1 mice, NZO.APP/PS1 exhibited increased amyloid plaque sizes alongside an increase in microhemorrhages. Overall, our work supports a model whereby MetS alters microglia-vascular interactions, compromising microglial plasticity. IntroductionMetabolic syndrome (MetS) is a combination of three or more metabolic impairments such as dyslipidemia, hyperglycemia, hypertension, and increased waist circumference (1). Patients displaying aspects of MetS are more at risk for neurodegenerative disorders including Alzheimers disease (AD) (2-6). Central to this risk may be the influence of MetS and Type 2 Diabetes (T2D) on peripheral and central immune cell states and function (7,8). Burgeoning evidence has implicated critical roles for microglia, central nervous system (CNS) resident macrophages, in neurodegeneration (9-15). Microglia play roles in pathogen surveillance, debris phagocytosis, synapse regulation, and recently have been shown to support the cerebral vasculature (16-21). Current efforts have uncovered remarkable heterogeneity of microglial responses through single cell RNA-sequencing (scRNA-seq) (9,11,15,22). Increased disease-associated microglia (DAM) (11,12) and interferon response microglia (IRM) states (23) have been documented in neurodegeneration. The abundance of these states is heavily dependent on additional factors including age, sex, and genetic context (15,22-25). However, the effect of MetS on microglial states has yet to be determined. To address this, we investigated MetS-induced changes in microglial transcriptional states using scRNA-seq. We utilized combinations of genetic and environmental models relevant to MetS contrasting New Zealand Obese (NZO/HlLtJ) mice, a polygenic model of obesity (26-28), with the commonly used C57BL/6J (B6J) mice fed a high fat/high sugar diet (HFD). Microglia scRNA-seq suggested MetS resulted in reduced microglial responsiveness in NZO mice relating to vascular interactions and function. In support of this, NZO but not B6J mice exhibited fibrin deposition within the cerebrovasculature. NZO mice also showed reduced microglia responses to an acute lipopolysaccharide (LPS) challenge compared to B6J mice, and larger amyloid-{beta} plaques and increased microhemorrhages in the presence of APP/PS1 transgenes (NZO.APP/PS1) compared to B6J.APP/PS1 mice. In summary, MetS appeared to impair microglial plasticity, which could potentially drive neurodegenerative disease pathology. ResultsO_ST_ABSMetabolic syndrome (MetS) caused subtle changes in abundances of microglia states.C_ST_ABSTo determine how MetS affects microglia, we fed NZO and B6J mice a HFD or a standard diet (SD) from 2-9 months of age (mo). NZO mice showed signs of MetS at 2mo (Figure 1A-E). At 9mo, NZO mice displayed increased age-and diet-associated metabolic impairments relative to B6J mice, including weight gain, dyslipidemia, high blood pressure, and hyperglycemia (Figure 1F-J). These data indicate that the NZO strain better models complex endophenotypes observed in humans with MetS compared to HFD-fed B6J mice. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/560877v1_fig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@12b3e4eorg.highwire.dtl.DTLVardef@783e5org.highwire.dtl.DTLVardef@1643ec9org.highwire.dtl.DTLVardef@563f76_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Strain-dependent effects of both high-fat diet and aging on characteristics of metabolic syndrome. a. Body weight at 2mo. b. Fasted cholesterol at 2mo. c. Fasted triglycerides at 2mo. d. Hemoglobin A1c (HbA1c) at 2mo. e. Blood pressure at 2mo. f. Body weight of mice fed either SD or HFD from 2-9mo. g. Fasted cholesterol at 9mo. h. Fasted triglycerides at 9mo. i. HbA1c at 9mo. j. Blood pressure at 8mo. In a-e: two-way ANOVA with post-hoc Tukeys test. In a, N=16 NZO (7M,9F), N=23 B6J (11M,12F) mice. In b-c, N=7 NZO (4M,3F), N=8 B6J (4M,4F) mice. In d, N=8 NZO (4/sex), N=9 B6J (4M,5F) mice. In e, N=4M mice/strain. In f, mixed effects model with repeated measures. N=7M NZO (4SD,3HFD), N=9F NZO (4SD,5HFD), N=11M B6J (5SD,6HFD), N=12F B6J (7SD,5HFD). In g-h, N=7M NZO (4SD,3HFD), N=9F NZO (4SD,5HFD), N=10M B6J (5SD,5HFD), N=12F B6J (7SD,5HFD). Dashed lines and represent values > 200mg/dL. In i, N=7M NZO (4SD,3HFD), N=9F NZO (4SD,5HFD), N=10M B6J (4SD,6HFD), N=11F B6J (6SD,5HFD). Red line indicates diabetic HbA1c, gray line indicates prediabetes. In j, N=13M NZO, N=9M B6J mice. All data shown are Mean{+/-}SEM. C_FIG To determine the effects of strain (NZO, B6J) and diet (SD, HFD) on myeloid cell transcriptional states, we performed scRNA-seq of CD11B+ cells (24,29) from brains of 2 and 9mo male and female B6J, B6J HFD, NZO, and NZO HFD mice (Figure 2A). Microglia represented [~]75% of all captured CD11B+ cells in B6J mice, and [~]88% in NZO mice, irrespective of diet (Figure S1A-D) (30,31). The remaining cells consisted mainly of monocytes, macrophages, NK cells, and neutrophils (Figure S1D). Re-clustering only microglia resulted in 18 clusters (Figure S1E-F) with further annotation as: homeostatic (H, clusters: 0-6,9, 13,14, and 16), proliferating (cluster 17), Hexb high (HexB, cluster 10), disease-associated (DAM, cluster 8), Ccl4 Ccl3 high DAM (Ccl4+ Ccl3+ DAM, cluster 7), major histocompatibility enriched (MHC, cluster 16), interferon responsive microglia (IRM, cluster 12) and Klf2 Tcim high (Klf2+ Tcim+, cluster 13) (Figure 2B-D, Figure S1E-F) (11,12,22,24). In contrast to amyloid-(11,12,24) and aging-related (22) studies, MetS (NZO strain and/or HFD) did not cause significant changes in the percentages of DAM, IRM or MHC clusters (Figures 2E, S2A). However, the abundance of DAM, MHC, and Klf2+ microglia changed with age, regardless of strain or diet (Figure 2E, Figure S2A-B). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/560877v1_fig2.gif" ALT="Figure 2"> View larger version (44K): org.highwire.dtl.DTLVardef@606354org.highwire.dtl.DTLVardef@1940d0org.highwire.dtl.DTLVardef@1ee5549org.highwire.dtl.DTLVardef@214d70_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 2.C_FLOATNO Profiling myeloid transcriptional states in a cohort of varying metabolic impairment. a. Experimental design scheme; created with BioRender.com. b. Dimensionality reduction plot (UMAP) of microglia colored by cluster. c. UMAPs of microglia colored by SCT-normalized expression of marker genes. d. Dot plot of marker genes associated with each annotated state. e. UMAP of microglia colored by annotated state. In a-e, 9mo mice: N=3M NZO/diet, N=4F NZO/diet, N=4M B6J/diet, N=3F B6J/diet; 2mo mice: N=3 NZO/sex, N=7 B6J (3M,4F). C_FIG High fat diet altered stress response and cell-cell communication gene expression in NZO, but not B6J microgliaDespite the lack of MetS-induced shifts in microglial states (Figure 2E), we reasoned that MetS may alter gene expression across all microglia. To determine this, we utilized a pseudobulking strategy followed by differential expression analyses, separately assessing the strain (NZOvB6J), and diet (HFDvSD) or aging (9v2mo) effect within each strain (Figure S3A-B)(32,33). First, we determined the HFD effect across both NZO and B6J mice and found that there were 141 differentially expressed genes (DEGs) in all microglia, which were enriched in gene sets associated with cell viability, migration, and proliferation (Figures 3A, S4A-C). However, when HFD effects were analyzed separately for each strain, B6J microglia did not show a substantial response to chronic HFD exhibiting only 2 DEGs in comparison with >300 DEGs in NZO microglia (Figure 3A,S4D). These NZO DEGs were associated with endothelial and immune cell signaling, and heat shock stress (Figure 3B-D). Together, these data suggest HFD caused a significant stress response in microglia in NZO, but not B6J mice. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/560877v1_fig3.gif" ALT="Figure 3"> View larger version (37K): org.highwire.dtl.DTLVardef@62f734org.highwire.dtl.DTLVardef@153b29eorg.highwire.dtl.DTLVardef@1f3a9e7org.highwire.dtl.DTLVardef@17b723_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 3.C_FLOATNO HFD promotes stress-responses and alters cellular communication pathways in NZO but not B6J microglia. a. Bar chart summarizing the number of DEGs associated with HFDvSD across all microglia, within NZO microglia alone, and within B6J microglia alone. b. IPA graphical summary of NZO HFDvSD microglia DEGs. c. Violin plots of selected HFDvSD NZO DEGs. d. Enrichment GO term plot for NZO HFDvSD microglia DEGs. In a-d, N=3M NZO/diet, N=4F NZO/diet, N=4M B6J/diet, N=3F B6J/diet mice. C_FIG Aging differentially effected NZO and B6J microgliaTo assess transcriptional programs in NZO compared to B6J, we first identified DEGs comparing NZO to B6J microglia at 2 or 9mo. DEGs identified at both ages included genes that have been implicated in regulating microglial and macrophage responses, such as Angptl7, Itgam, and Fcrls (Figure 4A-B,S5A-D) (22,34-36). Interestingly, Apoe, genetic variations in APOE increase risk for AD (37,38), was significantly greater in NZO compared to B6J microglia at both ages (Figure 4C). At 9mo, DEGs were associated with immune responses, vascular interactions, and cell migration (Figure 4D-E), suggesting these processes are perturbed in NZO but not B6 microglia. When microglia states were analyzed separately, differentially expressed genes detected in all microglia were primarily driven by homeostatic microglia, downsampling indicated this was independent of the number of microglia within each state (Figure S5A-B). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/560877v1_fig4.gif" ALT="Figure 4"> View larger version (55K): org.highwire.dtl.DTLVardef@f8758dorg.highwire.dtl.DTLVardef@1ed7c7aorg.highwire.dtl.DTLVardef@874a45org.highwire.dtl.DTLVardef@1d1d36_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 4.C_FLOATNO NZO and B6J microglia exhibit strain- and age- associated transcriptional differences. a. Venn diagram displaying overlap of NZOvB6J DEGs at 2 and 9mo. b. Violin plots of a subset of NZOvB6J 9mo DEGs. c. UMAP plots for NZO and B6J microglia. Colored by SCT-normalized Apoe expression. d. Enrichment GO term plot of 9mo NZOvB6J microglia DEGs. e. IPA graphical summary of 9mo NZOvB6J DEGs. f. Venn diagram of 9v2mo DEGs identified in NZO and B6J microglia. g. Violin plots of selected 9v2mo NZO microglia DEGs. Enrichment GO term plots for 9v2mo B6J (h) or NZO (i) microglia DEGs. 9mo mice: N=7 NZO (3M,4F), N=7 B6J (4M,3F). 2mo mice: N=6 NZO mice (3M,3F), N=7 (3M,4F) B6J mice. C_FIG Next, to better understand how transcriptional programs were influenced by aging, we compared 9 to 2mo microglia from either NZO or B6J mice (Figure 4F). Most aging related transcriptional changes were again detected within the homeostatic microglia state when analyzed separately (Figure S6A-B). Both strains showed aging-associated changes in Itga6, Ctss, Cd48, and antigen processing and presentation pathways (Figure 4F-I). However, NZO microglia exhibited more aging-associated DEGs than B6J including Sparc, Pfkfb3, and Irf8; which have been implicated in regulating synaptic function, glycolysis, and microglial identity respectively (Figure 4F- G) (39-41). In addition, NZO microglia exhibited age-dependent changes in cytotoxicity, wound healing, and circulation pathways (Figure 4I). Interestingly, Ingenuity Pathway Analysis (IPA) of upstream regulators predicted aging-associated upregulation of interferon signaling regulators in B6J microglia, but regulators associated with environmental stress and tissue repair in NZO microglia (Figure S6C-E). NZO microglia displayed increased association with blood vesselsThe expression of genes regulating myeloid-endothelial interactions including Itgam, Ccr1, P2ry12, and Ccr5 (16,35,36,42), was higher in NZO relative to B6J microglia (Figure 4). To probe this further, we performed immunohistochemistry to localize microglia and vasculature within the cortex and hippocampus of 9mo NZO and B6J mice fed SD or HFD. We found that while the numbers of TMEM119+DAPI+ microglia in the hippocampus or cortex did not differ across strains or diets, the percentage of CD31+ area covered by microglia was significantly higher in NZO relative to B6J mice (Figure 5A-E). HFD did not modulate this phenomenon (Figure 5B-E). scRNA-seq analyses predicted fibrin(ogen) to be an upstream regulator of aging-associated DEGs in NZO microglia (Figure 5F). Fibrin is absent in the healthy CNS but can deposit in the perivascular space and within vessels in conditions of stress (38,43,44). We found NZO vessels exhibited peri-vascular and vascular deposition of fibrin in the hippocampus and cortex, while B6J vessels did not (Figure 5G-I). Furthermore, many of these fibrin+ vessels had microglia juxtaposed (Figure 5G). Altogether, these data suggest that vessel stress signals may promote microglia-vascular interactions in NZO mice. O_FIG O_LINKSMALLFIG WIDTH=151 HEIGHT=200 SRC="FIGDIR/small/560877v1_fig5.gif" ALT="Figure 5"> View larger version (83K): org.highwire.dtl.DTLVardef@168d9d3org.highwire.dtl.DTLVardef@b4db8eorg.highwire.dtl.DTLVardef@1ba4f2dorg.highwire.dtl.DTLVardef@a41544_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 5.C_FLOATNO NZO vasculature displays fibrin deposition concomitant with increased coverage by microglia. a. Representative images of TMEM119 and CD31 staining in the hippocampus. Quantification of microglia in the hippocampus (b) and cortex (d). Quantification of the percentage of CD31 colocalized with TMEM119 in the hippocampus (c) and cortex (e). f. IPA upstream regulator analysis of 9v2mo NZO microglia DEGs. g. Representative images of TMEM119, CD31, and fibrin. Insets are high resolution confocal imaging of noted area. Quantification of the percentage of CD31 colocalized with fibrin in the hippocampus (h) and cortex (i). N=8 NZO (2/sex/diet) and N=8 B6J (2/sex/diet) mice. In b-c, independent two sample two-sided t test. In e, h-i, Mann-Whitney test. SD: closed circles, HFD: open circles. Data are presented as Mean{+/-}SEM. C_FIG NZO mice displayed a dampened response to LPSAs NZO microglia appeared to have a reduction in DEGs relevant to immune responses compared to B6J microglia (Figure 4,S5), we sought to probe the responsiveness of NZO microglia to an acute inflammatory challenge. LPS was administered to 4mo NZO and B6J mice, and bulk hemibrain RNA-seq (Figure 6A) was performed. As we suspected from the observed fibrin deposition, NZO animals exhibited altered CNS expression of vascular associated pathways and genes including Edn1, Angpt1, and Serpine1 (Figure 6B-C) even with PBS-treatment. LPS-treated NZO animals also displayed fewer DEGs than LPS-treated B6J mice (Figure 6D). The strain-dependent LPS effects suggested potential differences in microglial responses, as NZO animals displayed no change in Cx3cr1 expression with LPS treatment (Figure 6E). Furthermore, IPA upstream regulator and pathway analyses predicted strain-dependent differences in the LPS induction of genes involving macrophage recruitment, antigen processing and presentation, and aggregation of cells (Figure 6F). These data indicate that compared to B6J, NZO mice display altered microglial responses to not only HFD, but also to an acute insult such as LPS. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/560877v1_fig6.gif" ALT="Figure 6"> View larger version (39K): org.highwire.dtl.DTLVardef@704276org.highwire.dtl.DTLVardef@1b6decorg.highwire.dtl.DTLVardef@cdcb11org.highwire.dtl.DTLVardef@cecc94_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 6.C_FLOATNO NZO animals display increased expression of hemibrain vascular associated genes and an altered central nervous system response to an acute LPS treatment. a. Experimental schematic of CNS responsiveness to LPS. Created with BioRender.com. b. Bar chart of DEGs associated with each comparison. c. Top IPA regulatory effect for NZOvB6J DEGs. d. Venn diagram displaying DEGs associated with the LPS response in each strain. e. Volcano plot of the Strain-by-Treatment interaction effect. Top 10 genes by significance are labeled. Genes are colored by significance. f. IPA graphical summary of the Strain:LPS interaction effect. In a-f, N=3F mice/strain/treatment. C_FIG NZO.APP/PS1 mice displayed larger amyloid plaques and increased incidences of microhemorrhagesMetS has been demonstrated to increase risk for AD and related dementias (2,4,5,45). We hypothesized that this may be due to reduced responsiveness of microglia to AD-relevant insults such as amyloid deposition. To test this, we backcrossed the commonly used amyloid-inducing transgenes (APP/PS1) (46) from B6J to NZO resulting in [~]98.375% congenicity. Unexpectedly, male NZO.APP/PS1 mice exhibited pronounced weight loss, likely associated with exacerbation of T2D (Figure S7A-E). To avoid this confound, we primarily focused our analyses on amyloid-related microglia responses in female mice. At 8mo, in comparison to B6J.APP/PS1 mice, female NZO.APP/PS1 mice exhibited fewer, but larger, amyloid plaques in both the hippocampus and cortex, which were positive for the dystrophic neurite marker LAMP1 (Figure 7A-L). Similar results were observed in several male NZO.APP/PS1 mice that survived to 8mo (Figure S7F-G). There was a small but significant increase in total IBA1+ area in the hippocampus, but not the cortex of NZO.APP/PS1 compared to B6J.APP/PS1 mice (Figure 7F,K). However, the area of plaque covered by microglia were the same in both regions (Figure 7G, L). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/560877v1_fig7.gif" ALT="Figure 7"> View larger version (73K): org.highwire.dtl.DTLVardef@d92194org.highwire.dtl.DTLVardef@12ba91eorg.highwire.dtl.DTLVardef@16bfc74org.highwire.dtl.DTLVardef@8b3e0b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 7.C_FLOATNO NZO mice exhibit fewer, but larger neuritic amyloid plaques, without differences in microglia coverage of plaques. Representative images of 6E10 amyloid- staining with IBA1 (a) or regions of interest with LAMP1 co-staining (b) in the hippocampus. c. Quantification of 6E10+ area in the hippocampus (c) and cortex (h). Quantification of 6E10+ counts in the hippocampus (d) and cortex (i). Quantification of the average size of 6E10+ objects in the hippocampus (e) and cortex (j). Quantification of IBA1+ area in the hippocampus (f) and cortex (k). Quantification of the percentage of 6E10+ area colocalized with IBA1+ area in the hippocampus (g) and cortex (l). m. Representative images of Prussian blue staining with higher magnification image of the inset. n. Quantification of Prussian blue+ microhemorrhages per brain section. SD: closed circles, HFD: open circles. In a-l, N=4F NZO.APP/PS1, N=3F B6J.APP/PS1 mice. Independent two sample two-sided t test. In m-n, N=4/strain/diet WT mice and N=4F NZO, N=3F B6J APP/PS1 mice. Two-way ANOVA with post-hoc Tukeys test. Data are presented as Mean{+/-}SEM. C_FIG Previous studies have shown microglia depletion drives development of cerebral amyloid angiopathy (CAA) (14,47). We wondered whether the reduced responsiveness of NZO microglia would result in increased CAA. However, extensive CAA was not present in NZO.APP/PS1 or B6J.APP/PS1 mice (Figure 7A, S7F-G). Our previous findings of increased vascular stress in NZO (Figures 5-6) suggested blood-brain-barrier integrity may be more compromised in NZO.APP/PS1 compared to B6J.APP/PS1 mice. To assess this, we stained brain tissue with Prussian blue, which marks areas of iron deposition, and found that NZO.APP/PS1 mice displayed increased incidence of microhemorrhages throughout the brain relative to either B6J.APP/PS1 mice or their WT littermate controls (Figure 7M-N). Together, these data support that in a context of MetS, amyloid plaques are larger and the cerebrovasculature is more prone to microhemorrhages, outcomes which may increase risk for diseases such as AD. DiscussionOur work focused on the relationship between MetS and alterations in microglial responses using mice exhibiting varying aspects of MetS (26-28). Consistent with previous reports, NZO mice displayed most aspects of MetS, and this was exacerbated with HFD. However, HFD-fed B6J mice exhibited dysfunctional metabolic measures associated only with pre-diabetes and obesity, identifying NZO mice as a more appropriate model of MetS than B6J HFD mice. We profiled 83,757 microglia and unexpectedly, MetS did not significantly shift the proportion of previously identified microglial states (11,12,22,24). We noted an increase in MHC and Klf2+ transcriptional states and a decrease in both DAM populations between 2 and 9mo. This is consistent with previous reports of age-related alterations in MHC and DAM microglia populations (48). We identified significant strain-specific changes in gene expression programs across all microglia, and within specific states. Strikingly, when we analyzed differential expression within each state, cells within the homeostatic clusters exhibited the greatest number of DEGs even after downsampling. This suggests that these transcriptional changes, while not sufficient to alter state abundances, are potentially altering microglial function. Strain-specific differences in microglia are likely driven by the MetS endophenotypes exhibited by NZO as early as 2mo. NZO microglia exhibited significantly higher expression of Apoe compared to B6J or B6J HFD microglia. ApoE has been linked to the transition to a DAM (or activated response microglia) state (12,23), yet, there was no difference in the abundance of DAM between NZO and B6J mice. One explanation for this paradox may be that the increase in Apoe expression is in response to dyslipidemia, as ApoE has known roles in lipid metabolism (37,49,50). Furthermore, it is possible that NZO microglia are unable to fully transition to a DAM state yet still acquire DAM-like characteristics such as high Apoe expression. In addition to the aging-and HFD-independent strain differences, there were also significant aging-and HFD-dependent strain differences between NZO and B6J microglia. For instance, aging influenced NZO microglia more than B6J microglia through higher numbers of DEGs and a broader range of impacted pathways, including wound healing and cytotoxicity. One recent study suggested that NZO mice display enhanced aging-associated changes within peripheral immune populations --suggesting that NZO were a model of accelerated aging (51) -- and our data also support this possibility. A prevailing signature of both aging-and strain-associated analyses implicated microglia differential interactions with the vasculature. Further exploration through IHC highlighted that regardless of diet, NZO mice exhibited more perivascular microglia than B6J mice. Upstream regulator analysis predicted fibrin may a significant culprit behind aging-associated changes in NZO microglia and fibrin deposition was detected in NZO brains. Fibrin has previously been shown to be neurotoxic (35,36). Fibrin upregulates Hmox1 expression in microglia (36) and upstream regulator analysis predicted activation of HMOX1-dependent inflammatory response signaling pathways when comparing 9mo NZO and B6J microglia. Furthermore, MetS has been associated with increased CCL5, which can recruit immune cells via CCR1/CCR5 to vasculature (52-54). NZO mice exhibit peripheral vascular stress (55), and NZO microglia display higher expression of Ccr1 and Ccr5. Collectively, these data predict MetS may increase vascular stress and fibrin deposition in the CNS, resulting in recruitment of microglia to the cerebrovasculature. This primary endophenotype may than render microglia less responsive to a secondary insult. To test this, we first used an acute LPS treatment and performed bulk RNA-seq on hemibrains. We found increased expression of vascular associated genes in NZO hemibrains relative to B6J hemibrains including Serpine1, Edn1 and Angpt1 which mediate vascular stress and fibrin accumulation (56-58). Further, fewer DEGs were identified in LPS-treated NZO mice compared to LPS-treated B6J. This provides support for decreased responsiveness in NZO microglia. For example, Cx3cr1 did not change in LPS-treated NZO mice. Cx3cr1 expression has been shown to decrease in neurodegeneration (11,12). Interestingly, recently published data suggest aged microglia display a dampened response to LPS, supporting the concept that NZO mice may be a model of MetS-dependent accelerated aging (59). Following acute stimuli, we turned to a more chronic and disease-relevant inflammatory stimulus, amyloid deposition (12,14,23,60). We found that NZO.APP/PS1 mice displayed a significant increase in plaque size, independent of numbers of plaque-associated microglia. Increased plaque sizes may impact larger regions leading to increased likelihood of cognitive dysfunction. Patients displaying MetS have accelerated plaque deposition, and MetS blood biomarkers correlate with the rate of cognitive decline in patients with MCI and dementia (61,62). One possibility for the increased plaque size is in NZO.APP/PS1 compared to B6J.APP/PS1 mice is inefficient plaque compaction or clearance by NZO microglia - that would fit with the model of MetS-dependent reduced responsiveness. A second possibility may be altered activity of insulin degrading enzyme (IDE). In addition to insulin degradation, IDE also contributes to plaque degradation (63). Microglia-specific or brain-wide Ide expression was not changed between NZO and B6J mice, however, a MetS-dependent increase in insulin, requiring degrading by IDE in NZO mice, may result in reduced amyloid-{beta} degradation. Emerging work has implicated microglia in regulating blood flow and closure of injured vascular barriers (16,18,64). Therefore, given the vascular stress and changes to microglia-vascular interactions in NZO mice, we investigated whether NZO.APP/PS1 mice were susceptible to microhemorrhages. NZO.APP/PS1 mice presented with microhemorrhages, which were rare in B6J.APP/PS1 mice or WT littermate controls. Microhemorrhages are more common in AD patients than in control groups and were previously detected in Ob/Ob APP/PS1 mice (65-68). One possible mechanism driving the microhemorrhages in NZO.APP/PS1 mice relates to fibrin deposition. Fibrin is stabilized by amyloid-{beta} potentiating vascular damage and blood-brain-barrier breakdown (38,69). Recent evidence suggests the amount of fibrin coverage of vessels correlates to cerebral microbleeds in patients with CAA (70). Yet, we did not observe extensive CAA in NZO.APP/PS1 mice. It is possible microhemorrhages in NZO.APP/PS1 mice are a result of the inability of microglia to clear fibrin and/or amyloid-{beta} efficiently, alongside potential impairments in wound healing and vascular repair pathways may predispose NZO mice to increased microhemorrhages. Diabetes worsens wound healing and vascular repair pathways in a variety of pathological conditions (71) and NZO microglia exhibit age-associated alterations in wound healing pathways underscoring this possibility. These data provide further evidence that MetS reduces or dampens the responsiveness of microglia. In summary, we have found that MetS in NZO mice fundamentally altered microglia responses even when compared to microglia of HFD-fed B6J mice. NZO microglia displayed age-associated transcriptional changes concomitant with increased perivascular association. These changes were associated with abnormal responses to an acute LPS challenge and chronic amyloid pathology altogether suggesting MetS reduced microglial plasticity. Overall, this also supported the hypothesis of accelerated aging in NZO compared to B6J. This work provides the foundation to investigate the mechanisms by which MetS compromises microglial responses, leading to increased risk for neurodegenerative disease such as Alzheimers disease.

Microglia, which are productively infected by HIV-1, are critical for brain development and maturation, as well as synaptic plasticity. The pathophysiology of HIV-infected microglia and their role in the pathogenesis of HIV-1-associated neurocognitive and affective alterations, however, remains understudied. Three complementary aims were undertaken to critically address this knowledge gap. First, the predominant cell type expressing HIV-1 mRNA in the dorsolateral prefrontal cortex of postmortem HIV-1 seropositive individuals with HAND was investigated. Utilization of a combined RNAscope multiplex fluorescent and immunostaining assay revealed prominent HIV-1 mRNA in microglia of postmortem HIV-1 seropositive individuals with HAND. Second, measures of microglia proliferation and neuronal damage were evaluated in chimeric HIV (EcoHIV) rats. Eight weeks after EcoHIV innoculation, enhanced microglial proliferation was observed in the medial prefrontal cortex (mPFC) of EcoHIV rats, evidenced by an increased number of cells co-localized with both Iba1+ and Ki67+ relative to control animals. Neuronal damage in EcoHIV infected rats was evidenced by pronounced decreases in both synaptophysin and post synaptic density protein 95 (PSD-95), markers of pre-synaptic and post-synaptic damage, respectively. Third, regression analyses were conducted to evaluate whether microglia proliferation mechanistically underlies neuronal damage in EcoHIV and control animals. Indeed, microglia proliferation accounts for 42-68.6% of the variance in synaptic dysfunction. Collectively, microglia proliferation induced by chronic HIV-1 viral protein exposure may underlie the profound synaptodendritic alterations in HIV-1. Understanding how microglia are involved in the pathogenesis of HAND and HIV-1-associated affective disorders affords a key target for the development of novel therapeutics.

While males are more likely to suffer severe outcomes during acute COVID-19, a greater proportion of females develop post-acute sequalae of COVID-19 (PASC) despite similar rates of infection. To identify mechanisms of PASC, mice were infected with SARS-CoV-2 and viral, inflammatory, and behavioral outcomes were evaluated through 84 days post infection. Sex differences were not observed in virus replication or persistence of viral RNA in pulmonary or extrapulmonary tissues in acute or PASC phases. Following recovery from infection, female mice exhibited persistent neurocognitive and behavioral impairments, along with greater frequencies of inflammatory myeloid subsets, neuroinflammation, and dysregulated T cell subsets, including Tregs. Sex differences in inflammation and cognitive phenotypes during PASC were mediated by the presence of two X chromosomes. XX animals independent of chromosome Y presented with neuroinflammation and PASC along with infection-induced upregulation of the X-linked genes Xist and Tlr7 that regulate inflammation and chronic disease outcomes.

Glioblastoma multiforme (GBM), a highly aggressive brain tumor that accounts for approximately 60% of all gliomas and 48% of primary central nervous system malignancies, is incurable and poorly understood, with a median survival of only 15 months after diagnosis. Thus, there is an urgent need to understand GBM pathogenesis in order to develop an effective treatment. Recent research has revealed frequent Alzheimers disease (AD) pathology in the brains of patients with GBM, i.e., amyloid beta (A{beta}) and hyperphosphorylated tau (pTau), indicating that GBM and AD may share some unknown environmental risk. Since chronic periodontitis (CP), and specifically Porphyromonas gingivalis (P. gingivalis), a keystone bacterial pathogen in CP, have emerged as risk factors for both AD and GBM, we investigated whether P. gingivalis gingipain virulence factors could be identified in GBM tissue samples and whether P. gingivalis infection affects glioma cell behavior. Using immunohistochemistry on tissue microarrays (70 GBM cores from 35 patients; 34 cerebral tissue cores from 17 patients), we quantified the presence of arginine-gingipain B (RgpB) and lysine-gingipain (Kgp) antigens. Both gingipains showed significantly elevated staining in GBM samples compared to controls (**p < .01, ****p < .0001, respectively), with Kgp levels notably higher than RgpB within GBM tissue (****p < .0001). In functional assays using U251 glioma cells, P. gingivalis infection induced robust, dose-dependent IL-6 secretion (peaking at MOI 5), increased PD-L1 expression by 30% (*p = .036), and significantly enhanced cell invasiveness (**p < .01) in a viability-dependent manner. These findings demonstrate that P. gingivalis gingipains are present at elevated levels in GBM tissue and that P. gingivalis infection reprograms glioma cells to adopt an immunosuppressive, invasive phenotype through upregulation of the IL-6/PD-L1 axis, suggesting a potential microbial contribution to GBM pathogenesis and immune evasion. Key pointsO_LIGlioblastoma multiforme (GBM) patients have frequent Alzheimers disease (AD) neuropathological changes in the tumor-adjacent cortex, indicating that GBM tumors may share some environmental risk factors with AD. C_LIO_LIThis study identifies gingipain antigens in GBM tissue samples at significantly elevated levels compared to healthy controls, suggesting that P. gingivalis infection may be an environmental risk factor for both AD and GBM. C_LIO_LIIn in vitro experiments, P. gingivalis infection of the human glioma cell line U251 upregulated IL-6 secretion and PD-L1 expression, and significantly increased cell invasiveness compared to uninfected cells. C_LI

Microglia have been implicated in neurodegeneration, though their role remains unclear, as microglia can perform protective functions or promote neuroinflammation. Numerous studies have found that the transcriptome state of microglia can indicate where they lie along this continuum. To understand regulation of microglia transcriptome state, we considered the transcription factor PPAR8, because it is highly expressed in microglia and is a therapeutic target for Alzheimers disease, (AD) a neurodegenerative disorder characterized by progressive memory loss where microglia dysfunction is involved. When we delineated the microglia transcriptome in mice treated with PPAR8 agonist, we noted that PPAR8 activation blunted expression of inflammatory mediators and migration-enhancing genes, while boosting phagocytic genes. We then examined PPAR8 function in induced transcription factor (iTF) microglia-like cells, and confirmed PPAR8 agonism increases phagocyte function while reducing pro-inflammatory cytokines and migration. To understand PPAR8 regulation upon CNS insult, we exposed iTF-microglia to apoptotic neuron debris and defined six microglia transcriptome states as a function of PPAR8 activation, and observed PPAR8 agonism can shift microglia out of a homeostatic state to a primed, disease-associated microglia-like state. As PPAR8 agonism opposed gene expression favored by PU.1, a critical transcription factor in microglial inflammation and AD pathogenesis, we examined their relationship, documented a physical interaction, and found evidence for transrepression. Finally, we tested PPAR8 agonism in Huntingtons disease and tauopathy mice, and demonstrated PPAR8 could decrease neuroinflammation in vivo. These findings suggest that PPAR8 agonist therapy may mitigate microglial dysfunction by restoring beneficial functions, while suppressing detrimental inflammation.

Neurotropic alphaviruses such as Venezuelan equine encephalitis virus (VEEV) are critical human pathogens that continually expand to naive populations and for which there are no licensed vaccines or therapeutics. VEEV is highly infectious via the aerosol route and is a recognized weaponizable biothreat that causes neurological disease in humans. The neuropathology of VEEV has been attributed to an inflammatory immune response in the brain yet the underlying mechanisms and specific immune cell populations involved are not fully elucidated. This study uses single-cell RNA sequencing to produce a comprehensive transcriptional profile of immune cells isolated from the brain over a time course of infection in a mouse model of VEEV. Analyses reveal differentially activated subpopulations of microglia, including a distinct type I interferon-expressing subpopulation. This is followed by the sequential infiltration of myeloid cells and cytotoxic lymphocytes, also comprising subpopulations with unique transcriptional signatures. We identify a subpopulation of myeloid cells that form a distinct localization pattern in the hippocampal region whereas lymphocytes are widely distributed, indicating differential modes of recruitment, including that to specific regions of the brain. Altogether, this study provides a high-resolution analysis of the immune response to VEEV in the brain and highlights potential avenues of investigation for therapeutics that target neuroinflammation in the brain. Author SummaryVenezuelan equine encephalitis virus (VEEV) causes brain inflammation in both animals and humans when transmitted by mosquito bite or infectious aerosols. The mechanisms underlying disease caused by VEEV, including the role of the immune response in brain pathology, are not well understood. Here we performed a comprehensive assessment of the immune response to VEEV in the brain over time using two advanced sequencing techniques. Following infection, immune cells infiltrate the brain in a sequential fashion and display different activation profiles. Different types of immune cells also display strikingly different spatial patterns throughout the brain. This study provides the most comprehensive description of the immune response to VEEV in the brain performed to date and advances our understanding of immune-driven neuropathology and identification of therapeutic targets.

Traumatic brain injury (TBI) is a leading cause of long-term neurological dysfunction, with secondary neuroinflammation playing a pivotal role in disease progression. Microglia, the brains resident immune cells, respond to injury in a spectrum of activation states, influencing neuronal survival and cognitive recovery. While past studies have broadly examined neuroinflammatory responses following TBI, the influence of injury approach--specifically, anterior vs. posterior impact--on microglial activation and neuronal damage remains unexplored. This study investigated how the site of controlled cortical impact (CCI) in a moderate TBI model alters neuroinflammatory responses and neuronal vulnerability, providing key insights into coordinate-specific mechanisms of injury.

Neuroinflammaging, a moderate, chronic, and sterile inflammation in the hippocampus, contributes to age-related cognitive decline. Neuroinflammaging comprises the activation of the nucleotide-binding domain, leucine-rich repeat family, and pyrin domain-containing 3 (NLRP3) inflammasomes, and the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway that triggers type 1 interferon (IFN-1) signaling. Studies have shown that extracellular vesicles from human induced pluripotent stem cell-derived neural stem cells (hiPSC-NSC-EVs) contain therapeutic miRNAs that can alleviate neuroinflammation. Therefore, this study examined the effects of late middle-aged (18-month-old) male and female C57BL6/J mice receiving two intranasal doses of hiPSC-NSC-EVs on neuroinflammaging in the hippocampus at 20.5 months of age. Compared with animals receiving vehicle treatment, the hippocampus of animals receiving hiPSC-NSC-EVs exhibited reductions in astrocyte hypertrophy, microglial clusters, and oxidative stress, along with elevated expression of antioxidant proteins and genes that maintain mitochondrial respiratory chain integrity. Moreover, hiPSC-NSC-EVs therapy decreased the levels of various proteins involved in the activation of the NLRP3 inflammasome, p38/mitogen-activated protein kinase, cGAS-STING-IFN-1, and Janus kinase and signal transducer and activator of transcription signaling pathways. Furthermore, in vitro assays using genetically engineered RAW cells and hiPSC-NSC-EVs, with or without targeted depletion of specific miRNAs, demonstrated that miRNA-30e-3p and miRNA-181a-5p, both present in hiPSC-NSC-EVs, can significantly inhibit the activation of the NLRP3 inflammasome and the STING pathway, respectively. Additionally, single-cell RNA sequencing conducted 7 days post-treatment revealed that hiPSC-NSC-EVs induce widespread transcriptomic changes in microglia, including increased expression of numerous genes that enhance oxidative phosphorylation and reduced expression of abundant genes that drive multiple proinflammatory signaling pathways. These changes mediated by hiPSC-NSC-EVs were also associated with improved cognitive and memory function. Thus, intranasal hiPSC-NSC-EVs therapy in late middle age can effectively diminish proinflammatory microglial transcriptome and signaling cascades that drive neuroinflammaging in the hippocampus, contributing to better brain function in old age.

Parechovirus ahumpari 3 (HPeV-3), is among the main agents causing severe neonatal neurological infections such as encephalitis and meningitis. However, the underlying molecular mechanisms and changes to the host cellular landscape leading to neurological disease has been understudied. Through quantitative proteomic analysis of HPeV-3 infected neural organoids, we identified unique metabolic changes following HPeV-3 infection that indicate immunometabolic dysregulation. Protein and pathway analyses showed significant alterations in neurotransmission and potentially, neuronal excitotoxicity. Elevated levels of extracellular glutamate, lactate dehydrogenase (LDH), and neurofilament light (NfL) confirmed glutamate excitotoxicity to be a key mechanism contributing to neuronal toxicity in HPeV-3 infection and can lead to apoptosis induced by caspase signaling. These insights are pivotal in delineating the metabolic landscape following severe HPeV-3 CNS infection and may identify potential host targets for therapeutic interventions.

CommentaryElucidating how normal aging increases vulnerability to neurodegeneration remains a major gap in our understanding of disease risk and progression. The dorsal striatum serves as the primary input nucleus of the basal ganglia and is a key region implicated in multiple neurodegenerative diseases (NDDs) (1). In Colon et al. 2025 (2), we examined the impact of normal aging on neuroinflammatory signaling and perineuronal net (PNN) homeostasis within the dorsal striatum. We observed age-associated shifts in the inflammatory landscape and evidence of increased microglial activation, yet PNN homeostasis was largely preserved (2). PNNs are highly organized extracellular matrix (ECM) specializations that preferentially enwrap the soma and proximal dendrites of fast-spiking GABAergic parvalbumin (PV) interneurons, where they contribute to the regulation of synaptic plasticity and provide protection against oxidative stress (3,4). Building on these findings, we developed a working hypothesis to explain the apparent preservation of PNN homeostasis despite an aging-associated pro-inflammatory environment. The shift toward a pro-inflammatory milieu, together with increased gliosis and phagocytic activity, would be expected to impact the maintenance and integrity of perineuronal nets. The observed increase in phagocytosis-related markers may reflect microglia-directed activity as well as contributions from additional central nervous system (CNS) cell populations. Microglia are specialized embryonic-derived myeloid cells that serve as the resident immune cells of the brain and contribute to PNN homeostasis under physiological conditions (5). In Colon et al. 2025, we observed evidence of microgliosis (e.g., morphological changes, Iba1, Trem2) along with elevated expression of markers associated with phagocytosis (e.g., Cd68) and extracellular matrix-modifying proteases (e.g., Mmp9, Adam17) capable of cleaving key PNN components (2). Importantly, Cd68 expression is not exclusive to microglia and has been detected in brain infiltrating macrophages, reactive astrocytes, and neutrophils during inflammation (6-8). Thus, increased Cd68 levels may not solely reflect microglial phagocytic activation but may also reflect astrocyte reactivity and phagocytic phenotypes. Furthermore, astrocytes are the most abundant glial cell in the brain, and they play a major role in maintaining CNS homeostasis by regulating extracellular neurotransmitter concentrations, providing metabolic support, contributing to the synthesis and remodeling of PNN components, and modulating neuronal communication through their involvement in the tetrapartite synapse (9-12). Astrocytes can also phagocytosis microglial debris, myelin, and synapses (7). To better define the cellular source of phagocytic activity and its relationship to PNN remodeling in aging, we performed immunostaining for microglia (Iba1+), astrocytes (GFAP+), phagolysosomal activity (CD68+), and PNNs using Wisteria floribunda agglutinin (WFA+), enabling us to assess the spatial relationship between phagocytosis and PNN components.

SARS-CoV2, severe acute respiratory syndrome coronavirus 2, is frequently associated with neurological manifestations. Despite the presence of mild to severe CNS-related symptoms in a cohort of patients, there is no consensus whether the virus can infect directly brain tissue or if the symptoms in patients are a consequence of peripheral infectivity of the virus. Here, we use long-term human stem cell-derived cortical organoids to assess SARS-CoV2 infectivity of brain cells and unravel the cell-type tropism and its downstream pathological effects. Our results show consistent and reproducible low levels of SARS-CoV2 infection of astrocytes, deep projection neurons, upper callosal neurons and inhibitory neurons in 6 months human cortical organoids. Interestingly, astrocytes showed the highest infection rate among all infected cell populations that led to increased presence of reactive states. Further, transcriptomic analysis revealed overall changes in expression of genes related to cell metabolism, astrocyte activation and, inflammation and further, upregulation of cell survival pathways. Thus, local and minor infectivity of SARS-CoV2 in the brain may induce widespread adverse effects and may lead to resilience of dysregulated neurons and astrocytes within an inflammatory environment.

Glia antigen-presenting cells (APCs) are pivotal regulators of immune surveillance within the retina, maintaining tissue homeostasis and promptly responding to insults. The intricate mechanisms underlying their local coordination and activation remain unclear. Our study integrates an animal model of retinal injury, retrospective analysis of human retinas, and in vitro experiments to elucidate insights into the pivotal role of antigen presentation in neuroimmunology during retinal degeneration, uncovering the involvement of various glial cells, notably Muller glia, and microglia. Glial cells act as sentinels, detecting antigens released during degeneration and interacting with T-cells via MHC molecules, which are essential for immune responses. Microglia function as APCs via the MHC class II pathway, upregulating key molecules such as Csf1r and cytokines. In contrast, Muller cells act as atypical APCs through the MHC class I pathway, exhibiting upregulated antigen processing genes and promoting a CD8+ T-cell response. Distinct cytokine signaling pathways, including TNF- and IFN, contribute to the immune balance. Human retinal specimens corroborate these findings, demonstrating glial activation and MHC expression correlating with degenerative changes. In vitro assays also confirmed differential T-cell migration responses to activated microglia and Muller cells, highlighting their role in shaping the immune milieu within the retina. These insights emphasize the complex interplay between glial cells and T-cells, influencing the inflammatory environment and potentially modulating degenerative processes. In summary, our study emphasizes the involvement of retinal glial cells in modulating the immune response after insults to the retinal parenchyma. Thus, unraveling the intricacies of glia-mediated antigen presentation in retinal degeneration is essential for developing precise therapeutic interventions for retinal pathologies.

Group A Streptococcus (GAS) infections can cause neuropsychiatric sequelae in children due to post-infectious encephalitis. Multiple GAS infections induce migration of Th17 lymphocytes from the nose into the brain, which are critical for microglial activation, blood-brain barrier (BBB) and neural circuit impairment in a mouse disease model. How endothelial cells (ECs) and microglia respond to GAS infections, and which Th17-derived cytokines are essential for these responses are unknown. Using single-cell RNA sequencing and spatial transcriptomics, we found that ECs downregulate BBB genes and microglia upregulate interferon-response, chemokine and antigen-presentation genes after GAS infections. Several microglial-derived chemokines were elevated in patient sera. Administration of a neutralizing antibody against interleukin-17A (IL-17A), but not ablation of granulocyte-macrophage colony-stimulating factor (GM-CSF) in T cells, partially rescued BBB dysfunction and microglial expression of chemokine genes. Thus, IL-17A is critical for neuropsychiatric sequelae of GAS infections and may be targeted to treat these disorders.

Excess lipid droplet (LD) accumulation is associated with several pathological states, including Alzheimers disease (AD). However, the mechanism(s) by which changes in LD composition and dynamics contribute to pathophysiology of these disorders remains unclear. Apolipoprotein E (ApoE) is a droplet associated protein with a common risk variant (E4) that confers the largest increase in genetic risk for late-onset AD. E4 is associated with both increased neuroinflammation and excess LD accumulation. In the current study, we sought to quantitatively profile the lipid and protein composition of LDs between the neutral E3 and risk variant E4, to gain insight into potential LD-driven contributions to AD pathogenesis. Targeted replacement mice expressing human E3 or E4 were injected with saline or lipopolysaccharide (LPS), and after 24 hours, hepatic lipid droplets were isolated for proteomic and lipidomic analyses. Lipidomics revealed a shift in the distribution of glycerophospholipids in E4 LDs with a concomitant increase in phosphatidylcholine species, and overall, the baseline profile of E4 LDs resembled that of the LPS-treated groups. Quantitative proteomics showed that LDs from E4 mice are enriched for proteins involved in protein/vesicle transport but have decreased levels of proteins involved in fatty acid {beta}-oxidation. Interestingly, proteins associated with LDs showed substantial overlap with previously published lists of AD postmortem tissue and microglia omics studies, suggesting a potential role for LDs in modulating AD risk or progression. Given this, we exposed primary microglia from the same E3 or E4 mice to exogenous lipid, inflammatory stimulation, necroptotic N2A cells (nN2A), or a combination of treatments to evaluate LD formation and its impact on the cells immune state. Microglia from E4 mice accumulated more LDs in every condition tested - at baseline and following addition of fatty acids, LPS stimulation, or nN2As. E4 microglia also secreted significantly more cytokines (TNF, IL-1{beta}, IL-10) than E3 microglia in the control, oleic acid, and nN2A treatment conditions, yet showed a blunted response to LPS. In sum, these results suggest that E4 microglia accumulate more LDs compared to E3 microglia and that E4 is associated with a basal LD composition that resembles a pro-inflammatory cell. Together with the high overlap of the LD proteome with established AD-associated datasets, these data further support the idea that alterations in LD dynamics, particularly within microglia, may contribute to the increased risk for AD associated with APOE4.

IntroductionThe major histocompatibility complex class II (MHC-II) pathway is central to adaptive immunity and immune tolerance, and age-related erosion of these mechanisms is increasingly recognized as a driver of chronic neuroinflammation. The HLA-DRB1*15:01 allele--the strongest genetic risk factor for multiple sclerosis in Caucasians--has been implicated in shaping pathogenic CD4 T-cell responses and broader neuroimmune vulnerability, yet how this allele modulates age- and sex-dependent neuroimmune processes within the central nervous system (CNS) remains poorly defined. MethodsWe investigated the impact of HLA-DRB1*15:01 expression using a humanized mouse model (HLA mice) and wild-type (WT) controls. Male and female mice were analyzed at 6, 9, and 15 months of age, with endocrine stratification in females. Behavioral testing, flow cytometry, immunofluorescence, and multiplex cytokine analyses were used to assess cognitive performance, glial activation and oxidative stress, astrocyte-microglia IL-3/IL-3R signaling, endothelial activation, selective immune cell accumulation at CNS borders, tissue organization, and hippocampal cytokine profiles. ResultsHLA mice developed age- and sex-dependent cognitive impairment, most pronounced in aged females. HLA-DRB1*15:01 expression promoted progressive microglial activation, characterized by increased CD14 and CD68 expression, elevated mitochondrial oxidative stress, altered astrocyte phenotypes, and enhanced IL-3/IL-3R signaling. Hippocampal axonal and myelin organization was disrupted in aged HLA mice, and this disruption was spatially associated with increased microglial presence. At CNS interfaces, HLA mice exhibited selective immune remodeling, including increased accumulation of CD4 T cells and NK1.1CD3 natural killer T (NKT) cells, particularly in females, accompanied by endothelial activation, as evidenced by elevated ICAM-1 and E-selectin expression. Hippocampal cytokine profiling revealed selective, sex-biased alterations, including increased IL-12p70 and reduced IL-10 and IL-2, without broad induction of classical inflammatory cytokines. ConclusionTogether, these findings demonstrate that HLA-DRB1*15:01 drives a coordinated, age- and sex-dependent neuroinflammatory program linking behavioral dysfunction, glial activation and oxidative stress, selective immune cell recruitment, endothelial activation, tissue remodeling, and targeted cytokine imbalance. This integrated phenotype provides mechanistic insight into how this major MS risk allele confers vulnerability to chronic neuroinflammation during aging, with heightened impact in females.

People living with HIV suffer multiple comorbid conditions related to chronic inflammation at increased rates compared to the general population, even when on effective antiretroviral therapy. In particular, current data indicate that the increased incidence and severity of neurocognitive impairment (NCI) are associated with unresolved neuroinflammation. Attempts to treat NCI in people living with HIV by reducing inflammation have thus far been unsuccessful, suggesting that a more mechanistic understanding of inflammatory processes in the CNS during HIV is necessary. Here, we use iPSC-derived microglia (iMg) and astrocytes (iAst) to model HIV infection in the CNS. We show that our iMg robustly express markers associated with microglial identity and are susceptible to HIV infection, but exhibit lower HIV replication rates and weaker immune response to HIV challenge compared to monocyte-derived macrophages. Coculture of iAst with iMg leads to a much stronger pro-inflammatory immune response, and, surprisingly, a robust increase in rates of HIV replication. Increased replication in iMg/iAst cocultures is associated with higher levels of multiple pro-inflammatory cytokines, including TNF, which is produced by iAst upon exposure to HIV-infected iMg. Addition of exogenous TNF to iMg during HIV infection is also sufficient to increase rates of replication, and neutralization of TNF via adalimumab/Humira treatment in iMg/iAst cocultures reduces replication. Blocking NF-kB signaling with iKK inhibitor Bay-11-7082 (Bay-11) demonstrates that increased HIV replication in iMg/iAst cocultures is due to increased NF-kB activity. Finally, we show that in HIV-infected iMg there is movement of lysosomes to the periphery of the cell membrane and release of lysosomal content into the extracellular space, suggesting that this dysregulated lysosomal flux could further contribute to the pro-inflammatory microenvironment. We propose that this altered lysosomal trafficking and increased cytokine production drives a pro-inflammatory phenotype in glia and represents a potential source of unresolved neuroinflammation in people living with HIV.