HIV-1 infection of human microglia activates inflammatory pathways associated with HIV-associated neurocognitive disorders (HAND)

HIV-1 infection of human microglia activates inflammatory pathways associated with HIV-associated neurocognitive disorders (HAND) | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results HIV-1 infection of human microglia activates inflammatory pathways associated with HIV-associated neurocognitive disorders (HAND) Jason E. Hammonds , Roy Moscona , Kathleen Candor , View ORCID Profile Thomas Hagan , View ORCID Profile Paul Spearman doi: https://doi.org/10.1101/2025.02.13.638028 Jason E. Hammonds 1 Division of Infectious Diseases, Department of Pediatrics, Cincinnati Children’s Hospital and University of Cincinnati , Cincinnati, OH, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Roy Moscona 1 Division of Infectious Diseases, Department of Pediatrics, Cincinnati Children’s Hospital and University of Cincinnati , Cincinnati, OH, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kathleen Candor 2 Immunology Graduate Program, University of Cincinnati and Cincinnati Children’s Hospital, Cincinnati, OH, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Thomas Hagan 1 Division of Infectious Diseases, Department of Pediatrics, Cincinnati Children’s Hospital and University of Cincinnati , Cincinnati, OH, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Thomas Hagan For correspondence: paul.spearman{at}cchmc.org Paul Spearman 1 Division of Infectious Diseases, Department of Pediatrics, Cincinnati Children’s Hospital and University of Cincinnati , Cincinnati, OH, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Paul Spearman For correspondence: paul.spearman{at}cchmc.org Abstract Full Text Info/History Metrics Preview PDF Abstract HIV-associated neurocognitive disorders (HAND) cause significant dysfunction among people living with HIV. Microglia are the primary immune cells of the central nervous system and are readily infected by HIV. Microglia are thought to contribute to neuroinflammation and cognitive dysfunction in neurodegenerative diseases such as Alzheimer’s Disease and are likely to play an important role in the pathogenesis of HAND. In order to identify pathways that may contribute to neuropathogenesis in HAND, we infected induced pluripotent stem cell-derived microglia (iMG) with HIV and defined gene expression changes over an 8-day period. Monocyte-derived macrophages (MDMs) were studied in parallel in order to identify common pathways stimulated in myeloid cells versus the unique aspects of microglia infection. Infection of iMG led to the induction of a robust early inflammatory response triggered within hours of infection, a pattern that differed significantly from that seen in MDMs. Remarkably, gene expression changes in iMG reproduced many of the characteristic genetic signatures previously identified in brain tissues obtained from individuals clinically diagnosed with HAND. Inflammatory activation representing interferon-mediated signaling, TNF/NF-κB, and IL-6/JAK/STAT pathways were particularly prominent over the time course of infection. Interferon-mediated signaling led to enhanced expression of multiple HIV restriction factors, yet viral replication in iMG remained robust. These findings suggest that HIV infection of microglia is the key cellular driver of neuroinflammation in the CNS of HIV-infected individuals. Further studies of infected microglia are likely to aid in understanding the pathogenesis of HAND and in evaluating therapeutic strategies to limit or eliminate HIV-induced neuropathogenesis. Author Summary Persons living with HIV frequently develop debilitating problems with brain function that are collectively known as HIV-associated neurocognitive disorders (HAND). Treatment with antiretroviral drugs to control HIV has largely eliminated the most severe form of HIV-related brain dysfunction, HIV encephalitis, but a significant proportion of HIV-infected individuals receiving antiviral therapy still develop significant problems with cognitive function. Microglia are the major macrophage-like cells of the brain and are highly susceptible to HIV infection. Inflammation in the brain following infection contributes to damage to neurons and is the likely source of decline of brain function. This study used microglia that were derived from induced pluripotent stem cells, termed iMG, to study the kinetics of gene expression changes in HIV-infected microglia, and compared those changes to those seen in monocyte-derived macrophages (MDMs). iMG were found to be easily infected with a macrophage-tropic HIV strain and exhibited a rapid increase in gene expression associated with inflammatory signaling through interferon- and tumor necrosis factor/NF-kappa B-mediated pathways. Remarkably, the patterns of gene expression in microglia strongly overlapped with gene expression signatures derived from brain tissue samples of HIV patients with HAND. The rapid onset and magnitude of inflammation as well as induction of specific HIV restriction factors differed from that seen in infected macrophages/MDMs. These studies reinforce the central role of microglia in the pathogenesis of HAND and provide insights into HIV-microglia interactions that can help direct interventions to reduce inflammation and preserve brain function. Introduction HIV-1 infection remains a significant global health challenge, with an estimated 39.9 million people living with HIV-1 (PLWH) as of 2023 [ 1 ]. The availability and utilization of combination antiretroviral therapy (cART) has significantly lowered the morbidity and mortality associated with HIV infection, shifting HIV-1 infection from a fatal illness to a manageable chronic condition [ 2 – 5 ]. Despite this remarkable advance, cART fails to eradicate long-term tissue reservoirs harboring infectious virus, and HIV-related comorbidities remain a problem even in individuals who successfully control their plasma viral load. The central nervous system (CNS) has distinct biological and pharmacological properties that differentiate it from other tissues [ 6 ]. An important characteristic is the brain parenchyma’s relative isolation from peripheral immune surveillance. Immune defense within the parenchyma is primarily managed by microglia, specialized myeloid cells that reside permanently within brain tissue [ 7 ]. Myeloid cells, including microglia, are susceptible to and permissive for HIV replication, and contribute to tissue reservoirs for the virus [ 8 – 14 ]. HIV infects microglia in the brain at early times following acute infection, likely through infected peripheral blood monocytes and lymphocytes that cross the blood-brain barrier [ 15 ]. Following infection, microglia serve as a source of ongoing HIV replication and spread within the CNS, contributing to inflammation and neurodegeneration, in addition to serving as a long-lived latent reservoir. HIV infection of microglia and resident brain macrophages contributes to neurologic and neurocognitive complications, spanning from subtle neurocognitive difficulties to severe, debilitating dementia [ 16 – 18 ]. Collectively, these conditions are classified as HIV-associated neurocognitive disorders (HAND). The development of cART has significantly lowered the prevalence of HIV-associated dementia (HAD); however, its impact on the other primary forms of HIV-associated neurocognitive disorders (HAND), asymptomatic neurocognitive impairment (ANI) and mild neurocognitive disorder (MND), has been more limited [ 3 , 19 – 21 ]. Even with access to cART, an estimated 20-50% of HIV-infected individuals experience some degree of neurocognitive impairment [ 22 – 24 ]. Microglia normally perform neuroprotective roles by clearing apoptotic cells and releasing neurotrophic factors and growth hormones that support cellular health in brain parenchyma. In contrast, persistent inflammation imparted by chronically activated microglia has been shown to affect neural plasticity and memory degradation, contributing to the pathogenesis of many neurodegenerative disorders [ 25 , 26 ]. In cases of neuroinflammation and neurodegeneration, chronically activated microglia release a variety of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), reactive oxygen species (ROS), and chemokines such as CXCL10 and MCP-1 [ 25 ]. The HAND signature derived from brains of PLWH shares many features with other chronic neurocognitive diseases, including Alzheimer’s disease and multiple sclerosis [ 19 , 20 ]. Research on HIV-related neuropathology in humans has largely been restricted to analyzing brain tissues obtained postmortem. Due to sample degradation prior to collection, high-resolution analyses of microglia activation in response to HIV-infection have not been possible from infected brain tissues. In order to study in detail the response of microglia to HIV infection, we generated and infected induced pluripotent stem-derived microglia (iMG) [ 27 – 29 ] with an M-tropic HIV-1 primary isolate. To provide context, parallel analyses were performed using human whole-blood isolated, primary monocyte-derived macrophages (MDMs) [ 30 , 31 ]. We found marked differences between inflammatory pathway activation induced in iMG vs. MDMs, with an initial response in iMG that was marked by significant type 1 IFN, TNF, IL-6 and IL-1β production, and characterized by a broader and more potent induction of inflammatory pathways over time than that seen in MDMs. The signature elicited in iMG matched remarkably well with the signatures of HAND previously characterized from brains of subjects exhibiting this clinical disorder. Results Gene expression analysis of human iPSC-derived microglia using signature profiles from primary human microglia Human microglia (iMG) were generated following well-established methods first described by the Blurton-Jones laboratory and adapted by our laboratory [ 27 , 29 , 32 ]. Gene expression in iMG was quantified via RNA sequencing and compared against a core transcriptomic profile of primary human adult microglia (AMG) to confirm its relevance as a robust human microglia model [ 27 , 33 – 42 ]. As an initial step, we performed principal component analysis (PCA) of gene expression from iMG alongside AMG [ 33 ], CD14+ monocytes (CD14M) [ 26 , 28 ], MDMs [ 30 ], and an alternative iMG population from the Blurton-Jones laboratory (labelled as iMG_Abud) [ 27 , 28 , 31 ]. In this analysis, both sources of iMG exhibited greater similarity to AMG than the two primary myeloid cell types, MDMs and CD14+ monocytes, across both principal axes of variance ( Fig 1A ). The previously established iMG-Abud gene expression profile clustered with the iMG from the current study, indicating similarity in the transcriptional profiles of the distinct iMG lines. To further analyze similarities and differences in gene expression between microglia and macrophages, gene-level fold changes between both iMG and AMG against MDM were computed ( Fig 1B ). Among genes highly differentially expressed in adult microglia compared to macrophages, there was a strong correlation between expression in ex vivo brain-derived microglia and iMGs (R = 0.58, p < 2.2e−16), indicating a common transcriptional profile that is distinct from MDMs. The top right quadrant in Fig 1B represents genes upregulated in both types of microglia, while the bottom left quadrant shows genes upregulated in MDMs. Genes highly expressed in both iMG and AMG relative to MDMs include those identified in several signature human microglia studies, including OLFML3, SYT6, ACY3, SMARCA1, TAL1, WNT5A, GAREM1, and FCGBP ( Fig 1B )[ 33 , 34 , 36 , 37 , 42 , 43 ]. Despite the shared myeloid lineage of microglia and MDMs, our comparative analysis identified 43 distinct marker genes uniquely or highly differentially expressed (log2FC≥7) in microglia ( Fig 1C ). Hallmark microglia signature genes including P2RY12, OLFML3, CX3CR1, FCGBP and C3 were differentially expressed as expected, yet these genes also had a detectable level of expression in MDMs ( Fig 1C , genes shown in cluster 1). In contrast, we identified novel microglia markers that were distinct from MDMs, including genes related to GO ontology categories of somatodendritic compartment (KCNQ3, SYNDIG1, PCLO, IGF1, GABRA3), cell leading edge (NAV3, ANRTRX1, GABRA3, MCF2L) and postsynapse (SYNDIG1, PCLO, IGF1, GABRA3) ( Fig 1C , cluster 2). These microglia-specific genes are not present in the well-established microglia signatures reported to date, and may represent useful markers for future studies [ 33 , 34 , 36 ]. To better characterize differential gene expression common to microglia versus MDMs, microglia genes with high expression ( Fig 1B scatterplot quadrant 1 genes with log2FC>2), were subjected to over representation analysis (ORA). This analysis revealed several shared enriched GO ontological biological processes (BPs) associated with CNS development and immune function for microglia, which fell into four functional categories: synaptic activity, nervous system development, immune response, and cellular adhesion ( Fig 1D ). These results align with findings from prior foundational studies performed from ex vivo human microglia [ 34 – 36 , 44 ]. Taken together, these analyses support the authenticity of iMG as a model system that can be employed for studying HIV interactions. Download figure Open in new tab Fig 1. iMG resemble primary human microglia and exhibit a transcriptomic signature distinct from human MDMs. (A) Principal component analysis utilizing a core gene expression signature of human microglia [ 33 ]. iMG (pink), iMG_Abud (green)[ 28 ], human adult microglia (AMG, peach) [ 36 ], human monocyte-derived macrophage (MDM, purple), CD14 monocytes (CD14M, olive green) [ 27 ]. (B) Scatter plot of gene expression fold changes between iMG vs MDM and AMG vs MDM. Correlation coefficient shown for genes highly expressed (log2FC>2) in either AMG or iMG vs MDM (absolute log2FC ≥ ±2). (C) Heatmap of microglia marker genes identified by differential expression analysis between human adult microglia [ 36 ], iMG, and MDMs from our study. Microglia signature genes were defined as commonly differentially expressed (FDR<0.05) between both microglia cell types (AMG and iMG) and MDMs (FDR2 for both comparisons (iMG vs MDM and AMG vs MDM). Bars represent a significant level of P values. Red dashed line represents the significant threshold (P<0.05). Response of iMGs to HIV-1 Infection The results above establish a high transcriptional similarity between the iMG model and primary human microglia and distinguish both from the basal transcriptional profile of MDMs. We next sought to comprehensively define differences in gene expression between iMG and MDMs following HIV-1 infection. In order to establish the infection model, iMG and MDMs were infected with the R5 M-tropic HIV-1 isolate BaL at a multiplicity of infection (MOI) ranging from 0.05 to 0.5, and viral production monitored by measurement of supernatant p24 antigen over time. Infection was readily established in iMG, even at the lowest MOI ( Fig 2A ). A consistent release of virus was observed, representing continuous, low-level release commonly exhibited by HIV-1-infected myeloid cell populations. This trend closely mirrored the pattern observed in human MDMs infected with HIV-1 BaL ( Fig 2B ). For comparison of changes in gene expression for the remainder of these studies, an MOI of 0.25 was chosen as the input, with RNA samples harvested on days 0, 1, 2, 4, 6, and 8 for comprehensive gene expression analysis. The study of gene expression was concluded on day 8 in order to minimize effects resulting from virus-induced cytotoxicity that occurred at later timepoints [ 29 ]. As an additional readout of replication, sample reads that mapped to the HIV-1 BaL genome (GenBank: AY713409.1 ) were quantified. Logarithmic increases in normalized Gag and Env counts were observed between days 2 and 8 ( Fig 2C and 2D ), indicating comparable transcription and ongoing replication in iMG and MDMs. Download figure Open in new tab Fig 2. HIV replication drives increasing transcriptional changes in iMGs and MDMs, while iMGs display a distinct gene expression profile. ( A) HIV BaL growth curve spanning a log range of MOIs in iMG as measured by p24 ELISA from harvested cell culture supernatants. (B) HIV BaL growth curve at a range of MOIs from MDMs. Red “F” indicates time points in growth curve where media was replaced with fresh culture media. ( C) Number of reads mapped to env from the HIV BaL genome in iMG and MDM over the experimental time course. (D) Number of reads mapped to gag in iMG and MDMs. (E) Number of differentially expressed genes in iMG and MDM during the 8-day infection time course. (F) Spearman correlation matrix comparing gene-level fold changes in iMG and MDM during HIV infection time course. Global gene expression analysis of HIV-infected iMGs demonstrated an increasing number of differentially expressed genes (DEGs) throughout the infection time course ( Fig 2E ). The total number of infection-induced DEGs in iMG rose from 629 on day 1 to 4719 on day 8. In contrast, the number of DEGs for HIV-infected MDMs was markedly lower, with no DEGs identified on day 1 and a peak of 1060 on day 8 post-infection. As MDMs from repeated experiments represent primary cells derived from multiple donors, while iMG are isogenic, the lower number of DEGs identified in infected MDMs could simply result from increased variance in responses due to genetic diversity. To investigate this, we examined the variance and fold changes of infection-induced DEGs identified from iMG in both cell types. We found that while MDMs did have increased variance in their response (S1A Fig), they also exhibited decreased fold changes in these genes compared to iMG (S1B Fig). This indicates that the difference in DEGs was not solely due to genetic variance in MDMs, but that instead HIV triggers a more robust transcriptional response in iMG compared to MDMs. In order to compare the similarity in responses across cell types and time points post HIV-infection, a correlation matrix was generated via pairwise comparisons of gene-level infection-induced fold changes. The infection correlation matrix generated distinct clusters specific for iMG and MDMs ( Fig 2F ). The fold changes in iMG on day 1 (iMG_HIV d1) did not correlate with those from MDMs at any time point, indicating that responses on day one post-infection were unique to iMGs. The highest correlations between cell types occurred on days 6 and 8 post-infection, suggesting a more similar response at later time points. Furthermore, responses were largely cell type-dependent, rather than demonstrating a conserved temporal response to HIV infection common to both iMG and MDMs. Together, these findings establish that following infection with HIV, viral replication is similar in iMG and MDM cultures, while infection of iMG generates an accelerated transcriptional response that is of higher magnitude and distinct from that seen in MDMs. Genome wide RNA expression analysis of HIV-infected iMG and MDMs The remarkable differences in gene expression between iMG and MDMs following HIV infection were next examined via gene set enrichment analysis (GSEA) using the Hallmark gene set collection from the Molecular Signatures Database (MSigDB) [ 45 , 46 ] ( Fig 3A ). This analysis highlights the early upregulation of inflammatory pathways that is present on day 1 post-infection in iMG and absent in MDMs. Infected iMG upregulated pathways related to IFN responses, TNFα signaling via NF-κB, IL-6/Jak/Stat3 signaling, and inflammatory and complement responses. The early inflammatory response in iMG seen for TNFα, IL-6/Jak/Stat, and inflammatory response modules then diminished by day 2 before returning on day 6 or 8 ( Fig 3A ), suggesting a two-phase response to infection. In contrast, MDMs infected with HIV exhibited no positively enriched gene sets on day 1 post-infection, and their later responses (days 6 and 8) were primarily restricted to interferon signaling pathways. Among the downregulated pathways, it was notable that genes related to cell cycle, including E2F targets and G2M checkpoint, were significantly downregulated at all time points following infection in both iMG and in MDMs. To ensure that the early responses in iMG seen here were not an artifact of the iPSC line chosen for iMG development, a parallel comprehensive analysis using iMG derived from a second iPSC clone, referred to as iMG2, was performed. HIV-infection of iMG2 generated responses that were consistent with those observed in iMG by Hallmark gene set enrichment analysis (S2A Fig) and exhibited broad initial and later temporal upregulation of inflammatory genes consistent with similar kinetic changes that had been observed in iMG (S2B Fig). We conclude that the early inflammatory response in iMG, which differs markedly from inflammatory pathway changes in MDMs, is consistent across a second iPSC source and not merely due to cell selection. Furthermore, aging of cultures did not account for the observed changes. We compared DEGs in infected vs. uninfected iMG or MDM cultures of the same age, and there were no significant differences noted (S3 Fig). Download figure Open in new tab Fig 3. Temporal gene set enrichment analysis demonstrates an early induction of inflammatory responses in HIV-infected iMG. (A) Hallmark gene set enrichment analysis of HIV-infected iMG and MDM. Color represents normalized enrichment score (NES) in significantly enriched gene sets (FDR 2). (B) Temporal changes in expression of the most common genes (included in ≥ 25% of gene sets, log2 FC ≥ 1) from gene sets in cluster 3 of the Hallmark pathway enrichment analysis (panel A). (C) Circos plot of overlap in enriched blood transcriptional modules in HIV-infected iMG and MDMs. Each segment of the circle represents responses in either iMG or MDMs at a single timepoint post-infection. Bars in the outer ring represent significantly enriched modules (FDR < 0.05, absolute NES ≥ 2). Lines connect shared modules exhibiting positive enrichment (NES ≥ 2) in more than one cell type/time point. Inner circle and line colors represent the module functional groupings. The blood transcriptional modules were derived from large-scale network integration of publicly available human blood transcriptomes [ 2 ]. To conduct a more detailed analysis of the early and late inflammatory responses at the gene level, we examined expression changes over the infection time course of genes enriched in two or more pathways from cluster 3 in Fig 3A , and exhibiting an induction of log2 FC>1 in iMG ( Fig 3B ). Infected iMG broadly upregulated IFN-stimulated genes (ISGs) including IFITM1, USP18, ISG15, IFI27, IFI44L, IFIT2, IFIT3, MX1, OASL, SOCS1 AND SOCS3 on day 1 ( Fig 3B ). HIV infection of iMG also induced heightened expression of inflammatory chemokine genes (CCL2, CXCL1, CXCL10) together with multiple genes regulated by TNFα/NF-κB signaling (LAMP3, SOD2, TNFRSF9 AND TNFAIP6). On day 1, iMG genes with the most significant log2 FC increase in gene expression were TNFAIP6 (log2 FC 5.6), IFI44L (4.7), CD38 (4.7), IFI27 (4.6), and LAMP3 (4.3); while 26 additional genes achieved a log2 FC of 2 or greater. On day 8, 21 iMG genes achieved gene expression changes exceeding log2 FC of ≥2, with the greatest changes observed in CXCL10 (log2 FC 9.5), IFI44L (8.6), IFIT2 (7.6), USP18 (7.1) and OASL (6.5). In contrast, HIV infection of MDMs resulted in only a single gene with a log2 FC of 1 on day 1 post-infection (IL7, log2 FC = 1.0) and 16 genes that achieved a log2 FC of ≥2 on day 8 from the same cluster of hallmark pathways. By day 6-8, however, MDMs demonstrated upregulation of a number of IFN-related genes and TNFα/NF-κB-related genes ( Fig 3B , MDM heatmap). Together, these results demonstrate that HIV induces significant inflammatory responses in iMG that occur earlier in the time course and are greater in terms of both magnitude and breadth when compared to MDMs, highlighted by ISGs, inflammatory chemokines, and TNFα/NF-κB signaling. To further depict HIV-dependent temporal transcriptome changes in iMG and MDM, we next performed pathway enrichment using an alternative set of widely-used gene modules from human blood transcriptome datasets, known as blood transcriptional modules (BTMs) [ 47 , 48 ], and examined the overlap across cell types and timepoints via Circos plot ( Fig 3C ). The connections drawn represent only the upregulated modules/pathways, showing shared modules between iMG and MDMs following infection. Upregulated modules were found in common for iMG at day 1 and 2, with increased commonality between day 1 and days 6 and 8. A unique response in iMG is shown in green (signal transduction) and is in common between days 6 and 8. In contrast, no modules were shared between iMG and MDMs until day 6. Common pathways from this set of modules between infected iMG and MDMs on day 6 and day 8 included IFN/antiviral and myeloid activation pathways (pink and purple modules/connecting lines, Fig 3C ). Consistent with the hallmark GSEA, these results illustrate the early and robust hyperinflammatory reactivity of HIV-infected iMG, with a shared late response with MGMs highlighted by IFN-related pathways and pathways indicating myeloid activation. HIV infection induces gene expression changes in iMG that recreate HAND signatures HIV-infected microglia have been implicated in the pathogenesis of HAND. To investigate the relationship between the observed HIV-induced inflammatory responses in iMG and established signatures of HAND, we performed GSEA on the iMG transcriptional response using previously identified HAND genetic signatures defined from postmortem brains of PLWH showing HAND symptoms [ 49 , 50 ] and CNS tissues of SIV-infected non-human primates [ 51 ]. Modules containing genes upregulated in postmortem brain tissue of patients with HAND and HIV encephalitis (HIVE), as well as from brains of SIV-infected non-human primates, were consistently positively enriched in iMG following HIV infection in vitro ( Fig 4A , HAND cluster). The upregulation of genes associated with HAND was apparent at the earliest timepoint following HIV infection of iMG (day 1) and was also present at late time points. Some of the early responses in infected iMG were also positively enriched in published signatures from HIV-uninfected brains that are associated with neurocognitive disorders ( Fig 4A , neurocognitive cluster), including Alzheimers disease (AD>Control) and inflammaging (Aging_inflammatory)[ 52 , 53 ]. We then selected ‘core’ HAND signature genes that were shared by 2 or more HAND modules (a total of 79 genes) and analyzed their expression in HIV-infected iMG across the time course. This analysis provides a clear view of the peak in expression of HAND-related genes during the early (day 1) response, followed by a decline and then a gradual rise from day 4 to day 8 ( Fig 4B ). Shown in red are the ten most highly induced genes on day one from infected iMG. These represent key genes that have previously been indicated as biomarkers of HAND and/or HIVE, including IFI44L, IFI44, ISG15, STAT1, MX1, IFIT3, PARP9 and NLRC5, along with the IFN-induced HIV-1 attachment/transmission factor SIGLEC1, and are all type I ISGs. Download figure Open in new tab Fig 4. Genes linked to HAND are upregulated early and late in HIV-infected iMG. (A) Analysis of gene set enrichment in HIV-infected iMG across 8-day time course using curated microglia disease state modules from the MGEnrichment application [ 3 ] and HAND-associated modules [ 19 , 50 ]. Modules are listed in Supplementary Table 1. Color represents the NES of significantly enriched modules (FDR 2). (B) Line graph depicting the average fold changes of 79 genes found in two or more (≥2) HAND modules from HIV-infected iMG. The genes highlighted in red represent the top 10 genes included in two or more of the HAND modules on day one post-infection. (C) ORA comparing Hallmark pathway enrichment of HAND genes from Borjabad 2011 et al. [ 49 ] and DEGs from HIV-infected iMG. Bars depict the FDR significance levels, while the red dashed line denotes the threshold for significance (FDR < 0.05). (D) Scatter plot of fold changes in upregulated HAND genes from Borjabad et al. [ 49 ] that were also identified on days 1 (x-axis) and 8 (y-axis) post-HIV infection in iMG. HAND-associated genes identified in iMG were colored according to time post-infection at which they were differentially expressed (red = day 8 unique, green = day 1 unique, yellow = common). HAND-related genes were identified as DEGs in our study using FDR < 0.1 criteria. Venn diagram in bottom right corner displays quantification of unique DEGs to either day 1 or day 8 and common DEGs (45.2% common to both day 1 and day 8 post-infection). (E) Distinct expression changes of HAND genes from Borjabad 2011 following HIV infection of iMG. The heatmap illustrates genes that were uniquely upregulated on either day 1 (D1_Unique) or day 8 (D8_Unique) post-infection. Color represents log2 fold change. Next, we performed Hallmark pathway enrichment of the Borjabad HAND signature gene set [ 19 ] and the HIV-infected iMG DEGs from the current study. This analysis revealed multiple co-enriched pathways for HAND and HIV-infected iMG, including interferon alpha responses, complement activation, TNFα / NF-κB signaling, IL6/JAK/STAT3 signaling, hypoxia, inflammatory responses and apoptosis ( Fig 4C ). The striking similarity of signatures between infected iMG and the HAND signature was further assessed by intersecting the genes that were differentially expressed in both datasets. There was significant overlap between the DEGs in the two conditions, with 57% (138/243 genes) of the postmortem HAND signature also induced by HIV infection of iMG, and the overlap increased over time (S4 Fig). We then examined those shared upregulated HAND signature genes present on day 1 post-infection with those expressed later in infection (day 8). A comparison of these shared HAND genes revealed a strong correlation between the early and late expression of this inflammatory signature (R=0.72, Fig 4D ). Among the HAND genes upregulated in HIV infected iMG on day 1 or day 8, the greatest portion (61/127) were common to both timepoints. The initial response displayed a small subset (14/127) of uniquely induced HAND genes, while the later response had a greater unique response (52/127) ( Fig 4D , Venn diagram). Visualization of the expression of these common HAND-associated genes confirmed that while many HAND signature genes were induced in both stages of the response, distinct subsets were present in iMG exclusively at early vs. late times post-HIV infection (green vs red, Fig 4D ). Unique genes for day 1 and day 8 are shown in Fig 4E . The early response unique HAND DEGs included CD14, S100 proteins (SA100A10 and SA100A6), and PIM-1, a kinase involved in HIV-1 latency [ 54 ]. Late unique response genes included those encoding the inflammatory chemokine CXCL16; the cell surface death receptor gene FAS; OAS1, a gene often implicated in HAND pathogenesis [ 55 ]; TRIM22, an ISG that can inhibit HIV transcription and promote latency [ 56 ]; and HERC5, encoding an E3 ligase linked to inhibition of the late stages of HIV replication [ 57 ]. Common genes to the two time points included numerous ISGs, including ISG15, CCL8, IFI44L, MX2, IFITM1, GBP1, GBP2, and SIGLEC1, in addition to STAT1 and TNF superfamily member TNSF13B. Together, these results demonstrate that HIV infection of iMG in vitro was able to recapitulate, in large part, the HAND immune signature derived from postmortem human brain. iMG exhibit a rapid and bi-modal inflammatory reaction following HIV infection HIV infection of microglia may result in inflammatory responses that ultimately are damaging to surrounding glia and neurons and contribute to the development of HAND [ 58 , 59 ]. We therefore sought to better characterize the principle inflammatory pathways that are activated over the course of HIV infection in iMG. Four major pathways as defined in Cluster 3 of Fig 3A were chosen: Inflammatory Response, TNFα signaling via NF-κB, IFNα response, and IL6/JAK/STAT3 signaling. Visualization of the temporal patterns shown as average FCs of genes within each pathway in iMG and MDMs clearly demonstrated the early (day 1) response across all pathways in iMG that was lacking in MDMs ( Fig 5A ). Following the early response, there was a decline in expression of inflammatory genes on day 2-4, followed by a gradual increase that was seen on day 6 and 8 to levels exceeding the day 1 response ( Fig 5A ). In infected MDMs, upregulation of inflammatory pathways was seen only on day 6 or 8, and the magnitude and breadth of the change in expression remained well below that seen in iMG (grey curves, Fig 5A ). Download figure Open in new tab Fig 5. HIV-infected iMG develop rapid hyper-inflammatory responses. (A) Line graphs of average log2FC of all genes present in each inflammation-related pathway across 8-day HIV-infection time course in iMG. Shaded area represents the 95% confidence interval. Responses in MDMs are shown in grey. (B) Heatmap of temporal changes within iMG and MDMs in genes among selected inflammation-related Hallmark gene sets significantly enriched post-HIV infection as presented previously ( Fig 4A ). Top 8 genes with the greatest magnitude of expression changes on day 1 in iMG are represented from each pathway. (C) Bar graphs of gene expression changes for representative genes from each inflammation-related pathway quantified by RT-qPCR for iMG (solid black bars) and MDMs (white, black lined bars) at 1, 3, 6, 12 and 24-hours post-infection with HIV Ba-L. (D) Bar graphs of cell culture supernatant cytokine levels measured for iMG and MDMs at 0.5, 1, 2, 4, 6 and 8-days post-infection with HIV BaL using Luminex-based multiplex assay. Fig 5B depicts the most highly upregulated genes seen on day 1 post-infection from each of these pathways and demonstrates their change in expression over time following HIV infection using heatmaps. Elevation of TNF is a prominent feature of neuroinflammation seen in brain tissues from persons with HAND or HIVE [ 60 ]. NFκB gene expression itself was elevated in HIV-infected iMG, along with TNF receptor superfamily 9 message and the TNF-induced gene TNFAIP6 ( FIG 5B , TNF/NFκB). Elevation of IFNα-induced genes including OASL, MX1, and IFI44L ( Fig 5B , INFα) is consistent with findings from brain tissue from persons with HIV and HAND or HIV, and with elevation of IFNα levels found in SIV-infected brain tissues [ 55 , 61 – 63 ]. The IL-6/JAK/STAT3 signaling pathway ( Fig 5A-B , bottom panel) is a key driver of inflammatory and immune responses. In addition to the known role of STAT1 as already described, we note here that IL7 and IL1β gene products have been associated with neurological impairment in patients with HIV [ 60 , 64 ], and CD38 is frequently seen to be elevated in neuroinflammatory and neurodegenerative conditions although not shown to be causal [ 65 ]. We conclude that the gene clusters shown in Fig 5A and 5B from HIV-infected iMG recapitulate many findings seen in brain tissues from individuals with HIV-associated neurocognitive disorders. To validate the inflammatory signature detected via RNAseq and further delineate the kinetics of the rapid response observed in iMGs, we selected a single representative gene from each cluster to evaluate by RT-PCR (shown in Fig 5C ). Analysis here was performed at multiple times during day 1 post-infection and compared to day 0 (uninfected) RNA levels. From the inflammatory cluster, we found that CXCL10 was rapidly induced in iMG, peaking at 12 hours post-infection, and was greatly compared to infected MDMs. TNF RNA levels were very rapidly induced, as soon as 1-hour post-infection in iMG, while minor change was observed in MDMs. IFNB1 gene expression (representing type 1 IFN) rose and peaked at 6 hours in iMGs, and IL6 expression peaked at 3 hours post-infection ( Fig 5C ). Together, these results verify the difference seen in RNAseq studies in early responses between iMG and MDM, suggesting that there is a very rapid response in iMGs that is not seen in MDMs. Additional validation of the differences at early times post-HIV infection was then performed by measuring cytokine release by ELISA. Production of TNFα was rapid and robust, peaking at 12-24 hours post-infection in iMG ( Fig 5D , left). Protein levels remained higher than those seen in infected MDM over the 8-day sampling period. IL-6 secretion was similar, peaking in the first day and remaining elevated as compared with MDs throughout the time course ( Fig 5D , middle). IL-1β levels in the supernatant peaked on day 1, then were lower on days 2-4 before rising again on days 6 and 8 ( Fig 5D , right). Thus, RT-PCR and ELISA results confirm the early inflammatory response seen from RNAseq analysis in iMG that was lacking in HIV-infected MDM. Receptor and restriction factor expression is rapidly induced in iMG upon HIV infection Although our primary focus in this study was on the genes and pathways in HIV-infected microglia that are likely to provide insights into HAND, we sought also to define the expression of a panel of HIV-relevant genes following infection of iMG. We first analyzed basal expression of receptor CD4, coreceptors CCR5 and CXCR4 and a panel of restriction factor genes in iMG and MDMs ( Fig 6A ). Receptor and coreceptor messages were significant at baseline in both MDMs and iMG, with reduced levels of CXCR4 in iMG as compared with MDMs and compared with the published data for AMG ( Fig 6A ). Expression of CD4 and CCR5 in all cell types was consistent with prior results indicating that receptor and coreceptor levels do not limit infection of microglia with M-tropic, R5 HIV [ 29 ]. Upon HIV infection, HIV receptor and coreceptor genes increased in RNA expression from day 1 to day 8 in iMG and MDMs ( Fig 6B ). Siglec-1 has been shown to play a key role in HIV transmission through trans-infection from both DCs and macrophages to T cells, and contributes to the formation of the virus-containing compartment (VCC) in macrophages [ 31 , 66 , 67 ]. Interestingly, SIGLEC1 expression levels were elevated on day 1 in iMGs (FC of 2.0) but less so in MDMs. SIGLEC1 expression continued to rise in iMG to a log2FC of 4.1 on day 8 ( Fig 6B ). Download figure Open in new tab Fig 6. Infection of iMG induces rapid gene expression changes in HIV-related factors. (A) Steady state (day 0) gene expression levels for a panel of HIV-related factors in iMG, adult microglia (AMG) and MDMs. Color in heatmap represents log2 (normalized counts+1). (B) Gene expression changes in HIV-related factors across 8-day time course of infection at an MOI of 0.25 in iMG and MDMs. Color represents log2 fold change. (C) RT-qPCR quantitation of mRNA fold changes at 1, 3-, 6-, 12-and 24-hours post-infection for SIGLEC1, BST2, MX2 and IFITM3 (technical replicate shown, 2 times biological replicate performed with no magnitude or temporal differences). Restriction factors are innate cellular defense proteins that inhibit the replication of viruses for the benefit of the host [ 68 , 69 ]. We analyzed gene expression changes in a panel of myeloid-relevant restriction factors following infection of iMG and MDMs ( Fig 6A and B ). HIV infection caused a rapid induction of restriction factor gene expression in iMG on day 1, with six restriction factors exhibiting a log2FC greater than 1.5 ( Fig 6B ). Restriction factors exhibiting a log2FC of greater than 3 in infected iMG included MX2, IFITM1, GBP2, AND ISG15. In contrast, no restriction factors in infected MDMs were induced above a log2FC of 0.5 on day 1 or day 2. Expression of most restriction factors increased more gradually in MDMs, so that both iMG and MDMs demonstrated increases on day 6 and day 8 ( Fig 6B ). One of the differences observed between HIV-infected iMG and MDMs was seen with the IFN-inducible factor GBP2, encoding a protein that restricts Env protein processing and incorporation [ 70 ], which was expressed at low levels in iMG at baseline but was rapidly upregulated following infection. It is also notable that SAMHD1, a dNTPase that limits HIV-1 infection of macrophages and resting T cells [ 71 , 72 ], was highly expressed at baseline but rose following infection only in MDMs ( Fig 6B ). BST2 encodes the restriction factor tetherin, a type 2 transmembrane protein that imposes a late restriction on particle release [ 73 ]. BST2 was well expressed at baseline and upregulated at late time points following infection in both iMG and MDM ( Fig 6B ). These data indicate that HIV infection of iMG induces rapid expression of multiple restriction factors, whereas comparative induction in MDM is more delayed. The early induction of a subset of INF-stimulated restriction factor genes in iMG was confirmed by RT-PCR ( Fig 6C ). Increases in BST2 expression were comparable in both cell types, while early increases in iMG were confirmed for SIGLEC1, IFITM3, and MX2 and were lacking in MDMs ( Fig 6C ). Early induction of restriction factors may be important in some contexts, although in these in vitro experiments it did not appear to limit the magnitude of infection when compared to that of MDMs ( Fig 2 ). Discussion The pathogenesis of HAND remains incompletely understood. HIV can be detected in the CNS along with elevated inflammatory markers as early as 8 days following infection [ 74 ]. The initial entry of HIV into the CNS is likely to occur through transit of infected T cells and monocytes, followed by infection of brain resident cells such as perivascular macrophages or microglia [ 75 – 77 ]. Breakdown of the blood-brain barrier through the action of viral proteins has also been suggested to enhance HIV entry into the CNS [ 78 – 80 ], while alternatively transcellular migration of infected CD4+ T lymphocytes across an intact BBB has been reported to contribute to HIV neuropathogenesis [ 81 ]. Inflammation in the CNS is triggered soon after entry of HIV, and the detection of elevated inflammatory markers in CSF and in postmortem brain tissue is a hallmark feature of HAND [ 60 , 64 , 74 ]. Microglia are the major resident myeloid cells of the brain, and are highly susceptible to HIV infection. Infection of microglia has been detected at early time points following infection of rhesus macaques with SIV and resulted in inflammatory or damage-associated microglial phenotypes [ 82 , 83 ]. Microglia have been demonstrated to be infected in human brain even at early times post-infection, harbor replication-competent virus, and are thought to contribute to the latent viral reservoir [ 6 ]. Given the prominent role of microglia in early and chronic HIV infection and their potential to elicit the inflammatory responses that contribute to neuronal damage leading to HAND, we designed this study as a comprehensive analysis of the gene expression changes that occur in microglia following HIV infection, using an iPSC-derived microglial model system that has been previously established to closely represent authentic human microglia [ 27 , 29 , 32 ]. Gene expression studies were conducted in parallel using HIV-infected human MDMs in order to distinguish the features of microglia infection that are held in common with macrophages, and to identify those responses that are unique to microglia. A major finding from this study is the strong overlap of the inflammatory profiles seen in HIV-infected microglia following in vitro infection with an M-tropic HIV strain to those that have come from postmortem HIV-infected brain tissues derived from persons with HAND or HIVE. Beginning even within the first 24 hours of infection, iMG demonstrated upregulation of inflammatory genes in a signature that has been previously identified as characteristic of HAND. Prominent among the clusters of upregulated genes were those involved in IFN signaling, TNFα/NFκB signaling, and the IL-6/JAK/STAT3 pathway, as well as inflammatory chemokines including CXCL8 and CXCL10. While many of the individual genes upregulated in this study have been implicated as mediators of inflammation in HIV-infected brains or from CSF-based studies from PLWH with clinical findings of HAND, the significant overlap with the HAND signature profile is a new finding. The pattern of gene expression following HIV infection of iMG was distinctly different than that of macrophages. Infection of iMG produced a very early upregulation of inflammatory pathways, suggesting an early signaling event that was absent in MDMs, and the magnitude (FC) of differential expression of inflammatory genes was significantly higher than that seen in MDM. The early activation followed by a decline and later rise in expression was found in common to several inflammatory pathways, including IFN-mediated signaling, TNF/NFκB signaling, and IL-6/Jak/Stat pathways. IFN-mediated signaling and TNFα elevation are hallmark findings in HAND. IFNα has been found to be elevated in the CSF of HIV-infected persons with dementia, and has been suggested to have a role in the pathogenesis of HAD [ 84 – 87 ]. Elevated CSF IFNα has also been implicated in more subtle neurocognitive impairment in HIV-infected individuals [ 88 ]. Similarly, TNFα is a key mediator of inflammation that has been found to be consistently elevated in postmortem brain tissue from patients with HAND [ 60 ]. While the early and robust inflammatory profile and strong overlap with HAND/HIVE signatures following infection of iMG are new findings, previous infection studies performed in vitro in microglia have reported upregulation of some of the same inflammatory pathways we highlight in this work. Boreland and colleagues focused on the sustained type 1 IFN-mediated signaling that occurred following HIV infection of iMG [ 89 ]. These investigators reported the upregulation of ISGs IFIT3, MX2, ISG15, IRF7, OAS1, STAT1, CXCL10, IFITM3, and SIGLEC1, consistent with the work shown here. They also showed upregulation of CCL2, TNFα, and IL-1β, while not observing upregulation of IL6. Kong and colleagues examined HIV infection of microglia in a cerebral organoid model, and demonstrated enhanced production of CXCL10, CCL2, and a series of ISGs including MX1, ISG15, ISG20, IFI27, and IFITM3 [ 58 ]. In an iMG/neuron co-culture model, Akiyama and colleagues have shown that HIV infection upregulated CXCL10, CCL2, CCL7 and ISGs SIGLEC1 and RSAD2, and demonstrated that intron-containing RNA export into the cytosol was required for induction of proinflammatory responses [ 90 ]. In a humanized mouse model utilizing iMG and engrafted human PBMCs, Min and colleagues demonstrated elevation of TNFα, IL-6, and CD68 message following HIV infection [ 91 ]. A neuroinflammatory profile was also seen in a tri-culture method employing iMG with stem cell-derived neurons and astrocytes, including elevation of IL-1β, TNFα, and IL-8 [ 92 ]. Thus, there are clear and consistent results indicating that HIV infection of iMG stimulates inflammatory pathways, and that enhanced expression of ISGs and TNFα is a prominent feature. In the current study, we have presented a global picture of gene expression in HIV-1-infected iMG over a time course of acute infection, and utilized a myeloid comparator (MDM) to derive features unique to iMG. The extensive overlap at both early and later time points in HIV-infected iMG with gene expression studies from postmortem brain tissues of individuals with HAND or HIVE provides suggestive evidence that microglia represent the major source of neuroinflammation in humans with HAND, and confirming the relevance of this in vitro infection model. This study highlights the importance of IFN-stimulated pathways as well as TNF/NFκB and IL-6/JAK/STAT signaling in microglia, and demonstrates that inflammatory signaling pathways are triggered earlier and to higher degree than that seen in HIV-infected macrophages. This supports the idea that microglia are uniquely susceptible to HIV-induced inflammatory responses, as compared to tissue macrophages in other compartments, and this may help explain the prominence of HAND in HIV-infected individuals even following institution of cART. A heightened susceptibility to inflammation may require only transient or low levels of viral replication to elicit, leading to ongoing neural damage over time. Chronic inflammation is a major driver of the neuroaging phenotype, contributing to cognitive decline, synaptic dysfunction, and increased vulnerability to neurodegenerative diseases such as HAND and Alzheimer’s Disease. HIV infection of iMG showed significant enrichment for gene sets associated with microglia associated with both a neurodegnerative and senescent or aged phenotype [ 36 , 52 , 53 , 93 , 94 ]. Targeting inflammatory pathways and modulating microglial function may therefore represent promising therapeutic approaches to mitigating the detrimental effects of inflammaging and preserving cognitive function in PLWH. Data in this report also elucidates the expression of HIV-relevant genes in iMG and changes occurring following HIV infection. One of the interesting findings is that SIGLEC1 RNA expression is relatively high at baseline in iMG, and then is rapidly increased following HIV infection. Expression of SIGLEC1 is not merely an artifact of the iMG system, as similar levels have been documented from brain-derived AMG [ 28 ]. The expression of Siglec-1 at the protein level has been reported to be very low or undetectable in microglia within mouse brain outside of the choroid plexus and leptomeninges, but can be induced following damage to the blood-brain barrier [ 95 ]. SIGLEC1+ microglia have been seen in brains of humans with neuroinflammation from multiple sclerosis [ 96 ] and have been implicated in neuroinflammation in a murine model of ceroid lipofuscinosis [ 97 ]. The rapid upregulation of SIGLEC1 expression shown in the present study is of interest, not just as a potential marker of neuroinflammatory microglia phenotype, but also because this lectin plays a major role in HIV-myeloid cell interactions. In DCs and MDM, Siglec-1 specifically captures HIV-1 particles through interactions with gangliosides incorporated onto the viral lipid envelope, leading to coalescence of Siglec-HIV microdomains on the cell surface and subsequent internalization of viral particles to a compartment known as the virus-containing compartment or VCC [ 31 , 66 , 67 , 98 ]. The VCC functions as a holding compartment in these cell types, and can contribute to trans-infection of susceptible cells upon cell-cell contact. The high expression of Siglec-1 in HIV-infected microglia might similarly be expected to facilitate viral capture and spread, potentially contributing to the spread of HIV infection within the CNS. BST2/tetherin is an IFN-induced host restriction factor that arrests the final release of viral particles through the presence of two membrane anchors, a membrane-spanning domain and a GPI anchor, allowing attachment to both the viral lipid envelope and the plasma membrane [ 73 ]. The N-terminus of tetherin contains a hemiITAM motif that upon particle retention and dimerization of tetherin is phosphorylated, recruiting the spleen tyrosine kinase (Syk) and activating NF-κB signaling [ 99 ]. We found that tetherin/Bst2 is upregulated at late time points in HIV-infected microglia. Although the accessory protein Vpu of HIV downregulates tetherin and thus inhibits tetherin-mediated signaling, macrophage-tropic viral isolates including some derived from the CNS often are defective in vpu expression [ 100 ]. It will be interesting in future studies to decipher the contribution of tetherin-mediated signaling to neuroinflammation in HIV-infected microglia, as this may provide a direct link between active viral replication and signaling leading to activation of NF-κB. Results here together with those from other groups highlights the high susceptibity of iMG to HIV-1 infection [ 29 , 58 , 91 , 92 , 101 ]. We found that viral infection and subsequent replication were robust in iMGs even at low MOI, using an R5 M-tropic HIV-1 isolate, indicating that receptor CD4 and coreceptor CCR5 were not limiting. Indeed, at the RNA level and at the surface protein level expression of CD4 and CCR5 are robust in iMG [ 29 ]. AMG from human brain also indicate strong expression of CD4 and CCR5 [ 28 ]. CD4 levels in microglia have been reported by some to be undetectable in fresh brain tissue from HIV-negative individuals, while detectable at low levels in microglia from HIV-infected individuals [ 102 ]. However, others have shown that ex vivo brain-derived microglia express surface CD4 at levels comparable to that of MDM, and shown that CD4 and CCR5 levels are greatly upregulated in response to inflammatory cytokines including IL-1β, IL-6, and TNFα [ 103 ]. Some of the discrepancy in reported CD4 levels in microglia may come from how ex vivo microglia are handled, with activation following isolation potentially leading to higher CD4 expression. It is reasonable to postulate that initial infection of a limited number of microglia may lead to inflammatory responses that result in upregulation of CD4 in surrounding cells and thereby enhance susceptibility to the spread of infection. We found a robust induction of inflammatory signatures in iMG following acute HIV infection that differed from that in MDM. The sensor(s) responsible for the initial rapid induction of innate responses in iMG remain under evaluation. Innate immune sensing of HIV-1 in its target cells can occur through a variety of mechanisms. TGFβ-activated kinase (TAK1) activation can occur at early times following viral entry through TRIM5 interactions with the incoming capsid [ 104 ] or potentially through interaction of the cytoplasmic domain of gp41 [ 105 ]. RIG-I-dependent sensing of HIV-1 genomic RNA secondary structures in the cytosol of infected T cells and macrophages has been reported, leading to robust expression of ISGs [ 106 , 107 ]. cGAS has been shown to sense HIV cDNA in CD4+ T cells and myeloid DCs [ 108 – 110 ]. Plasmacytoid DCs initiate an early type I interferon response stimulated by incoming HIV-1 single-stranded RNA by TLR7 [ 111 , 112 ]. Importantly, McCauley and colleagues reported that viral integration followed by production and nuclear export of intron-containing RNA was required for activation of IFN and inflammatory pathways in T cells and macrophages [ 113 ]; this work was followed by evidence that MDA5 was required for sensing of the intron-containing RNA in myeloid DCs and macrophages [ 114 ]. Akiyama and coworkers reported that nuclear export of intron-containing RNA was required for induction of inflammatory responses in iMG, establishing this mechanism as an important one in this cell type [ 90 ]. The early inflammatory responses observed in our study, together with the data cited above suggest that there may be at least two sensing events in iMG from the present study, one during early stages following virus entry that results in an early (day 1) peak response, followed by a slower rise over the course of HIV replication that is most likely explained by production of intron-containing HIV RNA and sensing through MDA5 as previously reported. However, the sensors responsible for the kinetics of inflammatory responses observed here will need to be directly addressed in future studies. It will be of great interest to define how HIV alters transcriptional regulatory networks in infected microglia in future studies. The epigenetic changes induced by HIV following HIV sensing may lead to sustained changes in a subset of microglia that can contribute to neuroinflammation and neuronal damage during chronic infection or following institution of cART and suppression of viral replication. Inflammation may alter chromatin accessibility of regulatory regions of genes involved in inflammation, potentially leading to a sustained proinflammatory phenotype or a tendency to develop an enhanced response upon restimulation [ 115 ]. Understanding epigenetic changes will require combining comprehensive gene expression analysis with studies of chromatin accessibility and transcription factor binding in relevant infection models. While isolated microglia or iMG can reveal key aspects of the changes occurring following HIV infection, it will also be useful to perform this analysis within the context of 3D organoid models and relevant animal models, and to do so in the context of chronic infection. Materials and Methods Induced Pluripotent Stem Cell (iPSC) Culture and Maintenance iPSC_TFS (hPSCreg: TMOi001-A, female) and iPSC_72.3 (hPSCreg: CCHMCi001-A, male) were used in this study. iPSC_72.3 was derived from primary human foreskin fibroblasts (HFFs) cultured from neonatal human foreskin tissue. Tissues were obtained through the Department of Dermatology, University of Cincinnati. iPSC_72.3 was generated by the CCHMC Pluripotent Stem Cell Facility, approved by the CCHMC institutional review board, and previously characterized [ 116 , 117 ]. iPSC_72.3 demonstrated normal karyotype and differentiated into endoderm, mesoderm, and ectoderm lineages in an in vivo teratoma assay. iPSCs were cultured on Cultrex (R&D Systems) coated plasticware and maintained in mTeSR1 (STEM Cell Technologies) at 37 °C with 5% CO2. Cells were passaged using Gentle Cell Dissociation Reagent (STEM Cell Technologies) according to manufacturer’s instruction. Karyotype and mycoplasma contamination were routinely monitored. Production of Hematopoietic Progenitor Cells (HPCs) Differentiation of iPSCs to HPCs was performed using the STEMdiff Hematopoietic Kit (STEM Cell Technologies). On the day prior to differentiation, iPSCs were passaged with Gentle Cell Dissociation Reagent (STEM Cell Technologies) and aggregates were plated onto several 10 cm 2 dishes at a target density range of 5 – 10 aggregates per cm 2 . On the following day, plates containing between 80 and 100 total colonies (approximately 2 per cm 2 ) were chosen to proceed with differentiation protocol. On day 0, mTeSR1 media supplemented with 0.5 µM Thiazovivin was replaced with 8 mL of basal media A containing a 1:200 dilution of supplement A. On day 2, a half culture basal media A change was performed. On day 3, basal media A was completely removed and replaced with basal media B containing a 1:200 dilution of supplement B. Half media exchanges were performed with basal media B on days 5, 7, 9, 10, 12 and 14. On days 12, 14 and 16 non-adherent cells were collected and centrifuged for 5 min at 300 x g. Clarified supernatants were half media exchanged with basal media B on days 12 and 14 after harvesting HPCs. Pelleted cells were cryopreserved at a density of 1.5 – 2 x 10 6 HPCs per mL of CryoSTOR CS10 (STEM Cell Technologies). iPSC-derived microglia (iMG) Maturation and Culture Frozen HPCs were thawed rapidly by immersion in a 37 °C water bath and cultured immediately in cytokine supplemented iMG maturation media and plated onto 1 mg/mL growth factor reduced (GFR) Matrigel-coated 6-well plates at 1.5 x 10 5 cells per cm 2 or 2.5 x 10 5 per well. HPCs are cultured at a density of 1.5 × 10 5 per cm 2 onto 1 mg/mL GFR Matrigel-coated 6-well plates in 2 mL of iMG media per well (DMEM/F12, 2% B27, 0.5% N2, 2% insulin-transferrin-selenium, 1X MEM Non-Essential Amino Acids Solution, 1X GlutaMAX, 400 µM 1-thioglycerol and 5 µg/mL human insulin). Prior to use, iMG media was supplemented with 100 ng/mL human IL-34, 50 ng/mL TGF-β1 and 25 ng/mL M-CSF (Peprotech). On days 2, 4 and 6 media were supplemented with the addition of 1 mL per well of iMG media with cytokines. On day 8, media was removed leaving behind 1 mL per well of conditioned media. Cells were centrifuged for 5 min at 300×g, media aspirated, and cells resuspended in 1 mL of iMG media with cytokines prior to addition back to wells. On days 10, 12 and 14 media were again supplemented with addition of 1 mL per well of iMG media with cytokines. On day 16, media was removed leaving behind 1 mL per well of conditioned media. Cells were centrifuged for 5 min at 300×g, media aspirated, and cells resuspended in 1 mL of iMG media with cytokines prior to addition back to wells. On days 18, 20 and 22 media were again supplemented with addition of 1 mL per well of iMG media with cytokines. On day 24, cells were resuspended in iMG media supplemented with a five-cytokine cocktail consisting of 100 ng/mL human IL-34, 50 ng/mL TGF-β1, 25 ng/mL M-CSF, 100 ng/mL CD200 and 100 ng/mL CX3CL1 to facilitate final maturation into iMG. On days 26 and 28, cells were fed by the addition of 1 mL per well of iMG media supplemented with a five-cytokine cocktail. By day 28 iMG were considered mature and used for further characterization and RNA seq analyses. Cells were maintained for a maximum of 2 weeks following the 28-day cycle of maturation and conditioned media maintenance described above. Reagents and media formulations used to generate iMG are listed in S1 Table. Human Whole Blood-derived Monocyte Isolation and Monocyte-derived Macrophage (MDM) Maturation and Culture Human peripheral blood mononuclear cells (PMBCs) were isolated from fresh heparinized blood by Ficoll-Hypaque gradient centrifugation. Buffy coats were pooled, and platelets removed by washing repeatedly with phosphate-buffered saline (PBS). Monocyte enrichment was performed by indirect magnetic labeling using the Pan Monocyte Isolation Kit (Miltenyi Biotec) according to manufacturer’s protocol. Enriched monocytes were plated on poly-D-lysine coated plates (Corning) and type 1 rat tail collagen coated 35 mm MatTek dishes (MatTek). Monocytes were maintained in RPMI-1640 supplemented with 10% FBS (Lot No., R&D Systems), 100 µg/mL streptomycin, 100 U/mL penicillin, 2 mM GlutaMAX, and 5 ng/mL GM-CSF (Peprotech). Monocyte cultures were maintained in GM-CSF supplemented media for 7 days to mature cells to MDMs. Media was replaced every 3–4 days. Production of Cell-free Stocks of HIV-1 BaL in PBMC and Infection HIV-1 isolate BaL stocks were prepared as follows: Human peripheral blood mononuclear cells (PBMCs) were isolated from fresh heparinized blood by standard Ficoll-Hypaque gradient centrifugation methods. PBMCs were resuspended in RPMI 1640 supplemented with 20% heat-inactivated fetal bovine serum and 50 μg/mL gentamicin (RPMI 1640-GM). Primary HIV-1 isolates were propagated in PBMCs stimulated with 5 μg/mL phytohemagglutinin (PHA-P, Sigma-Aldrich) and 5% IL-2/T-cell Growth Factor (Hemagen Diagnostics). The IL-2/PHA-stimulated cells were infected using a high-titer seed stock of virus minimally passaged in PBMCs, starting from a viral stock obtained through the NIH AIDS Reagent Program (from Dr. Suzanne Gartner, Dr. Mikulas Popovic and Dr. Robert Gallo). One mL of virus was transferred to the flask containing freshly stimulated PBMCs and incubated overnight at 37 °C in 5% CO2. The cells were washed and resuspended in 30 mL of RPMI-GM supplemented with 5% IL-2/T-cell Growth Factor (Hemagen Diagnostics). Typically, the virus was harvested two times; the first harvest was on day 4 post-infection, with subsequent harvest on day 7. The virus-containing supernatants were collected, clarified by centrifugation, and filtered through a 0.45-μm filter. The virus was then aliquoted into 2 mL sterile screwcap cryovials and stored at − 80 °C. iMGs and MDMs were infected with primary HIV-1 isolate BaL at an MOI of 0.05, 0.1, 0.25 and 0.5 for generating multiple round growth curves over 14 days and an MOI of 0.25 for experiments involving subsequent RNA seq analyses. Titration of HIV-1 in TZM-bl Cells Infectivity of viral stocks were assayed for infectivity using TZM-bl indicator cells (obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH; from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc.). TZM-bl were incubated for 48 h with a serial dilution of HIV-1 BaL stocks, and 100 μL of supernatant was removed from each well prior to the addition of 100 μL of Britelite Plus substrate (Revvity). Measurement of infectivity involved transfer of 150 μL of cell/substrate mixture to black 96-well solid plates and measurement of luminescence. P24 ELISA P24 antigen content of infected cell supernatants were measured using a p24 antigen capture ELISA as described previously [ 1 ]. Briefly, murine anti-p24 CA183 hybridoma supernatants were coated onto MaxiSorp 96-well plates (Nunc) at a dilution of 1:800 in PBS and incubated overnight at 37 C. Plates were washed two times with PBS and blocked for 1 h at 37C with 5% fetal calf serum (FCS) in PBS. Samples were diluted in p24 ELISA sample diluent containing 5% FCS and 0.5% Triton X-100 in PBS and incubated for 2 h at 37 C. Plates were then washed four times with 0.1% Tween 20 in PBS. The detection of bound p24 was determined using HIV-Ig, obtained from NABI through the NIH AIDS Research and Reference Reagent Program, at a dilution of 1:20,000 in p24 ELISA sample diluent for 1 h at 37C. Plates were then washed four times with 0.1% Tween 20 in PBS and incubated with goat anti-Human IgG HRP (ThermoFisher, 31412) at a dilution of 1:5000 in p24 ELISA sample diluent. Plates were washed four times with 0.1% Tween 20 in PBS and colorimetric analysis was performed using the TMB Substrate Kit (Thermo Scientific). 30 min later or less reactions were stopped with100 µL of 4 N H2SO4 and absorbance immediately read at 450 nm using a microplate spectrophotometer. Recombinant p24 was used for the standard curve and sensitive to less than 40 pg of p24. RNA Isolation from iMG and MDM Cellular total RNA was isolated from 5 × 105 cells using the RNeasy Mini Kit (Qiagen, 74104). Briefly, cells were pelleted, washed, and lysed in RLT buffer prior to centrifugation through a QIAshredder cell-lysate homogenizer (Qiagen, 79654). Samples were further DNase treated according to manufacturer’s instructions. RNA quality control was performed using an Advanced Analytical Technologies, Inc. (AATI) Fragment Analyzer and integrity RIN/RQN values exceeded 9.5. For high throughput RNA sequencing, 450 ng of RNA per sample was used to construct RNA seq libraries employing Illumina TruSeq mRNA standard protocols. Each sample was subsequently sequenced using an Illumina NovaSeq 6000 apparatus. Quantitative Real-time PCR (RT-qPCR) RNA was isolated from cell pellets using a RNeasy Mini Kit (Qiagen). The RNA was quantified using a NanoDrop Microvolume Spectrophotometer (Thermo Fisher Scientific) and stored at – 80 °C. cDNA synthesis was performed using 1 µg of RNA and the SuperScript IV VILO Master Mix with ezDNase Enzyme (Thermo Fisher Scientific). qPCR amplification reaction was performed using TaqMan probes in a 10 µl reaction using an Applied Biosystems QuantStudio 3 Real-Time PCR System. The specific genes probed, and TaqMan Assay IDs are listed in supplementary table Reactions were performed in triplicate for all genes. Quantification was performed using the comparative CT method using formula 2 −ΔΔCT . Relative fold expression was normalized to levels in iMGs and MDMs for all genes assayed. Quantitation of Cytokines in Culture Supernatants by ELISA iMG and MDM culture supernatant concentrations of IFNγ, TNFα, IL-6, IL-1β and IL-12p70 were determined by enzyme-linked immunosorbent assay (ELISA) using a Luminex assay (R&D Systems) according to manufacturer’s protocol. Briefly, in a 96 well black plate, 50 µL sample in duplicate was incubated with 50 µL antibody coated beads for 2 hours RT on a plate shaker. Plates were then washed 3 times using the BioTek 405 TS (BioTek) and 50 µL of secondary antibody was added and incubated at room temperature for 1 hour on while shaking. Plates were then washed again, 3 times. Finally, 50 µL of streptavidin-RPE was added directly to the secondary antibody and incubated for 30 minutes at room temperature with shaking. Plates were then washed 3 more times and 100 µL of wash buffer was added. Plates were shaken for 5 minutes and then read using Luminex technology on the Milliplex Analyzer (Millipore Sigma). Concentrations were calculated from standard curves using recombinant proteins and expressed in pg/ml. Data analysis performed by Alyssa Sproles in the Cincinnati Children’s Medical Center Research Flow Cytometry Core. Library Preparation, Sequencing and Analysis of Bulk RNA seq Raw reads from purified RNA of cultured macrophages/microglia were processed using the nf-core/rnaseq pipeline version 3.4 [ https://dx.doi.org/10.1038/s41587-020-0439-x ]. Briefly, adapter sequences and low-quality reads were filtered and trimmed using FastQ and Trim Galore. Filtered reads were mapped to the human GRCh38 reference genome version 108 using STAR [10.1093/bioinformatics/bts635]. Duplicate reads were removed using MarkDuplicates. Next, Salmon [10.1038/nmeth.4197] was used to generate a count matrix from genes with mapped reads. The full description of the pipeline is available at https://nf-co.re/rnaseq . Gene-level counts were filtered to remove genes with a median expression less than 5. Differential gene expression pre- and post-infection timepoints were evaluated using the DESeq2 R package [ https://doi.org/10.1186/s13059-014-0550-8 ]. The fgsea R package [10.1101/060012] was used for gene enrichment analysis using the Hallmark geneset, using genes ranked by the Wald statistic as reported by DESeq2. Heatmaps, dotplots, and PCA plots were generated using ComplexHeatmap [10.1093/bioinformatics/btw313] and ggplot2 [isbn: 978-3-319-24277-4] R packages. Statistical tests were performed in R. A detailed quantification of the figure data is provided in S4 Table. All datasets generated in this study have been uploaded to the GEO database and are included in S5 Table. Published datasets from the GEO database that we utilized in our analysis are also listed in S5 Table. Ethics statement Human blood used for MDM preparation in this study was obtained from volunteer donors and de-identified before investigator handling. Participants provided informed consent under a protocol approved by the Cincinnati Children’s Hospital Institutional Review Board. Code availability A markdown file and the code used to create all applicable figures are available from the Hagan laboratory repository: httys://haganlab.org/hiv_hand/generic.html. The code is available under an open-source MIT license. Supporting Information Captions Table captions S1 Table. Reagents. Excel worksheet containing details of reagents, media formulations, and TaqMan qPCR probes utilized in this manuscript. S2 Table. RNAseq counts. Excel worksheet containing raw and normalized counts for samples analyzed in this manuscript. S3 Table. HIV Infection Time Course Differentially Expressed Genes (DEGs). Lists of differentially expressed genes (DEGs) for HIV-infected iMG and MDM across the infection time course. S4 Table. Figure Quantification. Excel worksheet containing figure quantification data, with individual sheets corresponding to the quantification results for each specific figure panel. S4 Table. GEO Accession Numbers. 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