Cognitive dysfunction persists in 30-50% of chronically HIV-infected individuals despite combination antiretroviral therapy (ART). Although monocytes are implicated in poor cognitive performance, distinct biological mechanisms associated with cognitive dysfunction in HIV infection are unclear. We previously showed that a regulatory region of the interferon regulatory factor-8 (IRF8) gene is hyper-methylated in HIV-infected individuals with cognitive impairment compared to those with normal cognition. Here, we investigated IRF-8 protein expression and assessed relationships with multiple parameters associated with brain health. Intracellular IRF-8 expression was measured in cryopreserved peripheral blood mononuclear cells from chronically HIV-infected individuals on ART using flow cytometry. Neuropsychological performance was assessed by generating domain-specific standardized (NPZ) scores, with a global score defined by aggregating individual domain scores. Regional brain volumes were obtained by magnetic resonance imaging and soluble inflammatory factors were assessed by immunosorbent assays. Non-parametric analyses were conducted and statistical significance was defined as p < 0.05. Twenty aviremic (HIV RNA<50 copies/ml) participants, 84% male, median age 51 [interquartile range (IQR) 46, 55], median CD4 count 548 [439, 700] were evaluated. IRF-8 expression was highest in plasmacytoid dendritic cells (pDCs). Assessing cognitive function, lower IRF-8 density in classical monocytes significantly correlated with worse NPZ_learning memory (LM; rho = 0.556) and NPZ_working memory (WM; rho = 0.612) scores, in intermediate monocytes with worse NPZ_LM (rho = 0.532) scores, and in non-classical monocytes, lower IRF-8 correlated with worse global NPZ (rho = 0.646), NPZ_LM (rho = 0.536), NPZ_WM (rho = 0.647), and NPZ_executive function (rho = 0.605) scores. In myeloid DCs (mDCs) lower IRF-8 correlated with worse NPZ_WM (rho = 0.48) scores and in pDCs with worse NPZ_WM (rho = 0.561) scores. Declines in IRF-8 in classical monocytes significantly correlated with smaller hippocampal volume (rho = 0.573) and in intermediate and non-classical monocytes with smaller cerebral white matter volume (rho = 0.509 and rho = 0.473, respectively). IRF-8 density in DCs did not significantly correlate with brain volumes. Among biomarkers tested, higher soluble ICAM-1 levels significantly correlated with higher IRF-8 in all monocyte and DC subsets. These data may implicate IRF-8 as a novel transcription factor in the neuropathophysiology of brain abnormalities in treated HIV and serve as a potential therapeutic target to decrease the burden of cognitive dysfunction in this population.

COVID-19 can present with lymphopenia and extraordinary complex multiorgan pathologies that can trigger long-term sequela.

Due to differences in human and murine angiotensin converting enzyme 2 (ACE-2) receptor, initially available SARS-CoV-2 isolates could not infect mice. Here we show that serial passaging of USA-WA1/2020 strain in mouse lungs results in "mouse-adapted" SARS-CoV-2 (MA-SARS-CoV-2) with mutations in S, M, and N genes, and a twelve-nucleotide insertion in the S gene. MA-SARS-CoV-2 infection causes mild disease, with more pronounced morbidity depending on genetic background and in aged and obese mice. Two mutations in the S gene associated with mouse adaptation (N501Y, H655Y) are present in SARS-CoV-2 variants of concern (VoCs). N501Y in the receptor binding domain of viruses of the B.1.1.7, B.1.351, P.1 and B.1.1.529 lineages (Alpha, Beta, Gamma and Omicron variants) is associated with high transmissibility and allows VoCs to infect wild type mice. We further show that S protein mutations of MA-SARS-CoV-2 do not affect neutralization efficiency by human convalescent and post vaccination sera.

Chronic HIV infection has long been associated with an increased risk for cardiovascular diseases. The metabolites of the renin−angiotensin system (RAS) such as angiotensin II (AngII) play an important role in regulating blood pressure and fluid dynamics. Cross-sectional analysis of HIV-positive individuals (n = 71, age > 40 years, stable ART > 3 months with HIV viral load < 50 copies/mL) were compared to a similar HIV seronegative group (n = 72). High-resolution B-mode ultrasound images of the right carotid bifurcation (RBIF) and right common carotid artery (RCCA) were conducted to measure the extent of carotid atherosclerotic vascular disease. Plasma RAS peptide levels were quantified using a liquid chromatography-mass spectrometry-based metabolomics assay. RAS peptide concentrations were compared between persons with HIV and persons without HIV, correlating their association with clinical and cardiac measures. Median precursor peptides (Ang(1-12) and AngI) were significantly higher in the HIV-positive group compared to the HIV-negative. Analyses of the patient subgroup not on antihypertensive medication revealed circulating levels of AngII to be four-fold higher in the HIV-positive subgroup. AngII and TNF-alpha levels were found to have a positive association with RCCA, and AngI/Ang(1-12) ratio and TNF-alpha levels were found to have a positive association with RBIF. In both predictive models, AngIII had a negative association with either RCCA or RBIF, which may be attributed to its ability to bind onto AT2R and thus oppose pro-inflammatory events. These results reveal systemic alterations in RAS as a result of chronic HIV infection, which may lead to the activation of inflammatory pathways associated with carotid thickening. RAS peptide levels and cytokine markers were associated with RCCA and RBIF measurements.

The CCR5-specific antibody Leronlimab is being investigated as a novel immunotherapy that can suppress HIV replication with minimal side effects. Here we studied the virological and immunological consequences of Leronlimab in chronically CCR5-tropic HIV-1 infected humans (n = 5) on suppressive antiretroviral therapy (ART) and in ART-naïve acutely CCR5-tropic SHIV infected rhesus macaques (n = 4). All five human participants transitioned from daily combination ART to self-administered weekly subcutaneous (SC) injections of 350 mg or 700 mg Leronlimab and to date all participants have sustained virologic suppression for over seven years. In all participants, Leronlimab fully occupied CCR5 receptors on peripheral blood CD4+ T cells and monocytes. In ART-naïve rhesus macaques acutely infected with CCR5-tropic SHIV, weekly SC injections of 50 mg/kg Leronlimab fully suppressed plasma viremia in half of the macaques. CCR5 receptor occupancy by Leronlimab occurred concomitant with rebound of CD4+ CCR5+ T-cells in peripheral blood, and full CCR5 receptor occupancy was found in multiple anatomical compartments. Our results demonstrate that weekly, self-administered Leronlimab was safe, well-tolerated, and efficacious for long-term virologic suppression and should be included in the arsenal of safe, easily administered, longer-acting antiretroviral treatments for people living with HIV-1. Trial Registration: ClinicalTrials.gov Identifiers: NCT02175680 and NCT02355184.

The host epigenetic landscape rapidly changes during SARS-CoV-2 infection, and evidence suggest that severe COVID-19 is associated with durable scars to the epigenome. Specifically, aberrant DNA methylation changes in immune cells and alterations to epigenetic clocks in blood relate to severe COVID-19. However, a longitudinal assessment of DNA methylation states and epigenetic clocks in blood from healthy individuals prior to and following test-confirmed non-hospitalized COVID-19 has not been performed. Moreover, the impact of mRNA COVID-19 vaccines upon the host epigenome remains understudied. Here, we first examined DNA methylation states in the blood of 21 participants prior to and following test-confirmed COVID-19 diagnosis at a median time frame of 8.35 weeks; 756 CpGs were identified as differentially methylated following COVID-19 diagnosis in blood at an FDR adjusted p-value < 0.05. These CpGs were enriched in the gene body, and the northern and southern shelf regions of genes involved in metabolic pathways. Integrative analysis revealed overlap among genes identified in transcriptional SARS-CoV-2 infection datasets. Principal component-based epigenetic clock estimates of PhenoAge and GrimAge significantly increased in people over 50 following infection by an average of 2.1 and 0.84 years. In contrast, PCPhenoAge significantly decreased in people fewer than 50 following infection by an average of 2.06 years. This observed divergence in epigenetic clocks following COVID-19 was related to age and immune cell-type compositional changes in CD4+ T cells, B cells, granulocytes, plasmablasts, exhausted T cells, and naïve T cells. Complementary longitudinal epigenetic clock analyses of 36 participants prior to and following Pfizer and Moderna mRNA-based COVID-19 vaccination revealed that vaccination significantly reduced principal component-based Horvath epigenetic clock estimates in people over 50 by an average of 3.91 years for those who received Moderna. This reduction in epigenetic clock estimates was significantly related to chronological age and immune cell-type compositional changes in B cells and plasmablasts pre- and post-vaccination. These findings suggest the potential utility of epigenetic clocks as a biomarker of COVID-19 vaccine responses. Future research will need to unravel the significance and durability of short-term changes in epigenetic age related to COVID-19 exposure and mRNA vaccination.

HIV-infected individuals have increased risk for cardiovascular disease (CVD) despite suppressive antiretroviral therapy (ART). This is likely a result of persistent immune activation and systemic inflammation. Extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication and may drive inflammation contributing to CVD. EVs were characterized in plasma from 74 HIV-infected individuals on combination antiretroviral therapy (cART) and 64 HIV-uninfected controls with paired carotid intima-media thickness (cIMT) assessment. EVs were profiled with markers reflecting lymphoid, myeloid, and endothelial origin. Seventeen plasma inflammatory biomarkers were also assessed. Human umbilical vein endothelial cell (HUVEC) apoptosis was quantified after EV exposure. A significant correlation was observed in HIV-infected participants between cIMT and EVs expressing CD16, and the monocyte-related markers CD4, CD14, and CX3CR1 showed a similar but nonsignificant association with cIMT. No significant correlation between cIMT measurements from HIV-uninfected individuals and EVs was observed. Levels of serum amyloid A, C-reactive protein, and myeloperoxidase significantly correlated with CD14+, CD16+, and CX3CR1+ EVs. No correlation was noted between cIMT and soluble inflammatory markers. HUVECs showed increased necrosis after exposure to the EV-containing fraction of plasma derived from HIV-infected individuals compared to uninfected controls. Our study reveals that EVs expressing monocyte markers correlated with cIMT in HIV-infected individuals on cART. Moreover, EV fractions derived from HIV-infected individuals lead to greater endothelial cell death via necrotic pathways. Collectively, EVs have potential as biomarkers of and therapeutic targets in the pathogenesis of CVD in the setting of treated HIV disease. IMPORTANCE HIV-infected individuals have a 2-fold-increased risk of cardiovascular disease compared with the general population, yet the mechanisms underlying this comorbidity are unclear. Extracellular vesicles have emerged as important mediators in cell-cell communication and, given what we know of their biology, may drive inflammation contributing to cardiovascular disease in this vulnerable population.

Coronavirus disease 2019 (COVID-19) is frequently associated with neurological deficits, but how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces these effects remains unclear. Here, we show that astrocytes are readily infected by SARS-CoV-2, but surprisingly, neuropilin-1, not angiotensin-converting enzyme 2 (ACE2), serves as the principal receptor mediating cell entry. Infection is further positively modulated by the two-pore segment channel 2 (TPC2) protein that regulates membrane trafficking and endocytosis. Astrocyte infection produces a pathological response closely resembling reactive astrogliosis characterized by elevated type I interferon (IFN) production, increased inflammation, and the decreased expression of transporters of water, ions, choline, and neurotransmitters. These combined events initiated within astrocytes produce a hostile microenvironment that promotes the dysfunction and death of uninfected bystander neurons. IMPORTANCE SARS-CoV-2 infection primarily targets the lung but may also damage other organs, including the brain, heart, kidney, and intestine. Central nervous system (CNS) pathologies include loss of smell and taste, headache, delirium, acute psychosis, seizures, and stroke. Pathological loss of gray matter occurs in SARS-CoV-2 infection, but it is unclear whether this is due to direct viral infection, indirect effects associated with systemic inflammation, or both. Here, we used induced pluripotent stem cell (iPSC)-derived brain organoids and primary human astrocytes from the cerebral cortex to study direct SARS-CoV-2 infection. Our findings support a model where SARS-CoV-2 infection of astrocytes produces a panoply of changes in the expression of genes regulating innate immune signaling and inflammatory responses. The deregulation of these genes in astrocytes produces a microenvironment within the CNS that ultimately disrupts normal neuron function, promoting neuronal cell death and CNS deficits.

Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) is a widely used approach to study DNA methylation genome-wide. Here, we developed a MeDIP-Seq protocol compatible with the Ion Torrent semiconductor-based sequencing platform that is low cost, rapid, and scalable. We applied this protocol to demonstrate MeDIP-Seq on the Ion Torrent platform provides adequate coverage of CpG cytosines, the methylation states of which we validated at single-base resolution on the Infinium HumanMethylation450 BeadChip array, and accurately identifies sites of differential DNA methylation. Furthermore, we applied an integrative approach to further investigate and confirm the role of DNA methylation in alternative splicing and to profile 5mC and 5hmC variants of DNA methylation in normal human brain tissue that is localized over distinct genomic regions. These applications of MeDIP-Seq on the Ion Torrent platform have broad utility and add to the current methodologies for profiling genome-wide DNA methylation states in normal and disease conditions.

Persistent inflammation and immune activation has been hypothesized to contribute to increased prevalence of subclinical atherosclerosis and cardiovascular disease (CVD) risk in patients with chronic HIV infection. In this study, we examined the correlation of peripheral monocyte subsets and soluble biomarkers of inflammation to coronary artery calcium (CAC) progression, as measured by cardiac computed tomography scan.

Abnormal cellular growth and organization have been characterized in postmortem tissue from brains of autistic individuals, suggestive of pathology in a critical neurogenic niche, the subventricular zone (SVZ) of the brain lateral ventricles (LV). We examined cellular organization, cell proliferation, and constituents of the extracellular matrix such as N-sulfated heparan sulfate (HS) and laminin (LAM) in postmortem brain tissue from the LV-SVZ of young to elderly individuals with autism (n=4) and age-matched typically developing (TD) individuals (n=4) using immunofluorescence techniques. Strong and systematic reductions in HS immunofluorescence were observed in the LV-SVZ of the TD individuals with increasing age. For young through mature, but not elderly, autistic pair members, HS was reduced compared to their matched TDs. Cellular proliferation (Ki67+) was higher in the autistic individual of the youngest age-matched pair. These preliminary data suggesting that HS may be reduced in young to mature autistic individuals are in agreement with previous findings from the BTBR T+tf/J mouse, an animal model of autism; from mice with genetic modifications reducing HS; and with genetic variants in HS-related genes in autism. They suggest that aberrant extracellular matrix glycosaminoglycan function localized to the subventricular zone of the lateral ventricles may be a biomarker for autism, and potentially involved in the etiology of the disorder.

Autism spectrum disorders (ASD) form a common group of neurodevelopmental disorders appearing to be under polygenic control, but also strongly influenced by multiple environmental factors. The brain mechanisms responsible for ASD are not understood and animal models paralleling related emotional and cognitive impairments may prove helpful in unraveling them. BTBR T+ tf/J (BTBR) mice display behaviors consistent with the three diagnostic categories for ASD. They show impaired social interaction and communication as well as increased repetitive behaviors. This review covers much of the data available to date on BTBR behavior, neuroanatomy and physiology in search for candidate biomarkers, which could both serve as diagnostic tools and help to design effective treatments for the behavioral symptoms of ASD.

Compared to healthy individuals, those with stably repressed HIV experience a higher risk of developing insulin resistance, a hallmark of pre-diabetes and a major determinant for cardiometabolic diseases. Although epigenetic processes, including in particular DNA methylation, appear to be dysregulated in individuals with insulin resistance, little is known about where these occur in the genomes of immune cells and the origins of these alterations in HIV-infected individuals. Here, we examined the genome-wide DNA methylation states of monocytes in HIV-infected individuals (n = 37) with varying levels of insulin sensitivity measured by the homeostatic model assessment of insulin resistance (HOMA-IR).

While the protective role of neutrophil extracellular traps (NETs) in limiting human immunodeficiency virus (HIV) spread to susceptible cells has been documented, there is comparatively little insight into whether NET formation is harmful in people living with HIV (PLWH). To gain insight into neutrophil dysregulation and the pathological role of NETs in HIV, we examined expressions of NET-associated markers [cell-free DNA (cfDNA) and citrullinated histone H3 (CitH3)] in the plasmas from a cohort of the Hawaii Aging with HIV-cardiovascular and HIV-seronegative (HIV-) individuals. In a subset of participants, circulating low-density granulocyte (LDG) levels and their maturation and activation status were analyzed via flow cytometry. We demonstrated higher plasma levels of CitH3 in PLWH compared to HIV- individuals. LDGs from PLWH had heightened CD66b, but reduced CD16 expression. The percentages and counts of CD10+ LDGs were significantly decreased in PLWH. In addition, the CD16Lo LDG subsets were enriched in PLWH, compared to HIV- group, indicating that immature LDGs are increased in PLWH. Moreover, LDGs from PLWH exhibited significantly higher NET forming capacity. In summary, our study presents evidence that LDGs from PLWH on ART display an immature and altered phenotype with increased NET formation. Among PLWH, plasma NET levels as well as LDG parameters correlated with blood markers for inflammation and coagulation, suggesting that neutrophil activation and NETs may exert proinflammatory and coagulation effects. Our data provide insights into the pathologic role of LDGs at least in part mediated through NET formation in PLWH.

Natural killer (NK) cells are critical modulators of HIV transmission and disease. Recent evidence suggests a loss of NK cell cytotoxicity during aging, yet analysis of NK cell biology and aging in people with HIV (PWH) is lacking. Herein, we perform comprehensive analyses of people aging with and without HIV to determine age-related NK phenotypic changes. Utilizing high-dimensional flow cytometry, we analyze 30 immune-related proteins on peripheral NK cells from healthy donors, PWH with viral suppression, and viremic PWH. NK cell phenotypes are dynamic across aging but change significantly in HIV and on antiretroviral drug therapy (ART). NK cells in healthy aging show increasing ⍺4β7 and decreasing CCR7 expression and a reverse phenomenon in PWH. These HIV-associated trafficking patterns could be due to NK cell recruitment to HIV reservoir formation in lymphoid tissue or failed mucosal signaling in the HIV-infected gut but appear to be tight delineators of age-related NK cell changes.

People with HIV (PWH) on antiretroviral therapy (ART) experience elevated rates of neurological impairment, despite controlling for demographic factors and comorbidities, suggesting viral or neuroimmune etiologies for these deficits. Here, we apply multimodal and cross-compartmental single-cell analyses of paired cerebrospinal fluid (CSF) and peripheral blood in PWH and uninfected controls. We demonstrate that a subset of central memory CD4+ T cells in the CSF produced HIV-1 RNA, despite apparent systemic viral suppression, and that HIV-1-infected cells were more frequently found in the CSF than in the blood. Using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), we show that the cell surface marker CD204 is a reliable marker for rare microglia-like cells in the CSF, which have been implicated in HIV neuropathogenesis, but which we did not find to contain HIV transcripts. Through a feature selection method for supervised deep learning of single-cell transcriptomes, we find that abnormal CD8+ T cell activation, rather than CD4+ T cell abnormalities, predominated in the CSF of PWH compared with controls. Overall, these findings suggest ongoing CNS viral persistence and compartmentalized CNS neuroimmune effects of HIV infection during ART and demonstrate the power of single-cell studies of CSF to better understand the CNS reservoir during HIV infection.

Transgender women (TGW) are disproportionally affected by HIV infection, with a global estimated prevalence of 19.9%, often attributed to behavioral risk factors, with less known about biological factors. We evaluated potential biological risk factors for HIV acquisition in TGW at the sites of viral entry by assessing immune parameters of the neovaginal surface and gut mucosa. The neovagina in TGW, compared with the vagina in cisgender women (CW), shows distinct cell composition and may pose a more inflammatory environment, evidenced by increased CD4+ T cell activation and higher levels of soluble markers of inflammation (C-reactive protein, soluble CD30). Increased inflammation may be driven by microbiome composition, as shown by a greater abundance of Prevotella and a higher Shannon Diversity Index. In addition, we have observed higher frequency of CD4+CCR5+ target cells and decreased DNA methylation of the CCR5 gene in the gut mucosa of TGW compared with CW and men who have sex with men, which was inversely correlated with testosterone levels. The rectal microbiome composition in TGW appears to favor a proinflammatory milieu as well as mucosal barrier disruption. Thus, it is possible that increased inflammation and higher frequencies of CCR5-expressing target cells at sites of mucosal viral entry may contribute to increased risk of HIV acquisition in TGW, with further validation in larger studies warranted.

Genetic variations affecting neural tube closure along the head result in malformations of the face and brain. Neural tube defects (NTDs) are among the most common birth defects in humans. We previously reported a mouse mutant called tuft that arose spontaneously in our wild-type 3H1 colony. Adult tuft mice present midline craniofacial malformations with or without an anterior cephalocele. In addition, affected embryos presented neural tube closure defects resulting in insufficient closure of the anterior neuropore or exencephaly. Here, through whole-genome sequencing, we identified a nonsense mutation in the Tet1 gene, which encodes a methylcytosine dioxygenase (TET1), co-segregating with the tuft phenotype. This mutation resulted in premature termination that disrupts the catalytic domain that is involved in the demethylation of cytosine. We detected a significant loss of TET enzyme activity in the heads of tuft embryos that were homozygous for the mutation and had NTDs. RNA-Seq transcriptome analysis indicated that multiple gene pathways associated with neural tube closure were dysregulated in tuft embryo heads. Among them, the expressions of Cecr2, Epha7 and Grhl2 were significantly reduced in some embryos presenting neural tube closure defects, whereas one or more components of the non-canonical WNT signaling pathway mediating planar cell polarity and convergent extension were affected in others. We further show that the recombinant mutant TET1 protein was capable of entering the nucleus and affected the expression of endogenous Grhl2 in IMCD-3 (inner medullary collecting duct) cells. These results indicate that TET1 is an epigenetic determinant for regulating genes that are crucial to closure of the anterior neural tube and its mutation has implications to craniofacial development, as presented by the tuft mouse.

There are limited data from resource-limited settings on antiretroviral resistance mutations that develop in patients failing second-line PI ART.

Cardiovascular disease (CVD) remains the leading cause of death worldwide despite improvements in the treatment of atherosclerosis, an inflammatory disease and major underlying cause of CVD. Monocytes, an innate immune cell type, are linked to CVD progression; however, given their heterogeneity, the association between distinct monocyte subsets and increased risk of CVD remains unclear. This study investigated the association between peripheral monocyte subpopulation numbers and carotid intima-media thickness (cIMT), a sensitive measure of CVD risk, in a cohort of adults recruited from the general population.