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OBJECTIVE: This study aimed to identify candidate host epigenetic biomarkers predicting latency reversal agents (LRA) efficacy and HIV-1 rebound kinetics during analytical treatment interruption (ATI). DESIGN: Retrospective longitudinal epigenetic profiling study from 13 people with HIV (PWH) on virologically suppressive antiretroviral therapy (ART) that participated in a LRA (HDAC inhibitor) clinical trial (NCT01680094) and a subsequent optional ATI to monitor for viral recrudescence after ART cessation. METHODS: Genome-wide DNA methylation (DNAm) in purified CD4+ T cells was measured at single-nucleotide resolution using the Infinium MethylationEPIC array. HIV-1 DNA and RNA measures were previously assessed by PCR-based methods and the association of DNAm levels at regulatory sites of the human genome were examined with reservoir size, responsiveness to LRA, and time to viral rebound following ATI. RESULTS: A distinct set of 15 candidate DNAm sites in purified CD4+ T cells at baseline pre-LRA and pre-ATI significantly correlated with time to viral rebound. Eight of these DNAm sites occurred in genes linked to HIV-1 replication dynamics including (SEPSECS, cg19113954), (MALT1, cg15968021), (CPT1C, cg14318858), (CRTAM, cg10977115), (B4GALNT4, cg04663285), (IL10, cg16284789), (TFPI2, cg19645693), and (LIFR, cg26437306); with the remaining sites at intergenic regions containing regulatory elements. Moreover, baseline DNAm states related to total HIV-1 DNA levels and the fold change in unspliced cell-associated HIV RNA following LRA treatment. CONCLUSION: Preexisting host epigenetic states may determine HIV-1 rebound kinetics and reservoir maintenance. These findings suggest integrating a suite of DNA methylation markers to improve optimal participant selection and drug regimen in future HIV cure clinical trials.

HIV-1 disrupts the host epigenetic landscape with consequences for disease pathogenesis, viral persistence, and HIV-associated comorbidities. Here, we examined how soon after infection HIV-associated epigenetic changes may occur in blood and whether early initiation of antiretroviral therapy (ART) impacts epigenetic modifications. We profiled longitudinal genome-wide DNA methylation in monocytes and CD4+ T lymphocytes from 22 participants in the RV254/SEARCH010 acute HIV infection (AHI) cohort that diagnoses infection within weeks after estimated exposure and immediately initiates ART. We identified monocytes harbored 22,697 differentially methylated CpGs associated with AHI compared to 294 in CD4+ T lymphocytes. ART minimally restored less than 1% of these changes in monocytes and had no effect upon T cells. Monocyte DNA methylation patterns associated with viral load, CD4 count, CD4/CD8 ratio, and longitudinal clinical phenotypes. Our findings suggest HIV-1 rapidly embeds an epigenetic memory not mitigated by ART and support determining epigenetic signatures in precision HIV medicine. Trial Registration:NCT00782808 and NCT00796146., Author summary The epigenetic marker, DNA methylation, plays a key role regulating the immune system during host-pathogen interactions. Using cell-type specific DNA methylation profiling, we explored whether epigenetic changes occurred soon after HIV infection and following early treatment with anti-HIV drugs. Acute infection was associated with early DNA methylation changes in purified monocytes and CD4+ T cells isolated from blood. In monocytes, rapid anti-HIV treatment minimally restored DNA methylation changes associated with infection and unexpectedly had no impact in CD4+ T cells. DNA methylation patterns before treatment informed long term clinical outcomes including CD4+ T cell counts and favorable clinical phenotypes. These findings identify candidates for consideration in epigenome editing approaches in HIV prevention, treatment, and cure strategies.

Background COVID‐19 can present with lymphopenia and extraordinary complex multiorgan pathologies that can trigger long‐term sequela. Aims Given that inflammasome products, like caspase‐1, play a role in the pathophysiology of a number of co‐morbid conditions, we investigated caspases across the spectrum of COVID‐19 disease. Materials & Methods We assessed transcriptional states of multiple caspases and using flow cytometry, the expression of active caspase‐1 in blood cells from COVID‐19 patients in acute and convalescent stages of disease. Non‐COVID‐19 subject presenting with various comorbid conditions served as controls. Results Single‐cell RNA‐seq data of immune cells from COVID‐19 patients showed a distinct caspase expression pattern in T cells, neutrophils, dendritic cells, and eosinophils compared with controls. Caspase‐1 was upregulated in CD4+ T‐cells from hospitalized COVID‐19 patients compared with unexposed controls. Post‐COVID‐19 patients with lingering symptoms (long‐haulers) also showed upregulated caspase‐1activity in CD4+ T‐cells that ex vivo was attenuated with a select pan‐caspase inhibitor. We observed elevated caspase‐3/7levels in red blood cells from COVID‐19 patients compared with controls that was reduced following caspase inhibition. Discussion Our preliminary results suggest an exuberant caspase response in COVID‐19 that may facilitate immune‐related pathological processes leading to severe outcomes. Further clinical correlations of caspase expression in different stages of COVID‐19 will be needed. Conclusion Pan‐caspase inhibition could emerge as a therapeutic strategy to ameliorate or prevent severe COVID‐19., This study assesses transcriptional states of multiple caspases and the expression of active caspase‐1 in blood cells from COVID‐19 patients in acute and convalescent stages of disease. Elevated caspase‐3/7 levels in red blood cells is observed in COVID‐19 patients compared to controls. Post‐COVID‐19 patients with lingering symptoms show up‐regulated caspase‐1 activity in CD4+ T‐cells that is attenuated ex vivo with a select pan‐caspase inhibitor. An exuberant caspase response in COVID‐19 that may facilitate immune‐related pathological processes leading to severe outcomes.

The global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is a highly pathogenic RNA virus causing coronavirus disease 2019 (COVID‐19) in humans. Although most patients with COVID‐19 have mild illness and may be asymptomatic, some will develop severe pneumonia, acute respiratory distress syndrome, multi‐organ failure, and death. RNA viruses such as SARS‐CoV‐2 are capable of hijacking the epigenetic landscape of host immune cells to evade antiviral defense. Yet, there remain considerable gaps in our understanding of immune cell epigenetic changes associated with severe SARS‐CoV‐2 infection pathology. Here, we examined genome‐wide DNA methylation (DNAm) profiles of peripheral blood mononuclear cells from 9 terminally‐ill, critical COVID‐19 patients with confirmed SARS‐CoV‐2 plasma viremia compared with uninfected, hospitalized influenza, untreated primary HIV infection, and mild/moderate COVID‐19 HIV coinfected individuals. Cell‐type deconvolution analyses confirmed lymphopenia in severe COVID‐19 and revealed a high percentage of estimated neutrophils suggesting perturbations to DNAm associated with granulopoiesis. We observed a distinct DNAm signature of severe COVID‐19 characterized by hypermethylation of IFN‐related genes and hypomethylation of inflammatory genes, reinforcing observations in infection models and single‐cell transcriptional studies of severe COVID‐19. Epigenetic clock analyses revealed severe COVID‐19 was associated with an increased DNAm age and elevated mortality risk according to GrimAge, further validating the epigenetic clock as a predictor of disease and mortality risk. Our epigenetic results reveal a discovery DNAm signature of severe COVID‐19 in blood potentially useful for corroborating clinical assessments, informing pathogenic mechanisms, and revealing new therapeutic targets against SARS‐CoV‐2., Graphical Abstract A distinct epigenetic signature associated with severe COVID‐19 exists in blood and is potentially useful for corroborating clinical assessments, informing pathogenic mechanisms, and revealing new therapeutic targets.

Currently, there is no effective vaccine and only one FDA approved early-stage therapy against SARS-CoV-2 infection as an indication to prevent disease progression. Cellular caspases play a role in the pathophysiology of a number of disorders that the co-morbid conditions seen in severe COVID-19 disease. In this study, we assessed transcriptional states of caspases in blood cells from COVID-19 patients. Gene expression levels of select caspases were increased inin vitroSARS-CoV-2 infection models and single cell RNA-Seq data of blood from COVID-19 patients showed a distinct caspase expression in T cells, neutrophils, and dendritic cells. Flow cytometric evaluation of CD4 T cells showed up-regulation of caspase-1 in hospitalized COVID-19 patients compared to unexposed controls. Convalescent COVID-19 patients with lingering symptoms (“long haulers”) showed persistent up-regulation of caspase-1 in CD4 T cells that was attenuatedex vivofollowing co-culture with a select pan-caspase inhibitor. Further, we observed elevated caspase-3 levels in red blood cells from COVID-19 patients compared to controls that were responsive to caspase inhibition. Our results expose an exuberant caspase response in COVID-19 that may facilitate immune-related pathological processes leading to severe outcomes. Pan-caspase inhibition could emerge as a therapeutic strategy to ameliorate or prevent severe COVID-19 outcomes.

Highlights • Treatment with the anti-CCR5 humanized monoclonal antibody restored depressed CD8 counts which inversely correlated with decreases in plasma viral load (pVL) which went to undetectable by Day 14 of treatment. • CCL5/RANTES is elevated 3–5 fold in mild to moderate COVID patients and over 100-fold in critical COVID patients. • First report of highly sensitive, quantitative pVL by ddPCR in COVID patients. • Statistically significant drop in IL-6 by Day 14 during treatment. • Single cell transcriptome analysis revealed decreased IL-6 in myeloid cells., Objective Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is now a global pandemic. Emerging results indicate a dysregulated immune response. Given the role of CCR5 in immune cell migration and inflammation, we investigated the impact of CCR5 blockade via the CCR5-specific antibody leronlimab on clinical, immunological and virological parameters in patients with severe COVID-19 disease. Methods In March 2020, ten terminally-ill, critical COVID-19 patients received two doses of leronlimab via individual emergency use indication (EIND). We analyzed changes in clinical presentation, immune cell populations, inflammation as well as SARS-CoV-2 plasma viremia before and 14 days after treatment. Results Over the 14 day study period 6/10 patients survived, 2 extubated, and 1 patient was discharged. We observed complete CCR5 receptor occupancy in all donors by day 7. Compared to baseline, we observed a concomitant statistically significant reduction of plasma IL-6, restoration of the CD4/CD8 ratio, and resolution of SARS-CoV2 plasma viremia (pVL) compared to controls. Further, the increase in CD8% was inversely correlated with reduction in pVL (r = −0.77, p = 0.0013). Conclusions While the current study design precludes clinical efficacy inferences, these results implicate CCR5 as a therapeutic target for COVID-19 and form the basis for ongoing randomized clinical trials.

Objectives The aim of this study was to examine the relationship between gut microbial communities in HIV-infected individuals on suppressive antiretroviral therapy (cART), and the peripheral HIV-Gag-specific CD8 T-cell responses before and after ex-vivo immune checkpoint blockade (ICB). Design Thirty-four HIV-seropositive, 10 HIV-seronegative and 12 HIV-seropositive receiving faecal microbiota transplant (FMT) participants were included. Gut microbial communities, peripheral and gut associated negative checkpoint receptors (NCRs) and peripheral effector functions were assessed. Methods Bacterial 16s rRNA sequencing for gut microbiome study and flow-based assays for peripheral and gut NCR and their cognate ligand expression, including peripheral HIV-Gag-specific CD8 T-cell responses before and after ex-vivo anti-PD-L1 and anti-TIGIT ICB were performed. Results Fusobacteria abundance was significantly higher in HIV-infected donors compared to uninfected controls. In HIV-infected participants receiving Fusobacteria-free FMT, Fusobacteria persisted up to 24 weeks in stool post FMT. PD-1 TIGIT and their ligands were expanded in mucosal vs. peripheral T cells and dendritic cells, respectively. PD-L1 and TIGIT blockade significantly increased the magnitude of peripheral anti-HIV-Gag-specific CD8 T-cell responses. Higher gut Fusobacteria abundance was associated with lower magnitude of peripheral IFN-γ+ HIV-Gag-specific CD8 T-cell responses following ICB. Conclusion The gut colonization of Fusobacteria in HIV infection is persistent and may influence anti-HIV T-cell immunity to PD-1 or TIGIT blockade. Strategies modulating Fusobacteria colonization may elicit a favourable mucosal immune landscape to enhance the efficacy of ICB for HIV cure.

Skeletal muscle handles ~80–90% of the insulin-induced glucose uptake. In skeletal muscle, insulin binding to its cell surface receptor triggers redistribution of intracellular glucose transporter GLUT4 protein to the cell surface, enabling facilitated glucose uptake. In adipocytes, the eight-protein exocyst complex is an indispensable constituent in insulin-induced glucose uptake, as it is responsible for the targeted trafficking and plasma membrane-delivery of GLUT4. However, the role of the exocyst in skeletal muscle glucose uptake has never been investigated. Here we demonstrate that the exocyst is a necessary factor in insulin-induced glucose uptake in skeletal muscle cells as well. The exocyst complex colocalizes with GLUT4 storage vesicles in L6-GLUT4myc myoblasts at a basal state and associates with these vesicles during their translocation to the plasma membrane after insulin signaling. Moreover, we show that the exocyst inhibitor endosidin-2 and a heterozygous knockout of Exoc5 in skeletal myoblast cells both lead to impaired GLUT4 trafficking to the plasma membrane and hinder glucose uptake in response to an insulin stimulus. Our research is the first to establish that the exocyst complex regulates insulin-induced GLUT4 exocytosis and glucose metabolism in muscle cells. A deeper knowledge of the role of the exocyst complex in skeletal muscle tissue may help our understanding of insulin resistance in type 2 diabetes.

Autism spectrum disorder (ASD) is a pervasive, multifactorial neurodevelopmental disorder diagnosed according to deficits in three behavioral domains: communication, social interaction, and stereotyped/repetitive behaviors. Mutations in Shank genes account for ~1% of clinical ASD cases with Shank3 being the most common gene variant. In addition to maintaining synapses and facilitating dendritic maturation, Shank genes encode master scaffolding proteins that build core complexes in the postsynaptic densities of glutamatergic synapses. Male mice with a deletion of the PDZ domain of Shank3 (Shank3B KO) were previously shown to display ASD-like behavioral phenotypes with reported self-injurious repetitive grooming and aberrant social interactions. Our goal was to extend these previous findings and use a comprehensive battery of highly detailed ASD-relevant behavioral assays including an assessment of mouse ultrasonic communication carried out on key developmental days and male and female Shank3B KO mice. We demonstrate that ASD-related behaviors, atypical reciprocal social interaction and indiscriminate repetitive grooming, are apparent in juvenile stages of development of Shank3B KO mice. Our findings underscore the importance of utilizing Shank mutant models to understand the impact of this gene in ASD etiology, which may enable future studies focusing on etiological gene-environment interactions in ASD.

Chemical modifications of DNA comprise epigenetic mechanisms that contribute to the maintenance of cellular activities and memory. Although the function of 5-methylcytosine (5-mC) has been extensively studied, little is known about the function(s) of relatively rarer and underappreciated cytosine modifications including 5-hydroxymethylcytosine (5-hmC). The discovery that ten-eleven translocation (Tet) proteins mediate conversion of 5-mC to 5-hmC, and other oxidation derivatives, sparked renewed interest to understand the biological role of 5-hmC. Studies examining total 5-hmC levels revealed the highly dynamic yet tissue-specific nature of this modification, implicating a role in epigenetic regulation and development. Intriguingly, 5-hmC levels are highest during early development and in the brain where abnormal patterns of 5-hmC have been observed in disease conditions. Thus, 5-hmC adds to the growing list of epigenetic modifications with potential utility in clinical applications and warrants further investigation. This review discusses the emerging functional roles of 5-hmC in normal and disease states, focusing primarily on insights provided by recent studies exploring the genome-wide distribution of this modification in mammals.