Neuroinflammation in the Dorsal Root Ganglia and Dorsal Horn Contributes to Persistence of Nociceptor Sensitization in SIV-Infected Antiretroviral Therapy-Treated Macaques.

Despite the development of antiretroviral therapy (ART), HIV-associated distal sensory polyneuropathy remains prevalent. Using SIV-infected rhesus macaques, this study examined molecular mechanisms of peripheral and central sensitization to infer chronic pain from HIV infection. Previous studies identified atrophy in nociceptive neurons during SIV infection, which was associated with monocyte infiltration into the dorsal root ganglia (DRG). However, the sensory signaling mechanism connecting this pathology to symptoms remains unclear, especially because pain persists after resolution of high viremia and inflammation with ART. We hypothesized that residual DRG and dorsal horn neuroinflammation contributes to nociceptive sensitization. Using three cohorts of macaques [uninfected (SIV-), SIV-infected (SIV+), and SIV infected with ART (SIV+/ART)], this study showed an increase in the cellular and cytokine inflammatory profiles in the DRG of SIV+/ART macaques compared with uninfected animals. It found significant increase in the expression of nociceptive ion channels, TRPV1, and TRPA1 among DRG neurons in SIV+/ART compared with uninfected animals. SIV-infected and SIV+/ART animals showed reduced innervation of the nonpeptidergic nociceptors into the dorsal horn compared with uninfected animals. Finally, there were a significantly higher number of CD68+ cells in the dorsal horn of SIV+/ART macaques compared with uninfected animals. In summary, these data demonstrate that neuroinflammation, characteristics of nociceptor sensitization, and central terminal atrophy persists in SIV+/ART animals.

Antiretroviral Therapy Ameliorates Simian Immunodeficiency Virus-Associated Myocardial Inflammation by Dampening Interferon Signaling and Pathogen Response in the Heart.

People with human immunodeficiency virus have an increased risk of developing cardiovascular disease. RNA-Seq was performed on hearts from simian immunodeficiency virus (SIV)-infected rhesus macaques with or without antiretroviral therapy (ART). SIV infection led to high plasma viral load with very little myocardial viral RNA. SIV infection promoted an inflammatory environment in the heart through interferon and pathogen signaling, in the absence of myocardial viral RNA. While ART dampened interferon and cytokine response in the heart, SIV-infected animals receiving ART had deficits in the expression of genes directly involved in fatty acid metabolism relative to SIV-uninfected animals.

Osteopontin Is an Integral Mediator of Cardiac Interstitial Fibrosis in Models of Human Immunodeficiency Virus Infection.

BACKGROUND: People with human immunodeficiency virus (HIV) have heightened incidence/risk of diastolic dysfunction and heart failure. Women with HIV have elevated cardiac fibrosis, and plasma osteopontin (Opn) is correlated to cardiac pathology. Therefore, this study provides mechanistic insight into the relationship between osteopontin and cardiac fibrosis during HIV infection. METHODS: Mouse embryonic fibroblasts (MEFs) modeled cardiac fibroblasts in vitro. Simian immunodeficiency virus (SIV)-infected macaques with or without antiretroviral therapy and HIV-infected humanized mice modeled HIV-associated cardiac fibrosis. RESULTS: Lipopolysaccharide-stimulated MEFs were myofibroblast-like, secreted cytokines, and produced Opn transcripts. SIV-infected animals had elevated plasma Opn at necropsy, full-length Opn in the ventricle, and ventricular interstitial fibrosis. Regression modeling identified growth differentiation factor 15, CD14+CD16+ monocytes, and CD163 expression on CD14+CD16+ monocytes as independent predictors of plasma Opn during SIV infection. HIV-infected humanized mice showed increased interstitial fibrosis compared to uninfected/untreated animals, and systemic inhibition of osteopontin by RNA aptamer reduced left ventricle fibrosis in HIV-infected humanized mice. CONCLUSIONS: Since Opn is elevated in the plasma and left ventricle during SIV infection and systemic inhibition of Opn reduced cardiac fibrosis in HIV-infected mice, Opn may be a potential target for adjunctive therapies to reduce cardiac fibrosis in people with HIV.

Increased Peripheral Inflammation Is Associated With Structural Brain Changes and Reduced Blood Flow in People With Virologically Controlled HIV.

BACKGROUND: While antiretroviral therapy (ART) has improved outcomes for people with HIV (PWH), brain dysfunction is still evident. Immune activation and inflammation remain elevated in PWH receiving ART, thereby contributing to morbidity and mortality. Previous studies demonstrated reduced functional and structural changes in PWH; however, underlying mechanisms remain elusive. METHODS: Our cohort consisted of PWH with ART adherence and viral suppression ( < 50 copies/mL; N = 173). Measurements included immune cell markers of overall immune health (CD4/CD8 T-cell ratio) and myeloid inflammation (CD16+ monocytes), plasma markers of inflammatory status (soluble CD163 and CD14), and structural and functional neuroimaging (volume and cerebral blood flow [CBF], respectively). RESULTS: Decreased CD4/CD8 ratios correlated with reduced brain volume, and higher levels of inflammatory CD16+ monocytes were associated with reduced brain volume in total cortex and gray matter. An increase in plasma soluble CD14-a marker of acute peripheral inflammation attributed to circulating microbial products-was associated with reduced CBF within the frontal, parietal, temporal, and occipital cortices and total gray matter. CONCLUSIONS: CD4/CD8 ratio and number of CD16+ monocytes, which are chronic immune cell markers, are associated with volumetric loss in the brain. Additionally, this study shows a potential new association between plasma soluble CD14 and CBF.

Transmitted/founder SHIV.D replicates in the brain, causes neuropathogenesis, and persists on combination antiretroviral therapy in rhesus macaques.

A biologically relevant non-human primate (NHP) model of HIV persistence in the central nervous system (CNS) is necessary. Most current NHP/SIV models of HIV infection fail to recapitulate viral persistence in the CNS without encephalitis or fail to employ viruses that authentically represent the ongoing HIV-1 pandemic. Here, we demonstrate viral replication in the brain and neuropathogenesis after combination antiretroviral therapy (ART) in rhesus macaques (RMs) using novel macrophage-tropic transmitted/founder (TF) simian-human immunodeficiency virus SHIV.D.191,859 (SHIV.D). Quantitative immunohistochemistry (IHC) and DNA/RNAscope in situ hybridization (ISH) were performed on three brain regions from six SHIV.D-infected RMs; two necropsied while viremic, two during analytical treatment interruptions, and two on suppressive ART. We demonstrated myeloid-mediated neuroinflammation, viral replication, and proviral DNA in the brain in all animals. These results demonstrate that TF SHIV.D models native HIV-1 CNS replication, pathogenesis, and persistence on ART in rhesus macaques.

Atrophy and Death of Nonpeptidergic and Peptidergic Nociceptive Neurons in SIV Infection.

HIV-associated sensory neuropathy is a common neurologic comorbidity of HIV infection and prevails in the post-antiretroviral therapy (ART) era. HIV infection drives pathologic changes in the dorsal root ganglia (DRG) through inflammation, altered metabolism, and neuronal dysfunction. Herein, we characterized specific neuronal populations in an SIV-infected macaque model with or without ART. DRG neuronal populations were identified by neurofilament H-chain 200, I-B4 isolectin (IB4), or tropomyosin receptor kinase A expression and assessed for cell body diameter, population size, apoptotic markers, and regeneration signaling. IB4+ and tropomyosin receptor kinase A-positive neurons showed a reduced cell body size (atrophy) and decreased population size (cell death) in the DRG of SIV-infected animals compared with uninfected animals. IB4+ nonpeptidergic neurons were less affected in the presence of ART. DRG neurons showed accumulation of cleaved caspase 3 (apoptosis) and nuclear-localized activating transcription factor 3 (regeneration) in SIV infection, which was significantly lower in uninfected animals and SIV-infected animals receiving ART. Nonpeptidergic neurons predominantly colocalized with cleaved caspase 3 staining. Nonpeptidergic and peptidergic neurons colocalized with nuclear-accumulated activating transcription factor 3, showing active regeneration in sensory neurons. These data suggest that nonpeptidergic and peptidergic neurons are susceptible to pathologic changes from SIV infection, and intervention with ART did not fully ameliorate damage to the DRG, specifically to peptidergic neurons.

Anti-Inflammatory Interleukin 10 Inversely Relates to Coronary Atherosclerosis in Persons With Human Immunodeficiency Virus.

Interleukin 10 (IL-10) is an anti-inflammatory cytokine that may be protective against coronary atherosclerosis. In an observational study of persons with human immunodeficiency virus (PWH) and uninfected controls, IL-10 was measured in serum samples by means of enzyme-linked immunosorbent assay, and coronary atherosclerosis was assessed using computed tomographic angiography. Among PWH, a 10-fold decrease in IL-10 was associated with a 2.6-fold increase in the odds of coronary plaque (P = .01), after controlling for traditional and nontraditional cardiovascular risk factors. IL-10 was also inversely associated with total coronary plaque (ρ = -0.19; P = .02) and noncalcified coronary plaque (ρ = -0.24; P = .004). Our findings suggest a role for IL-10 in mitigating atherosclerosis in PWH. Clinical Trials Registration. NCT00455793.

SIV-Mediated Synaptic Dysfunction Is Associated with an Increase in Synapsin Site 1 Phosphorylation and Impaired PP2A Activity.

Although the reduction of viral loads in people with HIV undergoing combination antiretroviral therapy has mitigated AIDS-related symptoms, the prevalence of neurological impairments has remained unchanged. HIV-associated CNS dysfunction includes impairments in memory, attention, memory processing, and retrieval. Here, we show a significant site-specific increase in the phosphorylation of Syn I serine 9, site 1, in the frontal cortex lysates and synaptosome preparations of male rhesus macaques infected with simian immunodeficiency virus (SIV) but not in uninfected or SIV-infected antiretroviral therapy animals. Furthermore, we found that a lower protein phosphatase 2A (PP2A) activity, a phosphatase responsible for Syn I (S9) dephosphorylation, is primarily associated with the higher S9 phosphorylation in the frontal cortex of SIV-infected macaques. Comparison of brain sections confirmed higher Syn I (S9) in the frontal cortex and greater coexpression of Syn I and PP2A A subunit, which was observed as perinuclear aggregates in the somata of the frontal cortex of SIV-infected macaques. Synaptosomes from SIV-infected animals were physiologically tested using a synaptic vesicle endocytosis assay and FM4-64 dye showing a significantly higher baseline depolarization levels in synaptosomes of SIV+-infected than uninfected control or antiretroviral therapy animals. A PP2A-activating FDA-approved drug, FTY720, decreased the higher synaptosome depolarization in SIV-infected animals. Our results suggest that an impaired distribution and lower activity of serine/threonine phosphatases in the context of HIV infection may cause an indirect effect on the phosphorylation levels of essential proteins involving in synaptic transmission, supporting the occurrence of specific impairments in the synaptic activity during SIV infection.SIGNIFICANCE STATEMENT Even with antiretroviral therapy, neurocognitive deficits, including impairments in attention, memory processing, and retrieval, are still major concerns in people living with HIV. Here, we used the rhesus macaque simian immunodeficiency virus model with and without antiretroviral therapy to study the dynamics of phosphorylation of key amino acid residues of synapsin I, which critically impacts synaptic vesicle function. We found a significant increase in synapsin I phosphorylation at serine 9, which was driven by dysfunction of serine/threonine protein phosphatase 2A in the nerve terminals. Our results suggest that an impaired distribution and lower activity of serine/threonine phosphatases in the context of HIV infection may cause an indirect effect on the phosphorylation levels of essential proteins involved in synaptic transmission.

Caspase-1 Activation Is Related With HIV-Associated Atherosclerosis in an HIV Transgenic Mouse Model and HIV Patient Cohort.

OBJECTIVE: Atherosclerotic cardiovascular disease (ASCVD) is an increasing cause of morbidity and mortality in people with HIV since the introduction of combination antiretroviral therapy. Despite recent advances in our understanding of HIV ASCVD, controversy still exists on whether this increased risk of ASCVD is due to chronic HIV infection or other risk factors. Mounting biomarker studies indicate a role of monocyte/macrophage activation in HIV ASCVD; however, little is known about the mechanisms through which HIV infection mediates monocyte/macrophage activation in such a way as to engender accelerated atherogenesis. Here, we experimentally investigated whether HIV expression is sufficient to accelerate atherosclerosis and evaluated the role of caspase-1 activation in monocytes/macrophages in HIV ASCVD. Approach and Results: We crossed a well-characterized HIV mouse model, Tg26 mice, which transgenically expresses HIV-1, with ApoE-/- mice to promote atherogenic conditions (Tg26+/-/ApoE-/-). Tg26+/-/ApoE-/- have accelerated atherosclerosis with increased caspase-1 pathway activation in inflammatory monocytes and atherosclerotic vasculature compared with ApoE-/-. Using a well-characterized cohort of people with HIV and tissue-banked aortic plaques, we documented that serum IL (interleukin)-18 was higher in people with HIV compared with non-HIV-infected controls, and in patients with plaques, IL-18 levels correlated with monocyte/macrophage activation markers and noncalcified inflammatory plaques. In autopsy-derived aortic plaques, caspase-1+ cells and CD (clusters of differentiation) 163+ macrophages correlated. CONCLUSIONS: These data demonstrate that expression of HIV is sufficient to accelerate atherogenesis. Further, it highlights the importance of caspase-1 and monocyte/macrophage activation in HIV atherogenesis and the potential of Tg26+/-/ApoE-/- as a tool for mechanistic studies of HIV ASCVD.

Suppression of Zika Virus Infection in the Brain by the Antiretroviral Drug Rilpivirine.

Zika virus (ZIKV) infection is associated with microcephaly in neonates and Guillain-Barré syndrome in adults. ZIKV produces a class of nonstructural (NS) regulatory proteins that play a critical role in viral transcription and replication, including NS5, which possesses RNA-dependent RNA polymerase (RdRp) activity. Here we demonstrate that rilpivirine (RPV), a non-nucleoside reverse transcriptase inhibitor (NNRTI) used in the treatment of HIV-1 infection, inhibits the enzymatic activity of NS5 and suppresses ZIKV infection and replication in primary human astrocytes. Similarly, other members of the NNRTI family, including etravirine and efavirenz, showed inhibitory effects on viral infection of brain cells. Site-directed mutagenesis identified 14 amino acid residues within the NS5 RdRp domain (AA265-903), which are important for the RPV interaction and the inhibition of NS5 polymerase activity. Administration of RPV to ZIKV-infected interferon-alpha/beta receptor (IFN-A/R) knockout mice improved the clinical outcome and prevented ZIKV-induced mortality. Histopathological examination of the brains from infected animals revealed that RPV reduced ZIKV RNA levels in the hippocampus, frontal cortex, thalamus, and cerebellum. Repurposing of NNRTIs, such as RPV, for the inhibition of ZIKV replication offers a possible therapeutic strategy for the prevention and treatment of ZIKV-associated disease.

Caspase-1-associated immune activation in an accelerated SIV-infected rhesus macaque model.

In the antiretroviral therapy (ART) era, chronic HIV infection is primarily associated with chronic inflammation driving comorbidities such as cardiovascular disease and neurocognitive impairment. Caspase-1 activation in leukocytes has been documented in HIV infection; however, whether caspase-1 activation and the downstream pro-inflammatory cytokines interleukin-1beta (IL-1ß) and interleukin-18 (IL-18) contribute to chronic inflammation in HIV comorbidities remains undetermined. The relationship between the caspase-1 cascade and persistent inflammation in HIV has not been investigated. Here, we used an accelerated simian immunodeficiency virus (SIV)-infected rhesus macaque model with or without ART to investigate the dynamics of caspase-1 and immune cell activation before infection, 21 days post infection (dpi), and necropsy. Caspase-1, IL-18, IL-1ß, and immune markers were measured both in the circulation and lymphoid tissues. We found a significant increase in caspase-1 and IL-18 in SIV infection that positively correlated with inflammatory monocytes and negatively correlated with CD4+ T cell counts. ART attenuated these effects at necropsy in the circulation. Further, lymph nodes from SIV+ or SIV+ART animals had increased activation of caspase-1 and potential upstream priming of the NF-κB pathway, indicating that tissue-specific immune activation persists with ART. Together, these results shed light on the interconnectedness of the caspase-1 pathway and peripheral immune activation and further indicate that ART is not sufficient for suppressing inflammation. The caspase-1 pathway may provide novel therapeutic targets to improve HIV-associated comorbidities and health outcomes in the context of viral suppression.

α4-Integrin Antibody Treatment Blocks Monocyte/Macrophage Traffic to, Vascular Cell Adhesion Molecule-1 Expression in, and Pathology of the Dorsal Root Ganglia in an SIV Macaque Model of HIV-Peripheral Neuropathy.

Traffic of activated monocytes into the dorsal root ganglia (DRG) is critical for pathology in HIV peripheral neuropathy. We have shown that accumulation of recently recruited (bromodeoxyuridine(+) MAC387(+)) monocytes is associated with severe DRG pathology and loss of intraepidermal nerve fibers in SIV-infected macaques. Herein, we blocked leukocyte traffic by treating animals with natalizumab, which binds to α4-integrins. SIV-infected CD8-depleted macaques treated with natalizumab either early (the day of infection) or late (28 days after infection) were compared with untreated SIV-infected animals sacrificed at similar times. Histopathology showed diminished DRG pathology with natalizumab treatment, including decreased inflammation, neuronophagia, and Nageotte nodules. Natalizumab treatment resulted in a decrease in the number of bromodeoxyuridine(+) (early), MAC387(+) (late), CD68(+) (early and late), and SIVp28(+) (late) macrophages in DRG tissues. The number of CD3(+) T lymphocytes in DRGs was not affected by natalizumab treatment. Vascular cell adhesion molecule 1, an adhesion molecule that mediates leukocyte traffic, was diminished in DRGs of all natalizumab-treated animals. These data show that blocking monocyte, but not T lymphocyte, traffic to the DRG results in decreased inflammation and pathology, supporting a role for monocyte traffic and activation in HIV peripheral neuropathy.

Loss of intraepidermal nerve fiber density during SIV peripheral neuropathy is mediated by monocyte activation and elevated monocyte chemotactic proteins.

BACKGROUND: Peripheral neuropathy (PN) continues to be a major complication of human immunodeficiency virus (HIV) infection despite successful anti-retroviral therapy. Human HIV-PN can be recapitulated in a CD8-depleted, simian immunodeficiency virus (SIV)-infected rhesus macaque animal model, characterized by a loss of intraepidermal nerve fiber density (IENFD) and damage to the dorsal root ganglia (DRG). Increased monocyte traffic to the DRG has previously been associated with severe DRG pathology, as well as a loss in IENFD. Here, we sought to characterize the molecular signals associated with monocyte activation and trafficking to the DRGs. METHODS: Eleven SIV-infected CD8-depleted rhesus macaques were compared to four uninfected control animals. sCD14, sCD163, sCD137, regulated on activation normal T cell expressed and secreted (RANTES), and monocyte chemoattractant protein 1 (MCP-1) were measured in plasma and the latter three proteins were also quantified in DRG tissue lysates. All SIV-infected animals received serial skin biopsies to measure IENFD loss as well as BrdU inoculations to measure monocyte turnover during the course of infection. The number of BrdU+ and CD14+ CD16+ peripheral blood monocytes was determined by flow cytometry. The number of MAC387+, CCR2+, CCR5+, and CD137+ cells in DRG tissue was quantified by immunohistochemistry. RESULTS: sCD14, sCD163, MCP-1, and sCD137 increased significantly in plasma from pre-infection to necropsy. Plasma sCD163 and RANTES inversely correlated with IENFD. Additionally, sCD137 in DRG tissue lysate was elevated with severe DRG pathology and associated with the recruitment of MAC387+ cells to DRG. Elevated numbers of CCR5+ and CCR2+ satellite cells in the DRG were found, suggesting a chemotactic role of their ligands, RANTES, and MCP-1 in recruiting monocytes to the tissue. CONCLUSIONS: We characterized the role of systemic (plasma) and tissue-specific (DRG) monocyte activation and associated cytokines in the pathogenesis of SIV-PN. We identified sCD163 and RANTES as potential biomarkers for HIV-PN, as these were associated with a loss of IENFD. Additionally, we identified CD137 signaling to play a role in MAC387+ cell traffic to DRG and possibly contribute to severe pathology. These studies highlight the role of monocyte activation and traffic in the pathogenesis of SIV-PN, while identifying specific signaling proteins for future pharmacological blockade.

Plasma osteopontin relates to myocardial fibrosis and steatosis and to immune activation among women with HIV.

OBJECTIVE: Women with HIV (WWH) have heightened heart failure risk. Plasma OPN (osteopontin) is a powerful predictor of heart failure outcomes in the general population. Limited data exist on relationships between plasma OPN and surrogates of HIV-associated heart failure risk. DESIGN: Prospective, cross-sectional. METHODS: We analyzed relationships between plasma OPN and cardiac structure/function (assessed using cardiovascular magnetic resonance imaging) and immune activation (biomarkers and flow cytometry) among 20 WWH and 14 women without HIV (WWOH). RESULTS: Plasma OPN did not differ between groups. Among WWH, plasma OPN related directly to the markers of cardiac fibrosis, growth differentiation factor-15 (ρ = 0.51, P = 0.02) and soluble interleukin 1 receptor-like 1 (ρ = 0.45, P = 0.0459). Among WWH (but not among WWOH or the whole group), plasma OPN related directly to both myocardial fibrosis (ρ = 0.49, P = 0.03) and myocardial steatosis (ρ = 0.46, P = 0.0487). Among the whole group and WWH (and not among WWOH), plasma OPN related directly to the surface expression of C-X3-C motif chemokine receptor 1 (CX3CR1) on nonclassical (CD14-CD16+) monocytes (whole group: ρ = 0.36, P = 0.04; WWH: ρ = 0.46, P = 0.04). Further, among WWH and WWOH (and not among the whole group), plasma OPN related directly to the surface expression of CC motif chemokine receptor 2 (CCR2) on inflammatory (CD14+CD16+) monocytes (WWH: ρ = 0.54, P = 0.01; WWOH: ρ = 0.60, P = 0.03), and in WWH, this held even after controlling for HIV-specific parameters. CONCLUSION: Among WWH, plasma OPN, a powerful predictor of heart failure outcomes, related to myocardial fibrosis and steatosis and the expression of CCR2 and CX3CR1 on select monocyte subpopulations. OPN may play a role in heart failure pathogenesis among WWH. CLINICALTRIALSGOV REGISTRATION: NCT02874703.

Isolation and Culture of Dorsal Root Ganglia (DRG) from Rodents.

Preparations of peripheral sensory neurons from rodents are essential for studying the molecular mechanism of neuronal survival and physiology. Although, isolating and culturing these neurons proves difficult, often these preparations are contaminated with nonneuronal proliferating cells. Here, we describe an isolation method using a Percoll gradient and an antimitotic reagent to significantly reduce the nonneuronal cell contamination while maintaining the integrity of the rodent sensory dorsal root ganglia (DRG) neurons.

Pro-Inflammatory Interleukin-18 is Associated with Hepatic Steatosis and Elevated Liver Enzymes in People with HIV Monoinfection.

People with HIV (PWH) are at an increased risk of developing nonalcoholic fatty liver disease (NAFLD). Interleukin (IL)-18 is regulated by inflammasomes in response to pathogens and danger signals and has been implicated in both the pathogenesis of NAFLD and HIV disease progression. We hypothesized that increased IL-18 may be associated with NAFLD and liver injury in PWH. This was an observational study of 125 PWH and 59 individuals without HIV in the Boston area. Participants with known hepatitis B, hepatitis C, and excessive alcohol use were excluded. IL-18 was measured in serum by enzyme-linked immunosorbent assay. Liver lipid content was assessed by liver-to-spleen computed tomography (CT) attenuation ratio. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and IL-18 levels were higher in PWH than in controls. In PWH, log10 IL-18 was associated with log10AST (r = 0.34, p = .0001), log10ALT (r = 0.33, p = .0002), log10HIV RNA (r = 0.29, p = .002), and inversely associated with liver-to-spleen ratio (r = -0.24, p = .02). In addition, log10 IL-18 was associated with log10 triglycerides (r = 0.26, p = .003), log10 MCP-1 (monocyte chemoattractant protein-1; r = 0.33, p = .0004), log10caspase-1 (r = 0.35, p < .0001), log10LPS (r = 0.28, p = .004), and inversely associated with high-density lipoprotein (r = -0.28, p = .002), and CD4+/CD8+ T cell ratio (r = -0.24, p = .007). In controls without HIV, log10 IL-18 was also associated with log10ALT (r = 0.44, p = .0005). After adjusting for potential confounders, the relationships between IL-18 and AST (p = .004) and ALT (p = .003) remained significant, and the relationship between IL-18 and liver-to-spleen ratio (p = .02). Increased inflammasome activation and subsequent monocyte recruitment in PWH may contribute to the development and progression of NAFLD. Clinical Trials Registration. NCT00455793.

Characterization of Nef expression in different brain regions of SIV-infected macaques.

OBJECTIVE: HIV-associated CNS dysfunction is a significant problem among people with HIV (PWH), who now live longer due to viral suppression from combined anti-retroviral therapy (ART). Over the course of infection, HIV generates toxic viral proteins and induces inflammatory cytokines that have toxic effects on neurons in the CNS. Among these viral proteins, HIV Nef has been found in neurons of postmortem brain specimens from PWH. However, the source of Nef and its impact on neuronal cell homeostasis are still elusive. METHODS AND RESULTS: Here, in using a simian immunodeficiency virus (SIV) infected rhesus macaque model of neuroHIV, we find SIV Nef reactivity in the frontal cortex, hippocampus and cerebellum of SIV-infected animals using immunohistochemistry (IHC). Interestingly, SIV-infected macaques treated with ART also showed frequent Nef positive cells in the cerebellum and hippocampus. Using dual quantitative RNAscope and IHC, we observed cells that were positive for Nef, but were not for SIV RNA, suggesting that Nef protein is present in cells that are not actively infected with SIV. Using cell specific markers, we observed Nef protein in microglia/macrophages and astrocytes. Importantly, we also identified a number of NeuN-positive neurons, which are not permissive to SIV infection, but contained Nef protein. Further characterization of Nef-positive neurons showed caspase 3 activation, indicating late stage apoptosis in the CNS neurons. CONCLUSIONS: Our results suggest that regardless of ART status, Nef is expressed in the brain of SIV infected macaques and may contribute to neurological complications seen in PWH.

CRISPR based editing of SIV proviral DNA in ART treated non-human primates.

Elimination of HIV DNA from infected individuals remains a challenge in medicine. Here, we demonstrate that intravenous inoculation of SIV-infected macaques, a well-accepted non-human primate model of HIV infection, with adeno-associated virus 9 (AAV9)-CRISPR/Cas9 gene editing construct designed for eliminating proviral SIV DNA, leads to broad distribution of editing molecules and precise cleavage and removal of fragments of the integrated proviral DNA from the genome of infected blood cells and tissues known to be viral reservoirs including lymph nodes, spleen, bone marrow, and brain among others. Accordingly, AAV9-CRISPR treatment results in a reduction in the percent of proviral DNA in blood and tissues. These proof-of-concept observations offer a promising step toward the elimination of HIV reservoirs in the clinic.

HIV-Associated Neurocognitive Impairment in the Modern ART Era: Are We Close to Discovering Reliable Biomarkers in the Setting of Virological Suppression?

The prevalence of the most severe forms of HIV-associated neurocognitive disorders (HAND) is decreasing due to worldwide availability and high efficacy of antiretroviral treatment (ART). However, several grades of HIV-related cognitive impairment persist with effective ART and remain a clinical concern for people with HIV (PWH). The pathogenesis of these cognitive impairments has yet to be fully understood and probably multifactorial. In PWH with undetectable peripheral HIV-RNA, the presence of viral escapes in cerebrospinal fluid (CSF) might explain a proportion of cases, but not all. Many other mechanisms have been hypothesized to be involved in disease progression, in order to identify possible therapeutic targets. As potential indicators of disease staging and progression, numerous biomarkers have been used to characterize and implicate chronic inflammation in the pathogenesis of neuronal injuries, such as certain phenotypes of activated monocytes/macrophages, in the context of persistent immune activation. Despite none of them being disease-specific, the correlation of several CSF cellular biomarkers to HIV-induced neuronal damage has been investigated. Furthermore, recent studies have been evaluating specific microRNA (miRNA) profiles in the CSF of PWH with neurocognitive impairment (NCI). The aim of the present study is to review the body of evidence on different biomarkers use in research and clinical settings, focusing on PWH on ART with undetectable plasma HIV-RNA.

Sequential LASER ART and CRISPR Treatments Eliminate HIV-1 in a Subset of Infected Humanized Mice.

Elimination of HIV-1 requires clearance and removal of integrated proviral DNA from infected cells and tissues. Here, sequential long-acting slow-effective release antiviral therapy (LASER ART) and CRISPR-Cas9 demonstrate viral clearance in latent infectious reservoirs in HIV-1 infected humanized mice. HIV-1 subgenomic DNA fragments, spanning the long terminal repeats and the Gag gene, are excised in vivo, resulting in elimination of integrated proviral DNA; virus is not detected in blood, lymphoid tissue, bone marrow and brain by nested and digital-droplet PCR as well as RNAscope tests. No CRISPR-Cas9 mediated off-target effects are detected. Adoptive transfer of human immunocytes from dual treated, virus-free animals to uninfected humanized mice fails to produce infectious progeny virus. In contrast, HIV-1 is readily detected following sole LASER ART or CRISPR-Cas9 treatment. These data provide proof-of-concept that permanent viral elimination is possible.