Asha R. Kallianpur

Antiretroviral therapy (ART) drugs vary in their distribution into cerebrospinal fluid (CSF), which can be estimated using the central nervous system (CNS) penetration effectiveness (CPE) score. Although higher CPE has been associated with lower CSF HIV RNA levels, its relationship to CSF inflammation is less clear. We investigated associations between CPE and three CSF immune biomarkers (CXCL10, TNF-α, and IL-6) in 275 virally suppressed people with HIV (PWH) on three-drug ART regimens using a training–validation design. Participants were randomized into training (TG, n = 144) and validation (VG, n = 131) groups with similar demographics, ART characteristics, and CPE scores. The CSF levels of the biomarkers were quantified by bead suspension array-based immunoassays. In both groups, higher CPE correlated with lower levels of CXCL10 (TG: r = −0.31, p < 0.001; VG: r = −0.30, p < 0.001) and TNF-α (TG: r = −0.19, p = 0.04; VG: r = −0.18, p = 0.03), with remarkably similar effect size. CPE did not correlate with IL-6 in either group. Multivariable models confirmed the associations between higher CPE and both lower CXCL10 (R² = 0.16, p < 0.001) and TNF-α (R² = 0.07, p = 0.02) in CSF, and supported the relative resistance of IL-6 to ART effects. During suppressive ART, regimens that achieve higher concentrations in the CNS may better reduce some indicators of CSF inflammation (CXCL10 and TNF-α, closely related to the interferon pathway), but they may not fully normalize the neuroimmune environment (IL-6). Distinct ART regimens may produce different neuroimmune signatures, potentially contributing to heterogeneous patterns of brain injury.

Ferroptosis is implicated in viral neuropathogenesis and may underlie HIV-associated neurocognitive impairment (NCI). Emerging data also suggest differences in brain iron transport by sex. We hypothesized that circulating ferritins that inhibit ferroptosis associate with neurocognitive function and NCI in people with HIV (PWH) in a sex-biased manner. Serum ferritin heavy-chain-1 (FTH1), ferritin light-chain (FTL), and urinary F₂-isoprostanes (uF₂-isoPs, specific lipid peroxidation marker) were quantified in 324 PWH (including 61 women) with serial global (NPZ-4) and domain-specific neurocognitive testing. Biomarker associations with neurocognitive test scores and NCIs were evaluated by multivariable regression; correlations with uF₂-isoPs were also assessed. Higher FTL and FTH1 levels were associated with less NCI in all PWH (adjusted odds ratios 0.53, 95% confidence interval (95% CI) 0.36–0.79 and 0.66, 95% CI 0.45–0.97, respectively). In women, higher FTL and FTH1 were also associated with better NPZ-4 (FTL adjusted beta (β) = 0.15, 95% CI 0.02–0.29; FTL-by-sex β_interaction = 0.32, p = 0.047) and domain-specific neurocognitive test scores. Effects on neurocognitive performance persisted for up to 5 years. Levels of both ferritins correlated inversely with uF₂-isoPs in women (FTL: rho = −0.47, p < 0.001). Circulating FTL and FTH1 exert sustained, sex-biased neuroprotective effects in PWH, possibly by protecting against iron-mediated lipid peroxidation (ferroptosis). Larger studies are needed to confirm the observed sex differences and further delineate the underlying mechanisms.

Ferroptosis is implicated in viral neuropathogenesis and may underlie HIV-associated neurocognitive impairment (NCI). Emerging data also suggest differences in brain iron transport by sex. We hypothesized that circulating ferritins that inhibit ferroptosis associate with neurocognitive function and NCI in people with HIV (PWH) in a sex-biased manner. Serum ferritin heavy-chain-1 (Fth-1), ferritin light-chain (Ftl), and urinary F2-isoprostanes (uF2-isoPs, specific lipid peroxidation marker) were quantified in 324 PWH (including 61 women) with serial global (NPZ4) and domain-specific neurocognitive testing. Biomarker associations with neurocognitive test scores and NCI were evaluated by multivariable regression; correlations with uF2-isoPs were also assessed. Higher Ftl and Fth-1 associated with less NCI in all PWH (adjusted odds ratios 0.53, 95% Confidence Interval (95% CI) 0.36-0.79 and 0.66, 95% CI 0.45-0.97, respectively). In women, higher Ftl and Fth-1 associated with better NPZ-4 (Ftl: adjusted beta (β)=0.15, 95% CI 0.02-0.29; Ftl-by-sex βinteraction=0.32, p=0.047) and domain-specific neurocognitive test scores. Effects on neurocognitive performance persisted for up to 5 years. Levels of both ferritins correlated inversely with uF2-isoPs in women (Ftl: rho=-0.47, p<0.001). Circulating Ftl and Fth-1 exert sustained, sex-biased neuroprotective effects in PWH, possibly by protecting against iron-mediated lipid peroxidation (ferroptosis). Larger studies are needed to confirm the observed sex differences and further delineate underlying mechanisms.

HIV-associated cognitive dysfunction during combination antiretroviral therapy (cART) involves mitochondrial dysfunction, but the impact of contemporary cART on chronic metabolic changes in the brain and in latent HIV infection is unclear. We interrogated mitochondrial function in a human microglia (hμglia) cell line harboring inducible HIV provirus and in SH-SY5Y cells after exposure to individual antiretroviral drugs or cART, using the MitoStress assay. cART-induced changes in protein expression, reactive oxygen species (ROS) production, mitochondrial DNA copy number, and cellular iron were also explored. Finally, we evaluated the ability of ROS scavengers or plasmid-mediated overexpression of the antioxidant iron-binding protein, Fth1, to reverse mitochondrial defects. Contemporary antiretroviral drugs, particularly bictegravir, depressed multiple facets of mitochondrial function by 20–30%, with the most pronounced effects in latently infected HIV+ hμglia and SH-SY5Y cells. Latently HIV-infected hμglia exhibited upregulated glycolysis. Increases in total and/or mitochondrial ROS, mitochondrial DNA copy number, and cellular iron accompanied mitochondrial defects in hμglia and SH-SY5Y cells. In SH-SY5Y cells, cART reduced mitochondrial iron–sulfur-cluster-containing supercomplex and subunit expression and increased Nox2 expression. Fth1 overexpression or pre-treatment with N-acetylcysteine prevented cART-induced mitochondrial dysfunction. Contemporary cART impairs mitochondrial bioenergetics in hμglia and SH-SY5Y cells, partly through cellular iron accumulation; some effects differ by HIV latency.

HIV-associated cognitive dysfunction during combination antiretroviral therapy (cART) involves mitochondrial dysfunction, but the impact of contemporary cART on chronic metabolic changes in the brain and in latent HIV infection is unclear. We interrogated mitochondrial function in a human microglia (hμglia) cell line harboring inducible HIV provirus, and in SH-SY5Y cells, after exposure to individual antiretroviral drugs or cART, using the MitoStress assay. cART-induced changes in protein expression, reactive oxygen species (ROS) production, mitochondrial DNA copy number, and cellular iron were also explored. Finally, we evaluated the ability of ROS scavengers or plasmid-mediated overexpression of the antioxidant iron-binding protein, Fth1, to reverse mitochondrial defects. Contemporary antiretroviral drugs, particularly bictegravir, depressed multiple facets of mitochondrial function by 20-30%, with most pronounced effects in latently infected-HIV+ hμglia and SH-SY5Y cells. Latently HIV-infected hμglia exhibited upregulated glycolysis. Increases in total and/or mitochondrial ROS, mitochondrial DNA copy number, and cellular iron accompanied mitochondrial defects in hμglia and SH-SY5Y cells. In SH-SY5Y cells, cART reduced mitochondrial iron-sulfur-cluster-containing supercomplex and subunit expression and increased Nox2 expression. Fth1 overexpression or pre-treatment with N-acetylcysteine prevented cART-induced mitochondrial dysfunction. Contemporary cART impairs mitochondrial bioenergetics in hμglia and SH-SY5Y cells, partly through cellular iron accumulation; some effects differ by HIV latency.

Coding and noncoding single-nucleotide variants (SNVs) of EIF2AK3 , which encodes an integrated stress response (ISR) kinase, may play a role in neurodegenerative disorders. We used a candidate gene approach to determine the correlation of EIF2AK3 SNVs with neurocognitive (NC) impairment (NCI), which can persist with viral suppression from antiretroviral therapy (ART) in people with HIV (PWH). This retrospective study of prospectively collected data included participants of the CNS HIV Anti-Retroviral Therapy Effects Research (CHARTER) cohort, after excluding participants with severe neuropsychiatric comorbidities. Genome-wide data previously obtained in the CHARTER cohort participants (n=1,047) were analyzed to interrogate the association of three noncoding EIF2AK3 SNVs with the continuous global deficit score (GDS) and global NCI (GDS≥0.5). Targeted sequencing (TS) was performed in 992 participants with available genomic DNA to determine the association of three coding EIF2AK3 SNVs with GDS and NCI. Analyses included univariable and multivariable methods such as analysis of variance and regression. Multivariable models covaried demographic, disease-associated, and treatment characteristics. The cohort characteristics were as follows: median age, 43.1 years; females, 22.8%; European ancestry, 41%; median CD4+ T cell counts, 175/µL (nadir) and 428/µL (current). At first assessment, 70.5% used ART and 68.3% of these had plasma HIV RNA ≤ 200 copies/mL. A minority of participants had at least one risk allele for rs6739095 (T,41.7%), rs1913671 (C,41.4%), and rs11684404 (C,39.4%). All three noncoding EIF2AK3 SNVs were associated with significantly worse GDS and more NCI (all p p 13 were independently associated with GDS and NCI ( p EIF2AK3 SNVs were specifically associated with worse performance in executive functioning, motor functioning, learning, and verbal fluency. Coding and non-coding SNVs of EIF2AK3 were associated with global NC and domain-specific performance. The effects were small-to-medium in size but were present in multivariable analyses. Specific SNVs in EIF2AK3 may be an important component of genetic vulnerability to NC complications in PWH. Identification of host factors that predict NCI could allow for earlier interventions, including those directly modulating the ISR, to improve NC outcomes.

HIV-associated neurocognitive disorder (HAND) remains prevalent despite antiretroviral therapy and involves white matter damage in the brain. Although iron is essential for myelination and myelin maintenance/repair, its role in HAND is largely unexplored. We tested the hypotheses that cerebrospinal fluid (CSF) heavy-chain ferritin (Fth1) and transferrin, proteins integral to iron delivery and myelination, are associated with neurocognitive performance in people with HIV (PWH). Fth1, transferrin, and the pro-inflammatory cytokines TNF-α and IL-6 were quantified in CSF at baseline (entry) in 403 PWH from a prospective observational study who underwent serial, comprehensive neurocognitive assessments. Associations of Fth1 and transferrin with Global Deficit Score (GDS)-defined neurocognitive performance at baseline and 30–42 months of follow-up were evaluated by multivariable regression. While not associated with neurocognitive performance at baseline, higher baseline CSF Fth1 predicted significantly better neurocognitive performance over 30 months in all PWH (p < 0.05), in PWH aged < 50 at 30, 36, and 42 months (all p < 0.05), and in virally suppressed PWH at all three visit time-points (all p < 0.01). Higher CSF transferrin was associated with superior neurocognitive performance at all visits, primarily in viremic individuals (all p < 0.05). All associations persisted after adjustment for neuro-inflammation. In summary, higher CSF Fth1 is neuroprotective over prolonged follow-up in all and virally suppressed PWH, while higher CSF transferrin may be most neuroprotective during viremia. We speculate that higher CSF levels of these critical iron-delivery proteins support improved myelination and consequently, neurocognitive performance in PWH, providing a rationale for investigating their role in interventions to prevent and/or treat HAND.

Iron homeostasis is essential for brain health, and iron is also required for HIV replication, but the role of iron is largely unexplored in HIV-associated neurocognitive disorders. These disorders remain extremely common in people with HIV, despite antiretroviral therapy capable of suppressing viral replication, and they involve damage to white matter tracts and synaptodendritic architecture in the brain. We hypothesized that cerebrospinal fluid (CSF) levels of iron and two proteins involved in iron delivery, mitochondrial function, and protection against iron-mediated oxidative stress (H-ferritin and transferrin) are associated with neurocognitive performance over time in adults with HIV. These CSF iron-related biomarkers were measured at baseline (entry) in 403 adults with HIV from a large, prospective observational study who underwent comprehensive neurocognitive assessments at 6-month intervals. All participants were assigned a Global Deficit Score (GDS) and neurocognitive change status ( improving/stable/declining ), compared to baseline, at each visit. Biomarker associations with change status, and GDS differences over 30, 36, and 42 months of follow-up, were evaluated by multivariable regression. GDS-defined neurocognitive impairment was present in 120 study participants at entry (29.8%); 73% were on antiretroviral therapy. Of 267 participants with longitudinal follow-up, 16% were improving neurocognitively, and 20% were declining at their last follow-up visit (median 36 months). In multivariable-adjusted analyses, higher baseline CSF H-ferritin predicted improving neurocognitive performance at the last assessment ( p =0.029), and a better GDS over at least 30 months. Higher H-ferritin levels were also associated with better GDS-defined neurocognitive performance over 30, 36, and 42 months in virally suppressed individuals ( p -values p -values p e . g ., viremia/aviremia) of HIV infection, and they may provide a rationale for the use of iron-modulating interventions to prevent or treat neurocognitive decline in people with HIV.

People with HIV (PWH) continue to experience sensory neuropathy and neuropathic pain in the combination antiretroviral therapy (cART) era for unclear reasons. This study evaluated the role of iron in a previously reported association of iron-loading hemochromatosis (HFE) gene variants with reduced risk of neuropathy in PWH who received more neurotoxic cART, since an iron-related mechanism also might be relevant to neuropathic symptoms in PWH living in low-resource settings today. This time-to-event analysis addressed the impact of systemic iron levels on the rapidity of neuropathy onset in PWH who initiated cART. Soluble transferrin receptor (sTFR), the sTFR-ferritin index of iron stores, and high-sensitivity C-reactive protein (hsCRP) levels were determined in stored baseline sera from participants of known HFE genotype from AIDS Clinical Trials Group (ACTG) Study 384, a multicenter randomized clinical trial that evaluated cART strategies. Associations with incident neuropathy were evaluated in proportional-hazards, time-to-event regression models, adjusting for potential confounders. Of 151 eligible participants with stored serum who were included in the original genetic study, 43 had cART-associated neuropathy; 108 had sufficient serum for analysis, including 30 neuropathy cases. Carriers of HFE variants had higher systemic iron (lower sTFR and sTFR-ferritin index) and lower hsCRP levels than non-carriers (all pG variant, and lower baseline hsCRP were associated with significantly delayed neuropathy in self-reported whites (n = 28; all p-valuesG variant, and lower hsCRP predicted delayed onset of neuropathy among self-reported white individuals initating cART. These findings require confirmation but may have implications for cART in HIV+ populations in areas with high endemic iron deficiency, especially those PWH in whom older, more neurotoxic antiretroviral drugs are occasionally still used.

Corrected sentence in Interactions between the Effects of Drug Use and HIV-1 Infection Leads to Accelerated Disease Progression: “White matter damage