Benjamin B. Gelman

Mycobacterium tuberculosis (Mtb) and HIV are leading infectious causes of death worldwide and act synergistically to worsen disease during co-infection. C-type lectin receptors (CLR) respond to pathogen-associated carbohydrates to activate downstream innate immunity and can be exploited for entry of intracellular pathogens. The macrophage (MΦ) galactose-type lectin (MGL, CLEC10A) is an immunomodulatory CLR associated with M2 MΦ. We previously described an immune role for a murine MGL homologue in an experimental model of tuberculosis (TB). Herein we extend these findings by identifying human MGL as an important member of the Mtb-responsive pathogen recognition receptor (PRR) repertoire that is activated in both M1 and M2 polarizing conditions. MΦ exposure to Mtb activates MGL expression and abundant MGL+ cells are present in TB granulomas of human lung and lymph node. Silencing of MGL permits greater Mtb replication in MΦ derived from human peripheral blood monocytes. Compared to healthy controls, MΦ and neutrophils of people with HIV (PWH) have reduced MGL and tissue MGL levels negatively correlate with viral load. Binding assays with recombinant MGL demonstrates direct interaction with Mtb, but not HIV. In vitro Mtb exposure of PBMC from PWH revealed potential recovery of the MGL defect as well as a differential activation of MGL compared to the DC-SIGN and MR CLRs. MGL is thus an important mechanism of innate immunity and potential target for host directed therapy in those with TB or TB-HIV.

L-arginine metabolism is strongly linked with immunity to mycobacteria, primarily through the antimicrobial activity of nitric oxide (NO). The potential to modulate tuberculosis (TB) outcomes through interventions that target L-arginine pathways are limited by an incomplete understanding of mechanisms and inadequate in vivo modeling. These gaps in knowledge are compounded for HIV and Mtb co-infections, where activation of arginase-1 due to HIV infection may promote survival and replication of both Mtb and HIV. We utilized in vitro and in vivo systems to determine how arginase inhibition using N^ω-hydroxy-nor-L-arginine (nor-NOHA) alters L-arginine pathway metabolism relative to immune responses and disease outcomes following Mtb infection. Treatment with nor-NOHA polarized murine macrophages (RAW 264.7) towards M1 phenotype, increased NO, and reduced Mtb in RAW macrophages. In Balb/c mice, nor-NOHA reduced pulmonary arginase and increased the antimicrobial metabolite spermine in association with a trend towards reduced Mtb CFU in lung. In humanized immune system (HIS) mice, HIV infection increased plasma arginase and heightened the pulmonary arginase response to Mtb. Treatment with nor-NOHA increased cytokine responses to Mtb and Mtb/HIV in lung tissue but did not significantly alter bacterial burden or viral load. Our results suggest that L-arginine pathway modulators may have potential as host-directed therapies to augment antibiotics in TB chemotherapy.

Structural brain abnormalities, including those in white matter (WM), remain common in people with HIV (PWH). Their pathogenesis is uncertain and may reflect multiple etiologies. Oxidative stress is associated with inflammation, HIV, and its comorbidities. The post-translational carbonylation of proteins results from oxidative stress, and circulating protein carbonyls may reflect this. In this cross-sectional analysis, we evaluated the associations between protein carbonyls and a panel of soluble biomarkers of neuronal injury and inflammation in plasma (N = 45) and cerebrospinal fluid (CSF, n = 32) with structural brain MRI. The volume of abnormal WM was normalized for the total WM volume (nAWM). In this multisite project, all regression models were adjusted for the scanner. The candidate covariates included demographics, HIV disease characteristics, and comorbidities. Participants were PWH on virally suppressive antiretroviral therapy (ART) and were mostly white (64.4%) men (88.9%), with a mean age of 56.8 years. In unadjusted analyses, more nAWM was associated with higher plasma protein carbonyls (p = 0.002) and higher CCL2 (p = 0.045). In the adjusted regression models for nAWM, the association with plasma protein carbonyls remained significant (FDR p = 0.018). Protein carbonyls in plasma may be a valuable biomarker of oxidative stress and its associated adverse health effects, including within the central nervous system. If confirmed, these findings would support the hypothesis that reducing oxidative stress could treat or prevent WM injury in PWH.

Background: HIV infection causes neuroinflammation and immune activation (NIIA) and systemic inflammation and immune activation (SIIA), which in turn drive neurodegeneration (ND). Cross-sectionally, higher levels of NIIA biomarkers correlate with increased biomarkers of ND. A more convincing confirmation would be a longitudinal demonstration. Methods: PWH in the US multisite CHARTER Aging project were assessed at a baseline visit and after 12 years using standardized evaluations. We measured a panel of 14 biomarkers of NIIA, SIIA, and ND in plasma and CSF at two time points and calculated changes from baseline to the 12-year visit. Factor analysis yielded simplified indices of NIIA, SIIA, and ND. Results: The CSF NIIA factor analysis yielded Factor1 loading on soluble tumor necrosis factor type-2 (sTNFR-II) and neopterin, and Factor2, loading on MCP1, soluble CD14, and IL-6. The SIIA factor analysis yielded Factor1 loading on CRP, D-dimer, and Neopterin; Factor2 loading on sTNFR-II. The ND analysis yielded Factor1 loading on Phosphorylated tau (p-tau) and Aβ42; Factor2 loading on NFL. NIIA Factor1, but not Factor2, correlated with increases in CSF NFL (r = 0.370, p = 0.0002). Conclusions: Increases in NIIA and SIIA in PWH were associated with corresponding increases in ND, suggesting that reducing neuro/systemic inflammation might slow or reverse neurodegeneration.

HIV establishes a persistent viral reservoir in the brain despite viral suppression in blood to undetectable levels on antiretroviral therapy (ART). The brain viral reservoir in virally suppressed HIV+ individuals is not well-characterized. In this study, intact, defective, and total HIV proviral genomes were measured in frontal lobe white matter from 28 virally suppressed individuals on ART using the intact proviral DNA assay (IPDA). HIV gag DNA/RNA levels were measured using single-copy assays and expression of 78 genes related to inflammation and white matter integrity was measured using the NanoString platform. Intact proviral DNA was detected in brain tissues of 18 of 28 (64%) individuals on suppressive ART. The median proviral genome copy numbers in brain tissue as measured by the IPDA were: intact, 10 (IQR 1–92); 3′ defective, 509 (225–858); 5′ defective, 519 (273–906); and total proviruses, 1063 (501–2074) copies/10⁶ cells. Intact proviral genomes accounted for less than 10% (median 8.3%) of total proviral genomes in the brain, while 3′ and 5′ defective genomes accounted for 44% and 49%, respectively. There was no significant difference in median copy number of intact, defective, or total proviruses between groups stratified by neurocognitive impairment (NCI) vs. no NCI. In contrast, there was an increasing trend in intact proviruses in brains with vs. without neuroinflammatory pathology (56 vs. 5 copies/10⁶ cells, p = 0.1), but no significant differences in defective or total proviruses. Genes related to inflammation, stress responses, and white matter integrity were differentially expressed in brain tissues with >5 vs. +5 intact proviruses/10⁶ cells. These findings suggest that intact HIV proviral genomes persist in the brain at levels comparable to those reported in blood and lymphoid tissues and increase CNS inflammation/immune activation despite suppressive ART, indicating the importance of targeting the CNS reservoir to achieve HIV cure.

HIV-encoded DNA, RNA and proteins persist in the brain despite effective antiretroviral therapy (ART), with undetectable plasma and cerebrospinal fluid viral RNA levels, often in association with neurocognitive impairments. Although the determinants of HIV persistence have garnered attention, the expression and regulation of antiretroviral host restriction factors (RFs) in the brain for HIV and SIV remain unknown. We investigated the transcriptomic profile of antiretroviral RF genes by RNA-sequencing with confirmation by qRT-PCR in the cerebral cortex of people who are uninfected (HIV[−]), those who are HIV-infected without pre-mortem brain disease (HIV[+]), those who are HIV-infected with neurocognitive disorders (HIV[+]/HAND) and those with neurocognitive disorders with encephalitis (HIV[+]/HIVE). We observed significant increases in RF expression in the brains of HIV[+]/HIVE in association with the brain viral load. Machine learning techniques identified MAN1B1 as a key gene that distinguished the HIV[+] group from the HIV[+] groups with HAND. Analyses of SIV-associated RFs in brains from SIV-infected Chinese rhesus macaques with different ART regimens revealed diminished RF expression among ART-exposed SIV-infected animals, although ART interruption resulted in an induced expression of several RF genes including OAS3, RNASEL, MX2 and MAN1B1. Thus, the brain displays a distinct expression profile of RFs that is associated with the neurological status as well as the brain viral burden. Moreover, ART interruption can influence the brain’s RF profile, which might contribute to disease outcomes.

Previous studies showed that persons living with HIV (PLWH) demonstrate higher brain prefrontal cortex neuroinflammation and immunoproteasome expression compared to HIV-negative individuals; these associate positively with HIV levels. Lower expression of the antioxidant enzyme heme oxygenase 1 (HO-1) was observed in PLWH with HIV-associated neurocognitive impairment (HIV-NCI) compared to neurocognitively normal PLWH. We hypothesized that similar expression patterns occur throughout cortical, subcortical, and brainstem regions in PLWH, and that neuroinflammation and immunoproteasome expression associate with lower expression of neuronal markers. We analyzed autopsied brains (15 regions) from 9 PLWH without HIV-NCI and 7 matched HIV-negative individuals. Using Western blot and RT-qPCR, we quantified synaptic, inflammatory, immunoproteasome, endothelial, and antioxidant biomarkers, including HO-1 and its isoform heme oxygenase 2 (HO-2). In these PLWH without HIV-NCI, we observed higher expression of neuroinflammatory, endothelial, and immunoproteasome markers in multiple cortical and subcortical regions compared to HIV-negative individuals, suggesting a global brain inflammatory response to HIV. Several regions, including posterior cingulate cortex, globus pallidus, and cerebellum, showed a distinct pattern of higher type I interferon (IFN)-stimulated gene and immunoproteasome expression. PLWH without HIV-NCI also had (i) stable or higher HO-1 expression and positive associations between (ii) HO-1 and HIV levels (CSF, plasma) and (iii) HO-1 expression and neuroinflammation, in multiple cortical, subcortical, and brainstem regions. We observed no differences in synaptic marker expression, suggesting little, if any, associated neuronal injury. We speculate that this may reflect a neuroprotective effect of a concurrent HO-1 antioxidant response despite global neuroinflammation, which will require further investigation.