Role of macrophages in HIV pathogenesis and cure: NIH perspectives

Journal of leukocyte biology

2022 Sep 08

Joseph, J;Daley, W;Lawrence, D;Lorenzo, E;Perrin, P;Rao, VR;Tsai, SY;Varthakavi, V;
PMID: 36073341 | DOI: 10.1002/JLB.4MR0722-619R

Macrophages play a significant role in HIV infection and contribute to pathogenesis of comorbidities as well as establishment of the viral reservoir in people living with HIV. While CD4+ T cells are considered the main targets of HIV infection, infected macrophages resist the cytopathic effects of infection, contributing to the persistent HIV reservoir. Furthermore, activated macrophages drive inflammation and contribute to the development of comorbidities, including HIV-associated CNS dysfunction. Better understanding the role of macrophages in HIV infection, persistence, and comorbidities can lead to development of innovative therapeutic strategies to address HIV-related outcomes in people living with HIV. In October 2021, the National Institute of Mental Health and the Ragon Institute of MGH, MIT, and Harvard conducted a virtual meeting on role of macrophages in HIV infection, pathogenesis, and cure. This review article captures the key highlights from this meeting and provides an overview of interests and activities of various NIH institutes involved in supporting research on macrophages and HIV.Published 2022. This article is a U.S. Government work and is in the public domain in the USA.

Implications of the accumulation of CXCR5+ NK cells in lymph nodes of HIV-1 infected patients

EBioMedicine

2021 Dec 29

Guo, AL;Jiao, YM;Zhao, QW;Huang, HH;Deng, JN;Zhang, C;Fan, X;Xu, RN;Zhang, JY;Zhen, C;Xie, ZM;Qin, YM;Xu, JQ;Yang, Y;Shi, M;Huang, L;Song, JW;Wang, FS;
PMID: 34973625 | DOI: 10.1016/j.ebiom.2021.103794

B cell follicles are immune-privileged sites where intensive HIV-1 replication and latency occur, preventing a permanent cure. Recent study showed that CXCR5+ NK cells in B cell follicles can inhibit SIV replication in African green monkeys, but this has not been reported in HIV-1 infected patients.Lymphocytes and tissue sections of lymph node were collected from 11 HIV-1 positive antiretroviral therapy (ART)-naive and 19 HIV-1 negative donors. We performed immunofluorescence and RNA-scope to detect the location of CXCR5+ NK cells and its relationship with HIV-1 RNA, and performed flow cytometry and RNA-seq to analyze the frequency, phenotypic and functional characteristics of CXCR5+ NK cells. The CXCL13 expression were detected by immunohistochemistry.CXCR5+ NK cells, which accumulated in LNs from HIV-1 infected individuals, expressed high levels of activating receptors such as NKG2D and NKp44. CXCR5+ NK cells had upregulated expression of CD107a and β-chemokines, which were partially impaired in HIV-1 infection. Importantly, the frequency of CXCR5+NK cells was inversely related to the HIV-1 viral burden in LNs. In addition, CXCL13-the ligand of CXCR5-was upregulated in HIV-1 infected individuals and positively correlated with the frequency of CXCR5+ NK cells.During chronic HIV-1 infection, CXCR5+ NK cells accumulated in lymph node, exhibit altered immune characteristics and underlying anti-HIV-1 effect, which may be an effective target for a functional cure of HIV-1.

MRP8/14 is a Molecular Signature Triggered by Dopamine in HIV Latent Myeloid Targets that Increases HIV Transcription and Distinguishes HIV+ Methamphetamine Users with Detectable CSF Viral Load and Brain Pathology

PREPRINT

2023 May 04

Basova, L;Lindsey, A;McGovern, A;Gaskill, P;Rosander, A;Delorme-Walker, V;ElShamy, W;Pendyala, V;Ellis, R;Cherner, M;Iudicello, J;Marcondes, M;
| DOI: 10.20944/preprints202305.0132.v1

There is a significant overlap between HIV infection and substance use disorders. Dopamine (DA) is the most abundantly upregulated neurotransmitter in Methamphetamine abuse, with receptors (DRD1-5) that are expressed by neurons as well as by a large diversity of cell types, including innate immune cells that are targets of HIV infection, making them responsive to the hyperdopaminergic environment that is characteristic of stimulant drugs. Therefore, the presence of high levels of dopamine may affect the pathogenesis of HIV, particularly in the brain. The stimulation of HIV-latently infected U1 promonocytes with DA significantly increased viral p24 levels in the supernatant at 24 hrs, suggesting effects on activation and replication. Using selective agonists to different DRDs, we found that DRD1 played a major role in activating viral transcription, followed by DRD4, which increased p24 with a slower kinetic. Transcriptome and systems biology analyses led to the identification of a cluster of genes responsive to DA, where S100A8 and S100A9 were most significantly correlated with the early increase of p24 levels following DA stimulation. Conversely, DA increased the expression of these genes’ transcripts at the protein level, respectively MRP8 and MRP14, which form a complex also known as Calprotectin. Interestingly, MRP8/14 was able to stimulate HIV transcription in latent U1 cells, and this occurred via binding to the receptor for advanced glycosylation end-product (RAGE). Using selective agonists, both DRD1 and DRD4 increased MRP8/14 on the surface and in the cytoplasm, as well as secreted in the supernatants. On the other hand, while DRD1/5 did not affect the expression of RAGE, DRD4 stimulation caused its downregulation, offering a mechanistic for the delayed effect via DRD4 on p24 increase. To cross-validate MRP8/14 as a DA signature with biomarker value, we tested its expression in HIV+ Meth users’ brains and peripheral cells. MRP8/14+ cells were more frequently identified in mesolimbic areas such as basal ganglia of HIV+ Meth+ cases compared to HIV+ non-Meth users or to controls. Likewise, MRP8/14+ CD11b+ monocytes were more frequent in HIV+ Meth users, particularly in participants with detectable viral load in the CSF. Overall, our results suggest that the MRP8 and MRP14 complex may serve as a signature to distinguish subjects using addictive substances in the context of HIV, and that this may play a role in aggravating HIV pathology by promoting viral replication in people with HIV who use Meth.

Microglia Proliferation Underlies Synaptic Dysfunction in the Prefrontal Cortex: Implications for the Pathogenesis of HIV-1-Associated Neurocognitive and Affective Alterations

bioRxiv : the preprint server for biology

2023 Jan 21

Li, H;McLaurin, KA;Mactutus, CF;Booze, RM;
PMID: 36711456 | DOI: 10.1101/2023.01.20.524942

Microglia, which are productively infected by HIV-1, are critical for brain development and maturation, as well as synaptic plasticity. The pathophysiology of HIV-infected microglia and their role in the pathogenesis of HIV-1-associated neurocognitive and affective alterations, however, remains understudied. Three complementary aims were undertaken to critically address this knowledge gap. First, the predominant cell type expressing HIV-1 mRNA in the dorsolateral prefrontal cortex of postmortem HIV-1 seropositive individuals with HAND was investigated. Utilization of a combined RNAscope multiplex fluorescent and immunostaining assay revealed prominent HIV-1 mRNA in microglia of postmortem HIV-1 seropositive individuals with HAND. Second, measures of microglia proliferation and neuronal damage were evaluated in chimeric HIV (EcoHIV) rats. Eight weeks after EcoHIV innoculation, enhanced microglial proliferation was observed in the medial prefrontal cortex (mPFC) of EcoHIV rats, evidenced by an increased number of cells co-localized with both Iba1+ and Ki67+ relative to control animals. Neuronal damage in EcoHIV infected rats was evidenced by pronounced decreases in both synaptophysin and post synaptic density protein 95 (PSD-95), markers of pre-synaptic and post-synaptic damage, respectively. Third, regression analyses were conducted to evaluate whether microglia proliferation mechanistically underlies neuronal damage in EcoHIV and control animals. Indeed, microglia proliferation accounts for 42-68.6% of the variance in synaptic dysfunction. Collectively, microglia proliferation induced by chronic HIV-1 viral protein exposure may underlie the profound synaptodendritic alterations in HIV-1. Understanding how microglia are involved in the pathogenesis of HAND and HIV-1-associated affective disorders affords a key target for the development of novel therapeutics.

HIV-1 Vpr suppresses expression of the thiazide-sensitive sodium chloride co-transporter in the distal convoluted tubule

PloS one

2022 Sep 21

Shrivastav, S;Lee, H;Okamoto, K;Lu, H;Yoshida, T;Latt, KZ;Wakashin, H;Dalgleish, JLT;Koritzinsky, EH;Xu, P;Asico, LD;Chung, JY;Hewitt, S;Gildea, JJ;Felder, RA;Jose, PA;Rosenberg, AZ;Knepper, MA;Kino, T;Kopp, JB;
PMID: 36129874 | DOI: 10.1371/journal.pone.0273313

HIV-associated nephropathy (HIVAN) impairs functions of both glomeruli and tubules. Attention has been previously focused on the HIVAN glomerulopathy. Tubular injury has drawn increased attention because sodium wasting is common in hospitalized HIV/AIDS patients. We used viral protein R (Vpr)-transgenic mice to investigate the mechanisms whereby Vpr contributes to urinary sodium wasting. In phosphoenolpyruvate carboxykinase promoter-driven Vpr-transgenic mice, in situ hybridization showed that Vpr mRNA was expressed in all nephron segments, including the distal convoluted tubule. Vpr-transgenic mice, compared with wild-type littermates, markedly increased urinary sodium excretion, despite similar plasma renin activity and aldosterone levels. Kidneys from Vpr-transgenic mice also markedly reduced protein abundance of the Na+-Cl- cotransporter (NCC), while mineralocorticoid receptor (MR) protein expression level was unchanged. In African green monkey kidney cells, Vpr abrogated the aldosterone-mediated stimulation of MR transcriptional activity. Gene expression of Slc12a3 (NCC) in Vpr-transgenic mice was significantly lower compared with wild-type mice, assessed by both qRT-PCR and RNAScope in situ hybridization analysis. Chromatin immunoprecipitation assays identified multiple MR response elements (MRE), located from 5 kb upstream of the transcription start site and extending to the third exon of the SLC12A3 gene. Mutation of MRE and SP1 sites in the SLC12A3 promoter region abrogated the transcriptional responses to aldosterone and Vpr, indicating that functional MRE and SP1 are required for the SLC12A3 gene suppression in response to Vpr. Thus, Vpr attenuates MR transcriptional activity and inhibits Slc12a3 transcription in the distal convoluted tubule and contributes to salt wasting in Vpr-transgenic mice.

Characterization of Macrophage-Tropic HIV-1 Infection of Central Nervous System Cells and the Influence of Inflammation

Journal of virology

2022 Aug 17

Woodburn, BM;Kanchi, K;Zhou, S;Colaianni, N;Joseph, SB;Swanstrom, R;
PMID: 35975998 | DOI: 10.1128/jvi.00957-22

HIV-1 infection within the central nervous system (CNS) includes evolution of the virus, damaging inflammatory cascades, and the involvement of multiple cell types; however, our understanding of how Env tropism and inflammation can influence CNS infectivity is incomplete. In this study, we utilize macrophage-tropic and T cell-tropic HIV-1 Env proteins to establish accurate infection profiles for multiple CNS cells under basal and interferon alpha (IFN-α) or lipopolysaccharide (LPS)-induced inflammatory states. We found that macrophage-tropic viruses confer entry advantages in primary myeloid cells, including monocyte-derived macrophage, microglia, and induced pluripotent stem cell (iPSC)-derived microglia. However, neither macrophage-tropic or T cell-tropic HIV-1 Env proteins could mediate infection of astrocytes or neurons, and infection was not potentiated by induction of an inflammatory state in these cells. Additionally, we found that IFN-α and LPS restricted replication in myeloid cells, and IFN-α treatment prior to infection with vesicular stomatitis virus G protein (VSV G) Envs resulted in a conserved antiviral response across all CNS cell types. Further, using RNA sequencing (RNA-seq), we found that only myeloid cells express HIV-1 entry receptor/coreceptor transcripts at a significant level and that these transcripts in select cell types responded only modestly to inflammatory signals. We profiled the transcriptional response of multiple CNS cells to inflammation and found 57 IFN-induced genes that were differentially expressed across all cell types. Taken together, these data focus attention on the cells in the CNS that are truly permissive to HIV-1, further highlight the role of HIV-1 Env evolution in mediating infection in the CNS, and point to limitations in using model cell types versus primary cells to explore features of virus-host interaction. IMPORTANCE The major feature of HIV-1 pathogenesis is the induction of an immunodeficient state in the face of an enhanced state of inflammation. However, for many of those infected, there can be an impact on the central nervous system (CNS) resulting in a wide range of neurocognitive defects. Here, we use a highly sensitive and quantitative assay for viral infectivity to explore primary and model cell types of the brain for their susceptibility to infection using viral entry proteins derived from the CNS. In addition, we examine the ability of an inflammatory state to alter infectivity of these cells. We find that myeloid cells are the only cell types in the CNS that can be infected and that induction of an inflammatory state negatively impacts viral infection across all cell types.

Paradoxically greater persistence of HIV RNA+ cells in lymphoid tissue when ART is initiated in the earliest stage of infection

The Journal of infectious diseases

2022 Mar 11

Kroon, E;Chottanapund, S;Buranapraditkun, S;Sacdalan, C;Colby, DJ;Chomchey, N;Prueksakaew, P;Pinyakorn, S;Trichavaroj, R;Vasan, S;Manasnayakorn, S;Reilly, C;Helgeson, E;Anderson, J;David, C;Zulk, J;de Souza, M;Tovanabutra, S;Schuetz, A;Robb, ML;Douek, DC;Phanuphak, N;Haase, A;Ananworanich, J;Schacker, TW;
PMID: 35275599 | DOI: 10.1093/infdis/jiac089

Starting antiretroviral therapy (ART) in Fiebig 1 acute HIV infection limits the size of viral reservoirs in lymphoid tissues, but does not impact time to virus rebound during a treatment interruption. To better understand why the reduced reservoir size did not increase the time to rebound we measured the frequency and location of HIV RNA+ cells in lymph nodes from participants in the RV254 acute infection cohort. HIV vRNA+ cells were detected more frequently and in greater numbers when ART was initiated in Fiebig 1 compared to later Fiebig stages and were localized to the T cell zone compared to the B cell follicle with treatment in later Fiebig stages. Variability of virus production in people treated during acute infection suggests that the balance between virus producing cells and the immune response to clear infected cells rapidly evolves during the earliest stages of infection.

CRISPR editing of CCR5 and HIV-1 facilitates viral elimination in antiretroviral drug-suppressed virus-infected humanized mice

Proceedings of the National Academy of Sciences of the United States of America

2023 May 09

Dash, PK;Chen, C;Kaminski, R;Su, H;Mancuso, P;Sillman, B;Zhang, C;Liao, S;Sravanam, S;Liu, H;Waight, E;Guo, L;Mathews, S;Sariyer, R;Mosley, RL;Poluektova, LY;Caocci, M;Amini, S;Gorantla, S;Burdo, TH;Edagwa, B;Gendelman, HE;Khalili, K;
PMID: 37126704 | DOI: 10.1073/pnas.2217887120

Treatment of HIV-1ADA-infected CD34+ NSG-humanized mice with long-acting ester prodrugs of cabotegravir, lamivudine, and abacavir in combination with native rilpivirine was followed by dual CRISPR-Cas9 C-C chemokine receptor type five (CCR5) and HIV-1 proviral DNA gene editing. This led to sequential viral suppression, restoration of absolute human CD4+ T cell numbers, then elimination of replication-competent virus in 58% of infected mice. Dual CRISPR therapies enabled the excision of integrated proviral DNA in infected human cells contained within live infected animals. Highly sensitive nucleic acid nested and droplet digital PCR, RNAscope, and viral outgrowth assays affirmed viral elimination. HIV-1 was not detected in the blood, spleen, lung, kidney, liver, gut, bone marrow, and brain of virus-free animals. Progeny virus from adoptively transferred and CRISPR-treated virus-free mice was neither detected nor recovered. Residual HIV-1 DNA fragments were easily seen in untreated and viral-rebounded animals. No evidence of off-target toxicities was recorded in any of the treated animals. Importantly, the dual CRISPR therapy demonstrated statistically significant improvements in HIV-1 cure percentages compared to single treatments. Taken together, these observations underscore a pivotal role of combinatorial CRISPR gene editing in achieving the elimination of HIV-1 infection.

Characterization and distribution of HIV-infected cells in semen

Emerging microbes & infections

2022 Dec 01

Gao, L;Jiao, YM;Ma, P;Sun, L;Zhao, H;Guo, AL;Fan, X;Zhang, C;Song, JW;Zhang, JY;Lu, F;Wang, FS;
PMID: 35253610 | DOI: 10.1080/22221751.2022.2049982

Semen is a known vector for both human immunodeficiency virus (HIV) infection and transmission. However, the distribution and characteristics of HIV-infected cells in semen remain unclear. Investigating the possibility of transmission through the spermatozoon in semen is of great clinical significance to improve the strategies for exposure prevention and assisted reproduction for HIV-infected partners. Twenty-six HIV-infected patients, including twelve treatment-naïve (TN) patients and fourteen antiretroviral treated (ART) patients, were enrolled in this study. HIV p24 protein in spermatozoa was detected using imaging flow cytometry and immunohistochemistry, and HIV RNA was identified using next-generation RNAscope in situ hybridization. Additionally, we described the rates of HIV-positive spermatozoon and CD4+ T lymphocytes in semen, and found that p24+ spermatozoon were mainly CD4 negative regardless of whether the patients received ART. Of note, p24-positive cells in semen are predominantly spermatozoa, and we confirmed that motile spermatozoa carried HIV into peripheral blood mononuclear cells of healthy men in vitro. Our findings provide evidence regarding the risk of HIV-infected spermatozoa.

Intact HIV proviruses persist in the brain despite viral suppression with ART

Annals of neurology

2022 Jul 22

Cochrane, CR;Angelovich, TA;Byrnes, SJ;Waring, E;Guanizo, AC;Trollope, GS;Zhou, J;Vue, J;Senior, L;Wanicek, E;Jamal Eddine, J;Gartner, MJ;Jenkins, TA;Gorry, PR;Brew, BJ;Lewin, SR;Estes, JD;Roche, M;Churchill, MJ;
PMID: 35867351 | DOI: 10.1002/ana.26456

Human Immunodeficiency Virus (HIV) persistence in blood and tissue reservoirs including the brain is a major barrier to HIV cure and possible cause of comorbid disease. However, the size and replication competent nature of the CNS reservoir is unclear. Here we used the intact proviral DNA assay (IPDA) to provide the first quantitative assessment of the intact and defective HIV reservoir in the brain of people with HIV (PWH).Total, intact and defective HIV proviruses were measured in autopsy frontal lobe tissue from viremic (n=18) or virologically suppressed (n=12) PWH. Total or intact/defective proviruses were measured by detection of HIV pol or the IPDA, respectively, through use of droplet digital PCR (ddPCR). HIV-seronegative individuals were included as controls (n=6).Total HIV DNA was present at similar levels in brain tissues from untreated viremic and antiretroviral (ART)-suppressed individuals (median: 22.3 vs 26.2 HIV pol copies/106 cells), reflecting a stable CNS reservoir of HIV that persists despite therapy. Furthermore, 8/10 viremic and 6/9 virally suppressed PWH also harbored intact proviruses in the CNS (4.63 vs 12.7 intact copies/106 cells). Viral reservoirs in CNS and matched lymphoid tissue were similar in the composition of intact and/or defective proviruses, albeit at lower levels in the brain. Importantly, CNS resident CD68+ myeloid cells in virally suppressed individuals harbored HIV DNA, directly showing the presence of a CNS resident HIV reservoir.Our results demonstrate the first evidence for an intact, potentially replication competent HIV reservoir in the CNS of virally suppressed PWH. This article is protected by

S100A8-mediated metabolic adaptation controls HIV-1 persistence in macrophages in vivo

Nature communications

2022 Oct 11

Real, F;Zhu, A;Huang, B;Belmellat, A;Sennepin, A;Vogl, T;Ransy, C;Revol, M;Arrigucci, R;Lombès, A;Roth, J;Gennaro, ML;Bouillaud, F;Cristofari, S;Bomsel, M;
PMID: 36220814 | DOI: 10.1038/s41467-022-33401-x

HIV-1 eradication is hindered by viral persistence in cell reservoirs, established not only in circulatory CD4+T-cells but also in tissue-resident macrophages. The nature of macrophage reservoirs and mechanisms of persistence despite combined anti-retroviral therapy (cART) remain unclear. Using genital mucosa from cART-suppressed HIV-1-infected individuals, we evaluated the implication of macrophage immunometabolic pathways in HIV-1 persistence. We demonstrate that ex vivo, macrophage tissue reservoirs contain transcriptionally active HIV-1 and viral particles accumulated in virus-containing compartments, and harbor an inflammatory IL-1R+S100A8+MMP7+M4-phenotype prone to glycolysis. Reactivation of infectious virus production and release from these reservoirs in vitro are induced by the alarmin S100A8, an endogenous factor produced by M4-macrophages and implicated in "sterile" inflammation. This process metabolically depends on glycolysis. Altogether, inflammatory M4-macrophages form a major tissue reservoir of replication-competent HIV-1, which reactivate viral production upon autocrine/paracrine S100A8-mediated glycolytic stimulation. This HIV-1 persistence pathway needs to be targeted in future HIV eradication strategies.

The Imbalance Between Intestinal Th17 and Treg Cells Is Associated with an Incomplete Immune Reconstitution During Long-Term Antiretroviral Therapy in Patients with HIV

Viral immunology

2023 May 15

Guo, YT;Guo, XY;Fan, LN;Wang, ZR;Qu, MM;Zhang, C;Fan, X;Song, JW;Yang, BP;Zhang, JY;Xu, R;Jiao, YM;Ma, P;Chen, YK;Wang, FS;
PMID: 37184871 | DOI: 10.1089/vim.2023.0017

Studies assessing the gut mucosal immune balance in HIV-infected patients using intestinal samples are scarce. In this study, we used intestinal mucosal specimens from the ileocecal region of seven immunological nonresponders (INRs), nine immunological responders (IRs), and six HIV-negative controls. We investigated T helper 17 (Th17) and T regulatory (Treg) cell counts and their ratio, zonula occludens-1 (ZO-1), intestinal fatty acid-binding protein (I-FABP), tumor necrosis factor-α, CD4+ T cell counts, HIV DNA, and cell-associated HIV RNA. The results showed that INRs had lower Th17 and higher Treg cell counts than IR, resulting in a significant difference in the Th17/Treg ratio between IRs and INRs. In addition, INRs had lower ZO-1 and higher I-FABP levels than IRs. The Th17/Treg ratio was positively associated with ZO-1 and negatively associated with I-FABP levels. There was a positive correlation between Th17/Treg ratio and CD4+ T cell counts and a negative correlation between the Th17/Treg ratio and HIV DNA in the intestine. Our study suggests that the imbalance of Th17/Treg in the intestine is a characteristic of incomplete immune reconstitution to antiretroviral therapy and is associated with intestinal damage.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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