Non-thermal plasma modulates cellular markers associated with immunogenicity in a model of latent HIV-1 infection

Effective control of infection by human immunodeficiency virus type 1 (HIV-1), the causative agent of the acquired immunodeficiency syndrome (AIDS), requires continuous and life-long use of anti-retroviral therapy (ART) by people living with HIV-1 (PLWH). In the absence of ART, HIV-1 reemergence from latently infected cells is ineffectively suppressed due to suboptimal innate and cytotoxic T lymphocyte responses. However, ART-free control of HIV-1 infection may be possible if the inherent immunological deficiencies can be reversed or restored. Herein we present a novel approach for modulating the immune response to HIV-1 that involves the use of non-thermal plasma (NTP), which is an ionized gas containing various reactive oxygen and nitrogen species (RONS). J-Lat cells were used as a model of latent HIV-1 infection to assess the effects of NTP application on viral latency and the expression of pro-phagocytic and pro-chemotactic damage-associated molecular patterns (DAMPs). Exposure of J-Lat cells to NTP resulted in stimulation of HIV-1 gene expression, indicating a role in latency reversal, a necessary first step in inducing adaptive immune responses to viral antigens. This was accompanied by the release of pro-inflammatory cytokines and chemokines including interleukin-1β (IL-1β) and interferon-γ (IFN-γ); the display of pro-phagocytic markers calreticulin (CRT), heat shock proteins (HSP) 70 and 90; and a correlated increase in macrophage phagocytosis of NTP-exposed J-Lat cells. In addition, modulation of surface molecules that promote or inhibit antigen presentation was also observed, along with an altered array of displayed peptides on MHC I, further suggesting methods by which NTP may modify recognition and targeting of cells in latent HIV-1 infection. These studies represent early progress toward an effective NTP-based ex vivo immunotherapy to resolve the dysfunctions of the immune system that enable HIV-1 persistence in PLWH.

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HIV-1 subverts the complement system in semen to enhance viral transmission

Mucosal Immunology ◽

10.1038/s41385-021-00376-9 ◽

2021 ◽

Author(s):

Bernadien M. Nijmeijer ◽

Marta Bermejo-Jambrina ◽

Tanja M. Kaptein ◽

Carla M. S. Ribeiro ◽

Doris Wilflingseder ◽

...

Keyword(s):

Ex Vivo ◽

Virus Transmission ◽

Sexual Contact ◽

Target Cells ◽

People Living With Hiv ◽

Lectin Receptor ◽

Pre Treatment ◽

Living With Hiv ◽

Hiv 1

AbstractSemen is important in determining HIV-1 susceptibility but it is unclear how it affects virus transmission during sexual contact. Mucosal Langerhans cells (LCs) are the first immune cells to encounter HIV-1 during sexual contact and have a barrier function as LCs are restrictive to HIV-1. As semen from people living with HIV-1 contains complement-opsonized HIV-1, we investigated the effect of complement on HIV-1 dissemination by human LCs in vitro and ex vivo. Notably, pre-treatment of HIV-1 with semen enhanced LC infection compared to untreated HIV-1 in the ex vivo explant model. Infection of LCs and transmission to target cells by opsonized HIV-1 was efficiently inhibited by blocking complement receptors CR3 and CR4. Complement opsonization of HIV-1 enhanced uptake, fusion, and integration by LCs leading to an increased transmission of HIV-1 to target cells. However, in the absence of both CR3 and CR4, C-type lectin receptor langerin was able to restrict infection of complement-opsonized HIV-1. These data suggest that complement enhances HIV-1 infection of LCs by binding CR3 and CR4, thereby bypassing langerin and changing the restrictive nature of LCs into virus-disseminating cells. Targeting complement factors might be effective in preventing HIV-1 transmission.

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Potential of the NKG2D/NKG2DL Axis in NK Cell-Mediated Clearance of the HIV-1 Reservoir

International Journal of Molecular Sciences ◽

10.3390/ijms20184490 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4490 ◽

Cited By ~ 2

Author(s):

Maria G. Desimio ◽

Daniela A. Covino ◽

Margherita Doria

Keyword(s):

T Cells ◽

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

Virus Reactivation ◽

Major Drawback ◽

Virus Eradication ◽

Latent Hiv ◽

Hiv 1

Viral persistency in latently infected CD4+ T cells despite antiretroviral therapy (ART) represents a major drawback in the fight against HIV-1. Efforts to purge latent HIV-1 have been attempted using latency reversing agents (LRAs) that activate expression of the quiescent virus. However, initial trials have shown that immune responses of ART-treated patients are ineffective at clearing LRA-reactivated HIV-1 reservoirs, suggesting that an adjuvant immunotherapy is needed. Here we overview multiple lines of evidence indicating that natural killer (NK) cells have the potential to induce anti-HIV-1 responses relevant for virus eradication. In particular, we focus on the role of the NKG2D activating receptor that crucially enables NK cell-mediated killing of HIV-1-infected cells. We describe recent data indicating that LRAs can synergize with HIV-1 at upregulating ligands for NKG2D (NKG2DLs), hence sensitizing T cells that exit from viral latency for recognition and lysis by NK cells; in addition, we report in vivo and ex vivo data showing the potential benefits and drawbacks that LRAs may have on NKG2D expression and, more in general, on the cytotoxicity of NK cells. Finally, we discuss how the NKG2D/NKG2DLs axis can be exploited for the development of effective HIV-1 eradication strategies combining LRA-induced virus reactivation with recently optimized NK cell-based immunotherapies.

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Ex Vivo Vitamin D Stimulation of Alveolar Macrophage Phagocytosis Index in People Living with HIV, Stratified by Smoking Status

10.1164/ajrccm-conference.2019.199.1_meetingabstracts.a5197 ◽

2019 ◽

Author(s):

J. Han ◽

S.K. Cribbs ◽

D.M. Guidot ◽

V. Tangpricha ◽

G.S. Martin ◽

...

Keyword(s):

Vitamin D ◽

Alveolar Macrophage ◽

Ex Vivo ◽

Smoking Status ◽

People Living With Hiv ◽

Macrophage Phagocytosis ◽

Phagocytosis Index ◽

Living With Hiv ◽

Stimulation Of

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Selective cell death in HIV-1-infected cells by DDX3 inhibitors leads to depletion of the inducible reservoir

Nature Communications ◽

10.1038/s41467-021-22608-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Shringar Rao ◽

Cynthia Lungu ◽

Raquel Crespo ◽

Thijs H. Steijaert ◽

Alicja Gorska ◽

...

Keyword(s):

T Cells ◽

Cell Death ◽

Cd4 T Cells ◽

Viral Rna ◽

People Living With Hiv ◽

Infected Cells ◽

Latent Hiv ◽

Apoptotic Pathways ◽

Induced Apoptosis ◽

Hiv 1

AbstractAn innovative approach to eliminate HIV-1-infected cells emerging out of latency, the major hurdle to HIV-1 cure, is to pharmacologically reactivate viral expression and concomitantly trigger intracellular pro-apoptotic pathways in order to selectively induce cell death (ICD) of infected cells, without reliance on the extracellular immune system. In this work, we demonstrate the effect of DDX3 inhibitors on selectively inducing cell death in latent HIV-1-infected cell lines, primary CD4+ T cells and in CD4+ T cells from cART-suppressed people living with HIV-1 (PLWHIV). We used single-cell FISH-Flow technology to characterise the contribution of viral RNA to inducing cell death. The pharmacological targeting of DDX3 induced HIV-1 RNA expression, resulting in phosphorylation of IRF3 and upregulation of IFNβ. DDX3 inhibition also resulted in the downregulation of BIRC5, critical to cell survival during HIV-1 infection, and selectively induced apoptosis in viral RNA-expressing CD4+ T cells but not bystander cells. DDX3 inhibitor treatment of CD4+ T cells from PLWHIV resulted in an approximately 50% reduction of the inducible latent HIV-1 reservoir by quantitation of HIV-1 RNA, by FISH-Flow, RT-qPCR and TILDA. This study provides proof of concept for pharmacological reversal of latency coupled to induction of apoptosis towards the elimination of the inducible reservoir.

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Humanized Mouse Model of HIV-1 Latency with Enrichment of Latent Virus in PD-1+and TIGIT+CD4 T Cells

Journal of Virology ◽

10.1128/jvi.02086-18 ◽

2019 ◽

Vol 93 (10) ◽

Cited By ~ 4

Author(s):

George N. Llewellyn ◽

Eduardo Seclén ◽

Stephen Wietgrefe ◽

Siyu Liu ◽

Morgan Chateau ◽

...

Keyword(s):

T Cells ◽

Mouse Model ◽

Ex Vivo ◽

Latent Reservoir ◽

Humanized Mouse ◽

Humanized Mouse Model ◽

Latently Infected ◽

Infected Cells ◽

Latent Hiv ◽

Hiv 1

ABSTRACTCombination anti-retroviral drug therapy (ART) potently suppresses HIV-1 replication but does not result in virus eradication or a cure. A major contributing factor is the long-term persistence of a reservoir of latently infected cells. To study this reservoir, we established a humanized mouse model of HIV-1 infection and ART suppression based on an oral ART regimen. Similar to humans, HIV-1 levels in the blood of ART-treated animals were frequently suppressed below the limits of detection. However, the limited timeframe of the mouse model and the small volume of available samples makes it a challenging model with which to achieve full viral suppression and to investigate the latent reservoir. We therefore used anex vivolatency reactivation assay that allows a semiquantitative measure of the latent reservoir that establishes in individual animals, regardless of whether they are treated with ART. Using this assay, we found that latently infected human CD4 T cells can be readily detected in mouse lymphoid tissues and that latent HIV-1 was enriched in populations expressing markers of T cell exhaustion, PD-1 and TIGIT. In addition, we were able to use theex vivolatency reactivation assay to demonstrate that HIV-specific TALENs can reduce the fraction of reactivatable virus in the latently infected cell population that establishesin vivo, supporting the use of targeted nuclease-based approaches for an HIV-1 cure.IMPORTANCEHIV-1 can establish latent infections that are not cleared by current antiretroviral drugs or the body’s immune responses and therefore represent a major barrier to curing HIV-infected individuals. However, the lack of expression of viral antigens on latently infected cells makes them difficult to identify or study. Here, we describe a humanized mouse model that can be used to detect latent but reactivatable HIV-1 in both untreated mice and those on ART and therefore provides a simple system with which to study the latent HIV-1 reservoir and the impact of interventions aimed at reducing it.

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Glycolysis downregulation is a hallmark of HIV-1 latency and sensitizes infected cells to oxidative stress

10.1101/2020.12.30.424810 ◽

2020 ◽

Author(s):

Iart Luca Shytaj ◽

Francesco Andrea Procopio ◽

Mohammad Tarek ◽

Irene Carlon-Andres ◽

Hsin-Yao Tang ◽

...

Keyword(s):

Pentose Phosphate ◽

Viral Reactivation ◽

People Living With Hiv ◽

Cellular Models ◽

Downstream Effectors ◽

Latently Infected ◽

Infected Cells ◽

Latent Hiv ◽

Living With Hiv ◽

Hiv 1

AbstractHIV-1 infects lymphoid and myeloid cells, which can harbor a latent proviral reservoir responsible for maintaining lifelong infection. Glycolytic metabolism has been identified as a determinant of susceptibility to HIV-1 infection, but its role in the development and maintenance of HIV-1 latency has not been elucidated. By combining transcriptomic, proteomic and metabolomic analysis, we here show that transition to latent HIV-1 infection downregulates glycolysis, while viral reactivation by conventional stimuli reverts this effect. Decreased glycolytic output in latently infected cells is associated with downregulation of NAD+/NADH. Consequently, infected cells rely on the parallel pentose phosphate pathway and its main product, the antioxidant NADPH, fueling antioxidant pathways maintaining HIV-1 latency. Of note, blocking NADPH downstream effectors, thioredoxin and glutathione, favors HIV-1 reactivation from latency in lymphoid and myeloid cellular models. This provides a “shock and kill effect” decreasing proviral DNA in cells from people-living-with-HIV/AIDS. Overall, our data show that downmodulation of glycolysis is a metabolic signature of HIV-1 latency that can be exploited to target latently infected cells with eradication strategies.

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Novel Role of UHRF1 in DNA methylation-mediated repression of latent HIV-1

10.1101/2021.06.15.448539 ◽

2021 ◽

Author(s):

Roxane Verdikt ◽

Sophie Bouchat ◽

Alexander O. Pasternak ◽

Lorena Nestola ◽

Gilles Darcis ◽

...

Keyword(s):

Dna Methylation ◽

Molecular Mechanisms ◽

Ex Vivo ◽

Proof Of Concept ◽

Methylation Inhibitor ◽

Dna Methylation Inhibitor ◽

Latent Hiv ◽

Hiv 1 ◽

Patient Cell

The multiplicity, heterogeneity and dynamic nature of HIV-1 latency mechanisms are reflected in the current lack of functional cure for HIV-1 and in the various reported ex vivo potencies of latency-reversing agents. Here, we investigated the molecular mechanisms underlying the potency of the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-AzadC) in HIV-1 latency reversal. Doing so, we uncovered specific demethylation CpG signatures induced by 5-AzadC in the HIV-1 promoter. By analyzing the binding modalities to these CpG, we revealed the recruitment of the epigenetic integrator UHRF1 to the HIV-1 promoter. We further demonstrated the role of UHRF1 in DNA methylation-mediated silencing of the latent HIV-1 promoter. As a proof-of-concept to this molecular characterization, we showed that pharmacological downregulation of UHRF1 in ex vivo HIV+ patient cell cultures resulted in potent reactivation of latent HIV-1. Together, we identify UHRF1 as a novel actor in HIV-1 gene silencing and highlight that it constitutes a new molecular target for HIV-1 curative strategies.

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Differentiation into an Effector Memory Phenotype Potentiates HIV-1 Latency Reversal in CD4+ T Cells

Journal of Virology ◽

10.1128/jvi.00969-19 ◽

2019 ◽

Vol 93 (24) ◽

Cited By ~ 17

Author(s):

Deanna A. Kulpa ◽

Aarthi Talla ◽

Jessica H. Brehm ◽

Susan Pereira Ribeiro ◽

Sally Yuan ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Ex Vivo ◽

T Cell Subsets ◽

Effector Memory ◽

Cell Surface Markers ◽

Cell Cycle Entry ◽

Latent Hiv ◽

Hiv 1

ABSTRACT During antiretroviral therapy (ART), human immunodeficiency virus type 1 (HIV-1) persists as a latent reservoir in CD4+ T cell subsets in central memory (TCM), transitional memory (TTM), and effector memory (TEM) CD4+ T cells. We have identified differences in mechanisms underlying latency and responses to latency-reversing agents (LRAs) in ex vivo CD4+ memory T cells from virally suppressed HIV-infected individuals and in an in vitro primary cell model of HIV-1 latency. Our ex vivo and in vitro results demonstrate the association of transcriptional pathways of T cell differentiation, acquisition of effector function, and cell cycle entry in response to LRAs. Analyses of memory cell subsets showed that effector memory pathways and cell surface markers of activation and proliferation in the TEM subset are predictive of higher frequencies of cells carrying an inducible reservoir. Transcriptional profiling also demonstrated that the epigenetic machinery (known to control latency and reactivation) in the TEM subset is associated with frequencies of cells with HIV-integrated DNA and inducible HIV multispliced RNA. TCM cells were triggered to differentiate into TEM cells when they were exposed to LRAs, and this increase of TEM subset frequencies upon LRA stimulation was positively associated with higher numbers of p24+ cells. Together, these data highlight differences in underlying biological latency control in different memory CD4+ T cell subsets which harbor latent HIV in vivo and support a role for differentiation into a TEM phenotype in facilitating latency reversal. IMPORTANCE By performing phenotypic analysis of latency reversal in CD4+ T cells from virally suppressed individuals, we identify the TEM subset as the largest contributor to the inducible HIV reservoir. Differential responses of memory CD4+ T cell subsets to latency-reversing agents (LRAs) demonstrate that HIV gene expression is associated with heightened expression of transcriptional pathways associated with differentiation, acquisition of effector function, and cell cycle entry. In vitro modeling of the latent HIV reservoir in memory CD4+ T cell subsets identify LRAs that reverse latency with ranges of efficiency and specificity. We found that therapeutic induction of latency reversal is associated with upregulation of identical sets of TEM-associated genes and cell surface markers shown to be associated with latency reversal in our ex vivo and in vitro models. Together, these data support the idea that the effector memory phenotype supports HIV latency reversal in CD4+ T cells.

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