Immunoglobulin G N-glycan markers of accelerated biological aging during chronic HIV infection.

People living with HIV (PLWH) experience increased vulnerability to premature aging and inflammation-associated comorbidities, even when HIV replication is suppressed by antiretroviral therapy (ART). However, the factors associated with this vulnerability remain uncertain. In the general population, alterations in the N-glycans on IgGs trigger inflammation and precede the onset of aging-associated diseases. Here, we investigate the IgG N-glycans in cross-sectional and longitudinal samples from 1214 women and men, living with and without HIV. PLWH exhibit an accelerated accumulation of pro-aging-associated glycan alterations and heightened expression of senescence-associated glycan-degrading enzymes compared to controls. These alterations correlate with elevated markers of inflammation and the severity of comorbidities, potentially preceding the development of such comorbidities. Mechanistically, HIV-specific antibodies glycoengineered with these alterations exhibit a reduced ability to elicit anti-HIV Fc-mediated immune activities. These findings hold potential for the development of biomarkers and tools to identify and prevent premature aging and comorbidities in PLWH.

Plasma Glycomic Markers of Accelerated Biological Aging During Chronic HIV Infection.

People with HIV (PWH) experience an increased vulnerability to premature aging and inflammation-associated comorbidities, even when HIV replication is suppressed by antiretroviral therapy (ART). However, the factors that contribute to or are associated with this vulnerability remain uncertain. In the general population, alterations in the glycomes of circulating IgGs trigger inflammation and precede the onset of aging-associated diseases. Here, we investigate the IgG glycomes of cross-sectional and longitudinal samples from 1,216 women and men, both living with virally suppressed HIV and those without HIV. Our glycan-based machine learning models indicate that living with chronic HIV significantly accelerates the accumulation of pro-aging-associated glycomic alterations. Consistently, PWH exhibit heightened expression of senescence-associated glycan-degrading enzymes compared to their controls. These glycomic alterations correlate with elevated markers of inflammatory aging and the severity of comorbidities, potentially preceding the development of such comorbidities. Mechanistically, HIV-specific antibodies glycoengineered with these alterations exhibit reduced anti-HIV IgG-mediated innate immune functions. These findings hold significant potential for the development of glycomic-based biomarkers and tools to identify and prevent premature aging and comorbidities in people living with chronic viral infections.

Repurposing sunscreen as an antibiotic: zinc-activated avobenzone inhibits methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major healthcare concern with associated healthcare costs reaching over ${\$}$1 billion in a single year in the USA. Antibiotic resistance in S. aureus is now observed against last line of defense antibiotics, such as vancomycin, linezolid, and daptomycin. Unfortunately, high throughput drug discovery approaches to identify new antibiotics effective against MRSA have not resulted in much tangible success over the last decades. Previously, we demonstrated the feasibility of an alternative drug discovery approach, the identification of metallo-antibiotics, compounds that gain antibacterial activity only after binding to a transition metal ion and as such are unlikely to be detected in standard drug screens. We now report that avobenzone, the primary active ingredient of most sunscreens, can be activated by zinc to become a potent antibacterial compound against MRSA. Zinc-activated avobenzone (AVB-Zn) potently inhibited a series of clinical MRSA isolates [minimal inhibitory concentration (MIC): 0.62-2.5 µM], without pre-existing resistance and activity without zinc (MIC: >10 µM). AVB-Zn was also active against clinical MRSA isolates that were resistant against the commonly used zinc-salt antibiotic bacitracin. We found AVB-Zn exerted no cytotoxicity on human cell lines and primary cells. Last, we demonstrate AVB-Zn can be deployed therapeutically as lotion preparations, which showed efficacy in a mouse wound model of MRSA infection. AVB-Zn thus demonstrates Zn-activated metallo-antibiotics are a promising avenue for future drug discovery.

Spatiotemporal resolution of germinal center Tfh cell differentiation and divergence from central memory CD4+ T cell fate.

Follicular helper T (Tfh) cells are essential for germinal center (GC) B cell responses. However, it is not clear which PD-1+CXCR5+Bcl6+CD4+ T cells will differentiate into PD-1hiCXCR5hiBcl6hi GC-Tfh cells and how GC-Tfh cell differentiation is regulated. Here, we report that the sustained Tigit expression in PD-1+CXCR5+CD4+ T cells marks the precursor Tfh (pre-Tfh) to GC-Tfh transition, whereas Tigit-PD-1+CXCR5+CD4+ T cells upregulate IL-7Rα to become CXCR5+CD4+ T memory cells with or without CCR7. We demonstrate that pre-Tfh cells undergo substantial further differentiation at the transcriptome and chromatin accessibility levels to become GC-Tfh cells. The transcription factor c-Maf appears critical in governing the pre-Tfh to GC-Tfh transition, and we identify Plekho1 as a stage-specific downstream factor regulating the GC-Tfh competitive fitness. In summary, our work identifies an important marker and regulatory mechanism of PD-1+CXCR5+CD4+ T cells during their developmental choice between memory T cell fate and GC-Tfh cell differentiation.

Lentiviral Nef Proteins Differentially Govern the Establishment of Viral Latency.

Despite the clinical importance of latent human immunodeficiency virus type 1 (HIV-1) infection, our understanding of the biomolecular processes involved in HIV-1 latency control is still limited. This study was designed to address whether interactions between viral proteins, specifically HIV Nef, and the host cell could affect latency establishment. The study was driven by three reported observations. First, early reports suggested that human immunodeficiency virus type 2 (HIV-2) infection in patients produces a lower viral RNA/DNA ratio than HIV-1 infection, potentially indicating an increased propensity of HIV-2 to produce latent infection. Second, Nef, an early viral gene product, has been shown to alter the activation state of infected cells in a lentiviral lineage-dependent manner. Third, it has been demonstrated that the ability of HIV-1 to establish latent infection is a function of the activation state of the host cell at the time of infection. Based on these observations, we reasoned that HIV-2 Nef may have the ability to promote latency establishment. We demonstrate that HIV-1 latency establishment in T cell lines and primary T cells is indeed differentially modulated by Nef proteins. In the context of an HIV-1 backbone, HIV-1 Nef promoted active HIV-1 infection, while HIV-2 Nef strongly promoted latency establishment. Given that Nef represents the only difference in these HIV-1 vectors and is known to interact with numerous cellular factors, these data add support to the idea that latency establishment is a host cell-virus interaction phenomenon, but they also suggest that the HIV-1 lineage may have evolved mechanisms to counteract host cell suppression. IMPORTANCE Therapeutic attempts to eliminate the latent HIV-1 reservoir have failed, at least in part due to our incomplete biomolecular understanding of how latent HIV-1 infection is established and maintained. We here address the fundamental question of whether all lentiviruses actually possess a similar capacity to establish latent infections or whether there are differences between the lentiviral lineages driving differential latency establishment that could be exploited to develop improved latency reversal agents. Research investigating the viral RNA/DNA ratio in HIV-1 and HIV-2 patients could suggest that HIV-2 indeed has a much higher propensity to establish latent infections, a trait that we found, at least in part, to be attributable to the HIV-2 Nef protein. Reported Nef-mediated effects on host cell activation thus also affect latency establishment, and HIV-1 vectors that carry different lentiviral nef genes should become key tools to develop a better understanding of the biomolecular basis of HIV-1 latency establishment.

Host T Cell Dedifferentiation Effects Drive HIV-1 Latency Stability.

The development of therapies to eliminate the latent HIV-1 reservoir is hampered by our incomplete understanding of the biomolecular mechanism governing HIV-1 latency. To further complicate matters, recent single-cell RNA sequencing (scRNA-seq) studies reported extensive heterogeneity between latently HIV-1-infected primary T cells, implying that latent HIV-1 infection can persist in greatly differing host cell environments. We show here that transcriptomic heterogeneity is also found between latently infected T cell lines, which allowed us to study the underlying mechanisms of intercell heterogeneity at high signal resolution. Latently infected T cells exhibited a dedifferentiated phenotype, characterized by the loss of T cell-specific markers and gene regulation profiles reminiscent of hematopoietic stem cells (HSC). These changes had functional consequences. As reported for stem cells, latently HIV-1-infected T cells efficiently forced lentiviral superinfections into a latent state and favored glycolysis. As a result, metabolic reprogramming or cell redifferentiation destabilized latent infection. Guided by these findings, data mining of single-cell RNA-seq data of latently HIV-1-infected primary T cells from patients revealed the presence of similar dedifferentiation motifs. More than 20% of the highly detectable genes that were differentially regulated in latently infected cells were associated with hematopoietic lineage development (e.g., HUWE1, IRF4, PRDM1, BATF3, TOX, ID2, IKZF3, and CDK6) or were hematopoietic markers (SRGN; hematopoietic proteoglycan core protein). The data add to evidence that the biomolecular phenotype of latently HIV-1-infected cells differs from that of normal T cells and strategies to address their differential phenotype need to be considered in the design of therapeutic cure interventions. IMPORTANCE HIV-1 persists in a latent reservoir in memory CD4 T cells for the lifetime of a patient. Understanding the biomolecular mechanisms used by the host cells to suppress viral expression will provide essential insights required to develop curative therapeutic interventions. Unfortunately, our current understanding of these control mechanisms is still limited. By studying gene expression profiles, we demonstrated that latently HIV-1-infected T cells have a dedifferentiated T cell phenotype. Software-based data integration allowed the identification of drug targets that would redifferentiate viral host cells and, by extension, destabilize latent HIV-1 infection events. The importance of the presented data lies within the clear demonstration that HIV-1 latency is a host cell phenomenon. As such, therapeutic strategies must first restore proper host cell functionality to accomplish efficient HIV-1 reactivation.

T-Cell Immune Dysregulation and Mortality in Women With Human Immunodeficiency Virus.

SUMMARY: In women with HIV, higher activation and exhaustion of CD4+ T cells were associated with risk of non-HIV-related mortality during a median of 13.3 years of follow-up, independent of baseline demographic, behavioral, HIV-related, and cardiometabolic factors and longitudinal HIV disease progression. BACKGROUND: Dysregulation of adaptive immunity is a hallmark of human immunodeficiency virus (HIV) infection that persists on antiretroviral therapy (ART). Few long-term prospective studies have related adaptive immunity impairments to mortality in HIV, particularly in women. METHODS: Among 606 women with HIV in the Women's Interagency HIV Study, peripheral blood mononuclear cells collected from 2002 to 2005 underwent multiparameter flow cytometry. Underlying cause of death was ascertained from the National Death Index up to 2018. We examined associations of CD4+ and CD8+ T-cell activation (%CD38+HLA-DR+), senescence (%CD57+CD28-), exhaustion (%PD-1+), and nonactivation/normal function (%CD57-CD28+) with natural-cause, HIV-related, and non-HIV-related mortality. RESULTS: At baseline, median participant age was 41, and 67% were on ART. Among 100 deaths during a median of 13.3 years follow-up, 90 were natural-cause (53 non-HIV-related, 37 HIV-related). Higher activation and exhaustion of CD4+ T cells were associated with risk of natural-cause and non-HIV-related mortality, adjusting for age, demographic, behavioral, HIV-related, and cardiometabolic factors at baseline. Additional adjustment for time-varying viral load and CD4+ T-cell count did not attenuate these associations. CD8+ T-cell markers were not associated with any outcomes adjusting for baseline factors. CONCLUSIONS: Persistent CD4+ T-cell activation and exhaustion may contribute to excess long-term mortality risk in women with HIV, independent of HIV disease progression.

Pyrazolyl Thioureas and Carbothioamides with an NNSN Motif against MSSA and MRSA.

A novel series of copper-activatable drugs intended for use against methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) were synthesized, characterized, and tested against the MSSA strain Newman and the MRSA Lac strain (a USA300 strain), respectively. These drugs feature an NNSN structural motif, which enables the binding of copper. In the absence of copper, no activity against MSSA and MRSA at realistic drug concentrations was observed. Although none of the novel drug candidates exhibits a stereocenter, sub-micromolar activities against SA Newman and micromolar activities against SA Lac were observed in the presence, but not in the absence, of bioavailable copper. Copper influx is a component of cellular response to bacterial infections, which is often described as nutritional immunity.

Extensive proteomic and transcriptomic changes quench the TCR/CD3 activation signal of latently HIV-1 infected T cells.

The biomolecular mechanisms controlling latent HIV-1 infection, despite their importance for the development of a cure for HIV-1 infection, are only partially understood. For example, ex vivo studies have recently shown that T cell activation only triggered HIV-1 reactivation in a fraction of the latently infected CD4+ T cell reservoir, but the molecular biology of this phenomenon is unclear. We demonstrate that HIV-1 infection of primary T cells and T cell lines indeed generates a substantial amount of T cell receptor (TCR)/CD3 activation-inert latently infected T cells. RNA-level analysis identified extensive transcriptomic differences between uninfected, TCR/CD3 activation-responsive and -inert T cells, but did not reveal a gene expression signature that could functionally explain TCR/CD3 signaling inertness. Network analysis suggested a largely stochastic nature of these gene expression changes (transcriptomic noise), raising the possibility that widespread gene dysregulation could provide a reactivation threshold by impairing overall signal transduction efficacy. Indeed, compounds that are known to induce genetic noise, such as HDAC inhibitors impeded the ability of TCR/CD3 activation to trigger HIV-1 reactivation. Unlike for transcriptomic data, pathway enrichment analysis based on phospho-proteomic data directly identified an altered TCR signaling motif. Network analysis of this data set identified drug targets that would promote TCR/CD3-mediated HIV-1 reactivation in the fraction of otherwise TCR/CD3-reactivation inert latently HIV-1 infected T cells, regardless of whether the latency models were based on T cell lines or primary T cells. The data emphasize that latent HIV-1 infection is largely the result of extensive, stable biomolecular changes to the signaling network of the host T cells harboring latent HIV-1 infection events. In extension, the data imply that therapeutic restoration of host cell responsiveness prior to the use of any activating stimulus will likely have to be an element of future HIV-1 cure therapies.

Sustained cellular immune dysregulation in individuals recovering from SARS-CoV-2 infection.

SARS-CoV-2 causes a wide spectrum of clinical manifestations and significant mortality. Studies investigating underlying immune characteristics are needed to understand disease pathogenesis and inform vaccine design. In this study, we examined immune cell subsets in hospitalized and nonhospitalized individuals. In hospitalized patients, many adaptive and innate immune cells were decreased in frequency compared with those of healthy and convalescent individuals, with the exception of an increase in B lymphocytes. Our findings show increased frequencies of T cell activation markers (CD69, OX40, HLA-DR, and CD154) in hospitalized patients, with other T cell activation/exhaustion markers (PD-L1 and TIGIT) remaining elevated in hospitalized and nonhospitalized individuals. B cells had a similar pattern of activation/exhaustion, with increased frequency of CD69 and CD95 during hospitalization followed by an increase in PD1 frequencies in nonhospitalized individuals. Interestingly, many of these changes were found to increase over time in nonhospitalized longitudinal samples, suggesting a prolonged period of immune dysregulation after SARS-CoV-2 infection. Changes in T cell activation/exhaustion in nonhospitalized patients were found to positively correlate with age. Severely infected individuals had increased expression of activation and exhaustion markers. These data suggest a prolonged period of immune dysregulation after SARS-CoV-2 infection, highlighting the need for additional studies investigating immune dysregulation in convalescent individuals.

Glycan Positioning Impacts HIV-1 Env Glycan-Shield Density, Function, and Recognition by Antibodies.

HIV-1 envelope (Env) N-glycosylation impact virus-cell entry and immune evasion. How each glycan interacts to shape the Env-protein-sugar complex and affects Env function is not well understood. Here, analysis of two Env variants from the same donor, with differing functional characteristics and N-glycosylation-site composition, revealed that changes to key N-glycosylation sites affected the Env structure at distant locations and had a ripple effect on Env-wide glycan processing, virus infectivity, antibody recognition, and virus neutralization. Specifically, the N262 glycan, although not in the CD4-binding site, modulated Env binding to the CD4 receptor, affected Env recognition by several glycan-dependent neutralizing antibodies, and altered site-specific glycosylation heterogeneity, with, for example, N448 displaying limited glycan processing. Molecular-dynamic simulations visualized differences in glycan density and how specific oligosaccharide positions can move to compensate for a glycan loss. This study demonstrates how changes in individual glycans can alter molecular dynamics, processing, and function of the Env-glycan shield.

The tetraspanin CD151 marks a unique population of activated human T cells.

Tetraspanins are a family of proteins with an array of functions that are well studied in cancer biology, but their importance in immunology is underappreciated. Here we establish the tetraspanin CD151 as a unique marker of T-cell activation and, in extension, an indicator of elevated, systemic T-cell activity. Baseline CD151 expression found on a subset of T-cells was indicative of increased activation of the MAPK pathway. Following TCR/CD3 activation, CD151 expression was upregulated on the overall T-cell population, a quintessential feature of an activation marker. CD151+ T-cell frequencies in the spleen, an organ with increased immune activity, were twice as high as in paired peripheral blood samples. This CD151+ T-cell frequency increase was not paralleled by an increase of CD25 or CD38, demonstrating that CD151 expression is regulated independently of other T-cell activation markers. CD151+ T-cells were also more likely to express preformed granzyme B, suggesting that CD151+ T cells are pro-inflammatory. To this end, HIV-1 patients on antiretroviral therapy who are reported to exhibit chronically elevated levels of immune activity, had significantly higher CD4+CD151+ T-cell frequencies than healthy controls, raising the possibility that proinflammatory CD151+ T cells could contribute to the premature immunological aging phenotype observed in these patients.

A copper-dependent compound restores ampicillin sensitivity in multidrug-resistant Staphylococcus aureus.

Multi-drug resistant Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), has become a worldwide, major health care problem. While initially restricted to clinical settings, drug resistant S. aureus is now one of the key causative agents of community-acquired infections. We have previously demonstrated that copper dependent inhibitors (CDIs), a class of antibiotics that are only active in the presence of copper ions, are effective bactericidal agents against MRSA. A second-generation CDI, APT-6K, exerted bactericidal activity at nanomolar concentrations. At sub-bactericidal concentrations, it effectively synergized with ampicillin to reverse drug resistance in multiple MRSA strains. APT-6K had a favorable therapeutic index when tested on eukaryotic cells (TI: > 30) and, unlike some previously reported CDIs, did not affect mitochondrial activity. These results further establish inhibitors that are activated by the binding of transition metal ions as a promising class of antibiotics, and for the first time, describe their ability to reverse existing drug resistance against clinically relevant antibiotics.

Breathing New Life into TRAIL for Breast Cancer Therapy: Co-Delivery of pTRAIL and Complementary siRNAs Using Lipopolymers.

Preclinical studies showed that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) therapy is safe and effective to combat cancers, but clinical outcomes have been less than optimal due to short half-life of TRAIL protein, insufficient induction of apoptosis, and TRAIL resistance displayed in many tumors. In this study, we explored co-delivery of a TRAIL expressing plasmid (pTRAIL) and complementary small interfering RNAs (siRNAs) (silencing Bcl2-like 12 [BCL2L12] and superoxide dismutase 1 [SOD1]) to improve the response of breast cancer cells against TRAIL therapy. It is desirable to co-deliver the pDNA along with siRNA using a single delivery agent, but this is challenging given different structures of long/flexible pDNA and short/rigid siRNA. Toward this goal, we identified an aliphatic lipid-grafted low-molecular weight polyethylenimine (PEI) that accommodated both pDNA and siRNA in a single complex. The co-delivery of pTRAIL with BCL2L12- or SOD1-specific siRNAs resulted more significant cell death in different breast cancer cells compared with separate delivery without affecting nonmalignant cells viability. Ternary complexes of lipopolymer with pTRAIL and BCL2L12 siRNA significantly retarded the growth of breast cancer xenografts in mice. The enhanced anticancer activity was attributed to increased in situ secretion of TRAIL and sensitization of breast cancer cells against TRAIL by the co-delivered siRNAs. The lipid-grafted PEIs capable of co-delivering multiple types of nucleic acids can serve as powerful carriers for more effective complementary therapeutics. Graphical Abstract [Figure: see text].

Bach2 Negatively Regulates T Follicular Helper Cell Differentiation and Is Critical for CD4+ T Cell Memory.

T follicular helper (Tfh) cells are essential for germinal center B cell responses. The molecular mechanism underlying the initial Tfh cell differentiation, however, is still incompletely understood. In this study, we show that in vivo, despite enhanced non-Tfh cell effector functions, the deletion of transcription factor Bach2 results in preferential Tfh cell differentiation. Mechanistically, the deletion of Bach2 leads to the induction of CXCR5 expression even before the upregulation of Ascl2. Subsequently, we have identified a novel regulatory element in the murine CXCR5 locus that negatively regulates CXCR5 promoter activities in a Bach2-dependent manner. Bach2 deficiency eventually results in a collapsed CD4+ T cell response with severely impaired CD4+ T cell memory, including Tfh cell memory. Our results demonstrate that Bach2 critically regulates Tfh cell differentiation and CD4+ T cell memory.

High-throughput screening and Bayesian machine learning for copper-dependent inhibitors of Staphylococcus aureus.

One potential source of new antibacterials is through probing existing chemical libraries for copper-dependent inhibitors (CDIs), i.e., molecules with antibiotic activity only in the presence of copper. Recently, our group demonstrated that previously unknown staphylococcal CDIs were frequently present in a small pilot screen. Here, we report the outcome of a larger industrial anti-staphylococcal screen consisting of 40 771 compounds assayed in parallel, both in standard and in copper-supplemented media. Ultimately, 483 had confirmed copper-dependent IC50 values under 50 µM. Sphere-exclusion clustering revealed that these hits were largely dominated by sulfur-containing motifs, including benzimidazole-2-thiones, thiadiazines, thiazoline formamides, triazino-benzimidazoles, and pyridinyl thieno-pyrimidines. Structure-activity relationship analysis of the pyridinyl thieno-pyrimidines generated multiple improved CDIs, with activity likely dependent on ligand/ion coordination. Molecular fingerprint-based Bayesian classification models were built using Discovery Studio and Assay Central, a new platform for sharing and distributing cheminformatic models in a portable format, based on open-source tools. Finally, we used the latter model to evaluate a library of FDA-approved drugs for copper-dependent activity in silico. Two anti-helminths, albendazole and thiabendazole, scored highly and are known to coordinate copper ions, further validating the model's applicability.

Pyrazolopyrimidinones, a novel class of copper-dependent bactericidal antibiotics against multi-drug resistant S. aureus.

The treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections poses a therapeutic challenge as even last resort drugs become increasingly ineffective. As the demand for antibiotics with novel modes of action is growing, new approaches are needed to probe a greater spectrum of antimicrobial activities for their potential efficacy against drug-resistant pathogens. The use of copper (Cu) by the innate immune system to mount an antimicrobial response against bacterial invaders has created an opportunity to explore a role for Cu in antimicrobial therapy. Here we describe pyrazolopyrimidinones (PZP) as novel copper-dependent inhibitors (CDI) of S. aureus. 5-Benzyl-3-(4-chlorophenyl)-2-methyl-4H,7H-pyrazolo[1,5-a]pyrimidin-7-one (PZP-915) showed potent bactericidal properties at sub-micromolar concentrations and activity against clinical MRSA isolates and biofilms cultures. This cupricidal activity is founded on the molecule's ability to coordinate Cu and induce accumulation of Cu ions inside S. aureus cells. We demonstrate that exposure to 915 + Cu led to an almost instantaneous collapse of the membrane potential which was accompanied by a complete depletion of cellular ATP, loss of cell-associated K+, a substantial gain of cell associated Na+, and an inability to control the influx of protons in slightly acidic medium, while the integrity of the cell membrane remained intact. These findings highlight PZP-915 as a novel membrane-directed metalloantibiotic against S. aureus that is likely to target a multiplicity of membrane associated protein functions rather than imposing physical damage to the membrane structure.

Antiretroviral therapy potentiates high-fat diet induced obesity and glucose intolerance.

OBJECTIVE: Breakthroughs in HIV treatment, especially combination antiretroviral therapy (ART), have massively reduced AIDS-associated mortality. However, ART administration amplifies the risk of non-AIDS defining illnesses including obesity, diabetes, and cardiovascular disease, collectively known as metabolic syndrome. Initial reports suggest that ART-associated risk of metabolic syndrome correlates with socioeconomic status, a multifaceted finding that encompasses income, race, education, and diet. Therefore, determination of causal relationships is extremely challenging due to the complex interplay between viral infection, ART, and the many environmental factors. METHODS: In the current study, we employed a mouse model to specifically examine interactions between ART and diet that impacts energy balance and glucose metabolism. Previous studies have shown that high-fat feeding induces persistent low-grade systemic and adipose tissue inflammation contributing to insulin resistance and metabolic dysregulation via adipose-infiltrating macrophages. Studies herein test the hypothesis that ART potentiates the inflammatory effects of a high-fat diet (HFD). C57Bl/6J mice on a HFD or standard chow containing ART or vehicle, were subjected to functional metabolic testing, RNA-sequencing of epididymal white adipose tissue (eWAT), and array-based kinomic analysis of eWAT-infiltrating macrophages. RESULTS: ART-treated mice on a HFD displayed increased fat mass accumulation, impaired glucose tolerance, and potentiated insulin resistance. Gene set enrichment and kinomic array analyses revealed a pro-inflammatory transcriptional signature depicting granulocyte migration and activation. CONCLUSION: The current study reveals a HFD-ART interaction that increases inflammatory transcriptional pathways and impairs glucose metabolism, energy balance, and metabolic dysfunction.

Regulatory CD4 T cells inhibit HIV-1 expression of other CD4 T cell subsets via interactions with cell surface regulatory proteins.

During chronic HIV-1 infection, regulatory CD4 T cells (Tregs) frequently represent the largest subpopulation of CD4 T cell subsets, implying relative resistant to HIV-1. When HIV-1 infection of CD4 T cells was explored in vitro and ex vivo from patient samples, Tregs possessed lower levels of HIV-1 DNA and RNA in comparison with conventional effector and memory CD4 T cells. Moreover, Tregs suppressed HIV-1 expression in other CD4 T cells in an in vitro co-culture system. This suppression was mediated in part via multiple inhibitory surface proteins expressed on Tregs. Antibody blockade of CTLA-4, PD-1, and GARP on Tregs resulted in increased HIV-1 DNA integration and mRNA expression in neighboring CD4 T cells. Moreover, antibody blockade of Tregs inhibitory proteins resulted in increased HIV-1 LTR transcription in co-cultured CD4 T cells. Thus, Tregs inhibit HIV-1 infection of other CD4 T cell subsets via interactions with inhibitory cell surface proteins.

The phosphatase PPM1A controls monocyte-to-macrophage differentiation.

Differentiation of circulating monocytes into tissue-bound or tissue-resident macrophages is a critical regulatory process affecting host defense and inflammation. However, the regulatory signaling pathways that control the differentiation of monocytes into specific and distinct functional macrophage subsets are poorly understood. Herein, we demonstrate that monocyte-to-macrophage differentiation is controlled by the Protein Phosphatase, Mg2+/Mn2+-dependent 1A (PPM1A). Genetic manipulation experiments demonstrated that overexpression of PPM1A attenuated the macrophage differentiation program, while knockdown of PPM1A expression accelerated the ability of monocytes to differentiate into macrophages. We identify imiquimod and Pam3CSK4 as two Toll-like receptor agonists that induce PPM1A expression, and show that increased expression of PPM1A at the onset of differentiation impairs cellular adherence, reduces expression of inflammatory (M1) macrophage-specific markers, and inhibits the production of inflammatory cytokines. Our findings reveal PPM1A as a negative threshold regulator of M1-type monocyte-to-macrophage differentiation, establishing it as a key phosphatase that orchestrates this program.