Inhibition of Adipose Tissue Beiging by HIV Integrase Inhibitors, Dolutegravir and Bictegravir, Is Associated with Adipocyte Hypertrophy, Hypoxia, Elevated Fibrosis, and Insulin Resistance in Simian Adipose Tissue and Human Adipocytes.

For people living with HIV, treatment with integrase-strand-transfer-inhibitors (INSTIs) can promote adipose tissue (AT) gain. We previously demonstrated that INSTIs can induce hypertrophy and fibrosis in AT of macaques and humans. By promoting energy expenditure, the emergence of beige adipocytes in white AT (beiging) could play an important role by limiting excess lipid storage and associated adipocyte dysfunction. We hypothesized that INSTIs could alter AT via beiging inhibition. Fibrosis and gene expression were measured in subcutaneous (SCAT) and visceral AT (VAT) from SIV-infected, dolutegravir-treated (SIVART) macaques. Beiging capacity was assessed in human adipose stromal cells (ASCs) undergoing differentiation and being exposed to dolutegravir, bictegravir, or raltegravir. Expression of beige markers, such as positive-regulatory-domain-containing-16 (PRDM16), were lower in AT of SIVART as compared to control macaques, whereas fibrosis-related genes were higher. Dolutegravir and bictegravir inhibited beige differentiation in ASCs, as shown by lower expression of beige markers and lower cell respiration. INSTIs also induced a hypertrophic insulin-resistant state associated with a pro-fibrotic phenotype. Our results indicate that adipocyte hypertrophy induced by INSTIs is involved via hypoxia (revealed by a greater hypoxia-inducible-factor-1-alpha gene expression) in fat fibrosis, beiging inhibition, and thus (via positive feedback), probably, further hypertrophy and associated insulin resistance.

The Endothelin Receptor Antagonist Macitentan Inhibits Human Cytomegalovirus Infection.

Human cytomegalovirus (HCMV) infection is an important cause of morbidity and mortality in immunocompromised patients and a major etiological factor for congenital birth defects in newborns. Ganciclovir and its pro-drug valganciclovir are the preferred drugs in use today for prophylaxis and treatment of viremic patients. Due to long treatment times, patients are at risk for developing viral resistance to ganciclovir and to other drugs with a similar mechanism of action. We earlier found that the endothelin receptor B (ETBR) is upregulated during HCMV infection and that it plays an important role in the life cycle of this virus. Here, we tested the hypothesis that ETBR blockade could be used in the treatment of HCMV infection. As HCMV infection is specific to humans, we tested our hypothesis in human cell types that are relevant for HCMV pathogenesis; i.e., endothelial cells, epithelial cells and fibroblasts. We infected these cells with HCMV and treated them with the ETBR specific antagonist BQ788 or ETR antagonists that are approved by the FDA for treatment of pulmonary hypertension; macitentan, its metabolite ACT-132577, bosentan and ambrisentan, and as an anti-viral control, we used ganciclovir or letermovir. At concentrations expected to be relevant in vivo, macitentan, ACT-132577 and BQ788 effectively inhibited productive infection of HCMV. Of importance, macitentan also inhibited productive infection of a ganciclovir-resistant HCMV isolate. Our results suggest that binding or signaling through ETBR is crucial for viral replication, and that selected ETBR blockers inhibit HCMV infection.

Metformin alleviates stress-induced cellular senescence of aging human adipose stromal cells and the ensuing adipocyte dysfunction.

Aging is associated with central fat redistribution and insulin resistance. To identify age-related adipose features, we evaluated the senescence and adipogenic potential of adipose-derived stromal cells (ASCs) from abdominal subcutaneous fat obtained from healthy normal-weight young (<25 years) or older women (>60 years). Increased cell passages of young-donor ASCs (in vitro aging) resulted in senescence but not oxidative stress. ASC-derived adipocytes presented impaired adipogenesis but no early mitochondrial dysfunction. Conversely, aged-donor ASCs at early passages displayed oxidative stress and mild senescence. ASC-derived adipocytes exhibited oxidative stress, and early mitochondrial dysfunction but adipogenesis was preserved. In vitro aging of aged-donor ASCs resulted in further increased senescence, mitochondrial dysfunction, oxidative stress, and severe adipocyte dysfunction. When in vitro aged young-donor ASCs were treated with metformin, no alteration was alleviated. Conversely, metformin treatment of aged-donor ASCs decreased oxidative stress and mitochondrial dysfunction resulting in decreased senescence. Metformin's prevention of oxidative stress and of the resulting senescence improved the cells' adipogenic capacity and insulin sensitivity. This effect was mediated by the activation of AMP-activated protein kinase as revealed by its specific inhibition and activation. Overall, aging ASC-derived adipocytes presented impaired adipogenesis and insulin sensitivity. Targeting stress-induced senescence of ASCs with metformin may improve age-related adipose tissue dysfunction.

Contribution of Adipose Tissue to the Chronic Immune Activation and Inflammation Associated With HIV Infection and Its Treatment.

White adipose tissue (AT) contributes significantly to inflammation - especially in the context of obesity. Several of AT's intrinsic features favor its key role in local and systemic inflammation: (i) large distribution throughout the body, (ii) major endocrine activity, and (iii) presence of metabolic and immune cells in close proximity. In obesity, the concomitant pro-inflammatory signals produced by immune cells, adipocytes and adipose stem cells help to drive local inflammation in a vicious circle. Although the secretion of adipokines by AT is a prime contributor to systemic inflammation, the lipotoxicity associated with AT dysfunction might also be involved and could affect distant organs. In HIV-infected patients, the AT is targeted by both HIV infection and antiretroviral therapy (ART). During the primary phase of infection, the virus targets AT directly (by infecting AT CD4 T cells) and indirectly (via viral protein release, inflammatory signals, and gut disruption). The initiation of ART drastically changes the picture: ART reduces viral load, restores (at least partially) the CD4 T cell count, and dampens inflammatory processes on the whole-body level but also within the AT. However, ART induces AT dysfunction and metabolic side effects, which are highly dependent on the individual molecules and the combination used. First generation thymidine reverse transcriptase inhibitors predominantly target mitochondrial DNA and induce oxidative stress and adipocyte death. Protease inhibitors predominantly affect metabolic pathways (affecting adipogenesis and adipocyte homeostasis) resulting in insulin resistance. Recently marketed integrase strand transfer inhibitors induce both adipocyte adipogenesis, hypertrophy and fibrosis. It is challenging to distinguish between the respective effects of viral persistence, persistent immune defects and ART toxicity on the inflammatory profile present in ART-controlled HIV-infected patients. The host metabolic status, the size of the pre-established viral reservoir, the quality of the immune restoration, and the natural ageing with associated comorbidities may mitigate and/or reinforce the contribution of antiretrovirals (ARVs) toxicity to the development of low-grade inflammation in HIV-infected patients. Protecting AT functions appears highly relevant in ART-controlled HIV-infected patients. It requires lifestyle habits improvement in the absence of effective anti-inflammatory treatment. Besides, reducing ART toxicities remains a crucial therapeutic goal.

LIPE-related lipodystrophic syndrome: clinical features and disease modeling using adipose stem cells.

OBJECTIVE: The term Multiple Symmetric Lipomatosis (MSL) describes a heterogeneous group of rare monogenic disorders and multifactorial conditions, characterized by upper-body adipose masses. Biallelic variants in LIPE encoding hormone-sensitive lipase (HSL), a key lipolytic enzyme, were implicated in three families worldwide. We aimed to further delineate LIPE-related clinical features and pathophysiological determinants. METHODS: A gene panel was used to identify pathogenic variants. The disease features were reviewed at the French lipodystrophy reference center. The immunohistological, ultrastructural, and protein expression characteristics of lipomatous tissue were determined in surgical samples from one patient. The functional impact of variants was investigated by developing a model of adipose stem cells (ASCs) isolated from lipomatous tissue. RESULTS: We identified new biallelic LIPE null variants in three unrelated patients referred for MSL and/or partial lipodystrophy. The hallmarks of the disease, appearing in adulthood, included lower-limb lipoatrophy, upper-body and abdominal pseudo-lipomatous masses, diabetes and/or insulin resistance, hypertriglyceridemia, liver steatosis, high blood pressure, and neuromuscular manifestations. Ophthalmological investigations revealed numerous auto-fluorescent drusen-like retinal deposits in all patients. Lipomatous tissue and patient ASCs showed loss of HSL and decreased expression of adipogenic and mature adipocyte markers. LIPE-mutated ASCs displayed impaired adipocyte differentiation, decreased insulin response, defective lipolysis, and mitochondrial dysfunction. CONSLUSIONS: Biallelic LIPE null variants result in a multisystemic disease requiring multidisciplinary care. Loss of HSL expression impairs adipocyte differentiation, consistent with the lipodystrophy/MSL phenotype and associated metabolic complications. Detailed ophthalmological examination could reveal retinal damage, further pointing to the nervous tissue as an important disease target.

SIV Infection and the HIV Proteins Tat and Nef Induce Senescence in Adipose Tissue and Human Adipose Stem Cells, Resulting in Adipocyte Dysfunction.

BACKGROUND: Aging is characterized by adipose tissue senescence, inflammation, and fibrosis, with trunk fat accumulation. Aging HIV-infected patients have a higher risk of trunk fat accumulation than uninfected individuals-suggesting that viral infection has a role in adipose tissue aging. We previously demonstrated that HIV/SIV infection and the Tat and Nef viral proteins were responsible for adipose tissue fibrosis and impaired adipogenesis. We hypothesized that SIV/HIV infection and viral proteins could induce adipose tissue senescence and thus lead to adipocyte dysfunctions. METHODS: Features of tissue senescence were evaluated in subcutaneous and visceral adipose tissues of SIV-infected macaques and in human adipose stem cells (ASCs) exposed to Tat or Nef for up to 30 days. RESULTS: p16 expression and p53 activation were higher in adipose tissue of SIV-infected macaques than in control macaques, indicating adipose tissue senescence. Tat and Nef induced higher senescence in ASCs, characterized by higher levels of senescence-associated beta-galactosidase activity, p16 expression, and p53 activation vs. control cells. Treatment with Tat and Nef also induced oxidative stress and mitochondrial dysfunction. Prevention of oxidative stress (using N-acetyl-cysteine) reduced senescence in ASCs. Adipocytes having differentiated from Nef-treated ASCs displayed alterations in adipogenesis with lower levels of triglyceride accumulation and adipocyte marker expression and secretion, and insulin resistance. CONCLUSION: HIV/SIV promotes adipose tissue senescence, which in turn may alter adipocyte function and contribute to insulin resistance.

The Integrase Inhibitors Dolutegravir and Raltegravir Exert Proadipogenic and Profibrotic Effects and Induce Insulin Resistance in Human/Simian Adipose Tissue and Human Adipocytes.

BACKGROUND: Although some integrase strand transfer inhibitors (INSTIs) promote peripheral and central adipose tissue/weight gain in people with human immunodeficiency virus (PHIV), the underlying mechanism has not been identified. Here, we used human and simian models to assess the impact of INSTIs on adipose tissue phenotype and function. METHODS: Adipocyte size and fibrosis were determined in biopsies of subcutaneous and visceral adipose tissue (SCAT and VAT, respectively) from 14 noninfected macaques and 19 PHIV treated or not treated with an INSTI. Fibrosis, adipogenesis, oxidative stress, mitochondrial function, and insulin sensitivity were assessed in human proliferating or adipocyte-differentiated adipose stem cells after long-term exposure to dolutegravir or raltegravir. RESULTS: We observed elevated fibrosis, adipocyte size, and adipogenic marker expression in SCAT and VAT from INSTI-treated noninfected macaques. Adiponectin expression was low in SCAT. Accordingly, SCAT and VAT samples from INSTI-exposed patients displayed higher levels of fibrosis than those from nonexposed patients. In vitro, dolutegravir and, to a lesser extent, raltegravir were associated with greater extracellular matrix production and lipid accumulation in adipose stem cells and/or adipocytes as observed in vivo. Despite the INSTIs' proadipogenic and prolipogenic effects, these drugs promoted oxidative stress, mitochondrial dysfunction, and insulin resistance. CONCLUSIONS: Dolutegravir and raltegravir can directly impact adipocytes and adipose tissue. These INSTIs induced adipogenesis, lipogenesis, oxidative stress, fibrosis, and insulin resistance. The present study is the first to shed light on the fat modifications observed in INSTI-treated PHIV.

Metabolic complications affecting adipose tissue, lipid and glucose metabolism associated with HIV antiretroviral treatment.

Introduction: Efficient antiretroviral-treatment (ART) generally allows control of HIV infection. However, persons-living-with-HIV (PLWH), when aging, present a high prevalence of metabolic diseases. Area covered: Altered adiposity, dyslipidemias, insulin resistance, diabetes, and their consequences are prevalent in PLWH and could be partly related to ART. Expert opinion: At first, personal and lifestyle factors are involved in the onset of these complications. The persistence of HIV in tissue reservoirs could synergize with some ART and enhance metabolic disorders. Altered fat repartition, diagnosed as lipodystrophy, has been related to first-generation nucleoside-reverse-transcriptase-inhibitors (NRTIs) (stavudine zidovudine) and some protease inhibitors (PIs). Recently, use of some integrase-inhibitors (INSTI) resulted in weight/fat gain, which represents a worrisome unresolved situation. Lipid parameters were affected by some first-generation NRTIs, non-NRTIs (efavirenz) but also PIs boosted by ritonavir, with increased total and LDL-cholesterol and triglycerides. Insulin resistance is common associated with abdominal obesity. Diabetes incidence, high with first-generation-ART (zidovudine, stavudine, didanosine, indinavir) has declined with contemporary ART close to that of the general population. Metabolic syndrome, a dysmetabolic situation with central obesity and insulin resistance, and liver steatosis are common in PLWH and could indirectly result from ART-associated fat gain and insulin resistance. All these dysmetabolic situations increase the atherogenic cardiovascular risk.

Impact of HIV/simian immunodeficiency virus infection and viral proteins on adipose tissue fibrosis and adipogenesis.

OBJECTIVE: HIV-infected patients receiving antiretroviral treatment (ART) often present adipose tissue accumulation and/or redistribution. adipose tissue has been shown to be an HIV/SIV reservoir and viral proteins as Tat or Nef can be released by infected immune cells and exert a bystander effect on adipocytes or precursors. Our aim was to demonstrate that SIV/HIV infection per se could alter adipose tissue structure and/or function. DESIGN: Morphological and functional alterations of subcutaneous (SCAT) and visceral adipose tissue (VAT) were studied in SIV-infected macaques and HIV-infected ART-controlled patients. To analyze the effect of Tat or Nef, we used human adipose stem cells (ASCs) issued from healthy donors, and analyzed adipogenesis and extracellular matrix component production using two dimensional (2D) and three-dimensional (3D) culture models. METHODS: Adipocyte size and index of fibrosis were determined on Sirius red-stained adipose tissue samples. Proliferating and adipocyte 2D-differentiating or 3D-differentiating ASCs were treated chronically with Tat or Nef. mRNA, protein expression and secretion were examined by RT-PCR, western-blot and ELISA. RESULTS: SCAT and VAT from SIV-infected macaques displayed small adipocytes, decreased adipogenesis and severe fibrosis with collagen deposition. SCAT and VAT from HIV-infected ART-controlled patients presented similar alterations. In vitro, Tat and/or Nef induced a profibrotic phenotype in undifferentiated ASCs and altered adipogenesis and collagen production in adipocyte-differentiating ASCs. CONCLUSION: We demonstrate here a specific role for HIV/SIV infection per se on adipose tissue fibrosis and adipogenesis, probably through the release of viral proteins, which could be involved in adipose tissue dysfunction contributing to cardiometabolic alterations of HIV-infected individuals.

The Frequencies of Immunosuppressive Cells in Adipose Tissue Differ in Human, Non-human Primate, and Mouse Models.

Although the metabolic properties of white adipose tissue have been extensively characterized, the tissue's immune properties are now attracting renewed interest. Early experiments in a mouse model suggested that white adipose tissue contains a high density of regulatory T cells (Tregs), and so it was assumed that all adipose tissue has an immunosuppressive profile-even though the investigation was limited to visceral body fat in relatively old male mice. This observation was also corroborated by high frequencies of other cell subsets with immunoregulatory properties, such as anti-inflammatory M2 macrophages, and regulatory B cells. Many studies have since evidenced the persistence of pathogens (trypanosomes, Mycobacterium tuberculosis, HIV, etc.) in adipose tissue. However, a recent report identified adipose tissue as a reservoir of memory T cells capable of protecting animals upon rechallenge. The immune potential of lean adipose tissue thus remains to be further investigated. Here, we compared the relative proportions of immune cells (and Tregs in particular) in lean adipose tissue collected from humans, a non-human primate (the cynomolgus macaque), and three mouse models. We demonstrated that the proportion of Foxp3+ Tregs in visceral adipose tissue was low in all models other than the C57Bl/6 mouse. These low values were not linked to correspondingly low proportions of effector cells because T lymphocytes (a main target of Treg suppression) were more frequent in cynomolgus macaques than in C57Bl/6 mice and (to a lesser extent) humans. In contrast, the proportions of macrophages and B cells were lower in cynomolgus macaques than in C57Bl/6 mice. We also observed a higher proportion of CD34+CD45- cells (which predominantly correspond to mesenchymal stem cells) in C57Bl/6 mouse and cynomolgus macaques than in humans and both for subcutaneous and visceral adipose tissues. Lastly, a microscopy analysis confirmed predominant proportion of adipocytes within adipose tissue, and highlighted a marked difference in adipocyte size among the three species studied. In conclusion, our study of lean, middle-aged, male individuals showed that the immune compartment of adipose tissue differed markedly in humans vs. mice, and suggesting the presence of a more inflammatory steady-state profile in humans than mice.

Specific Biological Features of Adipose Tissue, and Their Impact on HIV Persistence.

Although white AT can contribute to anti-infectious immune responses, it can also be targeted and perturbed by pathogens. The AT's immune involvement is primarily due to strong pro-inflammatory responses (with both local and paracrine effects), and the large number of fat-resident macrophages. Adipocytes also exert direct antimicrobial responses. In recent years, it has been found that memory T cells accumulate in AT, where they provide efficient secondary responses against viral pathogens. These observations have prompted researchers to re-evaluate the links between obesity and susceptibility to infections. In contrast, AT serves as a reservoir for several persistence pathogens, such as human adenovirus Ad-36, Trypanosoma gondii, Mycobacterium tuberculosis, influenza A virus, and cytomegalovirus (CMV). The presence and persistence of bacterial DNA in AT has led to the concept of a tissue-specific microbiota. The unexpected coexistence of immune cells and pathogens within the specific AT environment is intriguing, and its impact on anti-infectious immune responses requires further evaluation. AT has been recently identified as a site of HIV persistence. In the context of HIV infection, AT is targeted by both the virus and the antiretroviral drugs. AT's intrinsic metabolic features, large overall mass, and wide distribution make it a major tissue reservoir, and one that may contribute to the pathophysiology of chronic HIV infections. Here, we review the immune, metabolic, viral, and pharmacological aspects that contribute to HIV persistence in AT. We also evaluate the respective impacts of both intrinsic and HIV-induced factors on AT's involvement as a viral reservoir. Lastly, we examine the potential consequences of HIV persistence on the metabolic and immune activities of AT.