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Impact of HIV/simian immunodeficiency virus infection and viral proteins on adipose tissue fibrosis and adipogenesis
 
 
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Gorwood, Jennifera; Bourgeois, Christineb; Mantecon, Matthieua; Atlan, Michaela,c; Pourcher, Valéried; Pourcher, Guillaumee; Le Grand, Rogerb; Desjardins, Delphineb; Fève, Brunoa,f; Lambotte, Olivierb,g; Capeau, Jacquelinea; Béréziat, Véroniquea,*; Lagathu, Clairea,*
 
AIDS May 1 2019
 
We show here for the first time that HIV/SIV infection per se is associated with adipose tissue fibrosis and dysfunction that could, in turn, participate to the onset of cardiovascular and metabolic disorders commonly observed in ART-controlled HIV-infected patients.
 
-------In vitro, several HIV proteins, including Tat or Nef, can induce inflammation and senescence that could alter adipogenesis and affect mature adipocyte function [36-42]
 
------As adipose tissue is an HIV reservoir [9-11], we hypothesized that HIV-infected immune cells within adipose tissue could release viral proteins, even in the presence of ART, which could exert a direct effect on bystander cells, such as adipocytes or their precursors.
 
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.
 
Discussion

 
We show here that the morphological and functional analysis of SCAT and VAT from SIV-infected macaques and HIV-infected individuals revealed fibrosis, with increased deposition of collagens, together with a decreased adipocyte size. To go further, we addressed the specific relationship between ASCs, adipocytes and HIV/SIV infection. We show here, for the first time, that HIV proteins, Tat and Nef, promoted the acquisition of a profibrotic phenotype of human ASCs and to a lesser extent of adipocytes.
 
HIV-infected patients receiving current ART, and well controlled for HIV replication, often present trunk fat accumulation associated with metabolic and cardiovascular comorbidities [1]. Nonetheless, adverse effects of ART cannot explain all these metabolic alterations, and growing evidence suggest that HIV-infection per se could play a role. Importantly, adipose tissue has been shown to be an HIV/SIV reservoir, the virus being located in immune cells [9,10], suggesting that within adipose tissue, infected cells could release viral proteins, such as Tat and Nef [37,47], which in turn could represent key effectors of adipose tissue remodeling.
 
We observed that fibrosis was up to three-fold higher in SCAT and VAT from SIV-infected animals when compared with uninfected macaques [22]. Indeed, SIV infection was associated with fibrotic bundles and increased collagen 1 expression in both depots. These results were also confirmed in adipose tissue from obese HIV-infected patients treated with ART. In SIV-infected macaques, we observed an increased expression of collagen 6, fibronectin and of the profibrotic factor TGF-β in SCAT and to a lesser extent in VAT. These results were also confirmed in adipose tissues from obese HIV-infected patients treated with ART. An increased level of collagen 6 is often associated with pericellular fibrosis, whereas collagen 1 and collagen 3 are usually found in bundles of fibrosis [48]. Our results, regarding increased collagen 6 mainly in SCAT, may highlight different types of fibrosis in VAT or SCAT in both humans and macaques. The differences regarding collagen 6 in VAT between macaques and patients could result from species or sex differences and/or from the metabolic state and could also be related partly to ART in HIV-infected patients. Nonetheless, both our in-vitro results and the presence of an adipose tissue fibrosis in untreated SIV-infected macaques indicate a key role for SIV/HIV infection in collagen deposition and fibrosis.
 
Adipose precursors and other stroma-vascular cells of adipose tissue have been implicated in the onset of fibrosis [23-24]. Here, we showed that, in proliferating ASC, Tat and Nef altered ECM component production as shown by increased pro-fibrotic markers (TGF-β, fibronectin, and α-SMA). These results are in accordance with a myofibroblast-like phenotype of ASCs [27,29,30]. Similar patterns were observed in mesenchymal precursors in the case of muscle fibrosis [49]. This phenotype could result from interactions between ASCs and macrophages, the latter being a major regulator of fibrosis [20,27,31,50]. Furthermore, the impact of Tat and Nef in 3D-differentiated ASC are modest and suggest that mainly adipose precursor, and to a lesser extent adipocyte, participate to the onset of HIV-induced fibrosis. Of note, it would be interesting to study the impact on ECM in adipose tissue of other secreted HIV proteins as Vpr. It should be noted that ASCs were isolated from SCAT in accordance with HIV/SIV-induced collagen 6 production in SCAT rather than VAT.
 
In obese individuals, fibrosis negatively correlates with adipocyte size and has been suggested to limit adipocyte plasticity, therefore, contributing to the onset of metabolic alterations [20]. Accordingly, in our study, HIV/SIV infection was associated with smaller adipocytes in both SCAT and VAT and adipocyte mean size was negatively related to the size of fibrosis in SCAT of SIV-infected and control macaques. As previously described by the group of Villarroya [51], we observed a dramatic decrease in the adipogenic effector PPARγ suggesting that HIV infection itself could contribute to adipose tissue failure to expand, leading to fat redistribution. In line with this observation, we found that Nef can alter adipogenesis of ASCs as shown by reduced lipid accumulation and expression of adipogenic markers in both 2D and 3D models of differentiation. Nef has been shown to interact with PPARγ in hematopoietic progenitors, suggesting a possible mechanism whereby Nef alters adipogenesis [52]. Moreover, fibrosis could also have a negative effect on adipogenesis, through the action of the profibrotic factor TGF-β [53]. Hypoxia can also participate to the onset of fibrosis and altered adipocyte differentiation and represents a possible pathway, which remains to be investigated [54]. Finally, adipose tissue inflammation can lead to excessive synthesis of ECM proteins. HIV infection is associated with low-grade inflammation in adipose tissue [19], characterized by immune cells recruitment [55,56], which can directly participate to the onset of fibrosis in SCAT and VAT [27,50]. However, several studies on chronically SIV-infected macaques showed no increased inflammatory cytokine expression in adipose tissue [9,11] supporting the idea that HIV infection can be directly responsible for fibrosis in adipose tissue.
 
We acknowledge that our study has some limitations. First, our in-vivo data provide association and not causality. We have evaluated Tat and Nef but not the other HIV proteins that could also induce fibrosis. Several reasons could potentially explain the differences between human and macaques regarding collagen 6, including differences in species, sex (female patients vs. male macaques), metabolic state (obese vs. lean) and/or ART. Otherwise, results obtained in human adipose tissue samples were limited to women presenting morbid obesity and undergoing bariatric surgery and can neither be directly extrapolated to all people living with HIV, nor to men. The number of patients was low, nonetheless, obese HIV-infected patients were paired for sex, age and BMI to noninfected obese individuals, and all samples were withdrawn by surgical biopsies at similar locations. As well, a similar necropsy procedure was performed for adipose tissue samples from macaques.
 
Altogether, these results allow a better understanding of adipose tissue alteration in SIV-infected macaques and HIV-infected patients. We show here for the first time that HIV/SIV infection per se is associated with adipose tissue fibrosis and dysfunction that could, in turn, participate to the onset of cardiovascular and metabolic disorders commonly observed in ART-controlled HIV-infected patients.

 
 
 
 
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