The Role Of Adipose Stem Cells In Obesity-induced Adipose Tissue Inflammation

Obesity is a major public health issue worldwide.Obesity-associated chronic,low-grade inflammation leads to a spectrum of systemic abnormalities.Visceral adipose tissue（VAT）is prone to inflammation in response to obesity,and its inflammation plays a causative role in the development of obesity-related diseases.Therefore,understanding the mechanisms underlying obesity-induced immunocyte accumulation in adipose tissue will provide new insights into treating obesity-induced adipose inflammation and complications.During the development of obesity,inflammatory immunocyte,including CD8⁺T cell,Th1 cell,M1-like macrophage,neutrophil,and mast cell,accumulate in VAT.In contrast,the anti-inflammatory immunocytes,including Tregs,Th2 cells,M2-like macrophages,eosinophils,and group 2 innate lymphoid（ILC2）cells,are decreased in obese VAT.Besides adipocytes,adipose stem cells（ASCs）constitute the second largest non-immune cell components of VAT,constituting approximately 15%-30%of the stromal vascular fraction（SVF）in VAT of lean mice.ASCs show a biological feature of multipotency,immunomodulatory properties,and pro-angiogenesis capacities,through which ASCs influence adipose tissue homeostasis.However,the role of ASCs in obesity-induced adipose inflammation remains obscure.We aim to deepen our understanding of the roles of ASCs in adipose inflammation and the related mechanisms.In this study,we employed human VAT samples and several mice models,as well as the technologies on single-cell RNA sequencing（sc RNA-seq）analysis,bioinformatics,flow cytometry,biochemistry,and molecular biology.Here,we performed sc RNA-seq on the SVF from the epididymal white adipose tissue（e WAT）of normal chow diet（NCD）-fed and 12-week high fat diet（HFD）-fed mice.By comparing the ASCs from lean and obese mice at cellular and molecular levels,we found that ASCs from obese mice scored lower on the stemness scale and higher on the inflammation scale than did ASCs from lean mice,indicating a dramatic functional shift in ASCs during obesity.The downregulation of stemness score in obese ASCs led us to compare the capacity of multilineage differentiation,including adipogenesis,osteogenesis,chondrogenesis,and myogenesis between ASCs from lean and obese mice（lean and obese ASCs）.Our data from both in vitro and in vivo experiments demonstrate impaired multilineage differentiation and proliferative capacities of obese ASCs.To investigate the potential role of obese ASCs in adipose tissue inflammation and insulin resistance,red fluorescent dye-labeled lean and obese ASCs were transplanted into e WAT of lean mice.Mice injected with obese ASCs exhibited decreased insulin sensitivity and glucose tolerance compared with mice injected with lean ASCs.To determine the effects of obese ASCs on immunocyte components in adipose tissue,the immunocyte marker gene expressions were measured by RT-q PCR and the frequency of immunocytes was analyzed by flow cytometry.The m RNA levels of T cell marker genes（Cd3,Cd4,and Cd8）and Th1marker genes（Tbet and Ifng）were markedly higher in the e WAT of the obese ASCs group compared with the lean ASCs group.The flow cytometry data showed that the total number and frequency of T cells in the e WAT of the obese ASCs group were significantly higher than that of the lean ASCs.Our data highlight an important role for obese ASCs in adipose tissue inflammation and provide evidence that obese ASCs have the potency to participate in adipose tissue T cell accrual.Since the obese ASCs promote T cell migration into adipose tissue,we suppose that obese ASCs may secrete one or several chemokine（s）to attract T cells.To test this hypothesis,we collected the conditioned medium（CM）from lean and obese ASCs,and examined the effect of these CM on T cell migration by coculture experiments.CM from obese ASCs led to an increased migration of T cells in a dose-dependent manner,strongly suggesting that chemokines secreted from obese ASCs promoted T cell migration.To identify the chemokines promoting T cell migration from obese ASCs,we analyzed our sc RNA-seq data on lean and obese ASCs.Gene ontology analysis revealed that obese ASCs were enriched in numerous genes related to leukocyte migration and chemotaxis.Among these genes,CCL5 caught our attention as it has been reported to induce T cell migration into adipose tissue during obesity.Flow cytometry analysis and the ELISA analysis further confirmed that obese ASCs expressed higher secretion levels of CCL5 than lean ASCs,and induction of CCL5 occurred as early as 4 weeks of HFD feeding.Blockade of CCL5 by neutralizing antibody remarkedly inhibited the migration of T cells induced by CM from obese ASCs,suggesting that CCL5 secreted from obese ASCs are functional to induce T cell migration.In vivo,we transplanted the ASCs purified from 12-week HFD-fed wild type（WT）and Ccl5 knockout(Ccl5^-/-)mice into e WAT of C57BL/6J mice on a normal chow diet.There was a significant reduction of the T cell number in e WAT of the obese Ccl5^-/-ASCs transplanted group when compared with the obese WT group.In addition,we performed the bone marrow transplantation experiment to exclude the chemotaxis of CCL5 secreted by T cells in e WAT.We utilized 8-week-old NCD-fed WT and Ccl5^-/-mice and exposed the two groups to lethal irradiation to deplete their endogenous adipose tissue T cells and bone marrow populations.After irradiation,mice were transplanted with isolated whole bone marrow cells from GFP mice.The transplanted cells were allowed to engraft for 6 weeks,after which time these chimera mice were then fed on HFD for up to 12 weeks to induce adipose inflammation with T cell infiltration.Interestingly,under the HFD feeding condition,the Ccl5^-/-group exhibited lower body weight,more insulin sensitivity,and no changed glucose tolerance than the WT group.Both the marker genes and cell numbers of T cell subsets were notably lower in the e WAT of the Ccl5^-/-group than in the WT group.These results indicated that CCL5 secreted by obese ASCs contributes to HFD-induced adipose T cell accumulation and insulin resistance.To investigate the key factor that gives rise to the increased CCL5level in ASCs under obese conditions,we incubated ASCs with cytokines known to increase in obese e WAT.Among these cytokines,TNFαinduced CCL5 expression obviously.To determine whether the activation of the NF-κB signaling pathway in ASCs promotes the infiltration of T cells in e WAT during obesity,we derived a loss of function model in which P65can be inactivated in Pdgfrα⁺cells(Pdgfrα-Cre;P65flox/flox,herein,P65^PdgfrαKO mice).P65^PdgfrαKO and littermate WT mice（P65flox/flox）were fed on HFD for 12 weeks.Inhibition of the NF-κB signaling pathway was associated with a statistically significant reduction in CCL5m RNA levels as well as protein levels in ASCs from P65^PdgfrαKO mice.m RNA levels of inflammatory T cell-related genes were lower in the e WAT of P65^PdgfrαKO mice.Notably,both the frequency and cell number of T cells were decreased in the e WAT of P65^PdgfrαKO mice than those observed in WT mice.Collectively,these data suggest that the NF-κB signaling pathway is necessary for ASCs to drive sustained T cell accrual via CCL5 in obesity.Collectively,our data indicated that CCL5 secreted from ASCs is an essential driver for T cell infiltration in the early stage of obesity,highlighting an important role of ASCs in the initiation and maintenance of T cell-mediated adipose tissue inflammation and systemic insulin resistance.Our study provides a theoretical basis for the development of novel drugs targeting non immune cells to treat obesity and adipose tissue inflammation.