_:b437971163 . _:b437971164 . _:b437971165 . _:b437971166 . _:b8196184 "This persistent activation leads to the induction of reactive oxygen species [39], which can attenuate T cell responses [46], [>>47<<], and uncouple TCR signal transduction, resulting in lower cell surface expression of the TCR/CD3 complex [48]." . _:b437971167 . _:b8196179 . _:b437971152 . _:b437971153 . . _:b437971154 . . _:b437971155 . _:b437971156 . _:b437971157 . _:b437971158 . _:b437971206 . _:b437971159 . _:b437971144 . _:b437971145 . _:b8196135 . _:b437971146 . _:b437971147 . _:b437971148 . _:b437971149 . _:b437971150 . _:b437971129 . _:b437971121 . . _:b437971151 . _:b437971136 . _:b437971137 . _:b437971138 . _:b437971210 . _:b437971139 . _:b437971140 . _:b437971168 . _:b8196146 . _:b437971141 . _:b437971204 . _:b437971142 . _:b437971143 . . _:b437971177 . _:b8196170 "Nutrients, such as glucose, are critical for lymphocyte survival, proliferation, differentiation and function [>>41<<], [42]. Many growth factors, such as insulin, IGF-1 and members of the common \u03B3c cytokine family (IL-2, IL-4, IL-7, IL-15) increase glucose uptake and metabolism via signaling through the PI3K/Akt pathway [42]." . _:b8196105 . _:b8196139 "Epidermal sheets were isolated and stained as described previously [>>1<<], [37]. For in vitro stimulation assays, ears were removed from control db/+ animals, separated in half and floated on DMEM media (supplemented with 10% FBS) and treated with 10 \u00B5g/ml anti-CD3\u03B5 antibody or 100 ng/ml recombinant TNF\u03B1 (R&D" . _:b437971172 . _:b437971140 . _:b8196154 . _:b437971200 . . _:b8196191 . . _:b437971224 . _:b437971225 . _:b437971226 . _:b437971227 . _:b437971228 . _:b437971229 . _:b8196171 "Nutrients, such as glucose, are critical for lymphocyte survival, proliferation, differentiation and function [41], [>>42<<]. Many growth factors, such as insulin, IGF-1 and members of the common \u03B3c cytokine family (IL-2, IL-4, IL-7, IL-15) increase glucose uptake and metabolism via signaling through the PI3K/Akt pathway [42]." . _:b437971216 . _:b437971217 . _:b437971218 . _:b437971219 . _:b8196158 . _:b8196125 "Mice deficient in \u03B3\u03B4 T cells exhibit disrupted skin homeostasis, impaired barrier function and delayed wound healing [1], [15], [>>16<<], [17]. In humans, the epidermis consists of a mixed resident \u03B1\u03B2 and \u03B3\u03B4 T cell population [18]. Similar to observations in mice, skin-resident V\u03B41+ \u03B3\u03B4 T cells in humans produce cytokines and growth factors after activation and participate" . _:b437971220 . _:b437971221 . _:b437971222 . _:b437971223 . _:b437971208 . _:b437971209 . _:b437971214 . _:b437971210 . _:b437971211 . . _:b437971212 . _:b437971213 . _:b437971214 . _:b437971215 . _:b437971200 . _:b437971205 . _:b437971201 . _:b437971202 . _:b437971203 . _:b437971204 . _:b437971205 . _:b437971206 . _:b437971207 . . _:b437971145 . _:b437971131 . _:b437971179 . . _:b437971199 . . _:b437971124 . . . . . . _:b8196156 "Following injury and activation through their TCR, \u03B3\u03B4 T cells round-up at the wound edge and lose their dendritic morphology [>>1<<]. Cells distal to the wound site remain dendritic [1], confirming that this is a localized response to tissue damage. Full-thickness punch biopsy wounds were performed on obese 10- to 14-week old BKS db/db mice and skin \u03B3\u03B4 T cell" . . _:b437971209 . _:b437971162 . _:b437971189 . _:b437971143 . . _:b8196185 . . . . _:b8196131 "Even with medical treatment, these chronic non-healing wounds may ultimately result in amputation of extremities [>>21<<]. Recent work has focused on the initiation of chronic inflammation in adipose tissue in obesity. An increase in effector CD8+ T cells and a decrease in CD4+ and T regulatory cells in adipose tissue have been shown to correlate with" . _:b437971142 . _:b437971153 . _:b8196153 "Unlike the rapid turnover of epithelial keratinocytes [29], [30], [>>31<<], LC turnover is much slower, between 5 and 10% of cells proliferating per week [32], [33]." . _:b437971194 . _:b437971192 . _:b8196108 . _:b8196113 "In addition to their role in the innate immune response, \u03B3\u03B4 T cells regulate the subsequent recruitment of inflammatory cells to sites of injury and infection [6], [7], [>>8<<]. Murine skin resident T cells express a canonical V\u03B33V\u03B41 T cell receptor (TCR) and respond to a proposed, yet unknown, self antigen expressed by stressed or damaged keratinocytes [9], [10]. Skin \u03B3\u03B4 T cells display a dendritic morphology," . _:b8196166 "Increased plasma TNF\u03B1 levels correlate with obesity and insulin resistance in both humans and animals [>>38<<]. Our microarray data revealed that several members of the TNF\u03B1 signaling pathway were increased in skin \u03B3\u03B4 T cells isolated from obese db/db mice, including Traf2, Tradd and Ripk1 ( Figure 7A ), which lead to activation of NF-\u03BAB and Jun" . . . _:b8196108 . _:b8196109 . _:b8196110 . . . _:b8196131 . _:b8196111 . . _:b437971171 . _:b437971125 . . _:b8196174 "Similarly, both \u03B1\u03B2 T cells and B cells have been shown to exhibit reduced proliferation when exposed to elevated glucose concentrations in vitro [>>43<<]. This suggests that although glucose and other nutrients may be critical for lymphocyte homeostasis and function, a chronic overabundance of nutrients is detrimental to the maintenance of \u03B3\u03B4 T cells in the epidermis." . _:b8196105 . _:b437971124 . _:b8196117 . _:b8196106 . _:b8196126 "Mice deficient in \u03B3\u03B4 T cells exhibit disrupted skin homeostasis, impaired barrier function and delayed wound healing [1], [15], [16], [>>17<<]. In humans, the epidermis consists of a mixed resident \u03B1\u03B2 and \u03B3\u03B4 T cell population [18]. Similar to observations in mice, skin-resident V\u03B41+ \u03B3\u03B4 T cells in humans produce cytokines and growth factors after activation and participate in" . . _:b437971127 . _:b8196125 . _:b8196107 . . _:b437971126 . _:b8196116 . . _:b437971121 . _:b8196117 . . _:b8196140 "Epidermal sheets were isolated and stained as described previously [1], [>>37<<]. For in vitro stimulation assays, ears were removed from control db/+ animals, separated in half and floated on DMEM media (supplemented with 10% FBS) and treated with 10 \u00B5g/ml anti-CD3\u03B5 antibody or 100 ng/ml recombinant TNF\u03B1 (R&D" . _:b8196118 . _:b8196130 "Chronic wounds in obese and diabetic patients show diminished or altered levels of growth factors, impaired leukocyte infiltration and function and the absence of cell growth and migration over the wound [>>20<<]. Even with medical treatment, these chronic non-healing wounds may ultimately result in amputation of extremities [21]. Recent work has focused on the initiation of chronic inflammation in adipose tissue in obesity. An increase in" . _:b437971123 . _:b8196119 . _:b437971122 . _:b8196112 . _:b437971133 . _:b8196113 . _:b437971132 . _:b8196114 . . _:b437971135 . _:b8196115 . . _:b437971134 . _:b437971218 . _:b8196124 . _:b8196129 "In obesity and metabolic syndrome, the epidermal barrier is disrupted and skin complications can ultimately result in chronic and debilitating non-healing wounds and persistent infections [>>20<<]. Chronic wounds in obese and diabetic patients show diminished or altered levels of growth factors, impaired leukocyte infiltration and function and the absence of cell growth and migration over the wound [20]." . . _:b437971129 . . _:b8196125 . . . _:b8196179 "In addition to the production of cytokines by skin \u03B3\u03B4 T cells early in tissue damage, skin \u03B3\u03B4 T cells also produce growth factors which are critical to skin homeostasis [>>16<<]. We observed a decrease in homeostatic TGF\u03B21 production by skin \u03B3\u03B4 T cells and an inability to upregulate TGF\u03B21 following injury in obesity and metabolic disease. In the skin, the effects of TGF\u03B21 are broad and contribute to various" . _:b437971128 . _:b8196126 . . _:b437971131 . _:b437971196 . _:b8196127 . _:b437971130 . _:b8196120 . _:b437971141 . _:b8196121 . _:b8196176 . . _:b437971140 . _:b8196122 . . . _:b437971143 . . _:b8196123 . . _:b437971142 . . _:b8196168 . _:b8196132 . _:b437971137 . _:b8196122 "morphology, retract their dendrites following activation and are critical for epidermal homeostasis and wound repair through their production of cytokines and regulation of inflammatory cells [1], [6], [11], [12], [13], [14], [>>15<<]. Mice deficient in \u03B3\u03B4 T cells exhibit disrupted skin homeostasis, impaired barrier function and delayed wound healing [1], [15], [16], [17]." . . _:b8196175 . _:b8196133 . . _:b437971227 . . _:b437971136 . _:b8196134 . _:b437971139 . _:b8196135 . _:b8196147 "To determine the impact of obesity and metabolic disease on another skin-resident immune population, we examined LC numbers using anti-langerin and anti-CD45.2 antibodies to stain epidermal sheets [>>25<<]. Obese db/db mice had similar numbers of LC in the epidermis as compared to lean db/+ control mice at all ages tested ( Figure 1C and 1D ). Our data suggest that the early progression of obesity and metabolic syndrome are marked by a" . _:b437971138 . _:b8196111 . _:b8196128 . _:b8196129 . _:b437971149 . _:b8196129 . _:b8196130 . . _:b437971159 . _:b437971148 . _:b8196130 . . . _:b437971151 . _:b8196109 . _:b8196131 . _:b437971150 . _:b437971127 . _:b8196140 . _:b8196113 . _:b437971145 . _:b8196141 . _:b8196160 "Together these genes, which are necessary for the growth and function of \u03B3\u03B4 T cells [>>36<<], were altered in obese mice and reveal a breakdown in the normal signaling pathways required for skin \u03B3\u03B4 T cells homeostasis and function." . _:b8196171 . _:b437971144 . . _:b8196142 . _:b437971147 . _:b8196143 . . . _:b437971146 . _:b437971144 . . _:b8196136 . _:b437971157 . _:b8196137 . _:b437971156 . _:b8196132 "An increase in effector CD8+ T cells and a decrease in CD4+ and T regulatory cells in adipose tissue have been shown to correlate with exacerbated adipocyte inflammation and metabolic disease progression [>>22<<], [23], [24]." . _:b8196172 "Many growth factors, such as insulin, IGF-1 and members of the common \u03B3c cytokine family (IL-2, IL-4, IL-7, IL-15) increase glucose uptake and metabolism via signaling through the PI3K/Akt pathway [>>42<<]. For example, IL-7 signaling in lymphocytes results in STAT5 and PI3K/Akt activation-induced glucose uptake [43]. However, we report here that during the first phase of dysfunction, skin \u03B3\u03B4 T cells are highly susceptible to alterations in" . _:b8196138 . _:b8196172 . _:b437971159 . _:b8196139 . _:b8196115 "Murine skin resident T cells express a canonical V\u03B33V\u03B41 T cell receptor (TCR) and respond to a proposed, yet unknown, self antigen expressed by stressed or damaged keratinocytes [9], [>>10<<]. Skin \u03B3\u03B4 T cells display a dendritic morphology, retract their dendrites following activation and are critical for epidermal homeostasis and wound repair through their production of cytokines and regulation of inflammatory cells [1], [6]," . _:b8196120 . _:b437971158 . _:b8196173 "For example, IL-7 signaling in lymphocytes results in STAT5 and PI3K/Akt activation-induced glucose uptake [>>43<<]. However, we report here that during the first phase of dysfunction, skin \u03B3\u03B4 T cells are highly susceptible to alterations in glucose concentrations. Similarly, both \u03B1\u03B2 T cells and B cells have been shown to exhibit reduced proliferation" . _:b8196148 . _:b8196118 . _:b8196163 "a V\u03B33V\u03B41 TCR and constitutively elevated levels of the activation markers CD69 and CD25 (IL-2 receptor \u03B1), suggesting that they are primed to rapidly respond to TCR-mediated activation and growth factors, such as IL-2 [1], [36], [>>37<<]. No changes were observed in the expression of activation markers on skin \u03B3\u03B4 T cells isolated from 6-week old BKS db/+ and db/db mice (Figure S4A)." . _:b437971153 . _:b8196149 . _:b8196116 "Skin \u03B3\u03B4 T cells display a dendritic morphology, retract their dendrites following activation and are critical for epidermal homeostasis and wound repair through their production of cytokines and regulation of inflammatory cells [>>1<<], [6], [11], [12], [13], [14], [15]." . _:b437971152 . _:b8196150 . _:b437971155 . _:b437971185 . _:b8196151 . _:b437971225 . _:b437971154 . _:b8196144 . _:b437971156 . _:b437971165 . _:b8196144 "1 \u00B5l cDNA was amplified using PCR with primers directed against Ob-Ra and Ob-Rb for 35 cycles [56], leptin for 30 cycles [>>57<<] and \u03B2-actin controls [1]." . _:b8196145 . _:b8196123 "Mice deficient in \u03B3\u03B4 T cells exhibit disrupted skin homeostasis, impaired barrier function and delayed wound healing [>>1<<], [15], [16], [17]." . _:b437971164 . _:b8196146 . _:b437971167 . . _:b437971166 . _:b8196147 . . . _:b437971166 . _:b8196156 . _:b437971161 . _:b8196157 . _:b437971160 . . _:b8196158 . _:b437971163 . _:b8196159 . _:b8196161 . _:b437971162 . _:b8196152 . . _:b437971173 . _:b8196153 . _:b437971182 . _:b437971172 . _:b8196178 "Additionally, skin-resident T cells in chronic wounds isolated from human patients do not upregulate growth factor production, which may contribute to the inability of chronic non-healing wounds to resolve [>>19<<]. In addition to the production of cytokines by skin \u03B3\u03B4 T cells early in tissue damage, skin \u03B3\u03B4 T cells also produce growth factors which are critical to skin homeostasis [16]. We observed a decrease in homeostatic TGF\u03B21 production by skin" . _:b8196154 . _:b437971175 . _:b8196155 . _:b437971152 . _:b437971174 . _:b8196164 . . _:b437971169 . _:b8196192 "Furthermore, knockdown or deletion of mTORC2 complex molecules, including Rictor, Sin1 and Gbl, result in defective mTORC2 complex assembly and Akt activation [52], [>>53<<], [54]. Both mTORC1 and mTORC2 have been shown to be critical for skin \u03B3\u03B4 T cell homeostasis and in vivo wound healing response [36]. Chronic TNF\u03B1 stimulation of skin \u03B3\u03B4 T cells results in direct effects, including alterations in TCR" . _:b8196165 . _:b437971168 . _:b8196166 . _:b8196190 . _:b437971171 . _:b8196106 "Resident intraepithelial \u03B3\u03B4 T cells are responsible for maintaining epithelial integrity, regulating homeostasis and providing a first line of defense against pathogens and injury in mice and humans [>>1<<], [2], [3]. \u03B3\u03B4 T cells arise in the thymus during ontogeny and migrate, in waves, to epithelial tissues such as the skin, lung, intestine and reproductive tract where they populate these tissues for the life of the animal [4], [5]." . _:b8196167 . _:b437971207 . _:b437971170 . _:b8196162 "express a V\u03B33V\u03B41 TCR and constitutively elevated levels of the activation markers CD69 and CD25 (IL-2 receptor \u03B1), suggesting that they are primed to rapidly respond to TCR-mediated activation and growth factors, such as IL-2 [1], [>>36<<], [37]. No changes were observed in the expression of activation markers on skin \u03B3\u03B4 T cells isolated from 6-week old BKS db/+ and db/db mice (Figure S4A)." . _:b8196160 . _:b437971124 "6"^^ . _:b437971181 . _:b8196186 "In addition, chronic TNF\u03B1 and persistent NF-\u03BAB activation negatively impact other cell signaling pathways, including PI3K/Akt/mTOR signaling [>>36<<], [49], [50]. TNF\u03B1 and NF-\u03BAB suppress TSC1 inhibition of mTORC1, resulting in hyperactive mTORC1 activity, which contributes to insulin resistance [50]." . _:b8196161 . _:b437971127 "5"^^ . . _:b437971125 "6"^^ . _:b437971180 . _:b8196153 . _:b8196162 . _:b437971126 "5"^^ . _:b8196115 . _:b437971150 . _:b437971183 . _:b437971122 "8"^^ . _:b8196163 . _:b8196149 . _:b8196192 . _:b8196159 "array, we found that skin \u03B3\u03B4 T cells differentially express NR4A1 and NR4A3, two orphan nuclear receptors which have been shown to sensitize muscle to insulin and have been reported to be underexpressed in obesity and type 2 diabetes [>>35<<]. We observed reduced expression of both NR4A1 and NR4A3 in \u03B3\u03B4 T cells isolated from obese db/db mice ( Figure 4A ), suggesting that skin \u03B3\u03B4 T cells residing in db/db animals have decreased insulin sensitivity." . _:b437971182 . _:b437971123 "8"^^ . _:b8196172 . . _:b437971177 . _:b8196173 . . . _:b437971176 . _:b8196174 . _:b437971121 "9"^^ . _:b437971179 . _:b437971133 "4"^^ . _:b8196175 . . _:b8196177 . _:b8196155 "Unlike the rapid turnover of epithelial keratinocytes [29], [30], [31], LC turnover is much slower, between 5 and 10% of cells proliferating per week [32], [>>33<<]. To determine the rate of \u03B3\u03B4 T cell proliferation in the epidermis, control BKS db/+ mice were treated for one week with BrdU in the drinking water and skin \u03B3\u03B4 T cells were analyzed for BrdU incorporation at 6- and 10-weeks of age. Skin" . _:b437971178 . _:b437971132 "4"^^ . _:b8196168 . . _:b8196133 "An increase in effector CD8+ T cells and a decrease in CD4+ and T regulatory cells in adipose tissue have been shown to correlate with exacerbated adipocyte inflammation and metabolic disease progression [22], [>>23<<], [24]. However, the consequence of obesity and metabolic disease on the function of skin resident lymphocyte populations and how this contributes to skin complications associated with obesity and metabolic disease are unknown." . _:b437971189 . _:b8196121 "a dendritic morphology, retract their dendrites following activation and are critical for epidermal homeostasis and wound repair through their production of cytokines and regulation of inflammatory cells [1], [6], [11], [12], [13], [>>14<<], [15]. Mice deficient in \u03B3\u03B4 T cells exhibit disrupted skin homeostasis, impaired barrier function and delayed wound healing [1], [15], [16], [17]." . _:b437971135 "4"^^ . . _:b8196169 . _:b437971148 . _:b437971188 . _:b437971134 "4"^^ . _:b8196170 . _:b437971161 . . _:b8196142 . _:b437971191 . _:b8196171 . _:b437971130 . _:b437971129 "5"^^ . _:b437971190 . _:b8196180 . _:b437971128 "5"^^ . _:b437971185 . _:b437971170 . . _:b8196181 . _:b437971131 "4"^^ . _:b8196144 . _:b437971184 . _:b8196182 . _:b437971130 "5"^^ . _:b437971187 . _:b437971141 "3"^^ . _:b8196183 . _:b437971186 . _:b437971140 "3"^^ . . _:b8196176 . _:b437971219 . _:b8196187 . . _:b437971197 . _:b437971143 "3"^^ . _:b8196177 . _:b437971196 . _:b437971142 "3"^^ . _:b8196178 . _:b437971199 . . _:b437971137 "4"^^ . _:b8196179 . _:b437971198 . _:b437971198 . _:b437971136 "4"^^ . _:b8196188 . _:b8196170 . . _:b437971193 . _:b437971139 "3"^^ . _:b8196189 . _:b437971192 . _:b437971138 "3"^^ . _:b8196190 . . _:b437971195 . _:b437971149 "3"^^ . _:b8196191 . _:b437971155 . _:b437971194 . _:b437971148 "3"^^ . _:b8196184 . _:b437971157 . _:b437971151 . _:b437971151 "3"^^ . _:b8196185 . _:b437971158 . _:b437971150 "3"^^ . _:b8196186 . _:b437971145 "3"^^ . _:b437971154 . _:b8196187 . _:b8196141 . _:b437971144 "3"^^ . _:b437971147 "3"^^ . _:b437971208 . _:b8196124 . _:b437971164 . _:b437971146 "3"^^ . . . _:b8196146 "results" . . _:b437971157 "3"^^ . _:b8196122 . _:b8196137 . _:b437971180 . _:b437971156 "3"^^ . _:b8196192 . _:b437971159 "3"^^ . _:b8196193 . _:b8196139 . . _:b437971158 "3"^^ . _:b8196188 "In addition, chronic TNF\u03B1 and persistent NF-\u03BAB activation negatively impact other cell signaling pathways, including PI3K/Akt/mTOR signaling [36], [49], [>>50<<]. TNF\u03B1 and NF-\u03BAB suppress TSC1 inhibition of mTORC1, resulting in hyperactive mTORC1 activity, which contributes to insulin resistance [50]." . _:b8196194 . _:b437971160 . _:b437971153 "3"^^ . _:b8196183 "This persistent activation leads to the induction of reactive oxygen species [39], which can attenuate T cell responses [>>46<<], [47], and uncouple TCR signal transduction, resulting in lower cell surface expression of the TCR/CD3 complex [48]." . _:b437971152 "3"^^ . _:b437971155 "3"^^ . _:b8196167 "that several members of the TNF\u03B1 signaling pathway were increased in skin \u03B3\u03B4 T cells isolated from obese db/db mice, including Traf2, Tradd and Ripk1 ( Figure 7A ), which lead to activation of NF-\u03BAB and Jun N-terminal kinase (JNK) [>>39<<]. As shown in Figure 7B , downstream molecules contributing to survival, such as Birc5 (survivin), are increased in skin \u03B3\u03B4 T cells isolated from obese db/db animals." . _:b437971146 . _:b437971154 "3"^^ . _:b437971165 "2"^^ . _:b8196126 . _:b437971138 . _:b8196145 . _:b437971164 "2"^^ . _:b8196194 . _:b8196119 "cells display a dendritic morphology, retract their dendrites following activation and are critical for epidermal homeostasis and wound repair through their production of cytokines and regulation of inflammatory cells [1], [6], [11], [>>12<<], [13], [14], [15]. Mice deficient in \u03B3\u03B4 T cells exhibit disrupted skin homeostasis, impaired barrier function and delayed wound healing [1], [15], [16], [17]." . . _:b437971167 "2"^^ . _:b8196105 "introduction" . _:b8196109 "\u03B3\u03B4 T cells arise in the thymus during ontogeny and migrate, in waves, to epithelial tissues such as the skin, lung, intestine and reproductive tract where they populate these tissues for the life of the animal [>>4<<], [5]. In addition to their role in the innate immune response, \u03B3\u03B4 T cells regulate the subsequent recruitment of inflammatory cells to sites of injury and infection [6], [7], [8]. Murine skin resident T cells express a canonical V\u03B33V\u03B41 T" . _:b437971166 "2"^^ . . . . _:b437971161 "3"^^ . _:b8196157 . _:b437971160 "3"^^ . _:b8196148 . _:b8196156 . _:b437971163 "2"^^ . _:b8196117 "Skin \u03B3\u03B4 T cells display a dendritic morphology, retract their dendrites following activation and are critical for epidermal homeostasis and wound repair through their production of cytokines and regulation of inflammatory cells [1], [>>6<<], [11], [12], [13], [14], [15]. Mice deficient in \u03B3\u03B4 T cells exhibit disrupted skin homeostasis, impaired barrier function and delayed wound healing [1], [15], [16], [17]." . _:b437971162 "3"^^ . _:b437971221 . _:b437971173 "2"^^ . _:b8196167 . _:b437971213 . _:b437971172 "2"^^ . . _:b437971175 "2"^^ . . . . _:b437971174 "2"^^ . _:b8196152 . . _:b8196146 _:b8196156 . _:b8196146 _:b8196157 . _:b437971169 "2"^^ . _:b8196146 _:b8196158 . _:b8196146 _:b8196159 . _:b437971126 . _:b8196146 _:b8196152 . _:b8196146 _:b8196153 . _:b437971168 "2"^^ . _:b8196146 _:b8196154 . _:b8196146 _:b8196155 . . _:b437971228 . _:b8196146 _:b8196148 . _:b8196194 "Both mTORC1 and mTORC2 have been shown to be critical for skin \u03B3\u03B4 T cell homeostasis and in vivo wound healing response [>>36<<]. Chronic TNF\u03B1 stimulation of skin \u03B3\u03B4 T cells results in direct effects, including alterations in TCR expression, and effects on other signaling pathways, including mTOR and Akt. These alterations in signaling ultimately render epidermal T" . _:b8196146 _:b8196149 . _:b437971173 . _:b437971171 "2"^^ . _:b8196181 "In primary cells, TNF\u03B1 induces NF-\u03BAB but not cell death pathways, and chronic TNF\u03B1 would predictably result in chronic NF-\u03BAB activation, gene expression and survival [>>39<<]. This persistent activation leads to the induction of reactive oxygen species [39], which can attenuate T cell responses [46], [47], and uncouple TCR signal transduction, resulting in lower cell surface expression of the TCR/CD3 complex" . _:b8196146 _:b8196150 . _:b8196146 _:b8196151 . _:b8196164 "The epidermis of \u03B3\u03B4 T cell knockout mice (\u03B4\u2212/\u2212) lacks V\u03B33+ T cells but does have an \u03B1\u03B2 T cell population that takes up residence, however, these \u03B1\u03B2 T cells do not respond to keratinocyte damage [>>37<<]. No differences in breeding, litter size or growth of the animals were observed in the B6 \u03B4\u2212/\u2212 db/db mice as compared to B6 db/db animals. Both male and female B6 \u03B4\u2212/\u2212 db/db mice gained weight and became obese similar to B6 db/db mice (" . _:b437971170 "2"^^ . _:b8196146 _:b8196147 . _:b8196121 . . _:b437971181 "2"^^ . . _:b437971128 . _:b437971180 "2"^^ . . _:b437971223 . _:b437971183 "2"^^ . _:b437971182 "2"^^ . _:b437971177 "2"^^ . _:b437971134 . _:b437971176 "2"^^ . . _:b437971179 "2"^^ . . _:b437971178 "2"^^ . _:b8196182 . _:b437971189 "2"^^ . _:b8196169 "discussion" . _:b437971188 "2"^^ . _:b8196159 . _:b8196186 . _:b8196135 _:b8196144 . . . _:b8196135 _:b8196145 . _:b437971191 "2"^^ . _:b8196181 . _:b8196184 . _:b8196147 . _:b437971190 "2"^^ . _:b8196135 _:b8196136 . _:b8196135 _:b8196137 . _:b8196106 . _:b437971185 "2"^^ . _:b8196135 _:b8196138 . _:b8196135 _:b8196139 . _:b8196135 _:b8196140 . _:b8196135 _:b8196141 . _:b437971184 "2"^^ . _:b8196135 _:b8196142 . _:b437971193 . _:b8196135 _:b8196143 . _:b8196116 . _:b437971187 "2"^^ . . . _:b8196127 "In humans, the epidermis consists of a mixed resident \u03B1\u03B2 and \u03B3\u03B4 T cell population [>>18<<]. Similar to observations in mice, skin-resident V\u03B41+ \u03B3\u03B4 T cells in humans produce cytokines and growth factors after activation and participate in wound repair [19]." . . _:b437971186 "2"^^ . _:b8196160 . _:b437971197 "2"^^ . _:b8196146 _:b8196168 . _:b437971184 . _:b437971196 "2"^^ . _:b8196162 . . _:b8196146 _:b8196164 . _:b437971217 . . _:b8196146 _:b8196165 . _:b437971199 "2"^^ . _:b8196146 _:b8196166 . _:b8196146 _:b8196167 . _:b8196158 "Another characteristic feature of skin \u03B3\u03B4 T cell activation is the upregulation of several Th1-type proinflammatory cytokines, including TNF\u03B1 [>>34<<]. To determine if skin \u03B3\u03B4 T cells in obese mice have lost their ability to produce cytokines, we examined TNF\u03B1 production by \u03B3\u03B4 T cells located along the wound edge." . _:b8196146 _:b8196160 . _:b8196146 _:b8196161 . _:b8196110 "\u03B3\u03B4 T cells arise in the thymus during ontogeny and migrate, in waves, to epithelial tissues such as the skin, lung, intestine and reproductive tract where they populate these tissues for the life of the animal [4], [>>5<<]. In addition to their role in the innate immune response, \u03B3\u03B4 T cells regulate the subsequent recruitment of inflammatory cells to sites of injury and infection [6], [7], [8]. Murine skin resident T cells express a canonical V\u03B33V\u03B41 T cell" . _:b437971198 "2"^^ . _:b8196146 _:b8196162 . _:b8196146 _:b8196163 . _:b437971193 "2"^^ . _:b8196128 . _:b437971137 . _:b437971192 "2"^^ . . _:b437971195 "2"^^ . . "PMC0" . _:b8196166 . _:b8196112 "In addition to their role in the innate immune response, \u03B3\u03B4 T cells regulate the subsequent recruitment of inflammatory cells to sites of injury and infection [6], [>>7<<], [8]. Murine skin resident T cells express a canonical V\u03B33V\u03B41 T cell receptor (TCR) and respond to a proposed, yet unknown, self antigen expressed by stressed or damaged keratinocytes [9], [10]. Skin \u03B3\u03B4 T cells display a dendritic" . _:b437971194 "2"^^ . _:b437971174 . . _:b437971205 "2"^^ . _:b437971204 "2"^^ . _:b8196132 . _:b8196150 "Although long-lived, memory-like V\u03B32+ T cells in the periphery have been shown to have very slow turnover [>>28<<], the rate of V\u03B33+ T cell proliferation and homeostatic maintenance in the epidermis has yet to be defined." . _:b437971207 "2"^^ . _:b8196133 . _:b437971147 . . _:b8196123 . . _:b437971206 "2"^^ . _:b8196127 . _:b437971183 . _:b437971201 "2"^^ . _:b437971229 . _:b437971200 "2"^^ . _:b437971203 "2"^^ . _:b8196114 . _:b8196183 . . _:b437971202 "2"^^ . _:b437971213 "2"^^ . _:b437971212 "2"^^ . . _:b8196120 "display a dendritic morphology, retract their dendrites following activation and are critical for epidermal homeostasis and wound repair through their production of cytokines and regulation of inflammatory cells [1], [6], [11], [12], [>>13<<], [14], [15]. Mice deficient in \u03B3\u03B4 T cells exhibit disrupted skin homeostasis, impaired barrier function and delayed wound healing [1], [15], [16], [17]." . _:b437971215 "2"^^ . _:b437971214 "2"^^ . _:b437971209 "2"^^ . _:b437971208 "2"^^ . . _:b437971211 "2"^^ . _:b8196110 . _:b437971135 . . _:b8196134 . _:b437971210 "2"^^ . _:b437971190 . _:b437971221 "2"^^ . _:b437971220 "2"^^ . _:b437971141 . _:b8196189 "TNF\u03B1 and NF-\u03BAB suppress TSC1 inhibition of mTORC1, resulting in hyperactive mTORC1 activity, which contributes to insulin resistance [>>50<<]. Recently, mTORC1 has been shown to negatively inhibit mTORC2 signaling, a necessary complex for Akt activation, and may negatively inhibit growth factor signaling in pathways that don't require IRS-1 [51]. Furthermore, knockdown or" . _:b437971223 "2"^^ . _:b8196105 _:b8196106 . _:b8196105 _:b8196107 . _:b8196105 _:b8196108 . _:b8196105 _:b8196109 . . _:b437971222 "2"^^ . _:b8196105 _:b8196110 . _:b8196105 _:b8196111 . _:b8196105 _:b8196112 . _:b8196143 . _:b8196105 _:b8196113 . _:b437971217 "2"^^ . _:b8196105 _:b8196114 . _:b8196105 _:b8196115 . _:b8196105 _:b8196116 . _:b8196105 _:b8196117 . _:b437971216 "2"^^ . _:b8196105 _:b8196118 . _:b8196105 _:b8196119 . _:b8196155 . _:b8196105 _:b8196120 . _:b8196105 _:b8196121 . _:b437971219 "2"^^ . _:b8196105 _:b8196122 . _:b8196105 _:b8196123 . _:b8196105 _:b8196124 . _:b8196105 _:b8196125 . _:b437971218 "2"^^ . _:b8196105 _:b8196126 . _:b8196105 _:b8196127 . . _:b8196105 _:b8196128 . _:b8196105 _:b8196129 . . _:b437971229 "2"^^ . _:b8196105 _:b8196130 . _:b8196105 _:b8196131 . . _:b8196105 _:b8196132 . _:b8196105 _:b8196133 . _:b437971205 . _:b437971228 "2"^^ . _:b8196105 _:b8196134 . . _:b437971204 . . _:b437971207 . _:b8196180 "In the skin, the effects of TGF\u03B21 are broad and contribute to various aspects of wound healing including inflammation, angiogenesis, tissue remodeling and reepithelialization [>>45<<]. Altered TGF\u03B21 production by skin \u03B3\u03B4 T cells in obesity and metabolic disease may impact multiple phases of epidermal homeostasis and early and late stages of tissue repair." . _:b437971215 . _:b437971206 . _:b437971139 . _:b437971225 "2"^^ . _:b437971201 . _:b437971224 "2"^^ . _:b437971136 . _:b437971200 . _:b437971227 "2"^^ . _:b8196176 "The inability of glucose-treated \u03B3\u03B4 T cells to phosphorylate STAT5B in response to IL-2 points directly to an effect on proliferation as mice expressing a constitutively active STAT5B have an expanded \u03B3\u03B4 T cell population [>>44<<]. Additionally, the severity of loss of skin \u03B3\u03B4 T cells in BKS db/db mice correlates with a period of rapid expansion of \u03B3\u03B4 T cells in the epidermis at 6-weeks of age. The hyperglycemic conditions during this seeding are severe in BKS" . _:b437971187 . . _:b437971203 . _:b437971226 "2"^^ . . _:b437971202 . . _:b437971213 . _:b8196137 "Epidermal cells were isolated from mouse skin as described previously [>>1<<], [37] and rested at 37\u00B0C for 3\u201316 hours followed by antibody staining and flow cytometric analysis." . _:b8196175 "STAT5A/B signaling is critical to \u03B3\u03B4 T cells as mice deficient in STAT5A/B lack \u03B3\u03B4 T cells [>>27<<]. The inability of glucose-treated \u03B3\u03B4 T cells to phosphorylate STAT5B in response to IL-2 points directly to an effect on proliferation as mice expressing a constitutively active STAT5B have an expanded \u03B3\u03B4 T cell population [44]." . _:b437971212 . _:b8196118 "\u03B3\u03B4 T cells display a dendritic morphology, retract their dendrites following activation and are critical for epidermal homeostasis and wound repair through their production of cytokines and regulation of inflammatory cells [1], [6], [>>11<<], [12], [13], [14], [15]. Mice deficient in \u03B3\u03B4 T cells exhibit disrupted skin homeostasis, impaired barrier function and delayed wound healing [1], [15], [16], [17]." . _:b8196142 "Full-thickness biopsy punch wounds were performed on the dorsal surface and ears of mice as previously described [1], [>>14<<]. At the indicated time after wounding, mice were euthanized and wounds were harvested." . _:b437971215 . _:b437971214 . _:b8196128 "Similar to observations in mice, skin-resident V\u03B41+ \u03B3\u03B4 T cells in humans produce cytokines and growth factors after activation and participate in wound repair [>>19<<]." . . _:b437971211 . _:b437971209 . _:b8196187 "In addition, chronic TNF\u03B1 and persistent NF-\u03BAB activation negatively impact other cell signaling pathways, including PI3K/Akt/mTOR signaling [36], [>>49<<], [50]. TNF\u03B1 and NF-\u03BAB suppress TSC1 inhibition of mTORC1, resulting in hyperactive mTORC1 activity, which contributes to insulin resistance [50]." . _:b437971208 . _:b437971178 . _:b437971211 . . _:b437971210 . . _:b437971222 . . _:b437971221 . _:b8196141 "Full-thickness biopsy punch wounds were performed on the dorsal surface and ears of mice as previously described [>>1<<], [14]. At the indicated time after wounding, mice were euthanized and wounds were harvested." . _:b8196151 "Unlike the rapid turnover of epithelial keratinocytes [>>29<<], [30], [31], LC turnover is much slower, between 5 and 10% of cells proliferating per week [32], [33]." . _:b437971220 . . _:b437971223 . _:b437971202 . _:b437971222 . . _:b437971217 . . _:b437971167 . _:b437971216 . _:b437971195 . _:b8196178 . _:b437971175 . _:b437971219 . . . . _:b437971218 . . _:b8196143 "1 \u00B5l cDNA was amplified using PCR with primers directed against Ob-Ra and Ob-Rb for 35 cycles [>>56<<], leptin for 30 cycles [57] and \u03B2-actin controls [1]." . _:b437971229 . _:b437971191 . _:b437971228 . _:b8196145 "1 \u00B5l cDNA was amplified using PCR with primers directed against Ob-Ra and Ob-Rb for 35 cycles [56], leptin for 30 cycles [57] and \u03B2-actin controls [>>1<<]. A plasmid containing leptin cDNA was kindly provided by Dr. Luc Teyton (The Scripps Research Institute, La Jolla)." . _:b437971186 . _:b437971220 . _:b437971225 . _:b437971224 . _:b8196185 "persistent activation leads to the induction of reactive oxygen species [39], which can attenuate T cell responses [46], [47], and uncouple TCR signal transduction, resulting in lower cell surface expression of the TCR/CD3 complex [>>48<<]. This supports our observation that cell surface \u03B3\u03B4 TCR expression is decreased and downstream molecules regulating survival and negative feedback of NF-\u03BAB signaling were altered in skin \u03B3\u03B4 T cells isolated from obese mice." . _:b437971227 . _:b437971226 . . . . _:b8196173 . _:b8196161 "T cells express a V\u03B33V\u03B41 TCR and constitutively elevated levels of the activation markers CD69 and CD25 (IL-2 receptor \u03B1), suggesting that they are primed to rapidly respond to TCR-mediated activation and growth factors, such as IL-2 [>>1<<], [36], [37]. No changes were observed in the expression of activation markers on skin \u03B3\u03B4 T cells isolated from 6-week old BKS db/+ and db/db mice (Figure S4A)." . _:b437971216 . _:b437971181 . _:b8196138 "Epidermal cells were isolated from mouse skin as described previously [1], [>>37<<] and rested at 37\u00B0C for 3\u201316 hours followed by antibody staining and flow cytometric analysis." . . _:b8196174 . _:b8196191 "Furthermore, knockdown or deletion of mTORC2 complex molecules, including Rictor, Sin1 and Gbl, result in defective mTORC2 complex assembly and Akt activation [>>52<<], [53], [54]." . _:b8196165 "Although the epidermal \u03B1\u03B2 T cells identified in B6 \u03B4\u2212/\u2212 mice are not responsive to keratinocyte damage, they do express the activation markers CD69, CD25 and CD103 similar to \u03B3\u03B4 T cells in the skin [>>37<<]. Since expression of these molecules was diminished on \u03B3\u03B4 T cells in the obese environment, we determined whether activation markers on epidermal \u03B1\u03B2 T cells in the B6 \u03B4\u2212/\u2212 db/db mouse were similarly affected." . _:b8196190 "Recently, mTORC1 has been shown to negatively inhibit mTORC2 signaling, a necessary complex for Akt activation, and may negatively inhibit growth factor signaling in pathways that don't require IRS-1 [>>51<<]. Furthermore, knockdown or deletion of mTORC2 complex molecules, including Rictor, Sin1 and Gbl, result in defective mTORC2 complex assembly and Akt activation [52], [53], [54]. Both mTORC1 and mTORC2 have been shown to be critical for" . _:b8196124 "Mice deficient in \u03B3\u03B4 T cells exhibit disrupted skin homeostasis, impaired barrier function and delayed wound healing [1], [>>15<<], [16], [17]." . _:b8196163 . _:b8196119 . _:b8196193 "Furthermore, knockdown or deletion of mTORC2 complex molecules, including Rictor, Sin1 and Gbl, result in defective mTORC2 complex assembly and Akt activation [52], [53], [>>54<<]. Both mTORC1 and mTORC2 have been shown to be critical for skin \u03B3\u03B4 T cell homeostasis and in vivo wound healing response [36]. Chronic TNF\u03B1 stimulation of skin \u03B3\u03B4 T cells results in direct effects, including alterations in TCR expression," . _:b8196165 . . _:b8196169 _:b8196170 . . _:b8196164 . _:b8196169 _:b8196171 . _:b8196169 _:b8196172 . _:b8196169 _:b8196173 . _:b8196169 _:b8196174 . _:b437971133 . _:b8196169 _:b8196175 . _:b8196169 _:b8196176 . _:b8196169 _:b8196177 . _:b8196193 . _:b8196169 _:b8196178 . _:b437971169 . _:b8196169 _:b8196179 . _:b437971226 . _:b8196169 _:b8196180 . _:b8196169 _:b8196181 . _:b8196169 _:b8196182 . . _:b8196169 _:b8196183 . _:b8196169 _:b8196184 . . . _:b8196169 _:b8196185 . . _:b8196169 _:b8196186 . _:b8196169 _:b8196187 . . _:b8196169 _:b8196188 . . _:b8196169 _:b8196189 . _:b8196169 _:b8196190 . _:b8196169 _:b8196191 . _:b8196169 _:b8196192 . _:b8196169 _:b8196193 . _:b8196169 _:b8196194 . _:b8196107 . . _:b437971197 . . _:b8196177 "Skin \u03B3\u03B4 T cells are important mediators of inflammation and tissue repair as mice deficient in \u03B3\u03B4 T cells (\u03B4\u2212/\u2212 mice) exhibit delayed wound healing [>>1<<]. Additionally, skin-resident T cells in chronic wounds isolated from human patients do not upregulate growth factor production, which may contribute to the inability of chronic non-healing wounds to resolve [19]. In addition to the" . _:b8196134 "An increase in effector CD8+ T cells and a decrease in CD4+ and T regulatory cells in adipose tissue have been shown to correlate with exacerbated adipocyte inflammation and metabolic disease progression [22], [23], [>>24<<]. However, the consequence of obesity and metabolic disease on the function of skin resident lymphocyte populations and how this contributes to skin complications associated with obesity and metabolic disease are unknown." . _:b8196148 "IL-2 receptor binding results in Jak1 and Jak3 activation, phosphorylation of STAT5 and translocation of the STAT5 complex to the nucleus where it regulates gene transcription [>>26<<]. Following stimulation of untreated skin \u03B3\u03B4 T cells with IL-2, phosphorylation of STAT5A and STAT5B peaked at 30 minutes, followed by a rapid decrease in phosphorylation ( Figure 2D ). However, in glucose-treated \u03B3\u03B4 T cells, STAT5A was" . _:b437971132 . . _:b8196152 "Unlike the rapid turnover of epithelial keratinocytes [29], [>>30<<], [31], LC turnover is much slower, between 5 and 10% of cells proliferating per week [32], [33]." . . . . . _:b437971122 . _:b8196107 "Resident intraepithelial \u03B3\u03B4 T cells are responsible for maintaining epithelial integrity, regulating homeostasis and providing a first line of defense against pathogens and injury in mice and humans [1], [>>2<<], [3]. \u03B3\u03B4 T cells arise in the thymus during ontogeny and migrate, in waves, to epithelial tissues such as the skin, lung, intestine and reproductive tract where they populate these tissues for the life of the animal [4], [5]." . _:b437971212 . _:b437971165 . . . _:b437971125 . . _:b8196154 "Unlike the rapid turnover of epithelial keratinocytes [29], [30], [31], LC turnover is much slower, between 5 and 10% of cells proliferating per week [>>32<<], [33]. To determine the rate of \u03B3\u03B4 T cell proliferation in the epidermis, control BKS db/+ mice were treated for one week with BrdU in the drinking water and skin \u03B3\u03B4 T cells were analyzed for BrdU incorporation at 6- and 10-weeks of age." . . _:b8196111 "In addition to their role in the innate immune response, \u03B3\u03B4 T cells regulate the subsequent recruitment of inflammatory cells to sites of injury and infection [>>6<<], [7], [8]." . _:b437971149 . . . _:b8196136 "Fatty acids were prepared for cell culture assays as described elsewhere [>>55<<]. For analysis of phosphorylated STAT5A and STAT5B, 7\u201317 cells were pre-treated with starvation media for 4 hours, then placed into IL-2 containing growth media supplemented with 33.3 mM glucose for 24 hours." . . . _:b8196108 "Resident intraepithelial \u03B3\u03B4 T cells are responsible for maintaining epithelial integrity, regulating homeostasis and providing a first line of defense against pathogens and injury in mice and humans [1], [2], [>>3<<]. \u03B3\u03B4 T cells arise in the thymus during ontogeny and migrate, in waves, to epithelial tissues such as the skin, lung, intestine and reproductive tract where they populate these tissues for the life of the animal [4], [5]." . . _:b437971128 . _:b437971129 . . _:b437971130 . . _:b437971131 . _:b437971132 . _:b8196150 . _:b8196135 "materials and methods" . _:b437971133 . _:b437971134 . _:b437971135 . _:b8196157 "Cells distal to the wound site remain dendritic [>>1<<], confirming that this is a localized response to tissue damage." . _:b437971121 . _:b437971122 . _:b437971188 . _:b437971123 . _:b437971124 . _:b437971125 . _:b437971126 . _:b437971127 . _:b8196112 . . _:b8196114 "Murine skin resident T cells express a canonical V\u03B33V\u03B41 T cell receptor (TCR) and respond to a proposed, yet unknown, self antigen expressed by stressed or damaged keratinocytes [>>9<<], [10]. Skin \u03B3\u03B4 T cells display a dendritic morphology, retract their dendrites following activation and are critical for epidermal homeostasis and wound repair through their production of cytokines and regulation of inflammatory cells" . . . _:b437971203 . . . _:b8196182 "This persistent activation leads to the induction of reactive oxygen species [>>39<<], which can attenuate T cell responses [46], [47], and uncouple TCR signal transduction, resulting in lower cell surface expression of the TCR/CD3 complex [48]." . . . . . . _:b437971123 . _:b437971224 . _:b437971192 . _:b8196149 "Moreover, STAT5A/B signaling is critical to \u03B3\u03B4 T cell function as \u03B3\u03B4 T cells are absent in mice deficient in STAT5A/B [>>27<<]." . . _:b437971193 . _:b437971194 . _:b8196138 . . _:b437971195 . _:b437971196 . _:b8196169 . _:b437971197 . _:b437971198 . _:b8196189 . . _:b437971199 . _:b437971184 . _:b437971185 . _:b437971186 . _:b8196188 . _:b8196140 . _:b437971187 . _:b437971188 . _:b437971189 . _:b437971190 . _:b437971191 . _:b437971176 . _:b437971177 . _:b437971178 . "10.1371%2Fjournal.pone.0011422" . _:b8196136 . _:b437971179 . _:b437971163 . _:b437971180 . _:b437971181 . _:b8196168 "However, molecules that negatively regulate Ripk1 and Jnk signaling, such as Tnfaip3 (A20) and GADD45\u03B2 respectively [>>40<<], are decreased in \u03B3\u03B4 T cells isolated from obese db/db mice." . . _:b437971201 . _:b437971182 . _:b437971183 . _:b437971168 . . _:b8196180 . _:b437971169 . _:b437971170 . _:b8196151 . _:b437971171 . _:b437971176 . _:b437971172 . _:b437971173 . _:b437971174 . _:b437971175 . _:b437971160 . _:b437971161 . _:b437971162 .