Adipose‐tissue regulatory T cells: Critical players in adipose‐immune crosstalk

Obesity and type‐2 diabetes (T2D) are associated with metabolic defects and inflammatory processes in fat depots. FoxP3⁺ regulatory T cells (Tregs) control immune tolerance, and have an important role in controlling tissue‐specific inflammation. In this mini‐review we will discuss current insights into how cross‐talk between T cells and adipose tissue shapes the inflammatory environment in obesity‐associated metabolic diseases, focusing on the role of CD4⁺T cells and Tregs. We will also highlight potential opportunities for how the immunoregulatory properties of Tregs could be harnessed to control inflammation in obesity and T2D and emphasize the critical need for more research on humans to establish mechanisms that are conserved in both mice and humans.     

An update on immune dysregulation in obesity‐related insulin resistance

Obesity is associated with chronic low‐grade inflammation of the adipose tissue (AT) that might develop into systemic inflammation, insulin resistance (IR) and an increased risk of type 2 diabetes mellitus (T2DM) in severe obese rodents and humans. In the lean state, small normal adipocytes and AT macrophages interact with each other to maintain metabolic homeostasis but during obesity, enlarged adipocytes secrete inflammatory mediators and express immune receptors to recruit immune cells and aggravate the inflammation. The better understanding of the obesity‐related inflammatory milieu and the sequential events leading to IR could be helpful in designing new preventive and therapeutic strategies. The present review will discuss the cellular and molecular abnormalities participating in the pathogenesis of obesity in obese individuals as well as high‐fat diet (HFD)‐fed mice, a mouse model of obesity.     

Comparison of T1 relaxation times in adipose tissue of severely obese patients and healthy lean subjects measured by 1.5 T MRI

Subcutaneous (SAT) and visceral adipose tissue (VAT) differ in composition, endocrine function and localization in the body. VAT is considered to play a role in the pathogenesis of insulin resistance, type 2 diabetes, fatty liver disease, and other obesity‐related disorders. It has been shown that the amount, distribution, and (cellular) composition of adipose tissue (AT) correlate well with metabolic conditions. In this study, T₁ relaxation times of AT were measured in severely obese subjects and compared with those of healthy lean controls. Here, we tested the hypothesis that T₁ relaxation times of AT differ between lean and obese individuals, but also between VAT and SAT as well as superficial (sSAT) and deep SAT (dSAT) in the same individual. Twenty severely obese subjects (BMI 41.4 ± 4.8 kg/m²) and ten healthy lean controls matched for age (BMI 21.5 ± 1.9 kg/m²) underwent MRI at 1.5 T using a single‐shot fast spin‐echo sequence (short‐tau inversion recovery) at six different inversion times (T_I range 100–1000 ms). T₁ relaxation times were computed for all subjects by fitting the T_I‐dependent MR signal intensities of user‐defined regions of interest in both SAT and VAT to a model function. T₁ times in sSAT and dSAT were only measured in obese patients. For both obese patients and controls, the T₁ times of SAT (275 ± 14 and 301 ± 12 ms) were significantly (p < 0.01) shorter than the respective values in VAT (294 ± 20 and 360 ± 35 ms). Obese subjects also showed significant (p < 0.01) T₁ differences between sSAT (268 ± 11 ms) and dSAT (281 ± 19 ms). More important, T₁ differences in both SAT and VAT were highly significant (p < 0.001) between obese patients and healthy subjects. The results of our pilot study suggest that T₁ relaxation times differ between severely obese patients and lean controls, and may potentially provide an additional means for the non‐invasive assessment of AT conditions and dysfunction. Copyright © 2014 John Wiley & Sons, Ltd.     

Invariant NKT cells and CD1d+ cells amass in human omentum and are depleted in patients with cancer and obesity

Invariant NKT (iNKT) cells recognize lipid antigens presented by CD1d and respond rapidly by killing tumor cells and release cytokines that activate and regulate adaptive immune responses. They are essential for tumor rejection in various mouse models, but clinical trials in humans involving iNKT cells have been less successful, partly due to their rarity in humans compared with mice. Here we describe an accumulation of functional iNKT cells in human omentum, a migratory organ with healing properties. Analysis of 39 omental samples revealed that T cells are the predominant lymphoid cell type and of these, 10% expressed the invariant Vα24Jα18 TCR chain, found on iNKT cells, higher than in any other human organ tested to date. About 15% of omental hematopoietic cells expressed CD1d, compared with 1% in blood (p<0.001). Enriched omental iNKT cells killed CD1d⁺ targets and released IFN‐γ and IL‐4 upon activation. Omental iNKT‐cell frequencies were lower in patients with severe obesity (p=0.005), and with colorectal carcinoma (p=0.004) compared with lean healthy subjects. These data suggest a novel role for the omentum in immune regulation and tumor immunity and identify it as a potential source of iNKT cells for therapeutic use.     

Protective and Detrimental Roles for Regulatory T Cells in a Viral Model for Multiple Sclerosis

Multiple sclerosis (MS) has been proposed to be an immune‐mediated disease in the central nervous system (CNS) that can be triggered by virus infections. In Theiler's murine encephalomyelitis virus (TMEV) infection, during the first week (acute stage), mice develop polioencephalomyelitis. After 3 weeks (chronic stage), mice develop immune‐mediated demyelination with virus persistence, which has been used as a viral model for MS. Regulatory T cells (Tregs) can suppress inflammation, and have been suggested to be protective in immune‐mediated diseases, including MS. However, in virus‐induced inflammatory demyelination, although Tregs can suppress inflammation, preventing immune‐mediated pathology, Tregs may also suppress antiviral immune responses, leading to more active viral replication and/or persistence. To determine the role and potential translational usage of Tregs in MS, we treated TMEV‐infected mice with ex vivo generated induced Tregs (iTregs) on day 0 (early) or during the chronic stage (therapeutic). Early treatment worsened clinical signs during acute disease. The exacerbation of acute disease was associated with increased virus titers and decreased immune cell recruitment in the CNS. Therapeutic iTreg treatment reduced inflammatory demyelination during chronic disease. Immunologically, iTreg treatment increased interleukin‐10 production from B cells, CD4⁺ T cells and dendritic cells, which may contribute to the decreased CNS inflammation.     

NLRP3 inflammasome blockade reduces adipose tissue inflammation and extracellular matrix remodeling

The NLRP3-IL-1β pathway plays an important role in adipose tissue (AT)-induced inflammation and the development of obesity-associated comorbidities. We aimed to determine the impact of NLRP3 on obesity and its associated metabolic alterations as well as its role in adipocyte inflammation and extracellular matrix (ECM) remodeling. Samples obtained from 98 subjects were used in a case−control study. The expression of different components of the inflammasome as well as their main effectors and inflammation- and ECM remodeling-related genes were analyzed. The impact of blocking NLRP3 using siRNA in lipopolysaccharide (LPS)-mediated inflammation and ECM remodeling signaling pathways was evaluated. We demonstrated that obesity (P < 0.01), obesity-associated T2D (P < 0.01) and NAFLD (P < 0.05) increased the expression of different components of the inflammasome as well as the expression and release of IL-1β and IL-18 in AT. We also found that obese patients with T2D exhibited increased (P < 0.05) hepatic gene expression levels of NLRP3, IL1B and IL18. We showed that NLRP3, but not NLRP1, is regulated by inflammation and hypoxia in visceral adipocytes. We revealed that the inhibition of NLRP3 in human visceral adipocytes significantly blocked (P < 0.01) LPS-induced inflammation by downregulating the mRNA levels of CCL2, IL1B, IL6, IL8, S100A8, S100A9, TLR4 and TNF as well as inhibiting (P < 0.01) the secretion of IL1-β into the culture medium. Furthermore, blocking NLRP3 attenuated (P < 0.01) the LPS-induced expression of important molecules involved in AT fibrosis (COL1A1, COL4A3, COL6A3 and MMP2). These novel findings provide evidence that blocking the expression of NLRP3 reduces AT inflammation with significant fibrosis attenuation.     

The influence of obesity on cerebral blood flow in young adults using arterial spin labeling MRI

Obesity causes damage to several organs, including the brain. Recent studies have been focusing on understanding the mechanisms through which obesity affects brain structure and function using neuroimaging techniques. A functional biomarker, such as cerebral blood flow (CBF), is a powerful tool that can be used to explore neural dysfunction. However, there is currently limited information regarding the association between CBF and obesity. The study was conducted to investigate the potential effect of obesity on brain perfusion in a young cohort aged 20‐30 years. A total of 21 obese (body mass index (BMI) > 26 kg/m²) and 21 lean (BMI < 24 kg/m²) right‐handed volunteers were included in this study. CBF was acquired using the 2D single post‐labeling delay (PLD) arterial spin labeling (ASL) technique on a 3 T MRI scanner. A multiple regression analysis was performed to examine the difference in global and regional gray matter (GM) CBF between the groups. CBF value was assigned as the dependent variable, whereas age, sex, and group (obese or lean) were considered as the independent variables. Results showed that group‐related differences in CBF were homogeneous across brain regions, as obese subjects had significantly lower global GM CBF than lean subjects (P < 0.05). In the voxelwise analysis, obese individuals had significantly lower CBF in the left pulvinar of the thalamus and visual association areas, including Brodmann area (BA) 7, BA18, and BA19, than lean subjects. Although the signal‐to‐noise ratio was slightly compromised for 2D sequences and subject‐specific arterial transit time was not estimated due to a single PLD sequence, this study demonstrated alterations in CBF in obese subjects, particularly in regions of the pulvinar of the thalamus and its synchronously related areas such as visual association areas. These results suggest that ASL provides a potential platform for further obesity‐related research.     

Molecular screening of the microsomal triglyceride transfer protein: association between polymorphisms and both abdominal obesity and plasma apolipoprotein B concentration

Microsomal triglyceride transfer protein (MTP) plays a critical role in the assembly of lipoproteins. The aim of this study was first to seek new MTP gene variants and then to verify whether MTP gene polymorphisms were associated with plasma lipoprotein/lipid levels in men with visceral obesity. Molecular screening of the MTP gene revealed 11 polymorphisms. The carriers of the c.933A allele and c.1151C allele or –400A/A homozygotes were characterized by increased levels of abdominal visceral adipose tissue (AT) measured by computed tomography (P=0.02, P=0.04, P=0.03, respectively). After dividing each genotype group into subgroups using 130 cm² as a cutoff point for visceral AT, significantly higher low-density lipoprotein (LDL)–apolipoprotein B (apoB) concentrations were found in obese men bearing the c.891G allele, the –400 T allele, as well as for 282G/G homozygotes, 933C/C homozygotes, and 1151A/A homozygotes when compared to their lean counterparts. Haplotypes were not associated with phenotypes under study. In conclusion, some MTP gene polymorphisms in the French Canadian population are associated with the amount of abdominal visceral AT and plasma LDL–apoB concentrations.     

Antigen-specific prevention of type 1 diabetes in NOD mice is ameliorated by OX40 agonist treatment

Antigen-specific therapies are possibly the safest approach to prevent type 1 diabetes (T1D). However their clinical translation has yielded poor results and greater efforts need to be put into the development of novel strategies to ameliorate their clinical outcome. OX40 is a costimulatory molecule expressed by T cells after antigen recognition and has been implicated in the control effector but also regulatory T cells (Tregs) function in vivo. The activity of OX40 signal on Tregs function has been controversial. In this context we investigated whether an anti-OX40 agonist antibody treatment can ameliorate antigen-specific immune intervention for the prevention of T1D. We show that treatment of non-obese diabetic (NOD) mice with an OX40 agonistic antibody (OX86) reduced type 1 diabetes (T1D) incidence by inducing both CD4⁺CD25⁺Foxp3⁺ Tregs and CD4⁺Foxp3⁻ T cells expressing the latency-associated peptide (LAP). These OX86-induced CD4⁺Foxp3⁻LAP⁺ T cells also demonstrated suppressive activity in vitro. A significant increase in protection was observed when OX86 was combined with insulin B9:23 (insB9:23) peptide immunizations. Synergy resulted from an expansion of IL-10-expressing insB9:23-reactive Tregs which augmented the proportion of CD4⁺ T cells with in vivo suppressive activity. Consequently, CD4⁺ T cells purified from OX86/insB9:23 combination treatment prevented T1D development when adoptively transferred into recipient mice. These findings suggest that the requirement for OX40 signaling by antigen-induced Tregs can be dominant over its well-documented need for effector memory cell function and may have potentially important implications for improving the clinical translation of antigen-specific prevention of T1D and possibly other autoimmune disorders.     

Regulatory T-cell physiology and application to treat autoimmunity

Summary: Endowed with the ability to actively suppress an immune response, regulatory T cells (Tregs) hold the promise of halting ongoing pathogenic autoimmunity and restoring self‐tolerance in patients suffering from autoimmune diseases. Through many in vitro and in vivo studies, we have learned that Tregs can function in the lymph nodes as well as in the peripheral tissues. In vivo, Tregs act through dendritic cells to limit autoreactive T‐cell activation, thus preventing their differentiation and acquisition of effector functions. By limiting the supply of activated pathogenic cells, Tregs prevent or slow down the progression of autoimmune diseases. However, this protective mechanism appears insufficient in autoimmune individuals, likely because of a shortage of Tregs cells and/or the development and accumulation of Treg‐resistant pathogenic T cells over the long disease course. Thus, restoration of self‐tolerance in these patients will likely require purging of pathogenic T cells along with infusion of Tregs with increased ability to control ongoing tissue injury. In this review, we highlight advances in dissecting Treg function in vivo in autoimmune settings and summarize multiple studies that have overcome the limitations of the low abundance of Tregs and their hypoproliferative phenotype to develop Treg‐based therapies.     

TLR2 signaling improves immunoregulation to prevent type 1 diabetes

Signaling through TLR2 promotes inflammation and modulates CD4⁺CD25⁺ Tregs. We assessed mechanistically how this molecule would alter immunoregulation in type 1 diabetes (T1D). We also asked whether TLR2 may be involved in our recent discovery that viral infection can protect from autoimmune diabetes by expanding and invigorating Tregs. Treatment of prediabetic mice with a synthetic TLR2 agonist diminished T1D and increased the number and function of CD4⁺CD25⁺ Tregs, also conferring DCs with tolerogenic properties. TLR2 ligation also promoted the expansion of Tregs upon culture with DCs and ameliorated their capacity to prevent the disease. Protection from T1D by lymphocytic choriomeningitis virus (LCMV) infection depended on TLR2. LCMV increased the frequency of CD4⁺CD25⁺ Tregs and their production of TGF‐β more significantly in WT than TLR2‐deficient mice. Furthermore, LCMV infection in vivo or LCMV‐infected DCs in vitro rendered, via TLR2, CD4⁺CD25⁺ Tregs capable of diminishing T1D. We identify novel mechanisms by which TLR2 promotes immunoregulation and controls autoimmune diabetes in naïve or infected hosts. This work should help understand T1D etiology and develop novel immune‐based therapeutic interventions.     

Making regulatory T cells with defined antigen specificity: role in autoimmunity and cancer

Summary: There is increasing evidence that agonist ligand presentation either intrathymically or extrathymically plays a crucial if not essential role in the generation of regulatory T cells (Tregs). Thus, it is possible to induce Tregs of any desired specificity in vivo. The same goal can be achieved in vitro by expanding antigen‐specific CD4⁺ T cells and retrovirally transducing them. In contrast, in vitro expansion of Tregs is limited to antigens that have resulted in Treg generation in vivo. Antigen‐specific Tregs can be used in cellular therapy with the goal to prevent autoimmune disease or even to interfere with established autoimmunity. The latter requires that the Tregs can suppress effector cells that have already caused harm, which is possible because of the antigen‐dependent homing properties of Tregs, i.e. these cells can accumulate in antigen‐draining lymph nodes and exit into inflamed tissue. Generally, the in vivo interference is dependent on cytokines such as transforming growth factor‐β and interleukin‐10 that were dispensable in in vivo analysis of immunosuppression. The precise mechanisms of suppression remain enigmatic, however, but may be further elucidated by the molecular analysis of suppressed versus non‐suppressed T cells.     

Dendritic cells expand antigen-specific Foxp3+CD25+CD4+ regulatory T cells including suppressors of alloreactivity

Summary: Thymic derived naturally occurring CD25⁺CD4⁺ T regulatory cells (Tregs) suppress immune responses, including transplantation. Here we discuss the capacity of dendritic cells (DCs) to expand antigen‐specific Tregs, particularly polyclonal Tregs directed to alloantigens. Initial studies have shown that mature DCs are specialized antigen‐presenting cells (APCs) for expanding antigen‐specific CD25⁺ CD4⁺ Tregs from TCR transgenic mice. When triggered by specific antigen, these Tregs act back on immature DCs to block the upregulation of CD80 and CD86 costimulatory molecules. More recently, DCs have been used to expand alloantigen‐specific CD25⁺CD4⁺ Tregs from the polyclonal repertoire in the presence of interleukin‐2 (IL‐2). Allogeneic DCs are much more effective than allogeneic spleen cells for expanding CD25⁺CD4⁺ Tregs. The DC‐expanded Tregs continue to express high levels of Foxp3, even without supplemental IL‐2, whereas spleen cells poorly sustain Foxp3 expression. When suppressive activity is tested, relatively small numbers of DC‐expanded CD25⁺CD4⁺ Tregs exert antigen‐specific suppression in the mixed leukocyte reaction (MLR), blocking immune responses to the original stimulating strain 10 times more effectively than to third party stimulating cells. DC‐expanded Tregs also retard graft versus host disease (GVHD) across full major histocompatibility complex (MHC) barriers. In vitro and in vivo, the alloantigen‐specific CD25⁺CD4⁺ Tregs are much more effective suppressors of transplantation reactions than polyclonal populations. We suggest that the expansion of Tregs from a polyclonal repertoire via antigen‐presenting DCs will provide a means for antigen‐specific control of unwanted immune reactions.     

Interleukin‐10‐secreting type 1 regulatory T cells in rodents and humans

Summary: Interleukin‐10 (IL‐10)‐secreting T regulatory type 1 (Tr1) cells are defined by their specific cytokine production profile, which includes the secretion of high levels of IL‐10 and transforming growth factor‐β(TGF‐β), and by their ability to suppress antigen‐specific effector T‐cell responses via a cytokine‐dependent mechanism. In contrast to the naturally occurring CD4⁺CD25⁺ T regulatory cells (Tregs) that emerge directly from the thymus, Tr1 cells are induced by antigen stimulation via an IL‐10‐dependent process in vitro and in vivo. Specialized IL‐10‐producing dendritic cells, such as those in an immature state or those modulated by tolerogenic stimuli, play a key role in this process. We propose to use the term Tr1 cells for all IL‐10‐producing T‐cell populations that are induced by IL‐10 and have regulatory activity. The full biological characterization of Tr1 cells has been hampered by the difficulty in generating these cells in vitro and by the lack of specific marker molecules. However, it is clear that Tr1 cells play a key role in regulating adaptive immune responses both in mice and in humans. Further work to delineate the specific molecular signature of Tr1 cells, to determine their relationship with CD4⁺CD25⁺ Tregs, and to elucidate their respective role in maintaining peripheral tolerance is crucial to advance our knowledge on this Treg subset. Furthermore, results from clinical protocols using Tr1 cells to modulate immune responses in vivo in autoimmunity, transplantation, and chronic inflammatory diseases will undoubtedly prove the biological relevance of these cells in immunotolerance.     

Interleukin-33 (IL-33): A nuclear cytokine from the IL-1 family

Interleukin-33 (IL-33) is a tissue-derived nuclear cytokine from the IL-1 family abundantly expressed in endothelial cells, epithelial cells and fibroblast-like cells, both during homeostasis and inflammation. It functions as an alarm signal (alarmin) released upon cell injury or tissue damage to alert immune cells expressing the ST2 receptor (IL-1RL1). The major targets of IL-33 in vivo are tissue-resident immune cells such as mast cells, group 2 innate lymphoid cells (ILC2s) and regulatory T cells (Tregs). Other cellular targets include T helper 2 (Th2) cells, eosinophils, basophils, dendritic cells, Th1 cells, CD8⁺ T cells, NK cells, iNKT cells, B cells, neutrophils and macrophages. IL-33 is thus emerging as a crucial immune modulator with pleiotropic activities in type-2, type-1 and regulatory immune responses, and important roles in allergic, fibrotic, infectious, and chronic inflammatory diseases. The critical function of IL-33/ST2 signaling in allergic inflammation is illustrated by the fact that IL33 and IL1RL1 are among the most highly replicated susceptibility loci for asthma. In this review, we highlight 15 years of discoveries on IL-33 protein, including its molecular characteristics, nuclear localization, bioactive forms, cellular sources, mechanisms of release and regulation by proteases. Importantly, we emphasize data that have been validated using IL-33-deficient cells.     

Increased adipose tissue expression of lipocalin-2 in obesity is related to inflammation and matrix metalloproteinase-2 and metalloproteinase-9 activities in humans

Lipocalin-2 (LCN2) is a novel adipokine with potential roles in obesity, insulin resistance, and inflammation. The aim of the present work was to evaluate the effect of obesity on circulating concentrations and gene and protein expression levels of LCN2 in human visceral adipose tissue (VAT) as well as its involvement in inflammation. VAT biopsies from 47 subjects were used in the study. Real-time PCR and Western-blot analyses were performed to quantify levels of LCN2 in VAT as well as the association with other genes implicated in inflammatory pathways. Forty-four serum samples were used to analyze the circulating concentrations of LCN2. Zymography analysis was used to determine the activity of matrix metalloproteinase (MMP) in VAT. Obese patients exhibited increased mRNA (p < 0.0001) and protein (p = 0.017) expression levels of LCN2 compared to lean subjects. Although no differences in plasma LCN2 concentrations were observed, increased circulating LCN2/MMP-9 complex levels were found (p = 0.038) in the obese group. Moreover, obese individuals showed increased (p < 0.01) activity of MMP-2 and MMP-9/LCN2 complex, while a positive correlation (p < 0.01) between MMP-2 and MMP-9 activities and BMI was observed. Gene and protein expression levels of LCN2 in VAT were positively associated with inflammatory markers (p < 0.01). These findings represent the first observation that mRNA and protein levels of LCN2 are increased in human VAT of obese subjects. Furthermore, LCN2 is associated with MMP-2 and MMP-9 activities as well as with pro-inflammatory markers suggesting its potential involvement in the low-grade chronic inflammation accompanying obesity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.