The Roles Of CD4+T Subsets In The Development Of Obesity And Type2Diabetes And Its Associated Complications

The Prevalence of obesity, in parallel with type2diabetes (T2D), has expanded in an explosive manner and threaten human as a worldwide epidemic concern. Evidences from the past decade unequivocally indicate that the major underlying link between these two disorders is chronic, systemic low-grade inflammation. Inflammation has long been postulated to be the central link between T2D and its associated complications. However, up until recently, the specific immunologic sensors that are triggered in response to metabolic dysfunction to produce a state of inflammation was not identified. The study of how metabolic pressures impact the modulation of immune homeostasis and how the immune imbalance stirs up the chronic inflammation in return are now topics of intensive investigation. In fact, apart from the long list of metabolic disorders, obesity is frequently reported with an apparent increased prevalence of autoimmune and inflammatory disorders, e.g. asthma, rheumatic diseases, inflammatory bowel disease (IBD), etc. These clinical observations strengthen hints that immunologic imbalance may underlie or at least be closely linked to the aggravating metabolic disturbances resulting from nutrition load.Accumulation of adipose tissue macrophages (ATMs) was first well-described inflammatory participant in obesity, whose recruitment and pro-inflammatory polarization directly contribute to the inflammatory status. More recently, studies of Wu and Kintscher et al extended those observations to cells of Adaptive immunity. They suggested the accumulation of T-lymphocyte occurs in the adipose tissue prior to that of macrophages. Moreover, the infiltration of T cells accompanied the initiation of insulin resistance characterized by an impaired systemic insulin sensitivity and glucose tolerance. These works, taken together, indicates that T cell groups are important regulators of inflammation in both rodent models and patients of obesity and T2D. Previous work indicated that obesity was accompanied with a decreased frequency in Treg. However, it was further argued that the decrease in Treg frequency did not explain for the panorama of immunologic disturbance in obesity. An appropriate balance between pro-inflammatory and anti-inflammatory subsets of T cells is essential to maintenance of immune homeostasis and avoidance of inflammatory diseases. Elevation of Thl and Th17subsets and/or significant decrease of regulatory T cells (Tregs) could directly trigger activation of innate immunity and thus inflammation. Moreover,_van der Weerd K et al’s work even gave controversary results in a study restricted to obese but metabolically health (without none sign of hyperlipidmia, hyperglycemia or apparent insulin resistance) participants. Obese individuals often exhibit metabolic dysfunction, diabetes and elevated cardiovascular risk. However not all obese subjects are accompanied with cardiometabolic risk and that the "metabolically healthy obesity"(MHO) may exist. The above study in MHO suggested hypothetically an anti-inflammatory compensation and remained to be testified in further well-designed studies. Moreover, whether decompensated immunologic imbalance occurred in T2D and roles of immune disturbance in cardiometabolic outcomes in obesity are clearly new challenges. It is worth noting that besides the balance in frequencies/numbers, the function status of Th subpopulations would also provide indispensable clues in future.Sufferings and burdens of diabetes come mainly from onerous complications. Among them, coronary atherosclerotic heart disease (CHD) is the most perishing concern characterized by its high prevalence and overwhelming life threats. Diabetes correlates CHD in such an impressive manner that it is considered a cardiovascular disease equivalent. Chronic inflammation has been postulated to bridging the increased risk of cardiovascular disease and T2D and considered as the "common soil". Although some of the studies demonstrated an aberrant population of Th subsets in patients, systemic clinical data that could verified the contribution of these changes on CHD were so far absent, especially in diabetes which was characterized by an increase in baseline pro-inflammatory Th subsets levels. Clinical investigations would provide more indispensable clues for further exploration. And a better knowledge of the intricate interactions of metabolic pressure and immunologic disturbance could undoubtedly lead to valuable strategies for therapeutic intervention in obesity and T2D, as well as their associated complications.Our early work preliminarily suggests that Th22cells might be a potent participant in development of obesity and T2D. Th22cells, newly identified as a distinct lineage of CD4+T cells characterized by their production of interleukin-22(IL-22), is an emergent constitutor in host defense and inflammatory diseases. Receptors for IL-22(IL-22R) are absent on immune cells, but instead restricted to tissues, thus providing signal directionality from immune system to tissues. IL-22exerts its biological effects by binding with IL-22R, which is a heterodimer composed of an IL-10β and an IL-22Ra subunit. It is bi-functional with both pro-inflammatory and proliferative effects on tissues depending on the inflammatory context. Our study suggested a positive correlation of Th22with β cell damage. Considering that IL-22R1is expressed locally in the human pancreatic islets. What’s more, another independent group has reported of increased Th22in human T1D which is characterized by targeted immunologic destruction and subsequently failure of β cell. Consistent with our data, this clue raised questions that whether the increased Th22is causative/hazardous or compensatory/protective factor in β cell failure. Based on our clinical findings, the present study would further explore the exact effects of IL-22(with or with TNF-a) on β cell damage and proliferation as well as its underlying mechanisms.Objective:1To explore the alternations in frequencies of peripheral Th1/Th2, Th17/Treg, Th22cells, plasma cytokine profile and expression of specific transcription factors in MHO and T2D group; to investigate relevancies between each Th subsets and clinical parameters in patients;2To further compare the function status of Treg and Tresp among CTL, MHO and T2D group.3To comprehensively investigate the independent contributions of specific Th subsets to the prevalence of CHD in a diabetic population in this case-controlled study; to evaluate the potential predictive values of specific Th subsets in the incidence of CHD in T2D.4To further explore the effects of IL-22(with or with TNF-a) on β cell damage and proliferation as well as its molecular mechanisms.Materials and Methods:1Subjects:3groups includes:A. T2D group (clinical definite history of CHD or none-CHD); B. Metabolically healthy obesity,(BMI^30kg/m2)(MHO); C. heath control groups. Exclusion criteria included unclear diagnosis of CHD or any clues of autoimmune disorders, recent infections, tumors, fever of any causes and significantly elevated erythrocyte sedimentation rate, etc. All donors did not receive immunosuppressive or immunomodulatory drugs for at least3months when sampling. Our research was carried out in accordance with the Declaration of Helsinki (2008) of the World Medical Association. The study has been approved by the Medical Ethical Committee of Qilu Hospital, Shandong University. Each patient signed a consent document. This study was conducted in a blinded manner.2Parameters involved:A. Detailed clinical record was kept for each subject including history of disease, physical and laboratory examinations (including fasting serum levels of CRP, insulin and glucose).B. Peripheral frequencies of total CD4+T helper cells, pro-inflammatory Thl, Th17, Th22and anti-inflammatory Th2, Treg subsets were determined by flowcytometry.C. Fresh plasma levels of cytokines were quantified by ELISA or Flowcytomix.D. mRNA levels of pecific transcription factors in PBMCs were quantified by realtime RT-PCR.E. Tregs and Tresps were obtained by MACS sorting of PBMCs, and their viability and proliferating capacity were analysed by FCM; the cytotoxic function of Tresps and inhibitory function of Tregs were determined by Mix lymphocyte reactions in vitro.3Significance of differences was determined by ANOVA or Student’s t tests unless the data were apparently skewedly-distributed. The Pearson or Spearman correlation and Partial correlation test was used for correlation analysis depending on data distribution. Logistic regressions and Caterogical analysis (Chi-square test or Fisher’s exact test) were conducted to verify the independent contribution of specific Th subsets to disease risks.4In vitro experiment on INS-1:Cells were stimulated with single or combined administration of TNF-a, IL-22. A combined protocol of LDH and CKK-8assay were followed to detect the cytotoxicity and proliferation; activation of STAT-3pathway (pSTAT-3/STAT-3) were determined by western blot; siRNA interfere technique was utilized to verified the role of IL-22R-STAT-3pathway in inflammatory damage caused to INS-1.Results:1Increased circulating Th22cells are in concert with Thl/Thl7cells frequencies in both MHO and T2DM patients:A. Compared with CTLs(1.18±0.06%, n=28), the peripheral Th22cells frequency was significantly increased in both MHO(1.88±0.10%, n=30)(*P <0.0001) and T2D (2.247±0.10%, n=89)(*P<0.0001) patients. And even higher percentages of Th22cells were observed in T2D compared to MHO patients(*P<0.05)B. significant increase of Thl frequency in T2D patients (median,9.06%; range,6.01-14.02%; n=89) was shown compared with MHO patients(median,9.06%; range,6.01-14.02%; n=30)(*P<0.0001) and CTLs (median,9.06%; range,6.01-14.02%; n=28)(Fig.2B). While no significant difference was found in Thl cells frequency between MHO patients and CTLs.C. Th17cells expanded in peripheral blood of MHO(median,9.06%; range,6.01-14.02%; n=30) and T2D patients(median,9.06%; range,6.01-14.02%; n=89) compared with CTLs(median,9.06%; range,6.01-14.02%)(*P <0.0001). But our present research failed to show significant difference in Thl7frequency between T2D and MHO patients.D. Correlation between the percentages of Th22and Thl as well as Th17cells from all blood donors. Positive correlation was revealed among all the three subsets of helper T cells.E. In the sub-cohort of metabolically health subjects(n=58),the odds ratio was54.47(*P<0.0001). And in obese people who had an elevated Th22, diabetes might occur8.636times as often as in those with normal Th22frequency (*P=0.0148).2Elevated plasma levels of inflammatory cytokines in non-stimulated peripheral blood from MHO and T2D patients:A. there is a notable increase in plasma IL-22from T2D patients (median,47.56pg/ml; range,30.55-76.89pg/ml) compared with both MHO patients (median,36.65pg/ml; range,29.52-55.70pg/ml)(*P<0.0001) and CTLs (median,36.33pg/ml; range,31.93-40.62pg/ml, n=16)(*P<0.0001). No significant difference was observed between MHO and healthy controls.B. Significant higher IFN-γ were also observed in MHO(median,1.76pg/ml; range,0.33-4.10pg/ml)(*P<0.0001) and T2D(median,1.08pg/ml; range,0.36-3.43pg/ml)(*P<0.05) patients compared to CTLs(median,0.18pg/ml; range, lower limit-2.64pg/ml). No significant difference was found between MHO and T2D patients (*P>0.05).C. Significant higher IL-17were found in T2D patients(median,3.84pg/ml; range,0.81-7.78pg/ml) compared with both CTLs(median,2.06pg/ml; range,0.81-3.04pg/ml)(*P<0.0001) and MHO patients(median,1.91pg/ml; range,0.89-5.11pg/ml)(*P<0.0001).3Elevated plasma levels of IL-6and TNF-αand increased expressions of AhR gene in MHO and T2DM patients promoted Th22polarization.4Elevated Th22cells frequency is bound up with insulin resistance in both MHO and newly diagnosed T2D patients and the hyperactive Th22cells phenotype also negatively correlates with residual islets β-cell function in later stage of T2D5Tresp from MHO and T2D showed enhanced proliferating and cytotoxic capacity. However, the Tregs from patients had weaker inhibitory potentiation in vivo.6Increased peripheral pro-inflammatory Th subsets contribute to cardiovascular complications in T2D:A. Both peripheral frequencies and total numbers of Th1, Th17and Th22cells were further increased in diabetic patients with CHD(*P<0.05) and elevated Th subsets also correlated with increased CRP levels(with coefficency of0.249,0.257and0.303,0.454respectively;*P<0.05).B. After adjusted by age, sex and disease duration of diabetes, frequencies of Th1, Th17and Th22stil contributed to the increased prevalence of CHD. Among them the recently-identified Th22cells exerted the most remarkable impacts. The odd ratio of Th22frequencies (per1%) was as high as41364.708.C. Plasma levels of CRP, LDL-C, Ln(HOMA-IR) and Th22number were successively introduced into the stepwise regression model. Th22cells were proved to be the only independent participant in development of CHD (OR:1.102; CI:1.016-1.195; P=0.019) among the pro-inflammatory Th subsets involved.D. Patients with even moderately elevated (higher than the median) Thl frequency or Th22number already had a significantly increased risk (4.451and67.002folds respectively) of CHD. In further analysis in2x2tables, the frequency of Th1above the median20.145%represented an about5-fold (CI:2.158-11.82) increased risk of CHD, with a Positive Predictive Value (PPV) of0.7963(CI:0.6647-0.8937) and a Negative Predictive Value (NPV) of0.5636(CI:0.4232-0.6970).7The dual role of IL-22:A. Stimulation with IL-22alone promoted INS-1proliferation and cause damages.B. TNF-α alone cause damages to INS-1in a dose-dependent manner.C. Combined stimulation of IL-22and TNF-α with even half dose could cause apparent damage to INS-1 D. Knockdown of either IL-22R1or STAT-3with siRNA could both block effects of IL-22.Conclusions:1. Increased pro-inflammatory Th subsets occurred even in MHO group. Elevated Th22and IL-22also help distinguishes MHO from T2D patients. The notable correlation implicate that Th22may play a more determinant role in both insulin resistance and β-cells impairment. And Elevated plasma levels of IL-6and TNF-a and increased expressions of AhR gene in MHO and T2DM patients promoted Th22polarization.2. The immuno-suppressive function of Tregs was impaired in both obesity and T2D while the proliferation and function of Tresps were hyperactivated.3. Increased peripheral pro-inflammatory T helper subsets act in concert and contribute to the increased prevalence of diabetic cardiovasculopathy probably through promoting inflammation. The recently-identified Th22cells might play an independent role in CHD and represent a novel proxy for cardiovascular risks in diabetes.4. Th22may exert its effect in β cell damage with both IL-22and TNF-a. Cytotoxic effect of IL-22becomes dominant when acts in concert with TNF-a. And its cytotoxic effect is dependent of IL-22R1-STAT-3pathway.