type 2 diabetes mellitus ( t2 dm ) is one of the most common noncommunicable diseases characterized by insulin resistance and impaired insulin secretion [ 1 , 2 ] .
metabolic proinflammatory disorder including chronic hyperglycemia and increased levels of circulating cytokines suggests immunological disturbances [ 37 ] , which seriously affects the quality of life of the patients and imposes a large economic burden on the national health care system .
genetic and environmental factors are blamed for t2 dm and up to 25% of first - degree relatives of t2 dm patients may develop this disease .
the origin and development of t2 dm were involved in multiple risk factors . regulatory t cells ( treg ) and cytokines play important roles in the development of t2 dm .
treg is a subset of cd4 t cells that maintain peripheral tolerance and suppress antigen specific immune responses by secreting transforming growth factor- ( tgf- ) , interleukin-10 ( il-10 ) , and il-4 to inhibit autoimmunity .
it was found that the ratios of cd4cd25treg / th17 cells and cd4cd25treg / th1 cells were significantly decreased in t2 dm patients .
expression of foxp3 , a key player for the development and function of treg , correlates well with regulatory activity and number of treg .
indeed , foxp3 is exclusively expressed in cd4cd25treg [ 1316 ] . a positive correlation between cd4cd25foxp3treg and the enhanced expression of il-6 on cd4 t cells
il-10 , as a multifunctional cytokine and secretion of treg , plays a key role in the inflammatory response that is associated with insulin resistant states and t2 dm . increased levels of il-17
were found to protect against autoimmune mediated t1 dm in nonobese diabetic mice . on the other hand ,
loss of il-17 has been associated with disease susceptibility in part because it has been suggested that the absence of il-17 results in enhanced production of other proinflammatory cytokines .
tgf- is also a multifunctional cytokine circulating as a biologically inactive form in human plasma [ 21 , 22 ] .
the tgf- family includes multifunctional molecules that exert specific effects on cell proliferation , differentiation , migration , development , tissue remodeling , and repair .
tnf- inhibits the insulin signaling cascade through regulating several pivotal regulatory proteins , such as the insulin receptor substrate ( irs ) and akt substrate 160 in human skeletal muscle in vitro   and in vivo .
it has reported that polymorphism of immune genes such as tnf-   and tgf-   was associated with the development of t2 dm . intriguingly , increased renal production of tgf- was a distinct feature of diabetes [ 2831 ] .
within the past few years , many clinical studies have been focusing on the association of treg with proinflammatory and immunosuppressive cytokines in t2 dm . despite intensive research efforts ,
therefore , we performed this meta - analysis synthesizing the data from case - control studies to evaluate changes of treg , il-6 , il-10 , il-17 , tgf- , and tnf- in t2 dm patients .
our study was conducted according to the preferred reporting items for systematic reviews and meta - analyses ( prisma ) criteria .
we identified relevant studies of treg , il-6 , il-10 , il-17 , tgf- , and tnf- in t2 dm patients by systematically searching pubmed , wanfang database , chinese - cqvip , and cnki databases from february 1 , 1991 , to july 15 , 2016 .
il-17  ) or (  transforming growth factor beta  or  tgf-  ) or (  tumor necrosis factor alpha  or 
tnf-  ) or (  regulatory t cells  or  treg  or  cd4cd25 t cell  or  cd4cd25foxp3 t cell  ) and (  type 2 diabetes mellitus  or  type 2 diabetes  or  diabetes mellitus  or 
in addition , we also conducted an extensive literature search and articles were further identified in reference lists .
we reviewed all relevant articles using the following inclusion criteria : ( 1 ) the study should evaluate the relationship of cd4cd25foxp3treg , cd4cd25treg , il-6 , il-10 , il-17 , tgf- , or tnf- with t2 dm patients ; ( 2 ) the design had to be a case - control study ; ( 3 ) original data were displayed or could be converted to as mean  sd ; and ( 4 ) original report showed no duplicated data .
the data were extracted independently by two reviewers ( yong - chao qiao and jian shen ) by using predefined data extraction forms and the quality of all eligible studies was evaluated according to the newcastle - ottawa scale ( nos ) .
the following information was extracted : ( 1 ) name of the first author ; ( 2 ) date of publication ; ( 3 ) country of the study ; ( 4 ) study design ; ( 5 ) sample size of patients and controls ; ( 6 ) mean age of the sample ; and ( 7 ) mean  sd of patients and controls . in case of disagreement , a third investigator ( hai - lu zhao )
we presented the data ( sample size , mean  sd ) to illustrate the changes of treg , il-6 , il-10 , il-17 , tgf- , and tnf- in t2 dm patients versus healthy controls , and chi - squared q test and i statistics were used to assess heterogeneity .
when p < 0.1 or i > 50% , the heterogeneity was considered significant and a random effect model was used ; otherwise , a fixed - effect model was used .
considering the influence of diabetic complications , patients were divided into two groups ( t2 dm with complication and t2 dm without complication ) for subgroup analysis .
we performed sensitivity analysis by limiting the studies of nos score  7 or excluding studies with a high risk of bias .
publication bias was examined graphically by constructing egger 's test and p < 0.05 was considered to be representative of statistically significant publication bias .
the flow chart of the article search and inclusion process was displayed in figure 1 . based on the search strategy ,
a total of 5,064 articles were collected and 332 were removed after our initial screening . furthermore , 3,954 articles were excluded because they were not dm relevant , have no controls , or were animal studies or review articles .
then , we excluded 687 studies because of duplicated data , no original data , or original data expressed with figures . eventually , this meta - analysis included 91 articles involving 138 case - control studies of 5642 t2 dm patients and 7378 healthy controls : 13 for il-6 [ 3446 ] , 22 for tgf- [ 23 , 4767 ] , 7 for tnf- [ 3436 , 38 , 45 , 68 , 69 ] , 6 for cd4cd25foxp3treg [ 7075 ] , 15 for il-10 [ 7690 ] , 18 for cd4cd25treg [ 70 , 72 , 74 , 75 , 91104 ] , and 10 for il-17 [ 105114 ] .
t2 dm patients had significantly increased levels of serum il-6 ( smd , 1.28 ; 95% ci , 0.73 to 1.83 ; p < 0.001 ) ( figure 2 ) , tgf- ( smd , 2.88 ; 95% ci , 2.37 to 3.40 ; p < 0.001 ) ( figure 3 ) , and tnf- ( smd , 1.56 ; 95% ci , 1.10 to 2.02 ; p < 0.001 ) ( figure 4 ) but significantly decreased the percentage of cd4cd25foxp3treg ( smd , 0.47 ; 95% ci , 0.72 to 0.23 ; p < 0.001 ) ( figure 5 ) and the level of serum il-10 ( smd , 1.37 ; 95% ci , 2.32 to 0.42 ; p = 0.005 ) ( figure 6 ) .
changes in the percentage of cd4cd25treg ( smd , 0.24 ; 95% ci , 0.76 to 0.28 ; p = 0.360 ) ( figure 7 ) and il-17 ( smd , 0.51 ; 95% ci , 1.87 to 0.84 ; p = 0.459 ) ( figure 8) were not significant . some but not all the results of the meta - analysis displayed significant heterogeneity .
subgroup analysis was performed to explore the impact of diabetic complication on the changes in treg and cytokines . as shown in figures 28
, both t2 dm patients with complication and the patients without complication had significantly increased levels of serum il-6 ( figure 2 ) , tgf- ( figure 3 ) , and tnf- ( figure 4 ) , while not significant changes were found in the percentage of peripheral cd4cd25treg ( figure 7 ) and il-17 ( figure 8) .
intriguingly , t2 dm patients with complication showed lower percentage of peripheral cd4cd25foxp3treg ( p < 0.001 ) ( figure 5 ) , whereas patients without complication had decreased levels of serum il-10 ( p = 0.033 ) ( figure 6 ) .
the high heterogeneity existed in some subgroup analysis . in order to explore the source of heterogeneity , we further conducted regression analysis according to the complication as covariate .
the results were as follows : tgf- ( t = 4.08 ; p < 0.001 ; 95% ci , 1.23 to 3.65 ) , il-6 ( t = 0.09 ; p = 0.929 ; 95% ci , 1.09 to 1.18 ) , tnf- ( t = 0.34 ; p = 0.740 ; 95% ci , 1.23 to 1.67 ) , cd4cd25foxp3treg ( t = 2.04 ; p = 0.097 ; 95% ci , 1.55 to 0.18 ) , il-10 ( t = 0.36 ; p = 0.723 ; 95% ci , 5.33 to 3.77 ) , cd4cd25treg ( t = 0.63 ; p = 0.534 ; 95% ci , 0.96 to 1.81 ) , and il-17 ( t = 0.56 ; p = 0.586 ; 95% ci , 4.84 to 2.86 ) .
therefore , diabetic complication was a key influencing factor for the high heterogeneity in the meta - analysis of tgf- but not the others .
sensitivity analysis was used to assess the stability of the results by excluding studies with high risk of bias and no significant changes in the results were found .
we further conducted sensitivity analysis by including studies with high nos score ( 7 ) and found that all the results remained consistent .
egger 's test showed significant publication bias in the meta - analysis of tnf- but not the others ( figure 9 ) .
in this study , we found that the patients with t2 dm had increased serum levels of il-6 , tgf- , and tnf- but decreased percentage of peripheral cd4cd25foxp3treg and serum il-10 level .
furthermore , the percentage of peripheral cd4cd25foxp3treg and serum il-10 level were influenced by diabetic complication .
the expression of inflammatory and proinflammatory cytokines from peripheral blood t lymphocyte plays an important role in the development of diabetes and diabetic complications .
many studies have proved the maintenance of immunological self - tolerance by cd4cd25treg and cd4cd25foxp3treg .
the finding of decreased percentage of peripheral cd4cd25foxp3treg in t2 dm patients indicates that foxp3 might be a key player for the development and function of treg .
some researchers also considered that the differentiation and function maintenance of treg were dependent on the expression of the foxp3 , and , consequently , foxp3 is considered as the key transcriptional factor in treg cells [ 117119 ] .
il-10 and tgf- secreted by treg [ 116 , 120 ] are the biomarkers in t2 dm patients [ 2 , 116 ] .
previous studies suggested that il-10 could suppress the proliferation of t leukomonocyte and the secretion of cytokines , whereas tgf- may sustain the expression of foxp3 in cd4cd25treg to enhance immunosuppressive function [ 122 , 123 ] .
consistent with our findings , several studies have shown a significantly decreased level of serum il-10 in t2 dm patients [ 88 , 124 ] .
a recent investigation has showed that il-6 could enhance treg in mice . in the present meta - analysis of t2 dm patients , increased levels of serum il-6 , tgf- , and tnf- coexisted with decreased levels of il-10 and decreased percentage of cd4cd25foxp3treg .
this finding highlights that the cytokines and growth factors may originate from multiple sources such as macrophages , t cells , and other tissue cells rather than treg alone .
furthermore , chronic persistent activation of innate immunity and il-6 secretion occurring in t2 dm might inhibit the development of inducible treg cells .
th17 cells could produce il-17 , tnf- , and il-6 and induce inflammation in the pathogenesis of autoimmune diseases .
th17 cells are a major t cell subset implicated in the pathogenesis of multiple sclerosis , rheumatoid arthritis , and psoriasis .
a previous study has revealed that not only th1/th2 imbalance but also th17/treg imbalance can contribute to the pathogenesis of autoimmune diseases such as t1 dm as well as proinflammatory disorders and such as t2 dm . t2
dm patients have elevated serum levels of il-6 , il-1 , and tgf- , the cytokines known to induce th17 differentiation .
enhanced production of il-6 and tnf- and decreased levels of serum il-10 that occurred in t2 dm patients may suppress treg cells and ratios of treg to th17 and th1 cells [ 132 , 133 ] .
the immunocompromised effects on macrophages and lymphocytes likely drive an inflammatory state to contribute to the occurrence of diabetic complications . here
, in this study , no significant changes of foxp3treg cells and serum il-17 levels were found in t2 dm subjects without complication .
there is an intimate relationship of the differentiation of th17 cells with the relative abundance of peripheral cd4cd25foxp3treg cells and the serum levels of il-6 , il-10 , and tgf-. although changes of serum levels of il-17 were not significant in this meta - analysis of t2 dm patients versus controls , il-17 may be a clue to the possible involvement of th17 cells in t2 dm pathogenesis .
firstly , a decrease of treg cells might be accompanied by an increase of th17 cells . the study by guan et al .
has indicated the existence of a developmental switch between th1/th17 cells , on one hand , and th2/treg cells , on the other hand .
secondly , in the presence of high serum levels of il-6 and tgf- , as we reported here , differentiation of th17 cells might be favoured .
lastly , th17 cells might be , together with innate cells , a primary source of the increased il-6 levels and might be actively orchestrating the immunity - driven , chronic inflammation of target tissues and organs in t2 dm . in this systematic review ,
the studies examining the number of th17 cells in t2 dm were too scarce for being included in the meta - analysis .
future studies are required to focus on the role of th17/treg and products of the th17 cells in the pathogenesis of t2 dm and associated complications .
diabetic complications such as retinopathy , nephropathy , and cardiovascular disease affect immune cells and cytokines in type 2 diabetes [ 135 , 136 ] .
actually , urinary tgf- levels are elevated in the presence of microalbuminuria and overt proteinuria .
additionally , elevated plasma tgf- may reflect the state of hyperglycemia in t2 dm patients .
systemic inflammation in t2 dm is linked to the development of diabetic complications [ 138 , 139 ] . yet
, the mechanism of immune alteration in t2 dm and diabetic complication remains unclear . in this meta - analysis , diabetic complication
indeed has an impact on the percentage of peripheral cd4cd25foxp3treg and level of serum il-10 .
the percentage of treg cells and levels of cytokines in t2 dm may also depend on ethnicity , sex , weight , age , and disease duration .
the results of egger 's tests explain that no publication bias existed in all comparisons except for tnf-. the publication bias in this meta - analysis might be attributed to studies of small samples and positive results published more easily than negative reports .
firstly , we have selected random effect model to synthesize smd because of the high heterogeneity existing in some comparisons , but this selection may affect the accuracy of outcome .
secondly , we could not conduct further subgroup analysis of gender , weight , and disease duration because most of the included studies lack sufficient original data .
thirdly , articles published in chinese or english are included , while unpublished data and papers published in other languages are unknown .
in summary , t2 dm patients and the patients with diabetic complication have decreased immunosuppressive cd4cd25foxp3treg cells and increased proinflammatory il-10 , tgf- , and tnf-. the presence of diabetic complication has an impact on the compromised immunosuppression .
 
 objective . 
 the aim of this study was to investigate the changes of regulatory t cells ( treg ) , interleukin-6 ( il-6 ) , il-10 , transforming growth factor- ( tgf- ) , and tumor necrosis factor - alpha ( tnf- ) in patients with type 2 diabetes mellitus ( t2 dm ) . 
 methods . 
 we performed a comprehensive search up to july 2016 for all clinical studies about the changes of treg , il-6 , il-10 , il-17 , tgf- , and tnf- in t2 dm patients versus healthy controls . 
 results . 
 a total of 91 articles ( 5642 cases and 7378 controls ) were included for this meta - analysis . compared with the controls ( all p < 0.001 ) 
 , the patients had increased serum levels of il-6 , tgf- , and tnf- but decreased the percentage of peripheral cd4+cd25+foxp3+treg and serum il-10 level . 
 furthermore , the percentage of peripheral cd4+cd25+foxp3+treg ( p < 0.001 ) and serum il-10 level ( p = 0.033 ) were significantly lower in the patients with complication and in the patients without complication , respectively . 
 no significant changes about the percentage of cd4+cd25+treg ( p = 0.360 ) and serum il-17 level ( p = 0.459 ) were found in t2 dm patients . 
 conclusions . 
 t2 dm patients have decreased the percentage of peripheral cd4+cd25+foxp3+treg and levels of serum il-10 but elevated serum levels of il-6 , tgf- , and tnf-. presence of diabetic complications further lowers the peripheral cd4+cd25+foxp3+treg number . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions