Expression of Toll-Like receptors in metabolic syndrome: A systematic review

Introduction: Toll-Like Receptors (TLRs) of innate immune system have documented roles in the pathogenesis of metabolic disorders. This study aims to systematically review the expression of TLRs on metabolic syndrome (MetS). Materials and methods: We systematically searched PubMed/Medline, ISI web of Science, Scopus, Google Scholar, EMBASE, and OVID databases until February 2017. The terms ‘‘Metabolic Syndrome’’ OR ‘‘Mets’’ AND ‘‘Toll like receptor’’ OR ‘‘Toll like’’ OR ‘‘TLRs’’ OR ‘‘TLR’’ were used. “Expression” advertently was not used in our search and was considered in the selection process. Three steps for selecting the articles and then their qualification were conducted. Results: First, 1373 articles were found in the international databases. After removing duplicates, 963 papers remained and after two steps of selection, this number reached 410 and then 27, respectively. After full text screening and qualifying processes, we finally included 13 articles consisting of five animal and eight human studies. All human studies reported overexpression of TLRs (types 2, 4, 5, 9) in MetS, and most animal studies documented an increased TLRs expression. Conclusion: This systematic review provides evidence for the relation of innate immune system with MetS. Its findings regarding overexpression of special TLRs (e.g. types 2, 4, 5, 9) in MetS and their basic mechanisms and clinical implications might be investigated in further studies.


Introduction
Metabolic syndrome (MetS) is defined as coincidence of metabolic disorders including dyslipidemia, abdominal obesity, hypertension and insulin resistance/glucose intolerance (1). It has emerged as a worldwide epidemic and major public health care concern with an increasing prevalence rate (2). In addition to its high prevalence rate, the importance of MetS is because of its association with worldwide epidemics of diabetes, cardiovascular diseases and nonalcoholic fatty liver disease (NAFLD) (3, 4). Many studies have proposed that conditions as MetS, atherosclerotic cardiovascular disease, insulin resistance, and obesity are associated with the activation of innate immune system. Latest evidence suggests that much of this association can be traced to a unique family of pattern recognition receptors known as Toll-Like receptors (TLRs) (5-8).
TLRs are trans-membrane receptors that are widely expressed in immune, epithelial and endothelial cells. Most of these receptors are on the cell surface, except for TLR 3, -7, -8, and -9, which mainly are in the endosomes and lysosomes (9). Briefly, TLR activation promotes inflammatory signaling cascade, phagocytosis, oxidative burst and eventually insulin resistance (10,11). Important agonists that trigger TLR activity are saturated fatty acids, endotoxins, oxidized LDL, and damageassociated molecular patterns (12)(13)(14). It is suggested that targeting the TLRs pathway may be an effective method to help treating these disorders (15). Large body of evidence exists on the role of TLRs in each of the aforementioned disorders (16,17), but limited experience is available on pooled information about MetS. This systematic review aims to provide the most recent data about the TLR types expressed in the context of MetS.

Materials and methods
Outcome/ Measures definition: The primary outcome in the current review was the expression (qualitative and quantitative) of TLRs in every cell/tissue and at every level .

Search strategy:
We searched international databases until February 2017. We considered PubMed/Medline, ISI web of Science, Scopus, Google Scholar, EMBASE and OVID without limiting for age range, time, and language.
The following terms were used: ''Metabolic Syndrome'' OR ''Mets'' AND ''Toll like receptor'' OR ''Toll like'' OR ''TLRs'' OR ''TLR''. We did not include "expression" in the search terms, and we looked for it at screening stages. We used MeSH term for MetS in PubMed/Medline search, built search strategies and also looked at the reference list of retrieved articles. Search strategy in PubMed was as follows: (

Inclusion and exclusion criteria:
We included all experimental (in vivo, in vitro, interventional) and observational studies. We looked at baseline data of trials to find any expression data appropriate for extraction. We considered both human and animal studies. In the case of finding multiple publications from one study, we selected the more comprehensive one or considered them together. Selection process: At the first step, the retrieved titles were screened to find relevant articles. Next, the abstracts were screened, and finally the full texts of the recovered papers were screened. Quality assessment: Our study eligibility criteria, design of studies, sample sizes, measurement methods and estimates were considered to qualify the papers. Two independent reviewers (ZF and MM) qualified the articles and the poor-rated ones were excluded. Data extraction: The data related to authors, publications features, study population, methods, values of selected measures and main conclusions were extracted.

Results
The search algorithm displaying the number of initial search results and included studies is shown in Figure 1. At the first step, 1373 articles were found in the international databases. After the duplicate removal and further two steps of screening processes, 963, 410 and 27 articles were remained respectively. As presented in Table 1, after full text screening and qualifying processes, 13 articles were eligible to be included in final review. (6, [18][19][20][21][22][23][24][25][26][27][28][29] Altogether, these thirteen studies consisted of eight human studies (18)(19)(20)22, 25-28) with a total population of 732 patients (at least 70% females, with total age range of 15 years and above), as well as five animal studies (mice/murine models) (6, 21, 23, 24, 29). As presented in Table 2, all human studies displayed increased expression of one or more type of TLRs in the monocytes of MetS patients. The most prevalent types of the receptor with increased expression in humans were TLR 2, 4 and 9. Three studies on animal models of MetS showed a controlling/regulating role for TLRs especially TLR 9 (21) and TLR 5 (24, 29). Another two studies showed overexpression of TLR 2 (6) and increase in a related protein named Janus Kinase 3(JAK3) in signaling cascade of TLRs (23).

Discussion
Based on the results analyzed, our systematic review revealed uniformly increased expression of TLRs in the context of MetS both in humans and animal models. The main TLRs involved were the types 2, 4, 5 and 9 . Several studies have shown that TLRs are possible links between obesity /visceral fat accumulation and inflammation and their consequent effects on body tissues (30,31). Konner and colleagues reported that upon development of obesity, numerous molecular conditions might promote activation of stress kinases, causing peripheral insulin as well as central insulin and leptin resistance (10). Jang and colleagues in their study concluded that TLR-2 in vascular endothelium mediates some pro-inflammatory and "unfolding protein response",which results in impairment of vaso-dilatory effect of insulin and ends in endothelial dysfunction (32). Thompson et al. suggested that continued activation of TLR might result in vascular oxidative stress and thereby would aggravate the process of hypertension and heart failure (33). These links would ultimately result in cardio-metabolic disorders (8) which are common consequences of MetS. Our findings propose some molecular mediators in their pathways.
McMillan et al found that in the mice with over-expression of muscular TLR-4, highfat diet significantly decreases fatty acid oxidation in muscles in accordance with increased body weight and fat, glucose intolerance, and cellular oxidative damage (34). Frisard et al showed that activation of TLR-4 by lipo-polysaccharides would result in increased glucose consumption and reduced fatty acid oxidation in skeletal muscles (35). These findings suggest that TLR-4 plays an important role in the metabolic reactions in skeletal muscle. This might explain MetS related metabolic disturbances in muscles. Some lipopolysaccharide endotoxins, originating from gut microbiome, are secreted into blood, and by activating TLRs, lead to a chronic metabolic inflammation status called meta-inflammation (36)(37)(38). This is a core pathogenic process in the development of MetS. Our study refers to some TLRs involved in this situation. As is evident from the articles systematically reviewed in this study, many aspects of the relation between TLRs and metabolic/endocrine disorders are well documented. Himes et al showed that mice lacking TLR-2 are substantially protected from diet-induced adiposity, insulin resistance, hypercholesterolemia, and hepatic steatosis (6). Cuevas et al speculated that there is a positive feedback between the expression of TLR4, myeloid differentiation primary response gene 88(MYD88) and plasminogen activator inhibitor-1(PAI-1) in the adipose tissue of obese individuals that develop metabolic complications (18).  Monocytes and adipose tissue TLR4  Zwolak A, 2016 19 Jialal I, 2014 25 Cuevas AM, 2017 18 Endosomal, monocytes TLR9  with no changes in TLR3 Devaraj S, 2015 20 Adipose tissue TLR9 for regulating role Hong CP, 2015 21

Animal
Liver and Primary epididymal adipose tissue  (20). Hong et al. found a dramatic increase of macrophages as well as T helper cells in the adipose tissue of TLR-9-deficient mice compared to wild-type mice. They showed that TLR-9 signaling is involved in regulating the inflammation of adipose tissue and controlling obesity and the MetS (21). Jialaland colleagues showed a significant increase in Adipose Tissue TLR2 and TLR4 protein in MetS compared to controls (22). Mishraet al study demonstrates the essential role of JAK3 in promoting mucosal tolerance through suppressed expression and limiting activation of TLRs thereby preventing intestinal chronic low grade inflammation and associated obesity and MetS (23). Chassaingand coworkers showedTLR-5 null mice had Mets and were more prone to develop colitis compared to their sibling controls (24). Jialal et alstudy reflects increased monocyte TLR-4 protein and activity in nascent MetS (25). Orsatti and colleagues showed that TLR-2 and TLR-4 expressions were associated with increased pro-inflammatory cytokines, IL-6 and TNF-α, with no association with Several other disorders such as autoimmune thyroid and pancreatic diseases and septic dysregulation of the hypothalamic pituitary adrenal axis have been linked to TLR activation (39) and its gene polymorphism (40)(41)(42) as well. These receptors have also shown wide range relations with diabetes (43)(44)(45)(46) and nonalcoholic fatty liver disease (47)(48)(49)17). On the other hand, Zhang and colleagues reported that environmental circumstances may override the genetic aspects of TLRs and their interaction with gut microbime (50). Based on their suggestion, researchers should consider the environment-induced alterations of gut microbiota as a significant confounding factor in this relation.

Study limitations and strengths
This systematic review considered published data, and not the grey literature. A variety of tissues of interest and methods for observation existed in the included articles. There were no uniform quantitative data to conduct a metaanalysis. The strength of the study is its novelty as a systematic review in this field.

Conclusion
This systematic review provides thirteen citations which have worked on the expression of TLRs in MetS. From all the data stated, it is apparent that there is constant over-expression of TLR receptors 2, 4, 5 and 9 in the monocytes and some