Pathophysiology/Complications ORIGINAL ARTICLE

Increased Toll-Like (TLR) Activation and TLR Ligands in Recently Diagnosed Type 2 Diabetic Subjects

1 1 MOHAN R. DASU, PHD SAMUEL PARK, BS pathogenesis of , dia- 1 1,2 SRIDEVI DEVARAJ, PHD ISHWARLAL JIALAL, MD, PHD betes, and (5–7). Com- plimentary genetic studies link TLR2 and TLR4 polymorphisms to type 2 di- OBJECTIVE — Individuals with type 2 have a myriad of metabolic aberrations abetes, suggesting a casual relationship including increased inflammation, increasing their cardiovascular risk. Toll-like receptors between TLR function and diabetes and (TLRs) and their ligands play a key role in insulin resistance and atherosclerosis. However, there its complications (8). is a paucity of data examining the expression and activity of TLRs in type 2 diabetes. Thus, in the TLRs are evolutionarily preserved present study, we examined TLR2 and TLR4 mRNA and expression, their ligands, and pattern-recognition receptors (9), ex- signaling in of recently diagnosed type 2 diabetic patients. pressed on several types including monocytes, predominant cells of the in- RESEARCH DESIGN AND METHODS — TLR mRNA, protein expression, TLR li- nate that are pivotal in gands, and TLR signaling were measured in freshly isolated monocytes from healthy human diabetes and atherogenesis (10). TLRs control subjects (n ϭ 23) and type 2 diabetic subjects (n ϭ 23) using real-time RT-PCR, Western blot, and flow cytometric assays. play an important role in the activation and regulation of the innate immune sys- tem and inflammation (9). Each TLR RESULTS — Type 2 diabetic subjects had significantly increased TLR2, TLR4 mRNA, and protein in monocytes compared with control subjects (P Ͻ 0.05). Increased TLR2 and TLR4 family member recognizes a specific expression correlated with BMI, homeostasis model assessment–insulin resistance (HOMA-IR), component, upon activation, glucose, A1C, Nε-(carboxymethyl) lysine (CML), and free (FFA). Ligands of TLR2 and triggers a signaling cascade leading to cy- TLR4, namely, HSP60, HSP70, HMGB1, endotoxin, and hyaluronan levels, were elevated in type tokine production and adaptive immune 2 diabetic subjects and positively correlated with TLR2 and TLR4. Type 2 diabetic subjects response (9). Among the TLRs, TLR2 and showed increased MyD88, phosphorylated IRAK-1, , TICAM-1, IRF-3, and NF-␬B p65 TLR4 play a critical role in the pathogen- expression in monocytes compared with control subjects. Furthermore, TLR-MyD88-NF-␬B esis of insulin resistance, diabetes, and signaling resulted in elevated levels of (P Ͻ 0.05), but increased (IL)-1␤, ␥ atherosclerosis in both clinical and exper- interferon (IFN)- , and endotoxin were not significant when adjusted for BMI. imental conditions (5–7,11). Ligands for TLR2 and TLR4 include high-mobility CONCLUSIONS — In this comprehensive study, we make the novel observation that TLR2 group B1 protein (HMGB1), heat shock and TLR4 expression and their ligands, signaling, and functional activation are increased in protein (HSP) 60, HSP70, endotoxin, recently diagnosed type 2 diabetes and contribute to the proinflammatory state. hyaluronan, advanced glycation end Diabetes Care 33:861–868, 2010 products, and extracellular matrix com- ponents (12). TLR2 and TLR4 bind to components of the gram-positive and ype 2 diabetes constitutes a group of associations between elevated levels of -negative , respectively (12). metabolic aberrations including hy- circulating C-reactive protein, proinflam- TLR2 has broad recognition spec- T perglycemia, inflammation, and in- matory cytokines, and homeostasis ificity because of its ability to form het- sulin resistance that increase the risk of model assessment–insulin resistance erodimers with TLR1, TLR6, and (1). The mecha- (HOMA-IR) suggest that inflammation is sometimes with CD14 and CD36 (13). nisms by which these metabolic abnor- an important etiological factor in the de- TLR4 does not recognize endotoxin with- malities cause cardiovascular disease are velopment of both insulin resistance and out the cofactor MD-2 (14). Collectively, not clear. Considerable evidence indi- type 2 diabetes (4). Moreover, inflamma- these ligands and cofactors markedly in- cates that the harmful effects of elevated tory markers link the pathology of insulin crease TLR functionality and have not glucose are mediated by receptors leading resistance and type 2 diabetes. Activation been studied in type 2 diabetes. to increased inflammation and oxidative of the via toll-like TLR2 and TLR4 expression have been stress (2,3). Recent studies demonstrating receptors (TLRs) is implicated in the shown to be increased in conventional in- ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● sulin resistance target tissues like skeletal From the 1Laboratory for Atherosclerosis and Metabolic Research, University of California Davis Medical muscle and adipose tissue of type 2 dia- Center, Sacramento, California; and the 2VA Medical Center of Northern California, Mather Field, betic subjects (15,16). These important California. Corresponding author: Mohan R. Dasu, [email protected]. studies implicating TLRs in type 2 diabe- Received 24 September 2009 and accepted 6 January 2010. Published ahead of print at http://care. tes were derived using small sample size, diabetesjournals.org on 12 January 2010. DOI: 10.2337/dc09-1799. and the association with respective TLR2/ © 2010 by the American Diabetes Association. Readers may use this article as long as the work is properly TLR4 ligands, cofactors, downstream sig- cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons. naling, and functional activation remains org/licenses/by-nc-nd/3.0/ for details. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby to be properly addressed. Moreover, sys- marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. temic inflammation plays a significant care.diabetesjournals.org DIABETES CARE, VOLUME 33, NUMBER 4, APRIL 2010 861 TLR2 and TLR4 expression and activation in type 2 diabetes role in the etiology and comorbidities of intensity exercisers were excluded. rum of all study subjects. Nuclear extracts type 2 diabetes, and the role of TLR2 Informed consent was obtained from all were used to verify activation of NF-␬B and TLR4 in this setting is not clear. the subjects, and the study was approved (Active Motif) in study subjects, indicat- Experimental evidence in mice demon- by the University of California Davis In- ing increased inflammation, as described strates that TLR2 and TLR4 activation stitutional Review Board. After history previously (3,11). Intra- and interassay and downstream production and physical examination, fasting blood CV for all the assays were determined to lead to the development of diabetes (30 ml) was obtained. All the type 2 dia- be between 7 and 12% (21). All the re- (17,18). More recently, TLR4 has been betic patients had family history of diabe- agents used in the study were tested for indicated as a molecular link between tes. Of the subjects, 60% were Caucasian endotoxin (Ͻ100 ELISA unit [EU]/ml) free fatty acids (FFAs), inflammation, and the remaining 40% were Hispanic using a Limulus ameobocyte lysate assay and the innate immune system (5). and African American. (Lonza), allowing precise TLR expression Also, Song et al. (19) reported increased Complete blood count, and li- measurements devoid of endotoxin TLR4 mRNA expression in differentiat- poprotein profile, glucose, A1C, C- (3,11). ing adipose tissue of db/db mice. peptide, and C-reactive protein in all the While these important observations enrolled subjects were assayed by stan- Real-time RT-PCR from animal and human tissue data sug- dard laboratory techniques (11). FFA lev- RNA was isolated from monocytes using gest a role for TLR2 and TLR4 in type 2 els were assayed using reagents from TRI reagent (Invitrogen). cDNA was syn- diabetes, it remains unknown whether Wako Chemicals (Richmond). Fasting thesized using 1 ␮g total RNA, and 50 ng alterations in TLR2 and TLR4 pathway plasma insulin concentrations were mea- was amplified using primer probe sets for activation contribute to systemic in- sured using an enzyme-linked immunosor- TLR1, TLR2, TLR4, TLR6, MD-2, and 18s flammation in diabetic human subjects. bent assay (ELISA) (Linco Research). (SA Bioscience) following manufacturer’s Therefore, the purpose of this study was Insulin sensitivity was estimated by cycling parameters. Data are calculated Ϫ⌬⌬ to undertake a comprehensive analysis HOMA-IR (20). using the 2 CT method (3,11). of TLR2 and TLR4 activation, ligands, cofactors, and downstream signaling in Human monocytes monocytes isolated from recently diag- Human monocytes were isolated by gra- Western blots and nosed patients with type 2 diabetes and dient density centrifugation of peripheral coimmunoprecipitation matched control subjects. blood using Histopaque (Sigma) and cell lysates were subjected to magnetic separation (Miltenyi Biotech) as electrophoresis and transfer, as reported RESEARCH DESIGN AND reported previously (3,11). More than earlier (3,11). Blots were probed with hu- METHODS — Type 2 diabetic pa- 88% of cells were identified as monocytes man myeloid differentiation factor-88 tients (n ϭ 23) (with mean duration of by CD14 staining, and viability was found (MyD88), IL receptor–associated protein diabetes ϭ 29 months; age Ͼ35 years) to be Ͼ92%. Monocytes were examined kinase-1 (IRAK-1), toll–IL-1 receptor do- were recruited from the Diabetes and Pe- before and after activation with synthetic main (TIR)-containing adaptor molecule (TICAM-2), IFN regulatory factor-3 (IRF- diatric Clinics at University of California Pam3Cys-Ser-(Lys) 4 (TLR2 ␬ ␬ Davis Medical Center. None of the pa- ligand; Pam3CSK4, 170 ng/ml; Invitro- 3), NF- B (NF- Bp65; Santa Cruz Bio- technology, Santa Cruz, CA), TIR- tients were on , ACE gen) and (LPS) (TLR4 ␤ inhibitors, angiotensin receptor blockers, ligand; 160 ng/ml; E. coli 026:B6; Sigma) containing adapter-inducing IFN- (Trif; insulin, or statins, since they interfere overnight (12 h), with suitable control Abcam), and with respective secondary IgG antibodies and developed. with TLR expression and activation; six subjects (3,11). After treatment, cells ␤ type 2 diabetic subjects were on met- were incubated with TLR2, TLR4, or iso- In all assays, -actin and total nonphos- ϭ phorylated were used as internal formin. Healthy control subjects (n type-matched IgG (eBioscience) depend- ␮ 23), age Ͻ35 years, with normal com- ing on the cell treatment; 10,000 events control. Besides, cell lysates (100 g) plete blood count; no family history of were analyzed with the Becton-Dickinson were immunoprecipitated with TLR2 or diabetes or other chronic diseases; normal Fluorescence-Activated Cell Sorter Bio- TLR4 overnight at 4°C as re- kidney, liver, and thyroid function; and analyzer as described previously (11) af- ported previously (3) and blotted with fasting plasma glucose Ͻ100 mg/dl were ter gating for CD14. Intra-assay and TLR6, HMGB1, HSP60 (Santa Cruz), included in the study. Healthy control interassay coefficient of variation (CV) for and hyaluronan (Ray Biotech) antibod- subjects and type 2 diabetic patients were TLR2 and TLR4 expression was Ͼ8 and ies as depicted in Fig. 2F and G. Densi- matched for age (within 5–10 years), sex, Ͼ12%, respectively (3,11). tometric ratios were determined as and race. Subjects with mean A1C over Serum and cell supernatants were reported earlier from four independent the last year Ͼ10%; inflammatory disor- used for measuring interleukin (IL)-1␤, assays (3,11). ders; microvascular and macrovascular IL-6, IL-8, interferon (IFN)-␥, monocyte complications; abnormal liver, renal, or chemoattractant protein (MCP)-1, and Statistical analysis thyroid function; steroid therapy, anti- (TNF)-␣ with a Statistical analyses were performed using inflammatory, antihypertensive, or hypo- Multiplex assay (Millipore) as a functional SAS software (SAS Institute, Cary, NC). lipidemic drugs; antioxidant supplements readout of TLR activation. IFN-␤ (Antige- Data are expressed as means Ϯ SD for all in the past 3 months; pregnancy; smoking; nix America), HSP60, HSP70 (Assay De- data. Parametric data were analyzed using abnormal complete blood count; alcohol signs), HMGB1 (Shino-Test, Japan), paired, two-tailed t tests and nonparamet- ε consumption Ͼ1 oz/day; consumption of hyaluronan (Echelon), and N -(carboxy- ric data using Wilcoxon’s signed- omega-3 polyunsaturated fatty acid cap- methyl) lysine (CML) (CycLex, Japan) tests. Level of significance was set at P Ͻ sules (Ͼ1 g/day); and chronic high- were determined using ELISA in the se- 0.05. Spearman’s rank correlation was

862 DIABETES CARE, VOLUME 33, NUMBER 4, APRIL 2010 care.diabetesjournals.org Dasu and Associates

Table 1—Clinical and laboratory characteristics TLR2 dimerizes with TLR1 or TLR6 and results in receptor activation and down- Control Type 2 diabetes P stream signaling upon saturated fatty acid challenge. To determine whether TLR1 or n 23 23 TLR6 is required for the activation of Sex (M/F) 11/12 9/16 TLR2, we measured TLR1 and TLR6 Age (years) 46 Ϯ 12 51 Ϯ 10 NS mRNA expression in monocytes of type 2 BMI (kg/m2) 25 Ϯ 734Ϯ 9 0.001 diabetes and control subjects. TLR6 Fasting glucose (mmol/l) 4.9 Ϯ 0.5 8.9 Ϯ 3.1 0.0001 mRNA levels significantly increased in Fasting insulin (pmol/l) 79 Ϯ 17 190 Ϯ 120 0.018 type 2 diabetes (3.8 Ϯ 0.4 vs. 1.3 Ϯ 0.2 HOMA-IR 1.5 Ϯ 0.4 3.7 Ϯ 1.8 0.001 mRNA/18s ratio; P Ͻ 0.05) compared Fasting FFAs (mEq/l) 0.3 Ϯ 0.2 0.9 Ϯ 0.2 0.0001 with control subjects, whereas TLR1 A1C (%) 5.2 Ϯ 0.2 8 Ϯ 2.1 0.05 mRNA levels remained unchanged (con- C-peptide (ng/ml) 1.2 Ϯ 1 2.5 Ϯ 0.6 0.001 trol: 1.5 Ϯ 0.4, type 2 diabetes: 1.9 Ϯ 0.5 C-reactive peptide (mg/l) 2.9 Ϯ 4 4.7 Ϯ 4 0.002 mRNA/18s ratio), and coimmunoprecipi- Total cholesterol (mg/dl) 184 Ϯ 36 190 Ϯ 36 NS tation showed an increased TLR2/TLR6 LDL cholesterol (mg/dl) 115 Ϯ 26 120 Ϯ 29 NS association in type 2 diabetes (Fig. 2F). HDL cholesterol (mg/dl) 50 Ϯ 11 39 Ϯ 15 0.3 Further, MD-2 mRNA expression was Triglycerides (mg/dl) 100 Ϯ 62 145 Ϯ 67 0.45 also significantly increased (control 1.2 Ϯ CML (ng/ml) 1.9 Ϯ 1.1 5.4 Ϯ 3.1 0.001 0.2 vs. 2.9 Ϯ 0.3 mRNA/18s ratio) in type Data are means Ϯ SD. P values correspond to the differences between control and type 2 diabetes. 2 diabetes monocytes. These data are in line with our earlier observation that under hyperglycemic conditions, TLR2 het- computed to assess association between RT-PCR. Figure 1B and E depict the cor- erodimerizes with TLR6 in monocytes (3). variables. responding increased TLR2 and TLR4 mRNA expression in type 2 diabetes com- TLR2 and TLR4 activation results in RESULTS pared with control subjects. We further MyD88-dependent and -independent confirmed the increased monocyte TLR2 signaling in type 2 diabetes Baseline characteristics of the study and TLR4 protein content using Western monocytes subjects blot assay (Fig. 1C and F). We measured We examined the TLR-mediated MyD88- Subject characteristics are detailed in downstream functional readouts (IL-1␤, dependent signaling pathway using Table 1. There were no significant dif- IL-6, IL-8, and TNF-␣) of TLR2/4 ligation Western blot technique. TLR2 and TLR4 ferences in age and lipid profile between in resting and activated monocytes (Fig. both use MyD88 and activate NF-␬B, control and type 2 diabetes groups. 1G). There was a significant upregulation of common downstream signaling compo- Levels of glucose, A1C, FFAs, and proinflammatory cytokines in type 2 dia- nents for all TLRs except TLR3 (3,9). There- HOMA-IR were significantly higher in betic patients compared with control sub- fore, activation of MyD88-dependent and type 2 diabetic subjects than in control jects in both the basal and activated state. -independent pathways was used to inter- subjects. C-peptide and advanced gly- pret the activation of TLR2 and TLR4. cation end product and CML levels were TLR2 and TLR4 ligand and cofactor Phosphorylation of IRAK-1, MyD88, significantly increased in type 2 diabetic expression in type 2 diabetes TRIF, TECAM-2, IRF-3 protein expres- subjects compared with control sub- Identification of TLR2 and TLR4 ligands sion in cytoplasmic extract, and p65 jects. C-reactive protein, a prototypic and cofactors is a key element in TLR ac- protein levels in nuclear extracts of atherosclerotic marker, is increased in tivation and is unclear in type 2 diabetic monocytes were significantly increased in type 2 diabetic subjects compared with subjects. Beyond the established micro- type 2 diabetes compared with control control subjects. bial ligands of TLR2 and TLR4, several subjects, with no change in total protein molecules of endogenous origin have and ␤-actin levels (Fig. 2H), suggesting TLR2 and TLR4 mRNA and protein been suggested to act as TLR2 and TLR4 activation of TLR-mediated signaling cas- expression in type 2 diabetes ligands, notably HMGB1, HSP60, HSP70, cade (both MyD88-dependent and -inde- monocytes and hyaluronan fragments. Accordingly, pendent) in type 2 diabetes. In addition, We first determined the levels of TLR2 we measured concentration of endotoxin, densitometry ratios further corroborate and TLR4 expression by flow cytometric HMGB1, hyaluronan, HSP60, and HSP70 the data (Fig. 2H). analysis in control and type 2 diabetic in the serum of type 2 diabetic subjects subjects. Monocyte surface expression of and control subjects using ELISAs. Figure Increased TLR2 and TLR4 TLR2 and TLR4 was significantly elevated 2 shows the significant increased levels of expression results in increased in type 2 diabetes compared with control these ligands in type 2 diabetic subjects. inflammation mediated by NF-␬B in both the resting and activated state In addition, coimmunoprecipitation with To further investigate TLR2- and TLR4- (Pam3CSK4 or LPS) (Fig. 1A and D, P Ͻ TLR2 and TLR4 and blotting for HMGB1, mediated inflammation in type 2 diabe- 0.005). Next, to determine if the increase HSP60, and hyaluronan showed strong tes, we measured NF-␬B activity in the in monocyte TLR2 and TLR4 expression association with both TLR2 and TLR4 in nuclear extracts of type 2 diabetes and in type 2 diabetes resulted from increased type 2 diabetes compared with control control subject monocytes. Type 2 diabe- mRNA expression, we investigated the subjects (Fig. 2F and G). tes showed increased NF-␬B p65- TLR2 and TLR4 mRNA levels by real-time It has been previously shown that dependent DNA binding activity care.diabetesjournals.org DIABETES CARE, VOLUME 33, NUMBER 4, APRIL 2010 863 TLR2 and TLR4 expression and activation in type 2 diabetes

Figure 1—TLR protein and mRNA content. A: TLR2 surface protein expression was measured in monocytes after Pam3CSK4 (Pam) challenge in control (n ϭ 23) and type 2 diabetic (n ϭ 23) subjects by flow cytometry as described in RESEARCH DESIGN AND METHODS. Values are expressed as mean fluorescence intensity units (MFI)/105 cells. *P Ͻ 0.005 vs. control; †P Ͻ 0.05 versus CϩPam. C, control; T2DM, type 2 diabetes. B: Monocyte TLR2 mRNA expression ratios in control (n ϭ 23) and type 2 diabetes (n ϭ 23) subjects by real-time RT-PCR as described in RESEARCH DESIGN AND METHODS. 18s mRNA is used as the housekeeping . Values are expressed as mean ratio Ϯ SD. *P Ͻ 0.001 vs. control. C: Representative Western blot depicting the TLR2 protein expression in pooled resting monocytes from three control subjects and three type 2 diabetic subjects. ␤-Actin was used as a loading control as described in RESEARCH DESIGN AND METHODS. Each assay is repeated four times. D: TLR4 surface protein expression was measured in monocytes after LPS challenge in control (n ϭ 23) and type 2 diabetic (n ϭ 23) subjects by flow cytometry as described in RESEARCH DESIGN AND 5 METHODS. Values are expressed as mean fluorescence intensity units (MFI)/10 cells. *P Ͻ 0.005 vs. control; †P Ͻ 0.05 vs. C ϩ LPS. E: Monocyte TLR4 mRNA expression ratios in control (n ϭ 23) and type 2 diabetic (n ϭ 23) subjects by real-time RT-PCR as described in RESEARCH DESIGN AND METHODS. 18s mRNA is used as the housekeeping gene. Values are expressed as mean ratio Ϯ SD. *P Ͻ 0.005 vs. control. F: Representative Western blot depicting the TLR4 protein expression in pooled resting monocytes from three control and three type 2 diabetic subjects. ␤-Actin was used as a loading control as described in RESEARCH DESIGN AND METHODS. Each assay is repeated four times. G: Release of cytokines in resting and activated monocyte cell culture supernatants from control and type 2 diabetic subjects using Multiplex assay as described in RESEARCH DESIGN AND METHODS. Values are expressed as pg/mg cell protein. *P Ͻ 0.001 vs. untreated C; **P Ͻ 0.005 vs. control (healthy control). C, untreated; LPS, lipopolysac- charide, TLR4 ligand; Pam, Pam3CSK4, synthetic TLR2 ligand. compared with control subjects (P Ͻ Increased TLR expression correlates HOMA-IR (TLR2: r ϭ 0.64; TLR4, r ϭ 0.01) (Fig. 2I). Increased NF-␬B activity with BMI, glucose, HOMA-IR, and 0.54, P Ͻ 0.005), and A1C (TLR2: r ϭ corresponded to increased systemic in- inflammation 0.66; TLR4, r ϭ 0.62, P Ͻ 0.001). In ad- flammation. We measured IL-1␤, IL-6, Additionally, there was a significant cor- dition, HSP60, HSP70, HMGB1, and en- IL-8, MCP-1, IFN-␤, and TNF-␣ serum relation between BMI and TLR2 expres- dotoxin levels significantly correlated concentration in type 2 diabetes and con- sion (r ϭ 0.5; P Ͻ 0.05) and TLR4 with TLR2 expression (r ϭ 0.43, r ϭ trol subjects as a functional readout of expression (r ϭ 0.43; P Ͻ 0.01) and sig- 0.64, r ϭ 0.48, and r ϭ 0.16; P Ͻ 0.05) TLR activation. There was a significant in- nificant correlation between TLR expres- and TLR4 expression (r ϭ 0.43, r ϭ 0.65, crease in all the proinflammatory media- sion and glucose (TLR2: r ϭ 0.7; TLR4, r ϭ 0.41, and r ϭ 0.21; P Ͻ 0.001), re- tors in type 2 diabetic subjects compared r ϭ 0.64, P Ͻ 0.001), FFA levels (TLR2: spectively. There was a significant corre- with control subjects (Fig. 2J and K). r ϭ 0.66; TLR4, r ϭ 0.66, P Ͻ 0.05), lation between TLR2 expression and IL-

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sion than healthy control subjects, with no mechanistic details. In this context, our study fills in all the above gaps se- quentially: First, we provide data on both TLR2 and TLR4 mRNA/protein levels and critical downstream signaling events that follow their ligation in type 2 diabetes; second, we determined cofactors re- quired for TLR2/4 activation; third, we demonstrated ligands in use for TLR2/4 activation; fourth, notably, we and others have shown that statins, thiazolidinedio- nes, and angiotensin receptor blockers downregulate TLRs and our type 2 dia- betic patients are not on any of these drugs; fifth, our TLR data showed signif- icant correlation with major clinical esti- mates of type 2 diabetes, namely adjusted BMI, glucose, HOMA-IR, FFAs, ligands, cytokines; and finally, data are shown for Figure 1—Continued. both MyD88-dependent and -indepen- dent signaling pathways, which may be Ϫ Ϫ 1␤, TNF-␣ (r ϭ 0.5, r ϭ 0.5; P Ͻ 0.001), LDLR / background and MyD88 acting collectively. MCP-1, and NF-␬B(r ϭ 0.42, r ϭ 0.35; knockout mice show reduced lesion size, In addition to the well-characterized P Ͻ 0.05) and between TLR4 and IL-1␤, lipid content, and infiltra- microbial ligands, several molecules of TNF-␣ (r ϭ 0. 56, r ϭ 0.55, P Ͻ 0.001), tion in plaques and reduced inflammation the host origin (endogenous) have been IFN-␤, and NF-␬B(r ϭ 0.46, r ϭ 0.4, P Ͻ (22,23). proposed to act as ligands for TLR2 and 0.05). Because BMI was significantly dif- In two recent studies, we showed in- TLR4, including HMGB1, HSPs, and hya- ferent between control and type 2 diabe- creased TLR2 and TLR4 expression, TLR luronan (25), and were not examined in tes, additional statistical analyses using ligands, intracellular signaling, and TLR- type 2 diabetes. HMGB1 is considered as a BMI as a covariant were performed. TLR2, mediated inflammation in monocytes “late” proinflammatory mediator in sep- TLR4 (with and without Pam3CSK4 or with significant correlation to A1C levels sis. It induces activation of intracellular LPS), NF-␬B, endogenous ligands, CML, in type 1 diabetic patients (11,21). More- signaling pathways via TLR2, TLR4, and and cytokines were significantly higher in over, TLR2/4 expression and activation is the receptor for advanced glycation end type 2 diabetes compared with control increased in human monocytes under hy- products (RAGEs), thus acting as an ex- subjects (P Ͻ 0.001), whereas IL-1␤ (P ϭ perglycemia conditions (3). Reyna et al. tracellular alarmin. HSPs also act as en- 0.09), IFN-␥ (P ϭ 0.16), and endotoxin (15) have shown abnormal TLR4 expres- dogenous ligands of TLR2 and TLR4. (P ϭ 0.11) were no longer significant. sion in skeletal muscle of a small number HSP60 and HSP70 induce the production of insulin-resistant subjects, with little in- of proinflammatory cytokines via activa- CONCLUSIONS — The interactions formation on the TLR4-MyD88 signaling, tion of TLR2 and TLR4. The inflammatory among inflammation, hyperglycemia, in- levels of TLR4 cofactors (CD14/MD-2), effects of recombinant human HSP60 sulin resistance, and type 2 diabetes have TLR4 ligand (endotoxin), and its correla- were shown to be TLR4 dependent, sug- clear implications for atherosclerosis via tion to TLR4 expression. Moreover, the in gesting that HSP60 may be a TLR4 ligand. the innate immune system. Besides being vitro experiments in this article with myo- Low–molecular weight degradation prod- activators of inflammation under hyper- tubes and FFAs lacked appropriate endo- ucts of hyaluronan elicit proinflammatory glycemia and insulin resistance (3,5–7), toxin controls, and it is not clear if the responses in murine alveolar macro- both TLR2 and TLR4 are critical in study subjects were on any cholesterol- phages in rheumatoid arthritis models atherosclerosis (7). In this study, we lowering , adding complexity and other chronic inflammatory condi- provided key evidence for increased to the conclusions. Creely et al. (16) tions. Endotoxin is the most important monocyte TLR2 and TLR4 expression, ac- showed increased TLR2 expression in the ligand required for TLR4 activation (14), tivation, cofactor expression, ligands, and adipose tissue of type 2 diabetes with and its levels are significantly increased in downstream signaling contributing to strong correlates to endotoxin levels, with type 2 diabetic patients. In the present systemic inflammation seen in type 2 di- no change in TLR4 expression. This study study, we show that type 2 diabetic pa- abetic subjects. Our observations signifi- is inconclusive in terms of the lack of tients have high circulating levels of cantly add to the emerging role of TLRs in TLR4 expression, even under high endo- HMGB1, HSP60, HSP70, and hyaluro- atherosclerosis and diabetes and are con- toxin levels, smaller sample size, and min- nan, which could trigger TLR2 and TLR4 sistent with other studies in this context imal patient details. Du et al. activation, leading to a proinflammatory (3,7,11). Increased TLR2 and TLR4 ex- (24) in a descriptive study showed that state by the activation of TLR2/4, syner- pression is demonstrated in atheroscle- monocytes from LADA, (latent autoim- gistically with glucose, FFAs, and endo- rotic plaque and in animal mune diabetes in adults) type 2 diabetic toxin. Our coimmunoprecipitation models of atherosclerosis. TLR2/4 knock- patients on sulfonylurea therapy have sig- studies further suggest an association be- Ϫ Ϫ out mice on C57BL6, ApoE / , and nificantly higher TLR4 and CD14 expres- tween TLR2, TLR4, HMGB1, and HSP60. care.diabetesjournals.org DIABETES CARE, VOLUME 33, NUMBER 4, APRIL 2010 865 TLR2 and TLR4 expression and activation in type 2 diabetes

Figure 2—A: Endotoxin concentration in control (n ϭ 23) and type 2 diabetic (n ϭ 23) subjects was measured using Limulus Ameobocyte lysate assay as described in RESEARCH DESIGN AND METHODS. Values are expressed as EU/ml. *P Ͻ 0.05 vs. control. C, control; T2DM, type 2 diabetes. B: HMGB1 concentration in control (n ϭ 23) and type 2 diabetic (n ϭ 23) subjects was measured using ELISA as described in RESEARCH DESIGN AND METHODS. Values are expressed as ng/ml. *P Ͻ 0.0001 vs. control. C: Hyaluronan concentration in control (n ϭ 23) and type 2 diabetic (n ϭ 23) subjects was measured using ELISA as described in RESEARCH DESIGN AND METHODS. Values are expressed as ng/ml. *P Ͻ 0.0001 vs. control. D: HSP60 concentration in control (n ϭ 23) and type 2 diabetes (n ϭ 23) subjects was measured using ELISA as described in RESEARCH DESIGN AND METHODS. Values are expressed as ng/ml. *P Ͻ 0.0001 vs. control. E: HSP70 concentration in control (n ϭ 23) and type 2 diabetic (n ϭ 23) subjects was measured using ELISA as described in RESEARCH DESIGN AND METHODS. Values are expressed as ng/ml. *P Ͻ 0.0001 vs. control. F: Association of TLR2 with its ligands and TLR6. Representative Western blot showing enhanced expression of TLR6, HMGB1, hyaluronan, and HSP60 in basal control and type 2 diabetes monocyte cell lysates immunoprecipitated with TLR2 antibody as detailed in RESEARCH DESIGN AND METHODS. ␤-Actin was used as a loading control. Each assay is repeated four times. IP, immunoprecipitation. G: Association of TLR4 with its ligands. Representative Western blot showing enhanced expression of HMGB1, hyaluronan, and HSP60 in basal control and type 2 diabetes monocyte cell lysates immunoprecipitated with TLR4 antibody as detailed in RESEARCH DESIGN AND METHODS. ␤-Actin was used as a loading control. Each assay is repeated four times. H: Monocyte TLR signaling in the basal state. TLR downstream signaling proteins MyD88, pIRAK-1, Trif, IRF-3, TECAM-2, and NF-␬B p65 were performed using specific antibodies to the respective (phospho) proteins, as described in RESEARCH DESIGN AND METHODS using ␤-actin as a loading and internal control for MyD88, Trif, IRF-3, TECAM-2, and NF-␬B p65 and IRAK for pIRAK-1. Each blot is repeated four times with pooled monocytes from three subjects. Densitometric ratios corroborate the data. *P Ͻ 0.05 vs. control. I: The DNA binding activity of nuclear NF-␬B p65 in control (n ϭ 23) and type 2 diabetes (n ϭ 23) monocytes was assessed by ELISA as detailed in RESEARCH DESIGN AND METHODS in the basal state. Values are normalized to milligram nuclear protein and expressed as mean Ϯ SD. *P Ͻ 0.05 vs. control. J: Serum concentration of cytokines/chemokines in study subjects were measured using Multiplex assays as described in RESEARCH DESIGN AND METHODS. Values are expressed as picograms per milliliter. *P Ͻ 0.0001 vs. control. K: Serum concentration of cytokines/chemokines in study subjects were measured using Multiplex assays as described in RESEARCH DESIGN AND METHODS. Values are expressed as picograms per milliliter. *P Ͻ 0.0001 vs. control.

866 DIABETES CARE, VOLUME 33, NUMBER 4, APRIL 2010 care.diabetesjournals.org Dasu and Associates

Figure 2—Continued.

Dimerization is a major event in the TLR6 and MD-2 mRNA expression is in- induces IFN-␤. In two recent studies, we functional activation of TLRs and results creased in type 2 diabetic patients, sug- showed that activation of the MyD88 in cytokine production (3). TLR2 activity gesting their requirement for TLR ligation pathway is increased in monocytes ex- requires heterodimerization with TLR1 or in type 2 diabetic patients. posed to high glucose and in type 1 dia- TLR6 to recognize ligands. Using lucif- TLRs mainly signal through the betic patients (3,9,11). In the present erase reporter assays and real-time RT- adapter protein MyD88 via activation of study, we examined MyD88-dependent PCR, we showed that high glucose NF-␬B, resulting in the increased tran- and -independent TLR activation, by de- induces TLR2 and TLR6 heterodimeriza- scription of inflammation-related , termining IRF-3 and IFN-␤ levels as a bi- tion, resulting in NF-␬B activation and such as those encoding indicated cyto- ological indicator of MyD88-independent cytokine production (3). MD-2 is re- kines (3,9,11). In addition, a MyD88- activation. quired for TLR4 ligation with endotoxin independent pathway involving Trif is Taken together, the novel findings of (14). Here, we demonstrate that both essential to TLR3 and TLR4 signaling and this comprehensive study suggest that care.diabetesjournals.org DIABETES CARE, VOLUME 33, NUMBER 4, APRIL 2010 867 TLR2 and TLR4 expression and activation in type 2 diabetes there is significant elevation of TLR2 and 6. Wong FS, Wen L. Toll-like receptors and in and type 2 diabetes. Am J TLR4 protein, mRNA, endogenous ligands, diabetes. Ann N Y Acad Sci 2008;1150: Physiol Endocrinol Metab 2007;292: and cofactors in type 2 diabetic patients, 123–132 E740–E747 which, in concert with hyperglycemia, con- 7. Curtiss LK, Tobias PS. Emerging role of 17. Mohammad MK, Morran M, Slotterbeck tributes to the increase in TLR2 and TLR4 Toll-like receptors in atherosclerosis. J B, Leaman DW, Sun Y, Grafenstein H, Lipid Res 2009;50(Suppl.):S340–S345 Hong SC, McInerney MF. Dysregulated signaling that results in the proinflamma- 8. Bagarolli RA, Saad MJ, Saad ST. Toll-like tory state of type 2 diabetes. In future stud- Toll-like receptor expression and signal- receptor 4 and inducible nitric oxide syn- ing in bone marrow-derived macrophages ies, we will address the mechanism of thase gene polymorphisms are associated at the onset of diabetes in the non-obese synergistic effects of hyperglycemia, FFAs, with type 2 diabetes. J Diabetes Com- diabetic mouse. Int Immunol 2006;18: and endogenous ligands on TLR2 and TLR4 plications 2009 Apr 21 (Epub ahead of 1101–1113 expression and signaling. print) 18. Kim HS, Han MS, Chung KW, Kim S, Kim 9. Takeda K, Kaisho T, Akira S. Toll-like re- E, Kim MJ, Jang E, Lee HA, Youn J, Akira ceptors. Annu Rev Immunol 2003;21: S, Lee MS. Toll-like receptor 2 senses be- Acknowledgments— This study was sup- 335–376 ta-cell death and contributes to the initi- ported by American Diabetes Association 10. Hill H, Hogan N, Rallison M, Santos JI, ation of autoimmune diabetes. Immunity Grant 7-07-JF-16 to M.R.D. and National Charette RP, Kitahara M. Functional Institutes of Health Grant K24 AT 00596 and metabolic abnormalities of diabetic 2007;27:321–333 to I.J. S.D. was supported by NCRR-UL1- monocytes. Adv Expt Med Biol 1980;69: 19. Song MJ, Kim KH, Yoon JM, Kim JB. Ac- RR024146. 621–627 tivation of Toll-like receptor 4 is associ- No potential conflicts of interest relevant to 11. Devaraj S, Dasu MR, Rockwood J, Winter ated with insulin resistance in . this article were reported. W, Griffen SC, Jialal I. Increased toll-like Biochem Biophys Res Commun 2006; We thank all the study participants for their receptor (TLR) 2 and TLR4 expression in 346:739–745 time and efforts. We thank Rohan Jialal for monocytes from patients with type 1 dia- 20. Devaraj S, Siegel D, Jialal I. Simvastatin help with blood draws (phlebotomy). We betes: further evidence of a proinflamma- (40 mg/day), adiponectin levels, insulin thank Catherine Duncan-Staley for assistance tory state. J Clin Endocrinol Metab 2008; sensitivity, in subjects with the metabolic with HSP70 ELISA assay and Manpreet Kaur 93:578–583 syndrome. Am J Cardiol 2007;100:1397– for editorial assistance. 12. Tsan MF, Gao B. Endogenous ligands of 1399 Toll-like receptors. J Leukocyte Biol 2004; 21. Devaraj S, Dasu MR, Park S, Jialal I. In- 76:514–519 creased levels of ligands of TLR2 and References 13. 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