Adipose tissues were collected from patients with obesity (8 males, 40–50 years old, BMI>28) and age and sex matched lean controls (8 males, 40–50 years old, BMI<24) during abdominal surgery from the First Affiliated Hospital of Jinzhou Medical University (Jinzhou, China) from December 2016 to January 2017. All volunteers signed written informed consent forms prior to the study. All experiments were approved by the Medical Ethics Committee of the First Affiliated Hospital of Jinzhou Medical University.

Raw264.7 cells were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in glucose free 1640 medium (Thermo Fisher Scientific, Inc., Waltham. MA, USA) with 5 mM glucose, supplemented with 10% fetal bovine serum (FBS; Tiangen Biotech Co., Ltd., Beijing, China) and 1% penicillin-streptomycin (P/S). The medium was replaced at least once every two days. Cells were harvested for passaging when plates were 90% confluent.

TLR2^−/− or TLR4^−/− mice backcrossed on C57BL/6J from the Jackson Laboratory (Ben Harbor, ME, USA or Sacramento, CA, USA) were housed with C57BL/6J mice from Beijing HFK Bioscience Co., Ltd. (Beijing, China) to get TLR2 or 4^+/+ and TLR2 or 4^−/− sex and age matched littermate mice used for studies in vitro. A total number of 315 mice were used. Primary macrophages were extracted from abdominal cavities of TLR2^+/+ or TLR 4^+/+ and TLR2^−/− or TLR 4^−/− mice. Briefly, 10 ml bacteria free PBS was injected to the abdominal cavities multiple times in each fresh sacrificed mouse in order to generate macrophages, and then was pumped out following massaging for 5 min. Cells in the PBS extracted from abdominal cavities were centrifuged at 700 × g for 5 min at 20–25°C and cultured in the aforementioned 1640 medium. The medium was replaced following 12 h of culture to remove unadherent cells from macrophages. All animal studies were approved by the Ethics Committee for Experimental Research, Jinzhou Medical University (Jinzhou, China).

Cells (Raw264.7 cells and primary macrophages) were cultured in the 1640 medium as previously described with 5 mM glucose, 10% FBS and 1% P/S for two days, and then cultured in FBS free medium for IL29 and high glucose-activated assays. Activation was performed by treatment with IL29 at a concentration of 0, 10, 30 or 100 ng/ml or 25 mM glucose for 24 h (low dose group=5 mM and high dose group=30 mM). Antibodies against TLR2 or 4 (dilution, 1:100; cat no. 10472R; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China) were added to the medium at 37°C for 24 h to inhibit TLR2 or TLR4. Subsequently, cells were harvested with a cell scraper followed by centrifugation at 700 × g for 5 min at 20–25°C for further research.

Western blotting was performed according to a previously described protocol (18). Total proteins from adipose tissue were extracted using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) (with 1% phenylmethane sulfonyl fluoride or phenylmethylsulfonyl fluoride). Subsequently, a bicinchoninic acid assay was performed to measure protein concentration in middle extracting solution. Proteins were separated by SDS-PAGE on 10% gels, transferred to polyvinylidene fluoride membranes and blocked with skimmed milk for 2 h at room temperature. Rabbit-anti-mouse (or human) primary antibodies to TIMP1 (1:500; cat no. wl02342; Wanleibo Co., Ltd., Shanghai, China), myeloid differentiation primary response protein MyD88 (MyD88; 1:300; cat no. bs-1047R; Beijing Biosynthesis Biotechnology Co., Ltd.), IL29 (1:1,500; cat no. ab38569; Abcam, Cambridge, MA, USA) and β-actin (1:3,000; cat no. AB21800; Absci, Vancouver, WA, USA) were used to bind target proteins at 4°C overnight. Following incubation with goat-anti-rabbit secondary antibody (1:5,000; cat no. ABL3012-2; Absci, USA) for 2 h at 20–25°C, an Enhanced Chemiluminescence kit (Wanleibo Co., Ltd.) was used to detect protein expression.

Raw264.7 cells were collected by centrifugation (700 × g for 5 min at 20–25°C) and seeded on dish climbing glasses (https://item.taobao.com/item.htm?id=13540518880) in 24-well plate for 24 h. IL29 and high glucose-activated assays were performed as described previously, for 24 h. Raw264.7 cells were rinsed 3 times with PBS and fixed with 4% paraformaldehyde for 30 min at 20–25°C. Non-specific binding was blocked in PBS containing 5% normal goat serum (cat no. SL03B; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) at room temperature for 1 h. Cells were initially incubated with primary antibodies [rabbit anti-mouse TLR2 (cat no. 10472R; 1:300) or TLR4 (cat no. 1021R; 1:300) at 4°C on a shaker overnight]. Subsequently, climbing glasses with Raw264.7 cells were washed 3 times in PBS at room temperature prior to incubation with goat anti-rabbit fluorescein isothiocyanate (green; Wanleibo Co., Ltd., Shanghai, China; 1:200; cat no. WLA031a) or Alexa Fluor 594 (red; Thermo Fisher Scientific, Inc,. 1:300; cat no. A-11032) secondary antibodies in the dark at room temperature for 1 h. Following washing with PBS 3 times, TLR2 and 4 expression levels were observed under a fluorescence microscope.

TIMP1 in conditioned media was quantified using sandwich ELISA kits (cat no. MTM100) according to the manufacturers' protocol (R&D Systems, Inc., Minneapolis, MN, USA).

Total RNA was extracted from the Raw264.7 cell line using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) and dissolved in RNase free water with RNase inhibitor added. Prior to cDNA synthesis, RNA concentration was measured by Nanodrop 2000 (Thermo Fisher Scientific, Inc.) and modified to 100 µg/µl with diethyl pyrocarbonate (DEPC) water (Life Technologies; Thermo Fisher Scientific, Inc., cat no. AM9915G). All experiments were performed according to the manufacturers' protocol of PrimeScriptTM RT reagent kit with gDNA Eraser (Takara Bio, Inc., Otsu, Japan). DNA was removed in a 10 µl reaction with RNA sample (1 µl), 5X gDNA Eraser Buffer (2 µl), gDNA Eraser (1 µl) and RNase Free ddH₂O (6 µl) for 2 min at 42°C. The cDNA was synthesized in a 20 µl reaction with Primer Script RT Enzyme Mix (1 µl), RT Primer Mix (1 µl), 5X Primer Script Buffer (4 µl), RNase Free dH₂O (4 µl), and the 10 µl reaction mixture from the previous step. The RT was performed in 20 µl reactions at 25°C for 10 min followed by 37°C for 15 min and final denaturation at 85°C for 5 min. The cDNA was stored at −80°C until further use. The expression of IL-6 mRNA and tumor necrosis factor-α (TNF-α) mRNA was analyzed in 20 µl reactions with 2X GreenStar Master Mix (10 µl), forward primer (1 µl, 10 pmol/µl), reverse primer (1 µl, 10 pmol/µl), DEPC water (6 µl) and template DNA (2 µl) from reverse transcription. Primers were designed and synthesized by Sangon Biotech (Sangon Biotech Co., Ltd, Shanghai, China; Table I). qPCR experiments were performed according to the manufacturers' protocol of AccuPower^® 2X GreenStar qPCR Master Mix kit with gDNA Eraser (Bioneer Corporation, Daejeon, Korea). Reactions were performed in 20 µl volume and the following thermocycling conditions were used for PCR: Initial denaturation for 30 sec at 95°C; 45 cycles of 95°C for 5 sec and 60°C for 34 sec. Relative quantification of gene expression was performed using comparative 2^−ΔΔCq method. GAPDH was used as a reference gene.

Data are presented as the mean ± standard error of the mean, and analyzed by one-way analysis of variance between multiple groups, followed by the least significant difference post-hoc test. Interaction effects were analyzed by analysis of variance of factorial design. SPSS software (version 20.0; IBM SPSS, Armonk, NY, USA) was used for data analysis. P<0.05 was considered to indicate a statistically significant difference.

In this study, elevated levels of IL29 and TIMP1 in adipose tissue of obese individuals compared with lean individuals was observed (Fig. 1).

In the present study, IL29 and high glucose stimulated TLR2 and TLR4 expression respectively (Fig. 2A and B), as well as TIMP1 in a concentration-independent manner (Fig. 3). Upregulated MyD88 protein expression level was detected by western blotting in high glucose group compared with low glucose group (Fig. 3A). Moreover, MyD88 protein expression was upregulated in a IL29 concentration-dependent manner. mRNA expression levels of downstream inflammation factors TNF-α and IL-6 were also up-regulated (Fig. 3C and D).

TIMP1 expression pattern in primary macrophages was similar to that in Raw264.7 cells. IL29 and high glucose, activated TIMP1 expression in primary macrophages in a concentration independent manner (Fig. 3E). Indeed, it was found that TIMP1 expression was significantly increased in the 100 ng/ml group compared with the 0, 10 and 30 ng/ml groups.

Antibody-mediated inhibition of TLR2 signaling suppressed IL29-stimulated TIMP1 expression but did not affect high glucose activated TIMP1 release in the medium. In addition, high glucose-stimulated TIMP1 levels were inhibited by TLR4 antibody. However, TLR4 antibody could not suppress upregulated TIMP1 caused by IL29 (Fig. 4A).

Similar to TLR2 and 4 antibody-mediated inhibition studies, IL29 did not increase TIMP1 expression in primary macrophages extracted from TLR2^−/− mice compared with macrophages extracted from TLR2^+/+ mice. In addition, increased TIMP1 expression level in high glucose medium was inhibited by TLR4 gene knockout in primary macrophages (Fig. 4B).