The novel adipokine progranulin counteracts IL-1 and TLR4-driven inflammatory response in human and murine chondrocytes via TNFR1

Abstract

Progranulin (PGRN) is a recently identified adipokine that is supposed to have anti-inflammatory actions. The proinflammatory cytokine interleukin-1β (IL1β) stimulates several mediators of cartilage degradation. Toll like receptor-4 (TLR4) can bind to various damage-associated molecular patterns, leading to inflammatory condition. So far, no data exist of PGRN effects in inflammatory conditions induced by IL1β or lipopolysaccharide (LPS). Here, we investigated the anti-inflammatory potential of PGRN in IL1β- or LPS-induced inflammatory responses of chondrocytes. Human osteoarthritic chondrocytes and ATDC-5 cells were treated with PGRN in presence or not of IL1β or LPS. First, we showed that recombinant PGRN had no effects on cell viability. We present evidence that PGRN expression was increased during the differentiation of ATDC-5 cell line. Moreover, PGRN mRNA and protein expression is increased in cartilage, synovial and infrapatellar fat pad tissue samples from OA patients. PGRN mRNA levels are upregulated under TNFα and IL1β stimulation. Our data showed that PGRN is able to significantly counteract the IL1β-induced expression of NOS2, COX2, MMP13 and VCAM-1. LPS-induced expression of NOS2 is also decreased by PGRN. These effects are mediated, at least in part, through TNFR1. Taken together, our results suggest that PGRN has a clear anti-inflammatory function.

Figure 1. PGRN mRNA and protein expression during ATDC-5 differentiation after 7, 14, and 21 days.

Values are the mean ± SEM of at least 4 independent experiments (**P < 0.01 vs. Ctrl, ***P < 0.001 vs. Ctrl.). Cell lysates underwent Western blot analysis using PGRN antibody. GAPDH was used as a loading control. The blots were run under the same experimental conditions. The full blots are shown in Supplementary Fig. 2. Blots are representative of at least 3 independent experiments.

Figure 2. Determination of PGRN mRNA and protein in healthy and osteoarthritis human tissues.

PCR results were shown in fold change, where grey bars represent the PGRN mRNA expression in chondrocytes, infrapatellar fat pads (IPFPs) and synovial tissues obtained from osteoarthritis (OA) patients (*P < 0.05 vs. Ctrl.). PGRN protein expression was showed by representative western blots of 18 OA patients (age 52–73; mean BMI 28.4) and 6 healthy donors (age 23–50; mean BMI 23.4). GAPDH was used as loading control. Western blots have been run under the same experimental conditions. The full blots are shown in Supplementary Fig. 3.

Figure 3. Mouse PGRN mRNA expression after TNFα, IL1β, IL6 and LPS treatment.

(a) Cells were treated with TNFα 0.1, 1, 5 ng/ml, (b) IL1β 0.025, 0.05, 0.1 ng/ml, (c) IL6 0.1, 0.5, 1 ng/ml and (d) LPS 100, 250, 500 ng/ml for 24 h. Values are the mean ± SEM of at least 3 independent experiments (**P < 0.01 vs. Ctrl, ***P < 0.001 vs. Ctrl.).

Figure 4. PGRN suppressed IL1-β induction of chondrocyte catabolism.

(a) Cells were treated with 10 ng/ml of IL1β, in presence or absence of 200 ng/ml of PGRN for 48 h. Culture medium was subsequently analyzed for nitrite levels. NO concentration (μM) was determined using the Griess reaction. Values are the mean ± SEM of at least 3 independent experiments (**P < 0.01 vs. IL1β) (a, upper panel). Cell lysates underwent Western blotting analysis under the same condition (a, lower panel). After transferring the blots onto PVDF membranes, we cropped the targeted blots according to referenced indicating markers, and then targeted proteins were immunoblotted with NOS2, COX-2, MMP13 and VCAM-1 antibodies. GAPDH was used as a loading control. Blots are representative of at least 3 independent experiments. (b–e) Western blot densitometric analysis (n = 3; ***P < 0.001 PGRN + IL1β vs. IL1β).

Figure 5. PGRN counteracted IL1β and LPS-induced nitric oxide (NO) production and inhibited NOS2 expression in cultured ATDC-5 cells, at least in part, through TNFR1.

(a,b) Cells were treated with 0.1 ng/ml of IL1β (a) and 250 ng/ml of LPS (b) alone or in combination PGRN (200 ng/ml) during 48 h. Culture medium was subsequently analyzed for nitrite levels. NO concentration (μM) was determined using the Griess reaction. Values are the mean ± SEM of at least 3 independent experiments (a) **P < 0.01 PGRN + IL1β vs. IL1β; (b) **P < 0.01 PGRN + LPS vs. LPS). Cell lysates underwent Western blotting analysis using NOS2 antibody. GAPDH was used as a loading control. The blots were run under the same experimental conditions. The full blots are shown in Supplementary Fig. 3. Blots are representative of at least 3 independent experiments. (c,d) Cells were transfected with negative control siRNA or TNFR1-targeted siRNA (siTNFR1) and stimulated with 0.1 ng/ml of IL1β (c) and 250 ng/ml of LPS (d) in absence or presence of PGRN 200 ng/ml for 48 h. Relative mRNA levels of NOS2 were measure by RT-PCR. Values are the mean ± SEM of at least 3 independent experiments (c) **P < 0.01 siC- PGRN + IL1β vs. siC- IL1β; (d) **P < 0.01 siC- PGRN + LPS vs. siC-LPS).