Background/Purpose:

We have previously demonstrated that chondrocyte deficient in both TLR2 and TLR4 almost completely abolished pro-catabolic responses to rHMGB1 and LMW-HA, which are known to be present in OA joints. Since both TLR2 and TLR4 signaling can modulate autophagy, and autophagy plays an important role in cartilage homeostasis, we investigated the role of TLR2 and TLR4 in chondrocytes in vitro.

Methods:

Immature knee chondrocytes were isolated from TLR2/TLR4 double knockout (dKO) and the wild type (WT) mice. Microtubule-associated protein 1 light chain 3 (LC3) conversion (LC3-I to LC3-II) was examined by Western blotting. To monitor autophagic flux (the rate at which autophagic vacuoles are processed by lysosomes), the TLR2/TLR4 dKO and WT chondrocytes were transfected with a tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3). Green and red fluorescent proteins have different pH stability. The acidic environment (pH<5) inside lysosome quenches the fluorescent signal of EGFP. The transfected cells were treated with HBSS containing 1% DMEM high (for starvation to induce autophagy) or chloroquine (an inhibitor of autophagic flux by inhibition of fusion of autophagosome with lysosome) for 1-2 hours and then subjected to flow cytometry analysis for fluorescence intensity of GFP and RFP or Western blot analysis for GFP or RFP expression levels.

Results: Basal level of LC3 conversion was higher in TLR2/TLR4 dKO chondrocytes, and basal level of mean fluorescence intensity (MFI) of GFP and RFP in TLR2/TLR4 dKO chondrocytes was 2.3 and 1.9 fold higher than that in WT chondrocytes. Western blot analysis also showed significantly higher level of GFP in TLR2/TLR4 dKO chondrocytes compared to WT chondrocytes. As expected, the MFI of GFP and RFP was increased by 34.8% and 19.3% respectively by chloroquine, but was decreased by 12% and 18% respectively by starvation, compared to non-treatment control in WT chondrocytes, indicating that there was autophagic flux. In contrast, despite that chloroquine was still able to increase the MFI of GFP and RFP by 27.3% and 23% respectively, starvation did not decrease the MFI of GFP and RFP at all, compared to non-treatment control in TLR2/TLR4 dKO chondrocytes. These data suggested that autophagic flux was impaired in TLR2/TLR4 dKO chondrocytes.

Conclusion:

Chondrocytes deficient in TLR2 and TLR4 exhibit impairment of autophagic flux. Given that autophagy is a cellular homeostasis mechanism for the removal of dysfunctional organelles and macromolecules, and autophagic pathways are constitutively targets intracellular cytosolic components for lysosomal degradation, and is essential for maintaining cellular energy and metabolic homeostasis, the diminished efflux of products from autolysosomes is likely to induce a state of metabolic insufficiency and accumulates dysfunctional organelles and aggregates of macromolecules. The fact that TLR2 and TLR4 deficiency show little chondroprotection in mouse OA model may partially result from impairment of autophagic flux in chondrocytes.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/deficiency-of-tlr2-and-tlr4-impairs-autophagic-flux-in-chondrocytes/