Inhibition of Toll-Like Receptor 3 Relieves Osteoarthritis by Suppression of Cartilage Degradation,Nuclear Factor Kappa B–Mediated Inflammation,and Activation of Autophagy

Abstract

Objective:

To investigate the regulatory role of Toll-like receptor 3 (TLR3) in osteoarthritis (OA) progression, particularly its impacts on cartilage degradation, NF-κB-mediated inflammation, and autophagy activation.

Method:

1. Model Constuction: OA mouse model generated via anterior cruciate ligament transection (ACLT); LPS-induced inflammatory injury in murine ATDC5 chondrocytes; Histomorphological analysis of cartilage tissue using H&E and Safranine O staining. 2. Molecular Detection: TLR3 expression assessed by Western blot; Cartilage degradation markers (MMP-13, ADAMTS) and NF-κB pathway proteins analyzed via Western blot; Pro-inflammatory cytokine levels (IL-1β, TNF-α) quantified via RT-qPCR and Western blot. 3. Functional Assays: Cell viability examined via CCK-8 assay.

Results:

1. TLR3 Upregulation: TLR3 was highly expressed in OA cartilage and LPS-treated chondrocytes. 2. Cartilage Protection: TLR3 inhibition reduced cartilage erosion and proteoglycan loss in ACLT mice (confirmed by H&E and Safranine O staining); Downregulation of cartilage degradation markers (MMP-13, ADAMTS-5) observed in TLR3-knockdown models. 3. Anti-inflammatory Effects: TLR3 knockdown suppressed NF-κB activation, reducing IL-1β and TNF-α levels. 4. Autophagy Activation: Enhanced LC3-II/LC3-I ratio and Beclin-1 expression indicated TLR3 inhibition promotes autophagy.

Conclusion:

TLR3 drives OA progression through dual mechanisms: 1. Pro-inflammatory Pathway: Activates NF-κB signaling to amplify cytokine release and cartilage matrix breakdown. 2. Autophagy Suppression: Inhibits autophagy-related proteins, impairing cellular homeostasis. Targeting TLR3 may represent a therapeutic strategy to balance inflammation and autophagy, potentially slowing OA progression in multi-joint involvement cases.

Introduction

Osteoarthritis (OA) refers to the gradually degenerative process involving articular cartilage, synovium, and subchondral bone. ¹ According to worldwide estimates, around 250 million individuals were afflicted with symptomatic OA in 2019, the number of which may reach 400 million in China by 2030.^2,3 Clinically, OA is primarily manifested as chronic pain, joint instability, stiffness, joint deformity and narrowing of the joint space, raising considerable pain, joint movement disorder, and even disability. ⁴ Primary OA is commonly attributed to a combination of variable factors, such as obesity, heredity, aging, genetic factors, and joint injury. ⁵ The available agents for OA therapy are targeted at temporary symptom relief, ⁶ but no effect on physical function. ⁷

Pathologically, OA is marked by progressive erosion of articular cartilage. ⁸ Chondrocytes resident in the articular cartilage are responsible for maintaining cartilage homeostasis. ⁹ However, in response to external stimuli, chondrocyte function and survival are impaired, initiating the pathogenesis of OA. Hence, it is of ample significance to identify key genes involved in cartilage destruction and chondrocyte injury during OA that have therapeutic values for a precise and effective treatment.

Toll-like receptors (TLRs) comprise a class of conserved transmembrane pattern recognition receptors (PRRs) that recognize and respond to invading pathogens and pathogen-associated molecular patterns (PAMPs). ¹⁰ TLRs are expressed primarily on immune cells, where they activate downstream signaling cascades that induce the secretion of cytokines and chemokines, culminating in innate and adaptive immune responses. ¹⁰ Toll-like receptor 3 (TLR3) is recognized as a receptor for host-derived or viral double-stranded (ds) RNA, which is fundamental for antiviral responses. ¹¹ Emerging evidence has supported the anti-cancer efficacy of TLR3 agonists.^12,13 Particularly, dysregulation of TLR3 signaling extensively participates in autoimmune diseases.^14,15 The existing investigations have uncovered that TLR3 can serve as a diagnostic marker of OA ¹¹ and targeting the TLR3 signaling pathway can regulate macrophage polarization and joint degeneration in OA.^16,17

This research was undertaken to suggest an improved understanding of the effect of TLR3 on the biological events in the process of OA.

Materials and Methods

Animals

The 6- to 8-week-old mice with a C57BL/6J background weighing between 20 and 22 g were given 7 days of feeding and housing adaptation under a controlled temperature (23 ± 2°C) and humidity (55 ± 5%) exposed to a 12 h light/dark regimen. All operational procedures conformed to the standards and guidelines of ethnics committee of Jofunhwa Biotechnology (Nanjing) Co., Ltd.

Grouping, Model Preparation, and Processing

The mice were allocated to the Control group, OA group, OA+Lv-shRNA-NC group, and OA+Lv-shRNA-TLR3 group (6 mice per group). According to the previous protocol, ¹⁸ to induce mechanical instability-associated OA, the right joint capsule of mice in the OA group was exposed under anesthesia, followed by the transection of the anterior cruciate ligament. The skin of mice in the Control group was incised in the same position and sutured without patellar dislocation or ligament transection. The articular cavity of mice in the OA+Lv-shRNA-NC group and OA+Lv-shRNA-TLR3 group were individually injected with 1 × 10¹⁰ PFU/ml empty Lentivirus (shRNA-NC) or TLR3 shRNA Lentivirus (shRNA-TLR3) via the trans-patella tendon approach 2 days following the anterior cruciate ligament transection (ACLT) surgery. Eight weeks later, the mice were put to death and the cartilage tissues were harvested.

Hematoxylin-Eosin and Safranine O Staining

After being immersed in 4% paraformaldehyde for 48 h and de-calcified in 10% ethylene diamine tetraacetic acid (EDTA) for 8 weeks, the cartilage tissues were subjected to ethanol dehydration, paraffin embedding and sectioning. The obtained 4-μm-thick slices were processed with hematoxylin-eosin (H&E) or Safranine O for light microscopy.

Immunohistochemical Staining

The paraffin-embedded samples were deparaffinized and covered with 3% H₂O₂. After antigen repair and blockade of nonspecific IgG binding, the sections reacted with MMP-13 (#DF6494; 1/50; Affinity Biosciences; Dublin, Ireland), p-p65 (#AF3387; 1/50; Affinity Biosciences), or p65 primary antibody (#AF0874; 1/50; Affinity Biosciences) diluted in 5% bovine serum albumin (BSA) at 4°C overnight and the corresponding secondary antibody for half an hour. The sections were eventually double-dyed by DAB (3,3′-diaminobenzidine) and hematoxylin for light microscopy.

Cell Preparation, Intervention, and Transfection

Dulbecco’s modified Eagle medium (DMEM) media enhanced with 10% fetal bovine serum (FBS) was applied to cultivate murine ATDC5 chondrocytes. Cells were exposed to lipopolysaccharide (LPS) at concentrations of 1.25, 2.5, and 5 μg/ml for 12 h. ¹⁹ Then, cells were transfected with shRNA-TLR3 or shRNA-NC with HighGene transfection reagent.

Cell Viability Assay

After being distributed on a 96-well culture plate (4 × 10³ cells/well), ATDC5 chondrocytes were treated by LPS at varying concentrations (1.25, 2.5, and 5 μg/ml). After each well was exposed to 10 μL Cell Counting Kit-8 (CCK-8) buffer for a duration of 2 h, the OD value at 450 nm wavelengths was read with a microplate reader.

Immunofluorescence Staining

After being covered with 4% paraformaldehyde, ATDC5 chondrocytes were hatched with MMP-13 antibody (#DF6494; 1/100; Affinity Biosciences) diluted in 5% BSA overnight at 4°C. The secondary antibody was applied for 1 h at 37°C for secondary detection. Cell nuclei were identified following DAPI (4′,6-diamidino-2-phenylindole) staining. The immunofluorescence results were taken by a fluorescence microscope.

Reverse Transcription-Quantitative Polymerase Chain Reaction

With the aid of TRIzol reagent, total RNA was isolated, from which cDNA was then produced by means of the First Strand cDNA Synthesis Kit. Subsequently, the cDNA template was mixed with SYBR Green PCR Kit. Quantification was performed with the ^2−ΔΔCT approach.

Western Blot

After being harvested in radio-immunoprecipitation assay lysis buffer, total protein was quantitated based on BCA method. Proteins electrophoresed in 10% SDS-PAGE were blotted to PVDF membranes. After the exposure to primary antibodies against TLR3, matrix metallopeptidase 3 (MMP3), matrix metallopeptidase 13 (MMP13), A disintegrin and metalloproteinase with thrombospondin motifs type 4 (ADAMTS-4), Collagen II, p-p65, p65, p-inhibitor of kappa Balpha (IκBα), IκBα, interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), light chain 3 (LC3), p62, Beclin-1, and GAPDH overnight at 4°C and the matching secondary antibodies for 1 h at room temperature, the membranes pre-incubated in 5% BSA were soaked in the enhanced chemiluminescence (ECL) reagent for signal development.

Statistics

All collected data measured with PRISM5 software (GraphPad Software, La Jolla, CA) were represented as mean ± SD. Student’s t-test or one-way analysis of variance (ANOVA) analysis was applied when comparing continuous data from two or more groups. The significance of the difference was defined as a P-value below 0.05.

Results

Histomorphological Alternations and TLR3 Expression in the Cartilage Tissues of ACLT-Induced OA Mice

Through H&E staining, the severe cartilage damage was observed in ACLT-induced OA mice ( Fig. 1A ). As illustrated in Safranine O staining, extracellular matrix (ECM) degradation was markedly aggravated after ACLT ( Fig. 1B ). Intriguingly, TLR3 expression was discovered to be profoundly elevated in the OA group relative to the Control group ( Fig. 1C ).

Figure 1.

Histomorphological alternations and TLR3 expression in the cartilage tissues of ACLT-induced OA mice. (A) H&E and (B) Safranine O staining appraised histopathological characteristics of cartilage tissues. (C) Western blot examined TLR3 expression. ***P < 0.001 versus Control.

Interference with TLR3 Eased Cartilage Injury in ACLT-Induced OA Mice

To deplete TLR3 expression, TLR3 shRNA Lentivirus injected into the articular cavity of OA mice, and as expected, the distinctly upregulated TLR3 expression in the cartilage tissues of OA mice was repressed after infection of Lv-shRNA-TLR3 ( Fig. 2A ). As Figure 2B portrayed, the severe cartilage destruction stimulated by ACLT procedure was evidently alleviated after TLR3 was knocked down. Notably, ACLT surgery significantly resulted in the deterioration on ECM degradation in the mice cartilage tissues, which was then remarkably reverted by downregulation of TLR3 ( Fig. 2C ).

Figure 2.

Interference with TLR3 eased cartilage injury and suppressed ECM degradation in ACLT-induced OA mice. (A) Western blot examined TLR3 expression following lentivirus infection. (B) H&E and (C) Safranine O staining appraised histopathological characteristics of cartilage tissues. ***P < 0.001 versus Control; ###P < 0.001 versus OA+Lv-shRNA-NC.

Interference with TLR3 Suppressed Cartilage Degradation in ACLT-Induced OA Mice

As analyzed by Western blot and immunohistochemical staining, the impacts of TLR3 on cartilage degradation-related proteins were measured and the experimental data delineated that during ACLT operation, MMP3, MMP13, ADAMTS-4 expressions were noticeably raised whereas Collagen II expression was depleted in the cartilage tissues of OA mice. However, after TLR3 was interfered, MMP3, MMP13, ADAMTS-4 expressions were descending and Collagen II expression was ascending in the cartilage tissues of mice subjected to ACLT (Fig. 3A, B).

Figure 3.

Interference with TLR3 suppressed cartilage degradation in ACLT-induced OA mice. (A) Western blot determined the expressions of cartilage degradation-related proteins. (B) Immunohistochemical staining measured MMP-13 expression. ***P < 0.001 versus Control; #P < 0.05, ##P < 0.01, ###P < 0.001 versus OA+Lv-shRNA-NC.

Interference with TLR3 Suppressed NF-κB-Mediated Inflammatory Response and Promoted Autophagy in the Cartilage Tissues of ACLT-Induced OA Mice

In addition, the data from immunohistochemical staining and Western blot exposed that both p-p65 and p-IκBα expressions were both increased in the cartilage tissues of mice during OA modeling, which were both declined again when TLR3 was lowly expressed. Also, no profound changes were noticed on p65 and IκBα expressions among the Control, OA, OA+Lv-shRNA-NC, and OA+Lv-shRNA-TLR3 groups (Fig. 4A, B). Besides, the prominently boosted expressions and levels of pro-inflammatory cytokines including IL-1β, IL-6, TNF-α imposed by ACLT surgery in the cartilage tissues of OA mice were all eliminated (Fig. 4C, D). Moreover, relative to the Control group, autophagy-related LC3Ⅱ/LC3Ⅰ and Beclin-1 expressions were dramatically decreased, whereas p62 expression was promoted in the OA group. Under the circumstance, inhibition of TLR3 caused the increase on LC3Ⅱ/LC3Ⅰ and Beclin-1 expressions and the decline on p62 expression ( Fig. 4E ).

Figure 4.

Interference with TLR3 suppressed NF-κB-mediated inflammatory response and promoted autophagy in the cartilage tissues of ACLT-induced OA mice. (A) Immunohistochemical staining measured p-p65 and p65 expressions. (B) Western blot determined the expressions of NF-κB signaling-related proteins. (C) RT-qPCR and (D) Western blot evaluated the pro-inflammatory cytokine levels. (E) Western blot determined the expressions of autophagic proteins. ***P < 0.001 versus Control; #P < 0.05, ###P < 0.001 versus OA+Lv-shRNA-NC.

TLR3 Was Hyperexpressed upon LPS-Evoked Chondrocyte Inflammation Injury

Simultaneously, upon treatment with LPS at increasing concentrations, the viability of ATDC5 chondrogenic cells was forcefully diminished in a concentration-dependent manner. ( Fig. 5A ). On the contrary, TLR3 protein expression was gradually improved with the increasing dosages of LPS ( Fig. 5B ). In particular, the chondrocyte viability and TLR3 expression were, respectively, the most efficiently declined and enhanced by 5 μg/ml LPS which was then applied for the follow-up experiments.

Figure 5.

TLR3 was hyperexpressed upon LPS-evoked chondrocyte inflammation injury. (A) CCK-8 method appraised cell viability. (B) Western blot examined TLR3 expression. **P < 0.01, ***P < 0.001 versus Control.

Deletion of TLR3 Obstructed LPS-Evoked ECM Degradation in ATDC5 Chondrogenic Cells

Consistently, MMP3, MMP13, ADAMTS-4 expressions were augmented and Collagen II expression was declined in ATDC5 cells when challenged with LPS. Depletion of TLR3 overtly reduced MMP3, MMP13, ADAMTS-4 expressions and elevated Collagen II expression in LPS-treated ATDC5 cells (Fig. 6A, B).

Figure 6.

Deletion of TLR3 obstructed LPS-evoked ECM degradation in ATDC5 chondrogenic cells. (A) Western blot determined the expressions of cartilage degradation-related proteins. (B) Immmunofluorescence staining measured MMP-13 expression. ***P < 0.001 versus Control; ##P < 0.01, ###P < 0.001 versus LPS+shRNA-NC.

Deletion of TLR3 Ameliorated NF-κB-Mediated Inflammatory Response and Activated Autophagy in LPS-Challenged ATDC5 Chondrogenic Cells

Furthermore, absence with TLR3 lowered p-p65 and p-IκBα expressions that were both boosted in LPS-exposed ATDC5 cells ( Fig. 7A ). In addition, IL-1β, IL-6, TNF-α expressions and levels were notably elevated in ATDC5 cells in response to LPS, which were all significantly lessened when TLR3 was downregulated (Fig. 7B, C). Also, LPS challenge caused the downregulation on LC3Ⅱ/LC3Ⅰ and Beclin-1 expressions and the upregulation on p62 expression in ATDC5 cells. However, silencing of TLR3 accelerated LC3Ⅱ/LC3Ⅰ, Beclin-1 expressions and reduced p62 expression in LPS-treated ATDC5 cells ( Fig. 7D ).

Figure 7.

Deletion of TLR3 ameliorated NF-κB-mediated inflammatory response and activated autophagy in LPS-challenged ATDC5 chondrogenic cells. (A) Western blot determined the expressions of NF-κB signaling-related proteins. (B) RT-qPCR and (C) Western blot evaluated the pro-inflammatory cytokine levels. (D) Western blot determined the expressions of autophagic proteins. ***P < 0.001 versus Control; ##P < 0.01, ###P < 0.001 versus LPS+shRNA-NC.

Discussion

Articular cartilage regression is an essential hallmark of OA, for which the compromise of the functions of chondrocytes, the exclusive cellular constituents of articular cartilage, is reckoned as a primary contributing factor. ⁹ The preceding researches have demonstrated the significance of TLR3 signaling in OA through modulating macrophage polarization, joint degeneration.^16,17 Our present work introduced the inhibitory role of TLR3 downregulation in the cartilage degeneration, NF-κB-mediated inflammatory response and the promoting role in the autophagy in both ACLT-induced OA mice and LPS-exposed ATDC5 chondrocytes.

To begin with, ACLT operation was employed in mice. Consequently, the mice cartilage tissues were severely damaged and cartilage degradation occurred, implying the successful establishment of the experimental OA model. Recent evidence has mentioned that TLR3 signaling is activated in OA cartilage and its blockade can protect against OA-like cartilage changes. ¹⁷ Moreover, Li et al. ²⁰ have reported that TLR3 can be activated by dsRNA released from damaged cartilages. Herein, we also consistently illustrated the overexpression of TLR3 in the cartilage tissues of OA mice undergoing ACLT and the ameliorating effects of TLR3 interference on the pathological progression of OA via protecting against cartilage damage and degradation.

LPS is perceived as a pivotal pro-inflammatory factor associated with the pathogenesis of OA and LPS exposure can stimulate inflammatory injury in chondrocytes in vitro. ²¹ Also, the inflammatory injury was induced by LPS in ATDC5 chondrogenic cells in our present job and the cell viability was notably eliminated in response to increasing concentrations of LPS. Previous proof has claimed that TLR3 is expressed in chondrocytes. ¹⁷ Our present results elaborated that LPS treatment resulted in the concentration-dependent upregulation on TLR3 expression in ATDC5 cells, suggesting that TLR3 is activate upon the inflammatory state in chondrocytes.

ECM is a dynamic 3-dimensional network of interconnected macromolecules that provides mechanical support for cells and tissues. ²² In the context of OA, the synthesis and degradation of ECM are mediated by chondrocytes and the metabolic disturbance of cartilage ECM plays a significant role in the disease pathogenesis. ²³ ADAMTS proteases are secreted metalloproteinases that function as pivotal participators in ECM formation, homeostasis and remodeling. ²⁴ Matrix metalloproteinases (MMPs) are a class of enzymes that maintain the balance between cartilage ECM synthesis and degradation. ²⁵ Collagen II is a crucial organic component of the cartilage ECM, which can be degraded by ADAMTS4/5 and MMPs. Previous work has identified TLR3 activation as the main stimulus for MMP3 expression in vitro. ²⁰ During our research, in addition to the depleted MMP3 expression caused by TLR3 knockdown, TLR3 depletion also discovered to dampen ADAMTS-4, MMP13 expressions, and raise Collagen II expression in the cartilage tissues of OA mice and LPS-treated ATDC5 cells.

NF-κB is a versatile and multi-functional transcription factor that acts as a master regulator of the inflammatory reaction during OA.^26,27 As a member of the TLR family, TLR3 can interact with PAMPs and recruit a TIR domain-containing adapter inducing interferon (IFN)-β (TRIF), which initiates TLR signaling to activate NF-κB to induce innate immune responses and the production of pro-inflammatory cytokines. ²⁸ The phosphorylation of NF-κB p65 is the key element in the activation of the NF-κB-signaling pathway. Normally, NF-κB is localized in the cytoplasm and binds to the inhibitory κB (IκB) proteins. The activation of inhibitor of kappa B kinase (IKK) complex can bring about the phosphorylation of IκB proteins. Once IκBα is phosphorylated, NF-κB is freed and activated, translocating into the nucleus and activating the target genes. Mounting literatures have demonstrated that TLR3 can stimulate NF-κB activation. ²⁹ As innate immune cells, mast cells (MCs) and macrophages also play the important roles in the occurrence and progression of OA. Specifically, an increasing number of studies have found that MCs and MCs-secreted inflammatory mediators and cytokines are notably increased in the synovial fluid of OA patients, indicating a potential association between MCs and the onset and progression of synovial inflammation. ³⁰ Activated macrophages can be regulated by NF-κB and are polarized into either M1 or M2 subtypes in OA synovial tissues, synovial fluid, and peripheral blood. The activation state and the M1/M2 ratio are highly associated with OA severity.^31,32 Interestingly, MCs stimulated through engagement of TLR3 are potent regulators of T-cell activities. ³³ Our current work substantiated that p-p65, p-IκBα expressions, TNF-α, IL-1β, IL-6 levels, and expressions that were all elevated in both cartilage tissues of OA mice and LPS-treated ATDC5 cells were all depleted again when TLR3 was interfered, further underlining the anti-inflammatory role of TLR3 inhibition in the course of OA.

Interestingly, autophagy is closely linked with NF-κB activation.^34,35 Autophagy, a cellular homeostatic mechanism involving the clearance of damaged and dysfunctional macromolecules and organelles, has been reported as an adaptive response to exert cytoprotective effects for articular cartilage, and its level is decreased in the process of OA.^36,37 Besides, as an autophagy-positive regulatory protein, Beclin-1 upregulation reflects the enhancement of autophagy process, ³⁸ whereas the autophagy-negative regulatory protein, p62, indicates impaired autophagy. ³⁹ In addition, the increase of the ratio of LC3II to LC3I also indicates the upregulation of autophagy. ⁴⁰ Further research conducted by Gao et al. ⁴¹ have supported that TLR3 can contribute to persistent autophagy in mice after myocardial infarction. On the contrary, in both cartilage tissues of OA mice and LPS-treated ATDC5 cells, we demonstrated that autophagy was activated by deficiency of TLR3, as evidenced by the ascending LC3Ⅱ/LC3Ⅰ, Beclin-1 expressions and the lowered p62 expression.

Nevertheless, there are several limitations in the present study. TLR3 expression in clinical OA samples needs to be detected for further validation. Besides, although TLR3 knockdown was demonstrated to induce autophagy in the context of OA, the causal relationship between TLR3 inhibition and enhanced autophagy remains unclear. Also, considering the importance of MCs and macrophages in the development and severity of OA,^33,42 the specific types of inflammation in the peri-cartilage area caused by the involvement of MCs and macrophages need to be taken into account in our future studies. Hence, further investigations should be performed to evaluate the clinical importance of TLR3 in OA.

Conclusion

In summary, TLR3 inhibition exhibited chondroprotective activity in OA by suppressing ECM degradation, NF-κB-mediated inflammatory response and stimulating autophagy in vivo and in vitro. These findings might highlight the significance of TLR3 participating in the progression of OA and deepen the understandings regarding its relationship of TLR3 with inflammatory response and autophagy.