TLR4 contributes to the damage of cartilage and subchondral bone in discectomy‐induced TMJOA mice

Abstract The abundance of inflammatory mediators in injured joint indicates innate immune reactions activated during temporomandibular joint osteoarthritis (TMJOA) progression. Toll‐like receptor 4 (TLR4) can mediate innate immune reaction. Herein, we aimed to investigate the expression profile and effect of TLR4 in the cartilage and subchondral bone of the discectomy‐induced TMJOA mice. The expression of TLR4 and NFκB p65 in the synovium of TMJOA patients was measured by immunohistochemistry, Western blotting and RT‐PCR. H&E and Masson staining were utilized to assess the damage of cartilage and subchondral bone of the discectomy‐induced TMJOA mice. A TLR4 inhibitor, TAK‐242, was used to assess the effect of TLR4 in the cartilage and subchondral bone of the discectomy‐induced TMJOA mice by Safranin O, micro‐CT, immunofluorescence and immunohistochemistry. Western blotting was used to quantify the expression and effect of TLR4 in IL‐1β–induced chondrocytes. The expression of TLR4 and NFκB p65 was elevated in the synovium of TMJOA patients, compared with the normal synovium. TLR4 elevated in the damaged cartilage and subchondral bone of discectomy‐induced TMJOA mice, and the rate of TLR4 expressing chondrocytes positively correlated with OA score. Intraperitoneal injections of TAK‐242 ameliorate the extent of TMJOA. Furthermore, TLR4 promotes the expression of MyD88/NFκB, pro‐inflammatory and catabolic mediators in cartilage of discectomy‐induced TMJOA. Besides, TLR4 participates in the production of MyD88/NFκB, pro‐inflammatory and catabolic mediators in IL‐1β–induced chondrocytes. TLR4 contributes to the damage of cartilage and subchondral bone in discectomy‐induced TMJOA mice through activation of MyD88/NFκB and release of pro‐inflammatory and catabolic mediators.


| INTRODUC TI ON
Limited mouth opening, joint pain and noise are the primary performance of temporomandibular joint osteoarthritis (TMJOA) in clinical, affecting the living quality of patients greatly. 1 Although the aetiology of TMJOA remains unknown, excessive loading on joint, such as masticatory muscle overload, malocclusion and jaw asymmetry, has been recognized as one of the main causes. [2][3][4][5][6] Toll-like receptor 4 (TLR4) is a pattern recognition receptor (PRR) which lies on the cell membrane and can mediate innate immune reaction. 7 It is confirmed that the production of endogenous ligands induced by consistent burden or press strain on intervertebral disc cells, chondrocytes, kidney and heart activates TLR4 and promotes secretion of inflammatory mediators. [8][9][10][11] Moreover, up-regulated TLR2 and TLR4 were detected in articular cartilage lesions and synovium of knee OA. 12,13 Meanwhile, elevated expression of TLR2, TLR3, TLR4 and TLR5 was confirmed in human OA cartilage compared with normal cartilage, verifying the role of Tolllike receptors (TLRs) in the innate immune reactions during OA progression. 14 By means of the pathological examination, a couple of inflammatory mediators, including IL-1β, TNF-α and IL-6, have been detected in the tissues of TMJOA. 15 Although frequently appears in bacterial inflammation, including pneumonia, periodontitis and enteritis, the flooding of inflammatory mediators in injured joint indicates innate immune reactions are involved in the development of TMJOA progression. [16][17][18] Tissue damage and chronic inflammation both accounted for the activation of innate immune reactions during knee OA and TMJOA, while compared with the hyaline cartilages of knee joints, fibrocartilages in TMJ condyles display different characteristics regarding pressure-bearing capacities, nutrition supply and constituent. 19 Concerning that TLR4 can initiate immune reactions during the process of long-term burden, it is reasonable to explore the effect of TLR4 in TMJOA, which has been characterized by synovitis, condylar degeneration and osteophyte.
Indeed, recent studies reported that TLR4 elevated in the synovial membrane of temporomandibular joint (TMJ) synovitis induced by occlusal interference, and inhibition of TLR4 by TMJ injection of TAK-242 could alleviate the level of synovitis, suggesting TLR4 participates in TMJ synovitis. 20,21 Animal model is an efficacious approach to investigate the pathology and progression of TMJOA as it is difficult to obtain clinical samples. 15 Discectomy has been accepted as an effective strategy to mimic overloading in the TMJ because discs are responsible for dispersing intra-articular pressure, 22,23 and it is easy to manipulate and duplicate. 24,25 Herein, this study was aimed to detect the alteration of TLR4 in the synovium of TMJOA patients, and to investigate the expression profile of TLR4, its downstream pathways, pro-inflammatory and catabolic mediators in the cartilage as well as subchondral bone of the discectomy-induced TMJOA mice. Furthermore, a specific TLR4 inhibitor was utilized to investigate the effect of TLR4 in the progression of TMJOA both in vivo and in vitro.

| Discectomy-induced TMJOA mice
The discectomy-induced TMJOA model was employed in this study.
Fifty-four male C57BL/6J mice (8 weeks old) from the Experimental Animal Centre of Hubei Province were utilized. Three groups (discectomy, discectomy with TAK, and control) were established, and each was examined for three time points of 2, 4, and 6 weeks (n = 6).
For the discectomy groups, microsurgery was performed by unilateral discectomy of right TMJ according to Lan's method. 24 10 mg/ kg 27 of TAK-242 (HY-11109, MCE, NJ, USA), a specific TLR4 inhibitor interfering with the interactions between TLR4 and its intracellular adaptors, 28,29 was administered by an intraperitoneal injection before the discectomy and maintained twice a week post-surgery for the discectomy with TAK groups. Mice without any treatment were utilized as control. At 2, 4 or 6 weeks post-surgery, the mice in each group were killed, respectively (n = 6). All the procedures of animal experiment were subjected to approval by the Ethics Committee for Animal Research, School and Hospital of Stomatology, Wuhan University, China (protocol No. 00273385).

| Immunohistochemistry
After dewaxing and hydration, the tissue sections were micro-

| RT-PCR
After total mRNA of clinical samples was extracted using TRIzol Kit (Invitrogen) and reversely transcribed, polymerase chain reaction was manipulated to cDNA, with the SYBR Premix Ex Taq (TaKaRa).
Then, gene expression of mRNA was calculated using the cycle threshold method (2 −ΔΔCt ). The primer sequences were as follows:

| Western blotting
The collected and measured proteins were separated by SDS-PAGE electrophoresis and then electro-transferred onto a PVDF membrane. After blocking with non-fat milk, the membrane was incubated with different primary antibodies at 4°C overnight, respectively, including rabbit anti-TLR4 (1:1000), rabbit anti-MyD88 Proteintech). Then, HRP-conjugated secondary antibody was incubated with PVDF membrane, and a chemiluminescence ECL system (Advansta) was utilized to detect the immunoreactive proteins, as described previously. 34

| Elevation of TLR4 and NFκB p65 in the synovium of TMJOA patients
Compared with the normal synovium, the expression of TLR4 and NFκB p65 was enhanced in the synovium of TMJOA patients detected by immunohistochemistry ( Figure 1A), accompanied by the increased rate of TLR4-and NFκB p65-positive cells ( Figure 1B).
Western blotting results exhibited that the bands of TLR4 and NFκB p65 of TMJOA synovium appeared thicker compared with those of normal ( Figure 1C). Similarly, the gene expression of TLR4 and NFκB p65 detected by RT-PCR was significantly higher in TMJOA synovium, compared with that in normal ( Figure 1D) (**P < .01, ***P < .001).

| TLR4 accounts for the damage of cartilage and subchondral bone in discectomyinduced TMJOA mice
As shown in Figure 2, the discectomy groups revealed a timedependent cartilage thinning, unmineralized bone areas enlarging and chondrocytes decreasing in condyle, comparing with control. Immunofluorescence results exhibited that TLR4 expression was elevated gradually in the cartilage and subchondral bone from the discectomy groups ( Figure 2A). Moreover, the rate of TLR4 expressing cells in the cartilage was positively correlated with OA score ( Figure 2D) (*P < .05, **P < .01, ***P < .001).
Besides, to further explore the effect of TLR4 in the injured cartilage and subchondral bone of TMJOA, a specific TLR4 inhibitor, TAK-242, was administered intraperitoneally before F I G U R E 1 Elevation of TLR4 and NFκB p65 in the synovium of TMJOA patients. A and B, Representative sections of TMJOA or normal synovium were detected by immunohistochemistry (n = 6). TMJOA synovium showed significant and homogeneous positive staining of TLR4 and NFκB p65, while normal synovium exhibited inconspicuous staining (A). The rate of TLR4-or NFκB p65-positive cells increased in TMJOA groups compared with normal (B). C, Western blotting results exhibited that the bands of TLR4 and NFκB p65 of TMJOA synovium appeared thicker compared with those of normal. D, The expression of TLR4 or NFκB p65 gene was increased in the synovium of TMJOA compared with normal by RT-PCR (n = 4). Data were analyzed by Mann-Whitney U test (**P < .01, ***P < .001) (OA represents TMJOA) the discectomy and maintained twice a week in this study. By Safranin O staining, a time-dependent reduction of proteoglycans occurred in the cartilage of discectomy groups as compared with that of control, which maintained abundant and regular proteoglycans ( Figure 3A,B). Furthermore, TAK-242 injections significantly ameliorate the lessening of cartilage proteoglycans induced by TMJ discectomy (Figure 3A

| TLR4 promotes the expression of MyD88/ NFκB, pro-inflammatory and catabolic mediators in mice cartilage of discectomy-induced TMJOA
Contrasting with control, the expression of MyD88 and NFκB p65 increased in the cartilage of discectomy groups at all time points, with more positive cells found in the fibre layer ( Figure 4A,B).

F I G U R E 2
Elevation of TLR4 positively correlates with the damage of cartilage and subchondral bone in discectomy-induced TMJOA mice. A, Compared with control, there was a time-dependent cartilage thinning (two-way arrow), chondrocytes decreasing (black arrow) and unmineralized bone areas increasing (black arrowhead), as well as elevation of TLR4 in the cartilage and subchondral bone of discectomy groups. B, Comparison between control and discectomy groups regarding cartilage thickness (*: vs CON, horizontal solid lines, medians; box plots, 25th and 75th percentiles; horizontal bars, minimum and maximum). C, Gradually increased the percentages of unmineralized bone areas in the subchondral bone of discectomy mice comparing with control mice (*: vs CON). D, The expression of TLR4 was increased in the cartilage of discectomy groups gradually. The rate of TLR4 expressing cells in the cartilage was positively correlated with OA score (*: vs 0W). (Data were calculated at each time point using Student's t test, n = 6, *P < .05, **P < .01, ***P < .001). (CON represents control; OA represents TMJOA)

| D ISCUSS I ON
Activation of the innate immune system is involved in the initiation and perpetuation of tissue damage or chronic inflammation. As a classic innate immunity mediator, TLR4 has been currently found to be elevated in rat TMJ synovitis induced by occlusal interference, F I G U R E 3 TLR4 participates in the damage of cartilage and subchondral bone in discectomy-induced TMJOA mice. A, The arrangement of control cartilage was regular, and its matrix was abundant, in contrast, obvious disorder occurred in the cartilage of discectomy groups, while appeared mitigated after twice a week of TAK-242 injections. B, The percentage of proteoglycan gradually decreased in the discectomy groups compared with control, whereas TAK-242 injections significantly ameliorate the reducing of cartilage proteoglycans induced by discectomy alone (*: discectomy groups vs control; #: discectomy + TAK groups vs discectomy groups). C, Larger bone lesion of condyle (red arrows) and obvious atypical condyle shape were found in the micro-CT image of discectomy groups, while these phenomena were ameliorated after twice a week of TAK-242 injections. D-G, The remodelling of subchondral bone was assessed by histomorphometric parameters, including ratio of bone volume to tissue volume (BV/TV), trabecular thickness (Tb. Th), trabecular number (Tb. N) and trabecular space (Tb. Sp). (Data were analysed by Student's t test, n = 3-6, *P < .05, ***P < .001; #P < .05, ##P < .01, ###P < .001). (CON represents control; Discectomy + TAK represents discectomy combined with TAK-242 injection groups) and synovitis mitigated by TMJ injection of TAK-242. 20 In ex vivo experiments of LPS incubated TMJ synovial fibroblasts, pretreatment of TAK-242 could decline the elevated expression of inflammatory factors. 35 In the present study, TLR4 was highly expressed in the synovium of TMJOA patients, compared with condylar hypertrophy patients' synovium, which has been identified as a noninflammatory normal synovial specimens by our histochemical professionals according to the criteria of Murakami. 26 In terms of patients with condylar hypertrophy, mandible deviation is the striking feature in clinic, while these patients exhibit no pain and normal mouth opening, whose synovium has been verified and used as normal in our former study, 5 as normal synovium cannot be acquired from normal human beings. Meanwhile, an augmented expression of TLR4 occurred in mouse cartilage and subchondral bone of discectomy-induced TMJOA. During 6 weeks after the discectomy surgery, there was a time-dependent cartilage thinning and unmineralized bone areas enlarging, as well as cartilage OA score increasing, which was consistent with predecessor's observation. 24,36 However, regarding the synovitis induced by discectomy, there is no obvious inflammation in the synovium based on our observation (data not shown), speculating two reasons for this phenomenon. One is that discectomy cannot jeopardize synovium, and the other is that synovial inflammation has vanished before samples were harvested at 2 weeks. Notably, cartilage OA score was positively correlated with the elevation of TLR4. Furthermore, when TAK-242 injected intraperitoneally, the damage of mouse cartilage and subchondral bone induced by TMJ discectomy was remarkably attenuated. Therefore, these findings indicate that TLR4 contributes to synovitis, the degeneration of cartilage and breakdown of subchondral bone, which are key features of TMJOA.
Accompanied by the elevation of TLR4, the expression of NFκB p65 was also up-regulated in the synovium of TMJOA patients.
Former literatures reported that TLR4 binds to its corresponding ligands, followed by activating MyD88 and ultimately leading to the activation as well as nuclear translocation of the NFκB. 37,38 This prompted us to postulate that substantial TLR4 in the cartilage  It should be noted that TAK-242 administered by intraperitoneal injection can approach subchondral bone and synovium through blood transport. Undoubtedly, there is no vessel in normal cartilage.
However, under the circumstance of TMJOA, obvious vascular invasion occurs in the damaged cartilage. 53 Therefore, TAK242 can reach synovium, cartilage and subchondral bone of TMJOA mice by F I G U R E 5 TLR4 promotes the expression of cartilage pro-inflammatory and catabolic mediators in discectomy-induced TMJOA mice. A, The expression of IL-1β, TNF-α, ADAMTS5 and MMP13 was detected by immunohistochemistry in the cartilage of discectomy groups, discectomy with TAK-242 treatment groups at week 6, or in the control cartilage. B, The rate of IL-1β, TNF-α, ADAMTS5 or MMP13 positive cells in the cartilage was markedly increased in the discectomy groups, while markedly inhibited after treatment with TAK-242. (Data were analysed by Student's t test, n = 6, **P < .01, ***P < .001). (CON represents control; Discectomy + TAK represents discectomy combined with TAK-242 injection groups) blood transport, and target against TLR4 effectively. Meanwhile, it is reasonable to assume that catabolic mediators from synovial fluid are lessened because TAK242 inhibits the production of TLR4 and the downstream inflammatory mediators in the synovium, thereby resulting in the attenuation of the damage of cartilage and subchondral bone. Therefore, the above two reasons can explain our in vivo experiments using intraperitoneal injection of TAK-242.
In the present study, the elevation of TLR4 was accompanied by the augmentation of MyD88, NFκB p65 as well as pro-inflammatory and catabolic mediators both in discectomy-induced TMJOA mice and in IL-1β-induced chondrocytes, and vice versa. Although these findings cannot illustrate the explicit mechanism that how TLR4 manipulates TMJOA progression, it is strongly indicated that TLR4 contributes to the damage of cartilage and subchondral bone during TMJOA progression through activation of MyD88/NFκB to release the pro-inflammatory and catabolic mediators, as the role of TLR4/ MyD88/NFκB signal pathway in regulating inflammatory mediators has been specified in varied diseases. [54][55][56][57]

ACK N OWLED G EM ENTS
This research was supported by National Science Foundation of China (No. 81771100, No. 81870789).

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.  F I G U R E 6 TLR4 participates in the production of MyD88/NFκB, pro-inflammatory and catabolic mediators in IL-1β-induced chondrocytes. A, A dose-dependent augmentation of TLR4, MyD88 and NFκB p65 in rat chondrocytes was induced by IL-1β for 24 h. B, A time-dependent increase of TLR4, MyD88 and NFκB p65 in these cells was induced by 10 ng/mL of IL-1β. C and D, Cells were pre-treated with 10 μmol/L TAK-242 for 3 h, and then, 10 ng/mL of IL-1β was treated for 24 h. The production of TLR4, MyD88, NFκB p65, MMP3, MMP13, TNF-α and COX-2 was detected by Western blotting. (E) Proteins relative optical density normalized to GAPDH was calculated. IL-1β promoted the production of pro-inflammatory mediators (TNF-α, COX-2) and catabolic mediators (MMP3, MMP13) in chondrocytes, whereas pretreatment of TAK-242 significantly reduced the production of MyD88/NFκB and above mediators induced by IL-1β (#: IL-1β group vs Con group, *: IL-1β + TAK group vs IL-1β group. Data were analysed by Mann-Whitney U test, n = 3, ##P < .01, ###P < .001; *P < .05, **P < .01, ***P < .001)

DATA AVA I L A B I L I T Y S TAT E M E N T
The data supporting the findings of this study are available from the corresponding author upon reasonable request.