IL-1beta inhibits TGFbeta in the temporomandibular joint

Abstract

Similarly to humans, healthy, wild-type mice develop osteoarthritis, including of the temporomandibular joint (TMJ), as a result of aging. Pro-inflammatory cytokines, such as IL-1beta, IL-6, and TNFalpha, are known to contribute to the development of osteoarthritis, whereas TGFbeta has been associated with articular regeneration. We hypothesized that a balance between IL-1beta and TGFbeta underlies the development of TMJ osteoarthritis, whereby IL-1beta signaling down-regulates TGFbeta expression as part of disease pathology. Our studies in wild-type mice, as well as the Col1-IL1beta(XAT) mouse model of osteoarthritis, demonstrated an inverse correlation between IL-1beta and TGFbeta expression in the TMJ. IL-1beta etiologically correlated with joint pathology, whereas TGFbeta expression associated with IL-1beta down-regulation and improvement of articular pathology. Better understanding of the underlying inflammatory processes during disease will potentially enable us to harness inflammation for orofacial tissue regeneration.

Figure 1.

Wild-type mice spontaneously develop arthritis-like TMJ pathology. (A) Two-month-old C57Bl6 wild-type mice showed normal TMJ histology (score = 1). However, (B) when they were 6 mos of age, we observed histopathological changes, including loss of normal cyto-architecture, decreased articular cellularity, and chondrocyte cloning (score = 2). (C) The aforementioned joint pathology was partially ameliorated by 12 mos of age (score = 1). (D) Abrogation of IL-1 signaling following targeted deletion of the type I receptor for IL-1 (IL1RI^-/- knockout mice) normalized joint histology (score = 1). (E) IL-1β expression was evaluated by immunohistochemistry and was found to be low in 2-month-old mice, but (F) was found to be increased at 6 mos of age. Furthermore, (G) IL-1β immunoreactivity was found to be reduced at 12 mos of age. (H) Deletion of the IL1RI receptor did not affect IL-1β expression in 6-month-old mice. Moreover, (I) we observed elevated TGFβ expression at 2 mos of age, which (J) was reduced in 6-month-old mice. Moreover, (K) TGFβ immunoreactivity was restored at 12 mos of age. (L) Deletion of the IL1RI receptor restored the aforementioned TGFβ down-regulation in 6-month-old mice. Bar = 100 µm.

Figure 2.

IL-1β expression is associated with articular pathology and TGFβ down-regulation in the TMJ. (A) TMJ pathology was evaluated by a 1-to-4 scale introduced by Wilhelmi and Faust (1976) in the TMJs of wild-type C57/Bl mice over time. (B) We used articular cloning as an additional method of assessing arthritis-like changes in the TMJ. (C) The levels of IL-1β and TGFβ expression, presented as numbers of immunoreactive cells staining positive for these two antigens, are presented. Please note the inverse relationship of IL-1β and TGFβ expression levels in the TMJ, which changes with time. Furthermore, note how increased IL-1β temporally correlates with higher articular pathology levels in 6-month-old wild-type mice, whereas higher TGFβ levels temporally correlate with low articular pathology levels. In total, 26 Col1-IL1β^XAT transgenic mice (13 males and 13 females) were used in this study. Nine Col1-IL1β^XAT transgenic mice received FIV(Cre) injection (5 mice were killed at 2 mos and 4 mice at 6 mos after viral transduction), and 9 received FIV(gfp) in their TMJ (5 mice were killed at 2 mos and 4 mice at 6 mos after viral transduction). In addition, 8 (5 males, 3 females) Col1-IL1β^XAT;IL1RI^-/- compound mice were injected with FIV(Cre) in the TMJ (4 mice were killed at 2 mos and 4 mice at 6 mos after viral transduction). In addition, 13 wild-type mice (7 males, 6 females) were used in this study, with 5 mice killed at 2 mos, 4 mice at 6 mos, and 4 mice at 12 mos of age. An additional 13 IL1RI^-/- knockout mice (5 males, 8 females) were also included in the study: 5 mice were killed at 2 mos, 4 mice at 6 mos, and 4 mice at 12 mos of age. Mean (± SEM). *p < 0.05.

Figure 3.

IL-1β over-expression in the TMJ induces arthritis-like pathology and decreases TGFβ expression. Injection of FIV(Cre) into the TMJs of Col1-IL1β^XAT transgenic mice resulted (A) in the development of arthritis-like pathology, including normal cyto-architecture, decreased articular cellularity, and chondrocyte cloning (score = 3). With time, however, (B) the observed joint pathology ameliorated spontaneously (score = 1). In addition, (C) ablation of IL-1β signaling resulted in the prevention of joint pathology development, as observed in Col1-IL1β^XAT;IL1RI^-/- compound mice (score = 1). (D) Injection of the control vector FIV(gfp), encoding for the reporter gene green fluorescent protein, did not induce any articular changes in the Col1-IL1β^XAT transgenic mice (score = 1). IL-1β immunoreactivity correlated with the development of articular pathology, (E) was increased at the 2-month time-point, and (F) decreased at 6 mos due to transgene silencing. (G) Deletion of the IL1RI receptor did not alter IL-1β expression by articular chondrocytes. (H) Injection of the control vector FIV(gfp) did not IL-1β over-expression in the TMJ. Moreover, we evaluated TGFβ immunoreactivity in the TMJ during the aforementioned conditions and found (I) that TGFβ expression was decreased following the injection of FIV(Cre) into the TMJ of Col1-IL1β^XAT transgenic mice. Conversely, (J) TGFβ expression was normalized in TMJs experiencing transgene silencing 6 mos after FIV(Cre) injection into the TMJ of Col1-IL1β^XAT transgenic mice. (K) TGFβ expression was also restored in Col1-IL1β^XAT;IL1RI^-/- compound mice. (L) Col1-IL1β^XAT transgenic mice injected with the control vector FIV(gfp) showed normal levels of TGFβ immunoreactivity in the TMJ.

Figure 4.

Spontaneous amelioration of joint pathology following IL-1β over-expression in the TMJ of Col1-IL1β^XAT transgenic mice. Transgene activation and IL-1β induction in the TMJ of the Col1-IL1β^XAT mouse model resulted in the development of joint pathology 2 mos after viral transduction. At 6 mos after viral transduction, however, we observed a spontaneous amelioration of the joint pathology, concomitantly with a reduction in human hIL-1β expression in the TMJ, due to apparent transgene silencing. At the same time, the level of TGFβ expression was significantly increased at the 6-month time-point compared with the 2-month time-point (P < 0.001). Deletion of the IL1RI receptor in this mouse model prevented the development of joint pathology, despite the induction of transgenic IL-1β. Abrogation of IL-1 signaling allowed for increased TGFβ levels of expression. In total, 26 Col1-IL1β^XAT transgenic mice (13 males and 13 females) were used in this study. Nine Col1-IL1β^XAT transgenic mice received FIV(Cre) injection (5 mice were killed at 2 mos and 4 mice at 6 mos after viral transduction), and 9 received FIV(gfp) in their TMJs (5 mice were killed at 2 mos and 4 mice at 6 mos after viral transduction). In addition, 8 (5 males, 3 females) Col1-IL1β^XAT;IL1RI^-/- compound mice were injected with FIV(Cre) in the TMJ (4 mice were killed at 2 mos and 4 mice at 6 mos after viral transduction). In addition, 13 wild-type mice (7 males, 6 females) were used in this study, with 5 mice killed at 2 mos, 4 mice at 6 mos, and 4 mice at 12 mos of age. An additional 13 IL1RI^-/- knockout mice (5 males, 8 females) were also included in the study: 5 mice were killed at 2 mos, 4 mice at 6 mos, and 4 mice at 12 mos of age. Mean (± SEM).