Inhibition Of Inflammatory Response By Using IL-10 And TGF-β: A Biological Therapy For Intervertebral Disc Degeneration In A Beagle Model

Background and Purpose: Lower back pain(LBP) is a significant medical problem for over half of the adult population worldwide. A key factor that contributes to LBP is degenerative disc disease(DDD), which can result in debilitating pain and reduce the quality of life for LBP patients as well as inflicting significant financial burdens due to lost productivity and increased health care costs. The main cause of DDD is intervertebral disc degeneration(IDD). Effective clinical management of LBP caused by IDD is challenging, and current therapies for LBP caused by IDD only address symptomatic relief for the patient and not the underlying cause of the disease. A better understanding of the mechanisms that cause IDD are needed to enable the development of effective disease-modifying therapies that block the biochemical and pathophysiologic processes mediating this degeneration. Biological therapeutic approaches appear to be promising strategies to consider since proinflammatory cytokines play a role in the development of IDD. Mechanical stress can increase the release of various cytokines within the intravertebral disk(IVD) region. More importantly, inflammation contributes to IDD and the inflammatory cytokines produced are associated with progression of IDD. A successful therapeutic approach should promote anabolism and cell proliferation in addition to controlling inflammation and the resulting tissue damage. Tumor necrosis factor(TNF)-α significantly reduces anabolism in IVD and induces cell senescence. Interleukin(IL)-1β plays a crucial role in IDD by inducing proteoglycan breakdown and inhibiting matrix biosynthesis by IVD cells. IL-1β is produced in response to infection, injury. IL-1β also increases catabolic enzyme activity, and other studies in human systems have shown that IL-1β inhibitors can affect signaling pathways in models of IDD.IL-10 inhibits inflammatory cytokine synthesis and is has been reported that in vitro TNF-α secretion in cells is suppressed more than 90% after IL-10 treatment. IL-10 plays an important role in modulating the expression of immune effector molecules. Numerous studies have shown the protective effect of transforming growth factor(TGF)-β in IDD. TGF-β also has key anti-inflammatory properties, such as suppressing the ability of monocytes/macrophages to release inflammatory cytokines. Additionally, the transfer of plasmid DNA encoding TGF-β was shown to suppress inflammatory lesions in a rat model of arthritis. TGF-β also up-regulates IL-10 synthesis by mouse macrophages and rat hepatic stellate cells.Although IL-10 and TGF-β can inhibit synthesis of inflammatory cytokines, few studies have evaluated the therapeutic potential of IL-10 and TGF-β as anti-inflammatory mediators in IDD. We evaluated the potential of IL-10 and TGF-β to inhibit the release of TNF-α and IL-1β from degenerative NP cells to determine if combining these two biologics might be a promising approach for the treatment of IDD.Methods: Six healthy 1-year-old male beagle dogs(10–10.5 kg) were used. Digital radiography was performed to exclude animals with vertebral abnormalities in the spine. T2-weighted MRI of lumbar discs in the sagittal plane were obtained. The beagle model of IDD was performed. A surgical incision was made in each animal posterolateral from L2 to L5, followed by an annular puncture with a 16-gauge needle on the L2–L3, L3–L4, and L4–L5 discs. T2-weighted MRI studies were conducted on all animals after disc puncture and revealed substantial disc degeneration 6 weeks later. Similar changes were seen in all study animals at this time point. Once substantial disc degeneration was confirmed upon magnetic resonance imaging analyses. The L1–L2, L2–L3, L3–L4, and L4–L5 intervertebral discs were incised under aseptic conditions and NP tissues were obtained. L2–L3, L3–L4, and L4–L5 NP tissues were evenly mixed as integral degenerative NP tissues for further cell culture. The L1–L2 discs were used to obtain normal cells(no annular puncture). NP tissues were diced into 2-mm3 pieces and digested in 0.2% pronase for 60 min at 37 °C and then digested overnight. Cells were cultured in 75cm2 cell culture flasks in a culture medium. All NP cells were used within the first three passages. A total of 2 × 107 NP cells were obtained from the L1–L2 normal disc, and a total of 6 × 107 NP cells were obtained from the L2–L5 combined degenerative discs. Cultured degenerative NP cells were divided into four treatment groups(n = 6 per treatment group, one for each animal: untreated degenerative NP cell group; degenerative NP cells treated with IL-10(IL-10 group) or TGF-β(TGF-β group) or both(IL-10 + TGF-β group) for 6, 12, 24, or 48 h at a concentration of 20 ng/m L for each cytokine. Untreated normal NP cells from the L1–L2 disc(n = 6, one for each animal) served as the negative control group. To analyze the expression of intracellular inflammatory cytokines at the various time points after treatment with cytokines, cells from each group were counted and cell suspension was added to Eppendorf tubes. Fluorescein isothiocyanate-labeled anti-TNF-α monoclonal antibodies were added to the tubes and the contents mixed thoroughly. The mean fluorescence intensity was calculated to detect NP cells expressing TNF-α. After treatment with corresponding cytokines for 12, 24, and 48 h, IL-1β and TNF-α in the culture supernatants of each group were measured with enzyme-linked immunosorbent assay kits for quantification. The detection sensitivity limit was at least 5 pg/m L for IL-1β and TNF-α. To analyze the m RNA expression levels of IL-1β and TNF-α, RNA was extracted, and DNA was digested, then c DNA was synthesized. The transcripts were quantified with real-time PCR using an ABI PRISM 7500 Sequence Detector with Applied Biosystems predesigned Taq Man Gene Expression Assays and reagents according to the manufacturer’s instructions. For relative quantification, gene expression in the samples was normalized to ?-actin m RNA expression using the 2-??Ct method. The data were analyzed by using Stata 12.0 statistical software for single-factor ANOVA. Pairwise comparisons between each group were analyzed with the Bonferroni method, and the mean ± SD was determined.Results: The cell count and MFI of each group were determined by flow cytometry at different time points. Compared with untreated degenerative NP cells, the number of cells positive for TNF-α in the IL-10 and IL-10 + TGF-β treatment groups decreased sharply at 12 h after treatment. The expression of TNF-α in untreated normal NP cells as well as in IL-10 and IL-10 + TGF-β treated NP cells was significantly lower(p < 0.01) compared with the untreated degenerative NP cell group. After 24 h, the MFI values for TNF-α in the normal NP cells group and all three treatment groups were significantly lower(p < 0.01) relative to untreated degenerative NP cells. Treatment with both IL-10 and TGF-β resulted in significantly lower TNF-α expression(p < 0.01) than treatment with TGF-β or IL-10 alone, and TNF-α levels were similar to levels observed in the normal NP cells group. The number of cells positive for TNF-α in the TGF-β group and the IL-10 + TGF-β group reached their lowest levels 48 h after treatment, and the MFI values in these two groups were significantly lower(p < 0.01) compared with untreated degenerative NP cells, resulting in TNF-α levels that were similar to levels for untreated normal NP cells. Using ELISA, we measured IL-1β and TNF-α levels in the supernatant of normal NP cells in each treatment group at 12, 24, and 48 h. These levels were compared to the levels expressed by untreated degenerative NP cells. In the IL-10 + TGF-β group, TNF-α and IL-1β levels were significantly lower at 24 and 48 h, as compared with the untreated degenerative NP cells group. IL-1β levels were the lowest 24 h after treatment and slightly increased at 48 h. IL-1β and TNF-α levels in the IL-10 + TGF-β treatment group were significantly lower(p < 0.01) at 24 and 48 h, whereas the corresponding levels in the normal NP cells group were significantly lower(p < 0.01) at 12, 24 and 48 h. The expression of TNF-α and IL-1β m RNA was suppressed by treatment with IL-10 and TGF-β. After treatment with TGF-β, the expression of TNF-α and IL-1β m RNA was maximal at 6 h and then decreased gradually from the 6 to 24 h time points. Compared with untreated degenerative NP cells, TNF-α and IL-1β m RNA levels(2-ΔΔCt values) in untreated normal NP cells and the TGF-β treatment group were significantly lower(p < 0.01) at 12 and 24 h. In contrast, a rapid decrease in TNF-α and IL-1β m RNA levels in the IL-10 or IL-10 + TGF-β treatment groups was seen at 6 h and persisted up to the 24 h time point. Compared with untreated degenerative NP cells, TNF-α and IL-1β m RNA levels in untreated normal NP cells, IL-10, and IL-10 + TGF-β treatment groups were significantly lower(p < 0.01) at 6, 12, and 24 h.Conclusions: We succeed in puncture and induce intervertebral disc degeneration. In this study, TGF-β acted slowly, requiring 12 to 24 h to exert an inhibitory effect, which is consistent with suppression of the translation of cytokine m RNA. IL-10 acted at an early step in cytokine production and markedly suppressed TNF-α and IL-1β m RNA levels, having a more rapid effect than TGF-β, showing inhibition of cytokine expression as early as 6 h after treatment. The experiments described here demonstrated that TGF-β and IL-10 have anti-inflammatory properties in IDD, suppressing IL-1β and TNF-α production, and that there is an interaction between TGF-β and IL-10 since the combination of the two has greater effect than either as a single agent. The expression of inflammatory cytokines in degenerative IVDs can be blocked by using exogenous TGF-β and IL-10, which could have therapeutic benefit in this disorder. IL-10 and TGF-β treatment suppressed the expression of IL-1β and TNF-α and inhibited the development of inflammatory responses. IL-10 and TGF-β could be a therapeutic approaches for the treatment of IDD.