| 1 Background and PurposeTuberculosis(TB) represents a challenging public health problem across the world. According to the World Health Organization, one-third of the world’s population is believed to be infected with the causative bacterium Mycobacterium tuberculosis. Spinal TB is the most common form of bone TB in developing countries. Numerous articles have been published on spinal TB in recent decades. Spinal TB usually affects the intervertebral discs, leading to the destruction of spinal stabilization, adjacent vertebral bodies, and surrounding soft tissue. Although spinal TB is common, little information is available on the inflammatory and immune mechanisms involved in its development. In particular, it is unclear which cells and mediators are involved in the intervertebral disc destruction processes and whether resident immunocompetent cells orchestrate the development of an inflammatory response. The interleukin(IL) family plays important roles in inflammatory and immune responses to TB. The role of such factors in the pathogenesis of intervertebral disc tuberculosis destruction is particularly deserving of elucidation.The ILs comprise a large group of immunomodulatory proteins that elicit awide variety of responses in cells and tissues. ILs can exert both inflammatory and anti-inflammatory effects. A few members act as chemoattractants for helper T cells, paralleling the actions of chemokines. Others are intimately involved in the cellular response to viral pathogens, making them akin to interferons(IFNs). ILs are extremely important mediators of the physiological response to infection, and they also contribute significantly to the pathophysiology of a wide range of disorders.As such, they represent a group of proteins with potential importance as therapeutic targets.IFN-gamma, TNF, and matrix metalloproteinases are important inflammatory markers in the pathogenesis of TB. Some published reports investigated their role in the pathogenesis of TB.However,there are few published data on the role of ILs in the pathogenesis of tuberculous intervertebral discs, particularly in human samples. Based on the aforementioned background,the interleukins(ILs; IL-9, IL-10, IL-12, IL-13, IL-16, IL-17, IL-18, IL-26, and IL-27) selected in this study were chosen because they are the most commonly studied members of the IL family. ILs are assigned to each family based on sequence homology and receptor chain similarities or functional properties. Members of the major subgroups of ILs were included in the analysis, e.g, the IL-1 family(IL-18), the common γ chain family(IL-9), the IL-10 family(IL-10, IL-26), the IL-12 family(IL-12, IL-27), the Th2-like cytokines(IL-13), the ILs with chemokine activity(IL-16), and the IL-17 family(IL-17). Therefore, a quantitative molecular analysis of the transcript levels of a large number of ILs(IL-9, IL-10, IL-12, IL-13, IL-16, IL-17, IL-18, IL-26, and IL-27) will provide insight into the molecular mechanism that regulates the expression of ILs in human tuberculous intervertebral discs.2 Methods2.1 TB disc tissue samples were obtained from 70 patients that underwent surgery for spinal TB in our hospital from 1 Jan 2013 to 30 December 2014. These patients were assigned into the TB group. After excision, two experienced pathologists examined the tissue samples. Seventy herniated disc specimens were also collected from seventy patients that underwent surgery for intervertebral disc(ID) herniation. These patients were included in the ID herniation group. Ten fresh human cadaver intervertebral disc specimens were assigned to the control group. The specimens were obtained within 10 h after death. Patients with spinal diseases(e.g, ID herniation, spinal TB, etc.) were excluded from the control group. Samples were obtained after surgery, immediately frozen in liquid nitrogen, and maintained at-80°C until RNA extraction.2.2 Using RNA extraction technology and real-time fluorescence quantitative PCR(Real time fluorescent quantitative PCR, RT-PCR), research and analysis of ILs transcription level in tuberculous intervertebral disc tissue, degenerative disc and normal disc tissues and differences.2.3 The following data were collected for the three groups: age, gender, employment, smoking habits, pain intensity(visual analog scale score, VAS), level of ID, duration of symptoms,C-reactive protein(CRP), and erythrocyte sedimentation rate(ESR).3 Results3.1 IL m RNA expression IL-9, IL-13, and IL-26 were not amplified in the three groups. The IL-10, IL12, IL-16, IL-17, IL-18, and IL-27 amplification curves revealed that the cycle threshold(Ct) values were within an acceptable range. The melting curves illustrated the specificity of the primers. The standard curves were plotted as the Ct versus the concentration of the total RNA from serial dilutions.IL mRNA was not detected in all tissue samples, but the detection rates of the ILs examined in this study did not differ significantly among the three groups(Table 3). Significantly higher m RNA expression in the TB group than in the control group was detected for IL-10, IL-16, IL-17, IL-18, and IL-27(P < 0.05). The m RNA expression of IL-10, IL-16, IL-18, and IL-27 m RNA was higher in the TB group than in the ID herniation group(P < 0.05). Our data also demonstrated that patients with more severe disease had significantly higher IL-16, IL-17, and IL-18 expression and significantly lower IL-10 and IL-27 expression.3.2 Pairwise m RNA co-expression analysis Multiple positive correlations were found in the TB and ID herniation groups concerning the m RNA expression of ILs. Specifically, in the TB group, IL-18 transcript levels were positively correlated with IL-16 and IL-17 expression, whereas IL-10 and IL-27 transcript levels were negatively correlated with IL-16, IL-17, and IL-18 levels. Furthermore, IL-16 and IL-17 m RNA were also found to be co-expressed in the TB group. Similarly, IL-10 and IL-27 m RNA were observed to be co-expressed in the TB group.3.3 Correlation analyses Sex, employment, and the level of intervertebral disc herniation did not significantly affect the m RNA levels of the ILs included in the present study.Age was positively correlated with elevated IL-18 m RNA levels in the TB and ID herniation groups(P = 0.023 and P = 0.007, respectively). Smoking habits were found to be positively correlated with the m RNA levels of IL-17 and negatively correlated with the m RNA levels of IL-10 in the TB group(P = 0.008 and P = 0.041, respectively).Pain intensity as assessed by a VAS significantly affected the transcript levels of IL-10, IL-16, IL-17, IL-18, and IL-27(P = 0.009, P = 0.007, P = 0.034, P = 0.016, and P =0.008, respectively). In particular, patients with intense pain(VAS score between 8 and 10) exhibited higher IL-16, IL-17, and IL-18 m RNA levels than patients with mild pain(VAS score between 0 and 4). Meanwhile, patients with mild pain exhibited higher IL-10 and IL-27 m RNA levels than patients with intense pain.The data revealed significant correlations between the duration of symptoms and the m RNA levels of IL-10, IL-16, IL-17, IL-18, and IL-27 in the TB group(P = 0.014, P = 0.033, P = 0.037, P = 0.006, and P = 0.009, respectively). Specifically, tissue samples obtained from patients who experienced pain for 3–12 months exhibited significantly higher IL-16, IL-17, and IL-18 m RNA levels than those who experienced pain for <3 months. Conversely, patients who experienced pain for <3 months exhibited significantly higher IL-10 and IL-27 m RNA levels than those who experienced pain for 3–12 months.CRP levels and the ESR were significantly correlated with the m RNA levels of IL-10, IL-16, IL-17, and IL-27 in the TB group(CRP: P = 0.018, P = 0.007, P = 0.024, and P = 0.009, respectively; ESR: P = 0.037, P = 0.041, P = 0.003, and P = 0.008, respectively). The m RNA levels of IL-16 and IL-17 were significantly higher in patients with high CRP levels(>30 mg/L), whereas the m RNA levels of IL-10 and IL-27 were significantly higher in patients with low CRP levels(<10 mg/L). Significantly higher IL-16 and IL-17 m RNA levels were observed in tissue samples obtained from patients with high ESRs(>40 mm/h) compared to those in patients with low ESRs(<20 mm/h). The m RNA levels of IL-10 and IL-27 were significantly higher in patients with low ESRs(<20 mm/h).The data also illustrated significant associations among pain intensity, symptom duration, smoking, CRP levels, and ESRs. Specifically, CRP levels and ESRs were found to be positively correlated with the intensity of pain(P = 0.024 and P = 0.019, respectively). Furthermore, smoking was positively associated with pain intensity(P = 0.008).4 ConclusionIn conclusion, this study provides evidence of the molecular profile of ILs in tuberculous spinal intervertebral discs. IL-10, IL-16, IL-17, IL-18, and IL-27 are more strongly expressed in tuberculous spinal intervertebral disc tissue than in normal intervertebral disc tissue. This suggests that ILs play important roles in the intervertebral disc destruction associated with spinal TB. Multiple correlations among IL-10, IL-16, IL-17, IL-18, and IL-27 m RNA levels were found in tuberculous spinal disc tissue and imply a synergistic effect of the activity of these ILs in tuberculous spinal intervertebral discs. Smoking habits were found to have a positive correlation with the transcript levels of IL-17 and a negative correlation with the transcript levels of IL-10, suggesting multiple roles for smoking in the intervertebral discs destruction process of spinal TB. |
