The Role Of IL-15in The Pathogenesis Of Idiopathic Inflammatory Myopathies

Idiopathic inflammatory myopathies are a group of heterogeneous chronic inflammatory disorders clinically characterized by proximal skeletal muscle weakness and muscle fatigue, and histologically featured by mononuclear inflammatory cell infiltrates in the muscle tissues. Generally, they can be classified into three different subgroups:polymyositis, dermatomyositis and inclusion body myositis. The pathogenesis and etiology of these groups of diseases are yet not fully understood untill now, although different mechanisms have been suggested including the roles of inflammatory cells, cytokines, major histocompatibility complex class I et al. The treatment of these diseases is aimed to improve the muscle functions and enhance the living quality of the patients. High-dose glucocorticoids are still regarded as the most important and efficient therapy in these diseases. However, not every patient responds well to glucocorticoids. Therefore, the conventional immunosuppressive treatment is usually added in the treatment strategy. This combine treatment improved the clinical outcome of the patients and also benefits the patients by lowering the risk of side effects due to reducing the amount of glucocorticoids. But some patients are still resistant to this treatment, and it is highly demanded to develop more efficient and individual therapy in the future.The role of cytokines in the pathogenesis of patients with idiopathic inflammatory myopathies has been highlighted during the last decade mainly because of the finding of the overexpression of interleukin-la, interleukin-1β, tumor necrosis factor a, high mobility group box protein1in the muscle tissues of the patients. Moreover, the level of these cytokines was found correlated with the muscle dysfunction in the patients with idiopathic inflammatory myopathies. However, the biological treatments using these cytokines, such as Humira (anti-tumor necrosis factor a) or anakinra (interleukin-la receptor antagonist) which can neutralize the effects of tumor necrosis factor a or interleukin-1respectively, unfortunately, are not so impressive. The clinical outcomes of these treatments are variant and sometimes are contradictory with each other. Hence, it suggested that some other cytokines instead of these "classic" ones could be involved in the pathogenesis of the diseases.Interleukin-15belongs to the4a-helix bundle cytokine family with some structural homology to interleukin-2. Originally, it was described as a T-cell-activating factor sharing some similar functions with interleukin-2, but later its roles during development of different cells were revealed, such as natural killer cells, natural killer-T cells, and intestinal intraepithelial lymphocytes. Interleukin-15meditates its functions via interleukin-2Rα, common gamma chain (γc) and its unique receptor (interleukin-15receptor α) which has a very high affinity to interleukin-15.Both interleukin-15and interleukin-15receptor α have a widespread expression at transcriptional level in a variety of normal human tissues and cells including skeletal muscle. But due to strict posttranscriptional regulations, interleukin-15and interleukin-15receptor α are rarely detected at protein level. Recently, different groups have reported interleukin-15expression in different tissues of patients with autoimmune or inflammatory disorders, such as in rheumatoid arthritis where interleukin-15protein was demonstrated in synovial fluids and synovial membranes. In idiopathic inflammatory myopathies, one report based on three polymyositis and two dermatomyositis patients showed that interleukin-15was predominantly expressed in muscle fibers and interleukin-15was also expressed in myoblasts derived from these patients. High serum levels of interleukin-15in polymyositis and dermatomyositis patients have also been reported. All these observations suggest a potential role of interleukin-15in idiopathic inflammatory myopathies.The objective of our study in this thesis is to investigate the expression of interleukin-15and interleukin-15receptor alpha in muscle tissue from patients with polymyositis or dermatomyositis before and after conventional immunosuppressive treatment. And also to investigate the expression interleukin-15in the more differentiated myotubes.In order to investigate these hypotheses, we used muscle biopsy samples from seventeen patients with polymyositis or dermatomyositis before and after conventional immunosuppresive treatment. And as controls, muscle biopsy samples from four patients with muscle dystrophy and seven healthy individuals were also investigated by immunohistochemistry using antibodies against interleukin-15and Interleukin-15receptor α. The characteristics of the inflammatory cell infiltrates were also demonstrated by immunohistochemistry by the antibodies agaist CD68(a marker for total macrophages), CD163(a marker for activated macrophages), CD3(a marker for T cells), NKp46(a marker for natural killer cells). Quantification of these stainings was performed by computerized image analysis. Interleukin-15and interleukin-15receptor α was quantified by the percentage of the positive stained cells of total counter stained cells; while macrophages, T cells, natural killer cells were quantified by the percentage of total positive stained area of the total tissue area. Cellular localization of interleukin-15was determined by double staining using immunofluorescence. Clinical outcome was measured by the functional index, manual muscle test and serum creatine phosphokinase. Human myosatellite cells were separately from fresh human muscle tissues and culture into myoblasts first and then these myoblasts were further differentiated into myotubes. Later, interleukin-15staining was performed on these myotubes by immunocytochemistry.Our results demonstrated that interleukin-15was mainly observed in mononuclear inflammatory cells of muscle tissue from the patients with polymyositis or dermatomyositis, occationally interleukin-15was also found in the capillary and the endothelial cells of large vessels, Interleukin-15receptor a was localized to mononuclear inflammatory cells, capillaries and large vessels. Macrophages and T cells are observed in the muscle tissues in a large number, but natural killer cells are rarely observed. Double staining showed the localization of interleukin-15to CD163+macrophages. Immunofluorescent staining also showed that the interleukin-15positive cells are closely localized to CD3+T cells. The number of interleukin-15positive cells is correlated with T cells. Both interleukin-15and interleukin-15receptor a are also shown in the muscle tissues of patients with muscle dystrophy with a similar pattern as in patients with idiopathic inflammatory myopathies. Both interleukin-15and interleukin-15receptor a can be detected in the muscle tissues of the healthy individuals with an occasional level. An evidently significant larger number of interleukin-15and interleukin-15receptor a positive cell were shown in muscle tissue of patients compared to healthy individuals. The baseline interleukin-15expression correlated negatively with muscle function improvement. After conventional immunosuppressive treatment, a significantly lower number of interleukin-15and interleukin-15receptor a positive cells was found. Macrophages were also decreased after conventional immunosuppressive treatment but the level of T cells are sustainable although there is a trend there. However, compared to interleukin-15levels in healthy controls, eight out of seventeen patients still had much more interleukin-15positive cells in their muscle tissues and interestingly less muscle function improvement was shown in this group of patients both from short term observation and long term observations. Human differentiated myotubes were the dominated cells in our culture and they were stained with anti-interleukin-15antibody by immunocytochemistry and the result showed that myotubes were negatively stained for interleukin-15, while myoblasts were positively stained by anti-interleukin15anitbodies which is in line with the result as shown by the previous study. The novelty of this report compared to previously published studies is that:(a) interleukin-15and interleukin-15receptor a expression in different cell types was studied;(b) interleukin-15and interleukin-15receptor a expression were re-assessed after conventional immunosuppressive treatment in a repeat muscle biopsies; and (c) interleukin-15and interleukin-15receptor a expression were correlated with clinical findings and we found that patients with a high number of interleukin-15expressing cells in muscle tissue after a median of eight months with conventional immunosuppressive treatment had less improvement in muscle performance both in a short term and long term perspective.Interleukin-15expression in macrophages in muscle biopsies from myositis patients is consistent with observations in synovial tissues from rheumatoid arthritis patients. This is not specific for inflammatory myopathies as it was also detected in patients with muscle dystrophies. We could not detect any interleukin-15expression in T cells using double staining, arguing against the T cells being a source of interleukin-15in patients with idiopathic inflammatory myopathies. In contrast to a previous report we could not detect interleukin-15expressions in the muscle fibers neither in patients nor in healthy individuals. The difference may be explained by different patient selection, or due to different staining protocols. But the absence of interleukin-15expressions in fully differentiated myotubes supports our notion that differentiated muscle fibers do not express significant interleukin-15at protein level; thereby it is less likely that differentiated muscle fibers are a source of interleukin production in polymyositis and dematomyositis.Interleukin-15receptor a was expressed in mononuclear cells in muscle tissue of patients. Moreover, there was a strong correlation between the number of interleukin-15and interleukin-15receptor a expressing cells. We were not able to determine the phenotype of the mononuclear cells that expressed interleukin-15receptor a due to a non-specific staining when using double immunofluorescence. Interleukin-15receptor a was also expressed in capillaries and endothelial cells of large vessels in most patients with idiopathic inflammatory myopathies, which is in line with previous reports on interleukin-15receptor a in other tissues. It has been suggested that interleukin-15could promote angiogenesis by inducing neovascularization in vivo via interleukin-15receptor a. Whether interleukin-15receptor a mediates neovascularization in polymyositis and dermatomyositis could not be answered by our study but it is worth being investigated, especially in the patients with dematomyositis as the vascular damage has been shown more frequently. Interleukin-15may mediate its functions via trans-presentation, that is interleukin-15receptor a-producing cells bind interleukin-15and then present it to cells expressing interleukin-2receptor β and yc subunits. This trans-presentation is anticipated to be relevant for T cells as they express interleukin-2receptor β and yc subunits on their cell membrane. T cell infiltration in skeletal muscle is a characteristic histopathological feature of polymyositis and dermatomyositis. T cells usually maintain a homeostasis state even after conventional immunosuppressive treatment as shown by our data in this study and in a previous report. As the most important T cell-activating cytokine, interleukin-2, is rarely found in muscle tissues of myositis patients, there is a possibility that that interleukin-15might play a role in this context to sustain T cells activation and homeostasis instead of interleukin-2. Thus, the positive correlation between interleukin-15and CD3+T cells, their close localization as well as the sustained expression of interleukin-15in muscle tissue after immunosuppressive treatment in some patients might support this hypothesis.An inverse correlation between baseline interleukin-15expression and muscle function improvement after treatment was seen in our study. Moreover, the group of patients still with high interleukin-15expressions after treatment showed less functional improvement compared to the low interleukin-15expressing group after immunosuppressive treatment. Furthermore, after five year follow up, muscle performance as measured by manual muscle text was also lower in those with high interleukin-15expression after eight months'treatment which might indicate a role of interleukin-15in the cause of muscle weakness. However, the long-term follow up data were only available in a subpopulation and thus need to be interpreted with caution and need to be confirmed in larger studies. To reduce sampling error as an explanation of the changes between the two biopsies we confirmed consistency of inflammatory cells in the first and last tissue section of the biopsies. Thus our data might indicate that follow up biopsies may be used for prognosis and, furthermore, that interleukin-15expression in muscle tissue may serve as a prognostic biomarker.A limitation of our study is the inconsistency in clinical outcome measures. Manual muscle test was not used at the time when the study started as the International Myositis Assessment and Clinical Studies Group (IMACS) consensus recommendations on outcome measures were not available at that time. As manual muscle test is a recommended outcome measure for muscle performance by IMACS and data were available from most patients after five years' follow up we chose to include this as a measure for long term follow up. In conclusion, we demonstrated in this study that both interleukin-15and its unique receptor interleukin-15receptor a are expressed in muscle tissue of myositis patients. Moreover, we showed that interleukin-15expression was correlated with muscle function improvement. Thus, interleukin-15signal pathway is likely to play a role in the pathogenesis of myositis and could be a biological therapy target at least in a subgroup of polymyositis or dermatomyositis patients with high interleukin-15expression in the muscle tissue after conventional immunosuppressive treatment in the future.