Elsevier

Gene

Volume 591, Issue 1, 10 October 2016, Pages 137-147
Gene

Research paper
IL-15 up-regulates the MMP-9 expression levels and induces inflammatory infiltration of macrophages in polymyositis through regulating the NF-kB pathway

https://doi.org/10.1016/j.gene.2016.06.055Get rights and content

Highlights

  • It is the first time to investigate the relationship between IL-15, MMP-9 and NF-kB pathway in polymyositis.

  • Anti-IL-15 and IL-15Rα siRNA play protective effects in polymyositis.

  • ERK1/2 pathway plays important role in IL-15 regulating MMP-9 expression.

  • IKBα is a important downstream molecule of IL-15/MMP-9 pathway.

Abstract

This study was aimed to research the effects of IL-15 on inducing inflammatory infiltration of macrophages in polymyositis (PM) through the NF-kB pathway, and whether IL-15 was able to further regulate MMP-9 expression levels. Prepared PM cells, collected from the patients suffering from PM, were administered to SD rats. Also, a group of healthy SD rats was undergoing the same treatment as the control group. The test animals were treated with either anti-IL-15, IL-15, MMP-9 siRNA or ERK1/2 inhibitor. The blood toxicological parameters creatine kinase (CK) and CD163 were tested by using ELISA and immunohistochemistry assay. In addition, NF-kB expression in macrophages was measured by immunocytochemical assay. To measure the degree of cell infiltration the Transwell assay was performed. Lastly, western blot and zymography were carried out to compare MMP-9 and ERK expression levels between the two groups, both in vivo and in vitro. The results showed that S-CK, IL-15 and IL-15Rα levels increased rapidly after the conventional treatment was introduced to the PM infected SD rats. The PM model establishment and IL-15 treatment significantly increased the expressions of IL-15Rα, MMP-9, p-ERK and p-IKBα. However, the same effect can be suppressed by using anti-IL-15, MMP-9 siRNA or ERK1/2 inhibitor (P < 0.05). In addition, IL-15 is proved to increase cell migration and nucleus expression of NF-kB in the macrophages. IL-15 is able to significantly regulate the inflammatory infiltration of macrophages in PM patients through affecting the NF-kB pathway and MMP-9 expression levels.

Introduction

Polymyositis (PM) is a type of inflammatory disorder where muscle tissue inflames and deteriorates, causing the proximal weakness of muscles and limbs. PM is a cell immunity damage-based disease, and it is closely related to the emergence of T cells, macrophages and dendritic cells in muscle tissues (Milisenda et al., 2014a, Milisenda et al., 2014b). Prior immunohistochemistry experiments have indicated that inflammatory cell infiltration is also expressed in CD4+, CD8+ T cells and B cells for patients suffering from myositis muscle biopsies (Engel and Arahata, 1984a, Engel and Arahata, 1984b). Additionally, autoantibodies were observed with PM patients that are in direct connection to clinical phenotypes via molecule channels (Stone et al., 2007a, Stone et al., 2007b). Until today, PM etiology remains unclear and therefore more extensive research should be dedicated to this field.

Interleukin 15 (IL-15) is a pleiotropic cytokine and participates in the regulation of numerous biological activities (Waldmann, 2006a, Waldmann, 2006b). Interleukin regulates the growth and proliferation of the natural killer T cells the memory T cells as well as the CD8+ cells. Interleukin 15 (IL-15) is predominantly expressed as its high-affinity binding partner IL-15 receptor alpha (IL-15Rα) at the RNA level, and the expression of IL-15 is regulated multiple post-transcriptional regulations(Huang et al., 2015a, Huang et al., 2015b, Ruckert et al., 2009a, Ruckert et al., 2009b). IL-15 has been proved to stimulate myoblast differentiation, muscle wasting and muscle hypertrophy among cancer patients. In contrary, excessive IL-15 could cause skeletal muscle atrophy (Carbo et al., 2000a, Carbo et al., 2000b). For example, excessive IL-15 resulted from rheumatoid arthritis could stimulate monocyte/macrophages to activate the corresponding auto reactive CD4+ T cells (Ruckert et al., 2009a, Ruckert et al., 2009b). In addition, research has shown that the genetic ablation of the IL-15 gene leads to the alleviation of autoimmune myositis in mice (Huang et al., 2015a, Huang et al., 2015b). Also, it has been proven that PM patients show significantly higher concentrations of IL-15 compared to healthy controls. Nevertheless, the dynamic mechanism underlying PM is still unclear (Mielnik et al., 2012a, Mielnik et al., 2012b, Notarnicola et al., 2014a, Notarnicola et al., 2014b).

The nuclear factor kappa B (NF-kB) families are composed of critical transcription factors which are involved in a wide range of biological processes such as immune development, immuneresponses, cell survival, stress responses and the maturation of numerous cells. Although NF-kB is usually activated to protect organisms against environmental hazards, disordered NF-kB activity is often associated with several types of diseases such as chronic inflammation and cancer (Doyle et al., 2013a, Doyle et al., 2013b). The NF-kB signaling system responds to a number of stimuli and several distinct cellular reactions are caused by ligand-receptor engagement (Mitchell et al., 2016a, Mitchell et al., 2016b). It has been found that NF-kB signaling pathway plays a key role in IL-15 variation resulted from respiratory syndrome virus infection (Park et al., 2014a, Park et al., 2014b). Investigation in acute lymphoblastic leukemia indicated that IL-15 induces the signal transducer and activator of NF-kB phosphorylation (Williams et al., 2014a, Williams et al., 2014b).

MMPs are a large family of endopeptidases that play a significant role in the degradation, remodeling and formation of extracellular matrix (ECM). MMPs also regulate the activity of many important non-ECM molecules (Kousios et al., 2016a, Kousios et al., 2016b). MMP-9 is initially expressed by the invading T lymphocytes in patients who experienced some inflammatory myopathies. Since MMP-9 has been proved to be involved in the inflammatory process of muscle degeneration, there may be an association between MMP-9 and the emergence of PM (Fukushima et al., 2007a, Fukushima et al., 2007b). Apart from that, tumor necrosis factor-related weak inducer of apoptosis (TWEAK) is a newly identified muscle-wasting cytokine, which can regulate the level of MMP-9 by affecting the NF-kB signaling pathways (Li et al., 2009a, Li et al., 2009b). As a result of this, we predicted that IL-15 can be considered as a target for curing PM as it is able to regulate the NF-kB signaling pathway which further influences the expression of MMP-9.

Patients suffering from PM or dermatomyositis were included in this research. The corresponding clinical characteristics of these patients were recorded and biopsies were carried out. PM models established on rats were used to examine the corresponding levels of creatine phosphokinase (CK), IL-15 and IL-15Rα in serum. Immunohistochemistry, Transwell analysis, zymography and Western hybridization were conducted to examine the underlying mechanism of PM in which IL-15, NF-kB signaling pathway and MMP-9 may be involved.

Section snippets

Clinical samples

This research includes a total of twenty patients (male: female = 7:13), with an average age of 62 years old. All patients were either diagnosed with polymyositis (PM) or dermatomyositis (DM) according to the Bohan and Peter criteria (Troyanov et al., 2014a, Troyanov et al., 2014b). All patients were selected from Ningbo No.2 Hospital between March 2014 and October 2014. Besides that, these patients were treated with high dose glucocorticoids or other immunosuppressive substances including

Clinical assessments, muscle histopathology and effects of conventional hormone treatment

Positive staining of CD163 was detected in mononuclear inflammatory cells, specifically in macrophages. No CD163 expression was observed in the muscle samples collected from the healthy control group (Fig. 1C): neither in the tissues collected from patients prior to treatment (Fig. 1A) nor the tissues collected from patients after treatment (Fig. 1B). The stained area of macrophages with CD163 decreased after hormone treatment. The measured muscle performance of the infected rats was

Discussion

Polymyositis (PM), an autoimmune disease, is distinguished by inflammatory infiltrates that predominantly consists of macrophages and T lymphocytes (Dorph et al., 2006). Unfortunately, researchers are still unable to uncover the aetiology and pathogenesis of this disease. Further clarification of the molecular mechanism of PM not only can provide a credible and valuable early screening index for clinical diagnosis but also can help clinicians undertake targeted therapy, resulting in a positive

Conflicts of interest statements

There is no conflict of interest.

Acknowledgement

This research was supported by Projects of Medical and Health Technology Development Program in Zhejiang Province (NO. 2014kya198).

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