Sustained benefit from combined plasmapheresis and allogeneic mesenchymal stem cells transplantation therapy in systemic sclerosis

Systemic sclerosis (SSc) is a rare chronic autoimmune disease characterized by increased synthesis and deposition of extra-cellular matrix in skin and various internal organs. Skin involvement, important in diagnosis and classification of SSc, is an almost universal feature of SSc [1]. Depending on the extent of skin fibrosis, SSc can be classified as two main subtypes, the limited cutaneous form (lcSSc) and the diffuse cutaneous form (dcSSc). The extensive degree of skin involvement coincides with future severe internal organ manifestations, poor prognosis and mortality, at least in the early phase of dcSSc [2]. Although skin disease does not directly threaten life, the thick skin can lead to psychological stress and thus worsens the quality of life.

Many inflammatory cytokines and growth factors are associated with the onset and progression of fibrosis, such as transforming growth factor (TGF)-β, endothelin-1, interleukin (IL)-17, IL-23 and tumor necrosis factor (TNF)-α [3‐6]. In general, skin disease in SSc patients is treated with immunosuppressive agents such as methotrexate (MTX) or mycophenolate mofetil (MMF) [7, 8], but are not acceptable to all Chinese patients in terms of side effects and cost. There are also various novel therapies focusing on skin disease, including anti-inflammatory immunosuppressive agents such as imatinib or rituximab, and extracorporeal shock waves [9‐12]. However, assessment of their effects remains inconclusive. A few studies on plasmapheresis (PE) for treatment of SSc have demonstrated improvement in the modified Rodnan Skin Score (MRSS), decreased level of cytokines, soluble adhesion molecules and immunolaboratory markers after treatment [13‐15]. However, patients often received three repeated PE treatments every 2–3 months in these studies as a high frequency of PE would increase the risk of infection due to allogeneic blood transfusion. Thus, newer therapies are needed with enhanced efficacy and less toxicity in the treatment of skin disease in SSc.

Mesenchymal stem cells (MSCs) are a subset of multipotent adult somatic stem cells that have the ability to undergo self-renewal, proliferation and pluripotent differentiation. They can be obtained from different sources such as bone marrow, umbilical cord, and adipose tissue in the human body [16‐19]. Besides their multi-lineage differentiation potential [20‐22], MSCs also harbor immunosuppressive activities owing to their paracrine effects and interaction with different immune cells [23‐26], and limited immunogenicity with low human leukocyte antigen (HLA) I and no HLA II expression [27]. These properties of MSCs have offered a new strategy in the treatment of numerous autoimmune inflammatory diseases and demonstrated promising results in safety and efficacy. To date, MSC transplantation (MSCT) has been proved in our center to be effective in the treatment of refractory systemic lupus erythematosus, rheumatoid arthritis and inflammatory bowel disease [28‐31].

Recently studies have shown deficiency of MSCs in SSc patients. Compared with healthy controls, bone marrow MSCs (BM-MSCs) isolated from SSc patients display a more mature and myofibroblast-like phenotype, which re-programs these cells toward pro-angiogenic behavior [32]. MSCs from SSc patients also exhibit abnormal functional activities, such as increased expression of TGF-β and vascular endothelial growth factor (VEGF), and impairment of endothelial cell differentiation, which may play critical roles during the development of fibrosis in SSc [33, 34]. Based on these findings, allogeneic MSCT appears a promising therapy for SSc. Indeed, one animal experiment has shown that MSCT results in lower expression of fibrotic markers in both skin and lung, and decreased levels of anti-topoisomerase (anti-scl70) autoantibodies, suggesting systemic effect of MSCs [35]. Our previous pilot study in five SSc patients also showed that MSCT results in decreased anti-nuclear antibody (ANA) titer, and improvement in the MRSS and Health Assessment Questionnaire (HAQ) [36]. Thus, we aimed to evaluate the effectiveness and safety of combination therapy with PE and MSCT for treatment of SSc on the basis of their different mechanisms in treating SSc patients.

Skin involvement is the hallmark of SSc; improvement in skin thickening may be useful as a surrogate for improvement in survival in clinical trials [37]. Therefore, the treating skin symptoms have been the focus of investigation in many clinical trials. Recent studies have assessed different options for the treatment of skin thickness; however, most of these therapies did not show significant efficacy [38]. Herein, we proposed allogeneic MSCT combined with PE as a potential therapy for the diffuse cutaneous form of SSc. In this study, of the total of 14 patients, 11 only had diffuse sclerosis and thickening of the skin without internal organ involvement, including two newly diagnosed patients who were not receiving any treatment before and after PE + MSCT; the other 9 patients received small doses of glucocorticoid in combination with immunosuppresive agents such as MTX or MMF, etc., which 3 patients gradually stopped taking after the combined therapy, and the other 6 cases also had reduced dosage of glucocorticoids and immunosuppressants. The results indicate this combined therapy has a possible benefit in improving MRSS and reducing inflammatory markers including anti-Scl70 autoantibody titer, TGF-β and VEGF levels.

Fibrosis is the final step and is the basis of most prominent clinical manifestations in SSc patients, including skin thickness and tightness [39]. Two fundamental biological processes contribute to the development of skin fibrosis, including vasculopathy with perivascular inflammation and coagulation activation, and fibroblast activation with the excess accumulation of extracellular matrix components. Multiple factors and signaling pathways are involved in the development or persistence of skin involvement in SSc, such as TGF-β, IL-4, IL-6, platelet-derived growth factor (PDGF), IL-1, IL-13, IL-17, IL-5, monocyte chemoattractant protein (MCP)-1, VEGF and connective tissue growth factor (CTGF) [6]. Recently, some experimental studies have revealed MSC deficiency in SSc. MSCs in SSc have a different phenotype from healthy controls [32]. BM-MSCs isolated from SSc patients have upregulation of α-smooth muscle actin (SMA) and smooth muscle (SM)22α genes and reduced proliferative activity, displaying a more mature and myofibroblast-like phenotype. Cipriani P et al. have observed that BM-MSCs from SSc patients have increased senescence biomarkers, through increased activation of the IL-6 pathway [40]. Furthermore, MSCs have been proved to not only have the properties of reduced inflammatory and fibrotic processes, but also have the ability to differentiate into endothelial cells [41]. These findings supported the hypothesis that MSCs from SSc patients are structurally and functionally defective, and provide basis for allogeneic MSCT as a potential therapy for SSc patients. Our results showed that MSCT combined with PE downregulated serum levels of TGF-β, which is a major cytokine involved in early angiogenesis and latent collagen production leading to fibrosis [42]. In addition, the combined therapy was shown to reduce the levels of VEGF, which was elevated in SSc patients and could stimulate angiogenesis [6]. We also noted lower levels of anti-Scl70 antibodies after the combined treatment, suggesting reduced B cell activation.

Some studies have suggested that MSCs can exhibit a protective effect on skin tightness and thickness [36, 43]. PE is also reported to be able to improve the Rodnan skin score in small case series of SSc patients [13]. These two therapies were taken into account for combination in view of their seemingly complementary characteristics. PE could quickly remove serum pro-inflammatory substances such as inflammatory cytokines, antibodies, immunoglobulins and complements, which play major roles in the immune responses against normal skin and fibrosis. MSC could secrete anti-inflammatory cytokines, anti-fibrotic factors and trophic molecules, and differentiate into epithelial cells, which will all promote regeneration of skin. Moreover, the self-renewal capacity of MSCs would make the therapeutic effects last a long time. However, it is difficult to tell which treatment is important in the resulting clinical and laboratory test improvements. Future randomized clinical trials with larger sample sizes and long-term follow up are required to further verify the results of this study.

In addition to improving the skin lesions, the data also suggest that the combined therapy can improve internal function in these patients. ILD is a common visceral lesion in SSc patients. For more than 15 years, CTX has commonly been used in the treatment of SSc-ILD. CTX is a cytotoxic immunosuppressive agent that suppresses lymphokine production and modulates lymphocyte function. In a landmark study, the Scleroderma Lung Study Research Group noted that CTX achieved a modest but significant beneficial effect on lung function and patients’ quality of life. After 12 months of therapy, FVC increased in 49.3% of the patients who received the CTX treatment. However, unfortunately, it also provoked a serious adverse event [44]. In our study, there were three patients with different degrees of ILD, all of whom had received glucocorticoids and CTX for at least 3 ~ 6 months before the combined therapy, but without improvement in the pulmonary symptoms. However, lung function and CT images in these three patients all improved significantly, after 12 months of combined therapy, FVC increased from 65.0 ± 7.0% to 81.7 ± 8.5%. We have gradually reduced the dosage of glucocorticoids and CTX to maintaining treatment. Compared with glucocorticoids and immunosuppressive agents, this combined therapy has shown a low side reaction and good safety; only a few patients had mild upper respiratory tract infections and diarrhea.

Our study has limitations. First, we were aware of the limitation of the small sample size of this study population, thus this is an exploratory analysis. The adequacy of sample size is important to clinical trials but depends on the availability of patients. A lower prevalence of SSc explained the sample size in this trial to some extent. Second, the uncontrolled study design (automatically also not blinded) may result in a substantial overestimation of therapeutic effect.

This study indicates that MSCT combined with PE is a feasible treatment associated with possible clinical benefit in SSc patients. The true value and safety will require much more robust data from a controlled trial and longer-term follow up in many more SSc patients.