强直性脊柱炎患者骨髓间充质干细胞调控巨噬细胞的功能异常
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摘要
背景:强直性脊柱炎是一种伴有高炎性状态的自身免疫性疾病,但其具体发病机制不明确,目前尚缺乏有效的治疗方案。目的:体外探讨强直性脊柱炎患者来源骨髓间充质干细胞调控巨噬细胞的功能及正常人骨髓间充质干细胞在强直性脊柱炎治疗中的潜在作用。方法:提取25例强直性脊柱炎患者及21例健康志愿者的骨髓间充质干细胞进行体外培养扩增,以第4代细胞进行实验。体外诱导THP-1细胞系分化成为巨噬细胞。运用流式细胞术鉴定骨髓间充质干细胞及巨噬细胞的表面标志。ELISA检测健康志愿者/强直性脊柱炎患者骨髓间充质干细胞与巨噬细胞共培养体系上清中肿瘤坏死因子α及TSG-6蛋白水平,PCR检测巨噬细胞及骨髓间充质干细胞相关因子基因表达水平。结果与结论:(1)两种骨髓间充质干细胞均呈现典型间充质干细胞表型,巨噬细胞表型标志CD68为阳性。(2)强直性脊柱炎患者骨髓间充质干细胞与巨噬细胞共培养组中巨噬细胞分泌的肿瘤坏死因子α蛋白水平及肿瘤坏死因子α转录水平均高于健康志愿者骨髓间充质干细胞(P<0.05)。(3)强直性脊柱炎患者骨髓间充质干细胞组TSG-6基因的转录水平及蛋白分泌水平均低于健康志愿者骨髓间充质干细胞组(P<0.05)。结果提示:(1)强直性脊柱炎患者骨髓间充质干细胞抑制巨噬细胞肿瘤坏死因子α分泌的功能减弱,可能是导致强直性脊柱炎免疫异常的重要原因。(2)正常人骨髓间充质干细胞可分泌足量的TSG-6抑制强直性脊柱炎患者巨噬细胞的活化,降低其肿瘤坏死因子α的分泌而发挥治疗作用。
BACKGROUND: Ankylosing spondylitis is an autoimmune disease at high inflammatory state, and its pathogenesis is still unclear. Besides, there is a lack of entirely satisfactory curative strategies. OBJECTIVE: To explore the immunoregulation capability of bone marrow mesenchymal stem cells from ankylosing spondylitis patients on macrophages and the potential therapeutic use of bone marrow mesenchymal stem cells from healthy donors on ankylosing spondylitis. METHODS: Bone marrow mesenchymal stem cells were extracted from 21 healthy donors and 25 ankylosing spondylitis patients respectively, and passage 4 cells were used in subsequent experiments. A human monocytic cell line was induced to differentiate into macrophages. The phenotypic markers of bone marrow mesenchymal stem cells and macrophages were detected by flow cytometry. Expressions of tumor necrosis factor-α and tumor necrosis factor-α-stimulated gene 6(TSG-6) proteins in the supernatant of co-culture system were detected by ELISA. Quantitative real-time PCR was applied to detect the m RNA level of cytokines secreted by bone marrow mesenchymal stem cells and macrophages. RESULTS AND CONCLUSION: The typical mesenchymal stem cell surface markers were expressed in both bone marrow mesenchymal stem cells from healthy donors and patients with ankylosing spondylitis, and CD68 was detected positively in induced macrophages. The protein and m RNA levels of tumor necrosis factor-α secreted by macrophages co-cultured with bone marrow mesenchymal stem cells from patients with ankylosing spondylitis were obviously higher than those from healthy donors(P < 0.05). TSG-6 secreted by bone marrow mesenchymal stem cells from patients with ankylosing spondylitis was lower than that by bone marrow mesenchymal stem cells from healthy donors in both RNA transcriptional and protein levels(P < 0.05). Our study demonstrates that bone marrow mesenchymal stem cells from patients with ankylosing spondylitis shows abnormal immunoregulatory function on inhibiting the tumor necrosis factor-α secretion from macrophages, which reveals a mechanism of immune disorder in ankylosing spondylitis. The therapeutic mechanism of bone marrow mesenchymal stem cells from healthy donors may work by secreting enough TSG-6 to inhibit the activation of macrophages in patients with ankylosing spondylitis, and thereby to decrease the secretion of tumor necrosis factor-α.
BACKGROUND: Ankylosing spondylitis is an autoimmune disease at high inflammatory state, and its pathogenesis is still unclear. Besides, there is a lack of entirely satisfactory curative strategies. OBJECTIVE: To explore the immunoregulation capability of bone marrow mesenchymal stem cells from ankylosing spondylitis patients on macrophages and the potential therapeutic use of bone marrow mesenchymal stem cells from healthy donors on ankylosing spondylitis. METHODS: Bone marrow mesenchymal stem cells were extracted from 21 healthy donors and 25 ankylosing spondylitis patients respectively, and passage 4 cells were used in subsequent experiments. A human monocytic cell line was induced to differentiate into macrophages. The phenotypic markers of bone marrow mesenchymal stem cells and macrophages were detected by flow cytometry. Expressions of tumor necrosis factor-α and tumor necrosis factor-α-stimulated gene 6(TSG-6) proteins in the supernatant of co-culture system were detected by ELISA. Quantitative real-time PCR was applied to detect the m RNA level of cytokines secreted by bone marrow mesenchymal stem cells and macrophages. RESULTS AND CONCLUSION: The typical mesenchymal stem cell surface markers were expressed in both bone marrow mesenchymal stem cells from healthy donors and patients with ankylosing spondylitis, and CD68 was detected positively in induced macrophages. The protein and m RNA levels of tumor necrosis factor-α secreted by macrophages co-cultured with bone marrow mesenchymal stem cells from patients with ankylosing spondylitis were obviously higher than those from healthy donors(P < 0.05). TSG-6 secreted by bone marrow mesenchymal stem cells from patients with ankylosing spondylitis was lower than that by bone marrow mesenchymal stem cells from healthy donors in both RNA transcriptional and protein levels(P < 0.05). Our study demonstrates that bone marrow mesenchymal stem cells from patients with ankylosing spondylitis shows abnormal immunoregulatory function on inhibiting the tumor necrosis factor-α secretion from macrophages, which reveals a mechanism of immune disorder in ankylosing spondylitis. The therapeutic mechanism of bone marrow mesenchymal stem cells from healthy donors may work by secreting enough TSG-6 to inhibit the activation of macrophages in patients with ankylosing spondylitis, and thereby to decrease the secretion of tumor necrosis factor-α.
引文
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[4]Rashid T,Ebringer A.Ankylosing spondylitis is linked to Klebsiella--the evidence.Clin Rheumatol.2007;26(6):858-864.
[5]Rashid T,Wilson C,Ebringer A.The link between ankylosing spondylitis,Crohn's disease,Klebsiella,and starch consumption.Clin Dev Immunol.2013;2013:872632.
[6]Lou YJ,Jin J,Mai WY.Ankylosing spondylitis presenting with macrophage activation syndrome.Clin Rheumatol.2007;26(11):1929-1930.
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[9]van der Linden S.Issues in the treatment of ankylosing spondylitis with non-steroidal anti-inflammatory drugs.Wien Med Wochenschr.2008;158(7-8):195-199.
[10]Braun J,Sieper J.Therapy of ankylosing spondylitis and other spondyloarthritides:established medical treatment,anti-TNF-alpha therapy and other novel approaches.Arthritis Res.2002;4(5):307-321.
[11]Braun J,Baraliakos X,Heldmann F,et al.Tumor necrosis factor alpha antagonists in the treatment of axial spondyloarthritis.Expert Opin Investig Drugs.2014;23(5):647-659..
[12]Tong Q,Cai Q,de Mooij T,et al.Adverse events of anti-tumor necrosis factorαtherapy in ankylosing spondylitis.PLo S One.2015;10(3):e0119897.
[13]Le Blanc K,Mougiakakos D.Multipotent mesenchymal stromal cells and the innate immune system.Nat Rev Immunol.2012;12(5):383-396.
[14]Chinnadurai R,Ng S,Velu V,et al.Challenges in animal modelling of mesenchymal stromal cell therapy for inflammatory bowel disease.World J Gastroenterol.2015;21(16):4779-4787.
[15]阮光萍,姚翔,刘菊芬,等.人脐带间充质干细胞移植治疗系统性红斑狼疮[J].中国组织工程研究,2015,19(14):2172-2178.
[16]赖勤,余莲,邱永荣,等.脐血间充质干细胞移植治疗多发性肌炎/皮肌炎:Th细胞因子的变化[J].中国组织工程研究,2015,19(14):2186-2191.
[17]Ben-Ami E,Berrih-Aknin S,Miller A.Mesenchymal stem cells as an immunomodulatory therapeutic strategy for autoimmune diseases.Autoimmun Rev.2011;10(7):410-415.
[18]Wang P,Li Y,Huang L,et al.Effects and safety of allogenic mesenchymal stem cell intravenous infusion in active ankylosing spondylitis patients who failed NSAIDs:a 20-week clinical trial.Cell Transplant.2014;23(10):1293-303.
[19]Lee TH,Lee GW,Ziff EB,et al.Isolation and characterization of eight tumor necrosis factor-induced gene sequences from human fibroblasts.Mol Cell Biol.1990;10(5):1982-1988.
[20]Qi Y,Jiang D,Sindrilaru A,et al.TSG-6 released from intradermally injected mesenchymal stem cells accelerates wound healing and reduces tissue fibrosis in murine full-thickness skin wounds.J Invest Dermatol.2014;134(2):526-537.
[21]Wang N,Li Q,Zhang L,et al.Mesenchymal stem cells attenuate peritoneal injury through secretion of TSG-6.PLo S One.2012;7(8):e43768.
[22]Chen M,Li X,Zhang X,et al.The inhibitory effect of mesenchymal stem cell on blood-brain barrier disruption following intracerebral hemorrhage in rats:contribution of TSG-6.J Neuroinflammation.2015;12:61.
[23]Liu Y,Zhang R,Yan K,et al.Mesenchymal stem cells inhibit lipopolysaccharide-induced inflammatory responses of BV2microglial cells through TSG-6.J Neuroinflammation.2014;11:135.
[24]Oh JY,Lee RH,Yu JM,et al.Intravenous mesenchymal stem cells prevented rejection of allogeneic corneal transplants by aborting the early inflammatory response.Mol Ther.2012;20(11):2143-2152.
[25]Lee RH,Yu JM,Foskett AM,et al.TSG-6 as a biomarker to predict efficacy of human mesenchymal stem/progenitor cells(h MSCs)in modulating sterile inflammation in vivo.Proc Natl Acad Sci U S A.2014;111(47):16766-16771.
[26]Mahoney DJ,Swales C,Athanasou NA,et al.TSG-6 inhibits osteoclast activity via an autocrine mechanism and is functionally synergistic with osteoprotegerin.Arthritis Rheum.2011;63(4):1034-1043.
[27]Inoue I,Ikeda R,Tsukahara S.Current topics in pharmacological research on bone metabolism:Promyelotic leukemia zinc finger(PLZF)and tumor necrosis factor-alpha-stimulated gene 6(TSG-6)identified by gene expression analysis play roles in the pathogenesis of ossification of the posterior longitudinal ligament.J Pharmacol Sci.2006;100(3):205-210.
[28]Wang H,Wang X,Li X,et al.CD68(+)HLA-DR(+)M1-like macrophages promote motility of HCC cells via NF-κB/FAK pathway.Cancer Lett.2014;345(1):91-99.
[29]Daikh DI,Chen PP.Advances in managing ankylosing spondylitis.F1000Prime Rep.2014;6:78.
[30]M?rker-Hermann E.Therapy of psoriatic arthritis.Z Rheumatol.2013;72(8):784-790.
[31]Fran?ois RJ,Neure L,Sieper J,et al.Immunohistological examination of open sacroiliac biopsies of patients with ankylosing spondylitis:detection of tumour necrosis factor alpha in two patients with early disease and transforming growth factor beta in three more advanced cases.Ann Rheum Dis.2006;65(6):713-720.
[32]Gratacós J,Collado A,Filella X,et al.Serum cytokines(IL-6,TNF-alpha,IL-1 beta and IFN-gamma)in ankylosing spondylitis:a close correlation between serum IL-6 and disease activity and severity.Br J Rheumatol.1994;33(10):927-931.
[33]Yi H,Kang KY,Kim Y,et al.Human adipose-derived mesenchymal stem cells attenuate collagen antibody-induced autoimmune arthritis by inducing expression of FCGIIB receptors.BMC Musculoskelet Disord.2015;16:170.
[34]Guo Z,Zhou X,Li J,et al.Mesenchymal stem cells reprogram host macrophages to attenuate obliterative bronchiolitis in murine orthotopic tracheal transplantation.Int Immunopharmacol.2013;15(4):726-734.
[35]Davey-Ranasinghe N,Deodhar A.Osteoporosis and vertebral fractures in ankylosing spondylitis.Curr Opin Rheumatol.2013;25(4):509-516.
[2]Singh G,Lawrence A,Agarwal V,et al.Higher prevalence of extra-articular manifestations in ankylosing spondylitis with peripheral arthritis.J Clin Rheumatol.2008;14(5):264-266.
[3]Castro-Santos P,Gutiérrez MA,Díaz-Pe?a R.Genetics of ankylosing spondylitis.Rev Med Chil.2014;142(9):1165-1173.
[4]Rashid T,Ebringer A.Ankylosing spondylitis is linked to Klebsiella--the evidence.Clin Rheumatol.2007;26(6):858-864.
[5]Rashid T,Wilson C,Ebringer A.The link between ankylosing spondylitis,Crohn's disease,Klebsiella,and starch consumption.Clin Dev Immunol.2013;2013:872632.
[6]Lou YJ,Jin J,Mai WY.Ankylosing spondylitis presenting with macrophage activation syndrome.Clin Rheumatol.2007;26(11):1929-1930.
[7]De Keyser F,Van den Bosch F,Mielants H.Anti-TNF-alpha therapy in ankylosing spondylitis.Cytokine.2006;33(5):294-298.
[8]Sari?,?ztürk MA,Akko?N.Treatment of ankylosing spondylitis.Turk J Med Sci.2015;45(2):416-430.
[9]van der Linden S.Issues in the treatment of ankylosing spondylitis with non-steroidal anti-inflammatory drugs.Wien Med Wochenschr.2008;158(7-8):195-199.
[10]Braun J,Sieper J.Therapy of ankylosing spondylitis and other spondyloarthritides:established medical treatment,anti-TNF-alpha therapy and other novel approaches.Arthritis Res.2002;4(5):307-321.
[11]Braun J,Baraliakos X,Heldmann F,et al.Tumor necrosis factor alpha antagonists in the treatment of axial spondyloarthritis.Expert Opin Investig Drugs.2014;23(5):647-659..
[12]Tong Q,Cai Q,de Mooij T,et al.Adverse events of anti-tumor necrosis factorαtherapy in ankylosing spondylitis.PLo S One.2015;10(3):e0119897.
[13]Le Blanc K,Mougiakakos D.Multipotent mesenchymal stromal cells and the innate immune system.Nat Rev Immunol.2012;12(5):383-396.
[14]Chinnadurai R,Ng S,Velu V,et al.Challenges in animal modelling of mesenchymal stromal cell therapy for inflammatory bowel disease.World J Gastroenterol.2015;21(16):4779-4787.
[15]阮光萍,姚翔,刘菊芬,等.人脐带间充质干细胞移植治疗系统性红斑狼疮[J].中国组织工程研究,2015,19(14):2172-2178.
[16]赖勤,余莲,邱永荣,等.脐血间充质干细胞移植治疗多发性肌炎/皮肌炎:Th细胞因子的变化[J].中国组织工程研究,2015,19(14):2186-2191.
[17]Ben-Ami E,Berrih-Aknin S,Miller A.Mesenchymal stem cells as an immunomodulatory therapeutic strategy for autoimmune diseases.Autoimmun Rev.2011;10(7):410-415.
[18]Wang P,Li Y,Huang L,et al.Effects and safety of allogenic mesenchymal stem cell intravenous infusion in active ankylosing spondylitis patients who failed NSAIDs:a 20-week clinical trial.Cell Transplant.2014;23(10):1293-303.
[19]Lee TH,Lee GW,Ziff EB,et al.Isolation and characterization of eight tumor necrosis factor-induced gene sequences from human fibroblasts.Mol Cell Biol.1990;10(5):1982-1988.
[20]Qi Y,Jiang D,Sindrilaru A,et al.TSG-6 released from intradermally injected mesenchymal stem cells accelerates wound healing and reduces tissue fibrosis in murine full-thickness skin wounds.J Invest Dermatol.2014;134(2):526-537.
[21]Wang N,Li Q,Zhang L,et al.Mesenchymal stem cells attenuate peritoneal injury through secretion of TSG-6.PLo S One.2012;7(8):e43768.
[22]Chen M,Li X,Zhang X,et al.The inhibitory effect of mesenchymal stem cell on blood-brain barrier disruption following intracerebral hemorrhage in rats:contribution of TSG-6.J Neuroinflammation.2015;12:61.
[23]Liu Y,Zhang R,Yan K,et al.Mesenchymal stem cells inhibit lipopolysaccharide-induced inflammatory responses of BV2microglial cells through TSG-6.J Neuroinflammation.2014;11:135.
[24]Oh JY,Lee RH,Yu JM,et al.Intravenous mesenchymal stem cells prevented rejection of allogeneic corneal transplants by aborting the early inflammatory response.Mol Ther.2012;20(11):2143-2152.
[25]Lee RH,Yu JM,Foskett AM,et al.TSG-6 as a biomarker to predict efficacy of human mesenchymal stem/progenitor cells(h MSCs)in modulating sterile inflammation in vivo.Proc Natl Acad Sci U S A.2014;111(47):16766-16771.
[26]Mahoney DJ,Swales C,Athanasou NA,et al.TSG-6 inhibits osteoclast activity via an autocrine mechanism and is functionally synergistic with osteoprotegerin.Arthritis Rheum.2011;63(4):1034-1043.
[27]Inoue I,Ikeda R,Tsukahara S.Current topics in pharmacological research on bone metabolism:Promyelotic leukemia zinc finger(PLZF)and tumor necrosis factor-alpha-stimulated gene 6(TSG-6)identified by gene expression analysis play roles in the pathogenesis of ossification of the posterior longitudinal ligament.J Pharmacol Sci.2006;100(3):205-210.
[28]Wang H,Wang X,Li X,et al.CD68(+)HLA-DR(+)M1-like macrophages promote motility of HCC cells via NF-κB/FAK pathway.Cancer Lett.2014;345(1):91-99.
[29]Daikh DI,Chen PP.Advances in managing ankylosing spondylitis.F1000Prime Rep.2014;6:78.
[30]M?rker-Hermann E.Therapy of psoriatic arthritis.Z Rheumatol.2013;72(8):784-790.
[31]Fran?ois RJ,Neure L,Sieper J,et al.Immunohistological examination of open sacroiliac biopsies of patients with ankylosing spondylitis:detection of tumour necrosis factor alpha in two patients with early disease and transforming growth factor beta in three more advanced cases.Ann Rheum Dis.2006;65(6):713-720.
[32]Gratacós J,Collado A,Filella X,et al.Serum cytokines(IL-6,TNF-alpha,IL-1 beta and IFN-gamma)in ankylosing spondylitis:a close correlation between serum IL-6 and disease activity and severity.Br J Rheumatol.1994;33(10):927-931.
[33]Yi H,Kang KY,Kim Y,et al.Human adipose-derived mesenchymal stem cells attenuate collagen antibody-induced autoimmune arthritis by inducing expression of FCGIIB receptors.BMC Musculoskelet Disord.2015;16:170.
[34]Guo Z,Zhou X,Li J,et al.Mesenchymal stem cells reprogram host macrophages to attenuate obliterative bronchiolitis in murine orthotopic tracheal transplantation.Int Immunopharmacol.2013;15(4):726-734.
[35]Davey-Ranasinghe N,Deodhar A.Osteoporosis and vertebral fractures in ankylosing spondylitis.Curr Opin Rheumatol.2013;25(4):509-516.