Inhibitory Effect Of Umbilical Cord-derived Mesenchymal Stem Cells On Local Interleukin-23/Th17Axis In Spondyloarthritis Patients

PART I Local activation of the interleukin-23/Th17axis in patients with spondyloarthritisBackground and Objective. Interleukin-23(IL-23), a new member of IL-12family, is mainly produced by innate immune cells. It has been shown that HLA-B27misfolding leads to activation of the unfolded protein response, which significantly enhances the expression of IL-23in macrophages. Binding to IL-23receptor, IL-23favors the generation of Th17cells. Growing evidence shows that the IL-23/Th17axis may play an important role in the pathogenesis of several chronic immune-mediated inflammatory diseases, such as psoriasis, inflammatory bowel disease. In order to evaluate the pathogenic role of the IL-23/Th17axis in spondyloarthritis (SpA), we investigated the expression of the IL-23/Th17axis in involved joints of patients with SpA.Methods. SpA patients and osteoarthritis (OA) patients with knee effusion seen at outpatient clinic of Department of Rheumatology, Chinese PLA General Hospital between September2012and January2013, were recruited in the study. Synovial fluid mononuclear cells (SFMCs) were isolated from all subjects by density gradient centrifugation on Ficoll-paqueTM-Plus. CD14+monocytes were separated from SFMCs by fluorescence activated cell sorting. The frequencies of CD14+IL-23+cells and Th17cells were analyzed by intracellular staining and flow cytometry.Results.(1) Twenty-two SpA patients (19men and3women) and6knee OA patients (2men and4women) were enrolled in the study. All SpA patients had a mean (SD) age of32.4(8.4) years. The median disease duration of the SpA patients was27.0months ((interquartile range, IQR),6.8to60.0) and all patients’Bath Ankylosing Spondylitis Disease Activity Indices were≥4. OA patients with knee involvement had a mean (SD) age of58.3(7.9) years. Their median disease durations were43.0months (IQR,22.5to75.0). Sixteen out of22SpA patients were positive for HLA-B27, while none of6OA patients was HLA-B27positive.(2) SpA patients had a higher frequency of CD14+IL-23+cells in SF compared with OA patients (median1.49%in SpA patients vs0.68%in OA patients, P<0.05). Frequency of Th17cells from SF of patients with SpA was greater than that from OA patients (median4.15%in SpA patients vs1.11%in OA patients, P<0.05).(3) HLA-B27-positive SpA patients had a higher frequency of CD14+IL-23+cells (1.60%(IQR,1.24to2.11)) compared with HLA-B27-negative SpA patients (1.26%(IQR,0.88to1.59)), although the difference did not reach statistical significance (P>0.05).(4) There was a strong positive association between the frequency of CD14+IL-23+cells and Th17cells in SF of SpA patients.Conclusions. Our findings indicate that local activation of IL-23/Th17axis exists in SpA, suggesting that IL-23/Th17axis may contribute to the pathogenesis of SpA. PART Ⅱ Inhibitory effect of umbilical cord-derived mesenchymal stem cells on local interleukin-23/Th17axis in spondyloarthritis patientsBackground and Objective. Mesenchymal stem cells (MSCs) are adult multipotent non-hematopoietic stem cells which are now shown to reside in various tissues of normal human body. These cells possess not only multiple differentiation potential, but also potent immunoregulatory and anti-inflammatory properties. Recent studies have shown that MSCs have therapeutic potential in several autoimmune diseases and chronic inflammatory disease. SpA belong to a group of chronic immune-mediated inflammatory diseases. In order to explore the fesibility of MSCs transplantation applied to the treatment for SpA, we evaluated the effect of human umbilical cord-derived MSCs (UCMSCs) on local IL-23/Th17axis, which may play an important role in the pathogenesis of SpA.Methods. SpA patients with knee effusion seen at outpatient clinic of Department of Rheumatology, Chinese PL A General Hospital between September2012and January2013, were recruited in the study. After isolated from all subjects by density gradient centrifugation on Ficoll-paqueTM-Plus, SFMCs were divided into two subsets:CD14+monocytes and CD14+-depleted SFMCs by fluorescence activated cell sorting. CD14+monocytes or CD14+-depleted SFMCs were cultured in the absence or presence of UCMSCs. Celluar interactions between CD14+monocytes and CD14+-depleted SFMCs were investigated simultaneously. The frequencies of CD14+IL-23+cells and Th17cells were analyzed by intracellular staining and flow cytometry.Results.(1) Nine SpA patients (7men and2women) were enrolled in the study. All SpA patients had a mean (SD) age of31.1(8.3) years. The mean (SD) disease duration of the SpA patients was39.7(31.9) months and all patients’ Bath Ankylosing Spondylitis Disease Activity Indices were≥4.(2) UCMSCs significantly diminished the frequency of CD14+IL-23+cells (absence of UCMSC,1.55%(IQR,1.32,2.61) vs presence of UCMSC,1.06%(IQR,0.80,1.81), P<0.05). Moreover, the concentration of IL-23in cell supernatant of coculture of CD14+monocytes and UCMSCs were significantly lower than that of CD14+monocytes cultured alone (coculture of CD14+monocytes and UCMSCs,121.1pg/ml (IQR,111.1,195.0) vs CD14+monocytes cultured alone,205.6pg/ml (IQR,182.8,256.7), P<0.05).(3) The presence of untreated monocytes significantly increased the frequency of Th17cells in CD14+-depleted SFMCs (absence of monocytes,0.86%(IQR,0.22,0.94) vs presence of untreated monocytes,2.71%(IQR,0.65,3.31), P<0.05). The ability of monocytes to drive Th17cells generation was downregulated by UCMSCs treatment (untreated monocytes, 2.71%(IQR,0.65,3.31) vs UCMSCs-treated monocytes,2.26%(IQR,0.40,2.66), P<0.05).(4) In the absence of monocytes, the frequency of Thl7cells was diminished from2.71%(IQR,1.80,3.05) to1.59%(IQR,1.20,2.67) by the coculture of UCMSCs. In the presence of monocytes, UCMSCs diminished the frequency of Th17cells from3.10%(IQR,2.81,5.49) to2.20%(IQR,1.52,2.95). The inhibitory rate of UCMSCs on Th17cells generation was enhanced from18.0%(IQR,11.6,32.5) to40.3%(IQR,25.8,51.6) by the addition of monocytes (P<0.05).Conclusion. These results show that monocytes from SF of SpA patients have a enhanced ability to produce IL-23, which may contribute to excessive generation of Th17cells. IL-23/Th17axis that may play an important role in the pathogenesis of SpA, could be inhibited by UCMSCs, which may be applied to the treatment for SpA.