| BackgroundMyeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that consists of myeloid progenitor cells and immature myeloid cells (IMCs).IMCs that are generated in the bone marrow quickly differentiate into mature granulocytes, macrophages or dendritic cells (DCs) in healthy individuals. By contrast, in pathological conditions, such as cancer, various infection diseases, sepsis,bone marrow transplantation and some autoimmune diseases, a block in the differentiation of IMCs into mature myeloid cells results in the expansion of this population. The activation of IMCs in pathological conditions leads to the up-regulation of their expression of immune suppressive factor, such as arginase 1(ARG 1) and inducible nitric oxide synthase (iNOS), as well as an increase in their production of nitric oxide (NO) and reactive oxygen species (ROS).MDSCs are characterized by the co-expression Gr-1 (Ly6C and Ly6G) and CDllb in mice. Normal mouse bone marrow contains 50%?60% of cells with this phenotype,but these cells account for 2%?5% of spleen cells. In humans,MDSCs are commonly defined as CD14-CDllb+ cells. Strictly, these cells express the common myeloid and lymphoid cells, and of the MHC class ? molecule HLA-DR.Recently, the morphological heterogeneity of MDSCs has been defined more precisely in mice based on their expression of Gr-1. Granulocytic MDSCs have a phenotype CD11b+Ly6G+LyClow (G-MDSCs),whereas MDSCs with monocytic morphology are CD11b+Ly6G-Ly6Chi (M-MDSCs).MDSCs were originally described in the contexts of murine tumor models and cancer patients. And these cells will be the focus in autoimmune disease due to the establishment of murine models. There were already reported in experimental autoimmune encephalomyelitis (EAE), autoimmune hepatitis (AIH), inflammatory bowel disease (IBD), experimental autoimmune uveoretinitis (EAU) and systemic lupus erythematosus (SLE) mouse model. But there were fewer studies in rheumatoid arthritis (RA) and their mouse models,RA is a systemic autoimmune disease characterized by chronic inflammation of synovial tissue. In RA, the clinical symptoms including persistent and progressive inflammation of peripheric joints, pannus formation, and cartilage and bone destruction that ultimately cause the deformity and dysfunction. Therefore, inhibiting bone destruction is one of the most challenging goals in the treatment of RA.Over the past few years, the remarkable advances of treatment, including biologic or non-biologic disease-modifying anti-rheumatic drugs (DMARDs), have improved the symptoms, long-term outcomes and quality of life of RA patients. With the putting forward of the "treat-to-target" strategy and enhanced treatment of this disease, however, there remain a large number of patients who continue to suffer from severe bone destruction.Collagen-induced arthritis (CIA) model with multi-joints inflammation was inducing by type ? collagen (C?). Peripheric polyarthritis, usually affected hind legs, no vasculitis and no haunted articular symptoms were the main characteristics of this model. The pathogenesis contained that macrophages and T cells activated by C? infiltrated into synovium and released cytokines, then granulocytes invaded and together formed the pannus, finally bone loss was caused by OCs and collagenase.The mechanisms, clinical manifestation, pathologic histology and immunology of CIA model which all resembled RA, and currently it was considered to be an ideal mouse model for the study of pathogenesis and medicine of RA. In addition, bone destruction caused by OCs in the synovium, which was one of the prominent features.OCs were multinucleated cells responsible for bone resorption. Normally, OCs were generated and formed in bone marrow cavity. Oc precursors (OCPs) were arised from hematopoietic stem cells contained lymphoid and myeloid precursors. The common lymphoid progenitor cells gave rise to the T, B lymphocytes and natural killer cells (NK). B220+ pro-B cells could form OCs, but mature T or B lymphocytes couldn't, and they indirectly influenced OCs generation by receptor activator of NF-?B ligand, (RANKL) and osteoprotegerin (OPG). NK cells isolated from synovial fluid of RA patients were capable of inducing OCs differentiation from CD14+monocytes when co-cultured. The common myeloid progenitor cells gave rise to megakaryocyte, erythrocyte and granulocyte/macrophage progenitor cells.Clonogenic showed megakaryocyte and erythrocyte couldn't produce colony-forming units for granulocyte and macrophage (CFU-GM), which suggested that they haven't potential to form OCs. Like mature T or B lymphocytes, megakaryocyte indirectly regulated OCs differentiation by transforming growth factor-? (TGF- ? )and platelet-derived growth factor (PDGF). Granulocytes and macrophages could further differentiate into mature granulocytes, macrophages, DCs and OCs. In the presence of macrophage colony-stimulating factor (M-CSF) and RANKL, macrophages or DCs differentiated in OCs.Previous research demonstrated that the percentage of MDSCs was enhanced in the PB, SP and BM of CIA mice. These cells were also detected in the synovial tissue.And there was positive correlation between MDSCs and arthritis score. Then, what's the role of these augmented cells in CIA? Considering that MDSCs could differentiate in to macrophage and DCs, both of which were treated as OCPs, and could differentiate into OCs in the presence of M-CSF and RANKL. Together, we speculated that MDSCs could differentiate into OCs.MDSCs played an important role in immune microenviroment. Research focusing on all aspects and their function became more and more clear. The therapies targeting MDSCs constantly evolved. Those could divide into 4 sections, such as promotion differentiation, inhibition maturation,prevention accumulation in peripheral organs and regulation suppressive molecules. It was reported that IL-1? ,IL-6 and MMP-9 regulated maturation of MDSCs. In addition, cyclooxygenase 2(COX-2) was a key target which regulated suppressive function of MDSCs.RA belongs to the Traditional Chinese Medicine (TCM) "paralysis disease" or"arthralgia" category. The earliest documented in the "Neijing", called "paralysis.""Suwen· Bilum" called "paralysis" characterized by the syndromes of wind,cold or damp. And it caused by the effect of wind, cold or damp. The wind, cold or damp usually resulted in migratory arthralgia, pain or fixed impediment, respectively.Therapeutic principle of "paralysis" was dispelling wind and removing obstruction in the meridians. It was treated by the means of dispel the wind and cold,dehumidification, activation veins or assisting tonic to patients with longer duration.Sinomenine (SIN) is an alkaloid extracted from herbs, Sinonmenin acutun,which has multi-effects including anti-inflammation, analgesia and immunosuppression. It has been utilized to treat rheumatoid diseases over 1000 years.And mechanism research showed that it controlled synovitis by regulating cycle,proliferation or apoptisis of fibroblast-like synoviocytes (FLS), and suppressed the activation of DCs and their surface molecules or chemokine expression, and also inhibited the production of IL-1?,IL-6 or COX-2. Therefore,we postulated that SIN could arrest the maturation and function of MDSCs through suppression the expression of IL-1? ,IL-6 or COX-2.Objectives1. To explore the possible mechanism that MDSCs could participate in CIA model.2. To investigate the relationship between MDSCs and bone destruction of CIA after MDSCs differentiated into OCs in vitro.3. To examine the effect of SIN on the MDSCs and further confirm the significance between MDSCs and bone destruction of CIA.Methods1. The percentage, phenotype and suppression function of MDSCs in CIA mice.Male C57BL/6(H-2b) were 8?10 weeks old. 10 mM acetic acid dissolved in C?, stirred at 4? overnight. Equal volume dissolved C? and CFA mixed and emulsified using homogenizer in ice-bath. 1ml syringe with 26G needle aspirated C?/CFA emulsified and injected mice intradermally emulsion containing 100?l, using the loose skin at the base of the tail. 14 days after first immunization 100?l equal volume C?/IFA emulsion administered a boost injection and avoided first injection sites. After first immunization,we recorded the arthritis score 3 times every week.Mice were sacrificed by dislocation after ether anesthesia. Then collected the bilateral limbs, and which were processed routine decalcification, fixation, dehydration,transparency, waxdip, embedment, section and H&E staining.The percentage of PB, SP and BM MDSCs was detected by flow cytometry 4 weeks after immunization. Mice synovium Gr-1+cells infiltration were examined by immunofluoresence. MDSCs and their subsets G-MDSCs and M-MDSCs were sorted by MACS and flow cytometry. The effects of MDSCs and their subsets G-MDSCs and M-MDSCs on T cells proliferation were tested by 3H-TdR. The supernatant level of IFN-? was measured by ELISA.2. MDSCs differentiated into OCs.C57BL/6 mice were immunized with C?/CFA for 4 weeks. Naive or CIA splenic MDSCs were sorted by MACS. 2× 105cells were seeded in 400ul medium in a chamber sliders in the presence of 20 ng/ml M-CSF for 3 days. Thereafter, they were further incubated with 20ng/ml M-CSF and 100ng/ml RANKL for 6 days. After stimulation, cultures were fixed and stained for the presence of tartrate-resistant acid phosphatese (TRAP).C57BL/6 mice were immunized with C? /CFA for 4 weeks. CIA splenic G-MDSCs and M-MDSCs were sorted by flow cytometry. The cells were cultured according to the protocol above. After stimulation, cultures were also fixed and stained for TRAP.2×105MDSCs, M-MDSCs and G-MDSCs were seeded in 400ul alpha-minimal medium(containing 10%FBS) in a 96 wells osteo assay surface plate in the presence of 20ng/ml M-CSF for 3 days. Thereafter, they were further incubated with 20ng/mlM-CSF and 100ng/ml RANKL for 6 days. Then discarded the medium and added 100ul 10% bleach to each well to remove cells. Allow the plate to air dry completely at room temperature. Observe the pit formation under the microscope and randomly choose 6 fields to take pictures. The data was analyzed by NIS-Elements software and compared the difference of the pit areas.3. The effect of SIN on the MDSCs in CIA mice.Male C57BL/6(H-2b) were 8?10 weeks old. CIA model was induced by C?/CFA. Equal volume C?/IFA emulsion administered a boost injection 14 days after first immunization. The study was divided into experiment and control groups.100mg/kg SIN orally administered daily from day 35 to the end of experiment.PBS was given as negative control. The clinical scores were recorded 3 times every week after first immunization. Mice were sacrificed by dislocation after ether anesthesia.Then collected the bilateral limbs, and which processed routine decalcification,fixation, dehydration, transparency, waxdip, embedment, section and H&E staining.Histopathology scores of inflammation, cartilage demage and bone erosion were also analyzed. The frequency of PB and SP MDSCs and SP G-MDSCs and M-MDSCs were detected by flow cytometry. SIN or PBS orally administered and then collected the spleens of CIA mice. The expressions of IL-1?, IL-6, COX-2, TRAP, CTR and MMP-9 were detected by reverse transcription polymerase chain reaction.4. Statistical analysisAll data were analyzed using SPSS 13.0 statistical software. The data in the text represent the mean ± SD. Student t test was used for two group comparisons and One-Way or repeated measures analysis of variance. Bonferroni or Dunnett's T3 was used for multiple comparisons. And significant level of a =0.05 is desired.Two-side P value less than 0.05 were considered statistically significant.Results1. The percentage, phenotype and suppression function of MDSCs in CIA mice.1.1 Identification and evaluation of CIA modelThe joints of mice would become red or swelling on the 18th day after first immunization. The performance appeared interphalangeal joints first, and then foot pad, ankle. The presentation of swell and multi-arthritis would up to the peak on 28 days after first immunization. The incidence was 60% on 50 days, however there were any changes in control group.The pathology results showed: the synovium tissue of control mice was regularly arranged and there were no lymphocytes and plasma cells infiltration, and cartilage and bone destruction. However, the synovium tissue of CIA mice was severe proliferative and the layer of synovium became thicker. There were a large number of lymphocytes and neutrophils infiltration. Still the cartilage and bone destruction were observed.1.2 The accumulation of MDSCs and their subsets of G-MDSCs and M-MDSCsThe levels of PB,SP and BM MDSCs were detected by flow cytometry after immunization 4 weeks. Compared with control group, the levels of PB, SP and BM MDSCs were significantly elevated (t=-18.77, -9.48 and -8.48, P<0.05).The levels of splenic MDSCs subsets were detected by flow cytometry after immunization 4 weeks. Compared with control group, the levels of splenic G-MDSCs and M-MDSCs were significantly elevated (t=-5.18 and -4.90,P<0.05).The percentage of PB CD1lb+Gr-1+ MDSCs from CIA mice was dynamicly analyzed on day 0, 7, 14, 21, 28, 35 and 42. The results showed the percentage of PB CD11b+Gr-1+ MDSCs was increased along with days. The percentage was the highest on day 28 (43.34±4.30) % and then decreased from the 35th day (36.59±8.31) %.Thereafter, joints Gr-1+ cells recruitment were examined by immunofluorescence.The results displayed that abundant Gr-l+cells infiltrated into sunovial membrane.However, there was no Gr-1+ cells expression in the joints of control mice.1.3 The phenotypic characterization of MDSCs during CIA progressionThe phenotypic characterization of splenic MDSCs was detected by flow cytometry after immunization 4 weeks. The results indicated that these cells from control or CIA mice did not express CD11c, MHC-?, F4/80 and CD80. But these cells expressed cells surface markers,such as CD115, CCR2 and CD62L.More importantly, PB, SP and BM MDSCs from naive or CIA mice expressed OCs specific marker, CD51. And the expression levels of CD51 were higher in CIA mice than in control mice (60.61% vs 72.24%, 29.30% vs 38.80%, 52.66% vs 58.84%).1.4 MDSCs from CIA mice can suppress T cells activation which stimulate by CD3/CD281.4.1 MDSCs and their subsets influence T cell proliferationNaive SP stimulating by CD3 and CD28 mAb co-cultured with different ratio of CD11b+Gr-1+ MDSCs for 72h. Cell proliferation was tested by 3H-TdR. The results showed that the variances were heterogeneity tested by Levene (F=3.84, P=0.039).Multiple comparisons were needed based on Welch test (F=323.41, v=3, P=0.000).Comparing with no MDSCs group it could significantly suppress T cells proliferation when the ratio of MDSCs and SP was 1:2(P=0.001). Also comparing with no MDSCs group it could significantly suppress T cells proliferation when the ratio of MDSCs and SP was 1:1(P=0.000).Naive SP stimulating by CD3 and CD28 mAb co-cultured with different ratio of G-MDSCs for 72h. Cell proliferation was tested by 3H-TdR. The results showed that the variances were heterogeneity tested by Levene (F=5.127, P=O.016). Multiple comparisons were needed based on Welch test (F=1382.751, v=3, P=0.000).Comparing with no MDSCs group there was no significant di:fference when the ratio of G-MDSCs and SP was 1:2 or 1:1(P=0.074 and P=0.092).Naive SP stimulating by CD3 and CD28 mAb co-cultured with different ratio of M-MDSCs for 72h. Cell proliferation was tested by 3H-TdR. The results showed that the variances were heterogeneity tested by Levene (F=7.042, P=0.005). Multiple comparisons were needed based on Welch test (F=1382.751, v=3, P=0.000).Comparing with no MDSCs group it could significantly suppress T cells proliferation when the ratio of M-MDSCs and SP was 1:2(P=0.000). And likewise, comparing with no MDSCs group it could significantly suppress T cells proliferation when the ratio of M-MDSCs and SP was 1:1(P=0.000).1.4.2 The levels of IFN-? in cultural supernatantsNaive SP stimulating by CD3 and CD28 mAb co-cultured with different ratio of CD11b+Gr-1+ MDSCs for 56h and then collected the supernatants. The levels of IFN-Y were tested by ELISA. The results showed that the variances were heterogeneity tested by Levene ( F=3.491, P=0.05). Multiple comparisons were needed based on Welch test (F=1832.78, v=3, P=0.000). Comparing with no MDSCs group the level of IFN- Y was significantly decreased when the ratio of MDSCs and SP was 1:2(P=0.000). Similarly, comparing with no MDSCs group the level of IFN- ? was significantly reduced when the ratio of MDSCs and SP was 1:1(P=0.000).Naive SP stimulating by CD3 and CD28 mAb co-cultured with different ratio of G- MDSCs for 56h and then collected the supernatants. The levels of IFN- ? were tested by ELISA. The results showed that the variances were heterogeneity tested by Levene (F=12.586, P=0.001). Multiple comparisons were needed based on Welch test(F=26188.37, v=3, P=0.000). Comparing with no MDSCs group the level of IFN- Y was no significant difference when the ratio of G-MDSCs and SP was 1:2 or 1:1(P=0.115and P=0.795).Naive SP stimulating by CD3 and CD28 mAb co-cultured with different ratio of M- MDSCs for 56h and then collected the supernatants. The levels of IFN- Y were tested by ELISA. The results showed that the variances were heterogeneity tested by Levene (F=l 1.444, P=0.001). Multiple comparisons were needed based on Welch test(F=8320.55, v=3, P=0·000). Comparing with no MDSCs group the level of IFN- Y was significantly decreased when the ratio of MDSCs and SP was 1:2(P=0.000). In addition, comparing with no MDSCs group the level of IFN-? was significantly reduced when the ratio of MDSCs and SP was 1:1(P=0.000).2. MDSCs differentiated into OCs.2.1 CD11b+Gr-1? MDSCs differentiate into OCs in vitroLight microscope observed that OCs presented characteristics like this: big volume, irregular shape, wine red precipitates, many round acidophilia particles,multi-nuclear cells after TRAP staining. And nucleus presented dark black after counterstaining hematoxylin. The numbers of OCs generated in cultures of MDSCs derived from CIA mice (27.00±2.00) were higher than those from naive mice (8.33± 1.53). There was significant difference (t=-12.85, P=0.00).2.2 G-MDSCs and M-MDSCs differentiate into OCs in vitroSplenic G-MDSCs and M-MDSCs were sorted by flow cytometry after immunization 4 weeks. Cells were cultured according to the methods as mentioned above. The results showed that M-MDSCs from CIA mice could differentiate into OCs, but G-MDSCs couldn't. And the purities of splenic G-MDSCs and M-MDSCs from CIA mice sorted by flow cytometry were 99.2% and 98.6%, respectively.2.3 Pit formation assayAfter that we investigated whether MDSCs, G-MDSCs or M-MDSCs could differentiate into functional OCs or not. MDSCs, G-MDSCs or M-MDSCs were cultured and then discarded the medium and added 10Oul 10% bleach to each well to remove cells. Allow the plate to air dry at room temperature. The pits were observed under a microscope. The pit areas of MDSCs from CIA mice were higher than control group (t=-3.99, P=0.003). M-MDSCs from CIA mice could formed many pits, but G-MDSCs couldn't.3. The effect of SIN on the MDSCs in CIA mice.3.1 The effect of SIN on the levels of PB and SP MDSCs and SP G-MDSCs and M-MDSCsComparing with control group the percentage of PB or SP MDSCs was dropped after intervention by SIN. There was significant difference (t=9.87 and 4.23, P<0.001 and 0.05).The percentage of splenic G-MDSCs from CIA mice after SIN intervention was no significant difference comparing with control group (t=2.40, P>0.05). Whereas,the percentage of splenic M-MDSCs from CIA mice after SIN intervention was significant difference comparing with control group (t=3.24,P<0.05).3.2 The effect of SIN on the inflammation and bone destruction of CIA mice3.2.1 The effect of SIN on the clinical score of CIA miceThe joints of mice would become red or swelling on the 21St day after first immunization. The performance appeared feet first. The presentation of swell and multi-arthritis would up to the peak on 35 days after first immunization. 100mg/kg SIN orally administered daily from day 35 to the end of experiment.PBS was given as negative control. The arthritis score was no significant difference between treatment and control group on the 35th or 38th day (P=0.90 and P=0.16). But the arthritis score was reduced after SIN intervention comparing with control group on the 41st day(P=0.01). The arthritis score was no significant difference between the 35th day and the 38th days or between the 38th day and the 41th days (P=0.51 and P=0.10), however,the arthritis score was significantly reduced in treatment group (P=0.04). The data completely met the assumption of sphericity (W-0.939, P=0.975).Reapted factors among 3 different time points have significant difference (P<0.001). And there was an interaction between time and group (F=6.93, P=0.005). Pairwise comparisons results showed that the arthritis score was no significant difference between d3 5 and d38 (P=0.063); but the arthritis score was decreased between d35 and d41 or between d38 and d41 (P=0.000 and 0.004).3.2.2 The effect of SIN on the pathology score of CIA miceThe pathology score containing inflammatory cells infiltration, cartilage damage or bone erosion was lower than control group after SIN intervention (t=3.80, 5.58 and 6.33;P<0.05).3.3 The effect of SIN on the expressions of IL-1?,IL-6, COX-2, TRAP,CTR and MMP-9 mRNAComparing with control group the expressions of IL-1?, IL-6, COX-2, TRAP,CTR and MMP-9 mRNA were all depressed after SIN intervention.Conclusions1.A large number of CD11b+Gr-1+ MDSCs and their subsets(G-MDSCs,CD11b+Ly6G+Ly6Clow or M-MDSCs,CD11b+Ly6G-Ly6Chi) accumulate in vivo which may involve in the pathogenesis of CIA.2. Spenic MDSCs don't express F4/80, CD80, CD11c and MHC-?, which indicate these cells are immature or undifferentiated myeloid cells. However, these cells express inflammatory monocytic markers, such as CD 115, CCR2 and CD62L.3. PB, SP and BM CDllb+Gr-1+MDSCs express higher levels of CD51 which suggest that these cells may be a new cluster of OCPs in CIA.4. MDSCs and M-MDSCs both can suppress T cells proliferation and reduce IFN- Y secretion, which imply that these cells from CIA mice have suppression function,however insufficient suppression on T cells may lead to the emergency of CIA.5. MDSCs, especially M-MDSCs can differentiate into functional OCs, which confirm a new source of OCs.6. SIN can alleviate the severity of CIA by reducing MDSCs and their subsets M-MDSCs accumulation.7. SIN can decrease OCs formation and alleviate bone destruction by lessening IL-1?,IL-6, COX-2, MMP-9, CTR and TRAP mRNA levels and thus reducing MDSCs and their subsets M-MDSCs enrichment. |
PDF Full Text Request