| Background and Objectives: CD4+T helper(Th) cells play important roles in the immunopathogenesis of pulmonary sarcoidosis. Recently, Th cells are classified into four major lineages, Thl, Th2, Th17 and T regulatory (Treg) cells. Now, the role of Th17 and Treg cells in autoimmune diseases has been hot topics. And a theory of disturbance of Thl7/Treg cell in autoimmune is coming into being. During differentiation of Th17 cells,IL-23 plays an important role in expansion and stabilization of them. IL-17A is the key cytokine of Thl7 cells. So, some researchers draw a close attention to the role of the IL-23/IL-17 axis in autoimmune diseases. IL-10 and TGF-βare the primary cytokines involved in the formation of Treg cells. At the same time, Treg cells exert their suppressive activity partly through secretion of these two cytokines. Retinoic acid-related orphan receptors ?t (ROR?t) and Fork-head protein3(Foxp3) is the specific transcript factor for Th17 and Treg cells, respectively. In recent years, although several researchers pay attention to the role of Treg cells in the immunopathogenesis of sarcoidosis, the results are still ambiguous. Only one or two articles about the Th17 cells in sarcoidosis can be retrieved from the Pubmed. Then, how about the balance of Th17/Treg cells in sarcoidosis? In this study, the research objects are patients suffered with stage I to II pulmonary sarcoidosis and healthy controls. We investigate the percentage of Th17 and Treg cells through Flow Cytometry for them. The concentration of IL-23, IL-17 A, IL-10 and TGF-βare also detected by ELISA ananlysis. Finally, we compare the expression of ROR?t and Foxp3 before and after the treatment of glucocorticoids for sarcoidosis patients.Subjects and Methods SubjectsTen sarcoidosis patients are recruited from the Out-Patient Department of Respiratory at Peking Union Medical Hospital (PUMCH). Ten healthy controls(HCs) are recruited from volunteers with the same sex distinctions and age. Informed consents are obtained from all patients and HCs before collection of samples. All patients are diagnosed as sarcoidosis with positive pathological results. According to the guideline, all of the patients should accept treatment. After treatment with glucocorticoids for 4 to 6 weeks, we divide them into improved and unimproved subgroup according the improvement of clinical manifestations and the change of chest CT scans.Sample collectionAbout 14 ml peripheral blood are drawn from HCs,untreated sarcoidosis patients and after the patients taken glucocorticoids for 4 to 6 weeks. Peripheral blood mononuclear cells (PBMCs) are isolated by density gradient centrifugation. Bronchoalveolar lavage fluid(BALF) are collected from the sarcoidosis patients and the mononuclear cells are isolated by centrifugation.Methods Analysis by Flow Cy tome try Surface staining of CD4 as well as intracellular staining of IL-17A are used to identified Thl7 cells. Surface staining of CD4 and CD25(bright) as well as intracellular staining of Foxp3 are used to identified Treg cells. Flow cytometry are carried out to analysis the percentage of Th17 cells and Treg cells in PBMC from HCs, sarcoidosis patients(before and after treatment with glucocorticoids). The percentage of Th17 cells and Treg cells are also analysed in BALF mononuclear cells from untreated sarcoidosis patients. The mean fluorescence index(MFI) of IL-17A and Foxp3 are also detected.ELISA analysis for cytokinesThe plasma concentration of IL-23, IL-17A, IL-10 and TGF-B are detected by ELISA analysis for HCs and sarcoidosis patients(before and after treatment with glucocorticoids). The BALF concentration of these cytokines are also detected for untreated sarcoidosis patients. Real-time PCR Analysis for ROR?t and Foxp3 ExpressionExpression of ROR?t and Foxp3 in PBMC from sarcoidosis patients(before and after treatment with glucocorticoids) are analysed by Real-time PCR analysis, respectively.Results Subjects The average age was 51.9±7.6 years. There are nine females and one male. The common manifestation are fatigue(n=8), body weight loss(n=7,decreased 3-15kg),cough(n=5), exertional dyspnea(n=5),chest pain(n=3), rash(n=2),low-grade fever(n=1)and central paralysis of the facial nerve(n=1). No patient has superficial palpable lymph nodes, clubbings and Velcro rales. The ratio of CD4+T cells to CD8+Tcells are 6.54±3.82. Four patients are diagnosed as stage I pulmonary sarcoidosis, and 6 patients are stage II. The clinical manifestations and the reexamine of chest CT scan of five patients are improved after 4 to 6 weeks treatment with glucocoiticoids. And another five patients show no improvement after treatment. Analysis by Flow Cytometry1.The percentage of CD4+IL-17A+Th17 Cells are elevated in the peripheral blood(PB) of sarcoidosis patients compared to HCs'(P=0.0139). For the same patients, the percentage of Th17 Cells are elevated in the BALF compared to in PB(P=0.0016<0.05).But there is no difference of the MFI of IL-17A among the groups.2. The percentage of CD4+CD25bright+Foxp3+Treg cells are decreased in the PB of sarcoidosis patients compared to HCs'(P=0.0286), and the MFI of Foxp3 are lower in sarcoidosis patients compared to HCs'(P=0.0038). But for the same patient, the percentage of Treg cells are are elevated in the BALF compared to in PB(P=0.0038), and there is no difference of the MFI of Foxp3 between the two groups(P>0.05).3. There are an imbalance of Th17 and Treg cells in PBMC and BALF of sarcoidosis patients compared to HCs'.4. After treatment with glucocorticoids, the percentage of Th17 Cells and Treg cells and the MFI of IL-17A and Foxp3 of PBMC have no differences compared to the initial levels(P>0.05).5.Between different therapeutic effect subgroups, the percentage of Thl7 Cells and Treg cells and the MFI of IL-17A and Foxp3 in PB and BALF show no differences(P>0/05). Analysis by ELISA1.In contrast to HCs', plasma concentration of IL-10 is elevated in sarcoidosis patients (P=0.0406), But there are no differences of the plasma concentration of IL-23, IL-17A and TGF-βbetween them.2. To the same patient, the concentration of IL-17A and IL-23 are elevated in BALF compared to in PB(P=0.0347 and P<0.0001, respectively), and IL-10 is decreased in the BALF compared to in PB(P=0.0003). There is no difference of the concentration of TGF-βbetween in PB and BALF(P>0.05).3.After treatment with glucocorticoids, there are no differences of the plasma concentration of IL-23, IL-17A, IL-10 and TGF-βcompared to the initial's(P>0.05).4. Between different therapeutic effect subgroups, there are no differences of the concentration of IL-23, IL-17A, IL-10 and TGF-βbetween in PB and in BALF, respectively(P>0.05).Analysis by Real-time PCR1. After treatment with glucocorticoids, there is no difference of expression of ROR?t in PB compared to the initial level(P=0.057). But there is a obvious downward trend of ROR?t expression after treatment.2. After treatment with glucocorticoids, expression of Foxp3 is increased in PB compared to the initial level(P=0.0135).ConclusionsThere are significant disturbance of Th17/Treg cells ratio in PB and BALF of stage I toⅡpulmonary sarcoidosis patients. And there are obvious disturbance of the associated cytokines of Th17 and Treg cells. Those results may indicate the pathogenic role of Th17 and Treg cells in sarcoidosis. The immune cells and cytokines is a complex network. Every cytokine is not specific to a kind of cell. After treatment with glucocorticoids, there are no differences between the percentage of Thl7 and Treg cells and between the plasma concentration of their associated cytokines compared to the initial levels. But there is difference between the expression of Foxp3(specific transcript factor for Treg cell) and there is obvious downward trend of ROR?t (specific transcript factor for Th17 cell)expression compared to the initial levels. We may conclude that glucocorticoids may try to influence the imbalance of Thl7/Treg cells in sarcoidosis. But the therapeutic effect is not related directly to the percentage of Th17, Treg cells and the levels of their associated cytokines.
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