The Distribution And Roles Of Monocyte Subsets In Primary Immune Thrombocytopenia The Levels And Clinical Significance Of Interleukin-35in Primary Immune Thrombocytopenia

Background:Human monocytes are heterogeneous and play an important role in the innate immune, inflammatory and autoimmune response. With the aid of two color immunofluorence and flow cytometry, a new subset of CD16+monocytes was identified in1989. Based on higher expression of proflammatory cytokines and higher potency in antigen presentation, the CD16+monocytes were labeled ’proflammatory monocytes’ This new subset has been described to increase in many different types of diseases including infection of virus and bacterium, inflammatory diseases and autoimmune diseases such as rheumatoid arthritis and crohn disease. Nowadays, new evidence suggests that the CD16+monocytes could be further subdivided into the intermediate (CD14++CD16+) and the nonclassical (CD14+CD16++) group according to the recently published nomenclature. Primary immune thrombocytopenia (ITP) is an acquired autoimmune disease with many immune dysfunctions. The distribution and roles of monocyte subsets in ITP patients remain uncovered.Objective:To explore the distribution and roles of monocyte subsets in ITP patients.Methods:Frequencies of monocyte subpopulations were determined in71untreated active ITP patients,23ITP patients being treated with glucocorticoid and49age-matched controls by flow cytometry. The correlation between monocytes frequencies and platelet counts were analyzed. Cytokines both in sera and the cultured supernatant of monocyte subpopulations were measured by ELISA. The sorted monocyte subset was co-cultured with CD4+T cells and autologus platelet respectively. The proliferation of auto-reactive CD4+T cells was determined by Brdu ELISA. The levels of IFN-y in the coculture supernant were measured by ELISA.Results:The frequency of the nonclassical monocytes was significantly increased in untreated active ITP patients compared with controls (untreated ITP:5.44%[0.36%-22.29%] vs Ctrl:1.83%[0.12%-7.11%],p<0.001). The frequency of the intermediate monocytes was also increased in active patients (untreated ITP:10.54%[5.01%-45.15%] vs Ctrl:7.6%[1.6%-23.99%], p<0.001).The frequency of classical monocyte was decreased in active patients (untreated ITP:82.6%[51.23%-92.38%] vs Ctrl:90.75%[66.83%-95.92%],p=0.001). In addition, the frequencies of intermediate and non-classical monocyte showed negative correlation with platelet counts and the frequencies of classical monocyte showed positive correlation with platelet counts. In9patients, the monocyte subsets were also measured after complete remission and the frequency of nonclassical monocyte was decreased substantially (untreated:7.47%[0.57%-11.85%] vs CR:1.16%[0-8.78%],p=0.015).In the patients treated with GCs, the nonclassical monocytes were decreased quickly (p<0.001), but the frequency of intermediate monocyte remains stable and the frequency of classical monocyte was recovered during GCs therapy. Compared to the controls, serum TNF-a and IL-1β levels in ITP patients were elevated. After stimulation by LPS for24hours, the intermediate subset produced the most TNF-a and IL-1β.The nonclassical subset showed highest stimulation of platelet reactive T cell proliferation (p=0.066). Further studies demonstrated that the nonclassical monocytes promoted Thl porlarization.Conclusion:We concluded that the nonclassical and intermediate monocytes expanded dramatically in active ITP patients and were probably involved in the immune dysfunction of ITP. Background:Primary immune thrombocytopenia (ITP) is an autoimmune disease with complex dysregulation of the immune system. IL-35is a novel heterodimeric anti-inflammatory cytokine consist of Epstein-Barr virus-induced gene3(EBB) and the p35subunit of IL-12, which was shown to have the potency of inhibiting the CD4+effector T cells and alleviates autoimmune diseases. However, the levels of plasma IL-35and its potential role in ITP remain to be investigated.Methods:Plasma IL-35, TGF-β1and IL-10levels were measured in41active ITP patients,22patients in remission and30healthy controls by ELISA. The mRNA expression levels of P35and EBI3in peripheral blood mononuclear cells (PBMCs) of36ITP patients and21healthy controls were studied by real-time quantitative RT-PCR. The correlation between plasma cytokine levels and clinical data were analyzed.Results:Active ITP patients had significantly lower plasma IL-35levels than those of patients in remission (p=0.017) and normal controls (p<0.001). Furthermore, the plasma IL-35levels of patients in remission were still lower than normal controls (p<0.001). In active ITP patients, the plasma IL-35levels showed significantly positive correlation with platelet counts(r=0.5335, p<0.001). There is no difference between the untreated and treated group. P35mRNA expression levels in ITP patients were lower than patients in remission (P=0.033) and healthy controls (P=0.016). Conclusion:Our results showed a dramatically decreased IL-35levels in ITP patients, suggesting that IL-35may be involved in the pathogenesis of ITP.