Roles And Mechanisms Of Protein Tyrosine Phosphatase SHP2 In Eosinophil Differentiation In Allergic Airwav Inflammation

IntroductionAsthma is a chronic airway disease characterized by reversible hyper-reactivity and progressive airway inflammation, especially infiltration of eosinophils into the airway. Eosinophils appear to be the key effector cells in asthma, and there is a positive correlation between increased numbers and activation of eosinophils and the severity of asthma. Increased numbers of eosinophils have also been found in the bone marrow of patients with atopic asthma. Eosinophils develop in bone marrow, exit to the bloodstream and enter lung tissue in response to pro-inflammatory mediators such as eotaxin.Eosinophils develop from granulocyte/monocyte progenitors (GMPs) through intermediate eosinophil lineage-committed progenitors (EoPs) in mice, whereas human EoPs are derived from common myeloid progenitors (CMPs) or their upstream multipotent progenitors. Expression of the IL-5 receptor on EoPs is a result of commitment of GMPs to the eosinophil lineage, which means that IL-5 supports eosinophil development from EoPs rather than instructing GMPs to commit to the eosinophil lineage.The protein tyrosine phosphatase SHP2 is a ubiquitously expressed intracellular enzyme that contains two Src homology 2 domains and one catalytic protein tyrosine phosphatase domain. SHP2 integrates multiple signaling events and mediates a variety of physiological functions. Studies have shown that SHP2 participates in multiple signaling events by mediating T-cell development and function, and stimulating CEBPA gene expression to regulate cytokine-dependent granulopoiesis. Normal SHP2 function is critical in mediating the proper balance of embryonic stem cells and the maintenance of a functional hematopoietic stem cell/progenitor pool. Recent studies suggest an important role of SHP2 in the pathological changes associated with cigarette smoking-induced inflammation.In a previous study, we have shown that specific deletion of shp2 expression in mouse airway epithelia reduces TGF-β1 production and attenuates allergic airway remodeling. However, as SHP2 is expressed ubiquitously, it is of interest to know its possible roles in other cells or tissues during the development of asthma. As eosinophils are crucial for the pathogenesis of asthma, it is important to understand how eosinophil differentiation is regulated. Therefore, we investigated whether and how SHP2 affects eosinophil development. By using the SHP2 inhibitor PHPS-1, Ade-Cre-GFP and specifically deleting shp2 in myeloid cells, we found that inactivation or loss of SHP2 in these cells decreased the level of eosinophil recruitment to the airway, resulting in alleviation of lung inflammation and reduction of airway hyper-responsiveness, which were most likely through a direct inhibition of eosinophil differentiation. These results suggest that SHP2 may be a key regulator of eosinophil differentiation and thus can serve as a potential therapeutic target for the treatment of asthma.Part 1 PHPS-1 Alleviates Airway Inflammation and Decreases, Inhibits the Production of Eosinophils and Eops in Asthmatic MiceAim:The aim of this study is to investigate the protective effects of PHPS-1 in asthmatic mice, and explore the underlying regulatory mechanisms.Methods:6-8 week old male C57BL/6 mice were given the new ID by their weight from small to large. By using the random number table, the mice were randomly divided into four groups of SHAM+PBS, SHAM+PHPS-1, OVA+PBS and OVA+PHPS-1. The mice in SHAM+PBS group were sensitized and challenged with PBS; while the mice in SHAM+PHPS-1 group we sensitized with PBS, and treated with PHPS-1 (i.p.,5mg/kg) 1 hour before PBS challenge; The mice in OVA+PBS group were sensitized and challenged with OVA; while the mice in OVA+PHPS-1 group were sensitized with OVA, and treated with PHPS-1 (i.p.,5mg/kg) 1 hour before the OVA challenge. The mice were sacrificed 24 hours after the last OVA and PBS exposure. The BALF cells were harvested for counting the numbers of total cells and the classified cells as eosinophils, lymphcytes and neutrophils; the left lung lobes were removed for the pathological assay; we estimated the expression of Th2 inflammatory factors in lung by Q-PCR, and also detected the expression levels of cytokines of lung homogenate. What is more, the percentage of eosinophils and eosinophil prognitors were determined by using flow cytometry.Results:PHPS-1 led to a significant reduction in total cell number, and specifically, there was a remarkable decrease of eosinophil number in OVA/PHPS-1 mice compared with the OVA+PBS group. PHPS-1 also decreased AHR in asthmatic mice, reducing the levels to those in the PBS-treated control group. Pharmacological inhibition of SHP2 resulted in marked reduction of the mRNA levels of Il-4,Il-5 and Il-13.The protein levels of IL-4 and IL-13 in lung tissue were consistent with the mRNA levels. Lung histopathology showed considerably fewer inflammatory cells in the lungs of OVA+PHPS-1 mice than OVA+PBS mice. Periodic acid-Schiff (PAS) staining demonstrated less mucus production and fewer mucus- producing cells in the bronchioles and lungs of OVA+PHPS-1 mice than in OVA+PBS mice. The numbers of eosinophils in blood and bone marrow decreased when asthmatic mice were treated with PHPS-1, with no obvious change in sereum IL-5 and the apoptosis of eosinophils in bone marrow. EoPs and Eos-CFU were dramatic decreased in asthmatic mice treated with PHPS-1. However, the absolute number of GMPs was not significantly different in OVA+PHPS-1 and OVA+PBS mice. The mRNA level of GATA-1 in bone marrow was inhibited in OVA+PHPS-1 mice compared with OVA+PBS mice.Conclusion:PHPS-1 has a protective effect on ovalbumin induced pulmonary inflammation. The underlying mechanisms may be due to down-regulate the expression of inflammatory cytokines, decrease inflammatory cell infiltration, inhibit the number and functions of eosinophils and eosinophil progenitors, but not via regulating the level of IL-5 and the apoptisis level of eosinophil.Part 2 PHPS-1 Inhibits Eosinophil Differentiation in vitro Without Influence on the Apopotosis of EosinophilsAim:The aim of this study is to investigate the protective effects of SHP2 in vitro in the differentiation of eosinophils.Methods:Bone-marrow cells were cultured in vitro for the eosinophil differentiation and cells were enumerated and harvested for analysis of the expression of SiglecF and the mRNA levels of GATA-1 and MBP. NAMNCs from shp2flox/flox mice were transduced with Ad-Cre-GFP and Ad-GFP and cultured with rmIL-5 for the assay of bone marrow eosinophil CFUs.Results:SHP2 protein was induced by IL-5 in a time-dependent fashion. PHPS-1 (20 uM) administered together with IL-5 dramatically reduced the production of eosinophils. Interestingly, PHPS-1 failed to have any considerable effect on the apoptosis of eosinophils, indicating that PHPS-1 specifically inhibits the differentiation of eosinophils without affecting their survival. Real-time PCR analysis also revealed that the IL-5-induced mRNA levels of GATA-1 and MBP were markedly reduced by PHPS-1. Furthermore, the number of eosinophil CFUs was remarkably reduced when NAMNCs were treated with PHPS-1, especially those from asthmatic mice. Bone marrow NAMNCs in which shp2 was deleted showed remarkably reduced eosinophil percentages and numbers of eosinophil CFUs compared with controls. The CFU-eosinophils were much smaller in NAMNCs treated with Ad-Cre-GFP. We also measured the level of GATA-1 mRNA on day 8 of eosinophil differentiation, and found it was significantly reduced when NAMNCs were treated with Ad-Cre-GFP.Conclusion:PHPS-1 directly inhibits the differentiation of eosinophils in vitro. Genetic knockdown of shp2 in mouse bone marrow can also decrease eosinophil differentiation. These data further suggest that SHP2 indeed plays a key role in the differentiation of eosinophils.Part 3 Genetic Knockdown of shp2 in Mouse Bone Marrow Decreases Eosinophil Differentiation via p-Erk ActivationAim:The aim of this study is to investigate the protective effects of myeloid shp2 knockdown in mouse model, and explore the underlying regulatory mechanisms.Methods:Bone-marrow cells from LysMcreShp2flox/flox mice were cultured in vitro for the eosinophil differentiation and deteted the possible passway. Erk inhibitor U0126 was given in the eosinophil differentiation and eosinophils were determined by using flow cytometry.6-8 week old male LysMcreShp2flox/flox mice and Shp2flox/flox mice were used to establish the asthmatic model. Mice were randomly divided into four groups. The mice were sacrificed 24 hours after the last OVA and PBS exposure. The BALF cells were harvested for counting the numbers of total cells and the classified cells as eosinophils, lymphcytes and neutrophils; the left lung lobes were removed for the pathological assay; we estimated the expression of Th2 inflammatory factors in lung by Q-PCR, and also detected the expression levels of cytokines of lung homogenate.Results:The base level of eosinophils in bone marrow was remarkably decreased in the LysMcreShp2flox/flox mice. The number of eosinophils was dramtically decreased in LysMcreShp2flox/flox mice compared with Shp2flox/flox mice during eosinophil differentiation. SHP2 protein levels were markedly decreased in the bone marrow cells of LysMcreShp2flox/flox mice compared with Shp2flox/flox mice. Furthermore, knockdown of shp2 suppressed activation of the Erk signal. The percentage and number of eosinophils also decreased when the bone marrow cells were treated with U0126. Myeloid cell-specific shp2 knockout resulted in a significant reduction of total cell number in BLAF; in particular, the eosinophil numbers in LysMcreShp2flox/flox mice showed a marked decrease relative to Shp2flox/flox control mice. Moreover, AHR in LysMcreShp2flox/flox asthmatic mice was significantly reduced compared to OVA-treated Shp2flox/flox controls. Furthermore, myeloid cell-specific shp2 knockdown resulted in a remarkable reduction of the mRNA levels of Il-4,Il-5 and IL-13. The decreased pro-inflammatory cell infiltrates in mice were further demonstrated by a corresponding alleviation in the histopathology observed in these mice as reflected quantitatively by the inflammatory and PAS scores.Conclusion:Myeloid shp2 conditional knockout mice (LysMcreshp2flox/flox) displayed significantly attenuated allergic airway inflammation. SHP2 induced eosinophil differentiation via regulation of the extracellular signal-regulated kinase pathway. These data reveal SHP2 as a critical regulator of eosinophil differentiation, and inhibition of SHP2 specifically in myeloid cells alleviates allergic airway inflammation.