Xiaoxia Hou

Identification of the E3 Deubiquitinase Ubiquitin-specific Peptidase 21 (USP21) as a Positive Regulator of the Transcription Factor GATA3 *

Background: GATA3 is regulated both transcriptionally and post-translationally. GATA3 is important for the function of FOXP3+ Treg cells. Results: USP21 prevents the ubiquitination and degradation of GATA3. Conclusion: USP21 is a positive regulator of GATA3 expression. Significance: The identification of a molecular pathway where USP21 positively controls GATA3 expression at the post-translational level reveals USP21 as a potential drug target to manipulate the function of T cells. The expression of the transcription factor GATA3 in FOXP3+ regulatory T (Treg) cells is crucial for their physiological function in limiting inflammatory responses. Although other studies have shown how T cell receptor (TcR) signals induce the up-regulation of GATA3 expression in Treg cells, the underlying mechanism that maintains GATA3 expression in Treg cells remains unclear. Here, we show how USP21 interacts with and stabilizes GATA3 by mediating its deubiquitination. In a T cell line model, we found that TcR stimulation promoted USP21 expression, which was further up-regulated in the presence of FOXP3. The USP21 mutant C221A reduced its capacity to stabilize GATA3 expression, and its knockdown led to the down-regulation of GATA3 protein expression in Treg cells. Furthermore, we found that FOXP3 could directly bind to the USP21 gene promoter and activated its transcription upon TcR stimulation. Finally, USP21, GATA3, and FOXP3 were found up-regulated in Treg cells that were isolated from asthmatic subjects. In summary, we have identified a USP21-mediated pathway that promotes GATA3 stabilization and expression at the post-translational level. We propose that this pathway forms an important signaling loop that stabilizes the expression of GATA3 in Treg cells.

Histone deacetylase inhibitor regulates the balance of Th17/Treg in allergic asthma

The aim of this study is to investigate the expression pattern of histone deacetylase 9 in peripheral blood of patients with allergic asthma and its regulatory effect on the balance of Th17/Treg cells involved in the pathogenesis of asthma.

Critical roles of adenosine A2A receptor in regulating the balance of Treg/Th17cells in allergic asthma.

Deficiency of Treg cells and hyperactivity of Th17 cells together are involved in the immunological pathogenesis of asthma. The adenosine A2A receptor (A2AR) plays a critical role in the increased Foxp3 expression of Treg cells and the decreased Th17 generation.

Role of PIM2 in allergic asthma

T cell-associated inflammation, particularly type 2 inflammation, has an important role in asthma pathogenesis, which is suppressed by regulatory T cells (Tregs). Proviral integration site for Moloney murine leukemia virus 2 (PIM2), a member off the serine/threonine kinase family, promotes the growth and survival of T cells and influences the function of Treg cells. However, whether PIM2 affects asthma pathogenesis remains unclear. Peripheral blood mononuclear cells and Treg cells from asthmatic and healthy subjects were obtained, and the expression level of PIM2 was measured by reverse transcription-quantitative polymerase chain reaction and immunocytochemistry. In addition, BALB/c female mice sensitized and challenged by ovalbumin were used as an asthma model, and PIM2 inhibitor was injected during the challenge period to observe the effect of PIM2 on asthma. The asthma symptoms were recorded, and airway hyper-responsiveness (AHR), expression levels of cytokines in the serum or bronchoalveolar lavage fluid (BALF), and the number of BALF leukocytes were evaluated. In addition, hematoxylin and eosin staining and immunohistochemistry of lung tissues was performed. The results demonstrated that PIM2 was overexpressed in patients with asthma in natural Treg cells. Inhibition of PIM2 attenuated asthma symptoms, and improved AHR and airway inflammation compared with asthmatic mice without inhibition of PIM2. In addition, expression levels of interleukin (IL)-10 and forkhead box protein 3 (FOXP3) in BALF were increased following PIM2 inhibition (IL-10, 470.3±21.78 vs. 533.7±25.55 pg/ml, P<0.05; FOXP3, 259±4.68 vs. 279.3±3.68 pg/ml; asthma and PIM2 inhibition groups, respectively; P<0.05). In conclusion, PIM2 may exhibit an important role in asthma pathogenesis and exacerbate AHR, airway inflammation and asthma symptoms. These effects of PIM2 may be dependent on Treg cells and the secretion of IL-10 by Tregs. The results of the present study suggest that PIM2 may be a potential target molecule for asthma treatment.

4-1BB ligand-mediated imbalance of helper 17 T cells and regulatory T cells in patients with allergic asthma.

OBJECTIVES: To investigate the presence of 4-1BB ligand (4-1BBL) in the peripheral blood of patients with allergic asthma and evaluate its role in controlling the balance between helper 17 T (Th17) and regulatory T (Treg) cells. METHODS: Soluble 4-1BBL (s4-1BBL) was quantified by enzyme-linked immunosorbent assay in plasma from patients with asthma (n = 45) and from healthy control subjects (n = 35). The proportion of monocytes positive for membrane-bound 4-1BBL (m4-1BBL) was determined by flow cytometry. Peripheral blood mononuclear cells from patients with asthma were incubated with anti-4-1BB monoclonal antibody in vitro. Concentrations of interleukin (IL)-17 and transforming growth factor (TGF)-β1 in the culture supernatant were analysed. RESULTS: Plasma s4-1BBL concentrations and the proportion of m4-1BBL-positive monocytes were significantly lower in patients with asthma than in control subjects. The culture supernatant concentration of TGF-β1 was increased and that of IL-17 was decreased by incubation with anti-4-1BB monoclonal antibody. CONCLUSIONS: Both soluble and membrane-bound 4-1BBL were reduced in patients with allergic asthma compared with control subjects. 4-1BBL/4-1BB signalling may play an important role in allergic asthma by regulating the Th17/Treg balance.

Dephosphorylated Polymerase I and Transcript Release Factor Prevents Allergic Asthma Exacerbations by Limiting IL-33 Release

Background Asthma is a chronic inflammatory disease characterized by airway inflammation and airway hyperresponsiveness (AHR). IL-33 is considered as one of the most critical molecules in asthma pathogenesis. IL-33 is stored in nucleus and passively released during necrosis. But little is known about whether living cells can release IL-33 and how this process is regulated. Objective We sought to investigate the role of polymerase I and transcript release factor (PTRF) in IL-33 release and asthma pathogenesis. Methods Ovalbumin (OVA)-induced asthma model in PTRF+/− mice were employed to dissect the role of PTRF in vivo. Then, further in vitro experiments were carried out to unwind the potential mechanism involved. Results In OVA asthma model with challenge phase, PTRF+/− mice showed a greater airway hyper-reaction, with an intense airway inflammation and more eosinophils in bronchoalveolar lavage fluid (BALF). Consistently, more acute type 2 immune response in lung and a higher IL-33 level in BALF were found in PTRF+/− mice. In OVA asthma model without challenge phase, airway inflammation and local type 2 immune responses were comparable between control mice and PTRF+/− mice. Knockdown of PTRF in 16HBE led to a significantly increased level of IL-33 in cell culture supernatants in response to LPS or HDM. Immunoprecipitation assay clarified Y158 as the major phosphorylation site of PTRF, which was also critical for the interaction of IL-33 and PTRF. Overexpression of dephosphorylated mutant Y158F of PTRF sequestered IL-33 in nucleus together with PTRF and limited IL-33 extracellular secretion. Conclusion Partial loss of PTRF led to a greater AHR and potent type 2 immune responses during challenge phase of asthma model, without influencing the sensitization phase. PTRF phosphorylation status determined subcellular location of PTRF and, therefore, regulated IL-33 release.

The Imbalance of FOXP3/GATA3 in Regulatory T Cells from the Peripheral Blood of Asthmatic Patients

Background Treg cells play an important role in the pathogenic progress of asthma. Objective To address the alterations of Treg cells in asthma. Methods Proliferation-and function-associated markers of Treg cells along with the percentage of Treg cells producing some cytokine from asthmatics and healthy subjects were analyzed by flow cytometry. Besides, the expressions of USP21 and PIM2 in Treg cells were measured by cell immunochemistry after Treg cells were sorted. Results Treg cells from asthmatic patients showed lower proliferation activity and were more likely to be apoptotic. These cells expressed lower levels of GITR, CTLA-4, Nrp-1, and IL-10 compared to those from the healthy control. Th2-like Treg cells increased in asthmatic patients, while the percentage of IFN-r+ Treg cells was similar between two groups. Moreover, the percentage of IL-4+ Treg cells is related to the asthma control. Treg cells from asthmatic patients expressed more FOXP3 as well as GATA3; the expression level of GATA3 negatively correlated with FEV1%pred. Increased expressions of USP21 and PIM2 in Treg cells from asthmatic patients were found. Conclusion Treg cells decreased in asthmatic patients, with an impaired immunosupression function and a Th2-like phenotype, which may be due to overexpression of GATA3 and FOXP3, regulated by USP21 and PIM2, respectively.

Intratracheal administration of adipose derived mesenchymal stem cells alleviates chronic asthma in a mouse model

BackgroundAdipose-derived mesenchymal stem cell (ASCs) exerts immunomodulatory roles in asthma. However, the underlying mechanism remains unclear. The present study aimed to explore the effects and mechanisms of ASCs on chronic asthma using an ovalbumin (OVA)-sensitized asthmatic mouse model.MethodsMurine ASCs (mASCs) were isolated from male Balb/c mice and identified by the expression of surface markers using flow cytometry. The OVA-sensitized asthmatic mouse model was established and then animals were treated with the mASCs through intratracheal delivery. The therapy effects were assessed by measuring airway responsiveness, performing immuohistochemical analysis, and examining bronchoalveolar lavage fluid (BALF). Additionally, the expression of inflammatory cytokines and lgE was detected by CHIP and ELISA, respectively. The mRNA levels of serum indices were detected using qRT-PCR.ResultsThe mASCs grew by adherence with fibroblast-like morphology, and showed the positive expression of CD90, CD44, and CD29 as well as the negative expression of CD45 and CD34, indicating that the mASCs were successfully isolated. Administering mASCs to asthmatic model animals through intratracheal delivery reduced airway responsiveness, the number of lymphocytes (P < 0.01) and the expression of lgE (P < 0.01), IL-1β (P < 0.05), IL-4 (P < 0.001), and IL-17F (P < 0.001), as well as increased the serum levels of IL-10 and Foxp3, and the percentage of CD4 + CD25 + Foxp3+ Tregs in the spleen, and reduced the expression of IL-17 (P < 0.05) and RORγ.ConclusionsIntratracheal administration of mASCs alleviated airway inflammation, improved airway remodeling, and relieved airway hyperresponsiveness in an OVA-sensitized asthma model, which might be associated with the restoration of Th1/Th2 cell balance by mASCs.