| Background:Acute lung injury(ALI)and its severe form of acute respiratory distress syndrome(ARDS)are acute inflammatory reactions in the lungs caused by different causes.It is s a severe form of diffuse lung disease,and there is no spcific clinical treatment,and resulting in a high mortality rate of the disease.The early pathophysiological changes are mainly inflammatory exudation,and the late stage is characterized by progressive fibrosis.The histological hallmark of ALI/ARDS is classically differentiated into two major temporal phases:exudative(early)followed by fibroproliferative,(late)with the latterpotentially evolving into fibrosis.Fibroproliferation is part of the normal repair response to ALI/ARDS.However,aberrant or excessive fibroproliferation results in impaired lung repair following acute lung inflammation which is associated with worse clinical outcomes.we recently reported that CD4+CD25+regulatory T cells(Tregs)contributed to the recovery of patients with acute lung injury(Acute lung injury,ALI)by upregulating T cell immunoglobulin and mucin-domain containing-3(Tim-3).However,the molecular mechanism by which Tim-3 regulates Tregs' function in the resolution and fibro proliferation after ALI remains unknown.Methods:In this study,we chose Male C57BL/6 mice aged 6-8 weeks(16-18g),and adoptively transferred Tim-3+Tregs or Tim-3-Tregs into LPS-induced ALI mice model.BALF was analysed to measure the protein.Lung Histology and Immunohistochemistry was used to assess of pulmonary fibrosis quantitatively.Lung MPO activity was measured as a marker for neutrophil influx.In order to study the interaction between cells,we used isolation of lung mononuclear cells and cocultures experiments.We alse used the technology of the immunoblot analysis/quantification of Cytokines/Real-time PCR/flow cytometry to explore Tim-3 in vitro and in vivo by regulating Tregs cellular immune function,secreting IL-10 and IL-4 to induce the production of M2 macrophage cells,promoting inflammation regression,reducing the occurrence of fibrosis after ALI,and promoting the mechanism of repair.Result:1.Higher expression of Tim-3 on Tregs was associated with better clinical outcome in ALI patients.The data suggested that adoptively transferring of Tim-3+Tregs but not Tim-3-Tregs had the ability to reducethe injury after ALI/ARDS in murine models.Adoptive transfer of exogenous Tregs could directly modify the innate immune response in experimental indirect ALI/ARDS.2.Tim-3+Tregs transfer mitigated the LPS-induced fibroproliferation in ALI/ARDS mice3.Tim-3+Tregs transfer induced M2-like macrophage differentiation in the LPS-induced fibroproliferation.4.Tim-3+Tregs induced M2-like macrophage polarization in vitro.5.Tim-3+Tregs's capacity in the polarization of M2-like macrophage was partially through the STAT-3 pathway.Conclusions:Data demonstrated thatTim-3+Tregs not only decreased indices of lung inflammation and injury but also mitigated the lung fibrosis after ALI.Furthermore,we observed that the transfer of Tim-3+Tregs led to M2-like macrophages differentiation as demonstrated by significantly upregulated levels of M2-associated phenotypic markers as well as down regulated expressions of M1-related markers both in the pro-fibrotic lung tissue and sorted pulmonary monocytes after ALI.In addition,cytokines as interleukin(IL)-10 and IL-4 were also noticed upregulated in lung tissues after Tim-3+Tregs transferring.In vitro experiments further demonstrated that cell-contact co-cultures with Tregs lacking Tim-3 presented decreased polarization of M2-like macrophages partially mediated by a decreased expression and function of STAT-3.Therefore,these data demonstrate a previously unrecognized immune-modulatory function and mechanism of Tim-3 on Tregs in their ability to repress the fibroproliferation of ALI by inducing alternative macrophages polarization.Moreover,the data highlight that Tim-3+Tregs mediated induction of M2-like macrophagesmay be a novel treatment modality with transitional potential. |
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