Receptor Mechanism Of Regulatory T Cell Immune Response In Acute Lung Injury Mice Induced By High Mobility Group Box-1

Objective: Investigate the changes of regulatory T cell immune function and immunosuppressive activity after acute lung injury induced by high mobility group box-1(HMGB1) in mice with different TLR4 genotypes, investigate the interaction between regulatory T cells(Treg) and CD4+CD25-T cells, and research the receptor mechanism of Treg cell immune response in acute lung injury mice induced by HMGB1.Methods: Healthy clean grade C57BL/10 J and C57BL/10 Sc [TLR4 wild type(TLR4+/+) and knockout type(TLR4-/-)] mice were given HMGB1(20 μg/mouse) by intratracheal injection as the experimental group, and other wide type mice were given normal saline by intratracheal injection as the control group.(1) The mice were raised for 48 hours after establishment of model, and then sacrificed with eyeball extirpating,followed by collection of serum and bronchoalveolar lavage fluid(BALF). The pathology of lung was observed under light microscope, and the dry wet ratio of lung was measured. Immunomagnetic beads were used to separate spleen CD4+CD25+ Treg cells and CD4+CD25-T cells.(2) Flow cytometry was used to determine the expression of forkhead/winged helix transcription factor(FOXP3) and cytotoxic T lymphocyte antigen 4(CTLA-4) in CD4+CD25+Treg cells, and enzyme-linked immunosorbent assay(ELISA) was used to determine the content of interleukin-1(IL-1) and tumor necrosis factor-α(TNF-α) in BALF, as well as the content of interleukin-10(IL-10) and transforming growth factor-β(TGF-β) secreted by CD4+CD25+Treg cells. after the CD4+CD25+ Treg cells of each group were cultured in vitro for 24 hours,(3) The CD4+CD25+Treg cells of each group and the CD4+CD25-T cells of control group were co-cultured at a ratio of 1:1, coated with CD3 monoclonal antibody and soluble CD28 monoclonal antibody to promote activation. CCK-8 method was used to determine the inhibitory effect of CD4+CD25+Treg cells on the proliferation of CD4+CD25-T cells. ELISA was used to determine the content of interleukin-4(IL-4),IL-2 and interferon-γ(IFN-γ) in the co-culture system.Results:(1) Compared with the control group, the degree of lung injury in two experimental groups after intratracheal injection of HMGB1 was significantly severe, the dry and wet ratio of lung and the content of TNF-α and IL-1 in BALF were significantly increased,and the severity of wild type mice was higher than that of knockout mice.(2) Flow cytometry showed that purity of CD4+CD25+Treg cells sorted by immunomagnetic beads was above 90%; the expression of FOXP3 and CTLA-4 in wild type acute lung injury mice was lower than that in the control group and knockout type mice(P<0.05), while the expression in knockout mice was higher than that in the control group(P<0.05); the content of IL-10 and TGF-β secreted by Treg cells was lower in wild type mice of experimental group than in the control group(P<0.05),while the content in knockout mice was higher than that in the control group(P<0.05).(3) In the co-culture supernatant of CD4+CD25+Treg cells and effector T cells after the activation was promoted by CD3 and CD28, as well as the co-culture supernatant of CD4+CD25+Treg cells in wild type acute lung injury mice and effector T cells, the content of IL-2 and IFN-γ was higher than that in the control group(P<0.05), while the content of IL-4 was lower than that in the control group(P<0.05), and the ability of CD4+CD25+Treg cells to polarize towards Th2 cells was reduced, but they polarized towards Th1 cells; in the co-culture supernatant of CD4+CD25+Treg cells in knockout acute lung injury mice and effector T cells, the content of IL-2 and IFN-γ was lower than that in the control group(P<0.05), while the content of IL-4 was higher than that in the control group(P<0.05), and the immunosuppressive effect mediated by CD4+CD25+Treg cells caused Th1/Th2 shift towards Th2 polarization.Conclusion: 1. Intratracheal injection of HMGB1 can induce stable and reliable acute lung injury mouse model, which provides a compacted basis for follow-up in vivo experiment. 2. The CD4+CD25+Treg cells sorted by immunomagnetic beads have high purity, which can meet the requirement of follow-up experiment. 3. During acute lung injury, HMGB1 may regulate the expression of Foxp3 and CTLA-4 in Treg cells through TLR4 at least in part, thus reducing its immunosuppressive function. 4. During acute lung injury, HMGB1 may reduce the ability of CD4+CD25+Treg cells to polarize towards Th2 at least in part, making the cells polarize towards Th1 cells.