Qi-Dong-Huo-Xue-Yin balances the immune microenvironment to protect against LPS induced acute lung injury

Abstract

COVID-19 induces acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and leads to severe immunological changes that threatens the lives of COVID-19 victims. Studies have shown that both the regulatory T cells and macrophages were deranged in COVID-19-induced ALI. Herbal drugs have long been utilized to adjust the immune microenvironment in ALI. However, the underlying mechanisms of herbal drug mediated ALI protection are largely unknown. This study aims to understand the cellular mechanism of a traditional Chinese medicine, Qi-Dong-Huo-Xue-Yin (QD), in protecting against LPS induced acute lung injury in mouse models. Our data showed that QD intrinsically promotes Foxp3 transcription via promoting acetylation of the Foxp3 promoter in CD4⁺ T cells and consequently facilitates CD4⁺CD25⁺Foxp3⁺ Tregs development. Extrinsically, QD stabilized β-catenin in macrophages to expedite CD4⁺CD25⁺Foxp3⁺ Tregs development and modulated peripheral blood cytokines. Taken together, our results illustrate that QD promotes CD4⁺CD25⁺Foxp3⁺ Tregs development via intrinsic and extrinsic pathways and balanced cytokines within the lungs to protect against LPS induced ALI. This study suggests a potential application of QD in ALI related diseases.

Keywords: ALI; Foxp3; Qi-Dong-Huo-Xue-Yin; Tregs; macrophage; traditional Chinese medicine.

FIGURE 1

QD attenuates LPS induced ALI (A) H&E staining of lung tissues from control and ALI model mice treated with or without QD (×40 magnification). Ctrl stands for control. (B). Histopathological mean lung injury scores and pulmonary permeability index calculated from six control and ALI model mice treated with or without QD. (C). TGF-β level in BALF of control and ALI model mice treated with or without QD. (D). IL-17A, IL-23, and TNF-α levels in BALF of control and ALI model mice treated with or without QD.

FIGURE 2

QD induces CD4⁺CD25⁺Foxp3⁺ Tregs in the ALI mouse model (A). Analysis of CD4⁺CD25⁺Foxp3⁺ Tregs in the peripheral blood from control and ALI model mice treated with or without QD. (B). Quantification of peripheral blood Tregs from six control and six ALI model mice treated with or without QD. (C). mRNA levels of Foxp3 from whole blood extractions.

FIGURE 3

QD treatment leads to hyper acetylation of Histone H3 of the Foxp3 promoter in CD4⁺ cells (A). ChIP-qPCR analysis of the Foxp3 promoter region pulled down by acetylated Histone H3 or IgG antibody in CD4⁺ cells treated with or without QD and pretreated with LPS. (B). Western blot analysis of H3K9Ac in CD4⁺ cells treated with or without QD and pretreated with LPS. (C). Quantification of H3K9Ac levels in CD4⁺ cells treated with or without QD and pretreated with LPS. (D). Chromogenic assays of HDAC activity in CD4⁺ cells lysates. TSA stands for Trichostatin A, and is a histone deacetylase inhibitor.

FIGURE 4

QD regulates HDACs in CD4⁺ cells (A). Protein levels of HDACs in CD4⁺ cells treated with or without QD and pretreated with LPS. (B). mRNA levels of HDACs in CD4⁺ cells treated with or without QD and pretreated with LPS.

FIGURE 5

QD facilitates macrophage mediated Treg development Macrophages pretreated with or without QD were co-cultured with CD4⁺ cells treated with or without LPS. (A). Analysis of CD4⁺CD25⁺Foxp3⁺ Tregs after 24 h co-culture. (B). Quantification of CD4⁺CD25⁺Foxp3⁺ Tregs of co-cultured cells. (C). The TGF-β level in the supernatant of co-cultured cells. (D). qPCR analysis of Foxp3 and IL-17A from CD4⁺ cells in the co-culture.

FIGURE 6

QD upregulates β-Catenin in macrophages (A). Western blot analysis of PTEN and β-Catenin in macrophages treated with or without QD and pretreated with LPS. (B). Quantification of protein levels of PTEN and β-Catenin in macrophages treated with or without QD and pretreated with LPS. Ctrl stands for control. (C). Macrophages transfected with siRNA targeting β-Catenin were pretreated with or without QD and then co-cultured with CD4⁺ cells treated with or without LPS. (D). Analysis of CD4⁺CD25⁺Foxp3⁺ Tregs after 24 h co-culture. (E). The TGF-β level in the supernatant of co-cultured cells. (F). qPCR analysis of Foxp3, RORγt, and IL-17A from CD4⁺ cells in the co-culture.

FIGURE 7

Myeloid β-Catenin is required for QD function (A) H&E staining of lung tissue from QD treated ALI model mice established from control or myeloid β-Catenin deficient mice (×40 magnification). (B). Histopathological mean lung injury scores and myeloperoxidase (MPO) activity in lung homogenates of QD treated ALI model mice established from control or myeloid β-Catenin deficient mice. (C). TGF-β levels in the serum from QD treated or untreated ALI model mice established from control or myeloid β-Catenin deficient mice. (D). IL-17A levels in the serum from QD treated or untreated ALI model mice established from control or myeloid β-Catenin deficient mice. (E). mRNA expression levels of Foxp3, TGF-β, RORγt, IL-17A, TNF-α, and IL-1β in the CD4⁺ cells from peripheral blood of QD treated or untreated ALI model mice established from control or myeloid β-Catenin deficient mice. (F). Analysis of CD4⁺CD25⁺Foxp3⁺ Tregs in the peripheral blood from peripheral blood of QD treated or untreated ALI model mice established from control or myeloid β-Catenin deficient mice. (G). Quantification of CD4⁺CD25⁺Foxp3⁺ Tregs in the peripheral blood from peripheral blood of QD treated or untreated ALI model mice established from control or myeloid β-Catenin deficient mice.

FIGURE 8

QD inhibits β-Catenin phosphorylation (A). Western blot analysis of β-Catenin in macrophages treated with or without QD and pretreated with or without LPS. (B). Quantification of protein level of β-Catenin in macrophages treated with or without QD and pretreated with or without LPS. (C). Western blot analysis of β-Catenin in macrophages treated with CHX and pretreated with or without QD in the presence of LPS. (D). Western blot analysis of β-Catenin phosphorylation in macrophages treated with or without QD in the presence of LPS. (E). A diagram that illustrate the effects of QD has on Treg and macrophage which finally leads to the activation of Tregs and protect against LPS induced acute lung injury.