Baicalin restores innate lymphoid immune imbalance during exacerbation of COPD

Abstract

Chronic obstructive pulmonary disease (COPD) is characterized by immune dysregulation, including altered innate lymphoid cell (ILC) immune responses, particularly during exacerbations (ECOPD). Baicalin, a natural compound prevalent in various herbal medicines, has shown promise as a therapeutic candidate in ECOPD. However, its potential and molecular mechanism for addressing ILC immune imbalance during ECOPD remain poorly understood. First, this study conducted a cross-sectional analysis of ILC immune responses in stable COPD patients and those experiencing exacerbations. Then, clinical findings of skewed ILC immunity were validated in cigarette smoke and lipopolysaccharide-induced ECOPD mouse models. Lastly, the therapeutic effect of baicalin on restoring ILC immune homeostasis was investigated in experimental ECOPD mouse models. Significant downregulation of ILC2 immunity was observed during COPD exacerbations, accompanied by increased ILC1 and ILC3 responses, particularly in cases associated with bacterial infections. Notably, elevated IL-22 levels were observed in this group. Administration of recombinant IL-22 in ECOPD mouse models disrupted lung ILC homeostasis, specifically inhibiting the accumulation of ILC2. Proteomics and transcriptomics analyses suggested IL-22 as a mediator of type 2 immune suppression by creating a molecular environment that favors type 1 and type 3 immunity. Treatment with baicalin effectively restored ILC2 immunity by enhancing the recruitment and activation of lung ILC2 while suppressing ILC1 and ILC3 responses. Importantly, baicalin attenuated IL-22 production from lung ILC3, highlighting its potential as an IL-22 inhibitor. Baicalin demonstrates potential as a therapeutic strategy for addressing ILC immune imbalance in COPD exacerbations, particularly by restoring ILC2 immunity and partially inhibiting IL-22 production. Clinical registration The cross-sectional study was registered with the Chinese Clinical Trial Registry (ChiCTR2100050683).

Keywords: Baicalin; Exacerbation of chronic obstructive pulmonary disease; Immune imbalance; Innate lymphoid cells; Interleukin-22.

Fig. 1

ECOPD patients complicated with bacterial infections show blood and lung eosinopenia, accompanied with a downregulated ILC2 immune response and upregulated ILC1 and 3 immune responses. a Blood and lung eosinophil levels. b Proportion of blood ILC subsets. c Expression of the signature cytokines and transcription factors in blood ILC subsets. d Expression of signature cytokines in blood for each type of immunity. Data are mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. HC: healthy control; SCOPD: stable COPD; B-ECOPD: COPD exacerbation complicated with bacterial infection; NB-ECOPD: COPD exacerbation not complicated with bacterial infection; GMFI: geometric mean fluorescence intensity

Fig. 2

CS and LPS-induced ECOPD mice show lung eosinopenia, accompanied by increased inflammatory lesions in lungs. a Body weight and lung function indicators. b Lung inflammation lesion scores. c BALF cell classifications. Macrophages (black arrows), neutrophils (yellow arrows) and lymphocytes (green arrows). Data are mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. FRC: functional residual capacity; TLC: total lung capacity; FVC: forced vital capacity; FEV20: forced expiratory volume at 20 ms; FEV50: forced expiratory volume at 50 ms; FEV100: forced expiratory volume at 100 ms

Fig. 3

CS and LPS-induced ECOPD mice show a suppressed ILC2 immunity. a ILC2 numbers in lung tissues. b Expression of ILC2-related cytokines in BALF and lung tissues. Data are mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. LTH: lung tissue homogenate

Fig. 4

CS and LPS-induced ECOPD mice show an upregulation of IL- 22/Reg3γ axis. a Expression of IL- 22 in both protein and mRNA levels in BALF and lung tissues. b Expression of IL- 22, GATA3, and Reg3γ in lung tissues, assessed using immuohistochemical staining. c Expression of IL- 22, GATA3, and Reg3γ in lung tissues, assessed using western blotting. Data are mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. LTH: lung tissue homogenate

Fig. 5

CS and LPS-induced ECOPD mice show an increased number of IL- 22-producing ILC3 s in lung tissues. Data are mean ± SD. ^*P < 0.05

Fig. 6

IL- 22 suppressed the IL- 33/ILC2 axis in ECOPD mice. a Effect of IL- 22 on the expression of IL- 33 and the accumulation of eosinophils and ILC2 s in lungs. b Effect of IL- 22 on lung inflammatory lesions. c Effect of IL- 22 on GATA3 expression in lungs. Data are mean ± SD, ^*P < 0.05, ^***P < 0.001, ^****P < 0.0001. LTH: lung tissue homogenate

Fig. 7

Proteomics analysis shows a regulation effect of IL- 22 on the innate immune response. a The heat-map showing different expressed proteins between groups. b The volcano plot showing different expressed proteins between groups. c The GO enrichment bar plot showing the biological processes of different expressed proteins induced by IL- 22 treatment

Fig. 8

Transcriptomics analysis shows an immune-regulatory effect of IL- 22 on cytokine and chemokine to favors type 1 and type 3 immunity. a The heat-map showing different expressed genes between groups. b The volcano plot showing different expressed genes between groups. c The GO enrichment chord plot showing the relationship between the GO terms and IL- 22-regulated genes. d The KEGG enrichment chord plot showing the relationship between the KEGG terms and IL- 22-regulated genes. e The GO enrichment bar plot showing the biological processes of different expressed genes induced by IL- 22 treatment. f The KEGG enrichment bar plot showing the signaling pathways regulated by different expressed genes induced by IL- 22 treatment

Fig. 9

Baicalin alleviates lung inflammation, eosinopenia and ILC2 s immune suppression in ECOPD mice. a The effect of baicalin on body weight and pulmonary function indicators in ECOPD mice. b The effect of baicalin on lung inflammatory lesion scores and cell infiltrations in BALF in ECOPD mice. c The effect of baicalin on ILC subsets and immune responses in lung tissues of ECOPD mice. d The effect of baicalin on ILC2-related cytokines in ECOPD mice. Data are mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. BA: baicalin; LTH: lung tissue homogenate

Fig. 10

Baicalin inhibits the IL- 22/Reg3γ axis in ECOPD mice. a The effect of baicalin on the expression of IL- 22 in lung tissue in ECOPD mice, assessed using ELISA and qPCR. b The effect of baicalin on the expression of IL- 22, GATA3, and Reg3γ in lung tissue in ECOPD mice, assessed using immuohistochemical staining. c The effect of baicalin on the expression of IL- 22 and Reg3γ in lung tissue in ECOPD mice, assessed using western blotting. d The effect of baicalin on the number of IL- 22-producing ILC3 s in lung tissue in ECOPD mice. e The effect of baicalin on the production of IL- 22 in isolated human blood ILC3 s. Data are mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. BA: baicalin; LTH: lung tissue homogenate

Fig. 10

Baicalin inhibits the IL- 22/Reg3γ axis in ECOPD mice. a The effect of baicalin on the expression of IL- 22 in lung tissue in ECOPD mice, assessed using ELISA and qPCR. b The effect of baicalin on the expression of IL- 22, GATA3, and Reg3γ in lung tissue in ECOPD mice, assessed using immuohistochemical staining. c The effect of baicalin on the expression of IL- 22 and Reg3γ in lung tissue in ECOPD mice, assessed using western blotting. d The effect of baicalin on the number of IL- 22-producing ILC3 s in lung tissue in ECOPD mice. e The effect of baicalin on the production of IL- 22 in isolated human blood ILC3 s. Data are mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. BA: baicalin; LTH: lung tissue homogenate