Gut flora-derived succinate exacerbates Allergic Airway Inflammation by promoting protein succinylation

Abstract

Allergic airway inflammation (AAI) is a prevalent respiratory disorder that affects a vast number of individuals globally. There exists a complex interplay among inflammation, immune responses, and metabolic processes, which is of paramount importance in the pathogenesis of AAI. Metabolic dysregulation and protein translational modification (PTM) are well-recognized hallmarks of diseases, playing pivotal roles in the onset and progression of numerous ailments. However, the role of gut microbiota metabolites in the development of AAI, as well as their influence on PTM modifications within this disease context, have not been thoroughly explored and investigated thus far. In AAI patients, succinate was identified as a key metabolite, positively correlated with certain immune parameters and IgE levels, and having good diagnostic value. In AAI mice, gut bacteria were the main source of high succinate levels. Mendelian randomization showed succinate as a risk factor for asthma. Exogenous succinate worsened AAI in mice, increasing airway resistance and inflammatory factor levels. Protein succinylation in AAI mice lungs differed significantly from normal mice, with up-regulated proteins in metabolic pathways. FMT alleviated AAI symptoms by reducing succinate and protein succinylation levels. In vitro, succinate promoted protein succinylation in BEAS-2B cells, and SOD2 was identified as a key succinylated protein, with the K68 site crucial for its modification and enzyme activity regulation. Gut flora-derived succinate exacerbates AAI in mice by increasing lung protein succinylation, and FMT can reverse this. These findings offer new insights into AAI mechanisms and potential therapeutic targets.

Keywords: Allergic airway inflammation; Gut flora; Succinate; Succinylation.

Graphical abstract

Fig. 1

Characterization of metabolites in sera of AAI patients. A, PCA plot of the positive spectrum. B, OPLS-DA plot of the positive spectrum (NC vs AAI). C, Permutation test model of OPLS-DA (NC vs AAI) with positive spectrum; the times of permutation tests were n = 200. D, PCA plot of the negative spectrum. E, OPLS-DA plot of the negative spectrum (NC vs AAI). F, Permutation test model of OPLS-DA (NC vs AAI) with negative spectrum. G, Distribution of differential metabolites for NC vs AAI; each dot is a metabolite and grey dots indicate metabolites with no differences; Up, the latter group was adjusted upward compared to the former group; Down, the latter group was adjusted downward compared to the former group. H, The plot of differential metabolic pathways of HC vs AAI (Down). I, The plot of differential metabolic pathways of HC vs AAI (Up). J, Heatmap of correlation between 65 differential metabolites and clinical indicators in AAI patients. K, Metabolic network diagram of 65 differential metabolites involved in regulation. L-Y, Correlation analysis of core metabolites with IgE; the correlation analysis was done using the spearman method; Z, Diagnostic value of 14 core metabolites for AAI. NC group, 33 volunteers; AAI group,74 volunteers.

Fig. 2

Characterization of intestinal flora in AAI mice. A, Molding process for AAI mouse models. B, α diversity and β diversity of AAI mouse. C, Succinate levels in different sites. D, histogram of LDA values of dominant genera based on LEfSe analysis. E, Levels of enzymes related to succinate synthesis and metabolism in intestinal flora. F, Relative abundance of gut bacteria associated with succinate synthesis and metabolism in AAI mice. G, Correlation analysis of the abundance of intestinal bacteria with succinate levels in lung tissue and serum. Unless otherwise stated, there were 6 mice in each group and all data in this paper were presented as mean ± SD.

Fig. 3

The mRNA level, protein expression level and Western blotting analysis of tight junction proteins in intestinal and lung tissues of AAI mice. A, Relative mRNA expression levels of Occludin, Claudin-1 and ZO1 in lung and gut tissues. B, Representative Western-blot images of Occludin, Claudin1, and ZO1 proteins in gut and lung tissues with β-Actin as the internal reference. Statistical significance was determined by Student's t-test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 compared with the NC group.

Fig. 4

Two-sample MR analysis of succinate and associated flora with asthma. A, Results of the causal relationship between succinate & associated gut flora and asthma. B, Scatterplots of the causal relationship between succinate & associated gut flora and asthma using different MR methods.

Fig. 5

Exogenous succinate may exacerbate AAI development in mice. A, Flowchart of the model of succinate-exacerbated AAI mice. B, Results of airway resistance (RI) and dynamic lung compliance (Cdyn) in mice. C, Relative mRNA expression levels of cytokines in lung tissue. D and E, Cytokine levels in lung tissues and serum. F, Summary results of HE staining, PAS staining and immunohistochemical staining (MUC5AC). G, Western blot results of succinylation modifications in mouse lung tissue.

Fig. 6

Characterization of succinylated proteins in the lungs of AAI mice. A, Flowchart of succinylation modification histology of proteins. B, Principal component analysis scatter plot. C, Numbers of differentially modified proteins and loci. D, Functional classification of differentially modified proteins based on KEGG database. E, Subcellular structure annotation of differentially modified proteins. F, Functional enrichment analysis of differentially modified proteins based on KEGG database.

Fig. 7

FMT reduced levels of overall succinylation of lung proteins and alleviated AAI. A, Flowchart of FMT treatment of AAI mice. B, Succinate levels in different sites. C, Results of airway resistance (RI) and dynamic lung compliance (Cdyn) in FMT mice. D, Relative mRNA expression levels of cytokines in lung tissue. E and F, Cytokine levels in lung tissues and serum.G, Summary results of HE staining, PAS staining and immunohistochemical staining (MUC5AC). H, Western blot results of succinylation modifications in FMT mouse lung tissue.

Fig. 8

Succinate may regulate AAI by affecting succinylation modifications at the SOD2 K68 site. A, The maximum non-toxic concentration of succinate to BEAS-2B cells. B, The effect of different concentrations of succinate on protein succinylation in BEAS-2B cells. C, The protein-protein interactions of proteins undergoing succinylation modification. D, Effect of succinate on the enzyme activity of SOD2. E, The effect of succinate on SOD2 protein expression. F, Potential succinylation modification sites of SOD2 identified by MS/MS. G, Prediction results of the 3D structure of SOD2 protein and the 3D conformation of three modification sites based on the AlphaFold3 deep-learning model. H, Experimental results of succinylation modification at specific sites of SOD2 obtained by means of IP-succinylation technology. I, The effect of succinate on the enzymatic activity of SOD2 mutants. J, The conservation of specific sequences of SOD2 in different species.

Fig. 9

Gut-Lung axis: diagram of possible mechanisms of AAI exacerbation by gut flora-derived succinate (Created with BioRender.com). High levels of succinate in AAI are mainly produced by intestinal flora and exogenous succinate can exacerbate AAI by enhancing overall succinylation of lung proteins, and FMT reduced levels of succinate and overall succinylation of lung proteins and alleviated AAI.