Moonlighting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein of Lactobacillus gasseri attenuates allergic asthma via immunometabolic change in macrophages

Abstract

Background: The extra-intestinal effects of probiotics for preventing allergic diseases are well known. However, the probiotic components that interact with host target molecules and have a beneficial effect on allergic asthma remain unknown. Lactobacillus gasseri attenuates allergic airway inflammation through the activation of peroxisome proliferator- activated receptor γ (PPARγ) in dendritic cells. Therefore, we aimed to isolate and investigate the immunomodulatory effect of the PPARγ activation component from L. gasseri.

Methods: Culture supernatants of L. gasseri were fractionated and screened for the active component for allergic asthma. The isolated component was subjected to in vitro functional assays and then cloned. The crystal structure of this component protein was determined using X-ray crystallography. Intrarectal inoculation of the active component-overexpressing Clear coli (lipopolysaccharide-free Escherichia coli) and intraperitoneal injection of recombinant component protein were used in a house dust mite (HDM)-induced allergic asthma mouse model to investigate the protective effect. Recombinant mutant component proteins were assayed, and their structures were superimposed to identify the detailed mechanism of alleviating allergic inflammation.

Results: A moonlighting protein, glycolytic glyceraldehyde 3-phosphate dehydrogenase (GAPDH), LGp40, that has multifunctional effects was purified from cultured L. gasseri, and the crystal structure was determined. Both intrarectal inoculation of LGp40-overexpressing Clear coli and intraperitoneal administration of recombinant LGp40 protein attenuated allergic inflammation in a mouse model of allergic asthma. However, CDp40, GAPDH isolated from Clostridium difficile did not possess this anti-asthma effect. LGp40 redirected allergic M2 macrophages toward the M1 phenotype and impeded M2-prompted Th2 cell activation through glycolytic activity that induced immunometabolic changes. Recombinant mutant LGp40, without enzyme activity, showed no protective effect against HDM-induced airway inflammation.

Conclusions: We found a novel mechanism of moonlighting LGp40 in the reversal of M2-prompted Th2 cell activation through glycolytic activity, which has an important immunoregulatory role in preventing allergic asthma. Our results provide a new strategy for probiotics application in alleviating allergic asthma.

Keywords: Allergic asthma; GAPDH; Macrophages; Moonlighting protein; Probiotics.

Fig. 1

Identification of active ingredients, LGp40, from lysed L. gasseri components. a IL-12p40 levels of the sub-fraction IE1-1G1 to IE3-3G5 (sub-fractions identified on size-exclusion chromatography) stimulated mouse BMDC. b PPARγ expression of sub-fraction IE3-3G1 stimulated mouse BMDC. The production of IL-12p40 and PPARγ was detected with ELISA and Western blot assay, respectively (n = 3, *p < 0.05, one-way ANOVA with Bonferroni multiple comparison test). c The tetrameric assembly (biological assembly) of GAPDH of L. gasseri is shown (LGp40). The structure representing the tetrameric assembly of the LGp40 molecules through crystal symmetry is depicted. Individual protein subunits are represented as cartoon diagrams and painted in different colors. d The LGp40 molecule is represented as a cartoon diagram with green indicating the NAD(P)-binding domain (residues 3–156 and 321–338) and cyan indicating the C-terminal domain (residues 156–320). NAD⁺ molecules are shown as ball-and-stick models. The carbon, nitrogen, oxygen, and phosphate atoms of the NAD⁺ molecule are shown in magenta, blue, red, and orange, respectively. The covalent bonds between the atoms of the NAD⁺ molecule are shown in light gray

Fig. 2

Intrarectal administration of LGp40-overexpressing Clear coli alleviated HDM-induced asthma in mice. a Experimental scheme of Protocol 1. SDS-PAGE showed the quantities of LGp40 derived from IPTG-non-induced and IPTG-induced Clear coli. b Total IgE serum concentrations were measured using ELISA (n = 10 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). c Rrs for increasing dosages of aerosolized methacholine were measured using the flexiVent FX system (n = 10 mice, *p < 0.05 and **p < 0.01, two-way ANOVA with Bonferroni multiple comparison test). d Total cell, eosinophil, neutrophil, lymphocyte, and macrophage counts in the BALF were determined (n = 10 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). e TARC production in BALF was determined using ELISA (n = 10 mice, *p < 0.05, one-way ANOVA with Bonferroni multiple comparison test). f Lung histology. Lung sections were stained with hematoxylin and eosin (H&E; scale bar = 200 μm). Results comprise pooled data from two independent experiments

Fig. 3

Intraperitoneal administration of LGp40, rather than CDp40, suppressed HDM-induced asthma. a Experimental scheme of Protocol 2. b HDM-specific IgE serum concentrations were measured using ELISA (n = 15 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). c Ers for increasing dosages of aerosolized methacholine were measured using the flexiVent FX system (n = 15 mice, *p < 0.05 and **p < 0.01, two-way ANOVA with Bonferroni multiple comparison test). d Total cells, eosinophils, neutrophils, lymphocytes, and macrophages in the BALF were counted (n = 15 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). e TARC and CCL24 production in BALF were determined using an ELISA assay kit (n = 15 mice, *p < 0.05, one-way ANOVA with Bonferroni multiple comparison test). f Lung histology. Lung sections were stained with H&E (scale bar = 200 μm). Results comprise pooled data from three independent experiments

Fig. 4

Intraperitoneal administration of LGp40 inhibited M2 macrophage accumulation and promoted accumulation of M1 macrophages in the lung of mice with HDM-induced asthma. Lung sections were immunostained with a iNOS and b arginase-1 antibodies and then reacted with diaminobenzidine (scale bars = 200 and 100 μm). The iNOS-positive cells (blue arrows) were counted in every lobe per group, and the average numbers were calculated. The arginase-1-positive cells (red arrows) were counted in ×200 microscopic fields per group, and the average numbers were calculated (n = 6 mice, **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). M1 and M2 cell percentages c in single lung suspensions and d in peritoneal cavity were determined using flow cytometry analysis (n = 15 mice, *p < 0.05 and **p < 0.01, two-way ANOVA with Bonferroni multiple comparison test). e IL-4- and IL-13-stimulated BMDM were incubated with naïve Th cells in the presence of LGp40 and CDp40. IL-4⁺ Th2 cell populations were determined using flow cytometry analysis (n = 6 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). Results comprise pooled data from three independent experiments

Fig. 5

LGp40 failed to alleviate HDM-induced asthma when its enzymatic activity was lost. a Enzymatic activity of recombinant GAPDH proteins. b HDM-specific IgE serum concentrations were measured using ELISA (n = 5 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). c Ers for increasing dosages of aerosolized methacholine were measured using the flexiVent FX system (n = 5 mice, *p < 0.05 and **p < 0.01, two-way ANOVA with Bonferroni multiple comparison test). d Total cells, eosinophils, neutrophils, lymphocytes, and macrophages in the BALF were counted (n = 5 mice, *p < 0.05 and **p < 0.01, two-way ANOVA with Bonferroni multiple comparison test). e TARC production in BALF was determined using an ELISA assay kit (n = 5 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). f Lung histology. Lung sections were stained with H&E (scale bar = 200 μm)

Fig. 6

LGp40 failed to inhibit M2 macrophages in HDM-induced asthma when the enzymatic activity was lost. Lung sections were immunostained with a iNOS and b arginase-1 antibodies then reacted with diaminobenzidine (Scale bars = 200 and 100 μm). The iNOS-positive cells (blue arrows) were counted in every lobe per group, and the average numbers were calculated. The arginase-1-positive cells (red arrows) were counted in ×200 microscopic fields per group. The average numbers were calculated (n = 5 mice, **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). c IL-6, d IL-12p40, and e IL-10 production in cultured supernatants of IL-4- and IL-13-stimulated BMDM incubated with recombinant proteins were determined using an ELISA assay kit. f IL-4- and IL-13-stimulated BMDM were incubated with naïve Th cells in the presence of recombinant proteins. IL-4⁺ Th2 cell populations were determined using flow cytometry analysis (n = 6 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). Results comprise pooled data from three independent experiments

Fig. 7

LGp40, rather than CDp40, triggered glycolysis in allergic M2 macrophages. a Lactate production in cultured supernatants of IL-4- and IL-13-stimulated BMDM that were incubated with recombinant proteins were determined using an ELISA assay kit (n = 6 mice, *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni multiple comparison test). Results are pooled data from three independent experiments. b The superimposed structures of LGp40 (green) and CDp40 (cyan) in a schematic representation. The key residues surrounding the NAD-binding sites of LGp40 and CDp40 are indicated. The side chains of the residues around the NAD(P)-binding sites and NAD⁺ molecule are shown as ball-and-stick models. The carbon atoms of the LGp40, the CDp40, and NAD⁺ molecules are shown in yellow, light gray, and magenta, respectively. Oxygen atoms are shown in red, nitrogen in blue, and phosphate in orange. The residues marked at the front (A36, T123, C155, H182, T185, R236, and N318) are key residues that surround the NAD(P)-binding site of CDp40. The corresponding residues (D38, S125, C156, H183, T186, R239 and N320) in LGp40 are marked in back

Fig. 8

The diverse localization of LGp40 alters macrophage phenotypes with its high glycolytic activity. LGp40 adheres to plasminogen on macrophages and changes the metabolism of allergic M2 macrophage from oxidative phosphorylation to glycolysis, including macrophage polarization toward M1. The moonlighting LGp40 modulates macrophage reprogramming with dehydrogenase activities to alleviate the progression of allergic inflammation of airways. (The graph was generated using BioRender software.)