Dectin-1 aggravates neutrophil inflammation through caspase-11/4-mediated macrophage pyroptosis in asthma

Abstract

Background: The pattern recognition receptor Dectin-1 was initially discovered to play a pivotal role in mediating pulmonary antifungal immunity and promoting neutrophil-driven inflammation. Recent studies have revealed that Dectin-1 is overexpressed in asthma, but the specific mechanism remains elusive. Additionally, Dectin-1 has been implicated in promoting pyroptosis, a hallmark of severe asthma airway inflammation. Nevertheless, the involvement of the non-classical pyroptosis signal caspase-11/4 and its upstream regulatory mechanisms in asthma has not been completely explored.

Methods: House dust mite (HDM)-induced mice was treated with Dectin-1 agonist Curdlan, Dectin-1 inhibitor Laminarin, and caspase-11 inhibitor wedelolactone separately. Subsequently, inflammatory cells in bronchoalveolar lavage fluid (BALF) were analyzed. Western blotting was performed to measure the protein expression of caspase-11 and gasdermin D (GSDMD). Cell pyroptosis and the expression of chemokine were detected in vitro. The correlation between Dectin-1 expression, pyroptosis factors and neutrophils in the induced sputum of asthma patients was analyzed.

Results: Curdlan appeared to exacerbate neutrophil airway inflammation in asthmatic mice, whereas wedelolactone effectively alleviated airway inflammation aggravated by Curdlan. Moreover, Curdlan enhanced the release of caspase-11 activation fragments and N-terminal fragments of gasdermin D (GSDMD-N) stimulated by HDM both in vivo or in vitro. In mouse alveolar macrophages (MH-S cells), Curdlan/HDM stimulation resulted in vacuolar degeneration and elevated lactate dehydrogenase (LDH) release. In addition, there was an upregulation of neutrophil chemokines CXCL1, CXCL3, CXCL5 and their receptor CXCR2, which was suppressed by wedelolactone. In asthma patients, a positive correlation was observed between the expression of Dectin-1 on macrophages and caspase-4 (the human homology of caspase-11), and the proportion of neutrophils in induced sputum.

Conclusion: Dectin-1 activation in asthma induced caspase-11/4 mediated macrophage pyroptosis, which subsequently stimulated the secretion of chemokines, leading to the exacerbation of airway neutrophil inflammation.

Keywords: Asthma; Caspase-11; Dectin-1; Neutrophil; Pyroptosis.

Fig. 1

Curdlan mainly enhances pulmonary neutrophil inflammation in asthmatic mice. C57BL/6 mice were randomly divided into 4 groups, PBS, HDM, HDM + Curdlan and HDM + Laminarin group. Mice received either Curdlan (20 μg Curdlan in 50 μl PBS) or Laminarin (5 mg/kg, 100 μl) prior to HDM at days 0, 2, 4, 11 to 14. A The lung tissue of each group was stained with H&E and PAS (magnification: 25x). B, C Histopathological inflammatory score were assessed based on H&E staining and PAS staining. D Levels of HDM specific IgG1 (sIgG1) in BALF were quantitated using a mouse specific ELISA kit. E–I The counts of total cells and neutrophils, eosinophils, macrophages and lymphocytes in BALF of each mice group were detected by flow cytometry. J, K Detection of IL-4 and IL-5 cytokines in BALF of mice in each group by ELISA kit. L-M. mRNA expression of IL-6 and IL-17a in the lungs of mice measured using qRT-PCR (n = 6–8) (*P < 0.05; **P < 0.01;***P < 0.005; ****P < 0.001, one way ANOVA, error bars represent mean ± SEM)

Fig. 2

Caspase-11 and pyroptosis is activated in Dectin-1 agonist induced asthma mice. A Expression of pro caspase-11 and cleavage caspase-11 in mice lung tissue by western blot. B Densitometric quantification of immunoblots in (A). Levels of cleavage caspase-11 were normalized to β-tubulin. C Densitometric quantification of immunoblots in (D). D Expression of full length of gasdermin D (GSDMD-FL) and N-terminal fragments of gasdermin D (GSDMD-N) in mice lung tissue by western blot. B Levels of GSDMD-N were normalized to β-actin. E, F IL-1α and IL-1β mRNA expression in lung tissue of mice by qRT-PCR. (n = 4–6). (*P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001, one way ANOVA, error bars represent mean ± SEM)

Fig. 3

Caspase-11 inhibitor has therapeutic effect on Curdlan-induced asthma mice. C57BL/6 mice were divided into 4 groups, HDM, HDM + Wed (wedelolactone), HDM + Cur (Curdlan) and HDM + Cur + Wed group. Wedelolactone (20 mg/kg, 200 μl) was administered to mice before stimulation with HDM or HDM/Curdlan at days 0, 2, 4, 11 to 14. A The lung tissue of each group was stained with H&E and PAS (25x). B, C Inflammatory score of lung histopathology by H&E staining and PAS staining. D–H The counts of total cells and eosinophils, lymphocytes, neutrophils and macrophages and in BALF of each mice group were detected by flow cytometry. I–L mRNA expression of IL-1α, IL-1β, IL-6 and IL-17a in the lungs of mice by qRT-PCR. (n = 4–8). (*P < 0.05; **P < 0.01;***P < 0.005; ****P < 0.001, one way ANOVA, error bars represent mean ± SEM) Wed: wedelolactone

Fig. 4

Activation of Dectin-1 promotes pyroptosis and pro-inflammatory function in MH-S cells. A The mean fluorescence intensity (MFI) of Dectin-1 on inflammatory cells in BALF of mice in each group by flow cytometry. MH-S cells were cultured in vitro, stimulated with PBS (50 μg/ml), HDM (50 μg/ml), HDM plus Curdlan (200 μg/ml) or HDM plus Laminarin(1 μg/ml) for 24 h. B, C The MFI of Dectin-1 expression on MH-S cells in each stimulation group by flow cytometry. D Optical microscope images of cell morphology in each stimulation group. E LDH release in supernatant of cells in each stimulation group. F The mRNA expression level of iNOS, TNF-α, Arg-1, Fizz1 in each stimulation group by qRT-PCR. (*P < 0.05; **P < 0.01;***P < 0.005; ****P < 0.001, one way ANOVA, error bars represent mean ± SEM)

Fig. 5

Curdlan increases but wedelolactone downregulates caspase-11 activation and GSDMD cleavage in HDM-induced MH-S cells. MH-S cells were stimulated with PBS (50 μg/ml), HDM (50 μg/ml), HDM plus Laminarin (Lam) (1 μg/ml), HDM plus Curdlan (Cur) (200 μg/ml), or giving wedelolactone (Wed) (30 μM) after HDM/Curdlan for 24 h. A Total proteins were extracted and subjected, western blot was used to detect the expression of pro caspase-11 and cleavage caspase-11 in each stimulation group. B Densitometric quantification of immunoblots in (A). Levels of cleavage caspase-11 were normalized to GAPDH. C Expression of GSDMD-FL, GSDMD-N and NLRP3 in each stimulation group by western blot. D Densitometric quantification of immunoblots in (C). Levels of GSDMD-N were normalized to β-tubulin. E Densitometric quantification of immunoblots in (C). Levels of NLRP3 were normalized to β-tubulin. F Representative images of caspase-11 expression in each stimulation group detected by immune fluorescence microscopy. G, H The expression of IL-1α and IL-1β mRNA in the cells of each stimulation group by qRT-PCR. MH-S cells were stimulated with PBS (50 μg/ml), HDM (50 μg/ml), HDM plus Curdlan (200 μg/ml), or giving MCC950(10 μM) after HDM/Curdlan for 24 h, cleavage caspase-11 (I) and GSDMD-N (J) in each stimulation group by western blot. (*P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001, one way ANOVA, error bars represent mean ± SEM)

Fig. 6

Curdlan increase but wedelolactone downregulate neutrophil related chemokine CXCL1 in HDM-induced MH-S cells. A, B mRNA expression of CXCL1and CXCR2 in the lungs of mice by qRT-PCR. MH-S cells were stimulated with PBS (50 μg/ml), HDM (50 μg/ml), HDM plus Laminarin (Lam)(1 μg/ml), HDM plus Curdlan (Cur) (200 μg/ml), or giving wedelolactone (Wed) (30 μM) after HDM/Curdlan for 24 h. CXCL1 (C), CXCL3 (D), CXCL5 (E), CXCL15 (F), G-CSF (G) and CXCL2 (H) mRNA expression levels in each group by qRT-PCR. (*P < 0.05; **P < 0.01;***P < 0.005; ****P < 0.001, one way ANOVA, error bars represent mean ± SEM)

Fig. 7

Positive correlation between macrophage Dectin-1 and neutrophils in asthma patients. Induced sputum of 33 asthma patients and 10 healthy controls was collected. A Flow gating strategy for macrophages, neutrophils, eosinophils in sputum of asthma patients, and MFI of Dectin-1 on macrophages. B Calculation of the MFI of Dectin-1 on induced sputum macrophages of healthy and asthma groups. C–F The correlation between MFI of macrophage Dectin-1 and sputum Neu%, sputum EOS%, blood neu count, blood eos count of asthma group. G–J Analysis of the difference between low Dectin-1 and high Dectin-1 expression groups of asthma (Sputum Neu%: proportion of neutrophils in induced sputum; sputum EOS%: proportion of eosinophils in induced sputum; blood neu count: peripheral blood neutrophil count; blood eos count: peripheral blood eosinophil count). ( *P < 0.01,**P < 0.05, two-tailed paired Student’s t test, spearman rank correlation, error bars represent mean ± SD)

Fig. 8

Positive correlation between macrophage Dectin-1 and caspase-4, IL-1α, IL-1β mRNA expression in asthma patients. Total RNA of induced sputum from asthma patients was extracted for qRT-PCR detection. A–C The correlation between MFI of Dectin-1 on induced sputum macrophages and caspase-4, IL-1α, IL-1β mRNA. D, E The correlation between induced sputum caspase-4 mRNA and sputum Neu%, blood neu count in induced sputum of asthma patients (Spearman rank correlation)