Mincle-Mediated Neutrophil Extracellular Trap Formation by Regulation of Autophagy

Abstract

Background: Neutrophil extracellular traps (NETs) constitute antimicrobial function of neutrophils but have also been linked to perpetuation of inflammation. Despite this evident physiological relevance, mechanistic understanding of NET formation is poor. In this study, we examined the mechanism by which Mincle, a C-type lectin receptor, regulates NET formation.

Methods: NET formation, reactive oxygen species, autophagy activation and intracellular signaling pathways were analyzed in Mincle-sufficient and -deficient neutrophils stimulated in vitro with various stimuli and in vivo during Klebsiella infection.

Results: We found that Mincle mediates NET formation in response to several activation stimuli in vitro and in vivo during pneumoseptic infection with Klebsiella pneumoniae, indicating its regulatory role in NET formation. Mechanistically, we show that attenuated NET formation in Mincle-/- neutrophils correlates with an impaired autophagy activation in vitro and in vivo, whereas reactive oxygen species (ROS) formation in these neutrophils remained intact. The requirement of autophagy in Mincle-mediated NET formation was further supported by exogenous treatment with autophagy inducer tamoxifen, which rescued the NET formation defect in Mincle-/- neutrophils.

Conclusions: Our findings identify a previously unrecognized role of Mincle as a regulator of autophagy, which mediates NET formation without affecting ROS generation. Our study addresses a major challenge in the field by positing this pathway to be targeted for modulation of NETs while preserving ROS production, an important innate immune defense.

Keywords: Klebsiella pneumoniae; Mincle; autophagy; bacterial infection; innate immune response.; neutrophil extracellular traps (NETs); pneumonia; reactive oxygen species; sepsis.

Figure 1.

Mincle regulates neutrophil extracellular trap (NET) formation in response to phorbol-myristate-acetate (PMA) and N-formylmethionyl-leucyl-phenylalanine (fMLP) stimulation. A, Representative fluorescence images of wild-type (WT) and Mincle^-/- neutrophils unstimulated or stimulated with PMA (50 nM) or fMLP (1 uM) for 4 hours. Neutrophil extracellular traps were fixed and stained with Sytox Green as described in the Methods. Magnification = 200X. The bar graph shows quantitation of NET-forming WT and Mincle^-/- neutrophils. NS; no stimulation. Data presented in the bar graph is from 4 independent experiments. (*P < .05). B, Representative images of NETs in WT neutrophils transfected with control or siRNA targeted to Mincle 6 hours before stimulation with PMA. Neutrophil extracellular traps were stained with Sytox Green 4 hours after PMA stimulation. Magnification = 200X. Percentage of NET forming neutrophils ± SD from 4 independent experiments is shown in the bar graph. **P < .01. Abbreviations: fMLP, N-formylmethionyl-leucyl-phenylalanine; NET, neutrophil extracellular trap; NS, no stimulation; PMA, phorbol-myristate-acetate; WT, wild-type.

Figure 2.

Mincle-deficient neutrophils are fully competent in reactive oxygen species (ROS) generation. A, Reactive oxygen species were measured in wild-type (WT) and Mincle^-/- neutrophils 10 minutes after phorbol-myristate-acetate (PMA) stimulation using a fluoro H₂O₂ detection kit as described in Methods. Data from 4 independent experiments are shown. No statistically significant differences were found between the levels of ROS in WT and Mincle^-/- neutrophils. B, Reactive oxygen species measurement in WT neutrophils transfected with control or test siRNA targeted against Mincle 6 hours before stimulation with PMA for 10 minutes. Reactive oxygen species generation was measured in unstimulated or stimulated neutrophils. C, Quantitation of neutrophil extracellular trap (NET) formation by WT and Mincle^-/- neutrophils stimulated for 4 hours with PMA with or without treatment with NADPH oxidase inhibitor apocynin. Graph shows average ± SD from 3 independent experiments. *P < .05; **P < .01. D, Flow cytometry analysis of mitochondrial ROS (MitoSox) in unstimulated or PMA-stimulated WT and Mincle^-/- neutrophils. The numbers on contour plots represent percentages of MitoSox-positive cells. Bar graph shows average ± SD of MitoSox-positive cells from 5 independent experiments. Abbreviations: Apo, apocynin; NET, neutrophil extracellular trap; NS, not stimulated; PMA, phorbol-myristate-acetate; ROS, reactive oxygen species; WT, wild-type.

Figure 3.

Mincle-deficient neutrophils exhibit impaired autophagy activation. A, Representative immunofluorescence images showing the extent of LC3 punctation in wild-type (WT) and Mincle^-/- neutrophils 30 minutes after phorbol-myristate-acetate (PMA) stimulation. LC3 puncta (red) indicating activation of autophagy were visualized using a rabbit antimouse LC3 antibody followed by secondary goat antirabbit Alexa546 antibody. DAPI (blue) was used to stain the nuclei. Arrows indicate punctation of LC3-II in WT neutrophils after PMA stimulation. B, Western blot analysis to detect conversion of LC3-I to LC3-II in WT and Mincle^-/- neutrophils stimulated with PMA with or without rapamycin treatment. Immunoblotting of housekeeping protein GAPDH is shown as loading control. Blot shown is representative of 4 independent experiments. C, Quantitation of neutrophil extracellular trap (NET) formation by WT and Mincle^-/- neutrophils stimulated with PMA for 4 hours with or without rapamycin treatment. Average ± SD from 5 independent experiments is shown. *P < .05; ***P < .001. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; NS, not stimulated; P, phorbol-myristate-acetate alone; PMA, phorbol-myristate-acetate; R, rapamycin alone; Rapa, rapamycin; RP, rapamycin+PMA; WT, wild-type.

Figure 4.

Tamoxifen treatment rescues autophagy and neutrophil extracellular trap (NET) formation in Mincle^-/- neutrophils. A, Representative fluorescence images of wild-type (WT) and Mincle^-/- neutrophils unstimulated or stimulated with phorbol-myristate-acetate (PMA) with or without tamoxifen (TMX). Neutrophil extracellular traps were fixed and stained with Sytox Green as described in Methods. Magnification = 200X. B, The bar graph shows quantitation of NET-forming WT and Mincle^-/- neutrophils stimulated with PMA with or without TMX. Data presented are from 3 independent experiments. Average ± SD is shown. *P < .05; **P < .01; ***P < .001. C, Western blot analysis to detect conversion of LC3-I to LC3-II in WT and Mincle^-/- neutrophils stimulated with PMA with or without TMX treatment. Immunoblotting of housekeeping protein GAPDH is shown as loading control. Blot shown is representative of 3 independent experiments. Abbreviations: NS, not stimulated; P, phorbol-myristate-acetate alone; PMA, phorbol-myristate-acetate; TP, tamoxifen+PMA; TMX, tamoxifen; WT, wild-type.

Figure 5.

Mincle^-/- neutrophils show reduced neutrophil extracellular trap (NET) formation and defective autophagy but normal reactive oxygen species (ROS) in the lungs of Klebsiella pneumoniae (KPn)–infected pneumonic mice. A, Representative fluorescence images of the neutrophils isolated 3 days after infection from BAL fluid of wild-type (WT) and Mincle^-/- mice infected with KPn, and stained with Sytox Green to label NETs (green). Magnification = 200X. The bar graph shows quantitation of NET-forming neutrophils from 3 independent experiments with 3–4 mice per group in each experiment. B, Measurement of ROS in BAL neutrophils isolated 3 days after infection from WT and Mincle^-/- mice infected intranasally with KPn. Data from 4 independent experiments with 3–4 mice per group in each experiment are shown. No statistically significant differences were found between the levels of ROS in neutrophils from WT and Mincle^-/- mice. C, Representative immunofluorescence images showing the extent of LC3 punctation in BAL neutrophils isolated from KPn-infected WT and Mincle^-/- mice 3 days after infection. LC3 puncta (red) indicating activation of autophagy were visualized using a rabbit antimouse LC3 antibody followed by secondary goat antirabbit Alexa546 antibody. DAPI (blue) was used to stain the nuclei. Arrows indicate punctation of LC3 in WT neutrophils. Magnification = 400X. D, Western blot analysis to detect conversion of LC3-I to LC3-II and the levels of autophagy protein Beclin-1 in BAL neutrophils isolated from KPn-infected WT and Mincle^-/- mice 3 days after infection. Immunoblotting of housekeeping protein GAPDH is shown as loading control. Blots shown are representative of 4 independent experiments with 2–3 mice per group in each experiment. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; ROS, reactive oxygen species; WT, wild-type.

Figure 6.

Working model of Mincle-mediated neutrophil extracellular trap (NET) formation. Mincle-mediated activation of autophagy, occurring downstream of mTOR (target of rapamycin), is required for NET formation in response to pneumonic infection as well as phorbol-myristate-acetate stimulation. This autophagy activation precedes reactive oxygen species (ROS) effect on NET formation, because Mincle deficiency causes NET formation defect despite intact ROS. Tamoxifen reverses this defect in autophagy activation in the absence of Mincle and rescues NET formation in Mincle^-/- neutrophils. Abbreviations: mTOR, target of rapamycin; NET, neutrophil extracellular trap; PMA, phorbol-myristate-acetate; ROS, reactive oxygen species.