NLRP3 inflammasome activation by mitochondrial ROS in bronchial epithelial cells is required for allergic inflammation

S R Kim, D I Kim, S H Kim, H Lee, K S Lee, S H Cho, Y C Lee, S R Kim, D I Kim, S H Kim, H Lee, K S Lee, S H Cho, Y C Lee

Abstract

Abnormality in mitochondria has been suggested to be associated with development of allergic airway disorders. In this study, to evaluate the relationship between mitochondrial reactive oxygen species (ROS) and NLRP3 inflammasome activation in allergic asthma, we used a newly developed mitochondrial ROS inhibitor, NecroX-5. NecroX-5 reduced the increase of mitochondrial ROS generation in airway inflammatory cells, as well as bronchial epithelial cells, NLRP3 inflammasome activation, the nuclear translocation of nuclear factor-κB, increased expression of various inflammatory mediators and pathophysiological features of allergic asthma in mice. Finally, blockade of IL-1β substantially reduced airway inflammation and hyperresponsiveness in the asthmatic mice. These findings suggest that mitochondrial ROS have a critical role in the pathogenesis of allergic airway inflammation through the modulation of NLRP3 inflammasome activation, providing a novel role of airway epithelial cells expressing NLRP3 inflammasome as an immune responder.

Figures

Figure 1
Figure 1
Generation of Intracellular ROS and mitochondrial ROS in BAL cells and primary cultured tracheal epithelial cells from OVALPS-OVA mice. BAL cells were obtained at 48 h after the challenge in SAL-SAL mice administered with drug vehicle (SV), OVALPS-OVA mice administered with drug vehicle (OLV), OVALPS-OVA mice administered with 3 mg/kg Necrox-5 (OLN 3) or OVALPS-OVA mice administered with 30 mg/kg Necrox-5 (OLN 30). The experiment was performed with the primary cultured tracheal epithelial cells from SAL-SAL mice without any treatment (Control), OVALPS-OVA mice with no treatment (OL), or OVALPS-OVA mice at 90 min after the treatment with drug vehicle (OLV) or 10 μM Necrox-5 (OLN 10). Representative confocal laser immunofluorescence photomicrography of BAL cells (a) and primary cultured tracheal epithelial cells (e) showed the localization of mitochondrial ROS (the center panels, red fluorescence views) in the cells. The blue fluorescent DAPI stain was used for nuclear localization. The left and the right panels presented phase contrast views and the merger views, respectively. Bars indicate scale of 10 μm (a) or 20 μm (e). Histogram analysis for ROS generation in BAL cells (b) and primary cultured tracheal epithelial cells (f). (c) DCF fluorescence intensity of intracelluar ROS in BAL cells. (d) MitoTracker fluorescence intensity of mitochondrial ROS in BAL cells. The ROS levels are presented as the relative ratio of values in experimental groups to those in SV. The relative ratio of ROS levels in the BAL cells of SV arbitrarily presented as 100. Bars represent mean±S.E.M. from seven mice per group. #P<0.05 versus SV or control; *P<0.05 versus OLV
Figure 2
Figure 2
Effects of NecroX-5 on the integrity and content of mtDNA in lung tissues of OVALPS-OVA mice. Sampling was performed at 48 h after the last challenge in SV, OLV, OLN 3 or OLN 30. (a) Representative PCR analyses for both a long (8642-bp) mtDNA fragment and a short (316-bp) mtDNA fragment from the lung tissues of OVALPS-OVA mice. (b) The long fragment/short mtDNA fragment hybridization ratio in the lung tissues of OVALPS-OVA mice. The ratio of SV is arbitrarily presented as 1. (c) Changes of the content of mtDNA in the lung tissues of OVALPS-OVA mice. The levels of mtDNA were presented as ng/1 μg of protein/1 g of lung tissues. Data represent mean±S.E.M. from six mice per group. #P<0.05 versus SV; *P<0.05 versus OLV
Figure 3
Figure 3
Effects of mitochondrial ROS inhibitor, Necrox-5 on the protein levels of NLRP3, caspase-1 and IL-1β in primary cultured tracheal epithelial cells and lung tissues of OVALPS-OVA mice. Representative western blot analysis of NLRP3 (a), caspase-1 (b) and IL-1β (c) in primary cultured tracheal epithelial cells of OVALPS-OVA mice. Representative western blots of NLRP3 (d), caspase-1 (e) and IL-1β (f) in lung tissues of OVALPS-OVA mice. Densitometric analyses of the bands on films are presented as the relative ratio of NLRP3 (g), caspase-1 (h) or IL-1β (i) to actin. The relative ratio of each protein in the lung tissues of SV is arbitrarily presented as 1. Representative western blots of NLRP3 (j) and caspase-1 (k) in human BAL fluids from asthmatic patients and healthy subjects (n=3 per group). (l) Enzyme immunoassay of IL-1β in BAL fluids of OVALPS-OVA mice. Bars represent mean±S.E.M. from seven mice per group. #P<0.05 versus SV; *P<0.05 versus OLV. (m) Representative confocal laser immunofluorescence photomicrography of BAL cells of OVALPS-OVA mice showed the expression of IL-1β. The blue fluorescent DAPI stain was used for nuclear localization. The left and the right panels presented phase contrast views and the merger views, respectively. Bars indicate scale of 20 μm
Figure 4
Figure 4
Effects of Necrox-5 on total and differential cell counts in BAL fluids, histological changes, MPO activity, airway hyperresponsiveness and serum levels of OVA-specific immunoglobulins in OVALPS-OVA mice. All parameters were measured at 48 h after the last challenge in SV, OLV, OLN 3 and OLN 30. (a) Cellular changes in BAL fluids of OVALPS-OVA mice. Bars represent mean±S.E.M. from seven mice per group. (b–e) Representative H&E-stained sections of the lungs isolated from SV (b), OLV (c), OLN 3 (d) and OLN 30 (e). Bars indicate scale of 20 μm. (f) MPO activity in lung tissue homogenates of OVALPS-OVA mice. Airway responsiveness in OVALPS-OVA mice was assessed by both noninvasive (g, %Penh) and invasive (h, Rrs) measurements. (i–k) Changes of the serum levels of OVA-specific IgE (i), OVA-specific IgG1 (j) and OVA-specific IgG2a (k). Bars represent mean±S.E.M. from six or seven mice per group. ND, not detected, #P<0.05 versus SV; *P<0.05 versus OLV
Figure 5
Figure 5
Effects of NecroX-5 on the protein levels of various inflammatory mediators and the activation of NF-κB/IκBα in lung tissues of OVALPS-OVA mice. Sampling was performed at 48 h after the last challenge in SV, OLV, OLN 3 and OLN 30. Representative western blots of IL-4 (a), IL-5 (c), IL-13 (e), TNF-α (g), IFN-γ (i), IL-17 (k) and KC (m) in lung tissues. (b, d, f, h, j, l, n) Densitometric analysis of the bands on films is presented as the relative ratio of each protein to actin. (o) Representative western blot of NF-κB p65 in lung tissues. (p) Densitometric analyses of the bands on films are presented as the relative ratio of NF-κB p65 level in OLV, OLN 3 or OLN 30 to the level in SV. The relative ratio of each protein in the lung tissues of SV is arbitrarily presented as 1. Bars represent mean±S.E.M. from seven mice per group. #P<0.05 versus SV; *P<0.05 versus OLV
Figure 6
Figure 6
Effects of NecroX-5 on total and differential cell counts in BAL fluids, histological changes, airway hyperresponsiveness, protein levels of NLRP3, IL-1β and caspase-1, and generation of mitochondrial ROS in the lung of HDM-instilled mice. All parameters were measured at 48 h after the last challenge in saline-instilled mice administered drug vehicle (SV), HDM-instilled mice administered drug vehicle (HV), HDM-instilled mice administered 3 mg/kg of NecroX-5 (HN 3) and HDM-instilled mice administered 30 mg/kg of NecroX-5 (HN 30). (a) Cellular changes in BAL fluids of HDM-instilled mice. Bars represent mean±S.E.M. from six mice per group. (b–e) Representative H&E-stained sections of the lungs isolated from SV (b), HV (c), HN 3 (d) and HN 30 (e). Bars indicate scale of 20 μm. (f) Airway responsiveness in HDM-instilled mice was assessed by invasive measurement (Rrs). (g–l) Representative western blots for NLRP3 (g), caspase-1 (i) and IL-1β (k) in lung tissues and densitometric analysis. Bars represent mean±S.E.M. from six mice per group. (m) Representative confocal laser immunofluorescence photomicrography of BAL cells showed the localization of mitochondrial ROS (red fluorescence views) in the cells. The blue fluorescent DAPI stain was used for nuclear localization. The left and the right panels presented phase contrast views and the merger views, respectively. Bars indicate scale of 20 μm. #P<0.05 versus SV; *P<0.05 versus HV
Figure 7
Figure 7
Effects of blockade of IL-1β on total and differential cell counts in BAL fluids, histological changes and airway hyperresponsiveness in the lung of OVALPS-OVA mice and OVALPS-OVA IL-1R KO mice. All parameters were measured at 48 h after the last challenge in SAL-SAL mice administered with PBS (SP), OVALPS-OVA mice administered with isotype control antibody (OLC-Ab), OVALPS-OVA mice administered with an anti-IL-1β-neutralizing antibody (OLA-IL-1β Ab), SAL-SAL WT mice (WT CON), OVALPS-OVA WT mice (WT OL), SAL-SAL IL-1R KO mice (IL-1R KO CON) and OVALPS-OVA IL-1R KO mice (IL-1R KO OL). (a) Cellular changes in BAL fluids of OVALPS-OVA mice. Bars represent mean±S.E.M. from seven mice per group. (b-d) Representative H&E-stained sections of the lungs isolated from SP (b), OLC-Ab (c) and OLA-IL-1β Ab (d). Bars indicate scale of 20 μm. (e and f) Airway responsiveness in OVALPS-OVA mice was assessed by both noninvasive (e, %Penh) and invasive (f, Rrs) measurements. Bars represent mean±S.E.M. from seven mice per group. (g) Cellular changes in BAL fluids of IL-1R KO or WT mice. Bars represent mean±S.E.M. from five mice per group. (h–k) Representative H&E-stained sections of the lungs isolated from WT CON (h), WT OL (i), IL-1R KO CON (j) and IL-1R KO OL (k). Bars indicate scale of 50 μm. (l) Airway responsiveness in IL-1R KO or WT mice was assessed by invasive measurement (Rrs). Bars represent mean±S.E.M. from five mice per group. #P<0.05 versus SP or WT CON; *P<0.05 versus OLC-Ab or WT OL
Figure 8
Figure 8
Effects of blockade of IL-1β on the protein levels of various inflammatory mediators in lung tissues of OVALPS-OVA mice and OVALPS-OVA IL-1R KO mice. Sampling was performed at 48 h after the last challenge in SP, OLC-Ab, OLA-IL-1β Ab, WT CON, WT OL, IL-1R KO CON,and IL-1R KO OL. Representative western blots of IL-4 (a and o), IL-5 (c and q), IL-13 (e and s), TNF-α (g), IFN-γ (i), IL-17 (k and u) and KC (m and w) in lung tissues. (b, d, f, h, j, l, n, p, r, t, v, x) Densitometric analysis of the bands on films is presented as the relative ratio of each protein to actin. The relative ratio of each protein in the lung tissues of SP or WT CON is arbitrarily presented as 1. Bars represent mean±S.E.M. from five or seven mice per group. #P<0.05 versus SP or WT CON; *P<0.05 versus OLC-Ab or WT OL
Figure 9
Figure 9
Structure of NecroX-5

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