AIM2 nuclear exit and inflammasome activation in chronic obstructive pulmonary disease and response to cigarette smoke

Abstract

Introduction: The role inflammasomes play in chronic obstructive pulmonary disease (COPD) is unclear. We hypothesised that the AIM2 inflammasome is activated in the airways of COPD patients, and in response to cigarette smoke.

Methods: Lung tissue, bronchoscopy-derived alveolar macrophages and bronchial epithelial cells from COPD patients and healthy donors; lungs from cigarette smoke-exposed mice; and cigarette smoke extract-stimulated alveolar macrophages from healthy controls and HBEC30KT cell line were investigated. AIM2 inflammasome activation was assessed by multi-fluorescence quantitative confocal microscopy of speck foci positive for AIM2, inflammasome component ASC and cleaved IL-1β. Subcellular AIM2 localization was assessed by confocal microscopy, and immunoblot of fractionated cell lysates. Nuclear localization was supported by in-silico analysis of nuclear localization predicted scores of peptide sequences. Nuclear and cytoplasmic AIM2 was demonstrated by immunoblot in both cellular fractions from HBEC30KT cells.

Results: Increased cytoplasmic AIM2 speck foci, colocalized with cleaved IL-1β, were demonstrated in COPD lungs (n = 9) vs. control (n = 5), showing significant positive correlations with GOLD stages. AIM2 nuclear-to-cytoplasmic redistribution was demonstrated in bronchiolar epithelium in cigarette-exposed mice and in HBEC30KT cells post 24 h stimulation with 5% cigarette smoke extract. Alveolar macrophages from 8 healthy non-smokers responded to cigarette smoke extract with an > 8-fold increase (p < 0.05) of cytoplasmic AIM2 and > 6-fold increase (p < 0.01) of colocalized cleaved IL-1β speck foci, which were also localized with ASC.

Conclusion: The AIM2 inflammasome is activated in the airway of COPD patients, and in response to cigarette smoke exposure, associated with a nuclear to cytoplasmic shift in the distribution of AIM2.

Keywords: AIM2 inflammasome; AIM2 protein nuclear localization; COPD; Cigarette smoke.

Fig. 1

Nuclear and cytoplasmic AIM2. Representative confocal images revealing nuclear and cytoplasmic AIM2 detected with a rabbit polyclonal antibody (a, aa250–300/354, red by AF594) and a mouse monoclonal antibody (b, aa1–159, pseudogreen by AF647) in the same paraffin section of a human lung biopsies. Boxed areas are shown at magnification (right) to reveal cells having predominantly nuclear AIM2 (top box), or both nuclear and cytoplasmic AIM2 (bottom box). Blue is DAPI. Nuclear localization of AIM2 is indicated by the merged colors magenta in A and azure in B. Scale bars are in micrometers

Fig. 2

Amino acid sequence variants of human AIM2 and their Nuclear LocalizationPrediction score. NucPred: Nuclear Localization Prediction. NLS: Nuclear localization sequence signal

Fig. 3

Nuclear exit of AIM2 in lungs of COPD patients was coupled with IL-1β cleavage. a, b Representative confocal images of alveolar macrophages (a) and bronchiolar epithelium (b) in lung biopsies from non-COPD non-smoker controls (top) and COPD-III patients (bottom). Red: AIM2 by mouse mAb #1 (aa1–195); green: cleaved IL-1β; blue: DAPI. Magenta is the merged color of red and blue, indicating nuclear localization of AIM2. Yellow/orange is the merged color of red and green, indicating colocalization of AIM2 and cleaved IL-1β (short arrows). Scale bars are in micrometers

Fig. 4

Correlation analysis of AIM2 and cleaved IL-1β specks in lung biopsies. A, B: Increase of cytoplasmic AIM2 specks directly correlated with that of cleaved IL-1β, in either alveolar macrophages (a) or bronchiolar epithelium (b). c, d Positive correlations of AIM2 and cleaved IL-1β specks between alveolar macrophages and bronchiolar epithelium. Black dots in a-d represent data sets obtained from COPD patients; grey dots: non-COPD non-smoker controls. e - h: Levels of AIM2 and cleaved IL-1β specks in each cell type positively correlated with GOLD stages. COPD, n = 9; non-COPD non-smoker controls, n = 5

Fig. 5

AIM2 expression and localization in airway epithelial cells obtained from COPD patients and non-COPD non-smoker controls by upper airway brushing. a, b: Representative confocal images of AIM2 (red, Ab #1, aa250–300/354) and cleaved IL-1β (green) in cells from three different COPD patients (b) vs. three non-COPD non-smoker controls (a). Blue: DAPI. Scale bars in b are in micrometers and applied for the whole panel. C: Quantitation of bright AIM2 positive cells from COPD (n = 7) vs. non-COPD non-smoker control (n = 9)

Fig. 6

Cigarette smoke extract (CS) induced AIM2 inflammasome activation in non-COPD non-smoker control BAL derived alveolar macrophages. a, b Representative confocal images of AIM2 (red, Ab #1, aa250–300/354) and cleaved IL-1β (green) in alveolar macrophages exposed to cigarette smoke extract (B), vs. vehicle control (a). c, d Quantitation of AIM2 and cleaved IL-1β specks (PPC, number of particles per cell) in cells treated with cigarette smoke extract (CSE) vs. vehicle control for paired analysis (n = 8). e Colocalization of cleaved IL-1β with ASC in CSE-treated alveolar macrophages

Fig. 7

Nuclear-to-cytoplasmic translocation of AIM2 and increased cleaved IL-1β in bronchiolar epithelium of cigarette smoke-exposed mice. a, b Representative confocal images of AIM2 (red, Ab #1, aa250–300/354) and cleaved IL-1β (green) in bronchioles of mice exposed to cigarette smoke for 24 week (b), vs. sham control (a). c, d Line profile analysis revealing nuclear localization of AIM2 in control mice (c) and cytoplasmic in smoked mice (d). Pixels intensities measured along the line (inset) were plotted in y-axis, vs. their localization in x-axis. e, f Quantitation of AIM2 cytoplasmic specks and cleaved IL-1β in mouse bronchioles, cigarette smoke-exposed (n = 4) vs. sham control (n = 4)

Fig. 8

Nuclear-to-cytoplasmic redistribution of AIM2 in cigarette smoke extract- (CSE-) stimulated HBEC30KT cells. a: Representative Western blots analysis of AIM2 in whole cell lysate(W), cytoplasmic (c) and nuclear fractions (N) (n = 2). b Comparison of band abundance between CSE-stimulated and control. Band abundance was normalized to the corresponding band in the whole cell lysate and adjusted to GAPDH (cytoplasmic) or Lamin B1 (nuclear) expression. c Representative confocal images of HBEC30KT, control vs. CSE-stimulated (n = 2). Green: mouse mAb (Ab5); Red: rabbit pAb (Ab1). Blue: DAPI. Scale bars are in micrometers