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Additional file 1: Table S1. AIM2 antibodies used in the study and their immunoreactivities. Figure S1. The majority of tested AIM2 antibodies displayed both nuclear and cytoplasmic immunoreactivity. Figure S2. Nuclear and cytoplasmic localization of AIM2 expressed by different cell types in frozen sections of human lung biopsies. Shown is a representative confocal image of the same section labeled with a mouse monoclonal (Ab5, aa1–195, red) and a rabbit polyclonal (Ab1, aa250–300/354, green) antibodies, revealing similar nuclear and cytoplasmic patterns of subcellular localization. AM: alveolar macrophages, Avl: alveolar wall cells, Vs: microvessel. The inset is magnification of the boxed area, revealing a macrophage and an endothelial cell. Blue is DAPI. Merged colors magenta (left) and azure (right) indicate nuclear localization of AIM2. Scale bars are in micrometers. Figure S3. Immunolocalization of AIM2 (red) and cleaved IL-1β (green) in a representative turbinate biopsies. Arrowhead: strong AIM2 nuclear signal in surface epithelium. Arrow: reduced nuclear AIM2 in glandular epithelium associated with increased cleaved IL-1β (green). Figure S4. Nuclear AIM2 in undifferentiated THP-1 monocytes. Ab #2 (aa232–309). Top: Microphoto taken with conventional immunofluorescence microscope. Bottom: Line profile analysis showing nuclear exit of AIM2 in the second right cell. Figure S5. Line profile analysis of subcellular localization of AIM2 and cleaved IL-1β in a COPD II patient’s airway. Shown are 4 nuclei, having relatively high AIM2 which (N1) or reduced AIM2 (N2,3,4). A cytoplasmic cluster of AIM2 is colocalized with cleaved IL-1β (Cy). Lines in the graph are colored in red for AIM2, green for cleaved IL-1β, and blue for DAPI. Figure S6. Subcellular localization of AIM2 in frozen sections of lung biopsies from non-COPD control patients (A and B) and from COPD patients (C and D). AIM2 (red) was labeled with a mouse monoclonal antibody (aa1–195). Blue is DAPI. Scale bars are in micrometers. Additonal file Representative confocal image of cigarette smoke extract-treated alveolar macrophages, showing intracellular (short arrows) and extracellular (arrowheads) of cleaved IL-1β particles colocalized with AIM2. Blue is DAPI. Scale bars are in micrometers.

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 6-fold increase (p 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.

E-cigarettes are perceived as harmless; however, evidence of their safety is lacking. New data suggests E-cigarettes discharge a range of compounds capable of physiological damage to users. We previously established that cigarette smoke caused defective alveolar macrophage phagocytosis. The present study compared the effect E-cigarette of components; E-liquid flavors, nicotine, vegetable glycerine, and propylene glycol on phagocytosis, proinflammatory cytokine secretion, and phagocytic recognition molecule expression using differentiated THP-1 macrophages. Similar to CSE, phagocytosis of NTHi bacteria was significantly decreased by E-liquid flavoring (11.65–15.75%) versus control (27.01%). Nicotine also decreased phagocytosis (15.26%). E-liquid, nicotine, and E-liquid+ nicotine reduced phagocytic recognition molecules; SR-A1 and TLR-2. IL-8 secretion increased with flavor and nicotine, while TNFα, IL-1β, IL-6, MIP-1α, MIP-1β, and MCP-1 decreased after exposure to most flavors and nicotine. PG, VG, or PG:VG mix also induced a decrease in MIP-1α and MIP-1β. We conclude that E-cigarettes can cause macrophage phagocytic dysfunction, expression of phagocytic recognition receptors and cytokine secretion pathways. As such, E-cigarettes should be treated with caution by users, especially those who are nonsmokers. Refereed/Peer-reviewed

Our previous studies have shown that nutritional zinc restriction exacerbates airway inflammation accompanied by an increase in caspase-3 activation and an accumulation of apoptotic epithelial cells in the bronchioles of the mice. Normally, apoptotic cells are rapidly cleared by macrophage efferocytosis, limiting any secondary necrosis and inflammation. We therefore hypothesized that zinc deficiency is not only pro-apoptotic but also impairs macrophage efferocytosis. Impaired efferocytic clearance of apoptotic epithelial cells by alveolar macrophages occurs in chronic obstructive pulmonary disease (COPD), cigarette-smoking and other lung inflammatory diseases. We now show that zinc is a factor in impaired macrophage efferocytosis in COPD. Concentrations of zinc were significantly reduced in the supernatant of bronchoalveolar lavage fluid of patients with COPD who were current smokers, compared to healthy controls, smokers or COPD patients not actively smoking. Lavage zinc was positively correlated with AM efferocytosis and there was decreased efferocytosis in macrophages depleted of Zn in vitro by treatment with the membrane-permeable zinc chelator TPEN. Organ and cell Zn homeostasis are mediated by two families of membrane ZIP and ZnT proteins. Macrophages of mice null for ZIP1 had significantly lower intracellular zinc and efferocytosis capability, suggesting ZIP1 may play an important role. We investigated further using the human THP-1 derived macrophage cell line, with and without zinc chelation by TPEN to mimic zinc deficiency. There was no change in ZIP1 mRNA levels by TPEN but a significant 3-fold increase in expression of another influx transporter ZIP2, consistent with a role for ZIP2 in maintaining macrophage Zn levels. Both ZIP1 and ZIP2 proteins were localized to the plasma membrane and cytoplasm in normal human lung alveolar macrophages. We propose that zinc homeostasis in macrophages involves the coordinated action of ZIP1 and ZIP2 transporters responding differently to zinc deficiency signals and that these play important roles in macrophage efferocytosis. Refereed/Peer-reviewed