AIM2 localized to both the cytoplasm and nucleus
To best determine the cellular localization of AIM2, a panel of antibodies raised in different species against various epitope domains of human AIM2, summarized in Table S1 were compared for their immunoreactivity in paraffin and frozen sections of human lung biopsies, paraffin sections of mouse lungs, and human cell cytospins (Figure 1, Supplementary Figures S1 and S2). The panel of antibodies showed a similar pattern of immunoreactivity with clear staining of both cytoplasm and nucleus, independent of preservation technique, with the exception of the goat polyclonal antibody Ab4 (aa321-335) that showed conspicuous surface staining in both paraffin and frozen sections. This dual localization was detected in various cell types, including cells outside the lung (Supplementary Figures S2, S3, S4). Cytoplasmic AIM2 immunoreactivity was present in homogenous as well as punctate patterns, the latter could be quantified as specks using uniform confocal setting of fluorescence intensity threshold and size (Figures 1, 2). Nuclear staining was often increased at the nuclear margination (Figures 1, 2). The nuclear immunoreactivity was confirmed by Z-analysis and line profile analysis of AIM2-DAPI colocalization in confocal sections of both human and mouse lung tissue (Supplementary Data Figure S5, Movie S1).
Bio-informatic analysis was further carried out to assess nuclear localization prediction of the AIM2 protein. Ten variants of amino acid sequences of human AIM2 available from the GenBank were analyzed, confirming their difference by Nuclear Localization Prediction scores (Figure 1B). The highest Nuclear Localization Prediction score of 0.58 was obtained for the longest sequence (accession number AAH10940.1, 356 aa), while all the shorter sequences showed reduced scores between 0.39-0.1722. In keeping with this result, a predicted monopartile Nuclear Localization Signal (NLS, VIKAKKKKHRE, position 336) was present only in the full-length variant AAH10940.1 (Figure 2).
Increase of cytoplasmic AIM2 in airway cells of COPD patients was associated with cleavage of IL-1β
A panel of 14 paraffin embedded lung biopsies taken from non-tumour adjacent tissue (as assessed by a trained pathologist) of patients undergoing lobectomy for their cancers (9 having COPD, 5 non-COPD controls, Table 1) was available for examination of protein expression and subcellular localisation of AIM2/cleaved IL-1β, and quantitative analysis of their specks. As shown above, AIM2 immunoreactivity was localized to both the cytoplasm and the nucleus in alveolar macrophages and bronchiolar epithelial cells. Cytoplasmic specks of AIM2 were co-localized and positively correlated with cleaved IL-1β (Figures 3, 4). Importantly quantitative measurement of AIM2/ cleaved IL-1β showed significant positive correlations with GOLD stages in both alveolar macrophages and bronchiolar epithelium (Figure 4). Speck analysis revealed also a significant positive correlation between the two cell types, though the bulk of increase observed in the former cell type (Figure 4).
The results obtained from multiple labelling of paraffin sections were confirmed by analysis of cryosections of COPD (n=2) and non-COPD (n=2) lungs (Supplementary Online Figure S6).
To confirm and further investigate the presence of AIM2 inflammasome in epithelial cells, cytospins of bronchial epithelial cells obtained via bronchoscopy-assisted brushing from a panel of 16 donors (7 COPD patients and 9 healthy controls) were examined for expression/localization of AIM2 and cleaved IL-1β. While AIM2 immunofluorescence in epithelial cells from healthy controls was dull (Figure 5A, middle and right panels) or moderate (5A, left panel), there was a bright cytoplasmic staining for AIM2 in epithelial cells from COPD patients, often associated with specks of cleaved IL-1β (Figure 5B), which was hardly detected in control. In contrast to tissue sections and primary cultures of alveolar macrophages, cytospins of cells freshly obtained via contained a high proportion of cell debris which prevented quantitative particle analysis of fluorescence specks. Percentage of cells having high MFI were counted instead, showing a significant increase of AIM2 in COPD donors vs. control (p = 0.045; Figure 5C).
Cigarette smoke extract induced AIM2 inflammasome activation in human alveolar macrophages
Our observations in lobectomy biopsies indicate alveolar macrophages are the major cell type for the AIM2 inflammasome activation in the COPD airway. Therefore, BAL-derived alveolar macrophages obtained from 8 healthy non-smoker donors were further studied. The macrophages in primary cultures expressed low baseline levels of immunoreactivities for both AIM2 and cleaved IL-1β, followed by a significant induction of AIM2 and cleaved IL-1β immunofluorescence specks in response to exposure to cigarette smoke extract (Figure 6). Similar to the in vivo situation, AIM2 and cleaved IL-1β specks were colocalized near the cell surface but also in the extracellular space (Figure 6 and Supplementary Data Figure S7). To further support the presence of functional inflammasomes in macrophages, co-staining with ASC was investigated, revealing increased ASC staining colocalised with cleaved IL-1β-positive particles (Figure 6).
AIM2 nuclear-to-cytoplasmic exit in mouse model of chronic exposure to cigarette smoke
We next tested if cigarette smoke, the major risk factor for COPD, can reproduce the characteristic AIM2 patterns of subcellular localization in experimental models. Lung tissue of mice chronically exposed to cigarette smoke for 24 weeks were examined for AIM2 subcellular localization, and immunoreactivity for cleaved IL-1β as indicator of a functionally active inflammasome. The shift in AIM2 subcellular distribution was found most remarkable in the epithelial cell type, but not alveolar macrophages. While bronchiolar epithelium of control mice revealed predominant nuclear localization of AIM2 immunoreactivity and low levels of immunoreactivity for cleaved IL-1β, a distinctive nuclear-to-apical translocation of AIM2 was shown in smoked mice, associated often with an increase in cleaved IL-1β. Quantitative analysis in small numbers of animals (n = 4 per group) confirmed that cigarette smoke exposure induced significant increase of cytoplasmic AIM2. Change in cleaved IL-1β was however not statistically significant (Figure 7).
AIM2 nuclear-to-cytoplasmic exit in cigarette smoke extract-stimulated HBEC30KT epithelial cell line
Subcellular localization of AIM2 was further investigated by cell fractionation followed by immunoblotting, using the cell line HBEC30KT derived from human bronchial epithelium. The purity of the nuclear and cytoplasmic fractions was confirmed by single bands of Lamin B1 in the former, and GADPH in the latter. While immunoblotting of the total cell lysate detected multiple bands for AIM2, the lower molecular weight bands (~35; ~41 and ~46kDa) were more abundant in the cytoplasmic fraction, but the higher molecular weight band (~50kDa) was predominant in the nuclear fraction (Figure 8).
The four above mentioned molecular weight bands of AIM2 were compared for their abundance after normalization to the corresponding bands in the whole cell lysate and adjustment based on the GAPDH (cytoplasmic) or Lamin B1 (nuclear) expression. Data obtained from cigarette smoke extract-stimulated vs. control cells indicated that while there was only a modest change in the total, cigarette smoke extract-stimulated cells showed a clear shift of AIM2 out of the nucleus (Figure 8).
In line with data from immunoblotting analysis of cell fractions, confocal analysis demonstrated a distinctive nuclear-to-cytoplasmic redistribution of AIM2 protein, compared to vehicle control (Figure 8). No significant increase of cleaved IL-1β was however observed.