Hypoxia induces a dfoxo-dependent activation of the immune response in Drosophila airway epithelia.

A) Early third-instar larvae of the LDH-Gal4/UAS-nGFP genotype were subjected to a brief period of mild hypoxia (2 h, 5% oxygen). Nuclear expression of GFP was observed only in the airway epithelium (arrows). Background staining of the intestine is due to the autofluorescence of ingested material. B) qRT-PCR analysis of the transcript levels of the major antimicrobial peptide (AMP) gene levels in the trachea of larval Drosophila in response to a hypoxic episode. C, D) Hypoxia treatment of a drosomycin reporter strain (Drs-GFP) induced GFP expression only in the airway epithelium. C) In the overview, expression is restricted to the primary and secondary branches (yellow arrows), whereas the main trunks show no or little expression (red arrows). D) At higher magnification, expression in the primary and secondary branches is more evident. E-G) Using the same line, hypoxia-induced expression of the drosomycin reporter was also observed in the adult tracheal system. E) At low magnification, the peripheral tracheal system is visible through the cuticle (yellow arrows). F) At higher magnification, tracheal structures directly adjacent to the spiracular opening and G) within the legs are visible. dfoxo-gfp targeting the tracheal system under H) control and I) hypoxic conditions. J) Hypoxia-induced transcript levels of drosomycin in larval trachea of control (left) and dfoxo-deficient animals. Mean values ± S.D. ≥ 3, ** p<0.01.

Cellular localizations of the transcription factors dfoxo, relish, and dorsal in the airways of Drosophila larvae exposed to different stressors.

A-F) dfoxo-GFP was expressed in the airways by crossing the driver line btl-Gal4 with the corresponding responder line UAS-dfoxo-gfp. A) Under non-stressed conditions, fluorescence was restricted to the cytoplasm. B-F) Starvation (B, 12 h of nutritional deprivation), oxidative stress (C, exposure to 100 mM paraquat for 2 h), UV irradiation (D, 254 nm UVC rays), cold stress (E, 2 h at 4°C), and heat stress (F, 30 min at 41°C) induced nuclear translocation of dFoxo-GFP in epithelial cells of the tracheae. A’-F’’) Relish-eYFP and Dorsal-eGFP were expressed by crossing the driver line btl-Gal4 with the corresponding responder lines UAS-relish-eyfp and UAS-dorsal-egfp, respectively. A’, A’’) Under non-stressed conditions, the fluorescence of Relish-eYFP and Dorsal-eGFP was restricted to the cytoplasm. B’, B’’) Except for starvation (B’), the various stressors including C’) oxidative stress, D’) UV irradiation, E’) cold stress, F’) and heat stress did not induce nuclear translocation of Relish-eYFP. B’’-F’’) None of the applied stressors induced nuclear translocation of Dorsal-eGFP. G-K) ppk4-Gal4XUASdfoxo-gfp animals remained unstressed (G, control), H) experienced cold, I) heat, and K) hypoxia. G’-K’) Animals of the genotype ppk4-Gal4; UAS-bskDNXUAS-dfoxo-gfp were kept under control conditions. White arrowheads indicate nuclear translocation. Yellow arrows indicate no nuclear translocation. All images were acquired 2 h, or 12 h in the case of starvation after stress stimulation was initiated. Scale bars are 200 µm.

RNAseq analyses of dfoxo deficiency and dfoxo overexpression in the airways of Drosophila larvae.

Heatmap of differentially expressed genes >1.5 fold, (p<0.05) of airway samples taken from control and dfoxo21/21 animals. B) Red indicates upregulation, blue indicates downregulation. PCoA analysis of the individual biological replicates is shown on the right. C) Heatmap of differentially expressed genes between control airways and those experiencing dfoxo overexpression (dfoxooe) in the airways. D) PCoA analysis is shown on the right. E) Venn diagram analysis of significantly up- and downregulated genes of comparisons between controls and dfoxo21/21 as well as controls and dfoxo overexpression (oe). The significance of overlapping cohorts of genes was calculated with Fisher’s exact test (***p<0.001, *p<0.05). F) Significantly regulated KEGG pathways in the cohort of genes that are downregulated in dfoxo21/21. G) Significantly regulated KEGG pathways in the cohort of genes that are upregulated in dfoxo21/21 trachea. H) Genes that are associated with glutathione metabolism are downregulated in dfoxo21/21 airways (mean values ± S.D. are shown). I) Heat-shock protein genes whose transcription is differentially regulated in dfoxo21/21 airways.

Reduced stress resistance of dfoxo21/21 flies.

A) Survival of dfoxo-deficient (dfoxo21/21) and control adult flies exposed to cigarette smoke daily (n>10). B) Survival of dfoxo-deficient and control flies exposed to desiccation. C) Lawn-leaving assays of Drosophila 3rd instar larvae exposed to hypoxia. D) dfoxo-deficient and control flies were briefly anesthetized with N2 and the time taken to full recovery was measured. For A, B, N≥100, for C, D, N=30-110. Shown are mean values ± SEM. * means p<0.05, ** means p<0.01, *** means p<0.005.

Expression of hFOXO1, hFOXO3A, hFOXO4, and hFOXO6 in lung tissues of humans and response to hypoxia.

A) Using the human lung atlas (Vieira Braga et al., 2019), expression plots of the four different hFOXO genes were generated. Red dots represent positive cells. The assignment to specific epithelial cell types is shown on the left. B-D) Response to hypoxic treatment (5% O2) in human A549 cells. B) Control cells show a cytosolic localization of hFOXO1, whereas hypoxia induced an almost complete nuclear translocation (C). (D) Quantitative evaluation of hFOXO translocation in response to hypoxia as indicated by hFOXO1, hFOXO3A, and hFOXO4. E, F) Hypoxia-induced expression of cytokines with antimicrobial activities in a time-dependent manner. E) Expression levels of CCL20 in A549 cells treated for different times with hypoxia (5 % O2). F) The same type of analysis as in D was performed with CXCL8. Data are presented as the mean and SD (n≥7 per group). Statistical analyses were performed using a one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001.

Stress-induced nuclear translocation of FoxO factors in AECs is conserved between species.

A-F) Cultured A549 cells (human alveolar type II cells) were exposed to A) medium only (control) or different stressors, namely, B) oxidative stress induced by paraquat, C) starvation, D) UV light, E) cold stress, and F) heat stress. hFOXO1 was detected immunohistochemically. Scale bars are 25 µm. G-N) Nuclear translocation was quantified by determining the distribution of the antibody-linked fluorescence signal in the nucleus and cytoplasm of G) A549 and H) BEAS-2B cells. Cultured A549 and BEAS-2B (human bronchial epithelial cells) cells were exposed to medium only (ctrl), oxidative stress, starvation, heat stress, cold stress, and UV light. G, H) Immunofluorescence staining revealed the cellular distribution of hFOXO1, hFOXO3A, hFOXO4, and hFOXO6. The relative ratios of fluorescence intensities in the nucleus to those in the cytoplasm are shown. A ratio of 1 indicates that the fluorescence intensities in the cytoplasm and the nucleus are identical. The higher the ratio, the higher the fluorescence intensity in the nucleus. I, J) Semi-intact and freshly explanted mouse tracheae were exposed to medium (control) or paraquat (oxidative stress). Nuclei were stained with Hoechst 33258 (red) and mFoxo1 was immunohistochemically stained (green). I’, J’) The bottom images are merged. The scale bar is 10 µm. Asterisks indicate nuclear regions without mFoxo1 signal. Arrowheads indicate nuclear regions containing mFoxo1 signals. K) Quantification of nuclear translocation of mFoxo1 in murine tracheae exposed to paraquat. Points represent values obtained in independent experiments/individual animals (n=4). Statistical analysis was performed using the t-test. Significant differences compared with the control were calculated by a one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

Expression of mFoxo1, mFoxo3A, mFoxo4, and mFoxo6 in lung tissues of PBS-treated control mice and OVA-treated mice used as models of acute and chronic asthma.

A-D) Micro-dissected airways of PBS-treated control mice and OVA-treated mice (acute asthma model) were separated into distal and proximal parts. A) mFoxo1, B) mFoxo3A, C) mFoxo4, D) and mFoxo6 were expressed in both lung regions in both types of mice. In PBS-treated mice, expression was higher in proximal airways than in distal airways. Furthermore, expression in both distal and proximal airways was lower in OVA-treated mice than in control mice. However, this reduction was more apparent in proximal airways than in distal airways. In distal airways, expression of A) mFoxo1, B) and mFoxo3 did not significantly differ between control and OVA-treated mice. Data are presented as the mean and SD (n=5). Statistical analysis was performed using a one-way ANOVA. (E-H) Expression of the four mFoxo genes was monitored in lung tissues of PBS-treated control mice and OVA-treated mice used as models of acute and chronic asthma. Expression of E) mFoxo1, F) mFoxo3A, G) mFoxo4, H) and mFoxo6 was reduced in lung tissue of the acute asthma mouse model and was decreased to an even greater extent in lung tissue of the chronic asthma mouse model. Data are presented as the mean and SD (n=4 mice per group). Statistical analyses were performed using a one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

Transcript levels of hFOXO factors in sputum samples of asthmatic patients and primary AECs after Th1/Th2 priming.

A) Relative levels of hFOXO factors in sputum samples of asthmatic patients and non-asthmatic controls were quantified by mRNA microarray analyses (n≥3). Statistical analysis was performed by the Mann-Whitney U test. B) NHBEs of healthy individuals were cultured under control conditions or treated with IL-4 to induce Th1/Th2 priming. Relative hFOXO transcripts were quantified by mRNA microarray analyses (n=6). Statistical analysis was performed by the Mann-Whitney U test. *p<0.05 and **p<0.01. C) Scheme summarizing the function of FoxO factors in a physiological state (left) or in a disease-associated non-physiological state (right).