Fig. 5: OMVs from A. baumannii activate AHR.

a AHR activity reporter (XRE-Luc) assay in A549 cells exposed to OMVs (100 μg/mL for 3 h). Cells were transfected with the indicated siRNA 48 h before OMV treatment. b Schematic showing the key steps of OMV endocytosis and the corresponding inhibitors. Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en). c, d AHR activity reporter (XRE-Luc) assay in A549 cells exposed to OMVs (20 μg/mL for 3 h) in the presence of the following chemicals: DMSO (solvent control), CytB (cytochalasin B, inhibitor of actin polymerization), DYN (dynasore, inhibitor of dynamin) and BafA1 (bafilomycin A1, inhibitor of endolysosomal acidification). e Quantification of AHR target gene expression (CYP1A1, CYP1B1, AHRR, TIPARP and STC2) by qRT-PCR in A549 cells exposed to OMVs (100 μg/mL for 3 h). CH223191 (10 μM) was added to inhibit AHR. n  =  3 independent experiments. f The mouse infection challenge model. Wild-type BALB/c mice were injected i.p. with 3 ×10³ CFUs of A. baumannii clinical isolate #0280 followed by western blot analysis of organs for the detection of CYP1A1, with vinculin for normalization. 3 pairs of mock and infected animals were analyzed, and similar results were obtained. The illustration was created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en). g Schematic showing the function of LpxA in lipid A synthesis. OMVs produced by the ΔlpxA mutant strain of A. baumannii lack lipid A. Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en). h, i AHR activity reporter (XRE-Luc) assay in A549 (H) and HEK 293 T (I) cells exposed to OMVs (100 μg/mL for 3 h) isolated from ATCC 19606 or the A. baumannii ΔlpxA mutant (lipid A synthesis mutant). a, c, h, i n = 3. d n = 4. Cells were seeded in 3 or 4 different wells per group, Treatment and measurement were performed independently for each well. Experiments were repeated independently three times and similar results were obtained. a, c, d, e, h, i Data are presented as mean values +/- SD, P-values by unpaired two-tailed t-test. See also Supplementary Fig. 5. Source data are provided in the Source Data file.