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. 2017 Jun 9;7:3161. doi: 10.1038/s41598-017-03552-9

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Exposure to ROS generated by human neutrophils selected for the modA2 locked OFF subpopulation. (A) NTHI and activated neutrophils were physically separated by a 0.4 µM pore membrane to prevent physical contact or phagocytosis, yet permit the diffusion of ROS. Neutrophils were seeded into the lower chamber of a Transwell system and activated with 50 nM PMA for 10 minutes. NTHI were then added to the upper chamber of a Transwell and incubated for 30 min to allow exposure to ROS. (B) A mixed culture that contained an equal number of modA2 locked ON:GFP and modA2 locked OFF:mCherry was subjected to multiple rounds of exposure to neutrophil-derived ROS. The increased sensitivity of modA2 locked ON to ROS resulted in an enrichment of the modA2 locked OFF subpopulation. Selection was apparent after the first round of exposure to ROS. The population percentages, modA2 locked ON compared to modA2 locked OFF, were significantly different after the first round, *P < 0.005, unpaired t-test. This difference remained significant after each subsequent round of exposure to ROS, *P < 0.005, multiple t-tests. These data suggest that ROS due to infiltrating immune cells will contribute to selection for the modA2 OFF status.