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In response to several types of bacteria, as well as pharmacological agents, neutrophils produce extracellular vesicles (EVs) and release DNA in the form of neutrophil extracellular traps (NETs). However, it is unknown whether these two neutrophil products cooperate to modulate inflammation. Consistent with vital NETosis, neutrophils challenged with S. aureus , as well as those treated with A23187, released significantly more DNA relative to untreated or fMLF-treated neutrophils, with no lysis occurring for any condition. To test the hypothesis that EVs generated during NETosis caused macrophage inflammation, we isolated and characterized EVs from A23187-treated neutrophils (A23187-EVs). A23187-EVs associated with neutrophil granule proteins, histone H3, transcription factor A, mitochondrial (TFAM), and nuclear and mitochondrial DNA (mtDNA). We showed that DNA from A23187-EVs, when transfected into macrophages, led to production of IL-6 and IFN-α2, and this response was blunted by pre-treatment with the STING inhibitor H151. Next, we confirmed that A23187-EVs were engulfed by macrophages, and showed that they induced cGAS-STING-dependent IL-6 production. In contrast, neither EVs from untreated or fMLF-treated cells exhibited pro-inflammatory activity. Although detergent-mediated lysis of A23187-EVs diminished IL-6 production, removal of surface-associated DNA with DNase I treatment had no effect, and A23187-EVs did not induce IFN-α2 production. Given these unexpected results, we investigated whether macrophage mtDNA activated the cGAS-STING signaling axis. Consistent with mitochondrial outer membrane permeabilization (MOMP), a defined mechanism of mtDNA release, we observed macrophage mitochondrial membrane depolarization, a decrease in cytosolic Bax, and a decrease in mitochondrial cytochrome c, suggesting that macrophage mtDNA may initiate this EV-dependent signaling cascade. All together, these data demonstrate that A23187-EVs behave differently than transfected NET- or EV-DNA, and that neutrophil-derived EVs could be used as a model to study NF-κB-dependent STING activation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Allen, Whitefoot-Keliin, Palmatier, Mahon and Greenlee-Wacker.)

There is a critical need to develop superior influenza vaccines that provide broader protection. Influenza vaccines are traditionally tested in naive animals, although humans are exposed to influenza in the first years of their lives, but the impact of prior influenza exposure on vaccine immune responses has not been well studied. Pigs are an important natural host for influenza, are a source of pandemic viruses, and are an excellent model for human influenza. Here, we investigated the immunogenicity of the ChAdOx2 viral vectored vaccine, expressing influenza nucleoprotein, matrix protein 1, and neuraminidase in H1N1pdm09 pre-exposed pigs. We evaluated the importance of the route of administration by comparing intranasal, aerosol, and intramuscular immunizations. Aerosol delivery boosted the local lung T-cell and antibody responses, while intramuscular immunization boosted peripheral blood immunity. These results will inform how best to deliver vaccines in order to harness optimal protective immunity., Competing Interests: SG is co-founder of Vaccitech and named as an inventor on a patent covering use of ChAdOx2-vectored vaccines. TL is a consultant to Vaccitech. RM is an employee of Aerogen Limited and is a named inventor on inhaled vaccine delivery system patents. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Vatzia, Allen, Manjegowda, Morris, McNee, Martini, Kaliath, Ulaszewska, Boyd, Paudyal, Carr, Chrun, Maze, MacLoughlin, van Diemen, Everett, Lambe, Gilbert and Tchilian.)