Abstract 6209: Monocytes phagocytose neutrophil extracellular traps (NETs) and differentiate into pro-inflammatory macrophages

Background: Macrophages are multipurpose phagocytes with various functions. Neutrophil extracellular traps (NETs) are web-like structures ensnare microbes which were composed of dsDNA, histones, and antimicrobial peptides. NETs are generally degraded by DNAse and macrophages, however, it is unknown how the degradation of NETs affects the local immune response thereafter. Here, we investigated the interaction between NETs and peripheral blood monocytes (PBMo). Method: 5 × 105 Polymorphonuclear leukocytes(PMNs), PMNs irradiated with UV for 15 minutes (UV-PMN) and PMNs stimulated by PMA for 15min and extensively washed (PMA-PMN) were placed on 6 well plate and cultured for 2 hours and NETs formation was visualized by SYTOX green. Peripheral blood was obtained from healthy volunteers and CD14+ PBMo were purified by positive selection using the magnetic cell separation system. The PBMo were co-cultured with those PMNs at 37°C in 5% CO2 conditions for 3 days. The differentiation of macrophage was evaluated by the expression of M1/M2 markers by flowcytometry. Cytokine production was evaluated using the Cytokine Array system. The macrophages were co-cultured with autologous T cells stimulated with anti-CD3 mAb and the effect on T cell proliferation was evaluated with CFSE dilution assay. Results: PMA-PMN, but not PMN or UV-PMN, produced massive SYTOX(+) NETs within 2 hours. Co-culture of the NETs with PBMo stained with PKH26 showed NETs are phagocytosed by monocytes within 24 hours. PBMo co-cultured with PMA-PMN showed significantly enhanced expression of CD86, and reduced expression of CD163 as compared to those co-cultured with PMNs or UV-PMN. However, the expression of CD86 was suppressed if PMA-PMN were pretreated with 100 u/ml DNase I. The supernatant of the co-culture with PMA-PMN showed markedly increased levels of IL-1β and TNF-α. PBMo co-cultured with PMA-PMN strongly promote the proliferation of autologous T cells. However, the enhanced T cell growth were significantly suppressed by the pretreatment of NETs with DNase I. Conclusion: Monocytes appear to differentiate to proinflammatory macrophages after the phagocytosis of NETs. In tumor-bearing host, NETs are known to be increased in circulating blood, which may promote tumor associated inflammatory process. Citation Format: Akira Saito, Hideyuki Ozawa, Kazuya Takahashi, Yuki Kimura, Mineyuki Tojo, Yuko Kumagai, Rihito Kanamaru, Hidenori Tsukui, Naohiro Sata, Joji Kitayama. Monocytes phagocytose neutrophil extracellular traps (NETs) and differentiate into pro-inflammatory macrophages [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6209.