br BCG induced NETs are potent activators of
3.4. BCG-induced NETs are potent activators of PBMCs, in which NETs' protein components play a key role
To determine whether cellular immunity was induced, we exposed PBMCs to NETs prepared from the same donor neutrophils. NETs in-duced IFN-γ, IL-2, and TNF-α in a negative dose-dependent manner
(P < .05; Fig. 4A), achieving a maximum eﬀect using 0.5 × NETs and minimum induction or even inhibition with 2 × NETs. The changes in IL-12 were not statistically significant. Furthermore, heat-inactivation remarkably decreased the responses to near the level of no stimulation (P < .01), and DNase reduced part of the responses (Fig. 4B). Similar to the secretion profiles, NETs showed a positive eﬀect on PBMC sur-vival at a low dose, but inhibition at a high dose (Fig. 4C). Additionally, NETs at a high dose induced apoptosis and death of PBMCs (P < .05; Fig. 4D).
Unlike the above responses, diﬀerent doses of NETs promoted CD4 expression in co-cultured PBMCs (P < .001), with no significant dif-ference between high and low NETs (Fig. 4E). However, CD8 expression and the percentage of CD8+ cells were not significantly upregulated by 0.5 × NETs (P > .05; Fig. 4F).
3.5. BCG-induced NETs inhibit tumors in mice by attacking tumor cells and increasing immunocyte infiltration, and the eﬀects are mainly mediated by NETs proteins
Prepared NETs were applied to preliminary analyze the NETs only eﬀect on tumors. Subcutaneous tumors rather than an orthotopic model were suitable for administration of NETs. All tumor-bearing mice sur-vived with metastases until the mice were sacrifice. The average tumor weight and volume were significantly lower in NETs-administered mice than the shams (P < .01; Fig. 5A–C). Importantly, the eﬀect of NETs was nearly eliminated by inactivation of proteins, indicating that the protein components were mostly responsible for the inhibition (Fig. 5B). Moreover, NETs administration resulted in increased necrosis, and significant apoptosis or death of tumor cells (Fig. 5D–E). NETs strongly increased intra-tumor CD3+ and CD14+ infiltration (P < .001), which was reduced by boiling but not DNase pre-treatment of NETs (Fig. 5F–G). Together these results suggest a therapeutic eﬀect of NETs on ASP1517 cancer by direct damage and induction of the cel-lular immune cascade. r> 4. Discussion
Recent studies suggested that NETs might lead to tissue damage, [18,21] activation of lymphocytes , dendritic cells (DC) and mac-rophages , as well as autoimmune  and chronic inflammatory diseases [23,24]. Additionally, several studies have demonstrated that cancers predispose neutrophils to release NETs, [27,32] which are in-volved in tumor-associated thrombosis, then possibly contribute to tumor progression and metastases by degrading the extracellular matrix [25,33,34]. In addition, the cytotoxicity of NETs on endothelial, epi-thelial, and neuronal cells has been reported [19,23]. After NETs treatment, tumor cell proliferation was significantly inhibited in the current study, and a similar observation was noted with respect to cy-totoxicity. Both BC cell lines exhibited shrinkage and a decline in ad-herent cell numbers after NETs treatment, suggesting a non-specific mechanism underlying cell damage. Also, BCG-induced NETs promoted G0/G1 phase arrest and apoptosis of tumor cells in a dose- and time-dependent fashion. In addition, our study confirmed that BCG-induced NETs inhibited mouse tumor growth. More tissue destruction was ob-served in NETs-treated tumors than the sham treatment. Therefore, we
Fig. 4. NETs influenced PBMCs proliferation and activation in vitro.
A, in contrast to the control (no NETs), the production of IFN-γ, IL-12, and TNF-α were up-regulated in PBMCs exposed to 0.5 × and 1 × NETs. Interestingly, 2 × NETs increased the levels of IFN-γ, IL-2 and TNF-α at 24 h, but inhibited the levels of them after 24 or 48 h. Thus, increased Th1-type cytokines secretion from PBMCs showed a profound eﬀect with 0.5 × NETs, and a mild to moderate increase or even inhibition at higher doses, compared with the control. B, DNase-digestion somewhat reduced NETs-induced secretion; however, heat-inactivation remarkably decreased the responses to nearly the levels of no induction. C, 1 × 105 PBMCs significantly proliferated after stimulation with lower doses of NETs (0.5× and 1×), whereas no increase or even an obvious inhibition was observed when treated with a high dose (2×) of NETs. Thus, the proliferation percentage was negatively dependent on NETs dose, and declined with time. D, the representative results of FCM analysis indicated that the induced apoptosis and death of PBMCs was significantly higher in 2× NETs after a 24 h incubation. E, overnight NETs upregulated the mean fluorescence intensity (MFI [column]) of the markers CD4, but not the percentage of CD4+ cells (% [line]). F, the marker on cytotoxic lymphocytes, CD8 was non-significantly upregulated in MFI and the percentage of positive cells treated with 0.5 × NETs. *P < .05, **P < .01.