br E mail address drelsun com E Sun Kangkang

E-mail address: [email protected] (E. Sun). 1 Kangkang Liu, Erlin Sun and Mingde Lei contributed equally to this work.
(caption on next page)
Fig. 1. BCG induced NETs formation.
A, a significant morphologic change induced by BCG was shown in neutrophils after a 3-h incubation under an optical microscope. Cell membranes became matted, and resembled a burr-like morphology. B, Under SEM, many fibers with different diameters and lengths were produced from BCG-treated neutrophils. The fibers aggregated and nearly covered the entire surface. C, Under CLSM, an extracellular co-localization was displayed of DNA fibers (blue), cit-H3 (green), and NE (red). D, after pretreatment by DNAse I and proteases, NETs formation was inhibited significantly, which demonstrated that DNAs and proteins were important components of NETs (P < .001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
first reported that activated PMNs could release NETs to kill pathogenic bacteria [16]. Unlike apoptosis or necrosis, NETosis is a kind of cell death process that is triggered by a variety of bacteria, eukaryotic parasites, viruses, and pro-inflammatory factors [17–19]. NETs are in-volved in the innate immune response, and the excessive release of NETs can perpetuate sterile inflammation, [19] autoimmune activity and pathologic changes. [20–22] Moreover, NETs might injure alveoli, intestinal epithelium, and endothelium. [18,23,24] Some studies have suggested that protein components, particularly histones are re-sponsible for NETs-mediated cytotoxicity. Interestingly, a possible role for NETs released by tumor-associated neutrophils in pediatric Ewing sarcoma has been indicated [25], and microvascular NETs deposition may be involved in cancer progression and metastasis, and systemic infection [26,27]. Neutrophils have been reported to have a role in cancer progression or suppression in a context-specific manner [28]. Thus, we hypothesize that BCG induce NETs that mediate im-munoreactions during oncotherapy.
The objective of this study was to confirm NETs formation by neutrophils following BCG stimulation. We verified the role of NETs in in excercising antitumor effects in vitro and in a mouse model of Dexmedetomidine cancer. The findings in this study will help us understand the role of neutrophils in the early stages of BCG-related antitumor immunity better.
2. Materials and methods
2.1. Human neutrophil isolation
Neutrophils were isolated from fresh blood of healthy volunteers using the Ficoll-Dextran method [29]. The purity of neutrophils was > 95%, as confirmed using CD11b (561015) and CD66b antibodies (561927, BD Biosciences) on flow cytometry. The viability was > 95% for all preparations, as indicated by trypan blue exclusion. The cell preparations that had become activated during isolation were ruled out by inspecting cell morphology microscopically. All researches involving human samples were approved by the Ethics Committee of the Second Hospital of Tianjin Medical University (No: KY2016K010) and in ac-cordance with the Helsinki declaration. Informed consents were ob-tained from all individual participants included in our study.
The BCG Connaught substrain (No: ATCC35733) was obtained from the American Type Culture Collection (Manassas, VA). BCG suspension was cultured in Middlebrock 7H9 broth media (BD 271310), and 7H10 solid media (BD 262710, Difco Laboratories USA) for colony growth. The number of colony-forming units (CFUs) was routinely determined by plating and incubation on solid medium at 37 °C for 4 weeks. Viable BCG selected from the logarithmic growth phase were used for ex-periments.
2.3. Induction of NETs formation
Freshly isolated PMNs (5 × 105/mL) were gently added into culture plates on anti-peeling coverslips (Sigma-Aldrich) and cultured in com-plete RPMI supplemented with 2% FCS (Gibco BRL) at 37 °C in 5% CO2 for 2 h. PMNs were stimulated with 50 nM PMA (Sigma-Aldrich), BCG (MOI = 10), or left unstimulated respectively. As a positive control, 
2.4. Visualization of NETs by confocal laser scanning microscope (CLSM)
and scanning electron microscopy (SEM)
NETs specimens were gently fixed with 4% paraformaldehyde (P8430, Solarbio) and NETs-forming neutrophils were determined by staining DNA with DAPI (D9542-1MG, Sigma). To evaluate NETs composition, immuno-fluorescence assays were applied with primary antibodies against citrullinated Histone H3 (cit-H3) (ab219407) and NE (ab14188) or isotype IgG (ab27478), then observed by CLSM (FV1000, Olympus, Japan), as previously described [17,30]. In addition, the samples were fixed in 2.5% glutaraldehyde then 1% osmium tetroxide/ 1% tannic acid. After dehydration with an ethanol series and critical-point drying, the specimens were coated with platinum and visualized by SEM (Hitachi, CA).