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Inflammation is a hallmark of colorectal cancer (CRC). Neutrophils are well-known mediators in tumor biology but their role in solid tumors, including CRC, was redefined by neutrophil extracellular traps (NETs). Given that it was recently demonstrated that platelet-derived polyP primes neutrophils to release NETs, we examined surgical specimens from CRC to investigate the presence of polyP, as a possible NET inducer. Biopsies with adenomas, hyperplastic polyps, inflammatory bowel disease and healthy colon tissues were used as controls. In all cases, the presence of polyP was apparent, with the main source of polyP being the mast cells. In all CRC and all adenomas with high-grade dysplasia, a substantial number of mast cells, more than 50%, co-expressed intracellularly polyP with CD68 surface antigen (CD68+), but this was not the case in the other examined disorders. PolyP-expressing mast cells were detected in close proximity with tumor cells and neutrophils, suggesting polyP expression by CD68+ mast cells among the stimuli which prime neutrophils to release NETs, in CRC. Moreover, the detection of CD68+ polyP-expressing mast cells could represent a potential prognostic marker in colorectal adenomas and/or carcinomas.

BACKGROUND:The role of neutrophils in tumour biology is largely unresolved. Recently, independent studies indicated either neutrophil extracellular traps (NETs) or Tissue Factor (TF) involvement in cancer biology and associated thrombosis. However, their individual or combined role in colonic adenocarcinoma is still unexplored. METHODS:Colectomy tissue specimens and variable number of draining lymph nodes were obtained from ten patients with adenocarcinoma of the colon. NETs deposition and neutrophil presence as well as TF expression were examined by immunostaining. The effect of NETs on cancer cell growth was studied in in vitro co-cultures of Caco-2 cell line and acute myeloid leukemia primary cells. Proliferation and apoptosis/necrosis of cancer cells were analyzed by flow cytometry. RESULTS:TF-bearing NETs and neutrophil localization were prominent in tumour sections and the respective metastatic lymph nodes. Interestingly, neutrophil infiltration and NETs concentration were gradually reduced from the tumour mass to the distal margin. The in vitro-generated NETs impeded growth of cancer cell cultures by inducing apoptosis and/or inhibiting proliferation. CONCLUSIONS:These data support further the role of neutrophils and NETs in cancer biology. We also suggest their involvement on cancer cell growth.

BACKGROUND: Sepsis is associated with systemic inflammatory responses and induction of coagulation system. Neutrophil extracellular traps (NETs) constitute an antimicrobial mechanism, recently implicated in thrombosis via platelet entrapment and aggregation. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we demonstrate for the first time the localization of thrombogenic tissue factor (TF) in NETs released by neutrophils derived from patients with gram-negative sepsis and normal neutrophils treated with either serum from septic patients or inflammatory mediators involved in the pathogenesis of sepsis. Localization of TF in acidified autophagosomes was observed during this process, as indicated by positive LC3B and LysoTracker staining. Moreover, phosphatidylinositol 3-kinase inhibition with 3-MA or inhibition of endosomal acidification with bafilomycin A1 hindered the release of TF-bearing NETs. TF present in NETs induced thrombin generation in culture supernatants, which further resulted in protease activated receptor-1 signaling. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the involvement of autophagic machinery in the extracellular delivery of TF in NETs and the subsequent activation of coagulation cascade, providing evidence for the implication of this process in coagulopathy and inflammatory response in sepsis.

BACKGROUND: Gout is a prevalent inflammatory arthritis affecting 1-2% of adults characterized by activation of innate immune cells by monosodium urate (MSU) crystals resulting in the secretion of interleukin-1β (IL-1β). Since neutrophils play a major role in gout we sought to determine whether their activation may involve the formation of proinflammatory neutrophil extracellular traps (NETs) in relation to autophagy and IL-1β. METHODOLOGY/PRINCIPAL FINDINGS: Synovial fluid neutrophils from six patients with gout crisis and peripheral blood neutrophils from six patients with acute gout and six control subjects were isolated. MSU crystals, as well as synovial fluid or serum obtained from patients with acute gout, were used for the treatment of control neutrophils. NET formation was assessed using immunofluorescence microscopy. MSU crystals or synovial fluid or serum from patients induced NET formation in control neutrophils. Importantly, NET production was observed in neutrophils isolated from synovial fluid or peripheral blood from patients with acute gout. NETs contained the alarmin high mobility group box 1 (HMGB1) supporting their pro-inflammatory potential. Inhibition of phosphatidylinositol 3-kinase signaling or phagolysosomal fusion prevented NET formation, implicating autophagy in this process. NET formation was driven at least in part by IL-1β as demonstrated by experiments involving IL-1β and its inhibitor anakinra. CONCLUSIONS/SIGNIFICANCE: These findings document for the first time that activation of neutrophils in gout is associated with the formation of proinflammatory NETs and links this process to both autophagy and IL-1β. Modulation of the autophagic machinery may represent an additional therapeutic study in crystalline arthritides.