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Supplementary Figure S2 from Neutrophils Mediate Protection Against Colitis and Carcinogenesis by Controlling Bacterial Invasion and IL22 Production by γδ T Cells

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posted on 2024-04-02, 07:23 authored by Silvia Carnevale, Andrea Ponzetta, Anna Rigatelli, Roberta Carriero, Simone Puccio, Domenico Supino, Giovanna Grieco, Piera Molisso, Irene Di Ceglie, Francesco Scavello, Chiara Perucchini, Fabio Pasqualini, Camilla Recordati, Claudio Tripodo, Beatrice Belmonte, Andrea Mariancini, Paolo Kunderfranco, Giuseppe Sciumè, Enrico Lugli, Eduardo Bonavita, Elena Magrini, Cecilia Garlanda, Alberto Mantovani, Sebastien Jaillon

Figure S2. Neutrophil deficiency is associated with intestinal dysbiosis. Related to Figure 2 A) Representative histological images of H&E-stained colon section from Csf3r+/+(left) and Csf3r-/- (right) mice, after one cycle of DSS, showing the increased inflammatory infiltrate and the presence of bacterial accretions (indicated by black arrow) in Csf3r-/-. B-C) Shannon Index representing the evenness in the overall number of bacterial species between wild-type and neutrophil deficient mice untreated (B) and DSS-treated mice (C). D-E) Taxonomic analysis of the phyla that compose the microbiota of feces of untreated (D) and DSS-treated (E) Csf3r+/+ (untreated n=4, DSS-treated n=3) and Csf3r-/- (untreated n=4, DSS-treated n=4) mice. F) Microbiota depletion efficiency measured by qPCR for 16S gene: complete depletion is achieved after 20 days of antibiotic treatment. G) Body weight loss during DSS-induced acute colitis in Csf3r+/+(n=11) and Csf3r-/- (n=9) mice with and without cohousing (Csf3r+/+Cohoused n=5 and Csf3r-/-Cohoused n=5). H) Body weight loss of AOM/DSS treated Csf3r+/+(n=7) and Csf3r-/- (n=5) mice with and without cohousing (Csf3r+/+Cohoused n=5 and Csf3r-/-Cohoused n=5); I) Macroscopic polyp count at the experimental endpoint. A-I) One experiment. B-C) Unpaired Student’s t-Test. F, I) Multiple Student’s t-Test. G-H) Wilcoxon matched-pairs signed rank test. Data are mean ± SEM. *** p < 0.001 ** p < 0.01 * p < 0.05.

Funding

Ministero della Salute (Italy Ministry of Health)

Fondazione AIRC per la ricerca sul cancro ETS (AIRC)

Ministero dell'Università e della Ricerca (MUR)

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ARTICLE ABSTRACT

Neutrophils are the most abundant leukocytes in human blood and play a primary role in resistance against invading microorganisms and in the acute inflammatory response. However, their role in colitis and colitis-associated colorectal cancer is still under debate. This study aims to dissect the role of neutrophils in these pathologic contexts by using a rigorous genetic approach. Neutrophil-deficient mice (Csf3r−/− mice) were used in classic models of colitis and colitis-associated colorectal cancer and the role of neutrophils was assessed by histologic, cellular, and molecular analyses coupled with adoptive cell transfer. We also performed correlative analyses using human datasets. Csf3r−/− mice showed increased susceptibility to colitis and colitis-associated colorectal cancer compared with control Csf3r+/+ mice and adoptive transfer of neutrophils in Csf3r−/− mice reverted the phenotype. In colitis, Csf3r−/− mice showed increased bacterial invasion and a reduced number of healing ulcers in the colon, indicating a compromised regenerative capacity of epithelial cells. Neutrophils were essential for γδ T-cell polarization and IL22 production. In patients with ulcerative colitis, expression of CSF3R was positively correlated with IL22 and IL23 expression. Moreover, gene signatures associated with epithelial-cell development, proliferation, and antimicrobial response were enriched in CSF3Rhigh patients. Our data support a model where neutrophils mediate protection against intestinal inflammation and colitis-associated colorectal cancer by controlling the intestinal microbiota and driving the activation of an IL22-dependent tissue repair pathway.

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