Gating strategy for FACS analysis of HSCs and progenitors.

Figure S3. Neutrophils drive the expression of IL-23 by BMDMs. Related to Figure 3. A) Total number of ulcers in the colon of DSS-treated Csfr3+/+ (n=6) and Csf3r-/- (n=7) mice. B) IL-23 levels detected by ELISA in supernatants of BMDMs, neutrophils (NΦ) and BMDM-NΦ co-cultures after stimulation with GM-CSF+CpG. In indicated conditions neutrophils were pre-treated with diphenyleneiodonium (DPI) (10µM) or BMDM-NΦ were cultured in transwells (for all conditions n=6). C) Percentage of viable neutrophils (AnnexinV-/PI-) cultured alone or after co-culture with BMDMs. D) Schematic representation of the treatment schedule for anti-IL-22 treatment and neutrophil adoptive cell transfer during acute colitis. Days of treatment are indicated by red arrows. E) Percentage of body weight loss during DSS-induced acute colitis in Csf3r+/+(n=4), Csf3r-/-(n=4) and Csf3r-/- mice upon adoptive transfer of neutrophils treated with anti-IL-22 or isotype control (50µg/mouse) via i.p. injection (Csf3r-/- mice+Isotype n=3; Csf3r-/-+anti-IL-22 n=3). A) Representative data of five independent experiments. B) Representative data of three independent experiments. C) Representative of two independent experiments. E) One experiment. A) Unpaired Student’s t-Test. B-C) Multiple Student’s t-Test. E) Wilcoxon matched-pairs signed rank test. Data are mean ± SEM. *** p < 0.001 ** p < 0.01 * p < 0.05.

Figure S7. A) Frequency of CD4+ Foxp3+ regulatory T cells in colon LP of DSS-treated Csf3r+/+ and Csf3r-/-. Unpaired Student’s t-Test. Data are mean ± SEM. ns: not significant.

Supplementary Table 1. Colitis grade score.

Gating strategy for FACS analysis of myeloid and lymphoid cell populations.

Figure S5. γδ T cell subsets activation in DSS-treated mice. Related to Figure 4-5 A) Representative gating strategy for γδ T cell subsets in colon LP. B) Frequency of γδ T cell subsets from colon tissue of DSS-treated mice in colon LP. C) AhR expression in γδ T cells subset from colon LP of DSS-treated mice. D-E) Expression of IL-22 by γδ T cell subsets stimulated 4 hours with IL-23 plus IL-1β (D) and PMA plus ionomycin (E) analyzed by flow cytometry. F-G) Expression of IL-17 by γδ T cell subsets stimulated 4 hours with IL-23 plus IL-1β (F) and PMA plus ionomycin (G) analyzed by flow cytometry. H) RorγT expression in γδ T cell subsets from colon LP of DSS-treated mice. I) Body weight loss during DSS-induced acute colitis in Csf3r+/+(n=4), Csf3r-/- (n=4) and Csf3r-/- mice upon adoptive transfer of neutrophils (NΦ) (n=4) with and without antibiotic (ABX) oral treatment (Csf3r+/++ABX n=4 and Csf3r-/-+ABX n=4, Csf3r-/-+NΦ+ABX n=4). Red arrows indicated days of neutrophils transfer. A-H) Representative data of two independent experiments, Csf3r+/+(n=4), Csf3r-/- (n=4). I) One experiments. B-H) Multiple t-test. I) Wilcoxon matched-pairs signed rank test. Data are mean ± SEM. *** p < 0.001 ** p < 0.01 * p < 0.05.

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.

Enhanced emergency granulopoiesis in IL-1R2-deficient MN/MCA1-bearing mice.

Interleukin 1 (IL1) plays dual functions in cancer. It promotes cancer-related inflammation and progression but also influences leukocyte functional activation. IL1 receptor 2 (IL1R2) functions as an IL1 decoy receptor, inhibiting IL1 activity. In this study, we investigated the contribution of IL1R2 in tuning IL1-dependent effects in mouse models of cancer, including colorectal cancer, lung cancer, and primary and metastatic transplantable and chemically induced sarcoma. Even though the prominent role of IL1 is protumoral, IL1R2 deficiency was selectively associated with reduced sarcoma growth, whereas it was irrelevant in other preclinical models investigated. IL1R2 deficiency was associated with a massive infiltration of neutrophils in the tumor, neutrophilia, and increased extramedullary emergency granulopoiesis. Neutrophils were crucial for tumor control in IL1R2-deficient mice. Immunophenotypic and transcriptional profiling of sarcoma-infiltrating neutrophils revealed that IL1R2 deficiency was associated with higher expression of activation or maturation markers and gene expression reprogramming, with downregulation of pathways associated with protumoral functions. In patients with sarcoma, the IL1R2 deficiency gene signature correlated with better clinical outcomes. Thus, this study shows that IL1R2 tunes IL1-driven cancer-associated emergency granulopoiesis and neutrophil functional activation to an antitumor mode in sarcomas and reveals the antitumor potential of neutrophils in this tumor.

Transcriptional profiling analysis of sarcoma-infiltrating neutrophils.

Figure S1. Neutrophil deficiency is associated with increased lymphoid aggregates. Related to Figure 1.A) Number of total lymphoid structures and B) number of lymphoid structures with follicular dendritic cells detected by RNAscope staining for the follicular dendritic cell marker Mfge8 in colon tissue sections from Csf3r+/+(n=8) and Csf3r-/- (n=5) mice after AOM/DSS-induced CRC. C) Representative images of RNAscope analysis for Mfge8 in colon tissue sections from Csf3r-/- mice after AOM/DSS-induced CRC: aggregates with follicular structures are visible as Mfge8+ (red spot): on the top is visible a lymphoid aggregate at two different magnifications, with diffuse Mfge8+ cells; on the bottom right lymphoid microaggregate with Mfge8+ cells; on the bottom left aggregate without follicular structures (Mfge8+). D) Representative dot-plot of neutrophils frequency in blood (top panels) and colon LP (bottom panels) of Csf3r-/- mice and Csf3r-/- mice 4hrs and 18hrs after adoptive transfer of neutrophils. E-F) Frequency of neutrophils in blood (D) and colon LP (E) of DSS-treated Csf3r-/- mice (n=5) and Csf3r-/- mice 4hrs (n=5) and 18hrs (n=5) after adoptive transfer of neutrophils. A-B) Representative data of three independent experiments. C-E) One experiment. A-B, D-E) Unpaired Student’s t-Test. Data are mean ± SEM. *** p < 0.001 ** p < 0.01 * p < 0.05.

Interleukin-1 (IL-1) was the first interleukin to be identified and characterized as a major endogenous pyrogen and a potent proinflammatory and pleiotropic molecule involved in resistance to microbes and in injury. Since IL-1's discovery, the IL-1 system has impressively grown and now its ligands and receptors are recognized as large and complex families of molecules involved in host responses in infections and inflammation, as well as in the activation of innate and adaptive cells. Indeed, all cells of the innate immune system express and/or are affected by IL-1 family members, and IL-1 family members play a key role in the differentiation and function of polarized innate and adaptive cells. The IL-1 family includes ligands with agonist activity, receptor antagonists, and an anti-inflammatory cytokine. Members of the IL-1 receptor family include signaling receptor complexes, decoy receptors, and negative regulators. The IL-1 system is tightly controlled at different levels by antagonists, decoy receptors, scavengers, and dominant negative molecules. Indeed, the deregulated or excessive activation of the IL-1 system is the potential cause of dangerous and detrimental local or systemic inflammatory reactions, as well as autoimmune or allergic responses, and anti-IL-1 therapies have had a tremendous impact on inflammatory diseases.

Neutrophil phenotype in MN/MCA1-bearing mice.