Anti mac 1

Bone marrow was isolated from tibia and femur of mice and erythrocytes were lysed. To sort pre-progenitor cells staining for linage markers anti-CD3e (Cat # 45-0031-80, eBioscience, 1:50), anti-GR1 (Cat # 108428, BioLegend, 1:400), anti-Mac-1 (Cat # 101228, BioLegend, 1:400), anti-CD4 (Cat # 101228, BioLegend, 1:200), anti-CD8a (Cat # 100734, BioLegend, 1:100), anti-TCRb (Cat # 109228, BioLegend, 1:100), anti-NK1.1 (Cat # 45-5941-82, eBioscience, 1:100), anti-B220 (Cat # 103236, BioLegend, 1:100) and anti-CD19 (Cat # 115534, BioLegend, 1:200) was performed. Additionally anti-CD16/32 (Cat # 101305, BioLegend, 1:200), anti-CD41 (Cat # 133906, BioLegend, 1:100), anti-Sca-1 (Cat # 108114, BioLegend, 1:200), anti-c-kit (Cat # 47-1171-82, Invitrogen, 1:50), anti-CD105 (Cat # 120412, BioLegend, 1:50) and anti-CD150 (Cat # 115910, BioLegend, 1:400) were stained to further classify cells. To sort immature granulocytes, mature granulocytes and monocytes anti-Ly6G/Ly6C (Cat # 17-5931-82, Invitrogen, 1:100), anti-CD11b (Cat # 25-0112-82, eBioscience, 1:100) and anti-CD115 (Cat # 11-1031-85, eBioscience, 1:100) were used. Gating strategy was performed as shown in Supplementary Fig 5.

The murine colon samples were sectioned from the ileocecal valve to the anus, washed in ice-cold PBS, fixed in 10% neutral buffered formalin, embedded in paraffin, and the 4 μm sections stained with hematoxylin and eosin (H&E). IHC was performed on paraffin-embedded sections as described [38 ] using the indicated antibodies. GR-1 and Mac-1 positive cells were counted in 8 randomly chosen fields under a microscope (magnification: 40x objective and 10x ocular lens; 0.180 mm² per field). For the immunofluorescence analyses in human samples, 7 μm frozen colon samples were obtained and incubated with the primary antibodies overnight at 4 °C, the specific AlexaFluor secondary antibodies were applied for 1 h at RT. Nuclei were visualized with TO-PRO™-3. The following antibodies were used: anti-GR1 (cat. 108,401; clone RB6–8C5) from BioLegend (San Diego, CA); anti-Mac-1 (cat. ab259372), anti-CD163 (cat. Ab182422), anti HLA-DR (cat. Ab92511), anti-IL4R (cat. Ab203398) and anti-CD86 (cat. Ab239075) from Abcam (Cambridge, UK). TO-PRO™-3 (T3605) was from Invitrogen (Milan, Italy). Secondary AlexaFluor-conjugated antibodies were from Invitrogen (Milan, Italy), secondary HRP antibodies from Amersham (Milan, Italy).