Volume 45, Issue 1, Pages 172-184 (July 2016) Type 2 Interleukin-4 Receptor Signaling in Neutrophils Antagonizes Their Expansion and Migration during Infection and Inflammation  Janine Woytschak, Nadia Keller, Carsten Krieg, Daniela Impellizzieri, Robert W. Thompson, Thomas A. Wynn, Annelies S. Zinkernagel, Onur Boyman  Immunity  Volume 45, Issue 1, Pages 172-184 (July 2016) DOI: 10.1016/j.immuni.2016.06.025 Copyright © 2016 Elsevier Inc. Terms and Conditions

Immunity 2016 45, 172-184DOI: (10.1016/j.immuni.2016.06.025) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Neutrophil Influx during Skin Infection Is Modulated by IL-4 Signals 3 × 107 colony-forming units (CFUs) of Group A Streptococcus (GAS) M1 were injected subcutaneously into the shaved flank of C57BL/6 mice pretreated with PBS or IL-4-anti-IL-4 mAb complexes (IL-4cx) or given a neutralizing anti-IL-4 mAb throughout the experiment, starting 1 day prior to infection. (A) Skin lesion size was measured 48 hr (left) and 72 hr (right) postinfection. (B) Leg swelling of the infected and uninfected site was determined over 72 hr postinfection. (C) Quantification of flow cytometric analysis of CD11b+Ly6G+ cells per 0.1 g skin 6 hr postinfection. (D) Immunohistochemistry and quantification of Ly6G+ cells in skin 6 hr postinfection. Scale bars represent 100 μm. (E) CFUs in skin 72 hr postinfection normalized to PBS-treated mice. Data are pooled from two to three independent experiments with a total of 4 (D) and 8–12 mice per condition (A–C and E; 2–3 mice for uninfected) and represented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S1. Immunity 2016 45, 172-184DOI: (10.1016/j.immuni.2016.06.025) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Blood Neutrophilia upon Systemic Infection Is Suppressed by IL-4 (A and B) C57BL/6 mice received either PBS or 105 CFUs Lm intravenously (i.v.) with or without a neutralizing anti-G-CSF mAb (anti-G-CSF) on days −1 and 0 of infection. Shown are flow cytometric analysis 24 hr postinfection of CD3–CD11b+Ly6G+ neutrophil frequencies in blood (A) and neutrophil counts in blood and spleen (B). Please also refer to Figure S2. (C and D) C57BL/6 mice received either PBS or 105 CFUs Lm i.v. without or with pretreatment using IL-4cx on days −3 to −1 prior to infection, followed by analysis 24 hr postinfection. Shown are flow cytometric analysis of CD3–CD11b+Ly6G+ neutrophil frequencies in blood (C) and their quantification (D). Data are representative of one out of two independent experiments with a total of four to five animals per condition and are represented as mean ± SD; ns indicates not significant; ∗p < 0.05. Immunity 2016 45, 172-184DOI: (10.1016/j.immuni.2016.06.025) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 IL-4 Antagonizes G-CSF Effects on Neutrophils (A and B) C57BL/6 mice were treated with PBS, G-CSF-anti-G-CSF mAb complexes (G-CSFcx), IL-4cx, or G-CSFcx plus IL-4cx for 3 consecutive days. Bone marrow (BM), blood, and spleen were analyzed 16 hr after last injection. (A) Shown is expression of Ly6G versus CD11b in CD3– BM, blood, and spleen cells. (B) Quantification of CD11b+Ly6G+ neutrophils in BM, blood, and spleen. (C) C57BL/6 mice were treated with PBS, G-CSF, IL-4, or G-CSF plus IL-4 for 3 consecutive days. Shown is quantification of CD11b+Ly6G+ neutrophils in BM, blood, and spleen 16 hr after last injection. (D–F) C57BL/6 mice were pretreated for 3 days with PBS, G-CSFcx, IL-4cx, or G-CSFcx plus IL-4cx, followed by systemic (i.v.) infection with 105 CFUs Lm the next day. (D) Quantification of Lm CFUs in spleen and liver 24 and 72 hr postinfection. (E) Mice were assessed for weight change. (F) Mice were monitored for survival. Plots (A) are representative of one out of three independent experiments with two to three mice per group each; quantifications (B and C) are pooled from three independent experiments with a total of seven mice per group and are displayed as mean ± SEM. Data shown as mean ± SD are representative of one out of two independent experiments with a total of four animals per condition (D) or pooled from two experiments with a total of six mice per condition (E and F). ns indicates not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Immunity 2016 45, 172-184DOI: (10.1016/j.immuni.2016.06.025) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 IL-4 Acts Directly on Neutrophils via Type 2 IL-4R (A and B) Wild-type (WT) mice received three injections of PBS, G-CSFcx, or G-CSFcx plus IL-4cx. Histograms show IL-4 receptor (IL-4R) subunit expression compared to isotype control (gray shaded area) of mice receiving PBS (black line) or G-CSFcx (blue line) and quantification by mean fluorescence intensity (MFI) of IL-4Rα, common γ-chain (γc), and IL-13Rα1 expression on CD11b+Ly6G+ neutrophils isolated from BM (A) or spleen (B). (C) Stimulation of splenocytes from WT, IL-4Rα-deficient (Il4ra–/–), γc-deficient (Il2rg–/–), and IL-13Rα1-deficient (Il13ra1–/–) mice with PBS or IL-4 (500 ng/mL) for 15 min, followed by quantification of phosphorylated STAT6 (pSTAT6) in CD11b+Ly6G+ neutrophils. Shown is change in percentage of MFI of pSTAT6 compared to PBS. Please also refer to Figures S3 and S4. (D) Stimulation of WT splenocytes with titrated concentrations of IL-4 and IL-13, followed by quantification of pSTAT6 in CD11b+Ly6G+ neutrophils. Shown is change in percentage of MFI of pSTAT6 compared to PBS. (E) WT and Il4ra–/– mice were treated with PBS, G-CSFcx, or G-CSFcx plus IL-4cx for 3 consecutive days. Spleen and blood were analyzed 16 hr after last injection. Shown is expression of CD11b versus Ly6G in blood CD3– cells (left) and quantification of CD11b+Ly6G+ neutrophils in indicated organs (middle and right). (F) WT and Il2rg–/– were treated and assessed as in (E). (G) Immune cell-lineage-depleted BM cells of WT (CD45.1+) and Il4ra–/– (CD45.2+) mice were mixed at a 1:1 ratio and adoptively transferred to irradiated CD45.2+ Il4ra–/– hosts. After reconstitution, BM chimeric mice were injected for 3 consecutive days with PBS, G-CSFcx, or G-CSFcx plus IL-4cx and change in ratios of CD45.2+ to CD45.1+ cells within CD3–CD11b+Ly6G+ blood neutrophils were determined by flow cytometry 16 hr after last injection. (H) WT and Il4ra–/– mice were infected i.v. with 105 CFU Lm and monitored for survival. Data (A, B, D–G) are representative of one out of two independent experiments and shown as mean ± SD or are pooled from two to three independent experiments and given as mean ± SEM (C and H), with two to three animals per condition each. ns indicates not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Immunity 2016 45, 172-184DOI: (10.1016/j.immuni.2016.06.025) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 IL-4 Induces a BM-Resident Phenotype in Neutrophils WT mice were treated with PBS, G-CSFcx, or IL-4cx for 3 consecutive days. BM and spleen were analyzed 16 hr after last injection. (A) Quantification of geometric MFI of CXCR2 and CXCR4 in neutrophils from BM. (B) Histogram showing CXCR4 expression on neutrophils in BM. (C) Quantification of geometric MFI of CXCR2 and CXCR4 in neutrophils from spleen. (D) Histogram of CXCR2 expression on neutrophils in spleen. Data are representative of one out of two experiments with two to three mice per group each and are presented as mean ± SD; ns indicates not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001. Immunity 2016 45, 172-184DOI: (10.1016/j.immuni.2016.06.025) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 IL-4 Inhibits CXCR2-Mediated Migration In Vitro and In Vivo (A) Purified BM-derived CD11b+Ly6G+ neutrophils were pretreated with either PBS or IL-4 (30 ng/mL), followed by migration toward CXCL2 (100 ng/mL) over 240 min. (B) Purified BM neutrophils were pretreated with either PBS or IL-4 (30 ng/mL), followed by migration toward titrated concentrations of CXCL2 for 2 hr. (C and D) Purified BM neutrophils were pretreated titrated amounts of IL-4, followed by migration toward a fixed concentration of CXCL1 (100 ng/mL) (C) or CXCL2 (100 ng/mL) (D) for 2 hr. (E) Purified BM neutrophils were pretreated in vitro with PBS, IL-2, IL-4, IL-7, IL-13, or IL-15 followed by migration toward CXCL1 (100 ng/mL) for 2 hr. (F) Purified BM neutrophils from WT or Il4ra–/– mice were pretreated with either PBS or IL-4 (30 ng/mL), followed by migration toward to CXCL2 (100 ng/mL) for 2 hr. (G) WT and Cxcr2–/– mice harboring an airpouch received either PBS or monosodium urate crystals (MSU) into their airpouch and were analyzed the next day by flow cytometry for CD3–CD11b+Ly6G+ neutrophils in their airpouch (top and middle) and blood (bottom). (H) WT mice harboring an airpouch were treated i.v. with either PBS or IL-4cx, followed 15 min later by injection of PBS (control), MSU, or IL-1β into the airpouch. Shown are total CD3–CD11b+Ly6G+ neutrophils that migrated into the airpouch. Data are representative of at least two independent experiments and are displayed as mean ± SD (A–D), or are pooled from two to three independent experiments (E, F, and H) and shown as mean ± SEM, with two to three animals per group each. ns indicates not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Immunity 2016 45, 172-184DOI: (10.1016/j.immuni.2016.06.025) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 IL-4 Signaling Interferes with p38 MAPK-PI3K Crosstalk (A) CXCL1-induced migration of purified neutrophils was analyzed upon treatment in vitro with PBS, phosphoinositide 3-kinase (PI3K) inhibitor LY294002, or p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. (B) WT splenocytes were stimulated with either PBS (gray shaded area) or IL-4 (30 ng/mL) for 5 (orange line) or 15 min (red line), followed by assessment by flow cytometry of p38 MAPK phosphorylation in CD3–CD11b+Ly6G+ neutrophils. (C) WT splenocytes were stimulated with either PBS, IL-4 plus DMSO, or IL-4 plus p38 inhibitor SB203580, followed by assessment by flow cytometry of p38 MAPK phosphorylation in CD3–CD11b+Ly6G+ neutrophils. Shown are histograms of phosphorylated p38 MAPK expression in CD3–CD11b+Ly6G+ neutrophils (left) and quantification of MFI values of phosphorylated p38 MAPK (right). (D) CXCL1-induced migration of purified neutrophils incubated with PBS, IL-4 plus DMSO, IL-4 plus SB203580, or IL-4 plus LY294002. (E) Flow cytometric quantification of phosphorylated p38 MAPK in CD3–CD11b+Ly6G+ neutrophils 15 min after in vivo treatment with either PBS, IL-4, or IL-4 plus p38 inhibitor SB203580. (F and G) WT mice were treated with PBS, G-CSFcx, G-CSFcx plus IL-4cx, or G-CSFcx plus IL-4cx plus SB203580 for 3 days. Shown are dot plots (F) and frequencies (G) of CD3–CD11b+Ly6G+ neutrophils from blood 16 hr after last injection. Data are representative of two to three independent experiments with two to five mice per condition each and are represented as mean ± SD; ns indicates not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Immunity 2016 45, 172-184DOI: (10.1016/j.immuni.2016.06.025) Copyright © 2016 Elsevier Inc. Terms and Conditions