Type 2 Interleukin-4 Receptor Signaling in Neutrophils Antagonizes Their Expansion and Migration during Infection and Inflammation

Article

Type 2 Interleukin-4 Receptor Signaling in Neutrophils Antagonizes Their Expansion and Migration during Infection and Inflammation Graphical Abstract

Authors Janine Woytschak, Nadia Keller, Carsten Krieg, ..., Thomas A. Wynn, Annelies S. Zinkernagel, Onur Boyman

Correspondence [email protected]

In Brief Although neutrophils usually predominate acute inflammatory conditions, they are conspicuously absent in many allergic disorders. Boyman and colleagues demonstrate that the type 2 cell signature cytokine interleukin-4 (IL-4) curtails neutrophil recruitment and migration by signaling via type 2 IL-4 receptors that become upregulated on neutrophils upon inflammation and infection.

Highlights d

Cutaneous and systemic bacterial infections are exacerbated by IL-4 signaling

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IL-4 restricts neutrophil expansion in and migration from bone marrow to tissues

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IL-4 opposes G-CSF effects by direct action on neutrophils via type 2 IL-4Rs

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IL-4 interferes with CXCR2-CXCR4 and p38 MAPK-PI3K regulation in neutrophils

Woytschak et al., 2016, Immunity 45, 172–184 July 19, 2016 ª 2016 Elsevier Inc. http://dx.doi.org/10.1016/j.immuni.2016.06.025

Immunity

Article Type 2 Interleukin-4 Receptor Signaling in Neutrophils Antagonizes Their Expansion and Migration during Infection and Inflammation Janine Woytschak,1 Nadia Keller,2 Carsten Krieg,3 Daniela Impellizzieri,1 Robert W. Thompson,4 Thomas A. Wynn,4 Annelies S. Zinkernagel,2 and Onur Boyman1,* 1Department

of Immunology, University Hospital Zurich, University of Zurich, 8091 Zurich, Switzerland of Infectious Diseases, University Hospital Zurich, University of Zurich, 8091 Zurich, Switzerland 3Institute of Experimental Immunology, University of Zurich, 8057 Zurich, Switzerland 4Immunopathogenesis Section, Program in Barrier Immunity and Repair, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Disease, NIH, Bethesda, MD 20892-0425, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.immuni.2016.06.025 2Department

SUMMARY

Neutrophils are the first immune cells recruited to sites of inflammation and infection. However, patients with allergic disorders such as atopic dermatitis show a paucity of skin neutrophils and are prone to bacterial skin infections, suggesting that allergic inflammation curtails neutrophil responses. Here we have shown that the type 2 cell signature cytokine interleukin-4 (IL-4) hampers neutrophil expansion and migration by antagonizing granulocyte colonystimulating factor (G-CSF) and chemokine receptor-mediated signals. Cutaneous bacterial infection in mice was exacerbated by IL-4 signaling and improved with IL-4 inhibition, each outcome inversely correlating with neutrophil migration to skin. Likewise, systemic bacterial infection was worsened by heightened IL-4 activity, with IL-4 restricting G-CSF-induced neutrophil expansion and migration to tissues by affecting CXCR2-CXCR4 chemokine signaling in neutrophils. These effects were dependent on IL-4 acting through type 2 IL-4 receptors on neutrophils. Thus, targeting IL-4 might be beneficial in neutropenic conditions with increased susceptibility to bacterial infections.

INTRODUCTION The canonical response of the immune system to tissue damage and invasion by pathogens involves the activation of tissue-resident stromal and immune cells, as well as the fast mobilization of neutrophil granulocytes (neutrophils) from the bone marrow (BM) to the periphery (Borregaard, 2010; Kolaczkowska and Kubes, 2013; Nathan, 2006). In fact, neutrophils are the first innate immune cells to migrate to the site of action and rapidly exert several effector functions, including the secretion of cytokines and chemokines, thereby guiding and attracting additional innate and adaptive immune cells (Galli et al., 2011; Lim et al., 172 Immunity 45, 172–184, July 19, 2016 ª 2016 Elsevier Inc.

2015; Mantovani et al., 2011). The cunning efficacy of neutrophil recruitment hinges on a multistep process, including vigorous de novo generation, proliferation, and maturation of neutrophil precursors in the BM, followed by their release as mature neutrophils to the bloodstream. Once neutrophils are in the circulation, they patrol the body in search of molecular cues of tissue inflammation, which induce neutrophils to leave the blood vessel and infiltrate the affected tissue (Sadik et al., 2011). All these steps of neutrophil mobilization are driven by cytokines and chemokines, most notably granulocyte colony-stimulating factor (G-CSF) and the C-X-C chemokine receptor 2 (CXCR2)-binding chemokines CXCL1 and CXCL2, also termed keratinocyte chemoattractant (KC) and macrophage inflammatory protein-2 (MIP-2), respectively. G-CSF is synthesized by stromal and immune cells upon acute inflammation and infection and leads to expansion and mobilization of myeloid cells in the BM, most prominently neutrophils (Galli et al., 2011; Knudsen et al., 2011; Mantovani et al., 2011; Metcalf, 2008). Although G-CSF directly stimulates neutrophil precursors to proliferate, G-CSF’s influence on neutrophil mobilization relies on CXCR2 and CXCR4 (Eash et al., 2010; Ko¨hler et al., 2011). Notably, neutrophil release from the BM is controlled by signals from CXCR4 and its ligand CXCL12, which cause neutrophils to remain in their BM niche, whereas CXCR2 and its ligands CXCL1 and CXCL2 mediate neutrophil egress from the BM (Eash et al., 2010; Sadik et al., 2011). G-CSF signals tip this balance toward egress by favoring signals via CXCR2 over CXCR4. Thus, provision of recombinant G-CSF to humans or mice leads—via the above-mentioned effects—to prominent blood neutrophilia and subsequent neutrophil migration to tissues whereby neutrophils follow CXCR2-binding chemokines and ultimately so-called end-target chemoattractants (Kolaczkowska and Kubes, 2013; Metcalf, 2008). Strikingly, type 2 cell-mediated inflammation appears to be an exception to the above-mentioned pattern. Type 2 cell immune responses are characterized by the cytokines interleukin-4 (IL-4), IL-5, IL-9, IL-13, IL-25, IL-33, and thymic stromal lymphopoietin, which in turn are produced by, stimulate, and recruit type 2 immune cells, such as T helper 2 cells as well as different innate immune cells, including type 2 innate lymphoid cells, eosinophils, basophils, mast cells, and IL-4- and/or IL-13-activated

macrophages (Akdis, 2012; Allen and Maizels, 2011; Galli et al., 2008; Pulendran and Artis, 2012; Wynn, 2015). However, although these innate immune cells predominate during type 2 cell immune responses, neutrophils are conspicuously absent in type 2 cell inflammation (De Benedetto et al., 2009). Moreover, chronic type 2 cell-driven inflammatory disorders, such as atopic dermatitis, are often associated with recurrent bacterial infections that are usually contained by neutrophils (De Benedetto et al., 2009). Notably, marked neutropenia in target organs is a common finding in many allergic diseases. Thus, patients suffering from atopic dermatitis contain normal counts of blood neutrophils but show a paucity of neutrophils in skin lesions, even though their skin is colonized with bacteria known to induce neutrophil recruitment (Boguniewicz and Leung, 2011; De Benedetto et al., 2009; Miller and Cho, 2011). Not surprisingly, the skin of atopic dermatitis patients is characterized by a dominant type 2 cell immune signature with IL-4 being prominently expressed (Boguniewicz and Leung, 2011). Given that IL-4 is instrumental in initiating, polarizing, and maintaining type 2 immunity (Paul and Zhu, 2010; Wynn, 2015), these findings beg the question of whether IL-4 signals directly affect neutrophil expansion, migration, or function. In this study, we show that IL-4 receptor (IL-4R) stimulation on neutrophils reduces their expansion and egress from BM and dampens their recruitment to peripheral tissues. Together, these data demonstrate that type 2 IL-4R signaling can directly impair neutrophil recruitment during infection and inflammation. RESULTS Neutrophil Influx during Bacterial Skin Infection Is Modulated by IL-4 Signals As mentioned above, the skin of patients with atopic dermatitis usually expresses higher IL-4 concentrations, shows some degree of neutropenia, and is the target of recurrent bacterial infections (Boguniewicz and Leung, 2011; De Benedetto et al., 2009). In order to assess whether a similar correlation could be observed in mice, we took advantage of the well-established murine skin infection model using Group A Streptococcus (GAS) (Nizet et al., 2001; Zinkernagel et al., 2008). As previously published, subcutaneous inoculation of GAS led to a large skin lesion within 48 hr, with further progression after 72 hr (Figure 1A). This was accompanied by a prominent inflammatory swelling of the infected leg (Figure 1B), which was paralleled by a significant influx of CD11b+Ly6G+ cells (Figure 1C), with Ly6G being specific for neutrophils (Daley et al., 2008). Of note, increasing systemic IL-4 concentrations exacerbated the skin lesion. Thus, provision of recombinant mouse IL-4 in the form of IL-4-anti-IL-4 monoclonal antibody (mAb) complexes (IL-4cx) in order to prolong IL-4’s in vivo biological half-life (Boyman et al., 2006; Finkelman et al., 1993) increased skin lesion size to 165% of PBS at 48 hr (Figure 1A) and caused enhanced leg swelling at 24 hr throughout 72 hr after infection (Figure 1B). Despite this prominent inflammation, animals receiving IL-4cx demonstrated an attenuated neutrophil response to GAS skin infection by showing lower neutrophil influx into the skin (Figures 1C and 1D). Conversely, bacterial loads of GAS were 4–5 times higher in the skin of mice receiving IL-4cx (Figure 1E).

Consistent with the effects of elevating IL-4 concentrations, decreasing systemic IL-4 by the use of a neutralizing anti-IL-4 mAb reduced cutaneous bacterial loads and skin lesion size by half at 72 hr after infection (Figures 1A and 1E) and decreased leg swelling from as early as 6 hr after infection (Figure 1B), whereas influx of CD11b+Ly6G+ neutrophils into the skin was almost doubled upon IL-4 neutralization (Figures 1C and 1D). Altogether, these results indicate that neutrophil skin infiltration upon bacterial cutanenous infection is affected by systemic IL-4 concentration. IL-4 Suppresses Blood Neutrophilia after Systemic Infection Similar to skin infection by GAS, which also led to systemic effects (Figure S1), intravenous injection of Listeria monocytogenes (Lm) at a high dose of 105 colony-forming units (CFU) led to systemic infection causing prominent blood neutrophilia (Figures 2A and 2B). Lm infection-induced blood neutrophilia was dependent on G-CSF secretion. Thus, Lm infection caused a significant surge in G-CSF concentration, as measured using serum of Lm-infected mice to stimulate a G-CSF-sensitive cell line, the proliferation of which was abrogated by using a neutralizing anti-G-CSF mAb in vivo or in vitro (Figure S2). Moreover, administration of a neutralizing anti-G-CSF mAb prior and during Lm infection of mice significantly reduced blood and spleen neutrophilia to almost initial counts (Figures 2A and 2B). Pretreatment of Lm-infected mice with IL-4cx prevented blood neutrophilia and led to blood neutrophil frequencies as seen in uninfected mice (Figures 2C and 2D). We observed similar findings in the blood of GAS-infected animals (Figure S1). These results suggest that IL-4 signals oppose G-CSF action on neutrophils, although a role of other cytokines, including granulocyte macrophage colony-stimulating factor, cannot be excluded. IL-4 Antagonizes G-CSF Effects on Neutrophils To determine whether IL-4 mediated its effects on neutrophils during GAS and Lm infection by affecting G-CSF, or rather other factors produced upon infection, we induced ‘‘sterile’’ neutrophilia by injection of recombinant mouse G-CSF. As for most other soluble cytokines, G-CSF has a short in vivo half-life, unless it is coupled to polyethylene glycol or a particular anti-GCSF mAb, thus forming G-CSFcx with extended biological half-life (Rubinstein et al., 2013), similar to IL-4cx. Administration of G-CSFcx caused a prominent increase in CD11b+Ly6G+ neutrophils in BM, blood, and spleen (Figures 3A and 3B), as expected from the literature (Galli et al., 2011; Knudsen et al., 2011; Metcalf, 2008; Sadik et al., 2011). Notably, G-CSFcxmediated neutrophilia was abrogated by concomitant injection of IL-4cx (Figures 3A and 3B). Moreover, administration of IL4cx alone led to a slight decrease of neutrophil percentages and counts in the assessed compartments (Figures 3A and 3B). Similar results were obtained using unmodified cytokines, i.e., G-CSF and IL-4 not complexed with anti-G-CSF and antiIL-4 mAbs, respectively, although these unmodified cytokines had to be injected at higher doses to account for the cytokines’ short in vivo half-life (Figure 3C). We next assessed whether IL-4 was able to antagonize G-CSF during systemic infection. To this end, mice received a lethal dose of 105 CFU Lm intravenously, leading to high bacterial titers Immunity 45, 172–184, July 19, 2016 173

Figure 1. Neutrophil Influx during Skin Infection Is Modulated by IL-4 Signals 3 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 mm. (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.

in the spleen already 24 hr later and in the liver at 72 hr after infection (Figure 3D). This was accompanied by rapid weight loss and death of animals around day 4 after infection (Figures 3E and 3F). Notably, injection of G-CSFcx rapidly and significantly lowered bacterial loads in spleen and liver of infected mice (Figure 3D), reversed the weight loss, and prevented death of animals (Figures 3E and 3F). However, co-administration of IL-4cx completely abrogated the beneficial effects of G-CSFcx, and these mice succumbed to lethal Lm infection with a similar kinetic as phosphate-buffered saline (PBS)-treated control mice (Figures 3D–3F). Altogether, these data demonstrate that IL-4 signals dominantly suppress G-CSF-mediated neutrophil expansion in BM, blood, and spleen, during both homeostasis and systemic infection. IL-4 Acts Directly on Neutrophils via Type 2 IL-4R IL-4 exerts its pleiotropic effects by binding to two types of IL-4Rs, both of which subsequently lead to phosphorylation of signal transducer and activator of transcription 6 (STAT6). Heter174 Immunity 45, 172–184, July 19, 2016

odimerization of IL-4Ra (also termed CD124) with the common g-chain (gc, also known as CD132) forms the type 1 IL-4R, which is expressed typically by hematopoietic cells, including B and T cells (LaPorte et al., 2008; Nelms et al., 1999). Conversely, type 2 IL-4Rs are thought to be predominant on non-hematopoietic cells as well as macrophages and consist of heterodimers of IL-4Ra and IL-13Ra1. Notably, also IL-13 can signal via type 2 IL-4Rs by binding to IL-13Ra1 and subsequently IL-4Ra (LaPorte et al., 2008). CD11b+Ly6G+ neutrophils isolated from the BM of control mice showed low expression of IL-4Ra and background expression of gc and IL-13Ra1 (Figure 4A). Upon administration of G-CSFcx to mice, IL-4Ra significantly increased by 8.5-fold along with a marked 3.8-fold increase in expression of IL13Ra1, both as measured by mean fluorescence intensity (MFI), whereas gc remained unchanged (Figure 4A). The addition of IL-4cx to G-CSFcx treatment of mice did not alter the effects of G-CSFcx on upregulation of these receptor subunits (Figure 4A). In comparison to BM neutrophils, their splenic

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.

counterparts showed similar expression of IL-4Ra and gc during steady state, whereas IL-13Ra1 expression was more prominent already on resting neutrophils from spleen compared to BM (Figure 4B). Injection of G-CSFcx to animals led to the appearance of IL-4RahiCD11b+Ly6G+ neutrophils in the spleen and further increased IL-13Ra1 on splenic neutrophils, whereas a change in gc was not evident (Figure 4B). As with BM neutrophils, coadministration of G-CSFcx with IL-4cx did not lead to decreased expression of these receptor subunits but, if anything, addition of IL-4cx slightly enhanced IL-4Ra and IL-13Ra1 expression on

splenic neutrophils (Figure 4B). These results suggest that G-CSF signals, as occurring during inflammation and infection, dynamically upregulate type 2 IL-4Rs on BM and spleen neutrophils. Further evidence for the involvement of the type 2 IL-4R came from studies of STAT6 phosphorylation in mature neutrophils from spleen. Therefore, we stimulated splenocytes of wild-type (WT), IL-4Ra-deficient (Il4ra–/–), gc-deficient (Il2rg–/–), and IL13Ra1-deficient (Il13ra1–/–) mice for 15 min (corresponding to the time point of maximum STAT6 phosphorylation; Figure S3) with IL-4 to assess phospho-STAT6 expression. Phosphorylation of STAT6 in CD11b+Ly6G+ neutrophils occurred efficiently in WT and Il2rg–/– neutrophils, whereas Il4ra–/– and Il13ra1–/– neutrophils failed to phosphorylate STAT6 (Figure 4C). Although IL-13 is also able to bind the type 2 IL-4R, 10- to 100-fold higher doses of IL-13 were necessary to reach phospho-STAT6 expression in neutrophils seen with 5 ng/mL IL-4 (Figure 4D). The above-mentioned data demonstrate that functional type 2 IL-4Rs consisting of IL-4Ra and IL-13Ra1 are prominently expressed on BM and splenic neutrophils upon G-CSF signals, whereas the expression of type 1 IL-4Rs on neutrophils is negligible in this context. This suggests that IL-4 might—by triggering type 2 IL-4Rs directly, rather than indirectly via lymphocytes— act on neutrophils during inflammation and infection. Along these lines, IL-4-mediated inhibition of G-CSFcx-induced neutrophilia was independent of B and T cells, as demonstrated by using animals deficient in the recombination-activating gene 1 (Rag1) (Figure S4). Moreover, although the vigorous expansion of CD11b+Ly6G+ neutrophils in blood and spleen by treatment with G-CSFcx was antagonized by co-injection of IL-4cx in WT mice (Figures 4E, 4F, and 3), use of Il4ra–/– mice completely abrogated the effects of IL-4cx (Figure 4E). Conversely, IL-4cx were able to fully counteract the effects of G-CSFcx even in Il2rg–/– mice, similar to the extent seen in WT (Figure 4F). To obtain further evidence of direct IL-4R signaling on neutrophils in an IL-4R-proficient environment, we generated BM chimeras using a 1:1 mix of Ly5.2 (CD45.2)-congenic Il4ra–/– and Ly5.1 (CD45.1)-congenic WT BM adoptively transferred to lethally irradiated Il4ra–/– mice (Figure 4G). Upon reconstitution at week 3, BM-chimeric mice were treated using PBS, G-CSFcx, or G-CSFcx plus IL-4cx. Whereas the ratio of Ly5.2 Il4ra–/– to Ly5.1 WT CD11b+Ly6G+ neutrophils in blood was 1.25 in PBStreated animals and changed only minimally in mice receiving G-CSFcx, co-administration of G-CSFcx plus IL-4cx caused a marked change and led to a ratio of Ly5.2 Il4ra–/– to Ly5.1 WT neutrophils of 3 (Figure 4G), showing that IL-4 acted directly on neutrophils to inhibit their expansion. To explore the functional consequence of IL-4Ra deficiency during Lm infection, we intravenously injected 105 CFU Lm to mice, which was lethal for WT, whereas Il4ra–/– survived this dose of infection (Figure 4H). Altogether, these data demonstrate that IL-4 directly acts on neutrophils via type 2 IL-4Rs to induce STAT6 signaling and inhibit G-CSF-mediated neutrophil expansion during inflammation and infection. IL-4 Induces a BM-Resident, Non-migratory Phenotype of Neutrophils Our results so far suggest that, in the presence of cell-intrinsic type 2 IL-4R-mediated signals, neutrophil accumulation in the Immunity 45, 172–184, July 19, 2016 175

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. (legend continued on next page)

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blood, spleen, and peripheral sites such as the skin was reduced. As mentioned previously, neutrophil migration is controlled by the chemokine receptors CXCR2 and CXCR4, with CXCR2 mediating BM egress and migration to inflamed sites, whereas CXCR4 serves to keep neutrophils in the BM (Eash et al., 2010; Sadik et al., 2011). Surface staining for CXCR2 and CXCR4 on BM neutrophils revealed that injection of G-CSFcx to mice nearly doubled expression of CXCR2, whereas CXCR4 decreased to 28% of PBS (Figures 5A and 5B), thus favoring BM egress of neutrophils. In splenic neutrophils, G-CSFcx treatment did not significantly affect either of these two chemokine receptors (Figures 5C and 5D). Conversely, administration of IL-4cx increased CXCR4 in BM neutrophils, with CXCR2 remaining low or slightly decreasing (Figures 5A and 5B), thus promoting BM retention of neutrophils. Moreover, in splenic neutrophils, IL-4cx significantly reduced CXCR2 expression to 8% of PBS (Figures 5C and 5D), which disfavors migration of neutrophils toward the CXCR2-binding chemokines CXCL1 and CXCL2. IL-4 Inhibits CXCR2-Mediated Migration In Vitro and In Vivo We next investigated the effects of IL-4 during neutrophil migration by purifying CD11b+Ly6G+ BM neutrophils, followed by assaying their migration toward CXCL1 and CXCL2 in vitro. In the presence of IL-4, a significant decrease of neutrophil migration toward these chemokines was evident, which could not be overcome by shorter or longer times of migration or by increasing the dose of the chemokine (Figures 6A and 6B and data not shown). IL-4 decreased migration toward these chemokines in a dose-dependent manner (Figures 6C and 6D). In contrast to IL-4, other gc or T helper 2 cell-type cytokines, including IL-2, IL-7, IL-13, and IL-15, did not reduce CXCR2induced neutrophil migration (Figure 6E). Consistent with our IL-4R expression data (Figure 4), the inhibitory effect of IL-4 on neutrophil migration was abrogated in neutrophils from Il4ra–/– animals (Figure 6F). To assess the effects of IL-4 in a model of neutrophil migration in vivo, we used the well-established airpouch model (Perretti et al., 1995; Ryckman et al., 2003). Neutrophil migration to an airpouch containing monosodium urate crystals (MSU) was crucially dependent on CXCR2. Thus, in contrast to their WT counterparts, Cxcr2–/– neutrophils were unable to migrate and accumulate in the MSU airpouch, although Cxcr2–/– mice contained above-normal percentages of blood neutrophils compared to WT animals (Figure 6G). Having established that neutrophil migration to an MSU-containing airpouch relied on CXCR2, we treated animals with either PBS or IL-4cx, prior to assessing neutrophil migration to MSU or IL-1b (Perretti et al., 1995). In both experimental conditions, increased systemic IL-4 concentration significantly inhibited the accumulation of neutrophils in the airpouch (Figure 6H). Hence, IL-4 signals potently dampen migration of neutrophils toward CXCR2-binding chemokines.

IL-4 Signaling Interferes with p38 MAPK-PI3K Crosstalk Neutrophil chemotaxis is intracellularly regulated by two antagonistic signaling pathways, involving phosphoinositide 3-kinase (PI3K) and p38 mitogen-activated protein kinase (MAPK). CXCR2 binding activates PI3K to cause migration of neutrophils. Conversely, once neutrophils arrive to a site of inflammation and infection, so-called end-target chemoattractants, such as N-formyl-Met-Leu-Phe (fMLP) from bacteria or complement factor C5a, stimulate the p38 MAPK pathway, which overrides PI3Kmediated signals (Heit et al., 2002, 2008; Sadik et al., 2011). This in turn allows neutrophils to be guided away from CXCL1 and CXCL2 gradients toward end-target chemoattractants. Consistent with this notion, PI3K was crucial for in vitro chemotaxis of purified CD11b+Ly6G+ neutrophils toward CXCR2-binding chemokines, as shown by using PI3K inhibitor LY294002 (Figure 7A). In contrast, addition of the selective p38ab inhibitor SB203580 (Kuma et al., 2005) did not hamper neutrophil migration toward CXCR2-binding chemokines (Figure 7A). IL-4 signaling has previously been shown to activate p38 MAPK in human polymorphonuclear cells, including neutrophils (Ratthe´ et al., 2007). We therefore hypothesized that the inhibitory effect of IL-4 on neutrophil migration might rely on p38 MAPK-mediated interference with PI3K signaling. Stimulation of purified CD11b+Ly6G+ neutrophils with IL-4 in vitro caused phosphorylation of p38 MAPK within 5 min, which further increased after 15 min (Figure 7B). Concomitant use of the selective p38ab inhibitor SB203580 completely abrogated the appearance of phospho-p38 MAPK in neutrophils (Figure 7C). This indicates that IL-4 induces phosphorylation of the p38a MAPK family member in neutrophils, because only p38a and p38d have been found in neutrophils, whereas p38b was absent in these cells (Hale et al., 1999). In line with these findings, neutrophil migration in vitro toward CXCL1 was dampened by IL-4, and IL-4-mediated inhibition was abrogated by addition of p38ab inhibitor SB203580 (Figure 7D). Conversely, PI3K inhibitor LY294002 did not affect IL-4-induced inhibition of neutrophil migration (Figure 7D). Injection of IL-4 to mice also caused p38a MAPK phosphorylation of neutrophils 15 min after injection, which was inhibited by co-administration of p38ab inhibitor SB203580 (Figure 7E). Treatment of mice with G-CSFcx induced blood neutrophilia, which was antagonized by co-injection of IL-4cx (Figures 7F and 7G), as also shown in Figure 3. Notably, simultaneous administration of SB203580 was able to block IL-4’s inhibitory effect on neutrophils, thus allowing neutrophils to accumulate in the blood in response to G-CSFcx (Figures 7F and 7G). These data demonstrate that IL-4 signaling in neutrophils activates p38a MAPK, thereby overriding G-CSF-CXCR2-PI3K-mediated signals and inhibiting neutrophil recruitment and migration. DISCUSSION In this study, we have shown that IL-4 inhibited neutrophil recruitment and migration during bacterial infection and

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.

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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-4Ra, common g-chain (gc), and IL-13Ra1 expression on CD11b+Ly6G+ neutrophils isolated from BM (A) or spleen (B). (C) Stimulation of splenocytes from WT, IL-4Ra-deficient (Il4ra–/–), gc-deficient (Il2rg–/–), and IL-13Ra1-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. (legend continued on next page)

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WT mice were treated with PBS, G-CSFcx, or IL4cx 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.

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inflammation via directly interacting with type 2 IL-4Rs on neutrophils. Thus, increased G-CSF production upon local and systemic bacterial infection or ‘‘sterile’’ inflammation as mimicked by injection of long-lasting G-CSF usually expands BM neutrophils. Moreover, G-CSF is known to induce neutrophil egress from the BM by weakening CXCR4-mediated retention signals and augmenting the neutrophils’ sensitivity toward CXCR2-binding chemokines. As shown here, G-CSF signals also increased the expression of type 2 IL-4Rs, made of IL-4Ra and IL-13Ra1, rendering neutrophils more sensitive to IL-4. Accordingly, increasing IL-4 concentration, either physiologically during infection and inflammation or by administration of IL-4, directly bound to and activated in neutrophils STAT6 and p38 MAPK and led to upregulation of CXCR4 as well as downregulation of CXCR2. Hence, IL-4 signaling in neutrophils resulted in decreased G-CSF-mediated expansion and increased retention of neutrophils in the BM. Moreover, in addition to decreasing CXCR2 expression, IL-4 signals also inhibited neutrophil migration by opposing CXCR2-PI3K-mediated signals via the activation of the PI3K antagonist p38 MAPK. Thus, type 2 IL-4R-mediated signaling in neutrophils efficiently interferes with several central and peripheral mechanisms of neutrophil recruitment. In placing our data in the context of previous findings, the literature on the effects of IL-4 on neutrophils is rather controversial.

In freshly isolated human neutrophils in vitro, IL-4 prolongs survival; increases 0 10 3 10 4 10 5 phagocytosis and killing; facilitates lysoCXCR2 zyme production, b-glucuronidase secretion, and fMLP-mediated respiratory burst; and enhances migration toward zymosan-activated serum and IL-5 (Bober et al., 1995; Boey et al., 1989; Ratthe´ et al., 2007). The results pertaining to fMLP are actually in line with our proposed model and could possibly be explained by the finding that fMLP (and other end-target chemoattractants) activate p38 MAPK (Heit et al., 2002, 2008); thus, IL-4 could synergize with fMLP to stimulate p38 MAPK in neutrophils in this context. As for survival, in our hands, IL-4 did not alter the viability of freshly isolated murine BM neutrophils (data not shown). Conversely, another study shows that upon stimulation with interferon-g (IFN-g) or tumor necrosis factor-a, in vitro phagocytic activity of human neutrophils is inhibited by IL-4 (Bober et al., 2000). In light of our data, these findings raise the following questions. Do resting and cytokine-activated human and mouse neutrophils express different types of IL-4Rs? And do type 1 versus type 2 IL-4Rs on neutrophils mediate different effects? Notably, one of the studies shows that resting human neutrophils express type 1 IL-4Rs, but lack IL-13Ra1 (Ratthe´ et al., 2007). Our findings in mice demonstrate that IL-13Ra1 is found on BM and particularly splenic neutrophils and its expression is further enhanced by G-CSF, which also upregulates IL-4Ra on neutrophils. The above-mentioned data refer to the in vitro use of IL-4 on human neutrophils. The effects of IL-4 on neutrophils in vivo reported in the literature are difficult to interpret in terms of direct and indirect actions. In an a-galactosylceramide-induced mouse

(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.

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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-1b 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.

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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.

hepatitis model, early IL-4 production by natural killer T cells increases neutrophil infiltrates and inflammation in the liver, whereas hepatic neutrophil counts are curtailed by later IFN-g secretion by natural killer T cells (Wang et al., 2013), indicating that IL-4 signals enhanced neutrophil accumulation in the liver. Conversely, other findings suggest that IL-4 signals dampen neutrophil or myeloid cell recruitment, which is in line with our herein-described mechanism. Thus, in mice infected with Schistosoma japonicum, neutrophil recruitment to the liver is increased in animals lacking both IL-4 and IL-13 (Seki et al., 2012). Also, in animals sensitized with house dust mite extract, inhibition of IL-4 by using a neutralizing anti-IL-4 mAb leads to

increased neutrophil counts in the lungs (Choy et al., 2015). Moreover, intravenous injection of IL-4 is able to hamper IL1b-mediated recruitment of neutrophils to an airpouch, although the mechanism by which IL-4 exerted its effect has been unclear and IL-4 is unable to inhibit CXCL8-mediated neutrophil chemotaxis in this publication (Perretti et al., 1995). Furthermore, systemic administration of IL-4 improves joint inflammation in three different arthritis models, including the rat adjuvant arthritis model, collagen-induced arthritis model in mice, and K/BxNmediated joint inflammation in mice (Bober et al., 2000; Hemmerle et al., 2014; Wermeling et al., 2013), as well as curtailed delayed-type pleuritis in mice (Fine et al., 2003). However, Immunity 45, 172–184, July 19, 2016 181

because some of these models rely on a late readout between days 17 and 22 and a sensitization and challenge procedure similar to delayed-type hypersensitivity, it is conceivable that in the reported models, IL-4 mediates its effects via dampening T helper 1 cell responses (Powrie et al., 1993). Hence, the previously reported in vivo findings show a rather heterogeneous picture of the effects of IL-4 on neutrophils and leave the question unanswered as to whether IL-4 affected neutrophils directly or via its action on other immune cells. Of note, our results indicate that type 2 IL-4R signaling antagonizes some of the actions of G-CSF on BM neutrophils, particularly those effects of G-CSF pertaining to expansion and egress of BM neutrophils. Previous reports showed that G-CSF exerted both STAT3-dependent and STAT3-independent effects in neutrophils and also stimulated suppressor of cytokine signaling 3 (SOCS3), which in turn acts as an important negative feedback regulator of G-CSF receptor signaling by inhibiting STAT3 (Carow and Rottenberg, 2014; Nguyen-Jackson et al., 2010). Thus it is conceivable that type 2 IL-4R signaling might interfere directly, or indirectly via SOCS3, with one of the pathways downstream of the G-CSF receptor. The therapeutic implications of the herein described type 2 IL4R-p38 MAPK-CXCR2 axis are manifold. As mentioned previously, individuals suffering from allergic disorders show a relative paucity of neutrophils in the affected organs. For example, the skin of patients with atopic dermatitis contains more IL-4 and lower counts of neutrophils and is more susceptible to bacterial infections that are usually contained by neutrophils. Thus, therapeutic approaches targeting IL-4Ra, IL-13Ra1, or p38 MAPK might lower the risk of recurrent bacterial infections. In line with this suggestion, a recent clinical trial using dupilumab, a mAb targeting human IL-4Ra, in atopic dermatitis suggested that patients receiving dupilumab had fewer skin infections compared to placebo (Beck et al., 2014). Likewise, patients undergoing myeloablative chemotherapy go through a phase of critical neutropenia and severely increased risk of systemic infections. Currently, long-lasting G-CSF formulations, such as PEGylated G-CSF, are given to patients in order to stimulate production and recruitment of neutrophils (Bennett et al., 2013; Metcalf, 2008; Ratti and Tomasello, 2015). Based on our data, concomitant inhibition of type 2 IL-4R-p38 MAPK signaling might be considered for severe cases. IL-4 is well known to directly affect many immune cells, including macrophages, dendritic cells, B cells, and T cells, thereby driving type 2 cell-mediated immunity (Jenkins et al., 2013; Nelms et al., 1999; Paul and Zhu, 2010). By showing a direct action of IL-4 on neutrophils via type 2 IL-4Rs we extend the range of target cells of IL-4 and also demonstrate that type 2 IL-4Rs can become very prominent on immune cells and potently affect their responses during immunity and immunopathology.

Infections and In Vivo Treatments Mice were infected either systemically with 105 CFU Listeria monocytogenes (Lm) or subcutaneously with 3 3 107 CFU Group A Streptococcus (GAS) M1 as previously described (Nizet et al., 2001; Zinkernagel et al., 2008). Where indicated, animals received on 3 consecutive days daily injections of PBS, free cytokines (5 mg human G-CSF [Neupogen]; 7.5 mg mouse IL-4 [mIL-4, eBioscience]), or cytokine-anti-cytokine monoclonal antibody (mAb) complexes (1 mg human G-CSF complexed with 6 mg anti-human G-CSF mAb clone BVD11-37G10 [SouthernBiotech]; 1.5 mg mIL-4 complexed with 7.5 mg anti-mIL-4 mAb clone 11B11 [BioXcell]) prior to infection, as previously published (Boyman et al., 2006; Finkelman et al., 1993; Rubinstein et al., 2013). Also, where indicated, mice received daily intraperitoneal injections of 100 mg neutralizing mAb against mG-CSF (MAB414; R&D) or mIL-4 (11B11; BioXcell). p38 MAPK activity was blocked in vivo by administering mice three injections of 300 mg of SB203580 (Calbiochem) (Heit et al., 2002). Bacterial Load To determine Lm bacterial load, liver was flushed with cold PBS, and processed liver and spleen were incubated for 20 min with 0.05% Triton X-100 (Sigma). Serial dilutions were plated on brain heart infusion agar (Oxoid) plates and CFU were counted after 24 hr of incubation at 37 C. To determine GAS load, skin homogenates were centrifuged and serial dilutions of supernatants were plated on THY agar (Oxoid) plates. Immunohistochemistry Murine skin samples were embedded in O.C.T. Compound (Sakura) and stained with anti-Ly6G mAb (1A8; BioXcell). Sections were analyzed using ImageScope for image acquisition (Aperio Technologies). Flow Cytometry Single-cell suspensions of organs were prepared according to standard protocols and stained for analysis by flow cytometry, as previously published (Bouchaud et al., 2013), using fluorochrome-labeled mAbs directed against the following mouse antigens (from eBioscience, unless stated otherwise): Annexin V (BD Biosciences), CD3 (145-2C11; BD Biosciences), CD4 (GK1.5), CD11b (M1/70), CD45.1 (A20), CD45.2 (104), Ly6G (1A8), CD132 (TUGm2; BioLegend), CXCR2 (TAB2164P; R&D), CXCR4 (2B11; BD Biosciences), IL-4Ra (mIL4R-M1; BD Biosciences), IL-13Ra1 (13MOKA), phospho-STAT6 (pY641; BD Biosciences), and phospho-p38 (pT180, pY182; BD Biosciences). For in vitro phosphostainings, splenocytes were stimulated with cytokines for 15 min and subsequently fixed by addition of paraformaldehyde and ice-cold methanol. Skin was cut into small pieces and incubated for 1 hr at 37 C with an enzymatic cocktail consisting of 5 mg/mL Liberase (Roche), 1 mg/mL DNAase I (Sigma), and 5 mg/mL Dispase II (Roche) in RPMI media. Subsequently, cells were liberated by extensive pipetting and filtered. Cells were acquired on a BD FACSCanto II or BD LSR Fortessa flow cytometer and analyzed using FlowJo software (Tristar). Bone Marrow Chimeras Immune lineage-negative (Lin–) bone marrow (BM) cells of WT CD45.1-congenic and Il4ra–/– CD45.2-congenic mice were purified by negative selection using magnetic beads (StemCell Technologies) and biotinylated mAbs against CD19, CD3, MHC class II, NK1.1, and Ter119. Lin– BM cells from WT and Il4ra–/– mice were mixed at a 1:1 ratio and injected intravenously into irradiated (950 rad) Il4ra–/– CD45.2-congenic host mice. BM chimeric mice received 1 mg/mL sulfamethoxazol and 0.2 mg/mL trimethoprim in their drinking water for 2 weeks and were left for 3 weeks in order to allow for reconstitution of neutrophils before use.

EXPERIMENTAL PROCEDURES Animals C57BL/6, CD45.1 (Ly5.1)-congenic, Cxcr2–/–, Il2rg–/–, Rag1–/– (all on a C57BL/6 background), and BALB/c and Il4ra–/– (on a BALB/c background) were purchased from The Jackson Laboratory. Il13ra1–/– (on a BALB/c background) were provided by Regeneron Pharmaceuticals (Ramalingam et al., 2008). Experiments were approved by the Cantonal Veterinary Office and performed in accordance with the Swiss law.

182 Immunity 45, 172–184, July 19, 2016

Neutrophil Migration Assay CD3–CD11b+Ly6G+ neutrophils were obtained by positive selection using Ly6G microbeads (Miltenyi Biotec), yielding a purity of 92%–95%. 105 purified neutrophils were pretreated for 20 min with PBS or cytokines (30 ng/mL), including IL-2, IL-4, IL-7, IL-13, or IL-15 (from eBioscience and Peprotech), before seeding into the upper chamber of a 5 mm-transwell (Corning Costar). Subsequently, migration of neutrophils was determined for 2 hr toward CXCL1 or CXCL2 (both 100 ng/mL; PeproTech) given to the lower chamber.

Where indicated, SB203580 or Ly294002 (both 30 mmol; Calbiochem) was added 15 min before pretreatment. Airpouch Model An airpouch was generated in the back of mice by subcutaneous injection of 4 mL sterile air on days 0 and 3, as previously described (Perretti et al., 1995; Ryckman et al., 2003). On day 6, we administered intravenously PBS or IL-4cx, followed by injection into the airpouch of either 1 mg monosodium urate crystals (MSU) or 10 ng IL-1b (Peprotech) in 1 mL sterile PBS. Mice were left overnight and subsequently euthanized, followed by flushing of the airpouch with PBS plus 2 mM EDTA to collect cells within. The content of the airpouch was counted and analyzed by flow cytometry for leukocytes. Statistical Analysis Differences between groups were examined for statistical significance by using unpaired Student’s t test or one-way or two-way analysis of variance (ANOVA) with Bonferroni’s post-test correction. SUPPLEMENTAL INFORMATION Supplemental Information includes four figures and can be found with this article online at http://dx.doi.org/10.1016/j.immuni.2016.06.025. AUTHOR CONTRIBUTIONS J.W. designed, performed, and analyzed most experiments and wrote the manuscript; N.K. and A.S.Z. designed, performed, and analyzed experiments in the cutaneous infection model; C.K. gave scientific input; D.I. performed experiments on neutrophil phenotype; R.W.T. and T.A.W. gave scientific input and assisted in setting up experiments with Il13ra1–/– mice; and O.B. designed and analyzed experiments, supervised the study, and wrote the manuscript, with input from all authors. ACKNOWLEDGMENTS We thank Daniel Legler and the members of the O.B. laboratory for helpful discussions and critical reading of the manuscript. We thank Emerita Ammann Meier for excellent technical assistance and Sandra Seyfferth for help with typing the manuscript. This study was funded by Swiss National Science Foundation grants PP00P3-128421, PP00P3-150751 (both to O.B.), and 310030-146295 (to A.S.Z.), a grant of University of Zurich (to O.B.), a Candoc fellowship of University of Zurich (to J.W.), and by Hochspezialisierte Medizin Schwerpunkt Immunologie (HSM-2-Immunologie; to O.B.). Received: November 20, 2015 Revised: March 29, 2016 Accepted: June 23, 2016 Published: July 19, 2016

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