IL4Rα Signaling Abrogates Hypoxic Neutrophil Survival and Limits Acute Lung Injury Responses In Vivo

Rationale: Acute respiratory distress syndrome is defined by the presence of systemic hypoxia and consequent on disordered neutrophilic inflammation. Local mechanisms limiting the duration and magnitude of this neutrophilic response remain poorly understood. Objectives: To test the hypothesis that during acute lung inflammation tissue production of proresolution type 2 cytokines (IL-4 and IL-13) dampens the proinflammatory effects of hypoxia through suppression of HIF-1α (hypoxia-inducible factor-1α)-mediated neutrophil adaptation, resulting in resolution of lung injury. Methods: Neutrophil activation of IL4Ra (IL-4 receptor α) signaling pathways was explored ex vivo in human acute respiratory distress syndrome patient samples, in vitro after the culture of human peripheral blood neutrophils with recombinant IL-4 under conditions of hypoxia, and in vivo through the study of IL4Ra-deficient neutrophils in competitive chimera models and wild-type mice treated with IL-4. Measurements and Main Results: IL-4 was elevated in human BAL from patients with acute respiratory distress syndrome, and its receptor was identified on patient blood neutrophils. Treatment of human neutrophils with IL-4 suppressed HIF-1α–dependent hypoxic survival and limited proinflammatory transcriptional responses. Increased neutrophil apoptosis in hypoxia, also observed with IL-13, required active STAT signaling, and was dependent on expression of the oxygen-sensing prolyl hydroxylase PHD2. In vivo, IL-4Ra–deficient neutrophils had a survival advantage within a hypoxic inflamed niche; in contrast, inflamed lung treatment with IL-4 accelerated resolution through increased neutrophil apoptosis. Conclusions: We describe an important interaction whereby IL4Rα-dependent type 2 cytokine signaling can directly inhibit hypoxic neutrophil survival in tissues and promote resolution of neutrophil-mediated acute lung injury.

Acute respiratory distress syndrome (ARDS) is a clinical syndrome defined by the presence of bilateral pulmonary infiltrates and arterial hypoxemia. Although several causes are recognized including sepsis, trauma, and aspiration, ARDS is the consequence of a disordered inflammatory response in which accumulation of activated neutrophils within the lung interstitium and distal airspaces is associated with lung endothelial and epithelial injury. A fine balance therefore exists between an effective innate response that enables host pathogen control and a disordered one in which inappropriate neutrophil persistence and activation drives tissue injury. Production of local factors during the course of an inflammatory response together with changes in the physiologic environment, are crucial in regulating this balance.
Hypoxia frequently characterizes acute inflammatory sites (1) and is an important mediator of neutrophil survival and inflammatory function (2). In human disease states, hypoxia has been associated with elevated neutrophil numbers, reduced neutrophil apoptosis, and increased disease severity (3)(4)(5)(6). Thus, hypoxia can be regarded as a proinflammatory stimulus that facilitates the persistence of neutrophils at inflamed sites.
In vivo models have shown that enhanced neutrophil survival in hypoxia is dominantly mediated by stabilization of the transcription factor HIF-1a (hypoxiainducible factor-1a) (7)(8)(9). HIF-1a itself is regulated by a family of dioxygenases including the prolyl hydroxylase domain containing enzymes (PHD1-3) and FIH (factor inhibiting HIF) (10). Other studies have delineated oxygenindependent regulation of the HIF pathway, with bacterial activation of toll-like receptor signaling pathways leading to HIF-1a stabilization (11,12). The interaction between hypoxia and local factors in regulating HIF-1a signaling, and the consequences for inflammatory responses, are therefore of great interest. IL-4 has long been considered an important cytokine for tissue repair, counterbalancing the effects of proinflammatory type 1 cytokines on the macrophage compartment (13), but conversely for promoting allergic airway inflammation. Interestingly, mouse neutrophils express both IL4Ra and IL13Ra (14), the components of the type 2 IL-4 receptor complex, through which both IL-4 and IL-13 signal. IL4Ra is upregulated in myeloid cells after stimulation with proinflammatory mediators, including LPS, with neutrophils displaying the highest fold increase in receptor expression (15). Moreover, IL-4 is itself released by macrophages after LPS simulation during the acute response (16). With cell-specific consequences of IL-4 signaling, we questioned whether IL-4 could regulate neutrophilic inflammation during acute lung inflammation to promote inflammation resolution and restoration of tissue homeostasis. We provide evidence of activation of IL-4 pathways in the setting of acute lung injury (ALI) and demonstrate that IL-4 prevents neutrophil hypoxic HIF-1a induction, abrogates hypoxic survival of human and mouse neutrophils, dampens proinflammatory cytokine expression, and promotes resolution of neutrophilic inflammation in vivo.

At a Glance Commentary
Scientific Knowledge on the Subject: The role for type 2 cytokines, such as IL-4 and IL-13, in inflammation resolution has been extensively studied, but their precise effect on neutrophils in this context remains largely unknown. Neutrophils are key innate cells adapted to working under severe hypoxic conditions, an environment that drives their survival. Such conditions are found in the lungs of patients with acute respiratory distress syndrome.
What This Study Adds to the Field: In this article we identify a significant role for IL-4 and IL-13 as regulators of neutrophil hypoxic survival in both human and mouse cells using a combination of in vitro and in vivo studies. We demonstrate that IL-4 is present in the lungs of acute respiratory distress syndrome sufferers and in mouse models of lung injury. Furthermore, IL-4 abrogates neutrophil hypoxic survival in vitro in human and mouse neutrophils in a prolyl hydroxylase 2-dependent manner. Finally, through the careful use of animal models including chimera studies we demonstrate the therapeutic potential of manipulating this pathway in the lung during hypoxic neutrophilic lung inflammation.

Chimera Studies
To dissect the consequence of IL4Ra loss on bone marrow and circulating neutrophil populations in the context of hypoxia, we used an organ-protected chimeric approach in the liver (regional hypoxia) and a fractionated radiation strategy in the lung (systemic hypoxia). Inflammation outcomes were defined in a model of acute liver injury (CCl 4 ) and ALI (LPS) (see online supplement).

Statistical Analysis
For nonparametric data significance was determined by Mann-Whitney U test. Data shown as individual points with median and interquartile range values. For multiple comparisons with one variable we used one-way ANOVA with Kruskal-Wallis post hoc test and for two variables, two-way ANOVA with Holm-Sidak post hoc test analysis, and adjustment for multiplicity of tests. Data shown as mean 6 SEM. P less than 0.05 was considered significant. Individual data points represent individual mice or human samples.

IL-4 Receptor a Signaling Pathways
Are Present in the Airways of Patients with ARDS, Circulating Neutrophils, and in a Mouse Model of ARDS To determine whether patients with ARDS have evidence of local production of IL-4, we measured IL-4 levels in BAL samples of patients with ARDS compared with healthy control subjects. ARDS BAL fluid contained significantly more IL-4 than lavage samples obtained from healthy airspaces ( Figure 1A). Interestingly, preliminary data suggest that neutrophils from patients with ARDS may have higher levels of IL4Ra expression than healthy control subjects ( Figure 1B) supporting the relevance of this pathway to ARDS.
To confirm the ability of circulating neutrophils to respond to IL-4 in the context of physiologic hypoxia, we examined neutrophil expression of IL4Ra. Healthy human blood neutrophils demonstrated IL4Ra surface protein expression and transcript when cultured in normoxia (21% O 2 ) and hypoxia (1% O 2 ), with loss of surface protein and induction of mRNA after stimulation with IL-4 ( Figures 1C  and 1D).
To determine if the IL-4 pathway is activated in an ALI mouse model we examined IL-4 BAL levels from mice exposed to nebulized LPS. In keeping with the data from human ARDS BAL, IL-4 is produced in the airspace of mice exposed to LPS ( Figure 1E). We then determined if systemic hypoxia (10% FI O 2 ) altered the neutrophil inflammatory responses in vivo and found that in vivo lung neutrophils express the IL4Ra, more so in hypoxia than in normoxia, after LPS ( Figure 1F). In this setting alveolar macrophages also upregulate their IL4Ra expression in hypoxia after LPS stimulation ( Figure 1G) but T cells do not ( Figure 1H). Importantly, BAL inflammatory neutrophils cultured ex vivo in hypoxia with IL-4 had reduced hypoxic survival thus suggesting IL-4 can overcome the enhanced neutrophil survival observed in physiologic and pathophysiologic hypoxia ( Figure 1I).

IL-4 Abrogates Neutrophil Hypoxic Survival and Suppresses Proinflammatory Responses
We next sought to determine whether IL-4 modified human neutrophil apoptosis in vitro. In marked contrast to the prosurvival effects of IFN-g/LPS, IL-4 had no effect on constitutive apoptosis in normoxia, but inhibited hypoxic survival at both early (6 h) ( Figure 2A) and late (20 h) ( Figure 2B) time points, as determined by morphologic appearance (chromatin condensation and loss of characteristic lobes and bridges). Culture with IL-13, which also signals through IL4Ra, similarly resulted in loss of hypoxic neutrophil survival ( Figure 2C). Abrogation of hypoxic survival by IL-4 ( Figures 2D and 2F) and IL-13 ( Figures 2E and 2F) was further validated by flow cytometry. We confirmed that these effects were IL4Ra-dependent using an IL4Ra blocking antibody, which restored hypoxic survival in the presence of IL-4 and IL-13 ( Figures 2G and 2H). Abrogation of enhanced neutrophil survival was not confined to hypoxic responses, with IL-4 and IL-13 also partially reversing increased survival after LPS stimulation ( Figure 2I). In addition to its effects on neutrophil hypoxic survival, IL-4 suppressed LPS-induced expression of the proinflammatory genes CCL2, CCL3, TNF, and IL1B, previously linked to HIF-1a activity ( Figure 2J) (18). Conversely, IL-4 induced CCL17 ( Figure 2K), a chemokine associated with Treg recruitment, M2polarized macrophages, and tumorassociated neutrophils (19)(20)(21)(22). IL-4 did not influence baseline reactive oxidative species production or neutrophil respiratory burst after stimulation with N-formyl-met-leuphe under normoxic or hypoxic conditions ( Figure 2L).

IL-4 Suppresses Hypoxic Induction of Nuclear Factor-kB Protein Despite Preserved Metabolic Capacity
Given that hypoxia upregulates glycolysis (2) and IL-4 has been shown to affect macrophage metabolism (23) we sought to determine whether the effects of IL-4 on neutrophil survival were through alteration of metabolic pathways. GAPDH, PKM, PGK1, and PFKFB3 levels were, however, unaltered after IL-4 treatment in vitro ( Figure 3A). Although a modest decrease in glucose uptake was observed with IL-4 (see Figure E1A in the online supplement), overall neutrophil glycolytic capacity was preserved ( Figure 3B), in contrast to the described effects in macrophages (23).
In macrophages, polarization of phenotypic states has been linked to metabolic rewiring, with IL-4 favoring fatty acid oxidation (23). IL-4 did not affect neutrophil expression of PPARGC1B, a key regulator of b-oxidation (see Figure  E1B) or flux through fatty acid oxidation ( Figure 3B). Overall energy status (ATP/ADP ratio) was also not modified by IL-4 ( Figure 3B), suggesting that altered metabolism and/or a lack of ATP are not responsible for the reduction in in vitro neutrophil survival.
We have previously shown NF-kB (nuclear factor-kB) upregulation to be critical for enhanced neutrophil survival under hypoxic conditions (8). Hif1a 2/2 neutrophils have reduced expression of the NF-kB subunit Rela and of Ikka, a kinase that targets the NF-kB inhibitor IkB for degradation. We found that IL-4 diminished NF-kB RelA protein expression under hypoxic conditions ( Figure 3C). However, this regulation occurred at a posttranscriptional level, with the relative expression of RELA and CHUK (IKKa) mRNA unchanged by stimulation with IL-4 in the setting of hypoxia ( Figure 3D).    Figure E2A). STAT6-mediated enhancement of PPARg activity (27) and STAT3/6-mediated induction of transcripts for both PPARG and ALOX15 (encoding an enzyme that catalyses production of endogenous PPARg agonist) has previously been observed with IL-4 stimulation in macrophages (26). In contrast to macrophages, neutrophil ALOX15 transcript was downregulated by hypoxia and unaffected by IL-4 ( Figure 4C), but PPARG transcript was strongly upregulated by IL-4 in both normoxia and hypoxia ( Figure 4D). PPARg has been shown to induce gene expression of a "master regulator" of apoptosis, DNA damage inducible transcript 3 (DDIT3) (28,29). Although IL-4 had an effect on DDIT3 expression, this was only in addition to the effect seen in hypoxia ( Figure 4E). PPARg has also been directly linked to increases in PHD transcript and protein abundance during adipocyte differentiation (30). We questioned whether IL-4 stimulation in vitro could lead to a paradoxical increase in PHD activity and thereby suppress HIF. We observed that IL-4 selectively induced PHD2 ( Figure 4F), but not PHD1 or PHD3 transcript (see Figure E2B). Neither hypoxic conditions nor IL-4 affected neutrophil HIF1A transcript ( Figure 4G). However, in keeping with PHD-mediated degradation of HIF-1a, IL-4 dramatically reduced hypoxic stabilization of HIF1a protein ( Figure 4H). To directly address the role of PHD2 in the IL-4 response, airspace neutrophils from mice deficient in myeloid  PHD2 were isolated. PHD2-deficient neutrophils treated with IL-4 displayed preserved hypoxic survival confirming that PHD2 expression is essential for the IL-4induced effects on neutrophil hypoxic survival ( Figure 4I). An overview of our proposed mechanism for IL-4/IL-13-mediated reduction in hypoxic survival is shown in Figure 4J.

Direct IL-4 Signaling Reduces Neutrophil Survival in Damaged Liver
To address whether IL4Ra-deficient neutrophils have an intrinsic survival advantage in vivo, we created liverprotected competitive bone marrowchimeras. Recipient Cd45.2 1/2 Cd45.1 1/2 wild type (WT) mice were partially depleted of host bone marrow by hind-leg irradiation to retain resident liver immune cell populations, before receiving Cd45.2 1/1 Cd45.1 2/2 Il4ra 2/2 (Il4ra 2/2 ) or Cd45.2 1/1 Cd45.1 2/2 Il4ra 1/1 (WT) bone marrow ( Figure 5A). We used a CCl 4mediated acute liver injury model that causes hypoxic zone 3 hepatocytes to preferentially undergo necrosis (31) leading to neutrophil recruitment (32). We also investigated the role of exogenous IL-4 in neutrophil survival in this system. Although the percentage of blood neutrophils derived from donor CD45.1 2 bone marrow before injury ( Figure 5B) was marginally greater in mice that received Il4ra 2/2 cells compared with those that received WT ( Figure 5C), chimerism in blood neutrophils at Day 3 remained unchanged irrespective of treatment, demonstrating no additional competitive neutrophil survival advantage to IL4Ra expression in blood neutrophils after treatment ( Figure 5D). Liver neutrophil chimerism was equivalent to blood chimerism in all groups except in CCl 4injured mice treated with IL4c, in which Il4ra 2/2 donor neutrophils displayed a significant advantage compared with their WT counterparts ( Figure 5E). This change in chimerism was caused by an increased number of donor Il4ra 2/2 neutrophils in the liver of CCl 4 1 IL4c-treated mice ( Figure 5F), rather than a decrease in host WT cells (Figure 5G), thereby confirming the competitive advantage conferred on Il4ra 2/2 tissue neutrophils when present in a necrotic environment permeated by IL-4.

IL4Ra-Deficient Neutrophils Demonstrate a Selective Advantage in an LPS-induced Lung Inflammation Model with Systemic Hypoxia
Having observed a competitive advantage for Il4ra 2/2 neutrophils in a liver injury model in the presence of exogenous IL-4, we questioned whether endogenous cytokine could be sufficient to drive a selective advantage for IL4Ra-deficient neutrophils in vivo in the setting of systemic hypoxia combined with an LPS challenge. In keeping with previous reports of IL-4 release after LPS ( Figure 1F) (33,34), we also observe IL-4 release in the setting of hypoxia in our ALI model ( Figure 6A). Competitive chimeras were generated to determine if a survival advantage was conferred by neutrophil IL4Ra deficiency in this model of ALI. Cd45.2 1/2 Cd45.1 1/2 WT recipient mice were fractionally irradiated to deplete endogenous bone marrow cells (35) but protect lung alveolar macrophages (see Figure E3A). The mice were subsequently reconstituted with a 50:50 mix of Cd45.2 1/2 Cd45.1 1/2 Il4ra 1/1 WT and either Cd45.2 1/1 Cd45.1 2/2 Il4ra 2/2 (Il4ra 2/2 ) or Cd45.2 1/1 Cd45.1 2/2 Il4ra 1/1 (WT) marrow ( Figure 6B) to offer a direct comparison between WT and knockout donor cells. After a recovery period, mice were treated with nebulized LPS, placed in hypoxia (10% O 2 ), and chimerism within the Ly6G 1 neutrophil population determined by flow cytometry. Blood neutrophils at both time-points exhibited higher chimerism in il4ra 2/2recipient mice ( Figure 6C), an effect that was also seen in the recruited BAL neutrophils ( Figure 6D). In our model, CXCR4 expression in bone marrow neutrophils was not significantly different between donor groups, suggesting that CXCR4 was not responsible for the increased chimerism of il4ra 2/2 blood and BAL neutrophils in this setting ( Figure 6E).

IL-4 Promotes Inflammation Resolution in Hypoxia through Increased Neutrophil Apoptosis
To determine whether IL-4 could directly promote resolution of neutrophilic inflammation in the context of systemic hypoxia, we used the same model of hypoxic airway inflammation with addition of sustained local release of IL-4 using IL-4 complex (IL4c) at 6 hours after LPS to allow for neutrophil recruitment to occur ( Figure 6F). Bone marrow neutrophil expression of chemokines involved in bone marrow neutrophil retention (CXCR4) and release (CXCR2) (Figures 6G and 6H) (14) was initially explored. Although IL-4c did seem to increase the expression of bone marrow neutrophils expressing CXCR4 ( Figure 6G), it also significantly increased the expression of CXCR2-expressing neutrophils in vivo ( Figure 6H), with an equivalent proportion of circulating neutrophils observed between treatment groups ( Figure 6I). Furthermore, peak recruitment to the alveolar space was also equal between groups at 24 hours ( Figure 6J). During the resolution phase IL-4c treatment increased 24-hour BAL neutrophil apoptosis levels, with a subsequent reduction in BAL neutrophil counts at 48 hours ( Figures 6J and 6K), and a reduction in extracellular elastase release measured in BAL fluid samples ( Figure 6L).

Discussion
We show that IL-4 signaling in neutrophils can regulate hypoxia-induced proinflammatory survival programs and can be further manipulated in vivo to promote restoration of constitutive rates of neutrophil apoptosis and inflammation resolution. This work extends the previously defined roles of IL-4 in type 2 mediated alternative reprogramming of macrophages during allergic, parasitic, wound healing, and acute inflammatory responses to now include direct regulation of hypoxic neutrophilic inflammation.
We show that in ARDS, a condition characterized by systemic hypoxia and uncontrolled neutrophilic inflammation, the IL-4 axis is activated and airspace  neutrophils have the capacity to respond to this stimulus. Furthermore, we observe that IL-4, and the related cytokine IL-13, directly increase apoptosis of human neutrophils under hypoxic conditions and after LPS challenge, an effect that was replicated in murine inflammatory BAL neutrophils. Abrogation of hypoxic survival by IL-4 and IL-13 was dependent on the IL4Radownstream signaling mediators STAT3 and STAT6. PPARg, a known target gene of STAT3 and STAT6, and important for macrophage polarization to IL-4 (26,36), was also induced by IL-4 in healthy human neutrophils, both in normoxia, as described previously (37), and in hypoxia.
PPARg upregulation has previously been linked to changes in neutrophil function in patients with glycogen storage disease type Ib (38), where circulating neutropenia and defective respiratory burst are described, alongside increased levels of HIF-1a. In these patients' neutrophils, treatment with a PPARg antagonist partially rescued the functional defects. In contrast, we observe that in metabolically sufficient neutrophils, IL4Ra signaling, with induction of PPARg, results in downregulation of HIF-1a protein expression, associated with increased PHD2 expression. These findings are in line with a previous study describing effects of PPARg on PHD transcription and protein levels in adipocytes (30). Importantly, we show that neutrophil PHD2 expression is essential to reproduce the inhibitory effects of IL-4 on hypoxic neutrophil survival, because PHD2 2/2 neutrophils are resistant to IL-4induced hypoxic apoptosis.
In addition, we also observed a decrease in hypoxic NF-kB RelA protein levels after IL-4 treatment. We have previously shown that NF-kB acting downstream of HIF1a is crucial for neutrophil hypoxic survival (8), whereas inhibition of RelA transcriptional activity by PPARg has been implicated in apoptosis of monocyte-derived macrophages (39). NF-kB has been described as a critical transcriptional regulator of HIF-1a, but the changes in HIF-1a and NF-kB seen with the combination of hypoxia and IL-4 are limited to changes in protein abundance and thus post-translational in nature. This is of particular relevance given the overlap in post-transcriptional regulation of HIF-1a and NF-kB by asparaginyl and prolyl hydroxylation (40,41).
Previous in vivo studies have demonstrated endogenous IL-4 release in the lung in response to LPS (33,34) and upregulation of IL4Ra on myeloid cells in response to proinflammatory stimuli (15), raising the possibility that IL-4/IL4Ra signals are generated early in the course of inflammation to ensure timely resolution. Woytschak and colleagues (14) recently described a role for IL4c in limiting neutrophil recruitment during bacterial infections. In contrast, our work indicates an effect of IL-4 specifically on inflammation resolution, when IL4c is administered after onset of the inflammatory response. Using competitive chimeras, we show that in the inflamed liver, which is characterized by local hypoxia (31), il4ra 2/2 neutrophils have an advantage over their WT control subjects with increased numbers after treatment with IL-4c, demonstrating a resistance to the proresolution effects of this cytokine. Furthermore, in our model of hypoxic ALI, BAL neutrophil counts after IL4c treatment were similar to control subjects during the recruitment phase, but lower after 48 hours, whereas apoptosis rates were higher. In keeping with IL-4 driving apoptosismediated inflammation resolution (42), we observed an increase in BAL neutrophil apoptosis levels at 24 hours with a subsequent reduction in BAL neutrophil counts at 48 hours after IL4c treatment and a reduction in BAL elastase. Blood neutrophil counts as a percentage of total leukocytes were equivalent and neutrophils from IL4c-treated animals expressed high levels of CXCR2, favoring marrow release. These data suggest that, in the context of systemic hypoxia and LPS, the major effect of locally delivered exogenous IL-4 on neutrophils was not on migration.
These data thus extend the current understanding from IL-4 influencing inflammation resolution via alternative macrophage activation to a model in which IL-4 concurrently limits the acute neutrophilic response through direct inhibition of hypoxic neutrophil survival.
One key limitation of this work is the lack of evidence as to whether IL-4 supplementation, in addition to promoting accelerated inflammation resolution, can recover organ function. These are important future studies to determine the potential of therapies targeting IL-4. Such therapies would be limited to the site of tissue injury, where both IL-4 release and tissue hypoxia occur, and not compromise systemic neutrophil function or longevity. Further understanding of the broader importance of this pathway both to allergic airway inflammation and more chronic respiratory conditions typified by neutrophilic inflammation (e.g., chronic obstructive pulmonary disease) is also required particularly given the current therapeutic interest in blocking IL-4 signaling responses to limit disease progression. n Author disclosures are available with the text of this article at www.atsjournals.org.