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. Author manuscript; available in PMC: 2020 Oct 1.
Published in final edited form as: J Immunol. 2019 Aug 30;203(7):1952–1960. doi: 10.4049/jimmunol.1900454

Early life represents a vulnerable time window for IL-33-induced peripheral lung pathology

Li Y Drake *, Diane Squillace *, Koji Iijima *, Takao Kobayashi *, Masaru Uchida *, Gail M Kephart *, Rodney Britt #, Daniel R O'Brien †, Hirohito Kita **
PMCID: PMC6761009  NIHMSID: NIHMS1536572  PMID: 31471525

Abstract

IL-33, an IL-1 family cytokine, is constitutively expressed in mucosal tissues and other organs in healthy humans and animals, and expression levels increase in inflammatory conditions. Although IL-33-mediated promotion of type 2 immune responses has been well established, a gap in our knowledge regarding the functional diversity of this pleiotropic cytokine remains. To address this gap, we developed a new IL-33 transgenic mouse model in which overexpression of full-length IL-33 is induced in lung epithelial cells under conditional control. In adult mice, an approximately 3-fold increase in the steady-state IL-33 levels produced no pathologic effects in the lungs. When exposed to airborne allergens, adult transgenic mice released more IL-33 extracellularly and exhibited robust type 2 immune responses. In neonatal transgenic mice, up to postnatal day 14, a similar increase in steady-state IL-33 levels resulted in increased mortality, enlarged alveolar spaces resembling bronchopulmonary dysplasia, and altered expression of genes associated with tissue morphogenesis. Processed 25-kDa IL-33 protein was detected in bronchoalveolar lavage fluids without any exogenous stimuli, and pathologic changes were abolished in mice deficient in the IL-33 receptor ST2. These findings suggest that adult lungs are relatively resistant to IL-33 overexpression unless they encounter environmental insults, whereas developing lungs are highly susceptible, with IL-33 overexpression resulting in detrimental and pathologic outcomes.

INTRODUCTION

IL-33, a member of the IL-1 cytokine family, is constitutively and abundantly expressed by a variety of cell types in healthy humans and animals, including epithelial cells, fibroblasts, and endothelial cells (1, 2). IL-33 normally localizes in the cell nucleus, where it is bound tightly to chromatin and prevented from mediating pro-inflammatory responses in the extracellular milieu (3, 4). Under various pathologic conditions, IL-33 expression by tissue cells increases, and this is often accompanied by extracellular release of the cytokine (5-9). IL-33 activates a variety of cells involved in type 2 immune responses and promotes inflammation and tissue remodeling (10, 11). IL-33 is also involved in tissue homeostasis and host immunity to viruses, microbes, and neoplasms (11). However, major gaps in our knowledge regarding the molecular control and diverse functionality of this cytokine remain. In particular, because IL-33 is normally expressed constitutively by various types of cells without detrimental consequences, a fundamental question remains as to whether increased expression of IL-33 per se produces pathologic outcomes.

Lung epithelial cells play important roles in tissue homeostasis and innate immunity (12). Under both steady-state and allergic inflammatory conditions, epithelial cells are a major source of IL-33 in the respiratory mucosa (8, 13). Furthermore, IL-33 expression is increased in the nasal and lung epithelia in patients with allergic asthma, allergic rhinitis, and chronic obstructive pulmonary disease (COPD) (14-17), suggesting that IL-33 plays a pivotal role in the pathogenesis of airway diseases. Indeed, systemic overexpression of IL-33 driven by the CMV promoter was shown to cause robust inflammatory and pathologic changes in the lungs of mice (18). In addition, systemic deletion of the chromatin-binding domain of IL-33 via an endogenous knock-in approach caused lethal eosinophilic inflammation in various organs in mice, including the lungs (19). These results provide evidence of strong pro-inflammatory activities of IL-33 and the critical roles the chromatin-binding domain plays in regulating its biological activities. However, a major question remains regarding physiologic relevance of the models, because IL-33 expression was driven systemically even in cell types that would not normally produce the cytokine and because IL-33 overexpression was induced throughout the lives of the animals.

To address whether overexpression of IL-33 specifically in the lung epithelium is associated with pathologic changes and whether there are any critical time windows during development, we established a novel transgenic mouse model in which expression of full-length IL-33 is driven in lung epithelial cells. An inducible system was employed to enable control of IL-33 expression timing. We observed no pathologic consequences of lung-specific overexpression of IL-33 in adult mice. On the other hand, IL-33 overexpression in neonatal mice was detrimental, as these mice exhibited spontaneous pathologic changes resembling bronchopulmonary dysplasia (BPD) in humans. Our data suggest that the impact of IL-33 differs with age.

MATERIALS AND METHODS

Generation of CCSP-Il33tg mice

The pTre-IL-13-hGH construct was kindly provided by Dr. Jack Elias (Yale School of Medicine, New Haven, CT) (20). Murine IL-33 cDNA was purchased from Open Biosystems and cloned into pTre-IL-13-hGH in place of the IL-13 gene. The final pTre-IL-33-hGH construct was digested with NotI and XhoI. The transgene cassette was purified and microinjected into fertilized eggs of FVB mice by the Mayo Clinic Transgenic Core Facility (Rochester, MN). IL-33 transgene–positive offsprings were identified by PCR using tail DNA. The primers used for PCR were 5’-GATCCAGCCTCCGCCGCCGCCGCCATG-3’ and 5’-AGGATCCTTAGATTTTCGAGAGCTTAAAC-3’. A total of three Tre-IL-33-hGH transgenic founders were identified, and they had similar IL-33 protein expression levels. Two of the three founders were used to generate experimental mice for this study. To generate CCSP-Il33tg mice, Tre-IL-33-hGH founders were bred with CCSP-rtTA transgenic mice (FVB.Cg-Tg(Scgb1a1-rtTA)1Jaw/J) purchased from Jackson Laboratory. The non-transgenic or single-transgenic littermates were used as controls for all experiments. To generate ST2−/−CCSP-rtTA-IL-33 mice, Tre-IL-33-hGH and CCSP-rtTA transgenic mice were bred with ST2−/−BALB/c mice (provided by Dr. Andrew McKenzie, Medical Research Council Laboratory of Molecular Biology, Cambridge, U.K.), followed by breeding of ST2−/−Tre-IL-33-hGH and ST2−/−CCSP-rtTA transgenic mice.

To induce transgene expression, mice were treated with Dox (Mayo Pharmacy, Mayo Clinic, Rochester, MN) via drinking water (Dox dissolved in 2.5% sucrose water and administered at 0.5 mg/ml) for the indicated periods. Dox-containing water was prepared fresh and changed twice weekly. Mice were observed daily, and both dead mice at the time of observation and those moribund and deemed to require euthanasia were considered dead. All animal experiments and handling procedures were approved by the Mayo Clinic Institutional Animal Care and Use Committee and performed according to established guidelines.

Airway allergen exposure models

Mice were lightly anesthetized with isoflurane prior to intranasal administration of 20 μg/dose of bromelain (Sigma, St. Louis, MO) or 50 μg/dose of Alternaria extract (Greer Laboratories, Lenoir, NC) in 40 μl of endotoxin-free PBS. Control mice received 40 μl of PBS only. Three different allergen exposure models were used. Mice were euthanized 1 hour (for IL-33 ELISA) or 4.5 hours (for IL-5 and IL-13 ELISA) after one-time Alternaria exposure. Alternatively, mice were euthanized 3 hours after one-time bromelain exposure. Finally, mice were exposed to bromelain on days 0, 2, and 4 and euthanized 24 hours after the last exposure. All mice were euthanized by intraperitoneal injection of pentobarbital, and BAL fluid as well as lung tissues and blood samples were collected. The number of cells in BAL fluid was determined, and cell differentials were determined using cytospin slides stained with Wright-Giemsa. Lung tissues were homogenized in 1 ml of PBS. Cytokine levels in the supernatants of BAL fluids and lung homogenates were determined as described below.

Immunoassays

The levels of cytokines in BAL fluids and lung homogenates were analyzed using Quantikine ELISA kits (R&D Systems) and a multiplex ELISA kit (Millipore), following the manufacturers’ instructions. For measurement of serum immunoglobulin levels, ELISA plates were coated with antibodies to mouse IgG1, IgM, IgA, or IgE (BD Pharmingen), blocked with BSA, and incubated with diluted serum samples. After washing, the IgG1 and IgM plates were incubated with HRP-conjugated anti–mouse IgG1 or IgM (BD Pharmingen) and peroxidase substrate. The IgA and IgE plates were incubated with biotinylated anti–mouse IgA or IgE (BD Pharmingen), followed by streptavidin and peroxidase substrate. In all assays, the absorbance was read using a microplate autoreader after stopping the reaction by addition of HCl.

Lung histology and immunofluorescence analysis

Lungs were fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). To quantitate the size of alveoli, five randomly selected non-overlapping photomicrographs were taken for each slide at 160× magnification. The pictures were digitally incorporated into the ImageJ software (NIH), and the alveolar size from ten different areas from two mice was determined using the tools available in the software.

For IL-33 staining, sections of formalin-fixed, paraffin-embedded mouse lung tissue were deparaffinized and rehydrated. Antigen retrieval was performed by heating the slides in citrate buffer (0.01 M, pH 6.0; Sigma) in a steamer for 30 minutes. The slides were then stained with polyclonal goat anti-mouse IL-33 (R&D Systems, AF3626) at 0.5 μg/ml, followed by AF488 rabbit F(ab’)2 anti-goat IgG (Abcam, ab169344; 1:500 dilution). After washing, the slides were mounted with Vectashield mounting medium with the DNA-binding dye DAPI (Vector Laboratories, Burlingame, CA). Fluorescent images were captured using an Olympus AX70 fluorescence microscope (Olympus Imaging America Inc.) and an Olympus DP71 microscope digital camera equipped with Olympus DP Manager Software.

Western blot analysis of IL-33 protein

To analyze IL-33 protein by Western blotting, lung tissues were homogenized in RIPA buffer containing protease inhibitor cocktail (Roche) and then centrifuged. Alternatively, supernatants of BAL fluids were used for analysis. The supernatant protein concentration was determined using a Pierce BCA protein assay kit (Thermo Scientific). Supernatants containing 2.5 mg of protein were incubated with a polyclonal goat anti-mouse IL-33 antibody (R&D Systems) overnight at 4°C, followed by the addition of protein A Dynabeads (DAKO, Denmark) for 1 hour. The beads were washed with PBS plus 0.02% Tween-20. Protein bound to the beads was eluted using protein sample buffer, separated over a 14% tris-glycine gel (Invitrogen), and transferred onto a nitrocellulose membrane. After protein transfer, nitrocellulose membranes were blocked in 5% milk buffer and incubated with goat anti-mouse IL-33 (1:1000), followed by HRP-conjugated donkey anti-goat IgG (R&D Systems). Blots were developed using SuperSignal West Pic (ThermoFisher), with exposure to BioMax film (Kodak, Rochester, NY).

RNA expression analysis

Isolated lung epithelial cells and total lung cells were used for RNA expression analyses. Lungs from 2 to 4 mice were pooled, incubated with collagenase (Roche Diagnostics) and DNase I (StemCell Technologies), and processed into single-cell suspensions using a GentleMACS dissociator (Miltenyi Biotec). Cells were stained with fluorescent-labeled antibodies to CD45 and EpCam (eBioscience). Epithelial cells were isolated as the CD45−EpCamhi cell population by sorting on a fluorescence-activated cell sorter (FACS, BD FACSAria®). Total RNA was purified from sorted lung epithelial cells and sequenced by the Mayo Medical Genome Facility. MAP-RSeq (21) was used to process each sample’s reads, and Tophat (22) was used for read alignments. The resulting alignment file was then examined using Feature Counts (23) to quantify gene expression in each sample. Targets with a minimum normalized gene count value of <25 across the two sets of paired samples were removed from the analysis. EdgeR (24) was used to normalize and quantify log fold-changes, p-values, and false-discovery rates across the paired samples for the remaining targets. Autosomal genes with an absolute log2 fold change >1 or with a false-discovery rate of <1 were then selected for pathway analysis. Qiagen’s Ingenuity Pathway Analysis software and KEGG pathways (25) were used to explore the pathways and functional groups of significance. The results of RNA sequencing have been submitted to a public database (NCBI GEO SRP198258, www.ncbi.nlm.nih.gov). Alternatively, RNA expression in isolated lung epithelial cells or total lung cells was analyzed by real-time RT-PCR, which was performed using Taqman Universal PCR master mix and Retnla and housekeeping gene 18S rRNA primers (Applied Biosystems). Retnla mRNA expression was normalized to the expression of 18S rRNA.

Statistical analysis

Data are presented as the mean ± SEM, as indicated in the figure legends. The statistical significance of differences was assessed using the Student’s t test; p<0.05 was considered significant.

RESULTS

Generation of CCSP–IL-33 transgenic mice

To study the function of IL-33 in the lungs, we developed a novel transgenic mouse model in which full-length IL-33 is overexpressed in lung epithelial cells under conditional control. Expression of the reverse tetracycline-controlled transactivator (rtTA) protein, which is driven under control of the rat club-cell secretory protein (CCSP) gene promoter, induces expression of transgenes in alveolar type 2 epithelial (AT2) cells of transgenic mice administered doxycycline (Dox) (26). In this study, we employed the same strategy and bred CCSP-rtTA transgenic mice with Tre-IL-33-hGH transgenic mice to create double-transgenic mice in which expression of the gene encoding full-length IL-33 is induced by Dox-sensitive rtTA protein driven by the rat CCSP promoter (i.e., CCSP-Il33tg mice) (Figure 1A).

Figure 1.

Figure 1.

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Generation and characterization of CCSP-Il33tg mice. (A) Design of the Tre-IL-33-hGH construct and generation of CCSP-Il33tg mice. (B and C) CCSP-Il33tg mice or control mice (6 to 8 weeks old) were treated with Dox for 1 week. IL-33 protein in lung homogenates was analyzed by ELISA (B) and Western blot (C). rmIL-33, a commercially available recombinant mouse IL-33 (a.a. 109-266). (D) Mice were treated with Dox for 4 weeks. Lung sections were stained with anti-IL-33 and examined under a fluorescence microscope. Original magnification, 600×. (E) Mice were treated with Dox for 1 week. IL-33 protein levels in homogenates of the lungs, spleen, and ear skin were analyzed by ELISA. (F) Mice were treated with Dox for 1 week (Dox on) and then switched over to normal water for 2 days (Dox off). IL-33 protein levels in lung homogenates were analyzed by ELISA. n.d., not determined. Data are presented as means ± SEMs (n=5 in each group). *p<0.05 and **p<0.01 between the groups indicated by horizontal lines. Data are representative of two to three independent experiments.

CCSP-Il33tg mice exhibited no breeding-related or developmental abnormalities when parents and pups were given access to normal drinking water without Dox. Comparable amounts of IL-33 were detected in the lungs of naïve adult CCSP-Il33tg mice (i.e., 6 to 10 weeks old) and their littermate controls (i.e., CCSP-rtTA mice, Tre-IL-33 mice, and non-transgenic mice), consistent with constitutive expression of IL-33 by airway epithelial cells in a steady-state condition (8). When administered Dox via drinking water for 1 week, adult CCSP-Il33tg mice showed an approximate 3-fold increase in lung levels of IL-33 protein as compared to control mice (Figure 1B). In Western blot analyses, lung lysates of Dox-treated control mice showed a single band with a molecular mass of approximately 30 kDa (Figure 1C), corresponding to full-length mouse IL-33 (27). Dox-treated CCSP-Il33tg adult mice showed increased density of the 30-kDa band together with a faint 25-kDa band. Commercial recombinant mouse IL-33 (a.a. 109–266) showed a molecular mass of 18 kDa. By immunofluorescence, IL-33 protein was localized in the nuclei of cuboid-shaped cells in the lung parenchyma of control mice, consistent with AT2 cells (Figure 1D). The intensity of IL-33 staining in AT2 cells, as well as the number of AT2 cells expressing IL-33, increased in Dox-treated CCSP-Il33tg mice, but no other cell types appeared positive for IL-33. Expression of IL-33 protein was upregulated only in the lungs and not in spleen or ear skin in CCSP-Il33tg mice (Figure 1E). Finally, expression of IL-33 was reversible, as IL-33 protein levels in Dox-treated CCSP-Il33tg mice returned to the levels of control mice within 2 days of cessation of Dox administration (Figure 1F). Altogether, these findings suggest that Dox administration led to a transient increase in the expression of full-length IL-33 protein in a lung-specific manner in CCSP-Il33tg mice.

Increased lung expression of IL-33 in adults does not lead to pathology

In animals infected with helminthes or exposed to airborne allergens, increased production and/or release of IL-33 in the lungs can lead to type 2 airway inflammation and lung pathology (11). To examine whether increased production of IL-33 in the lungs per se causes lung pathology, 6- to 8-week-old adult CCSP-Il33tg mice were treated with Dox for 4 weeks. Although Dox administration led to a 3-fold increase in lung levels of IL-33 protein in CCSP-Il33tg mice, no IL-33 protein was detectable in bronchoalveolar lavage (BAL) fluids (Figure 2A). Likewise, an analysis of BAL fluids revealed no signs of inflammation (Figure 2B), and no increase in the levels of type 2 cytokines, including IL-5 and IL-13, was observed in BAL fluids (Figure 2C). Histologic analysis of the lungs showed no signs of inflammation (Figure 2D). Serum levels of immunoglobulins were comparable between Dox-treated CCSP-Il33tg mice and control mice (Figure 2E). Adult CCSP-Il33tg mice that had been treated with Dox for a shorter period (i.e., 1 or 2 weeks) also showed no signs of inflammation (data not shown). Collectively, these findings suggest that increased lung expression of IL-33 in adults does not lead to IL-33 release or pathologic outcomes.

Figure 2.

Figure 2.

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Increased expression of IL-33 does not produce pathologic outcomes in adult mice. CCSP-Il33tg mice or control mice (6 to 8 weeks old) were treated with Dox for 4 weeks. (A) IL-33 protein levels in lung homogenates and BAL fluid supernatants were analyzed by ELISA. (B) Cell numbers and differentials in BAL fluids were determined. (C) Cytokine levels in BAL fluid supernatants were determined by ELISA. (D) Lung sections were stained with H&E. Original magnification, 160×. (E) Serum levels of immunoglobulins were quantified by ELISA. Data are presented as means ± SEMs (n=4 in each group). *p<0.05 between the groups indicated by horizontal line. Data are representatives of three independent experiments.

Increased IL-33 expression induces robust type 2 immune responses to inhaled allergens

The above findings suggest that IL-33 protein is sequestered from the extracellular milieu under steady-state conditions even if the expression level increases, consistent with a recent in vitro study demonstrating strict regulation of IL-33 activity by chromatin binding (4). IL-33 is considered an “alarm” molecule that is released as a result of tissue injury or cellular necrosis (27). Therefore, to examine the potential impact of airborne allergens, we exposed adult mice intranasally (i.n.) to an extract of a natural fungal allergen, Alternaria. Alternaria extract has been used extensively to examine type 2 immune responses in the airways (28). Adult CCSP-Il33tg mice or their littermate controls were treated with Dox for 1 week and then exposed i.n. to Alternaria extract. One hour after Alternaria exposure, IL-33 protein was detectable in BAL fluids of control mice, suggesting extracellular release of IL-33 protein (Figure 3A). Significantly higher amounts of IL-33 protein were detected in BAL fluids of Alternaria-exposed CCSP-Il33tg mice as compared to control mice (p<0.05). When re-examined 4.5 hours later, Alternaria-exposed control mice showed elevated lung levels of IL-5 and IL-13 as compared to PBS-exposed control mice (Figure 3B). CCSP-Il33tg mice showed significantly higher lung levels of IL-5 and IL-13; however, IFN-γ levels remained minimal in both CCSP-Il33tg mice and control mice.

Figure 3.

Figure 3.

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Increased expression of IL-33 promotes type 2 immunity in response to inhaled allergens in adult mice. CCSP-Il33tg mice or control mice (6 to 8 weeks old) were treated with Dox for 1 week. (A and B) Mice were exposed intranasally (i.n.) once to PBS or Alternaria extract (50 μg/dose). The levels of cytokines in BAL fluids (A) and lung homogenates (B) were determined 1 hour (A) or 4.5 hours (B) later by ELISA. (C and D) Mice were exposed i.n. to PBS or bromelain (20 μg/dose) once (C) or three times over 5 days (D). (C) Three hours later, cytokine levels in lung homogenates were determined by ELISA. (D) Twenty-four hours after the last exposure, cell numbers and differentials in BAL fluids were determined. Data are presented as means ± SEMs (n=5 in each group). *p<0.05 and **p<0.01 between the groups indicated by horizontal line. Data are representatives of two or three independent experiments.

Because Alternaria extract is a complex mixture of proteins and carbohydrates that can affect a variety of airway cell types (28), we examined the effect of the cysteine protease bromelain as another allergen model (29, 30). In response to a single exposure to bromelain, Dox-treated CCSP-Il33tg mice showed higher levels of IL-5 and IL-13 as compared to similarly treated control mice (Figure 3C). Furthermore, when exposed repeatedly to bromelain for 5 days, CCSP-Il33tg mice showed a significant increase in the number of eosinophils in BAL fluids as compared to control mice (Figure 3D). Collectively, these findings suggest that although increased expression of full-length IL-33 in the lungs per se does not cause airway pathology, it does lead to enhanced type 2 immune responses and airway inflammation upon exposure to inhaled allergens.

Increased expression of IL-33 in the perinatal period causes lung pathology

Given that constitutive systemic overexpression of full-length IL-33 can produce severe lung pathology (18), we speculated that there might be a time window during which animals are vulnerable to increased lung expression of IL-33. To address this hypothesis, we administered Dox to breeding CCSP-rtTA and Tre-IL-33 transgenic parents during the entire pregnancy and lactation period until postnatal day (PN)21. After weaning on PN21, Dox was administered continuously to the weaned pups born to these parents until PN42. Using this strategy, CCSP-Il33tg double-transgenic pups or control Tre-IL-33 or CCSP-rtTA single-transgenic pups were treated continuously with Dox from embryonic day (E)0 to PN42. From PN0 to PN5, no apparent mortality or abnormalities in gross appearance were observed in CCSP-Il33tg pups and control pups that had been treated with Dox (Figure 4A). However, CCSP-Il33tg pups began to die on PN6, and 61% had died by PN14 when the death rate appeared to plateau. Similar death of pups was not observed in control Tre-IL-33 or CCSP-rtTA single-transgenic mice. The lungs of dead CCSP-Il33tg pups (n=21) showed macroscopic abnormalities, including hemorrhage (80% of mice) (Figure 4B) and fewer lung lobes (i.e., ≤4 lung lobes, 19% of mice). Massive lung hemorrhaging was confirmed histologically (Figure 4C). Among the CCSP-Il33tg mice that survived until 6 weeks of age (n=21), none showed hemorrhage and 38% showed fewer lung lobes. The other mice showed a simplified alveolar structure and enlarged alveolar space, consistent with the lung pathology of BPD in humans (Figure 4D). The significant increase in alveolar size was confirmed by morphometric analysis (p<0.01).

Figure 4.

Figure 4.

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Increased expression of IL-33 causes lung pathology and increased mortality in neonatal mice without any exogenous stimuli. Eight litters of CCSP-Il33tg mice or control mice were treated with Dox from embryonic day (E)0 to postnatal day (PN)42. (A) Survival of pups was monitored daily after birth until PN21. N=41 and 40 for CCSP-Il33tg mice and control mice, respectively. (B) Photographs of lungs from a CCSP-Il33tg mouse and a control mouse are shown. (C and D) Photomicrographs from representative CCSP-Il33tg and control mice are shown. Original magnification, 160×. Alveolar size was determined by digital analysis. Data are presented as means ± SEMs (n=10 randomly selected areas from two mice in each group). **p<0.01 between the groups indicated by horizontal line.

To more clearly define the critical time window for development of lung pathology in neonates, we administered Dox to mothers (and therefore to pups via nursing) over four different perinatal intervals: E0–21, PN1–7, PN8–14, and PN15–21. Lungs of all mice were examined on PN21 or as soon as they were found dead or moribund. The developmental stage of lungs in PN20 mice is considered comparable to that of 3-year old child (25). The survival rate was lowest when pups were treated with Dox over PN1–7, whereas all pups survived when Dox was administered after PN15 (Figure 5A). In contrast, when Dox was administered during the embryonic stage, all dead pups showed signs of lung hemorrhaging (Figure 5B). Similarly, lung hemorrhaging was observed more often when Dox was given to younger pups (i.e., PN1–7). The number of mice with fewer lung lobes increased when Dox was given during PN1–7 or PN8–14 (Figure 5C). Importantly, none of the pups showed macroscopic lung abnormalities, such as hemorrhaging or fewer lung lobes, when Dox was given on or after PN15.

Figure 5.

Figure 5.

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The first 2-week period after birth provides a critical time window for developing lung pathology. Mothers and litters of CCSP-Il33tg mice or control mice were treated with Dox for four different time periods: E0-E21, PN0-7, PN8-14, or PN15-21. Four to eight litters were studied, and mice were analyzed at the time of death or after euthanasia on PN21. (A) Survival of pups was monitored daily after birth until PN21. (B) Proportion of pups with lung hemorrhage at the time of their death (left panel) and proportion of pups with less lung lobes within all CCSP-Il33tg pups (right panel) were determined.. (C) Photomicrographs from representative CCSP-Il33tg and control mice are shown. Original magnification, 160×. **p<0.01 between the groups indicated by horizontal line.

Histologically, when CCSP-Il33tg pups were administered Dox during either PN1–7 or PN8–14, they developed enlarged alveolar air spaces (Figure 5D) roughly comparable to those in mice administered Dox for a prolonged period from E0 to PN42 (Figure 4D). In contrast, when mice were administered Dox over PN15–21, no apparent pathologic changes were observed. These findings suggest that increased expression of IL-33 in lung epithelial cells during the neonatal period causes lung pathology and that the mice are more vulnerable during the first 2 weeks of life.

IL-33 protein is released extracellularly in neonatal CCSP-Il33tg mice

IL-33 was retained within airway epithelial cells in Dox-treated adult CCSP-Il33tg mice (Figure 2A). We speculated that IL-33 protein is released extracellularly in neonatal mice, providing a mechanism for the development of lung pathology. When administered Dox from E0 to PN5, lung levels of IL-33 protein in CCSP-Il33tg mice were approximately 5-times higher than those in controls (Figure 6A). Furthermore, when neonatal mice were treated with Dox from PN0 to PN10, IL-33 protein was detectable by ELISA in the airway lumen (i.e., BAL fluids) of CCSP-Il33tg mice but not that of control mice (Figure 6A). Western blot analysis of BAL fluids from neonatal mice showed a single band of IL-33 protein with a molecular mass of 25 kDa, which is apparently smaller than the 30-kDa molecular mass of full-length mouse IL-33 (Figure 6B). The 25-kDa molecular mass was comparable to IL-33 protein that was detected in BAL fluids from adult CCSP-Il33tg mice exposed to Alternaria extract.

Figure 6.

Figure 6.

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Extracellular IL-33 released from neonatal mice is likely responsible for lung pathology. (A) CCSP-IL33tg mice or control mice (n=10/group) were treated with Dox from E0 to PN5 (left panel) or from PN0 to PN10 (right panel). IL-33 protein levels in lung homogenates on PN5 (left panel) or BAL supernatants on PN10 (right panel) were determined by ELISA. **p<0.01 between the groups indicated by horizontal line. (B) IL-33 protein was analyzed by Western blotting. Lane 1, lysate of 293T cells transfected with full-length mouse IL-33 cDNA; lane 2, Pooled BAL fluids from PN10 CCSP-Il33tg mice treated with Dox water from PN0 to PN10; and lane 3, BAL fluids from an adult CCSP-Il33tg mouse treated with Dox and exposed i.n. to Alternaria extract for 4.5 h. (C through E) ST2−/−CCSP-Il33tg mice or ST2−/− control mice were treated with Dox from E0 to PN42. (C) Lung levels of IL-33 in ST2−/−CCSP-Il33tg mice and ST2−/− control mice were examined on Day 42 (n=10/group). (D) Survival of pups from six litters of ST2−/−CCSP-Il33tg mice and ST2−/− control mice were monitored until PN42 (n=27-30 mice in each group). (E) Photomicrographs from representative ST2−/−CCSP-Il33tg mice and ST2−/− control mice. Original magnification, 160×. **p<0.01 between the groups indicated by horizontal line.

The activity of IL-33 is mediated via binding to the cell surface receptor ST2 (3). To examine the involvement of ST2, we bred CCSP-Il33tg mice with ST2−/− mice and generated ST2−/−CCSP-Il33tg mice. Administration of Dox to ST2−/−CCSP-Il33tg mice from E0 to PN42 induced an approximately 4-fold increase in lung levels of IL-33 protein as compared to non-transgenic ST2−/− mice (i.e., ST2−/−CCSP-rtTA mice and ST2−/−Tre–IL-33 mice) (Figure 6C). Nonetheless, none of the ST2−/−CCSP-Il33tg mice died within the first 6 weeks of life (Figure 6D). When euthanized on PN42, ST2−/−CCSP-IL33tg mice showed no signs of pathologic changes in the lungs (Figure 6E). Collectively, these findings suggest that, unlike the case in adults, processed IL-33 is released extracellularly in neonatal CCSP-Il33tg mice without any exogenous stimuli, and this IL-33 likely induces lung pathology via its receptor, ST2.

Gene expression in lung epithelial cells is altered in neonatal mice

Because extracellular IL-33 promotes innate and adaptive type 2 immune responses in adult mice (11), we analyzed the lungs of neonatal mice treated with Dox from E0 to PN5 for evidence of inflammation. A multiplex assay revealed no significant differences in levels of specific cytokines between CCSP-Il33tg and control mice at PN5 (Figure 7A). Similarly, analyses of BAL fluids from CCSP-Il33tg mice revealed no increase or only minimal increases in the number of eosinophils and other inflammatory cells (Figure 7B).

Figure 7.

Figure 7.

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Increased IL-33 expression in the lungs modulates gene expression in the lungs and lung epithelial cells in neonates. (A) CCSP-Il33tg mice or control mice (n=10/group) were treated with Dox from E0 to PN5. Cytokine levels in lung homogenates on PN5 were analyzed by a multiplex assay. (B) Mice were treated with Dox from PN0 to PN10. Cell numbers and differentials in BAL fluids were analyzed on PN10. Each dot represents an individual mouse. Horizontal lines represent means. n.s., not significant. (C through E) Mice were treated with Dox from E0 to PN7. Lung epithelial cells were isolated by sorting on PN7. (C) Cells were analyzed by whole-genome RNA sequencing. Heat map shows genes with differential expression in pairwise comparisons between CCSP-IL33tg mice and control mice from two separate experiments. (D) Results of pathway analyses of the differentially expressed genes. (E) Expression of Retnla mRNA was analyzed by RT-PCR. (F) Neonatal mice or adult mice (7 week old) were treated with Dox for 1 week prior to euthanasia. Expression of Retnla mRNA in total lung cells from neonates (left panel) and adults (right panel) was examined by RT-PCR (n=5/group). *p<0.05 and **p<0.01 between the groups indicated by horizontal line.

To further investigate the molecular mechanism underlying lung pathology in neonatal CCSP-Il33tg mice, we examined gene expression in lung epithelial cells. CD45−EpCamhigh lung epithelial cells were isolated by sorting from PN7 pups treated with Dox from E0 to PN7. Whole-genome RNA sequencing revealed significant differences in the expression of 52 genes between CCSP-Il33tg mice and control mice, with 45 genes upregulated and 7 genes downregulated in CCSP-Il33tg mice (Figure 7C, Supplemental Table). Pathway analyses revealed that these genes are associated with immune cell trafficking, hematologic system development and function, tissue morphology, and tissue development (Figure 7D). For example, expression of Retnla (which encodes resistin-like molecule alpha) is induced during hypoxia and allergic airway inflammation, and this gene is reportedly involved in regulation of neuronal innervation, pulmonary arterial pressure, and vascular resistance (31, 32). Real-time RT-PCR analyses showed a 2-fold increase in Retnla mRNA expression in lung epithelial cells from CCSP-Il33tg mice on PN7 as compared to control mice (Figure 7E). Upregulation of Retnla mRNA expression was also observed in total lung cells from Dox-treated CCSP-Il33tg neonatal mice but not those from Dox-treated CCSP-Il33tg adult mice (Figure 7F). Together, these observations suggest that increased lung expression of IL-33 in neonates alters gene expression in lung epithelial cells, which may explain the lung pathology observed in these animals.

DISCUSSION

IL-33 expression is increased in the lungs of asthma and COPD patients (14-17). In mice, IL-33 expression is upregulated by allergen exposure (8, 9) and viral and nematode infections (5, 6). A major finding of the present study is that increased IL-33 expression per se is not pathologic in adult animals. This result contrasts with those of previous studies that showed lung pathology and bone marrow abnormalities in mice in which IL-33 expression was driven by a CMV promoter (18, 33). Differences in the cell type and timing of IL-33 expression could explain this discrepancy. In the present study, overexpression of IL-33 was induced in lung epithelial cells, which are a natural source of IL-33 (8, 13), whereas in previous studies, IL-33 was induced systemically. The machinery involved in IL-33 production, retention, and/or release might be regulated differently among different cell types. In addition, IL-33 overexpression was transiently induced in the present study, whereas previous studies involved constitutive expression of the IL-33 transgene throughout the lives of the experimental animals. The lung pathology observed in previous studies could reflect the effects of IL-33 during early life, as we also found that increased lung expression of IL-33 in neonates results in pathologic outcomes.

Lung epithelial cells constitutively express high levels of IL-33 under steady-state conditions and store the protein in the nucleus (2). The absence of pathologic effects despite increased lung levels of IL-33 in adult mice is likely due to sequestration of IL-33 protein from the extracellular milieu. Indeed, ST2-dependent IL-33 activity is regulated strictly by binding to chromatin (4). Elimination of the IL-33 nuclear localization domain leads to release of IL-33, resulting in multi-organ inflammation involving eosinophils and neutrophils (19). Therefore, adult lung epithelial cells likely have a reservoir capacity that enables them to accommodate an increase in IL-33 protein of ~3-fold, thereby protecting the host from any detrimental consequences of extracellular IL-33. Nonetheless, the CCSP-Il33tg mice examined in the present study released more IL-33 and exhibited robust type 2 immune responses once they were exposed to proteolytic allergens (Figure 3). Thus, in adults, a “hit” is likely required for IL-33 to promote inflammation and tissue pathology, even if its expression levels increase considerably.

In contrast to adult mice, neonatal mice were highly susceptible to overexpression of IL-33 in the present study, which resulted in lung hemorrhaging, enlarged alveolar spaces, and increased mortality, even without exogenous stimuli. Importantly, the detrimental effects of IL-33 were observed only up to PN14, and the animals became resistant thereafter (Figure 5). The final stage of lung development and maturation of the lung epithelium (i.e., the postnatal “alveolar” phase) occurs during the late prenatal and early postnatal periods, up to PN20 in mice and 3 years in humans (34). Therefore, developing lungs, perhaps those in infants to child up to 2–3 years old, are likely to be vulnerable to overexpression of IL-33. To ensure normal lung development, IL-33 production likely needs to be controlled strictly at the homeostatic condition during the postnatal “alveolar” phase. The peripheral lung pathology observed in these mice resembles BPD (35), a chronic lung disease often observed in premature infants. BPD patients typically show alveolar simplification and pulmonary capillary system malformation, which may have long-lasting consequences (36). Although the molecular mechanisms involved in BPD are not fully understood, inflammatory cytokines have implicated, including IL-1β and IFN-γ (37-39). The results of the present study suggest that dysregulation of IL-33 production during the neonatal period is also involved in the development of BPD. Indeed, oxidative stress due to inhalation of high concentrations of oxygen during the early postnatal period in mice enhances IL-33 production, which induces pathologic changes (40).

Lung development and maturation is a complex process involving interactions among a number of cell types and molecules (34, 41). By RNA sequencing and RT-PCR analyses, we found that the expression of several genes associated with tissue morphogenesis and development, including Retnla, is upregulated in lung epithelial cells from neonatal CCSP-Il33tg mice. The Retnla-encoded protein is involved in pulmonary inflammation and regulates lung vascular homeostasis and angiogenesis (31, 32). A major question remains as to which of these genes are critically involved in the development of BPD-like lung pathology. Another question concerns the mechanism leading to upregulation of these genes in epithelial cells in neonatal CCSP-Il33tg mice. Released IL-33 was detected in BAL fluids, and ST2-deficient mice were protected from the pathologic outcomes, suggesting the involvement of extracellular IL-33 interacting with its receptor, ST2. The products of type 2 immune responses triggered by IL-33 (e.g., cytokines, growth factors, lipid mediators) could impair growth, differentiation and/or homeostasis of neonatal epithelial cells (11). Indeed, a minimal but detectable increase in the number of eosinophils was observed in several CCSP-Il33tg mice (Figure 7). Alternatively, IL-33 could directly affect the differentiation of epithelial progenitors, as recently shown in gastrointestinal (42) and airway (43, 44) epithelial cells. Further studies are required to determine whether pathologic outcomes in neonatal lungs are caused by direct and/or indirect effects of IL-33 on the lung epithelium. Increased expression of IL-33 was observed mainly in AT2 cells of CCSP-Il33tg mice in this study (Figure 1), which likely explains the peripheral lung pathology. Therefore, further studies are also necessary to determine whether IL-33 promotes the pathology and inflammation of the conducting airways in neonates when the hosts are exposed to microbes or allergens.

In summary, we demonstrated age-dependent differences in sensitivity to increased IL-33 expression in the lungs of mice. Recent data in mice suggest that the process of birth is sufficient to enhance IL-33 production in lung epithelial cells (45, 46). Likewise, respiratory syncytial virus infection was shown to enhance lung IL-33 production in neonates but not adults (47). The results of our study suggest that IL-33 protein is more readily released extracellularly in neonates than adults, leading to ST2-associated lung pathology. Dynamic changes in the immune system occur during development in neonates, and early life exposures are key determinants of certain immune-mediated diseases later in life (48, 49). Therefore, further elucidation of the age-specific regulation of IL-33 production, retention, and extracellular release will enhance understanding of the pathophysiology of pulmonary diseases and the impact of early life events on disease development later in life.

Supplementary Material

1

KEY POINTS.

  • In adults, increased IL-33 expression in the lungs per se is not pathologic.

  • In neonates, increased IL-33 results in severe peripheral lung pathology.

  • Pathologic effects of IL-33 in the lungs are age-dependent.

ACKNOWLEDGMENTS

The authors thank Dr. Jack Elias for providing the pTre-IL-13-hGH construct, Dr. Andrew N. McKenzie for providing the Il1rl1−/− mice and LuRaye S. Eischens for secretarial assistance.

This work was supported by grants from the National Institute of Health (R01 AI128729) and the Mayo Foundation.

Abbreviations

AT2:

alveolar type 2 epithelial

BAL:

bronchoalveolar lavage

BPD:

bronchopulmonary dysplasia

CCSP:

club-cell secretory protein

COPD:

chronic obstructive pulmonary disease

Dox:

doxycycline

E:

embryonic day

hGH:

human growth hormone

i.n.:

intranasal

PN:

postnatal day

rtTA:

reverse tetracycline-controlled transactivator

Tg:

transgenic

Footnotes

Conflict of interest statement: All authors declare no conflict of interest.

REFERENCES

  • 1.Moussion C, Ortega N, and Girard JP. 2008. The IL-1-like cytokine IL-33 is constitutively expressed in the nucleus of endothelial cells and epithelial cells in vivo: a novel ‘alarmin’? PLoS One. 3:e3331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Pichery M, Mirey E, Mercier P, Lefrancais E, Dujardin A, Ortega N, and Girard JP. 2012. Endogenous IL-33 is highly expressed in mouse epithelial barrier tissues, lymphoid organs, brain, embryos, and inflamed tissues: in situ analysis using a novel Il-33-LacZ gene trap reporter strain. J. Immunol. 188:3488–3495. [DOI] [PubMed] [Google Scholar]
  • 3.Carriere V, Roussel L, Ortega N, Lacorre DA, Americh L, Aguilar L, Bouche G, and Girard JP. 2007. IL-33, the IL-1-like cytokine ligand for ST2 receptor, is a chromatin-associated nuclear factor in vivo. Proc. Natl. Acad. Sci. U. S. A. 104:282–287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Travers J, Rochman M, Miracle CE, Habel JE, Brusilovsky M, Caldwell JM, Rymer JK, and Rothenberg ME. 2018. Chromatin regulates IL-33 release and extracellular cytokine activity. Nat Commun. 9:3244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Chang YJ, Kim HY, Albacker LA, Baumgarth N, McKenzie AN, Smith DE, Dekruyff RH, and Umetsu DT. 2011. Innate lymphoid cells mediate influenza-induced airway hyper-reactivity independently of adaptive immunity. Nat. Immunol. 12:631–638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Yasuda K, Muto T, Kawagoe T, Matsumoto M, Sasaki Y, Matsushita K, Taki Y, Futatsugi-Yumikura S, Tsutsui H, Ishii KJ, Yoshimoto T, Akira S, and Nakanishi K. 2012. Contribution of IL-33-activated type II innate lymphoid cells to pulmonary eosinophilia in intestinal nematode-infected mice. Proc. Natl. Acad. Sci. U. S. A. 109:3451–3456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Hsu CL, and Bryce PJ. 2012. Inducible IL-33 expression by mast cells is regulated by a calcium-dependent pathway. J. Immunol. 189:3421–3429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Hardman CS, Panova V, and McKenzie AN. 2013. IL-33 citrine reporter mice reveal the temporal and spatial expression of IL-33 during allergic lung inflammation. Eur. J. Immunol. 43:488–498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Iijima K, Kobayashi T, Hara K, Kephart GM, Ziegler SF, McKenzie AN, and Kita H. 2014. IL-33 and thymic stromal lymphopoietin mediate immune pathology in response to chronic airborne allergen exposure. J Immunol. 193:1549–1559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Schmitz J, Owyang A, Oldham E, Song Y, Murphy E, McClanahan TK, Zurawski G, Moshrefi M, Qin J, Li X, Gorman DM, Bazan JF, and Kastelein RA. 2005. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines. Immunity. 23:479–490. [DOI] [PubMed] [Google Scholar]
  • 11.Molofsky AB, Savage AK, and Locksley RM. 2015. Interleukin-33 in Tissue Homeostasis, Injury, and Inflammation. Immunity. 42:1005–1019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Holtzman MJ, Byers DE, Alexander-Brett J, and Wang X. 2014. The role of airway epithelial cells and innate immune cells in chronic respiratory disease. Nat. Rev. Immunol. 14:686–698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Nakanishi W, Yamaguchi S, Matsuda A, Suzukawa M, Shibui A, Nambu A, Kondo K, Suto H, Saito H, Matsumoto K, Yamasoba T, and Nakae S. 2013. IL-33, but not IL-25, is crucial for the development of house dust mite antigen-induced allergic rhinitis. PLoS One. 8:e78099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Prefontaine D, Nadigel J, Chouiali F, Audusseau S, Semlali A, Chakir J, Martin JG, and Hamid Q. 2010. Increased IL-33 expression by epithelial cells in bronchial asthma. J. Allergy Clin. Immunol. 125:752–754. [DOI] [PubMed] [Google Scholar]
  • 15.Kurowska-Stolarska M, Stolarski B, Kewin P, Murphy G, Corrigan CJ, Ying S, Pitman N, Mirchandani A, Rana B, van Rooijen N, Shepherd M, McSharry C, McInnes IB, Xu D, and Liew FY. 2009. IL-33 amplifies the polarization of alternatively activated macrophages that contribute to airway inflammation. J. Immunol. 183:6469–6477. [DOI] [PubMed] [Google Scholar]
  • 16.Byers DE, Alexander-Brett J, Patel AC, Agapov E, Dang-Vu G, Jin X, Wu K, You Y, Alevy Y, Girard JP, Stappenbeck TS, Patterson GA, Pierce RA, Brody SL, and Holtzman MJ. 2013. Long-term IL-33-producing epithelial progenitor cells in chronic obstructive lung disease. J. Clin. Invest. 123:3967–3982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Kamekura R, Kojima T, Takano K, Go M, Sawada N, and Himi T. 2012. The role of IL-33 and its receptor ST2 in human nasal epithelium with allergic rhinitis. Clin. Exp. Allergy. 42:218–228. [DOI] [PubMed] [Google Scholar]
  • 18.Zhiguang X, Wei C, Steven R, Wei D, Wei Z, Rong M, Zhanguo L, and Lianfeng Z. 2010. Over-expression of IL-33 leads to spontaneous pulmonary inflammation in mIL-33 transgenic mice. Immunol. Lett. 131:159–165. [DOI] [PubMed] [Google Scholar]
  • 19.Bessa J, Meyer CA, de Vera Mudry MC, Schlicht S, Smith SH, Iglesias A, and Cote-Sierra J. 2014. Altered subcellular localization of IL-33 leads to non-resolving lethal inflammation. J. Autoimmun. 55:33–41. [DOI] [PubMed] [Google Scholar]
  • 20.Zheng T, Zhu Z, Wang Z, Homer RJ, Ma B, Riese RJ Jr., Chapman HA Jr., Shapiro SD, and Elias JA. 2000. Inducible targeting of IL-13 to the adult lung causes matrix metalloproteinase- and cathepsin-dependent emphysema. J. Clin. Invest. 106:1081–1093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Kalari KR, Nair AA, Bhavsar JD, O’Brien DR, Davila JI, Bockol MA, Nie J, Tang X, Baheti S, Doughty JB, Middha S, Sicotte H, Thompson AE, Asmann YW, and Kocher JP. 2014. MAP-RSeq: Mayo Analysis Pipeline for RNA sequencing. BMC Bioinformatics. 15:224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Trapnell C, Pachter L, and Salzberg SL. 2009. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 25:1105–1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Liao Y, Smyth GK, and Shi W. 2014. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 30:923–930. [DOI] [PubMed] [Google Scholar]
  • 24.Robinson MD, McCarthy DJ, and Smyth GK. 2010. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 26:139–140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Kanehisa M, and Goto S. 2000. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28:27–30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Tichelaar JW, Lu W, and Whitsett JA. 2000. Conditional expression of fibroblast growth factor-7 in the developing and mature lung. J. Biol. Chem. 275:11858–11864. [DOI] [PubMed] [Google Scholar]
  • 27.Cayrol C, and Girard JP. 2018. Interleukin-33 (IL-33): A nuclear cytokine from the IL-1 family. Immunol. Rev. 281:154–168. [DOI] [PubMed] [Google Scholar]
  • 28.Bartemes KR, and Kita H. 2018. Innate and adaptive immune responses to fungi in the airway. J. Allergy Clin. Immunol. 142:353–363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Sokol CL, Barton GM, Farr AG, and Medzhitov R. 2008. A mechanism for the initiation of allergen-induced T helper type 2 responses. Nat. Immunol. 9:310–318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Hara K, Iijima K, Elias MK, Seno S, Tojima I, Kobayashi T, Kephart GM, Kurabayashi M, and Kita H. 2014. Airway uric acid is a sensor of inhaled protease allergens and initiates type 2 immune responses in respiratory mucosa. J. Immunol. 192:4032–4042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Holcomb IN, Kabakoff RC, Chan B, Baker TW, Gurney A, Henzel W, Nelson C, Lowman HB, Wright BD, Skelton NJ, Frantz GD, Tumas DB, Peale FV Jr., Shelton DL, and Hebert CC. 2000. FIZZ1, a novel cysteine-rich secreted protein associated with pulmonary inflammation, defines a new gene family. EMBO J. 19:4046–4055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Teng X, Li D, Champion HC, and Johns RA. 2003. FIZZ1/RELMalpha, a novel hypoxia-induced mitogenic factor in lung with vasoconstrictive and angiogenic properties. Circ. Res. 92:1065–1067. [DOI] [PubMed] [Google Scholar]
  • 33.Talabot-Ayer D, Martin P, Vesin C, Seemayer CA, Vigne S, Gabay C, and Palmer G. 2015. Severe neutrophil-dominated inflammation and enhanced myelopoiesis in IL-33-overexpressing CMV/IL33 mice. J. Immunol. 194:750–760. [DOI] [PubMed] [Google Scholar]
  • 34.Rackley CR, and Stripp BR. 2012. Building and maintaining the epithelium of the lung. J. Clin. Invest. 122:2724–2730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Coalson JJ 2003. Pathology of new bronchopulmonary dysplasia. Semin. Neonatol. 8:73–81. [DOI] [PubMed] [Google Scholar]
  • 36.Kwinta P, and Pietrzyk JJ. 2010. Preterm birth and respiratory disease in later life. Expert Rev. Respir. Med. 4:593–604. [DOI] [PubMed] [Google Scholar]
  • 37.Bry K, Whitsett JA, and Lappalainen U. 2007. IL-1beta disrupts postnatal lung morphogenesis in the mouse. Am. J. Respir. Cell Mol. Biol. 36:32–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Harijith A, Choo-Wing R, Cataltepe S, Yasumatsu R, Aghai ZH, Janer J, Andersson S, Homer RJ, and Bhandari V. 2011. A role for matrix metalloproteinase 9 in IFNgamma-mediated injury in developing lungs: relevance to bronchopulmonary dysplasia. Am. J. Respir. Cell Mol. Biol. 44:621–630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Vicencio AG, Lee CG, Cho SJ, Eickelberg O, Chuu Y, Haddad GG, and Elias JA. 2004. Conditional overexpression of bioactive transforming growth factor-beta1 in neonatal mouse lung: a new model for bronchopulmonary dysplasia? Am. J. Respir. Cell Mol. Biol. 31:650–656. [DOI] [PubMed] [Google Scholar]
  • 40.Cheon IS, Son YM, Jiang L, Goplen NP, Kaplan MH, Limper AH, Kita H, Paczesny S, Prakash YS, Tepper R, Ahlfeld SK, and Sun J. 2018. Neonatal hyperoxia promotes asthma-like features through IL-33-dependent ILC2 responses. J. Allergy Clin. Immunol. 142:1100–1112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Schittny JC 2017. Development of the lung. Cell Tissue Res. 367:427–444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Mahapatro M, Foersch S, Hefele M, He GW, Giner-Ventura E, McHedlidze T, Kindermann M, Vetrano S, Danese S, Gunther C, Neurath MF, Wirtz S, and Becker C. 2016. Programming of Intestinal Epithelial Differentiation by IL-33 Derived from Pericryptal Fibroblasts in Response to Systemic Infection. Cell Rep. 15:1743–1756. [DOI] [PubMed] [Google Scholar]
  • 43.Ishinaga H, Kitano M, Toda M, D’Alessandro-Gabazza CN, Gabazza EC, Shah SA, and Takeuchi K. 2017. Interleukin-33 induces mucin gene expression and goblet cell hyperplasia in human nasal epithelial cells. Cytokine. 90:60–65. [DOI] [PubMed] [Google Scholar]
  • 44.Tan QY, and Cheng ZS. 2018. TGFbeta1-Smad Signaling Pathway Participates in Interleukin-33 Induced Epithelial-to-Mesenchymal Transition of A549 Cells. Cell. Physiol. Biochem. 50:757–767. [DOI] [PubMed] [Google Scholar]
  • 45.Saluzzo S, Gorki AD, Rana BMJ, Martins R, Scanlon S, Starkl P, Lakovits K, Hladik A, Korosec A, Sharif O, Warszawska JM, Jolin H, Mesteri I, McKenzie ANJ, and Knapp S. 2017. First-Breath-Induced Type 2 Pathways Shape the Lung Immune Environment. Cell Rep. 18:1893–1905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.de Kleer IM, Kool M, de Bruijn MJ, Willart M, van Moorleghem J, Schuijs MJ, Plantinga M, Beyaert R, Hams E, Fallon PG, Hammad H, Hendriks RW, and Lambrecht BN. 2016. Perinatal Activation of the Interleukin-33 Pathway Promotes Type 2 Immunity in the Developing Lung. Immunity. 45:1285–1298. [DOI] [PubMed] [Google Scholar]
  • 47.Saravia J, You D, Shrestha B, Jaligama S, Siefker D, Lee GI, Harding JN, Jones TL, Rovnaghi C, Bagga B, DeVincenzo JP, and Cormier SA. 2015. Respiratory Syncytial Virus Disease Is Mediated by Age-Variable IL-33. PLoS Pathog. 11:e1005217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Olin A, Henckel E, Chen Y, Lakshmikanth T, Pou C, Mikes J, Gustafsson A, Bernhardsson AK, Zhang C, Bohlin K, and Brodin P. 2018. Stereotypic Immune System Development in Newborn Children. Cell. 174:1277–1292 e1214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Zhang X, Zhivaki D, and Lo-Man R. 2017. Unique aspects of the perinatal immune system. Nat. Rev. Immunol. 17:495–507. [DOI] [PubMed] [Google Scholar]

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Supplementary Materials

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