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 and Hirohito Kita
J Immunol October 1, 2019, 203 (7) 1952-1960; DOI: https://doi.org/10.4049/jimmunol.1900454

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.

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 ∼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

Interleukin-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 proinflammatory 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 (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 proinflammatory 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. In contrast, 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 club-cell secretory protein–Il33tg mice

The pTre-IL-13-hGH construct was kindly provided by Dr. J. 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 club-cell secretory protein (CCSP)–Il33tg mice, Tre-IL-33-hGH founders were bred with CCSP–reverse tetracycline–controlled transactivator (rtTA) transgenic mice (FVB.Cg-Tg(Scgb1a1-rtTA)1Jaw/J) purchased from The Jackson Laboratory. The nontransgenic 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. A. 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 doxycycline (Dox) (Mayo Pharmacy, Mayo Clinic, Rochester, MN) via drinking water (Dox was 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-Aldrich, 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 h (for IL-33 ELISA) or 4.5 h (for IL-5 and IL-13 ELISA) after one-time Alternaria exposure. Alternatively, mice were euthanized 3 h after one-time bromelain exposure. Finally, mice were exposed to bromelain on days 0, 2, and 4 and euthanized 24 h after the last exposure. All mice were euthanized by i.p. injection of pentobarbital, and bronchoalveolar lavage (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 Ig levels, ELISA plates were coated with Abs 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 H&E. To quantitate the size of alveoli, five randomly selected nonoverlapping photomicrographs were taken for each slide at ×160 magnification. The pictures were digitally incorporated into the ImageJ software (National Institutes of Health), and the alveolar size from 10 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. Ag retrieval was performed by heating the slides in citrate buffer (0.01 M, pH 6; Sigma-Aldrich) in a steamer for 30 min. The slides were then stained with polyclonal goat anti-mouse IL-33 (AF3626; R&D Systems) at 0.5 μg/ml, followed by AF488 rabbit F(ab′)2 anti-goat IgG (ab169344, 1:500 dilution; Abcam). 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) 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 radioimmunoprecipitation assay buffer containing protease inhibitor mixture (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 Fisher Scientific). Supernatants containing 2.5 mg of protein were incubated with a polyclonal goat anti-mouse IL-33 Ab (R&D Systems) overnight at 4°C, followed by the addition of protein A Dynabeads (DAKO, Denmark) for 1 h. 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 (Thermo Fisher Scientific), 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 two to four 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 Abs to CD45 and EpCam (eBioscience). Epithelial cells were isolated as the CD45−EpCamhi cell population by sorting on FACS (BD FACSAria). Total RNA was purified from sorted lung epithelial cells and sequenced by the Mayo Medical Genome Facility. The Mayo Analysis Pipeline for RNA Sequencing (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 (National Center for Biotechnology Information Gene Expression Omnibus [www.ncbi.nlm.nih.gov], accession number SRP198258). 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 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 rtTA protein, which is driven under control of the rat CCSP gene promoter, induces expression of transgenes in alveolar type 2 epithelial (AT2) cells of transgenic mice administered 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) (Fig. 1A).

FIGURE 1.
FIGURE 1.

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 (6–8 wk old) were treated with Dox for 1 wk. IL-33 protein in lung homogenates was analyzed by ELISA (B) and Western blot (C). rmIL-33, a commercially available recombinant mouse IL-33 (aa 109–266). (D) Mice were treated with Dox for 4 wk. 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 wk. 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 wk (Dox on) and then switched over to normal water for 2 d (Dox off). IL-33 protein levels in lung homogenates were analyzed by ELISA. Data are presented as means ± SEMs (n = 5 in each group). *p < 0.05, **p < 0.01 between the groups, indicated by horizontal lines. Data are representative of two to three independent experiments. n.d., not determined.

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 naive adult CCSP-Il33tg mice (i.e., 6–10 wk old) and their littermate controls (i.e., CCSP-rtTA mice, Tre-IL-33 mice, and nontransgenic 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 wk, adult CCSP-Il33tg mice showed an approximate 3-fold increase in lung levels of IL-33 protein as compared with control mice (Fig. 1B). In Western blot analyses, lung lysates of Dox-treated control mice showed a single band with a molecular mass of ∼30 kDa (Fig. 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 (aa 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 (Fig. 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 (Fig. 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 d of cessation of Dox administration (Fig. 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-wk-old adult CCSP-Il33tg mice were treated with Dox for 4 wk. 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 BAL fluids (Fig. 2A). Likewise, an analysis of BAL fluids revealed no signs of inflammation (Fig. 2B), and no increase in the levels of type 2 cytokines, including IL-5 and IL-13, was observed in BAL fluids (Fig. 2C). Histologic analysis of the lungs showed no signs of inflammation (Fig. 2D). Serum levels of Igs were comparable between Dox-treated CCSP-Il33tg mice and control mice (Fig. 2E). Adult CCSP-Il33tg mice that had been treated with Dox for a shorter period (i.e., 1 or 2 wk) 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.

Increased expression of IL-33 does not produce pathologic outcomes in adult mice. CCSP-Il33tg mice or control mice (6–8 wk old) were treated with Dox for 4 wk. (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 Igs 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 representative of three independent experiments. Eos, eosinophils; Lym, lymphocytes; Mac, macrophages; Neut, neutrophils.

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 wk 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 (Fig. 3A). Significantly higher amounts of IL-33 protein were detected in BAL fluids of Alternaria-exposed CCSP-Il33tg mice as compared with control mice (p < 0.05). When reexamined 4.5 h later, Alternaria-exposed control mice showed elevated lung levels of IL-5 and IL-13 as compared with PBS-exposed control mice (Fig. 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.

Increased expression of IL-33 promotes type 2 immunity in response to inhaled allergens in adult mice. CCSP-Il33tg mice or control mice (6–8 wk old) were treated with Dox for 1 wk. (A and B) Mice were exposed 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 h (A) or 4.5 h (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 d (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, **p < 0.01 between the groups, indicated by horizontal line. Data are representative 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 with similarly treated control mice (Fig. 3C). Furthermore, when exposed repeatedly to bromelain for 5 d, CCSP-Il33tg mice showed a significant increase in the number of eosinophils in BAL fluids as compared with control mice (Fig. 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 (Fig. 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) (Fig. 4B) and fewer lung lobes (i.e., ≤4 lung lobes, 19% of mice). Massive lung hemorrhaging was confirmed histologically (Fig. 4C). Among the CCSP-Il33tg mice that survived until 6 wk of age (n = 21), none showed hemorrhage and 38% showed fewer lung lobes. All of the other mice showed a simplified alveolar structure and enlarged alveolar space, consistent with the lung pathology of BPD in humans (Fig. 4D). The significant increase in alveolar size was confirmed by morphometric analysis (p < 0.01).

FIGURE 4.
FIGURE 4.

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 E0 to PN42. (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) H&E staining of lung sections from a control mouse and a CCSP-Il33tg mouse with lung hemorrhage. 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 a 3-y-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 (Fig. 5A). In contrast, when Dox was administered during the embryonic stage, all dead pups showed signs of lung hemorrhaging (Fig. 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 (Fig. 5B). 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.

The first 2-wk 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) H&E staining of lung sections 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 (Fig. 5C) roughly comparable to those in mice administered Dox for a prolonged period from E0 to PN42 (Fig. 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 wk 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 (Fig. 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 five times higher than those in controls (Fig. 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 (Fig. 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 (Fig. 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.

Extracellular IL-33 released from neonatal mice is likely responsible for lung pathology. (A) CCSP-Il33tg mice or control mice (n = 10 per 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–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 per group). (D) Survival of pups from six litters of ST2−/−CCSP-Il33tg mice and ST2−/− control mice was monitored until PN42 (n = 27–30 mice in each group). (E) H&E staining of lung sections 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 ∼4-fold increase in lung levels of IL-33 protein as compared with nontransgenic ST2−/− mice (i.e., ST2−/−CCSP-rtTA mice and ST2−/−Tre-IL-33 mice) (Fig. 6C). Nonetheless, none of the ST2−/−CCSP-Il33tg mice died within the first 6 wk of life (Fig. 6D). When euthanized on PN42, ST2−/−CCSP-Il33tg mice showed no signs of pathologic changes in the lungs (Fig. 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 (Fig. 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 (Fig. 7B).

FIGURE 7.
FIGURE 7.

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 per 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. (C–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 wk old) were treated with Dox for 1 wk 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 per group). *p < 0.05, **p < 0.01 between the groups, indicated by horizontal line. n.s., not significant.

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 (Fig. 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 (Fig. 7D). For example, expression of Retnla (which encodes resistin-like molecule α) 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 with control mice (Fig. 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 (Fig. 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 chronic obstructive pulmonary disease 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 current 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 current 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 current 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 multiorgan 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 current study released more IL-33 and exhibited robust type 2 immune responses once they were exposed to proteolytic allergens (Fig. 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 current 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 (Fig. 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 y in humans (34). Therefore, developing lungs, perhaps those in infants to children up to 2–3 y 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 alveolarization 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 been implicated, including IL-1β and IFN-γ (37–39). The results of the current 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 (Fig. 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 (Fig. 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.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We 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.

Footnotes

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

  • The sequencing data presented in this article have been submitted to the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number SRP198258.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    AT2
    alveolar type 2 epithelial
    BAL
    bronchoalveolar lavage
    BPD
    bronchopulmonary dysplasia
    CCSP
    club-cell secretory protein
    Dox
    doxycycline
    E
    embryonic day
    i.n.
    intranasally
    PN
    postnatal day
    rtTA
    reverse tetracycline–controlled transactivator.

  • Received April 22, 2019.
  • Accepted July 29, 2019.

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