Unique functions of the type II interleukin 4 receptor identified in mice lacking the interleukin 13 receptor α1 chain

Generation of Il13ra1^−/− mice

We used VelociGene technology¹⁰ to create a targeting vector in which exon 2 (except for its first 17 nucleotides) through exon 4 were replaced with the transmembrane lacZ reporter gene and a loxP-flanked neomycin selection cassette (Supplementary Fig. 1a online). We assigned scores for correct gene targeting in F₁H₄ (C57BL/6 × 129 hybrid) embryonic stem cell clones by the loss-of-native- allele assay¹⁰ and determined the genotypes of Il13ra1^+/+, Il13ra1^+/− and Il13ra1^−/− offspring by PCR analysis (Supplementary Fig. 1b). Il13ra1^−/− mice were healthy and fertile and manifested no physical impairment. Naive Il13ra1^−/− mice showed no fundamental anomalies in the lymphoid compartment; thymi, lymph nodes and spleens were of a size and cellularity similar to that of their Il13ra1^+/+ littermates (Supplementary Table 1 online). CD4⁺, CD8⁺ and CD4⁺CD8⁺ populations were present at the expected frequencies in the thymus (Fig. 1). B cells, CD4⁺ and CD8⁺ T cells, and natural killer cells were also present in wild-type proportions and numbers in the spleen and lymph nodes.

IL-13Rα1 in macrophage and fibroblast activation

Alternative macrophage activation is regulated by an IL-4Rα–STAT6– dependent mechanism¹¹; however, the function of type I versus type II IL-4R signaling in the development of classically and alternatively activated macrophages has remained unclear. To elucidate the contribution of the type II IL-4R, we generated bone marrow–derived macrophages (BMDMs) from Il13ra1^+/+ and Il13ra1^−/− mice. We then examined the ability of IL-4 and IL-13 to regulate a variety of responses, including phosphorylation of STAT6, expression of STAT6-regulated genes (Chi3l3 and Arg1), elaboration of arginase activity, and interferon-γ (IFN-γ)–induced nitric oxide production.

As expected, both IL-4 and IL-13 stimulated STAT6 phosphorylation in Il13ra1^+/+ BMDMs (Fig. 2a). In contrast, IL-4 but not IL-13 triggered STAT6 phosphorylation in Il13ra1^−/− BMDMs. Il13ra1^−/− mice showed no IL-13-induced increase in Chi3l3 or Arg1 mRNA (Fig. 2b) and, unlike Il13ra1^+/+ BMDMs, did not show substantial arginase activity after IL-13 simulation (Fig. 2c). We obtained similar results with thioglycollate-elicited macrophages, although Il13ra1^+/+ thioglycollate-elicited macrophages showed greater arginase activity at baseline and after IL-13 stimulation than did Il13ra1^+/+ BMDMs (Fig. 2d). Finally, IL-13 potently inhibited IFN-γ-induced nitric oxide production (classical macrophage activation) in Il13ra1^+/+ macrophages but had no effect on nitric oxide production by Il13ra1^−/− macrophages, even at very high concentrations (Fig. 2e). However, in contrast to IL-13, IL-4 exerted similar effects on Il13ra1^+/+ and Il13ra1^−/− macrophages.

IL-4 and IL-13 also regulate the function of nonhematopoietic mesenchymal cells, including fibroblasts¹². Nevertheless, it has remained unclear whether IL-4 and IL-13 exploit mainly type I or type II IL-4R signaling pathways in fibroblast activation^7,13,14. To address this, we generated primary fibroblasts from Il13ra1^+/+ and Il13ra1^−/− mice and monitored STAT6 phosphorylation and STAT6-dependent gene expression (Chi3l3, Retnla and Il13ra2) after stimulating the cells with IL-4 and IL-13. As expected, we found no detectable Il13ra1 message in Il13ra1^−/− fibroblasts. In contrast, transcripts encoding the common γ-chain (Il2rg) and IL-4Rα were readily detectable in the mutant cells; Il13ra1^+/+ fibroblasts expressed transcripts encoding all three receptor subunits (Supplementary Fig. 2a online). Expression of the common γ-chain by nonhematopoietic cells, although unexpected, has been shown before in myofibroblasts¹⁴ and human bronchial epithelial cells¹⁵. Consistent with those findings, there was IL-13-induced STAT6 phosphorylation in Il13ra1^+/+ but not Il13ra1^−/− fibroblasts (Supplementary Fig. 2b). The considerable increase in expression of Chi3l3, Retnla and Il13ra2 mRNA in IL-13-stimulated Il13ra1^+/+ fibroblasts was also abolished in Il13ra1^−/− fibroblasts, thus confirming the functional ablation of type II IL-4R signaling (Supplementary Fig. 2c). In contrast, IL-4 stimulated robust STAT6 phosphorylation and induction of Chi3l3 and Retnla mRNA in Il13ra1^+/+ and Il13ra1^−/− fibroblasts. Notably, we found consistently less IL-4-induced Il13ra2 mRNA expression in the Il13ra1^−/− fibroblasts, which suggested that at least a subset of IL-4-inducible genes depend on type II IL-4R signaling for optimal induction (Supplementary Fig. 2c). In contrast to Il13ra2 expression, expression of genes encoding the other receptor subunits was not substantially regulated by IL-4 or IL-13 (Supplementary Fig. 2d). Thus, the type II IL-4R signaling pathway was functionally disabled in Il13ra1^−/− mice and was dispensable for fibroblast and alternative macrophage activation.

IL-13Rα1 in S. mansoni–induced T_H2 responses

In addition to modulating the activation of macrophages and fibroblasts, IL-4 and IL-13 regulate B cell proliferation and survival and antibody class switching^16,17. To elucidate the function of the type II IL-4R in B cells, we infected Il13ra1^+/+ and Il13ra1^−/− mice with S. mansoni and assessed the parasite-specific antibody response in acutely infected mice (8–9 weeks) and chronically infected mice (12 weeks). Unexpectedly, although IL-13 has been postulated to promote B cell survival and antibody class switching¹⁸, in general, the infected Il13ra1^+/+ and Il13ra1^−/− mice developed similar antibody responses (Fig. 3). In fact, Il13ra1^−/− mice had more immunoglobulin A (IgA) at week 8 and slightly more IgG1 and IgG3 at week 12. IgE was the only isotype that was much lower in Il13ra1^−/− serum, but this defect was restricted to chronically infected mice. Thus, type II IL-4R signaling is minimally involved in the development of humoral responses after infection with S. mansoni.

Although it is well known that IL-4 functions as the main inducer of CD4⁺ T_H2 cell responses, the function of IL-13 in the development of T_H2 responses is controversial^16,19,20. Although IL-13 receptors are not expressed on T cells²¹, many studies with Il13^−/− mice have suggested that IL-13 has a critical albeit indirect function in the development of T_H2 cell–mediated responses in vivo²⁰. However, disruptions in Il13 can affect expression of the closely linked Il4 gene, perhaps complicating the conclusions of such studies¹⁹. Because the type II IL-4R functions as the main signaling receptor for IL-13, Il13ra1^−/− mice provide an ideal tool for investigating the function of IL-13 and, more specifically, the contribution of type II IL-4R–mediated signaling in T_H2 responses in vivo.

We infected mice with S. mansoni cercariae and examined liver lymphocyte production of IL-4, IL-5, IL-13 and IFN-γ ex vivo by intracellular cytokine staining at 9 and 12 weeks after infection. As expected, we detected many IL-4-, IL-5- and IL-13-producing CD4⁺ T cells in Il13ra1^+/+ livers at 9 weeks after infection (Fig. 4). However, the percentage of T_H2 cytokine–producing CD4⁺ T cells was significantly higher in Il13ra1^−/− livers at both the acute and chronic time points. These data suggest that IL-13Rα1 exerts a negative effect on T_H2 response development in vivo, in contrast to its T_H2-skewing effect on neonatal CD4⁺ cells²². Notably, we found no change in the number of IFN-γ-producing CD4⁺ T cells at week 9 and a small but consistent increase at week 12 in Il13ra1^−/− mice (Fig. 4). Thus, the greater frequency of CD4⁺ T_H2 cells did not result from a diminished counter-regulatory T_H1 response. These data were reproducible on both the BALB/c and C57BL/6 genetic backgrounds (data not shown).

IL-13Rα1 in schistosomiasis

Next we examined the granulomatous response in the liver at 9 and 12 weeks after infection. In general, we noted no considerable differences in granuloma development in the Il13ra1^+/+ and Il13ra1^−/− mice at any time point, suggesting that type II IL-4R signaling has little effect on the overall inflammatory response (Fig. 5a). However, we noted a modestly higher frequency of eosinophils in granulomatous lesions in Il13ra1^−/− mice (Fig. 5b), which was consistent with the higher IL-5 production in these mice (Fig. 4). Nevertheless, despite their wild-type inflammatory response, the development of hepatic fibrosis in the Il13ra1^−/− mice was significantly lower at both the acute and chronic time points (Fig. 5c). Notably, the attenuated fibrotic response was not attributed to differences in parasite burden (Supplementary Table 2 online).

To explore the mechanisms by which IL-13Rα1 regulates the pathogenesis of schistosomiasis, we isolated liver RNA at 9 and 12 weeks after infection and used real-time PCR to quantify the expression of genes associated with T_H2 responses (Fig. 6a), alternative macrophage activation (Fig. 6b) and extracellular matrix deposition (Fig. 6c). In agreement with the intracellular cytokine staining data, Il13ra1^−/− mice had much higher expression of transcripts encoding IL-13, IL-4 and IL-10; these findings confirmed the conclusion that the type II IL-4R functions as a negative regulator of T_H2 responses in vivo (Fig. 6a). We also noted much greater abundance of transcripts encoding tumor necrosis factor and near ablation of expression of eotaxin 1 (Ccl11) mRNA in Il13ra1^−/− mice, which suggested that type II IL-4R signaling regulates several distinct cytokine signals in the liver.

However, in contrast to the influence of IL-13Rα1 deficiency on the T_H2 response, we found no evidence that IL-13Rα1 regulated the expression of genes that characterize AAMϕ development in vivo (Fig. 6b), consistent with our in vitro functional studies with BMDMs (Fig. 2). Indeed, expression of genes encoding the mannose receptor, YM1, FIZZ1, AMCase and inducible nitric oxide synthase were significantly higher in Il13ra1^+/+and Il13ra1^−/− livers after infection. Thus, IL-13Rα1 is not critically involved in alternative macrophage activation in vivo.

Finally, we monitored the expression of several matrix-associated genes, including those encoding transforming growth factor-β1 (Tgfb1), matrix metalloproteinase 9 (Mmp9) and procollagen VI (Col6a1), which are all induced in the liver after S. mansoni infection^8,23. Consistent with the diminished fibrosis, Col6a1 expression was much lower in Il13ra1^−/− livers (Fig. 6c). In contrast, expression of Tgfb1 and Mmp9 was much higher in Il13ra1^−/− livers, particularly in the chronically infected mice. Thus, activation of the profibrotic cytokine TGF-β1 by MMP9 could have contributed to the residual increase in fibrosis noted in the Il13ra1^−/− mice.

Because the construction of the Il13ra1^−/− mouse included an in-frame insertion of a lacZ reporter gene, we measured β-galactosidase activity as a surrogate of Il13ra1 expression. We detected β-galactosidase activity in the villi, muscularis mucosae and muscularis externa of the gut in naive mice (Supplementary Fig. 3 online). However, the pattern of β-galactosidase expression in the gut changed very little after S. mansoni infection. In contrast, we noted little β-galactosidase activity in the liver before infection, although when we viewed sections under high power, we detected modest staining along the epithelial lining of the biliary tracts (Supplementary Fig. 3). Nevertheless, β-galactosidase expression was much higher in the liver after infection, with substantial staining concentrated in fibroblast-dense areas surrounding the granulomas.

Lower mortality in Il13ra1^−/− mice

We did survival studies with the S. mansoni model to determine the consequences of type II IL-4R deficiency during a chronic T_H2-driven inflammatory response. We included mice lacking both IL-4 and IL-13 (double-knockout mice) as controls, as published studies have shown that mice with deficiencies in both type I and type II IL-4R signaling pathways are highly susceptible to S. mansoni infection^24–27. As expected, by week 10, over 75% of the double-knockout mice succumbed to infection (Fig. 7a). In contrast, the wild-type group showed only 50% mortality by week 18. However, over 80% of the Il13ra1^−/− mice survived through week 22, demonstrating that the type II IL-4R was highly pathogenic during S. mansoni infection. The enhanced survival of the chronically infected Il13ra1^−/− mice was also associated with much less liver fibrosis, as determined by hydroxyproline assay (Fig. 7b) and by microscopy (Fig. 7c). In addition, markers of hepatocellular damage and biliary obstruction (alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase) were also significantly lower in the serum of chronically infected Il13ra1^−/− mice (Fig. 7d).

IL-13Rα1 deficiency impairs the expulsion of N. brasiliensis

To determine whether the type II IL-4R was critical for the development of immunity in the gut, we also infected Il13ra1^−/− mice with the gastrointestinal nematode parasite N. brasiliensis. In this model, infective-stage larvae are injected subcutaneously, immature parasites transit through the lungs, and adult parasites mature in the jejunum 5–6 d after infection. Immunity to N. brasiliensis depends on IL-4R and STAT6 (ref. ²⁸), and although IL-13 seems to be more important than IL-4 (ref. ²⁸), exogenous treatment with IL-4 can induce expulsion in the absence of IL-13 (ref. ¹). Ex vivo intracellular cytokine staining of mesenteric lymph node cells 12 d after infection showed that Il13ra1^−/− mice had twofold more IL-5-and IL-13-producing CD4⁺ cells than did their Il13ra1^+/+ littermates (Fig. 8a). However, despite showing stronger T_H2 responses, Il13ra1^−/− mice failed to expel the parasites, emphasizing the importance of the type II IL-4R in this process (Fig. 8b).

IL-13Rα1 is required for allergic airway hyperreactivity

Finally, we examined the function of the type II IL-4R in a mouse model of asthma²⁹. Il13ra1^+/+ mice primed and challenged intratracheally with S. mansoni egg antigen (SEA), a strong T_H2 stimulus, developed robust T_H2 responses in the lungs, as shown by quantitative PCR analysis of cytokine gene expression (Fig. 9a). However, as noted in the infection studies, Il13ra1^−/−mice developed enhanced lung T_H2 responses. Il13ra1^−/− mice showed no defect in induction of Arg1 expression (Fig. 9a), a marker of alternative macrophage activation; this finding suggests that the type II IL-4R is not critical for the development of Arg1-expressing cells in the lung, in agreement with the S. mansoni infection studies (Fig. 6b). Furthermore, analysis of bronchoalveolar lavage fluid suggested that IL-13Rα1 had little effect on the inflammatory response (Fig. 9b), although close examination of lung histology indicated that there were fewer eosinophils and more macrophages in the lungs of Il13ra1^−/− mice (Fig. 9c).

Nevertheless, despite developing substantial inflammation and stronger T_H2 responses, Il13ra1^−/− mice showed little to no increase in the expression of several important asthma-associated genes, including Ccl11, Clca3 and Muc5ac (Fig. 9a). IgE titers were also much lower in the serum of SEA-challenged Il13ra1^−/− mice (Fig. 9d). Most notable, however, was the complete absence of airway hyper-reactivity (Fig. 9e) and mucus hypersecretion (Fig. 9f,g) in Il13ra1^−/− mice, which indicated that the type II IL-4R was critically required for the development of allergic airway disease.

Generation of Il13ra1^−/− mice

VelociGene technology was used to generate the Il13ra1^−/− mice¹⁰ (Supplementary Fig. 1a). Heterozygous female offspring were repeatedly backcrossed to the C57BL/6 and BALB/c backgrounds. Because the Il13ra1 gene is located on the X chromosome, all male offspring carry either the mutant (50%) or wild-type (50%) allele. DNA obtained from tail tissue was genotyped (primers, Supplementary Table 3 online). Unless otherwise specified, all experiments used male wild-type and mutant littermates at the N₈ backcross on the BALB/c background. Mice were housed in a specific pathogen–free animal facility at National Institute of Allergy and Infectious Diseases. All experimental protocols were approved by the animal care and use committee of the National Institute of Allergy and Infectious Diseases.

Parasites and experimental infections

Mice were infected percutaneously with about 35 S. mansoni cercariae as described⁴⁶. Schistosome experiments used mice of generations N₄–N₈, with appropriate littermate controls of the same generation. Mice of these generations were indistinguishable in terms of each phenotype examined. N. brasiliensis larvae (L3) were prepared as described⁴⁷. Wild-type and mutant littermates (N₈; C57BL/6 background) were inoculated by subcutaneous injection of 500 L3.

Hepatic leukocyte isolation

About 200 mg of granulomatous liver was disrupted into a single-cell suspension by being ground through a 100-µm nylon mesh. Leukocytes were separated on a 34% (vol/vol) Percoll gradient (350g for 20 min). After being washed twice in RPMI medium, liver leukocytes were treated for 2 min with 2 ml ACK (ammonium chloride–potassium bicarbonate) lysis buffer to lyse erythrocytes.

Intracellular cytokine staining

Leukocytes isolated from granuloma or mesenteric lymph nodes were stimulated for 3 h with phorbol 12-myristate 13-acetate (10 ng/ml), ionomycin (1 µg/ml) and brefeldin A (10 µg/ml). Cells surfaces were stained with phycoerythrin–indodicarbocyanine–conjugated antibody to CD4 (anti-CD4; H129.19), were fixed for 20 min at 25 °C in 2% (wt/vol) formaldehyde, were made permeable for 30 min with 0.1% saponin buffer and were further stained with fluorescein isothiocyanate–conjugated anti-IFN-γ (XMG1.2) phycoerythrin-conjugated anti-IL-13 (C531; Centocor), Alexa Fluor 647–conjugated anti-IL-4 (11B11) and allophycocyanin-conjugated anti-IL-5 (TRFK5) before being analyzed on a FACSCalibur (Beckton Dickinson). Antibodies were from BD Pharmingen except where noted otherwise.

RNA isolation and real-time PCR

About 100 mg of liver tissue was preserved at −80 °C in 500 µl of RNAlater (Ambion). The sample was homogenized in 1 ml TRIzol reagent (Invitrogen) and total RNA was extracted and analyzed by real-time PCR as described²⁹. Primers were designed with Primer Express software (version 2.0; Applied Biosystems), PrimerBank (http://pga.mgh.harvard.edu/primerbank/) or ProbeFinder (version 2.3; https://www.roche-applied-science.com/sis/rtpcr/upl/adc.jsp). Primers for hypoxanthine guanine phosphoribosyl transferase, IL-4, IL-13, IL-10 (ref. ⁴²), IL-13Rα2 (ref. ⁸), YM1, FIZZ1 and IFN-γ⁴⁸ have been published (full list of primers, Supplementary Table 3).

Enzyme-linked immunosorbent assay

Immulon 2HB plates (Thermo) were coated overnight with SEA (10 µg/ml in PBS). After plates were blocked with 5% (wt/vol) nonfat dry milk (Carnation), serum was added at various dilutions beginning with a 1:10 dilution. The following secondary antibodies (Southern Biotech) were used for the detection of the corresponding isotypes: anti-IgG1 (H143.225.8), anti-IgG2b (LO-MG2b), anti-IgG3 (LO-MG3), anti-IgA (11–44.2), anti-IgE (23G3) and anti-IgM (1B4B1). Total serum IgE was measured with capture antibody to mouse IgE (R35-72) and biotinylated detection antibody to mouse IgE (R35-118), with a recombinant IgE standard curve (27–74).

Fibrosis, histopathology and β-galactosidase staining

Tissue were fixed in Bouin-Hollande fixative and were embedded in paraffin for sectioning. Sizes of hepatic granulomas and composition of lung inflammation were determined on histological sections stained with Wright’s Giemsa stain as described²⁹. Hepatic collagen was measured as hydroxyproline after hydrolysis of 200 mg liver in 5 ml of 6 N HCl. Goblet cells were stained with Alcian blue–periodic acid Schiff and were assigned scores of 1 through 4, where ‘0’ is no PAS⁺ staining and ‘4’ is the maximum staining noted. A published protocol was used for β-galactosidase staining⁴⁹. The same person assigned scores for all histological features and had no knowledge of the experimental design.

Immunoblot

Tyrosine-phosphorylated STAT6 were measured by immunoblot as described⁵⁰. After cytokine treatment, cells were washed three times with Dulbecco’s PBS and whole-cell lysates were prepared. Total STAT6 protein was immunoprecipitated with rabbit anti-STAT6 (SC-621; Santa Cruz Biotechnology). Immunoprecipitated proteins were resolved by 8% SDS-PAGE (Invitrogen) and then were transferred to polyvinylidene difluoride membranes. Tyrosine-phosphorylated STAT6 or total STAT6 was visualized by enhanced chemiluminescence with rabbit antibody to STAT6 phosphorylated at Tyr641 (93645; Cell Signaling Technology) or rabbit anti-STAT6, respectively.

Macrophage and fibroblast cultures

These cultures are described in the Supplementary Methods online.

Arginase activity and nitrite assays

BMDMs or thioglycollate-elicited macro-phages (day 4) were cultured in 48-well tissue culture plates and were stimulated with IL-4 or IL-13 (20 ng/ml). After being stimulated, cells were washed with PBS and were lysed with 0.1% (wt/vol) Triton X-100 containing protease inhibitor (Roche Diagnostics). Lysates were transferred to a 96-well PCR plate and were incubated for 10 min at 55 °C with 10 mM MnCl₂ and 50 mM Tris HCl, pH 7.5, for enzyme activation. Then, 25 µl lysate was removed and was added to 25 µl of 1 M arginine, pH 9.7, in a new PCR plate, followed by incubation for 20 h at 37 °C. A portion (5 µl) of each sample was added in duplicate to a 96-well enzyme-linked immunosorbent assay plate along with 5 µl of each standard, diluted in the same assay conditions, beginning with 100 mg/dl. The urea determination reagent from the BioAssay Systems Quantichrome Urea Assay Kit was used according to the manufacturer’s protocol. The concentration of nitrite in culture supernatants was determined by spectrophotometry with the Griess reagent.

Allergic airway inflammation and hyperreactivity

Mice were primed and boosted by intraperitoneal injection of 10 µg SEA in PBS on days 0 and 7, respectively. On days 14, 16 and 18, SEA-primed mice were anesthetized with a ketamine-xylazine ‘cocktail’ and were challenged by intratracheal instillation of 10 µg of SEA in 30 µl PBS. Then, 24 h after the last challenge, the airway hyperreactivity of unrestrained mice in response to aerosolized methacholine (3–25 mg/ml in PBS; Sigma-Aldrich) was measured by noninvasive whole-body plethysmography (Buxco systems). Each dose of methacholine was aerosolized for 2 min, followed by 4 min of data collection during which enhanced-pause measurements were collected and averaged for the entire dose period. Mice were killed by pentobarbital overdose and broncholveoloar lavage was done with PBS and an Insyte venous catheter (BD) to cannulate the trachea.

Statistics

All data were analyzed with Prism (Version 4; GraphPad). Data were considered statistically significant for P values less than 0.05, obtained with a two-tailed t-test.

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