Immunity Mediators in Intestinal Mucosa Hyperplasia and Up-Regulation of Innate IL-9 Promotes IL-13-Dependent Paneth Cell

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IL-9 Promotes IL-13-Dependent Paneth Cell Hyperplasia and Up-Regulation of Innate Immunity Mediators in Intestinal Mucosa

This information is current as Valérie Steenwinckel, Jamila Louahed, Muriel M. Lemaire, of September 29, 2021. Caroline Sommereyns, Guy Warnier, Andrew McKenzie, Frank Brombacher, Jacques Van Snick and Jean-Christophe Renauld J Immunol 2009; 182:4737-4743; ;

doi: 10.4049/jimmunol.0801941 Downloaded from http://www.jimmunol.org/content/182/8/4737

References This article cites 52 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/182/8/4737.full#ref-list-1 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-9 Promotes IL-13-Dependent Paneth Cell Hyperplasia and Up-Regulation of Innate Immunity Mediators in Intestinal Mucosa1

Vale´rie Steenwinckel,*† Jamila Louahed,*† Muriel M. Lemaire,*† Caroline Sommereyns,*† Guy Warnier,* Andrew McKenzie,‡ Frank Brombacher,§ Jacques Van Snick,*† and Jean-Christophe Renauld2*†

IL-9 contributes to lung inflammatory processes such as asthma, by promoting mast cell differentiation, B cell activation, eosinophilia, and mucus production by lung epithelial cells. The observation that IL-9 overexpressing mice show increased mast cell numbers in the intestinal mucosa suggests that this cytokine might also play a role in intestinal inflammation. In colons from IL-9 transgenic mice, the expression of Muc2, a major intestinal mucin gene, was up-regulated, together with that of CLCA3 chloride Downloaded from channel and resistin like ␣, which are goblet cell-associated genes. Additional IL-9 up-regulated genes were identified and included innate immunity genes such as angiogenin 4 and the PLA2g2a phospholipase A2, which are typical Paneth cell markers. Histo- chemical staining of Paneth cells by phloxine/tartrazine showed that IL-9 induces Paneth cell hyperplasia in Lieberku¨hn glands of the small intestine, and in the colonic mucosa, where this cell type is normally absent. Expression of Paneth cell markers, including angiogenin 4, PLA2g2a, and cryptdins, was induced in the colon of wild-type mice after two to four daily administrations /of IL-9. By crossing IL-9 transgenic mice with IL-13؊/؊ mice, or by injecting IL-9 into IL-4R؊/؊ mice, we showed that IL-13 was http://www.jimmunol.org required for the up-regulation of these Paneth cell-specific genes by IL-9. Taken together, our data indicate that Paneth cell hyperplasia and expression of their various antimicrobial products contribute to the immune response driven by TH2 cytokines, such as IL-9 and IL-13 in the intestinal mucosa. The Journal of Immunology, 2009, 182: 4737–4743.

he lung and gastrointestinal mucosae are both com- including mucus production, which is a hallmark of nematode posed of a single layer of interconnected epithelial infections and allergic asthma. Such conditions can be mim- cells, which form a ﬁrst line of defense against external icked by overexpression of TH2 cytokines such as IL-9 and T 3 aggressions by outer microorganisms. Depending on further dif- IL-13 in the lungs. Indeed, IL-9 transgenic (Tg) and IL-13 Tg by guest on September 29, 2021 ferentiation, epithelial cells directly contribute to mucosal im- mice show increased mucus production by lung goblet cells (1, munity either mainly by establishing a physical barrier, or by 2) and airway hyperresponsiveness (2–4). Further studies of producing a large spectrum of bioactive molecules. Goblet cells IL-9 Tg mice showed that IL-9 activities on lung epithelial cells are responsible for mucus production, which confers a physical are actually mediated by up-regulation of IL-13 expression by protection by trapping pathogens and interfering with their ad- T and mast cells (5–7). hesion to the mucosa. In contrast, Paneth cells, which are In the gastrointestinal mucosa, IL-13 and IL-9 production sim- mainly located in the small intestine, are specialized in the pro- ilarly contribute to the resistance against nematode infections (8– duction of bactericidal peptides (␣ defensins or cryptdins, ly- 10). Although the actual effector mechanisms by which TH2-po- sozyme, phospholipase A2, and angiogenin 4). larized immune responses allow for parasite expulsion are not Like most effector cells from the innate immune system, lung completely elucidated, epithelial cells seem to be actively involved and intestinal epithelial cells act as an efferent arm of adaptive in this process, for instance as a result of accelerated cell turnover immune responses. In particular, cytokine production by TH2 (11). Paneth cell hyperplasia is also associated with nematode in- lymphocytes is well known to affect epithelial cell physiology, fections (12, 13). Although there is no evidence that intestinal epithelial cells respond to IL-9, the similarities between the lung and intestinal mucosae, as well as the fact that IL-9-induced mas- *Ludwig Institute for Cancer Research, Brussels Branch, Brussels, Belgium; †Exper- imental Medicine Unit, de Duve Institute, Universite´Catholique de Louvain, Brus- tocytosis seemed mainly restricted to these tissues, prompted us to sels, Belgium; ‡Medical Research Council Laboratory of Molecular Biology, Hills investigate the potential activity of IL-9 on colonic epithelial cells Road, Cambridge, United Kingdom; and §University of Cape Town, Health Science Faculty, Institute of Infectious Disease and Molecular Medicine, Cape Town, South in vivo. In this study, we show that IL-9 up-regulates the expres- Africa sion of innate immunity genes including Paneth cell markers such

Received for publication June 16, 2008. Accepted for publication February 3, 2009. as angiogenin 4, cryptdins, and phospholipase A2 genes, and that The costs of publication of this article were defrayed in part by the payment of page this activity requires IL-13. Histochemical staining confirmed that charges. This article must therefore be hereby marked advertisement in accordance IL-9 induces Paneth cell hyperplasia in small intestine and in with 18 U.S.C. Section 1734 solely to indicate this fact. colon. 1 This work was supported in part by the Belgian Federal Service for Scientific, Technical, and Cultural Affairs, by the Actions de Recherche Concerteés of the Com- munaute´Française de Belgique and the Ope´ration Te´le´vie. 3 Abbreviations used in this paper: Tg, transgenic; WT, wild type; PLA2, phospho- 2 Address correspondence and reprint requests to Dr. Jean-Christophe Renauld, Lud- lipase A2; mMCP, mouse MCP; mCLCA3, mouse calcium-activated chloride channel wig Institute for Cancer Research and Experimental Medicine Unit, Universite´ 3; Ang4, angiogenin 4; Retnla, resistin-like ␣. catholique de Louvain, Avenue Hippocrate 74, Brussels, Belgium. E-mail address: [email protected] Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0801941 4738 IL-9-INDUCED PANETH CELL EXPANSION IN INTESTINAL MUCOSA

Materials and Methods Mouse MCP (mMCP)-1 ELISA Mice Serum mMCP-1 concentrations were measured in duplicate on 96-well IL-9-Tg mice, obtained in the FVB/N background and expressing high microtiter plates using a commercial mMCP-1 kit (Moredun Scientific) levels of IL-9 in all organs, were described previously (14). IL-13-deficient according to the manufacturer’s instructions. mice (129 ϫ C57BL/6 genetic background) were obtained by insertion of RT-PCR a cassette containing a LacZ reporter gene and a neomycin resistance gene into exon 1 of the IL-13 locus, as described previously (15). IL-9 Tg mice Distal colons and small intestine were removed and submerged in RNA Ϫ/Ϫ were bred with IL-13 knock-out mice (IL-13 ) to generate F1 mice that later (Ambion) between 12 or 48 h. Lungs were frozen in liquid nitrogen. were backcrossed for one generation with IL-13Ϫ/Ϫ mice. Mice were Total RNA was isolated from tissues using TriPure isolation reagent screened by PCR on genomic DNA for the IL-13 gene and with a TS1 (Roche) according to the manufacturer’s instructions. Reverse transcription bioassay for circulating IL-9. The sequences of the primers were: IL-13 was performed on 2 ␮g of total RNA with an oligo(dT) primer (Roche) and WT: 5Ј-GGGTGACTGCAGTCCTGGCT-3Ј (forward) and 5Ј-GTTGCTC M-MLV RT (Invitrogen). PCR amplifications were performed from cDNA AGCTCCTCAATAAGC-3Ј (reverse); IL-13 Ϫ/Ϫ:5Ј-GGCGGATGAGC corresponding to 20 ng of total RNA. Quantitative PCR were performed GGCATTTTCCGTG-3Ј (forward) and 5Ј-GCCGAAAGGCGCGGTGC using primers sets corresponding to murine angiogenin 4 (Ang4), phos- Ј ␣ Ϫ/Ϫ ␣ CGCTGGC-3 (reverse). The IL-4R -deficient (IL-4R ) and IL-4 pholipase A2 g4c (PLA2g4c), PLA2g2a, resistin-like (Retnla), cryptdins, deficient (IL-4Ϫ/Ϫ) mice were generated as described (16), and have been mMCPs, and ␤-actin with qPCR Mastermix for SYBR Green I (Eurogen- backcrossed to C57BL/6 for nine generations. IL-9RϪ/Ϫ mice were gen- tec). The sequences of the primers (final concentration: 300 nM) were: erated in the C57BL/6 background by deletion of the exons 2–6 of the Ang4: 5Ј-CTCTGGCTCAGAATGTAAGGTACGA-3Ј (forward) and 5Ј- IL-9R␣-chain, corresponding to the extracellular part of the receptor as GAAATCTTTAAAGGCTCGGTACCC-3Ј (reverse); PLA2g4c: 5Ј-CAC described in Ref. 5. For the current study, IL-9RϪ/Ϫ mice were backcrossed TCCCTATCCACTTGTTCTGCC-3Ј (forward) and 5Ј-GAACAGAGTAA for five generations onto BALB/c background. FVB and IL-9Tg mice were AGTGCATGACAACAG-3Ј (reverse); PLA2g2a: 5Ј-GCTGACAGCAT Ј Ј Ј Downloaded from bred with BALB/c mice to obtain F1 mice (respectively designated FVB/c GAAGGTCCTC-3 (forward) and 5 -CATTCAGCGGCGGCTTTA-3 and IL-9 Tg/c) used as cell donors for transfer experiments. (reverse); Retnla: 5Ј-GCCCCAGGATGCCAACTTTGA-3Ј (forward) and 5Ј-CTCCATTCTGGATCTCCCAAG-3Ј (reverse); cryptdins (primers de- Microarray and subtractive hybridization experiments signed to simultaneously amplify all 17 cryptdins except cryptdin 4 and 5): 5Ј-GAGGAATCGTTGAGAGATCTGGTATG-3Ј (forward) and 5Ј-CCA Total RNA was extracted from the distal colon of four FVB or IL-9 Tg TGTTCAGCGACAGCAGAGC Ϫ3Ј (reverse); mMCPs (primers designed mice using TriPure isolation reagent (Roche). Four micrograms of total to amplify mMCP-1, Ϫ2, and Ϫ4): 5Ј-GCTGGAGCTGAGGAGATT-3Ј Ј Ј ␤

RNA, pooled for each mouse group, were amplified according to the One- (forward) and 5 -GGTGAAGACTGCAGGGG-3 (reverse); and -actin: http://www.jimmunol.org/ cycle cDNA Synthesis Kit from Affymetrix. The expression of 14,000 5Ј-TCCTGAGCGCAAGTACTCTGT-3Ј (forward) and 5Ј-CTGATCCAC genes was assessed on the GeneChip mouse expression Array 430A. Stain- ATCTGCTGGAAG-3Ј (reverse). Samples were first heated 10 min at ing, washing, and scanning procedures were conducted as described in the 95°C. cDNA was amplified as follows: 40 cycles of a two-step PCR pro- GeneChip Expression Analysis technical manual (Affymetrix). All inten- gram at 95°C for 15 s and 60°C for 1 min (63°C for PLA2g4c), except for sity values were scaled to an average value of 100 per GeneChip according mMCPs for which the hybridization and elongation steps were performed to the method of global scaling, or normalization, provided in the Af- respectively at 65°C for 30 s and at 72°C during 1 min. Melting point fymetrix Microarray Suite software, version 5.0 (MAS5.0). Differential analysis was conducted by heating the amplicon from 60–65°C to 95°C. expression between two arrays was then calculated with the GeneChip Muc2 qPCR was performed with the Premix Ex Taq from TaKaRa using Operating software from Affymetrix. Complete microarray data have been SYBR Green I (Eurogentec). The sequences of the primers (final concen- deposited in the GEO public repository under the series record GSE14249. tration: 300 nM) were: 5Ј-TGAGAAGAACGATGCCTACA-3Ј (forward)

For representational difference analysis experiments, pooled total RNA and 5Ј-AGGCTTGTTGATCTTCTGCA-3Ј (reverse). The samples were by guest on September 29, 2021 was prepared from colons of five FVB and five IL-9 Tg mice, using gua- first heated at 95°C for 30 s and then amplified with 45 cycles of a three- nidium isothiocyanate lysis and CsCl gradient centrifugation. Polyadenyl- step PCR program at 95°C for 10 s, 60°C for 15 s, and 72°C for 15 s, ated RNA was purified from total RNA with oligo(dT) cellulose columns followed by a melting point analysis. (Pharmacia). Double-stranded cDNA was generated from 5 ␮g poly(A)ϩ Semiquantitative PCR for IL-13 and ␤-actin were performed with the RNA using oligo(dT) primer and the SuperScript Choice System for cDNA TaKaRa Mix for PCR. The sequences of the primers (final concentration: synthesis, according to the manufacturer’s recommendations (Life Tech- 200 nM) were: ␤-actin: 5Ј-ATGGATGACGATATCGCTGC-3Ј (forward) nologies). Representational difference analysis was performed as described and 5Ј-GCTGGAAGGTGGACAGTGAG Ϫ3Ј (reverse) and IL-13: 5Ј-TGG by Hubank and Schatz (17). After three rounds of subtraction, final differ- GTGACTGCAGTCCTGGCT-3Ј (forward) and 5Ј-GTTGCTTTGTGTA ence products were digested with DpnII and cloned into the BamHI site of GCTGAGCA-3Ј (reverse). cDNA was amplified as follows: 18 cycles (␤- pTZ19R. Double-stranded plasmid DNA was prepared and sequenced with actin) or 36 cycles (IL-13) of a three-step PCR program at 95°C for 1 min, a Thermo-sequenase Sequencing kit (Amersham Biosciences). Sequence 57°C for 1 min, and 72°C for 2 min. comparisons with GenBank and EMBL databases were performed with the BLAST search program (National Center for Biotechnology Information). Northern blot Tissue expression of mCLCA3/gob5 was determined by Northern blot In vivo treatments analysis of RNA extracted from colons using the TriPure isolation reagent ϩ Ϫ Ϫ Ϫ Ϫ (Roche). Five micrograms of poly(A) mRNA were fractionated by elec- C57BL/6, IL-4 / , and IL-4R / mice were injected i.p. with IL-9 (200 trophoresis in 1.3% agarose gels containing 2.2 mol/L formaldehyde and ng), which had been purified from the serum of IL-9 Tg mice using a rat blotted onto Hybond-C Extra membranes (Amersham Biosciences). Filters anti-mouse IL-9 Ab and ion exchange chromatography. Four days later, the were hybridized to mCLCA3/gob5 probes, 32P-labeled by using Rediprime mice were given a second i.p. injection (200 ng) and were sacrificed the II labeling kit (Amersham Biosciences). ␤-Actin probes were used as con- following day. Control mice received the same volume of buffer (PBS plus trols for equal loading. Phosphorimager or InstantImager quantifications 1% mouse serum) as used for cytokine injections. In the experiments with were used to determine the level of signal. FVB mice, the animals received two or four daily injections, and were sacrificed the day following the last injection. Histochemical staining Cell transfer Distal colons were fixed with buffered formalin and embedded in paraffin. For Paneth cell-specific staining, 7-␮m sections were cut, dewaxed, and Bone marrow was aseptically flushed from femora and tibias of FVB/c or rehydrated. Nuclei were stained with hemalun for 45 s. After a brief wash IL-9 Tg/c 10- to 12-wk-old mice. Cells were resuspended and filtered in water, sections were stained in phloxine solution (0.5 g of phloxine B through a 70-␮m nylon cell strainer. Red cells were removed by osmotic and 0.5 g of calcium chloride in 100 ml of distilled water) for 20 min, shock. Hematopoietic cells were washed, counted, and resuspended in successively rinsed in water and in Cellosolve (2-ethoxy ethanol, Aldrich), DMEM for injection. For cell transfer experiments, IL-9RϪ/Ϫ recipient and differentiated with a saturated tartrazine solution (2.5 g of tartrazine in mice were exposed to two doses of 550 rad of ionizing radiation, given 3 h 100 ml of Cellosolve) for 4–6 min. After rinsing and mounting, sections apart, followed by i.v. injection of 4 ϫ 106 bone marrow cells isolated from were evaluated by light microscopy wherein Paneth cells granules appear mice. Cells were injected in a volume of 100 ␮l of sterile DMEM. Control in red. To assess mucus production, slides were stained with Alcian blue (5 recipient mice received an equivalent volume of medium. Mice were killed min), periodic acid (10 min), Schiff reagent (20 min), and counterstained 3 wk after the transfer. with hematoxylin (30 s). The Journal of Immunology 4739 Downloaded from

FIGURE 1. A functional IL-13 gene is required for IL-9-induced Muc2 and mCLCA3 expression in the colon. Distal colon total RNA was ex-

tracted either from IL-9 Tg or from FVB mice (A) or from IL-9 Tg, IL- http://www.jimmunol.org/ 13Ϫ/Ϫ, IL-9Tg/IL13Ϫ/Ϫ, and control littermates (B and C). A, RT-PCR for IL-13 and ␤-actin. Each lane corresponds to one mouse. B, Quantitative RT-PCR for Muc2 normalized with ␤-actin. Results were expressed as mean values and SEM (n ϭ 4–5). C, Northern blot analysis of mCLCA3/ gob5 expression was quantified by InstantImager analysis of blots hybrid- FIGURE 2. Angiogenin 4 basal expression and induction by IL-9 re- ized with a mCLCA3 probe, before reprobing with ␤-actin as a control for quire IL-13. A, Quantitative RT-PCR for Ang4 was performed on colon -p Յ 0.0286; Mann-Whitney U test. total RNA from IL-9Tg, IL-13 Ϫ/Ϫ, IL-9Tg/IL13Ϫ/Ϫ, and control litter ,ء .equal loading mates. B, Total RNA was extracted from colon and small intestine of WT and IL-13Ϫ/Ϫ mice, and Ang4 expression was measured by quantitative by guest on September 29, 2021 For immunostaining, euthanized mice were perfused with PBS. Freshly RT-PCR. C, IL-4Ϫ/Ϫ and IL-4R␣Ϫ/Ϫ mice received two i.p. injections of collected colons were immersed in buffered formaldehyde for 24 h at room IL-9 (200 ng) or sterile PBS, 5 days and 1 day before the analysis of Ang4 temperature and embedded in paraffin. Tissue sections of 7 ␮m in thickness expression by quantitative RT-PCR on colon and small intestine total RNA were cut, placed on SuperFrost Plus slides, dried at 37°C overnight, and (upper and middle panel). Results were normalized with ␤-actin. Serum processed by standard methods for immunohistochemistry. In brief, sec- MMCP-1 concentrations were measured by ELISA (lower panel). D, tions were deparaffinized, permeabilized for 10 min in PBS containing Quantitative RT-PCR for Ang4 was performed on small intestine of total 0.1% Triton X-100, treated for 5 min in PBS containing 1.5% H O to 2 2 Ϫ/Ϫ ϫ 6 block endogenous peroxidase activity, and washed in PBS. Sections were RNA from irradiated IL-9R mice reconstituted with 4 10 bone mar- treated for 90 min at 97°C in sodium citrate buffer 0.01 M (pH 5.8) to row cells from WT or IL-9 Tg mice. Mice were sacrificed 3 wk after the unmask Ags. Blocking was performed by incubating sections for 30 min transfer of cells. Mean values and SEM were calculated from at least four .p Յ 0.05 ,ء .with TNB (PerkinElmer, TSA Fluorescein system, NEL70). Immunolabel- mice of each group ing was done in blocking solution containing the Abs. PLA2g2a was detected with a goat Ab at a dilution of 1/50 (Santa Cruz Biotechnology, sc-14472). The secondary Ab (at 1/800) was a donkey anti-goat Ab IgG- is a major component of the intestinal mucus, in colons from IL-9 HRP (Santa Cruz Biotechnology, sc-2033). The immonustaining was re- Tg and IL-9 Tg/IL-13 knockout mice. As shown in Fig. 1B, IL-9 vealed with DAB substrate reaction (Brown staining) (Roche, ref.1 718 up-regulates Muc2 expression in colons from heterozygous but not 096), and the sections were stained with Hemalun (45 s, blue staining). from IL-13Ϫ/Ϫ mice, in line with IL-9 effect on the lung Muc5AC Statistical analysis mucin gene (5). A similar induction was observed for mCLCA3 Statistical significance was analyzed using the Instat3 program. Mann- (Fig. 1C), a gene whose expression correlates with mucus produc- Whitney U test or unpaired Student’s t test were run to determine the tion in the lungs (18). p value when comparing two groups. Differences with p Ͻ 0.05 were consid- ered statistically significant. Values were presented as means Ϯ SEM. IL-9 induces angiogenin 4 expression in an IL-13-dependent way To identify additional intestinal epithelial genes regulated by IL-9, we Results performed a representational difference analysis experiment using IL-9 increases IL-13 and mucus production in the intestinal RNA from the colon of IL-9 Tg or normal mice. After three rounds of mucosa cDNA subtraction, four genes were identified, including two mast IL-9 overexpression was previously shown to induce mucus pro- cell-specific genes, namely mMCP-2 and Fc␧RI␣, and two epithelial duction and to up-regulate the expression of various mucus-asso- genes, namely IgGFc Binding Protein (Fcgbp) and angiogenin 4 ciated genes by lung epithelial cells, and this effect of IL-9 was (Ang4). Fcgbp, a protein containing cystein-rich mucin-like domains, mediated by IL-13 (5–7). As shown in Fig. 1A, IL-13 expression is expressed by mucin-secreting cells (19) and was recently shown to was also up-regulated in the colons from IL-9 transgenic mice. To be induced by IL-13 in lung epithelial cells (20). In line with this determine whether mucus production was similarly up-regulated observation, IL-9 administration up-regulated Fcgbp expression in the by IL-9 in this tissue, we analyzed the expression of Muc2, which colon of wild-type (WT), but not IL-4RϪ/Ϫ mice (data not shown). 4740 IL-9-INDUCED PANETH CELL EXPANSION IN INTESTINAL MUCOSA

FIGURE 3. Induction of PLA2g4c and PLA2g2a by IL-9 in the colon. Quantitative RT-PCR was performed on colon total RNA from WT and IL-9 Tg mice for PLA2g4c (A) or for PLA2g2a (B). Results were normalized with ␤-actin. The data were expressed as mean values and SEM (n ϭ 4–8). Differences between IL-9 Tg and wild-type mice were statistically signiﬁcant (p Յ 0.0286; Mann-Whitney U test).

Ang4 is mainly produced by Paneth cells, belongs to the RNase A superfamily, and has angiogenic and bactericidal activities (21–23). As shown on Fig. 2A, Ang4 expression in the colon is up-regulated in Downloaded from IL-9 Tg mice in an IL-13-dependent way. Interestingly, Ang4 basal expression in colon, but not in small bowel, was dramatically lower in IL-13Ϫ/Ϫ mice as compared with wild-type mice, even in the absence of IL-9 overexpression (Fig. 2B). This observation indicates that IL-13 is required for constitutive expression of Ang4 in colon, prob- ably in response to commensal microﬂora, which was previously http://www.jimmunol.org/ shown to induce the expression of this gene (23). Similar results were observed with mice deﬁcient in IL-4R␣, which is involved in responses to IL-4 and IL-13. These mice showed a decrease of basal Ang4 expression in the colon, but not in the small bowel, and no up-regulation of Ang4 in response to IL-9 administration (Fig. 2C). mMCP-1 concentrations were measured as a positive control for IL-9 FIGURE 5. IL-9 induces hyperplasia of Paneth cells in intestinal mucosa. A–D, Representative sections of colon (B and D) and duodenum (A and C) from control FVB (WT) and IL-9 Tg mice stained with hemalun, phloxine B,

and tartrazine. Sections were examined by light microscopy (ϫ40 objective). by guest on September 29, 2021 Paneth cells granules are stained in red, and are indicated by arrows when they are located at the base of the crypts or by arrowheads when located nearby the apex of the glands. E–H, PLA2g2a expression detected in colon by immunohistochemistry in wild-type (E and F) or in IL-9 Tg mice (G and H) (magni- ﬁcation, ϫ20). E and G, Longitudinal sections. F and H, Transversal sections.

activity in IL-4RϪ/Ϫ mice as IL-9 acts directly on mast cells (24). By contrast, in IL-4Ϫ/Ϫ mice, IL-9 up-regulated Ang4 expression both in the colon and small bowel. Taken together, these observations confirm that IL-13, but not IL-4, regulates Ang4 expression via the IL- 4R␣-chain, and that IL-13 is required for basal Ang4 expression only in colon, but for Ang4 induction by IL-9 both in colon and small bowel. To confirm that the effect of IL-9 on Ang4 expression is in- direct and does not require any IL-9R expression by epithelial cells, adoptive transfer experiments of IL-9 Tg or wild-type bone marrow cells were performed into IL-9RϪ/Ϫ mice. As shown in Fig. 2D, Ang4 expression was markedly increased upon transfer of IL-9-producing cells into IL-9R-deficient animals, indicating that IL-9 acts primarily on hematopoietic cells and that expression of its receptor by epithelial cells is not required for Ang4 induction.

IL-13 is required for phospholipase A2 induction by IL-9 A microarray experiment was performed to further identify differ- FIGURE 4. Role of IL-13 in PLA2g4c and PLA2g2a basal expression entially expressed genes in the colon of IL-9 Tg or wild-type mice. and induction by IL-9. WT and IL-4R␣Ϫ/Ϫ mice received i.p. injections of Beside mast cell-specific genes such as mMCP-1, Ϫ2, Ϫ4, and IL-9 (200 ng) or sterile PBS, 5 and 1 day before analysis of PLA2g4c ␧ (upper panel) and PLA2g2a (middle panel) expression by quantitative RT- Fc RI, B cell-specific genes such as the Ig heavy and light chains, PCR on colon total RNA. Results were normalized with ␤-actin. Serum CD19, and CD79, two members of the phospholipase A2 (PLA2) MMCP-1 concentrations were measured by ELISA (lower panel). Mean family were found to be up-regulated by IL-9, namely PLA2g2a and PLA2g4c. PLA2s hydrolyze glycerophospholipids to produce ,ء .values and SEM were calculated from four to five mice of each group p Յ 0.03; Mann-Whitney U test. free fatty acids, such as arachidonic acid and lysophospholipids The Journal of Immunology 4741

FIGURE 7. Lung expression of IL-9-induced PLA2g4c is IL-13-dependent. Total RNA was extracted from lungs of control littermates, IL-9Tg, IL- 13Ϫ/Ϫ, and IL-9Tg/IL13Ϫ/Ϫ mice and quantitative RT-PCR was performed on RNA for PLA2g4c. Results were normalized with ␤-actin and expressed as .p Յ 0.0286; Mann-Whitney U test ,ء .(mean values and SEM (n ϭ 4–5

PLA2G2A Abs showed a very similar pattern of expression and correlated nicely with phloxine/tartrazine staining (Fig. 5, E–H).

Kinetics of Paneth cell marker up-regulation by IL-9 Downloaded from To determine whether a short-term exposure to IL-9 is sufficient to induce the up-regulation of Paneth cell markers in colon, we injected 200 ng of murine IL-9 to wild-type mice daily, and sacrificed them after 2 or 4 days of injections. Whereas Ang4 is clearly induced in the colon after two IL-9 injections (Fig. 6), PLA2g2a and cryptdins up-regulation required four IL-9 injections. The up- http://www.jimmunol.org/ regulation of these Paneth cell-specific genes correlated with that FIGURE 6. Kinetics of Ang4, PLA2g2a, and cryptdin induction by of mast cell specific genes such as mMCPs. IL-9. Normal FVB mice received i.p. daily injections of IL-9 (200 ng) or sterile PBS, for 2 or 4 days. Mice were sacrificed the day after the last PLA2g4c and resistin-like ␣, but not Paneth cell markers, are injection. Quantitative RT-PCR was performed for Ang4, PLA2g2a, crypt- similarly regulated by IL-9 in lung and intestinal mucosae dins, and mMCPs on colon total RNA as described in Materials and Meth- ods. Results were normalized with ␤-actin. Mean values and SEM were To compare the effects of IL-9 on the intestinal and lung mucosae, we calculated from two to three mice of each group. analyzed the expression of the IL-9-induced genes in lungs from IL-9 Tg mice. As shown in Fig. 7, PLA2g4c expression was up-regulated by guest on September 29, 2021 in IL-9 transgenic lungs, and this effect was dependent on IL-13 as previously observed in colon. By contrast, PLA2g2a and Ang4 (25). PLA2g2a is a small secreted protein released by Paneth cells, and has antimicrobial activity against Gram-positive bacteria prob- ably conferred by the highly cationic nature of this protein and its preference for cleaving anionic phosphatidylglycerol which is highly represented in bacterial membranes (26, 27). By contrast, PLA2g4c is a cytosolic, membrane-associated protein that contributes to arachidonic acid metabolism and phospholipid remodeling in response to inflammatory stimuli, such as IL-1 (28).

As illustrated on Fig. 3, both PLA2s were up-regulated in the colon of IL-9 Tg. IL-9 administration induced the expression of these genes in the colon of wild-type, but not of IL-4RϪ/Ϫ mice, whereas IL-9 similarly increased mMCP-1 expression by mast cells in both strains of mice (Fig. 4). In line with this observation, IL-9 overexpression failed to up-regulate the expression of these B two genes in IL-13Ϫ/Ϫ mice (data not shown), conﬁrming that IL-13 is required for this activity of IL-9.

IL-9 induces Paneth cell hyperplasia in duodenum and colon The up-regulation of Ang4 and PLA2g2a prompted us to assess the number of Paneth cells in IL-9 Tg mice, by using phloxine/tartrazine staining, which specifically stains their granular eosinophilic material in red (29). In wild-type mice, these cells were confined at the base of ␣ the Lieberku¨hn crypts in the small intestine (Fig. 5A), and absent from FIGURE 8. Colon expression of Resistin-like is induced by IL-9 in an IL-13-dependent way. A, Quantitative RT-PCR for Retnla on colon total colon (Fig. 5B). By contrast, Paneth cells accumulated in small intes- Ϫ Ϫ Ϫ Ϫ RNA from IL-9Tg, IL-13 / , IL-9Tg/IL13 / , and control littermates. B, tine of IL-9 Tg mice, not only at the bottom of the crypt but also IL-4Ϫ/Ϫ and IL-4R␣ Ϫ/Ϫ mice received two i.p. injections of IL-9 (200 ng) nearby the apex of the glands (Fig. 5C). Similar observations were or sterile PBS, 5 and 1 day before the analysis of Retnla expression by done in the jejunum and ileum (data not shown). Most strikingly, quantitative RT-PCR on colon total RNA. Results were normalized with ;p Յ 0.03 ,ء .(Paneth cell expansion was also observed in colonic glands from IL-9 ␤-actin and expressed as mean values and SEM (n ϭ 4 Tg mice (Fig. 5D). Immunohistochemistry staining with anti- Mann-Whitney U test. 4742 IL-9-INDUCED PANETH CELL EXPANSION IN INTESTINAL MUCOSA mRNAs were not detectable in lungs (data not shown), in line with the was recently shown to provoke membrane pore formation in fact that these genes are specific Paneth cell markers. Trypanosoma cruzi parasites (42). Ang4 belongs to the RNase A The resistin-like molecule family contains a series of other an- family and has both angiogenic and microbicidal activities (22, timicrobial proteins secreted by epithelial cells in an IL-13-depen- 23). Ang4 preferentially kills Gram-positive bacteria by unknown dent way, and playing a role in defenses against nematode infec- mechanisms and its activity on nematodes has not been tested. tions (30–32). We have previously shown that Retnla was induced However, another member of this RNase family, eosinophil cat- by IL-9 in an IL-13-dependent way in lungs (5). We therefore ionic protein or RNase 3, has been characterized as a membrane- analyzed the colonic expression of Retnla in our model. As illus- disruptive cationic toxin with nonspecific toxicity to bacteria, hel- trated in Fig. 8A, Retnla was up-regulated in IL-9 Tg mice through minthes, and various eukaryotic cellular targets (43). an IL-13-dependent mechanism. In line with this observation, IL-9 PLA2g2a also shows antimicrobial properties (27, 44). administration failed to increase Retlna expression in IL-4RϪ/Ϫ PLA2g2a catalyzes the hydrolysis of glycerophospholipids but has mice, whereas its effect was maintained in IL-4Ϫ/Ϫ mice (Fig. 8B). a preference for anionic phosphatidylglycerol, conferring specific- Incidentally, basal expression of Retnla also seems to be partially ity for the bacterial cell membranes, usually rich in phosphatidyl- IL-13 dependent, as illustrated by the lower expression of this gene glycerol (26, 45). The large number of cationic residues of in untreated IL-4RϪ/Ϫ mice or in nontransgenic IL-13Ϫ/Ϫ mice, as PLA2g2a confers to this enzyme a global positive charge, allowing compared with IL-4Ϫ/Ϫ and to IL-13ϩ/Ϫ mice, respectively. its penetration into the negatively charged bacterial cell wall (27).

Another function of proteins from the PLA2 family consists in Discussion producing free fatty acids, such as arachidonic acid and lysophos-

We have previously shown that IL-9 promotes mucus production pholipids, which are the respective precursors of eicosanoids Downloaded from by lung epithelial cells via IL-13 induction in hematopoietic cells. and platelet-activating factor. Before being secreted, PLA2g2a can In this study, we focused on the activities of IL-9 on the intestinal promote the release of such mediators of inﬂammation (46–48). mucosa and found that IL-9 up-regulates not only the expression of However, this function is mainly attributed to cytosolic PLA2s goblet cell-associated genes, such as Muc2, mCLCA3/gob5, and from group IV subfamily, such as PLA2g4c, which is also up- Retnla, but also that of Paneth cell markers, such as Ang4, regulated by IL-9 in the intestinal mucosa (28). So, both PLA2g2a

PLA2g2a, and cryptdins. These activities required the presence of and PLA2g4c can mediate the production of eicosanoid progeni- http://www.jimmunol.org/ a functional IL-13 or IL-4RA gene, as previously shown for IL-9 tors in response to IL-9 and IL-13. In contrast, PLA2g4c could activities on lung epithelial cells (5). These observations suggest contribute to the exocytosis of granules containing antimicrobial that Paneth cell hyperplasia represents one of the efferent arms of products by its lysophospholipase and transacylase properties, the TH2-driven immune response against intestinal pathogens such which mediate phospholipid remodeling and vesicular trafﬁcking as nematode worms. (49–52). Finally, PLA2g4c might participate in PLA2g2a induc-

Several reports have highlighted the key role of IL-9 and IL-13 tion like another cytosolic PLA2, PLA2g4a, which was shown to during nematode infections. Inhibition of IL-9 impaired Trichuris be required for PLA2g2a up-regulation by TGF-␣ and IL-1 in rat muris clearance (10), whereas IL-9 overexpression accelerated ex- gastric mucosal cells (48). pulsion of T. muris or Trichinella spiralis (9, 33). Similarly, mice Kinetics experiments show that a few days of IL-9 exposure are by guest on September 29, 2021 treated with anti-IL-13 Abs or deficient in IL-13 fail to expel T. sufficient to induce an up-regulation of Paneth cell markers in the muris (8, 34). However, the mechanism involved in worm clear- colon mucosa, and that this observation correlates with IL-9-in- ance remains controversial, and might depend on the nematode duced mast cell expansion in this organ. As IL-9 induces IL-13 species. For T. muris, mast cells and eosinophils seem dispensable, expression by mast cells and that this cell population is expanded since anti-c-kit treatment and ablation of IL-5 have no effect on in the intestinal mucosa of IL-9 transgenic mice (5–7), it is likely development of protective immunity (35). By contrast, mast cells that these cells are at least partly responsible for this IL-13-depen- are required for T. spiralis expulsion. In this model, mast cells dent activity of IL-9 on Paneth cells. T lymphocytes represent increase epithelial permeability by producing proteases such as another source of IL-9-induced IL-13 in these mice, although this mMCP-1, a process promoted by IL-9 (24, 36). Other anti- population was not expanded in the intestinal mucosa of IL-9 nematode effector mechanisms modulated by IL-9 or IL-13 include transgenic mice (data not shown). Further studies will be increased epithelial cell turnover acting like an epithelial escalator needed to unravel the molecular mechanisms by which IL-13 to expel Trichuris parasites (11) and jejunal muscle hypercontrac- promotes Paneth cell differentiation and hyperplasia. tility (37). Paneth cell hyperplasia has also been reported during T. spiralis infection, and this process seemed T cell-dependent (13), Acknowledgments but the role of IL-9 and IL-13 in this process had not been de- We thank Dr. Ivan Theate for help and expert advice on histological stain- scribed so far. The up-regulation of Paneth cell-derived antimicro- ing and slide preparation. bial products, including cryptdins, Ang4, and PLA2g2a might therefore directly contribute to the anti-parasite response. Interest- Disclosures ingly, Retnla can also participate to nematode clearance, although The authors have no financial conflict of interest. this potential antimicrobial protein is preferentially produced by goblet cells and is up-regulated by IL-9 and IL-13 in both lungs References and colon. This gene is also up-regulated during helminth infec- 1. Louahed, J., M. Toda, J. Jen, Q. Hamid, J. C. Renauld, R. C. Levitt, and tions (38), and could participate to the immune response by its N. C. Nicolaides. 2000. Interleukin-9 upregulates mucus expression in the air- ways. Am. J. Respir. Cell Mol. Biol. 22: 649–656. profibrotic activity and tissue remodeling or by increasing smooth 2. Zhu, Z., R. J. Homer, Z. Wang, Q. Chen, G. P. Geba, J. Wang, Y. Zhang, and muscle cell proliferation (31, 39, 40). J. A. Elias. 1999. Pulmonary expression of interleukin-13 causes inflammation, So far, there is no direct evidence that Paneth cell-derived prod- mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J. Clin. Invest. 103: 779–788. ucts such as cryptdins, Ang4, and PLA2g2a possess nematode kill- 3. McLane, M. P., A. Haczku, M. van de Rijn, C. Weiss, V. Ferrante, ing activity. Cryptdins are particularly potent against bacteria such D. MacDonald, J. C. Renauld, N. C. Nicolaides, K. J. Holroyd, and R. C. Levitt. 1998. Interleukin-9 promotes allergen-induced eosinophilic inflammation and air- as Escherichia coli (41), but their potential cytotoxicity against way hyperresponsiveness in transgenic mice. Am. J. Respir. Cell Mol. Biol. 19: parasites has not been assessed. However, another human defensin 713–720. The Journal of Immunology 4743

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