IL-33 Drives Eosinophil Infiltration and Pathogenic Type 2 Helper T-Cell Immune Responses Leading to Chronic Experimental Ileitis
2016; Elsevier BV; Volume: 186; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2015.11.028
ISSN1525-2191
AutoresCarlo De Salvo, Xiao-Ming Wang, Luca Pastorelli, Benedetta Mattioli, Sara Omenetti, Kristine A. Buela, Saleem Chowdhry, Rekha Garg, Wendy A. Goodman, Alexander Rodriguez‐Palacios, Dirk E. Smith, Derek W. Abbott, Fabio Cominelli, Giorgos Bamias, Wei Xin, James J. Lee, Maurizio Vecchi, Theresa T. Pizarro,
Tópico(s)Eosinophilic Esophagitis
ResumoAlthough a clear association has been established between IL-33 and inflammatory bowel disease, mechanistic studies to date, primarily using acute murine models of colitis, have yielded contradicting results, demonstrating both pathogenic and protective roles. We used a well-characterized, spontaneous model of inflammatory bowel disease [ie, SAMP1/YitFc (SAMP) mice] to investigate the role of IL-33 during chronic intestinal inflammation. Our results showed marked eosinophil infiltration into the gut mucosa with increased levels of eotaxins and type 2 helper T-cell (Th2) cytokines as disease progressed and became more severe, which could be reversed upon either eosinophil depletion or blockade of IL-33 signaling. Exogenous IL-33 administration recapitulated these effects in ilea of uninflamed (parental) control AKR/J mice. Human data supported these findings, showing colocalization and up-regulation of IL-33 and eosinophils in the colonic mucosa of inflammatory bowel disease patients versus noninflamed controls. Finally, colonization of commensal flora by fecal material transplantation into germ-free SAMP and the presence of the gut microbiome induced IL-33, subsequent eosinophil infiltration, and mounting of Th2 immune responses, leading to exacerbation of chronic intestinal inflammation characteristic of SAMP mice. These data demonstrate a pathogenic role for IL-33–mediated eosinophilia and activation of Th2 immunity in chronic intestinal inflammation that is dependent on the gut microbiome. Targeting IL-33 may represent a novel therapeutic approach to treat patients with inflammatory bowel disease. Although a clear association has been established between IL-33 and inflammatory bowel disease, mechanistic studies to date, primarily using acute murine models of colitis, have yielded contradicting results, demonstrating both pathogenic and protective roles. We used a well-characterized, spontaneous model of inflammatory bowel disease [ie, SAMP1/YitFc (SAMP) mice] to investigate the role of IL-33 during chronic intestinal inflammation. Our results showed marked eosinophil infiltration into the gut mucosa with increased levels of eotaxins and type 2 helper T-cell (Th2) cytokines as disease progressed and became more severe, which could be reversed upon either eosinophil depletion or blockade of IL-33 signaling. Exogenous IL-33 administration recapitulated these effects in ilea of uninflamed (parental) control AKR/J mice. Human data supported these findings, showing colocalization and up-regulation of IL-33 and eosinophils in the colonic mucosa of inflammatory bowel disease patients versus noninflamed controls. Finally, colonization of commensal flora by fecal material transplantation into germ-free SAMP and the presence of the gut microbiome induced IL-33, subsequent eosinophil infiltration, and mounting of Th2 immune responses, leading to exacerbation of chronic intestinal inflammation characteristic of SAMP mice. These data demonstrate a pathogenic role for IL-33–mediated eosinophilia and activation of Th2 immunity in chronic intestinal inflammation that is dependent on the gut microbiome. Targeting IL-33 may represent a novel therapeutic approach to treat patients with inflammatory bowel disease. IL-33 (alias IL-1F11) is the newest member of the IL-1 family. IL-33 represents a protein with dual function that can act as both a signaling cytokine and an intracellular nuclear factor.1Schmitz J. Owyang A. Oldham E. Song Y. Murphy E. McClanahan T.K. Zurawski G. Moshrefi M. Qin J. Li X. Gorman D.M. Bazan J.F. Kastelein R.A. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines.Immunity. 2005; 23: 479-490Abstract Full Text Full Text PDF PubMed Scopus (2825) Google Scholar, 2Carriere V. Roussel L. Ortega N. Lacorre D.A. Americh L. Aguilar L. Bouche G. Girard J.P. IL-33, the IL-1-like cytokine ligand for ST2 receptor, is a chromatin-associated nuclear factor in vivo.Proc Natl Acad Sci U S A. 2007; 104: 282-287Crossref PubMed Scopus (781) Google Scholar IL-33 is widely distributed throughout various organ systems, primarily in nonhematopoietic cells, including fibroblasts, adipocytes, smooth muscle cells, endothelial cells, and bronchial and intestinal epithelial cells (IECs), as well as in cells of hematopoietic origin, particularly in restricted populations of professional antigen-presenting cells.1Schmitz J. Owyang A. Oldham E. Song Y. Murphy E. McClanahan T.K. Zurawski G. Moshrefi M. Qin J. Li X. Gorman D.M. Bazan J.F. Kastelein R.A. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines.Immunity. 2005; 23: 479-490Abstract Full Text Full Text PDF PubMed Scopus (2825) Google Scholar, 3Pastorelli L. Garg R.R. Hoang S.B. Spina L. Mattioli B. Scarpa M. Fiocchi C. Vecchi M. Pizarro T.T. Epithelial-derived IL-33 and its receptor ST2 are dysregulated in ulcerative colitis and in experimental Th1/Th2 driven enteritis.Proc Natl Acad Sci U S A. 2010; 107: 8017-8022Crossref PubMed Scopus (314) Google Scholar IL-33 was initially associated with the development of type 2 helper T-cell (Th2) immunity, on the basis of the expression of its cell-bound receptor, ST2L (IL-1R4), on polarized Th2 lymphocytes1Schmitz J. Owyang A. Oldham E. Song Y. Murphy E. McClanahan T.K. Zurawski G. Moshrefi M. Qin J. Li X. Gorman D.M. Bazan J.F. Kastelein R.A. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines.Immunity. 2005; 23: 479-490Abstract Full Text Full Text PDF PubMed Scopus (2825) Google Scholar and, more recently, on innate lymphoid cells,4Neill D.R. Wong S.H. Bellosi A. Flynn R.J. Daly M. Langford T.K. Bucks C. Kane C.M. Fallon P.G. Pannell R. Jolin H.E. McKenzie A.N. Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity.Nature. 2010; 464: 1367-1370Crossref PubMed Scopus (1618) Google Scholar as well as its ability to potently induce Th2 cytokine production (eg, IL-4, IL-5, and IL-13). In regard to disease pathogenesis, IL-33 is primarily involved in Th2-mediated disorders, such as airway inflammation and allergic reactions5Kurowska-Stolarska M. Kewin P. Murphy G. Russo R.C. Stolarski B. Garcia C.C. Komai-Koma M. Pitman N. Li Y. Niedbala W. McKenzie A.N. Teixeira M.M. Liew F.Y. Xu D. IL-33 induces antigen-specific IL-5+ T cells and promotes allergic-induced airway inflammation independent of IL-4.J Immunol. 2008; 181: 4780-4790Crossref PubMed Scopus (392) Google Scholar; however, IL-33 has also been described to exacerbate arthritis, widely considered a Th1/Th17-mediated pathology.6Xu D. Jiang H.R. Kewin P. Li Y. Mu R. Fraser A.R. Pitman N. Kurowska-Stolarska M. McKenzie A.N. McInnes I.B. Liew F.Y. IL-33 exacerbates antigen-induced arthritis by activating mast cells.Proc Natl Acad Sci U S A. 2008; 105: 10913-10918Crossref PubMed Scopus (401) Google Scholar, 7Palmer G. Talabot-Ayer D. Lamacchia C. Toy D. Seemayer C.A. Viatte S. Finckh A. Smith D.E. Gabay C. Inhibition of interleukin-33 signaling attenuates the severity of experimental arthritis.Arthritis Rheum. 2009; 60: 738-749Crossref PubMed Scopus (260) Google Scholar One of the original observations and prominent functions of IL-33 is its ability to activate and induce eosinophil (EOS) infiltration into mucosal organs exposed to the external environment, including the respiratory and gastrointestinal tracts.1Schmitz J. Owyang A. Oldham E. Song Y. Murphy E. McClanahan T.K. Zurawski G. Moshrefi M. Qin J. Li X. Gorman D.M. Bazan J.F. Kastelein R.A. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines.Immunity. 2005; 23: 479-490Abstract Full Text Full Text PDF PubMed Scopus (2825) Google Scholar In fact, IL-33 has emerged as an essential mediator in the development of EOS-mediated allergic inflammation and asthma,8Stolarski B. Kurowska-Stolarska M. Kewin P. Xu D. Liew F.Y. IL-33 exacerbates eosinophil-mediated airway inflammation.J Immunol. 2010; 185: 3472-3480Crossref PubMed Scopus (238) Google Scholar and plays a pivotal role in EOS recruitment and helminth expulsion after parasitic hookworm infection.9Hung L.Y. Lewkowich I.P. Dawson L.A. Downey J. Yang Y. Smith D.E. Herbert D.R. IL-33 drives biphasic IL-13 production for noncanonical Type 2 immunity against hookworms.Proc Natl Acad Sci U S A. 2013; 110: 282-287Crossref PubMed Scopus (157) Google Scholar EOS development and recruitment depends on IL-5, which represents the primary factor for EOS maturation and differentiation, but is also important in EOS activation and recruitment.10Mishra A. Hogan S.P. Lee J.J. Foster P.S. Rothenberg M.E. Fundamental signals that regulate eosinophil homing to the gastrointestinal tract.J Clin Invest. 1999; 103: 1719-1727Crossref PubMed Scopus (332) Google Scholar, 11Hogan S.P. Rosenberg H.F. Moqbel R. Phipps S. Foster P.S. Lacy P. Kay A.B. Rothenberg M.E. Eosinophils: biological properties and role in health and disease.Clin Exp Allergy. 2008; 38: 709-750Crossref PubMed Scopus (627) Google Scholar Chemokine ligand (CCL) 11 and CCL24 (alias eotaxin-1 and eotaxin-2, respectively) are EOS-specific chemokines that bind to the chemokine receptor, CCR3, expressed on the surface of EOS, and are critical for EOS recruitment.10Mishra A. Hogan S.P. Lee J.J. Foster P.S. Rothenberg M.E. Fundamental signals that regulate eosinophil homing to the gastrointestinal tract.J Clin Invest. 1999; 103: 1719-1727Crossref PubMed Scopus (332) Google Scholar, 11Hogan S.P. Rosenberg H.F. Moqbel R. Phipps S. Foster P.S. Lacy P. Kay A.B. Rothenberg M.E. Eosinophils: biological properties and role in health and disease.Clin Exp Allergy. 2008; 38: 709-750Crossref PubMed Scopus (627) Google Scholar A growing body of evidence also supports the importance of EOS in the pathogenesis of inflammatory bowel disease (IBD), a disorder previously attributed to dysregulated and overly aggressive T-effector cell responses. This paradigm has been challenged in recent years by the concept that the primary cause of IBD resides in dysfunction of host innate immunity and aberrant interactions with the gut microbiome, which together shape downstream adaptive immune responses. In regard to EOS, although their numbers are elevated in both ulcerative colitis (UC) and Crohn's disease (CD), two of the main etiopathogenic forms of IBD, most studies report the prevalence of EOS/EOS activity in UC compared with CD.12Lampinen M. Ronnblom A. Amin K. Kristjansson G. Rorsman F. Sangfelt P. Safsten B. Wagner M. Wanders A. Winqvist O. Carlson M. 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Fang M. Interleukin-33 ameliorates experimental colitis through promoting Th2/Foxp3(+) regulatory T-cell responses in mice.Mol Med. 2012; 18: 753-761Crossref PubMed Scopus (145) Google Scholar, 25Groß P. Doser K. Falk W. Obermeier F. Hofmann C. IL-33 attenuates development and perpetuation of chronic intestinal inflammation.Inflamm Bowel Dis. 2012; 18: 1900-1909Crossref PubMed Scopus (91) Google Scholar functions. To date, the IL-33/ST2 axis has not been mechanistically evaluated in a chronic, immunologically mediated model of IBD. The SAMP1/YitFc (SAMP) mouse strain represents a chronic model of Th1/Th2-mediated ileitis and provides an excellent system to study the initiation and progression of chronic intestinal inflammation.26Pizarro T.T. Pastorelli L. Bamias G. Garg R.R. Reuter B.K. Mercado J.R. Chieppa M. Arseneau K.O. Ley K. Cominelli F. SAMP1/YitFc mouse strain: a spontaneous model of Crohn's disease-like ileitis.Inflamm Bowel Dis. 2011; 17: 2566-2584Crossref PubMed Scopus (120) Google Scholar Because SAMP were derived from brother-sister mating of wild-type AKR/J (AKR; parental strain), the phenotype occurs spontaneously, as in the human condition, without chemical, genetic, or immunological manipulation. Relevant to the present study, we have previously reported elevated levels of serum and ileal IL-33 in SAMP compared with AKR mice.3Pastorelli L. Garg R.R. Hoang S.B. Spina L. Mattioli B. Scarpa M. Fiocchi C. Vecchi M. Pizarro T.T. Epithelial-derived IL-33 and its receptor ST2 are dysregulated in ulcerative colitis and in experimental Th1/Th2 driven enteritis.Proc Natl Acad Sci U S A. 2010; 107: 8017-8022Crossref PubMed Scopus (314) Google Scholar Herein, we used the SAMP model to mechanistically investigate whether IL-33 served any role in the development of chronic intestinal inflammation mediated by EOS. Our results show the following: i) EOS increase with the progression and severity of chronic intestinal inflammation (ie, reversed upon EOS depletion); ii) exogenous IL-33 administration induces EOS infiltration into the gut and a potent mucosal Th2 immune response, whereas IL-33 blockade reduces these effects and diminishes the overall severity of disease; and iii) the commensal flora is essential for the induction of IL-33, subsequent EOS infiltration, and production of Th2 cytokines, which exacerbates ileitis characteristic of SAMP mice. Together, these data demonstrate an important pathogenic role for IL-33 during chronic, spontaneous intestinal inflammation that is dependent on the gut microbiome and downstream Th2 immune responses. As such, targeting the IL-33/ST2 axis for therapeutic purposes may prove to be beneficial for the treatment of patients with IBD. SAMP and AKR mice were propagated at Case Western Reserve University (CWRU; Cleveland, OH), with SAMP founders provided by S. Matsumoto (Yakult Central Institute for Microbiological Research, Tokyo, Japan).27Matsumoto S. Okabe Y. Setoyama H. Takayama K. Ohtsuka J. Funahashi H. Imaoka A. Okada Y. Umesaki Y. Inflammatory bowel disease-like enteritis and caecitis in a senescence accelerated mouse P1/Yit strain.Gut. 1998; 43: 71-78Crossref PubMed Scopus (232) Google Scholar, 28Kosiewicz M.M. Nast C.C. Krishnan A. Rivera-Nieves J. Moskaluk C.A. Matsumoto S. Kozaiwa K. Cominelli F. Th1-type responses mediate spontaneous ileitis in a novel murine model of Crohn's disease.J Clin Invest. 2001; 107: 695-702Crossref PubMed Scopus (217) Google Scholar Mice were maintained under specific pathogen-free (SPF) conditions, fed standard laboratory chow (Harlan Teklad, Indianapolis, IN), and kept on 12-hour light/dark cycles. Original AKR were purchased from The Jackson Laboratory (Bar Harbor, ME). Germ-free (GF)-SAMP were maintained at Taconic Farms (Albany, NY) and shipped to CWRU in GF vessels. All procedures were approved by the Institutional Animal Care and Use Committee at CWRU and followed the American Association for Laboratory Animal Care guidelines. GF-SAMP recipients were maintained at CWRU's Animal Resource Center inside high-efficiency particulate air–filtered pressurized isolators and feeding on a double-irradiated diet (Irradiated Prolab Isopro RMH 3000; Lab Diet, St. Louis, MO) designed for maintenance and breeding of GF mice. On the day of transplantation, fresh fecal samples (single, approximately 100-mg pellet per mouse), collected from a representative cohort of SPF-raised SAMP (n = 6 per sex), were used to prepare donor inoculum. In brief, fecal pellets were homogenized using 200 mg of 1.4-mm-diameter sterile ceramic beads in 6 mL prechilled phosphate-buffered saline (PBS) by gentle agitation for 20 seconds using a vortex mixer. Recipient mice were orally gavaged with 100-μm filtered homogenates (108 to 109 colony-forming units in 300 μL) within 60 minutes of inoculum preparation. Transplanted mice were maintained following strict aseptic techniques, following GF standards for food, water, and nonpalatable bedding on the basis of aspen wood shavings. Prolab Isopro RMH 3000 diet was autoclaved, and a special isolation facility used to prevent mouse-mouse cross-contamination (1 to 2 mice per cage). Mice were sacrificed 6 weeks after fecal material transplantation (FMT), and ilea were harvested for later assays. Mucosal biopsy specimens were obtained during colonoscopy of adult UC patients and uninflamed controls. Diagnosis of UC was established by clinical, macroscopic, and histological criteria. Endoscopies were performed at the Endoscopy Unit of the first Department of Internal Medicine, Propaedeutic, at Laikon Hospital (Athens, Greece) and at the Gastroenterology and Endoscopy Unit of IRCCS Policlinico San Donato, San Donato Milanese (Milano, Italy). In most cases, samples were taken from the same individual, from both normal-appearing mucosa (noninvolved) and areas with evident macroscopic inflammation (involved). Individuals undergoing screening colonoscopy, who did not display endoscopic and histopathological mucosal abnormalities, served as controls. Specimens were maintained at 4°C for 1 to 2 days in RNAlater Solution (Ambion, Life Technologies, Austin, TX) until later total RNA isolation. Full-thickness colonic surgical specimens were obtained from UC patients, as well as noninflammatory controls, who were admitted to Case Medical Center/University Hospitals and underwent therapeutic bowel resection for either inflammatory disease or malignant and other nonmalignant, noninflammatory conditions. Tissues were subsequently processed, as previously described,3Pastorelli L. Garg R.R. Hoang S.B. Spina L. Mattioli B. Scarpa M. Fiocchi C. Vecchi M. Pizarro T.T. Epithelial-derived IL-33 and its receptor ST2 are dysregulated in ulcerative colitis and in experimental Th1/Th2 driven enteritis.Proc Natl Acad Sci U S A. 2010; 107: 8017-8022Crossref PubMed Scopus (314) Google Scholar and evaluated for EOS and IL-33 expression. All diagnoses were confirmed by clinical, macroscopic, and histological criteria. All studies were approved by the Ethics Committee of Laikon Hospital and IRCCS Policlinico San Donato, as well as by the Internal Review Board of Case Medical Center. Experimental mice were sacrificed, and terminal ilea were removed, opened longitudinally, and rinsed in PBS. Gut specimens were divided in half along the longitudinal plane, with one part placed in RNAlater Solution and maintained at 4°C until later total RNA extraction, and the other submerged in Bouin's solution (LabChem, Inc., Pittsburgh, PA). Bouin's fixed tissues were processed, as previously described,3Pastorelli L. Garg R.R. Hoang S.B. Spina L. Mattioli B. Scarpa M. Fiocchi C. Vecchi M. Pizarro T.T. Epithelial-derived IL-33 and its receptor ST2 are dysregulated in ulcerative colitis and in experimental Th1/Th2 driven enteritis.Proc Natl Acad Sci U S A. 2010; 107: 8017-8022Crossref PubMed Scopus (314) Google Scholar sectioned (3 μm thick), stained with hematoxylin and eosin, and histologically evaluated by a trained gastrointestinal pathologist (W.X.) in a blinded manner, using a validated semiquantitative scoring system.3Pastorelli L. Garg R.R. Hoang S.B. Spina L. Mattioli B. Scarpa M. Fiocchi C. Vecchi M. Pizarro T.T. Epithelial-derived IL-33 and its receptor ST2 are dysregulated in ulcerative colitis and in experimental Th1/Th2 driven enteritis.Proc Natl Acad Sci U S A. 2010; 107: 8017-8022Crossref PubMed Scopus (314) Google Scholar In addition, EOS counts were performed (W.X.) on major basic protein (MBP)–stained slides with 10 randomly selected areas per section evaluated under 40× high-power field (Olympus superwide eyepiece; 0.53-mm diameter; BX41 Laboratory Microscope; Olympus America, Inc., Melville, NY). Only intact MBP+ cells were counted, with an average number of EOS calculated and reported. Four-, 12-, and 20-week-old SAMP and age-matched AKR were used to evaluate baseline inflammation and progression of disease. EOS depletion was performed by i.p. injection (twice per week for 6 weeks) of 20-week-old SAMP with monoclonal antibodies against mouse IL-5 and CCR3 (GS2-19-4 and TRFK-5, respectively; Mayo Clinic Arizona, Scottsdale, AZ), both at 5 mg/kg, administered either alone or in combination. Control mice were treated with an isotype mouse IgG antibody (IR-MS-GF-ED; Innovative Research, Novi, MI), using the same dose and treatment schedule. For IL-33 administration experiments, 4- and 12-week-old SAMP and AKR were injected i.p. (33 μg/kg, daily for 1 week) with either murine recombinant IL-33 (ALX-522-101; Enzo Life Sciences, Farmingdale, NY) or PBS (vehicle controls). IL-33 blockade was achieved using 4- and 14-week-old SAMP treated with a murinzied rat IgG1 antibody against mST2, and controls were administered an isotype mouse IgG1 antibody (i.p., twice per week for 6 weeks; 5 mg/kg; Amgen, Seattle, WA). Total RNA was isolated from patient endoscopic biopsy specimens and mouse ilea using the RNeasy Mini Kit (Qiagen, Germantown, MD) and reverse transcribed (RNA-to-cDNA kit; Applied Biosystems, Forest City, CA), both according to the manufacturer's instructions. Quantitative RT-PCR was performed, as previously described, using primers for mouse IL-33,3Pastorelli L. Garg R.R. Hoang S.B. Spina L. Mattioli B. Scarpa M. Fiocchi C. Vecchi M. Pizarro T.T. Epithelial-derived IL-33 and its receptor ST2 are dysregulated in ulcerative colitis and in experimental Th1/Th2 driven enteritis.Proc Natl Acad Sci U S A. 2010; 107: 8017-8022Crossref PubMed Scopus (314) Google Scholar IL-4, IL-5, IL-13,29Bamias G. Martin C. Mishina M. Ross W.G. Rivera-Nieves J. Marini M. Cominelli F. Proinflammatory effects of TH2 cytokines in a murine model of chronic small intestinal inflammation.Gastroenterology. 2005; 128: 654-666Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar CCL11 (forward, 5′-TGTCTCCCTCCACCATGCA-3′; reverse, 5′-GATCTTCTTACTGGTCATGATAAAGCA-3′), CCL24 (forward, 5′-TGCATCTCCCCATAGATTCTGT-3′; reverse, 5′-ACTCGGTTTTCTGGAATTTTCTTG-3′), and β-actin (forward, 5′-CAGGGTGTGATGGGAATG-3′; reverse, 5′-GTAGAAGGTGTGGTGCCAGAT-3′), and primers for human IL-33,3Pastorelli L. Garg R.R. Hoang S.B. Spina L. Mattioli B. Scarpa M. Fiocchi C. Vecchi M. Pizarro T.T. Epithelial-derived IL-33 and its receptor ST2 are dysregulated in ulcerative colitis and in experimental Th1/Th2 driven enteritis.Proc Natl Acad Sci U S A. 2010; 107: 8017-8022Crossref PubMed Scopus (314) Google Scholar IL-5 (PPH00692A-200), CCL11 (PPH0057B-200), CCL24 (PPH01162B-200), CCL26 (PPH0163E-200), and β-actin (330001 PPH00073E-200) (all from Qiagen), and on an Applied Biosystems Step Plus machine (Applied Biosystems). Reaction mixture consisted of 15% volume first-strand synthesis in 20 μL total volume that included Power SYBR Green core reagents (Applied Biosystems) and 500 nmol/L final concentration of primers. Thermal cycling conditions were 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Expression of all target mRNA transcripts was normalized to β-actin, and reported as relative fold difference among groups within an experiment, with control group set arbitrarily at 1. Cytokine protein levels were measured in cell culture supernatants [mouse Cytokine Screen-IR 16-plex (Quansys Biosciences, Logan, UT) and mouse IL-13 Ready-Set-Go ELISA kit, 88-7137-22 (eBioscience, San Diego, CA)] for EOS depletion and IL-33 administration experiments. Ready-Set-Go ELISA kits (eBioscience) were used for measuring levels of IL-4 (88-7044-22), IL-5 (88-7054-22), and IL-13 (88-7137-22) for IL-33 neutralization experiments. Immunohistochemical (IHC) staining was performed using a polyclonal goat anti-mouse IL-33 IgG at 1:100 (AF3626; R&D Systems, Minneapolis, MN), a goat anti-human IL-33 IgG at 1:50 (AF3625; R&D Systems), and monoclonal rat anti-mouse MBP IgG (clone MT-14.7) or mouse anti-human EOS protein X IgG antibodies (clone MM25-82.2), both at 1:500 (J.J. Lee, Mayo Clinic, Scottsdale, AZ). Paraffin-embedded tissues were sectioned (3 to 4 μm thick), placed on Superfrost Plus glass slides (Thermo Scientific, Logan, UT), deparaffinized, and incubated in normal serum for non-specific blocking. Samples were blocked for endogenous peroxidase activity using 1.75% H2O2, and antigen retrieval was performed on human samples by microwave exposure (twice for 5 minutes), while submerged in unmasking solution (Vector Labs, Burlingame, CA). After incubation with primary antibodies at 4°C, slides were rinsed in PBS, incubated with appropriate biotinylated secondary antibodies (Vector Labs), rinsed in PBS, and further assa
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