Allergy-driven Alternative Splicing of Interleukin-13 Receptor 2 Yields Distinct Membrane and Soluble Forms Yasuhiro Tabata, Weiguo Chen, Manoj R. Warrier, Aaron M. Gibson, Michael O. Daines, and Gurjit K. Khurana Hershey Division of Allergy & Immunology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio IL-13: a Central Mediator of Allergic Inflammation Collagen, Fibrosis eot axin-3 Fibroblast Kerat inocyt e Chemokine expression Mucus Hypersecret ion Goblet cell met aplasia Respirat ory Epit helium VCAM expression Chemokine expression Vascular Endot helium IL-1 3 Smoot h Muscle Smoot h Muscle Proliferat ion Cholinergic-induced cont ract ions Recruit ment Act ivat ion Eosinophil IgE Product ion Ag Ag B Cell Modulat ion of FcRI IgE priming CD23 expression Class II MHC expression Inhibit ion of proinflammat ory gene expression Macrophage/ monocyt e Mast Cell IL-13 binds to two distinct IL-13 receptors IL-13 IL-13 ? IL-13 IL-13R1 TYK2 Soluble form IL-4R IL-13R2 ? JAK1 Membrane form IL-13 receptor / Type II IL-4 receptor STAT6 P P STAT6 STAT6 P STAT6 P STAT6 Cytoplasm P STAT6 P STAT6 Transcription Nucleus Background • IL-13R2 knockout mice have high serum IgE and enhanced IL-13 responsiveness. (Wood et al. JEM 197:703,2003) • Soluble IL-13R2 has been measured at high levels in mouse serum (10 ~ 20 ng/ml at baseline). (Morimoto et al. JI 176:491,2006) • Soluble IL-13R2 regulates serum and tissue levels of IL-13, and inhibits IL-13-induced liver fibrosis. (Chiaramonte et al. JEM 197:687,2003) • Soluble IL-13R2 is induced by parasitic infection in both mouse and human serum. (Mentink-Kane et al. PNAS 101:586,2004) Hypothesis Alternative splicing may yield a secreted form of IL-13R2 3’-RACE revealed two distinct transcripts for mouse IL-13R2 Mouse IL-13R2 gene ATG TAA 1 Exon: 2 3 4 5 6 7 8 9 10 11 99bp 2803bp 108bp 2841bp 109bp 1623bp 152bp 931bp 154bp 121bp 3295bp 185bp 146bp 1304bp 145bp 3655bp 116bp 752bp 200bp Intron: 568bp 1030bp Untranslated region Signal peptide Extracellular domain Transmembrane domain Intracellular domain F1 F2 Tail adaptor Mouse spleens express two transcripts of IL-13R2 (nested RT-PCR result) M 1 2 a (387 bp) b (271 bp) The shorter transcript lacks exon 10, which encodes transmembrane motif Full-length transcript ATG TAA TM 3 4 5 6 7 8 9 10 11 Ex10 transcript ATG TGA (from position 952 to 1152) exon9 GAA TGG AGT GAA E W S E TAC ACA GGG CCA Y T G P ATA GTA CCA GTT exon10 I V P V CTT TTG TTA CTT L L L L AAG GAA GAA CCT K E E P exon11 CAT GTG GAT CTG H V D L TAT GAA GAT ACC Y E D T GAG E GAC D TGT C CTT L GAA E AAC N CTC L GAA E TCA S CTT L TGC C CCC P AAA K TGT C TGT C AAG K TTC F CTT L ACA T GAA E TAA * TGG W ATT I TTT F ATT I TTG L GTG V GAA E ATT I ATA I GTG V AGC S TGT C GGT G TTC F TTC F GAG E CTC L GCT A 3 4 5 6 7 8 9 11 (from position 952 to 1036) exon9 GAA TGG AGT GAA GAG GAA TGT TGG GAA GAG E W S E E E C W E E exon11 CCT CCA TGT GGA TCT GAA CAA AGA AGT GTG P P C G S E Q R S V TGC TTA TGA AGA TAC CCT CTG TTA A C L * Full-length transcript encodes membrane IL-13R2 Surface IL-13R2 Untransfected Full-length Ex10 Surface IL-13 binding Untransfected Full-length Ex10 Ex10 transcript encodes soluble IL-13R2 Ex10 transfectants secreted high levels of IL-13R2 in supernatants. Ex10 transcript encodes biologically active soluble IL-13R2 UT: Supernant from untransfected cells DE: Supernant from Ex10 transfectants IL-4 : murine IL-4 5ng/ml IL-13 : murine IL-13 5ng/ml Distribution of two IL-13R2 transcripts in mouse organs at baseline a: full-length transcript b: Ex10 transcript (496 bp) (380 bp) House dust mite (HDM) challenge results in allergic reaction in a mouse asthma model Day: 0 7 14 21 25 26 Intraperitoneal sensitization HDM 10g Intraperitoneal sensitization HDM 10g Intratracheal challenge HDM 100g Intratracheal challenge HDM 100g BAL AHR Measurement Airway hyperresponsiveness (AHR) Eosinophil counts in BALF Allergen challenge increased soluble IL-13R2 in BALF and serum in the mouse asthma model Soluble IL-13R2 in BALF Soluble IL-13R2 in serum Expression of full-length and Ex10 transcripts in the mouse asthma model. Full-length Ex10 Day 1 Day 5 Ex10 decreased in spleen but not in bone marrow in the mouse asthma model 1 day after the challenge Full-length Ex10 Spleen Bone marrow Summary • Mouse IL-13R2 gene has two transcripts: longer full-length and shorter Ex10 transcript lacking the transmembrane motif. • The full-length transcript encodes functional membrane IL-13R2, which can bind to IL-13. • The Ex10 transcript encodes biologically active soluble IL-13R2, which can effectively block IL-13-dependent STAT6 DNA binding activity. • Expression of the two IL-13R2 transcripts was differentially regulated in vivo in the mouse asthma model. • The alternatively spliced variants of IL-13R2 may have distinct biologic functions in vivo. Thank You Confirmation of two sequences from HDM-treated mouse lungs exon9 exon10 exon9 exon11 IL-13R2 derived from Ex10 transcript has 2.3-fold higher IL-13 binding ability compared with that from full-length transcript.