were analysed for overall 96 hours, in a 24 hour interval. Additionally, we generated organoidsfrom SurviviniΔIEC and control mice. Survivin deletion was performed by addition oftamoxifen. Specimens were investigated by quantitative PCR, immunohistochemistry andwestern blot analysis. Results: Time course analysis of gene deletion of survivin in IECsrevealed an initial pulse of apoptosis at the crypt bottom 48 hours after tamoxifen injection.This did not led to death of SurviviniΔIEC animals. Histological analysis at later time pointsrevealed aberrant chromosome segregation in IECs and the formation of multinucleatedcells starting from the crypt bottom. Importantly, these cells did not show signs of cell deathbut of DNA damage, as confirmed by phopho-H2AX and phospho-p53 positive nuclei.Quantitative PCR showed elevated gene expression levels of p21 and ddit3 (dna-damage-inducible-transcript 3) in SurviviniΔIEC mice, suggesting mitotic catastrophe in IEC depletedof survivin. Conclusions: Although survivin belongs to the IAP family, we could not seeextensive apoptosis after survivin deletion in IECs. Our data demonstrates that the loss ofsurvivin initially leads to the death of apoptosis prone IECs, followed by the formation ofmultinucleated cells with signs of DNA damage in apoptosis reluctant IECs. This leads toan alternative cell death pathway, known as mitotic catastrophe.

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AMPK Positively Regulates Neurotensin Secretion Through Inhibition ofmTORC1 SignalingJun Song, Jing Li, Heidi L. Weiss, Tianyan Gao, Courtney M. Townsend, B. Mark Evers

Neurotensin (NT) is a gut peptide produced and stored in N cells of the distal smallbowel. NT has been implicated in the regulation of gastrointestinal functions, including thestimulation of intestinal fat absorption, inhibition of gastric acid secretion and gastroduodenalmotility. AMPK and mTORC1 are two important signaling pathways regulating energybalance. Previously, we have shown that mTORC1 inhibits NT gene expression and peptidesecretion through feedback inhibition of ERK1/2. We also found that AMPK activationincreases NT secretion. The purpose of the present study was to investigate whether NTsecretion is regulated by a mechanistic crosstalk between AMPK and mTORC1 signaling.METHODS. i) The human endocrine cell lines, BON (pancreatic carcinoid) and QGP-1(pancreatic somatostatinoma), were used. AMPK was activated by Aicar (1 mM) or 2-DG(10 mM) or inhibited by compound C (CC) (10 μM). To confirm findings obtained in celllines, experiments were performed using a murine primary intestinal crypt model. Theisolated crypts were cultured in serum-free bronchial epithelial growth medium, whichenriches for neuroendocrine (NE) cells and inhibits fibroblast growth. In addition, micewere injected with saline (vehicle) or Aicar and blood samples collected for measurementof plasma NT. ii) Expression of TSC2, an mTORC1 negative regulator activated by AMPKphosphorylation, was decreased by RNA interference (RNAi). iii) To detect the involvementof ERK1/2, cells were treated with PD98059 (10 μM), an MEK inhibitor. RESULTS. NTmRNA expression was increased in BON cells treated with Aicar in a time-dependent fashion(0, 3 and 24 h). Enriched NT-positive cells were noted in isolated primary intestinal cryptsby immunofluorescent microscopic analysis; 2-DG treatment for 3 h increased NT secretionfrom primary intestinal crypts. Plasma NT was also increased in mice injected with Aicar(500 mg/kg body weight) for 1 h compared to saline injection (n=5 mice per group).Phosphorylation of S6K1 was decreased by Aicar treatment for 3 h which was reversed byCC. Consistently, RNAi-mediated inhibition of TSC2 expression decreased Aicar-stimulatedNT release. Furthermore, Aicar-stimulated NT release was attenuated by PD98059 treatment.Aicar treatment resulted in ERK1/2 translocation from cytosol to the nucleus, which wasinhibited by CC. CONCLUSIONS. AMPK plays a positive role in the regulation of NTsecretion through inhibition of mTORC1. These findings suggest that AMPK and mTORC1are important signaling pathways in the control of NT gene expression and peptide releasein response to low or high energy status in endocrine cells.

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Intestinal Epithelial Deletion of Sphingosine Kinase 1 Suppresses CryptRegeneration Following Radiation InjuryJung Wan Choe, Beom Jae Lee, Moon Kyung Joo, Hyo jung Kim, Jong-Jae Park, Jae SeonKim, Young-Tae Bak, Dae Sik Yang

Background: Genotoxins such as γ-irradiation lead to major increases in crypt apoptosis andthe loss of crypt stem cells. Sphingosine kinase 1(Sphk1) is a key enzyme in the sphingolipidpathway to produce the shingolipid metabolite, sphingosine-1-phosphate. Sphk1 is overex-pressed in many solids tumors, including prostate, stomach, kidney and oral squamouscancer. Overexpressed Sphk1 in these cancers is associated with radiation resistance. Weinvestigated the role of intestinal epithelial Sphk1 on crypt regeneration and the expressionof stem cell markers after radiation injury Methods: The Sphk1 was conditionally deletedfrom intestinal epithelial cells in mice by crossing Sphk1 flox/flox mice with villin-cretransgenic mice to generate Villin-cre Sphk1 flox/flox (Sphk1 KO mice). Sphk1 flox/floxmice were used for control. Mice were irradiated at a single dose of 12 Gy. To examineSphk1/S1P pathway during radiation injury, Sphk1/2, S1PR1-3 were assessed by qPCR at0hr, 24hr, 84hr and 120 hr after 12 Gy whole body radiation (WBR). IEC apoptosis in thesmall bowel was assessed by Tunnel and H&E staining. To determine crypt survival andregeneration, microcolony assay was done at 84hrs after 12 Gy WBR. Quantitative RT-PCRwas performed with specific primers for mice Lgr5, Bmi1, Msi1, Hopx, Oflm4, Defa5, Pla2v5,lysozyme, MMP-7, cyclin D1 and c-myc at each time point. Immunohistochemistry forDCAMKL-1 was done at 120hrs after irradiation. Results: Expression of Sphk1 m RNAbegan to increase at after irradiation and peak level was observed at 120hrs after 12 GyWBR. Sphk1/Sphk2 ratio was significantly increased at this time point. In Sphk1 KO mice,epithelial cell apoptosis in the crypt of small intestine was significantly increased in bothH&E staining and Tunnel activity. In microcolony assay, crypt survival and regenerationwas significantly decreased 2 fold in Sphk1 KO mice compared with control(p<0.05).Quantitative RT-PCR revealed significantly reduced stem cell markers (Lgr5, Olfm4, Bmi1,Msi1) and paneth cell markers (Defa5, Lysozyme, MMP-7) in Sphk1 KO mice comparedwith control mice at 120hr after irradiation. Conclusion These data suggest that epithelialSphk1 plays an important role in the intestinal epithelial cell regeneration after radiation

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injury. Sphk1 in the intestinal epithelial cells might protect adult stem cell population fromradiation injury.

406

A Self-Reinforcing Pathway of Protective Mucosal Immunity Mediated byEpithelial CD1dTorsten Olszak, Joana F. Neves, Marie C. Dowds, Kristi Baker, Jonathan Glickman,Nicholas Davidson, Christian Jobin, Stephan Brand, Werner Muller, Koichiro Wada,Kazufumi Katayama, Atsushi Nakajima, Hiroyuki Mizuguchi, Kunito Kawasaki, KazuhiroNagata, Stefan Schreiber, Arthur Kaser, Sebastian Zeissig, Richard S. Blumberg

Background & Aim: The mechanisms by which homeostasis at mucosal surfaces is maintainedis of central importance to the pathogenesis of inflammatory bowel disease (IBD). Criticalto these processes is the intestinal epithelial cell (IEC), which regulates the responses ofimmune cells to environmental factors. CD1d presents lipid antigens to natural killer T(NKT) cells, which are critical for the pathogenesis of ulcerative colitis (UC), a major formof IBD. Since CD1d cross-linking on model IEC lines results in the production of barrierprotective cytokines, we aimed to characterize the role of epithelial CD1d in IBD andits contribution to intestinal inflammation. Methods: Villin(V)-CreERT2xIl10fl/fl mice (IL-10ΔIEC), V-CreERT2xCd1d1fl/fl (CD1dΔIEC), and V-CreERT2xMttpfl/fl (MTPΔIEC) weregenerated to allow for tamoxifen-induced, IEC-specific deletion of IL-10, CD1d, and microso-mal triglyceride transfer protein (MTP), a lipid transfer protein critical for CD1d function.Oxazolone-induced colitis was investigated in the presence or absence of anti-CD1d- oranti-IL-10R blockade as well as using adenoviruses which express heat-shock protein 110(HSP110). RNA and protein expression by IECs were assessed using microarrays, qPCR,ELISA, Western blot and immunohistochemistry. Results: We show that oxazolone colitisleads to rapid mortality and morbidity of MTPΔIEC, CD1dΔIEC and IL-10ΔIEC micecompared to wildtype littermates. Bone marrow chimera experiments with HSP110-deficientmice demonstrated that epithelial deficiency of HSP110 phenocopied observations made forepithelial deficiency of MTP, CD1d, and IL-10. Mechanistically, we demonstrate that CD1d,HSP110, and IL-10 participate in a self-reinforcing epithelial pathway, which converges on,and is mediated by, STAT3. As such, STAT3 silencing inhibited constitutive expression ofepithelial CD1d, HSP110, and IL-10 and prevented HSP110-induced upregulation of IL-10and CD1d. In accordance with these findings, adenoviral reconstitution of HSP110 in vivorestored epithelial IL-10 expression and protected from inflammation-associated morbidityand mortality in the oxazolone model in an IL-10 receptor-dependent manner. Conclusion:These results indicate a critical role for epithelial MTP, CD1d, HSP110 and IL-10 in a self-reinforcing pathway, which governs mucosal homeostasis and protects from intestinal inflam-mation.

407

Mitochondrial Unfolded Protein Responses Control Epithelial Stem CellProliferation in the IntestineEmanuel Berger, Detian Yuan, Nadine Waldschmitt, Eva Rath, Michael Allgäuer, OriStaszewski, Mark V. Boekschoten, Michael Müller, Marco Prinz, Achim Weber, MarkusGerhard, Klaus-Peter Janssen, Mathias Heikenwälder, Dirk Haller

Background and aim: Heat shock protein 60 (HSP60), a mitochondrial unfolded proteinresponse (mtUPR)-associated chaperone, is highly expressed in intestinal epithelial cells(IEC) of mouse models of chronic inflammation and in patients with inflammatory boweldisease (IBD). This study investigates the role of HSP60 in epithelial homeostasis using noveltissue-specific knockout mouse models. Methods and Results: Generation of IEC-specificHsp60 knockout mice (Hsp60flox/floxXVillinCre) antagonized embryonic development at dayE11.5 and induced embryonic lethality. Postnatal induction of HSP60 deficiency (Hsp60flox/

floxXVillinCreERT2) caused massive aberrations in the villus-crypt architecture of the smallintestine associated with symptoms of severe wasting and mortality within one week. Sporadicfailure of Cre-mediated Hsp60 deletion resulted in the generation of highly proliferativeHSP60-positive escaper cells expressing the stem cell marker olfactomedin 4 (OLFM4). Atthe macroscopic level, the colonic tissue was free of any pathology, despite an increasedinfiltration of macrophages. Independent of hyperproliferative crypt foci, HSP60-deficientcrypts in colon and small intestine revealed hallmarks of mtUPR, associated with an abrogatedexpression of Ki67, Lgr5 and Olfm4, indicating a significant loss of epithelial stemness.Consistently, tamoxifen-induced deletion of Hsp60 ex vivo reduced growth of small intestinalcrypt organoids. Microarray analysis of HSP60-deficient colonic epithelium identified 2,512differentially expressed genes (q<0.01), including a set of distinct C/EBP-homologous protein(CHOP) target genes. Comparative analysis with the transcriptional profile of epithelial cell-specific CHOP transgenic (ChopIEC Tg/Tg) mice identified 165 shared genes, emphasizingon metabolic processes. Mitochondria in the HSP60-deficient epithelium showed alteredmorphology, as well as decreased expression of functional markers like the mitochondrialencoded cytochrome c oxidase I (mtCoxI) subunit located in the respiratory chain. Conclusion:Tissue-specific deletion of Hsp60 disrupts epithelial cell homeostasis in both the pre- andpostnatal gut, independent of an inflammation-associated pathology. Hsp60 deletion in theintestinal epithelium triggers mtUPR and alterations in mitochondrial function, leading toan impaired proliferative response and a loss of stemness.

408

Epithelial Derived MMP9 Exhibits Tumor Suppressive Role in ColitisAssociated CancerNoopur Bhatnagar, Lewins Walter, Hamed Laroui, Pallavi Garg

Background and Aims: Individuals with Inflammatory Bowel Disease (IBD) have increasedrisk of developing colorectal cancer (CRC). Colitis-associated cancer (CAC) a subtype ofCRC is unique as it develops to adenocarcinoma via ‘inflammation-dysplasia-carcinoma'sequence compared to ‘adenoma-carcinoma sequence' of CRC. Matrix metalloproteinases

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s(MMPs) are zinc-dependent neutral endopeptidases that participate in degradation of extra-cellular matrix proteins involved in a variety of pathological processes including inflammationand cancer. MMP9 protein expression and activity is undetectable in most healthy adulttissues including the intestine but is highly expressed in inflammatory state. We have shownthat despite being a mediator of acute inflammation MMP9 plays a protective role in thedevelopment of CAC. Aim of the present study is to determine the necessity and sufficiencyof epithelial derived MMP9 mediated p53 activation via Notch1 signaling for its tumorsuppressive function in CAC, by using MMP9 transgenic mice (Tg-villin-MMP9) that specifi-cally overexpresses MMP9 in the colonic epithelium. Methods: Age and gender matchedTg-villin-MMP9 and their wild type littermates (WT) mice were used for in vivo experimentsand embryonic fibroblasts (MEFs) isolated from WT and MMP9-/- mice were used for invitro experiments. Mice were exposed to dextran sulfate sodium (DSS, 3% in drinking water)and were given 3 DSS cycles of one week each and with two weeks apart and were sacrificedat day 75. Colons were processed for inflammation and polyps. Swiss rolls of colonic tissueswere histologically examined. Western blot (WB) analysis was done with the colonic mucosalstripping. Results: Tg-villin-MMP9 mice were more resistant to CAC as evidenced bydecreased tumor burden. In addition, decreased erosion of mucosal layer, infiltration ofneutrophils and dysplasia were seen in the polyps of Tg-villin-MMP9 mice compared toWTs both induced with CAC. WB analysis indicated significantly increased protein expressionof NICD (active Notch1), p53, p21WAF1/Cip1, Bax-1 and caspase-3 compared to WTsmice both induced with CAC. TUNEL staining showed increased apoptosis in the colonsof Tg-villin-MMP9 mice compared to WTs mice both induced with CAC. MMP9 overexpres-sion (by stable transfection) in MMP9-/- MEFs resulted in significantly increased proteinexpression of NICD, p53 and p21WAF1/Cip1 expressions compared to non-transfectedMMP9-/- MEFs, and were comparable to WT MEFs. Conclusion: Together, the data showthat constitutive expression of MMP9 in colonic epithelium modulates apoptosis and cellcycle arrest, and thereby acts as a tumor suppressor in CAC. This study elucidates the noveltumor suppressive role of MMP9 in malignant transformation of the colonic epithelium incontext of chronic inflammation which may provide new diagnostic and therapeuticapproaches.

409

The Colonic Adherent-Invasive Escherichia coli Strain HM605 Induces Anti-Apoptotic Responses in Intestinal Epithelial Cells, Reduces Barrier Integrityand Worsens Experimental ColitisTrevor Darby, Emma Smith, Aoife Quinlan, Grainne Hurley, David Clarke, FergusShanahan, Kenneth Nally, Silvia Melgar

Background: Adherent-invasive Escherichia coli (AIEC) are commonly found in lesions ofCrohn's Disease (CD) patients. AIEC adhere, grow and induce cytokine production inmacrophages and intestinal epithelial cells (IECs) suggesting AIEC may play a role in theimmunopathology of CD. ATG16L1 is a member of the ATG5-ATG12 autophagy complexand a CD-susceptibility gene. Aim: To investigate functional responses induced by the colonicAIEC strain HM605 in intestinal epithelial cells deficient in ATG16L1 expression and in anin vivo model of colitis. Methods: Monolayers of confluent C2B cells stably transduced withshRNA targeting ATG16L1 (ATG16L1KD) or non-targeting shRNA (NT) were infected withHM605 (MOI-10:1; 3 hours), washed with antibiotics and cultured for up to 21h. Functionalanalysis included barrier function [trans-epithelial resistance (TER)], bacterial uptake, West-ern blots and PCR analysis on autophagy, apoptosis and signalling pathways. Male C57BL/6 mice were treated with streptomycin 24h before orally gavaged with 1x108 HM605,followed by 5 days of 2.5% DSS exposure and 3 days of water or non-DSS-treatment (n=4-8 mice/group). On day 3 post-DSS, mice were orally gavaged with FITC-Dextran andblood collected 4h later for in vivo intestinal permeability analysis. On day 8, mice weresacrificed and colonic tissue processed for cytokine analysis. Body weight and health conditionwere monitored daily. Results: HM605-infected ATG16L1KD cells displayed significantlyhigher bacteria invasion and cytokine expression accompanied by a reduced autophagyresponse and decreased barrier function when compared to NT-infected cells. Componentsof the mTOR and MAPK/ERK-signaling pathways were expressed at similar levels in infected-ATG16L1KD and infected-NT cells. In contrast, there was a delayed PI3K-independent AKT-activation in infected ATG16L1KD cells compared to NT-infected cells. Inhibition of AKTrecovered the autophagy response in the infected-ATG16L1KD cells to a level similar to theinfected-NT cells. Also, HM605 induced an anti-apoptotic response in IECs independent ofATG16L1-expression. In mice, HM605 colonised the cecum and colon and when supple-mented with DSS, mice presented significantly higher plasma FITC-Dextran levels accompa-nied by worsening symptoms of colitis and significantly elevated levels of colonic IL-6 andmKC. Conclusions: Our findings suggest that altered ATG16L1 function in IECs contributesto a persistent AIEC-infection and sustained AKT-activation accompanied by impaired barrierfunction and induction of anti-apoptotic responses. We also show that in vivo colonisationof a colonic AIEC strain results in impaired barrier integrity and greater colitis severity.Collectively, these model systems can be used as tools to investigate host-bacteria interactionsfor the potential identification of new therapeutic targets in CD.

410

Impaired Epithelial Barrier for Macromolecules in Ulcerative Colitis Is Causedby Downregulation of the Tricellular Tight Junction Protein Tricellulin,Mediated by the Interleukin-13 Receptor α2-Activated PathwaySusanne M. Krug, Christian Bojarski, Petra Dames, Jerrold R. Turner, Michael Fromm,Joerg D. Schulzke

The tight junction protein tricellulin (TRIC) is predominantly located in tricellular tightjunctions, the meeting points of three cells. Here it regulates the barrier for macromoleculepassage across the intestinal epithelium. We have found that TRIC expression is downregu-lated in biopsies from patients suffering from ulcerative colitis (UC), but not in Crohn'sdisease (CD). Aim of the present study was to discover the regulative mechanisms of thiseffect. Methods. The human colon cell line HT-29/B6 was treated with cytokines connectedto CD and UC development. TRIC expression was analyzed by Western blot while claudin-2 expression served as control for the cytokine effects. Fluxes of 4 KDa-FITC-dextran were

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measured to validate the barrier loss for macromolecules after reduction of TRIC abundance.Involved signaling pathways were studied employing different inhibitors (e.g. Baricitinib,AS1517499, SAHA, U0126, SP600125) and specific inhibitory antibodies . Results. In HT-29/B6 monolayers, addition of interleukin-13 (IL-13, 100 ng/ml, 48 h) reduced the expressionof TRIC by 35%. By that also the permeability for 4K-FITC-dextran was increased from 60± 10 to 150 ± 14 × 10-9 cm/s. Inhibitors and inhibitory antibodies of the IL13Rα2-activatedpathway were able to block TRIC reduction, but those affecting the IL13Rα1-activatedpathway had no effect. In contrast, this route was regulating expression of claudin-2.Antibodies directed against the respective receptor confirmed these findings and were ableto inhibit the effects on either claudin-2 or TRIC. Conclusion. TRIC expression is downregu-lated by IL-13, the key cytokine in UC, via the pathways activated by its interaction with thereceptor IL13Rα2, accompanied by an impaired epithelial barrier for macromolecule passage.

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Intestinal Organoids: A Model of Intestinal FibrosisLaura A. Johnson, Eva Rodansky, Sha Huang, Jason R. Spence, Peter D. Higgins

Purpose: To develop a multi-cellular, 3-dimensional organoid model of intestinal fibrosis.Background: Intestinal fibrosis is a critical complication of Crohn's disease. Current in vitrointestinal myofibroblast models of fibrosis which are activated by cytokine stimulation (e.g.TGFβ) or the physical environment (mechanical stress) do not fully recapitulate the complexmulticellular 3-dimensional (3D) intestinal architecture while in vivo models (rat TNBS,mouse (S. typhimurium) have limited utility for large-scale screening of anti-fibrotic com-pounds or analyzing the mechanistic contributions of multiple genetic loci. Here, we exploitrecent advances in 3D in vitro growth of intestinal tissue as a new human model to studyintestinal fibrosis associated with IBD. Materials and Methods : Human pluripotent stemcells were differentiated into human intestinal organoids (HIOs), which are 3D structurescomprised of a lumen, intestinal epithelium and mesenchyme. In order to identify myofibro-blasts, which are key effectors of fibrosis, 45-day old HIOs were stained for a-smooth muscleactin (αSMA), vimentin, and desmin by immunofluorescence. TGFβ responsiveness wasassayed by treatment of organoids with increasing amounts of TGFβ for 48 hrs or 2ng/mlof TGFβ for 96 hr. Fibrotic response was assayed by expression of pro-fibrotic genes (col1A1,ACTA2, Fn1, MYLK, and MKL1). Results: Immunofluorescent staining of organoids fromhealthy controls identified a population of myofibroblasts (αSMA+/vimentin+/desmin- cells),which were identified as a discrete population of cells within the intestinal mesenchyme,located adjacent to the intestinal epithelium. Healthy control organoids were TGFβ-respon-sive as increasing concentrations of TGFb for 48hr induced a dose-dependent pro-fibroticresponse with increased Fn1 and MKL1 gene expression. Longer exposure (96 hr) to TGFβ(2 ng/ml) triggered a more robust fibrotic response with significant 2 to ~4-fold increasesobserved in col1A1, ACTA2, MYLK, Fn1, and MKL1 gene expression. Conclusions: Intestinalorganoids derived from healthy tissue develop a myofibroblast layer and respond to pro-fibrotic stimuli, in a manner that is consistent with myofibroblasts isolated from normaland IBD patients. Given that our current understanding of the etiology of fibrosis in IBDpatients is severely lacking, generation and use of HIOs from both healthy and IBD patientswill enable elucidation of the cellular, molecular and genetic differences between the healthyand IBD states. HIOs could also advance the screening of anti-fibrotic therapies forCrohn's disease.

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A BiPhasic IL-1 Response Mediates Postoperative Ileus via Enteric GliaActivationKristof J. Hupa, Burkhard Stoffels, Mariola Lysson, Joerg C. Kalff, Wouter de Jonge, SvenWehner

INTRODUCTION: Abdominal surgery commonly leads to a transient postoperative intestinaldysmotility, known as postoperative ileus (POI). We have previously shown that entericglia mediate innate immune responses and contribute to POI in an IL1 receptor type Idependent manner. Herein, we studied the role of the two IL1 receptor agonists IL-1α andIL1β in POI development. AIMS & METHODS: C57BL6 wildtype, IL-1α -/- and IL1β -/-,ASC-/- and Caspase-1-/- mice underwent a standardized intestinal manipulation (IM). Chi-meric mice were generated by bone marrow transplantation between wildtype and IL-1knockout mouse strains. After 3 and 24 hours, cytokine expression, leukocyte infiltrationand geometric center (GC) of gastrointestinal transit (GIT) were assessed. Myenteric plexuscell cultures were analyzed for IL-1 mediated cell activation. Results: In wildtype animals,IM led to a significant induction of chemokine expression (Ccl-2: IM 368+/-56-fold vs. C1.1+/-0.2 ) and neutrophil influx (805+/-184 vs. 2+/-1 cells/mm2) into the muscularis externacompared to sham-operated controls (C). GIT was also significantly decelerated by IM (GC:IM 3.4+/-0.5 vs. C 9.3+/-0.3). Early phase CCl2 gene expression was dependent on IL-1α(114.8+/-38 but not IL-1β (534+/-58). Mice deficient for IL-1α in radio-resistant cells butnot in hematopoietic cells were protected from POI while IL-1β deficiency only provedprotective in hematopoietic cells but not resident cells. Immunofluorescence analysis identi-fied IL-1α expression in resident F4/80+ macrophages of untreated mice while IL1β expres-sion was only induced by surgery in infiltrating monocytes in an ASC and Caspase-1dependent manner. Exogenous i.p. administration of IL-1α induced a 10-fold higher induc-tion of Ccl2 chemokine expression compared to IL1β. Conclusion: Our results show thatendogenous IL-1 signaling in POI is a biphasic response with IL-1α acting as a potentinducer of glia-derived chemokines in the initial phase of POI. IL1β expression occurs inlate phase POI and is dependent on inflammasome activation. We conclude that IL-1antagonism could be helpful in prevention (via IL-1α) and treatment (via IL1β) of exist-ing POI.