Enteroviruses cause a range of human and animal diseases, some life-threatening, but there remain no licenced anti-enterovirus drugs. However, a benzene-sulfonamide derivative and related compounds have been shown recently to block infection of a range of enteroviruses by binding the capsid at a positively-charged surface depression conserved across many enteroviruses. It has also been established that glutathione is essential for the assembly of many enteroviruses, interacting with the capsid proteins to facilitate the formation of the pentameric assembly intermediate, although the mechanism is unknown. Here we show, by high resolution structure analyses of enterovirus F3, that reduced glutathione binds to the same interprotomer pocket as the benzene-sulfonamide derivative. Bound glutathione makes strong interactions with adjacent protomers, thereby explaining the underlying biological role of this druggable binding pocket and delineating the pharmacophore for potential antivirals. Helen Duyvesteyn et al. present a high-resolution structural analysis of gluthathione binding to an enterovirus capsid. They show that glutathione binds to a surface cavity, promoting assembly of the viral capsid, and providing a potential therapeutic target. Enterovirus (EV) is the most populous genus of the Picornaviridae family of small, single-stranded RNA viruses. Enteroviruses are responsible for a range of animal and human diseases. These non-enveloped viruses are constructed of an icosahedral protein capsid composed of 60 copies each of viral proteins VP1–4. VP1–3 are similar to each other (comprising a β-barrel with extended surface loops and termini) and form the capsid surface. Whilst a handful of enterovirus vaccines are available, including for poliovirus (PV) and for EV-A71 4, there are as yet no licenced anti-enterovirus drugs, despite evidence for efficacy in clinical trials and reports of extremely high potency for compounds targeting a pocket internal to capsid protein VP1 (termed here the P site)6. However, a cavity on the capsid surface at an inter-protomer interface, where we previously reported extra electron density7, has recently been identified as potentially druggable, with micro-molar binding for a benzene-sulfonamide derivative8. This compound, and a number of related compounds, are inhibitors of Coxsackie B viruses and a range of other enteroviruses including rhinoviruses, and have been shown to act synergistically with P site binders8.The genus 9. Disruption of the GSH/GSSG balance is associated with several enterovirus infections, including EV-A7110, CV-A16 and CV-B313. Furthermore, depletion of GSH, using l-butathione sulfoximate (BSO)15 and TP21916, blocks the assembly of protomeric units into pentamers17. GSH-independence can be conferred by mutations at the protomer interface, consistent with GSH binding to protomers driving the formation of pentameric units critical to assembly17. However, there are no published structural data for GSH binding to an enterovirus capsid.Cells contain glutathione in both reduced (GSH) and oxidised (GSSG) forms, whose balance is crucial for maintaining cellular redox potential22. We firstly show that EV-F3 is dependent on GSH and that GSH stabilises the capsid. We then determine high-resolution structures of complexes of GSH and its initial breakdown product Cys-Gly (CG) bound to EV-F3 and by a competition binding study demonstrate that GSH binds more strongly. We find that the binding site of both compounds is identical to that for a similar molecule that remains naturally attached to the virus7, and to the binding site observed for the benzene-sulfonamide derivative in complex with Coxsackievirus B38, identifying the biological role for this binding site.Here we study EV-F3, a relatively benign bovine enterovirus which is our model system of choice23, we next used the PaSTRy assay24 to demonstrate that GSH also shows a modest dose-dependent stabilisation of EV-F3  site. There is also an additional weak binding site nestled between the VP1 and VP3 C-termini at a non-conserved site, but this is probably not biologically significant  that GSH binds more strongly than CG , residues 75–79, 95–98 and 227–231 respectively, a fourth is from the C-terminus of VP3 (protomer A), and the fifth and sixth are from residues 150–155 and 216 of VP1 of protomer B  and, indirectly, R227 (protomer A) at the inter-protomer interface 25, chlorine 26, glycerol 27 and glutamic acid 7. These previous interpretations sometimes fail to fully explain the observed density  that is thought to be a precursor to uncoating and infection29. In line with this, the antiviral activity of compound-17 is not impeded by increased intracellular glutathione levels, consistent with inhibition of a stage of the infection process which occurs before virus assembly in the cytoplasm, such as uncoating in the endosome. Compounds that increase the stability of the virus capsid may also be useful as vaccine-stabilising additives23. Indeed it has been found that oral poliovirus vaccine is stabilised by GSH23, and thus developing a more potent binder may be useful for the formulation of future virus-like-particle-based vaccines, including, but not limited to, PV30.Pan-enterovirus conservation of key residues within the I site suggests that this protomer glue is widely used, with natural molecules remaining associated at this site for many purified enteroviruses. Together with the recent studies on benzene sulphonamide derivatives28, it is clear that site I presents a druggable pocket, and our high-resolution results provide a firm basis for structure-led drug discovery.In summary, our structures explain how GSH facilitates initial enteroviral assembly into pentamers. The structural conservation of the × g. A clear band at approximately 30% sucrose concentration was observed, extracted and the presence of EV-F3 was confirmed by SDS-Page gel electrophoresis.BHK-21 cells were grown to 80% confluency before infection with EV-F3 virus stocks in Glasgow modified Eagle’s medium , supplemented with 2% fetal horse serum (Thermo Fisher Scientific) and minimal essential medium non-essential amino acids . Cells were incubated at 37 °C for 3 days before harvesting. Virus-infected media was first clarified, before overnight PEG-precipitation of virus particles. PEG-precipitated virus was then re-suspended in 50 mM HEPES pH 8.0, 200 mM NaCl, 0.5% NP40 buffer, and particles pelleted on a 30% sucrose cushion. The resulting pellet was re-suspended in minimal 50 mM HEPES pH 8.0, 200 mM NaCl, applied to a 15–45% w/v sucrose gradient and subjected to ultracentrifugation at 4 °C for 3 h, 105,000 −1 increments for 30 s in an expanding saw-tooth profile, a refinement of a previously described protocol24. Fluorescence changes were monitored at 25 °C with excitation and emission wavelengths of 492 and 517 nm, respectively.Solutions of EV-F3 and additive at either 100× or 1000× molar excesses  were aliquoted into a 96-well PCR plate containing SYTO9 dye (Thermo Fisher Scientific) in triplicate with 50 mM HEPES, pH 8.0, 200 mM NaCl buffer, before centrifugation for 3 min at 4 °C. The assay was performed by heating samples in a Mx3005p qPCR machine  from 25 to 97 °C in 1 °C min2. Wells were visually scored for virus growth at 17 h post infection  and MEM NEAA (Gibco) seeded into 96-well plates. For BSO treatment, cells were GSH starved for 48 h before washing and addition of EV-F3 and further 0.4 mM BSO (Sigma). Control plates were also seeded 48 h prior to infection. One hundred microlitres of tenfold serial dilutions of EV-F3 in serum-free G-MEM was added to washed, confluent cells. For BSO-treatment conditions, additional BSO was added to each of the EV-F3 dilutions. CPE could clearly be seen after 12 h (compared with negative controls). Eight replicates of each dilution were plated and were monitored over 2 days at 37 °C and 5% CO32. Briefly, sitting drop crystallisations were set up in 96-well trays using a Cartesian robot with a reservoir solution of 1.5 M ammonium sulfate and 0.1 M Tris at pH 8.5, each crystallisation drop containing 100 nL each of protein and reservoir solutions.Crystals were grown using previously reported protocolsCrystals of approximately 50–80 μm diameter were soaked overnight with either (1) GSH; (2) CG; or (3) GSH/CG at 10 mM  and then cryo-protected using sequential addition of glycerol from 10 to 25% (v/v). The compound to be soaked was maintained during the cryo-protection process.2. A 0.0075 s or 0.01 s exposure time per 0.03 or 0.05° image with 100% beam transmission, under cryogenic conditions, enabled the collection of up to 180° of data before radiation damage was observed. Data from a single crystal were used for experiment (2) whilst data from two crystals were merged for experiments (1) and (3) , with an X-ray wavelength of 0.976 Å. Diffraction images of 0.03° or 0.05° rotation were recorded on either a Pilatus3 6M, or Eiger2 X 16M detector 37. Structures were initially determined by rigid body fitting of the EV-F3 XFEL structure (PDB: 5OSN)7 using increasing resolution cut-offs in CNS38. Some unit cell shrinkage was observed relative to the room-temperature XFEL structure, consistent with these data being collected under cryogenic conditions7. Cyclic positional and B-factor refinement with strict NCS constraints was then performed with CNS, and models were improved with COOT40. For experiments 1, 2 and 3, respectively, the percentages of residues in the favoured region of the Ramachandran plot are 94.5%, 95.1% and 94.9%. If the allowed regions are included, these percentages rise to 98.6%, 99.2% and 99.2%, respectively. Ramachandran and rotamer outliers for experiment 2 are shown in Supplementary Fig. 35.Data were indexed and integrated using either the xia2-dials pipeline (experiment (3))Infectivity assays were performed with eight repeats. PaSTRy assays were performed in triplicate, with mean values and standard deviations shown in Supplementary Fig. Further information on research design is available in the Supplementary InformationReporting Summary


Osteoarthritis is characterized by articular cartilage breakdown, and emerging evidence suggests that dysregulated innate immunity is likely involved. Here, we performed proteomic, transcriptomic, and electron microscopic analyses to demonstrate that mast cells are aberrantly activated in human and murine osteoarthritic joint tissues. Using genetic models of mast cell deficiency, we demonstrate that lack of mast cells attenuates osteoarthritis in mice. Using genetic and pharmacologic approaches, we show that the IgE/FcεRI/Syk signaling axis is critical for the development of osteoarthritis. We find that mast cell-derived tryptase induces inflammation, chondrocyte apoptosis, and cartilage breakdown. Our findings demonstrate a central role for IgE-dependent mast cell activation in the pathogenesis of osteoarthritis, suggesting that targeting mast cells could provide therapeutic benefit in human osteoarthritis.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (ddressed . Osteoarthritis, characterized by progressive degeneration of articular cartilage in the joints, is a major cause of disability and the most common form of arthritis . CurrentMast cells are sentinels of the innate immune system, poised to rapidly respond to exogenous pathogens and to endogenous danger signals . A wide Several studies have documented the presence of mast cells and their mediators in the synovium and synovial fluids of individuals with osteoarthritis . RecentlGuided by knowledge that mast cells are present in osteoarthritic synovium , we analKIT and IL3RA), protease processing and/or stabilization , and Fc receptor subunits  were significantly upregulated in the synovium of both early- and end-stage osteoarthritis compared to the healthy synovium  were likewise upregulated in osteoarthritic as compared to healthy synovial membranes  mice  mice, a c-kit-independent model of mast cell deficiency    in mice sh) mice , which hficiency . Deficieficiency . Twenty 6J mice) . We vali surgery .KitW-sh/W-sh and Cpa3-Cre;Mcl-1fl/fl mice have phenotypic abnormalities in addition to their mast cell deficiencies. For example, KitW-sh/W-sh mice have increased levels of circulating neutrophils and basophils, while Cpa3-Cre;Mcl-1fl/fl mice have reduced numbers of basophils , a drug that potently inhibits several receptor tyrosine kinases, including c-kit , a cruciHaving established a pathogenic role for mast cells in osteoarthritis and because levels of the activated form of mast cell-derived tryptase are significantly elevated in the synovial fluids of individuals with osteoarthritis, a finding in agreement with previous reports , we nextAs tryptase has been shown to promote pathogenic properties in human rheumatoid arthritis-derived synovial fibroblasts , we exam-/-Igh7) mice and IgE-sufficient littermate controls (+/+Igh7) to DMM. Twenty weeks after DMM surgery, IgE-deficient mice exhibited markedly diminished cartilage damage . We found that mice deficient in FcεRIα, which as a consequence cannot transduce IgE signals, were significantly protected against osteoarthritic development as compared to wild-type controls . To further define a role for FcεRI in the pathogenesis of osteoarthritis, we performed DMM surgeries in mice deficient in FcεRIα  as well as FcεRI at levels similar to those seen in rheumatoid arthritis , we propWhile it is well documented that IgE is critical for initiating and sustaining chronic allergic inflammation, emerging evidence suggests that IgE-mediated cellular activation directly contributes to tissue remodeling . Indeed,Previous reports found that IgE levels are not elevated in the synovial fluids of osteoarthritic joints , and in While many classical IgE-mediated allergic diseases including asthma, allergic rhinitis and eczema exhibit comorbidities , we are Mechanical instability and stresses likely contribute to the pathogenesis of osteoarthritis in a significant subset of patients. We do not believe that a role for mechanical stresses is inconsistent with a role for IgE and mast cells, and it is possible that mechanical stresses produce cartilage breakdown products and/or cellular responses that promote IgE-dependent mast cell activation.In addition to IgE-mediated activation of mast cells, a wide range of physical, biological, and chemical triggers can contribute to mast cell activation, including products of complement activation, platelet-activating factor, damage-associated molecular patterns (DAMPs), and a number of endogenous peptides  . We prevThe development and survival of mast cells is critically dependent on signaling via the stem cell factor (SCF) receptor c-kit (CD117), a member of the receptor tyrosine kinase family. While c-kit is widely expressed by hematopoietic progenitor and germ cells, in the context of the mature immune system only mast cells robustly express c-kit, and in the synovial compartment c-kit is predominantly expressed by mast cells . ImatiniMast cells could participate in osteoarthritis through multiple mechanisms, including release of pre-formed mediators such as tryptases and de novo synthesis of cytokines that could further propagate inflammation, promoting a vicious cycle of inflammation and tissue damage. Because we found high levels of active tryptase in osteoarthritic synovial fluids and previous studies have shown tryptases can induce cartilage aggrecanolysis in vitro , and proAlthough we have not formally demonstrated that mast cells are the source of tryptases that promote the pathogenesis of osteoarthritis, our data demonstrate that: (i) mast cells comprise 1–3% of synovial cells in synovial linings from both osteoarthritic joints and joints with prior injury but no radiographic osteoarthritis, (ii) mast cells are actively degranulating to release tryptase in human osteoarthritic synovial linings, and (iii) pharmacologic inhibition of typtase prevents the development of osteoarthritis in mice. However, mast cells are the predominant producer of tryptases, and to a much lesser extent basophils . TogetheThere are multiple potential limitations to this study. First, it was previously shown that mast cells are associated with joint pain in murine osteoarthritis  and thatTogether, our results demonstrate that IgE/FcεRI/Syk axis-activated mast cells promote the development of osteoarthritis following mechanical injury (DMM) in mice. As the DMM model is most representative of osteoarthritis development following traumatic joint injury (PTOA) in humans, these findings suggest that PTOA arises as result of activation of the IgE/FcεRI/Syk axis. It is possible that non-traumatic osteoarthritis, calcium pyrophosphate crystal-associated osteoarthritis, or other subsets of osteoarthritis may arise from activation of other molecular pathways.We propose a model wherein IgE-mediated mast cell activation via FcεRI and Syk which results in mast cell degranulation and the release of pro-inflammatory and degradative mediators, including tryptase, leads to cartilage and joint breakdown. This results in a vicious cycle of tissue damage, inflammation, and unchecked mast cell activation, and thus causes the development and progression of osteoarthritis. Our findings demonstrate a central role for IgE-mediated mast cell activation in the pathogenesis of osteoarthritis, and provide the rationale for targeting mast cells or tryptase as a disease-modifying therapeutic strategy for osteoarthritis.All human samples were obtained and studied under protocols that included written informed consent and consent to publish and that were approved by the Stanford University Institutional Review Board (IRB)  and the University of Padova IRB . Osteoarthritic synovial membranes were obtained at the time of total joint replacement from individuals with end-stage osteoarthritis at the VA Palo Alto Health Care System. Synovial fluids were obtained from individuals with varying degrees of osteoarthritis severity as assessed by K-L score. Synovial membranes and synovial fluids from individuals undergoing arthroscopic anterior cruciate ligament reconstruction surgery who had no arthroscopic evidence of articular cartilage loss were used as controls.Synovial membranes from five osteoarthritic knees and five non-osteoarthritic control knees with prior joint trauma >6 months ago but no radiographic osteoarthritis were analyzed, and after staining with immuno-gold labeled anti-tryptase  or isotype-matched control antibody the entire post-etch-embedded section for each sample was scanned by TEM. The number of mast cells and degranulated mast cells was determined by an examiner blinded to the experimental group of each sample. Mast cells were identified based on the presence of electron dense granules containing gold-labelled tryptase particles. Degranulation was determined by assessment of fusion of granule plasma membranes, fusion of granule membranes with cell membranes, exteriorization of granules, and presence of tryptase particles outside the exteriorized granule. The percent of degranulating mast cells per total mast cells was calculated for each sample. Multiple independent experiments were performed, and representative images and results are presented.3 pieces and returned to fresh fixative for 16–24 hr at 4°C. The specimens were washed with 100 mM cacodylate buffer, fixed with 1% osmium tetroxide for 1 hr, washed with excess distilled water then en bloc stained with 1% aqueous uranyl acetate overnight at 4°C. Samples were then dehydrated in a series of ethanol washes, propylene oxide, and embedded in resin. We picked up 75–90 nm sections on formvar/Carbon-coated slot Cu grids, stained them for 30 s in 3.5% uranyl acetate in 50% acetone followed by staining them in 0.2% lead citrate for 3 min. Post-embedding immunolabelling was carried out by micro etching in 10% periodic acid, followed by treatment with 10% sodium meta-periodate. Sections were blocked and stained with anti-human mast cell tryptase antibody , or an isotype-matched IgG1 control antibody (Abcam), followed by incubation with a goat anti-mouse antibody conjugated with 10 nm Gold particles (British Biocell). Sections were then observed in a JEOL JEM-1400 120kV transmission electron microscope (JEOL USA) and images captured using a Gatan Orius 4k × 4 k digital camera.Synovial lining tissue was fixed in Karnovsky’s fixative containing 2% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium cacodylate. After an initial ~30 min fixation, the specimens were cut into ~1 mmLevels of active tryptase were measured with the tosyl-gly-pro-lys-pNA substrate assay  according to the manufacturer's protocols.We downloaded publicly available data from the US National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO accession codes GSE32317) comparing gene expression profiles of synovial membranes obtained from patients with early- or end-stage osteoarthritis and from individuals with prior joint trauma >6 months ago but no radiographic osteoarthritis . All microarray analyses were restricted to putative mast cell- and mast cell activation-related genes. Unsupervised and supervised hierarchical clustering analyses were performed on the microarray data by using Cluster and TreeView software. Significance Analysis of Microarrays (SAM) analyses were used for determining statistical significance with a q-value cutoff set at 0.05. Paired or unpaired student’s t-tests were employed where appropriate and p<0.05 was considered statistically significant.Cpa3-Cre;Mcl-1fl/fl (B6-+/+), Igh7-/- (Igh7+/+), and Fcer1a-/- (Fcer1a+/+).Cpa3-Cre;Mcl-1 A fully congenic KitW-sh/W-sh mouse strain on a C57BL6/J genetic background (Stock No. 012861) and age-matched C57BL/6J (Stock No. 000664) were obtained from The Jackson Laboratory. Destabilization of the medial meniscus (DMM) was performed as described previously  and VA Palo Alto Health Care System Institutional Animal Care and Use Committees . Littermate controls were used for eviously . Five toKitW-sh/W-sh mice and Cpa3-Cre;Mcl-1fl/fl (Hello Kitty) mice intravenously (i.v.) with 107 wild-type bone marrow-derived cultured mast cells  into the stifle joints. Age-matched mast cell-deficient littermate mice injected both i.v. and i.a. with PBS  and mast cell-sufficient mice injected both i.v. and i.a. with PBS  were used as controls. DMM was then performed at 16 weeks of age. Mast cell engraftment was assessed by toluidine blue staining of stifle joint sections from mice sacrificed 20 weeks after DMM.To generate mast cell-engrafted mice, we injected 4-week-old, male, mast-cell deficient escribed ), and 8 Twenty-week-old wild-type C57BL/6J mice were randomized by cage to receive vehicle (water), 100 mg/kg/day imatinib mesylate (LC laboratories) or 5 mg/kg/day APC366 (Tocris) divided between two daily doses or 75 mg/Kg/day PRT062607 (synnovator) once daily by oral gavage for 12 weeks (beginning 24 hr after DMM surgery). Two of eight mice treated with 100 mg/kg/day imatinib were excluded from analyses due to inadequate histology. Similarly, for anti-IgE treatment, twenty-week-old wild-type C57BL/6J mice were administered 2.5 mg/Kg mouse anti-IgE antibody or IgG1κ isotype (Crown Biosciences) i.p. twice a week for 12 weeks. Mouse anti-mouse IgE was made using the sequences derived from the rat hybridoma (R1E4) that specifically binds to the region of mouse IgE known to bind FcεRI . Mice weStifle joints were harvested 12 or 20 weeks after DMM and fixed in 10% neutral buffered formalin followed by decalcification in formic acid for 48 hr. Joints were then embedded in paraffin, and 6 μm sections cut from three separate levels of the joint and stained with Safranin-O for assessment of cartilage damage; H and E for assessment of synovial thickening (synovitis) and osteophyte formation; and toluidine blue for the assessment of mast cells. Cartilage degeneration, synovitis, and osteophyte formation were evaluated by two blinded observers using a modified version of a described scoring system  as we prSynovial sections were fixed, decalcified, blocked, and stained with a biotinylated anti-tryptase antibody , followed by avidin-HRP, then TMB substrate, and microscopy performed to determine if tryptase-positive mast cells were present.3 primary synovial fibroblasts were stimulated with media alone (Alpha MEM) or with 1 μg/ml tryptase for 30 min and then lysates were run on SDS PAGE gel and western blot analysis was performed with anti-Erk1/2, anti-phosphoErk1/2 or anti-β-actin antibodies.Primary synovial fibroblasts were derived from synovium of individuals with end-stage osteoarthritis by enzymatic digestion with 2 mg/ml Collagenase Type IV for 24 hr at 37°C. Passage 3 (P3) fibroblasts were serum starved overnight in 1% fetal bovine serum and then stimulated with media alone (Alpha MEM) or with 0.2 μg/ml tryptase in the presence or absence of 100 μM of the tryptase-selective inhibitor APC366. Their mRNA was isolated, and mRNA levels of pro-inflammatory mediators were measured by qPCR and normalized to those of 18 s. Taqman probes were obtained from Applied Biosystems. Proinflammatory cytokine and chemokine secretion was measured by multiplexed, fluorescent bead-based immunoassay (Luminex) by using the human cytokine 27-plex assay (Bio-Rad). For analysis of Erk activation, 10t test or Mann-Whitney U test following tests for variance homogeneity. Multiple comparisons were performed using a one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc test.For analyses involving a single comparison, statistical comparisons were performed using either a two-tailed Student’s All human samples were obtained under protocols approved by the Stanford Institutional Review Board (IRB) or the University of Padova IRB, and written informed consent was received from participants prior to inclusion in the study. Participants were identified by numbers, never by name, in this study. All mouse breeding and osteoarthritis studies were performed under protocols approved by the Stanford Committee of Animal Research and in accordance with National Institutes of Health guidelines. In the interests of transparency, eLife includes the editorial decision letter, peer reviews, and accompanying author responses.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed.]eLife, provided that you will address some minor issues.Thank you for submitting the revised manuscript, entitled "IgE-mediated mast cell activation promotes inflammation and cartilage destruction in osteoarthritis". We are pleased to inform that your article is basically acceptable for publication in As you can see below, one reviewer had some concerns regarding the correlation between IgE level and severeity of osteoarthritis. Regarding this concern, I judge the Ig-7KO, FceRI KO, and anti-IgE data to provide convincing genetic evidence, and as mentioned in your letter, that authors' explanation of why the IgE level did not significantly change is reasonable. Moreover, the authors carefully mentioned, for instance, "using genetic and pharmacological approaches" in the Abstract.Please address the minor concerns of Reviewer #2 outlined below.Reviewer #1:The authors have answered a good part of the concerns of the reviewers. The manuscript is improved and I recommend that it will be accepted for publication.Reviewer #2:The authors proposed that the mast cell activation mediated by IgE/FceRI/Syk signaling is responsible for pathogenesis of OA. The authors corrected some statements in the revised manuscript. However, as I mentioned below, the fundamental problems of this manuscript have not yet been solved. In addition, they did not perform additional experiments to strengthen their conclusion.eLife.The authors showed that bulk IgE level in serum has no correlation with severity of osteoarthritis. ELISA analysis also demonstrated that synovial fluid IgE level in patients with OA was lower than that of patients with RA and PsA.  These results made me feel that it is very unlikely that IgE is related to development of OA. The authors also referred to the possibility that antigen specificity of IgE rather than the amount of IgE may be involved in development of OA by showing the preliminary data. However, the data are too preliminary to convince us of the surprising finding that OA is an autoimmune disease caused by IgE against join antigen(s) and mast cells. The proposed mechanism is not well based on evidence. Taken together, I cannot recommend this manuscript for the publication in 1) In general, OA is induced by long lasting mechanical stresses and can be accompanied by chronic inflammation, whereas IgE mediates antigen-specific acute and drastic inflammatory responses in allergic diseases. Thus, it is not easy to imagine that IgE-mediated immune responses are implicated in OA pathogenesis and induction of chronic inflammation that accompanies OA. The authors have to give convincing explanation.2) Authors showed that IgE level does not corelate with OA onset or severity. I think that this is contradictory to authors' conclusion. Authors tried to explain this by the antigen-specific IgE, but it is unbelievable that OA is an autoimmune disease caused by IgE against join antigen(s). If so, they should identify the specific antigens recognized by IgE from the synovial tissues.3) The authors could not analyze synovial derived mast cells by flow cytometry because enzymatic dissociation of synovium physically destroys mast cells. But many researchers succeeded in cell preparation from human and mouse synovial tissue. In particular, FACS analysis of mast cells in synovial tissues was previously shown . Therefore, the authors must show the accurate number of mast cell by FACS in Figure 1 to 5 and Figure 2—figure supplement 1.4) The authors cannot conclude that mast cell-derived tryptases promote osteoarthritis-associated pathology, since it remained unclear whether tryptase secreted by mast cells is sufficient to induce the destruction of the synovial cartilage. As I pointed out in the comment #6, addition of huge amount of tryptase is too artificial. The authors have to perform the co-culture experiment of mast cells with human cartilage explants. Furthermore, the authors have to show the evidence that chondrocyte undergoes apoptosis as I mentioned in comment #8. Major concerns:1) Functional assessment of the joint such as gait function or evaluating pain in mice OA model is required.The primary goal of our study was to demonstrate that mast cells play a crucial role in the pathogenesis of osteoarthritis, and specifically in the mechanisms that result in cartilage and joint destruction. Although it was previously reported that mast cells contribute to osteoarthritis joint pain in mice  and that synovial mast cell numbers are associated with the degree of synovitis in osteoarthritic joints in humans , mast cells have not previously been demonstrated to directly contribute to cartilage and joint tissue breakdown in osteoarthritis. In this manuscript, we demonstrate that IgE-mediated mast cell activation plays a critical role in cartilage degeneration, joint destruction and the development of osteoarthritis.We and others previously established that the cartilage degeneration and joint destruction that develop following DMM in mice are associated with abnormal gait and increased pain . Although we recognize the importance of pain and functional outcomes, we believe that histologic assessment of cartilage degeneration and joint destruction are the most definitive outcomes in mouse DMM osteoarthritis studies. Due to the large number of different murine experiments performed as part of the studies described in this manuscript, our analyses were focused on the definitive histologic outcome measures and we did not perform pain and function assessments. We now address this limitation in the Discussion:“There are multiple potential limitations to this study. First, it was previously shown that mast cells are associated with joint pain in murine osteoarthritis  and that synovial mast cell numbers are associated with the degree of synovitis in human osteoarthritis . We and others previously demonstrated that following DMM in mice development of histologic osteoarthritis is associated with poor functional outcomes including pain and abnormal gait . The studies presented in this manuscript demonstrate that mast cells and dysregulated mast cell activation contribute to cartilage and joint degeneration following DMM, and based on this prior work  such pathologic changes are anticipated to result in pain and abnormal gait. Future studies will be needed to further characterize the role of mast cells in osteoarthritis-associated pain and joint dysfunction…”2) The relationship between IgE-derived mast cell activation and complement activation in the OA model should be, at least, properly discussed.We agree with the reviewers that the connection between mast cell activation by IgE and complement should be discussed. We previously demonstrated that complement components are dysregulated in osteoarthritis and contribute to osteoarthritis pathogenesis, and it is possible that activated complement components stimulate mast cell recruitment and/or activation. We amended the Discussion section to address these possibilities:“We previously demonstrated that complement plays a critical role in the pathogenesis of osteoarthritis . Activation of the complement system results in the production of C3a and C5a which serve as anaphylatoxins and activators of mast cells . It is therefore possible that complement regulates the recruitment and activation of mast cells in the synovium to promote the pathogenesis of osteoarthritis. We previously demonstrated that the cartilage breakdown product, fibromodulin, can activate the complement system , and it is possible that mast cell tryptase-mediated cartilage degeneration produces fibromodulin and other cartilage breakdown products that reciprocally activate complement in the synovia in osteoarthritis. It will be important to define the temporal relationship between mast cell activation/degranulation and complement activation in the development and progression of osteoarthritis, including how activation of one affects the other and vice versa. Further, complement is capable of stimulating mast cells alone, and can also enhance IgE-dependent mast cell activation and degranulation . Future studies will be needed to further define the roles and relationships of complement activation, IgE, and mast cell activation/degranulation in the development and progression of osteoarthritis.”3) If IgE is implicated in the pathogenesis of human OA, it is reasonable to speculate the relathinship between IgE level and higher OS risk in human. Thus, these epidemiological data should be examined and provided.We appreciate the reviewers’ comment. However, previous data from others, as well as new data that we present in this response, suggest that levels of total IgE in either human sera or synovial fluids are not associated with osteoarthritis risk or severity. Previous reports found that IgE levels are not elevated in the synovial fluids of osteoarthritis joints . In addition, while increased serum levels of IgE can be detected in some atopic diseases, this is also not always the case . Further, in allergic diseases it is antigen-specific IgE, and not bulk IgE, that plays a critical role in activating mast cells. Thus, it is possible that antigen-specific IgE, rather than bulk IgE, might play a role in the pathogenesis of osteoarthritis.To identify potential correlations of bulk IgE with osteoarthritis in humans, we analyzed the relationship between serum IgE levels and the presence and severity of osteoarthritis. In collaboration with David Felson and using serum samples from 1,200+ individuals in the Framingham Osteoarthritis Study , we measured levels of serum IgE and performed statistical analyses to determine whether bulk serum IgE was associated with the presence of osteoarthritis and/or osteoarthritis severity. At certain serum IgE thresholds (e.g. >200 ng/mL), we detected trends towards more severe osteoarthritis in individuals with increased serum IgE, but these results were not statistically significant ; p=0.08.We also used ELISA to measure bulk IgE levels in osteoarthritis (OA), rheumatoid arthritis (RA), and psoriatic arthritis (PsA) synovial fluids . In thesIt remains possible that antigen-specific IgE, rather than bulk IgE levels, will be associated with the development of osteoarthritis. To address this possibility, we examined whether osteoarthritis synovial fluid IgE is able to bind cartilage extracts. We found that osteoarthritic joint synovial fluid IgE can bind both damaged “lesional” cartilage and “non-lesional” cartilage from osteoarthritic joints .These data suggest that antigen-specific IgE might play a role in the pathogenesis of osteoarthritis. Nevertheless, we believe these findings are too preliminary to include in the main manuscript. Further work will be needed to validate these preliminary findings, to define the role of antigen-specific IgE, and to characterize the antigen targets of IgE, in osteoarthritis.We added the following text to the Discussion to address our preliminary findings and these possibilities:“Previous reports found that IgE levels are not elevated in the synovial fluids of osteoarthritis joints , and in preliminary studies we observed only trends towards an association of increased bulk IgE in serum with osteoarthritis in humans. It remains possible that antigen-specific IgE contributes to mast cell activation in osteoarthritis, and future studies will be needed to investigate the relative contributions of antigen-specific IgE and other mechanisms of mast cell activation in the pathogenesis of osteoarthritis.”4) In vivo relationship between mast cell deficiency and cytokines/proteases/fibrobalstic proliferation/solubleglycoaminoglycans in the synovial tissue is required.5) In vivo relationship between IgE/FceRI/Syk axis and tryptase activity is required.6) In vivo relationship between Syk in mast cells, DMM-induced cartilage destruction, the related signatures is required.We appreciate the reviewers’ suggestions in these three interrelated questions. To provide further mechanistic data regarding the role of Syk in mast cell activation, we performed new in vivo experiments to examine the expression of a panel of mast cell mediators during treatment with the Syk inhibitor PRT062607 following DMM. We treated mice with either PRT062607 or vehicle for 6-weeks following DMM, and then harvested DMM joint synovial tissue and used qPCR to analyze expression levels of mRNAs encoding pro-inflammatory cytokines and proteases. We found that expression of multiple pro-inflammatory cytokines, chemokines and proteases were reduced in DMM joint tissues derived from mice receiving pharmacologic treatment with the Syk inhibitor as compared to vehicle. We now include these data in new Figure 5I. The following text was added to the “IgE signaling through FcRI promotes pathogenesis of osteoarthritis” sub-section of the Results:“We used qPCR to analyze the levels of mRNAs encoding pro-inflammatory cytokines and degradative enzymes known to be produced by mast cells. Six-weeks following DMM, transcriptional expression of multiple pro-inflammatory cytokines and proteases including IL-1, CCL2, ADAMTS4 and MMP13 were significantly reduced in DMM joint tissues derived from mice treated with the Syk-inhibitor PRT062607 as compared to vehicle .”Together, the data in our manuscript demonstrate a critical role for mast cells and mast cell-derived tryptases in promoting cartilage degeneration and joint destruction following DMM in mice. We specifically demonstrate in DMM mice that mast cell-deficiency , treatment with inhibitors of mast cell survival or signaling , deficiency or depletion of IgE , and treatment with the APC366 inhibitor of mast cell tryptase , all reduce cartilage destruction  and synovitis . We appreciate the reviewer’s suggestion for inclusion of additional data linking mast cells and mast cell activation pathways to the production of tryptase and other mast cell and inflammatory mediators, but believe our data provide compelling evidence for a critical role for mast cells and mast cell tryptases in the pathogenesis of osteoarthritis. Further, the DMM model requires a protracted timeline – mice must be aged to 18-20 weeks prior to surgical DMM, followed by a 4 month in-life period following surgical DMM prior to harvesting of joint tissues, and then 1+ month is necessary for histologic processing of joint tissues – making it not logistically possible for us to perform the additional experiments necessary to address all of the reviewers’ suggestion in the time allotted. As a result, we now address the need for further investigation of the role of mast cells in promoting these changes as a limitation of our study in the Discussion:“There are multiple potential limitations to our study… Second, while our findings demonstrate a critical role for mast cells, mast cell activation pathways, and the mast cell product tryptase in the development of osteoarthritis following DMM, further investigation is needed to characterize the relationship between IgE-mediated mast cell activation and the presence of pro-inflammatory cytokines and proteases implicated in synovitis and cartilage degradation in osteoarthritis.”7) In regard to the data presented in this manuscript, both reviewers require more high quality, more quantification, and incorporation of suitable controls.We revised the manuscript to address these concerns from the reviewers. The findings from our new experiments further strengthen our conclusions that IgE-mediated mast cell activation promotes the pathogenesis of osteoarthritis. The following changes and/or new data were added:1) We replaced the previous images from our electron microscopy analysis of mast cell degranulation and tryptase release in synovial tissues from humans with osteoarthritis in Figure 1C with new higher-quality versions of these images.2) In response to comment #7 from reviewer #2, we now provide additional mechanistic data implicating mast cell-derived tryptases in the pathogenesis of osteoarthritis by demonstrating that in DMM synovial tissue mRNA levels of multiple pro-inflammatory cytokines and degradative enzymes are reduced by pharmacologic inhibition of tryptase . We also now demonstrate that treatment of synovial fibroblasts with tryptase promotes pro-inflammatory signaling through activation of the MAPK/ERK pathway .3) In response to Concern #6 from the Reviewing Editor and comment #11 from reviewer 2, we now provide mechanistic data supporting a key role for Syk signaling in mast cells in the pathogenesis of osteoarthritis by showing that pharmacologic inhibition of Syk reduces mRNA levels of multiple pro-inflammatory cytokines and degradative enzymes in DMM synovial tissues .KitW-sh/W-sh and -1fl/flCpa3-Cre;MCl), including their levels following mast cell reconstitution . We also quantified the numbers of toluidine blue-positive mast cells in synovial tissues of IgE-sufficient (+/+Igh7) and IgE-deficient (-/-Igh7) mice following DMM .4) In response to comment #4 from reviewer #2, we now present data quantifying the numbers of mast cells present in mouse joint synovia from multiple experiments presented in the manuscript. Specifically, we quantified the numbers toluidine blue-positive mast cells in the synovia of DMM joints from two strains of mast cell-deficient mice (5) We performed new immunostains for mast cells in synovial samples from human osteoarthritis and post-trauma non-osteoarthritis joints, and demonstrate that similar numbers of mast cells are present in both conditions . Although there are similar numbers of mast cells in human osteoarthritis and post-trauma non-osteoarthritis synovial tissue samples, we demonstrate that the mast cells in the osteoarthritis synovium are actively degranulating  and releasing activated tryptase into the synovial fluid .6) We now include additional controls demonstrating that the observed downregulation of these cytokines and proteases is specific and not a general response .Separate reviews (please respond to each point):Reviewer #1:The authors present convincing evidence of the involvement of mast cells and the IgE/FceRI/Syk axis in inflammation and cartilage destruction in experimental Osteoarthritis (OA) in mice. They also described increased mast cell degranulation, mast cell tryptase release, and the upregulation of mast-cell related genes, in joints from osteoarthritis patients than in joints from non-OA trauma patients. Their findings support a role for IgE-mediated mast cell activation in OA.Their thorough experimental approach used a variety of genetically modified mice, cell transfers, neutralizing antibodies and pharmacological treatments to assess the role of mast cells in OA. They found that the deficiency of mast cells, IgE or FceRI, and the inhibition of Syk, decreased pathology in mice OA. They also demonstrated a pathogenic activity of mast-cell tryptase in promoting inflammation and cartilage destruction in in vitro and in vivo.What I found missing in the analysis of mice with OA is the functional assessment of the disease joint in the various experimental conditions, as they did for example in a previous publication by measuring gait function , or by evaluating pain .We appreciate the reviewer’s suggestion. Nevertheless, the primary objective of this study was to identify molecular mechanisms underlying structural changes in osteoarthritis. As previously demonstrated in both Wang et al., (2011) and Huesa et al., (2016), the cartilage degeneration and joint breakdown that occurs following DMM results in both abnormal gait and increased pain. We believe that histologic demonstration of cartilage degeneration, joint breakdown, and synovitis is the most definitive outcome in the DMM model, and as a result this was the focus of our studies. Additionally, a prior study implicated mast cells in human osteoarthritis pain , but did not demonstrate that they play a direct role in the pathogenesis of osteoarthritis. In this manuscript, we provide extensive evidence based on histologic outcomes that mast cells and IgE play critical roles in the cartilage breakdown, joint tissue destruction, and synovitis that are characteristic of osteoarthritis. Given that we and others previously demonstrated that histologic osteoarthritis pathology is associated with pain and gait dysfunction in mice , we do not believe that assessment of gait function or pain would significantly advance the conclusions of our study. We now address this potential limitation of our study in the Discussion:“There are multiple potential limitations to this study. First, it was previously shown that mast cells are associated with joint pain in murine osteoarthritis  and that synovial mast cell numbers are associated with the degree of synovitis in human osteoarthritis . We and others previously demonstrated that following DMM in mice development of histologic osteoarthritis is associated with poor functional outcomes including pain and abnormal gait . The studies presented in this manuscript demonstrate that mast cells and dysregulated mast cell activation contribute to cartilage and joint degeneration following DMM, and based on this prior work  such pathologic changes are anticipated to result in pain and abnormal gait. Future studies will be needed to further characterize the role of mast cells in osteoarthritis-associated pain and joint dysfunction…”I wonder if the authors have explored the relationship between mast cell activation and complement activation in OA. Some years ago they described a central role of complement activation in human and mouse OA (Nature Medicine 2011). Since the activation of complement produces anaphylotoxins that can recruit and activate mast cells, it is possible that complement regulates mast cells in the synovia. And reciprocally, products of the degradation of cartilage by mast-cell tryptase may activate complement in the synovia.It will be interesting to know what is temporal relationship during disease development between mast activation/degranulation and complement activation, and how deficiency or hyper-activation of one of these two mechanisms affect the other. The relationship between these two pathogenic mechanisms in human OA could also be explored, they may go together or define different disease phenotypes.We appreciate the reviewer’s comments in these two related questions. We agree that the known connection between complement and mast cells makes it possible that complement regulates mast cell recruitment and activation in osteoarthritis synovia. We also agree that understanding the temporal relationship between complement and mast cell activation in the development and progression of osteoarthritis is an important question. We now address these possibilities in the following new text added to the Discussion:“We previously demonstrated that complement plays a critical role in the pathogenesis of osteoarthritis . Activation of the complement system results in the production of C3a and C5a which serve as anaphylatoxins and activators of mast cells . It is therefore possible that complement regulates the recruitment and activation of mast cells in the synovium to promote the pathogenesis of osteoarthritis. We previously demonstrated that the cartilage breakdown product, fibromodulin, can activate the complement system , and it is possible that mast cell tryptase-mediated cartilage degeneration produces fibromodulin and other cartilage breakdown products that reciprocally activate complement in the synovia in osteoarthritis. It will be important to define the temporal relationship between mast cell activation/degranulation and complement activation in the development and progression of osteoarthritis, including how activation of one affects the other and vice versa. Further, complement is capable of stimulating mast cells alone, and can also enhance IgE-dependent mast cell activation and degranulation . Future studies will be needed to further define the roles and relationships of complement activation, IgE, and mast cell activation/degranulation in the development and progression of osteoarthritis.”Minor Comments:Histological analysis should be described in more detail, how many sections and microscope field were analyzed, and the criteria for grading.We now include a detailed description of the histological analysis and methods of scoring cartilage degradation, osteophyte formation and synovitis. The following text was added to the “Histologic assessment of osteoarthritic development in mice” sub-section of the Materials and methods:“Stifle joints were harvested 12 or 20 weeks after DMM and fixed in 10% neutral buffered formalin followed by decalcification in formic acid for 48 hours. Joints were then embedded in paraffin, and 6-μm sections cut from 3 separate levels of the joint and stained with Safranin-O for assessment of cartilage damage; H&E for assessment of synovial thickening (synovitis) and osteophyte formation; and toluidine blue for the assessment of mast cells. Cartilage degeneration, synovitis, and osteophyte formation were evaluated by two blinded observers using a modified version of a described scoring system  as we previously described . In brief: Cartilage degeneration was calculated by depth of cartilage degeneration (score of 0–4) × width of cartilage degeneration  in each third of the femoral-medial and tibial-medial condyles. The scores for the six regions were then summed. Synovitis scores were calculated as previously described : 0, no changes compared to normal joints; 1, thickening of the synovial lining and some influx of inflammatory cells; 2, thickening of the synovial lining and intermediate influx of inflammatory cells; and 3, profound thickening of the synovial lining (more than four cell layers) and maximal observed influx of inflammatory cells. Scores for synovitis were recorded for the femoral-medial and the tibial-medial condyles, and the scores for the two regions summed. Osteophyte formation was scored according to a previously described scoring system : 0, none; 1, formation of cartilage-like tissues; 2, increase of cartilaginous matrix; 3, endochondral ossification. Mast cells were quantified by a blinded examiner who determined the number of toluidine blue-positive mast cells per high power field of the joint sections.”Reviewer #2:Osteoarthritis (OA) is a degenerative joint disease and is the most common form of arthritis. In this manuscript, the authors found abnormally degranulated mast cells in human and murine osteoarthritic joint tissue. Disruption of IgE/FceRI/Syk signaling axis ameliorated development of osteoarthritis. This signaling, along with histamine-releasing factor (HFR) promoted releases of tryptases from mast cell and induced inflammation and cartilage destruction in synovial tissues. Therefore, the authors concluded that the aberrant mast cell activation is critical for pathogenesis of OA. Despite potential importance of mast cell in OA, the present study has several concerns.Major concerns:In general, OA is induced by long lasting mechanical stresses and can be accompanied by chronic inflammation, whereas IgE mediates antigen-specific acute and drastic inflammatory responses in allergic diseases. Therefore, the authors need to provide very careful experimental data to show that IgE-mediated immune responses are implicated in OA pathogenesis and induction of chronic inflammation that accompanies OA. Also, antigen specificity of the IgE involved in the pathogenesis of OA is unclear. Thus, the authors have to give convincing explanation by clarifying the mechanism by which mechanical stress induces IgE production, and identifying the antigen specificity of the IgE.1) If IgE is implicated in the pathogenesis of OA, diseases related to high level of IgE  should be associated with the onset and severity of OA. Therefore, the authors should provide epidemiological evidence on this point.We appreciate the reviewer’s suggestion. The possibility of osteoarthritis being associated with allergic diseases is an interesting question. While an epidemiological analysis could provide important new insights, we believe an epidemiological analysis is beyond the scope of our current study. We now address these possibilities in the following text added to the Discussion:“While many classical IgE-mediated allergic diseases including asthma, allergic rhinitis and eczema exhibit comorbidities , we are not aware of evidence of a clear link between osteoarthritis and allergic diseases. There is significant evidence that these classic allergic diseases are caused by antigen-specific IgE-dependent activation of mast cells . If the role of mast cells in osteoarthritis pathogenesis is dependent on antigen-specific IgE, the target antigens could potentially be exogenous allergens. Another possibility is that in osteoarthritis the IgE target antigens are bone or cartilage breakdown products that give rise to neoantigens following joint injury or instability. Although we are not aware of evidence that osteoarthritis is associated with classic allergic and/or IgE-dependent diseases, an epidemiological analysis that addresses this important question is warranted. Further studies are also needed to characterize the antigen targets of IgE in osteoarthritis, which could reveal shared or distinct targets as compared to classical allergic diseases as well as provide insights into the mechanistic basis for the development of osteoarthritis.”2) A mechanical damage induced OA model does not always reflect all the features of human OA. To propose the surprising concept like IgE involvement, one model of OA is not sufficient. The authors should verify the observation as described in the manuscript by another OA model.We appreciate the reviewer’s comment. While no mouse model perfectly mimics osteoarthritis pathogenesis in humans, we chose to use the DMM model because it is highly representative of a significant subset of human osteoarthritis and is also the gold standard in the field for experimental investigation of osteoarthritis . We, however, note that the DMM model is most representative of osteoarthritis development following traumatic joint injury (PTOA) in humans. It is possible that non-traumatic osteoarthritis, calcium pyrophosphate crystal-associated osteoarthritis, or other subsets of osteoarthritis may arise from activation of other molecular pathways. Thus, IgE-dependent mast cell activation may not contribute to development of all subsets of osteoarthritis disease.-/-Fcer1) on the development of osteoarthritis in the MM mouse model. Following MM, -/-Fcer1 mice were protected from cartilage degeneration as compared to wild-type mice  and destabilization of the medial meniscus (DMM) mechanical injury mouse models , indicating that similar molecular mechanisms related to the complement system underlie osteoarthritis pathogenesis in both models. To test whether IgE activation similarly promotes osteoarthritis in multiple mouse models, we have now analyzed the effect of genetic deficiency of the high-affinity IgE receptor, Fcer1  in mice. As the DMM model is most representative of osteoarthritis development following traumatic joint injury (PTOA) in humans, these findings suggest that PTOA arises as result of activation of the IgE/FceRI/Syk axis. It is possible that non-traumatic osteoarthritis, calcium pyrophosphate crystal-associated osteoarthritis, or other subsets of osteoarthritis may arise from activation of other molecular pathways.”3) The quality of electron microscopy is too poor to distinguish the mast cell granules. The authors should provide better quality immune-electron microscopic images as shown in Figure 1.We now included higher-quality versions of images from our election microscopy analysis of tryptase in synovial tissues from osteoarthritis and healthy individuals in Figure 1B.4) The quantification of the infiltrated mast cells is only based on the unclear images . Likewise, mast cell quantification in destabilization of the medial meniscus (DMM)-induced mice were also determined by histological analysis in Figure 2—figure supplement 3 and is not performed in Figure 3, 4 and 5. The authors have to quantify the number of mast cells infiltrated into synovial tissues regarding patient with OA in Figure 1 and DMM-induce mice in Figure 2—figure supplement 3, Figure 3, 4 and 5 by use of FACS. In addition, morphology of the FACS-sorted mast cells from synovial tissues must be examined by appropriate staining.The images provided in Figure 1B are representative from the overall experiment presented as well as from 2 additional independent experiments that utilized independent osteoarthritis and non-osteoarthritis samples. In the legend for Figure 1, we indicate that for the representative experiment presented in the manuscript, mast cells and degranulation were quantified in samples from 5 individuals with osteoarthritis and 5 non-osteoarthritis individuals. Quantitation of the blinded scoring of mast cell degranulation in human osteoarthritis vs. post-traumatic non-osteoarthritis synovium is presented in Figure 1C (P <0.01). Further, it is important to note that we also demonstrated increased levels of activated tryptase in human osteoarthritis as compared to non-osteoarthritis synovial fluids , showing active and ongoing degranulation of mast cells in human osteoarthritis synovium, given the half-life of activated tryptase is approximately 2 hours.In regard to the quantification of mast cell numbers in humans, others reported that synovial mast cell numbers are (i) elevated in osteoarthritis as compared to healthy synovium , and (ii) associated with the degree of synovitis in human osteoarthritis .Performance of FACS on synovial cells requires enzymatic digestion and dissociation of the synovium into single cell suspensions. We, and the NIH Accelerating Medicines Partnerships consortium of which we are a participating member, have found that such enzymatic dissociation of synovium  physically destroys mast cells, thereby limiting their recovery. Such makes it technically difficult and likely inaccurate to quantitate synovial mast cells through FACS, and we believe electron microscopy and immunostaining represent more appropriate methods. To further assess mast cell levels in human osteoarthritis synovial tissues, we performed new immunostains for mast cells on synovium from joints with osteoarthritis (OA) and from joints sustaining traumatic joint injury but exhibiting no radiographic evidence of osteoarthritis . These immunostains demonstrate similar levels of mast cells in the synovium from both groups . We have not compared the levels of mast cells observed in osteoarthritis with normal synovium, nor with the degree of synovitis in osteoarthritis synovium, as reported by others . We note that our studies reported in this manuscript are focused on mast cell activation and degranulation in osteoarthritis, and we provide evidence of ongoing mast cell activation and degranulation in human osteoarthritis through both electron microscopy demonstration of actively degranulating mast cells in osteoarthritis synovium and ELISA demonstration of increased activated mast cell tryptase (a mast cell degranulation product) in osteoarthritis synovial fluids .KitW-sh/W-sh and -1fl/flCpa3-Cre;MClmast cell-deficient mice as compared to both wild-type mice and the mast cell engrafted mice . Toluidine blue staining of mast cells in DMM synovium was confirmed by anti-tryptase immunostaining . We also demonstrate that imatinib-mediated protection against osteoarthritis following DMM was associated with reduced mast cell numbers in mouse DMM synovium . Further, we now provide new data quantifying the numbers of mast cells in the IgE-sufficient (+/+Igh7) and IgE-deficient (-/-Igh7) mice from our DMM experiments in Figure 4A-D. These data demonstrate decreased numbers of mast cells in -/-Igh7 as compared to +/+Igh7 mice .We now also provide new data quantifying the numbers of mast cells present in synovial tissues derived from our DMM experiments in Figure 2 and Figure 2—figure supplement 1 for which images are presented in Figure 2—figure supplement 3. For this quantitation, a blinded examiner determined the number of toluidine blue-positive mast cells per high power field in synovium derived from each of the mice included in the experiments presented in Figure 2 . This analysis demonstrates statistically decreased mast cells in both the 5) It also remains unclear whether DMM induces accumulation of mast cell at the synovial tissues. The author should compare the number of synovial mast cells in the DMM-induced mice with those of sham operation.We agree with the reviewer that this is an important experiment. We now quantify the numbers of mast cells present in synovial tissues of wild-type mice 20-weeks following either sham or DMM surgery. For this quantitation, a blinded examiner determined the number of toluidine blue-positive mast cells per high power section for each of the mice in this experiment. These data demonstrate a trend toward increased numbers of mast cells in DMM synovial tissues as compared to Sham synovial tissues, however this difference is not statistically significant .6) The results shown in Figure 3 are not enough to support authors' conclusion because the experiment in which human cartilage explants are cultured in the presence of recombinant tryptase is too artificial. To prove whether the mast cell derived tryptases destroy synovial cartilage, the authors have to culture human cartilage explants with mast cells and examine the all parameters as shown in Figure 3 A-F as well as soluble glycosaminoglycans.We agree with the reviewer that the in vitro human cartilage explant experiments represent a highly artificial experimental system, and as a result have now removed these experiments and the resulting data from the manuscript . Figures 3G and H, in which treatment of DMM mice with the tryptase inhibitor, APC366, preserves cartilage in vivo, show that tryptases promote cartilage degradation in osteoarthritis. We have now moved the mouse DMM experiment with treatment of APC366  to Figures 3A-D. We also added new experimental data demonstrating that pharmacologic inhibition of tryptase results in decreased transcriptional expression of inflammatory and degradative mast cell mediators in DMM joint tissues .7) There is no evidence whether the mast cell derived tryptases are involved in the induction of proinflammatory cytokines, proteases expression, fibroblast proliferation and soluble glycosaminoglycans in vivo. The authors have to check whether mast cell deficiency and APC366 administration reduce the induction of proinflammatory cytokines, proteases expression, fibroblast proliferation and soluble glycosaminoglycans in the synovial tissues under the DMM-induced condition.We agree with the reviewer that analysis of pharmacologic inhibition of tryptase on the expression of pro-inflammatory mast cell mediators would further strengthen our findings. To address this, we performed new experiments measuring the mRNA levels in DMM joint tissues of pro-inflammatory cytokines, chemokines and proteases following treatment with the tryptase inhibitor APC366. Six-week following DMM, transcriptional expression of multiple pro-inflammatory cytokines, chemokines and proteases was reduced in DMM joint tissues derived from mice treated with the tryptase inhibitor APC366 as compared to vehicle. We now include this analysis as new Figure 3E.We further investigated the role of tryptase in promoting pro-inflammatory signaling in synovial fibroblasts by treating cultured primary synovial fibroblasts from individuals with osteoarthritis with purified tryptase. Western blot analysis shows that phosphorylation of Erk1/2 was stimulated following treatment with tryptase. We now include this analysis as new Figures 3J and K.Based on the changes to Figure 3, we revised the “Mast cell-derived tryptases promote osteoarthritis-associated pathology” sub-section of the Results:MMP3 was significantly reduced in DMM synovial tissues derived from tryptase treated as compared to vehicle treated mice .“Having established a pathogenic role for mast cells in osteoarthritis and because levels of the activated form of mast cell-derived tryptase are significantly elevated in the synovial fluids of individuals with osteoarthritis, a finding in agreement with previous reports , we next investigated mechanisms by which tryptase might promote the pathogenesis of osteoarthritis. We first tested whether selectively inhibiting the protease activity of tryptase with APC366  – an oral, selective tryptase small-molecule inhibitor previously used to alleviate allergic, inflammatory and fibrotic responses in multiple mouse models  – could effectively attenuate the progression and/or severity of osteoarthritis in mice. We found that following DMM, treatment with APC366 for 12 weeks significantly reduced cartilage damage , osteophyte formation  and synovitis  compared to treatment with the vehicle-control mice, suggesting that tryptase inhibition can prevent the development of osteoarthritis in mice. We, additionally, measured the expression of pro-inflammatory and degradative mediators known to be produced by mast cells in DMM joints following treatment with the tryptase inhibitor APC366. Six-weeks after DMM, transcriptional expression of multiple mediators including IL-1, IL-6, IL-8, CCL2, CCL5, ADAMTS4 and MMP3 and ADAMTS4 , increased the secretion of cytokines IL-1β , IFN , and increased synovial fibroblast proliferation in vitro, as demonstrated by increased expression of the activation marker Ki-67 by fibroblasts . in vitro treatment of synovial fibroblasts with tryptase also promoted phosphorylation of Erk1/2, indicating that tryptase can activate pro-inflammatory signaling pathways in synovial fibroblasts . Further, in vitro inhibition of tryptase activity with APC366 abrogated the pro-inflammatory and proliferative responses of synovial fibroblasts .”As tryptase has been shown to promote pathogenic properties in human rheumatoid arthritis-derived synovial fibroblasts , we examined whether tryptase could also induce pro-inflammatory and proliferative responses in primary synovial fibroblasts derived from remnant osteoarthritic joint tissue. Indeed, tryptase significantly increased the expression of the pro-inflammatory cytokine IL-1 and degradative enzymes 8) The authors should show the evidence that chondrocyte undergoes apoptosis.Based on this reviewer’s comment #6, we have now removed the data from our in vitro experiments treating cultured chondrocytes with tryptase . As a result, this suggestion is no longer relevant.9) There is no evidence as to whether disruption of the IgE/FceRI/Syk signaling suppresses tryptases production in vivo. The authors have to measure the tryptase activity and soluble glycosaminoglycans in the synovial fluids in Figure 4 and 5.We appreciate the reviewer’s comment and believe this analysis is important. However, the mouse experiments required to analyze the relationship between IgE/Syk signaling and tryptase production require significant amounts of time (>6 months). As a result, we were unable to perform new experiments to address this question within the timeframe provided to respond to the reviewers’ critiques. Nevertheless, we demonstrate that genetic deficiency in IgE , depletion of IgE with anti-IgE antibody , or pharmacologic inhibition of Syk , all protect mice against development of osteoarthritis following DMM. We now address this limitation in the Discussion of our manuscript:Limitations paragraph: “There are multiple potential limitations to this study… Additionally, demonstration in vivo that IgE/Syk signaling is associated with increased tryptase production would further strengthen our findings.”10) The author must examine whether DMM induces increases in IgE concentration in synovial fluids in comparison to those of sham operation. If so, is it sufficient to stimulate synovial mast cells?We agree with the reviewer that it would be interesting to compare IgE levels in DMM and sham-operated mouse joints. Prior data indicate that levels of bulk  IgE are not elevated in the synovial fluids of human osteoarthritic joints . In addition, as described in our response to the Reviewing Editor’s comment #3 (above), in preliminary studies we found that bulk IgE levels in both synovial fluids and sera are not significantly elevated in individuals with osteoarthritis. As described above, we did observe a trend towards increased levels of bulk IgE in sera derived from individuals with osteoarthritis as compared to individuals with healthy joints. Further, our preliminary data suggest that antigen-specific IgE might be present in human osteoarthritis synovial fluid and thereby might play a role in osteoarthritis pathogenesis .While we agree that further investigation of IgE levels in mice is an interesting question, obtaining synovial fluids from mice is technically difficult – particularly from sham operated joints (e.g. mouse joints with no osteoarthritis) in which there is not significant accumulation of excess synovial fluid. As a result, we were not able to perform these experiments. As outlined above, we believe investigation of the role of antigen-specific IgE in human osteoarthritis represents a more promising line of investigation . We now address the need for further investigation of the role of IgE and antigen-specific IgE in the Discussion:“Previous reports found that IgE levels are not elevated in the synovial fluids of osteoarthritis joints , and in preliminary studies we observed only trends towards an association of increased bulk IgE in serum with osteoarthritis in humans. It remains possible that antigen-specific IgE contributes to mast cell activation in osteoarthritis, and future studies will be needed to investigate the relative contributions of antigen-specific IgE and other mechanisms of mast cell activation in the pathogenesis of osteoarthritis.”11) The authors must examine whether the DMM-induced cartilage destruction as well as the related signatures  are canceled in the Cpa3-Cre Syk-flox mice.Given that FcR1 signals via Syk , the experiment recommended above would further confirm a role for the FcR1 /Syk pathway in the DMM model of cartilage destruction. Based on our demonstration that both genetic deficiency of FcR1  and pharmacologic inhibition of Syk  prevent cartilage degradation in DMM mice, we believe that this additional experiment is redundant and would not significantly advance the current findings. Nevertheless, we now provide new data on the transcriptional expression of multiple mast cell mediators, including pro-inflammatory cytokines and proteases, in the joints of DMM mice following treatment with the Syk-inhibitor PRT062607. Six-weeks following DMM, transcriptional expression of IL-1, CCL2, CCL5, IL-6, TNF, MMP13, and ADAMTS4 was significantly reduced in the joints of PRT062607-treated as compared to vehicle-treated mice. These data are now included as new Figure 5I (see response above to Major Concern #6 from the Reviewing Editor).As you can see below, one reviewer had some concerns regarding the correlation between IgE level and severeity of osteoarthritis. Regarding this concern, I judge the Ig-7KO, FceRI KO, and anti-IgE data to provide convincing genetic evidence, and as mentioned in your letter, that authors' explanation of why the IgE level did not significantly change is reasonable. Moreover, the authors carefully mentioned, for instance, "using genetic and pharmacological approaches" in the Abstract.Please address the minor concerns of Reviewer #2 outlined below.Reviewer #1:The authors have answered a good part of the concerns of the reviewers. The manuscript is improved and I recommend that it will be accepted for publication.Reviewer #2:The authors proposed that the mast cell activation mediated by IgE/FceRI/Syk signaling is responsible for pathogenesis of OA. The authors corrected some statements in the revised manuscript. However, as I mentioned below, the fundamental problems of this manuscript have not yet been solved. In addition, they did not perform additional experiments to strengthen their conclusion.As part of our prior response to review, we performed multiple additional experiments including new in vivo experiments that provided further mechanistic data demonstrating a key role for IgE/FceRI/Syk signalling in mast cells in the pathogenesis of OA.First, following DMM, we treated mice with either the Syk inhibitor PRT062607 or vehicle for 6-weeks, and then harvested DMM joint synovial tissue and used qPCR to analyze expression levels of mRNAs encoding pro-inflammatory cytokines and proteases. We found that expression of multiple pro-inflammatory cytokines, chemokines and proteases were reduced in DMM joint tissues derived from mice receiving pharmacologic treatment with the Syk inhibitor as compared to vehicle. These data are included in new Figure 5I.-/-Fcer1), on the development of osteoarthritis following medial meniscectomy (MM). Following MM, -/-Fcer1 mice were protected from cartilage degeneration as compared to wild-type mice . This data recapitulates our results in the DMM model , indicating that IgE-signaling promotes osteoarthritis pathogenesis in multiple mechanical injury models.Second, we provided new in vivo data demonstrating a key role for the high-affinity IgE receptor, Fcer1  (P = 0.08). We previously added the following text to the Discussion to address this preliminary finding as well as possible explanations:“Previous reports found that IgE levels are not elevated in the synovial fluids of osteoarthritis joints , and in preliminary studies we observed only trends towards an association of increased bulk IgE in serum with osteoarthritis in humans. It remains possible that antigen-specific IgE contributes to mast cell activation in osteoarthritis, and future studies will be needed to investigate the relative contributions of antigen-specific IgE and other mechanisms of mast cell activation in the pathogenesis of osteoarthritis.”ELISA analysis also demonstrated that synovial fluid IgE level in patients with OA was lower than that of patients with RA and PsA.  These results made me feel that it is very unlikely that IgE is related to development of OA.As discussed above and in our prior response to review, we and others found that levels of total IgE in either sera or synovial fluids are not associated with osteoarthritis risk or severity in humans. While increased serum levels of IgE can be detected in some atopic diseases, this is also not always the case  – demonstrating that even in diseases well established to be mediated by IgE (e.g. antigen-specific IgE), elevations in bulk IgE are not always present. Importantly, in such allergic diseases it is antigen-specific IgE, and not bulk IgE, that plays a critical role in activating mast cells. Thus, it is possible that antigen-specific IgE, rather than bulk IgE, might play a role in the pathogenesis of osteoarthritis; or that antigen-independent mechanisms are responsible for IgE-mediated mast cell activation in osteoarthritis. Thus, we are unsure of the significance of differences in bulk IgE levels between different diseases. We do not believe these potential differences are relevant to the mechanisms by which IgE contributes to the pathogenesis of osteoarthritis.We believe our conclusions are already well supported by our data. Specifically, we believe that our Ig-7 KO, FceR1 KO, and anti-IgE data provide convincing genetic and pharmacologic evidence that IgE plays a critical role in the pathogenesis of osteoarthritis.The authors also referred to the possibility that antigen specificity of IgE rather than the amount of IgE may be involved in development of OA by showing the preliminary data. However, the data are too preliminary to convince us of the surprising finding that OA is an autoimmune disease caused by IgE against join antigen(s) and mast cells. The proposed mechanism is not well based on evidence. Taken together, I cannot recommend this manuscript for the publication in eLife.As we stated in our prior response to review, we believe our initial data on antigen-specific IgE is too preliminary to include in the manuscript and as a result we have not included it – we agree with the reviewer on this point.However, we respectfully disagree that our findings suggest that osteoarthritis is an autoimmune disease caused by IgE against joint antigens(s). A role for antigen-specific IgE, either against allergens or cartilage/synovial tissue breakdown products, is one of several potential mechanisms by which IgE could contribute to mast cell activation in synovial tissues and thereby the development of OA. We have now added to the following paragraph in the Discussion to address this consideration:“While many classical IgE-mediated allergic diseases including asthma, allergic rhinitis and eczema exhibit comorbidities , we are not aware of evidence of a clear link between osteoarthritis and allergic diseases. There is significant evidence that these classic allergic diseases are caused by antigen-specific IgE-dependent activation of mast cells . If the role of mast cells in osteoarthritis pathogenesis is dependent on antigen-specific IgE, the target antigens could potentially be exogenous allergens. Another possibility is that the IgE target antigens are bone or cartilage breakdown products that give rise to neoantigens following joint injury or instability. Although we are not aware of evidence that osteoarthritis is associated with classic allergic and/or IgE-dependent diseases, an epidemiological analysis that addresses this important question is warranted. Antigen-specific antibody responses can also be generated in autoimmune responses, however OA synovial linings do not exhibit histologic features consistent with an adaptive autoimmune response and we do not believe our findings suggest the presence of an classical adaptive autoimmune response. Future studies are needed to determine if IgE targets specific antigens, and to further characterize the role of IgE, in osteoarthritis.”1) In general, OA is induced by long lasting mechanical stresses and can be accompanied by chronic inflammation, whereas IgE mediates antigen-specific acute and drastic inflammatory responses in allergic diseases. Thus, it is not easy to imagine that IgE-mediated immune responses are implicated in OA pathogenesis and induction of chronic inflammation that accompanies OA. The authors have to give convincing explanation.We agree that long-lasting mechanical stresses likely contribute to the pathogenesis of osteoarthritis in a significant subset of patients, and added the following text to the Discussion to address this point: “Mechanical instability and stresses likely contribute to the pathogenesis of osteoarthritis in a significant subset of patients. We do not believe that a role for mechanical stresses is inconsistent with a role for IgE and mast cells, and it is possible that mechanical stresses produce cartilage breakdown products and/or cellular responses that promote IgE-dependent mast cell activation.”2) Authors showed that IgE level does not corelate with OA onset or severity. I think that this is contradictory to authors' conclusion. Authors tried to explain this by the antigen-specific IgE, but it is unbelievable that OA is an autoimmune disease caused by IgE against join antigen(s). If so, they should identify the specific antigens recognized by IgE from the synovial tissues.As described above, there are examples of IgE-mediated allergic diseases in which bulk IgE levels do not correlate with disease severity, and this may also be the case in osteoarthritis. As described in our responses above, we do not believe our findings suggest that osteoarthritis is an autoimmune disease and added several sentences to the Discussion to address this possibility (see above for full response and new text added). We believe definitive characterization of the potential targets of IgE in osteoarthritis, and/or if other mechanisms promote IgE-dependent mast cell activation in osteoarthritis, is beyond the scope of the current manuscript.3) The authors could not analyze synovial derived mast cells by flow cytometry because enzymatic dissociation of synovium physically destroys mast cells. But many researchers succeeded in cell preparation from human and mouse synovial tissue. In particular, FACS analysis of mast cells in synovial tissues was previously shown . Therefore, the authors must show the accurate number of mast cell by FACS in Figure 1 to 5 and Figure 2—figure supplement 1.We demonstrate that mast cells are present in human OA synovial lining by (i) immunostaining , and (ii) electron microscopy . In New Figure 1—figure supplement 2 we quantitate mast cells in human OA synovial linings and demonstrate that they represent approximately 1% of cells in the synovial lining.The work of Lee et al. is already cited in the following sentence in the Introduction of our manuscript: “Several studies have documented the presence of mast cells and their mediators in the synovium and synovial fluids of individuals with osteoarthritis .” We appreciate that the reviewer comments on the findings of Lee et al. , as their findings as well as the findings of the other groups cited all demonstrate the presence of mast cells in human osteoarthritis synovial linings and thereby support the findings presented in our manuscript. Further, these prior studies used multiple different experimental approaches and methods to demonstrate the presence of mast cells in human OA synovial linings – thus, these prior observations are consistent with and further support the findings reported in our manuscript. Even if technically feasible using other tissue digestion methods, we do not believe additional flow cytometry studies to analyze synovial mast cells would meaningfully strengthen or add to the findings presented in our manuscript.4) The authors cannot conclude that mast cell-derived tryptases promote osteoarthritis-associated pathology, since it remained unclear whether tryptase secreted by mast cells is sufficient to induce the destruction of the synovial cartilage. As I pointed out in the comment #6, addition of huge amount of tryptase is too artificial. The authors have to perform the co-culture experiment of mast cells with human cartilage explants. Furthermore, the authors have to show the evidence that chondrocyte undergoes apoptosis as I mentioned in comment #8.We appreciate the reviewer’s questions, and now add the following text to the Discussion to address these considerations. “Although we have not formally demonstrated that mast cells are the source of tryptases that promote the pathogenesis of OA, our data demonstrate that: (i) mast cells comprise 1 – 3% of synovial cells in both OA and PT non-OA synovial linings, (ii) mast cells are actively degranulating to release tryptase in human OA synovial linings , and pharmacologic inhibition of typtase prevents the development of OA in mice . Mast cells are the predominant producer of tryptases, and to a lesser extent basophils , and histologic examination of human and mouse OA synovial linings and tissues for basophils did not reveal basophils. Together, these data suggest that mast cells are the cellular source of tryptase in human and murine OA.”MMP3.Regarding this reviewer’s prior comment #6, we agree with the reviewer that the in vitro human cartilage explant experiments represent a highly artificial experimental system. As a result, in the prior revision we removed the data and figure panels in which relatively large amounts of tryptase were incubated with cartilage explants . We note that in vivo treatment of DMM mice with the tryptase inhibitor, APC366, preserves cartilage  – demonstrating that tryptases do promote cartilage degradation in osteoarthritis. In addition, as part of the prior revision, we performed new in vivo experiments that demonstrated that pharmacologic inhibition of tryptases results in decreased transcriptional expression of inflammatory and degradative mast cell mediators in DMM joint tissues . Specifically, we demonstrated that 6 weeks following DMM that treatment of mice with the tryptase inhibitor APC366 reduced the transcriptional expression of multiple inflammatory and degradative mediators including IL-1, IL-6, IL-8, CCL2, CCL5, ADAMTS4 and


Understanding the relationship between environmental factors and vital rates is an important step in predicting a species’ response to environmental change. Species associated with sea ice are of particular concern because sea ice is projected to decrease rapidly in polar environments with continued levels of greenhouse gas emissions. The relationship between sea ice and the vital rates of the Spectacled Eider, a threatened species that breeds in Alaska and Russia and winters in the Bering Sea, appears to be complex. While severe ice can impede foraging for benthic prey, ice also suppresses wave action and provides a platform on which eiders roost, thereby reducing thermoregulation costs. We analyzed a 23‐year mark‐recapture dataset for Spectacled Eiders nesting on Kigigak Island in western Alaska, and tested survival models containing different ice and weather‐related covariates. We found that much of the variation in eider survival could be explained by the number of days per year with >95% sea ice concentration at the Bering Sea core wintering area. Furthermore, the data supported a quadratic relationship with sea ice rather than a linear one, indicating that intermediate sea ice concentrations were optimal for survival. We then used matrix population models to project population trajectories using General Circulation Model (GCM) outputs of daily sea ice cover. GCMs projected reduced sea ice at the wintering area by year 2100 under a moderated emissions scenario (RCP 4.5) and nearly ice‐free conditions under an unabated emissions scenario (RCP 8.5). Under RCP 4.5, stochastic models projected an increase in population size until 2069 coincident with moderate ice conditions, followed by a decline in population size as ice conditions shifted from intermediate to mostly ice‐free. Under RCP 8.5, eider abundance increased until 2040 and then decreased to near extirpation toward the end of the century as the Bering Sea became ice‐free. Considerable uncertainty around parameter estimates for survival in years with minimal sea ice contributed to variation in stochastic projections of future population size, and this uncertainty could be reduced with additional survival data from low‐ice winters. AIC scores were adjusted for overdispersion using an overdispersion parameter (ĉ), taken as the ratio of deviance between real and simulated data. We estimated ĉ using the median ĉ method in Program MARK φ(year + age). We modeled p as a function of year, age, nest success, year + age, year*age, age + nest success, and as a constant  because this was confounded with age and year, and individual birds often had both types of tags within their lifetime.A maximum likelihood approach using Cormack‐Jolly‐Seber models was used to analyze the mark‐recapture data and estimate apparent survival (https://www.beringclimate.noaa.gov/data/index.php). The four 25 × 25 km cells defining the core wintering area were originally identified by  and Pacific Decadal Oscillation (PDO). Annual indices of AO, PDO, mean winter sea surface temperature, and a measure of winter North‐South wind anomalies in the Bering Sea were downloaded from the National Oceanic and Atmospheric Administration's Bering Climate data portal  and number of minimal sea ice days (days with <15% ice cover) at the Spectacled Eider wintering area from November through April each year. We chose the 95% cutoff to represent days with contiguous ice cover, which limits access by eiders to benthic foraging grounds , and sσ as the mean root‐square distance between all model pairs . Years that met this threshold had a covariate value for number of extreme ice days, and years that did not meet this threshold had the covariate value set at 0. Similarly, for minimal sea ice conditions, we used QAICc to select the model with the appropriate threshold of minimal sea ice days from 40 to 110 days per year with sea ice concentrations <15%. Years that met this threshold had a covariate value for number of days with low ice concentrations, and years that did not meet this threshold had the covariate value set at 0. Thus, extreme and minimal ice days became pseudo‐dummy variables in that no year would have a positive covariate value for both. Using this method, we were able to create a single model that estimated the relationship between survival and each end of the spectrum of ice concentrations. We then used parameter estimates from this model to estimate survival under projected future sea ice conditions.We projected future population size of female Spectacled Eiders on Kigigak Island using stochastic matrix population models Caswell, . We used3p was used in subsequent models of φ From 1992 to 2015, we obtained encounter histories for 1,328 birds, of which 649 were initially captured as ducklings. We observed slight overdispersion in the data, and ĉ was estimated at 1.326. For detection probability, the model that received the most support allowed detection to vary by year and age, and the relative likelihood of this model compared to other models was 1.0 , the best approximating model allowed survival to vary annually for adults but not first‐year birds, for which survival was modeled as a constant . The third best‐supported model indicated a quadratic relationship between survival and number of extreme ice days per year (days with >95% sea ice cover).. For F = 7.14, p = 0.005, Table F = 1.20, p = 0.47). Parameter estimates from our model indicated that survival increased with number of extreme ice days until approximately 52 days, beyond which survival flattened out and then began to decrease at approximately 80 days . The quadratic term for extreme ice days explained 44% of total deviance based on ANODEV test results  than for minimal ice days . However, caution must be used in interpreting projected survival rates because 95% confidence intervals around the parameter estimate for minimal ice days slightly overlapped zero. Using this relationship to project survival rates, we found that survival under moderate sea ice conditions  was relatively high at 0.88. These conditions were projected to occur early in the century, prior to 2049 for RCP 4.5, and prior to 2040 for RCP 8.5  females in 2069 coincident with moderate ice conditions early and mid‐century  is substantially greater than having no contact  to approximately 100 at the end of the century under RCP 4.5 and 175 days under RCP 8.5. These results agree with a CMIP5 GCM study of future sea ice cover in the Alaskan Arctic  and prey size (12–30 mm) relative to other locations in the Arctic , https://doi.org/10.5061/dryad.s1c5m5k.All survival and ice data used for this publication is publicly available in Dryad Digital Repository ( Click here for additional data file.