Results and discussion
In addition to immune cells, IL-33R is also expressed by sensory neurons.
We confirmed expression of IL-33R (ST2, Il1rl1) in mouse DRG (Fig 1, A), and using calcium imaging, we found that IL-33 activated 2.1% of DRG neurons (Fig 1, B and C). Further, we found that 52% and 63% of IL-33–responsive mouse DRG neurons also responded to histamine and the TRPV1 agonist capsaicin (Cap), respectively (Fig 1, D). Similarly, we found IL-33R was expressed by human DRG (Fig 1, E), and 6.6% of human DRG neurons were responsive to IL-33 (Fig 1, F and G). Of these neurons, 60% also responded to Cap (Fig 1, H). Together, these findings suggest that IL-33 can directly activate sensory neurons.
While these findings suggest that neuronal IL-33R signaling may be a critical itch pathway, the DRG contains a diversity of other cell types. The expression of IL-33R in the DRG beyond sensory neurons remains to be fully assessed. To address this, we analyzed a single-cell RNA-Seq data set of naive mouse DRG (Fig 1, I).
We found that Il1rl1 was indeed expressed by another cell type: DRG macrophages (Fig 1, J). Similarly, analysis of other neuronally expressed itch-associated cytokine receptors, such as Il4ra,
revealed expression across numerous cell types (Fig 1, K). Taken together, these data underscore that targeted, lineage-specific approaches are likely required to determine the precise contribution of a distinct cell type to itch development. Therefore, the consequence of disrupting IL-33R signaling specifically in sensory neurons remains unknown.
generating mice that conditionally lacked IL-33R in sensory neurons (IL-33RΔneuron mice). We confirmed the selective loss of Il1rl1 in sensory neurons, and not immune cells, isolated from IL-33RΔneuron mice (Fig 2, B). These mice exhibited normal motor function (Fig 2, C), thermal pain behavior (Fig 2, D), and acute itch response to the classical pruritogens histamine (Fig 2, E), chloroquine (CQ) (Fig 2, F), and serotonin (Fig 2, G), indicating that the mice had no gross developmental motor or sensory abnormalities.
Given the ability of IL-33 to promote type 2 inflammation,
there is considerable interest in the therapeutic potential of anti–IL-33 monoclonal antibodies in AD.
Several studies have found elevated levels of IL-33 in both the skin and blood of patients with AD.
In support, we found that patients with moderate to severe AD (n = 11, 5.17 ± 1.37) had increased IL-33 in their plasma compared to healthy control subjects (n = 11, 3.93 ± 1.20) (Fig 3, A and B, and see Table E1 in the Online Repository available at www.jacionline.org). We next utilized a model of AD-like disease, where mice are treated with MC903 (Fig E1, A). MC903-treated wild-type (WT) mice developed robust AD-like skin inflammation (Fig 3, C).
Indeed, analyzing our previously published RNA-Seq data set,
we found increased expression of Il33, along with transcripts for a number of other pruritogens, in the skin of MC903-treated WT mice compared to controls (Fig 3, D). However, while it is well known that IL-33 is dysregulated in both human and murine AD-associated inflammation, whether IL-33 directly engages the sensory neurons to elicit itch remains unclear. When we induced AD-like disease in IL-33RΔneuron mice, there were no notable differences in clinical or histopathologic presentation (Fig 3, E), ear thickness (Fig 3, F), or scratching bouts (Fig 3, G) compared to littermate controls. Thus, our findings suggest that neuronal IL-33R is dispensable for AD-like disease.
While patients with AD present with scaly, raised rashes (Fig 4, A), chronic itch in CPUO develops in the absence of overt cutaneous inflammation. Additionally, CPUO disproportionately occurs in aged individuals.
A key pathogenic factor of CPUO is skin barrier dysfunction, which frequently manifests as dry skin (Fig 4, B). The histopathology of CPUO often resembles control skin (Fig 4, C), while AD lesional skin exhibits a number of characteristic inflammatory features including irregular epidermal hyperplasia and robust dermal inflammatory infiltrate (Fig 4, D). In contrast, CPUO pruritic skin generally exhibits a relatively normal epidermis and mild dermal infiltrate (Fig 4, E). We found that patients with CPUO (n = 8, 6.22 ± 2.54) had significantly higher levels of IL-33 compared to healthy controls (n = 11, 3.93 ± 1.20) (Fig 4, F and G, Table E1). Thus, we hypothesized that IL-33 may be a key factor in itch physiology associated with CPUO.
We have previously utilized the AEW mouse model to identify novel therapeutic approaches that led to proof-of-concept studies in CPUO.
In contrast to other mouse models of chronic itch, AEW-elicited itch develops in the absence of notable cutaneous inflammation, similar to CPUO. Indeed, the frequency of cutaneous immune cells (Fig 5, B), including mast cells (Fig 5, C) and group 2 innate lymphoid cells (Fig 5, D), were comparable between WT mice that were treated with AEW and water-only controls, despite significantly increased itch behavior in AEW-treated mice (Fig 5, E). Notably, Il33 was elevated in the skin of AEW-treated mice compared to controls (Fig 5, F).
Taken together, our findings demonstrate that AEW-induced itch is associated with IL-33 dysregulation and minimal cutaneous inflammation, similar to CPUO.
However, how IL-33 drives the development of dry skin itch is poorly understood. Indeed, whether IL-33 can promote itch through a mechanism independent of canonical immune circuits remains unknown. Mast cells, and more recently basophils, have been implicated as key mediators of itch.
To test if these cell types contribute to dry skin itch, we used mast cell–specific enhancer-mediated toxin receptor–mediated conditional cell knockout (MasTRECK) mice, which allow for diphtheria toxin–mediated depletion of mast cells and basophils (see Fig E2 in the Online Repository at www.jacionline.org). However, AEW-induced scratching bouts were comparable between diphtheria toxin–treated littermate control and MasTRECK mice (Fig 5, G). IL-33 also potently activates both group 2 innate lymphoid cells and T cells to modulate the skin immune responses.
However, we found no difference in itch between AEW-treated lymphocyte-deficient Rag2/Il2rg−/− mice and controls (Fig 5, H). Finally, we generated mice that conditionally lacked IL-33R in immune cells by crossing the IL-33Rflox mice with the VavCre line (IL-33RΔimmune). After AEW-treatment, there was no difference in the number of scratching bouts between littermate control and IL-33Δimmune mice (Fig 5, I). Collectively, these data suggest that immune cells are largely dispensable for the induction of dry skin itch and instead implicate sensory neurons as the potential primary target of IL-33 for itch development.
This led us to hypothesize that IL-33 may instead sensitize sensory neurons. Indeed, it has been shown that AEW-treated mice exhibit enhanced responsiveness to exogenous pruritogens like CQ.
However, the mechanisms underlying these observations are not well understood. Using calcium imaging, in a proof-of-concept experiment, we found that IL-33 treatment of DRG neurons increased the number of cells responding to CQ (Fig 5, L and M). Thus, although CQ is not a native endogenous pruritogen in dry skin, these studies represent one example by which IL-33 may amplify responses to pruritogens in order to promote chronic itch. Future studies will be required to determine the precise molecular mechanisms by which IL-33 may enhance itch in this manner.
Together, these findings may help explain why anti–IL-33 monoclonal antibodies (eg, etokimab) have failed to meet their primary end points or have been discontinued after recent phase 2 clinical trials in AD (NCT03533751, NCT03736967). In contrast, IL-33 may be an important therapeutic target in dry skin itch and CPUO.
while the diagnosis of CPUO was based on Kim et al.
Healthy control and patient demographics are included in Table E1. DRG samples were obtained from deidentified US transplant donors under an institutional review board–exempt protocol at the University of Cincinnati. Control skin sections for hematoxylin and eosin (H&E) staining were obtained from the skin of patients who sought care at the hospital either for an amputation due to chronic ulcers or for cancer resection. There had to be a clear margin for sectioning (at least 10 cm for samples from amputations and 1 cm from tumor resections), which was reviewed again by a board-certified pathologist.
All animal experiments were conducted using protocols approved by the Washington University institutional animal care and use committee. Mice were maintained in standard husbandry conditions (social housing, 12-hour light–dark cycle, 23°C, food and water ad libitum). WT C56BL/6J were purchased from The Jackson Laboratory (Bar Harbor, Me). Rag2/Il2rg−/− double knockout mice were purchased from Taconic Biosciences (Germantown, NY). MasTRECK mice were donated by Dr Seiji Nishino (Stanford University). SNSCre mice were donated by Dr Rohini Kuner (Heidelberg University). IL-33Rflox were generated by Cyagen Biosciences (Santa Clara, Calif) on a C57BL6/J background. IL-33RΔneuron mice were generated by crossing SNSCre with IL-33Rflox mice. Experiments were conducted with independent cohorts of male and female mice that were 8 to 12 weeks old except for calcium imaging experiments, where 4- to 7-week-old mice were used. No phenotypic differences based on sex were observed.
Chronic itch mouse models
Ear thickness measurements were performed with dial calipers as previously described.
Percentage change was calculated from baseline (day 0).
At least 2 days before the first treatment, we shaved the nape or cheek skin. On treatment days, a 1:1 ratio of acetone (Sigma-Aldrich, St Louis, Mo) and diethyl ether (Sigma-Aldrich) was applied using a cotton pad for 15 seconds to the shaved skin (cheek or nape) followed immediately by application of a cotton pad soaked in distilled water for 30 seconds (Fig E1, B). Cotton pads used for water treatment were never reused. Mice received treatments twice daily (∼8 hours apart) for 5 days.
Itch behavior assessment (chronic and acute)
For assessment of itch behavior, mice were acclimated to the test chambers 2 days before the initiation of the experiment. Mice were additionally acclimated for at least 5 minutes before each recording. For chronic itch models, we recorded the mice in the morning (before they received any treatment). To assess acute itch responses, acclimated mice were provided an intradermal injection of 20 μL of either histamine (1 mg/mL, Sigma-Aldrich), CQ (100 μg, Thermo Fisher Scientific, Waltham, Mass), serotonin (1 mmol/L, Sigma-Aldrich), recombinant mouse (rm)IL-33 (50, 300 or 1000 ng; R&D Systems, Minneapolis, Minn), or saline control into their right cheek (shaved 2 days prior), with itch behavior immediately recorded. Video recordings were manually scored for the number of scratching bouts in a 30-minute period. A scratching bout was defined as a continuous scratching motion by the hind paw that ended when the mouse placed its paw on the floor or in its mouth. Data for acute itch model only contain cheek-directed scratching bouts (site of pruritogen administration).
Pain behavior assessment (thermal sensitivity)
Thermal sensitivity was assessed using the hot plate assay. Hot plate temperature was set to 50°C. Latency was measured as time from mouse being placed on the hot plate and removal when either flicking/licking of its hind paw or jumping was observed. Data were averaged across 2 trials taken over 2 days.
To test for potential defects in coordinated motor activity, mice were tested using a rotarod system (Ugo Basile, Lombardy, Italy) where mice were placed on a rotating treadmill that was accelerated from 5 rpm to 40 rpm over 5 minutes (built-in program of the apparatus). Time was recorded from acceleration initiation until mice fell from the rod or completed 1 passive rotation (time to failure). Training occurred over 3 days before testing day, with 3 trials conducted per day with a 10-minute break between each trial. On testing day, 3 trials were completed with a 10-minute break between each trial, and data were averaged across all trials.
Basophil and mast cell depletion
Briefly, MasTRECK and littermate (LM) mice were treated daily with intraperitoneal injections of diphtheria toxin (250 ng in 100 μL of phosphate-buffered saline; Sigma-Aldrich) for 5 consecutive days immediately before initiation of AEW treatments. Depletion was verified by flow cytometry (Fig E2).
Plasma cytokine measurement
Plasma was isolated after Ficoll gradient separation of peripheral blood drawn from patients with AD, patients with CPUO, or healthy control subjects (Table E1). Before the detection assay, plasma was diluted (1:1 in assay diluent), loaded onto protein L–coated plates (Thermo Fisher Scientific), and incubated on an orbital shaker at room temperature for 90 minutes. The FLEXMAP 3-dimensional system (Thermo Fisher Scientific) was used to collect the data.
RNA-Seq data analysis
under accession number GSE90883. Full methods on sample processing are available in Oetjen et al.
For our reanalysis, duplicate genes were removed, and genes were filtered for protein coding designation. Using row mean (counts), the top 12,000 genes were selected for additional downstream analysis. Differential gene expression was calculated using the limma R package (https://www.r-project.org/) with the online Phantasus software (https://artyomovlab.wustl.edu/phantasus/) along with hierarchical clustering (1 minus Pearson correlation) and heat map generation. Only the top 1300 most differentially expressed genes are displayed in Fig 3 (based on t value).
Single cell RNA-Seq data analysis
under the accession number GSE158892. Full methods on sample processing are available in Avraham et al.
Data analysis and processing were performed using commercial code from Partek Flow (https://www.partek.com/partek-flow/). Processed data are publicly available (https://mouse-drg-injury.cells.ucsc.edu/).
Mouse RNA isolation and PCR
For RNA isolation from the whole DRG, DRG were collected from naive WT mice and homogenized in 1 mL TRIzol reagent (Life Technologies, Carlsbad, Calif) with a bead homogenizer (BioSpec Products, Bartlesville, Okla). RNA was extracted using the RNeasy Mini Kit (Qiagen, Germantown, Md), and DNA was digested using the Turbo DNA-Free Kit (Invitrogen, Thermo Fisher Scientific) following the manufacturer’s instructions.
Briefly, DRG was incubated in Ca2+/Mg2+-free Hanks balanced salt solution containing collagenase type I (342 U/mL; Gibco, Thermo Fisher Scientific) and Dispase II (3.8 U/mL; Gibco) at 37°C on a rotator for 30 to 40 minutes. The sample was triturated, then transferred into MACS buffer (0.5% bovine serum albumin in Dulbecco PBS). Sensory neurons were negatively selected from the DRG using the MACS Neuron Isolation kit (130-115-389; Miltenyi Biotec, San Diego, Calif) as previously described by Thakur et al.
RNA was extracted using the Nucleospin RNA XS kit (Takara, Kyoto, Japan) according to the manufacturer’s instructions.
For RNA isolation from immune cells, lymph nodes were collected (inguinal, mesenteric, and superficial cervical) from naive LM control and IL-33RΔneuron mice before being manually homogenized through a 70 μm cell strainer and washed (5% fetal bovine serum in Dulbecco modified Eagle medium). RNA was extracted using the Nucleospin RNA kit (Takara) according to the manufacturer’s instructions.
For RNA isolation from skin, water control- or AEW-treated cheek skin from WT mice were collected 4 hours after the last treatment of AEW on day 4 of AEW mouse model. Skin samples were stored in RNAlater (Invitrogen) at 4°C before transfer to −80°C. After tissue homogenization with a bead homogenizer (BioSpec Products) in 350 μL of RNA lysis buffer (Nucleospin RNA, Takara), RNA was isolated using the Nucleospin RNA kit (Takara) according to the manufacturer’s instructions.
Products from the Il1rl1 qPCR reaction were run on a 2% agarose gel with 1 mg/mL of ethidium bromide at 140 V.
Human RNA isolation and qPCR
Samples were kept in RNAlater (Sigma-Aldrich) at −80°C. Half of a single DRG was homogenized in 1 mL of TRIzol reagent for total RNA extraction. For genomic DNA elimination and cDNA synthesis, the Maxima H Minus First Strand cDNA Synthesis kit with dsDNase (Thermo Fisher Scientific) was used with the IL1RL1 primer set (forward: CAG GGA GCG GCA GGA ATG T, reverse: CTT GCA TTT ATC AGC CTC CAG AGA A; MilliporeSigma, Burlington, Mass) in accordance with the manufacturer’s instructions. For gel electrophoresis, qPCR product was loaded onto a 2% agarose gel with 1 mg/mL of ethidium bromide at 100 V.
Calcium imaging of mouse DRG neurons
After humanely killing the animals via CO2 inhalation, the DRG were collected. Nerve fibers were trimmed, and connective tissue was removed from the DRGs. For enzymatic dissociation, DRGs were incubated in 1 mL of Ca2+/Mg2+-free Hanks balanced salt solution containing collagenase type I (342 U/mL; Worthington Biochemical, Lakewood, NJ) and Dispase II (4 U/mL; Gibco) at 37°C on a rotator for 30 to 40 minutes. DRGs were then triturated to generate a single cell suspension. Dissociated DRG neurons were then seeded on 8 mm glass precoated with poly-d-lysine (20 mg/mL, Thermo Fisher Scientific) and laminin (20 mg/mL, Sigma-Aldrich). Cells were cultured overnight at 37°C with 5% CO2 in Neurobasal A culture medium (Gibco) supplemented with nerve growth factor (100 ng/mL; Sigma-Aldrich), glial cell–derived neurotrophic factor (20 ng/mL; Sigma-Aldrich), B-27 (2%; Gibco), penicillin (100 U/mL; Sigma-Aldrich), streptomycin (100 mg/mL; Sigma-Aldrich), and 10% fetal bovine serum (Sigma-Aldrich).
The next day, the cultured DRG neurons were loaded with the calcium indicator dye Fura-2 AM (4 μmol; Invitrogen) for 20 to 45 minutes, then washed with calcium imaging buffer (130 mmol/L NaCl, 3 mmol/L KCl, 2.5 mmol/L CaCl2, 0.6 mmol/L MgCl2, 10 mmol/L HEPES, 10 mmol/L glucose, 1.2 mmol/L NaHCO3). Slides were imaged with alternating 340 and 380 nm excitation wavelengths using an inverted Nikon Ti-S microscope, CoolSNAP CCD camera (Photometrics, Tucson, Ariz), and NIS-Elements software (Nikon, Tokyo, Japan). Cap (300 nmol/L; Sigma-Aldrich), histamine (50 μmol/L, Sigma-Aldrich), and KCl (100 mmol/L; Sigma-Aldrich) were applied to DRG neurons via perfusion. rmIL-33 (1 μg/mL; R&D Systems) and vehicle control (0.1% bovine serum albumin (Sigma-Aldrich) in phosphate-buffered saline) were manually loaded by gently pipetting the solution into the recording chamber. Cells were washed with calcium imaging buffer for at least 3 minutes between stimuli. For sensitization experiments, we evaluated the response of DRG neurons to CQ (1 mmol/L; MP Biomedicals, Solon, Ohio) immediately after stimulation with vehicle control (0.1% bovine serum albumin) or rmIL-33 (4 μg/mL) with no wash between exposure to vehicle or rmIL-33 and CQ. Fluorescence ratios (340/380) were normalized to baseline. A change in the fluorescence ratio (340/380) of >10% was considered to indicate a cellular response.
Calcium imaging of human DRG neurons
After DRG dissociation, cells were plated on glass cover slips and incubated for 3 days in Neurobasal A media supplemented with B-27, penicillin (100 U/mL) plus streptomycin, GlutaMAX (2 mmol/L; Gibco), and fetal bovine serum (5%) at 37°C with 5% CO2. Cells were loaded with Fura-2 AM (3 μg in 3 μL dimethyl sulfoxide in 1 mL of media) before imaging on an Olympus BX51 microscope with Rolera Bolt camera (Q-Imaging, Olympus, Tokyo, Japan) and a CoolLED pE-4000 (365/385) illumination system controlled via MetaFluor software (Molecular Devices, Sunnyvale, Calif). Recombinant human IL-33 (1 μg/mL; R&D Systems), Cap (250 nmol/L), and KCl (60 mmol/L) were administered to the bath. A change in the fluorescence ratio (340/380) of >10% was considered to indicate a cellular response.
Samples were collected on day 5 of the AEW mouse model. For skin digestion, tissue was minced, then incubated in 500 μL of Liberase TL (0.25 mg/mL; Roche, Basel, Switzerland) in Dulbecco modified Eagle medium (Sigma-Aldrich) at 37°C and 5% CO2 for 90 minutes. To obtain a single cell suspension, skin and spleen samples were then manually homogenized through a 100 μm cell strainer. To lyse erythrocytes in spleen samples, samples were resuspended in 2 mL of Red Blood Cell Lysis Buffer Hybri-Max (Sigma-Aldrich) and incubated for 5 minutes. To test for cellular viability, all cells were stained with Zombie UV dye (1:500; BioLegend, San Diego, Calif) for 20 minutes at room temperature. Blocking solution (anti-mouse CD16/CD32-2.4G2 clone; 2 μg/mL; Bio X Cell) was applied to cells for 10 minutes (4°C) before cells were stained with primary antibodies diluted in BD Horizon Brilliant Stain Buffer for 30 minutes (4°C). Cells were then stained with secondary streptavidin-conjugated antibodies for 15 minutes (4°C). Finally, cells were fixed overnight at 4°C with BD Cytoperm/Cytofix reagent before sample acquisition on a LSR Fortessa X-20 (BD Biosciences, San Jose, Calif). Data were analyzed with FlowJo 10 (Treestar, Ashland, Ore). Immune cells were defined as live CD45+ cells.
Group 2 innate lymphoid cells (ILC2s) were defined as live IL7R+KLRG1+ST2+ cells that were negative for the following lineage (Lin) markers: CD11b, CD11c, NK1.1, CD19, FcεR1α, CD3ε, and CD4. Thus, cells were stained with the following antibodies to determine ILC2 frequencies: CD11b PerCP/Cy5.5 (M1/70; eBioscience, San Diego, Calif), CD11c PerCP/Cy5.5 (N418; eBioscience), NK1.1 PerCP/Cy5.5 (PK136; eBioscience), CD19 PerCP/Cy5.5 (1D3; eBioscience), FcεR1α PerCP/Cy5.5 (MAR-1; BioLegend), CD3ε Pe/Cy7 (145-2C11; BioLegend), CD4 BV421 (GK1.5; BioLegend), IL7Rα (CD127) BV650 (A7R34; BioLegend), KLRG1 PE/Daz (MAFA; BioLegend), ST2 biotin (RMST2-2; eBioscience), and streptavidin FITC (BioLegend).
Basophils were defined as live FcεR1α/IgE+CD49b+ cells that were negative for c-KIT, Siglec-F, and the Lin markers CD5, CD11c, CD19, and NK1.1. Mast cells were defined as live c-KIT+FcεR1α/IgE+ cells that were negative for SiglecF and the Lin markers CD5 (or alternatively CD3ε), CD11c, CD19, and NK1.1. Thus, cells were stained with the following antibodies to evaluate basophil and mast cell frequencies: CD5 PerCP/Cy5.5 (53-7.3; eBioscience) or CD3e PerCP/Cy5.5 (145-2C11; eBioscience), CD11c PerCP/Cy5.5 (N418; eBioscience), CD19 PerCP/Cy5.5 (1D3; eBioscience), NK1.1 PerCP/Cy5.5 (PK136; eBioscience), Siglec-F BV421 (E50-2440; BD Bioscience), c-KIT (CD117) Pe/Cy7 (2B8, eBioscience), or c-KIT BV605 (ACK2; BioLegend), FcεR1α FITC (MAR-1; eBioscience), IgE FITC (23G3; eBioscience), and CD49b APC (DX5; eBioscience).
Graphical results and statistical testing for RNA-Seq were conducted by Phantasus (https://artyomovlab.wustl.edu/phantasus/). scRNA-S analysis was generated by the Partek Flow package (https://www.partek.com/partek-flow/). Outliers were identified by using the ROUT method (Q = 1%; GraphPad Prism 7, GraphPad Software, La Jolla, Calif). The remainder of graphs were generated and the statistical analyses performed by GraphPad Prism 7. Not significant (NS), ∗P P P
Published online: September 20, 2021
Received in revised form:
In Press Journal Pre-Proof
Research in the Kim laboratory is supported by the Celgene Corporation , Doris Duke Charitable Foundation , LEO Pharma , and National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) ( K08AR065577 , R01AR070116 , R01AR077007 , and R21AI167047 ) (to B.S.K.). A.M.T. and M.R.M. are supported by National Institute of Allergy and Infectious Diseases (NIAID) ( T32AI007163 ). A.M.T. and L.K.O. are supported by National Heart, Lung, and Blood Institute (NHLBI) ( T32HL007317 ). A.M.T. is supported by NIAID ( F30AI154912 ). Research in the Gereau laboratory involving human dorsal root ganglia research is supported by National Institute of Neurological Disorders and Strokes (NINDS) ( R01NS042595 ) (to R.W.G.). Research in the Alexander-Brett laboratory is supported by NHLBI ( R01HL152245 ) and the Burroughs Welcome Fund ( 1014685 ) (to J.A.B). Research in the Cavalli laboratory is supported by the McDonnell Center for Cellular and Molecular Neurobiology and NINDS (R01NS111719) (to V.C.). O.A. is supported by the postdoctoral fellowship from the McDonnell Center for Cellular and Molecular Neurobiology. Research in the Davidson laboratory is supported by NINDS ( RF1NS113881 ) (to S.D.). Research in the Hu laboratory is supported by National Institute for Alcohol Abuse and Alcoholism (NIAAA) ( R01AA027065 ), NIAMS ( R01AR077183 ), and National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) ( R01DK103901 ) (to H.H.). Additional support was provided by the Washington University School of Medicine Digestive Disease Research Core Center (NIDDK, P30DK052574). Research in the Liu laboratory is supported by NIAID (R01AI125743), Brain Research Foundation Fay/Frank Seed Grant, and Pew Scholar Award (to Q.L.). Support with flow cytometry and Luminex was provided by the Bursky Center for Human Immunology & Immunotherapy Programs at Washington University, Immunomonitoring Laboratory (IML). The IML is a shared resource of the Alvin J. Siteman Cancer Center (Washington University School of Medicine and Barnes-Jewish Hospital in St Louis), which is supported in part by the National Cancer Institute (P30CA091842).
Disclosure of potential conflict of interest: B. S. Kim has served as a consultant for AbbVie, Almirall, AstraZeneca, Cara Therapeutics, Daewoong Pharmaceutical, Incyte Corporation, LEO Pharma, Lily, Maruho, OM Pharma, Pfizer, and Third Rock Ventures; has participated on advisory boards for Almirall, Boehringer Ingelheim, Cara Therapeutics, Kiniksa Pharmaceuticals, Regeneron Pharmaceuticals, Sanofi Genzyme, and Trevi Therapeutics; is a stockholder of Locus Biosciences; and serves on the scientific advisory boards for Abrax Japan, Granular Therapeutics, Recens Medical, National Eczema Association, and Cell Reports Medicine. The rest of the authors declare that they have no relevant conflicts of interest.
© 2021 The Authors. Published by Elsevier Inc. on behalf of the American Academy of Allergy, Asthma & Immunology.