Interleukin-4 (IL-4) and IL-13 are cytokines known to drive inflammation and play a role in atopic dermatitis (AD) pathogenesis. Both enhance neuronal itch via the Type 2 receptor. Eblasakimab, a high affinity IgG4 monoclonal antibody, which binds IL-13Rα1 and blocks signaling of IL-4 and IL-13 via the Type 2 receptor, is being developed for the treatment of moderate-to-severe AD. This study evaluated 1) whether IL-4 and IL-13 exert redundant or distinct functions in human sensory neurons, and whether eblasakimab can 2) attenuate cytokine-enhanced neuronal responses to itch and 3) reduce spontaneous neuronal activity. Human dorsal root ganglia neurons were treated with IL-4, IL-13, or their combination with or without eblasakimab and subsequently either challenged with pruritogens (BAM8-22 [bovine adrenal medulla 8–22 peptide] and PAMP-20 [pro-adrenomedullin peptide 1-20]) or tested for spontaneous neuronal activity. Neuronal responses to pruritogens and spontaneous neuronal activity were measured via live-cell calcium imaging. Treatment with IL-4, IL-13, and their combination enhanced neuronal responses to BAM8-22. Only IL-13 treatment increased neuronal responses to PAMP-20 by amplifying activity of the itch-specific receptor MRGPRX2 (Mas-related G-protein coupled receptor X2). This suggests a novel neuroimmune pathway besides the receptor's mast cell specific function. Eblasakimab significantly reduced cytokine-enhanced itch responses to both pruritogens (p<0.0001 BAM8-22; p<0.05, PAMP-20). Spontaneous neuronal activity was not impacted by IL-13 treatment but was increased with IL-4 treatment (p<0.05 vs vehicle), which was also effectively reduced by eblasakimab (p<0.05). These results reveal that IL-4 and IL-13 exert nonredundant neuronal function in enhancing itch and inducing spontaneous neuronal activity. They also demonstrate the ability of eblasakimab to block these cytokine-mediated effects. Together, these data provide a mechanistic basis for the reduction of itch observed in moderate-to-severe AD patients treated with eblasakimab in a phase 1b clinical trial.

Atopic dermatitis (AD) is an inflammatory skin disease characterized by dysregulated type 2 inflammation. Interleukins (IL)-4 and IL-13 are key cytokines mediating type 2 inflammation in AD, with IL-4 signaling through the type I receptor (composed of IL-4Rα and the common gamma chain) and IL-4 and IL-13 signaling through the type II receptor (composed of IL-4Rα and IL-13Rα1). The most effective way to inhibit type 2 inflammation remains unknown. In this study, we treated peripheral blood mononuclear cells (PBMCs) isolated from 10 AD patients with either an anti-IL-4Rα antibody to block both type I and II receptors or eblasakimab, a monoclonal antibody that binds IL-13Rα1, to block only the type II receptor. We investigated the downstream effects of blocking these receptors on cytokines involved in type 2 inflammation and other immune axes utilizing the Meso Scale Discovery panel in the PBMC supernatants. We find that treatment with IL-13Rα1 blockade as compared to IL-4Rα blockade resulted in statistically significant decreases in the release of key cytokines implicated in Th2 inflammation, including TARC (p=0.0001), IL-13 (p<0.0001), IL-4 (p<0.0001), IL-9 (p=0.0047), and MCP-4 (p<0.0001). Furthermore, our data demonstrate that IL-13Rα1 blockade did not induce an upregulation of the Th1 immune response as observed with anti-IL-4Rα therapy. Specifically, treatment with eblasakimab as compared to anti-IL-4Ra therapy did not lead to an increase in pro-inflammatory Th1 cytokines, including TNF-α (p=0.0319), IL-2 (p<0.0001), GM-CSF (p=0.0011), IL-12p70 (p<0.0001), and IP-10 (p=0.0017). Taken together, our findings suggest that selective targeting of the IL-13Rα1 receptor in AD may lead to a more potent reduction in type 2 inflammation while not activating the Th1 pathway.

Alopecia areata (AA) is an inflammatory hair disorder characterized by immune privilege (IP) collapse of the hair follicle (HF) bulb and intra/peri-bulbar T-cell infiltrate, which leads to premature transition to the catagen stage, HF dystrophy, and hair loss. IP collapse, marked among others, by elevated major histocompatibility complex (MHC) I and II expression and an upregulation of MHCII+ cells, is mainly mediated by increased IFNγ levels and a Th1-mediated inflammatory response. Interestingly, T-cell proliferation, Th1-cell differentiation, and IFNγ production are dependent on activity of the enzyme dihydroorotate dehydrogenase (DHODH) in proliferating T-cells. An innovative AA model was developed, in which microdissected healthy human scalp HFs were treated ex vivo with an antibody cocktail containing anti-CD3/CD28 to activate intra- and peri-follicular resident T-cells via the T-cell receptor (TCR) to induce key features of AA, including IP collapse. This model tested whether DHODH inhibition by farudodstat could be beneficial for the treatment of AA. Experimentally induced TCR activation resulted in increased numbers of proliferative T-cells in the HF epithelium and mesenchyme, as shown by CD3/Ki-67 immunofluorescence staining. Significant upregulation of MHC I and II protein expression in the bulb epithelium and mesenchyme of anagen VI HFs was observed, suggesting loss of IP. DHODH inhibition by farudodstat protected HFs from the increase in proliferative T-cells, induction of MHC I and II proteins, and upregulation of MHC II+ cells. Importantly, DHODH inhibition did not induce cytotoxicity or catagen promotion, and did not impact hair matrix keratinocyte proliferation or IP markers. Preliminary results show that anti-CD3/CD28 treatment in this ex vivo model can successfully stimulate T-cell proliferation, which subsequently induces key features of AA, including stimulation of MHC I and II expression in the bulb. Additionally, our data suggest that farudodstat might protect HFs from IP collapse and offer a novel therapeutic approach for AA.

Atopic dermatitis (AD) is an inflammatory skin disease characterized by dysregulated Th2-driven inflammation. Interleukins (IL)-4 and IL-13 are key cytokines mediating Th2-driven inflammation in AD that signal through the Type 1 receptor (composed of IL-4Rα and the common gamma chain) and Type 2 receptor (composed of IL-4Rα and IL-13Rα1). The most effective way to inhibit Th2-driven inflammation remains unknown. In this study, we treated peripheral blood mononuclear cells (PBMCs) isolated from 10 AD patients with either an anti-IL-4Rα antibody (R&D systems, MAB230) to block both Type 1 and 2 receptors or eblasakimab, a monoclonal antibody that binds IL-13Rα1, to block only the Type 2 receptor. We then investigated the downstream effects of blocking these receptors on cytokines involved in Th2-driven inflammation and other immune axes utilizing the Meso Scale Discovery panel from PBMC media. We find that treatment with IL-13Rα1 blockade with eblasakimab as compared to IL-4Rα blockade resulted in lower levels of key cytokines implicated in Th2-driven inflammation, including thymus and activation-regulated chemokine (TARC; p=0.0001), IL-13 (p<0.0001), IL-4 (p<0.0001), and monocyte chemotactic protein-4 (MCP-4; p<0.0001). Furthermore, our data demonstrate that IL-13Rα1 blockade prevents subsequent expression changes of Th1 cytokines as observed with anti-IL-4Rα therapy. Specifically, treatment with eblasakimab as compared to anti-IL-4Rα therapy suppressed a paradoxical increase of Th1 cytokines, including tumor necrosis factor alpha (TNF-α; p=0.0319), IL-2 (p<0.0001), granulocyte-macrophage colony-stimulating factor (GM-CSF; p=0.0011), IL-12p70 (p<0.0001), and interferon gamma-induced protein-10 (IP-10; p=0.0017). These results suggest that targeting different subunits of the same molecular pathway can lead to different downstream effects and subsequent expression of Th1- and Th2-associated cytokines. Eblasakimab may offer a differentiated therapeutic approach to treat moderate to severe AD with the potential to spare the Type 1 receptor and the effects seen with targeting IL-4Rα.

Interleukin-4 (IL-4) and IL-13 are cytokines known to drive inflammation and play a role in atopic dermatitis (AD) pathogenesis. Both enhance neuronal itch via the type 2 receptor. Eblasakimab, a high affinity IgG4 monoclonal antibody, which binds IL-13Rα1 and blocks signaling of IL-4 and IL-13 via the type 2 receptor, is being developed for the treatment of moderate-to-severe AD. This study evaluated 1) whether IL-4 and IL-13 exert redundant or distinct functions in human sensory neurons, and whether eblasakimab can 2) attenuate cytokine-enhanced neuronal responses to itch and 3) reduce spontaneous neuronal activity. Human dorsal root ganglia neurons were treated with IL-4, IL-13, or their combination with or without eblasakimab and subsequently either challenged with pruritogens (BAM8-22 and PAMP-20) or tested for spontaneous neuronal activity. Neuronal responses to pruritogens and spontaneous neuronal activity were measured via live-cell calcium imaging. Treatment with IL-4, IL-13, and their combination enhanced neuronal responses to BAM8-22. Only IL-13 treatment increased neuronal responses to PAMP-20 through amplifications of the activity of MRGPRX2, suggesting a novel neuroimmune pathway besides its mast cell specific function. Eblasakimab significantly reduced cytokine-enhanced itch responses to both pruritogens (p<0.0001 BAM8-22; p<0.01, PAMP-20). Spontaneous neuronal activity was not impacted by IL-13 treatment but was increased with IL-4 treatment (p<0.05 vs vehicle), which was also effectively reduced by eblasakimab (p<0.05). These results reveal that IL-4 and IL-13 exert nonredundant neuronal function and demonstrate the ability of eblasakimab to block these effects. This indicates that direct impact on neuronal responses may contribute to reduction of chronic itch demonstrated in AD patients treated with eblasakimab.

Chronic itch is a cardinal feature of multiple type-2 driven skin disorders exemplified by atopic dermatitis (AD). The signaling of itch in AD has been recently postulated to be amplified by the inflammatory cytokines present within the skin. In inflammatory skin diseases, cytokines exacerbate the immune responses, disrupt the skin barrier, and drive the disease pathology. The direct neuronal activation by type-2 canonical cytokines was first described with Interleukin-31 (IL-31). Recently, it has been shown that IL-13 acts as a neuronal enhancer for a multitude of different itch pathways in human neurons. Our objective is to understand the relevance of targeting the IL-13 receptor alpha 1 (IL-13R α1), on human sensory neurons and how this might result in cellular and intracellular changes altering neuronal activity. To study these phenomena, we used eblasakimab, a monoclonal human IgG4 antibody, which binds to the human IL-13Rα1 with nanomolar affinity. By binding to the receptor, eblasakimab prevents signaling of IL-4 and IL-13 through the type-2 receptor, which is expressed on a multitude of different immune and non-immune cells except for T-cells. Using an ex-vivo human neuronal model system, human dorsal root ganglia (hDRG) neurons were treated with IL-4 or IL-13 alone, or in combination, and were subsequently subjected to pruritogens. Neuronal responses were captured by live cell calcium imaging. Our data with human sensory neurons pre-stimulated with IL-4, IL-13 and their combination showed a neuronal enhancer effect for IL-4, and IL-13 with no obvious synergy or combined additive enhancer effects on pruritic pathways. Eblasakimab significantly reduced neuronal responses to IL-4, IL-13, and their combination by more than 40% to control conditions (p = 0.0001). Moreover, our finding that eblasakimab treatment reduced neuronal responses below vehicle group suggests that IL-13Rα1 has an additional role in neuro-immune modulation beyond the cytokine-related neuronal itch sensitization in inflammatory diseases.

Atopic dermatitis (AD) is a chronic, inflammatory, relapsing skin disease with a preponderance of type 2 immune cells, which release cytokines (i.e., IL-4, IL-13, and IL-31) that orchestrate the multi-faceted downstream effects of the disease. A clinical hallmark is chronic, persistent, and highly prevalent severe itch impacting the quality of life of AD patients. Our key objective is to understand the mechanistic basis of chronic itch and gain insight into the efficacy of lebrikizumab, a monoclonal investigational anti-IL-13 antibody that is in development for the treatment of moderate-to-severe AD. To ascribe a laboratory surrogate to chronic itch, we employed a primary human dorsal root ganglion (hDRG) tissue culture model and stimulated these sensory neurons with IL-13 along with different pruritic as well as other inflammatory agents (with or without lebrikizumab). Live-cell calcium measurements demonstrate that acute as well as prolonged exposure of sensory neurons to IL-13 amplifies the neuronal responses to a multitude of signals. These arrays of neuronal potentiation elicited by IL-13 were attenuated by lebrikizumab. Additional studies with electric field stimulation suggest that acute and prolonged exposure of IL-13 increases neuronal excitability in the DRG, which is reversed by lebrikizumab underlining a direct neuro-modulatory role and may be complementary to potentiation of itch responses. To highlight the possible molecular basis of neuronal activity, we measured the downstream transcriptional targets of IL-13 using RNA Seq. Dominant transcripts that were differentially regulated by IL-13 include immune-regulatory and neuroinflammatory genes. These IL-13 mediated changes were reversed by lebrikizumab highlighting lebrikizumabs’ ability to counteract the IL-13 driven neuroactive effects in this hDRG culture model.

Chronic itch is a prevalent and debilitating symptom in atopic dermatitis (AD) patients causing ongoing demand in finding effective treatments. Recent research has advanced our understanding how inflammatory immune dysregulation and neuronal dysfunction drive the pathophysiology of itch in AD. Type 2 cytokines, including IL-13, IL-4, IL-5 and IL-31 are upregulated in AD skin lesions. IL-31 was shown to directly activate dorsal root ganglia (DRG) to elicit itch. Recently, IL-4, another well-described cytokine in the pathophysiology of AD, was reported to activate mouse and human neurons. The objective of this study was to test the effects of IL-13 in human DRG to mediate itch. Human DRGs were treated with IL-13 alone or in combination with different pruritogens that represent histaminergic and non-histaminergic itch. Live cell imaging with calcium measurement via fluorescence detection was used to measure activation of neurons. Our data showed direct activation of hDRGs by IL-13 and additionally an extensive enhancement of serotonin-induced responses. A similar sensitization effect was observed for histamine-induced activation when neurons were incubated with IL-13. Lebrikizumab, a high affinity antibody against IL-13, significantly reduced sensitization of serotonin-triggered neuronal responses. In conclusion, our data suggests that IL-13 is a neuronal enhancer in different itch pathways and is potentially involved in chronic itch in AD.

Background—Although the cytokine, interleukin-31 (IL-31), has been implicated in inflammatory and lymphoma-associated itch, the cellular basis for its pruritic action is yet unclear. Objective—To determine whether immune cell-derived IL-31 directly stimulates sensory neurons, and to identify the molecular basis of IL-31-induced itch. Methods—We used immunohistochemistry and qRTPCR to determine IL-31 expression levels in mice and humans. Immunohistochemistry, immunofluorescence, qRTPCR, in vivo pharmacology, western blotting, single cell calcium and electrophysiology were used to examine © 2013 American Academy of Allergy, Asthma and Immunology. Published by Mosby, Inc. All rights reserved. #Addresses for correspondence: Martin Steinhoff, M.D., Ph.D., Departments of Dermatology and Surgery, University of California, San Francisco, 513 Parnassus Ave, Room S-1268, San Francisco, CA, 94143 USA, Phone: +1 415 476 6978, FAX: +1 415 476 0936, SteinhoffM@derm.ucsf.edu. Allan. I. Basbaum, Ph.D., Department of Anatomy, University of California, San Francisco, 1550 4th Street, San Francisco, CA, USA, Phone: +1 415 476 5270, FAX: +1 415 476 1974, Allan.Basbaum@ucsf.edu. Bernhard Homey, M.D.. Department of Dermatology. University Hospital Duesseldorf, Duesseldorf, Germany, Phone: +49 211 811 7600, FAX: +49 211 811 7316, bernhard.homey@uni-duesseldorf.de. *contributed equally to this work; §Co-senior authors; Author contribution: F. C.: conducted most of the experiments, designed the study, wrote manuscript. X. W.: conducted in vivo and morphological experiments with F.C. T.A: performed single cell calcium measurement and electrophysiology recordings under supervision of E.C; T. S.: designed the study for the in vivo mouse models of AD under supervision of H.A; A.A, M.F.: performed human staining experiments of skin tissue and qPCR of cells under supervision of B.H.; C. K.: performed western blotting and wrote part of the manuscript; G. K.: performed human staining experiments of skin tissue and qPCR of cells; A. I.: assisted in cheek model assay; T. B.: stained human DRG for IL-31RA; H. A.: supervised the murine AD study; S. D.: supervised vivo mouse studies; E. C.: supervised electrophysiology study; B. H.: designed, supervised human IL-31 studies and mouse atopy models, and wrote manuscript; A.I.B.: designed, supervised the neuronal experiments, and wrote manuscript; M.S.: designed, supervised all experiments, analyzed data, and wrote manuscript. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author Manuscript J Allergy Clin Immunol. Author manuscript; available in PMC 2015 February 01. Published in final edited form as: J Allergy Clin Immunol. 2014 February ; 133(2): 448–460.e7. doi:10.1016/j.jaci.2013.10.048. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript the distribution, functionality and cellular basis of the neuronal IL-31 receptor (IL-31RA) in mice and humans. Results—Among all immune and resident skin cells examined, IL-31 was predominantly produced by TH2 and to a significantly lesser extend by mature dendritic cells. Cutaneous and intrathecal injections of IL-31 evoked intense itch, and its concentration increased significantly in murine atopic-like dermatitis skin. Both human and mouse DRG neurons express IL-31RA, largely in neurons that co-express TRPV1. IL-31-induced itch was significantly reduced in TRPV1and TRPA1-deficient mice, not c-kit or PAR-2 mice. In cultured primary sensory neurons, IL-31 triggered Ca2+-release and ERK1/2 phosphorylation, Inhibition of which blocked IL-31 signaling in vitro and reduced IL-31-induced scratching in vivo. Conclusion—IL-31RA is a functional receptor expressed by a small subpopulation of IL-31RA+/TRPV1+/TRPA1+ neurons, and is a critical neuro-immune link between TH2 cells and sensory nerves for the generation of T cell-mediated itch. Thus, targeting neuronal IL-31RA may be effective in the management of TH2-mediated itch, including atopic dermatitis and cutaneous T cell lymphoma.