Neuronal FcεRⅠα Mediates Ocular Itch Via IgE-Immune Complex In A Mouse Model Of Allergic Conjunctivitis

Background and ObjectiveAllergic conjunctivitis(ACJ)which approximately afflicts 15-20%of the population worldwide actually contains a group of diseases affecting the ocular surface.Ocular itch is the pathognomonic symptom of ACJ,and significantly reduces the quality of patient’s life.Preclinical evidence indicated a direct contribution of immunoglobulin E(IgE)activated mast cells to both the early-phase allergic reaction and late-phase inflammation during ACJ.The action of mast cell-driven pruritogens such as histamine and protease to itch-related receptors in sensory neurons is regarded as the classic peripheral mechanism of itch.However,available treatments targeting mast cells and related inflammatory factors such as histamine receptor antagonist,mast cell stabilizer and steroid are often unsatisfying for ocular itch symptom and/or exhibit adverse side effects.Thus,additional mechanisms besides mast cell-associated inflammation probably contribute to ACJ itch,which remain largely unexplored.The ACJ patients usually have an elevated antigenspecific immunoglobulin(Ig)level,especially IgE in the serum and tears.IgE,a subtype of immunoglobulin associated with allergic diseases,is produced by B cells after immune rearrangement.IgE-immune complex(IgE-IC)formed by allergen and specific IgE can directly activate Fc-epsilon receptors(FcεRs),especially the high-affinity activating IgE receptor FcεRⅠ,and further elicit anaphylactic reaction.FcεRI is defined structurally as polymeric complex comprising α-subunit(FcεRⅠα,the IgE binding chain),β-subunit(FcεRIβ,signal amplification),and γ-subunit(FcεRⅠγ,signal transduction).FcεRⅠα plays a central role in allergic diseases through linking IgE and responsive cells such as mast cells and basophils.Omalizumab,the humanized monoclonal IgE antibody,neutralizes the free circulating IgE,thereby preventing the binding of IgE to FcεRⅠ.Clinical evidence showed favorable effects of omalizumab on antihistamines and mast-cell stabilizers unresponsive ocular itch symptom in ACJ.Therefore,IgE-FcεRⅠ might give rise to ACJ itch in a mast cell and inflammation independent manner.Moreover,the FcεRI is also expressed in peripheral sensory neurons besides the immune cells like mast cells and basophils.This neuronal receptor is functional since antigen application could directly induce neuronal activation after sensitized with antigen-specific IgE.However,the in vivo evidence that neuronal FcεRⅠ contributes to ocular itch has not been explored.Further study should be performed to further determine whether FcεRⅠ is expressed in conjunctiva sensory neurons or whether IgE-IC acts directly on conjunctiva pruriceptors through neuronal FcεRⅠ to induce ocular itch.In this study,we aim to determine the role of neuronal FcεRⅠα in allergic ocular itch.Materials and Methods1.Conjunctival microinjection with Wheat germ agglutinin(WGA)was used to detect the expression of retrograde labeled FcεRⅠα in the trigeminal ganglion.2.Calcium imaging was applied to detect neural responses of trigeminal ganglion neurons isolated from FcεRⅠα+/+and FcεRIα-/-mice to IgE-IC.3.The mice were eye-dropped with OVA antigen,IgE antibody and IgE-IC immune complex,respectively,and the pruritus and pain related behavior towards the eyes was observed.4.Application IgE-IC to FcεRⅠα-/-and c-KitW-sh/W-sh mast cell deficient mice,and the effect of FcεRⅠα and mast cell on IgE-IC associated acute ocular itch was observed.5.The FcεRⅠα of trigeminal sensory neurons was specifically knocked down by trigeminal injection of adeno-associated virus(AAV9-Pirt-shFcsRⅠα-EGFP)containing Pirt promoter(sensory neuron-specific promoter)and shRNA sequence that interferes with FcεRIα.The acute ocular pruritus induced by IgE-IC was further detected.6.To detect the expression of FcεRⅠin trigeminal sensory neurons under allergic condition,we constructed an OVA-mediated ACJ mouse model.We further detected the expression of FcεRⅠα in conjunctiva-WGA retrograde labeled trigeminal sensory neurons in ACJ mice.7.In mice injected with AAV9-Pirt-shFcsRⅠα-EGFP virus,the ACJ model was further constructed to observe the effect of FcεRⅠα knockdown on ocular pruritus induced by antigen exposure.8.The acute isolated trigeminal ganglion neurons were cultured in the medium containing IgE,and the effects of IgE on the mRNA and protein expression of FcεRⅠαwere detected.The changes of neuronal responses to IgE-IC were also detected.9.The incremental sensitization model was constructed by sensitizing mice different times.The changes of FcεRⅠα expression in trigeminal ganglion from mice sensitized different times were detected,and the effects of increased OVA sensitization on allergic pruritus towards the eyes after antigen exposure were observed.Results1.FcsRIa is expressed in mouse conjunctiva-innervating TG neurons.Moreover,FcεRⅠαwas expressed in the nerve fibers of conjunctiva,and FcεRⅠα was expressed in nociceptive sensory neurons and MrgprA3+ itch-specific neurons.2.IgE-IC directly activates TG neurons through FcεRIα.3.Local instillation of IgE-IC elicits acute ocular itch without obvious inflammation in conjunctiva and trigeminal ganglion.4.FcεRⅠα mediated acute ocular itch induced by IgE-IC.5.Neuronal FcεRⅠα mediated acute ocular itch induced by IgE-IC.6.ACJ upregulated the expression of FcεRⅠα in TG innervating the conjunctiva.7.Neuronal FcεRⅠα mediated allergic ocular itch in ACJ model.8.IgE upregulated the neural expression of FcεRⅠα with elevated responses of trigeminal neurons to IgE-IC.9.Incremental sensitization upregulated the expression of FcεRⅠα in TG neurons and enhanced allergic ocular itch in mice.ConclusionThis study demonstrates that FcεRⅠα in sensory neurons innervating conjunctiva directly mediates IgE-IC induced acute ocular itch.Furthermore,ACJ upregulates FcεRⅠα in sensory neurons innervating conjunctiva,which mediates ACJ-related ocular itch as antigen exposure.In addition,IgE upregulates FcεRⅠα protein in sensory neurons and enhances neuronal responses to IgE-IC.These findings reveal a novel neuroimmune axis in allergic itch condition,which highlights the immunosensory capabilities of the itchy neurons.