Connections between Immune-Derived Mediators and Sensory Nerves for Itch Sensation

Although histamine is a well-known itch mediator, histamine H1-receptor blockers often lack efficacy in chronic itch. Recent molecular and cellular based studies have shown that non-histaminergic mediators, such as proteases, neuropeptides and cytokines, along with their cognate receptors, are involved in evocation and modulation of itch sensation. Many of these molecules are produced and secreted by immune cells, which act on sensory nerve fibers distributed in the skin to cause itching and sensitization. This understanding of the connections between immune cell-derived mediators and sensory nerve fibers has led to the development of new treatments for itch. This review summarizes current knowledge of immune cell-derived itch mediators and neuronal response mechanisms, and discusses therapeutic agents that target these systems.


Introduction
Itch (or pruritus) is an unpleasant sensation inducing the desire to scratch [1], as well as being a major and distressing symptom of many skin and systemic diseases. Chronic itch represents a significant clinical problem resulting from renal [2], liver [3], and bowel diseases [4], as well as several serious skin diseases, such as atopic dermatitis (AD). Histamine is one of the best-evaluated itch mediators. If an itch is caused by histamine, antihistamines (histamine H 1 -receptor blockers) can be used to control it. However, recent studies have suggested that histamine-independent pathways are involved in chronic itch, making antihistamines ineffective in the treatment of these patients [5][6][7]. Thus, the mechanisms of itch development and enhancement other than through histamine remain to be determined. Analyses of the interactions between immune cells and sensory neurons have shown that cytokines produced by immune cells during inflammation enhance itch, and that they act directly on sensory nerve fibers to induce and/or sensitize itch sensation.
This review focuses on immune cell-derived itch mediators and describes the mechanisms by which they connect to sensory nerves to produce and enhance itch.

Subtype of Sensory Neurons
Generally, itch sensation is generated by the binding of itch-inducing substances (pruritogens) to their cognate receptors (pruriceptors) on peripheral sensory afferents, especially unmyelinated C-fibers [8]. Single-cell RNA-seq has classified the sensory neuron system into five neurofilament (NF)-containing clusters, two peptidergic (PEP) nociceptor clusters, a tyrosine hydroxylase (TH)-containing cluster and three non-peptidergic (NP) nociceptor clusters [9]. The NF clusters were shown to express neurofilament heavy chain (Nefh) and parvalbumin (Pvalb), molecules previously associated with myelinated dorsal

Itch Mediators and Modulators from Immune Cells
Tables 1 and 2 summarize the immune cell-derived itch mediators and modulators, and the therapeutic agents that target them. This section describes the itch mediators and modulators produced by immune cells. As detailed above, the primary sensory nerves associated with itch have been classified into at least three subtypes, each of which has its own response profile. Based on the subtypes of nerve cells, the itch mediators and modulators derived from immune cells are also summarized ( Figure 2).
A H 3 R inverse agonist was found to induce strong itch in mice. This H 3 R inverse agonist induced pruritus but could be completely blocked by combined treatment with an H 1 R and an H 4 R antagonist, whereas the H 2 R antagonist failed to inhibit the scratch response. The decreased threshold in response to H 3 R antagonism is thought to activate H 1 R and H 4 R on sensory neurons, leading to the excitation of histamine-sensitive afferents and eliciting a sensation of itch [29].

Substance P
Substance P (SP) is a short neuropeptide of the tachykinin family, consisting of 11 amino acids (Arg-Pro-Lys-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH 2 ), and is one of most potent pruritogens identified to date [72,73]. SP is expressed by many cell types, including sensory neurons, astrocytes, microglia, epithelial cells, endothelial cells and immune cells, including T cells, macrophages, DCs and eosinophils [11,20,74]. SP binds to neurokinin 1 receptor (NK-1R) and another class of receptors involved in itch signaling, consisting of mouse MrgprA1, mouse MrgprB2 and human MrgprX2. NK-1R is a tachykinin receptor belonging to the GPCR family and expressed in the CNS, keratinocytes, fibroblasts and mast cells [72,73]. In humans, SP promotes degranulation by binding to mast cell NK-1R, releasing histamine and LTB 4 and causing itch [22,73]. In mice, SP induces itch through direct action on primary sensory neurons, as well as by release of nitric oxide (NO) and LTB 4 from keratinocytes, rather than by mast cell degranulation [22,75].

IL-2
Interleukin (IL)-2 is a 15.5 kDa cytokine secreted by antigen-activated CD4 + T cells and mast cells [83][84][85][86]. It was first described as a T cell growth factor and later also found to have the ability to act on natural killer (NK) cells and NKT cells, to activate B cells, and to induce the proliferation of regulatory T cells (Tregs), innate lymphoid cells (ILCs) and effector T cells. IL-2 has three receptors, each of which is composed of three subunits: IL-2 receptor α (IL-2Rα, CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132). IL-2Rα is expressed by several types of immune cells, including Tregs, ILC2, activated CD4 + and CD8 + T cells, B cells, CD56 hi NK cells, mature DCs, and endothelial cells. IL-2Rβ is mainly expressed by multiple lymphoid populations, such as Tregs, memory CD8 + T cells, NK cells, and NKT cells, and to some extent, by monocytes and neutrophils. IL-2Rγ is expressed mostly by hematopoietic cells [83,86,87]. The binding of IL-2 to its receptors induces trans-phosphorylation of Janus kinase (JAK) 1 and JAK3. This, in turn, activates the JAK/signal transducer and activator of transcription (STAT), phosphoinositide (PI) 3-kinase and MAPK signaling pathways [86,87]. Intravenous IL-2 treatment has been approved for the treatment of patients with metastatic melanoma and renal cell carcinoma, with beneficial results in a subset of patients, although severe pruritus is a known side effect [83,[86][87][88][89]. Moreover, intradermal injection of IL-2 in either healthy subjects or patients with AD induced pruritus and erythema [89,90]. The calcineurin inhibitor cyclosporine A has been shown to downregulate IL-2 synthesis, reducing pruritus in patients with treatment resistant Sezary syndrome, as well as in patients with AD [89].

IL-4
IL-4 is a type 2 cytokine produced by T helper (Th) 2 cells, lymph node T follicular helper (Tfh) cells, mast cells, basophils, eosinophils and ILC2 [91][92][93][94]. IL-4 has two receptors, IL-4Rα (CD124) and the more common IL-4Rγ, with IL-4 having higher affinity to IL-4Rα [95]. IL-4 signals through the IL-4Rα/γC complex in hematopoietic cells, such as lymphocytes and DCs. IL-4 binds IL-4Rα/γC and activates the downstream signaling molecules JAK1/JAK3 and then STAT6. Non-hematopoietic cells including keratinocytes also express IL-4Rα/IL-13Rα1 complex, which binds both IL-4 and IL-13, resulting in the downstream activation of JAK1/TYK2/JAK2 and then STAT6/STAT3 [93]. IL-4-evoked mouse DRG neurons respond to calcium, and deletion of IL-4Ra on sensory neurons was found to disrupt scratching behavior in a mouse model of AD. Moreover, IL-4 has been suggested as a modulator of itch because it enhances itch by increasing the neural responses induced by histamine, chloroquine, TSLP, and IL-31 [13,91]. Intradermal administration of IL-4 has also been reported to induce itching and alloknesis [96,97]. Dupilumab, a monoclonal antibody that binds specifically to the shared alpha chain subunit of the IL-4 and IL-13 receptors, was associated with improvements in clinical end points, including reduced pruritus in AD [98].
To date, the role of IL-13Rα2 in itch has been unclear. However, a recent study showed that the expression of IL-13Rα2 is upregulated in the skin of patients with AD, but not in the skin of patients with psoriasis, in a disease activity-dependent manner. In keratinocytes, IL-13 regulated IL-13Rα2 expression level and promoted IL-13Rα2 signaling. In addition, TLR2 activation was found to increase IL-13 mediated itch by potentiating IL-13Rα2, suggesting that IL-13Rα2 signaling promotes AD symptoms including itch [100]. Monoclonal antibodies that target and neutralize IL-13, Tralokinumab and Lebrikizumab, both improved AD symptoms including itch [28].

IL-17
IL-17A, also called IL-17, is produced by various cell types of T cells, including the Th17 subset of CD4 + T cells, CD8 + T cells, γδ T cells, and NKT cells, as well as by immune cells such as lymphoid tissue inducer (LTi)-like cells and neutrophils, and nonimmune cells such as Paneth cells. IL-17 has two receptors, IL-17RA and IL-17RC, which form a heterodimer. Binding of IL-17A or an IL-17F heterodimer to IL-17R induces Act1 activation, which, in turn, activates multiple signaling cascades that operate through different TNF receptor-associated factor (TRAF) proteins. Subsequently, the complex associates with TRAF6, leading to the activation of NF-kB, MAPK-AP-1, and C/EBP. ERK1/2 mediates the phosphorylation of C/EBPβ at Thr188, with the CBAD of IL-17R also required for IL-17-mediated inducible phosphorylation of C/EBPβ at Thr179 through GSK3β. IL-17 can also induce different feedback regulatory responses by inducing and/or recruiting deubiquitinase enzymes (A20 and USP25) or kinases (TBK1) [101,102]. Three randomized, controlled, phase 3 trials reported that brodalumab, an IL-17 receptor A antagonist, is safe and effective in treating moderate-to-severe psoriasis. In addition, brodalumab demonstrated improved itch responses in psoriasis [103]. These results suggest that IL-17 may act as an itch mediator and/or modulator. Other studies, however, have reported that IL-17 is neither a mediator nor a modulator of itching, [104] leading to the need for additional research.

IL-23
IL-23 belongs to the IL-12 family of proinflammatory cytokines. IL-23 is heterodimeric, being composed of IL-12p40 and p19 molecules. It is produced by activated DCs and macrophages in response to microbial pathogens, with its production enhanced by interactions between the costimulatory molecule CD40 and its ligand. IL-23 signals via IL-12Rβ1 and IL-23R and mediates the phosphorylation of STAT3 and STAT4 by JAK2 and Tyk2 [105,106]. Intradermal injection of IL-23 did not induce scratching behavior, but calcium imaging showed that about 5% of DRG neurons in mice responded to IL-23. IL-23 was also found to attenuate histamine-induced itch, suggesting that this cytokine may function as a desensitizer [104]. In addition, IL-23 might play a role in regulating histaminergic itch by modulating TRPV1 activity [104].

PAF
Platelet-activating factor (PAF) is produced and released by mast cells, basophils, neutrophils, eosinophils, monocytes, macrophages, fibroblasts, platelets, endothelial cells, and cardiac muscle cells, all of which play important roles in inflammatory and thrombotic diseases. PAF is an inflammatory factor and has important functions in acute and chronic inflammation [117,118]. PAF receptor (PAFR) has been found in a host of cell membranes, including those of platelets, neutrophils, macrophages, mononuclear leukocytes, and eosinophils, as well as on hippocampal nerves, microglia, astrocytes, and oligodendrocyte progenitor cells [118]. Intradermal PAF injection evoked scratching behavior [35,119] and induced histamine release through degranulation of mast cells, contributing to itch accompanied by flare and wheal reactions [120].

LTC 4 LTC 4 is a cysteinyl LT produced and released by mast cells, basophils, and eosinophils
3.6. Others 3.6.1. IL-33 IL-33, a member of the IL-1 cytokine family, is considered important for host defenses and allergy by inducing Th2 cytokine production via the IL-33 receptor. This receptor is a heterodimer composed of IL-1 receptor-like 1 (IL-1RL1; also called ST2) and IL-1 receptor accessory protein (IL-1RAcP) molecules. IL-33 was first identified as a nuclear protein expressed in endothelial cell nuclei and was shown to be constitutively expressed in the nuclei of various cell types, such as endothelial and epithelial cells [134,135]. IL-33 was also recently shown to be constitutively expressed in other cells, including DCs, macrophages, mast cells, fibroblasts, smooth muscle cells, platelets and megakaryocytes [135,136]. ST2 expressing cells include basophils, mast cells, eosinophils, macrophages, DCs, NK cells, NKT cells, Th2 cells, cytotoxic T cells, Tregs, B cells, ILCs, microglia, astrocytes, neurons, epithelial cells, endothelial cells, and fibroblasts [135,137,138]. Treatment of AD model mice with anti-IL-33 antibody improved AD-like symptoms, including scratching behavior [139]. Moreover, IL-33/ST2 signaling was found to mediate chronic itch in a mouse model of contact hypersensitivity through the astrocytic JAK2/STAT3 cascade [140]. IL-33 was also shown to evoke calcium responses in neurons, with enhanced CQ evoking calcium responses [138]. Taken together, these findings suggested that IL-33 also functions as a modulator to enhance itch.

Therapeutic Drugs for Amines
As described above, conventional treatments such as anti-histamines are often ineffective in patients with chronic pruritus. Therapeutic drugs other than antihistamines that target histamine consist of topical or systemic anti-inflammatory and immunomodulatory agents (e.g., cyclosporine A, pimecrolimus, tacrolimus and corticosteroids) [6]. Serotonintargeted itch treatments include sertraline [41], but the clinical application of existing drugs such as sarpogrelate may also expand in the future.

Therapeutic Drugs for Proteases
Protease-targeted therapies for itch are thought to be similar to histamine [6]. Furthermore, the selective chymase inhibitor ONO-WH-236 and the cathepsin S inhibitor LHVS were found to suppress scratching behavior [64,70]. In the future, protease inhibitors may become a more established method of treating itch.

Therapeutic Drugs for Peptides
Gabapentin, pregabalin and capsaicin are effective for the treatment of neuropathic itch [6]. A phase II randomized clinical trial showed that a NK-1R (a receptor for SP) inhibitor was effective for treating itch in patients with psoriasis [151].

Therapeutic Drugs for Cytokines
More recently, a variety of monoclonal antibodies have been shown to be effective in the treatment of itch. For example, dupilumab was found to improve AD symptoms and itch [152]. Most cytokines are activated via JAK/STAT signaling. Recently, a JAK inhibitor, delgocitinib, was reported to improve symptoms and itching of AD and was approved in Japan [153]. Moreover, Baricitinib, which inhibits JAK1 and JAK2, and Abrocitinib, which inhibits JAK1, improved AD symptoms including itch [28]. JAK inhibitors will be used for the treatment of itch in AD in the future.

Therapeutic Drugs for Lipid Mediators
CMHVA, a LTB 4 receptor antagonist, was found to improve itch [130], suggesting it may be targeted as a lipid mediator to treat itch in the future.

Conclusions
This review presents recent knowledge regarding immune cell-derived mediators and modulators of itch. Many of these mediators cause nerve firing via their respective receptors expressed on sensory nerves, affecting the induction and modulation of itch. The variety of immune-derived itch mediators alone suggests that the mechanisms of itch are diverse. Although it is practical to focus on a common molecule such as JAK as a therapeutic target for itch, in fact, the development of therapeutic agents that target individual itch mediators and their receptors is ongoing. Thus, in clinical practice, however, in the future, due to the diverse molecules involved, a combination of therapies may be required to treat itch. It would be ideal to develop a system to test for itch mediators in each individual patient to determine the best treatment or appropriate combination therapy for each individual patient.