Histamine, the most well-known pruritogen, is produced by mast cells, basophils and keratinocytes [14,15,16,17,18,19,20,21]. Histamine evokes itch via histamine H₁ and H₄ receptors [19,22]. Histamine H₁ receptor (H₁R) is a G protein-coupled receptor (GPCR) [20,23,24,25], a class of receptors globally expressed in various tissues, including sensory nerves [17,21]. Histamine H₄ receptor (H₄R) is also a GPCR [20,24,25] and is mainly expressed in immunocompetent cells, including mast cells, eosinophils, neutrophils, monocytes, dendritic cells (DCs) and T cells; as well as in intestinal epithelia, spleen, lung, synovial tissue, the central nervous system (CNS), sensory neurons, and cancer cells [21,24,26]. H₁R and H₄R on histaminergic nerves bind histamine and then activate transient receptor potential vanilloid (TRPV) 1 [17,27]. The H₄R antagonist, ZPL-3893787, improved AD symptoms including itch [28].

A H₃R inverse agonist was found to induce strong itch in mice. This H₃R inverse agonist induced pruritus but could be completely blocked by combined treatment with an H₁R and an H₄R antagonist, whereas the H₂R antagonist failed to inhibit the scratch response. The decreased threshold in response to H₃R antagonism is thought to activate H₁R and H₄R on sensory neurons, leading to the excitation of histamine-sensitive afferents and eliciting a sensation of itch [29].

Serotonin (5-hydroxytryptamine; 5-HT), which is produced by mast cells, basophils and platelets [15,30,31,32,33], evokes scratching in rodents via the 5-HT₂ receptor, which is TRPV4-dependent [34,35,36,37]. The 5-HT₂ receptor is expressed in immunocompetent cells, including macrophages, DCs, Langerhans cells, CD3⁺ T cells, melanocytes, vascular smooth muscle cells, endothelial cells, central and peripheral neurons including primary sensory neurons (DRG/trigeminal ganglion cells) [31,38,39,40]. Activation of the 5-HT₂ receptor, which belongs to the GPCR super-family and is coupled to the Gq/11 protein, leads to increases in inositol trisphosphate (IP3) and diacylglycerol (DG) levels, generating an antinociceptive effect [38,40]. Sertraline, a selective serotonin reuptake inhibitor, has been found to be effective in treating serotonin-targeted itch [41]. In addition, existing drugs, such as the selective 5-HT₂ receptor antagonist sarpogrelate, may have expanded future clinical application in the treatment of itch.

Tryptase, a serine protease with trypsin-like specificity, consists of seven distinct isoforms, α, βI, βII, βIII, δ, ε and γ, encoded by a set of protease genes clustered together on chromosome 16p13.3 [42,43,44,45]. The tryptase best characterized to date is β-tryptase, and the term “tryptase” is often used as a synonym for this molecule [45]. Tryptase is expressed in mast cells and basophils [45,46,47,48,49]. Intradermal injection of tryptase elicits scratching in mice [50]. Proteases, including tryptase, activate protease-activated receptors (PARs) by cleaving a part of their extracellular domain. PARs are GRCRs, characterized by a unique mechanism of self-activation following specific proteolytic cleavage of their extracellular domains. To date, four PARs have been identified, PAR-1, PAR-2, PAR-3, and PAR-4, which are encoded by the F2R, F2RL1, F2RL2, and F2RL3 genes, respectively [51,52]. PAR-2 is activated by trypsin-like serine proteases and is widely distributed throughout the mammalian body. In the skin, PAR-2 is expressed by almost all cell types, especially by keratinocytes. In addition, endothelial cells, fibroblasts, sensory neurons, and inflammatory cells such as mast cells, T lymphocytes, eosinophils, neutrophils, monocytes, macrophages, and DCs have been reported to express functional PAR-2 [52]. Tethered ligands, such as the PAR-2 agonist SLIGRL-NH₂, have been shown to elicit scratching in mice, but not rats [53]. Activated PAR-2 coactivates TRPV1 channels stimulating the release of the neuropeptides SP and CGRP from nerve terminals [54,55]. In addition, SLIGRL-NH₂ enhances CQ- and BAM8-22-induced itch and acts as a modulator [56].

Chymase is a chymotrypsin-like serine endopeptidase stored in mast cell secretory granules [18]. Human chymase, encoded by the CMA1 gene located on chromosome 14q11.2, co-localizes with clusters formed by cathepsin G, granzyme B and granzyme C/H [46,57,58]. In rats, the chymase-encoding gene is located on chromosome 15p12/13, and in mice on chromosome 14C1/2 [58,59,60,61,62]. Chymase also activates PAR-2 [63,64]. The chymase specific inhibitor Y-40613 was found to suppress scratching behavior in a mouse model of pruritus [65]. In the eyes, chymase also induced scratching behavior, which was suppressed by the selective chymase inhibitor ONO-WH-236 [64].

Cathepsin S is a cysteine protease produced by DCs, macrophages, basophils and keratinocytes [19,66,67]. Cathepsin S activates PAR-2, PAR-4 and MrgprC11 to produce itch [68,69,70]. Intradermal injection of the selective PAR-4 agonist AYPGKF-NH₂ (AYP) elicited scratching behavior in mice [56,71], which was prevented by the selective PAR-4 antagonist (pepducin P4pal-10) [71]. AYP-induced itch was reduced by gastrin-releasing peptide (GRP), NK-1, TRPV1 and a TRPA1 antagonist. These results indicated that PAR-4-activated itch is induced via TRPV1/TRPA1 in mice [71]. Moreover, touch-evoked scratching (alloknesis) was observed following intradermal injection of AYP, but not PAR-2 [56]. Cathepsin S also evoked a calcium response in mouse DRG neurons, which is reduced by PAR-2 antagonists and in TRPV1-/-or TRPA1-/-mouse-derived DRGs. In addition, intradermal injection of cathepsin S induced scratching behavior, which was inhibited by the cathepsin S inhibitor LHVS [70].

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-NH₂), 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 LTB₄ 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 LTB₄ from keratinocytes, rather than by mast cell degranulation [22,75].

Endothelin (ET)-1 is a 21 amino-acid peptide member of the endothelin family and a potent pruritogen that can elicit scratching at low concentration (10–400 pmol/site) [76,77,78]. ET-1 is produced by mast cells, endothelial cells and keratinocytes in the skin [54,76,77,78]. ETs have two active receptors, ET_A and ET_B, which belong to the GPCR superfamily [78,79,80]. Endothelin receptors are widely expressed in all tissues [81], and ET-1-evoked scratching is mediated by ET_A [76]. In addition, the endothelin receptor antagonist bosentan inhibited symptoms including itch in AD model mice [82].

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 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].

IL-13 is another type 2 cytokine produced by Th2, ILC2, mast cells, basophils, and eosinophils [91,92,93,94]. It has two receptors, IL-13Rα1 (CD213α1) and IL-13Rα2 (CD213α2). IL-13Rα1 alone binds IL-13 with low affinity, but when paired with IL-4Rα it binds IL-13 with high affinity and forms a functional IL-13 receptor that signals and results in activation of STAT3/6 [93,99]. Similar to IL-4, intradermal administration of IL-13 has been reported to induce itching and alloknesis [96,97].

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].

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].

Leukotrienes (LTs) are eicosanoid lipid mediators generated upon activation of both immune and structural cells such as epithelial cells and endothelial cells. LTB₄, a 5-lipoxygenase metabolite, is increased in the skin of AD model mice [121]. This molecule is produced and released by various types of immune cells, including mast cells, basophils, eosinophils, and macrophages [122,123,124,125]. LTB₄ has two receptors, BLT1 and BLT2, which are GPCR and present on cell surfaces, with BLT1 having higher affinity and activity than BLT2. BLT1 is mainly expressed by leukocytes and DRG neurons, whereas BLT2 is expressed on many tissues [126,127,128]. LTB₄-induced DRG neurons respond to calcium, an effect inhibited by the LTB₄ antagonist ONO-4057 [128]. Intradermal LTB₄ injection induces scratching via TRPA1 and TRPV1 [129]. Moreover, the LTB₄ receptor antagonist CMHVA attenuated IL-31-induced scratching [130].

LTC₄ is a cysteinyl LT produced and released by mast cells, basophils, and eosinophils [131,132,133]. Its receptors, CysLTR1 and CysLTR2, are widely expressed by hematopoietic and structural cells [12]. Basophils have been shown to release LTC₄ upon stimulation with antigen-specific IgE, which binds to CysLTR2 expressed on sensory nerve fibers (mainly NP3 nerves), evoking acute severe itch (itch flares) of AD [132]. Moreover, the LTC4/CysLTR2 pathway was shown to contribute not only to acute but also to chronic itch [12].

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.

3.6.2. TSLP
Thymic stromal lymphopoietin (TSLP) is a IL-7 like cytokine belonging to the IL-2 cytokine family [110,141]. It is primarily produced by epithelial cells, including keratinocytes, fibroblasts and stromal cells, as well as by DCs, mast cells, and basophils [110,142]. Its receptor, TSLPR, is expressed on monocytes/macrophages, T cells, B cells, mast cells, eosinophils, NK cells, DCs, keratinocytes and sensory neuronal endings [143–148]. TSLPR is activated upon binding of TSLP, which activates JAK1/2 and STAT1/3/4/5/6 [149,150]. Intradermal injection of TSLP evoked scratching behavior. This is initiated by the binding of TSLP to TSLPR expressed on sensory nerve fibers. The TSLP-induced itch also required TRPA1, with the expression and release of keratinocyte-derived TSLP depending on the ORAI1/NFAT calcium signaling pathway [148]. Epithelial cell-derived cytokines, including TSLP and IL-33, strongly activate ILC2 and recruit Th2 cells into the skin. ILC2 and Th2 cells are rich sources of type 2 cytokines, which can initiate and perpetuate allergic skin inflammation, including itch, by recruiting basophils and eosinophils [91].