H4R 在体外和体内均显示参与 AD 发病机制的特征机制。3在 AD 选择性 H4R 拮抗剂患者中进行的两项临床概念试验证明，瘙痒减少和/或湿疹改善。2，S1我们最近描述了 H2R 作为 AD 中炎症 II 型反应的可能放大器的新作用，导致单核细胞和巨噬细胞中的趋化因子 CCL17 上调。8
Dupilumab 是第一个被批准用于治疗 AD S2 的单克隆抗体，也是针对 JAK/TYK 通路的小分子，在治疗患者炎症性疾病或临床试验中显示出积极的结果。9
我们对阻断 IL-4R I 型和 IL-4/IL-13R II 型受体亚基以及下游信号分子，特别是转录因子 AP-1 的特定物质进行的实验显示了导致防止人类 M2 巨噬细胞中 H2R 的上调和最显着的 H4R 上调。
我们的发现应该有助于更好地理解可能与 AD 相关的组胺受体的调节以及干扰过敏性皮肤病（如 AD）中 II 型炎症反应的物质的相关作用模式。
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Expression of histamine receptors H2R and H4R are predominantly regulated via the IL-4/IL-13 receptor type II on human M2 macrophages
To The Editor,
The pathophysiology of allergic skin diseases such as atopic dermatitis (AD) is characterized by complex interactions between susceptibility genes of the disease, a defect skin barrier and aberrant immune responses. Among other immune cells, monocytes differentiating to macrophages are attracted into inflamed skin and are exposed to Th2 cytokines and to histamine detectable in high concentrations in AD patients' skin contributing to immunomodulation and pruritus.1, 2 Increasing expression levels of functional histamine receptors, in particular of the histamine H4 receptor (H4R), on immune cells within the inflammatory infiltrate of eczematous skin lesions may exacerbate inflammation by mediating cytokine and chemokine release in contact to local histamine.3 Knowledge of these mechanisms may result in therapeutic strategies to alleviate inflammation.
Two specific antagonists of the H4R were studied in phase II studies with AD patients.2,S1 A number of other substances, particularly dupilumabS2, currently approved for treatment of type II inflammatory diseases, have shown meaningful reduction of pruritic symptoms and clinical AD severity scores.
Since little is known about the cross talk of type II cytokines and histamine receptors on macrophages known to be increased in the dermis of inflamed skin in AD S3, we investigated in this study if the Th2 cytokines IL-4 and IL-13 influence the histamine receptor mRNA expression levels in human monocyte-derived M2 macrophages differentiated in the presence of M-CSF for 10 days (see additional information in the Material and Methods section and Figure S1 in this article's Online Repository).
As shown previously, the H1R-, H2R- and H4R- but not H3R mRNA expressions are detectable in fully differentiated human M2 macrophages.4
The stimulation of M2 macrophages with IL-4 led to an up-regulation of H2R and H4R, whereas IL-13 selectively up-regulated the H4R at mRNA level in M2 macrophages (Figure 1B,C,E,F). The H1R mRNA expression was neither up-regulated by IL-4 nor by IL-13 (Figure 1A,D).
FIGURE 1
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Regulation of the H1R, H2R, and H4R mRNA expression in response to Th2 cytokines in human M2 macrophages. In the presence of M-CSF differentiated macrophages from residual blood samples from platelet apheresis A-F or from peripheral blood from healthy controls or from atopic dermatitis patients G and H were stimulated with IL-4 (20 ng/ml) or IL-13 (15 ng/ml) for 48 h. The mRNA expression of A, H1R, B, H2R, and C, H4R was measured in fully differentiated and in IL-4- or D, H1R, E, H2R, F, H4R in IL-13-activated M2 macrophages. G, H2R and H, H4R constitutive mRNA expression was measured in fully differentiated M2 macrophages (10 days) from healthy controls compared with cells from patients with atopic dermatitis. Significant differences, as determined by the Wilcoxon matched-pairs signed rank test A-F and the Mann Whitney test G and H, are indicated as follows: *p < .05; **p < .01; ***p < .001; medians are shown in the graphs. A, (n = 12 independent donors and experiments), B, (n = 13 independent donors and experiments), C, (n = 8 independent donors and experiments), D, (n = 9 independent donors and experiments), E, (n = 14 independent donors and experiments), F, (n = 10 independent donors and experiments), G, (n = 8 independent donors and experiments), H, (n = 6 independent donors and experiments), NS = non stimulated, HC = healthy controls, AD = atopic dermatitis, tgt/ref ratio = target/reference ratio
Macrophage M2 marker CD206, also known as mannose receptor C-type I, expressing cells were detected in the dermis of AD patients pointing to a possible role for macrophages in disease pathologyS3. Interestingly, M2 macrophages generated from monocytes from patients with moderate-to-severe AD showed higher constitutive expression levels of the H4R mRNA when compared to healthy controls, which was not seen for the H2R (Figure 1G,H). Since it is suggested that serum IgE levels correlate with disease severity of ADS4, we also measured total IgE levels in the serum of respective patients by ImmunoCap, Thermo Fisher Scientific. AD patients with elevated H4R mRNA expression levels showed also high total IgE levels in their serum as shown in Table S1 in this article´s Online Repository. These data obtained in human macrophages strongly fit observations in human eosinophils in a recently published work.5 In human eosinophils, H4R mRNA expression was also up-regulated by IL-4 and IL-13 whereas up-regulation of H2R mRNA expression was, just as in macrophages, mediated via IL-4 only.5 Similar to M2 macrophages, eosinophils from AD patients constitutively express high levels of H4R when compared to cells from healthy controls.5
The up-regulation of H4R mRNA expression also in response to IL-13 as well as the high levels of constitutive mRNA expression of the receptor shown in eosinophils5 and in this work in M2 macrophages from AD patients appoint to a relevant role of the H4R in the pathophysiology of AD. Under these conditions, the H4R is also referred to as a pathological H4R. The observation of a significant up-regulation of IL-13 mRNA expression in subacute and chronic AD skin lesions, which was not detected for IL-4S5, fosters this hypothesis.
Both cytokines IL-4 and IL-13 determine potent type 2 inflammatory processes by signaling through their cell surface receptor heterodimers.1 Heterodimerisation of type I or II receptor subunits by IL-4 or IL-13 leads to catalytic activation of associated Janus/Tyrosine kinases (JAK/TYK comprising of JAK1, JAK2, JAK3, and TYK2). Subsequent phosphorylation of tyrosine residues in the receptors takes place thereby signal transducer and activator of transcription 6 (STAT6) was activated and translocated to the nucleus to induce transcription of cytokine-induced genesS6 (Figure 2A).
FIGURE 2
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The IL-4- or IL-13-induced up-regulation of H2R and H4R mRNA expression in human M2 macrophages is mainly mediated via the IL-4/IL-13 receptor type II. A, The binding of IL-4 and IL-13 to their cognate receptors (type I and type II) triggers the activation of receptor subunit associated signaling adaptor molecules which induce the recruitment and phosphorylation of signal transducer and activator of transcription 6 (STAT6) that can cooperate with activator protein-1 (AP-1) to activate specific gene transcription programs (simplified diagram, created with BioRender.com.). B -G, In the presence of M-CSF differentiated macrophages were stimulated with IL-4 (20 ng/ml) or IL-13 (15 ng/ml) for 48 h. 20 min before stimulation, the subunits of IL-4R type I and IL-4/IL-13R type II cell surface receptors were either selectively blocked by specific antibodies (Ab): Dupilumab (10 µM), a monoclonal IgG4 antibody which blocks the IL-4Rα subunit, a polyclonal goat IgG antibody was used to block the common γ chain/IL-2Rγ (20 µM), the IL-13Rα1, and IL-13-Rα2 subunits were blocked by monoclonal antibodies (10 µM) each, or their downstream signaling adaptor molecules were blocked by selective inhibitors (Inh.): T-5224 for AP-1 (10 µM), PF 06651600 malonate (10 µM) for JAK3 and TC JL 37 (10 µM) for TYK2. B and C, H2R mRNA expression in IL-4 stimulated M2 macrophages, D and E, H4R mRNA expression in IL-4 stimulated M2 macrophages, F and G, H4R mRNA expression in IL-13 stimulated M2 macrophages. Significant differences, as determined by Friedman Dunn's Multiple Comparison test selected pairs and as determined by the Wilcoxon matched-pairs signed rank test (NS versus IL-4 or IL-13) are indicated as follows: *p < .05; **p < .01; ***p < .001; ****p < .0001; medians are shown in the graphs. NS = non-stimulated; B, (n = 11 independent donors and experiments), C, (n = 16 independent donors and experiments), D, (n = 13 independent donors and experiments), E, (n = 14 independent donors and experiments), F, (n = 13 independent donors and experiments), G, (n = 16 independent donors and experiments). NS = non-stimulated
We investigated the signaling pathways of IL-4 and IL-13 in order to find the checkpoints for H2R and H4R up-regulation in human M2 macrophages.
Therefore, IL-4 or IL-13 stimulated (48 h) M2 macrophages were pre-incubated with antibodies targeting the IL-4 and IL-13 receptor complex subunits: Type I (IL-4) receptor composed of the IL-4 receptor alpha (IL-4Rα) (dupilumab) and the common γ chain/IL-2Rγ, and type II (IL-4/IL-13) receptor composed of the IL-4Rα and IL-13Rα1 subunit.6 The binding of IL-13 to the IL-13Rα2 was also blocked by a specific antibody. Inhibitors of the downstream regulators JAK3, TYK2 and of the activator protein-1 (AP-1) were also added to the cells before Th2 cytokine stimulation. We did not block JAK1 signaling because JAK1 is associated with both the type I and type II receptor and thus does not differentiate between bothS6 (dose-response experiments for antibodies and inhibitors see Figure S2 in this article's Online Repository).
Dupilumab, a humanized IgG4 monoclonal antibody targeting the IL-4Rα subunit, significantly inhibited the IL-4-mediated H2R (Figure 2B) as well as the IL-4- and IL-13-mediated H4R mRNA up-regulation. Targeting the other IL-4 type I or IL-4/IL-13 type II receptor subunits by specific antibodies was less effective (Figure 2D,F). A possible explanation for this is offered by the following hypothesis: If the common γ chain/IL-2Rγ is exclusively blocked, the IL-13Rα1 subunit can be recruited to form heterodimers with IL-4Rα leading to signal transduction. A corresponding result would be seen vice versa when IL-13Rα1 is blocked alone. In our study, an anti-IL-13Rα1 antibody was able to inhibit the IL-13-induced H4R up-regulation by trend (Figure 2F).
Interestingly, the IL-4-induced up-regulation of H2R mRNA expression was also reversed by an antibody directed against the IL-13Rα2 subunit (Figure 2B). The mechanisms of the IL-13Rα2 subunit are not clearly understood until yet. Some reports describe that the IL-13Rα2 acts as a decoy receptor and does not transduce for IL-4/IL-13 signaling into the cellS7,S8. However, some data support the hypothesis that the cytoplasmic tail of IL-13Rα2 may function as a regulator of IL-4 signaling by physical interactions with the IL-4 Rα7,S8 which may fit our observation.
Regarding the selectively associated JAK3 or TYK2 signaling molecules for type I and type II receptors, respectively, we observed significant inhibition of IL-4-induced H2R mRNA up-regulation using the selective TYK2 inhibitor (Figure 2C).
The IL-4-induced H4R mRNA expression was blocked by a JAK3 inhibitor which is selective for type I receptor (IL-4R), and even stronger by the inhibitor of TYK2 which is selective for type II (IL-4/IL-13R). Inhibition of TYK2 also prevents the IL-13-induced up-regulation of H4R mRNA expression (Figure 2E,G). These results set the type II receptor in the focus of H2R and H4R regulation.
Blocking the transcription factor AP-1 by small molecules inhibited the IL-4-mediated up-regulation of H4R mRNA expression only (Figure 2E). The AP-1 family proteins play a critical part in several severe as well as in inflammatory disorders by regulating transcription of specific genes involved in production of cytokines, chemokines, proliferation, and differentiation of immune cellsS9. Remarkably, it has been demonstrated that the IL-4 dependent induction of germline ɛ transcripts, an essential step preceding Ig isotype switching to IgE in mice requires the interaction of AP-1 proteins Jun/Fos and STAT6S10.
For human M2 macrophages, we show here for the first time a novel function of AP-1 regulating the IL-4-dependent up-regulation of the H4R mRNA expression.
In parallel, we investigated the phosphorylation of STAT6 at tyrosine 641, which is considered to be a major pathway involved in IL-4 or IL-13 responsiveness, by flow cytometry.6 After respective blocking of the IL-4/IL-13 pathways by antibodies and inhibitors of the signaling molecules, inhibition of the phosphorylation of STAT6 mostly corresponded to the picture of the inhibition of IL-4- and IL-13-induced H4R mRNA up-regulation. The phosphorylation of STAT6 at tyrosine 641 was blocked by dupilumab, by an antibody against IL-13Rα1, and by JAK3 and TYK2 inhibitors (see Figure S3 in this article's Online Repository) pointing to a crucial role of STAT6 in the regulation of H4R gene transcription.
The H4R was shown in vitro and in vivo to be involved in mechanisms which are characteristic for the pathogenesis of AD.3 In two clinical proof of concept trials in patients with AD selective H4R antagonists showed reduction of pruritus and/or improvement of eczema.2,S1 A novel role of the H2R as a possible amplifier of inflammatory type II responses in AD resulting in up-regulating the chemokine CCL17 in monocytes and macrophages was recently described by us.8
Dupilumab, the first monoclonal antibody approved for the treatment of ADS2 and also small molecules targeting the JAK/TYK pathways showed positive outcomes in the treatment of inflammatory diseases of patients or in clinical trials.9
Our experiments with specific substances blocking the IL-4R type I and IL-4/IL-13R type II receptor subunits as well as the downstream signaling molecules, and in particular the transcription factor AP-1, showed novel mechanisms of action leading in the prevention of the up-regulation of H2R and most pronounced of H4R in human M2 macrophages.
Our findings should contribute to an improved understanding of the regulation of histamine receptors with possible relevance for AD and the related mode of action of substances interfering with type II inflammatory responses in allergic skin diseases such as AD.
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