Phosphodiesterase 4 Inhibitors, Basophils, and Atopic Dermatitis

Raif S. GehaView Large Image Figure ViewerDownload Hi-res image Download (PPT)Clinical Implications•Phosphodiesterase 4 (PDE4) is highly expressed in basophils, and PDE4 inhibitors block cyclic adenosine 3, 5'-monophosphate (cAMP) degradation but also block cAMP-driven extracellular signal–regulated kinase 1/2 activation•The latter results in the inhibition of IL4 gene expression in basophils and thereby the attenuation of allergic skin inflammation. •Phosphodiesterase 4 (PDE4) is highly expressed in basophils, and PDE4 inhibitors block cyclic adenosine 3, 5'-monophosphate (cAMP) degradation but also block cAMP-driven extracellular signal–regulated kinase 1/2 activation•The latter results in the inhibition of IL4 gene expression in basophils and thereby the attenuation of allergic skin inflammation. Atopic dermatitis (AD) is the most common chronic skin inflammatory disorder. It affects 20% of children and 5% of adults globally. It is characterized by barrier dysfunction, epidermal hyperplasia, dermal infiltration by T cells and eosinophilia, and type 2–dominated local and systemic immune responses. The beneficial effect of IL-4Ra blockade in AD demonstrates the critical roles of IL-4 and IL-13 in AD (Beck et al., 2014Beck L.A. Thaçi D. Hamilton J.D. Graham N.M. Bieber T. Rocklin R. et al.Dupilumab treatment in adults with moderate-to-severe atopic dermatitis.N Engl J Med. 2014; 371: 130-139Crossref PubMed Scopus (1108) Google Scholar). The enzyme phosphodiesterase 4 (PDE4) breaks down cyclic adenosine 3, 5'-monophosphate (cAMP) in cells (Figure 1). PDE4 has 4 subtypes—PDE4A, PDE4B, PDE4C, and PDE4D—all highly specific for cAMP degradation. cAMP is involved in the pathophysiology of allergic diseases, including asthma and AD. Before the advent of inhalation therapy, theophylline, an inhibitor of phosphodiesterase, was the mainstay of asthma treatment prior to the era of therapy with β-adrenergic agonists. β-Adrenergic agonists drive a rise in cAMP by activating adenylate cyclase (AC), which converts adenosine triphosphate (ATP) into cAMP (Figure 1). PDE4 is expressed in peripheral mononuclear cells and in inflamed skin, which has made it an attractive therapeutic target in AD. The usefulness of topical PDE4 inhibitors in AD is now well-established (Guttman-Yassky et al., 2019Guttman-Yassky E. Hanifin J.M. Boguniewicz M. Wollenberg A. Bissonnette R. Purohit V. et al.The role of phosphodiesterase 4 in the pathophysiology of atopic dermatitis and the perspective for its inhibition.Exp Dermatol. 2019; 28: 3-10Crossref PubMed Scopus (54) Google Scholar). However, the mechanisms underlying this therapeutic are not well-known. In their study, Takahashi et al., 2023Takahashi K. Miyake K. Ito J. Shimamura H. Suenaga T. Karasuyama H. et al.Topical application of a PDE4 inhibitor ameliorates atopic dermatitis through inhibition of basophil IL-4 production.J Invest Dermatol. 2024; 144: 1048-1057.e8Abstract Full Text Full Text PDF Scopus (1) Google Scholar uncover the cellular and molecular mechanisms of difamilast, a novel PDE4 inhibitor, using an oxazolone (OXA)-induced mouse model of allergic skin inflammation that mimics AD. cAMP is one of the most versatile cellular second messengers, and it regulates important biological processes, including cell migration, activation, proliferation, and survival. The activation of membrane receptors triggers cAMP production, mainly G protein-coupled receptors that activate cellular AC, which converts ATP to cAMP. Inhibition of PDE4 elevates the intracellular level of cAMP, thereby modulating the inflammatory responses and maintaining immune balance. cAMP transduces signal-encoded information by acting through several cellular effectors. These include cAMP-activated protein kinase A (PKA) and a family of 2 exchange proteins directly activated by cAMP1 (EPAC1) and cAMP2 (EPAC2) that act as nucleotide-gated ion channels. cAMP causes the release of the catalytic subunit of PKA from its regulatory subunit (Figure 1). This results in PKA-driven phosphorylation and activation of extracellular signal–regulated kinase (ERK) 1/2 (Figure 1) and of several transcription factors that include CREB protein, ATF-1, and cAMP-responsive element modulator, which recruits the CREB-binding protein. In T cells, ERK1/2 physically associates with a promoter element on the IL4 gene after antigen receptor crosslinking. The proximally localized ERK then facilitates the recruitment of the key transcription factors necessary for initiating IL4 gene transcription (Figure 1) (Tripathi et al., 2012Tripathi P. Sahoo N. Ullah U. Kallionpää H. Suneja A. Lahesmaa R. et al.A novel mechanism for ERK-dependent regulation of IL4 transcription during human Th2-cell differentiation.Immunol Cell Biol. 2012; 90: 676-687Crossref PubMed Scopus (31) Google Scholar). In addition to the canonical cAMP–PKA–CREB pathway, cAMP activates the small guanine nucleotide–binding protein RAP-1 through EPAC1/2. This promotes sustained ERK1/2 activation by PKA (Guttman-Yassky et al., 2019Guttman-Yassky E. Hanifin J.M. Boguniewicz M. Wollenberg A. Bissonnette R. Purohit V. et al.The role of phosphodiesterase 4 in the pathophysiology of atopic dermatitis and the perspective for its inhibition.Exp Dermatol. 2019; 28: 3-10Crossref PubMed Scopus (54) Google Scholar). Inhibition of PDE4 exerts regulatory activities in macrophages, neutrophils, monocytes, and dendritic cells. Notably, PDE4 inhibition has critical effects on TCR-induced activation of T cells, with a reduction of cytokine release from T helper (Th) cells, including Th1, Th2, and Th17 cells (Guttman-Yassky et al., 2019Guttman-Yassky E. Hanifin J.M. Boguniewicz M. Wollenberg A. Bissonnette R. Purohit V. et al.The role of phosphodiesterase 4 in the pathophysiology of atopic dermatitis and the perspective for its inhibition.Exp Dermatol. 2019; 28: 3-10Crossref PubMed Scopus (54) Google Scholar). Takahashi et al., 2023Takahashi K. Miyake K. Ito J. Shimamura H. Suenaga T. Karasuyama H. et al.Topical application of a PDE4 inhibitor ameliorates atopic dermatitis through inhibition of basophil IL-4 production.J Invest Dermatol. 2024; 144: 1048-1057.e8Abstract Full Text Full Text PDF Scopus (1) Google Scholar demonstrate that topical application of 1% ointment of the PDE4 inhibitor difamilast improves allergic skin inflammation, including ear swelling, lichenification, and neutrophil but not eosinophil infiltration in a mouse model of AD in which allergic skin inflammation is elicited by repeated hapten ear challenges of mice previously sensitized with OXA. Notably, the researchers further showed that difamilast treatment both before and during the OXA challenge improves allergic inflammation, indicating that the inhibition is beneficial in a setting of already triggered inflammation. Difamilast treatment reduced the level of IL-4 but not of IL-13 in the skin lesion. The investigators show that IL-4–deficient mice had reduced but measurable allergic skin inflammation. Difamilast treatment caused little or no change in the reduced allergic skin inflammation in these mice. Basophils are rare granulocytes representing <1% of peripheral blood leukocytes. Basophils, upon activation, secrete histamine, Th2 cytokines (eg, IL-4, IL-13, and IL-31), proteases, and autocoids such as leukotriene and prostaglandins. Patients with AD often show infiltration of basophils in lesional skin. Basophils can display both beneficial and detrimental roles in AD. In a mouse model of AD elicited by topical application of MC903 and in which inflamed skin resembles the acute lesions of patients with AD, TSLP triggers basophil recruitment into skin and skin-draining lymph nodes. In this model, basophils are the initial and main source of IL-4 in the skin (Leyva-Castillo et al., 2013Leyva-Castillo J.M. Hener P. Michea P. Karasuyama H. Chan S. Soumelis V. et al.Skin thymic stromal lymphopoietin initiates Th2 responses through an orchestrated immune cascade.Nat Commun. 2013; 4: 2847Crossref PubMed Scopus (132) Google Scholar). Furthermore, basophil-derived IL-4 promotes Th2 differentiation, implying their significant role in AD pathogenesis. In contrast to this pathogenic role, basophils promote repair and remodeling at sites of allergic skin inflammation. Indeed, basophil-derived macrophage colony-stimulating factor inhibits the accumulation of proinflammatory cells in the skin and promotes the expansion and function of M2-like macrophages, which are important for skin repair and remodeling (Pellefigues et al., 2021Pellefigues C. Naidoo K. Mehta P. Schmidt A.J. Jagot F. Roussel E. et al.Basophils promote barrier dysfunction and resolution in the atopic skin.J Allergy Clin Immunol. 2021; 148: 799-812.e10Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Takahashi et al., 2023Takahashi K. Miyake K. Ito J. Shimamura H. Suenaga T. Karasuyama H. et al.Topical application of a PDE4 inhibitor ameliorates atopic dermatitis through inhibition of basophil IL-4 production.J Invest Dermatol. 2024; 144: 1048-1057.e8Abstract Full Text Full Text PDF Scopus (1) Google Scholar showed that application of difamilast ointment caused little to no change in the allergic skin inflammation in mice depleted of basophils by treatment with anti-CD200R3 antibody and in Mcpt8DTR mice in which treatment with diphtheria toxin selectively kills basophils. The researchers further showed that ILl4 expression is reduced in basophils from difamilast-treated mice compared with that in basophils from control mice. Difamilast treatment showed no further reduction in allergic skin inflammation in Mctp8creIl4fl/fl mice with selective deletion of IL-4 in basophils. Difamilast inhibits preferentially the enzymatic activity of the isoform PDE4B. Notably, among the 4 subtypes of PDE4, the expression of Pde4b was highest in resting basophils and was upregulated further when basophils were stimulated in vitro with either cytokine (IL-3 + IL-33) or IgE/allergen complexes. Using the publicly available single-cell RNA-sequencing datasets, the researchers showed that Pde4b expression was upregulated in basophils isolated from the OXA-challenged and inflamed skin compared with that in basophils isolated from control-treated skin. Using bone marrow–derived basophils (BMBAs), the investigators showed that difamilast directly suppressed the basophils' activation and production of IL-4. To examine the possible mechanisms through which difamilast inhibits the IL-4 production from activated basophils, the researchers performed bulk RNA-sequencing analysis of BMBAs treated with difamilast. Gene enrichment analysis revealed that difamilast treatment downregulated the genes associated with the ERK signaling cascade in cytokine-stimulated basophils. In contrast, the expression of dual specificity phosphatases, such as Dusp1, Dusp5, and Dusp16, which are known to inhibit ERK phosphorylation, was significantly upregulated by difamilast treatment. Finally, the researchers showed that treatment with ERK1/2-specific inhibitor suppressed IL-4 production from activated BMBAs. Together, these results indicate that PDE4 inhibition, on the one hand, elevates cAMP levels but, on the other hand, blocks cAMP-dependent ERK1/2 activation, favoring the inhibition of PKA-driven ERK1/2-dependent IL4 gene transcription. The study by Takahashi et al., 2023Takahashi K. Miyake K. Ito J. Shimamura H. Suenaga T. Karasuyama H. et al.Topical application of a PDE4 inhibitor ameliorates atopic dermatitis through inhibition of basophil IL-4 production.J Invest Dermatol. 2024; 144: 1048-1057.e8Abstract Full Text Full Text PDF Scopus (1) Google Scholar demonstrates that difamilast, a PDE4 inhibitor, exerts its therapeutic effects in a mouse model of AD through suppression of basophil IL-4 production, likely through the inhibition of ERK phosphorylation in the pathogenesis of AD, specifically in the mouse model of OXA-mediated allergic skin inflammation. Similar to all exciting studies, it also raises questions. What are the underlying mechanisms through which basophil-derived IL-4 inhibits neutrophil recruitment in the case of difamilast treatment? Our recent findings demonstrate that basophil-derived IL-4 promotes Staphylococcus aureus colonization in AD-like skin inflammation in mice. Superficial infection of S aureus to tape-stripped skin causes cutaneous basophil recruitment and enhanced IL-4 expression. Importantly, basophil-derived IL-4 inhibited IL-23 production by keratinocytes, blocked IL-17A production by TCR gamma delta cells, and suppressed IL-17A–driven induction of neutrophil-attracting chemokines and antimicrobial peptides in keratinocytes, thereby promoting superficial skin infection with S aureus (Leyva-Castillo et al., 2021Leyva-Castillo J.M. Das M. Kane J. Strakosha M. Singh S. Wong D.S.H. et al.Basophil-derived IL-4 promotes cutaneous Staphylococcus aureus infection.JCI Insight. 2021; 6e149953Crossref PubMed Scopus (16) Google Scholar). Crisaborole, another PDE4 inhibitor, reduced itch and pain by inhibiting neutrophil infiltration in a mouse model of MC903-induced AD (Pavlenko et al., 2023Pavlenko D. Todurga Seven Z. Bystrom L. Markan A. Verpile R. Ishida H. et al.Crisaborole inhibits itch and pain by preventing neutrophil infiltration in a mouse model of atopic dermatitis.Acta Derm Venereol. 2023; 103: adv13382Crossref PubMed Scopus (1) Google Scholar). Is this a common mechanism through which PDE4 inhibitors suppress inflammation and itch in different mouse models of AD and patients with AD? Notably, the recruitment of neutrophils is associated with itching in AD (Walsh et al., 2019Walsh C.M. Hill R.Z. Schwendinger-Schreck J. Deguine J. Brock E.C. Kucirek N. et al.Neutrophils promote CXCR3-dependent itch in the development of atopic dermatitis.ELife. 2019; 8e48448Crossref Scopus (88) Google Scholar). On the other hand, a recent report highlights the crucial role of basophil-derived LTC4 in acute itch flares in patients with AD (Wang et al., 2021Wang F. Trier A.M. Li F. Kim S. Chen Z. Chai J.N. et al.A basophil-neuronal axis promotes itch.Cell. 2021; 184: 422-440.e17Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Do PDE4 inhibitors also suppress basophil-derived LTC4 through the inhibition of ERK phosphorylation? Finally, are the findings in the hapten-driven mouse model of allergic skin inflammation used by Takahashi et al., 2023Takahashi K. Miyake K. Ito J. Shimamura H. Suenaga T. Karasuyama H. et al.Topical application of a PDE4 inhibitor ameliorates atopic dermatitis through inhibition of basophil IL-4 production.J Invest Dermatol. 2024; 144: 1048-1057.e8Abstract Full Text Full Text PDF Scopus (1) Google Scholar applicable to the more physiologic model of antigen-driven inflammation (Yang et al., 2023Yang B. Wilkie H. Das M. Timilshina M. Bainter W. et al.The IL-4Rα Q576R polymorphism is associated with increased severity of atopic dermatitis and exaggerates allergic skin inflammation in mice.J Allergy Clin Immunol. 2023; 151: 1296-1306.e7Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar), and more importantly, are the results applicable to AD in which T cells are the major source of IL-4 in established skin lesions? Addressing these questions would potentially enhance the therapeutic usage of PDE4 inhibitors in patients with AD. The authors state no conflict of interest. RSG is the guarantor for this work. This work was supported by the National Institutes of Health grant AI114588. RSG is the guarantor for this work. Topical Application of a PDE4 Inhibitor Ameliorates Atopic Dermatitis through Inhibition of Basophil IL-4 ProductionJournal of Investigative DermatologyVol. 144Issue 5PreviewPhosphodiesterase 4 inhibitors have been approved for the treatment of atopic dermatitis. However, the cellular and molecular mechanisms underlying their therapeutic effect remain to be fully elucidated. In this study, we addressed this unsolved issue by analyzing the action of difamilast, a novel phosphodiesterase 4 inhibitor, on an oxazolone-induced skin allergic inflammation commonly used as a mouse model of atopic dermatitis. Topical application of difamilast ameliorated skin inflammation in association with reduced IL-4 expression even when the treatment commenced 4 days after the initiation of oxazolone challenge, showing its therapeutic effect on atopic dermatitis. Full-Text PDF Open Access