JAK1/2 inhibitor but not IL‐4 receptor alpha antibody suppresses allergen‐mediated activation of human basophils in vitro
Authors: Wenming Peng, Said Benfadal, Chunfeng Yu, Jörg Wenzel, Johannes Oldenburg, Natalija Novak
Journal: Allergy (2022)

Atopic dermatitis (AD) is one of the most common inflammatory skin diseases characterized by chronic and relapsing eczematous lesions with intense pruritus.1 Accumulating evidence demonstrates basophils are important mediators of itch and inflammation in AD skin.2, 3 Basophils express the high-affinity IgE receptor (FcεRI), which binds allergen-specific IgE in sensitized allergic subjects. Cross-linking of FcεRI by allergen stimulation triggers rapid activation and degranulation of basophils, which promote Th2 polarization and allergic inflammation. Although the percentage of basophils in the peripheral blood is low, they infiltrate the skin of AD patients and therein promote skin inflammation upon activation.2 In addition, skin-infiltrating basophils contribute to itch via different signaling pathways.3 Substantive progress has been achieved recently in the treatment of AD by using a monoclonal antibody against the IL-4Rα (dupilumab) or by using different Janus-kinase (JAK) inhibitors such as baricitinib.4 Dupilumab suppresses type 2 immune responses by blocking IL-4- and IL-13-mediated signaling. The JAK1/2 inhibitor baricitinib specifically suppresses the activation of JAK1 and JAK2 molecules, which are essential for activation of the JAK–STAT pathway. Although both IL-4Rα antagonist and JAK1/2 inhibitors have been investigated for the treatment of moderate-to-severe AD in clinical trials,4 their suppressive effects on basophils and allergen activation of basophils in vitro are still unclear. Therefore, we aimed to evaluate the suppressive function of dupilumab and baricitinib on FcεRI-mediated human basophil activation in vitro. In line with previous reports, significant basophil infiltration was detected in the lesional skin of AD patients but not in the skin of healthy donors (Figure 1A). Upon allergen stimulation, sensitized basophils express the activation and degranulation marker CD63.5 We could show that baricitinib significantly suppressed allergen-mediated basophil activation and degranulation in a dose-dependent manner in vitro (Figure 1B and C, Figure S1, Table S1). However, dupilumab did not suppress allergen-mediated basophil activation in vitro (Figure 1B and C). Aggregation of FcεRI on basophils triggers phosphorylation of ITAMs of the FcεRI subunits and the SH2-domains of kinases Syk, which further activates phosphorylation of multiple downstream molecules, including protein kinase C (PKC), to promote intracellular calcium flux and basophil degranulation.6 Results from flow cytometric intracellular phosphoprotein staining demonstrated that the JAK1/2 inhibitor, but not dupilumab, specifically suppressed the activation of STAT3, an essential molecule in the JAK-STAT pathway. Also, the JAK1/2 inhibitor suppressed the FcεRI-mediated activation of Syk (Figure S3). Western blot analysis confirmed that the JAK1/2 inhibitor suppressed the activation of STAT3 as well as FcεRI-mediated activation of Syk and PKC, indicating the JAK1/2 inhibitor suppresses basophil activation and degranulation via FcεRI-mediated signaling (Figure 1D). In the lesional skin of AD patients, activated basophils release histamine and inflammatory cytokines to promote skin inflammation and itch.3 We pre-incubated freshly isolated human basophils (Figure S2) with baricitinib or dupilumab before the cells were activated by anti-human IgE antibody to perform aggregation of FcεRI-bound IgE. FcεRI-activation changed basophil morphology from round spherical to irregular shape, which was suppressed by baricitinib but not dupilumab (Figure 2A). Furthermore, the inflammatory mediator analysis of supernatants of activated basophils demonstrated that FcεRI-activated basophils did not produce detectable IL-1β, IL-6, IL-10, IL-17A, IL-17E, IL-17F, IL-21, IL-23, TGF-β1, CCL20, IFN-γ, and IL-31 (data not shown). FcεRI-activated basophils produced a high amount of histamine and a moderate level of IL-4 (Figure 2B). Baricitinib but not dupilumab significantly suppressed the production of both histamine and IL-4 of FcεRI-activated basophils (Figure 2B). Basophils are vital players in mediating the pathogenesis of AD by IL-4 production to promote Th2 polarization on the one handside and release of histamine to favor local skin inflammation on the other handside.2, 7 Our study confirmed skin infiltration of basophils in the lesional skin of AD patients. Furthermore, our data demonstrated that the JAK1/2 inhibitor baricitinib but not dupilumab suppresses the IgE receptor and allergen-mediated degranulation and cytokine synthesis of basophils in allergic inflammation. Although the JAK1/2 inhibitor suppresses FcεRI-mediated basophil activation by downregulating Syk and PKC activation, the suppressive effect on FcεRI-mediated signaling is lower than its specific inhibitory effect on JAK-STAT signaling (Figure S3). Our results favor the concept which combine JAK inhibitors with specific inhibitors which target FcεRI signaling in order to control allergic inflammation. As both histamine and IL-4 are important mediators involved in skin pruritus, our results suggested that baricitinib may exert its anti-pruritic function by suppressing the activation of skin-infiltrating basophils in AD patients. Although the IL-4Rα antagonist did not display its suppressive function on basophil activation in our in vitro experiments, it is still possible that the IL-4Rα antagonistic biologics exert an indirect suppressive function on basophils through suppression of the activation of Th2 cells in vivo. The suppressive function of the JAK1/2 inhibitor baricitinib on activated basophils demonstrated here in vitro might be responsible for the fast and strong effect of JAK inhibitors including baricitinib on allergic skin inflammation and skin lesions in AD patients observed in clinical studies. We thank all the patients for their willingness to participate in the study. We thank Ms. Lisa Nauroth for her excellent technical support. This work was supported by a BONFOR Grant of the University of Bonn and by the Cluster of Excellence ImmunoSensation (EXC 1023 and EXC 2151) and funded by the German Research Foundation (DFG). Natalija Novak reports grants and personal fees from Abbvie, Alk Abello, Bencard Allergy Therapeutics, Blueprint, HAL Allergy, Leo Pharma, Leti Pharma, Lilly, Lofarma, Novartis, Pfizer, Sanofi Genzyme, Stallergenes, Streamend up. The other authors have declared no financial or commercial conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.