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Propranolol Is an Effective Topical and Systemic Treatment Option for Experimental Epidermolysis Bullosa Acquisita

Open ArchivePublished:May 22, 2020DOI:https://doi.org/10.1016/j.jid.2020.04.025
      Propranolol is an ADRB2 blocker that regulates heart muscle contractions, smooth muscle relaxation, and glycogenolysis. In addition, an increasing number of applications in dermatology have been described, most prominently, the use as a first-line treatment for infantile hemangiomas. We here show that propranolol enhances IL-8–induced neutrophil chemotaxis and reduces the release of ROS after immune complex stimulation. To obtain further molecular insights into the modulatory effects of propranolol in activated neutrophils, we performed RNA sequencing of immune complex–stimulated neutrophils in the absence and presence of the drug. We identified the transcriptomic signature of propranolol and demonstrated an ADR2-independent immunomodulatory effect. To determine if the anti-inflammatory transcriptomic signature of propranolol also translates into clinical effects, we next evaluated the impact of propranolol in a prototypical neutrophil-dependent skin disease, specifically, antibody transfer–induced epidermolysis bullosa acquisita in mice. To validate the identified propranolol gene signature obtained in human neutrophils, we analyzed a selection of genes by RT-PCR in mouse epidermolysis bullosa acquisita skin and confirmed TNF, among others, to be differentially regulated by propranolol treatment. Our data clearly indicate that, based on its molecular impact on immune complex–activated neutrophils, propranolol is a potential treatment option for neutrophil-mediated inflammatory skin diseases.

      Abbreviations:

      ADR (adrenoreceptor), EBA (epidermolysis bullosa acquisita), IC (immune complex), IH (infantile hemangioma), PD (pemphigoid disease), PMN (polymorphonuclear), RNA-Seq (RNA sequencing)

      Introduction

      Adrenoceptors (ADRs) are targeted by neurotransmitters such as noradrenaline and adrenaline. They regulate several physiological functions, such as the regulation of heart frequency, vasoconstriction, and smooth muscle relaxation. Besides these functions, the sympathetic nervous system significantly contributes to the brain–immune system cross-talk, mainly because ADRs are also expressed also on immune cells. However, the precise functions of ADR expression on immune cells is only incompletely understood (
      • Scanzano A.
      • Cosentino M.
      Adrenergic regulation of innate immunity: a review.
      ). ADRs are a class of G protein-coupled receptors that mediate actions of the sympathetic nervous system. There are two main groups of ADRs, α and β, with nine subtypes in total. ADRA1 couples with Gq protein, which results in increased intracellular calcium concentration and subsequent smooth muscle contraction. However, ADRA2 couples with the inhibitory Gi protein, which causes a decrease in neurotransmitter release, as well as a decrease in cAMP activity resulting in smooth muscle contraction. ADRBs generally couple with stimulatory Gs proteins (with the exception of ADRB2, which couples with Gi and Gs) and increase intracellular cAMP activity, resulting in heart muscle contraction, smooth muscle relaxation, and glycogenolysis, for example (
      • Kum J.J.
      • Khan Z.A.
      Mechanisms of propranolol action in infantile hemangioma.
      ). Depending on their affinity to the distinct ADRs, many subtype-specific medications like beta blockers, ADRB2 agonists, and ADRA2 agonists, are widely used to treat high blood pressure and asthma, for example.
      In addition to these effects, an increasing number of applications of ADRB blockers, such as propranolol and timolol, in dermatology have been described over the last 10 years (
      • Michel M.C.
      • Bond R.A.
      • Summers R.J.
      Adrenoceptors-new roles for old players.
      ,
      • Oberlin K.E.
      Expanding uses of propranolol in dermatology.
      ). Perhaps most prominently, propranolol is used as a first-line treatment for infantile hemangioma (IH), the most common benign vascular tumor of infancy (
      • Léauté-Labrèze C.
      • Dumas de la Roque E.
      • Hubiche T.
      • Boralevi F.
      • Thambo J.B.
      • Taïeb A.
      Propranolol for severe hemangiomas of infancy.
      ,
      • Luo Y.
      • Zeng Y.
      • Zhou B.
      • Tang J.
      A retrospective study of propranolol therapy in 635 infants with infantile hemangioma.
      ). This clinical use is based on randomized controlled trials in IH with a 60% successful treatment rate compared with 4% for patients receiving placebo (
      • Léauté-Labrèze C.
      • Hoeger P.
      • Mazereeuw-Hautier J.
      • Guibaud L.
      • Baselga E.
      • Posiunas G.
      • et al.
      A randomized, controlled trial of oral propranolol in infantile hemangioma.
      ,
      • Léauté-Labrèze C.
      • Voisard J.J.
      • Moore N.
      Oral propranolol for infantile hemangioma.
      ). In addition, both oral and topical propranolol treatments have been shown to be safe and effective in children, as well as adults, with IH (
      • Mashiah J.
      • Kutz A.
      • Rabia S.H.
      • Ilan E.B.
      • Goldberg I.
      • Sprecher E.
      • et al.
      Assessment of the effectiveness of topical propranolol 4% gel for infantile hemangiomas.
      ,
      • Wang Y.
      • Zhang X.
      • Yang Y.
      • Zhang J.
      • Yang Y.
      • Lu Y.
      Efficacy and safety of 2% topical propranolol cream for the treatment of proliferating infantile strawberry hemangiomas.
      ,
      • Zanela da Silva Marques T.
      • Santos-Oliveira R.
      • Betzler de Oliveira de Siqueira L.
      • Cardoso V.D.S.
      • de Freitas Z.M.F.
      • Barros R.C.D.S.A.
      • et al.
      Development and characterization of a nanoemulsion containing propranolol for topical delivery.
      ). Overall, adverse effects of oral propranolol treatment are rare and include sleep disturbances, acrocyanosis, hypotension, and hypoglycemia (
      • Drolet B.A.
      • Frommelt P.C.
      • Chamlin S.L.
      • Haggstrom A.
      • Bauman N.M.
      • Chiu Y.E.
      • et al.
      Initiation and use of propranolol for infantile hemangioma: report of a consensus conference.
      ,
      • El Hachem M.
      • Gesualdo F.
      • Diociaiuti A.
      • Berti I.
      • Vercellino N.
      • Boccaletti V.
      • et al.
      Safety and effectiveness of oral propranolol for infantile hemangiomas started before 5 weeks and after 5 months of age: an Italian multicenter experience.
      ;
      • Marqueling A.L.
      • Oza V.
      • Frieden I.J.
      • Puttgen K.B.
      Propranolol and infantile hemangiomas four years later: a systematic review.
      ,
      • Püttgen K.B.
      Evidence and nuances of propranolol safety.
      ,
      • Sagi L.
      • Zvulunov A.
      • Lapidoth M.
      • Ben Amitai D.
      Efficacy and safety of propranolol for the treatment of infantile hemangioma: a presentation of ninety-nine cases.
      ). The side effects of topical propranolol, such as irritation, redness, and scaling, are restricted to the site of application. No systemic adverse effects have been reported for topical treatment of IH, even when applied to large areas of the skin (
      • Mashiah J.
      • Kutz A.
      • Rabia S.H.
      • Ilan E.B.
      • Goldberg I.
      • Sprecher E.
      • et al.
      Assessment of the effectiveness of topical propranolol 4% gel for infantile hemangiomas.
      ).
      In addition to its effect on IH, case reports and series suggest that propranolol has beneficial effects in pyogenic granulomas and periorbital and ulcerated hemangiomas (
      • El Hachem M.
      • Gesualdo F.
      • Diociaiuti A.
      • Berti I.
      • Vercellino N.
      • Boccaletti V.
      • et al.
      Safety and effectiveness of oral propranolol for infantile hemangiomas started before 5 weeks and after 5 months of age: an Italian multicenter experience.
      ,
      • Neri I.
      • Baraldi C.
      • Balestri R.
      • Piraccini B.M.
      • Patrizi A.
      Topical 1% propranolol ointment with occlusion in treatment of pyogenic granulomas: an open-label study in 22 children.
      ,
      • Tiwari P.
      • Pandey V.
      • Gangopadhyay A.N.
      • Sharma S.P.
      • Gupta D.K.
      Role of propranolol in ulcerated haemangioma of head and neck: a prospective comparative study.
      ,
      • Xue L.
      • Sun C.
      • Xu D.P.
      • Liu Z.M.
      • Wang X.K.
      Clinical outcomes of infants with periorbital hemangiomas treated with oral propranolol.
      ), as well as limiting the recurrence of primary melanoma (
      • De Giorgi V.
      • Grazzini M.
      • Benemei S.
      • Marchionni N.
      • Botteri E.
      • Pennacchioli E.
      • et al.
      Propranolol for off-label treatment of patients with melanoma: results from a cohort study.
      ,
      • Jean Wrobel L.
      • Bod L.
      • Lengagne R.
      • Kato M.
      • Prévost-Blondel A.
      • Le Gal F.A.
      Propranolol induces a favourable shift of anti-tumor immunity in a murine spontaneous model of melanoma.
      ;
      • Killock D.
      Skin cancer: propranolol limits melanoma recurrence.
      ).
      Although it has been known since 2008 that propranolol is effective for the treatment of IH, the exact mechanism has not been completely elucidated. The mechanistic role of propranolol in these lesions is surmised to be due to vasoconstriction, decreased angiogenesis through the inhibition of both VEGF and basic fibroblast growth factor (
      • Léauté-Labrèze C.
      • Dumas de la Roque E.
      • Hubiche T.
      • Boralevi F.
      • Thambo J.B.
      • Taïeb A.
      Propranolol for severe hemangiomas of infancy.
      ). In addition, propranolol inhibits the growth or stimulates apoptosis of hemangioma-initiating cells such as endothelial cells, hemangioma-derived stem cells, or pericytes. (
      • Kum J.J.
      • Khan Z.A.
      Mechanisms of propranolol action in infantile hemangioma.
      ,
      • Léauté-Labrèze C.
      • Dumas de la Roque E.
      • Hubiche T.
      • Boralevi F.
      • Thambo J.B.
      • Taïeb A.
      Propranolol for severe hemangiomas of infancy.
      ). The effects of propranolol on these hemangioma-initiating cells may differ because of specific ADRB expression patterns. Apoptosis is stimulated by propranolol only in endothelial cells and other mature cell types through alterations in ADRB1 signaling, suggesting a constitutively active ADRB2 pathway that leads to cell cycle arrest and growth inhibition (
      • Kum J.J.
      • Khan Z.A.
      Mechanisms of propranolol action in infantile hemangioma.
      ). Further unexpected and probably ADR-independent effects have been described in animal experiments. First, propranolol in combination with dopamine has a synergistic action in intensifying and prolonging cutaneous analgesia in rats (
      • Chen Y.W.
      • Chiu C.C.
      • Wei Y.L.
      • Hung C.H.
      • Wang J.J.
      Propranolol combined with dopamine has a synergistic action in intensifying and prolonging cutaneous analgesia in rats.
      ). Second, low-dose propranolol improves cutaneous wound healing in burn-injured rats (
      • Romana-Souza B.
      • Nascimento A.P.
      • Monte-Alto-Costa A.
      Low-dose propranolol improves cutaneous wound healing of burn-injured rats.
      ). Propranolol reduces the local inflammatory response and improves subsequent healing phases by inhibiting cellular proliferation, myofibroblast density, collagen deposition, and active matrix metalloproteinase-2 levels. Third, conflicting results on the impact of propranolol on neutrophil functions have been described; propranolol inhibits superoxide generation in stimulated neutrophils through the attenuation of phosphatidic acid and diacylglycerol generation (
      • English D.
      • Taylor G.S.
      Divergent effects of propranolol on neutrophil superoxide release: involvement of phosphatidic acid and diacylglycerol as second messengers.
      ). Other groups have shown that the effects of propranolol are mediated by membrane stabilization (
      • Anderson R.
      • Ramafi G.
      • Theron A.J.
      Membrane stabilizing, anti-oxidative interactions of propranolol and dexpropranolol with neutrophils.
      ). In contrast to these inhibitory effects of propranolol on neutrophil activation, the drug has been shown to increase neutrophil motility (
      • Anderson R.
      • van Rensburg A.J.
      The in vitro effects of propranolol and atenolol on neutrophil motility and post-phagocytic metabolic activity.
      ). Finally, an inhibitory effect of propranolol on the regulation of spontaneous apoptosis in lipopolysaccharide-treated neutrophils has also been shown (
      • Frolov V.A.
      • Moiseeva E.G.
      • Pasechnik A.V.
      Pathophysiological aspects of functional modulation of human peripheral blood neutrophils with propranolol.
      ).
      Uncontrolled neutrophil activation is a pathogenic factor driving several chronic inflammatory diseases, including pemphigoid diseases (PDs) (
      • Chiriac M.T.
      • Roesler J.
      • Sindrilaru A.
      • Scharffetter-Kochanek K.
      • Zillikens D.
      • Sitaru C.
      NADPH oxidase is required for neutrophil-dependent autoantibody-induced tissue damage.
      ,
      • Ludwig R.J.
      • Vanhoorelbeke K.
      • Leypoldt F.
      • Kaya Z.
      • Bieber K.
      • McLachlan S.M.
      • et al.
      Mechanisms of autoantibody-induced pathology.
      ,
      • Soehnlein O.
      • Steffens S.
      • Hidalgo A.
      • Weber C.
      Neutrophils as protagonists and targets in chronic inflammation.
      ). PDs are a group of well-defined autoimmune disorders that are characterized by autoantibodies against structural proteins of the dermal-epidermal junction and, clinically, by tense blisters and erosions on skin or mucous membranes close to the skin surface. The most common of these diseases is bullous pemphigoid; others include mucous membrane pemphigoid and epidermolysis bullosa acquisita (EBA) (
      • Schmidt E.
      • Zillikens D.
      The diagnosis and treatment of autoimmune blistering skin disorders.
      ). EBA is a potentially life-threatening, neutrophil-driven, autoimmune skin blistering disease characterized by the presence of antibodies against COL7, a major anchor protein of the dermal-epidermal junction (
      • Schmidt E.
      • Zillikens D.
      Pemphigoid diseases.
      ,
      • Witte M.
      • Koga H.
      • Hashimoto T.
      • Ludwig R.J.
      • Bieber K.
      Discovering potential drug-targets for personalized treatment of autoimmune disorders - what we learn from epidermolysis bullosa acquisita.
      ). Binding of these antibodies induces an influx and subsequent activation of neutrophils and other immune cells into the skin. Neutrophils are the main effector cells in EBA (
      • Chiriac M.T.
      • Roesler J.
      • Sindrilaru A.
      • Scharffetter-Kochanek K.
      • Zillikens D.
      • Sitaru C.
      NADPH oxidase is required for neutrophil-dependent autoantibody-induced tissue damage.
      ,
      • Sezin T.
      • Krajewski M.
      • Wutkowski A.
      • Mousavi S.
      • Chakievska L.
      • Bieber K.
      • et al.
      The leukotriene B4 and its receptor BLT1 act as critical drivers of neutrophil recruitment in murine bullous pemphigoid-like epidermolysis bullosa acquisita.
      ). A contribution of macrophages to the effector phase in vivo was not shown so far (
      • Koga H.
      • Prost-Squarcioni C.
      • Iwata H.
      • Jonkman M.F.
      • Ludwig R.J.
      • Bieber K.
      Epidermolysis bullosa acquisita: the 2019 update.
      ). A marginal influence of T cells (regulatory T cells, NK T cells, and γδ T cells) was shown in the model of antibody-induced EBA (
      • Bieber K.
      • Sun S.
      • Witte M.
      • Kasprick A.
      • Beltsiou F.
      • Behnen M.
      • et al.
      Regulatory T cells suppress inflammation and blistering in pemphigoid diseases.
      ,
      • Bieber K.
      • Witte M.
      • Sun S.
      • Hundt J.E.
      • Kalies K.
      • Dräger S.
      • et al.
      T cells mediate autoantibody-induced cutaneous inflammation and blistering in epidermolysis bullosa acquisita.
      ). Collectively, so far, only effector mechanisms of neutrophils have been shown to directly contribute to clinical disease manifestation. This effect is mediated by the release of matrix metalloproteinases as well as ROS that cause subepidermal blistering and widespread skin inflammation (
      • Iwata H.
      • Vorobyev A.
      • Koga H.
      • Recke A.
      • Zillikens D.
      • Prost-Squarcioni C.
      • et al.
      Meta-analysis of the clinical and immunopathological characteristics and treatment outcomes in epidermolysis bullosa acquisita patients.
      ,
      • Koga H.
      • Prost-Squarcioni C.
      • Iwata H.
      • Jonkman M.F.
      • Ludwig R.J.
      • Bieber K.
      Epidermolysis bullosa acquisita: the 2019 update.
      ,
      • Schmidt E.
      • Zillikens D.
      Pemphigoid diseases.
      ,
      • Witte M.
      • Koga H.
      • Hashimoto T.
      • Ludwig R.J.
      • Bieber K.
      Discovering potential drug-targets for personalized treatment of autoimmune disorders - what we learn from epidermolysis bullosa acquisita.
      ). Corticosteroids and conventional immunosuppressants, which are associated with severe adverse events, insufficient treatment responses, and/or relapse after stopping treatment, remain the backbone of EBA and PD treatment (
      • Koga H.
      • Prost-Squarcioni C.
      • Iwata H.
      • Jonkman M.F.
      • Ludwig R.J.
      • Bieber K.
      Epidermolysis bullosa acquisita: the 2019 update.
      ,
      • Lamberts A.
      • Yale M.
      • Grando S.A.
      • Horváth B.
      • Zillikens D.
      • Jonkman M.F.
      Unmet needs in pemphigoid diseases: an international survey amongst patients, clinicians and researchers.
      ).
      Unrelated to PD, conflicting results have been reported regarding the impact of the ADRB antagonist propranolol on neutrophil functions (
      • Anderson R.
      • Ramafi G.
      • Theron A.J.
      Membrane stabilizing, anti-oxidative interactions of propranolol and dexpropranolol with neutrophils.
      ,
      • Anderson R.
      • van Rensburg A.J.
      The in vitro effects of propranolol and atenolol on neutrophil motility and post-phagocytic metabolic activity.
      ,
      • Chen Y.W.
      • Chiu C.C.
      • Wei Y.L.
      • Hung C.H.
      • Wang J.J.
      Propranolol combined with dopamine has a synergistic action in intensifying and prolonging cutaneous analgesia in rats.
      ,
      • English D.
      • Taylor G.S.
      Divergent effects of propranolol on neutrophil superoxide release: involvement of phosphatidic acid and diacylglycerol as second messengers.
      ,
      • Romana-Souza B.
      • Nascimento A.P.
      • Monte-Alto-Costa A.
      Low-dose propranolol improves cutaneous wound healing of burn-injured rats.
      ). To clarify the impact of propranolol on immune complex (IC)-induced neutrophil activation and to identify novel treatment options for PD, we systematically evaluated the impact of propranolol on neutrophil activation in vitro and in a preclinical animal model.
      To address these seemingly conflicting results, we systemically investigated the impact of propranolol on neutrophil features, including IC-induced ROS release, chemotaxis, and CD62L shedding, and we studied the transcriptomic signature of propranolol by RNA sequencing (RNA-Seq) In addition, we analyzed the effect of propranolol on clinical disease manifestation in experimental EBA, a neutrophil-dependent autoimmune skin blistering disease (
      • Koga H.
      • Prost-Squarcioni C.
      • Iwata H.
      • Jonkman M.F.
      • Ludwig R.J.
      • Bieber K.
      Epidermolysis bullosa acquisita: the 2019 update.
      ). This severe skin blistering disease is characterized by the presence of autoantibodies against the dermal-epidermal junction inducing complement activation and recruitment and activation of neutrophils (
      • Koga H.
      • Prost-Squarcioni C.
      • Iwata H.
      • Jonkman M.F.
      • Ludwig R.J.
      • Bieber K.
      Epidermolysis bullosa acquisita: the 2019 update.
      ,
      • Schmidt E.
      • Zillikens D.
      Pemphigoid diseases.
      ,
      • Witte M.
      • Koga H.
      • Hashimoto T.
      • Ludwig R.J.
      • Bieber K.
      Discovering potential drug-targets for personalized treatment of autoimmune disorders - what we learn from epidermolysis bullosa acquisita.
      ). Treatment of PDs is challenging and commonly relies on high-dose and prolonged corticosteroid therapy. The adverse effects of long-term corticosteroid use (often in combination with other potent immunosuppressive drugs) contribute to the high morbidity and mortality of patients (
      • Joly P.
      • Roujeau J.C.
      • Benichou J.
      • Picard C.
      • Dreno B.
      • Delaporte E.
      • et al.
      A comparison of oral and topical corticosteroids in patients with bullous pemphigoid.
      ). Therefore, the development of safe and effective treatments for PD is urgently needed (
      • Lamberts A.
      • Yale M.
      • Grando S.A.
      • Horváth B.
      • Zillikens D.
      • Jonkman M.F.
      Unmet needs in pemphigoid diseases: an international survey amongst patients, clinicians and researchers.
      ).

      Results

      ADRB blockade reduces neutrophil, but not lymphocyte, activation

      Although it was postulated that the sympathetic nervous system plays an important role in the brain–immune system axis, only scant information is available on the sympathoadrenergic regulation of the innate immune system (
      • Scanzano A.
      • Cosentino M.
      Adrenergic regulation of innate immunity: a review.
      ). We therefore investigated the impact of ADR inhibition on neutrophils. To exclude functions of ADR inhibitors on adaptive immune cells, we additionally performed in vitro stimulation of T cells and B cells. General inhibition of ADRAs was performed by tolazoline and inhibition of ADRBs by propranolol. Regarding neutrophils, human polymorphonuclear (PMN) leukocytes were stimulated with ICs consisting of human COL7 and anti–human COL7 IgG1 for 2 hours, and the release of ROS, as a hallmark of neutrophil activation, was measured by chemiluminescence (Figure 1a). Propranolol treatment reduced the ROS release in PMN cells by approximately 50%, whereas the ADRA inhibitor tolazoline had no effect. Next, human T cells were stimulated with anti-CD3 and anti-CD28, and proliferation was measured by BrdU incorporation. During the 3-day culture, the ADR inhibitors were added. No effect of either propranolol or tolazoline on anti-CD3– or anti-CD28–induced T-cell activation was observed (Figure 1b). Human B cells were stimulated with IL-21 and anti-CD40L for a total of 5 days in the presence or absence of propranolol or tolazoline, and BrdU incorporation was measured (Figure 1c). Tolazoline enhanced B-cell proliferation, whereas propranolol did not alter IL-21– and anti-CD40L–induced B-cell proliferation. Based on these results, we sought to analyze the effects of propranolol on PMN cells in more detail. Because of the risk of severe side effects of tolazoline treatment on B-cell stimulation in autoimmune diseases, tolazoline was excluded from further analysis (
      • Van Dijk H.
      • Rapis M.
      • Jacobse-Geels H.E.
      • Willers J.M.
      Histamine 2 receptor-mediated immunomodulation in the mouse. I. Immunomodulation by the H2 agonist tolazoline.
      ).
      Figure thumbnail gr1
      Figure 1Propranolol selectively impairs PMN but not lymphocyte activation. To analyze the effect of ADR inhibition on several immune cells, we stimulated T cells, B cells, and neutrophils in vitro. (a) Human PMN cells were stimulated with ICs, and the release of ROS was measured in the absence or presence of 10 μmol/liter propranolol or tolazoline. The average cumulative ROS release, expressed as AUC, is depicted. (b) Human T cells were stimulated with anti-CD3 and anti-CD28 in the presence of propranolol, tolazoline, or vehicle, and their proliferation was measured by BrdU incorporation. (c) Human B cells were stimulated in the presence or absence of ADR inhibitors, and BrdU incorporation was measured. All data were normalized to those of stimulated cells without any treatment. n = 3–4/group,∗∗P < 0.01. ADR, adrenoreceptor; AUC, area under the curve; IC, immune complex; PMN, polymorphonuclear.

      Propranolol reduces ROS release from PMN cells after IC activation

      We next evaluated whether propranolol has a dose-dependent effect on ROS release from IC-stimulated PMN cells. Propranolol treatment inhibited IC-induced ROS release at 10 μM, whereas lower doses had no significant effect (Figure 2a and b). An effect of high propranolol concentrations (>20 μM) on anion release was described previously by the use of phorbol myristate acetate–stimulated PMN cells (
      • Sozzani S.
      • Agwu D.E.
      • McCall C.E.
      • O'Flaherty J.T.
      • Schmitt J.D.
      • Kent J.D.
      • et al.
      Propranolol, a phosphatidate phosphohydrolase inhibitor, also inhibits protein kinase C.
      ). Our effects were not because of any toxic effects of the drug (Figure 2c and d). To determine whether propranolol affects PMN cell chemotaxis, we analyzed the effect of propranolol on CD62L (L-selectin) shedding as an additional marker of neutrophil activation (
      • Ivetic A.
      A head-to-tail view of L-selectin and its impact on neutrophil behaviour.
      ,
      • Ng L.G.
      • Ostuni R.
      • Hidalgo A.
      Heterogeneity of neutrophils.
      ,
      • Rosales C.
      Neutrophil: a cell with many roles in inflammation or several cell types?.
      ). IC stimulation of PMN cells increased the amount of shed CD62L (
      • Coxon A.
      • Cullere X.
      • Knight S.
      • Sethi S.
      • Wakelin M.W.
      • Stavrakis G.
      • et al.
      Fc gamma RIII mediates neutrophil recruitment to immune complexes. A mechanism for neutrophil accumulation in immune-mediated inflammation.
      ), but propranolol treatment did not alter CD62L shedding (Figure 2e and f). We also analyzed the effect of propranolol on chemotaxis in a Boyden chamber assay induced by several stimuli. Propranolol enhanced the chemotactic properties of PMN cells using IL-8 or N-formylmethionine-leucyl-phenylalanine as chemoattractant (Figure 2g and h). In contrast, no effect of propranolol was shown in LTB4-induced chemotaxis.
      Figure thumbnail gr2
      Figure 2Propranolol reduces IC-stimulated ROS release and increases IL-8– or fmLP-induced chemotaxis of PMN cells. Human PMN cells were stimulated with ICs in the presence or absence of propranolol. (a) ROS release was measured by chemiluminescence. As negative control, antigens or antibodies alone were used. The average cumulative ROS release (AUC) is shown. (b) Representative result of a total of five independent experiments. (c) To exclude toxicity by propranolol, the amount of PI- and Annexin V–positive cells after IC stimulation was identified. (d) Representative FACS images of Annexin V/PI staining in unstimulated (negative control), IC-stimulated, and IC-stimulated and 10 μmol/liter propranolol-treated cells. (e) The expression of CD62L-negative (activated) neutrophils is independent of propranolol. (f) Representative FACS images of CD15/CD62L staining in unstimulated, IC-stimulated, and IC-stimulated and propranolol-treated PMN cells. (g) Boyden chamber chemotaxis assay of PMN cells after stimulation with 6–12 nM IL-8, (h) 15 nM fmLP, or (i) 8 nM LTB4. Data were normalized to stimulated cells. ∗P < 0.05, ∗∗∗P < 0.001. AUC, area under the curve; fmLP, N-formylmethionine-leucyl-phenylalanine; hCOL7, human COL7; IC, immune complex; PI, propidium iodide; PMN, polymorphonuclear.
      Our data indicate that propranolol reduces IC-induced ROS release in human PMN cells but increases chemotaxis to IL-8 and N-formylmethionine-leucyl-phenylalanine. Because the half maximal inhibitory concentration of propranolol for ADRB binding is 12 nmol/liter, we assumed an off-target effect of propranolol on PMN cells. Indeed, we found that ADRB2 is expressed on PMN cells, but none of the open-source Kyoto Encyclopedia of Genes and Genomes signaling pathways (www.kegg.jp) induced by ADRB overlapped with the signaling pathways induced by propranolol in IC-induced human PMN cells (Supplementary Figure S1, Supplementary Tables S2 and S3).

      Comparison of the RNA transcriptome in human IC-stimulated PMN cells after treatment with propranolol identified more than 100 differentially regulated genes

      To unravel the transcriptomics and pathways that are involved in the effects of propranolol on IC-stimulated PMN cells, we performed RNA-Seq. For this purpose, PMN cells were stimulated for 4 hours with IC in the presence or absence of 10 μM propranolol. We identified 168 genes that are differentially expressed by propranolol treatment in IC-stimulated neutrophils. Among the genes, we found genes such as HSPA1 and TNF that are important for pathogenesis of neutrophil-dependent murine EBA (Figure 3a, Supplementary Figure S2, Supplementary Tables S1 and S2) (
      • Hirose M.
      • Kasprick A.
      • Beltsiou F.
      • Dieckhoff Schulze K.
      • Schulze F.S.
      • Samavedam U.K.
      • et al.
      Reduced skin blistering in experimental epidermolysis bullosa acquisita after anti-TNF treatment.
      ;
      • Tukaj S.
      • Hellberg L.
      • Ueck C.
      • Hänsel M.
      • Samavedam U.
      • Zillikens D.
      • et al.
      Heat shock protein 90 is required for ex vivo neutrophil-driven autoantibody-induced tissue damage in experimental epidermolysis bullosa acquisita.
      ). The involvement of differentially expressed genes in different signaling pathways revealed by Kyoto Encyclopedia of Genes and Genomes pathway analysis is shown in Figure 3b and Supplementary Figure S3; the analysis revealed that the FoxO pathway is a major pathway of propranolol action in IC-stimulated PMN cells.
      Figure thumbnail gr3
      Figure 3Propranolol reverses the IC-induced differential gene expression in human neutrophils. Human PMN cells were stimulated with ICs, and RNA-Seq was performed (n = 3/group). A total of 168 inversely differentially expressed genes were identified in the comparison between unstimulated and IC-stimulated versus IC-stimulated and IC-stimulated with propranolol treatment. (a) The top 50 of these genes are shown in the heat map, and the complete list of all differentially expressed genes is provided in . The gene name given is the official gene symbol, and the adjusted P-values are corrected for multiple testing using Bonferroni correction. (b) KEGG pathways altered by propranolol treatment in IC-stimulated PMNs. IC, immune complex; KEGG, Kyoto Encyclopedia of Genes and Genomes; PMN, polymorphonuclear; RNA-Seq, RNA sequencing.

      Treatment with propranolol diminishes the extent of skin inflammation in experimental neutrophil-dependent antibody transfer–induced EBA

      To determine whether the modulatory effects of propranolol are of potential therapeutic relevance, we next evaluated the impact of propranolol in a PMN activation-dependent murine skin inflammation model, specifically EBA. In EBA, autoantibodies bind to COL7, which is a major constituent of the anchoring fibrils. These ICs, through a series of events, attract and activate PMN cells in the skin (
      • Ludwig R.J.
      • Vanhoorelbeke K.
      • Leypoldt F.
      • Kaya Z.
      • Bieber K.
      • McLachlan S.M.
      • et al.
      Mechanisms of autoantibody-induced pathology.
      ). In this model, PMN cells are required to induce tissue damage, as their depletion completely protects mice from disease induction by the transfer of anti-COL7 IgG (
      • Chiriac M.T.
      • Roesler J.
      • Sindrilaru A.
      • Scharffetter-Kochanek K.
      • Zillikens D.
      • Sitaru C.
      NADPH oxidase is required for neutrophil-dependent autoantibody-induced tissue damage.
      ,
      • Sezin T.
      • Krajewski M.
      • Wutkowski A.
      • Mousavi S.
      • Chakievska L.
      • Bieber K.
      • et al.
      The leukotriene B4 and its receptor BLT1 act as critical drivers of neutrophil recruitment in murine bullous pemphigoid-like epidermolysis bullosa acquisita.
      ). Experimental EBA was induced by a single injection of anti–mouse COL7 IgG into the mouse ear base (Figure 4a, b, d, and e). In this model, systemic propranolol treatment dose-dependently impaired the induction of experimental EBA (Figure 4f). Injection of 100 μg normal rabbit IgG into the ear does not induce symptoms (Figure 4c). In addition, propranolol was well tolerated (Supplementary Tables S4 and S5). The therapeutic effects of propranolol were independent of IgG and C3 deposition along the dermal-epidermal junction (Figure 4a and b). Sections of comparable lesional skin are characterized in both groups by a strong infiltration of Ly6G+ cells that are positive for MPO (activated neutrophils), a key regulator of oxidative burst (
      • Khan A.A.
      • Alsahli M.A.
      • Rahmani A.H.
      Myeloperoxidase as an active disease biomarker: recent biochemical and pathological perspectives.
      ,
      • Nakazato T.
      • Sagawa M.
      • Yamato K.
      • Xian M.
      • Yamamoto T.
      • Suematsu M.
      • et al.
      Myeloperoxidase is a key regulator of oxidative stress mediated apoptosis in myeloid leukemic cells.
      ). To investigate whether the activation of neutrophils in lesional skin is different in propranolol-treated mice, we compared the mRNA expression of the neutrophil marker Ly6g (Figure 6f ), as well as the neutrophil activation markers Mpo, Mmp9, Il1b, and Elane (Figure 6g–j). All markers are significantly upregulated in lesional skin. No differences in the comparison of Ly6g were detected in lesional sections of control- and propranolol-treated mice of comparable size, but the expression of the activation marker Mpo is significantly reduced.
      Figure thumbnail gr4
      Figure 4Systemic propranolol treatment impairs clinical disease manifestation in experimental EBA. (a, b) Anti-mCOL7 IgG was injected into the ears of C57BL/6J mice, and the mice were i.p. treated with either 4 mg/kg propranolol or vehicle (PBS). (c) As control, 100 μg NR-IgG was injected in the ear; no visible clinical symptoms, infiltration of immune cells, and IgG binding and C3 deposition is visible. (a–c) Left panel: representative clinical images on day 2 after antibody injection. Middle panel: Histology. Right panel: staining with anti-rabbit IgG-FITC, anti-murine C3-Fluorescin antibodies, and double staining with anti-murine LyG (red) and anti-murine MPO. (d) The mean affected ear surface area and ear thickness were recorded after 2 days and were reduced in propranolol-treated mice. (e) The development of the affected ear surface area and the cumulative ear thickness increase was decreased by propranolol. (f) Oral treatment with propranolol shows dose-dependency. (d–f) n = 10/group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Bar = 50 μm. EBA, epidermolysis bullosa acquisita; i.p., intraperitoneally; mCOL7, mouse COL7; NR-IgG, normal rabbit IgG.
      Figure thumbnail gr5
      Figure 5Topical treatment of propranolol impairs clinical disease manifestation in experimental EBA. The ear base of C57BL/6J mice was injected with anti-mCOL7 IgG at day 0. Starting from day −1, the mice were treated daily with 1, 2, and 5% propranolol or vehicle (German Pharmaceutics Codex base cream). The mean affected ear surface area and ear thickness was recorded after 2 days and was reduced in propranolol-treated mice. (a) Representative clinical images obtained on day 2 of the experiment. The yellow circle indicate affected ear surface regions. (b) The development of the affected ear surface area and (c) the cumulative ear thickness increase (from day 0 to day 2) was decreased by propranolol treatment. (b, c) n = 10/group. ∗∗P < 0.01, ∗∗∗P < 0.001. EBA, epidermolysis bullosa acquisita; mCOL7, mouse COL7.
      Figure thumbnail gr6
      Figure 6Propranolol modulates gene expression in experimental EBA. (a) To assess whether differentially expressed genes in human PMN cells are important for the effect of propranolol on antibody-induced EBA, we searched for genes that are upregulated in murine EBA (dataset taken from
      • Samavedam U.K.
      • Mitschker N.
      • Kasprick A.
      • Bieber K.
      • Schmidt E.
      • Laskay T.
      • et al.
      Whole-genome expression profiling in skin reveals SYK as a key regulator of inflammation in experimental epidermolysis bullosa acquisita.
      ) and in parallel downregulated by propranolol in IC-stimulated human PMN cells (). We analyzed eight differentially expressed genes. The mRNA expression of these genes was analyzed in the EBA model after systemic treatment with propranolol using TaqMan gene expression assays with Gapdh as a housekeeping gene. (b) The expression of Tnf and (c) Aoah is upregulated in EBA and downregulated by propranolol. (d) The expression of Gadd45b and (e) Nfkbie is downregulated by propranolol treatment. (f) Ly6g, (g) Mpo, (h) Mmp9, (i) IL1b, and (j) Elane are upregulated in experimental EBA, but only the neutrophil activation maker Mpo is downregulated by propranolol. n = 8/group. ∗P < 0.05, ∗∗P < 0.01. EBA, epidermolysis bullosa acquisita; IC, immune complex; PMN, polymorphonuclear.
      Because high-dose propranolol may lead to hypotension (
      • Drolet B.A.
      • Frommelt P.C.
      • Chamlin S.L.
      • Haggstrom A.
      • Bauman N.M.
      • Chiu Y.E.
      • et al.
      Initiation and use of propranolol for infantile hemangioma: report of a consensus conference.
      ,
      • El Hachem M.
      • Gesualdo F.
      • Diociaiuti A.
      • Berti I.
      • Vercellino N.
      • Boccaletti V.
      • et al.
      Safety and effectiveness of oral propranolol for infantile hemangiomas started before 5 weeks and after 5 months of age: an Italian multicenter experience.
      ,
      • Marqueling A.L.
      • Oza V.
      • Frieden I.J.
      • Puttgen K.B.
      Propranolol and infantile hemangiomas four years later: a systematic review.
      ,
      • Püttgen K.B.
      Evidence and nuances of propranolol safety.
      ,
      • Sagi L.
      • Zvulunov A.
      • Lapidoth M.
      • Ben Amitai D.
      Efficacy and safety of propranolol for the treatment of infantile hemangioma: a presentation of ninety-nine cases.
      ), we next evaluated whether topical propranolol application, such as in IH, has similar effects to oral administration. For this purpose, we diluted propranolol in German Pharmaceutics Codex base cream and applied it daily to the ears of the EBA model mice. Consistent with the systemic treatment, topical propranolol was well tolerated (Supplementary Tables S4 and S5), and all tested concentrations reduced the skin inflammation induced by anti-COL7C injection (Figure 5a), as indicated by improvement of the area of affected ear skin (Figure 4b) and reduction in ear swelling (Figure 5c).

      Validation of Tnf and Aoah as EBA- and propranolol-regulated genes

      To assess whether the genes differentially expressed in IC-stimulated human PMN cells (Figure 3) are important for the effect of propranolol in murine antibody-induced EBA, we compared the expression profile of propranolol-treated human IC-stimulated PMN cells with the expression profile induced by inflammatory events in experimental EBA (induced by six 5-mg subcutaneous injections of rabbit anti-COL7C-IgG) that was recently published (
      • Samavedam U.K.
      • Mitschker N.
      • Kasprick A.
      • Bieber K.
      • Schmidt E.
      • Laskay T.
      • et al.
      Whole-genome expression profiling in skin reveals SYK as a key regulator of inflammation in experimental epidermolysis bullosa acquisita.
      ). According to previously published data in comparison with the data analyzed here, we found eight homolog genes that were differentially expressed in murine EBA and differentially expressed in IC-stimulated human PMN cells in response to propranolol treatment (Figure 6a, Supplementary Table S6). These genes were analyzed in the localized EBA model (induced by one single subcutaneous injection of 100 μg rabbit anti-COL7C-IgG into the ear base) after systemic treatment with propranolol. We found that two of these genes were also upregulated in the localized EBA model (Tnf and Aoah) and that the expression of four of the investigated genes (Tnf, Aoah, Gadd45b, and Nfkbie) was inhibited by propranolol treatment (Figure 6).

      Discussion

      The aim of the study was to investigate the impact of propranolol on IC-induced neutrophil activation and to identify novel treatment options for PD. We here systematically evaluated the impact of propranolol on neutrophil activation in vitro and in preclinical animal models of an autoantibody-mediated, neutrophil-dependent inflammatory skin disease.
      Here, we document the specific inhibitory activity of propranolol on IC-induced neutrophil ROS release in vitro. The activation of other immune cells remained unaltered despite the presence of propranolol. Hence, in vitro, propranolol selectively targets IC-induced ROS release in neutrophils, which is a key pathogenic event in PD (
      • Chiriac M.T.
      • Roesler J.
      • Sindrilaru A.
      • Scharffetter-Kochanek K.
      • Zillikens D.
      • Sitaru C.
      NADPH oxidase is required for neutrophil-dependent autoantibody-induced tissue damage.
      ). From these data, propranolol would be expected to induce few adverse events, such as infections, because it does not affect other immune cell types and other neutrophil functions. In addition, propranolol had no toxic effect on human neutrophils and did not alter the expression of CD62L, a key regulator of chemotaxis. Propranolol had no influence on LTB4-mediated chemotaxis but significantly increased IL-8–induced chemotaxis. In human PD, IL-8 is produced by keratinocytes and acts as a chemoattractant for neutrophils in vitro (
      • Schmidt E.
      • Ambach A.
      • Bastian B.
      • Bröcker E.B.
      • Zillikens D.
      Elevated levels of interleukin-8 in blister fluid of bullous pemphigoid compared with suction blisters of healthy control subjects.
      ,
      • Schmidt E.
      • Reimer S.
      • Kruse N.
      • Bröcker E.B.
      • Zillikens D.
      The IL-8 release from cultured human keratinocytes, mediated by antibodies to bullous pemphigoid autoantigen 180, is inhibited by dapsone.
      ). Despite the lack of a gene coding for IL-8, the mouse species possesses a receptor homologous to human CXCR2 that is able to mediate neutrophil chemotaxis in response to human IL-8, in addition to MIP-2 and KC (
      • Bozic C.R.
      • Gerard N.P.
      • von Uexkull-Guldenband C.
      • Kolakowski Jr., L.F.
      • Conklyn M.J.
      • Breslow R.
      • et al.
      The murine interleukin 8 type B receptor homologue and its ligands. Expression and biological characterization.
      ,
      • Rovai L.E.
      • Herschman H.R.
      • Smith J.B.
      The murine neutrophil-chemoattractant chemokines LIX, KC, and MIP-2 have distinct induction kinetics, tissue distributions, and tissue-specific sensitivities to glucocorticoid regulation in endotoxemia.
      ). In murine EBA, the IL-8 homolog CXCL1 is also involved in disease progression, as inhibition of the receptors CXCR1/2 slightly reduces EBA clinical disease manifestation (
      • Hirose M.
      • Brandolini L.
      • Zimmer D.
      • Götz J.
      • Westermann J.
      • Allegretti M.
      • et al.
      The allosteric CXCR1/2 inhibitor DF2156A improves experimental epidermolysis bullosa acquisita.
      ).
      In contrast to the murine IL-8 homologs, LTB4 acts as a key player in murine EBA, as blocking LTB4R1 completely abolished the disease and the number of invaded neutrophils in the skin (
      • Sezin T.
      • Krajewski M.
      • Wutkowski A.
      • Mousavi S.
      • Chakievska L.
      • Bieber K.
      • et al.
      The leukotriene B4 and its receptor BLT1 act as critical drivers of neutrophil recruitment in murine bullous pemphigoid-like epidermolysis bullosa acquisita.
      ). LTB4 is also abundant in the skin blister fluid of patients with bullous pemphigoid and therefore may also play a role in human PD (
      • Grando S.A.
      • Glukhenky B.T.
      • Drannik G.N.
      • Epshtein E.V.
      • Kostromin A.P.
      • Korostash T.A.
      Mediators of inflammation in blister fluids from patients with pemphigus vulgaris and bullous pemphigoid.
      ,
      • Kawana S.
      • Ueno A.
      • Nishiyama S.
      Increased levels of immunoreactive leukotriene B4 in blister fluids of bullous pemphigoid patients and effects of a selective 5-lipoxygenase inhibitor on experimental skin lesions.
      ). This idea is further supported by the efficacy of the antibiotic dapsone in the treatment of PDs (
      • Schmidt E.
      • Zillikens D.
      Pemphigoid diseases.
      ), but its mode of action has remained elusive. In vitro, dapsone inhibits LTB4 release from neutrophils as well as neutrophil migration toward LTB4 gradients (
      • Wozel G.
      • Blasum C.
      • Winter C.
      • Gerlach B.
      Dapsone hydroxylamine inhibits the LTB4-induced chemotaxis of polymorphonuclear leukocytes into human skin: results of a pilot study.
      ,
      • Wozel G.
      • Lehmann B.
      Dapsone inhibits the generation of 5-lipoxygenase products in human polymorphonuclear leukocytes.
      ). Because LTB4 seems to be a more potent stimulator of neutrophil migration during experimental EBA, the slightly chemotaxis-inducing ability of propranolol (dependent on the stimulus) in human PMN cells should not negatively affect the inhibitory properties of propranolol in experimental EBA. To further investigate whether the inhibitory function of propranolol on in vitro stimulated human PMN cells can also be reflected in murine EBA, we compared lesional skin of control- and propranolol-treated mice owing to the expression of neutrophil activation markers. The expression of the general neutrophil marker Ly6g remains unchanged in both groups, but the expression of the neutrophil activation marker Mpo is significantly reduced in propranolol-treated mice. As Mpo (
      • Khan A.A.
      • Alsahli M.A.
      • Rahmani A.H.
      Myeloperoxidase as an active disease biomarker: recent biochemical and pathological perspectives.
      ,
      • Nakazato T.
      • Sagawa M.
      • Yamato K.
      • Xian M.
      • Yamamoto T.
      • Suematsu M.
      • et al.
      Myeloperoxidase is a key regulator of oxidative stress mediated apoptosis in myeloid leukemic cells.
      ) is a key regulator in oxidative burst, these data implicate that the effect of propranolol on neutrophil activation by reduced ROS release in vitro in human cells can be of importance also in murine models of EBA. Therefore, the use of propranolol in PD may even be beneficial, because in bullous pemphigoid, the most frequent PD (
      • Hübner F.
      • Recke A.
      • Zillikens D.
      • Linder R.
      • Schmidt E.
      Prevalence and age distribution of pemphigus and pemphigoid diseases in Germany.
      ), cardiovascular morbidity and mortality is high (
      • Bech R.
      • Kibsgaard L.
      • Vestergaard C.
      Comorbidities and treatment strategies in bullous pemphigoid: an appraisal of the existing litterature.
      ). The results of systemic or topical propranolol application in experimental EBA, where profound and dose-dependent effects of propranolol on clinical disease manifestations were observed, further support the use of propranolol in PD.
      Regarding the mode of action, the inhibitory effect of propranolol on neutrophils seems to be an off-target effect. Our RNA-Seq data from IC-activated neutrophils show a low ADRB2 expression level, but the pathways involved in ADRB signaling do not overlap with the gene expression network of ADRB2. ADRB1 and ADRB3 were not detectable in human neutrophils. To evaluate the possible and known off-target effects of propranolol, we evaluated the expression of VEGF in propranolol-treated EBA mice, but the expression in both EBA and control groups was comparable (data not shown). Furthermore, the binding of propranolol to the serotonin (5-hydroxytryptamine) receptors 5HT1A and 5HT1B has also been described (
      • Pierson M.E.
      • Lyon R.A.
      • Titeler M.
      • Schulman S.B.
      • Kowalski P.
      • Glennon R.A.
      Design and synthesis of propranolol analogues as serotonergic agents.
      ), but our RNA-Seq data show that both receptors are not expressed in human neutrophils. However, on-target effects of propranolol cannot be fully excluded because of the presence of unknown gene expression networks induced by ADRB2 in neutrophils.
      To obtain insights into the molecular mechanisms of the observed inhibitory effect of propranolol on IC activation of neutrophils, we performed RNA-Seq. Here, we contrasted the mRNA expression of several hub genes, such as Tnf, Hsp70, Icam1, and Il1r, that have been shown to be important in the pathogenesis of EBA (
      • Hirose M.
      • Kasprick A.
      • Beltsiou F.
      • Dieckhoff Schulze K.
      • Schulze F.S.
      • Samavedam U.K.
      • et al.
      Reduced skin blistering in experimental epidermolysis bullosa acquisita after anti-TNF treatment.
      ,
      • Sadeghi H.
      • Gupta Y.
      • Möller S.
      • Samavedam U.K.
      • Behnen M.
      • Kasprick A.
      • et al.
      The retinoid-related orphan receptor alpha is essential for the end-stage effector phase of experimental epidermolysis bullosa acquisita.
      ,
      • Sadeghi H.
      • Lockmann A.
      • Hund A.C.
      • Samavedam U.K.S.R.L.
      • Pipi E.
      • Vafia K.
      • et al.
      Caspase-1-independent IL-1 release mediates blister formation in autoantibody-induced tissue injury through modulation of endothelial adhesion molecules.
      ,
      • Tukaj S.
      • Bieber K.
      • Kleszczyński K.
      • Witte M.
      • Cames R.
      • Kalies K.
      • et al.
      Topically applied Hsp90 Blocker 17AAG inhibits autoantibody-mediated blister-inducing cutaneous inflammation.
      ,
      • Tukaj S.
      • Hellberg L.
      • Ueck C.
      • Hänsel M.
      • Samavedam U.
      • Zillikens D.
      • et al.
      Heat shock protein 90 is required for ex vivo neutrophil-driven autoantibody-induced tissue damage in experimental epidermolysis bullosa acquisita.
      ).
      As experimental EBA manifestation strictly depends on neutrophil activation (
      • Chiriac M.T.
      • Roesler J.
      • Sindrilaru A.
      • Scharffetter-Kochanek K.
      • Zillikens D.
      • Sitaru C.
      NADPH oxidase is required for neutrophil-dependent autoantibody-induced tissue damage.
      ) and our in vitro results pointed toward an inhibitory effect of propranolol on these cells, we hypothesized that the observed inhibitory effect of propranolol is due to modulation of neutrophil functions. Based on these results, we next aimed to validate the differential expression of the identified genes in vivo in the EBA mouse model. Our data clearly showed an effect of propranolol on EBA. To confirm that this effect is dependent on the effect of propranolol on neutrophils, we confirmed by RT-PCR that propranolol inhibits the expression of Tnf and Aoah that were upregulated in the antibody transfer–induced EBA model. Consistent with these findings, TNF has been shown to be relevant for disease progression in EBA (
      • Hirose M.
      • Kasprick A.
      • Beltsiou F.
      • Dieckhoff Schulze K.
      • Schulze F.S.
      • Samavedam U.K.
      • et al.
      Reduced skin blistering in experimental epidermolysis bullosa acquisita after anti-TNF treatment.
      ).
      In a relevant preclinical model system of an autoantibody-induced, neutrophil-dependent disease, we show an inhibitory effect of both topical and systemic propranolol treatment. RNA-Seq data indicate that the effects are independent of ADRBs. Based on the RNA-Seq data and subsequent validation, we conclude that the anti-inflammatory effects of propranolol are mediated by the modulation of TNF and the inhibition of ROS release in neutrophils. Our observation could also have implications for the treatment of other (partially) neutrophil-dependent diseases like allergic skin inflammation, psoriasis, sweet disease, and anaphylactoid purpura (
      • Akiyama M.
      • Takeichi T.
      • McGrath J.A.
      • Sugiura K.
      Autoinflammatory keratinization diseases: an emerging concept encompassing various inflammatory keratinization disorders of the skin.
      ,
      • Gurung P.
      • Kanneganti T.D.
      Autoinflammatory skin disorders: the inflammasomme in focus.
      ,
      • Karp N.A.
      • Mason J.
      • Beaudet A.L.
      • Benjamini Y.
      • Bower L.
      • Braun R.E.
      • et al.
      Prevalence of sexual dimorphism in mammalian phenotypic traits.
      ,
      • Navarini A.A.
      • Satoh T.K.
      • French L.E.
      Neutrophilic dermatoses and autoinflammatory diseases with skin involvement--innate immune disorders.
      ).

      Materials and Methods

      A more detailed description of the Materials and Methods section can be found in the Supplementary Material.

      Experiments with human biomaterials

      For isolation of PMN cells and peripheral blood mononuclear cells, normal human blood was obtained. Healthy controls gave their written informed consent before study participation. All of the experiments using human samples were approved by the local ethics committee (University of Lübeck, Lübeck, Germany) and were performed in accordance with the Declaration of Helsinki.

      Animal experimentation

      C57BL/6J mice (Charles River, Sulzfeld, Germany) were bred in a specific pathogen–free environment and provided standard mouse chow and acidified drinking water ad libitum. Sex-matched mice were used for experimental EBA models at the age of 8 to 10 weeks. Animal experiments were approved by local authorities of the Animal Care and Use Committee (Kiel, Germany) and performed by certified personnel (AZ122.5 [108/08-15]) following the ARIVE guidelines.

      B- and T-cell culture and stimulation

      For the analysis of proliferation in B- and T-cell cultures, peripheral blood mononuclear cells were isolated by Ficoll Paque Plus (GE Healthcare, Chicago, IL) gradient following the manufacturer's instructions and further isolated using MACS Pan T or B cell isolation kits, respectively (Miltenyi Biotec, Bergisch Gladbach, Germany). B cells (2 × 104 cells/200 μl) were stimulated in 96-well plates with 1 μg/ml anti–human CD40 (clone 82111, R&D systems, Minneapolis, MN) and 100 ng/ml IL-21 (Cell Sciences, Newburyport, MA) for 5 days. T cells (5 × 104 cells/200 μl) were stimulated with 1 μg/ml anti–human CD3 (clone UCHT1, BioLegend, San Diego, CA) and anti–human CD28 (clone CD28.2, BioLegend) for 3 days. Cell proliferation was quantified by a BrdU incorporation assay (Roche, Basel, Switzerland) according to the manufacturer’s instructions.

      ROS release assay

      Human PMN leukocytes were isolated from whole blood samples using a PolymorphPrep (Progen, Heidelberg, Germany) gradient according to the manufacturer's instructions. ROS production was stimulated with ICs consisting of 2.5 μg/ml human COL7E-F antigen and 1.8 μg/ml anti–human COL7 IgG1 antibody as described previously (
      • Recke A.
      • Trog L.M.
      • Pas H.H.
      • Vorobyev A.
      • Abadpour A.
      • Jonkman M.F.
      • et al.
      Recombinant human IgA1 and IgA2 autoantibodies to type VII collagen induce subepidermal blistering ex vivo.
      ). The chemiluminescence resulting from the ROS production was measured by a luminol-based reaction as described in detail elsewhere (
      • Recke A.
      • Trog L.M.
      • Pas H.H.
      • Vorobyev A.
      • Abadpour A.
      • Jonkman M.F.
      • et al.
      Recombinant human IgA1 and IgA2 autoantibodies to type VII collagen induce subepidermal blistering ex vivo.
      ).

      Chemotaxis assay

      PMN cells were isolated from normal human blood using Ficoll (
      • Skoog W.A.
      • Beck W.S.
      Studies on the fibrinogen, dextran and phytohemagglutinin methods of isolating leukocytes.
      ). Chemotaxis was measured using a 48-well Boyden chamber (NeuroProbe Inc, Cabin John, MD) as described previously (
      • Ludwig A.
      • Schiemann F.
      • Mentlein R.
      • Lindner B.
      • Brandt E.
      Dipeptidyl peptidase IV (CD26) on T cells cleaves the CXC chemokine CXCL11 (I-TAC) and abolishes the stimulating but not the desensitizing potential of the chemokine.
      ).

      Flow cytometry

      For a complete list of antibodies used for FACS analysis of CD62L shedding or toxicity on IC-stimulated peripheral blood mononuclear cells, see Supplementary Table S8. The CD15posCD45pospropidium iodidenegAnnexin VnegLy6GposCD62Lneg cells are CD62L negative (defined as activated PMN cells). CD45+ and CD15+ gates were further analyzed for double-positive staining of propidium iodide and Annexin V for the measurement of toxicity and survival (CD15posCD45pospropidium iodidenegAnnexin Vneg cells). Measurements were performed on a Miltenyi MacsQuant10, and data were analyzed with the MACSQuantify Software (Version 2.11).

      RNA-Seq and bioinformatics analysis

      To identify gene expression differences in the neutrophil samples after 4-hour IC stimulation in the presence or absence of 10 μmol/liter propranolol, total RNA was extracted by a Qiagen RNeasy Mini Kit, and RNA-Seq was performed with NextSeq 500/550 v2 kits (supplied by Illumina, San Diego, CA) on a NextSeq 550 (Illumina). The data were compared with those of the human reference genome using the RNA-Seq aligner tool STAR (
      • Dobin A.
      • Davis C.A.
      • Schlesinger F.
      • Drenkow J.
      • Zaleski C.
      • Jha S.
      • et al.
      STAR: ultrafast universal RNA-seq aligner.
      ).

      Real-time PCR

      Real-time PCR analysis of lesional (n = 8) and nonlesional (n = 8) skin from ear skin sections was performed as previously described (
      • Bieber K.
      • Sun S.
      • Witte M.
      • Kasprick A.
      • Beltsiou F.
      • Behnen M.
      • et al.
      Regulatory T cells suppress inflammation and blistering in pemphigoid diseases.
      ). The analyzed TaqMan assays (Supplementary Table S7) were selected because these genes were differentially expressed in murine EBA and in response to propranolol treatment of human IC-stimulated PMN cells.

      Induction of antibody transfer–induced experimental EBA

      Specific anti–mouse COL7C IgG from immune serum was isolated as previously described (
      • Bieber K.
      • Koga H.
      • Nishie W.
      In vitro and in vivo models to investigate the pathomechanisms and novel treatments for pemphigoid diseases.
      ,
      • Bieber K.
      • Witte M.
      • Sun S.
      • Hundt J.E.
      • Kalies K.
      • Dräger S.
      • et al.
      T cells mediate autoantibody-induced cutaneous inflammation and blistering in epidermolysis bullosa acquisita.
      ,
      • Kasprick A.
      • Bieber K.
      • Ludwig R.J.
      Drug discovery for pemphigoid diseases.
      ,
      • Sitaru C.
      • Mihai S.
      • Otto C.
      • Chiriac M.T.
      • Hausser I.
      • Dotterweich B.
      • et al.
      Induction of dermal-epidermal separation in mice by passive transfer of antibodies specific to type VII collagen.
      ). Mice were injected in the ear base once with 30–100 μg rabbit anti–mouse COL7 IgG. Propranolol was administered intraperitoneally or topically onto the ear one day before the initial anti–mouse COL7C IgG injection and was performed daily.

      Histopathology and direct immunofluorescence staining

      Biopsies of lesional and perilesional skin were obtained on day 2 of the antibody transfer–induced EBA and prepared for examination by histopathology and immunofluorescence microscopy, as described previously (
      • Bieber K.
      • Sun S.
      • Witte M.
      • Kasprick A.
      • Beltsiou F.
      • Behnen M.
      • et al.
      Regulatory T cells suppress inflammation and blistering in pemphigoid diseases.
      ,
      • Kovacs B.
      • Tillmann J.
      • Freund L.C.
      • Nimmerjahn F.
      • Sadik C.D.
      • Bieber K.
      • et al.
      Fcγ receptor IIB controls skin inflammation in an active model of epidermolysis bullosa acquisita.
      ;
      • Sitaru C.
      • Mihai S.
      • Otto C.
      • Chiriac M.T.
      • Hausser I.
      • Dotterweich B.
      • et al.
      Induction of dermal-epidermal separation in mice by passive transfer of antibodies specific to type VII collagen.
      ).

      Statistical analysis

      Unless otherwise noted, data are presented as mean ± SD or Tukey’s box-and-whisker plots or presented as medians (black line), 25th and 75th percentiles (boxes), and maximum and minimum values (error bars); the dots represent actual results for each sample. For comparisons of two groups, a t-test or Mann-Whitney rank sum test was used whenever appropriate. For comparisons of more than two groups, ANOVA was used. For equally distributed data, one-way ANOVA followed by a Bonferroni t-test for multiple comparisons was used; if the data were nonparametric, ANOVA on ranks (Kruskal-Wallis) was applied, followed by a Bonferroni t-test for multiple comparisons. In all tests, P < 0.05 was considered indicative of significance. All statistical analyses were performed using SigmaPlot 13.0 (Systat Software, Erkrath, Germany).

      Conflict of Interest

      During the last three years, RJL has received research funding from Miltenyi Biotec, Biogen, Biotest, Almirall, True North Therapeutics, UCB Pharma, ArgenX, TxCell, Topadur, Incyte, and Admirx and fees for consulting or speaking from ArgenX, Immunogenetics, Novartis, and Lilly. All other authors state no conflict of interest.

      Acknowledgments

      This study was supported by the following grants: Excellence Cluster “Inflammation at Interfaces” (DFG EXC306/2), the Research Training Group “Modulation of Autoimmunity” (GRK 1727/1-2), and the Clinical Research Unit “Pemphigoid Diseases” ( KFO 303/1-2, projects: LU 877/11-1, BI 1820/2-2), all from the Deutsche Forschungsgemeinschaft . The authors thank Claudia Kauderer, Astrid Fischer, and Petra Lau for their excellent technical assistance.

      Author Contributions

      Conceptualization: RJL, KB; Data Curation: YG; Funding Acquisition: RJL; Investigation: PS, KSD, GK, WV, RV, SG, KM, MK, VH, FP; Methodology: RJL, KB; Project Administration: RJL; Resources: RJL, SK, RV, KK; Supervision: RJL, KB; Writing - Original Draft Preparation: RJL, KB

      Supplementary Material

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      Linked Article

      • Propranolol Off-Target: A New Therapeutic Option in Neutrophil-Dependent Dermatoses?
        Journal of Investigative DermatologyVol. 140Issue 12
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          Epidermolysis bullosa acquisita (EBA) is a rare subepidermal blistering dermatosis characterized by autoantibodies targeting collagen VII (COL7), an essential component of the anchoring fibrils, located in the sublamina densa of the dermal‒epidermal junction. In EBA, tissue-bound autoantibodies cause the recruitment and subsequent activation of neutrophils, which eventually lead to subepidermal blistering through the release of proteases and ROS. Thus, targeting either pathogenic IgG autoantibodies or neutrophil recruitment or activation has shown efficacy in experimental murine EBA models and patients with EBA.
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