ABSTRACT Mast cells (MCs) are tissue-resident sentinels of the innate immune system that play pivotal roles in host defense and inflammation. Perivascular MCs exert a particularly strong influence on the onset and dynamics of inflammation through the rapid, directional release of proinflammatory mediators into the circulation. Yet, the mechanisms governing their attachment to the vessel wall – a prerequisite for intravascular degranulation – remain poorly defined. Using a conditional knockout of integrin β1 (Itgb1) in MCs, we investigated how perivascular positioning, degranulation, and vasoactive function contribute to inflammatory responses. In vivo imaging revealed that Itgb1 is essential for positioning MCs within the perivascular niche, particularly around arterioles. The absence of Itgb1 markedly reduced directional MC degranulation into blood vessels during skin inflammation. In vitro , Itgb1-deficient MCs displayed impaired degranulation kinetics together with altered SHIP1/PI3K-AKT signaling and calcium influx upon P2X7 ligation by ATP. During contact hypersensitivity, mice lacking Itgb1 in MCs exhibited strongly diminished ear swelling and reduced recruitment of multiple leukocyte subsets. Mechanistically, disordered MC positioning and attenuated degranulation impaired endothelial activation, resulting in decreased leukocyte adhesion and extravasation. These findings uncover a dual role for Itgb1 in regulating MC responsiveness and pro-inflammatory vasoactive function, establishing Itgb1-mediated perivascular MC positioning as a key prerequisite for effective leukocyte recruitment. Abstract Figure

The prevalence of allergic diseases, including asthma, continues to rise in industrialized societies, yet the mechanisms sustaining pathogenic T helper 2 (Th2) responses remain incompletely understood. Here, we show that patients with allergic asthma exhibit elevated lipophilic volatile organic compounds in exhaled air and altered fatty acid–metabolism gene expression in sputum-derived Th2 cells. Using a mouse model of house dust mite–induced allergic airway inflammation, we find that the lipid transporter CD36 is dispensable for T follicular helper and germinal center B cell responses but is critical for maintaining lung-resident memory Th2 cells. CD36 regulates GATA3 and PPARγ expression in lung-resident memory Th2 cells and their interaction with type-2 conventional dendritic cells during airway inflammation. In human T cells, pharmacological inhibition of CD36 does not impair initial activation but blocks terminal Th2 differentiation. These findings identify CD36 as a metabolic checkpoint that sustains Th2 effector function and tissue residency, and establish lipid metabolism as a yet unrecognized therapeutic target in allergic asthma. Summary Allergic asthma is marked by rising prevalence yet the drivers of persistent T helper 2 (Th2) immunity remain unclear. We show that asthma patients exhibit altered fatty acid–metabolism signatures in sputum Th2 cells and elevated lipophilic volatile organic compounds in exhaled air. In a mouse model of house dust mite–induced airway inflammation, the lipid transporter CD36 was dispensable for germinal center responses but essential for lung-resident memory Th2 cells, controlling GATA3 and PPARγ expression and promoting cDC2 interactions. Pharmacological inhibition of CD36 in human T cells preserved activation but blocked terminal Th2 differentiation. These findings identify CD36 as a metabolic checkpoint that sustains Th2 effector function and tissue residency, and nominate it as a therapeutic target in allergic asthma. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes

BACKGROUND:Macrophages exhibit high heterogeneity and plasticity, which is essential for their multifaceted roles in host defense and tissue regeneration. Mast cells (MCs) respond rapidly to injury or infection by releasing intact secretory granules, thereby initiating and potentiating innate and adaptive immunity. OBJECTIVE:Because MCs reside in close proximity to macrophages in the skin, we decoded the impact of exocytosed MC granules (MCGs) on macrophage phenotype and function. METHODS:We tracked the fate of MCGs and studied their specific effects on macrophage plasticity and functional characteristics in vivo and in vitro in mouse and human samples using advanced imaging, functional assays, and transcriptomic profiling. RESULTS:We found that intact MCGs are engulfed by macrophages in vivo in murine models and in vitro. MCG ingestion boosted macrophage functional capacities and resulted in an atypical plasticity that contains both alternatively activated and classically activated macrophage features, suggesting increased efficiency in their multifaceted roles. In addition, the engulfment of intact MCGs by macrophages led to a specific transcriptome reprogramming. Importantly, both the process of MCG ingestion and its functional impact was confirmed with human MCs and macrophages in vitro and in situ in healthy human skin explants and in psoriatic patient lesional skin. CONCLUSION:MCs enhance the function of macrophages and drive them to atypical polarization through granule-mediated intercellular communication. Our results suggest that beyond acute inflammation, MCs act as important controllers of host defense and integrity, supporting their emerging relevance as therapeutic targets.

Abstract Background Neutrophils play a crucial role in inflammation and in the increased thrombotic risk in myeloproliferative neoplasms (MPNs). We have investigated how neutrophil-specific expression of JAK2-V617F or CALRdel re-programs the functions of neutrophils. Methods Ly6G-Cre JAK2-V617F and Ly6G-Cre CALRdel mice were generated. MPN parameters as blood counts, splenomegaly and bone marrow histology were compared to wild-type mice. Megakaryocyte differentiation was investigated using lineage-negative bone marrow cells upon in vitro incubation with TPO/IL-1β. Cytokine concentrations in serum of mice were determined by Mouse Cytokine Array. IL-1α expression in various hematopoietic cell populations was determined by intracellular FACS analysis. RNA-seq to analyse gene expression of inflammatory cytokines was performed in isolated neutrophils from JAK2-V617F and CALR-mutated mice and patients. Bioenergetics of neutrophils were recorded on a Seahorse extracellular flux analyzer. Cell motility of neutrophils was monitored in vitro (time lapse microscopy), and in vivo (two-photon microscopy) upon creating an inflammatory environment. Cell adhesion to integrins, E-selectin and P-selection was investigated in-vitro. Statistical analysis was carried out using GraphPad Prism. Data are shown as mean ± SEM. Unpaired, two-tailed t-tests were applied. Results Strikingly, neutrophil-specific expression of JAK2-V617F, but not CALRdel, was sufficient to induce pro-inflammatory cytokines including IL-1 in serum of mice. RNA-seq analysis in neutrophils from JAK2-V617F mice and patients revealed a distinct inflammatory chemokine signature which was not expressed in CALR-mutant neutrophils. In addition, IL-1 response genes were significantly enriched in neutrophils of JAK2-V617F patients as compared to CALR-mutant patients. Thus, JAK2-V617F positive neutrophils, but not CALR-mutant neutrophils, are pathogenic drivers of inflammation in MPN. In line with this, expression of JAK2-V617F or CALRdel elicited a significant difference in the metabolic phenotype of neutrophils, suggesting a stronger inflammatory activity of JAK2-V617F cells. Furthermore, JAK2-V617F, but not CALRdel, induced a VLA4 integrin-mediated adhesive phenotype in neutrophils. This resulted in reduced neutrophil migration in vitro and in an inflamed vessel. This mechanism may contribute to the increased thrombotic risk of JAK2-V617F patients compared to CALR-mutant individuals. Conclusions Taken together, our findings highlight genotype-specific differences in MPN-neutrophils that have implications for the differential pathophysiology of JAK2-V617F versus CALR-mutant disease.

Tissue resident mast cells (MCs) rapidly initiate neutrophil infiltration upon inflammatory insult, yet the molecular mechanism is still unknown. Here, we demonstrated that MC-derived tumor necrosis factor (TNF) was crucial for neutrophil extravasation to sites of contact hypersensitivity-induced skin inflammation by promoting intraluminal crawling. MC-derived TNF directly primed circulating neutrophils via TNF receptor-1 (TNFR1) while being dispensable for endothelial cell activation. The MC-derived TNF was infused into the bloodstream by directional degranulation of perivascular MCs that were part of the vascular unit with access to the vessel lumen. Consistently, intravenous administration of MC granules boosted neutrophil extravasation. Pronounced and rapid intravascular MC degranulation was also observed upon IgE crosslinking or LPs challenge indicating a universal MC potential. Consequently, the directional MC degranulation of pro-inflammatory mediators into the bloodstream may represent an important target for therapeutic approaches aimed at dampening cytokine storm syndromes or shock symptoms, or intentionally pushing immune defense.

Although mast cells (MCs) are well known as key effector cells of IgE-mediated type I allergy, there is growing evidence that MCs are also essential players in innate and adaptive immunity, and in many inflammatory or autoimmune diseases.1Dudeck A. Köberle M. Goldmann O. Meyer N. Dudeck J. Lemmens S. et al.Mast cells as protectors of health.J Allergy Clin Immunol. 2019; 144: S4-S18Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar IgE crosslinking, but also IgE-independent mechanisms of MC activation, result in degranulation of MC secretory granules containing preformed proinflammatory mediators including histamine, cytokines, and proteases. By studying hapten-induced contact hypersensitivity (CHS) responses in novel transgenic mouse lines of MC deficiency or conditional gene knockout, we have previously demonstrated that MCs are of critical impact in the pathomechanism of allergic contact dermatitis. More specifically, we have shown that MCs enhance the migration of dendritic cells (DCs) to draining lymph nodes upon hapten sensitization, thereby promoting effector T-cell expansion.2Dudeck A. Dudeck J. Scholten J. Petzold A. Surianarayanan S. Kohler A. et al.Mast cells are key promoters of contact allergy that mediate the adjuvant effects of haptens.Immunity. 2011; 34: 973-984Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar,3Dudeck J. Ghouse S.M. Lehmann C.H. Hoppe A. Schubert N. Nedospasov S.A. et al.Mast-cell-derived TNF amplifies CD8(+) dendritic cell functionality and CD8(+) T cell priming.Cell Rep. 2015; 13: 399-411Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar Upon challenge (second contact with the same hapten), MCs are required for efficient skin homing of T cells and thereby facilitate the T-cell–driven skin inflammation. In our previous study, longitudinal intravital imaging of the immune events after hapten contact had surprisingly revealed an immediate MC-triggered vascular response, happening irrespectively of previous sensitization and being crucial for subsequent T-cell–driven skin inflammation.2Dudeck A. Dudeck J. Scholten J. Petzold A. Surianarayanan S. Kohler A. et al.Mast cells are key promoters of contact allergy that mediate the adjuvant effects of haptens.Immunity. 2011; 34: 973-984Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar However, this IgE-independent mode of MC activation during allergic contact dermatitis is not yet clarified. In this study, we demonstrate that epicutaneous 2,4-dinitrofluorobenzene (DNFB) administration caused a pronounced vasodilatation of dermal blood vessels within the very first minutes, which was followed by a massive leakage of the intravascular dye due to increased vessel permeability (Fig 1, A). The edema formation could be measured as an early ear swelling response peaking at 2 hours post-DNFB (Fig 1, B) and was almost completely abolished in MC-depleted mice (Fig 1, A2). In sensitized mice, the early ear swelling response was followed by a second T-cell–driven edema at 24 hours after DNFB. However, the amplitude and kinetics of the early ear swelling was similar in nonsensitized mice (in contrast to the 24-hour ear swelling), indicating its innate nature. The hapten-induced innate response further comprised an MC-driven neutrophil infiltration. The early ear swelling response was impeded by treatment with the MC silencer cromolyn or with pyrilamine, a histamine receptor antagonist, before DNFB administration, suggesting a mechanism dependent on MC degranulation and histamine release (Fig 1, C). In contrast, the adaptive skin inflammation (at 24 hours post-DNFB) was significantly reduced in cromolyn-treated mice compared with pyrilamine-treated mice, indicating additional granule-embedded MC mediators beside histamine (Fig 1, C). Given the fast MC degranulation within only few minutes post-DNFB, we questioned the underlying mode of MC activation. Previous reports demonstrated that the absence of TLR signaling in DCs prevented DC-mediated sensitization, effector T-cell expansion, and subsequent CHS responses.4Martin S.F. Dudda J.C. Bachtanian E. Lembo A. Liller S. Dürr C. et al.Toll-like receptor and IL-12 signaling control susceptibility to contact hypersensitivity.J Exp Med. 2008; 205: 2151-2162Crossref PubMed Scopus (184) Google Scholar Therefore, we studied the innate CHS response to DNFB in TLR2/4−/− mice and found the MC-driven early vascular response to not be affected by concomitant absence of TLR2 and TLR4; although it confirmed previous findings, that the adaptive skin inflammation at 24 hours was markedly reduced (see Fig E1 in this article’s Online Repository at www.jacionline.org). Previous reports demonstrated that epicutaneous hapten treatment results in a rapid release of extracellular ATP by keratinocytes that is subsequently sensed by DCs via the purinergic receptor P2X7.5Weber F.C. Esser P.R. Müller T. Ganesan J. Pellegatti P. Simon M.M. et al.Lack of the purinergic receptor P2X(7) results in resistance to contact hypersensitivity.J Exp Med. 2010; 207: 2609-2619Crossref PubMed Scopus (167) Google Scholar In addition, IL-33, an alarmin of the IL-1 family being released upon cell stress or necrotic cell death, has been shown to have an impact on DNFB-induced CHS.6Imai Y. Yasuda K. Sakaguchi Y. Futatsugi-Yumikura S. Yoshimoto T. Nakanishi K. et al.Immediate-type contact hypersensitivity is reduced in interleukin-33 knockout mice.J Dermatol Sci. 2014; 74: 159-161Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar Questioning whether danger signaling might be involved in the rapid hapten-induced MC activation, we found a prominent surface expression of the ATP receptor P2X7 as well as the IL-33 receptor ST2 on a fraction of about 40% and 65%, respectively, of mouse ear skin MCs under healthy conditions (Fig 1, D). Given that expression, we assessed whether antagonizing the P2X7 ligand binding or neutralizing IL-33 affects the amplitude of the MC-mediated early ear swelling response and neutrophil infiltration. We could show that inhibiting the ATP/P2X7 signaling axis using the antagonist KN62 markedly reduced the innate ear swelling while not significantly affecting the adaptive response at 24 hours and neutrophil infiltration (Fig 1, E and F). In contrast, neutralizing IL-33 signaling by either an anti–IL-33 antibody or a soluble IL-33 receptor not only dramatically reduced the innate ear swelling but also significantly prevented the adaptive inflammation and neutrophil recruitment to the challenged skin (Fig 1, G and H). These data could be confirmed using P2X7−/− mice showing a reduced innate and adaptive edema formation upon hapten treatment (Fig 1, I). To discern P2X7 function in MCs, we used Mcpt5-Cre P2X7FL/FL mice, which lack P2X7, specifically in connective tissue–type MCs. Importantly, Mcpt5-Cre+P2X7FL/FL (referred to as MCΔP2X7) showed a marked reduction in innate and adaptive ear swelling compared with Cre−P2X7FL/FL littermates, but neutrophil infiltration to challenged ear skin was not affected (Fig 1, J and K). These findings suggest that MCs sense extracellular ATP released upon DNFB-induced cell stress or damage by secreting the vasoactive compound histamine. Because, in CHS, MCs trigger both the immediate vascular response and neutrophil infiltration,2Dudeck A. Dudeck J. Scholten J. Petzold A. Surianarayanan S. Kohler A. et al.Mast cells are key promoters of contact allergy that mediate the adjuvant effects of haptens.Immunity. 2011; 34: 973-984Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar we speculated that IL-33 signaling might play an additional role in cell stress–related MC activation. Hence, we studied CHS in ST2−/− mice lacking the IL-33 receptor, and detected a diminished innate and adaptive ear swelling (Fig 1, L). Specific IL-33 sensing by MCs via ST2 was proven using MC-deficient Mcpt5-Cre+R-DTA mice, which were reconstituted, locally in the ear skin, with in vitro expanded peritoneal MCs from either ST2−/− mice (referred to as ΔMC + MCΔST2) or wild-type (wt) mice (ΔMC + MCwt) (Fig 1, M). ΔMC + MCΔST2 mice showed decreased innate and adaptive ear swelling post-DNFB, as well as a significant reduction in neutrophil numbers infiltrating the challenged ear skin, compared with ΔMC + MCwt mice (Fig 1, N). Translation of extracellular ATP sensing by MCs into histamine-driven early vascular responses was confirmed by intradermal injection of ATP into P2X7−/− mice (Fig 2, A and B), MCΔP2X7 mice (Fig 2, C and D), and MC-deficient Mcpt5-Cre R-DTA (ΔMC) mice (Fig 2, E and F). Intradermal injection of ATP into mouse ear pinnae resulted in a short-term, self-resolving ear swelling, peaking at 1 hour postinjection, whereas a late ear swelling and neutrophil infiltration at 24 hours was not significantly induced. Importantly, the early ear swelling response was significantly reduced in MCΔP2X7 mice, but completely abolished in ΔMC mice, suggesting additional ATP sensing via other receptors (Fig 2, C and E). In contrast, intradermal injection of IL-33 resulted not only in a prominent early ear swelling, peaking at 2 hours postinjection, but also in pronounced skin inflammation at 24 hours, accompanied by massive neutrophil infiltration (Fig 2, G and H). All these events were significantly reduced in ST2−/− mice and hence, directly induced by IL-33/ST2 binding. Confirming the in vivo findings, we found that MC stimulation with ATP in vitro resulted in prominent degranulation and histamine release but not in TNF production (Fig 2, I-K), whereas in contrast, IL-33 has been demonstrated to induce TNF production in vitro and MC degranulation in vivo but not in vitro.7Drube S. Kraft F. Dudeck J. Müller A.L. Weber F. Göpfert C. et al.MK2/3 are pivotal for IL-33-induced and mast cell-dependent leukocyte recruitment and the resulting skin inflammation.J Immunol. 2016; 197: 3662-3668Crossref PubMed Scopus (35) Google Scholar Our data indicate that IL-33 signaling by MCs and amplification of stress responses by MC degranulation may be worth studying in more detail in the context of human inflammatory skin diseases. The observed mechanisms should have an impact on therapeutic decisions in cases of contact sensitivity which may be difficult to manage. Here, for instance, a local treatment with antihistamines and MC stabilizers as cromolyn glycates, or even local neutralization of IL-33 or TNF, might be a conceivable strategy. Indeed, the IL-33/ST2 signaling axis has been suggested to contribute to the pathomechanism of atopic dermatitis and currently, first clinical trials targeting IL-33 as a novel therapeutic strategy yield promising results.8Savinko T. Matikainen S. Saarialho-Kere U. Lehto M. Wang G. Lehtimäki S. et al.IL-33 and ST2 in atopic dermatitis: expression profiles and modulation by triggering factors.J Invest Dermatol. 2012; 132: 1392-1400Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar,9Renert-Yuval Y. Guttman-Yassky E. New treatments for atopic dermatitis targeting beyond IL-4/IL-13 cytokines.Ann Allergy Asthma Immunol. 2020; 124: 28-35Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar Collectively, we herein demonstrate that, upon hapten encounter, MCs rapidly initiate innate vascular responses including vasodilatation, vessel permeabilization, and neutrophil influx by prompt degranulation. This MC activation in the context of hapten-induced CHS is a consequence of sensing cell stress or damage by concomitant signaling via the IL-33/ST2 and ATP/P2X7 axis. Although ATP sensing by MCs is sufficient for inducing the histamine-triggered edema formation, additional IL-33 sensing is required for efficient induction of neutrophil infiltration. We cordially thank A. Roers (Dresden, Germany) for providing the Mcpt5-Cre mouse line and for his support for this project, S. Martin (Freiburg, Germany) for the TLR2/4−/− line, M. Idzko (Freiburg, Germany) for providing P2X7−/− and P2X7FL/FL mice, and D. Voehringer (Erlangen, Germany) for the R-DTA line for breedings. Expert technical assistance by C. Haase, T. Häring, and A. Karutz is gratefully acknowledged. All animal experiments were conducted in accordance with German guidelines and were approved by the relevant local authorities in Magdeburg and Dresden. Mcpt5-Cre miceE1Scholten J. Hartmann K. Gerbaulet A. Krieg T. Müller W. Testa G. et al.Mast cell-specific Cre/loxP-mediated recombination in vivo.Transgenic Res. 2008; 17: 307-315Crossref PubMed Scopus (152) Google Scholar were provided by A. Roers (Dresden, Germany), R-DTA miceE2Voehringer D. Liang H.E. Locksley R.M. Homeostasis and effector function of lymphopenia-induced “memory-like” T cells in constitutively T cell-depleted mice.J Immunol. 2008; 180: 4742-4753Crossref PubMed Scopus (168) Google Scholar by D. Voehringer (Erlangen, Germany), TLR2/4−/− miceE3Martin S.F. Dudda J.C. Bachtanian E. Lembo A. Liller S. Dürr C. et al.Toll-like receptor and IL-12 signaling control susceptibility to contact hypersensitivity.J Exp Med. 2008; 205: 2151-2162Crossref PubMed Scopus (185) Google Scholar by S. Martin (Freiburg, Germany), and P2X7−/− and P2X7FL/FL miceE4Stachon P. Heidenreich A. Merz J. Hilgendorf I. Wolf D. Willecke F. et al.P2X7 deficiency blocks lesional inflammasome activity and ameliorates atherosclerosis in mice.Circulation. 2017; 135: 2524-2533Crossref PubMed Scopus (57) Google Scholar by M. Idzko (Freiburg, Germany). C57BL/6 wt mice were purchased from Janvier (Le Genest Saint Isle, France). In all experiments, mice were housed under specific pathogen-free conditions and were used at the age of 8 to 16 weeks. Littermates were used as controls. DNFB, ATP, hyaluronidase, KN-62, A23187, IL-33, and propidium iodide were purchased from Sigma Aldrich (St Louis, Mo). DNAse and Liberase were obtained from Roche (Basel, Switzerland). The goat polyclonal IL-33 blocking antibody (AF3626) was purchased from R&D Systems (Wiesbaden, Germany).E5Bruhs A. Proksch E. Schwarz T. Schwarz A. Disruption of the epidermal barrier induces regulatory T cells via IL-33 in mice.J Investig Dermatol. 2018; 138: 570-579Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar mAbs for flow cytometry analysis directed against the following marker proteins were purchased from eBioscience (ThermoFisher Scientific, Waltham, Mass): CD45 (30-F11), F4/80 (Bm8), CD11b (M1/70), CD117 (2B8), and FcεRIα (MAR-1). Antibodies directed against Ly6G, P2X7R (1F11), and IL33R (DIH4) were purchased from BioLegend (San Diego, Calif). Recombinant Mouse ST2/IL-33R Fc Chimera Protein was obtained from R&D Systems. Longitudinal and noninvasive intravital 2-photon microscopy of mouse ear skin was performed as previously described.E6Dudeck A. Dudeck J. Scholten J. Petzold A. Surianarayanan S. Köhler A. et al.Mast cells are key promoters of contact allergy that mediate the adjuvant effects of haptens.Immunity. 2011; 34: 973-984Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, E7Dudeck J. Froebel J. Kotrba J. Lehmann C.H.K. Dudziak D. Speier S. et al.Engulfment of mast cell secretory granules on skin inflammation boosts dendritic cell migration and priming efficiency.J Allergy Clin Immunol. 2019; 143: 1849-1864Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Mice were sensitized with 100 μL 0.5% DNFB (v/v) in acetone/olive oil (4:1) and challenged 6 days later with 20 μL 0.2% DNFB at the ear (10 μL each side of the ear). Vehicle controls were sensitized and challenged with the acetone/olive oil solvent only.E6Dudeck A. Dudeck J. Scholten J. Petzold A. Surianarayanan S. Köhler A. et al.Mast cells are key promoters of contact allergy that mediate the adjuvant effects of haptens.Immunity. 2011; 34: 973-984Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar Ear thickness was measured before and at the indicated time points after challenge with an engineer’s micrometer (Mitutoyo). Ear swelling was determined as the percent increase compared with prechallenge ear thickness. Twenty-four hours after challenge ear skin samples were used for flow cytometry analysis. For analysis of cell infiltration, ear skin samples were cut into small pieces and digested in medium containing 20 mM HEPES, 0.025 mg/mL Liberase, 396 U/mL DNAse I, and 0.5 mg/mL hyaluronidase for 1 hour at 37°C and 1400 rpm on a thermo mixer. Cell suspensions were passed through a 40-μm sieve, washed twice with PBS/2% BSA, and stained in 100 μL PBS/2% BSA with the respective antibodies for 30 minutes at 4°C. Neutrophil granulocytes were assessed as CD45+Ly6G+CD11b+F4/80− cells. Infiltrated cells were quantified using Miltenyi MACS Quant or BD fortessa flow cytometer and MACS Quant Analysis software or FlowJo. Pyrilamine (10 mg/g bodyweight) and cromolyn (25 mg/g bodyweight) were injected intravenously 1 hour before challenge.E6Dudeck A. Dudeck J. Scholten J. Petzold A. Surianarayanan S. Köhler A. et al.Mast cells are key promoters of contact allergy that mediate the adjuvant effects of haptens.Immunity. 2011; 34: 973-984Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar Twenty microliter of KN-62 at a final concentration of 178.5 μg/mL was injected intradermally (i.d.) into the ear pinna 4 hours before challenge; control mice received an injection of 20 μL of saline.E8Weber F.C. Esser P.R. Müller T. Ganesan J. Pellegatti P. Simon M.M. et al.Lack of the purinergic receptor P2X(7) results in resistance to contact hypersensitivity.J Exp Med. 2010; 207: 2609-2619Crossref PubMed Scopus (169) Google Scholar One microgram of anti–IL-33 blocking antibodyE5Bruhs A. Proksch E. Schwarz T. Schwarz A. Disruption of the epidermal barrier induces regulatory T cells via IL-33 in mice.J Investig Dermatol. 2018; 138: 570-579Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar or soluble IL-33 receptorE9Fu A.K. Hung K.W. Yuen M.Y. Zhou X. Mak D.S. Chan I.C. et al.IL-33 ameliorates Alzheimer’s disease-like pathology and cognitive decline.Proc Natl Acad Sci U S A. 2016; 113: E2705-E2713Crossref PubMed Scopus (197) Google Scholar, E10Drube S. Heink S. Walter S. Löhn T. Grusser M. Gerbaulet A. et al.The receptor tyrosine kinase c-Kit controls IL-33 receptor signaling in mast cells.Blood. 2010; 115: 3899-3906Crossref PubMed Scopus (97) Google Scholar in 20 μL of saline was injected i.d. into the ear pinna 1 hour before challenge; control mice received an injection of 20 μL saline only. Twenty microliter of ATP at a final concentration of 826.7 μg/mL (1.5 μM) was injected i.d. in the ear pinna. Ten microliter of IL-33 (500 ng) was injected i.d. into the ear pinnae. Ear swelling was measured directly after the injection at the indicated time points and flow cytometry analysis were performed at 24 hours postinjection as described above. MC-deficient Mcpt5-Cre+R-DTA mice were locally reconstituted with in vitro expanded peritoneal MCs (PMCs) isolated from wt or ST2−/− mice by i.d. injection of 1 × 106 PMCs in 20 μL sterile saline into the ear pinnae as previously proven to result in MC numbers comparable to healthy skin.E11Drube S. Kraft F. Dudeck J. Müller A.L. Weber F. Göpfert C. et al.MK2/3 are pivotal for IL-33-induced and mast cell-dependent leukocyte recruitment and the resulting skin inflammation.J Immunol. 2016; 197: 3662-3668Crossref PubMed Scopus (39) Google Scholar Following experiments were conducted at least 2 weeks after reconstitution. PMCs were isolated and cultured as previously described.E12Dudeck A. Suender C.A. Lopez Kostka S. von Stebut E. Maurer M. Mast cells promote Th1 and Th17 responses by modulating dendritic cell maturation and function.Eur J Immunol. 2011; 41: 1883-1893Crossref PubMed Scopus (108) Google Scholar In brief, PMCs were isolated by flushing the peritoneal cavity with 5 mL ice-cold PBS. Cells were pelleted for 5 minutes at 4°C and 1200 rpm; resuspended in 5 mL RPMI containing 10% FCS, 1% penicillin/streptomycin, 1% l-Ala-l-Glu, 1% sodiumpyruvate; supplemented with 10 ng/mL IL-3 and 10-30 ng/mL SCF; and cultured for 8 days at 37°C and 5% CO2. If required, PMCs were enriched using CD117 MicroBeads (Miltenyi, Bergisch-Gladbach, Germany). Purity for reconstitution or following in vitro experiments was 95% or more as determined by flow cytometry using antibodies against CD117 and FcεRIα. PMCs were plated in a 96-well plate at a density of 2 × 105 cells/200 μL/well, and stimulated with 500 μM ATP in nonconditioned RPMI. Supernatant was collected after 10 minutes and 24 hours. PMCs stimulated with the calcium ionophore A23187 (500 ng/mL) served as a positive control, and PMCs cultured in RPMI alone served as negative control. Histamine (DRG) and TNF (TNF-α ELISA Max Standard from BioLegend) release was quantified using respective ELISAs according to the manufacturer’s instructions. MC degranulation was assessed by means of β-hexosaminidase assay. Twenty-five microliter cell supernatant and cell lysate, respectively, was mixed with 25 μL 1 mM p-nitrophenyl-N-acetyl β-d-glucosamine in citrate buffer (0.1 mol; pH 4.5) and incubated for 1 hour at 37°C. The reaction was stopped with 200 μL carbonate buffer (0.1 mol Na2CO3; 0.1 mol NaHCO3; pH 9) and OD was measured at 405 nm. Release of β-hexosaminidase was calculated as percent release of total content (100x amount supernatant/amount supernatant + cell lysate). Data are shown as mean ± SD. Data are pooled from 1 (Fig 1, B, D, G, H, and L), 2 (Fig 1, C, E, F, M, and N; Fig 2, A, B, F, G, and H), or 3 (Fig 1, I, J, and K; Fig 2, C, D, E, I, J, and K; Fig E1) independent experiments. Statistical analysis was performed with nonparametric 1-way ANOVA for all the graphs that contain more than 2 groups and 2-way ANOVA for the ear swelling curves. For the ear swelling curves, unless stated otherwise in the figure legend, the difference depicted is between the red and the black curves. #P < .001, ∗∗P < .01, and ∗P < .05.

Tissue resident mast cells (MCs) rapidly initiate neutrophil infiltration upon inflammatory insult, yet the molecular mechanism is still unknown. Here, we demonstrate that MC-derived TNF is crucial for neutrophil extravasation to sites of sterile skin inflammation by promoting intraluminal adhesion and crawling. Strikingly, MC-TNF directly primes circulating neutrophils via TNFR1, while being dispensable for chemokine production and endothelial cell activation. The MC-TNF is fused into the bloodstream by directional degranulation of perivascular MCs that are part of the vascular unit with access to the vessel lumen. Consistently, intravenous administration of MC granules can boost neutrophil extravasation. Consequently, the directional MC degranulation of pro-inflammatory mediators into the bloodstream may represent an important target for therapeutic approaches aimed at dampening cytokine storm syndromes or shock symptoms, or intentionally pushing immune defense.

Although mast cells (MCs) are known as key drivers of type I allergic reactions, there is increasing evidence for their critical role in host defense. MCs not only play an important role in initiating innate immune responses, but also influence the onset, kinetics, and amplitude of the adaptive arm of immunity or fine-tune the mode of the adaptive reaction. Intriguingly, MCs have been shown to affect T-cell activation by direct interaction or indirectly, by modifying the properties of antigen-presenting cells, and can even modulate lymph node-borne adaptive responses remotely from the periphery. In this review, we provide a summary of recent findings that explain how MCs act as a link between the innate and adaptive immunity, all the way from sensing inflammatory insult to orchestrating the final outcome of the immune response.

Mast cells (MCs), which are well known for their effector functions in T(H)2-skewed allergic and also autoimmune inflammation, have become increasingly acknowledged for their role in protection of health. It is now clear that they are also key modulators of immune responses at interface organs, such as the skin or gut. MCs can prime tissues for adequate inflammatory responses and cooperate with dendritic cells in T-cell activation. They also regulate harmful immune responses in trauma and help to successfully orchestrate pregnancy. This review focuses on the beneficial effects of MCs on tissue homeostasis and elimination of toxins or venoms. MCs can enhance pathogen clearance in many bacterial, viral, and parasitic infections, such as through Toll-like receptor 2-triggered degranulation, secretion of antimicrobial cathelicidins, neutrophil recruitment, or provision of extracellular DNA traps. The role of MCs in tumors is more ambiguous; however, encouraging new findings show they can change the tumor microenvironment toward antitumor immunity when adequately triggered. Uterine tissue remodeling by alpha-chymase (mast cell protease [MCP] 5) is crucial for successful embryo implantation. MCP-4 and the tryptase MCP-6 emerge to be protective in central nervous system trauma by reducing inflammatory damage and excessive scar formation, thereby protecting axon growth. Last but not least, proteases, such as carboxypeptidase A, released by Fc epsilon RI-activated MCs detoxify an increasing number of venoms and endogenous toxins. A better understanding of the plasticity of MCs will help improve these advantageous effects and hint at ways to cut down detrimental MC actions.

High numbers of mast cells populate the stroma of many types of neoplasms, including human papilloma virus-induced benign and malignant tumors in man and mouse. Equipped with numerous pattern recognition receptors and capable of executing important pro-inflammatory responses, mast cells are considered innate sentinels that significantly impact tumor biology. Mast cells were reported to promote human papilloma virus (HPV)-induced epithelial hyperproliferation and neo-angiogenesis in an HPV-driven mouse model of skin cancer. We analyzed HPV-induced epithelial hyperplasia and squamous cell carcinoma formation, as well as growth of tumors inoculated into the dermis, in mice lacking skin mast cells. Unexpectedly, the absence of mast cells had no effect on HPV-induced epithelial growth or angiogenesis, on growth kinetics of inoculated tumors, or on the immunological tumor micro-milieu. Thus, the conspicuous recruitment of mast cells into tumor tissues cannot necessarily be equated with important mast cell functions in tumor growth.

Background: Mast cells (MCs) are best known as key effector cells of allergic reactions, but they also play an important role in host defense against pathogens. Despite increasing evidence for a critical effect of MCs on adaptive immunity, the underlying mechanisms are poorly understood. Objective: Here we monitored MC intercellular communication with dendritic cells (DCs), MC activation, and degranulation and tracked the fate of exocytosed mast cell granules (MCGs) during skin inflammation. Methods: Using a strategy to stain intracellular MCGs in vivo, we tracked the MCG fate after skin inflammation-induced MC degranulation. Furthermore, exogenous MCGs were applied to MC-deficient mice by means of intradermal injection. MCG effects on DC functionality and adaptive immune responses in vivo were assessed by combining intravital multiphoton microscopy with flow cytometry and functional assays. Results: We demonstrate that dermal DCs engulf the intact granules exocytosed by MCs on skin inflammation. Subsequently, the engulfed MCGs are actively shuttled to skin-draining lymph nodes and finally degraded inside DCs within the lymphoid tissue. Most importantly, MCG uptake promotes DC maturation and migration to skin-draining lymph nodes, partially through MCderived TNF, and boosts their T-cell priming efficiency. Surprisingly, exogenous MCGs alone are sufficient to induce a prominent DC activation and T-cell response. Conclusion: Our study highlights a unique feature of peripheral MCs to affect lymphoid tissue-borne adaptive immunity over distance by modifying DC functionality through delivery of granule-stored mediators.

Mast cells (MCs) and dendritic cells (DCs) are essential innate sentinels populating host-environment interfaces. Using longitudinal intravital multiphoton microscopy of DCGFP/MCRFP reporter mice, we herein provide in vivo evidence that migratory DCs execute targeted cell-to-cell interactions with stationary MCs before leaving the inflamed skin to draining lymph nodes. During initial stages of skin inflammation, DCs dynamically scan MCs, whereas at a later stage, long-lasting interactions predominate. These innate-to-innate synapse-like contacts ultimately culminate in DC-to-MC molecule transfers including major histocompatibility complex class II (MHCII) proteins enabling subsequent ex vivo priming of allogeneic T cells with a specific cytokine signature. The extent of MHCII transfer to MCs correlates with their T cell priming efficiency. Importantly, preventing the cross talk by preceding DC depletion decreases MC antigen presenting capacity and T cell-driven inflammation. Consequently, we identify an innate intercellular communication arming resident MCs with key DC functions that might contribute to the acute defense potential during critical periods of migration-based DC absence.

The neurobeachin-like 2 protein (Nbeal2) belongs to the family of beige and Chediak-Higashi (BEACH) domain proteins. Loss-of-function mutations in the human NBEAL2 gene or Nbeal2 deficiency in mice cause gray platelet syndrome, a bleeding disorder characterized by macrothrombocytopenia, splenomegaly, and paucity of α-granules in megakaryocytes and platelets. We found that in mast cells, Nbeal2 regulates the activation of the Shp1-STAT5 signaling axis and the composition of the c-Kit/STAT signalosome. Furthermore, Nbeal2 mediates granule formation and restricts the expression of the transcription factors, IRF8, GATA2, and MITF as well as of the cell-cycle inhibitor p27, which are essential for mast cell differentiation, proliferation, and cytokine production. These data demonstrate the relevance of Nbeal2 in mast cells above and beyond granule biosynthesis.

Mast cells, important sensor and effector cells of the immune system, may influence bone metabolism as their number is increased in osteoporotic patients. They are also present during bone fracture healing with currently unknown functions. Using a novel c-Kit-independent mouse model of mast cell deficiency, we demonstrated that mast cells did not affect physiological bone turnover. However, they triggered local and systemic inflammation after fracture by inducing release of inflammatory mediators and the recruitment of innate immune cells. In later healing stages, mast cells accumulated and regulated osteoclast activity to remodel the bony fracture callus. Furthermore, they were essential to induce osteoclast formation after ovariectomy. Additional in vitro studies revealed that they promote osteoclastogenesis via granular mediators, mainly histamine. In conclusion, mast cells are redundant in physiologic bone turnover but exert crucial functions after challenging the system, implicating mast cells as a potential target for treating inflammatory bone disorders. © 2017 American Society for Bone and Mineral Research.

The IL-1R family member IL-33R mediates Fcε-receptor-I (FcεRI)-independent activation of mast cells leading to NF-κB activation and consequently the production of cytokines. IL-33 also induces the activation of MAPKs, such as p38. We aimed to define the relevance of the p38-targets, the MAPK-activated protein kinases 2 and 3 (MK2 and MK3) in IL-33-induced signaling and the resulting mast cell effector functions in vitro and in vivo. We demonstrate that the IL-33-induced IL-6 and IL-13 production strongly depends on the MK2/3-mediated activation of ERK1/2 and PI3K signaling. Furthermore, in the presence of the stem cell factors, IL-33 did induce an MK2/3-, ERK1/2- and PI3K-dependent production of TNF-α. In vivo, the loss of MK2/3 in mast cells decreased the IL-33-induced leukocyte recruitment and the resulting skin inflammation. Therefore, the MK2/3-dependent signaling in mast cells is essential to mediate IL-33-induced inflammatory responses. Thus, MK2/3 are potential therapeutic targets for suppression of IL-33-induced inflammation skin diseases such as psoriasis.

Mast cells are critical promoters of adaptive immunity in the contact hypersensitivity model, but the mechanism of allergen sensitization is poorly understood. Using Mcpt5-CreTNFFL/FL mice, we show here that the absence of TNF exclusively in mast cells impaired the expansion of CD8+ T cells upon sensitization and the T-cell-driven adaptive immune response to elicitation. T cells primed in the absence of mast cell TNF exhibited a diminished efficiency to transfer sensitization to naive recipients. Specifically, mast cell TNF promotes CD8+ dendritic cell (DC) maturation and migration to draining lymph nodes. The peripherally released mast cell TNF further critically boosts the CD8+ T-cell-priming efficiency of CD8+ DCs, thereby linking mast cell effects on T cells to DC modulation. Collectively, our findings identify the distinct potential of mast cell TNF to amplify CD8+ DC functionality and CD8+ T-cell-dominated adaptive immunity, which may be of great importance for immunotherapy and vaccination approaches.