Projects
Research Immunoregulation
Figure 1. Intravital 2-photon microscopy provides us the unique opportunity to study mast cell (MC) distribution in the live mouse ear skin. Here, perivascular MCs (white) are in close proximity to blood vessels. The specific staining of venules with endomucin (green) allows the discrimination from endomucin- arterioles (red); orange, merge. (See Dudeck et al., 2021)
Figure 1. Intravital 2-photon microscopy provides us the unique opportunity to study mast cell (MC) distribution in the live mouse ear skin. Here, perivascular MCs (white) are in close proximity to blood vessels. The specific staining of venules with endomucin (green) allows the discrimination from endomucin- arterioles (red); orange, merge. (See Dudeck et al., 2021)
Figure 2. Notably, perivascular mast cells (MCs) actively sample the blood stream via intraluminal protrusions. Pervascular MCs (white) can pass the endothelial vessel wall (red) and can take up intravenously applied avidin (green) which specifically stains MC granules. (See Dudeck et al., 2021)
For movies and more images, see Dudeck et al., 2021
Research Profile
Inflammatory skin diseases are major causes of occupational disability and are thereby a high burden for the patients and have a high socioeconomic relevance, too. On the one hand, allergic contact dermatitis (ACD) is a T-cell mediated chronic inflammatory skin diseases, triggered by small organic or inorganic environmental allergens, (haptens). On the other hand, atopic dermatitis (AD) is driven by IgE-mediated type I allergic responses.
Mast cells (MCs) are well known as key effector cells of IgE-dependent immediate type I allergy, but their role in chronic inflammatory skin diseases such as ACD and AD is far less understood. Using the contact hypersensitivity (CHS) mouse model, we have substantially contributed to the current understanding of MC functions in ACD. MCs show intense interaction with skin dendritic cells (DCs) and, moreover, MCs orchestrate the rapid recruitment of neutrophils to the site of hapten encounter independent of prior sensitization. Recently, we have shown that perivascular MCs can cross the endothelial barrier and degranulate directionally into the bloodstream, which provides rapid serum levels of pro-inflammatory mediators and directly primes circulating neutrophils.
​
Figure 3. Mast cells (MCs) have a pivotal role in the contact hypersensitivity (CHS) model. Upon administration of the hapten DNFB onto the skin, MCs sense the stress molecules ATP and IL-33 released by damaged keratinocytes (sensitization). Following, MCs release active mediators stored their granules, leading to the migration of immature dermal dendritic cells (DCs) to the draining lymph node. There, T cells get primed, leading to the specific inflammatory immune response upon the second encounter of the same hapten (elicitation). By that, and by their intense interaction with adjacent cells, MCs are crucial for the onset and kinetics of skin inflammation.
Research Projects
Project 1 - Identification of the role of TNF in the initiation and resolution of skin inflammation
Figure 4. Mast cell-derived tumor necrosis factor (TNF) is sensed by dendritic cells and thereby initiates the inflammation, leading to CD8+ T cells priming, monocyte recruitment and extracellular matrix degradation. Created with BioRender.com
Project addressed
by Martin Voss.
During contact hypersensitivity (CHS) induced skin inflammation, MCs initiate the innate and adaptive immune response. In detail, MC-derived TNF promotes CD8+ DC maturation and migration and consequently CD8+ T cell priming in the sensitization phase. In the absence of MC-derived TNF, skin infiltrating inflammatory monocyte and CD8+ T cell numbers are reduced. At later time points, we could highlight the importance of MC-derived TNF on tissue recovery. Importantly, MC-derived TNF is required for a sufficient tissue recovery by promoting the priming of CD8+ T cells, which subsequently guide monocyte recruitment. Intriguingly, the treatment of WT mice with an anti-TNF antibody at the beginning of CHS led to the same defect in resolving the ear swelling as seen in absence of MC-TNF, thereby suggesting a potential detrimental effect of anti-inflammatory TNF therapies in tissue recovery.
Project 2 - Characterization of the the perivascular immune cell niche
Figure 5. Along the perivascular cell niche, mast cells (MCs) interact with adjacent immune cells, such as macrophages and dendritic cells. In contrast to interstitial MCs, directional intraluminal degranulation by perivascular MCs is crucial for the onset and kinetics of the immune response. Created with BioRender.com
Project addressed
by Aaron Hoffmann.
Mast cells (MCs) are known to interact with other cells along the blood vessels, such as dendritic cells or macrophages. Hence, our objective is to study the organization of the perivascular immune cell niche. We aim to investigate the morphological and functional characteristics of perivascular MCs in contrast to interstital MCs. To address this topic, we utilize novel mouse lines with specific knockouts of surface receptors that reduce the alignment and directional intravascular degranulation of perivascular MCs. Applying in vivo approaches such as intravital 2-photon fluorescence microscopy, we are able to analyze the intercellular network along the vascular system. Collectively, we aim to ascertain the mechanistic effects of MC degranulation on tissue homeostasis and gene expression of transcription factor and cytokines.
Project 3 - Investigation of the Effect of Mast Cell-derived RANKL on Lymphocyte Recruitment
Figure 6. Besides tumor necrosis factor (TNF), mast cells (MCs) release receptor activator of NFκB (RANKL) upon sensitization in the contact hypersensitivity model. Mediated by a target cell, MC-derived RANKL thus is crucial for the migration of lymphocytes out of the blood circulation to the site of allergic skin inflammation. Created with BioRender.com
Project addressed
by Konstantinos Katsoulis-Dimitriou.
We recently identified MCs as a prominent source of receptor activator of NFκB (RANKL), which is mostly known for being involved in bone resorption, with more information coming to light about its qualities as an immune regulator. However, the relevance of MC-derived RANKL in skin inflammation is completely unknown. Using a conditional RANKL knockout in connective tissue type MCs mouse line, we identified a crucial role of MC-derived RANKL in contact hypersensitivity. We could reveal that RANKL release by peripheral skin MCs upon DNFB challenge exerts a long-distance effect that is a prerequisite for the timely lymphocyte egress from remote LNs. This shows that MC-derived RANKL is critical for the onset and severity of allergic skin inflammation.
Project 4 - Mast cell function in allergic contact dermatitis
Figure 7. Upon skin inflammation, mast cells (MCs) are activated and release intact MC granules (MCG). MCG are subsequently taken up by dermal dendritic cells (DCs), enhancing their migration and maturation. Thereby, MCG-bearing DCs promote and modulate the lymph node-borne adaptive immunity by acting on the priming of CD4+ T cells. Created with BioRender.com
Project addressed
by Johanna Kotrba.
In 2019, our group could show that dermal dendritic cells (DCs) engulf intact mast cell granules (MCG) exocytosed by mast cells (MCs) upon skin inflammation (Dudeck et al., 2021). Consequently, MCG-bearing DCs show an enhanced maturation and migratory capacity, in line with a boosted T cell priming compared to MCG-negative DCs. To determine the molecular mechanisms underlying the MCG uptake by DCs, we use in vitro and ex vivo/in vivo co-culture systems. Additionally, different conditional and conventional mouse strains are used during hapten-induced skin inflammation and house dust mite (HDM)-induced allergic airway inflammation. Finally, we aim to identify potential targets to either dampen elevated immune responses by disrupting the MCG uptake by DCs or intentionally boost adaptive immunity by targeted MC degranulation or strategic administration of MCG/MCG-based artificial vesicle.
Project 5 - Study of the mast cell-macrophage interactions upon skin inflammation
Figure 8. In the skin, macrophages are able to take up mast cell granules (MCG) upon mast cell (MC) activation during skin inflammation. This process leads to a atypical, non-classical macrophage activation, resulting in a distinct phagocytic behaviour during skin infection. Created with BioRender.com
Project addressed
by James Patrick Koch.
In the skin, mast cells and macrophages reside in high density and close proximity, particularly at the blood vessels. However, the functional consequences of mast cell-macrophage interactions on the respective cell type have been poorly studied. Therefore, we developed a method to fluorescently stain mast cell granules (MCG) in vivo to localize the fate of MCG and the communication of mast cells and macrophages during tissue inflammation. We found that intact MCG, released upon mast cell activation into the extracellular matrix, are engulfed by tissue resident macrophages, resulting in an atypical, non-classical macrophage activation phenotype. This distinct response by macrophages may support the emerging view that mast cells are particularily important regulators of the resolution of inflammation and healing.
Exemplary Research Findings
Figure 9. Immature dendritic cells (imDCs) take up mast cell granules (MCG) in vitro. imDCs (green) derived from CD11c-GFP reporter mice were treated in vitro with isolated and fluorescently labeled MCG (red) and subsequently analyzed by confocal microscopy.​ Arrowheads indicate DCs that engulfed no (white), few (blue) or many MCG (yellow). Data generated by Johanna Kotrba.
Figure 10. High-resolution imaging of mast cell granules (MCG) in a peritoneal-cell derived mast cell (PCMC). MCs are densely packed with MCG (green) where preformed, fully active mediators are stored. Nucleus (blue), F-actin (red). Data generated by Aaron Hoffmann.
Figure 11. Dermal dendritic cells (DCs) engulf intact mast cell granules (MCG) exocytosed in skin inflammation in vivo. Overview showing several DCs (eGFP, green) in mouse ear skin, aarrowheads indicate DCs with engulfed MCGs (red). Fluorescence emission spectra (Lambda scan) of TexasRed-avidin–stained MCGs inside MCs (red curve, open circles) and DCs (red curve, solid circles) in comparison with DC-intrinsic eGFP (green curve). (See Dudeck et al., 2019).
Figure 12. Whole-organ clearing and imaging with light sheet fluorescence microscopy of a brain from a Mcpt5-Cre tdTomato mouse. Following an ethyl cinnamate clearing protocol with staining for a Nissl dye (NeuroTrace, green) and a nuclear stain (TO-PRO-3 iodide, blue), the whole brain as an intact organ can be imaged using light sheet fluorescence microscopy. Applied to Mcpt5-Cre tdTomato mice (mast cells (MCs) express the reporter protein tdTomoato, red), spatial analysis of MC morphology and localization in the whole brain can be performed. Data generated by Hanna Edler.
Figure 13. Light sheet fluorescence microscopy of a whole mouse brain. Blood vessels (red), nuclei (cyan) were stained in mice expressing EYFP (white) in the brain. Data generated by Hanna Edler.
Figure 14. Reduced immune response in RANKL-deficient MC mice. Ear swelling of sensitized mice was quantified as a measure of skin inflammation upon re-exposure in the contact hypersensitivity model. Data generated by Konstantinos Katsoulis-Dimitriou.