Protease- and cell type-specific activation of protease-activated receptor 2 in cutaneous inflammation.

BACKGROUND: Protease-activated receptor 2 (PAR2) signaling controls skin barrier function and inflammation, but the roles of immune cells and PAR2-activating proteases in cutaneous diseases are poorly understood. OBJECTIVE: To dissect PAR2 signaling contributions to skin inflammation with new genetic and pharmacological tools. METHODS/RESULTS: We found markedly increased numbers of PAR2+ infiltrating myeloid cells in skin lesions of allergic contact dermatitis (ACD) patients and in the skin of contact hypersensitivity (CHS) in mice, a murine ACD model for T cell-mediated allergic skin inflammation. Cell type-specific deletion of PAR2 in myeloid immune cells as well as mutation-induced complete PAR2 cleavage insensitivity significantly reduced skin inflammation and hapten-specific Tc1/Th1 cell response. Pharmacological approaches identified individual proteases involved in PAR2 cleavage and demonstrated a pivotal role of tissue factor (TF) and coagulation factor Xa (FXa) as upstream activators of PAR2 in both the induction and effector phase of CHS. PAR2 mutant mouse strains with differential cleavage sensitivity for FXa versus skin epithelial cell-expressed proteases furthermore uncovered a time-dependent regulation of CHS development with an important function of FXa-induced PAR2 activation during the late phase of skin inflammation. CONCLUSIONS: Myeloid cells and the TF-FXa-PAR2 axis are key mediators and potential therapeutic targets in inflammatory skin diseases.

Myeloid cell populations and fibrogenic parameters in bleomycin- and HOCl-induced fibrosis.

Mouse models resembling systemic sclerosis can be chemically induced by application of bleomycin or hypochloric acid (HOCl). To date, little is known about inflammatory cells and their potential role in scleroderma (Scl)-related fibrosis. Therefore, we compared both Scl models to define the early immune cell subsets in relation to fibrosis-related parameters. Both agents induced a significant increase in dermal thickness and collagen deposition after 4 weeks, as hallmarks of Scl. However, clinical skin thickness, densely packed, sirius red-stained collagen bundles and collagen cross-links were more pronounced in HOCl-induced Scl. In parallel, there was a significant upregulation of procollagen α1(I), α-SMA and TGF-ß transcripts in HOCl animals, whereas IL-1ß and MMP-13 mRNA levels were significantly increased in bleomycin-treated mice. Flow cytometric analysis of the Scl skin demonstrated an early cellular infiltrate containing mainly CD19+ B cells, CD4+ T cells, CD11c+ DC and CD11b+ myeloid cells, the latter ones being significantly more prominent after HOCl injection. Subanalysis revealed that Scl mice exhibited a significant increase of inflammatory myeloid CD11b+  Ly6Clow-high  CD64low-high cells (HOCl>bleomycin). In particular, in the HOCl model, activated dermal macrophages (CCR2low  MHCIIhigh ) and monocyte-derived DC (CCR2high MHCIIhigh ) predominated over less activated CD11b+ myeloid cells. In conclusion, the two models differ in certain aspects of the murine and human scleroderma but in the HOCl model, myeloid CD11b+  MHCIIhigh cells correlate with some fibrosis-related parameters. Therefore, analysis of both models is suggested to cover a comprehensive profile of Scl symptoms but with focus on the HOCl model when the role of early myeloid immune cells will be evaluated.

Epicutaneous and Oral Low-Zone Tolerance Protects from Colitis in Mice.

Tolerance to environmental antigens that encounter the organism at interfaces like skin or gut prevents deleterious systemic immune responses. The aim of this study was to analyze whether and how low doses of haptens, by entry through the skin or gastrointestinal tract, affect the outcome of the predominantly Th1/Th17-mediated 2,4,6-trinitro-benzenesulfonic acid-induced colitis, which mimics an autoimmune bowl disease in man. Epicutaneous and oral applications of low doses of the allergen resulted in the induction of low-zone tolerance (LZT) and protected from colitis development, demonstrated by a significantly reduced inflammatory response of the gut in vivo. In line with this observation, we found a significantly diminished Th1/Th17-mediated T cell response and reduced T cell proliferation after both tolerance regimes, indicating that epicutaneous LZT is just as well efficient as oral tolerance in prevention of a gut-associated inflammatory immune response. Use of a second, unrelated hapten for LZT induction revealed an antigen-specific tolerance mechanism. Intriguingly, in the absence of hapten-activated CD4(+)CD25(+)Foxp3(+) regulatory T cells and IL-10, epicutaneous and oral LZT failed to abrogate the development of the intestinal inflammation. In conclusion, this study highlights in particular epicutaneous immunotherapies in the form of LZT through activation of CD4(+)CD25(+)Foxp3(+) regulatory T cells as treatment strategies for inflammatory, allergic, or autoimmune diseases.

Dendritic cell motility and T cell activation requires regulation of Rho-cofilin signaling by the Rho-GTPase activating protein myosin IXb.

Directed migration of stimulated dendritic cells (DCs) to secondary lymphoid organs and their interaction with Ag-specific T cells is a prerequisite for the induction of primary immune responses. In this article, we show that murine DCs that lack myosin IXB (Myo9b), a motorized negative regulator of RhoA signaling, exhibit increased Rho signaling activity and downstream acto-myosin contractility, and inactivation of the Rho target protein cofilin, an actin-depolymerizing factor. On a functional level, Myo9b(-/-) DCs showed impaired directed migratory activity both in vitro and in vivo. Moreover, despite unaltered Ag presentation and costimulatory capabilities, Myo9b(-/-) DCs were poor T cell stimulators in vitro in a three-dimensional collagen matrix and in vivo, associated with altered DC-T cell contact dynamics and T cell polarization. Accordingly, Myo9b(-/-) mice showed an attenuated ear-swelling response in a model of contact hypersensitivity. The impaired migratory and T cell stimulatory capacity of Myo9b(-/-) DCs was restored in large part by pharmacological activation of cofilin. Taken together, these results identify Myo9b as a negative key regulator of the Rho/RhoA effector Rho-kinase [Rho-associated coiled-coil-forming kinase (ROCK)]/LIM domain kinase signaling pathway in DCs, which controls cofilin inactivation and myosin II activation and, therefore may control, in part, the induction of adaptive immune responses.

Crosstalk of regulatory T cells and tolerogenic dendritic cells prevents contact allergy in subjects with low zone tolerance.

BACKGROUND: Allergic contact dermatitis is one of the most common occupational diseases. A main protective mechanism in those who do not develop allergic contact dermatitis is tolerance induction by repeated exposure to low doses of contact allergen, which is termed low zone tolerance (LZT). The mechanisms that determine the tolerance induction in subjects with LZT are still elusive. OBJECTIVE: We performed analysis of the role of CD4(+)CD25(+) forkhead box protein 3 (FOXP3)-positive regulatory T (Treg) cells and dendritic cells (DCs) in mice with LZT. METHODS: Mechanisms of tolerance induction were analyzed in a murine model of LZT by using FOXP3 and IL-10 reporter mice, as well as mice that allow the selective depletion of Treg cells or DCs. RESULTS: Depletion of CD4(+)CD25(+)FOXP3(+) Treg cells during tolerance induction completely abolishes the development of LZT, resulting in a pronounced contact hypersensitivity response. Adoptive transfer experiments, depletion studies, and use of cell type-specific deficient mice revealed that IL-10 production is critical for the suppressor function of Treg cells in mice with LZT and that tolerogenic CD8(+)CD11c(+) DCs located in the skin-draining lymph nodes are essential for LZT. In the absence of Treg cells, DCs did not develop tolerogenic functions, indicating that activated IL-10(+) Treg cells might imprint the tolerogenic DC phenotype. Cell communication analysis revealed that the education of tolerogenic DCs might involve a direct interaction with Treg cells mediated by gap junctions. Subsequently, induction of tolerogenic CD11c(+) DCs leads to the generation of hapten-specific CD8(+) Treg cells, which protect against contact hypersensitivity. CONCLUSIONS: Our data demonstrate critical interactions between CD4(+)CD25(+)FOXP3(+) Treg cells and tolerogenic CD8(+)CD11c(+) DCs during the induction of LZT.

T cell killing by tolerogenic dendritic cells protects mice from allergy.

It is well established that allergy development can be prevented by repeated low-dose exposure to contact allergens. Exactly which immune mechanisms are responsible for this so-called low zone tolerance (LZT) is not clear, although CD8⁺ suppressor T cells are known to have a role. Here, we show that TNF released by tolerogenic CD11⁺CD8⁺ DCs located in skin-draining lymph nodes is required and sufficient for development of tolerance to contact allergens in mice. DC-derived TNF protected mice from contact allergy by inducing apoptosis in allergen-specific effector CD8⁺ T cells via TNF receptor 2 but did not contribute to the generation and function of the regulatory T cells associated with LZT. The TNF-mediated killing mechanism was induced in an allergen-specific manner. Activation of tolerogenic DCs by LZT CD8⁺ suppressor T cells and enhanced TNF receptor 2 expression on contact allergen-specific CD8⁺ effector T cells were required for LZT. Our findings may explain how tolerance protects from allergic diseases, which could allow for the development of new strategies for allergy prevention.

Investigation of Sudan IV staining areas in aortas of infants and children: possible prelesional stages of atherogenesis.

OBJECTIVES/METHODS: Although atherosclerosis in infants and children is generally acknowledged, the temporal and spatial sequence of LDL insudation, modification and intimal monocyte accumulation has not been systematically studied. We have investigated herein very early stages of lesion formation in human aortas of individuals up to the age of 15 years. Aortic specimens from 61 cases (37 male, 24 female) were examined. 34 cases were <1 year old, 16 cases were between 1 and 5 years old, and 11 cases were between 6 and 15 years old. Areas preselected under a dissection microscope after Sudan IV staining were investigated in depth by immunohistochemical staining for apolipoprotein B, monocytes/macrophages, smooth muscle cells (SMCs), enzymatically and oxidatively modified lipoproteins, C-reactive protein and complement components. RESULTS: (i) Lipoprotein accumulation in the intima before macrophage infiltration, (ii) virtually no extracellular lipoprotein modification, either enzymatic or oxidative, within intimal lesions in infancy (<1 year), (iii) onset of extracellular enzymatic modification of low-density lipoprotein (LDL) in the age group between 6 and 15 years and (iv) no coincidence of lipoprotein accumulation in the intima with activation of the terminal complement cascade as known from early atherosclerotic lesions in adults. CONCLUSIONS: The present study indicates the existence of hitherto undescribed prelesional stages in atherogenesis characterized by 'inert' lipoprotein insudation in individuals <1 year of age without lipoprotein modification, monocyte/macrophage infiltration and/or inflammation on the one hand and the onset of extracellular enzymatic rather than oxidative lipoprotein modification in individuals between 6 and 15 years of age on the other hand. Further investigations of these stages should advance understanding of events underlying initiation, progression and regression of intimal lesions developing in early atherogenesis.