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.

Intervention of Inflammatory Monocyte Activity Limits Dermal Fibrosis.

Monocytes and monocyte-derived cells are important players in the initiation, progression, and resolution of inflammatory skin reactions. As inflammation is a prerequisite for fibrosis development, we focused on the role of monocytes in cutaneous fibrosis, the clinical hallmark of patients suffering from systemic sclerosis. Investigating the function of monocytes in reactive oxygen species-induced dermal fibrosis, we observed that early monocyte depletion partially reduced disease severity. Low numbers of inflammatory Ly6Chigh monocytes, as well as inhibition of CCR2 and CCL2 in wild type animals by a specific L-RNA aptamer, mitigated disease parameters, indicating a pivotal role for CCR2+ inflammatory monocytes and the CCR2/CCL2 axis in fibrosis development. Of note, mice lacking splenic reservoirs failed to recruit monocytes to the skin and developed less fibrosis. Furthermore, enforced monocyte conversion into noninflammatory, patrolling Ly6Clow monocytes by a nuclear receptor Nur77-agonist also resulted in significantly impaired cutaneous inflammation and dermal fibrosis. Most evident, pronounced monocyte conversion in interferon stimulated gene 12-deficient mice with pronounced nuclear Nur77 signaling completely protected from dermal fibrosis. Our study shows that inflammatory monocytes that are recruited from splenic reservoirs play a key role in the development of skin fibrosis and can be therapeutically challenged by forced conversion via the Nur77/interferon stimulated gene 12 axis.

Regulatory T cell deficient scurfy mice exhibit a Th2/M2-like inflammatory response in the skin.

BACKGROUND: Scurfy mice have a functional defect in regulatory T cells (Treg), which leads to lethal multi-organ inflammation. The missing Treg function results in uncontrolled autoimmune cellular and humoral inflammatory responses. We and others have previously shown that during the course of disease scurfy mice develop severe skin inflammation and autoantibodies including anti-nuclear autoantibodies (ANA). OBJECTIVE: Autoimmune skin inflammation and ANA are hallmarks for the diagnosis of autoimmune connective tissue diseases; therefore we analyzed scurfy mice for typical signs of these diseases. METHODS: Indirect immunofluorescence was used to specify the ANA pattern in scurfy mice. Skin fibrosis was assessed by cutaneous collagen accumulation (Goldeners trichrome staining), collagen crosslinking/disorganization (Sirus red polarimetry) and quantitative PCR for fibrosis-related transcripts. The cellular components of the inflammatory infiltrates in scurfy skin were analyzed by flow cytometry and intracellular cytokine staining. RESULTS: The majority of scurfy mice developed ANA with a predominant AC-5 pattern typical for mixed connective tissue disease, especially scleroderma. Scurfy mice showed higher skin collagen content compared to WT controls with a significant tendency in upregulation of TIMP-1. CD3+CD4+ T cells in scurfy skin exhibited a strong Th2 deviation with a significant increase of IL-4, IL-5 and IL-13, and M2-polarized CD11b+MHCII+ macrophages compared to WT mice. CONCLUSION: We show that Scurfy mice show a predominant AC-5 ANA pattern typical for mixed connective tissue disease as in scleroderma. The autoimmune inflammation in scurfy skin mainly consists of CD4+ T cells with Th2 differentiation and alternatively-activated (M2) macrophages as it is found in scleroderma with advanced fibrosis.

Early inflammatory players in cutanous fibrosis.

Systemic sclerosis (SSc) is one of the most complex systemic autoimmune diseases with multi-organ involvement and heterogeneous clinical manifestations. The exact etiology of SSc is still unknown. However, identified target structures are components of endothelial cells, the innate/adaptive immune systems and fibroblasts, resulting in the hallmarks of the disease in form of inflammation/autoimmunity, vasculopathy and fibrosis of the skin and internal organs. There has been a large body of evidence that the adaptive immune system with autoreactive T and B cells producing autoantibodies plays a central role in the pathogenesis of SSc but the role of earlier pathogenic processes involving the innate immune system, is far from being understood. There is strong evidence that a dysregulation of innate lymphoid cells and myeloid cells critically contributes to early pathogenic events in SSc. As disruption of vascular homeostasis and a fibroproliferative vasculopathy are hallmarks of early SSc, intravascular processes including platelet activation and interaction with neutrophils and monocytes, may even be upstream of innate immune deviation. Therefore, further studies of the dysregulated innate immune system may provide insights into novel and potentially curative treatments of SSc. In this review, we highlight the most relevant findings regarding the involvement of innate immune cells during the early stage of cutaneous fibrosis in SSc, with emphasis on the role of neutrophils, myeloid cells and innate lymphoid/NK cells in the pathogenesis of SSc and their potential as therapeutic targets for this difficult-to-treat autoimmune disease.

DNA damage response curtails detrimental replication stress and chromosomal instability induced by the dietary carcinogen PhIP.

PhIP is an abundant heterocyclic aromatic amine (HCA) and important dietary carcinogen. Following metabolic activation, PhIP causes bulky DNA lesions at the C8-position of guanine. Although C8-PhIP-dG adducts are mutagenic, their interference with the DNA replication machinery and the elicited DNA damage response (DDR) have not yet been studied. Here, we analyzed PhIP-triggered replicative stress and elucidated the role of the apical DDR kinases ATR, ATM and DNA-PKcs in the cellular defense response. First, we demonstrate that PhIP induced C8-PhIP-dG adducts and DNA strand breaks. This stimulated ATR-CHK1 signaling, phosphorylation of histone 2AX and the formation of RPA foci. In proliferating cells, PhIP treatment increased the frequency of stalled replication forks and reduced fork speed. Inhibition of ATR in the presence of PhIP-induced DNA damage strongly promoted the formation of DNA double-strand breaks, activation of the ATM-CHK2 pathway and hyperphosphorylation of RPA. The abrogation of ATR signaling potentiated the cell death response and enhanced chromosomal aberrations after PhIP treatment, while ATM and DNA-PK inhibition had only marginal effects. These results strongly support the notion that ATR plays a key role in the defense against cancer formation induced by PhIP and related HCAs.

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.