Activated Protein C Protects against Murine Contact Dermatitis by Suppressing Protease-Activated Receptor 2

Abstract

Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with excessive inflammation and defective skin barrier function. Activated protein C (APC) is a natural anticoagulant with anti-inflammatory and barrier protective functions. However, the effect of APC on AD and its engagement with protease activated receptor (PAR)1 and PAR2 are unknown. Methods: Contact hypersensitivity (CHS), a model for human AD, was induced in PAR1 knockout (KO), PAR2KO and matched wild type (WT) mice using 2,4-dinitrofluorobenzene (DNFB). Recombinant human APC was administered into these mice as preventative or therapeutic treatment. The effect of APC and PAR1KO or PARKO on CHS was assessed via measurement of ear thickness, skin histologic changes, inflammatory cytokine levels, Th cell phenotypes and keratinocyte function. Results: Compared to WT, PAR2KO but not PAR1KO mice displayed less severe CHS when assessed by ear thickness; PAR1KO CHS skin had less mast cells, lower levels of IFN-γ, IL-4, IL-17 and IL-22, and higher levels of IL-1β, IL-6 and TGF-β1, whereas PAR2KO CHS skin only contained lower levels of IL-22 and IgE. Both PAR1KO and PAR2KO spleen cells had less Th1/Th17/Th22/Treg cells. In normal skin, PAR1 was present at the stratum granulosum and spinosum, whereas PAR2 at the upper layers of the epidermis. In CHS, however, the expression of PAR1 and PAR2 were increased and spread to the whole epidermis. In vitro, compared to WT cells, PAR1KO keratinocytes grew much slower, had a lower survival rate and higher para permeability, while PAR2KO cells grew faster, were resistant to apoptosis and para permeability. APC inhibited CHS as a therapeutic but not as a preventative treatment only in WT and PAR1KO mice. APC therapy reduced skin inflammation, suppressed epidermal PAR2 expression, promoted keratinocyte growth, survival, and barrier function in both WT and PAR1KO cells, but not in PAR2KO cells. Conclusions: APC therapy can mitigate CHS. Although APC acts through both PAR1 and PAR2 to regulate Th and mast cells, suppression of clinical disease in mice is achieved mainly via inhibition of PAR2 alone. Thus, APC may confer broad therapeutic benefits as a disease-modifying treatment for AD.

Tissue factor-dependent colitogenic CD4+ T cell thrombogenicity is regulated by activated protein C signalling

“…Several studies have highlighted coagulation dysregulation in both IBD patients and preclinical models of IBD [12][13][14]26 . This disruption is particularly evident in models of innate immunemediated colitis, characterised by enhanced procoagulant TF activity, microparticle release and diminished PC pathway activity in the gut epithelia 11,14,16 .…”

“…APC is an important regulator of TF-mediated thrombin generation 24 , IBD pathophysiology [14][15][16] , and T cell inflammatory responses [25][26][27][28][29][30] . Therefore we next sought to investigate a potential role for APC anti-inflammatory signalling in regulating TF + CD4 + T cell procoagulant activity.…”

A recombinant signalling‐selective activated protein C that lacks anticoagulant activity is efficacious and safe in cutaneous wound preclinical models

“…57 We have also previously shown that PAR-1 is not required for wound healing in mice, but APC mediated reepithelialisation is PAR-2 dependent. 13,58,59 Thus, it is feasible that 3K3A-APC may signal at least part of its cytoprotective effects through PAR-3 and/or PAR-2 in the pig to induce reepithelialisation and collagen remodelling.…”

The protein C pathways

“…Direct stimulation with APC has a similar effect on effector T-cell responses, with reduced pathogenicity and the adoption of a Treg phenotype [56][57][58]. These effects require EPCR and various PAR receptors [57][58][59][60], which are expressed by both human and mouse T cells [57]. Recombinant APC ameliorates graft-versus-host disease in preclinical models by inhibiting allogeneic T-cell generation, instead redirecting lymphocytes toward a regulatory Treg lineage via PAR2/3-dependent signaling [57].…”

“…EPCR deficient (PROCR low ) mice exhibit enhanced proinflammatory Th17 cell generation and exacerbated disease in experimental autoimmune encephalomyelitis; Significantly, administration of APC to T cells isolated from PROCR low mice inhibits their differentiation to Th17 cells ex vivo [58]. Similarly, APC administration reduces Th1, Th17 and Treg populations in a preclinical atopic dermatitis model via PAR2dependent signaling [60] and dampens systemic inflammatory cytokine levels and Th1 and Th17 populations in collagen-induced arthritis models [61,62].…”