PAR-1 signaling on macrophages is required for effective in vivo delayed-type hypersensitivity responses

Abstract

Delayed-type hypersensitivity (DTH) responses underpin chronic inflammation. Using a model of oxazolone-induced dermatitis and a combination of transgenic mice, adoptive cell transfer, and selective agonists/antagonists against protease activated receptors, we show that that PAR-1 signaling on macrophages by thrombin is required for effective granuloma formation. Using BM-derived macrophages (BMMs) in vitro, we show that thrombin signaling induced (a) downregulation of cell membrane reverse cholesterol transporter ABCA1 and (b) increased expression of IFNg receptor and enhanced co-localization within increased areas of cholesterol-rich membrane microdomains. These two key phenotypic changes combined to make thrombin-primed BMMs sensitive to M1 polarization by 1000-fold less IFNg, compared to resting BMMs. We confirm that changes in ABCA1 expression were directly responsible for the exquisite sensitivity to IFNg in vitro and for the impact on granuloma formation in vivo. These data indicate that PAR-1 signaling plays a hitherto unrecognized and critical role in DTH responses.

Manipulation of tissue factor-mediated basal PAR-2 signalling on macrophages determines sensitivity for IFNγ responsiveness and significantly modifies the phenotype of murine DTH

“…Although there was marked intra-experiment variation, MCSF-derived bone marrow macrophages (BMM) cultured with the PAR-2 agonist 2-Furoyl-LIGRLO-amide appeared to have a blunted response to increasing concentrations of IFNγ, as measured by the upregulation of iNOS, when compared to untreated cells ( Figures 1A, B ). To clarify the effect of the PAR-2 agonist, we first incubated cells with thrombin, which significantly enhanced iNOS expression after exposure to low concentrations of IFNγ, as previously shown ( 9 ). This responsiveness was now very obviously blunted by prior incubation with a PAR-2 agonist, which reduced iNOS expression down to levels statistically insignificant from controls ( Figure 1C ).…”

“…In both models, we also reported that IV administration of a novel thrombin inhibitor called PTL060 (Thrombalexin) ( 11 – 13 ), could induce the same phenotype in ApoE-/- and WT mice respectively as that seen in transgenic mice ( 9 , 10 ). Importantly, PTL060 binds rapidly to circulating cells after IV administration, including monocytes, on which we showed it exerts an extended pharmacological effect.…”

“…We have previously demonstrated that thrombin, signalling through PAR-1, primes macrophages to be exquisitely sensitive to low concentrations of IFNγ (and TLR-4 stimuli) via reduced expression of the reverse cholesterol transporter ABCA1 and consequent increase in the number of membrane cholesterol-rich micro-domains, into which enhanced numbers of IFNγR (and TLR4) are recruited ( 9 ). We showed this mechanism was highly relevant in two in vivo murine models not usually associated with thrombosis: established atherosclerosis in ApoE-/- mice fed a high fat diet ( 10 ) and cutaneous delayed type hypersensitivity (DTH) ( 9 ).…”

“…We have previously demonstrated that thrombin, signalling through PAR-1, primes macrophages to be exquisitely sensitive to low concentrations of IFNγ (and TLR-4 stimuli) via reduced expression of the reverse cholesterol transporter ABCA1 and consequent increase in the number of membrane cholesterol-rich micro-domains, into which enhanced numbers of IFNγR (and TLR4) are recruited ( 9 ). We showed this mechanism was highly relevant in two in vivo murine models not usually associated with thrombosis: established atherosclerosis in ApoE-/- mice fed a high fat diet ( 10 ) and cutaneous delayed type hypersensitivity (DTH) ( 9 ). In both models, inhibiting thrombin-mediated signalling through PAR-1 on myeloid cells by transgenic expression of a tethered thrombin inhibitor led to profound phenotypic changes, including clearance of established atherosclerotic plaques in ApoE-/- mice and near complete inhibition of swelling and granuloma formation in DTH responses, through with preservation of T cell sensitisation.…”

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

“…Although both diseases are characterized by epidermal hyperplasia and excessive inflammation, chronic AD is marked by the activation of Th1 cells alongside Th2 and Th22 cells, and IL-17 has a limited role; psoriasis however is a Th17-centric disease, with Th1 probably being a bystander [ 32 ]. In addition, PAR1 signaling on macrophages is required for oxazolone-induced CHS in mice [ 33 ]. The different roles of PAR1 in these two CHS models may be attributed to the different involvement of immune cells in disease pathogenesis.…”