BACKGROUND AND PURPOSEIndependent studies in experimental models of Trypanosoma cruzi appointed different roles for endothelin-1 (ET-1) and bradykinin (BK) in the immunopathogenesis of Chagas disease. Here, we addressed the hypothesis that pathogenic outcome is influenced by functional interplay between endothelin receptors (ETAR and ETBR) and bradykinin B2 receptors (B2R). EXPERIMENTAL APPROACHIntravital microscopy was used to determine whether ETR/B2R drives the accumulation of rhodamine-labelled leucocytes in the hamster cheek pouch (HCP). Inflammatory oedema was measured in the infected BALB/c paw of mice. Parasite invasion was assessed in CHO over-expressing ETRs, mouse cardiomyocytes, endothelium (human umbilical vein endothelial cells) or smooth muscle cells (HSMCs), in the presence/absence of antagonists of B2R (HOE-140), ETAR (BQ-123) and ETBR (BQ-788), specific IgG antibodies to each GPCRs; cholesterol or calcium-depleting drugs. RNA interference (ETAR or ETBR genes) in parasite infectivity was investigated in HSMCs. KEY RESULTSBQ-123, BQ-788 and HOE-140 reduced leucocyte accumulation in HCP topically exposed to trypomastigotes and blocked inflammatory oedema in infected mice. Acting synergistically, ETAR and ETBR antagonists reduced parasite invasion of HSMCs to the same extent as HOE-140. Exogenous ET-1 potentiated T. cruzi uptake by HSMCs via ETRs/B2R, whereas RNA interference of ETAR and ETBR genes conversely reduced parasite internalization. ETRs/B2R-driven infection in HSMCs was reduced in HSMC pretreated with methyl-b-cyclodextrin, a cholesterol-depleting drug, or in thapsigargin-or verapamil-treated target cells. CONCLUSIONS AND IMPLICATIONSOur findings suggest that plasma leakage, a neutrophil-driven inflammatory response evoked by trypomastigotes via the kinin/endothelin pathways, may offer a window of opportunity for enhanced parasite invasion of cardiovascular cells. LINKED ARTICLE

Previous analysis of the endogenous innate signals that steer T cell-dependent immunity in mice acutely infected by the protozoan Trypanosoma cruzi revealed that bradykinin (BK) or lysyl-BK, i.e., the short-lived peptides excised from plasma-borne kininogens through the activity of cruzipain, induces dendritic cell maturation via BK B(2) receptors (B(2)R). Here, we used the s.c. model of T. cruzi infection to study the functional interplay of TLR2, CXCR2, and B(2)R in edema development. Using intravital microscopy, we found that repertaxin (CXCR2 antagonist) blocked tissue-culture trypomastigotes (TCT)-induced plasma leakage and leukocyte accumulation in the hamster cheek pouch topically exposed to TCT. Furthermore, we found that TCT-evoked paw edema in BALB/c mice was blocked by repertaxin or HOE-140 (B(2)R antagonist), suggesting that CXCR2 propels the extravascular activation of the kinin/B(2)R pathway. We then asked if TLR2-mediated sensing of TCT by innate sentinel cells could induce secretion of CXC chemokines, which would then evoke neutrophil-dependent plasma leakage via the CXCR2/B(2)R pathway. Consistent with this notion, in vitro studies revealed that TCT induce robust secretion of CXC chemokines by resident macrophages in a TLR2-dependent manner. In contrast, TLR2(+/+) macrophages stimulated with insect-derived metacyclic trypomastigotes or epimastigotes, which lack the developmentally regulated TLR2 agonist displayed by TCT, failed to secrete keratinocyte-derived chemokine/MIP-2. Collectively, these results suggest that secretion of CXC chemokines by innate sentinel cells links TLR2-dependent recognition of TCT to the kinin system, a proteolytic web that potently amplifies vascular inflammation and innate immunity through the extravascular release of BK.

During the course of Chagas disease, infectious forms of Trypanosoma cruzi are occasionally liberated from parasitized heart cells. Studies performed with tissue culture trypomastigotes (TCTs, Dm28c strain) demonstrated that these parasites evoke neutrophil/CXCR2-dependent microvascular leakage by activating innate sentinel cells via toll-like receptor 2 (TLR2). Upon plasma extravasation, proteolytically derived kinins and C5a stimulate immunoprotective Th1 responses via cross-talk between bradykinin B2 receptors (B2Rs) and C5aR. Awareness that TCTs invade cardiovascular cells in vitro via interdependent activation of B2R and endothelin receptors [endothelin A receptor (ETAR)/endothelin B receptor (ETBR)] led us to hypothesize that T. cruzi might reciprocally benefit from the formation of infection-associated edema via activation of kallikrein–kinin system (KKS). Using intravital microscopy, here we first examined the functional interplay between mast cells (MCs) and the KKS by topically exposing the hamster cheek pouch (HCP) tissues to dextran sulfate (DXS), a potent “contact” activator of the KKS. Surprisingly, although DXS was inert for at least 30 min, a subtle MC-driven leakage resulted in factor XII (FXII)-dependent activation of the KKS, which then amplified inflammation via generation of bradykinin (BK). Guided by this mechanistic insight, we next exposed TCTs to “leaky” HCP—forged by low dose histamine application—and found that the proinflammatory phenotype of TCTs was boosted by BK generated via the MC/KKS pathway. Measurements of footpad edema in MC-deficient mice linked TCT-evoked inflammation to MC degranulation (upstream) and FXII-mediated generation of BK (downstream). We then inoculated TCTs intracardiacally in mice and found a striking decrease of parasite DNA (quantitative polymerase chain reaction; 3 d.p.i.) in the heart of MC-deficient mutant mice. Moreover, the intracardiac parasite load was significantly reduced in WT mice pretreated with (i) cromoglycate (MC stabilizer) (ii) infestin-4, a specific inhibitor of FXIIa (iii) HOE-140 (specific antagonist of B2R), and (iv) bosentan, a non-selective antagonist of ETAR/ETBR. Notably, histopathology of heart tissues from mice pretreated with these G protein-coupled receptors blockers revealed that myocarditis and heart fibrosis (30 d.p.i.) was markedly and redundantly attenuated. Collectively, our study suggests that inflammatory edema propagated via activation of the MC/KKS pathway fuels intracardiac parasitism by generating infection-stimulatory peptides (BK and endothelins) in the edematous heart tissues.

IntroductionPulmonary fibrosis is a destructive, progressive disease that dramatically reduces life quality of patients, ultimately leading to death. Therapeutic regimens for pulmonary fibrosis have shown limited benefits, hence justifying the efforts to evaluate the outcome of alternative treatments.MethodsUsing a mouse model of bleomycin (BLM)-induced lung fibrosis, in the current work we asked whether treatment with pro-resolution molecules, such as pro-resolving lipid mediators (SPMs) could ameliorate pulmonary fibrosis. To this end, we injected aspirin-triggered resolvin D1 (7S,8R,17R-trihydroxy-4Z,9E,11E,13Z,15E19Z-docosahexaenoic acid; ATRvD1; i.v.) 7 and 10 days after BLM (intratracheal) challenge and samples were two weeks later.Results and discussionAssessment of outcome in the lung tissues revealed that ATRvD1 partially restored lung architecture, reduced leukocyte infiltration, and inhibited formation of interstitial edema. In addition, lung tissues from BLM-induced mice treated with ATRvD1 displayed reduced levels of TNF-α, MCP-1, IL-1-β, and TGF-β. Of further interest, ATRvD1 decreased lung tissue expression of MMP-9, without affecting TIMP-1. Highlighting the beneficial effects of ATRvD1, we found reduced deposition of collagen and fibronectin in the lung tissues. Congruent with the anti-fibrotic effects that ATRvD1 exerted in lung tissues, α-SMA expression was decreased, suggesting that myofibroblast differentiation was inhibited by ATRvD1. Turning to culture systems, we next showed that ATRvD1 impaired TGF-β-induced fibroblast differentiation into myofibroblast. After showing that ATRvD1 hampered extracellular vesicles (EVs) release in the supernatants from TGF-β-stimulated cultures of mouse macrophages, we verified that ATRvD1 also inhibited the release of EVs in the bronco-alveolar lavage (BAL) fluid of BLM-induced mice. Motivated by studies showing that BLM-induced lung fibrosis is linked to angiogenesis, we asked whether ATRvD1 could blunt BLM-induced angiogenesis in the hamster cheek pouch model (HCP). Indeed, our intravital microscopy studies confirmed that ATRvD1 abrogates BLM-induced angiogenesis. Collectively, our findings suggest that treatment of pulmonary fibrosis patients with ATRvD1 deserves to be explored as a therapeutic option in the clinical setting.