Abstract
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.
Highlights
The complement system is a classic example of a proteolytic cascade that stimulates immunity through the generation of proinflammatory peptides, studies in different infectious disease models extended this concept to the kallikrein–kinin system (KKS) [1]
intravital microscopy (IVM) in the hamster cheek pouch (HCP) seemed to be an appropriate system to address this question because (i) mast cells (MCs) are predominantly positioned along arterioles of the HCP and respond to topically applied stimuli such as 48/80 [51], (ii) the pathogens are topically applied to the HCP [33], dispensing the use of needles—a procedure that causes bleeding and spurious KKS activation, and (iii) hamsters are susceptible to T. cruzi infection and exhibit a dilated chronic cardiomyopathy that closely resembles the human heart disease [52]
Before checking whether T. cruzi-induced inflammation involved activation of the MC/KKS pathway, we performed a series of mechanistic studies in a simplified model involving topic application of dextran sulfate (DXS) (500 kDa)—a classical activator of the contact system [53], here used to simulate the effect of MC-derived triggers, such as heparin and polyP, both of which stored in MC granules [11, 12]
Summary
The complement system is a classic example of a proteolytic cascade that stimulates immunity through the generation of proinflammatory peptides, studies in different infectious disease models extended this concept to the kallikrein–kinin system (KKS) [1]. Feedback cycles of activation between PK/FXII lead to FXIIa-mediated cleavage of FXIa. Further downstream, FXIa generates FIXa—the effector of fibrin formation via the intrinsic pathway of coagulation. PK activates the proinflammatory KKS by proteolytically excising bradykinin (BK) from an internal moiety of high molecular weight kininogen (HK). A GPI-linked carboxypeptidase M removes the C-terminal arginine from the primary kinin, converting the B2R agonist into a high-affinity ligand for bradykinin B1 receptor (B1R) [6], a G protein-coupled receptor (GPCR) subtype that is transcriptionally upregulated in injured tissues by proinflammatory cues, such as IL-1β, TNF-α [7, 8], or by prooxidative polypeptides, e.g., angiotensin II and endothelin-1 (ET-1) [9]
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