The parasitic protozoan Trypanosoma cruzi employs multiple molecular strategies to invade a broad range of nonphagocytic cells. Here we demonstrate that the invasion of human primary umbilical vein endothelial cells (HUVECs) or Chinese hamster ovary (CHO) cells overexpressing the B₂ type of bradykinin receptor (CHO-B₂R) by tissue culture trypomastigotes is subtly modulated by the combined activities of kininogens, kininogenases, and kinin-degrading peptidases. The presence of captopril, an inhibitor of bradykinin degradation by kininase II, drastically potentiated parasitic invasion of HUVECs and CHO-B₂R, but not of mock-transfected CHO cells, whereas the B₂R antagonist HOE 140 or monoclonal antibody MBK3 to bradykinin blocked these effects. Invasion competence correlated with the parasites9 ability to liberate the short-lived kinins from cell-bound kininogen and to elicit vigorous intracellular free calcium ([Ca²⁺]_i) transients through B₂R. Invasion was impaired by membrane-permeable cysteine proteinase inhibitors such as Z-(SBz)Cys-Phe-CHN₂ but not by the hydrophilic inhibitor 1-trans-epoxysuccinyl-l-leucyl-amido-(4-guanidino) butane or cystatin C, suggesting that kinin release is confined to secluded spaces formed by juxtaposition of host cell and parasite plasma membranes. Analysis of trypomastigote transfectants expressing various cysteine proteinase isoforms showed that invasion competence is linked to the kinin releasing activity of cruzipain, herein proposed as a factor of virulence in Chagas9 disease.

Lysosomal cysteine proteases from mammalian cells and plants are regulated by endogenous tight-binding inhibitors from the cystatin superfamily. The presence of cystatin-like inhibitors in lower eukaryotes such as protozoan parasites has not yet been demonstrated, although these cells express large quantities of cysteine proteases and may also count on endogenous inhibitors to regulate cellular proteolysis. Trypanosoma cruzi, the causative agent of Chagas’ heart disease, is a relevant model to explore this possibility because these intracellular parasites rely on their major lysosomal cysteine protease (cruzipain) to invade and multiply in mammalian host cells. Here we report the isolation, biochemical characterization, developmental stage distribution and subcellular localization of chagasin, an endogenous cysteine protease inhibitor in T. cruzi. We used high temperature induced denaturation to isolate a heat-stable cruzipain-binding protein (apparent molecular mass, 12 kDa) from epimastigote lysates. This protein was subsequently characterized as a tight-binding and reversible inhibitor of papain-like cysteine proteases. Immunoblotting indicated that the expression of chagasin is developmentally regulated and inversely correlated with that of cruzipain. Gold-labeled antibodies localized chagasin to the flagellar pocket and cytoplasmic vesicles of trypomastigotes and to the cell surface of amastigotes. Binding assays performed by probing living parasites with fluorescein (FITC)-cruzipain or FITC-chagasin revealed the presence of both inhibitor and protease at the cell surface of amastigotes. The intersection of chagasin and cruzipain trafficking pathways may represent a checkpoint for downstream regulation of proteolysis in trypanosomatid protozoa.

We have previously reported that exogenous bradykinin activates immature dendritic cells (DCs) via the bradykinin B₂ receptor (B₂R), thereby stimulating adaptive immunity. In this study, we show that these premises are met in a model of s.c. infection by Trypanosoma cruzi, a protozoan that liberates kinins from kininogens through its major protease, cruzipain. Intensity of B₂R-dependent paw edema evoked by trypomastigotes correlated with levels of IL-12 produced by CD11c⁺ dendritic cells isolated from draining lymph nodes. The IL-12 response induced by endogenously released kinins was vigorously increased in infected mice pretreated with inhibitors of angiotensin converting enzyme (ACE), a kinin-degrading metallopeptidase. Furthermore, these innate stimulatory effects were linked to B₂R-dependent up-regulation of IFN-γ production by Ag-specific T cells. Strikingly, the trypomastigotes failed to up-regulate type 1 immunity in TLR2^−/− mice, irrespective of ACE inhibitor treatment. Analysis of the dynamics of inflammation revealed that TLR2 triggering by glycosylphosphatidylinositol-anchored mucins induces plasma extravasation, thereby favoring peripheral accumulation of kininogens in sites of infection. Further downstream, the parasites generate high levels of innate kinin signals in peripheral tissues through the activity of cruzipain. The demonstration that the deficient type 1 immune responses of TLR2^−/− mice are rescued upon s.c. injection of exogenous kininogens, along with trypomastigotes, supports the notion that generation of kinin “danger” signals is intensified through cooperative activation of TLR2 and B₂R. In summary, we have described a s.c. infection model where type 1 immunity is vigorously up-regulated by bradykinin, an innate signal whose levels in peripheral tissues are controlled by an intricate interplay of TLR2, B₂R, and ACE.

Although the concept that dendritic cells (DCs) recognize pathogens through the engagement of Toll-like receptors is widely accepted, we recently suggested that immature DCs might sense kinin-releasing strains of Trypanosoma cruzi through the triggering of G-protein-coupled bradykinin B₂ receptors (B₂R). Here we report that C57BL/6.B₂R^−/− mice infected intraperitoneally with T. cruzi display higher parasitemia and mortality rates as compared to B₂R^+/+ mice. qRT-PCR revealed a 5-fold increase in T. cruzi DNA (14 d post-infection [p.i.]) in B₂R^−/− heart, while spleen parasitism was negligible in both mice strains. Analysis of recall responses (14 d p.i.) showed high and comparable frequencies of IFN-γ-producing CD4⁺ and CD8⁺ T cells in the spleen of B₂R^−/− and wild-type mice. However, production of IFN-γ by effector T cells isolated from B₂R^−/− heart was significantly reduced as compared with wild-type mice. As the infection continued, wild-type mice presented IFN-γ-producing (CD4⁺CD44⁺ and CD8⁺CD44⁺) T cells both in the spleen and heart while B₂R^−/− mice showed negligible frequencies of such activated T cells. Furthermore, the collapse of type-1 immune responses in B₂R^−/− mice was linked to upregulated secretion of IL-17 and TNF-α by antigen-responsive CD4⁺ T cells. In vitro analysis of tissue culture trypomastigote interaction with splenic CD11c⁺ DCs indicated that DC maturation (IL-12, CD40, and CD86) is controlled by the kinin/B₂R pathway. Further, systemic injection of trypomastigotes induced IL-12 production by CD11c⁺ DCs isolated from B₂R^+/+ spleen, but not by DCs from B₂R^−/− mice. Notably, adoptive transfer of B₂R^+/+ CD11c⁺ DCs (intravenously) into B₂R^−/− mice rendered them resistant to acute challenge, rescued development of type-1 immunity, and repressed T_H17 responses. Collectively, our results demonstrate that activation of B₂R, a DC sensor of endogenous maturation signals, is critically required for development of acquired resistance to T. cruzi infection. Antibodies and IFN-γ-producing effector T cells are essential for the immune control of infection by Trypanosoma cruzi, the intracellular protozoa that causes human Chagas disease. Despite the potency of anti-parasite immunity, the parasites are not cleared from their intracellular niches. Instead, a low grade chronic infection prevails, provoking severe immunopathology in the myocardium. Although it is well established that innate sentinel cells sense T. cruzi through receptors for microbial structures, such as Toll-like receptors, it remained unclear whether endogenous inflammatory signals also contribute to the development of adaptive immunity. The present study was motivated by awareness that T. cruzi trypomastigotes (extracellular infective forms) are equipped with proteases that liberate the pro-inflammatory bradykinin peptide from an internal segment of kininogens. Here we demonstrate that splenic dendritic cells (DCs), the antigen-presenting cells that coordinate the adaptive branch of immunity in lymphoid tissues, are potently activated via G-protein-coupled bradykinin B₂ receptors (B₂R). Analysis of the outcome of infection in B₂R-knockout mice revealed that the mutant mice developed a typical susceptible phenotype, owing to impaired development of IFN-γ-producing effector T cells. Notably, the immune dysfunction of B₂R-knockout mice was corrected upon cell transfer of wild-type DCs, thus linking development of protective T cells to DCs' sensing of endogenous danger signals (kinins) released by trypomastigotes.

BACKGROUND AND PURPOSE Independent 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 (ET_AR and ET_BR) and bradykinin B₂ receptors (B₂R).EXPERIMENTAL APPROACH Intravital 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), ET_AR (BQ‐123) and ET_BR (BQ‐788), specific IgG antibodies to each GPCRs; cholesterol or calcium‐depleting drugs. RNA interference (ET_AR or ET_BR genes) in parasite infectivity was investigated in HSMCs.KEY RESULTS BQ‐123, BQ‐788 and HOE‐140 reduced leucocyte accumulation in HCP topically exposed to trypomastigotes and blocked inflammatory oedema in infected mice. Acting synergistically, ET_AR and ET_BR 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 ET_AR and ET_BR genes conversely reduced parasite internalization. ETRs/B₂R‐driven infection in HSMCs was reduced in HSMC pretreated with methyl‐β‐cyclodextrin, a cholesterol‐depleting drug, or in thapsigargin‐ or verapamil‐treated target cells.CONCLUSIONS AND IMPLICATIONS Our 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 This paper is commented on by D'Orléans‐Juste et al., pp. 1330–1332 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476‐5381.2011.01636.x

Porphyromonas gingivalis, a Gram-negative bacterium that causes periodontitis, activates the kinin system via the cysteine protease R-gingipain. Using a model of buccal infection based on P. gingivalis inoculation in the anterior mandibular vestibule, we studied whether kinins released by gingipain may link mucosal inflammation to T cell-dependent immunity through the activation of bradykinin B₂ receptors (B₂R). Our data show that P. gingivalis W83 (wild type), but not gingipain-deficient mutant or wild-type bacteria pretreated with gingipain inhibitors, elicited buccal edema and gingivitis in BALB/c or C57BL/6 mice. Studies in TLR2^−/−, B₂R^−/−, and neutrophil-depleted C57BL/6 mice revealed that P. gingivalis induced edema through the sequential activation of TLR2/neutrophils, with the initial plasma leakage being amplified by gingipain-dependent release of vasoactive kinins from plasma-borne kininogens. We then used fimbriae (Fim) Ag as a readout to verify whether activation of the TLR2→PMN→B₂R axis (where PMN is polymorphonuclear neutrophil) at early stages of mucosal infection had impact on adaptive immunity. Analyzes of T cell recall responses indicated that gingipain drives B₂R-dependent generation of IFN-γ-producing Fim T cells in submandibular draining lymph nodes of BALB/c and C57BL/6 mice, whereas IL-17-producing Fim T cells were generated only in BALB/c mice. In summary, our studies suggest that two virulence factors, LPS (an atypical TLR2 ligand) and gingipain, forge a trans-cellular cross-talk between TLR2 and B₂R, thus forming an innate axis that guides the development of Fim-specific T cells in mice challenged intrabuccally by P. gingivalis. Ongoing research may clarify whether kinin-driven modulation of T cell responses may also influence the severity of chronic periodontitis.