Erik Svensjö

Cooperative Activation of TLR2 and Bradykinin B2 Receptor Is Required for Induction of Type 1 Immunity in a Mouse Model of Subcutaneous Infection by Trypanosoma cruzi

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.

Trypanosoma cruzi invades host cells through the activation of endothelin and bradykinin receptors: a converging pathway leading to chagasic vasculopathy

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 (ETAR and ETBR) and bradykinin B2 receptors (B2R).

Kinin Danger Signals Proteolytically Released by Gingipain Induce Fimbriae-Specific IFN-γ- and IL-17-Producing T Cells in Mice Infected Intramucosally with Porphyromonas gingivalis

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.

Proteolytic generation of kinins in tissues infected by Trypanosoma cruzi depends on CXC chemokine secretion by macrophages activated via Toll-like 2 receptors

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 B2 receptors (B2R). Here, we used the s.c. model of T. cruzi infection to study the functional interplay of TLR2, CXCR2, and B2R 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 (B2R antagonist), suggesting that CXCR2 propels the extravascular activation of the kinin/B2R 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/B2R 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.

Kininogens coordinate adaptive immunity through the proteolytic release of bradykinin, an endogenous danger signal driving dendritic cell maturation.

Strategically positioned in peripheral tissues, immune sentinel cells sense microbes and/or their shed products through different types of pattern‐recognition receptors. Upon secretion, pre‐formed pro‐inflammatory mediators activate the microvasculature, inducing endothelium/neutrophil adherence and impairing endothelium barrier function. As plasma proteins enter into peripheral tissues, short‐lived proinflammatory peptides are rapidly generated by limited proteolysis of complement components and the kininogens (i.e. kinin‐precursor proteins). While much emphasis has been placed on the studies of the vascular functions of kinins, their innate effector roles remain virtually unknown. A few years ago, we reported that exogenous bradykinin (BK) potently induces dendritic cell (DC) maturation, driving IL‐12‐dependent Th1 responses through the activation of G‐protein‐coupled BK B2 receptors (B2R). The premise that immature DC might sense kinin‐releasing pathogens through B2R was demonstrated in the subcutaneous mouse model of Trypanosoma cruzi infection. Analysis of the dynamics of parasite‐evoked inflammation revealed that activation of TLR2/neutrophils drives the influx of plasma proteins, including kininogens, into peripheral tissues. Once associated to cell surfaces and/or extracellular matrices, the surface‐bound kininogens are cleaved by T. cruzi cysteine proteases. Acting as short‐lived ‘danger’ signals, kinins activate DC via B2R, converting them into Th1 inducers. Fine tuned control of the extravascular levels of these natural peptide adjuvants is exerted by kinin‐degrading metallopeptidases, e.g. Angiotensin converting enzyme (ACE/CD143). In summary, the studies in the subcutaneous model of T. cruzi infection revealed that the peripheral levels of BK, a DC maturation signal, are controlled by TLR2/neutrophils and ACE, respectively characterized as positive and negative modulators of innate/adaptive immunity.

Vascular permeability increase and plasma volume loss induced by endotoxin was attenuated by hypertonic saline with or without dextran.

Endotoxin given intravenously is known to cause plasma leakage and subsequent loss of circulating plasma volume. Hypertonic saline resuscitation has been successfully applied in hemorrhagic and traumatic shock, but its application for the treatment or prevention of septic or endotoxin shock is less well studied. Our aim was to investigate the effects of endotoxin on plasma leakage in hamsters when administered in two different ways: applied locally to the hamster cheek pouch microcirculation or systemically by i.v. injection. The cheek pouch was studied by intravital microscopy using FITC-labeled dextran as a tracer of plasma leakage. Escherichia coli lipopolysaccharide (LPS) was continuously added into the superfusion buffer of the cheek pouch preparation during 120 min in two control groups (each n = 6) and two further groups (each n = 6) treated with either hypertonic saline (HS) or hypertonic saline and dextran (HSD). Treatment was given as an i.v. injection 0.35 mL NaCl 7.5%/100 g b.w. during 4 min starting 15 min prior to the start of endotoxin application. Endotoxin caused a reversible increase in the number of postcapillary venular leaks with a maximal response at 70 min after start of endotoxin application. The maximal responses were reduced to 36% in the HS-treated and to 37% in the HSD-treated group in comparison to what was seen in the control groups. In the second part of the study endotoxin was given i.v. 0.3 mg/kg to anesthetized hamsters (n = 41) and arterial blood samples were collected at 0, 60, 120, and 180 min after endotoxin injection for measurement of hematocrit and plasma FITC-dextran concentration. Hamsters were divided into seven groups: untreated control group (n = 6); HSC control group given only an i.v. injection of hypertonic saline (n = 6); LPS group given endotoxin 0.3 mg/kg during 1 min (n = 9); HSp group given hypertonic saline (NaCl 7.5%) 10 min prior to i.v. endotoxin (n = 6); HSa group given hypertonic saline 10 min after i.v. endotoxin (n = 6); HSD group given hypertonic saline with dextran 40, 10 min prior to i.v. endotoxin (n = 6); HSD control group given only i.v. hypertonic saline + dextran and no endotoxin (n = 2). Injection of endotoxin caused a significant increase in hematocrit, which was counteracted by hypertonic saline treatment, with or without dextran, probably due to reduced extravasation of plasma in postcapillary venules.

The kallikrein-kinin system in experimental Chagas disease: a paradigm to investigate the impact of inflammatory edema on GPCR-mediated pathways of host cell invasion by Trypanosoma cruzi

Chronic chagasic myocarditis (CCM) depends on Trypanosoma cruzi persistence in the myocardium. Studies of the proteolytic mechanisms governing host/parasite balance in peripheral sites of T. cruzi infection revealed that tissue culture trypomastigotes (TCTs) elicit inflammatory edema and stimulate protective type-1 effector T cells through the activation of the kallikrein-kinin system. Molecular studies linked the proinflammatory phenotype of Dm28c TCTs to the synergistic activities of tGPI, a lipid anchor that functions as a Toll-like receptor 2 (TLR2) ligand, and cruzipain, a kinin-releasing cysteine protease. Analysis of the dynamics of inflammation revealed that TCTs activate innate sentinel cells via TLR2, releasing CXC chemokines, which in turn evoke neutrophil/CXCR2-dependent extravasation of plasma proteins, including high molecular weight kininogen (HK), in parasite-laden tissues. Further downstream, TCTs process surface bound HK, liberating lysyl-BK (LBK), which then propagates inflammatory edema via signaling of endothelial G-protein-coupled bradykinin B2 receptors (BK2R). Dm28 TCTs take advantage of the transient availability of infection-promoting peptides (e.g., bradykinin and endothelins) in inflamed tissues to invade cardiovascular cells via interdependent signaling of BKRs and endothelin receptors (ETRs). Herein we present a space-filling model whereby ceramide-enriched endocytic vesicles generated by the sphingomyelinase pathway might incorporate BK2R and ETRs, which then trigger Ca2+-driven responses that optimize the housekeeping mechanism of plasma membrane repair from cell wounding. The hypothesis predicts that the NF-κB-inducible BKR (BK1R) may integrate the multimolecular signaling platforms forged by ceramide rafts, as the chronic myocarditis progresses. Exploited as gateways for parasite invasion, BK2R, BK1R, ETAR, ETBR, and other G protein-coupled receptor partners may enable persistent myocardial parasitism in the edematous tissues at expense of adverse cardiac remodeling.

Angiotensin-converting enzyme limits inflammation elicited by Trypanosoma cruzi cysteine proteases: a peripheral mechanism regulating adaptive immunity via the innate kinin pathway.

Abstract Tissue injury by pathogens induces a stereotyped inflammatory response that alerts the innate immune system of the potential threat to host integrity. Here, we review knowledge emerging from investigations of the role of the kinin system in the mechanisms that link innate to the adaptive phase of immunity. Progress in this field started with results demonstrating that bradykinin is an endogenous danger signal that induces dendritic cell (DC) maturation via G protein-coupled bradykinin B2 receptors (B2R). The immunostimulatory role of kinins was recently confirmed in two different mouse models of Trypanosoma cruzi infection, a parasitic protozoan equipped with kinin-releasing cysteine proteases (cruzipain). Infection by the intraperitoneal route showed that DCs from B2R-/- mice (susceptible phenotype) failed to sense kinin ‘danger’ signals proteolytically released by parasites, explaining why these mutant mice display lower frequencies of interferon-γ-producing effector T-cells. Studies of the dynamics of inflammation in the subcutaneous model of infection revealed that the balance between cruzipain and angiotensin-converting enzyme, respectively acting as kinin-generating and degrading enzymes, governs extent of DC maturation and TH1 development via the B2R-dependent innate pathway. Studies of the kinin role in immunity may shed light on the relationship between proteolytic networks and the cytokine circuits that guide T-cell development.

Maxadilan, the Lutzomyia longipalpis vasodilator, drives plasma leakage via PAC1–CXCR1/2-pathway

Experiments were designed to determine if the vasodilatory peptides maxadilan and pituitary adenylate cyclase-activating peptide (PACAP-38) may cause plasma leakage through activation of leukocytes and to what extent these effects could be due to PAC1 and CXCR1/2 receptor stimulation. Intravital microscopy of hamster cheek pouches utilizing FITC-dextran and rhodamine, respectively, as plasma and leukocyte markers was used to measure arteriolar diameter, plasma leakage and leukocyte accumulation in a selected area (5mm(2)) representative of the hamster cheek pouch microcirculation. Our studies showed that the sand fly vasodilator maxadilan and PACAP-38 induced arteriolar dilation, leukocyte accumulation and plasma leakage in postcapillary venules. The recombinant mutant of maxadilan M65 and an antagonist of CXCR1/2 receptors, reparixin, and an inhibitor of CD11b/CD18 up-regulation, ropivacaine, inhibited all these effects as induced by maxadilan. Dextran sulfate, a complement inhibitor with heparin-like anti-inflammatory effects, inhibited plasma leakage and leukocyte accumulation but not arteriolar dilation as induced by maxadilan and PACAP-38. In vitro studies with isolated human neutrophils showed that maxadilan is a potent stimulator of neutrophil migration comparable with fMLP and leukotriene B(4) and that M65 and reparixin inhibited such migration. The data suggest that leukocyte accumulation and plasma leakage induced by maxadilan involves a mechanism related to PAC1- and CXCR1/2-receptors on leukocytes and endothelial cells.

Salivary Gland Homogenates of Lutzomyia longipalpis and Its Vasodilatory Peptide Maxadilan Cause Plasma Leakage via PAC1 Receptor Activation

Objectives: Experiments were designed to determine if salivary gland homogenates (SGH) of the sand fly Lutzomyia longipalpis, the vasodilatory peptides maxadilan and pituitary adenylate cyclase-activating peptide (PACAP-38) may cause plasma leakage and to what extent these effects could be due to PAC1 receptor stimulation. Methods: Using FITC-dextran as a plasma marker, intravital microscopy of the hamster cheek pouch (HCP) and a digital camera were used to assess arteriolar diameter and fluorescence of a selected area (5 mm2) representative of the HCP microcirculation. Results: Cheek pouches prepared for intravital microscopy and exposed to topical application of SGH, maxadilan or PACAP-38 developed maximal dilation of arterioles in the range of 20–60 μm within 10 min, and this effect lasted for 30–90 min. The increase in fluorescence intensity induced by each of these compounds was due to plasma leakage from postcapillary venules. The mutant peptide of maxadilan (M-65), a PAC1 receptor antagonist, inhibited both dilation and plasma leakage induced by SGH or maxadilan. Plasma leakage induced by SGH was modestly inhibited by the bradykinin B2 receptor antagonist HOE-140, but not by the antihistamine mepyramine or the nitric oxide synthase inhibitor L-NA. Conclusions: SGH of L. longipalpis and its vasodilatory peptide maxadilan caused long-lasting arteriolar dilation and plasma leakage in the cheek pouch via PAC1 receptor activation.