RESUMEN
Subverting the host immune response to inhibit inflammation is a key virulence strategy of Yersinia pestis. The inflammatory cascade is tightly controlled via the sequential action of lipid and protein mediators of inflammation. Because delayed inflammation is essential for Y. pestis to cause lethal infection, defining the Y. pestis mechanisms to manipulate the inflammatory cascade is necessary to understand this pathogen's virulence. While previous studies have established that Y. pestis actively inhibits the expression of host proteins that mediate inflammation, there is currently a gap in our understanding of the inflammatory lipid mediator response during plague. Here we used the murine model to define the kinetics of the synthesis of leukotriene B4 (LTB4), a pro-inflammatory lipid chemoattractant and immune cell activator, within the lungs during pneumonic plague. Furthermore, we demonstrated that exogenous administration of LTB4 prior to infection limited bacterial proliferation, suggesting that the absence of LTB4 synthesis during plague contributes to Y. pestis immune evasion. Using primary leukocytes from mice and humans further revealed that Y. pestis actively inhibits the synthesis of LTB4. Finally, using Y. pestis mutants in the Ysc type 3 secretion system (T3SS) and Yersinia outer protein (Yop) effectors, we demonstrate that leukocytes recognize the T3SS to initiate the rapid synthesis of LTB4. However, several Yop effectors secreted through the T3SS effectively inhibit this host response. Together, these data demonstrate that Y. pestis actively inhibits the synthesis of the inflammatory lipid LTB4 contributing to the delay in the inflammatory cascade required for rapid recruitment of leukocytes to sites of infection.
Asunto(s)
Peste , Yersinia pestis , Humanos , Animales , Ratones , Yersinia pestis/metabolismo , Peste/microbiología , Sistemas de Secreción Tipo III/metabolismo , Leucotrieno B4/metabolismo , Leucocitos/metabolismo , Inflamación , Proteínas Bacterianas/metabolismoRESUMEN
Chronic inflammation is the underlying cause of many diseases, including type 1 diabetes, obesity, and non-alcoholic fatty liver disease. Macrophages are continuously recruited to tissues during chronic inflammation where they exacerbate or resolve the pro-inflammatory environment. Although leukotriene B4 receptor 2 (BLT2) has been characterized as a low affinity receptor to several key eicosanoids and chemoattractants, its precise roles in the setting of inflammation and macrophage function remain incompletely understood. Here we used zebrafish and mouse models to probe the role of BLT2 in macrophage function during inflammation. We detected BLT2 expression in bone marrow derived and peritoneal macrophages of mouse models. Transcriptomic analysis of Ltb4r2-/- and WT macrophages suggested a role for BLT2 in macrophage migration, and studies in vitro confirmed that whereas BLT2 does not mediate macrophage polarization, it is required for chemotactic function, possibly mediated by downstream genes Ccl5 and Lgals3. Using a zebrafish model of tailfin injury, we demonstrated that antisense morpholino-mediated knockdown of blt2a or chemical inhibition of BLT2 signaling impairs macrophage migration. We further replicated these findings in zebrafish models of islet injury and liver inflammation. Moreover, we established the applicability of our zebrafish findings to mammals by showing that macrophages of Ltb4r2-/- mice have defective migration during lipopolysaccharide stimulation in vivo. Collectively, our results demonstrate that BLT2 mediates macrophage migration during inflammation, which implicates it as a potential therapeutic target for inflammatory pathologies.
Asunto(s)
Movimiento Celular , Macrófagos , Receptores de Leucotrieno B4 , Animales , Ratones , Inflamación/genética , Inflamación/metabolismo , Leucotrieno B4/genética , Leucotrieno B4/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Receptores de Leucotrieno B4/genética , Receptores de Leucotrieno B4/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Pneumocystis pneumonia (PCP) is a fungal pulmonary disease with high mortality in immunocompromised patients. Neutrophils are essential in defending against fungal infections; however, their role in PCP is controversial. Here we aim to investigate the effects of neutrophil extracellular traps (NETs) on Pneumocystis clearance and lung injury using a mouse model of PCP. Intriguingly, although neutrophils play a fundamental role in defending against fungal infections, NETs failed to eliminate Pneumocystis, but instead impaired the killing of Pneumocystis. Mechanically, Pneumocystis triggered Leukotriene B4 (LTB4)-dependent neutrophil swarming, leading to agglutinative NET formation. Blocking Leukotriene B4 with its receptor antagonist Etalocib significantly reduced the accumulation and NET release of neutrophils in vitro and in vivo, enhanced the killing ability of neutrophils against Pneumocystis, and alleviated lung injury in PCP mice. This study identifies the deleterious role of agglutinative NETs in Pneumocystis infection and reveals a new way to prevent NET formation, which provides new insights into the pathogenesis of PCP.
Asunto(s)
Trampas Extracelulares , Leucotrieno B4 , Neutrófilos , Pneumocystis , Neumonía por Pneumocystis , Trampas Extracelulares/inmunología , Animales , Ratones , Neutrófilos/inmunología , Neumonía por Pneumocystis/inmunología , Leucotrieno B4/metabolismo , Leucotrieno B4/inmunología , Pneumocystis/inmunología , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , HumanosRESUMEN
Treatment of tuberculosis (TB) is faced with several challenges including the long treatment duration, drug toxicity and tissue pathology. Host-directed therapy provides promising avenues to find compounds for adjunctively assisting antimycobacterials in the TB treatment regimen, by promoting pathogen eradication or limiting tissue destruction. Eicosanoids are a class of lipid molecules that are potent mediators of inflammation and have been implicated in aspects of the host response against TB. Here, we have explored the blood transcriptome of pulmonary TB patients to understand the activity of leukotriene B4, a pro-inflammatory eicosanoid. Our study shows a significant upregulation in the leukotriene B4 signalling pathway in active TB patients, which is reversed with TB treatment. We have further utilized our in-house network analysis algorithm, ResponseNet, to identify potential downstream signal effectors of leukotriene B4 in TB patients including STAT1/2 and NADPH oxidase at a systemic as well as local level, followed by experimental validation of the same. Finally, we show the potential of inhibiting leukotriene B4 signalling as a mode of adjunctive host-directed therapy against TB. This study provides a new mode of TB treatment along with mechanistic insights which can be further explored in pre-clinical trials.
Asunto(s)
Leucotrieno B4 , Mycobacterium tuberculosis , Transducción de Señal , Tuberculosis Pulmonar , Humanos , Leucotrieno B4/metabolismo , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/tratamiento farmacológico , Tuberculosis Pulmonar/metabolismo , Mycobacterium tuberculosis/inmunología , Antituberculosos/uso terapéutico , Antituberculosos/farmacología , Masculino , Femenino , Adulto , Persona de Mediana Edad , NADPH Oxidasas/metabolismo , Interacciones Huésped-PatógenoRESUMEN
Invasive fungal infections are life-threatening, and neutrophils are vital cells of the innate immune system that defend against them. The role of LTA4H-LTB4-BLT1 axis in regulation of neutrophil responses to fungal infection remains poorly understood. Here, we demonstrated that the LTA4H-LTB4-BLT1 axis protects the host against Candida albicans and Aspergillus fumigatus, but not Cryptococcus neoformans infection, by regulating the antifungal activity of neutrophils. Our results show that deleting Lta4h or Blt1 substantially impairs the fungal-specific phagocytic capacity of neutrophils. Moreover, defective activation of the spleen tyrosine kinase (Syk) and extracellular signal-related kinase (ERK1/2) pathways in neutrophils accompanies this impairment. Mechanistically, BLT1 regulates CR3-mediated, ß-1,3-glucan-induced neutrophil phagocytosis, while a physical interaction with CR3 with slight influence on its dynamics is observed. Our findings thus demonstrate that the LTA4H-LTB4-BLT1 axis is essential for the phagocytic function of neutrophils in host antifungal immune response against Candida albicans and Aspergillus fumigatus.
Asunto(s)
Antifúngicos , Neutrófilos , Antifúngicos/farmacología , Leucotrieno B4/metabolismo , Receptores de Leucotrienos/metabolismo , Receptores de Leucotrieno B4/metabolismo , Antígeno CD11b/metabolismoRESUMEN
BACKGROUND: Cardiac allograft vasculopathy (CAV) remains the leading cause of long-term graft failure and mortality after heart transplantation. Effective preventive and treatment options are not available to date, largely because underlying mechanisms remain poorly understood. We studied the potential role of leukotriene B4 (LTB4), an inflammatory lipid mediator, in the development of CAV. METHODS: We used an established preclinical rat CAV model to study the role of LTB4 in CAV. We performed syngeneic and allogeneic orthotopic aortic transplantation, after which neointimal proliferation was quantified. Animals were then treated with Bestatin, an inhibitor of LTB4 synthesis, or vehicle control for 30 days post-transplant, and evidence of graft CAV was determined by histology. We also measured serial LTB4 levels in a cohort of 28 human heart transplant recipients with CAV, 17 matched transplant controls without CAV, and 20 healthy nontransplant controls. RESULTS: We showed that infiltration of the arterial wall with macrophages leads to neointimal thickening and a rise in serum LTB4 levels in our rat model of CAV. Inhibition of LTB4 production with the drug Bestatin prevents development of neointimal hyperplasia, suggesting that Bestatin may be effective therapy for CAV prevention. In a parallel study of heart transplant recipients, we found nonsignificantly elevated plasma LTB4 levels in patients with CAV, compared to patients without CAV and healthy, nontransplant controls. CONCLUSIONS: This study provides key evidence supporting the role of the inflammatory cytokine LTB4 as an important mediator of CAV development and provides preliminary data suggesting the clinical benefit of Bestatin for CAV prevention.
Asunto(s)
Biomarcadores , Trasplante de Corazón , Leucotrieno B4 , Animales , Trasplante de Corazón/efectos adversos , Leucotrieno B4/sangre , Leucotrieno B4/metabolismo , Ratas , Masculino , Biomarcadores/metabolismo , Biomarcadores/sangre , Humanos , Modelos Animales de Enfermedad , Aloinjertos , Persona de Mediana Edad , Ratas Endogámicas Lew , Femenino , Neointima/patologíaRESUMEN
Abstract Purpose To evaluate the kinetics of apical periodontitis development in vivo , induced either by contamination of the root canals by microorganisms from the oral cavity or by inoculation of bacterial lipopolysaccharide (LPS) and the regulation of major enzymes and receptors involved in the arachidonic acid metabolism. Methodology Apical periodontitis was induced in C57BL6 mice (n=96), by root canal exposure to oral cavity (n=48 teeth) or inoculation of LPS (10 µL of a suspension of 0.1 µg/µL) from E. coli into the root canals (n= 48 teeth). Healthy teeth were used as control (n=48 teeth). After 7, 14, 21 and 28 days the animals were euthanized and tissues removed for histopathological and qRT-PCR analyses. Histological analysis data were analyzed using two-way ANOVA followed by Sidak's test, and qRT-PCR data using two-way ANOVA followed by Tukey's test (α=0.05). Results Contamination by microorganisms led to the development of apical periodontitis, characterized by the recruitment of inflammatory cells and bone tissue resorption, whereas inoculation of LPS induced inflammatory cells recruitment without bone resorption. Both stimuli induced mRNA expression for cyclooxygenase-2 and 5-lipoxygenase enzymes. Expression of prostaglandin E 2 and leukotriene B 4 cell surface receptors were more stimulated by LPS. Regarding nuclear peroxisome proliferator-activated receptors (PPAR), oral contamination induced the synthesis of mRNA for PPARδ, differently from inoculation of LPS, that induced PPARα and PPARγ expression. Conclusions Contamination of the root canals by microorganisms from oral cavity induced the development of apical periodontitis differently than by inoculation with LPS, characterized by less bone loss than the first model. Regardless of the model used, it was found a local increase in the synthesis of mRNA for the enzymes 5-lipoxygenase and cyclooxygenase-2 of the arachidonic acid metabolism, as well as in the surface and nuclear receptors for the lipid mediators prostaglandin E2 and leukotriene B4.
Asunto(s)
Animales , Masculino , Periodontitis Periapical/microbiología , Dinoprostona/metabolismo , Lipopolisacáridos/metabolismo , Leucotrieno B4/metabolismo , Cavidad Pulpar/microbiología , Periodontitis Periapical/metabolismo , Periodontitis Periapical/patología , Factores de Tiempo , Resorción Ósea/metabolismo , Resorción Ósea/microbiología , Araquidonato 5-Lipooxigenasa/análisis , Araquidonato 5-Lipooxigenasa/metabolismo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Dinoprostona/análisis , Distribución Aleatoria , Expresión Génica , Leucotrieno B4/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cavidad Pulpar/metabolismo , Cavidad Pulpar/patología , Ciclooxigenasa 2/análisis , Ciclooxigenasa 2/metabolismo , Ratones Endogámicos C57BLRESUMEN
Endotoxic responses to bacterial lipopolysaccharide (LPS) are triggered by Toll-like receptor 4 (TLR4) and involve the production of inflammatory mediators, including interleukin-6 (IL-6), by macrophages. The detailed mechanism of IL-6 production by macrophages in response to LPS has remained unclear, however. We now show that LPS induces IL-6 synthesis in mouse peritoneal macrophages via the leukotriene B4 receptor BLT2. Our results suggest that TLR4-MyD88 signaling functions upstream of BLT2 and that the generation of reactive oxygen species (ROS) by NADPH oxidase 1 (Nox1) and consequent activation of the transcription factor nuclear factor (NF)-kappaB function downstream of BLT2 in this response. These results suggest that a TLR4-MyD88-BLT2-Nox1-ROS-NF-kappaB pathway contributes to the synthesis of IL-6 in LPS-stimulated mouse macrophages.
Asunto(s)
Animales , Ratones , Línea Celular , Interleucina-6/biosíntesis , Leucotrieno B4/metabolismo , Ligandos , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Macrófagos Peritoneales/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de Leucotrieno B4/metabolismo , Transducción de SeñalRESUMEN
A variety of benzylidenethiazole analogs have been demonstrated to inhibit 5-lipoxygenase (5-LOX). Here we report the anti-atherogenic potential of 5-(4-hydroxy-2,3,5-trimethylbenzylidene) thiazolidin-2,4-dione (HMB-TZD), a benzylidenethiazole analog, and its potential mechanism of action in LDL receptor-deficient (Ldlr-/-) mice. HMB-TZD Treatment reduced leukotriene B4 (LTB4) production significantly in RAW264.7 macrophages and SVEC4-10 endothelial cells. Macrophages or endothelial cells pre-incubated with HMB-TZD for 2 h and then stimulated with lipopolysaccharide or tumor necrosis factor-alpha (TNF-alpha) displayed reduced cytokine production. Also, HMB-TZD reduced cell migration and adhesion in accordance with decreased proinflammatory molecule production in vitro and ex vivo. HMB-TZD treatment of 8-week-old male Ldlr-/- mice resulted in significantly reduced atherosclerotic lesions without a change to plasma lipid profiles. Moreover, aortic expression of pro-atherogenic molecules involved in the recruitment of monocytes to the aortic wall, including TNF-alpha , MCP-1, and VCAM-1, was downregulated. HMB-TZD also reduced macrophage infiltration into atherosclerotic lesions. In conclusion, HMB-TZD ameliorates atherosclerotic lesion formation possibly by reducing the expression of proinflammatory molecules and monocyte/macrophage recruitment to the lesion. These results suggest that HMB-TZD, and benzylidenethiazole analogs in general, may have therapeutic potential as treatments for atherosclerosis.