RESUMEN
BACKGROUND AND PURPOSE: Sepsis-surviving adult individuals commonly develop immunosuppression and increased susceptibility to secondary infections, an outcome mediated by the axis IL-33/ILC2s/M2 macrophages/Tregs. Nonetheless, the long-term immune consequences of paediatric sepsis are indeterminate. We sought to investigate the role of age in the genesis of immunosuppression following sepsis. EXPERIMENTAL APPROACH: Here, we compared the frequency of Tregs, the activation of the IL-33/ILC2s axis in M2 macrophages and the DNA methylation of epithelial lung cells from post-septic infant and adult mice. Likewise, sepsis-surviving mice were inoculated intranasally with Pseudomonas aeruginosa or by subcutaneous inoculation of the B16 melanoma cell line. Finally, blood samples from sepsis-surviving patients were collected and the concentration of IL-33 and Tregs frequency were assessed. KEY RESULTS: In contrast to 6-week-old mice, 2-week-old mice were resistant to secondary infection and did not show impairment in tumour controls upon melanoma challenge. Mechanistically, increased IL-33 levels, Tregs expansion, and activation of ILC2s and M2-macrophages were observed in 6-week-old but not 2-week-old post-septic mice. Moreover, impaired IL-33 production in 2-week-old post-septic mice was associated with increased DNA methylation in lung epithelial cells. Notably, IL-33 treatment boosted the expansion of Tregs and induced immunosuppression in 2-week-old mice. Clinically, adults but not paediatric post-septic patients exhibited higher counts of Tregs and seral IL-33 levels. CONCLUSION AND IMPLICATIONS: These findings demonstrate a crucial and age-dependent role for IL-33 in post-sepsis immunosuppression. Thus, a better understanding of this process may lead to differential treatments for adult and paediatric sepsis.
Asunto(s)
Interleucina-33 , Sepsis , Humanos , Ratones , Animales , Niño , Inmunidad Innata , Linfocitos/metabolismo , Linfocitos/patología , Terapia de InmunosupresiónRESUMEN
Severe thrombocytopenia can be a determinant factor in the morbidity of Plasmodium vivax, the most widespread human malaria parasite. Although immune mechanisms may drive P. vivax-induced severe thrombocytopenia (PvST), the current data on the cytokine landscape in PvST is scarce and often conflicting. Here, we hypothesized that the analysis of the bidirectional circuit of inflammatory mediators and their regulatory miRNAs would lead to a better understanding of the mechanisms underlying PvST. For that, we combined Luminex proteomics, NanoString miRNA quantification, and machine learning to evaluate an extensive array of plasma mediators in uncomplicated P. vivax patients with different degrees of thrombocytopenia. Unsupervised clustering analysis identified a set of PvST-linked inflammatory (CXCL10, CCL4, and IL-18) and regulatory (IL-10, IL-1Ra, HGF) mediators. Among the mediators associated with PvST, IL-6 and IL-8 were critical to discriminate P. vivax subgroups, while CCL2 and IFN-γ from healthy controls. Supervised machine learning spotlighted IL-10 in P. vivax-mediated thrombocytopenia and provided evidence for a potential signaling route involving IL-8 and HGF. Finally, we identified a set of miRNAs capable of modulating these signaling pathways. In conclusion, the results place IL-10 and IL-8/HGF in the center of PvST and propose investigating these signaling pathways across the spectrum of malaria infections.
Asunto(s)
Malaria Vivax , MicroARNs , Trombocitopenia , Humanos , Mediadores de Inflamación , Plasmodium vivaxRESUMEN
BACKGROUND: Low molecular weight carrageenan (Cg) is a seaweed-derived sulfated polysaccharide widely used as inflammatory stimulus in preclinical studies. However, the molecular mechanisms of Cg-induced inflammation are not fully elucidated. The present study aimed to investigate the molecular basis involved in Cg-induced macrophages activation and cytokines production. METHODS: Primary culture of mouse peritoneal macrophages were stimulated with Kappa Cg. The supernatant and cell lysate were used for ELISA, western blotting, immunofluorescence. Cg-induced mouse colitis was also developed. RESULTS: Here we show that Cg activates peritoneal macrophages to produce pro-inflammatory cytokines such as TNF and IL-1ß. While Cg-induced TNF production/secretion depends on TLR4/MyD88 signaling, the production of pro-IL-1ß relies on TLR4/TRIF/SYK/reactive oxygen species (ROS) signaling pathway. The maturation of pro-IL1ß into IL-1ß is dependent on canonical NLRP3 inflammasome activation via Pannexin-1/P2X7/K+ efflux signaling. In vivo, Cg-induced colitis was reduced in mice in the absence of NLRP3 inflammasome components. CONCLUSIONS: In conclusion, we unravel a critical role of the NLRP3 inflammasome in Cg-induced pro-inflammatory cytokines production and colitis, which is an important discovery on the pro-inflammatory properties of this sulfated polysaccharide for pre-clinical studies. Video abstract Carrageenan (Cg) is one the most used flogistic stimulus in preclinical studies. Nevertheless, the molecular basis of Cg-induced inflammation is not totally elucidated. Herein, Lopes et al. unraveled the molecular basis for Cg-induced macrophages production of biological active IL-1ß. The Cg-stimulated macrophages produces pro-IL-1ß depends on TLR4/TRIF/Syk/ROS, whereas its processing into mature IL-1ß is dependent on the canonical NLRP3 inflammasome.
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Carragenina/inmunología , Citocinas/inmunología , Activación de Macrófagos , Macrófagos Peritoneales/inmunología , Animales , Células Cultivadas , Inflamasomas/inmunología , Inflamación/inmunología , Interleucina-1beta/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Uric acid is a damage-associated molecular pattern (DAMP), released from ischemic tissues and dying cells which, when crystalized, is able to activate the NLRP3 inflammasome. Soluble uric acid (sUA) is found in high concentrations in the serum of great apes, and even higher in some diseases, before the appearance of crystals. In the present study, we sought to investigate whether uric acid, in the soluble form, could also activate the NLRP3 inflammasome and induce the production of IL-1ß. We monitored ROS, mitochondrial area and respiratory parameters from macrophages following sUA stimulus. We observed that sUA is released in a hypoxic environment and is able to induce IL-1ß release. This process is followed by production of mitochondrial ROS, ASC speck formation and caspase-1 activation. Nlrp3-/- macrophages presented a protected redox state, increased maximum and reserve oxygen consumption ratio (OCR) and higher VDAC protein levels when compared to WT and Myd88-/- cells. Using a disease model characterized by increased sUA levels, we observed a correlation between sUA, inflammasome activation and fibrosis. These findings suggest sUA activates the NLRP3 inflammasome. We propose that future therapeutic strategies for renal fibrosis should include strategies that block sUA or inhibit its recognition by phagocytes.
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Inflamasomas/metabolismo , Enfermedades Renales/metabolismo , Riñón/patología , Macrófagos/fisiología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Ácido Úrico/metabolismo , Animales , Caspasa 1/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Fibrosis , Interleucina-1beta/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismoRESUMEN
BACKGROUND: Given the increasing evidence of Plasmodium vivax infections associated with severe and fatal disease, the identification of sensitive and reliable markers for vivax severity is crucial to improve patient care. Circulating nucleic acids (CNAs) have been increasingly recognized as powerful diagnostic and prognostic tools for various inflammatory diseases and tumors as their plasma concentrations increase according to malignancy. Given the marked inflammatory status of P. vivax infection, we investigated here the usefulness of CNAs as biomarkers for malaria morbidity. METHODS AND FINDINGS: CNAs levels in plasma from twenty-one acute P. vivax malaria patients from the Brazilian Amazon and 14 malaria non-exposed healthy donors were quantified by two different methodologies: amplification of the human telomerase reverse transcriptase (hTERT) genomic sequence by quantitative real time PCR (qPCR), and the fluorometric dsDNA quantification by Pico Green. CNAs levels were significantly increased in plasma from P. vivax patients as compared to healthy donors (p<0.0001). Importantly, plasma CNAs levels were strongly associated with vivax morbidity (p<0.0001), including a drop in platelet counts (pâ=â0.0021). These findings were further sustained when we assessed CNAS levels in plasma samples from 14 additional P. vivax patients of a different endemic area in Brazil, in which CNAS levels strongly correlated with thrombocytopenia (pâ=â0.0072). We further show that plasma CNAs levels decrease and reach physiological levels after antimalarial treatment. Although we found both host and parasite specific genomic sequences circulating in plasma, only host CNAs clearly reflected the clinical spectrum of P. vivax malaria. CONCLUSIONS: Here, we provide the first evidence of increased plasma CNAs levels in malaria patients and reveal their potential as sensitive biomarkers for vivax malaria morbidity.
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Malaria Vivax/sangre , Malaria Vivax/epidemiología , Ácidos Nucleicos/sangre , Adulto , Anciano , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Secuencia de Bases , Brasil/epidemiología , Femenino , Genoma/genética , Humanos , Malaria Vivax/complicaciones , Malaria Vivax/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Plasmodium vivax/efectos de los fármacos , Plasmodium vivax/genética , Trombocitopenia/sangre , Trombocitopenia/complicaciones , Adulto JovenRESUMEN
Excessive release of proinflammatory cytokines by innate immune cells is an important component of the pathogenic basis of malaria. Proinflammatory cytokines are a direct output of Toll-like receptor (TLR) activation during microbial infection. Thus, interference with TLR function is likely to render a better clinical outcome by preventing their aberrant activation and the excessive release of inflammatory mediators. Herein, we describe the protective effect and mechanism of action of E6446, a synthetic antagonist of nucleic acid-sensing TLRs, on experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA. We show that in vitro, low doses of E6446 specifically inhibited the activation of human and mouse TLR9. Tenfold higher concentrations of this compound also inhibited the human TLR8 response to single-stranded RNA. In vivo, therapy with E6446 diminished the activation of TLR9 and prevented the exacerbated cytokine response observed during acute Plasmodium infection. Furthermore, severe signs of ECM, such as limb paralysis, brain vascular leak, and death, were all prevented by oral treatment with E6446. Hence, we provide evidence that supports the involvement of nucleic acid-sensing TLRs in malaria pathogenesis and that interference with the activation of these receptors is a promising strategy to prevent deleterious inflammatory responses that mediate pathogenesis and severity of malaria.
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Hidrocarburos Aromáticos/farmacología , Malaria Cerebral/prevención & control , Malaria Cerebral/terapia , Ácidos Nucleicos/metabolismo , Receptores Toll-Like/antagonistas & inhibidores , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/patología , Humanos , Hidrocarburos Aromáticos/química , Inflamación/complicaciones , Inflamación/patología , Lipopolisacáridos/farmacología , Malaria Cerebral/inducido químicamente , Malaria Cerebral/parasitología , Ratones , Ratones Endogámicos C57BL , Plasmodium chabaudi/efectos de los fármacos , Plasmodium chabaudi/fisiología , Choque Séptico/inducido químicamente , Choque Séptico/complicaciones , Receptores Toll-Like/metabolismoRESUMEN
BACKGROUND: In the last few years, the study of microparticles (MPs)--submicron vesicles released from cells upon activation or apoptosis--has gained growing interest in the field of inflammation and in infectious diseases. Their role in the human malaria parasite Plasmodium vivax remains unexplored. Because acute vivax malaria has been related to pro-inflammatory responses, the main hypothesis investigated in this study was that Plasmodium vivax infection is associated with elevated levels of circulating MPs, which may play a role during acute disease in non-immune patients. METHODS: Plasma MPs were analysed among thirty-seven uncomplicated P. vivax infections from an area of unstable malaria transmission in the Brazilian Amazon. The MP phenotype was analysed by flow cytometry using the classical MP marker, annexin, and fluorochrome-labeled monoclonal antibodies against specific cell surface markers. The frequencies of plasma MPs in P. vivax patients (n=37) were further compared to malaria-unexposed controls (n=15) and ovarian carcinoma patients (n=12), a known MPs-inducing disease non-related to malaria. RESULTS: The frequencies of plasma circulating MPs were markedly increased in P. vivax patients, as compared to healthy age-matched malaria-unexposed controls. Although platelets, erythrocytes and leukocytes were the main cellular sources of MPs during vivax malaria, platelet derived-MPs (PMPs) increased in a linear fashion with the presence of fever at the time of blood collection (ß=0.06, p<0.0001) and length of acute symptoms (ß=0.36, p<0.0001). Finally, the results suggest that plasma levels of PMPs diminish as patient experience more episodes of clinical malaria (ß=0.07, p<0.003). CONCLUSIONS: Abundant circulating MPs are present during acute P. vivax infection, and platelet derived-MPs may play a role on the acute inflammatory symptoms of malaria vivax.
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Micropartículas Derivadas de Células/química , Malaria Vivax/patología , Plasma/química , Plasma/parasitología , Adolescente , Adulto , Anciano , Anexinas/análisis , Brasil , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Malaria-induced sepsis is associated with an intense proinflammatory cytokinemia for which the underlying mechanisms are poorly understood. It has been demonstrated that experimental infection of humans with Plasmodium falciparum primes Toll-like receptor (TLR)-mediated proinflammatory responses. Nevertheless, the relevance of this phenomenon during natural infection and, more importantly, the mechanisms by which malaria mediates TLR hyperresponsiveness are unclear. Here we show that TLR responses are boosted in febrile patients during natural infection with P. falciparum. Microarray analyses demonstrated that an extraordinary percentage of the up-regulated genes, including genes involving TLR signaling, had sites for IFN-inducible transcription factors. To further define the mechanism involved in malaria-mediated "priming," we infected mice with Plasmodium chabaudi. The human data were remarkably predictive of what we observed in the rodent malaria model. Malaria-induced priming of TLR responses correlated with increased expression of TLR mRNA in a TLR9-, MyD88-, and IFNgamma-dependent manner. Acutely infected WT mice were highly susceptible to LPS-induced lethality while TLR9(-/-), IL12(-/-) and to a greater extent, IFNgamma(-/-) mice were protected. Our data provide unprecedented evidence that TLR9 and MyD88 are essential to initiate IL12 and IFNgamma responses and favor host hyperresponsiveness to TLR agonists resulting in overproduction of proinflammatory cytokines and the sepsis-like symptoms of acute malaria.
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Inmunidad Innata , Interferón gamma/inmunología , Interleucina-12/inmunología , Malaria/inmunología , Factor 88 de Diferenciación Mieloide/inmunología , Receptores Toll-Like/inmunología , Animales , Citocinas , Fiebre , Perfilación de la Expresión Génica , Humanos , Inflamación , Ratones , Plasmodium chabaudi , Plasmodium falciparum , Sepsis/parasitología , Sepsis/patología , Receptor Toll-Like 9/inmunología , Receptores Toll-Like/genética , Factores de Transcripción , Regulación hacia Arriba/genéticaRESUMEN
Toll-like receptors (TLRs) are important to initiate the innate immune response to a wide variety of pathogens. The protective role of TLRs during infection with protozoan parasites has been established. In this regard, malaria represents an exception where activation of TLRs seems to be deleterious to the host. In this article, we review the recent findings indicating the contrasting role of Myeloid Differentiation Primary-Response gene 88 (MyD88) and TLRs during malaria and infection with other protozoa. These findings suggest that MyD88 may represent an Achilles' heel during Plasmodium infection.
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Citocinas/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Malaria/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Infecciones por Protozoos/inmunología , Receptores Toll-Like/metabolismo , Animales , Citocinas/inmunología , Glicosilfosfatidilinositoles/inmunología , Humanos , Malaria/metabolismo , Factor 88 de Diferenciación Mieloide/inmunología , Infecciones por Protozoos/metabolismo , Transducción de Señal , Receptores Toll-Like/agonistas , Receptores Toll-Like/inmunologíaRESUMEN
We investigated the role of different TLRs and MyD88 in host resistance to infection and malaria pathogenesis. TLR2(-/-), TLR4(-/-), TLR6(-/-), TLR9(-/-) or CD14(-/-) mice showed no change in phenotypes (parasitemia, body weight and temperature) when infected with Plasmodium chabaudi chabaudi (AS). MyD88(-/-) mice displayed comparable ability to wild type animals in controlling and clearing parasitemia. Importantly, MyD88(-/-) mice exhibited impaired production of TNF-alpha and IFN-gamma as well as attenuated symptoms, as indicated by changes in body weight and temperature during parasitemia. Consistently, CD11b(+) monocytes and CD11c(+) dendritic cells from infected MyD88(-/-) mice were shown impaired for production of pro-inflammatory cytokines, and in initiating CD4(+) T cell responses. Importantly, the inhibition of T cell activation with anti-CD134L, mostly inhibited IFN-gamma, partially inhibited TNF-alpha production, and protected the animals from malaria symptoms. Our findings suggest that MyD88 and possibly its associated TLRs expressed by dendritic cells play an important role in pro-inflammatory responses, T cell activation, and pathogenesis of malaria, but are not critical for the immunological control of the erythrocytic stage of P. chabaudi.
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Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/inmunología , Malaria/inmunología , Factor 88 de Diferenciación Mieloide/inmunología , Plasmodium chabaudi/inmunología , Receptores Toll-Like/inmunología , Animales , Citocinas/inmunología , Citometría de Flujo , Inmunofenotipificación , Activación de Linfocitos/inmunología , Malaria/parasitología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Parasitemia/inmunología , Bazo/inmunologíaRESUMEN
Effect of Aedes fluviatilis saliva on the development of Plasmodium gallinaceum experimental infection in Gallus (gallus) domesticus was studied in distinct aspects. Chickens subcutaneously infected with sporozoites in the presence of the mosquito salivary gland homogenates (SGH) showed higher levels of parasitaemia when compared to those ones that received only the sporozoites. However, the parasitaemia levels were lower among chickens previously immunized by SGH or non-infected mosquito bites compared to the controls, which did not receive saliva. High levels of anti-saliva antibodies were observed in those immunized chickens. Moreover, 53 and 102 kDa saliva proteins were recognized by sera from immunized chickens. After the sporozoite challenge, the chickens also showed significant levels of anti-sporozoite antibodies. However, the ability to generate anti-sporozoites antibodies was not correlated to the saliva immunization. Our results suggest that mosquito saliva components enhance P. gallinaceum parasite development in naive chickens. However, the prior exposure of chickens to salivary components controls the parasitemia levels in infected individuals.
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Animales , Femenino , Aedes , Anticuerpos Antiprotozoarios , Malaria Aviar , Plasmodium gallinaceum , Glándulas Salivales , Pollos , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Parasitemia , EsporozoítosRESUMEN
Effect of Aedes fluviatilis saliva on the development of Plasmodium gallinaceum experimental infection in Gallus (gallus) domesticus was studied in distinct aspects. Chickens subcutaneously infected with sporozoites in the presence of the mosquito salivary gland homogenates (SGH) showed higher levels of parasitaemia when compared to those ones that received only the sporozoites. However, the parasitaemia levels were lower among chickens previously immunized by SGH or non-infected mosquito bites compared to the controls, which did not receive saliva. High levels of anti-saliva antibodies were observed in those immunized chickens. Moreover, 53 and 102 kDa saliva proteins were recognized by sera from immunized chickens. After the sporozoite challenge, the chickens also showed significant levels of anti-sporozoite antibodies. However, the ability to generate anti-sporozoites antibodies was not correlated to the saliva immunization. Our results suggest that mosquito saliva components enhance P. gallinaceum parasite development in naive chickens. However, the prior exposure of chickens to salivary components controls the parasitemia levels in infected individuals.