RESUMEN
Cytokine responses to malaria play important roles in both protective immunity development and pathogenesis. Although the roles of cytokines such as TNF-α, IL-12, IFN-γ, and IL-10 in immunity and pathogenesis to the blood stage malaria are largely known, the role of IL-4 remains less understood. IL-4 targets many cell types and induces multiple effects, including cell proliferation, gene expression, protection from apoptosis, and immune regulation. Accordingly, IL-4 has been exploited as a therapeutic for several inflammatory diseases. Malaria caused by Plasmodium falciparum manifests in many organ-specific fatal pathologies, including cerebral malaria (CM), driven by a high parasite load, leading to parasite sequestration in organs and consequent excessive inflammatory responses and endothelial damage. We investigated the therapeutic potential of IL-4 against fatal malaria in Plasmodium berghei ANKA-infected C57BL/6J mice, an experimental CM model. IL-4 treatment significantly reduced parasitemia, CM pathology, and mortality. The therapeutic effect of IL-4 is mediated through multiple mechanisms, including enhanced parasite clearance mediated by upregulation of phagocytic receptors and increased IgM production, and decreased brain inflammatory responses, including reduced chemokine (CXCL10) production, reduced chemokine receptor (CXCR3) and adhesion molecule (LFA-1) expression by T cells, and downregulation of cytotoxic T cell lytic potential. IL-4 treatment markedly reduced the infiltration of CD8+ T cells and brain pathology. STAT6, PI3K-Akt-NF-κB, and Src signaling mediated the cellular and molecular events that contributed to the IL-4-dependent decrease in parasitemia. Overall, our results provide mechanistic insights into how IL-4 treatment mitigates experimental CM and have implications in developing treatment strategies for organ-specific fatal malaria.
Asunto(s)
Antimaláricos/uso terapéutico , Encéfalo/patología , Interleucina-4/uso terapéutico , Malaria Cerebral/inmunología , Plasmodium falciparum/fisiología , Linfocitos T Citotóxicos/inmunología , Animales , Citotoxicidad Inmunológica , Humanos , Inflamación , Malaria Cerebral/terapia , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Parasitemia , Transducción de SeñalRESUMEN
Persistent high levels of proinflammatory and Th1 responses contribute to cerebral malaria (CM). Suppression of inflammatory responses and promotion of Th2 responses prevent pathogenesis. IL-4 commonly promotes Th2 responses and inhibits inflammatory and Th1 responses. Therefore, IL-4 is widely considered as a beneficial cytokine via its Th2-promoting role that is predicted to provide protection against severe malaria by inhibiting inflammatory responses. However, IL-4 may also induce inflammatory responses, as the result of IL-4 action depends on the timing and levels of its production and the tissue environment in which it is produced. Recently, we showed that dendritic cells (DCs) produce IL-4 early during malaria infection in response to a parasite protein and that this IL-4 response may contribute to severe malaria. However, the mechanism by which IL-4 produced by DCs contributing to lethal malaria is unknown. Using Plasmodium berghei ANKA-infected C57BL/6 mice, a CM model, we show here that mice lacking IL-4Rα only in CD8α+ DCs are protected against CM pathogenesis and survive, whereas WT mice develop CM and die. Compared with WT mice, mice lacking IL-4Rα in CD11c+ or CD8α+ DCs showed reduced inflammatory responses leading to decreased Th1 and cytotoxic CD8+ T cell responses, lower infiltration of CD8+ T cells to the brain, and negligible brain pathology. The novel results presented here reveal a paradoxical role of IL-4Rα signaling in CM pathogenesis that promotes CD8α+ DC-mediated inflammatory responses that generate damaging Th1 and cytotoxic CD8+ T cell responses.
Asunto(s)
Antígenos CD8/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Malaria Cerebral/inmunología , Plasmodium berghei/inmunología , Receptores de Superficie Celular/inmunología , Transducción de Señal/inmunología , Células TH1/inmunología , Animales , Antígenos CD8/genética , Linfocitos T CD8-positivos/patología , Células Dendríticas/patología , Interleucina-4/genética , Interleucina-4/inmunología , Malaria Cerebral/genética , Malaria Cerebral/patología , Ratones , Ratones Noqueados , Plasmodium berghei/genética , Receptores de Superficie Celular/genética , Transducción de Señal/genética , Células TH1/patología , Células Th2/inmunología , Células Th2/patologíaRESUMEN
Dendritic cells (DC) and cytokines produced by DC play crucial roles in inducing and regulating pro-/anti-inflammatory and Th1/Th2 responses. DC are known to produce a Th1-promoting cytokine, interleukin (IL)-12, in response to malaria and other pathogenic infections, but it is thought that DC do not produce Th2-promoting cytokine, IL-4. Here, we show that a protein factor of malaria parasites induces IL-4 responses by CD11chiMHCIIhiCD3ϵ-CD49b-CD19-FcϵRI- DC via PI3K-Akt-NF-κB signaling independent of TLR-MyD88/TRIF. Malaria parasite-activated DC induced IL-4 responses by T cells both in vitro and in vivo, favoring Th2, and il-4-deficient DC were unable to induce IL-4 expression by T cells. Interestingly, lethal parasites, Plasmodium falciparum and Plasmodium berghei ANKA, induced IL-4 response primarily by CD8α- DC, whereas nonlethal Plasmodium yoelii induced IL-4 by both CD8α+ and CD8α- DC. In both P. berghei ANKA- and P. yoelii-infected mice, IL-4-expressing CD8α- DC did not express IL-12, but a distinct CD8α- DC subset expressed IL-12. In P. berghei ANKA infection, CD8α+ DC expressed IL-12 but not IL-4, whereas in P. yoelii infection, CD8α+ DC expressed IL-4 but not IL-12. These differential IL-4 and IL-12 responses by DC subsets may contribute to different Th1/Th2 development and clinical outcomes in lethal and nonlethal malaria. Our results for the first time demonstrate that a malaria protein factor induces IL-4 production by DC via PI3K-Akt-NF-κB signaling, revealing signaling and molecular mechanisms that initiate and promote Th2 development.
Asunto(s)
Células Dendríticas/inmunología , Interleucina-4/metabolismo , Malaria/inmunología , Plasmodium yoelii/inmunología , Proteínas Protozoarias/metabolismo , Células Th2/inmunología , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Citocinas/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/parasitología , Interleucina-4/fisiología , Malaria/metabolismo , Malaria/parasitología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Protozoarias/genética , Transducción de Señal , Células TH1/inmunología , Células TH1/metabolismo , Células TH1/parasitología , Células Th2/metabolismo , Células Th2/parasitología , Receptor Toll-Like 2/fisiología , Receptor Toll-Like 4/fisiología , Receptor Toll-Like 9/fisiologíaRESUMEN
Malaria infections cause several systemic and severe single- or multi-organ pathologies, killing hundreds of thousands of people annually. Considering the existing widespread resistance of malaria parasites to anti-parasitic drugs and their high propensity to develop drug resistance, alternative strategies are required to manage malaria infections. Because malaria is a host immune response-driven disease, one approach is based on gaining a detailed understanding of the molecular and cellular processes that modulate malaria-induced innate and adaptive immune responses. Here, using a mouse cerebral malaria model and small-molecule inhibitors, we demonstrate that inhibiting MEK1/2, the upstream kinases of ERK1/2 signaling, alters multifactorial components of the innate and adaptive immune responses, controls parasitemia, and blocks pathogenesis. Specifically, MEK1/2 inhibitor treatment up-regulated B1 cell expansion, IgM production, phagocytic receptor expression, and phagocytic activity, enhancing parasite clearance by macrophages and neutrophils. Further, the MEK1/2 inhibitor treatment down-regulated pathogenic pro-inflammatory and helper T cell 1 (Th1) responses and up-regulated beneficial anti-inflammatory cytokine responses and Th2 responses. These inhibitor effects resulted in reduced granzyme B expression by T cells, chemokine and intracellular cell adhesion molecule 1 (ICAM-1) expression in the brain, and chemokine receptor expression by both myeloid and T cells. These bimodal effects of the MEK1/2 inhibitor treatment on immune responses contributed to decreased parasite biomass, organ inflammation, and immune cell recruitment, preventing tissue damage and death. In summary, we have identified several previously unrecognized immune regulatory processes through which a MEK1/2 inhibitor approach controls malaria parasitemia and mitigates pathogenic effects on host organs.
Asunto(s)
Antimaláricos/uso terapéutico , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Malaria Cerebral/tratamiento farmacológico , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Inmunidad Adaptativa/efectos de los fármacos , Animales , Antimaláricos/farmacología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/parasitología , Células de la Médula Ósea/patología , Células Cultivadas , Técnicas de Cocultivo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/parasitología , Células Dendríticas/patología , Femenino , Flavonoides/farmacología , Flavonoides/uso terapéutico , Inmunidad Innata/efectos de los fármacos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/parasitología , Células Asesinas Naturales/patología , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/metabolismo , Malaria Cerebral/inmunología , Malaria Cerebral/parasitología , Malaria Cerebral/patología , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Malaria Falciparum/patología , Masculino , Ratones Endogámicos C57BL , Carga de Parásitos , Parasitemia/prevención & control , Fagocitosis/efectos de los fármacos , Plasmodium falciparum/enzimología , Plasmodium falciparum/inmunología , Plasmodium falciparum/aislamiento & purificación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , Piridonas/farmacología , Piridonas/uso terapéutico , Pirimidinonas/farmacología , Pirimidinonas/uso terapéutico , Análisis de SupervivenciaRESUMEN
In malaria, CD36 plays several roles, including mediating parasite sequestration to host organs, phagocytic clearance of parasites, and regulation of immunity. Although the functions of CD36 in parasite sequestration and phagocytosis have been clearly defined, less is known about its role in malaria immunity. Here, to understand the function of CD36 in malaria immunity, we studied parasite growth, innate and adaptive immune responses, and host survival in WT and Cd36-/- mice infected with a non-lethal strain of Plasmodium yoelii Compared with Cd36-/- mice, WT mice had lower parasitemias and were resistant to death. At early but not at later stages of infection, WT mice had higher circulatory proinflammatory cytokines and lower anti-inflammatory cytokines than Cd36-/- mice. WT mice showed higher frequencies of proinflammatory cytokine-producing and lower frequencies of anti-inflammatory cytokine-producing dendritic cells (DCs) and natural killer cells than Cd36-/- mice. Cytokines produced by co-cultures of DCs from infected mice and ovalbumin-specific, MHC class II-restricted α/ß (OT-II) T cells reflected CD36-dependent DC function. WT mice also showed increased Th1 and reduced Th2 responses compared with Cd36-/- mice, mainly at early stages of infection. Furthermore, in infected WT mice, macrophages and neutrophils expressed higher levels of phagocytic receptors and showed enhanced phagocytosis of parasite-infected erythrocytes than those in Cd36-/- mice in an IFN-γ-dependent manner. However, there were no differences in malaria-induced humoral responses between WT and Cd36-/- mice. Overall, the results show that CD36 plays a significant role in controlling parasite burden by contributing to proinflammatory cytokine responses by DCs and natural killer cells, Th1 development, phagocytic receptor expression, and phagocytic activity.
Asunto(s)
Antígenos CD36/inmunología , Inmunidad Humoral , Malaria/inmunología , Parasitemia/inmunología , Plasmodium yoelii/inmunología , Animales , Antígenos CD36/genética , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Interferón gamma/genética , Interferón gamma/inmunología , Células Asesinas Naturales/inmunología , Macrófagos/inmunología , Malaria/genética , Ratones , Ratones Noqueados , Neutrófilos/inmunología , Parasitemia/genética , Fagocitosis/genética , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
Much research has been conducted on the morphological characteristics of the Chinese. However, very few facial measurements have been documented, especially of the side face. This study uses geometirc morphometric method to analyze the contour and variations of the side face in Bai and Yi ethnic minorities from Yunnan province, China. The mark collection proves that for the Bai ethnicity, the variations of the nose are comparatively large, while the forehead variations are small. Variations around the lips and the chin are the largest. For the Yi ethnicity, the forehead also witnesses small variations and the nose again has large variations. The area around the glabella has large variations. Through the comparisons, the area around the glabella tends to extrude more in males both in Bai and Yi. The situation, however, is much more different when it comes to the trichion landmark collection where we see an apparent intrusion in males. For the trichion, Yi people are more intruded than the Bai. Similarities between Bai and Yi are demonstrated by principal component analysis: one can roughly set the males apart from the females using the vertical axis. Profile at the end of horizontal axis suggests that the female facial profile has the following features: the nose is not so prominent as the male, the forehead and the nose are linked by an noticeable arc, the forehead is comparatively steep and is almost in a vertical plane with the lips and the chin. By comparison, the male has a flatter forehead, a more prominent nose, an obvious sellion, and an intruded chin. The common morphologic features of the Chinese face may be reflected through these similarities.
Asunto(s)
Pueblo Asiatico/etnología , Cefalometría/métodos , Cara/anatomía & histología , Adulto , Mentón/anatomía & histología , China , Femenino , Frente/anatomía & histología , Humanos , Labio/anatomía & histología , Masculino , Persona de Mediana Edad , Nariz/anatomía & histología , FotograbarRESUMEN
Inflammatory cytokines produced at the early stages of malaria infection contribute to shaping protective immunity and pathophysiology. To gain mechanistic insight into these processes, it is important to understand the cellular origin of cytokines because both cytokine input and cytokine-producing cells play key roles. Here, we determined cytokine responses by monocytes, macrophages, and dendritic cells (DCs) to purified Plasmodium falciparum and Plasmodium berghei ANKA, and by spleen macrophages and DCs from Plasmodium yoelii 17NXL-infected and P. berghei ANKA-infected mice. The results demonstrate that monocytes and macrophages do not produce inflammatory cytokines to malaria parasites and that DCs are the primary source early in infection, and DC subsets differentially produce cytokines. Importantly, blocking of phagosomal acidification by inhibiting vacuolar-type H(+)-ATPase enabled macrophages to elicit cytokine responses. Because cytokine responses to malaria parasites are mediated primarily through endosomal Toll-like receptors, our data indicate that the inability of macrophages to produce cytokines is due to the phagosomal acidification that disrupts endosomal ligand-receptor engagement. Macrophages efficiently produced cytokines to LPS upon simultaneously internalizing parasites and to heat-killed Escherichia coli, demonstrating that phagosomal acidification affects endosomal receptor-mediated, but not cell surface receptor-mediated, recognition of Toll-like receptor agonists. Enabling monocytes/macrophages to elicit immune responses to parasites by blocking endosomal acidification can be a novel strategy for the effective development of protective immunity to malaria. The results have important implications for enhancing the efficacy of a whole parasite-based malaria vaccine and for designing strategies for the development of protective immunity to pathogens that induce immune responses primarily through endosomal receptors.
Asunto(s)
Inmunidad Adaptativa , Citocinas/inmunología , Células Dendríticas/inmunología , Malaria/inmunología , Fagosomas/inmunología , Plasmodium/inmunología , Animales , Células Dendríticas/patología , Humanos , Concentración de Iones de Hidrógeno , Inflamación/inmunología , Inflamación/patología , Malaria/patología , Ratones , Fagosomas/patología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/inmunologíaRESUMEN
Malaria caused by the Plasmodium parasites continues to be an enormous global health problem owing to wide spread drug resistance of parasites to many of the available antimalarial drugs. Therefore, development of new classes of antimalarial agents is essential to effectively treat malaria. In this study, the efficacy of naturally occurring diterpenoids, dehydroabietylamine and abietic acid, and their synthetic derivatives was assessed for antimalarial activity. Dehydroabietylamine and its N-trifluoroacetyl, N-tribromoacetyl, N-benzoyl, and N-benzyl derivatives showed excellent activity against P. falciparum parasites with IC50 values of 0.36 to 2.6 µM. Interestingly, N-dehydroabietylbenzamide showed potent antimalarial activity (IC50 0.36), and negligible cytotoxicity (IC50 >100 µM) to mammalian cells; thus, this compound can be an important antimalarial drug. In contrast, abietic acid was only marginally effective, exhibiting an IC50 value of ~82 µM. Several carboxylic group-derivatives of abietic acid were moderately active with IC50 values of ~8.2 to ~13.3 µM. These results suggest that a detailed understanding of the structure-activity relationship of abietane diterpenoids might provide strategies to exploit this class of compounds for malaria treatment.
Asunto(s)
Abietanos/farmacología , Antimaláricos/farmacología , Plasmodium falciparum/efectos de los fármacos , Abietanos/química , Benzamidas , Benzotiazoles , Línea Celular , Línea Celular Tumoral , Diaminas , Relación Dosis-Respuesta a Droga , Eritrocitos/parasitología , Fibroblastos/efectos de los fármacos , Colorantes Fluorescentes , Humanos , Concentración 50 Inhibidora , Compuestos Orgánicos , Plasmodium falciparum/crecimiento & desarrollo , Quinolinas , Relación Estructura-ActividadRESUMEN
A new strategy was developed to synthesize 1,2-disubstituted 4-quinolones in good yield starting from 1,3-bisaryl-monothio-1,3-diketone substrates. The synthesized compounds were evaluated for antimalarial activity using Plasmodium falciparum strains. All compounds, except for two, showed good activity. Of these, seven compounds exhibited an excellent antimalarial activity (IC50, <2 µM). More importantly, all seven compounds were equally effective in inhibiting the growth of both chloroquine-sensitive and chloroquine-resistant strains. The cytotoxicity assessment using carcinoma and non-carcinoma human cell lines revealed that almost all synthesized compounds were minimally cytotoxic (IC50, >50 µM).
Asunto(s)
4-Quinolonas/síntesis química , 4-Quinolonas/farmacología , Antimaláricos/síntesis química , Antimaláricos/farmacología , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , 4-Quinolonas/química , Antimaláricos/química , Línea Celular , Línea Celular Tumoral , HumanosRESUMEN
Effective resolution of malaria infection by avoiding pathogenesis requires regulated pro- to anti-inflammatory responses and the development of protective immunity. TLRs are known to be critical for initiating innate immune responses, but their roles in the regulation of immune responses and development of protective immunity to malaria remain poorly understood. In this study, using wild-type, TLR2(-/-), TLR4(-/-), TLR9(-/-), and MyD88(-/-) mice infected with Plasmodium yoelii, we show that TLR9 and MyD88 regulate pro/anti-inflammatory cytokines, Th1/Th2 development, and cellular and humoral responses. Dendritic cells from TLR9(-/-) and MyD88(-/-) mice produced significantly lower levels of proinflammatory cytokines and higher levels of anti-inflammatory cytokines than dendritic cells from wild-type mice. NK and CD8(+) T cells from TLR9(-/-) and MyD88(-/-) mice showed markedly impaired cytotoxic activity. Furthermore, mice deficient in TLR9 and MyD88 showed higher Th2-type and lower Th1-type IgGs. Consequently, TLR9(-/-) and MyD88(-/-) mice exhibited compromised ability to control parasitemia and were susceptible to death. Our data also show that TLR9 and MyD88 distinctively regulate immune responses to malaria infection. TLR9(-/-) but not MyD88(-/-) mice produced significant levels of both pro- and anti-inflammatory cytokines, including IL-1ß and IL-18, by other TLRs/inflammasome- and/or IL-1R/IL-18R-mediated signaling. Thus, whereas MyD88(-/-) mice completely lacked cell-mediated immunity, TLR9(-/-) mice showed low levels of cell-mediated immunity and were slightly more resistant to malaria infection than MyD88(-/-) mice. Overall, our findings demonstrate that TLR9 and MyD88 play central roles in the immune regulation and development of protective immunity to malaria, and have implications in understanding immune responses to other pathogens.
Asunto(s)
Malaria/prevención & control , Factor 88 de Diferenciación Mieloide/fisiología , Receptor Toll-Like 9/fisiología , Animales , Células Cultivadas , Técnicas de Cocultivo , Inmunidad Innata/genética , Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/fisiología , Malaria/inmunología , Malaria/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Factor 88 de Diferenciación Mieloide/deficiencia , Parasitemia/inmunología , Parasitemia/patología , Parasitemia/prevención & control , Plasmodium berghei/inmunología , Plasmodium yoelii/inmunología , Tasa de Supervivencia , Receptor Toll-Like 9/deficienciaRESUMEN
Most researchers believe that anatomically modern humans (AMH) first appeared in Africa 160-190 ka ago, and would not have reached eastern Asia until â¼50 ka ago. However, the credibility of these scenarios might have been compromised by a largely inaccurate and compressed chronological framework previously established for hominin fossils found in China. Recently there has been a growing body of evidence indicating the possible presence of AMH in eastern Asia ca. 100 ka ago or even earlier. Here we report high-precision mass spectrometric U-series dating of intercalated flowstone samples from Huanglong Cave, a recently discovered Late Pleistocene hominin site in northern Hubei Province, central China. Systematic excavations there have led to the in situ discovery of seven hominin teeth and dozens of stone and bone artifacts. The U-series dates on localized thin flowstone formations bracket the hominin specimens between 81 and 101 ka, currently the most narrow time span for all AMH beyond 45 ka in China, if the assignment of the hominin teeth to modern Homo sapiens holds. Alternatively this study provides further evidence for the early presence of an AMH morphology in China, through either independent evolution of local archaic populations or their assimilation with incoming AMH. Along with recent dating results for hominin samples from Homo erectus to AMH, a new extended and continuous timeline for Chinese hominin fossils is taking shape, which warrants a reconstruction of human evolution, especially the origins of modern humans in eastern Asia.
Asunto(s)
Evolución Biológica , Cuevas/química , Fósiles , Sedimentos Geológicos/química , China , Cronología como Asunto , Humanos , Espectrometría de Masas , Datación Radiométrica , Diente/química , Uranio/químicaRESUMEN
Malaria infection causes multiple organ-specific lethal pathologies, including cerebral malaria, and severe liver and lung pathologies by inducing strong inflammatory responses. Gene polymorphism studies suggest that TLR4 and TLR2 contribute to severe malaria, but the roles of these signaling molecules in malaria pathogenesis remain incompletely understood. We hypothesize that danger-associated molecular patterns produced in response to malaria activate TLR2 and TLR4 signaling and contribute to liver and lung pathologies. By using a mouse model of Plasmodium berghei NK65 infection, we show that the combined TLR2 and TLR4 signaling contributes to malaria liver and lung pathologies and mortality. Macrophages, neutrophils, natural killer cells, and T cells infiltrate to the livers and lungs of infected wild-type mice more than TLR2,4-/- mice. Additionally, endothelial barrier disruption, tissue necrosis, and hemorrhage were higher in the livers and lungs of infected wild-type mice than in those of TLR2,4-/- mice. Consistent with these results, the levels of chemokine production, chemokine receptor expression, and liver and lung pathologic markers were higher in infected wild-type mice than in TLR2,4-/- mice. In addition, the levels of HMGB1, a potent TLR2- and TLR4-activating danger-associated molecular pattern, were higher in livers and lungs of wild-type mice than TLR2,4-/- mice. Treatment with glycyrrhizin, an immunomodulatory agent known to inhibit HMGB1 activity, markedly reduced mortality in wild-type mice. These results suggest that TLR2 and TLR4 activation by HMGB1 and possibly other endogenously produced danger-associated molecular patterns contribute to malaria liver and lung injury via signaling mechanisms distinct from those involved in cerebral malaria pathogenesis.
Asunto(s)
Proteína HMGB1 , Malaria Cerebral , Animales , Ratones , Receptor Toll-Like 2/metabolismo , Proteína HMGB1/metabolismo , Receptor Toll-Like 4/metabolismo , Malaria Cerebral/patología , Pulmón/patología , Transducción de Señal/genética , Hígado/metabolismo , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Plasmodium falciparum glycosylphosphatidylinositols (GPIs) have been proposed as malaria pathogenic factors based on their ability to induce proinflammatory responses in macrophages and malaria-like symptoms in mice. Parasite GPIs induce the production of inflammatory cytokines by activating the mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways. Importantly, inhibition of the extracellular-signal-regulated kinase (ERK) pathway upregulates a subset of cytokines, including IL-12. We investigated the role of nuclear transcription factor, IκB-ζ, in the GPI-induced dysregulated expression of IL-12 on inhibition of the ERK pathway. GPIs efficiently induced the expression of IκB-ζ in macrophages regardless of whether cells were pretreated or untreated with ERK inhibitors, indicating that ERK has no role in IκB-ζ expression. However, on ERK inhibition followed by stimulation with GPIs, NF-κB binding to Il12b promoter κB site was markedly increased, suggesting that the ERK pathway regulates Il12b transcription. Knockdown of IκB-ζ using siRNA markedly reduced the GPI-induced IL-12 production and abrogated the dysregulated IL-12 production in ERK inhibited cells. Together these results demonstrate that ERK modulates IL-12 expression by regulating IκB-ζ-dependent binding of NF-κB transcription factors to Il12b gene promoter. Additionally, our finding that IκB-ζ can be knocked down efficiently in primary macrophages is valuable for studies aimed at gaining further insights into IκB-ζ function.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Antígenos de Protozoos/inmunología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Ligadas a GPI/inmunología , Subunidad p40 de la Interleucina-12/genética , Malaria Falciparum/genética , Proteínas Nucleares/fisiología , Plasmodium falciparum/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Antígenos de Protozoos/farmacología , Células Cultivadas , Técnicas de Cultivo , Citocinas/genética , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Proteínas Ligadas a GPI/farmacología , Expresión Génica , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Subunidad p40 de la Interleucina-12/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Malaria Falciparum/inmunología , Malaria Falciparum/metabolismo , Ratones , FN-kappa B/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Interferencia de ARNRESUMEN
Dendritic cells (DCs) play a crucial role in the development of protective immunity to malaria. However, it remains unclear how malaria parasites trigger immune responses in DCs. In this study, we purified merozoites, food vacuoles, and parasite membrane fragments released during the Plasmodium falciparum schizont burst to homogeneity and tested for the activation of bone marrow-derived DCs from wild-type and TLR2(-/-), TLR4(-/-), TLR9(-/-), and MyD88(-/-) C57BL/6J mice. The results demonstrate that a protein-DNA complex is the exclusive parasite component that activates DCs by a TLR9-dependent pathway to produce inflammatory cytokines. Complex formation with proteins is essential for the entry of parasite DNA into DCs for TLR9 recognition and, thus, proteins convert inactive DNA into a potent immunostimulatory molecule. Exogenous cationic polymers, polylysine and chitosan, can impart stimulatory activity to parasite DNA, indicating that complex formation involves ionic interactions. Merozoites and DNA-protein complex could also induce inflammatory cytokine responses in human blood DCs. Hemozoin is neither a TLR9 ligand for DCs nor functions as a carrier of DNA into cells. Additionally, although TLR9 is critical for DCs to induce the production of IFN-gamma by NK cells, this receptor is not required for NK cells to secret IFN-gamma, and cell-cell contact among myeloid DCs, plasmacytoid DCs, and NK cells is required for IFN-gamma production. Together, these results contribute substantially toward the understanding of malaria parasite-recognition mechanisms. More importantly, our finding that proteins and carbohydrate polymers are able to confer stimulatory activity to an otherwise inactive parasite DNA have important implications for the development of a vaccine against malaria.
Asunto(s)
ADN Protozoario/fisiología , Células Dendríticas/inmunología , Células Dendríticas/parasitología , Inmunidad Innata , Plasmodium falciparum/inmunología , Proteínas Protozoarias/fisiología , Animales , Comunicación Celular/inmunología , Línea Celular , ADN Protozoario/sangre , Células Dendríticas/metabolismo , Humanos , Interferón gamma/biosíntesis , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/parasitología , Vacunas contra la Malaria/inmunología , Malaria Falciparum/sangre , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Merozoítos/crecimiento & desarrollo , Merozoítos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/sangre , Receptor Toll-Like 9/sangre , Receptor Toll-Like 9/deficiencia , Receptor Toll-Like 9/genéticaRESUMEN
The ligand specificity of TLRs and the details of signaling pathways that are activated by ligand-receptor engagements have been studied extensively. However, it is not known whether the signaling events initiated by defined doses of ligand are uniformly effective in producing various cytokines. In this study, we investigated the dose requirement for the saturated production of representative inflammatory cytokines, TNF-α, IL-6 and IL-12, by DCs stimulated with Plasmodium falciparum merozoites/protein-DNA complex or a CpG ODN TLR9 ligand. The data demonstrate that the ligand doses required for the maximal expression of TNF-α and IL-6 are substantially higher than those required for the maximal production of IL-12. The data also demonstrate that the uptake capacity of malaria parasite by plasmacytoid DCs is markedly lower than that of myeloid DCs, and that, like myeloid DCs, plasmacytoid DCs produce significant levels of TNF-α and IL-12 when the uptake of malarial DNA is facilitated by carrier molecules such as polylysine or cationic lipids. These results have implications for enhancing the effectiveness of vaccine against malaria by modulating the innate immune responses of plasmacytoid DCs to malaria parasites.
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Citocinas/biosíntesis , Merozoítos/metabolismo , Plasmodium falciparum/metabolismo , Animales , ADN Protozoario/aislamiento & purificación , Interleucina-12/biosíntesis , Interleucina-6/biosíntesis , Ligandos , Merozoítos/inmunología , Ratones , Ratones Endogámicos C57BL , Plasmodium falciparum/genética , Plasmodium falciparum/inmunología , Transducción de Señal , Organismos Libres de Patógenos Específicos , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Malaria caused by the Plasmodium family of parasites, especially P.falciparum and P. vivax, is a major health problem in many countries in the tropical and subtropical regions of the world. The disease presents a wide array of systemic clinical conditions and several life-threatening organ pathologies, including the dreaded cerebral malaria. Like many other infectious diseases, malaria is an inflammatory response-driven disease, and positive outcomes to infection depend on finely tuned regulation of immune responses that efficiently clear parasites and allow protective immunity to develop. Immune responses initiated by the innate immune system in response to parasites play key roles both in protective immunity development and pathogenesis. Initial pro-inflammatory responses are essential for clearing infection by promoting appropriate cell-mediated and humoral immunity. However, elevated and prolonged pro-inflammatory responses owing to inappropriate cellular programming contribute to disease conditions. A comprehensive knowledge of the molecular and cellular mechanisms that initiate immune responses and how these responses contribute to protective immunity development or pathogenesis is important for developing effective therapeutics and/or a vaccine. Historically, in efforts to develop a vaccine, immunity to malaria was extensively studied in the context of identifying protective humoral responses, targeting proteins involved in parasite invasion or clearance. The innate immune response was thought to be non-specific. However, during the past two decades, there has been a significant progress in understanding the molecular and cellular mechanisms of host-parasite interactions and the associated signaling in immune responses to malaria. Malaria infection occurs at two stages, initially in the liver through the bite of a mosquito, carrying sporozoites, and subsequently, in the blood through the invasion of red blood cells by merozoites released from the infected hepatocytes. Soon after infection, both the liver and blood stage parasites are sensed by various receptors of the host innate immune system resulting in the activation of signaling pathways and production of cytokines and chemokines. These immune responses play crucial roles in clearing parasites and regulating adaptive immunity. Here, we summarize the knowledge on molecular mechanisms that underlie the innate immune responses to malaria infection.
Asunto(s)
Interacciones Huésped-Parásitos/inmunología , Inmunidad Innata , Vacunas contra la Malaria/inmunología , Malaria/inmunología , Plasmodium/inmunología , Animales , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/metabolismo , Culicidae/parasitología , Vectores de Enfermedades , Hepatocitos/inmunología , Hepatocitos/parasitología , Humanos , Hígado/citología , Hígado/inmunología , Hígado/parasitología , Malaria/sangre , Malaria/parasitología , Malaria/prevención & control , Vacunas contra la Malaria/administración & dosificación , Merozoítos/inmunología , Merozoítos/patogenicidad , Plasmodium/genética , Plasmodium/metabolismo , Plasmodium/patogenicidad , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , Esporozoítos/inmunología , Esporozoítos/patogenicidadRESUMEN
The scavenger receptor CD36 plays important roles in malaria, including the sequestration of parasite-infected erythrocytes in microvascular capillaries, control of parasitemia through phagocytic clearance by macrophages, and immunity. Although the role of CD36 in the parasite sequestration and clearance has been extensively studied, how and to what extent CD36 contributes to malaria immunity remains poorly understood. In this study, to determine the role of CD36 in malaria immunity, we assessed the internalization of CD36-adherent and CD36-nonadherent Plasmodium falciparum-infected red blood cells (IRBCs) and production of pro-inflammatory cytokines by DCs, and the ability of DCs to activate NK, and T cells. Human DCs treated with anti-CD36 antibody and CD36 deficient murine DCs internalized lower levels of CD36-adherent IRBCs and produced significantly decreased levels of pro-inflammatory cytokines compared to untreated human DCs and wild type mouse DCs, respectively. Consistent with these results, wild type murine DCs internalized lower levels of CD36-nonadherent IRBCs and produced decreased levels of pro-inflammatory cytokines than wild type DCs treated with CD36-adherent IRBCs. Further, the cytokine production by NK and T cells activated by IRBC-internalized DCs was significantly dependent on CD36. Thus, our results demonstrate that CD36 contributes significantly to the uptake of IRBCs and pro-inflammatory cytokine responses by DCs, and the ability of DCs to activate NK and T cells to produce IFN-γ. Given that DCs respond to malaria parasites very early during infection and influence development of immunity, and that CD36 contributes substantially to the cytokine production by DCs, NK and T cells, our results suggest that CD36 plays an important role in immunity to malaria. Furthermore, since the contribution of CD36 is particularly evident at low doses of infected erythrocytes, the results imply that the effect of CD36 on malaria immunity is imprinted early during infection when parasite load is low.
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Antígenos CD36/inmunología , Citocinas/inmunología , Células Dendríticas/inmunología , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Parásitos/inmunología , Linfocitos T/inmunología , Animales , Células CHO , Cricetulus , Humanos , Inflamación/inmunología , Interferón gamma/inmunología , Malaria Falciparum/inmunología , Ratones , Ratones Endogámicos C57BL , Plasmodium falciparum/inmunologíaRESUMEN
The systemic clinical symptoms of Plasmodium falciparum infection such as fever and chills correspond to the proinflammatory cytokines produced in response to the parasite components released during the synchronized rupture of schizonts. We recently demonstrated that, among the schizont-released products, merozoites are the predominant components that activate dendritic cells (DCs) by TLR9-specific recognition to induce the maturation of cells and to produce proinflammatory cytokines. We also demonstrated that DNA is the active constituent and that formation of a DNA-protein complex is essential for the entry of parasite DNA into cells for recognition by TLR9. However, the nature of endogenous protein-DNA complex in the parasite is not known. In this study, we show that parasite nucleosome constitute the major protein-DNA complex involved in the activation of DCs by parasite nuclear material. The parasite components were fractionated into the nuclear and non-nuclear materials. The nuclear material was further fractionated into chromatin and the proteins loosely bound to chromatin. Polynucleosomes and oligonucleosomes were prepared from the chromatin. These were tested for their ability to activate DCs obtained by the FLT3 ligand differentiation of bone marrow cells from the wild type, and TLR2(-/-), TLR9(-/-) and MyD88(-/-) mice. DCs stimulated with the nuclear material and polynucleosomes as well as mono- and oligonucleosomes efficiently induced the production of proinflammatory cytokines in a TLR9-dependent manner, demonstrating that nucleosomes (histone-DNA complex) represent the major TLR9-specific DC-immunostimulatory component of the malaria parasite nuclear material. Thus, our data provide a significant insight into the activation of DCs by malaria parasites and have important implications for malaria vaccine development.
Asunto(s)
ADN Protozoario/metabolismo , Células Dendríticas/inmunología , Histonas/metabolismo , Nucleosomas/metabolismo , Plasmodium falciparum/citología , Plasmodium falciparum/inmunología , Receptor Toll-Like 9/metabolismo , Animales , Células de la Médula Ósea/citología , Diferenciación Celular/inmunología , Línea Celular Tumoral , Citocinas/biosíntesis , Citocinas/inmunología , Células Dendríticas/citología , Células Dendríticas/metabolismo , Células Dendríticas/parasitología , Humanos , Inflamación/metabolismo , Proteínas de la Membrana/metabolismo , Merozoítos/inmunología , Ratones , Proteínas Protozoarias/metabolismo , Bazo/citologíaRESUMEN
Proinflammatory responses induced by Plasmodium falciparum glycosylphosphatidylinositols (GPIs) are thought to be involved in malaria pathogenesis. In this study, we investigated the role of MAPK-activated protein kinase 2 (MK2) in the regulation of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-12, two of the major inflammatory cytokines produced by macrophages stimulated with GPIs. We show that MK2 differentially regulates the GPI-induced production of TNF-alpha and IL-12. Although TNF-alpha production was markedly decreased, IL-12 expression was increased by 2-3-fold in GPI-stimulated MK2(-/-) macrophages compared with wild type (WT) cells. MK2(-/-) macrophages produced markedly decreased levels of TNF-alpha than WT macrophages mainly because of lower mRNA stability and translation. In the case of IL-12, mRNA was substantially higher in MK2(-/-) macrophages than WT. This enhanced production is due to increased NF-kappaB binding to the gene promoter, a markedly lower level expression of the transcriptional repressor factor c-Maf, and a decreased binding of GAP-12 to the gene promoter in MK2(-/-) macrophages. Thus, our data demonstrate for the first time the role of MK2 in the transcriptional regulation of IL-12. Using the protein kinase inhibitors SB203580 and U0126, we also show that the ERK and p38 pathways regulate TNF-alpha and IL-12 production, and that both inhibitors can reduce phosphorylation of MK2 in response to GPIs and other toll-like receptor ligands. These results may have important implications for developing therapeutics for malaria and other infectious diseases.