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1.
Nat Immunol ; 18(8): 899-910, 2017 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-28604719

RESUMEN

Mammalian autophagy-related 8 (Atg8) homologs consist of LC3 proteins and GABARAPs, all of which are known to be involved in canonical autophagy. In contrast, the roles of Atg8 homologs in noncanonical autophagic processes are not fully understood. Here we show a unique role of GABARAPs, in particular gamma-aminobutyric acid (GABA)-A-receptor-associated protein-like 2 (Gabarapl2; also known as Gate-16), in interferon-γ (IFN-γ)-mediated antimicrobial responses. Cells that lacked GABARAPs but not LC3 proteins and mice that lacked Gate-16 alone were defective in the IFN-γ-induced clearance of vacuolar pathogens such as Toxoplasma. Gate-16 but not LC3b specifically associated with the small GTPase ADP-ribosylation factor 1 (Arf1) to mediate uniform distribution of interferon-inducible GTPases. The lack of GABARAPs reduced Arf1 activation, which led to formation of interferon-inducible GTPase-containing aggregates and hampered recruitment of interferon-inducible GTPases to vacuolar pathogens. Thus, GABARAPs are uniquely required for antimicrobial host defense through cytosolic distribution of interferon-inducible GTPases.


Asunto(s)
Factor 1 de Ribosilacion-ADP/inmunología , Autofagia/inmunología , Proteínas Portadoras/inmunología , Interferón gamma/inmunología , Proteínas Asociadas a Microtúbulos/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Factor 1 de Ribosilacion-ADP/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Familia de las Proteínas 8 Relacionadas con la Autofagia , Sistemas CRISPR-Cas , Proteínas Portadoras/metabolismo , Simulación por Computador , Proteínas del Citoesqueleto/inmunología , Proteínas del Citoesqueleto/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , GTP Fosfohidrolasas/inmunología , GTP Fosfohidrolasas/metabolismo , Edición Génica , Immunoblotting , Inmunoprecipitación , Interferón gamma/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo
2.
Immunity ; 37(2): 302-13, 2012 Aug 24.
Artículo en Inglés | MEDLINE | ID: mdl-22795875

RESUMEN

Interferon-γ (IFN-γ) is essential for host defense against intracellular pathogens. Stimulation of innate immune cells by IFN-γ upregulates ∼2,000 effector genes such as immunity-related GTPases including p65 guanylate-binding protein (Gbp) family genes. We show that a cluster of Gbp genes was required for host cellular immunity against the intracellular parasite Toxoplasma gondii. We generated mice deficient for all six Gbp genes located on chromosome 3 (Gbp(chr3)) by targeted chromosome engineering. Mice lacking Gbp(chr3) were highly susceptible to T. gondii infection, resulting in increased parasite burden in immune organs. Furthermore, Gbp(chr3)-deleted macrophages were defective in IFN-γ-mediated suppression of T. gondii intracellular growth and recruitment of IFN-γ-inducible p47 GTPase Irgb6 to the parasitophorous vacuole. In addition, some members of Gbp(chr3) restored the protective response against T. gondii in Gbp(chr3)-deleted cells. Our results suggest that Gbp(chr3) play a pivotal role in anti-T. gondii host defense by controlling IFN-γ-mediated Irgb6-dependent cellular innate immunity.


Asunto(s)
Proteínas de Unión al GTP/inmunología , Inmunidad Innata/inmunología , Interferón gamma/inmunología , Macrófagos/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Animales , Cromosomas de los Mamíferos/genética , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Humanos , Inmunidad Celular/inmunología , Interferón gamma/antagonistas & inhibidores , Interferón gamma/metabolismo , Listeria monocytogenes/inmunología , Mediciones Luminiscentes , Macrófagos/metabolismo , Macrófagos/ultraestructura , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Óxido Nítrico/metabolismo , Toxoplasma/metabolismo
3.
FASEB J ; 33(10): 10668-10679, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31268747

RESUMEN

PLC-ß exerts biologic influences through GPCR. GPCRs are involved in regulating glucose-stimulated insulin secretion (GSIS). Previous studies have suggested that PLC-ßs might play an important role in pancreatic ß cells. However, because of a lack of the specific inhibitors of PLC-ß isozymes and appropriate genetic models, the in vivo function of specific PLC-ß isozymes in pancreatic ß cells and their physiologic relevance in the regulation of insulin secretion have not been studied so far. The present study showed that PLC-ß1 was crucial for ß-cell function by generation of each PLC-ß conditional knockout mouse. Mice lacking PLC-ß1 in ß cells exhibited a marked defect in GSIS, leading to glucose intolerance. In ex vivo studies, the secreted insulin level and Ca2+ response in Plcb1f/f; pancreas/duodenum homeobox protein 1 (Pdx1)-Cre recombinase-estrogen receptor T2 (CreERt2) islets was lower than those in the Plcb1f/f islets under the high-glucose condition. PLC-ß1 led to potentiate insulin secretion via stimulation of particular Gq-protein-coupled receptors. Plcb1f/f; Pdx1-CreERt2 mice fed a high-fat diet developed more severe glucose intolerance because of a defect in insulin secretion. The present study identified PLC-ß1 as an important molecule that regulates ß cell insulin secretion and can be considered a candidate for therapeutic intervention in diabetes mellitus.-Hwang, H.-J., Yang, Y. R., Kim, H. Y., Choi, Y., Park, K.-S., Lee, H., Ma, J. S., Yamamoto, M., Kim, J., Chae, Y. C., Choi, J. H., Cocco, L., Berggren, P.-O., Jang, H.-J., Suh, P.-G. Phospholipase Cß1 potentiates glucose-stimulated insulin secretion.


Asunto(s)
Glucosa/metabolismo , Secreción de Insulina/fisiología , Fosfolipasa C beta/metabolismo , Animales , Línea Celular , Dieta Alta en Grasa/efectos adversos , Intolerancia a la Glucosa/genética , Intolerancia a la Glucosa/metabolismo , Intolerancia a la Glucosa/patología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Técnicas In Vitro , Secreción de Insulina/genética , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Isoenzimas/deficiencia , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfolipasa C beta/deficiencia , Fosfolipasa C beta/genética , Receptores Acoplados a Proteínas G/metabolismo , Transactivadores/genética , Transactivadores/metabolismo
4.
Proc Natl Acad Sci U S A ; 112(33): E4581-90, 2015 Aug 18.
Artículo en Inglés | MEDLINE | ID: mdl-26240314

RESUMEN

IFN-γ orchestrates cell-autonomous host defense against various intracellular vacuolar pathogens. IFN-γ-inducible GTPases, such as p47 immunity-related GTPases (IRGs) and p65 guanylate-binding proteins (GBPs), are recruited to pathogen-containing vacuoles, which is important for disruption of the vacuoles, culminating in the cell-autonomous clearance. Although the positive regulation for the proper recruitment of IRGs and GBPs to the vacuoles has been elucidated, the suppressive mechanism is unclear. Here, we show that Rab GDP dissociation inhibitor α (RabGDIα), originally identified as a Rab small GTPase inhibitor, is a negative regulator of IFN-γ-inducible GTPases in cell-autonomous immunity to the intracellular pathogen Toxoplasma gondii. Overexpression of RabGDIα, but not of RabGDIß, impaired IFN-γ-dependent reduction of T. gondii numbers. Conversely, RabGDIα deletion in macrophages and fibroblasts enhanced the IFN-γ-induced clearance of T. gondii. Furthermore, upon a high dose of infection by T. gondii, RabGDIα-deficient mice exhibited a decreased parasite burden in the brain and increased resistance in the chronic phase than did control mice. Among members of IRGs and GBPs important for the parasite clearance, Irga6 and Gbp2 alone were more frequently recruited to T. gondii-forming parasitophorous vacuoles in RabGDIα-deficient cells. Notably, Gbp2 positively controlled Irga6 recruitment that was inhibited by direct and specific interactions of RabGDIα with Gbp2 through the lipid-binding pocket. Taken together, our results suggest that RabGDIα inhibits host defense against T. gondii by negatively regulating the Gbp2-Irga6 axis of IFN-γ-dependent cell-autonomous immunity.


Asunto(s)
GTP Fosfohidrolasas/metabolismo , Regulación Enzimológica de la Expresión Génica , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Interferón gamma/inmunología , Toxoplasma/patogenicidad , Toxoplasmosis/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Chlorocebus aethiops , Cartilla de ADN/genética , Femenino , Fibroblastos/metabolismo , Inflamación/inmunología , Lípidos/química , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Unión Proteica , Homología de Secuencia de Aminoácido , Células Vero
5.
Proc Natl Acad Sci U S A ; 109(13): 5010-5, 2012 Mar 27.
Artículo en Inglés | MEDLINE | ID: mdl-22403066

RESUMEN

Adequate activation of CD4(+) T lymphocytes is essential for host defense against invading pathogens; however, exaggerated activity of effector CD4(+) T cells induces tissue damage, leading to inflammatory disorders such as inflammatory bowel diseases. Several unique subsets of intestinal innate immune cells have been identified. However, the direct involvement of innate immune cell subsets in the suppression of T-cell-dependent intestinal inflammation is poorly understood. Here, we report that intestinal CX(3)C chemokine receptor 1(high) (CX(3)CR1(high)) CD11b(+) CD11c(+) cells are responsible for prevention of intestinal inflammation through inhibition of T-cell responses. These cells inhibit CD4(+) T-cell proliferation in a cell contact-dependent manner and prevent T-cell-dependent colitis. The suppressive activity is abrogated in the absence of the IL-10/Stat3 pathway. These cells inhibit T-cell proliferation by two steps. Initially, CX(3)CR1(high) CD11b(+) CD11c(+) cells preferentially interact with T cells through highly expressed intercellular adhesion molecule-1/vascular cell adhesion molecule-1; then, they fail to activate T cells because of defective expression of CD80/CD86. The IL-10/Stat3 pathway mediates the reduction of CD80/CD86 expression. Transfer of wild-type CX(3)CR1(high) CD11b(+) CD11c(+) cells prevents development of colitis in myeloid-specific Stat3-deficient mice. Thus, these cells are regulatory myeloid cells that are responsible for maintaining intestinal homeostasis.


Asunto(s)
Colitis/inmunología , Colitis/patología , Intestinos/inmunología , Intestinos/patología , Células Mieloides/inmunología , Receptores de Quimiocina/metabolismo , Linfocitos T/inmunología , Animales , Antígenos CD11/metabolismo , Receptor 1 de Quimiocinas CX3C , Proliferación Celular , Colitis/complicaciones , Colitis/prevención & control , Inflamación/complicaciones , Inflamación/inmunología , Inflamación/patología , Inflamación/prevención & control , Interleucina-10/metabolismo , Ratones , Membrana Mucosa/inmunología , Membrana Mucosa/patología , Factor de Transcripción STAT3/deficiencia , Factor de Transcripción STAT3/metabolismo
6.
PLoS Pathog ; 8(5): e1002714, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22693446

RESUMEN

Specific intestinal microbiota has been shown to induce Foxp3(+) regulatory T cell development. However, it remains unclear how development of another regulatory T cell subset, Tr1 cells, is regulated in the intestine. Here, we analyzed the role of two probiotic strains of intestinal bacteria, Lactobacillus casei and Bifidobacterium breve in T cell development in the intestine. B. breve, but not L. casei, induced development of IL-10-producing Tr1 cells that express cMaf, IL-21, and Ahr in the large intestine. Intestinal CD103(+) dendritic cells (DCs) mediated B. breve-induced development of IL-10-producing T cells. CD103(+) DCs from Il10(-/-), Tlr2(-/-), and Myd88(-/-) mice showed defective B. breve-induced Tr1 cell development. B. breve-treated CD103(+) DCs failed to induce IL-10 production from co-cultured Il27ra(-/-) T cells. B. breve treatment of Tlr2(-/-) mice did not increase IL-10-producing T cells in the colonic lamina propria. Thus, B. breve activates intestinal CD103(+) DCs to produce IL-10 and IL-27 via the TLR2/MyD88 pathway thereby inducing IL-10-producing Tr1 cells in the large intestine. Oral B. breve administration ameliorated colitis in immunocompromised mice given naïve CD4(+) T cells from wild-type mice, but not Il10(-/-) mice. These findings demonstrate that B. breve prevents intestinal inflammation through the induction of intestinal IL-10-producing Tr1 cells.


Asunto(s)
Bifidobacterium/inmunología , Colon/microbiología , Interleucina-10/metabolismo , Lacticaseibacillus casei/inmunología , Probióticos/administración & dosificación , Linfocitos T Reguladores/citología , Traslado Adoptivo , Animales , Infecciones por Bifidobacteriales/inmunología , Infecciones por Bifidobacteriales/microbiología , Infecciones por Bifidobacteriales/terapia , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/trasplante , Diferenciación Celular , Técnicas de Cocultivo , Colitis/inmunología , Colitis/microbiología , Colitis/terapia , Colon/inmunología , Células Dendríticas/citología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Interacciones Huésped-Patógeno , Huésped Inmunocomprometido , Interleucina-10/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo
7.
Life Sci Alliance ; 4(7)2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34078740

RESUMEN

Interferon-inducible GTPases, such as immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs), are essential for cell-autonomous immunity against a wide variety of intracellular pathogens including Toxoplasma IRGs comprise regulatory and effector subfamily proteins. Regulatory IRGs Irgm1 and Irgm3 play important roles in anti-Toxoplasma immunity by globally controlling effector IRGs and GBPs. There is a remaining regulatory IRG, called Irgm2, which highly accumulates on parasitophorous vacuole membranes (PVMs). Very little is known about the mechanism of the unique localization on Toxoplasma PVMs. Here, we show that Irgm2 is important to control parasite killing through recruitment of Gbp1 and Irgb6, which does not require Irgm2 localization at Toxoplasma PVMs. Ubiquitination of Irgm2 in the cytosol, but not at the PVM, is also important for parasite killing through recruitment of Gbp1 to the PVM. Conversely, PVM ubiquitination and p62/Sqstm1 loading at later time points post-Toxoplasma infection require Irgm2 localization at the PVM. Irgm2-deficient mice are highly susceptible to Toxoplasma infection. Taken together, these data indicate that Irgm2 selectively controls accumulation of anti-Toxoplasma effectors to the vacuole in a manner dependent or independent on Irgm2 localization at the Toxoplasma PVM, which mediates parasite killing.


Asunto(s)
Proteínas de Unión al GTP/metabolismo , Inmunidad Celular/inmunología , Toxoplasma/metabolismo , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Fibroblastos/metabolismo , GTP Fosfohidrolasas/metabolismo , Interacciones Huésped-Parásitos/inmunología , Inmunidad Innata/inmunología , Interferón gamma/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Unión al GTP Monoméricas , Toxoplasma/patogenicidad , Toxoplasmosis/metabolismo , Toxoplasmosis/parasitología , Vacuolas
8.
Parasitol Int ; 83: 102335, 2021 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-33771680

RESUMEN

Sporozoites of the etiological agent of malaria, Plasmodium, form parasitophorous vacuoles (PVs) in hepatocytes. The PV membranes (PVM) are coated with a well-known host autophagy marker LC3 and parasite-derived protein called Upregulated in infective sporozoites 3 (UIS3), which has been shown to interact with LC3 and inhibit LC3-mediated autophagic disruption at the PV. Although uis3(-) sporozoites cannot proliferate in wild-type cells, they can replicate efficiently in cells defective in autophagy due to the lack of Atg proteins such as Atg3, Atg5 and Atg7, since these Atg proteins are essential for processing of LC3. However, it remains to be seen whether other Atg proteins participate in the restriction of uis3(-) parasite growth. Here we show that, despite essential roles of Atg9 and Atg14 in autophagy, both proteins are dispensable for the restriction of uis3(-) parasite growth. Moreover, we found that cells lacking LC3 proteins are also able to restrict uis3(-) parasite growth. In sharp contrast, GABARAPs, another subfamily of mammalian Atg8, participated in suppression of uis3(-) parasite growth. Taken together, contrary to a previous model in which UIS3 avoids host LC3- and autophagy-dependent parasite elimination program, our data demonstrate a role of GABARAPs for suppression of uis3(-) parasite growth in a manner independent on autophagy.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Autofagia , Malaria/parasitología , Proteínas Asociadas a Microtúbulos/genética , Plasmodium berghei/fisiología , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Interacciones Huésped-Parásitos , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/metabolismo
9.
J Exp Med ; 218(7)2021 07 05.
Artículo en Inglés | MEDLINE | ID: mdl-33970189

RESUMEN

Because of their common signaling molecules, the main T cell receptor (TCR) signaling cascades in CD4+ and CD8+ T cells are considered qualitatively identical. Herein, we show that TCR signaling in CD8+ T cells is qualitatively different from that in CD4+ T cells, since CD8α ignites another cardinal signaling cascade involving phospholipase C ß4 (PLCß4). TCR-mediated responses were severely impaired in PLCß4-deficient CD8+ T cells, whereas those in CD4+ T cells were intact. PLCß4-deficient CD8+ T cells showed perturbed activation of peripheral TCR signaling pathways downstream of IP3 generation. Binding of PLCß4 to the cytoplasmic tail of CD8α was important for CD8+ T cell activation. Furthermore, GNAQ interacted with PLCß4, mediated double phosphorylation on threonine 886 and serine 890 positions of PLCß4, and activated CD8+ T cells in a PLCß4-dependent fashion. PLCß4-deficient mice exhibited defective antiparasitic host defense and antitumor immune responses. Altogether, PLCß4 differentiates TCR signaling in CD4+ and CD8+ T cells and selectively promotes CD8+ T cell-dependent adaptive immunity.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Fosfolipasa C beta/inmunología , Transducción de Señal/inmunología , Animales , Línea Celular , Citoplasma/inmunología , Células HEK293 , Humanos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Fosforilación/inmunología , Receptores de Antígenos de Linfocitos T/inmunología
10.
Nitric Oxide ; 23(3): 214-9, 2010 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-20547236

RESUMEN

Macrophage-derived nitric oxide (NO) plays an important role in protection against microbial infection in immune responses. Overproduction of NO by inducible nitric synthase (iNOS) is known to be closely correlated with the pathology of a variety of diseases and inflammations. In this study, we investigated the inhibitory effect of polyethylene glycol coated gold nanoparticles (GNP) on NO production and its molecular mechanism in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. It was found that GNP inhibited LPS-induced NO production and iNOS expression in RAW264.7 cells. Furthermore, GNP suppressed LPS-induced activation of NF-kappaB through the inhibition of Akt activity. GNP also inhibited LPS-induced phosphorylation of signal transducer and activator of transcription 1 (STAT1) via down-regulation of interferon-beta (IFN-beta) expression. Our results suggest that GNP inhibits NO production and iNOS expression through blocking the activation of NF-kappaB and STAT1 in LPS-stimulated RAW264.7 cells.


Asunto(s)
Oro/farmacología , Interferón beta/antagonistas & inhibidores , Nanopartículas del Metal/química , FN-kappa B/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Factor de Transcripción STAT1/antagonistas & inhibidores , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Oro/química , Interferón beta/metabolismo , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Ratones , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Polietilenglicoles/química , Polietilenglicoles/farmacología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT1/metabolismo
11.
Parasitol Int ; 75: 102049, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-31901434

RESUMEN

Interferon-γ (IFN-γ) is important for host defense against various intracellular organisms including a protozoan pathogen Toxoplasma gondii. Various immune cells are recently shown to produce IFN-γ in T. gondii infection, however, it remains elusive which cell types are important for anti-T. gondii host defense so far. Here we generate a new IFN-γ reporter "GREVEN" mouse line in which a fusion protein of Venus and NanoLuc to analyze IFN-γ producing cells during T. gondii infection and find that CD4+, CD8+, γδ T cells and natural killer cells express Venus in a time dependent manner. Furthermore, Lck-Cre/Ifngfl/fl mice are highly susceptible to T. gondii infection. Taken together, our results demonstrate that T cell-derived IFN-γ plays an important role in anti-T. gondii host defense.


Asunto(s)
Resistencia a la Enfermedad/inmunología , Interferón gamma/inmunología , Linfocitos T/inmunología , Toxoplasma/inmunología , Toxoplasmosis Animal/inmunología , Animales , Interferón gamma/genética , Ratones , Toxoplasmosis/inmunología
12.
Front Immunol ; 11: 561948, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33042141

RESUMEN

Sepsis is a life-threating multi-organ disease induced by host innate immunity to pathogen-derived endotoxins including lipopolysaccharide (LPS). Direct sensing of LPS by caspase-11 activates inflammasomes and causes lethal sepsis in mice. Inhibition of caspase-11 inflammasomes is important for the prevention of LPS-induced septic shock; however, whether a caspase-11 inflammasome-specific suppressive mechanism exists is unclear. Here we show that deficiency of GABARAP autophagy-related proteins results in over-activation of caspase-11 inflammasomes but not of canonical inflammasomes. Gate-16-/-Gabarap-/- macrophages exhibited elevated guanylate binding protein 2 (GBP2)-dependent caspase-11 activation and inflammatory responses. Deficiency of GABARAPs resulted in formation of GBP2-containing aggregates that promote IL-1ß production. High mortality after low dose LPS challenge in Gate-16-/-Gabarap-/- mice primed with poly(I:C) or polymicrobial sepsis was ameliorated by compound GBP2 deficiency. These results reveal a critical function of Gate-16 and Gabarap to suppress GBP2-dependent caspase-11-induced inflammation and septic shock.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/deficiencia , Familia de las Proteínas 8 Relacionadas con la Autofagia/deficiencia , Caspasas Iniciadoras/metabolismo , Proteínas Asociadas a Microtúbulos/deficiencia , Choque Séptico/inmunología , Choque Séptico/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Proteínas de Unión al GTP/deficiencia , Inmunidad Innata , Inflamasomas/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Interleucina-1beta/metabolismo , Lipopolisacáridos/efectos adversos , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/genética , Piroptosis/genética , Choque Séptico/inducido químicamente , Transducción de Señal/genética
13.
Life Sci Alliance ; 3(1)2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31852733

RESUMEN

Toxoplasma gondii is an obligate intracellular protozoan parasite capable of infecting warm-blooded animals by ingestion. The organism enters host cells and resides in the cytoplasm in a membrane-bound parasitophorous vacuole (PV). Inducing an interferon response enables IFN-γ-inducible immunity-related GTPase (IRG protein) to accumulate on the PV and to restrict parasite growth. However, little is known about the mechanisms by which IRG proteins recognize and destroy T. gondii PV. We characterized the role of IRG protein Irgb6 in the cell-autonomous response against T. gondii, which involves vacuole ubiquitination and breakdown. We show that Irgb6 is capable of binding a specific phospholipid on the PV membrane. Furthermore, the absence of Irgb6 causes reduced targeting of other effector IRG proteins to the PV. This suggests that Irgb6 has a role as a pioneer in the process by which multiple IRG proteins access the PV. Irgb6-deficient mice are highly susceptible to infection by a strain of T. gondii avirulent in wild-type mice.


Asunto(s)
Interacciones Huésped-Parásitos/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Fosfolípidos/metabolismo , Toxoplasma/citología , Toxoplasmosis/metabolismo , Vacuolas/metabolismo , Animales , Células Cultivadas , Femenino , Fibroblastos/metabolismo , Interacciones Huésped-Parásitos/efectos de los fármacos , Interacciones Huésped-Parásitos/inmunología , Inmunidad Innata , Interferón gamma/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Unión al GTP Monoméricas/deficiencia , Proteínas de Unión al GTP Monoméricas/genética , Unión Proteica , Proteínas Recombinantes/farmacología , Toxoplasmosis/parasitología , Ubiquitina/metabolismo , Ubiquitinación/efectos de los fármacos , Ubiquitinación/genética
14.
Artículo en Inglés | MEDLINE | ID: mdl-31119110

RESUMEN

Toxoplasma gondii is an important human and animal pathogen that causes life-threatening toxoplasmosis. The host immune system produces interferon-γ (IFN-γ) to inhibit T. gondii proliferation. IFN-γ-inducible indole-2,3-dioxygenase 1 (IDO1), which mediates tryptophan degradation, has a major role in anti-T. gondii immune responses in various human cells. In response to the host's immune system, T. gondii secretes many virulence molecules into the host cells to suppress IFN-γ-dependent antiparasitic immune responses. The GRA15-induced proparasitic mechanism for suppressing IDO1-dependent immune responses has previously been tested only in human hepatocyte and monocyte co-cultures. Thus, whether human cells other than hepatocytes contain this virulence mechanism remains unclear. Here, we show that the GRA15-dependent virulence mechanism for suppressing the IDO1-dependent anti-T. gondii response operates in human neuronal cell lines and primary human neurons. Analysis of various human cell lines revealed that IL-1ß-induced iNOS-dependent reduction of IDO1 mRNA expression occurred in brain cell lines (A172; glioblastoma, IMR-32; neuroblastoma, and T98G; glioblastoma) and liver cell lines (Huh7 and HepG2), but not in other cell lines. Moreover, co-culturing type II T. gondii-infected THP-1 human monocytes with the brain cell lines inhibited the IDO1-mediated anti-T. gondii response in a GRA15-dependent manner. These data suggest that a GRA15-dependent virulence mechanism antagonizes the IDO1-dependent host immune response in human brain cells.


Asunto(s)
Antígenos de Protozoos/metabolismo , Antiparasitarios/metabolismo , Interferón gamma/metabolismo , Neuronas/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Toxoplasmosis/inmunología , Antígenos de Protozoos/inmunología , Antiparasitarios/farmacología , Línea Celular , Hepatocitos/inmunología , Interacciones Huésped-Parásitos/inmunología , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/farmacología , Interferón gamma/inmunología , Interleucina-1beta/metabolismo , Monocitos/inmunología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Proteínas Protozoarias/inmunología , ARN Mensajero/metabolismo , Toxoplasma/efectos de los fármacos , Toxoplasma/inmunología , Virulencia
15.
J Exp Med ; 216(8): 1733-1748, 2019 08 05.
Artículo en Inglés | MEDLINE | ID: mdl-31189656

RESUMEN

The liver stage of the etiological agent of malaria, Plasmodium, is obligatory for successful infection of its various mammalian hosts. Differentiation of the rod-shaped sporozoites of Plasmodium into spherical exoerythrocytic forms (EEFs) via bulbous expansion is essential for parasite development in the liver. However, little is known about the host factors regulating the morphological transformation of Plasmodium sporozoites in this organ. Here, we show that sporozoite differentiation into EEFs in the liver involves protein kinase C ζ-mediated NF-κB activation, which robustly induces the expression of C-X-C chemokine receptor type 4 (CXCR4) in hepatocytes and subsequently elevates intracellular Ca2+ levels, thereby triggering sporozoite transformation into EEFs. Blocking CXCR4 expression by genetic or pharmacological intervention profoundly inhibited the liver-stage development of the Plasmodium berghei rodent malaria parasite and the human Plasmodium falciparum parasite. Collectively, our experiments show that CXCR4 is a key host factor for Plasmodium development in the liver, and CXCR4 warrants further investigation for malaria prophylaxis.


Asunto(s)
Hepatocitos/metabolismo , Malaria/metabolismo , Plasmodium berghei/crecimiento & desarrollo , Plasmodium falciparum/crecimiento & desarrollo , Receptores CXCR4/metabolismo , Animales , Sistemas CRISPR-Cas , Calcio/metabolismo , Línea Celular Tumoral , Humanos , Hígado/metabolismo , Malaria/parasitología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Receptores CXCR4/genética , Esporozoítos/metabolismo , Transfección
16.
Front Immunol ; 9: 2073, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30283439

RESUMEN

Toxoplasma gondii is an important human and animal pathogen that causes life-threatening toxoplasmosis. Interferon-γ (IFN-γ) is critical for anti-T. gondii cell-autonomous immunity in both humans and mice. To proliferate efficiently within the hosts, virulent strains of T. gondii can suppress IFN-γ-dependent immunity. During parasite infection, it is well-characterized that various virulence effectors are secreted to transcriptionally or post-translationally target IFN-γ-inducible GTPases, which are essential for anti-parasite responses in mice. However, the role of IFN-γ-inducible GTPases in anti-T. gondii responses in human cells is controversial since they are non-functional or absent in humans. Instead, IFN-γ-induced tryptophan degradation by indole-2,3-dioxygenase (IDO) is important for the anti-T. gondii human response. To date, the T. gondii virulent mechanism targeting IDO in human cells remains elusive. Here we show that although humans possess two IDO isozymes, IDO1 and IDO2, human cells of various origins require IDO1 but not IDO2 for IFN-γ-induced cell-autonomous immunity to T. gondii. T. gondii secretes an effector TgIST to inhibit IDO1 mRNA expression. Taken together, the data suggests that T. gondii possesses virulence programs operated by TgIST to antagonize IFN-γ-induced IDO1-mediated anti-parasite cell-autonomous immunity in human cells.


Asunto(s)
Inmunidad Celular/inmunología , Indolamina-Pirrol 2,3,-Dioxigenasa/inmunología , Interferón gamma/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Animales , Autofagia/genética , Autofagia/inmunología , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/inmunología , Proteínas Relacionadas con la Autofagia/metabolismo , Línea Celular Tumoral , Células Cultivadas , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/inmunología , GTP Fosfohidrolasas/metabolismo , Células HeLa , Humanos , Inmunidad Celular/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interferón gamma/metabolismo , Ratones Noqueados , Toxoplasma/patogenicidad , Toxoplasmosis/enzimología , Toxoplasmosis/parasitología , Virulencia/genética , Virulencia/inmunología
17.
mBio ; 9(5)2018 10 09.
Artículo en Inglés | MEDLINE | ID: mdl-30301855

RESUMEN

Although Toxoplasma virulence mechanisms targeting gamma interferon (IFN-γ)-induced cell-autonomous antiparasitic immunity have been extensively characterized in mice, the virulence mechanisms in humans remain uncertain, partly because cell-autonomous immune responses against Toxoplasma differ markedly between mice and humans. Despite the identification of inducible nitric oxide synthase (iNOS) as an anti-Toxoplasma host factor in mice, here we show that iNOS in humans is a pro-Toxoplasma host factor that promotes the growth of the parasite. The GRA15 Toxoplasma effector-dependent disarmament of IFN-γ-induced parasite growth inhibition was evident when parasite-infected monocytes were cocultured with hepatocytes. Interleukin-1ß (IL-1ß), produced from monocytes in a manner dependent on GRA15 and the host's NLRP3 inflammasome, combined with IFN-γ to strongly stimulate iNOS expression in hepatocytes; this dramatically reduced the levels of indole 2,3-dioxygenase 1 (IDO1), a critically important IFN-γ-inducible anti-Toxoplasma protein in humans, thus allowing parasite growth. Taking the data together, Toxoplasma utilizes human iNOS to antagonize IFN-γ-induced IDO1-mediated cell-autonomous immunity via its GRA15 virulence factor.IMPORTANCEToxoplasma, an important intracellular parasite of humans and animals, causes life-threatening toxoplasmosis in immunocompromised individuals. Gamma interferon (IFN-γ) is produced in the host to inhibit the proliferation of this parasite and eventually cause its death. Unlike mouse disease models, which involve well-characterized virulence strategies that are used by Toxoplasma to suppress IFN-γ-dependent immunity, the strategies used by Toxoplasma in humans remain unclear. Here, we show that GRA15, a Toxoplasma effector protein, suppresses the IFN-γ-induced indole-2,3-dioxygenase 1-dependent antiparasite immune response in human cells. Because NLRP3-dependent production of IL-1ß and nitric oxide (NO) in Toxoplasma-infected human cells is involved in the GRA15-dependent virulence mechanism, blocking NO or IL-1ß production in the host could represent a novel therapeutic approach for treating human toxoplasmosis.


Asunto(s)
Interferón gamma/farmacología , Óxido Nítrico Sintasa de Tipo II/inmunología , Proteínas Protozoarias/inmunología , Toxoplasma/inmunología , Factores de Virulencia/inmunología , Animales , Antígenos de Protozoos/inmunología , Sistemas CRISPR-Cas , Línea Celular , Técnicas de Cocultivo , Hepatocitos/efectos de los fármacos , Hepatocitos/inmunología , Hepatocitos/parasitología , Interacciones Huésped-Parásitos/inmunología , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interleucina-1beta/metabolismo , Ratones , Ratones Endogámicos C57BL , Monocitos/inmunología , Monocitos/parasitología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Toxoplasma/patogenicidad
18.
Mol Brain ; 9(1): 100, 2016 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-27998287

RESUMEN

The transition from wakefulness to a nonrapid eye movement (NREM) sleep state at the onset of sleep involves a transition from low-voltage, high-frequency irregular electroencephalography (EEG) waveforms to large-amplitude, low-frequency EEG waveforms accompanying synchronized oscillatory activity in the thalamocortical circuit. The thalamocortical circuit consists of reciprocal connections between the thalamus and cortex. The cortex sends strong excitatory feedback to the thalamus, however the function of which is unclear. In this study, we investigated the role of the thalamic metabotropic glutamate receptor 1 (mGluR1)-phospholipase C ß4 (PLCß4) pathway in sleep control in PLCß4-deficient (PLCß4-/-) mice. The thalamic mGluR1-PLCß4 pathway contains synapses that receive corticothalamic inputs. In PLCß4-/- mice, the transition from wakefulness to the NREM sleep state was stimulated, and the NREM sleep state was stabilized, which resulted in increased NREM sleep. The power density of delta (δ) waves increased in parallel with the increased NREM sleep. These sleep phenotypes in PLCß4-/- mice were consistent in TC-restricted PLCß4 knockdown mice. Moreover, in vitro intrathalamic oscillations were greatly enhanced in the PLCß4-/- slices. The results of our study showed that thalamic mGluR1-PLCß4 pathway was critical in controlling sleep architecture.


Asunto(s)
Fosfolipasa C beta/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Sueño/fisiología , Tálamo/metabolismo , Animales , Corteza Cerebral/fisiología , Ritmo Delta/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfolipasa C beta/deficiencia , Tálamo/fisiología
19.
Cell Rep ; 13(2): 223-33, 2015 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-26440898

RESUMEN

Also known as Sqstm1, p62 is a selective autophagy adaptor with a ubiquitin-binding domain. However, the role of p62 in the host defense against Toxoplasma gondii infection is unclear. Here, we show that interferon γ (IFN-γ) stimulates ubiquitin and p62 recruitment to T. gondii parasitophorous vacuoles (PVs). Some essential autophagy-related proteins, but not all, are required for this recruitment. Regardless of normal IFN-γ-induced T. gondii clearance activity and ubiquitination, p62 deficiency in antigen-presenting cells (APCs) and mice diminishes the robust IFN-γ-primed activation of CD8(+) T cells that recognize the T. gondii-derived antigen secreted into PVs. Because the expression of Atg3 and Irgm1/m3 in APCs is essential for PV disruption, ubiquitin and p62 recruitment, and vacuolar-antigen-specific CD8(+) T cell activation, IFN-γ-mediated ubiquitination and the subsequent recruitment of p62 to T. gondii are specifically required for the acquired immune response after PV disruption by IFN-γ-inducible GTPases.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Presentación de Antígeno , Antígenos de Protozoos/inmunología , Proteínas de Choque Térmico/inmunología , Interferón gamma/metabolismo , Toxoplasma/inmunología , Vacuolas/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Células Presentadoras de Antígenos/inmunología , Autofagia , Linfocitos T CD8-positivos/inmunología , Línea Celular , Células Cultivadas , Proteínas de Choque Térmico/genética , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Proteína Sequestosoma-1 , Ubiquitina/metabolismo , Ubiquitinación
20.
J Exp Med ; 211(10): 2013-32, 2014 Sep 22.
Artículo en Inglés | MEDLINE | ID: mdl-25225460

RESUMEN

Toxoplasma gondii infection results in co-option and subversion of host cellular signaling pathways. This process involves discharge of T. gondii effector molecules from parasite secretory organelles such as rhoptries and dense granules. We report that the T. gondii polymorphic dense granule protein GRA6 regulates activation of the host transcription factor nuclear factor of activated T cells 4 (NFAT4). GRA6 overexpression robustly and selectively activated NFAT4 via calcium modulating ligand (CAMLG). Infection with wild-type (WT) but not GRA6-deficient parasites induced NFAT4 activation. Moreover, GRA6-deficient parasites failed to exhibit full virulence in local infection, and the treatment of WT mice with an NFAT inhibitor mitigated virulence of WT parasites. Notably, NFAT4-deficient mice displayed prolonged survival, decreased recruitment of CD11b(+) Ly6G(+) cells to the site of infection, and impaired expression of chemokines such as Cxcl2 and Ccl2. In addition, infection with type I parasites culminated in significantly higher NFAT4 activation than type II parasites due to a polymorphism in the C terminus of GRA6. Collectively, our data suggest that GRA6-dependent NFAT4 activation is required for T. gondii manipulation of host immune responses to maximize the parasite virulence in a strain-dependent manner.


Asunto(s)
Antígenos de Protozoos/metabolismo , Regulación de la Expresión Génica/fisiología , Factores de Transcripción NFATC/metabolismo , Proteínas Protozoarias/metabolismo , Transducción de Señal/fisiología , Toxoplasma/patogenicidad , Toxoplasmosis/fisiopatología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Antígenos de Protozoos/genética , Proteínas Bacterianas , Western Blotting , Femenino , Citometría de Flujo , Inmunoprecipitación , Luciferasas , Proteínas Luminiscentes , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Factores de Transcripción NFATC/antagonistas & inhibidores , Proteínas Protozoarias/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Toxoplasma/metabolismo
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