RESUMEN
Several members of the NLR family of sensors activate innate immunity. In contrast, we found here that NLRC3 inhibited Toll-like receptor (TLR)-dependent activation of the transcription factor NF-κB by interacting with the TLR signaling adaptor TRAF6 to attenuate Lys63 (K63)-linked ubiquitination of TRAF6 and activation of NF-κB. We used bioinformatics to predict interactions between NLR and TRAF proteins, including interactions of TRAF with NLRC3. In vivo, macrophage expression of Nlrc3 mRNA was diminished by the administration of lipopolysaccharide (LPS) but was restored when cellular activation subsided. To assess biologic relevance, we generated Nlrc3(-/-) mice. LPS-treated Nlrc3(-/-) macrophages had more K63-ubiquitinated TRAF6, nuclear NF-κB and proinflammatory cytokines. Finally, LPS-treated Nlrc3(-/-) mice had more signs of inflammation. Thus, signaling via NLRC3 and TLR constitutes a negative feedback loop. Furthermore, prevalent NLR-TRAF interactions suggest the formation of a 'TRAFasome' complex.
Asunto(s)
FN-kappa B/inmunología , Receptores Acoplados a Proteínas G/inmunología , Transducción de Señal/inmunología , Factor 6 Asociado a Receptor de TNF/inmunología , Receptores Toll-Like/inmunología , Secuencia de Aminoácidos , Animales , Retroalimentación Fisiológica , Células HEK293 , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Acoplados a Proteínas G/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor 6 Asociado a Receptor de TNF/metabolismo , Receptores Toll-Like/metabolismoRESUMEN
Plants evolved intracellular immune receptors that belong to the NOD-like receptor (NLR) family to recognize the presence of pathogen-derived effector proteins. NLRs possess an N-terminal Toll-like/IL-1 receptor (TIR) or a non-TIR domain [some of which contain coiled coils (CCs)], a central nucleotide-binding (NB-ARC) domain, and a C-terminal leucine-rich repeat (LRR). Activation of NLR proteins results in a rapid and high-amplitude immune response, eventually leading to host cell death at the infection site, the so-called hypersensitive response. Despite their important contribution to immunity, the exact mechanisms of NLR activation and signaling remain unknown and are likely heterogenous. We undertook a detailed structure-function analysis of the plasma membrane (PM)-localized CC NLR Resistance to Pseudomonas syringae pv. maculicola 1 (RPM1) using both stable transgenic Arabidopsis and transient expression in Nicotiana benthamiana We report that immune signaling is induced only by activated full-length PM-localized RPM1. Our interaction analyses demonstrate the importance of a functional P-loop for in planta interaction of RPM1 with the small host protein RPM1-interacting protein 4 (RIN4), for constitutive preactivation and postactivation self-association of RPM1 and for proper PM localization. Our results reveal an additive effect of hydrophobic conserved residues in the CC domain for RPM1 function and RPM1 self-association and their necessity for RPM1-RIN4 interaction. Thus, our findings considerably extend our understanding of the mechanisms regulating NLR activation at, and signaling from, the PM.
Asunto(s)
Proteínas de Arabidopsis/inmunología , Proteínas de Arabidopsis/metabolismo , Inmunidad de la Planta/inmunología , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Inmunidad Innata/inmunología , Péptidos y Proteínas de Señalización Intracelular , Proteínas NLR/inmunología , Enfermedades de las Plantas/inmunología , Unión Proteica , Pseudomonas syringae/fisiología , Receptores Inmunológicos/metabolismo , Transducción de Señal , Nicotiana/metabolismoRESUMEN
The nucleotide binding domain and leucine-rich repeat-containing (NLR) family of proteins is known to activate innate immunity, and the inflammasome-associated NLRs are prime examples. In contrast, the concept that NLRs can inhibit innate immunity is still debated, and the impact of such inhibitory NLRs in diseases shaped by adaptive immune responses is entirely unexplored. This study demonstrates that, in contrast to other NLRs that activate immunity, NLRX1 plays a protective role in experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. When compared with wild-type controls, Nlrx1(-/-) mice have significantly worsened clinical scores and heightened CNS tissue damage during EAE. NLRX1 does not alter the production of encephalitogenic T cells in the peripheral lymphatic tissue, but Nlrx1(-/-) mice are more susceptible to adoptively transferred myelin-reactive T cells. Analysis of the macrophage and microglial populations indicates that NLRX1 reduces activation during both active and passive EAE models. This work represents the first case of an NLR that attenuates microglia inflammatory activities and protects against a neurodegenerative disease model caused by autoreactive T cells.
Asunto(s)
Encefalomielitis Autoinmune Experimental/metabolismo , Macrófagos/metabolismo , Microglía/metabolismo , Proteínas Mitocondriales/metabolismo , Esclerosis Múltiple/metabolismo , Linfocitos T/metabolismo , Animales , Células Cultivadas , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Inflamación , Macrófagos/inmunología , Macrófagos/patología , Ratones , Ratones Noqueados , Microglía/inmunología , Microglía/patología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/inmunología , Esclerosis Múltiple/genética , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
Plants deploy intracellular innate immune receptors to recognize pathogens and initiate disease resistance. These nucleotide-binding, leucine-rich repeat (NB-LRR) proteins are activated by pathogen effector proteins that are delivered into the host cell to suppress host defense responses. Little is known about the sites and mechanisms of NB-LRR activation, but some NB-LRR proteins can function inside the plant nucleus. We demonstrate that RPM1 is activated on the plasma membrane and does not relocalize to the nucleus. An autoactive RPM1(D505V) allele that recapitulates key features of normal RPM1 activation also resides on the plasma membrane. There is no detectable relocalization of activated RPM1 to the nucleus. Hindering potential nuclear entry of RPM1-Myc did not affect either its effector-triggered hypersensitive-response (HR) cell death or its disease resistance functions, further suggesting that nuclear translocation is not required for RPM1 function. RPM1 tethered onto the plasma membrane with a dual acylated N-terminal epitope tag retained the ability to mediate HR, consistent with this RPM1 function being activated on the plasma membrane. Plant NB-LRR proteins can thus function at various locations in the cell.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Membrana Celular/metabolismo , Inmunidad Innata/genética , Enfermedades de las Plantas/inmunología , Proteínas/metabolismo , Arabidopsis/inmunología , Proteínas de Arabidopsis/inmunología , Vectores Genéticos/genética , Proteínas Repetidas Ricas en Leucina , Microscopía Confocal , Proteínas/inmunologíaRESUMEN
The type III effector protein encoded by avirulence gene B (AvrB) is delivered into plant cells by pathogenic strains of Pseudomonas syringae. There, it localizes to the plasma membrane and triggers immunity mediated by the Arabidopsis coiled-coil (CC)-nucleotide binding (NB)-leucine-rich repeat (LRR) disease resistance protein RPM1. The sequence unrelated type III effector avirulence protein encoded by avirulence gene Rpm1 (AvrRpm1) also activates RPM1. AvrB contributes to virulence after delivery from P. syringae in leaves of susceptible soybean plants, and AvrRpm1 does the same in Arabidopsis rpm1 plants. Conditional overexpression of AvrB in rpm1 plants results in leaf chlorosis. In a genetic screen for mutants that lack AvrB-dependent chlorosis in an rpm1 background, we isolated TAO1 (target of AvrB operation), which encodes a Toll-IL-1 receptor (TIR)-NB-LRR disease resistance protein. In rpm1 plants, TAO1 function results in the expression of the pathogenesis-related protein 1 (PR-1) gene, suggestive of a defense response. In RPM1 plants, TAO1 contributes to disease resistance in response to Pto (P. syringae pathovars tomato) DC3000(avrB), but not against Pto DC3000(avrRpm1). The tao1-5 mutant allele, a stop mutation in the LRR domain of TAO1, posttranscriptionally suppresses RPM1 accumulation. These data provide evidence of genetically separable disease resistance responses to AvrB and AvrRpm1 in Arabidopsis. AvrB activates both RPM1, a CC-NB-LRR protein, and TAO1, a TIR-NB-LRR protein. These NB-LRR proteins then act additively to generate a full disease resistance response to P. syringae expressing this type III effector.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Arabidopsis/microbiología , Proteínas Bacterianas/metabolismo , Enfermedades de las Plantas/inmunología , Proteínas/metabolismo , Pseudomonas syringae/fisiología , Receptores Toll-Like/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Hidrolasas de Éster Carboxílico/metabolismo , Regulación de la Expresión Génica de las Plantas , Inmunidad Innata/inmunología , Proteínas Repetidas Ricas en Leucina , Datos de Secuencia Molecular , Mutación/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Receptores Toll-Like/genética , Transcripción GenéticaRESUMEN
Burn patients suffer from immunological dysfunction for which there are currently no successful interventions. Similar to previous observations, we find that burn shock patients (≥15% Total Burn Surface Area (TBSA) injury) have elevated levels of the innate immune cytokines Interleukin-6 (IL-6) and Monocyte Chemoattractant Protein-1 (MCP-1)/CC-motif Chemokine Ligand 2(CCL2) early after hospital admission (0-48 Hours Post-hospital Admission (HPA). Functional immune assays with patient Peripheral Blood Mononuclear Cells (PBMCs) revealed that burn shock patients (≥15% TBSA) produced elevated levels of MCP-1/CCL2 after innate immune stimulation ex vivo relative to mild burn patients. Interestingly, treatment of patient PBMCs with the Nuclear Factor-Erythroid-2-Related Factor 2 (NRF2) agonist, CDDO-Me(bardoxolone methyl), reduced MCP-1 production but not IL-6 or Interleukin-10 (IL-10) secretion. In enriched monocytes from healthy donors, CDDO-Me(bardoxolone methyl) also reduced LPS-induced MCP1/CCL2 production but did not alter IL-6 or IL-10 secretion. Similar immunomodulatory effects were observed with Compound 7, which activates the NRF2 pathway through a different and non-covalent Mechanism Of Action (MOA). Hence, our findings with CDDO-Me(bardoxolone methyl) and Compound 7 are likely to reflect a generalizable aspect of NRF2 activation. These observed effects were not specific to LPS-induced immune responses, as NRF2 activation also reduced MCP-1/CCL2 production after stimulation with IL-6. Pharmacological NRF2 activation reduced Mcp-1/Ccl2 transcript accumulation without inhibiting either Il-6 or Il-10 transcript levels. Hence, we describe a novel aspect of NRF2 activation that may contribute to the beneficial effects of NRF2 agonists during disease. Our work also demonstrates that the NRF2 pathway is retained and can be modulated to regulate important immunomodulatory functions in burn patient immune cells.
Asunto(s)
Quemaduras/inmunología , Quemaduras/metabolismo , Citocinas/biosíntesis , Inmunidad Innata , Linfocitos/inmunología , Linfocitos/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Adulto , Quemaduras/mortalidad , Quemaduras/terapia , Quimiocina CCL2/biosíntesis , Estudios de Cohortes , Femenino , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana EdadRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0184164.].
RESUMEN
The possibility of engineering the immune system in a targeted fashion using biomaterials such as nanoparticles has made considerable headway in recent years. However, little is known as to how modulating the spatial presentation of a ligand augments downstream immune responses. In this report we show that geometric manipulation of phosphatidylserine (PS) through fabrication on rod-shaped PLGA nanoparticles robustly dampens inflammatory responses from innate immune cells while promoting T regulatory cell abundance by impeding effector T cell expansion. This response depends on the geometry of PS presentation as both PS liposomes and 1 micron cylindrical PS-PLGA particles are less potent signal inducers than 80 × 320 nm rod-shaped PS-PLGA particles for an equivalent dose of PS. We show that this immune tolerizing effect can be co-opted for therapeutic benefit in a mouse model of multiple sclerosis and an assay of organ rejection using a mixed lymphocyte reaction with primary human immune cells. These data provide evidence that geometric manipulation of a ligand via biomaterials may enable more efficient and tunable programming of cellular signaling networks for therapeutic benefit in a variety of disease states, including autoimmunity and organ rejection, and thus should be an active area of further research.
Asunto(s)
Tolerancia Inmunológica , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Animales , Linfocitos T CD4-Positivos/inmunología , Citocinas/metabolismo , Células Dendríticas/patología , Regulación hacia Abajo , Humanos , Inflamación/patología , Activación de Linfocitos/inmunología , Ratones Endogámicos C57BL , Vaina de Mielina/metabolismo , Nanotubos , Material Particulado/químicaRESUMEN
In plants, many of the innate immune receptors or disease resistance (R) proteins contain a NB-LRR (Nucleotide-binding site, Leucine-rich repeat) structure. The recent findings regarding NB-LRR signaling are summarized in this article. An emerging theme is that two NB-LRRs can function together to mediate disease resistance against pathogen isolates. Also, recent results delineate the NB-LRR protein fragments that are sufficient to initiate defense signaling. Importantly, distinct fragments of different NB-LRRs are sufficient for function. Finally, we describe the new roles of accessory proteins and downstream host genes in NB-LRR signaling.
Asunto(s)
Nucleótidos/metabolismo , Proteínas de Plantas/metabolismo , Secuencias Repetitivas de Aminoácido , Transducción de Señal , Animales , Sitios de Unión , Humanos , Enfermedades de las Plantas/inmunología , Proteínas de Plantas/química , Proteínas de Plantas/inmunologíaRESUMEN
A tapetum-specific gene, RTS, has been isolated by differential screening of a cDNA library from rice panicles. RTS is a unique gene in the rice genome. RNA blot analysis and in situ hybridization indicates that this gene is predominantly expressed in the anther's tapetum during meiosis and disappears before anthesis. RTS has no introns and encodes a putative polypeptide of 94 amino acids with a hydrophobic N-terminal region. The nucleotide and deduced amino acid sequence of the gene do not show significant homology to any known sequences. However, a sequence in the promoter region, GAATTTGTTA, differs only by one or two nucleotides from one of the conserved motifs in the promoter region of two pollen-specific genes of tomato. Several other sequence motifs found in other anther-specific promoters were also identified in the promoter of the RTS gene. Transgenic and antisense RNA approaches revealed that RTS gene is required for male fertility in rice. The promoter region of RTS, when fused to the Bacillus amyloliquefaciens ribonuclease gene, barnase, or the antisense of the RTS gene, is able to drive tissue-specific expression of both genes in rice, creeping bentgrass (Agrostis stolonifera L.) and Arabidopsis, conferring male sterility to the transgenic plants. Light and near-infrared confocal microscopy of cross-sections through developing flowers of male-sterile transgenics shows that tissue-specific expression of barnase or the antisense RTS genes interrupts tapetal development, resulting in deformed non-viable pollen. These results demonstrate a critical role of the RTS gene in pollen development in rice and the versatile application of the RTS gene promoter in directing anther-specific gene expression in both monocotyledonous and dicotyledonous plants, pointing to a potential for exploiting this gene and its promoter for engineering male sterility for hybrid production of various plant species.