RESUMEN
Elevated expression of the X-linked inhibitor of apoptosis protein (XIAP) has been frequently reported in malignant melanoma suggesting that XIAP renders apoptosis resistance and thereby supports melanoma progression. Independent of its anti-apoptotic function, XIAP mediates cellular inflammatory signalling and promotes immunity against bacterial infection. The pro-inflammatory function of XIAP has not yet been considered in cancer. By providing detailed in vitro analyses, utilising two independent mouse melanoma models and including human melanoma samples, we show here that XIAP is an important mediator of melanoma neutrophil infiltration. Neutrophils represent a major driver of melanoma progression and are increasingly considered as a valuable therapeutic target in solid cancer. Our data reveal that XIAP ubiquitylates RIPK2, involve TAB1/RIPK2 complex and induce the transcriptional up-regulation and secretion of chemokines such as IL8, that are responsible for intra-tumour neutrophil accumulation. Alteration of the XIAP-RIPK2-TAB1 inflammatory axis or the depletion of neutrophils in mice reduced melanoma growth. Our data shed new light on how XIAP contributes to tumour growth and provides important insights for novel XIAP targeting strategies in cancer.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis , Melanoma , Infiltración Neutrófila , Neoplasias Cutáneas , Proteína Inhibidora de la Apoptosis Ligada a X , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Apoptosis/genética , Apoptosis/inmunología , Modelos Animales de Enfermedad , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/inmunología , Interleucina-8/biosíntesis , Melanoma/genética , Melanoma/inmunología , Ratones , Infiltración Neutrófila/genética , Infiltración Neutrófila/inmunología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/inmunología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunología , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/inmunología , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
BACKGROUND: Cardiac hypertrophy is a key biological response to injurious stresses such as pressure overload and, when excessive, can lead to heart failure. Innate immune activation by danger signals, through intracellular pattern recognition receptors such as nucleotide-binding oligomerization domain 1 (Nod1) and its adaptor receptor-interacting protein 2 (RIP2), might play a major role in cardiac remodeling and progression to heart failure. We hypothesize that Nod1/RIP2 are major contributors to cardiac hypertrophy, but may not be sufficient to fully express the phenotype alone. METHODS: To elucidate the contribution of Nod1/RIP2 signaling to cardiac hypertrophy, we randomized Nod1-/-, RIP2-/-, or wild-type mice to transverse aortic constriction or sham operations. Cardiac hypertrophy, fibrosis, and cardiac function were examined in these mice. RESULTS: Nod1 and RIP2 proteins were upregulated in the heart after transverse aortic constriction, and this was paralleled by increased expression of mitochondrial proteins, including mitochondrial antiviral signaling protein (MAVS). Nod1-/- and RIP2-/- mice subjected to transverse aortic constriction exhibited better survival, improved cardiac function, and decreased cardiac hypertrophy. Downstream signal transduction pathways that regulate inflammation and fibrosis, including NF (nuclear factor) κB and MAPK (mitogen-activated protein kinase)-GATA4/p300, were reduced in both Nod1-/- and RIP2-/- mice after transverse aortic constriction compared with wild-type mice. Coimmunoprecipitation of extracted cardiac proteins and confocal immunofluorescence microscopy showed that Nod1/RIP2 interaction was robust and that this complex also included MAVS as an essential component. Suppression of MAVS expression attenuated the complex formation, NF κB signaling, and myocyte hypertrophy. Interrogation of mitochondrial function compared in the presence or ablation of MAVS revealed that MAVS serves to suppress mitochondrial energy output and mediate fission/fusion related dynamic changes. The latter is possibly linked to mitophagy during cardiomyocytes stress, which may provide an intriguing link between innate immune activation and mitochondrial energy balance under stress or injury conditions. CONCLUSIONS: We have identified that innate immune Nod1/RIP2 signaling is a major contributor to cardiac remodeling after stress. This process is critically joined by and regulated through the mitochondrial danger signal adapter MAVS. This novel complex coordinates remodeling, inflammatory response, and mitochondrial energy metabolism in stressed cardiomyocytes. Thus, Nod1/RIP2/MAVS signaling complex may represent an attractive new therapeutic approach toward heart failure.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Cardiomegalia/inmunología , Metabolismo Energético/fisiología , Inmunidad Innata/fisiología , Proteína Adaptadora de Señalización NOD1/inmunología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Animales Recién Nacidos , Cardiomegalia/metabolismo , Cardiomegalia/patología , Femenino , Humanos , Células Madre Pluripotentes Inducidas/inmunología , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Ratones , Ratones Noqueados , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Transducción de Señal/fisiologíaRESUMEN
We demonstrate that female C57BL/6J mice are susceptible to a transient lower genital tract infection with MmuPV1 mouse papillomavirus and display focal histopathological abnormalities resembling those of human papillomavirus (HPV) infection. We took advantage of strains of genetically deficient mice to study in vivo the role of innate immune signaling in the control of papillomavirus. At 4 months, we sacrificed MmuPV1-infected mice and measured viral 757/3139 spliced transcripts by TaqMan reverse transcription-PCR (RT-PCR), localization of infection by RNAscope in situ hybridization, and histopathological abnormities by hematoxylin and eosin (H&E) staining. Among mice deficient in receptors for pathogen-associated molecular patterns, MyD88-/- and STING-/- mice had 1,350 and 80 copies of spliced transcripts/µg RNA, respectively, while no viral expression was detected in MAVS-/- and Ripk2-/- mice. Mice deficient in an adaptor molecule, STAT1-/-, for interferon signaling had 46,000 copies/µg RNA. Among mice with targeted deficiencies in the inflammatory response, interleukin-1 receptor knockout (IL-1R-/-) and caspase-1-/- mice had 350 and 30 copies/µg RNA, respectively. Among mice deficient in chemokine receptors, CCR6-/- mice had 120 copies/µg RNA, while CXCR2-/- and CXCR3-/- mice were negative. RNAscope confirmed focal infection in MyD88-/-, STAT1-/-, and CCR6-/- mice but was negative for other gene-deficient mice. Histological abnormalities were seen only in the latter mice. Our findings and the literature support a working model of innate immunity to papillomaviruses involving the activation of a MyD88-dependent pathway and IL-1 receptor signaling, control of viral replication by interferon-stimulated genes, and clearance of virus-transformed dysplastic cells by the action of the CCR6/CCL20 axis.IMPORTANCE Papillomaviruses infect stratified squamous epithelia, and the viral life cycle is linked to epithelial differentiation. Additionally, changes occur in viral and host gene expression, and immune cells are activated to modulate the infectious process. In vitro studies with keratinocytes cannot fully model the complex viral and host responses and do not reflect the contribution of local and migrating immune cells. We show that female C57BL/6J mice are susceptible to a transient papillomavirus cervicovaginal infection, and mice deficient in select genes involved in innate immune responses are susceptible to persistent infection with variable manifestations of histopathological abnormalities. The results of our studies support a working model of innate immunity to papillomaviruses, and the model provides a framework for more in-depth studies. A better understanding of mechanisms of early viral clearance and the development of approaches to induce clearance will be important for cancer prevention and the treatment of HPV-related diseases.
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Interacciones Huésped-Patógeno/inmunología , Factor 88 de Diferenciación Mieloide/inmunología , Papillomaviridae/inmunología , Infecciones por Papillomavirus/inmunología , ARN Mensajero/inmunología , ARN Viral/inmunología , Receptores Tipo I de Interleucina-1/inmunología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Empalme Alternativo , Animales , Caspasa 1/deficiencia , Caspasa 1/genética , Caspasa 1/inmunología , Cuello del Útero/inmunología , Cuello del Útero/virología , Femenino , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Humanos , Inmunidad Innata , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/deficiencia , Factor 88 de Diferenciación Mieloide/genética , Papillomaviridae/crecimiento & desarrollo , Papillomaviridae/metabolismo , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/virología , ARN Mensajero/genética , ARN Viral/genética , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/deficiencia , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/genética , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/inmunología , Receptores CCR6/deficiencia , Receptores CCR6/genética , Receptores CCR6/inmunología , Receptores CXCR3/deficiencia , Receptores CXCR3/genética , Receptores CXCR3/inmunología , Receptores Tipo I de Interleucina-1/deficiencia , Receptores Tipo I de Interleucina-1/genética , Receptores de Interleucina-8B/deficiencia , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/inmunología , Factor de Transcripción STAT1/deficiencia , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/inmunología , Transducción de Señal , Vagina/inmunología , Vagina/virologíaRESUMEN
Exposure to house dust mite (HDM) is highly associated with the development of allergic asthma. The adaptive immune response to HDM is largely Th2 and Th17 dominant, and a number of innate immune receptors have been identified that recognize HDM to initiate these responses. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is a cytosolic sensor of peptidoglycan, which is important for Th2 and Th17 polarization. NOD2 mediates its signaling through its downstream effector kinase, receptor-interacting serine/threonine protein kinase 2 (RIP2). We have previously shown that RIP2 promotes HDM-associated allergic airway inflammation and Th2 and Th17 immunity, acting early in the HDM response and likely within airway epithelial cells. However, the consequences of inhibiting RIP2 during this critical period has not yet been examined. In this study, we pharmacologically inhibited RIP2 activity during the initial exposure to allergen in an acute HDM model of asthma and determined the effect on the subsequent development of allergic airway disease. We show that early inhibition of RIP2 was sufficient to reduce lung histopathology and local airway inflammation while reducing the Th2 immune response. Using a chronic HDM asthma model, we demonstrate that inhibition of RIP2, despite attenuating airway inflammation and airway remodeling, was insufficient to reduce airway hyperresponsiveness. These data demonstrate the potential of pharmacological targeting of this kinase in asthma and support further development and optimization of RIP2-targeted therapies.
Asunto(s)
Aminoquinolinas/farmacología , Asma/tratamiento farmacológico , Pyroglyphidae/inmunología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/antagonistas & inhibidores , Sulfonas/farmacología , Inmunidad Adaptativa , Animales , Asma/inmunología , Citocinas/metabolismo , Femenino , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Pulmón/patología , Masculino , Ratones Endogámicos C57BL , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/inmunología , Células Th17/efectos de los fármacos , Células Th17/inmunología , Células Th2/efectos de los fármacos , Células Th2/inmunologíaRESUMEN
The interplay between NOD2 and TLR2 following recognition of components of the bacterial cell wall peptidoglycan is well-established, however their role in redirecting metabolic pathways in myeloid cells to degrade pathogens and mount antigen presentation remains unclear. We show NOD2 and TLR2 mediate phosphorylation of the deubiquitinase ataxin-3 via RIPK2 and TBK1. In myeloid cells ataxin-3 associates with the mitochondrial cristae protein MIC60, and is required for oxidative phosphorylation. Depletion of ataxin-3 leads to impaired induction of mitochondrial reactive oxygen species (mROS) and defective bacterial killing. A mass spectrometry analysis of NOD2/TLR2 triggered ataxin-3 deubiquitination targets revealed immunometabolic regulators, including HIF-1α and LAMTOR1 that may contribute to these effects. Thus, we define how ataxin-3 plays an essential role in NOD2 and TLR2 sensing and effector functions in myeloid cells.
Asunto(s)
Ataxina-3/inmunología , Células Mieloides/inmunología , Células Mieloides/metabolismo , Proteína Adaptadora de Señalización NOD2/inmunología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/inmunología , Receptor Toll-Like 2/inmunología , Ataxina-3/metabolismo , Respiración de la Célula , Células HEK293 , Humanos , Inmunidad Innata , Mitocondrias/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Transducción de Señal , Células THP-1 , Receptor Toll-Like 2/metabolismoRESUMEN
An octamer consisting of two copies of histones H2A, H2B, H3 and H4 is the nucleosome core. It is well established that histone derived antimicrobial peptides (AMPs) have anti-microbial properties in various invertebrate and vertebrate species. Different from well-known histone H2A-derived AMPs, the antimicrobial properties of the complete histone H2A are rather limited. In the present study, we report the functional characterization of the complete histone H2A from zebrafish. The expression of zebrafish histone H2A was higher in embryos than in larvae, and inducible in response to bacterial infection. Furthermore, the expression of zebrafish histone H2A was decreased by RIP2 deficiency with and/or without bacterial infection. During Edwardsiella piscicida infection, the overexpression of zebrafish histone H2A inhibited bacterial proliferation and increased the survival rate of zebrafish larvae. The overexpression of zebrafish histone H2A demonstrated an increased transcription of many antibacterial genes and MHC related genes, which was dependent on RIP2, an adaptor protein for signal propagation of the NLRs-mediated antibacterial immune response. In line with this, zebrafish histone H2A cooperated with RIP2 to induce the transcription of many antibacterial genes and MHC related genes. All together, these results firstly demonstrate the antibacterial property of the complete histone H2A against gram-negative bacteria E. piscicida in vivo and the correlation between zebrafish histone H2A and RIP2 adaptor protein on the transcriptional regulation of antibacterial genes and MHC related genes.
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Proteínas de Peces/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Histonas/metabolismo , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Pez Cebra/fisiología , Animales , Infecciones por Enterobacteriaceae/inmunología , Proteínas de Peces/inmunología , Histonas/inmunología , Complejo Mayor de Histocompatibilidad/fisiología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/inmunologíaRESUMEN
Previous studies have shown that inhibition of receptor-interacting serine/threonine kinase (RICK) (also known as RIP2) results in amelioration of experimental colitis. This role has largely been attributed to nucleotide-binding oligomerization domain 2 (NOD2) signaling since the latter is considered a major inducer of RICK activation. In this study, we explored the molecular mechanisms accounting for RICK-mediated inhibition of inflammatory bowel disease (IBD). In an initial series of studies focused on trinitrobenzene sulfonic acid (TNBS)-colitis and dextran sodium sulfate (DSS)-colitis we showed that down-regulation of intestinal RICK expression in NOD2-intact mice by intra-rectal administration of a plasmid expressing RICK-specific siRNA was accompanied by down-regulation of pro-inflammatory cytokine responses in the colon and protection of the mice from experimental colitis. Somewhat surprisingly, intra-rectal administration of RICK-siRNA also inhibited TNBS-colitis and DSS-colitis in NOD2-deficient and in NOD1/NOD2-double deficient mice. In complementary studies of humans with IBD we found that expression of RICK, cellular inhibitor of apoptosis protein 2 (cIAP2) and downstream signaling partners were markedly increased in inflamed tissue of IBD compared to controls without marked elevations of NOD1 or NOD2 expression. In addition, the increase in RICK expression correlated with disease activity and pro-inflammatory cytokine responses. These studies thus suggest that NOD1- or NOD2-independenent activation of RICK plays a major role in both murine experimental colitis and human IBD.
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Inflamación/metabolismo , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Animales , Humanos , Inflamación/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/metabolismo , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/inmunología , Transducción de Señal/inmunologíaRESUMEN
House dust mites (HDMs) are one of the most significant environmental allergens in the establishment of the so-called "Atopic March." It is known that the immune response to HDM is Th2 dominant, but the innate mechanisms leading to HDM-induced type 2 responses are still not completely understood. A number of innate immune receptors have been implicated in the response to HDM including toll-like receptors, C-type lectin receptors, and protease activated receptors. NOD2 is a member of the NOD-like receptor family, which has been reported to be involved in the establishment of type 2 immunity and in blocking respiratory tolerance. NOD2 mediates its effects through its downstream effector kinase, receptor interacting protein (RIP2). It has not been shown if RIP2 is involved in the innate response to HDM and in the resulting generation of type 2 immunity. Furthermore, the role of RIP2 in modulating allergic airway inflammation has been controversial. In this study, we show that RIP2 is activated in airway epithelial cells in response to HDM and is important for the production of CCL2. Using a murine HDM asthma model, we demonstrate that lung pathology, local airway inflammation, inflammatory cytokines, HDM-specific IgG1 antibody production, and HDM-specific Th2 responses are all reduced in RIP2 knockout mice compared to WT animals. These data illustrate that RIP2 can be activated by a relevant allergic stimulus and that such activation can contribute to allergic airway inflammation. These findings also suggest that RIP2 inhibitors might have some efficacy in down-regulating the inflammatory response in type 2 dominated diseases.
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Asma/inmunología , Hipersensibilidad/inmunología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/inmunología , Células A549 , Animales , Dermatophagoides farinae , Femenino , Humanos , Inflamación/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Th2/inmunologíaRESUMEN
RIP2 is an adaptor protein which is essential for the activation of NF-κB and NOD1- and NOD2-dependent signaling. Although NOD-RIP2 axis conservatively existed in the teleost, the function of RIP2 was only reported in zebrafish, goldfish, and rainbow trout in vitro. Very little is known about the role and mechanisms of piscine NOD-RIP2 axis in vivo. Our previous study showed the protective role of zebrafish NOD1 in larval survival through CD44a-mediated activation of PI3K-Akt signaling. In this study, we examined whether RIP2 was required for larval survival with or without pathogen infection, and determined the signaling pathways modulated by RIP2. Based on our previous report and the present study, our data demonstrated that NOD1-RIP2 axis was important for larval survival in the early ontogenesis. Similar to NOD1, RIP2 deficiency significantly affected immune system processes. The significantly enriched pathways were mainly involved in immune system, such as "Antigen processing and presentation" and "NOD-like receptor signaling pathway" and so on. Furthermore, both transcriptome analysis and qRT-PCR revealed that RIP2 was a critical regulator for expression of NLRs (NOD-like receptors) and those genes involved in MHC antigen presentation. Different from NOD1, the present study showed that NOD1, but not RIP2 deficiency significantly impaired protein levels of MAPK pathways. Although RIP2 deficiency also significantly impaired the expression of CD44a, the downstream signaling of CD44a-Lck-PI3K-Akt pathway remained unchanged. Collectively, our works highlight the similarity and discrepancy of NOD1 and RIP2 in the regulation of immune signaling pathways in the zebrafish early ontogenesis, and confirm the crucial role of RIP2 in NLRs signaling and MHC antigen presentation, but not for MAPK and PI3K/Akt pathways.
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Proteína Adaptadora de Señalización NOD1/inmunología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/inmunología , Proteínas de Pez Cebra/inmunología , Animales , Presentación de Antígeno , Edwardsiella , Infecciones por Enterobacteriaceae/inmunología , Infecciones por Enterobacteriaceae/veterinaria , Enfermedades de los Peces/inmunología , Antígenos de Histocompatibilidad/inmunología , Larva , Proteínas Quinasas Activadas por Mitógenos/inmunología , Fosfatidilinositol 3-Quinasas/inmunología , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/genética , Transducción de Señal , Pez CebraRESUMEN
Being a key adaptor protein in NOD1/2 and NF-κB signaling pathways, receptor-interacting serine/threonine kinase 2 (RIP2) plays an important role in innate immune response in vertebrates. In this study, we identified and characterized the Paralichthys olivaceus RIP2 gene (PoRIP2). Phylogenetic, alignment, and genomic analysis of PoRIP2 were conducted to determine its conservation and evolutionary relationship with other RIP2 in vertebrates. qRT-PCR results showed that the expression of PoRIP2 was high in the spleen and head kidney. Meanwhile, embryonic development expression profile revealed that it was high in the early developmental stages and hatching stage. In vivo, we examined the expression pattern in different tissues after being challenged with Edwardsiella tarda. PoRIP2 was up-regulated in tissues at different time points. In vitro, the expression of PoRIP2 was also increased after treatment with Poly I:C, PGN, and E. tarda. Transfection and overexpression experiments indicated that PoRIP2 was located in the cytoplasm of the FG-9307â¯cell line. The pro-inflammatory cytokines, IL-1ß, IL-6, and IL-8, could be activated and up-regulated by PGN stimulation in PoRIP2 overexpressed cells. The inhibitory action was obvious in PoRIP2 overexpressed cells, and the quantity of E. tarda decreased. These findings highlight the important role of PoRIP2 in regulating innate immune in P. olivaceus. Our results indicated that PoRIP2 might be involved in immune response and the activation of the NF-κB signaling pathways. Our study can improve the knowledge on the immune system of fish and provide a theoretical basis for the study of prevention and treatment of fish diseases.
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Enfermedades de los Peces/inmunología , Peces Planos/genética , Peces Planos/inmunología , Inmunidad Innata/genética , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/genética , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/inmunología , Transducción de Señal , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Edwardsiella tarda/fisiología , Infecciones por Enterobacteriaceae/inmunología , Femenino , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Perfilación de la Expresión Génica/veterinaria , Masculino , Filogenia , Distribución Aleatoria , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/química , Alineación de Secuencia/veterinariaRESUMEN
The innate immune system is critical for clearing infection, and is tightly regulated to avert excessive tissue damage. Nod1/2-Rip2 signaling, which is essential for initiating the innate immune response to bacterial infection and ER stress, is subject to many regulatory mechanisms. In this study, we found that LRRK2, encoded by a gene implicated in Crohn's disease, leprosy and familial Parkinson's disease, modulates the strength of Nod1/2-Rip2 signaling by enhancing Rip2 phosphorylation. LRRK2 deficiency markedly reduces cytokine production in macrophages upon Nod2 activation by muramyl dipeptide (MDP), Nod1 activation by D-gamma-Glu-meso-diaminopimelic acid (iE-DAP) or ER stress. Our biochemical study shows that the presence of LRRK2 is necessary for optimal phosphorylation of Rip2 upon Nod2 activation. Therefore, this study reveals that LRRK2 is a new positive regulator of Rip2 and promotes inflammatory cytokine induction through the Nod1/2-Rip2 pathway.
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Citocinas/inmunología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/inmunología , Proteína Adaptadora de Señalización NOD1/inmunología , Proteína Adaptadora de Señalización NOD2/inmunología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/inmunología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Transducción de Señal/inmunología , Animales , Citocinas/genética , Células HEK293 , Humanos , Inmunidad Innata/genética , Inflamación/genética , Inflamación/inmunología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Ratones , Ratones Noqueados , Proteína Adaptadora de Señalización NOD1/genética , Proteína Adaptadora de Señalización NOD2/genética , Fosforilación/genética , Fosforilación/inmunología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Barettin is a marine natural compound with reported anti-inflammatory and antioxidant properties. The combination of these effects led us to explore barettin further as an inhibitor of atherosclerosis development. METHODS: The effect of barettin on MCP-1 and IL-10 secretion from activated immune cells was detected by ELISA. Determination of cell viability of oxidized low-density lipoprotein (oxLDL) and barettin exposed HUVEC cells were investigated by using CellTiter 96® AQ(ueous) One Solution. The kinase inhibition assays were performed using a radioactive ((33)P-ATP) filter binding assay at the University of Dundee, UK. RESULTS: Barettin reduces the secretion of monocyte chemotactic protein-1 (MCP-1) from LPS-stimulated monocytes, but was not able to prevent oxLDL-induced cell death in HUVEC. Barettin has inhibitory activity against two protein kinases related to inflammation, namely the receptor-interacting serine/threonine kinase 2 (RIPK2) and calcium/calmodulin-dependent protein kinase 1α (CAMK1α). We also demonstrate that barettin reduce the production of the anti-inflammatory cytokine interleukin-10 (IL-10) in a dose and time-dependent manner, possibly by inhibiting CAMK1α. CONCLUSIONS: The anti-inflammatory activity of barettin is exerted through the regulation of inflammatory mediators such as MCP-1 and IL-10, possibly via inhibition of kinases.
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Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/inmunología , Células Endoteliales de la Vena Umbilical Humana/inmunología , Factores Inmunológicos/farmacología , Péptidos Cíclicos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/inmunología , Antiinflamatorios/farmacología , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Línea Celular Tumoral , Quimiocina CCL2/inmunología , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Interleucina-10/inmunología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/antagonistas & inhibidoresRESUMEN
BACKGROUND/AIMS: Nucleotide binding oligomerization domain 1 (NOD1) signal pathway and human ß defensins (hBDs) play crucial roles in innate immune. Cigarette smoke has been confirmed to dampen innate immune in some human tissues, such as oral mucosa. The aim of this study was to evaluate potential effects of smoking on NOD1 signaling and hBDs expression in oral mucosa. METHODS: Tissue specimens of normal oral mucosa were collected from donors undergoing routine surgical treatment. All 20 participants were classified equally as two groups: non-smokers and smokers. By using Western blotting and immunohistochemistry, we investigated differential expression of crucial molecules in NOD1 signal pathway, hBD-1, -2, and -3 in oral mucosa tissues between non-smokers and smokers. Immortalized human oral mucosal epithelial (Leuk-1) cells were treated with various concentrations of cigarette smoke extract (CSE) for 24h. Western blotting and immunofluorescence assays were performed to study CSE-induced alteration of protein expression. Leuk-1 cells were treated with 4% CSE, iE-DAP (NOD1 agonist), CSE + iE-DAP, BAY 11-7082 (NF-κB inhibitor), 4% CSE + BAY 11-7082, respectively. Real-time PCR and ELISA were performed to detect the mRNA levels and secretion of hBD-1, -2, and -3, respectively. RESULTS: The levels of NOD1, NF-κB, hBD-1 and hBD-3 significantly reduced in oral mucosa tissues of smokers compared with non-smokers. The levels of RIP2 (receptor-interacting protein 2), phospho-NF-κB (P-NF-κB) and hBD-2 remarkably enhanced in oral mucosal tissues of smokers. CSE treatment suppressed NOD1 and NF-κB expression and activated RIP2 and P-NF-κB expression in Leuk-1 cells. The mRNA and secretory levels of hBD-1 and -3 were down-regulated by CSE, while the mRNA and secretory level of hBD-2 were up-regulated by CSE. The iE-DAP or BAY 11-7082 treatment reversed the regulatory effects of CSE on levels of hBDs. CONCLUSION: The present study indicated that cigarette smoke could potentially modulate the expression of crucial molecules of NOD1 signal pathway and hBDs in human oral mucosal epithelium. NOD1 signal pathway could play an important role in the regulatory effects of CSE on hBDs levels in oral mucosal epithelial cells.
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Mucosa Bucal/inmunología , Proteína Adaptadora de Señalización NOD1/inmunología , Transducción de Señal , Fumar/inmunología , beta-Defensinas/inmunología , Adulto , Línea Celular , Supervivencia Celular , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Mucosa Bucal/metabolismo , Mucosa Bucal/patología , FN-kappa B/análisis , FN-kappa B/genética , FN-kappa B/inmunología , Proteína Adaptadora de Señalización NOD1/análisis , Proteína Adaptadora de Señalización NOD1/genética , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/análisis , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/genética , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/inmunología , Fumar/genética , Fumar/patología , Adulto Joven , beta-Defensinas/análisis , beta-Defensinas/genéticaRESUMEN
Innate immunity is considered to be critical in the pathogenesis of fungal keratitis. Pattern recognition receptors (PRRs) recognize conserved microbial structures called pathogen-associated molecular patterns (PAMPS), thereby initiating the innate immunity. Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (leucine-rich repeat-containing receptors, NLRs) are two major PRR families. The crosstalk between TLR2 and NOD2 is not completely understood, and their interrelationship in Aspergillus fumigates keratitis is still unclear. To our surprise, we found herein that NOD2 and TLR2 were increased by A. fumigatus conidia in immortalized human corneal epithelial cells (HCECs). In addition, NOD2 expression was up-regulated by its agonist muramyl dipeptide (MDP), along with receptor interacting protein 2 (RIP2), nuclear factor κB (NFκB)-p65, inhibitor of NFκB (IκB)-α, and multiple inflammatory cytokines, including interleukin-6 (IL-6), IL-8 and tumor necrosis factor α (TNF-α). Interestingly, zymosan, a TLR2 agonist, promoted the expression of NOD2 and RIP2 in a TLR2-dependent manner. Furthermore, we demonstrated that the increased expression of NOD2 and RIP2 caused by A. fumigatus conidia occurred in part through a TLR2-dependent pathway. However, zymosan pretreatment decreased NOD2 and RIP2 expression along with the MDP induced secretion of inflammatory cytokines in HCECs. In agreement, NOD2 knockdown by small interfering RNA (siRNA) reduced the release of IL-6, IL-8 and TNF-α induced by A. fumigatus conidia. These findings suggest the existence of complex interactions between TLR2 and NOD2 in HCECs inflammatory response against A. fumigatus infection.
Asunto(s)
Aspergillus fumigatus/inmunología , Células Epiteliales/metabolismo , Proteína Adaptadora de Señalización NOD2/inmunología , Receptor Cross-Talk , Esporas Fúngicas/inmunología , Receptor Toll-Like 2/inmunología , Acetilmuramil-Alanil-Isoglutamina/farmacología , Línea Celular Transformada , Córnea/inmunología , Córnea/metabolismo , Córnea/microbiología , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Interleucina-8/genética , Interleucina-8/inmunología , Inhibidor NF-kappaB alfa , Proteína Adaptadora de Señalización NOD2/agonistas , Proteína Adaptadora de Señalización NOD2/antagonistas & inhibidores , Proteína Adaptadora de Señalización NOD2/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/genética , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/inmunología , Transducción de Señal , Receptor Toll-Like 2/agonistas , Receptor Toll-Like 2/antagonistas & inhibidores , Receptor Toll-Like 2/genética , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Zimosan/farmacologíaRESUMEN
We report on the functional characterization of RIP2 of the goldfish. Quantitative expression analysis of goldfish RIP2 revealed the greatest mRNA levels in the spleen, monocytes and splenocytes. We generated a recombinant form of the molecule (rgRIP2) and determined that anti-human RIP2 polyclonal antibody specifically recognized recombinant goldfish RIP2 (rgRIP2). Goldfish RIP2 activity was inhibited by the p38 MAPK pathway inhibitor SB203580. Treatment of goldfish macrophages with LPS, PGN, MDP, Poly I:C, heat-killed and live Mycobacterium marinum, and heat-killed Aeromonas salmonicida differentially changed the expression of RIP2 at both mRNA and protein levels. Co-immunoprecipitation assays indicated that RIP2 interacted with Nod1 and Nod2 receptors in eukaryotic cells. The results of dual luciferase reporter assay revealed that RIP2 over-expression caused the activation of the NF-κB signal pathway. In addition, RIP2 was involved in the regulation of the production of TNFα-2 and IL-1ß1 in goldfish macrophages exposed to M. marinum.
Asunto(s)
Carpa Dorada/inmunología , Inflamación/inmunología , Macrófagos/inmunología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/inmunología , Proteínas Recombinantes/inmunología , Acetilmuramil-Alanil-Isoglutamina/farmacología , Aeromonas salmonicida/inmunología , Animales , Anticuerpos/inmunología , Imidazoles/farmacología , Inflamasomas/inmunología , Interleucina-1beta/biosíntesis , Lipopolisacáridos/farmacología , Monocitos/metabolismo , Mycobacterium marinum/inmunología , FN-kappa B/metabolismo , Neutrófilos/inmunología , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Peptidoglicano/farmacología , Poli I-C/farmacología , Piridinas/farmacología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/antagonistas & inhibidores , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/genética , Proteínas Recombinantes/genética , Bazo/citología , Bazo/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
The intracellular innate immune receptor NOD1 detects Gram-negative bacterial peptidoglycan (PG) to induce autophagy and inflammatory responses in host cells. To date, the intracellular compartment in which PG is detected by NOD1 and whether NOD1 directly interacts with PG are two questions that remain to be resolved. To address this, we used outer membrane vesicles (OMVs) from pathogenic bacteria as a physiological mechanism to deliver PG into the host cell cytosol. We report that OMVs induced autophagosome formation and inflammatory IL-8 responses in epithelial cells in a NOD1- and RIP2-dependent manner. PG contained within OMVs colocalized with both NOD1 and RIP2 in EEA1-positive early endosomes. Further, we provide evidence for direct interactions between NOD1 and PG. Collectively, these findings demonstrate that NOD1 detects PG within early endosomes, thereby promoting RIP2-dependent autophagy and inflammatory signaling in response to bacterial infection.
Asunto(s)
Autofagia , Endosomas/inmunología , Infecciones por Helicobacter/inmunología , Helicobacter pylori/inmunología , Proteína Adaptadora de Señalización NOD1/inmunología , Peptidoglicano/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/inmunología , Receptores Inmunológicos/inmunología , Animales , Línea Celular , Endosomas/microbiología , Infecciones por Helicobacter/enzimología , Infecciones por Helicobacter/genética , Helicobacter pylori/fisiología , Humanos , Ratones , Proteína Adaptadora de Señalización NOD1/genética , Unión Proteica , Infecciones por Pseudomonas/enzimología , Infecciones por Pseudomonas/genética , Pseudomonas aeruginosa/fisiología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/genética , Receptores Inmunológicos/genética , Transducción de SeñalRESUMEN
Acinetobacter baumannii is a major extensively drug-resistant lethal human nosocomial bacterium. However, the host innate immune mechanisms controlling A. baumannii are not well understood. Although viewed as an extracellular pathogen, A. baumannii can also invade and survive intracellularly. However, whether host innate immune pathways sensing intracellular bacteria contribute to immunity against A. baumannii is not known. Here, we provide evidence for the first time that intracellular antibacterial innate immune receptors Nod1 and Nod2, and their adaptor Rip2, play critical roles in the sensing and clearance of A. baumannii by human airway epithelial cells in vitro. A. baumannii infection upregulated Rip2 expression. Silencing of Nod1, Nod2, and Rip2 expression profoundly increased intracellular invasion and prolonged the multiplication and survival of A. baumannii in lung epithelial cells. Notably, the Nod1/2-Rip2 axis did not contribute to the control of A. baumannii infection of human macrophages, indicating that they play cell type-specific roles. The Nod1/2-Rip2 axis was needed for A. baumannii infection-induced activation of NF-κB but not mitogen-activated protein kinases. Moreover, the Nod1/2-Rip2 axis was critical to induce optimal cytokine and chemokine responses to A. baumannii infection. Mechanistic studies showed that the Nod1/2 pathway contributed to the innate control of A. baumannii infection through the production of ß-defensin 2 by airway epithelial cells. This study revealed new insights into the immune control of A. baumannii and may contribute to the development of effective immune therapeutics and vaccines against A. baumannii.
Asunto(s)
Infecciones por Acinetobacter/inmunología , Acinetobacter baumannii/inmunología , Inmunidad Innata/inmunología , Proteína Adaptadora de Señalización NOD1/genética , Proteína Adaptadora de Señalización NOD2/genética , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/genética , Infecciones por Acinetobacter/genética , Infecciones por Acinetobacter/microbiología , Línea Celular , Quimiocinas/genética , Quimiocinas/inmunología , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Células HEK293 , Humanos , Inmunidad Innata/genética , Pulmón/inmunología , Pulmón/microbiología , Macrófagos/inmunología , Macrófagos/microbiología , FN-kappa B/genética , FN-kappa B/inmunología , Proteína Adaptadora de Señalización NOD1/inmunología , Proteína Adaptadora de Señalización NOD2/inmunología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunología , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunología , beta-Defensinas/genética , beta-Defensinas/inmunologíaRESUMEN
In recent years, genome wide association studies have discovered a large number of gene loci that play a functional role in innate and adaptive immune pathways associated with leprosy susceptibility. The immunological control of intracellular bacteria M. leprae is modulated by NOD2-mediated signaling of Th1 responses. In this study, we investigated 211 clinically classified leprosy patients and 230 ethnically matched controls in Indian population by genotyping four variants in NOD2 (rs9302752A/G), LRRK2 (rs1873613A/G), RIPK2 (rs40457A/G and rs42490G/A). The LRRK2 locus is associated with leprosy outcome. The LRRK2 rs1873613A minor allele and respective rs1873613AA genotypes were significantly associated with an increased risk whereas the LRRK2 rs1873613G major allele and rs1873613GG genotypes confer protection in paucibacillary and leprosy patients. The reconstructed GA haplotypes from RIPK2 rs40457A/G and rs42490G/A variants was observed to contribute towards increased risk whereas haplotypes AA was observed to confer protective role. Our results indicate that a possible shared mechanisms underlying the development of these two clinical forms of the disease as hypothesized. Our findings confirm and validates the role of gene variants involved in NOD2-mediated signalling pathways that play a role in immunological control of intracellular bacteria M. leprae.
Asunto(s)
Predisposición Genética a la Enfermedad , Lepra/genética , Mycobacterium leprae , Proteína Adaptadora de Señalización NOD2/genética , Proteínas Serina-Treonina Quinasas/genética , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/genética , Transducción de Señal/genética , Alelos , Femenino , Haplotipos/genética , Haplotipos/inmunología , Humanos , India/epidemiología , Lepra/epidemiología , Lepra/inmunología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Masculino , Proteína Adaptadora de Señalización NOD2/inmunología , Polimorfismo Genético/genética , Polimorfismo Genético/inmunología , Proteínas Serina-Treonina Quinasas/inmunología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/inmunología , Transducción de Señal/inmunología , Células TH1/inmunologíaRESUMEN
Toll-like receptor 5 (TLR5) and nucleotide-binding oligomerization domain 2 (Nod2) are two important pattern recognition receptors involved in innate immunity to invading pathogens. Flagellin, recognized by TLR5, is Salmonella's dominant pro-inflammatory determinant in intestinal epithelial cells (IECs). Nod2 has played a pivotal role in protecting against intestinal bacterial infection. Therefore the aim of the study is to investigate regulation of Salmonella flagellin-induced interleukin (IL)-8 (IL-8) in IECs by Nod2 agonist, muramyl dipeptide (MDP). We found that MDP by itself induced only a weak IL-8 secretion in Caco-2 cells. However, it did show synergistic enhancement on flagellin-induced IL-8 production in Caco-2 cells, possibly caused by flagellin-mediated enhanced Nod2 recruitment into cell membrane. By Western blot and siRNA, we showed ERK and NF-κB, Nod2 and Rip2 were involved in the synergistic effect of MDP. These findings suggested that the cooperation of TLR5 and Nod2 in IECs regulates inflammatory response to Salmonella infection.
Asunto(s)
Acetilmuramil-Alanil-Isoglutamina/farmacología , Células Epiteliales/efectos de los fármacos , Flagelina/farmacología , Interleucina-8/biosíntesis , Proteína Adaptadora de Señalización NOD2/genética , Salmonella typhimurium/química , Receptor Toll-Like 5/genética , Células CACO-2 , Células Cultivadas , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Expresión Génica , Humanos , Inmunidad Innata , Interleucina-8/genética , Interleucina-8/inmunología , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , FN-kappa B/genética , FN-kappa B/inmunología , FN-kappa B/metabolismo , Proteína Adaptadora de Señalización NOD2/inmunología , Proteína Adaptadora de Señalización NOD2/metabolismo , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/genética , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/inmunología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Salmonella typhimurium/inmunología , Transducción de Señal , Receptor Toll-Like 5/inmunología , Receptor Toll-Like 5/metabolismoRESUMEN
Understanding the mechanisms by which pathogens induce vascular inflammation and dysfunction may reveal novel therapeutic targets in sepsis and related conditions. The intracellular receptor NOD1 recognises peptidoglycan which features in the cell wall of gram negative and some gram positive bacteria. NOD1 engagement generates an inflammatory response via activation of NFκB and MAPK pathways. We have previously shown that stimulation of NOD1 directly activates blood vessels and causes experimental shock in vivo. In this study we have used an ex vivo vessel-organ culture model to characterise the relative contribution of the endothelium in the response of blood vessels to NOD1 agonists. In addition we present the novel finding that NOD1 directly activates human blood vessels. Using human cultured cells we confirm that endothelial cells respond more avidly to NOD1 agonists than vascular smooth muscle cells. Accordingly we have sought to pharmacologically differentiate NOD1 and TLR4 mediated signalling pathways in human endothelial cells, focussing on TAK1, NFκB and p38 MAPK. In addition we profile novel inhibitors of RIP2 and NOD1 itself, which specifically inhibit NOD1 ligand induced inflammatory signalling in the vasculature. This paper is the first to demonstrate activation of whole human artery by NOD1 stimulation and the relative importance of the endothelium in the sensing of NOD1 ligands by vessels. This data supports the potential utility of NOD1 and RIP2 as therapeutic targets in human disease where vascular inflammation is a clinical feature, such as in sepsis and septic shock.