RESUMEN
Autism spectrum disorder (ASD) is characterized by impaired social interactions and communication. The pathogenesis of ASD is not known, but it involves activation of microglia. We had shown that the peptide neurotensin (NT) is increased in the serum of children with ASD and stimulates cultured adult human microglia to secrete the proinflammatory molecules IL-1ß and CXCL8. This process is inhibited by the cytokine IL-37. Another cytokine, IL-38, has been reported to have antiinflammatory actions. In this report, we show that pretreatment of cultured adult human microglia with recombinant IL-38 (aa3-152, 1-100 ng/mL) inhibits (P < 0.0001) NT-stimulated (10 nM) secretion of IL-1ß (at 1 ng/mL) and CXCL8 (at 100 ng/mL). In fact, IL-38 (aa3-152, 1 ng/mL) is more potent than IL-37 (100 ng/mL). Here, we report that pretreatment with IL-38 (100 ng/mL) of embryonic microglia (HMC3), in which secretion of IL-1ß was undetectable, inhibits secretion of CXCL8 (P = 0.004). Gene expression of IL-38 and its receptor IL-36R are decreased (P = 0.001 and P = 0.04, respectively) in amygdala from patients with ASD (n = 8) compared to non-ASD controls (n = 8), obtained from the University of Maryland NeuroBioBank. IL-38 is increased (P = 0.03) in the serum of children with ASD. These findings indicate an important role for IL-38 in the inhibition of activation of human microglia, thus supporting its development as a treatment approach for ASD.
Asunto(s)
Amígdala del Cerebelo/inmunología , Trastorno del Espectro Autista/inmunología , Interleucinas/inmunología , Microglía/inmunología , Adolescente , Trastorno del Espectro Autista/sangre , Células Cultivadas , Niño , Preescolar , Humanos , Interleucina-16/sangre , Interleucina-16/inmunología , Interleucina-1beta/sangre , Interleucina-1beta/inmunología , Interleucina-8/inmunología , Interleucinas/sangre , Masculino , Neurotensina/sangre , Neurotensina/inmunologíaRESUMEN
Autism spectrum disorder (ASD) does not have a distinct pathogenesis or effective treatment. Increasing evidence supports the presence of immune dysfunction and inflammation in the brains of children with ASD. In this report, we present data that gene expression of the antiinflammatory cytokine IL-37, as well as of the proinflammatory cytokines IL-18 and TNF, is increased in the amygdala and dorsolateral prefrontal cortex of children with ASD as compared to non-ASD controls. Gene expression of IL-18R, which is a receptor for both IL-18 and IL-37, is also increased in the same brain areas of children with ASD. Interestingly, gene expression of the NTR3/sortilin receptor is reduced in the amygdala and dorsolateral prefrontal cortex. Pretreatment of cultured human microglia from normal adult brains with human recombinant IL-37 (1 to 100 ng/mL) inhibits neurotensin (NT)-stimulated secretion and gene expression of IL-1ß and CXCL8. Another key finding is that NT, as well as the proinflammatory cytokines IL-1ß and TNF increase IL-37 gene expression in cultured human microglia. The data presented here highlight the connection between inflammation and ASD, supporting the development of IL-37 as a potential therapeutic agent of ASD.
Asunto(s)
Amígdala del Cerebelo/metabolismo , Trastorno del Espectro Autista/metabolismo , Interleucina-1/metabolismo , Microglía/metabolismo , Neurotensina/metabolismo , Corteza Prefrontal/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Células Cultivadas , Niño , Humanos , Interleucina-18/metabolismo , Subunidad alfa del Receptor de Interleucina-18/metabolismo , Interleucina-1beta/biosíntesis , Interleucina-8/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Mast cells are critical for allergic and inflammatory responses in which the peptide substance P (SP) and the cytokine IL-33 are involved. SP (0.01-1 µM) administered together with IL-33 (30 ng/mL) to human cultured LAD2 mast cells stimulates a marked increase (P < 0.0001) in secretion of the proinflammatory cytokine IL-1ß. Preincubation of LAD2 (30 min) with the SP receptor (NK-1) antagonists L-733,060 (10 µM) or CP-96345 (10 µM) inhibits (P < 0.001) secretion of IL-1ß stimulated by either SP (1 µM) or SP together with IL-33 (30 ng/mL). Surprisingly, secretion of IL-1ß stimulated by IL-33 is inhibited (P < 0.001) by each NK-1 antagonist. Preincubation with an antibody against the IL-33 receptor ST2 inhibits (P < 0.0001) secretion of IL-1ß stimulated either by IL-33 or together with SP. The combination of SP (1 µM) with IL-33 (30 ng/mL) increases IL-1ß gene expression by 90-fold in LAD2 cells and by 200-fold in primary cultured mast cells from human umbilical cord blood. The combination of SP and IL-33 increases intracellular levels of IL-1ß in LAD2 by 100-fold and gene expression of IL-1ß and procaspase-1 by fivefold and pro-IL-1ß by twofold. Active caspase-1 is present even in unstimulated cells and is detected extracellularly. Preincubation of LAD2 cells with the natural flavonoid methoxyluteolin (1-100 mM) inhibits (P < 0.0001) secretion and gene expression of IL-1ß, procaspase-1, and pro-IL-1ß. Mast cell secretion of IL-1ß in response to SP and IL-33 reveals targets for the development of antiinflammatory therapies.
Asunto(s)
Interleucina-1beta/metabolismo , Interleucina-33/farmacología , Luteolina/farmacología , Mastocitos/metabolismo , Sustancia P/farmacología , Compuestos de Bifenilo/farmacología , Caspasa 1/metabolismo , Línea Celular Tumoral , Humanos , Mastocitos/citología , Piperidinas/farmacologíaRESUMEN
The peptide substance P (SP) and the cytokine tumor necrosis factor (TNF) have been implicated in inflammatory processes. Mast cells are recognized as important in inflammatory responses. Here, we report that IL-33 (30 ng/mL), a member of the IL-1 family of cytokines, administered in combination with SP (1 µM), markedly increase (by 1,000-fold) TNF gene expression in cultured human LAD2 and primary mast cells derived from umbilical cord blood. SP (0.01-1 µM) and IL-33 (1-100 ng/mL) in combination also greatly stimulate TNF secretion (by 4,500-fold). Pretreatment of LAD2 cells with two different neurokinin-1 (NK-1) receptor antagonists and siRNA inhibits TNF secretion by 50% (P < 0.001) when stimulated by SP and IL-33. Pretreatment of LAD2 cells with a neutralizing antibody for IL-33 receptor, ST2, inhibits TNF secretion by 50% (P < 0.001), and ST2 siRNA decreases TNF secretion by 30% (P < 0.05), when stimulated by SP and IL-33. Surprisingly, NK-1 antagonists also inhibit 50% of TNF secretion (P < 0.001) when stimulated only by IL-33, and ST2 receptor reduction also decreases SP-stimulated TNF secretion by 30% (P < 0.05), suggesting an interaction between NK-1 and ST2 receptors. Moreover, IL-33 increases NK-1 gene and surface protein expression, as well as IKß-α phosphorylation. Pretreatment of LAD2 cells with 5,7,3',4'-tetramethoxyflavone (methoxyluteolin) (1-100 µM) inhibits (P < 0.001) TNF gene expression (98%) and secretion (64%) at 50 µM and phosphorylation of p-IKB-α at 1 µM when stimulated by SP and IL-33. These findings identify a unique amplification process of TNF synthesis and secretion via the interaction of NK-1 and ST2 receptors inhibitable by methoxyluteolin.
Asunto(s)
Interleucina-33/metabolismo , Luteolina/química , Mastocitos/metabolismo , Sustancia P/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Compuestos de Bifenilo , Línea Celular Tumoral , Humanos , Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Luteolina/metabolismo , Sistema de Señalización de MAP Quinasas , FN-kappa B/metabolismo , Piperidinas , Receptores de Neuroquinina-1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We had reported elevated serum levels of the peptide neurotensin (NT) in children with autism spectrum disorders (ASD). Here, we show that NT stimulates primary human microglia, the resident immune cells of the brain, and the immortalized cell line of human microglia-SV40. NT (10 nM) increases the gene expression and release (P < 0.001) of the proinflammatory cytokine IL-1ß and chemokine (C-X-C motif) ligand 8 (CXCL8), chemokine (C-C motif) ligand 2 (CCL2), and CCL5 from human microglia. NT also stimulates proliferation (P < 0.05) of microglia-SV40. Microglia express only the receptor 3 (NTR3)/sortilin and not the NTR1 or NTR2. The use of siRNA to target sortilin reduces (P < 0.001) the NT-stimulated cytokine and chemokine gene expression and release from human microglia. Stimulation with NT (10 nM) increases the gene expression of sortilin (P < 0.0001) and causes the receptor to be translocated from the cytoplasm to the cell surface, and to be secreted extracellularly. Our findings also show increased levels of sortilin (P < 0.0001) in the serum from children with ASD (n = 36), compared with healthy controls (n = 20). NT stimulation of microglia-SV40 causes activation of the mammalian target of rapamycin (mTOR) signaling kinase, as shown by phosphorylation of its substrates and inhibition of these responses by drugs that prevent mTOR activation. NT-stimulated responses are inhibited by the flavonoid methoxyluteolin (0.1-1 µM). The data provide a link between sortilin and the pathological findings of microglia and inflammation of the brain in ASD. Thus, inhibition of this pathway using methoxyluteolin could provide an effective treatment of ASD.
RESUMEN
The purpose of this study was to elucidate the role of nucleotide binding oligomerization domain-containing protein 2 (NOD2) signaling in atherosclerosis and periodontal bone loss using an Apolipoprotein E(-/-) (ApoE(-/-)) mouse model based on the proposed role of NOD2 in inflammation. NOD2(-/-)ApoE(-/-) and ApoE(-/-) mice fed a standard chow diet were given an oral gavage of Porphyromonas gingivalis for 15 wk. NOD2(-/-)ApoE(-/-) mice exhibited significant increases in inflammatory cytokines, alveolar bone loss, cholesterol, and atherosclerotic lesions in the aorta and the heart compared with ApoE(-/-) mice. In contrast, ApoE(-/-) mice injected i.p. with Muramyl DiPeptide (MDP) to stimulate NOD2 and given an oral gavage of P. gingivalis displayed a reduction of serum inflammatory cytokines, alveolar bone loss, cholesterol, and atherosclerotic lesions in the aorta and aortic sinus compared with ApoE(-/-) mice orally challenged but injected with saline. A reduction in body weight gain was observed in ApoE(-/-) mice fed a high-fat diet (HFD) and injected with MDP compared with ApoE(-/-) mice fed a high-fat diet but injected with saline. MDP treatment of bone marrow-derived macrophages incubated with P. gingivalis increased mRNA expressions of NOD2, Toll-like receptor 2, myeloid differentiation primary response gene 88, and receptor-interacting protein-2 but reduced the expressions of inhibitor of NF-κB kinase-ß, NF-κB, c-Jun N-terminal kinase 3, and TNF-α protein levels compared with saline control, highlighting pathways involved in MDP antiinflammatory effects. MDP activation of NOD2 should be considered in the treatment of inflammatory processes affecting atherosclerosis, periodontal bone loss ,and possibly, diet-induced weight gain.
Asunto(s)
Pérdida de Hueso Alveolar/patología , Aterosclerosis/patología , Infecciones por Bacteroidaceae/complicaciones , Inflamación/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Porphyromonas gingivalis , Acetilmuramil-Alanil-Isoglutamina/administración & dosificación , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Pérdida de Hueso Alveolar/etiología , Análisis de Varianza , Animales , Apolipoproteínas E/genética , Aterosclerosis/etiología , Peso Corporal , Citocinas/sangre , Dieta Alta en Grasa , Inflamación/etiología , Ratones , Ratones Noqueados , Proteína Adaptadora de Señalización NOD2/deficiencia , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Bone morphogenetic protein 9 (BMP9) promotes the acquisition of the cholinergic phenotype in basal forebrain cholinergic neurons (BFCN) during development and protects these neurons from cholinergic dedifferentiation following axotomy when administered in vivo. A decline in BFCN function occurs in patients with Alzheimer's disease (AD) and contributes to the AD-associated memory deficits. We infused BMP9 intracerebroventricularly for 7 d in transgenic AD model mice expressing green fluorescent protein specifically in cholinergic neurons (APP.PS1/CHGFP) and in wild-type littermate controls (WT/CHGFP). We used 5-mo-old mice, an age when the AD transgenics display early amyloid deposition and few cholinergic defects, and 10-mo-old mice, by which time these mice exhibit established disease. BMP9 infusion reduced the number of Aß42-positive amyloid plaques in the hippocampus and cerebral cortex of 5- and 10-mo-old APP.PS1/CHGFP mice and reversed the reductions in choline acetyltransferase protein levels in the hippocampus of 10-mo-old APP.PS1/CHGFP mice. The treatment increased cholinergic fiber density in the hippocampus of both WT/CHGFP and APP.PS1/CHGFP mice at both ages. BMP9 infusion also increased hippocampal levels of neurotrophin 3, insulin-like growth factor 1, and nerve growth factor and of the nerve growth factor receptors, tyrosine kinase receptor A and p75/NGFR, irrespective of the genotype of the mice. These data show that BMP9 administration is effective in reducing the Aß42 amyloid plaque burden, reversing cholinergic neuron abnormalities, and generating a neurotrophic milieu for BFCN in a mouse model of AD and provide evidence that the BMP9-signaling pathway may constitute a therapeutic target for AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Amiloidosis/metabolismo , Neuronas Colinérgicas/metabolismo , Factor 2 de Diferenciación de Crecimiento/farmacología , Análisis de Varianza , Animales , Neuronas Colinérgicas/efectos de los fármacos , Femenino , Factor 2 de Diferenciación de Crecimiento/administración & dosificación , Factor 2 de Diferenciación de Crecimiento/metabolismo , Inmunoensayo , Immunoblotting , Inmunohistoquímica , Masculino , Ratones , Microscopía FluorescenteRESUMEN
Enteritis caused by Clostridium difficile toxin (Tx) is a nosocomial disease of increasing clinical concern, but the local mediators of C. difficile TxA inflammation are unknown. The potent vasodilator calcitonin gene-related peptide mediates neurogenic inflammation via the calcitonin receptor-like receptor (CLR). Here we examined the ileum-specific effects of reducing CLR on TxA ileitis by local preinjection of double-stranded RNAs. Treatment with CLR dsRNA for 7 d decreased CLR immunoreactivity, whereas treatment with non-CLR dsRNA did not. Subsequent injection of TxA in the same location increased CLR in rats treated with non-CLR dsRNA but not in rats treated with CLR dsRNA, documenting that local injection of dsRNA is effective in preventing the increase in CLR immunoreactivity in response to local TxA. After non-CLR dsRNA pretreatment, TxA induced robust intestinal secretion, myeloperoxidase activity, and histopathologic indications of inflammation including epithelial damage, congestion, neutrophil infiltration, loss of mucin from goblet cells, and increase in mast cell numbers. After CLR dsRNA pretreatment, TxA-induced changes in intestinal secretion and histopathologic inflammation were improved, including normal mucin staining and fewer resident mast cells. Loss of CLR prevented TxA-mediated activation of NF-κB and concomitant increases in pERK1/2 and TNF-α mRNA. Locally produced CLR plays a proinflammatory role in TxA ileitis via MAPK signaling and TNF-α. The results reported here strongly suggest that a local injection of dsRNA targeting CLR could be an effective local therapeutic approach at the inflammation site in the treatment of a growing, clinically relevant hospital-acquired disease, C. difficile infection.
Asunto(s)
Toxinas Bacterianas/toxicidad , Proteína Similar al Receptor de Calcitonina/metabolismo , Enterotoxinas/toxicidad , Ileítis/inducido químicamente , Ileítis/tratamiento farmacológico , ARN Bicatenario/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Western Blotting , Proteína Similar al Receptor de Calcitonina/administración & dosificación , Proteína Similar al Receptor de Calcitonina/inmunología , Células Caliciformes/efectos de los fármacos , Masculino , Mastocitos/efectos de los fármacos , Microscopía Fluorescente , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mucinas/metabolismo , FN-kappa B/metabolismo , Infiltración Neutrófila/efectos de los fármacos , Peroxidasa/metabolismo , Interferencia de ARN , ARN Bicatenario/administración & dosificación , ARN Bicatenario/metabolismo , Ratas , Ratas Sprague-Dawley , Estadísticas no Paramétricas , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Our previous data have linked obesity with immune dysfunction. It is known that physical exercise with dietary control has beneficial effects on immune function and the comorbidities of obesity. However, the mechanisms underlying the improvement of immune function in obesity after physical exercise with dietary control remain unknown. Here we show that moderate daily exercise with dietary control restores the impaired cytokine responses in diet-induced obese (DIO) mice and improves the resolution of Porphyromonas gingivalis-induced periodontitis. This restoration of immune responses is related to the reduction of circulating free fatty acids (FFAs) and TNF. Both FFAs and TNF induce an Akt inhibitor, carboxyl-terminal modulator protein (CTMP). The expression of CTMP is also observed increased in bone marrow-derived macrophages (BMMΦ) from DIO mice and restored after moderate daily exercise with dietary control. Toll-like receptor 2 (TLR2), which increases CTMP induction by FFAs, is inhibited in BMMΦ from DIO mice or after either FFA or TNF treatment, but unexpectedly is not restored by moderate daily exercise with dietary control. Furthermore, BMMΦ from DIO mice display reduced histone H3 (Lys-9) acetylation and NF-κB recruitment to TNF, IL-10, and TLR2 promoters after P. gingivalis infection. However, moderate daily exercise with dietary control restores these defects at promoters for TNF and IL-10, but not for TLR2. Thus, metabolizing FFAs and TNF by moderate daily exercise with dietary control improves innate immune responses to infection in DIO mice via restoration of CTMP and chromatin modification.
Asunto(s)
Infecciones por Bacteroidaceae/metabolismo , Dieta/efectos adversos , Inmunidad Innata/inmunología , Obesidad/inmunología , Condicionamiento Físico Animal/fisiología , Porphyromonas gingivalis/inmunología , Transducción de Señal/inmunología , Acetilación , Pérdida de Hueso Alveolar/microbiología , Análisis de Varianza , Animales , Western Blotting , Proteínas Portadoras/metabolismo , Inmunoprecipitación de Cromatina , Citocinas/metabolismo , Cartilla de ADN/genética , Ensayo de Inmunoadsorción Enzimática , Ácidos Grasos no Esterificados/sangre , Histonas/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Obesidad/etiología , Palmitoil-CoA Hidrolasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 2/metabolismo , Factor de Necrosis Tumoral alfa/sangreRESUMEN
LPS-induced TNF-α factor (LITAF) mediates cytokine expression in response to endotoxin challenge. Previously, we reported that macrophage-specific LITAF-deficient (macLITAF-/-) mice exposed to LPS have a delayed onset in the serum levels of proinflammatory cytokines and prolonged persistence of anti-inflammatory cytokines, but only partial protection from endotoxic shock. We postulated that greater protection might be achieved if LITAF were deleted from all LITAF-producing cells, including macrophages. Using a Cre-loxP system, we engineered a tamoxifen-induced recombination mouse [tamLITAF(i)-/-] that resulted in whole-body LITAF deficiency. Our findings demonstrate that (i) tamLITAF(i)-/- mice are more resistant to systemic Escherichia coli LPS-induced lethality than our previous macLITAF-/- mice, providing evidence that LITAF-producing cells other than LysMCre-positive cells play an important role in mediating endotoxic shock; (ii) tamLITAF(i)-/- mice show a similar pattern of cytokine expression with decreased proinflammatory and prolonged anti-inflammatory mediators compared with WT mice; and (iii) tamLITAF(i)-/- mice, compared with WT mice, display a significant reduction in bone resorption and inflammation associated with a local chronic inflammatory disease--namely, collagen antibody-induced arthritis. Our findings offer a unique model to study the role of LITAF in systemic and chronic local inflammatory processes, and pave the way for anti-LITAF therapeutic approaches for the treatment of TNF-mediated inflammatory diseases.
Asunto(s)
Artritis Experimental/inmunología , Artritis Experimental/terapia , Citocinas/metabolismo , Regulación de la Expresión Génica/inmunología , Inmunoterapia/métodos , Proteínas Nucleares/deficiencia , Choque Séptico/inmunología , Choque Séptico/terapia , Factores de Transcripción/deficiencia , Análisis de Varianza , Animales , Artritis Experimental/genética , Western Blotting , Cruzamientos Genéticos , Citocinas/inmunología , Cartilla de ADN/genética , Proteínas de Unión al ADN , Escherichia coli , Ingeniería Genética/métodos , Inmunohistoquímica , Lipopolisacáridos/toxicidad , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , Reacción en Cadena de la Polimerasa , Recombinación Genética/efectos de los fármacos , Choque Séptico/genética , Tamoxifeno/farmacología , Factores de Transcripción/genéticaRESUMEN
Patients with chronic ulcerative colitis (UC) are at high risk for developing colorectal cancer. In this study, archival formalin-fixed paraffin-embedded colonic tissue from patients with UC who developed carcinoma (CA) or high-grade dysplasia (HGD) was examined for changes in expression of the proinflammatory and mitogenic neurokinin-1 receptor (NK-1R). Laser capture microscopy was used to microdissect epithelia from areas of colons that showed histologic evidence of CA, HGD, and epithelia that were not dysplastic or cancerous but did contain evidence of prior inflammation (quiescent colitis). mRNA was extracted from the dissected tissue, and PCR array analysis was performed on extracted mRNA. Two antibodies were necessary to separately estimate the protein levels of the truncated (tr-NK-1R) and full-length (fl-NK-1R) receptors by immunohistochemistry. mRNA expression of tr-NK-1R increased 14-fold (P = 0.02) when comparing the HGD and CA groups. In contrast, the fl-NK-1R transcript showed no significant differences among groups. The protein levels of the total NK-1R increased by 40% (P = 0.02) in HGD and 80% (P = 0.0007) in CA compared with quiescent colitis. There were no significant changes in protein levels of the fl-NK-1R. We conclude that the increase in total NK-1R protein in HGD and CA is attributable to an increase in tr-NK-1R, suggesting there may be a functional role for tr-NK-1R in malignant transformation in colitis-associated cancer. The tr-NK-1R could prove useful as a diagnostic marker to identify patients at risk for neoplasia and may serve as a useful therapeutic target in the treatment of colitis-associated cancer.
Asunto(s)
Colitis Ulcerosa/metabolismo , Neoplasias Colorrectales/metabolismo , Receptores de Neuroquinina-1/metabolismo , Empalme Alternativo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Colitis Ulcerosa/complicaciones , Colitis Ulcerosa/genética , Colitis Ulcerosa/patología , Colon/metabolismo , Colon/patología , Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ligandos , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Receptores de Neuroquinina-1/química , Receptores de Neuroquinina-1/genética , Sustancia P/metabolismoRESUMEN
Recent research links diet-induced obesity (DIO) with impaired immunity, although the underlying mechanisms remain unclear. We find that the induction of inducible NO synthase (iNOS) and cytokines is suppressed in mice with DIO and in bone marrow macrophages (BMMPhi) from mice with DIO exposed to an oral pathogen, Porphyromonas gingivalis. BMMPhi from lean mice pre-treated with free fatty acids (FFAs) and exposed to P. gingivalis also exhibit a diminished induction of iNOS and cytokines. BMMPhi from lean and obese mice exposed to P. gingivalis and analyzed by a phosphorylation protein array show a reduction of Akt only in BMMPhi from mice with DIO. This reduction is responsible for diminished NF-kappaB activation and diminished induction of iNOS and cytokines. We next observed that Toll-like receptor 2 (TLR2) is suppressed in BMMPhi from DIO mice whereas carboxy-terminal modulator protein (CTMP), a known suppressor of Akt phosphorylation, is elevated. This elevation stems from defective TLR2 signaling. In BMMPhi from lean mice, both FFAs and TNF-alpha--via separate pathways--induce an increase in CMTP. However, in BMMPhi from DIO mice, TLR2 can no longer inhibit the TNF-alpha-induced increase in CTMP caused by P. gingivalis challenge. This defect can then be restored by transfecting WT TLR2 into BMMPhi from DIO mice. Thus, feeding mice a high-fat diet over time elevates the CTMP intracellular pool, initially via FFAs activating TLR2 and later when the defective TLR2 is unable to inhibit TNF-alpha-induced CTMP. These findings unveil a link between obesity and innate immunity.
Asunto(s)
Inmunidad Innata/inmunología , Macrófagos/inmunología , Obesidad/inmunología , Transducción de Señal/inmunología , Animales , Western Blotting , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/microbiología , Proteínas Portadoras/metabolismo , Línea Celular , Dieta , Ácidos Grasos no Esterificados/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Interacciones Huésped-Patógeno , Humanos , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Inmunológicos , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Obesidad/etiología , Obesidad/metabolismo , Palmitoil-CoA Hidrolasa , Fosforilación , Porphyromonas gingivalis/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismoRESUMEN
BACKGROUND: Mast cells derive from hematopoietic cell precursors and participate in tissue allergic, immune, and inflammatory processes. They secrete many mediators, including preformed TNF, in response to allergic, neuropeptide, and environmental triggers. However, regulation of mast cell degranulation is not well understood. OBJECTIVE: We investigated the role of mitochondrial dynamics in degranulation of human cultured mast cells. METHODS: Human umbilical cord blood-derived mast cells (hCBMCs) and Laboratory of Allergic Diseases 2 (LAD2) mast cells were examined by confocal and differential interference contrast microscopy during activation by IgE/antigen and substance P (SP). Mast cells in control and atopic dermatitis (AD) skin were evaluated by transmission electron microscopy. LAD2 cells were pretreated with mitochondrial division inhibitor, a dynamin-related protein 1 (Drp1) inhibitor, and small interfering RNA for Drp1, which is necessary for mitochondrial fission and translocation. Calcineurin and Drp1 gene expression was analyzed in stimulated LAD2 cells and AD skin biopsies. RESULTS: Stimulation of hCBMCs with IgE/antigen or LAD2 cells with SP leads to rapid (30 minutes) secretion of preformed TNF. Degranulation is accompanied by mitochondrial translocation from a perinuclear location to exocytosis sites. Extracellular calcium depletion prevents these effects, indicating calcium requirement. The calcium-dependent calcineurin and Drp1 are activated 30 minutes after SP stimulation. Reduction of Drp1 activity by mitochondrial division inhibitor and decrease of Drp1 expression using small interfering RNA inhibit mitochondrial translocation, degranulation, and TNF secretion. Mitochondrial translocation is also evident by transmission electron microscopy in skin mast cells from AD biopsies, in which gene expression of calcineurin, Drp1, and SP is higher than in normal skin. CONCLUSION: Human mast cell degranulation requires mitochondrial dynamics, also implicated in AD.
Asunto(s)
Degranulación de la Célula/fisiología , Dermatitis Atópica/fisiopatología , Mastocitos/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Adolescente , Adulto , Antígenos/administración & dosificación , Transporte Biológico Activo , Calcineurina/genética , Calcineurina/metabolismo , Calcio/metabolismo , Estudios de Casos y Controles , Degranulación de la Célula/efectos de los fármacos , Degranulación de la Célula/inmunología , Células Cultivadas , Niño , Dermatitis Atópica/genética , Dermatitis Atópica/inmunología , Dinaminas , Exocitosis/fisiología , Femenino , GTP Fosfohidrolasas/antagonistas & inhibidores , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Humanos , Inmunoglobulina E/administración & dosificación , Masculino , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Mastocitos/ultraestructura , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Persona de Mediana Edad , Mitocondrias/fisiología , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , ARN Interferente Pequeño/genética , Sustancia P/administración & dosificación , Sustancia P/genética , Adulto JovenRESUMEN
The neurokinin-1 receptor (NK1R) has two naturally occurring forms that differ in the length of the carboxyl terminus: a full-length receptor consisting of 407 aa and a truncated receptor consisting of 311 aa. We examined whether there are differential signaling properties attributable to the carboxyl terminus of this receptor by using stably transfected human embryonic kidney (HEK293) cell lines that express either full-length or truncated NK1R. Substance P (SP) specifically triggered intracellular calcium increase in HEK293 cells expressing full-length NK1R but had no effect in the cells expressing the truncated NK1R. In addition, in cells expressing full-length NK1R, SP activated NF-kappaB and IL-8 mRNA expression, but in cells expressing the truncated NK1R, SP did not activate NF-kappaB, and it decreased IL-8 mRNA expression. In cells expressing full-length NK1R, SP stimulated phosphorylation of PKCdelta but inhibited phosphorylation of PKCdelta in cells expressing truncated NK1R. There are also differences in the timing of SP-induced ERK activation in cells expressing the two different forms of the receptor. Full-length NK1R activation of ERK was rapid (peak within 1-2 min), whereas truncated NK1R-mediated activation was slower (peak at 20-30 min). Thus, the carboxyl terminus of NK1R is the structural basis for differences in the functional properties of the full-length and truncated NK1R. These differences may provide important information toward the design of new NK1R receptor antagonists.
Asunto(s)
Receptores de Neuroquinina-1/genética , Transducción de Señal , Calcio/metabolismo , Línea Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Interleucina-8/genética , Proteínas Mutantes , Proteína Quinasa C-delta/metabolismo , ARN Mensajero/análisis , Receptores de Neuroquinina-1/química , Sustancia P/metabolismo , TransfecciónRESUMEN
To study the role of NMDA receptors in dopamine signaling of the striatum, the brain area that receives glutamatergic inputs from various cortical areas and most dopaminergic inputs, we generated striatum-specific NMDA receptor-deficient mice. The mutant pups showed reduced food intake and retarded growth starting at the second postnatal week and died on approximately postnatal day 20 (P20). The time course of postnatal lethality is similar to that of compound mutant, double knockout of dopamine D1/D2 receptors, or genetically engineered dopamine-deficient mouse. In vivo electrophysiological recordings in the mutant pups showed that frequencies in the range of gamma oscillation were reduced in the striatal circuits. Moreover, the number of functional dopamine receptors in the striatum as measured by D1- and D2-binding experiments was greatly diminished in the mutants as compared with control animals. A consequence of diminished dopamine binding in the striatum manifested in an increase of locomotor activity. The administration of D1/D2 agonists paradoxically reduced the hyperactivity of the mutant mice as compared with an increase in locomotor activity in control mice. These results demonstrate that the NMDA receptor plays an essential role in the integration of dopamine signaling in the striatum and that is required in behavioral function.
Asunto(s)
Eliminación de Gen , Neostriado/patología , Receptores de N-Metil-D-Aspartato/genética , Potenciales de Acción , Animales , Animales Recién Nacidos , Electrofisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora , Especificidad de Órganos , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D2/metabolismo , Transmisión SinápticaRESUMEN
Cleavage and release (shedding) of membrane proteins is a critical regulatory step in many normal and pathological processes. Evidence suggests that the antiaging transmembrane protein Klotho (KL) is shed from the cell surface by proteolytic cleavage. In this study, we attempted to identify the enzymes responsible for the shedding of KL by treating KL-transfected COS-7 cells with a panel of proteinase inhibitors and measuring cleavage products by Western blot. We report that metalloproteinase inhibitors, including EDTA, EGTA, and TAPI-1, inhibit the shedding of KL, whereas insulin increases shedding. The effects of the inhibitors in KL-transfected COS-7 cells were repeated in studies on rat kidney slices ex vivo, which validates the use of COS-7 cells as our model system. Tissue inhibitor of metalloproteinase (Timp)-3 effectively inhibits KL cleavage, whereas Timp-1 and Timp-2 do not, a profile that indicates the involvement of members of the A Desintegrin and Metalloproteinase (ADAM) family. Cotransfection of KL with either ADAM10 or ADAM17 enhances KL cleavage, whereas cotransfection of KL with small interference RNAs specific to ADAM10 and ADAM17 inhibits KL secretion. These results indicate that KL shedding is mediated mainly by ADAM10 and ADAM17 in KL-transfected COS-7 cells. The effect of insulin is abolished when ADAM10 or ADAM17 are silenced. Furthermore, we demonstrate that the effect of insulin on KL shedding is inhibited by wortmannin, showing that insulin acts through a PI3K-dependent pathway. Insulin enhances KL shedding without increasing ADAM10 and ADAM17 mRNA and protein levels, suggesting that it acts by stimulating their proteolytic activities.
Asunto(s)
Proteínas ADAM/metabolismo , Glucuronidasa/metabolismo , Insulina/farmacología , Proteínas ADAM/genética , Animales , Células COS , Membrana Celular/enzimología , Chlorocebus aethiops , Glucuronidasa/genética , Proteínas Klotho , ARN Interferente Pequeño/genética , Ratas , Transducción de Señal/efectos de los fármacos , Inhibidores Tisulares de Metaloproteinasas/metabolismoRESUMEN
BACKGROUND: Current methods to prevent intraabdominal adhesions are not uniformly effective. We recently showed in rats that a neurokinin-1 receptor (NK-1R) antagonist is capable of reducing adhesion formation. To determine the clinical feasibility of using an NK-1R antagonist to reduce adhesions, this study examined the time dependence for the effectiveness of NK-1R antagonist administration and its effects on wound healing. METHODS: Adhesions were surgically induced in rats receiving a single intraperitoneal infusion of the NK-1R antagonist, CJ-12,255, during or 1, 5, 12, or 24 hours after surgery. Adhesion formation was assessed 7 days later. In a subset of animals, tissue plasminogen activator (tPA) activity, which is a measure of peritoneal fibrinolytic activity, was determined in peritoneal fluid 24 hours after surgery (48 hours for animals infused at 24 hours). The tPA activity was also determined in nonoperated animals 24 hours after peritoneal injection of the NK-1R antagonist. Colonic burst pressures were measured 7 days after creation of anastomoses in rats that were administered the antagonist at surgery. RESULTS: The NK-1R antagonist significantly reduced (P=.003) intraabdominal adhesions when administered during or 1 hour after surgery, only moderately reduced (P=.08) adhesions when administered at 5 hours, and had no effect at 12 or 24 hours. Peritoneal tPA activity was significantly increased (P<.05) in peritoneal fluid 24 hours after administration of the NK-1R antagonist regardless of the surgical procedure. The NK-1R antagonist did not alter colonic anastomotic healing. CONCLUSIONS: These data show that some of the events critical to adhesion formation occur within the first 5 hours following an abdominal operation in this model. The fact that the NK-1R antagonist does not impair colonic anastomotic healing enhances its usefulness as a therapeutic agent to inhibit adhesion formation.