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1.
Food Funct ; 10(9): 5768-5778, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31454000

RESUMO

In this study, we aimed at investigating the antiinflammatory activity of the freeze-dried fruit powder of Actinidia arguta (FAA) on dextran sulphate sodium (DSS)-induced ulcerative colitis (UC) in mice and the effect of its extract on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. For pharmacodynamic studies, the oral administration of FAA (300 or 600 mg kg-1) could decrease the disease activity index (DAI), reduce the incidence of colon and spleen edemas (caused by inflammation), and alleviate the pathological changes in UC. For research involving biochemical indicators, FAA could decrease the expression of inflammatory markers (such as myeloperoxidase (MPO)) and attenuate the oxidative stress levels. ELISA results revealed that the expressions of proinflammatory cytokines (IL-1ß, IL-6, and TNF-α) were downregulated by FAA. Furthermore, the expression levels of the inflammation-induced activation of p38, JNK, and ERK were decreased by FAA. Hence, it was concluded that FAA could alleviate the UC symptoms in mice and the inflammatory response of macrophages via the MAPK signal pathway. Overall, FAA might have the potential to treat UC when used as a dietary supplement.


Assuntos
Actinidia/química , Anti-Inflamatórios/metabolismo , Colite Ulcerativa/dietoterapia , Colite Ulcerativa/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Preparações de Plantas/metabolismo , Actinidia/metabolismo , Animais , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/imunologia , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Feminino , Frutas/química , Frutas/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Preparações de Plantas/química , Pós/química , Pós/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
2.
J Agric Food Chem ; 67(28): 7855-7868, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31274310

RESUMO

Bee pollen (BP) collected from different floras possesses various potential bioactivities, but the mechanism-related research on anti-inflammatory effects is limited. Here, three types of BP originating from Camellia sinensis L. (BP-Cs), Nelumbo nucifera Gaertn. (BP-Nn), and Brassica campestris L. (BP-Bc) were assessed using molecular and metabolomics methods to determine their anti-inflammatory effects. The differences in polyphenolic abundance of three types of BP extracts were determined by HPLC-DAD/Q-TOF-MS. In vitro anti-inflammatory effects of three BP extracts were evaluated in a lipopolysaccharide (LPS)-induced RAW 264.7 cells model. BP-Cs extract with the most abundant polyphenols was found to be the most effective in reducing inflammation by downregulating inflammatory-related genes expression and blocking the activation of MAPK and NF-κB signaling pathways. Polyphenol-rich BP-Cs was further evaluated for their in vivo anti-inflammatory effect in a LPS-induced acute lung injury mouse model. An UPLC-Q-TOF/MS-based metabolomics approach was applied to analyze metabolite changes in mouse serum. Weshowed that the pretreated BP-Cs extract alleviated inflammation and regulated glycerophospholipid metabolism significantly. Our findings provide a foundation for developing and justifying BP as a potential anti-inflammatory ingredient in functional foods or nutraceutical formulations.


Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Anti-Inflamatórios/administração & dosagem , Extratos Vegetais/administração & dosagem , Pólen/química , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/imunologia , Animais , Anti-Inflamatórios/química , Abelhas , Brassica/química , Camellia sinensis/química , Cromatografia Líquida de Alta Pressão , Humanos , Lipopolissacarídeos/efeitos adversos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos ICR , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Nelumbo/química , Extratos Vegetais/química , Polifenóis/administração & dosagem , Polifenóis/química , Células RAW 264.7
3.
J Agric Food Chem ; 67(22): 6169-6176, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31117553

RESUMO

Dietary choline and its containing foods are biotransformed to trimethylamine (TMA) via gut microbial metabolism. Subsequently, as an intermediate molecule, TMA is quickly transported and oxidized in the liver by hepatic flavin monooxygenases to form trimethylamine oxide (TMAO). TMAO was treated as a waste byproduct from choline metabolism, but recent convincing evidence demonstrated the association between the small molecule TMAO and inflammation-related diseases, including blood vessel inflammation and vascular diseases. The scope of this study is to investigate the preventive effect of nobiletin on TMAO-induced blood vessel inflammation. Our results from Western blot showed that the inhibition of TMAO-induced cardiovascular inflammation was correlated with nobiletin-mediated inhibitory effects on NF-κB and MAPK/ERK related pathways. More specifically, nobiletin prevented the oxidative damage of vascular sites (proximal aorta), inhibited the activity of MAPK/ERK, reduced the expression of NF-κB p65 and phospho-NF-κB p65, and consequently decreased the inflammatory response. Flow cytometry analyses showed that nobiletin decreased TMAO-induced apoptosis of HUVEC cells and counteracted TMAO-induced HUVEC cell proliferation. Results from HE staining and immunohistochemical results also showed that nobiletin reduced the degree of inflammation of the proximal aorta in Sprague-Dawley rats. In summary, nobiletin significantly reduced TMAO-induced vascular inflammation via inhibition of the NF-κB/MAPK pathways.


Assuntos
Flavonas/administração & dosagem , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Fator de Transcrição RelA/imunologia , Doenças Vasculares/prevenção & controle , Animais , Aorta/efeitos dos fármacos , Aorta/imunologia , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Fígado/efeitos dos fármacos , Fígado/imunologia , Masculino , Metilaminas/efeitos adversos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Ratos , Ratos Sprague-Dawley , Fator de Transcrição RelA/genética , Doenças Vasculares/induzido quimicamente , Doenças Vasculares/genética , Doenças Vasculares/imunologia
4.
J Agric Food Chem ; 67(18): 5122-5134, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-30995031

RESUMO

Neuroinflammation has been intensively demonstrated to be related to various neurodegenerative diseases including Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Alzheimer's disease (AD). A natural polymethoxylated flavone, nobiletin (NOB) has been reported to alleviate oxidative stress, insulin resistance, and obesity. In this study, we evaluated the protection effects of NOB on neuroinflammation and memory deficit. Three-month mice were administrated with NOB by oral gavage every day for 6 weeks (100 mg/kg/day); subsequently mice were injected intraperitoneally with lipopolysaccharide (LPS) for 7 days. Results of behavioral tests revealed that NOB dramatically ameliorated LPS-triggered memory deficit regarding synaptic dysfunctions and neuronal loss. Also, NOB suppressed the microglial activation and proinflammatory cytokine secretion, such as COX-2, IL-1ß, TNF-α, and iNOS. Similarly, upon LPS stimulation, pretreatment NOB diminished the secretion of the proinflammatory cytokines in BV-2 microglia cells by exposure to LPS via modulating MAPKs, PI3K/AKT, and NF-κB signaling pathways. In addition, NOB alleviated LPS-amplified redox imbalance, disturbance of mitochondrial membrane potential (MMP), and dampening of the expression of protein related to mitochondrial respiration. The present study provides compelling evidence that NOB decreased LPS-stimulated neuroinflammation and memory impairment through maintaining cellular oxidative balance and blocking the NF-κB transcriptional pathway, illustrating that the nutritional compound NOB may serve as a potential approach to alleviate neuroinflammation-related diseases.


Assuntos
Flavonas/administração & dosagem , Inflamação/complicações , Transtornos da Memória/prevenção & controle , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , NF-kappa B/imunologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/imunologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/imunologia , Humanos , Inflamação/genética , Inflamação/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Masculino , Transtornos da Memória/etiologia , Transtornos da Memória/genética , Transtornos da Memória/imunologia , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/imunologia , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , NF-kappa B/genética , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
5.
J Agric Food Chem ; 66(3): 571-580, 2018 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-29336147

RESUMO

Emerging data suggest that urolithins, gut microbiota metabolites of ellagitannins, contribute toward multiple health benefits attributed to ellagitannin-rich foods, including walnuts, red raspberry, strawberry, and pomegranate. However, there is limited data on whether the potential neuroprotective effects of these ellagitannin-rich foods are mediated by urolithins. Herein, we evaluated the potential mechanisms of antineuroinflammatory effects of urolithins (urolithins A, B, and C; 8-methyl-O-urolithin A; and 8,9-dimethyl-O-urolithin C) in BV2 murine microglia in vitro. Nitrite analysis and qRT-PCR suggested that urolithins A and B reduced NO levels and suppressed mRNA levels of pro-inflammatory genes of TNF-α, IL-6, IL-1ß, iNOS, and COX-2 in LPS-treated microglia. Western blot revealed that urolithins A and B decreased phosphorylation levels of Erk1/2, p38 MAPK, and Akt, prevented IκB-α phosphorylation and degradation, and inhibited NF-κB p65 subunit phosphorylation and nuclear translocation in LPS-stimulated microglia. Our results indicated that urolithins A and B attenuated LPS-induced inflammation in BV2 microglia, which may be mediated by inhibiting NF-κB, MAPKs (p38 and Erk1/2), and Akt signaling pathway activation. The antineuroinflammatory activities of urolithins support their role in the potential neuroprotective effects reported for ellagitannin-rich foods warranting further in vivo studies on these ellagitannin gut microbial derived metabolites.


Assuntos
Cumarínicos/farmacologia , Taninos Hidrolisáveis/farmacologia , Lipopolissacarídeos/efeitos adversos , Microglia/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Fármacos Neuroprotetores/farmacologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Cumarínicos/química , Taninos Hidrolisáveis/química , Camundongos , Microglia/imunologia , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética
6.
J Leukoc Biol ; 102(5): 1249-1259, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28877954

RESUMO

Microorganisms are known to devise various strategies to thwart protective responses by the host. One such strategy is to incorporate sequences and domains in their genes/proteins that have similarity to various domains of the host proteins. In this study, we report that Mycobacterium tuberculosis protein Rv3529c exhibits significant similarity to the death domain of the TLR pathway adaptor protein MyD88. Incubation of macrophages with Rv3529c specifically inhibited TLR2-mediated proinflammatory responses. This included attenuated oxidative burst, reduced phosphorylation of MAPK-ERK, reduced activation of transcription factor NF-κB and reduced secretion of proinflammatory cytokines IFN-γ, IL-6, and IL-17A with a concomitant increased secretion of suppressor cytokines IL-10 and TGF-ß. Importantly, Rv3529c significantly inhibited TLR2-induced association of MyD88 with IRAK1 by competitively binding with IRAK1. Further, Rv3529c mediated inhibition of apoptosis and phagosome-lysosome fusion. Lastly, incubation of macrophages with Rv3529c increased bacterial burden inside macrophages. The data presented show another strategy evolved by M. tuberculosis toward immune evasion that centers on incorporating sequences in proteins that are similar to crucial proteins in the innate immune system of the host.


Assuntos
Proteínas de Bactérias/farmacologia , Evasão da Resposta Imune , Macrófagos/microbiologia , Mycobacterium tuberculosis/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Carga Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Regulação da Expressão Gênica , Interferon gama/genética , Interferon gama/imunologia , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Lisossomos/efeitos dos fármacos , Lisossomos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Fusão de Membrana/efeitos dos fármacos , Fusão de Membrana/imunologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Mimetismo Molecular , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/patogenicidade , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Fagossomos/efeitos dos fármacos , Fagossomos/imunologia , Cultura Primária de Células , Domínios Proteicos , Explosão Respiratória/imunologia , Transdução de Sinais , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 2 Toll-Like/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologia
7.
Food Funct ; 8(10): 3737-3744, 2017 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-28952621

RESUMO

Osteoarthritis (OA) is the most common form of joint disease and is widespread in the elderly population and is characterized by erosion of articular cartilage, subchondral bone sclerosis and synovitis. Oleuropein (OL), a secoiridoid, is considered as the most prevalent phenolic component in olive leaves and seeds, pulp and peel of unripe olives and has been shown to have potent anti-inflammatory effects. However, its effects on OA have not been clearly elucidated. This study aimed to assess the effect of OL on human OA chondrocytes. Human OA chondrocytes were pretreated with OL (10, 50 and 100 µM) for 2 h and subsequently stimulated with IL-1ß for 24 h. The production of NO, PGE2, MMP-1, MMP-13, and ADAMTS-5 was evaluated by the Griess reaction and ELISA assays. The messenger RNA (mRNA) expression of COX-2, iNOS, MMP-1, MMP13, ADAMTS-5, aggrecan, and collagen-II was measured by using real-time PCR. The protein expressions of COX-2, iNOS, p65, IκB-α, JNK, p-JNK, ERK, p-ERK, p38, and p-p38 were tested by using western blot. We found that OL significantly inhibited the IL-1ß-induced production of NO and PGE2; expression of COX-2, iNOS, MMP-1, MMP-13, and ADAMTS-5; and degradation of aggrecan and collagen-II. Furthermore, OL dramatically suppressed IL-1ß-stimulated NF-κB and MAPK activation. Immunofluorescence staining demonstrated that OL could suppress IL-1ß-induced phosphorylation of p65 nuclear translocation. These results indicate that the therapeutic effect of OL on OA is accomplished through the inhibition of both NF-κB and MAPK signaling pathways. Altogether, our findings provide the evidence to develop OL as a potential therapeutic agent for patients with OA.


Assuntos
Anti-Inflamatórios/administração & dosagem , Condrócitos/efeitos dos fármacos , Mediadores da Inflamação/imunologia , Interleucina-1beta/imunologia , Iridoides/administração & dosagem , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , NF-kappa B/imunologia , Osteoartrite/tratamento farmacológico , Condrócitos/imunologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/imunologia , Feminino , Humanos , Interleucina-1beta/genética , Masculino , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/imunologia , Pessoa de Meia-Idade , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , NF-kappa B/genética , Osteoartrite/genética , Osteoartrite/imunologia , Transdução de Sinais/efeitos dos fármacos
8.
Food Funct ; 8(3): 1299-1312, 2017 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-28251195

RESUMO

The aim of this study was to investigate the molecular mechanism underlying the immunomodulatory effect of the purified Artemisia sphaerocephala Krasch seed polysaccharide (ASKP-1) in RAW264.7 macrophages. Chemical characteristic analysis revealed that ASKP-1 consisted of 14.1% mannose, 56.9% glucose and 19.6% galactose with the average molecular weight of 9.08 × 105 Da and the mixed glycan backbone structure containing 1→4)-Glcp (39.8%), 1→6)-Galp (18.8%), 1→3,6)-Manp (19.6%), 1→)-Glcp (10.8%), 2→6)-Manp (4.0%) and 2→3,5)-Araf (7.0%). In vitro studies showed that ASKP-1 markedly induced the release of cytotoxic molecules (NO and ROS) and secretion of the cytokines (TNF-α, INF-ß, and IL-6) and significantly enhanced the phagocytosis of RAW264.7 macrophages. Furthermore, TLR4 was found to be a recognized target of ASKP-1 and its related mitogen-activated protein (MAPK) and phosphoinositide 3-kinase (PI3K)/Akt, including phosphorylated ERK, JNK, p38 and Akt, were rapidly activated by ASKP-1 in RAW264.7 macrophages. Moreover, ASKP-1 was found to cause the nuclear translocation of the nuclear factor NF-κB subunit p65 and the degradation of IκB-α in RAW264.7 macrophages. All these findings suggest that MAPK, PI3K/Akt and NF-κB pathways are involved in ASKP-1-induced macrophage activation, and ASKP-1 is a potential immunomodulating function food.


Assuntos
Artemisia/química , Ativação de Macrófagos/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , NF-kappa B/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Extratos Vegetais/farmacologia , Polissacarídeos/farmacologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Animais , Interleucina-6/genética , Interleucina-6/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , NF-kappa B/genética , Fosfatidilinositol 3-Quinases/genética , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Proteínas Proto-Oncogênicas c-akt/genética , Células RAW 264.7 , Sementes/química , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
9.
J Immunol ; 197(5): 1852-63, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27439514

RESUMO

The Toll-like and IL-1 family receptors play critical roles in innate and adaptive immunity against intracellular pathogens. Although previous data demonstrated the importance of TLRs and IL-1R signaling events for the establishment of an effective immune response to mycobacteria, the possible function of the adaptor molecule IL-1R-associated kinase (IRAK)-4 against this pathogen has not been addressed. In this study, we determined the role of IRAK-4 in signaling pathways responsible for controlling mycobacterial infections. This kinase is important for the production of IL-12 and TNF-α by macrophages and dendritic cells exposed to mycobacteria. Moreover, Mycobacterium bovis-infected IRAK-4-knockout macrophages displayed impaired MAPK and NF-κB activation. IL-1ß secretion and caspase-1 activation were also dependent on IRAK-4 signaling. Mice lacking IRAK-4 showed increased M. bovis burden in spleen, liver, and lungs and smaller liver granulomas during 60 d of infection compared with wild-type mice. Furthermore, 80% of IRAK-4(-/-) mice succumbed to virulent M. tuberculosis within 100 d following low-dose infection. This increased susceptibility to mycobacteria correlated with reduced IFN-γ/TNF-α recall responses by splenocytes, as well as fewer IL-12p70-producing APCs. Additionally, we observed that IRAK-4 is also important for the production of IFN-γ by CD4(+) T cells from infected mice. Finally, THP-1 cells treated with an IRAK-4 inhibitor and exposed to M. bovis showed reduced TNF-α and IL-12, suggesting that the results found in mice can be extended to humans. In summary, these data demonstrate that IRAK-4 is essential for innate and adaptive immunity and necessary for efficient control of mycobacterial infections.


Assuntos
Quinases Associadas a Receptores de Interleucina-1/deficiência , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Células Th1/patologia , Tuberculose/imunologia , Imunidade Adaptativa , Animais , Carga Bacteriana , Caspase 1/genética , Caspase 1/metabolismo , Linhagem Celular , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Humanos , Imunidade Inata , Interleucina-12/metabolismo , Interleucina-1beta/metabolismo , Fígado/microbiologia , Fígado/patologia , Pulmão/microbiologia , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/microbiologia , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium bovis/imunologia , Mycobacterium bovis/patogenicidade , NF-kappa B/metabolismo , Transdução de Sinais , Baço/microbiologia , Células Th1/imunologia , Tuberculina/imunologia , Tuberculose/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
10.
J Agric Food Chem ; 64(18): 3564-73, 2016 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-27087645

RESUMO

The antiallergic potential of Arctium lappa L. was investigated in Sprague-Dawley rats, ICR mice, and RBL-2H3 cells. Ethanol extract (90%) of A. lappa (ALE, 100 µg/mL) inhibited the degranulation rate by 52.9%, determined by the level of ß-hexosaminidase. ALE suppressed passive cutaneous anaphylaxis (PCA) in rats and attenuated anaphylaxis and histamine release in mice. To identify the active compound of ALE, we subsequently fractionated and determined the level of ß-hexosaminidase in all subfractions. Oleamide was identified as an active compound of ALE, which attenuated the secretion of histamine and the production of tumor necrosis factor (TNF)-α and interleukin-4 (IL-4) in cells treated with compound 48/80 or A23187/phorbol myristate acetate (PMA). Oleamide suppressed FcεRI-tyrosine kinase Lyn-mediated pathway, c-Jun N-terminal kinases (JNK/SAPK), and p38 mitogen-activated protein kinases (p38-MAPKs). These results showed that ALE and oleamide attenuated allergic reactions and should serve as a platform to search for compounds with antiallergic activity.


Assuntos
Antialérgicos/administração & dosagem , Arctium/química , Hipersensibilidade/tratamento farmacológico , Ácidos Oleicos/administração & dosagem , Extratos Vegetais/administração & dosagem , Receptores de IgE/imunologia , Animais , Antialérgicos/isolamento & purificação , Linhagem Celular , Histamina/imunologia , Humanos , Hipersensibilidade/imunologia , Interleucina-4/imunologia , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos ICR , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Ácidos Oleicos/isolamento & purificação , Extratos Vegetais/isolamento & purificação , Raízes de Plantas/química , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/imunologia
11.
BMC Plant Biol ; 15: 298, 2015 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-26700161

RESUMO

BACKGROUND: Mitogen-activated protein kinase (MAPK) cascades, which consist of three functionally associated protein kinases, namely MEKKs, MKKs and MPKs, are universal signaling modules in all eukaryotes and have been shown to play critical roles in many physiological and biochemical processes in plants. However, little or nothing is known about the MPK and MKK families in watermelon. RESULTS: In the present study, we performed a systematic characterization of the ClMPK and ClMKK families including the identification and nomenclature, chromosomal localization, phylogenetic relationships, ClMPK-ClMKK interactions, expression patterns in different tissues and in response to abiotic and biotic stress and transient expression-based functional analysis for their roles in disease resistance. Genome-wide survey identified fifteen ClMPK and six ClMKK genes in watermelon genome and phylogenetic analysis revealed that both of the ClMPK and ClMKK families can be classified into four distinct groups. Yeast two-hybrid assays demonstrated significant interactions between members of the ClMPK and ClMKK families, defining putative ClMKK2-1/ClMKK6-ClMPK4-1/ClMPK4-2/ClMPK13 and ClMKK5-ClMPK6 cascades. Most of the members in the ClMPK and ClMKK families showed differential expression patterns in different tissues and in response to abiotic (e.g. drought, salt, cold and heat treatments) and biotic (e.g. infection of Fusarium oxysporum f. sp. niveum) stresses. Transient expression of ClMPK1, ClMPK4-2 and ClMPK7 in Nicotiana benthamiana resulted in enhanced resistance to Botrytis cinerea and upregulated expression of defense genes while transient expression of ClMPK6 and ClMKK2-2 led to increased susceptibility to B. cinerea. Furthermore, transient expression of ClMPK7 also led to hypersensitive response (HR)-like cell death and significant accumulation of H2O2 in N. benthamiana. CONCLUSION: We identified fifteen ClMPK and six ClMKK genes from watermelon and analyzed their phylogenetic relationships, expression patterns and protein-protein interactions and functions in disease resistance. Our results demonstrate that ClMPK1, ClMPK4-2 and ClMPK7 positively but ClMPK6 and ClMKK2-2 negatively regulate the resistance to B. cinerea when transiently expressed in N. benthamiana and that ClMPK7 functions as a regulator of HR-like cell death through modulating the generation of H2O2.


Assuntos
Citrullus/enzimologia , Regulação da Expressão Gênica de Plantas , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Proteínas Quinases Ativadas por Mitógeno/imunologia , Proteínas de Plantas/imunologia , Sequência de Aminoácidos , Citrullus/classificação , Citrullus/genética , Citrullus/microbiologia , Resistência à Doença , Quinases de Proteína Quinase Ativadas por Mitógeno/química , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/genética , Dados de Sequência Molecular , Filogenia , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Alinhamento de Sequência
12.
Biochem Biophys Res Commun ; 458(3): 667-673, 2015 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-25684183

RESUMO

Lipopolysaccharide (LPS), one of the most prominent pathogen-associated molecular patterns (PAMPs), activates macrophages, causing release of toxic cytokines (i.e. tumor necrosis factor (TNF)-α) that may provoke inflammation and endotoxin shock. Here, we tested the potential role of trametinib, a novel and highly potent MAPK/ERK kinase (MEK) inhibitor, against LPS-induced TNF-α response in monocytes, and analyzed the underlying mechanisms. We showed that trametinib, at nM concentrations, dramatically inhibited LPS-induced TNF-α mRNA expression and protein secretion in transformed (RAW 264.7 cells) and primary murine macrophages. In ex-vivo cultured human peripheral blood mononuclear cells (PBMCs), this MEK inhibitor similarly suppressed TNF-α production by LPS. For the mechanism study, we found that trametinib blocked LPS-induced MEK-ERK activation in above monocytes, which accounted for the defective TNF-α response. Macrophages or PBMCs treated with a traditional MEK inhibitor PD98059 or infected with MEK1/2-shRNA lentivirus exhibited a similar defect as trametinib, and nullified the activity of trametinib. On the other hand, introducing a constitutively-active (CA) ERK1 restored TNF-α production by LPS in the presence of trametinib. In vivo, mice administrated with trametinib produced low levels of TNF-α after LPS stimulation, and these mice were protected from LPS-induced endotoxin shock. Together, these results show that trametinib inhibits LPS-induced TNF-α expression and endotoxin shock probably through blocking MEK-ERK signaling.


Assuntos
Lipopolissacarídeos/imunologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Piridonas/uso terapêutico , Pirimidinonas/uso terapêutico , Choque Séptico/tratamento farmacológico , Fator de Necrose Tumoral alfa/imunologia , Adulto , Animais , Linhagem Celular , Células Cultivadas , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Choque Séptico/imunologia , Adulto Jovem
13.
Exp Biol Med (Maywood) ; 240(7): 946-54, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25361770

RESUMO

Cynanchum wilfordii has been traditionally used in eastern Asia for the treatment of various diseases such as gastrointestinal diseases and arteriosclerosis. Cynandione A (CA), an acetophenone, is one of major constituents from roots of C. wilfordii. In the present study, the anti-inflammatory activities of CA were investigated in lipopolysaccharide (LPS)-treated RAW264.7 macrophages and LPS-administered C57BL/6 N mice. CA significantly decreased LPS-induced production of nitric oxide and prostaglandin E2 in a dose-dependent manner, while CA up to 200 µM did not exhibit cytotoxic activity. Our data also showed that CA significantly attenuated expression of iNOS and COX-2 in LPS-stimulated macrophages. CA inhibited phosphorylation of IκB-α and MAP kinases such as ERK and p38. Furthermore, we demonstrated that CA inhibited translocation of NF-κB to the nucleus, transcription of the NF-κB minimal promoter and NF-κB DNA binding activity. Administration of CA significantly decreased the plasma levels of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1ß in LPS-injected mice and improved survival of septic mice with lethal endotoxemia. These results demonstrate that CA has effective inhibitory effects on production of inflammatory mediators via suppressing activation of NF-κB and MAPK signaling pathways, suggesting that CA may be used as a potential anti-inflammatory agent for the prevention and treatment of inflammatory diseases.


Assuntos
Anti-Inflamatórios/farmacologia , Compostos de Bifenilo/farmacologia , Mediadores da Inflamação/imunologia , Macrófagos/efeitos dos fármacos , Choque Séptico/imunologia , Animais , Western Blotting , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/toxicidade , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Quinases de Proteína Quinase Ativadas por Mitógeno/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , NF-kappa B/efeitos dos fármacos , NF-kappa B/imunologia , NF-kappa B/metabolismo , Extratos Vegetais/farmacologia , Raízes de Plantas , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Choque Séptico/metabolismo
14.
Brain Behav Immun ; 45: 233-44, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25486089

RESUMO

The adolescent brain undergoes important dynamic and plastic cell changes, including overproduction of axons and synapses, followed by rapid pruning along with ongoing axon myelination. These developmental changes make the adolescent brain particularly vulnerable to neurotoxic and behavioral effects of alcohol. Although the mechanisms of these effects are largely unknown, we demonstrated that ethanol by activating innate immune receptors toll-like receptor 4 (TLR4), induces neuroinflammation and brain damage in adult mice. The present study aims to evaluate whether intermittent ethanol treatment in adolescence promotes TLR4-dependent pro-inflammatory processes, leading to myelin and synaptic dysfunctions, and long-term cognitive impairments. Using wild-type (WT) and TLR4-deficient (TLR4-KO) adolescent mice treated intermittently with ethanol (3.0g/kg) for 2weeks, we show that binge-like ethanol treatment activates TLR4 signaling pathways (MAPK, NFκB) leading to the up-regulation of cytokines and pro-inflammatory mediators (COX-2, iNOS, HMGB1), impairing synaptic and myelin protein levels and causing ultrastructural alterations. These changes were associated with long-lasting cognitive dysfunctions in young adult mice, as demonstrated with the object recognition, passive avoidance and olfactory behavior tests. Notably, elimination of TLR4 receptors prevented neuroinflammation along with synaptic and myelin derangements, as well as long-term cognitive alterations. These results support the role of the neuroimmune response and TLR4 signaling in the neurotoxic and behavioral effects of ethanol in adolescence.


Assuntos
Transtornos Relacionados ao Uso de Álcool/genética , Depressores do Sistema Nervoso Central/farmacologia , Transtornos Cognitivos/genética , Cognição/efeitos dos fármacos , Etanol/farmacologia , Bainha de Mielina/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Receptor 4 Toll-Like/genética , Animais , Depressores do Sistema Nervoso Central/efeitos adversos , Transtornos Cognitivos/induzido quimicamente , Ciclo-Oxigenase 2/efeitos dos fármacos , Ciclo-Oxigenase 2/imunologia , Etanol/efeitos adversos , Proteína HMGB1/efeitos dos fármacos , Proteína HMGB1/imunologia , Camundongos , Camundongos Knockout , Quinases de Proteína Quinase Ativadas por Mitógeno/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Proteínas da Mielina/efeitos dos fármacos , Proteínas da Mielina/metabolismo , Bainha de Mielina/genética , Bainha de Mielina/ultraestrutura , NF-kappa B/efeitos dos fármacos , NF-kappa B/imunologia , Óxido Nítrico Sintase Tipo II/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/imunologia , Transdução de Sinais/efeitos dos fármacos , Sinapses/genética , Sinapses/ultraestrutura
15.
PLoS Pathog ; 10(7): e1004243, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25010425

RESUMO

Multivesicular bodies (MVBs) play essential roles in many cellular processes. The MVB pathway requires reversible membrane association of the endosomal sorting complexes required for transports (ESCRTs) for sustained protein trafficking. Membrane dissociation of ESCRTs is catalyzed by the AAA ATPase SKD1, which is stimulated by LYST-interacting protein 5 (LIP5). We report here that LIP5 is a target of pathogen-responsive mitogen-activated protein kinases (MPKs) and plays a critical role in plant basal resistance. Arabidopsis LIP5 interacts with MPK6 and MPK3 and is phosphorylated in vitro by activated MPK3 and MPK6 and in vivo upon expression of MPK3/6-activating NtMEK2DD and pathogen infection. Disruption of LIP5 has little effects on flg22-, salicylic acid-induced defense responses but compromises basal resistance to Pseudomonas syringae. The critical role of LIP5 in plant basal resistance is dependent on its ability to interact with SKD1. Mutation of MPK phosphorylation sites in LIP5 does not affect interaction with SKD1 but reduces the stability and compromises the ability to complement the lip5 mutant phenotypes. Using the membrane-selective FM1-43 dye and transmission electron microscopy, we demonstrated that pathogen infection increases formation of both intracellular MVBs and exosome-like paramural vesicles situated between the plasma membrane and the cell wall in a largely LIP5-dependent manner. These results indicate that the MVB pathway is positively regulated by pathogen-responsive MPK3/6 through LIP5 phosphorylation and plays a critical role in plant immune system likely through relocalization of defense-related molecules.


Assuntos
Arabidopsis/imunologia , Resistência à Doença/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/fisiologia , Pseudomonas syringae/imunologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Resistência à Doença/genética , Sistema de Sinalização das MAP Quinases/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/imunologia , Mutação , Pseudomonas syringae/genética
16.
PLoS Genet ; 10(5): e1004389, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24830651

RESUMO

Mitogen-activated protein (MAP) kinase signaling cascades play important roles in the regulation of plant defense. The Raf-like MAP kinase kinase kinase (MAPKKK) EDR1 negatively regulates plant defense responses and cell death. However, how EDR1 functions, and whether it affects the regulation of MAPK cascades, are not well understood. Here, we showed that EDR1 negatively regulates the MKK4/MKK5-MPK3/MPK6 kinase cascade in Arabidopsis. We found that edr1 mutants have highly activated MPK3/MPK6 kinase activity and higher levels of MPK3/MPK6 proteins than wild type. EDR1 physically interacts with MKK4 and MKK5, and this interaction requires the N-terminal domain of EDR1. EDR1 also negatively affects MKK4/MKK5 protein levels. In addition, the mpk3, mkk4 and mkk5 mutations suppress edr1-mediated resistance, and over-expression of MKK4 or MKK5 causes edr1-like resistance and mildew-induced cell death. Taken together, our data indicate that EDR1 physically associates with MKK4/MKK5 and negatively regulates the MAPK cascade to fine-tune plant innate immunity.


Assuntos
Proteínas de Arabidopsis/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Imunidade Vegetal/genética , Arabidopsis , Proteínas de Arabidopsis/imunologia , Regulação da Expressão Gênica de Plantas , Sistema de Sinalização das MAP Quinases/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Fosforilação , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Plantas Geneticamente Modificadas/genética
17.
PLoS Pathog ; 10(4): e1004057, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24763622

RESUMO

Genome sequences of several economically important phytopathogenic oomycetes have revealed the presence of large families of so-called RXLR effectors. Functional screens have identified RXLR effector repertoires that either compromise or induce plant defense responses. However, limited information is available about the molecular mechanisms underlying the modes of action of these effectors in planta. The perception of highly conserved pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs), such as flg22, triggers converging signaling pathways recruiting MAP kinase cascades and inducing transcriptional re-programming, yielding a generic anti-microbial response. We used a highly synchronizable, pathogen-free protoplast-based assay to identify a set of RXLR effectors from Phytophthora infestans (PiRXLRs), the causal agent of potato and tomato light blight that manipulate early stages of flg22-triggered signaling. Of thirty-three tested PiRXLR effector candidates, eight, called Suppressor of early Flg22-induced Immune response (SFI), significantly suppressed flg22-dependent activation of a reporter gene under control of a typical MAMP-inducible promoter (pFRK1-Luc) in tomato protoplasts. We extended our analysis to Arabidopsis thaliana, a non-host plant species of P. infestans. From the aforementioned eight SFI effectors, three appeared to share similar functions in both Arabidopsis and tomato by suppressing transcriptional activation of flg22-induced marker genes downstream of post-translational MAP kinase activation. A further three effectors interfere with MAMP signaling at, or upstream of, the MAP kinase cascade in tomato, but not in Arabidopsis. Transient expression of the SFI effectors in Nicotiana benthamiana enhances susceptibility to P. infestans and, for the most potent effector, SFI1, nuclear localization is required for both suppression of MAMP signaling and virulence function. The present study provides a framework to decipher the molecular mechanisms underlying the manipulation of host MAMP-triggered immunity (MTI) by P. infestans and to understand the basis of host versus non-host resistance in plants towards P. infestans.


Assuntos
Proteínas de Arabidopsis/imunologia , Arabidopsis/imunologia , Sistema de Sinalização das MAP Quinases/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Peptídeos/imunologia , Phytophthora infestans/imunologia , Imunidade Vegetal/fisiologia , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Lycopersicon esculentum/imunologia , Lycopersicon esculentum/microbiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Peptídeos/genética , Phytophthora infestans/genética
18.
Cell Immunol ; 288(1-2): 15-23, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24561310

RESUMO

Dendritic cells (DCs) link the sensing of the environment by the innate immune system to the initiation of adaptive immune responses. Accordingly, DCs are considered to be a major target in the development of immunomodulating compounds. In this study, the effect of niclosamide, a Food and Drug Administration-approved antihelminthic drug, on the activation of lipopolysaccharide (LPS)-stimulated murine bone marrow-derived DCs was examined. Our experimental results show that niclosamide reduced the pro-inflammatory cytokine and chemokine expression of LPS-activated DCs. In addition, niclosamide also affected the expression of MHC and costimulatory molecules and influenced the ability of the cells to take up antigens. Therefore, in mixed cell cultures composed of syngeneic OVA-specific T cells and DCs, niclosamide-treated DCs showed a decreased ability to stimulate T cell proliferation and IFN-γ production. Furthermore, intravenous injection of niclosamide also attenuated contact hypersensitivity (CHS) in mice during sensitization with 2,4-dinitro-1-fluorobenzene. Blocking the LPS-induced activation of MAPK-ERK, JNK and NF-κB may contribute to the inhibitory effect of niclosamide on DC activation. Collectively, our findings suggest that niclosamide can manipulate the function of DCs. These results provide new insight into the immunopharmacological role of niclosamide and suggest that it may be useful for the treatment of chronic inflammatory disorders or DC-mediated autoimmune diseases.


Assuntos
Anti-Helmínticos/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Hipersensibilidade/prevenção & controle , Niclosamida/farmacologia , Linfócitos T/efeitos dos fármacos , Animais , Anti-Helmínticos/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/citologia , Células Dendríticas/imunologia , Dinitrofluorbenzeno/administração & dosagem , Dinitrofluorbenzeno/imunologia , Feminino , Regulação da Expressão Gênica , Hipersensibilidade/imunologia , Imunização , Imunomodulação/efeitos dos fármacos , Injeções Intravenosas , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Niclosamida/imunologia , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/imunologia
19.
PLoS Genet ; 9(12): e1004015, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24348271

RESUMO

Network robustness is a crucial property of the plant immune signaling network because pathogens are under a strong selection pressure to perturb plant network components to dampen plant immune responses. Nevertheless, modulation of network robustness is an area of network biology that has rarely been explored. While two modes of plant immunity, Effector-Triggered Immunity (ETI) and Pattern-Triggered Immunity (PTI), extensively share signaling machinery, the network output is much more robust against perturbations during ETI than PTI, suggesting modulation of network robustness. Here, we report a molecular mechanism underlying the modulation of the network robustness in Arabidopsis thaliana. The salicylic acid (SA) signaling sector regulates a major portion of the plant immune response and is important in immunity against biotrophic and hemibiotrophic pathogens. In Arabidopsis, SA signaling was required for the proper regulation of the vast majority of SA-responsive genes during PTI. However, during ETI, regulation of most SA-responsive genes, including the canonical SA marker gene PR1, could be controlled by SA-independent mechanisms as well as by SA. The activation of the two immune-related MAPKs, MPK3 and MPK6, persisted for several hours during ETI but less than one hour during PTI. Sustained MAPK activation was sufficient to confer SA-independent regulation of most SA-responsive genes. Furthermore, the MPK3 and SA signaling sectors were compensatory to each other for inhibition of bacterial growth as well as for PR1 expression during ETI. These results indicate that the duration of the MAPK activation is a critical determinant for modulation of robustness of the immune signaling network. Our findings with the plant immune signaling network imply that the robustness level of a biological network can be modulated by the activities of network components.


Assuntos
Proteínas de Arabidopsis/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Imunidade Vegetal/genética , Ácido Salicílico/metabolismo , Transdução de Sinais/genética , Arabidopsis/genética , Arabidopsis/imunologia , Proteínas de Arabidopsis/imunologia , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/imunologia , Redes Reguladoras de Genes/imunologia , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/genética , Fatores de Transcrição/metabolismo
20.
Exp Lung Res ; 39(9): 399-409, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24102366

RESUMO

The fungal allergen Alternaria alternata is associated with development of asthma, though the mechanisms underlying the allergenicity of Alternaria are largely unknown. The aim of this study was to identify whether the MAP kinase homologue Fus3 of Alternaria contributed to allergic airway responses. Wild-type (WT) and Fus3 deficient Alternaria extracts were given intranasal to mice. Extracts from Fus3 deficient Alternaria that had a functional copy of Fus3 introduced were also administered (CpFus3). Mice were challenged once and levels of BAL eosinophils and innate cytokines IL-33, thymic stromal lymphopoeitin (TSLP), and IL-25 (IL-17E) were assessed. Alternaria extracts or protease-inhibited extract were administered with (OVA) during sensitization prior to ovalbumin only challenges to determine extract adjuvant activity. Levels of BAL inflammatory cells, Th2 cytokines, and OX40-expressing Th2 cells as well as airway infiltration and mucus production were measured. WT Alternaria induced innate airway eosinophilia within 3 days. Mice given Fus3 deficient Alternaria were significantly impaired in developing airway eosinophilia that was largely restored by CpFus3. Further, BAL IL-33, TSLP, and Eotaxin-1 levels were reduced after challenge with Fus3 mutant extract compared with WT and CpFus3 extracts. WT and CpFus3 extracts demonstrated strong adjuvant activity in vivo as levels of BAL eosinophils, Th2 cytokines, and OX40-expressing Th2 cells as well as peribronchial inflammation and mucus production were induced. In contrast, the adjuvant activity of Fus3 extract or protease-inhibited WT extract was largely impaired. Finally, protease activity and Alt a1 levels were reduced in Fus3 mutant extract. Thus, Fus3 contributes to the Th2-sensitizing properties of Alternaria.


Assuntos
Alérgenos/genética , Alternaria/genética , Alternaria/imunologia , Asma/etiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/imunologia , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Alérgenos/metabolismo , Alternaria/patogenicidade , Animais , Asma/imunologia , Asma/microbiologia , Modelos Animais de Doenças , Feminino , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Humanos , Imunidade Inata , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Ovalbumina/imunologia , Células Th2/imunologia , Células Th2/patologia
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