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1.
Front Immunol ; 15: 1409333, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38919608

RESUMO

Introduction: Therapeutic antibodies have become a major strategy to treat oncologic diseases. For chronic lymphocytic leukemia, antibodies against CD20 are used to target and elicit cytotoxic responses against malignant B cells. However, efficacy is often compromised due to a suppressive microenvironment that interferes with cellular immune responses. To overcome this suppression, agonists of pattern recognition receptors have been studied which promote direct cytotoxicity or elicit anti-tumoral immune responses. NOD2 is an intracellular pattern recognition receptor that participates in the detection of peptidoglycan, a key component of bacterial cell walls. This detection then mediates the activation of multiple signaling pathways in myeloid cells. Although several NOD2 agonists are being used worldwide, the potential benefit of these agents in the context of antibody therapy has not been explored. Methods: Primary cells from healthy-donor volunteers (PBMCs, monocytes) or CLL patients (monocytes) were treated with versus without the NOD2 agonist L18-MDP, then antibody-mediated responses were assessed. In vivo, the Eµ-TCL1 mouse model of CLL was used to test the effects of L18-MDP treatment alone and in combination with anti-CD20 antibody. Results: Treatment of peripheral blood mononuclear cells with L18-MDP led to activation of monocytes from both healthy donors and CLL patients. In addition, there was an upregulation of activating FcγR in monocytes and a subsequent increase in antibody-mediated phagocytosis. This effect required the NF-κB and p38 signaling pathways. Treatment with L18-MDP plus anti-CD20 antibody in the Eµ-TCL model of CLL led to a significant reduction of CLL load, as well as to phenotypic changes in splenic monocytes and macrophages. Conclusions: Taken together, these results suggest that NOD2 agonists help overturn the suppression of myeloid cells, and may improve the efficacy of antibody therapy for CLL.


Assuntos
Leucemia Linfocítica Crônica de Células B , Macrófagos , Proteína Adaptadora de Sinalização NOD2 , Receptores de IgG , Proteína Adaptadora de Sinalização NOD2/agonistas , Proteína Adaptadora de Sinalização NOD2/metabolismo , Proteína Adaptadora de Sinalização NOD2/imunologia , Animais , Humanos , Receptores de IgG/metabolismo , Receptores de IgG/imunologia , Camundongos , Macrófagos/imunologia , Macrófagos/metabolismo , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Feminino , Camundongos Endogâmicos C57BL , Transdução de Sinais , Fagocitose , Rituximab/farmacologia , Rituximab/uso terapêutico
2.
Int J Mol Sci ; 25(10)2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38791357

RESUMO

The lung is prone to infections from respiratory viruses such as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). A challenge in combating these infections is the difficulty in targeting antiviral activity directly at the lung mucosal tract. Boosting the capability of the respiratory mucosa to trigger a potent immune response at the onset of infection could serve as a potential strategy for managing respiratory infections. This study focused on screening immunomodulators to enhance innate immune response in lung epithelial and immune cell models. Through testing various subfamilies and pathways of pattern recognition receptors (PRRs), the nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) family was found to selectively activate innate immunity in lung epithelial cells. Activation of NOD1 and dual NOD1/2 by the agonists TriDAP and M-TriDAP, respectively, increased the number of IL-8+ cells by engaging the NF-κB and interferon response pathways. Lung epithelial cells showed a stronger response to NOD1 and dual NOD1/2 agonists compared to control. Interestingly, a less-pronounced response to NOD1 agonists was noted in PBMCs, indicating a tissue-specific effect of NOD1 in lung epithelial cells without inducing widespread systemic activation. The specificity of the NOD agonist pathway was confirmed through gene silencing of NOD1 (siRNA) and selective NOD1 and dual NOD1/2 inhibitors in lung epithelial cells. Ultimately, activation induced by NOD1 and dual NOD1/2 agonists created an antiviral environment that hindered SARS-CoV-2 replication in vitro in lung epithelial cells.


Assuntos
COVID-19 , Células Epiteliais , Pulmão , Proteína Adaptadora de Sinalização NOD1 , SARS-CoV-2 , Humanos , Células A549 , Antivirais/farmacologia , COVID-19/imunologia , COVID-19/virologia , Tratamento Farmacológico da COVID-19 , Ácido Diaminopimélico/análogos & derivados , Ácido Diaminopimélico/farmacologia , Células Epiteliais/virologia , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Imunidade Inata/efeitos dos fármacos , Interleucina-8/metabolismo , Pulmão/imunologia , Pulmão/virologia , Pulmão/metabolismo , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD1/agonistas , Proteína Adaptadora de Sinalização NOD2/agonistas , Proteína Adaptadora de Sinalização NOD2/metabolismo , SARS-CoV-2/fisiologia , SARS-CoV-2/imunologia , Transdução de Sinais/efeitos dos fármacos
3.
Eur J Med Chem ; 271: 116439, 2024 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-38691886

RESUMO

Nucleotide-binding oligomerization domain 2 (NOD2) is a receptor of the innate immune system that is capable of perceiving bacterial and viral infections. Muramyl dipeptide (MDP, N-acetyl muramyl L-alanyl-d-isoglutamine), identified as the minimal immunologically active component of bacterial cell wall peptidoglycan (PGN) is recognized by NOD2. In terms of biological activities, MDP demonstrated vaccine adjuvant activity and stimulated non-specific protection against bacterial, viral, and parasitic infections and cancer. However, MDP has certain drawbacks including pyrogenicity, rapid elimination, and lack of oral bioavailability. Several detailed structure-activity relationship (SAR) studies around MDP scaffolds are being carried out to identify better NOD2 ligands. The present review elaborates a comprehensive SAR summarizing structural aspects of MDP derivatives in relation to NOD2 agonistic activity.


Assuntos
Acetilmuramil-Alanil-Isoglutamina , Proteína Adaptadora de Sinalização NOD2 , Proteína Adaptadora de Sinalização NOD2/metabolismo , Proteína Adaptadora de Sinalização NOD2/agonistas , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Acetilmuramil-Alanil-Isoglutamina/química , Relação Estrutura-Atividade , Humanos , Animais , Estrutura Molecular
4.
Innate Immun ; 29(6): 122-131, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37545346

RESUMO

Consumption of diets high in fat has been linked to the development of obesity and related metabolic complications. Such associations originate from the enhanced, chronic, low-grade inflammation mediated by macrophages in response to translocated bacteria, bacterial products, or dietary constituents such as fatty acids (FAs). Nucleotide-binding Oligomerization Domain 2 (NOD2) senses muramyl dipeptide (MDP), a component of bacterial peptidoglycan. The inability to sense peptidoglycan through NOD2 has been demonstrated to lead to dysbiosis, increased bacterial translocation, inflammation and metabolic dysfunction. Currently, it is unknown how consumption of HFDs with different FA compositions might influence NOD2-dependent responses. In this study, we subjected WT mice to a control diet or to HFDs comprised of various ratios of unsaturated to saturated fats and determined the macrophage response to TLR4 and NOD2 agonists. A HFD with equal ratios of saturated and unsaturated fats enhanced subsequent responsiveness of macrophages to LPS but not to MDP. However, a high-unsaturated fat diet (HUFD) or a high-saturated fat diet (HSFD) both decreased the responsiveness to NOD2 agonists compared to that observed in control diet (CD) fed mice. These data suggest that dietary fatty acid composition can influence the subsequent macrophage responsiveness to bacterial products.


Assuntos
Gorduras na Dieta , Macrófagos , Proteína Adaptadora de Sinalização NOD2 , Receptor 4 Toll-Like , Animais , Camundongos , Acetilmuramil-Alanil-Isoglutamina , Dieta Hiperlipídica , Gorduras na Dieta/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Proteína Adaptadora de Sinalização NOD2/agonistas , Peptidoglicano/metabolismo , Receptor 4 Toll-Like/agonistas
5.
Nature ; 609(7927): 590-596, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36002575

RESUMO

Bacterial cell wall components provide various unique molecular structures that are detected by pattern recognition receptors (PRRs) of the innate immune system as non-self. Most bacterial species form a cell wall that consists of peptidoglycan (PGN), a polymeric structure comprising alternating amino sugars that form strands cross-linked by short peptides. Muramyl dipeptide (MDP) has been well documented as a minimal immunogenic component of peptidoglycan1-3. MDP is sensed by the cytosolic nucleotide-binding oligomerization domain-containing protein 24 (NOD2). Upon engagement, it triggers pro-inflammatory gene expression, and this functionality is of critical importance in maintaining a healthy intestinal barrier function5. Here, using a forward genetic screen to identify factors required for MDP detection, we identified N-acetylglucosamine kinase (NAGK) as being essential for the immunostimulatory activity of MDP. NAGK is broadly expressed in immune cells and has previously been described to contribute to the hexosamine biosynthetic salvage pathway6. Mechanistically, NAGK functions upstream of NOD2 by directly phosphorylating the N-acetylmuramic acid moiety of MDP at the hydroxyl group of its C6 position, yielding 6-O-phospho-MDP. NAGK-phosphorylated MDP-but not unmodified MDP-constitutes an agonist for NOD2. Macrophages from mice deficient in NAGK are completely deficient in MDP sensing. These results reveal a link between amino sugar metabolism and innate immunity to bacterial cell walls.


Assuntos
Acetilmuramil-Alanil-Isoglutamina , Proteína Adaptadora de Sinalização NOD2 , Fosfotransferases (Aceptor do Grupo Álcool) , Acetilmuramil-Alanil-Isoglutamina/química , Acetilmuramil-Alanil-Isoglutamina/imunologia , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Animais , Bactérias/química , Bactérias/imunologia , Parede Celular/química , Hexosaminas/biossíntese , Imunidade Inata , Macrófagos/enzimologia , Macrófagos/imunologia , Camundongos , Proteína Adaptadora de Sinalização NOD2/agonistas , Proteína Adaptadora de Sinalização NOD2/metabolismo , Peptidoglicano/química , Peptidoglicano/imunologia , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo
6.
Bull Exp Biol Med ; 172(2): 175-179, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34853967

RESUMO

In 3-month bone marrow transplants of CBA mice from bone marrow donors receiving single injections of TLR-4 ligand (LPS) or NOD-2 ligand (muramyl dipeptide, MDP) 24 h before transplantation, an increase in the total number of MSCs (by 2.6 and 1.9 times, respectively), as well as a slight increase in the number of nuclear cells and the mass of bone capsules (by 1.3 and 1.2 times) were observed. After combined administration of MDР and LPS to donors, the total content of MSCs in the grafts was higher by 1.6 times in comparison with the total result of their isolated administration (and by 7.2 times in comparison with the control). At the same time, the concentration of osteogenic MSCs in the grafts of all groups was almost the same and corresponded to the control level. The number of nuclear cells and the mass of bone capsules of the grafts after combined administration of LPS and MDP were close (~80%) to the sum of the results of their isolated administration. These findings suggest that activation of the stromal tissue and the success of bone marrow transplantation depend on the intensity of innate immune responses. These data can be useful for the development of optimal methods of tissue transplantation.


Assuntos
Acetilmuramil-Alanil-Isoglutamina/administração & dosagem , Células da Medula Óssea/efeitos dos fármacos , Transplante de Medula Óssea , Lipopolissacarídeos/administração & dosagem , Doadores de Tecidos , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Combinação de Medicamentos , Lipopolissacarídeos/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos CBA , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/efeitos dos fármacos , Proteína Adaptadora de Sinalização NOD2/agonistas , Receptor 4 Toll-Like/agonistas
7.
J Med Chem ; 64(11): 7809-7838, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34043358

RESUMO

We report on the design, synthesis, and biological evaluation of a series of nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) desmuramylpeptide agonists with improved in vitro and in vivo adjuvant properties. We identified two promising compounds: 68, a potent nanomolar in vitro NOD2 agonist, and the more lipophilic 75, which shows superior adjuvant activity in vivo. Both compounds had immunostimulatory effects on peripheral blood mononuclear cells at the protein and transcriptional levels, and augmented dendritic-cell-mediated activation of T cells, while 75 additionally enhanced the cytotoxic activity of peripheral blood mononuclear cells against malignant cells. The C18 lipophilic tail of 75 is identified as a pivotal structural element that confers in vivo adjuvant activity in conjunction with a liposomal delivery system. Accordingly, liposome-encapsulated 75 showed promising adjuvant activity in mice, surpassing that of muramyl dipeptide, while achieving a more balanced Th1/Th2 immune response, thus highlighting its potential as a vaccine adjuvant.


Assuntos
Acetilmuramil-Alanil-Isoglutamina/química , Adjuvantes Imunológicos/química , Proteína Adaptadora de Sinalização NOD2/agonistas , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Adjuvantes Imunológicos/metabolismo , Adjuvantes Imunológicos/farmacologia , Animais , Formação de Anticorpos/efeitos dos fármacos , Linhagem Celular , Desenho de Fármacos , Humanos , Imunoglobulina G/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Lipossomos/química , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Proteína Adaptadora de Sinalização NOD2/metabolismo , Ovalbumina/imunologia , Relação Estrutura-Atividade , Células Th1/citologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/citologia , Células Th2/imunologia , Células Th2/metabolismo
8.
J Cell Mol Med ; 25(8): 3785-3792, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33609010

RESUMO

Severe ionizing radiation causes the acute lethal damage of haematopoietic system and gastrointestinal tract. Here, we found CL429, the novel chimeric TLR2/NOD2 agonist, exhibited significant radioprotective effects in mice. CL429 increased mice survival, protected mice against the lethal damage of haematopoietic system and gastrointestinal tract. CL429 was more effective than equivalent amounts of monospecific (TLR2 or NOD2) and combination (TLR2 + NOD2) of molecules in preventing radiation-induced death. The radioprotection of CL429 was mainly mediated by activating TLR2 and partially activating NOD2. CL429-induced radioprotection was largely dependent on the activation of TLR2-MyD88-NF-κB signalling pathway. In conclusion, the data suggested that the co-activation of TLR2 and NOD2 could induce significant synergistic radioprotective effects and CL429 might be a potential high-efficiency selective agent.


Assuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Síndrome Aguda da Radiação/prevenção & controle , Sistema Hematopoético/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Proteína Adaptadora de Sinalização NOD2/agonistas , Protetores contra Radiação/farmacologia , Receptor 2 Toll-Like/agonistas , Irradiação Corporal Total/efeitos adversos , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Síndrome Aguda da Radiação/etiologia , Síndrome Aguda da Radiação/patologia , Animais , Sistema Hematopoético/efeitos da radiação , Intestinos/lesões , Intestinos/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL
9.
Med Sci Monit ; 26: e920325, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32006420

RESUMO

BACKGROUND Cerebral ischemia-reperfusion injury (CIRI) remains a serious health problem. Centella asiatica formulations are used to treat central nervous system disorders. In the present study, asiaticoside, an extract of the plant Centella asiatica, was investigated in CIRI in vivo and vitro. MATERIAL AND METHODS We made a CIRI model in vivo in SD rats treated by middle cerebral artery occlusion, and a cell model of ischemia-reperfusion injury was made in PC12 cells treated by deprivation of oxygen and glucose/restoration. CIRI in vivo was assessed by scores of neurological functions, encephaledema, and cerebral infarction area. Inflammation level and oxidative stress level were detected by the appropriate kits. TUNEL assay was performed for assessment of cell apoptosis and Western blot analysis was performed to assess protein expression levels. CCK8 assay was performed for evaluation of cell survival and flow cytometer was used to detect cell apoptosis in vitro. RESULTS Nervous function injury, brain edema, cell apoptosis, infarct size, apoptosis-related protein expressions, and protein expressions of the NOD2/MAPK/NF-kappaB signaling pathway in the CIRI model were all reversed by asiaticoside in rats. The cell apoptosis, inflammation level, and oxidative stress level in the model of cerebral ischemia-reperfusion injury were reduced by asiaticoside. The effects of asiaticoside on CIRI were reversed by NOD 2 agonists. CONCLUSIONS Asiaticoside showed a protective effect against cerebral ischemia-reperfusion injury via the NOD2/MAPK/NF-kappaB signaling pathway. These findings are vital for future research on use of asiaticoside in CIRI, providing a new avenue for alleviating CIRI.


Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais , Triterpenos/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Edema Encefálico/tratamento farmacológico , Edema Encefálico/patologia , Edema Encefálico/fisiopatologia , Infarto Encefálico/tratamento farmacológico , Infarto Encefálico/patologia , Infarto Encefálico/fisiopatologia , Sobrevivência Celular/efeitos dos fármacos , Inflamação/patologia , Proteína Adaptadora de Sinalização NOD2/agonistas , Proteína Adaptadora de Sinalização NOD2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Células PC12 , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/fisiopatologia , Triterpenos/farmacologia
10.
J Biol Chem ; 295(10): 3099-3114, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32005665

RESUMO

Upon activation with pathogen-associated molecular patterns, metabolism of macrophages and dendritic cells is shifted from oxidative phosphorylation to aerobic glycolysis, which is considered important for proinflammatory cytokine production. Fragments of bacterial peptidoglycan (muramyl peptides) activate innate immune cells through nucleotide-binding oligomerization domain (NOD) 1 and/or NOD2 receptors. Here, we show that NOD1 and NOD2 agonists induce early glycolytic reprogramming of human monocyte-derived macrophages (MDM), which is similar to that induced by the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide. This glycolytic reprogramming depends on Akt kinases, independent of mTOR complex 1 and is efficiently inhibited by 2-deoxy-d-glucose (2-DG) or by glucose starvation. 2-DG inhibits proinflammatory cytokine production by MDM and monocyte-derived dendritic cells activated by NOD1 or TLR4 agonists, except for tumor necrosis factor production by MDM, which is inhibited initially, but augmented 4 h after addition of agonists and later. However, 2-DG exerts these effects by inducing unfolded protein response rather than by inhibiting glycolysis. By contrast, glucose starvation does not cause unfolded protein response and, in normoxic conditions, only marginally affects proinflammatory cytokine production triggered through NOD1 or TLR4. In hypoxia mimicked by treating MDM with oligomycin (a mitochondrial ATP synthase inhibitor), both 2-DG and glucose starvation strongly suppress tumor necrosis factor and interleukin-6 production and compromise cell viability. In summary, the requirement of glycolytic reprogramming for proinflammatory cytokine production in normoxia is not obvious, and effects of 2-DG on cytokine responses should be interpreted cautiously. In hypoxia, however, glycolysis becomes critical for cytokine production and cell survival.


Assuntos
Citocinas/metabolismo , Glicólise/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Proteína Adaptadora de Sinalização NOD1/agonistas , Receptor 4 Toll-Like/agonistas , Animais , Carboxiliases/metabolismo , Hipóxia Celular , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Desoxiglucose/farmacologia , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Monócitos/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/agonistas , Proteína Adaptadora de Sinalização NOD2/metabolismo , Oligomicinas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor 4 Toll-Like/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos
11.
J Mol Neurosci ; 70(4): 600-609, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31907866

RESUMO

The purpose of the study was studying the influence of different NOD agonists on the morphological phenotype of primary murine microglia and to examine their influence on characteristic cytokines. Primary CD11b-positive cells were isolated from the brain of neonatal mice. The microglial phenotype of the cells was examined by ionized calcium-binding adapter molecule (Iba)1 staining. After14 days in culture, these cells were stimulated by iE-DAP, L18-MDP, or M-TriDAP as NOD1, NOD2, and NOD1/2 agonists, respectively. The cellular morphology was recorded and compared to the phenotype of cells cultured in medium alone or after LPS stimulation. The cells developed a specific phenotype only after treatment with the NOD2 agonist L18-MDP. These cells were characterized by straight extensions carrying tiny spikes and had a high ramification index. This was in sharp contrast to all other treatments, which always resulted in an amoeboid phenotype typically shown by activated microglia in vivo and by cultured microglia in vitro. The staining intensity of IL-6 and TNF-α did not reveal any clear difference independent of the NOD agonist treatment. In contrast, an increased staining intensity was observed for IL-10 after L18-MDP treatment. The NOD2 agonist L18-MDP induced a morphologically distinct phenotype characterized by microspike-decorated dendritiform extensions and a high degree of ramification in primary murine microglia. Increased ramification index and elevated staining intensity of anti-inflammatory IL-10 as hallmarks suggest that a M2-like phenotype of microglia was induced.


Assuntos
Acetilmuramil-Alanil-Isoglutamina/farmacologia , Adjuvantes Imunológicos/farmacologia , Ácido Diaminopimélico/análogos & derivados , Microglia/efeitos dos fármacos , Proteína Adaptadora de Sinalização NOD1/agonistas , Proteína Adaptadora de Sinalização NOD2/agonistas , Fenótipo , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Forma Celular , Extensões da Superfície Celular/efeitos dos fármacos , Células Cultivadas , Ácido Diaminopimélico/farmacologia , Interleucina-6/genética , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microglia/citologia , Microglia/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
12.
PLoS One ; 14(11): e0224738, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31697716

RESUMO

Tissue-type plasminogen activator (tPA) is a major activator of fibrinolysis, which also attenuates the pro-inflammatory activity of lipopolysaccharide (LPS) in bone marrow-derived macrophages (BMDMs) and in vivo in mice. The activity of tPA as an LPS response modifier is independent of its proteinase activity and instead, dependent on the N-methyl-D-aspartate Receptor (NMDA-R), which is expressed by BMDMs. The major Toll-like receptor (TLR) for LPS is TLR4. Herein, we show that enzymatically-inactive (EI) tPA blocks the response of mouse BMDMs to selective TLR2 and TLR9 agonists, rapidly reversing IκBα phosphorylation and inhibiting expression of TNFα, CCL2, interleukin-1ß, and interleukin-6. The activity of EI-tPA was replicated by activated α2-macroglobulin, which like EI-tPA, signals through an NMDA-R-dependent pathway. EI-tPA failed to inhibit cytokine expression by BMDMs in response to agonists that target the Pattern Recognition Receptors (PRRs), NOD1 and NOD2, providing evidence for specificity in the function of EI-tPA. Macrophages isolated from the peritoneal space (PMs), without adding eliciting agents, expressed decreased levels of cell-surface NMDA-R compared with BMDMs. These cells were unresponsive to EI-tPA in the presence of LPS. However, when PMs were treated with CSF-1, the abundance of cell-surface NMDA-R increased and the ability of EI-tPA to neutralize the response to LPS was established. We conclude that the anti-inflammatory activity of EI-tPA is selective for TLRs but not all PRRs. The ability of macrophages to respond to EI-tPA depends on the availability of cell surface NMDA-R, which may be macrophage differentiation-state dependent.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/patologia , Ativador de Plasminogênio Tecidual/farmacologia , Receptores Toll-Like/antagonistas & inibidores , Animais , Citocinas/metabolismo , Humanos , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Testes de Neutralização , Proteína Adaptadora de Sinalização NOD1/agonistas , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/agonistas , Proteína Adaptadora de Sinalização NOD2/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Toll-Like/metabolismo
13.
Int J Mol Sci ; 20(17)2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31480368

RESUMO

THP-1 cells express high levels of native functional nucleotide-binding oligomerization domain 1 (NOD1), NOD2, and Toll-like receptor 4 (TLR4) receptors, and have often been used for investigating the immunomodulatory effects of small molecules. We postulated that they would represent an ideal cell-based model for our study, the aim of which was to develop a new in vitro tool for functional characterization of NOD antagonists. NOD antagonists were initially screened for their effect on NOD agonist-induced interleukin-8 (IL-8) release. Next, we examined the extent to which the selected NOD antagonists block the NOD-TLR4 synergistic crosstalk by measuring the effect of NOD antagonism on tumor necrosis factor-α (TNF-α) secretion from doubly activated THP-1 cells. Overall, the results obtained indicate that pro-inflammatory cytokine secretion from THP-1 provides a valuable, simple and reproducible in vitro tool for functional characterization of NOD antagonists.


Assuntos
Citocinas/biossíntese , Mediadores da Inflamação/metabolismo , Proteína Adaptadora de Sinalização NOD1/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD2/antagonistas & inibidores , Sobrevivência Celular , Humanos , Proteína Adaptadora de Sinalização NOD1/agonistas , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/agonistas , Proteína Adaptadora de Sinalização NOD2/metabolismo , Células THP-1 , Receptor 4 Toll-Like/metabolismo
14.
J Cell Physiol ; 234(11): 21294-21306, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31054162

RESUMO

Injury-induced by ionizing radiation (IR) severely reduces the quality of life of victims. The development of radiation protectors is regarded as one of the most resultful strategies to alleviate damages caused by IR exposure. In the present study, we investigated the radioprotective effects of the agonist of nucleotide-binding-oligomerization-domain-containing proteins 2 called murabutide (MBD) and clarified the potential mechanisms. Our results showed that the pretreatment with MBD effectively protected cultured cells and mice against IR-induced toxicity and the pretreatment with MBD in vitro and in vitro also inhibited apoptosis caused by IR exposure. The downregulation of γ-H2AX and the upregulation of ATR signaling pathways by MBD treatment indicated that the radioprotective effects of MBD were due to the stimulation of DNA damage response (DDR) pathway to repair DNA double-strand breaks caused by IR exposure. As the radioprotective effects of MBD were diminished by the ATR selective inhibitor rather than the ATM inhibitor, ATR pathway was confirmed to be a more crucial checkpoint pathway in mediating the stimulation of DDR pathway by MBD. Taken together, our data provide a novel and effective protector to relieve the injury induced by IR exposure.


Assuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína Adaptadora de Sinalização NOD2/agonistas , Lesões por Radiação/metabolismo , Protetores contra Radiação/farmacologia , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
15.
Bull Exp Biol Med ; 166(4): 473-476, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30788737

RESUMO

In 24 h after combined administration of ligands of NOD2 (muramyl dipeptide) and TLR4 (LPS) receptors to CBA mice, a synergistic increase (by 10 times compared to the intact control) in cloning efficiency and content of multipotent stromal cells was observed in the bone marrow in comparison with the total effects of their individual administration (by 2.1 and 4.1 times, respectively). A similar effect was also observed in the peritoneal exudate. When ligands were administered simultaneously, the concentration of osteogenic multipotent stromal cells in the bone marrow decreased to a greater extent than in case of individual injections of the ligands, but did not drop below 7% of the control, which is apparently indicative of a decline threshold. In 3 h after simultaneous addition of the ligands in vitro to 12-day primary cultures of mouse bone marrow stromal cells, a synergistic increase in TNFα concentration was observed (32-fold increase from the level of intact control), while IL-10 concentration did not differ from the control, which is indicative of the proinflammatory nature of the process and the absence of immunosuppressive effect. These results suggest that activation of the stromal tissue depends on the intensity of innate immunity reactions.


Assuntos
Acetilmuramil-Alanil-Isoglutamina/farmacologia , Lipopolissacarídeos/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Proteína Adaptadora de Sinalização NOD2/agonistas , Receptor 4 Toll-Like/agonistas , Animais , Sinergismo Farmacológico , Masculino , Camundongos
16.
PLoS One ; 11(8): e0160784, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27513337

RESUMO

Muropeptides are fragments of peptidoglycan that trigger innate immune responses by activating nucleotide-binding oligomerization domain (NOD) 1 and NOD2. Muropeptides from Gram-negative bacteria contain a meso-diaminopimelic acid (meso-DAP) residue in either a terminal or a non-terminal position. While the former ones are known to be recognized by NOD1, much less is known about recognition of muropeptides with non-terminal meso-DAP, which are most abundant moieties of Gram-negative peptidoglycans. Here, we developed a novel system to assess biological activity of muropeptides, based on CRISPR/Cas9-mediated knockout (KO) of NOD1 and NOD2 genes in modified HEK293T cells. Using NOD1/NOD2 knockout and overexpression systems, as well as human monocytes and macrophages, we refine the current view of muropeptide recognition. We show that NOD2 can recognize different natural muropeptides containing a meso-DAP residue (preferably in a non-terminal position), provided they are present at micromolar concentrations. NOD2 accepts muropeptides with long and branched peptide chains and requires an intact N-acetylmuramyl residue. Muropeptides with non-terminal meso-DAP can activate NOD1 as well, but, in this case, probably require peptidase pre-processing to expose the meso-DAP residue. Depending on NOD1/NOD2 ratio in specific cell types, meso-DAP-containing muropeptides can be recognized either primarily via NOD2 (in monocytes) or via NOD1 (in monocyte-derived macrophages and HEK293T-derived cells). The dual NOD1/NOD2 agonism of meso-DAP-containing muropeptides should be taken into account when assessing cellular responses to muropeptides and designing muropeptide immunostimulants and vaccine adjuvants.


Assuntos
Ácido Diaminopimélico/farmacologia , Imunidade Inata/efeitos dos fármacos , Macrófagos/imunologia , Monócitos/imunologia , Proteína Adaptadora de Sinalização NOD1/agonistas , Proteína Adaptadora de Sinalização NOD2/agonistas , Adjuvantes Imunológicos/farmacologia , Células Cultivadas , Citocinas/metabolismo , Células HEK293 , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo
17.
Nat Med ; 22(5): 524-30, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27064448

RESUMO

Cholera toxin (CT) is a potent adjuvant for inducing mucosal immune responses. However, the mechanism by which CT induces adjuvant activity remains unclear. Here we show that the microbiota is critical for inducing antigen-specific IgG production after intranasal immunization. After mucosal vaccination with CT, both antibiotic-treated and germ-free (GF) mice had reduced amounts of antigen-specific IgG, smaller recall-stimulated cytokine responses, impaired follicular helper T (TFH) cell responses and reduced numbers of plasma cells. Recognition of symbiotic bacteria via the nucleotide-binding oligomerization domain containing 2 (Nod2) sensor in cells that express the integrin CD11c (encoded by Itgax) was required for the adjuvanticity of CT. Reconstitution of GF mice with a Nod2 agonist or monocolonization with Staphylococcus sciuri, which has high Nod2-stimulatory activity, was sufficient to promote robust CT adjuvant activity, whereas bacteria with low Nod2-stimulatory activity did not. Mechanistically, CT enhanced Nod2-mediated cytokine production in dendritic cells via intracellular cyclic AMP. These results show a role for the microbiota and the intracellular receptor Nod2 in promoting the mucosal adjuvant activity of CT.


Assuntos
Adjuvantes Imunológicos/farmacologia , Toxina da Cólera/farmacologia , Vida Livre de Germes/imunologia , Imunidade nas Mucosas/efeitos dos fármacos , Microbiota/efeitos dos fármacos , Proteína Adaptadora de Sinalização NOD2/efeitos dos fármacos , Infecções Estafilocócicas/imunologia , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Animais , Antibacterianos/farmacologia , Antígeno CD11c/metabolismo , AMP Cíclico/imunologia , Citocinas/imunologia , Células Dendríticas/imunologia , ELISPOT , Citometria de Fluxo , Imunidade nas Mucosas/imunologia , Camundongos , Camundongos Knockout , Microbiota/imunologia , Mucosa/imunologia , Mucosa Nasal/imunologia , Proteína Adaptadora de Sinalização NOD2/agonistas , Proteína Adaptadora de Sinalização NOD2/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Baço/citologia , Staphylococcus
18.
J Oral Pathol Med ; 45(4): 262-7, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26332444

RESUMO

OBJECTIVES: Microbial Pattern-recognition receptors (PRRs), such as nucleotide-binding oligomerization domains (NODs), are essential for mammalian innate immune response. This study was designed to determine the effect of NOD1 and NOD2 agonist on innate immune responses and antitumor activity in oral squamous cell carcinoma (OSCC) cells. MATERIALS AND METHODS: NODs expression was examined by RT-PCR, and IL-8 production by NODs agonist was examined by ELISA. Western blot analysis was performed to determine the MAPK activation in response to their agonist. Cell proliferation was determined by MTT assay. Flow cytometry and Western blot analysis were performed to determine the MDP-induced cell death. RESULTS: The levels of NODs were apparently expressed in OSCC cells. NODs agonist, Tri-DAP and MDP, led to the production of IL-8 and MAPK activation. NOD2 agonist, MDP, inhibited the proliferation of YD-10B cells in a dose-dependent manner. Also, the ratio of Annexin V-positive cells and cleaved PARP was increased by MDP treatment in YD-10B cells, suggesting that MDP-induced cell death in YD-10B cells may be owing to apoptosis. CONCLUSIONS: Our results indicate that NODs are functionally expressed in OSCC cells and can trigger innate immune responses. In addition, NOD2 agonist inhibited cell proliferation and induced apoptosis. These findings provide the potential value of MDP as novel candidates for antitumor agents of OSCC.


Assuntos
Acetilmuramil-Alanil-Isoglutamina/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/tratamento farmacológico , Ácido Diaminopimélico/análogos & derivados , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias Bucais/tratamento farmacológico , Proteína Adaptadora de Sinalização NOD2/agonistas , Oligopeptídeos/farmacologia , Antineoplásicos/farmacologia , Apoptose/fisiologia , Western Blotting , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ácido Diaminopimélico/farmacologia , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Imunidade Inata/efeitos dos fármacos , Interleucina-8/biossíntese , Interleucina-8/metabolismo , Proteínas Quinases Ativadas por Mitógeno/biossíntese , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias Bucais/imunologia , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Proteína Adaptadora de Sinalização NOD1/agonistas , Proteína Adaptadora de Sinalização NOD1/biossíntese , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/biossíntese , Proteína Adaptadora de Sinalização NOD2/genética , RNA Mensageiro/biossíntese , Carcinoma de Células Escamosas de Cabeça e Pescoço
19.
J Immunol ; 195(1): 217-26, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-26019273

RESUMO

Proper regulation of microbial-induced cytokines is critical to intestinal immune homeostasis. Acute stimulation of nucleotide-binding oligomerization domain 2 (NOD2), the Crohn's disease-associated sensor of bacterial peptidoglycan, induces cytokines. However, chronic NOD2 stimulation in macrophages decreases cytokines upon pattern recognition receptor (PRR) restimulation; cytokine attenuation to PRR stimulation is similarly observed in intestinal macrophages. The role for the transcriptional repressors Twist1 and Twist2 in regulating PRR-induced cytokine outcomes is poorly understood and has not been reported for NOD2. We found that Twist1 and Twist2 were required for optimal cytokine downregulation during acute and, particularly, chronic NOD2 stimulation of human macrophages. Consistently, Twist1 and Twist2 expression was increased after chronic NOD2 stimulation; this increased expression was IL-10 and TGF-ß dependent. Although Twist1 and Twist2 did not coregulate each other's expression, they cooperated to enhance binding to cytokine promoters after chronic NOD2 stimulation. Moreover, Twist1 and Twist2 contributed to enhance expression and promoter binding of the proinflammatory inhibitor c-Maf and the transcriptional repressor Bmi1. Restoring c-Maf and Bmi1 expression in Twist-deficient macrophages restored NOD2-induced cytokine downregulation. Furthermore, with chronic NOD2 stimulation, Twist1 and Twist2 contributed to the decreased expression and cytokine promoter binding of the transcriptional activators activating transcription factor 4, C/EBPα, Runx1, and Runx2. Knockdown of these transcriptional activators in Twist-deficient macrophages restored cytokine downregulation after chronic NOD2 stimulation. Finally, NOD2 synergized with additional PRRs to increase Twist1 and Twist2 expression and Twist-dependent pathways. Therefore, after chronic NOD2 stimulation Twist1 and Twist2 coordinate the regulation of both transcriptional activators and repressors, thereby mediating optimal cytokine downregulation.


Assuntos
Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Proteína Adaptadora de Sinalização NOD2/imunologia , Proteínas Nucleares/imunologia , Proteínas Repressoras/imunologia , Proteína 1 Relacionada a Twist/imunologia , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Fator 4 Ativador da Transcrição/antagonistas & inibidores , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/imunologia , Anticorpos Neutralizantes/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/antagonistas & inibidores , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/imunologia , Subunidade alfa 1 de Fator de Ligação ao Core/antagonistas & inibidores , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/imunologia , Subunidade alfa 2 de Fator de Ligação ao Core/antagonistas & inibidores , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/imunologia , Regulação da Expressão Gênica , Humanos , Interleucina-10/antagonistas & inibidores , Interleucina-10/genética , Interleucina-10/imunologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Proteína Adaptadora de Sinalização NOD2/agonistas , Proteína Adaptadora de Sinalização NOD2/genética , Proteínas Nucleares/genética , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/imunologia , Cultura Primária de Células , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-maf/genética , Proteínas Proto-Oncogênicas c-maf/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Proteínas Repressoras/genética , Transdução de Sinais , Transcrição Gênica , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologia , Proteína 1 Relacionada a Twist/genética
20.
Mol Immunol ; 64(2): 235-43, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25549945

RESUMO

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.


Assuntos
Aspergillus fumigatus/imunologia , Células Epiteliais/metabolismo , Proteína Adaptadora de Sinalização NOD2/imunologia , Receptor Cross-Talk , Esporos Fúngicos/imunologia , Receptor 2 Toll-Like/imunologia , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Linhagem Celular Transformada , Córnea/imunologia , Córnea/metabolismo , Córnea/microbiologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Inibidor de NF-kappaB alfa , Proteína Adaptadora de Sinalização NOD2/agonistas , Proteína Adaptadora de Sinalização NOD2/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD2/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/imunologia , Transdução de Sinais , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 2 Toll-Like/genética , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Zimosan/farmacologia
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