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1.
Nat Commun ; 12(1): 2281, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863879

RESUMO

Interleukin (IL)-11 is a member of the IL-6 family of cytokines and is involved in multiple cellular responses, including tumor development. However, the origin and functions of IL-11-producing (IL-11+) cells are not fully understood. To characterize IL-11+ cells in vivo, we generate Il11 reporter mice. IL-11+ cells appear in the colon in murine tumor and acute colitis models. Il11ra1 or Il11 deletion attenuates the development of colitis-associated colorectal cancer. IL-11+ cells express fibroblast markers and genes associated with cell proliferation and tissue repair. IL-11 induces the activation of colonic fibroblasts and epithelial cells through phosphorylation of STAT3. Human cancer database analysis reveals that the expression of genes enriched in IL-11+ fibroblasts is elevated in human colorectal cancer and correlated with reduced recurrence-free survival. IL-11+ fibroblasts activate both tumor cells and fibroblasts via secretion of IL-11, thereby constituting a feed-forward loop between tumor cells and fibroblasts in the tumor microenvironment.


Assuntos
Adenoma/imunologia , Colite/patologia , Neoplasias Colorretais/imunologia , Fibroblastos/imunologia , Interleucina-11/metabolismo , Recidiva Local de Neoplasia/epidemiologia , Adenoma/genética , Adenoma/mortalidade , Adenoma/cirurgia , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Colite/induzido quimicamente , Colite/imunologia , Colo/citologia , Colo/imunologia , Colo/patologia , Colo/cirurgia , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/cirurgia , Sulfato de Dextrana/administração & dosagem , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Intervalo Livre de Doença , Feminino , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Genes Reporter , Proteínas de Fluorescência Verde/genética , Humanos , Interleucina-11/genética , Subunidade alfa de Receptor de Interleucina-11/genética , Subunidade alfa de Receptor de Interleucina-11/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/imunologia , Organoides , Cultura Primária de Células , Estudos Retrospectivos , Transcriptoma/imunologia , Microambiente Tumoral/imunologia
2.
Nat Commun ; 12(1): 2343, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33879788

RESUMO

In healthy joints, synovial fibroblasts (SFs) provide the microenvironment required to mediate homeostasis, but these cells adopt a pathological function in rheumatoid arthritis (RA). Carbohydrates (glycans) on cell surfaces are fundamental regulators of the interactions between stromal and immune cells, but little is known about the role of the SF glycome in joint inflammation. Here we study stromal guided pathophysiology by mapping SFs glycosylation pathways. Combining transcriptomic and glycomic analysis, we show that transformation of fibroblasts into pro-inflammatory cells is associated with glycan remodeling, a process that involves TNF-dependent inhibition of the glycosyltransferase ST6Gal1 and α2-6 sialylation. SF sialylation correlates with distinct functional subsets in murine experimental arthritis and remission stages in human RA. We propose that pro-inflammatory cytokines remodel the SF-glycome, converting the synovium into an under-sialylated and highly pro-inflammatory microenvironment. These results highlight the importance of glycosylation in stromal immunology and joint inflammation.


Assuntos
Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Ácidos Siálicos/metabolismo , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Animais , Artrite Experimental/patologia , Artrite Reumatoide/imunologia , Linhagem Celular , Citocinas/metabolismo , Regulação para Baixo , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Glicosilação , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Masculino , Redes e Vias Metabólicas , Camundongos , Camundongos Endogâmicos DBA , Fenótipo , RNA-Seq , Sialiltransferases/genética , Sialiltransferases/metabolismo , Membrana Sinovial/imunologia , Transcriptoma
3.
Int J Mol Sci ; 22(4)2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33669239

RESUMO

Irregular inflammatory responses are a major contributor to tissue dysfunction and inefficient repair. Skin has proven to be a powerful model to study mechanisms that regulate inflammation. In particular, skin wound healing is dependent on a rapid, robust immune response and subsequent dampening of inflammatory signaling. While injury-induced inflammation has historically been attributed to keratinocytes and immune cells, a vast body of evidence supports the ability of non-immune cells to coordinate inflammation in numerous tissues and diseases. In this review, we concentrate on the active participation of tissue-resident adipocytes and fibroblasts in pro-inflammatory signaling after injury, and how altered cellular communication from these cells can contribute to irregular inflammation associated with aberrant wound healing. Furthering our understanding of how tissue-resident mesenchymal cells contribute to inflammation will likely reveal new targets that can be manipulated to regulate inflammation and repair.


Assuntos
Adipócitos Brancos/imunologia , Derme/citologia , Derme/lesões , Fibroblastos/imunologia , Cicatrização/imunologia , Envelhecimento/imunologia , Envelhecimento/metabolismo , Animais , Comunicação Celular/imunologia , Polaridade Celular/imunologia , Citocinas/metabolismo , Diabetes Mellitus/imunologia , Diabetes Mellitus/metabolismo , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Células-Tronco Mesenquimais/metabolismo , Transdução de Sinais/imunologia
4.
Nat Commun ; 12(1): 1940, 2021 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-33782411

RESUMO

Metabolic enzymes and metabolites display non-metabolic functions in immune cell signalling that modulate immune attack ability. However, whether and how a tumour's metabolic remodelling contributes to its immune resistance remain to be clarified. Here we perform a functional screen of metabolic genes that rescue tumour cells from effector T cell cytotoxicity, and identify the embryo- and tumour-specific folate cycle enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2). Mechanistically, MTHFD2 promotes basal and IFN-γ-stimulated PD-L1 expression, which is necessary for tumourigenesis in vivo. Moreover, IFN-γ stimulates MTHFD2 through the AKT-mTORC1 pathway. Meanwhile, MTHFD2 drives the folate cycle to sustain sufficient uridine-related metabolites including UDP-GlcNAc, which promotes the global O-GlcNAcylation of proteins including cMYC, resulting in increased cMYC stability and PD-L1 transcription. Consistently, the O-GlcNAcylation level positively correlates with MTHFD2 and PD-L1 in pancreatic cancer patients. These findings uncover a non-metabolic role for MTHFD2 in cell signalling and cancer biology.


Assuntos
Aminoidrolases/genética , Antígeno B7-H1/genética , Carcinogênese/genética , Regulação Neoplásica da Expressão Gênica , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Enzimas Multifuncionais/genética , Neoplasias Pancreáticas/genética , Processamento de Proteína Pós-Traducional , Linfócitos T Citotóxicos/imunologia , Aminoidrolases/antagonistas & inibidores , Aminoidrolases/imunologia , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Carcinogênese/imunologia , Carcinogênese/patologia , Linhagem Celular Tumoral , Embrião de Mamíferos , Fibroblastos/imunologia , Fibroblastos/patologia , Ácido Fólico/imunologia , Ácido Fólico/metabolismo , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/imunologia , Metilenotetra-Hidrofolato Desidrogenase (NADP)/antagonistas & inibidores , Metilenotetra-Hidrofolato Desidrogenase (NADP)/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Enzimas Multifuncionais/antagonistas & inibidores , Enzimas Multifuncionais/imunologia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Transdução de Sinais , Linfócitos T Citotóxicos/patologia , Carga Tumoral , Evasão Tumoral , Uridina Difosfato N-Acetilglicosamina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33540711

RESUMO

The macroscopic and microscopic anatomy of the oral cavity is complex and unique in the human body. Soft-tissue structures are in close interaction with mineralized bone, but also dentine, cementum and enamel of our teeth. These are exposed to intense mechanical and chemical stress as well as to dense microbiologic colonization. Teeth are susceptible to damage, most commonly to caries, where microorganisms from the oral cavity degrade the mineralized tissues of enamel and dentine and invade the soft connective tissue at the core, the dental pulp. However, the pulp is well-equipped to sense and fend off bacteria and their products and mounts various and intricate defense mechanisms. The front rank is formed by a layer of odontoblasts, which line the pulp chamber towards the dentine. These highly specialized cells not only form mineralized tissue but exert important functions as barrier cells. They recognize pathogens early in the process, secrete antibacterial compounds and neutralize bacterial toxins, initiate the immune response and alert other key players of the host defense. As bacteria get closer to the pulp, additional cell types of the pulp, including fibroblasts, stem and immune cells, but also vascular and neuronal networks, contribute with a variety of distinct defense mechanisms, and inflammatory response mechanisms are critical for tissue homeostasis. Still, without therapeutic intervention, a deep carious lesion may lead to tissue necrosis, which allows bacteria to populate the root canal system and invade the periradicular bone via the apical foramen at the root tip. The periodontal tissues and alveolar bone react to the insult with an inflammatory response, most commonly by the formation of an apical granuloma. Healing can occur after pathogen removal, which is achieved by disinfection and obturation of the pulp space by root canal treatment. This review highlights the various mechanisms of pathogen recognition and defense of dental pulp cells and periradicular tissues, explains the different cell types involved in the immune response and discusses the mechanisms of healing and repair, pointing out the close links between inflammation and regeneration as well as between inflammation and potential malignant transformation.


Assuntos
Polpa Dentária/patologia , Periodontite Periapical/patologia , Tecido Periapical/patologia , Pulpite/patologia , Animais , Antígenos de Neoplasias/imunologia , Carcinogênese/imunologia , Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/fisiopatologia , Quimiocinas/metabolismo , Proteínas do Sistema Complemento/metabolismo , Cárie Dentária/fisiopatologia , Polpa Dentária/microbiologia , Dentina/irrigação sanguínea , Dentina/inervação , Dentina/metabolismo , Fibroblastos/imunologia , Fibroblastos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Células-Tronco Mesenquimais/fisiologia , Neoplasias Bucais/etiologia , Neoplasias Bucais/imunologia , Neoplasias Bucais/fisiopatologia , Rede Nervosa/fisiologia , Neuropeptídeos/metabolismo , Óxido Nítrico/fisiologia , Odontoblastos/fisiologia , Granuloma Periapical/etiologia , Granuloma Periapical/patologia , Tecido Periapical/microbiologia , Cisto Radicular/etiologia , Cisto Radicular/fisiopatologia
6.
Nat Immunol ; 22(3): 312-321, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33510463

RESUMO

Mitochondrial abnormalities have been noted in lupus, but the causes and consequences remain obscure. Autophagy-related genes ATG5, ATG7 and IRGM have been previously implicated in autoimmune disease. We reasoned that failure to clear defective mitochondria via mitophagy might be a foundational driver in autoimmunity by licensing mitochondrial DNA-dependent induction of type I interferon. Here, we show that mice lacking the GTPase IRGM1 (IRGM homolog) exhibited a type I interferonopathy with autoimmune features. Irgm1 deletion impaired the execution of mitophagy with cell-specific consequences. In fibroblasts, mitochondrial DNA soiling of the cytosol induced cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-dependent type I interferon, whereas in macrophages, lysosomal Toll-like receptor 7 was activated. In vivo, Irgm1-/- tissues exhibited mosaic dependency upon nucleic acid receptors. Whereas salivary and lacrimal gland autoimmune pathology was abolished and lung pathology was attenuated by cGAS and STING deletion, pancreatic pathology remained unchanged. These findings reveal fundamental connections between mitochondrial quality control and tissue-selective autoimmune disease.


Assuntos
Doenças Autoimunes/metabolismo , Autoimunidade , Fibroblastos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Mitocôndrias/metabolismo , Mitofagia , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Células Cultivadas , Fibroblastos/imunologia , Fibroblastos/patologia , Proteínas de Ligação ao GTP/deficiência , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Macrófagos/imunologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Mitocôndrias/imunologia , Mitocôndrias/patologia , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Transdução de Sinais , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo
7.
Mol Med ; 26(1): 95, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-33054759

RESUMO

Pulmonary fibrosis arises from the repeated epithelial mild injuries and insufficient repair lead to over activation of fibroblasts and excessive deposition of extracellular matrix, which result in a mechanical stretched niche. However, increasing mechanical stress likely exists before the establishment of fibrosis since early micro injuries increase local vascular permeability and prompt cytoskeletal remodeling which alter cellular mechanical forces. It is noteworthy that COVID-19 patients with severe hypoxemia will receive mechanical ventilation as supportive treatment and subsequent pathology studies indicate lung fibrosis pattern. At advanced stages, mechanical stress originates mainly from the stiff matrix since boundaries between stiff and compliant parts of the tissue could generate mechanical stress. Therefore, mechanical stress has a significant role in the whole development process of pulmonary fibrosis. The alveoli are covered by abundant capillaries and function as the main gas exchange unit. Constantly subject to variety of damages, the alveolar epithelium injuries were recently recognized to play a vital role in the onset and development of idiopathic pulmonary fibrosis. In this review, we summarize the literature regarding the effects of mechanical stress on the fundamental cells constituting the alveoli in the process of pulmonary fibrosis, particularly on epithelial cells, capillary endothelial cells, fibroblasts, mast cells, macrophages and stem cells. Finally, we briefly review this issue from a more comprehensive perspective: the metabolic and epigenetic regulation.


Assuntos
Infecções por Coronavirus/imunologia , Epigênese Genética/imunologia , Fibrose Pulmonar Idiopática/imunologia , Mecanotransdução Celular/imunologia , Pneumonia Viral/imunologia , Embolia Pulmonar/imunologia , Insuficiência Respiratória/imunologia , Células Epiteliais Alveolares/imunologia , Células Epiteliais Alveolares/patologia , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Fenômenos Biomecânicos , Infecções por Coronavirus/genética , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Citocinas/genética , Citocinas/imunologia , Células Endoteliais/imunologia , Células Endoteliais/patologia , Fibroblastos/imunologia , Fibroblastos/patologia , Humanos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/virologia , Pulmão/irrigação sanguínea , Pulmão/imunologia , Pulmão/patologia , Macrófagos/imunologia , Macrófagos/patologia , Mecanotransdução Celular/genética , Pandemias , Pneumonia Viral/genética , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Embolia Pulmonar/genética , Embolia Pulmonar/patologia , Embolia Pulmonar/virologia , Insuficiência Respiratória/genética , Insuficiência Respiratória/patologia , Insuficiência Respiratória/virologia , Estresse Mecânico
8.
Proc Natl Acad Sci U S A ; 117(36): 22113-22121, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32843346

RESUMO

RNA polymerase (Pol) III has a noncanonical role of viral DNA sensing in the innate immune system. This polymerase transcribes viral genomes to produce RNAs that lead to induction of type I interferons (IFNs). However, the genetic and functional links of Pol III to innate immunity in humans remain largely unknown. Here, we describe a rare homozygous mutation (D40H) in the POLR3E gene, coding for a protein subunit of Pol III, in a child with recurrent and systemic viral infections and Langerhans cell histiocytosis. Fibroblasts derived from the patient exhibit impaired induction of type I IFN and increased susceptibility to human cytomegalovirus (HCMV) infection. Cultured cell lines infected with HCMV show induction of POLR3E expression. However, induction is not restricted to DNA virus, as sindbis virus, an RNA virus, enhances the expression of this protein. Likewise, foreign nonviral DNA elevates the steady-state level of POLR3E and elicits promoter-dependent and -independent transcription by Pol III. Remarkably, the molecular mechanism underlying the D40H mutation of POLR3E involves the assembly of defective initiation complexes of Pol III. Our study links mutated POLR3E and Pol III to an innate immune deficiency state in humans.


Assuntos
Citomegalovirus/fisiologia , Fibroblastos/imunologia , Fibroblastos/virologia , RNA Polimerase III/metabolismo , Animais , Chlorocebus aethiops , Citomegalovirus/imunologia , Células Dendríticas , Regulação Enzimológica da Expressão Gênica , Humanos , Mutação , RNA Polimerase III/genética , Células Vero
9.
Nature ; 583(7815): 296-302, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32612232

RESUMO

The mammalian immune system implements a remarkably effective set of mechanisms for fighting pathogens1. Its main components are haematopoietic immune cells, including myeloid cells that control innate immunity, and lymphoid cells that constitute adaptive immunity2. However, immune functions are not unique to haematopoietic cells, and many other cell types display basic mechanisms of pathogen defence3-5. To advance our understanding of immunology outside the haematopoietic system, here we systematically investigate the regulation of immune genes in the three major types of structural cells: epithelium, endothelium and fibroblasts. We characterize these cell types across twelve organs in mice, using cellular phenotyping, transcriptome sequencing, chromatin accessibility profiling and epigenome mapping. This comprehensive dataset revealed complex immune gene activity and regulation in structural cells. The observed patterns were highly organ-specific and seem to modulate the extensive interactions between structural cells and haematopoietic immune cells. Moreover, we identified an epigenetically encoded immune potential in structural cells under tissue homeostasis, which was triggered in response to systemic viral infection. This study highlights the prevalence and organ-specific complexity of immune gene activity in non-haematopoietic structural cells, and it provides a high-resolution, multi-omics atlas of the epigenetic and transcriptional networks that regulate structural cells in the mouse.


Assuntos
Endotélio/imunologia , Células Epiteliais/imunologia , Fibroblastos/imunologia , Regulação da Expressão Gênica/imunologia , Sistema Imunitário/citologia , Sistema Imunitário/imunologia , Especificidade de Órgãos/imunologia , Imunidade Adaptativa , Animais , Cromatina/genética , Cromatina/metabolismo , Endotélio/citologia , Epigênese Genética/imunologia , Epigenoma/genética , Células Epiteliais/citologia , Feminino , Fibroblastos/citologia , Regulação da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/imunologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Sistema Imunitário/virologia , Imunidade Inata , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/imunologia , Masculino , Camundongos , Especificidade de Órgãos/genética , Transcrição Genética/imunologia , Transcriptoma/genética
10.
Nat Cell Biol ; 22(6): 663-673, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32393887

RESUMO

The linear ubiquitin chain assembly complex (LUBAC), which consists of HOIP, SHARPIN and HOIL-1L, promotes NF-κB activation and protects against cell death by generating linear ubiquitin chains. LUBAC contains two RING-IBR-RING (RBR) ubiquitin ligases (E3), and the HOIP RBR is responsible for catalysing linear ubiquitination. We found that HOIL-1L RBR plays a crucial role in regulating LUBAC. HOIL-1L RBR conjugates monoubiquitin onto all LUBAC subunits, followed by HOIP-mediated conjugation of linear chains onto monoubiquitin, and these linear chains attenuate the functions of LUBAC. The introduction of E3-defective HOIL-1L mutants into cells augmented linear ubiquitination, which protected the cells against Salmonella infection and cured dermatitis caused by reduced LUBAC levels due to SHARPIN loss. Our results reveal a regulatory mode of E3 ligases in which the accessory E3 in LUBAC downregulates the main E3 by providing preferred substrates for autolinear ubiquitination. Thus, inhibition of HOIL-1L E3 represents a promising strategy for treating severe infections or immunodeficiency.


Assuntos
Proteínas de Transporte/fisiologia , Morte Celular , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Dermatite de Contato/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Salmonelose Animal/imunologia , Ubiquitina-Proteína Ligases/fisiologia , Ubiquitina/metabolismo , Animais , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Dermatite de Contato/metabolismo , Dermatite de Contato/patologia , Embrião de Mamíferos/imunologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Salmonella/patogenicidade , Salmonelose Animal/metabolismo , Salmonelose Animal/patologia , Índice de Gravidade de Doença , Transdução de Sinais , Ubiquitinação
11.
Vet Res Commun ; 44(2): 83-88, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32440968

RESUMO

OBJECTIVES: The faecal-oral route is a predominant mode of infectious disease transmission and yet the immunology of the bovine oral cavity is poorly understood. The objectives of this study were to develop an in vitro cell model of bovine salivary gland cells and to characterize the role of vitamin D on the expression of innate immune genes induced by stimulation with bacterial and viral pathogen-associated molecular patterns (PAMPs). METHODS: Submandibular glandular tissue was excised post-mortem, processed, cells isolated and cultured until confluency after which cells were incubated with the active form of vitamin D (1,25(OH)D) for 18 h before stimulation with lipopolysaccharide (LPS µg/ml), lipoteichoic acid (LTA µg/ml) or polyinosinic:polycytidylic acid (poly I:C-20 µg/ml) PAMPs for 6 h and immune gene expression was assessed by Quantitative Real-Time PCR (RT-qPCR). RESULTS: RT-qPCR analysis of vimentin expression in cells derived from the bovine submandibular gland shows that cultured cells were fibroblast in origin. These cells significantly induce the pro-inflammatory cytokine IL1B, ß-defensin and cathelicidin genes but these were not significantly altered in response to 1,25(OH)D. In contrast, 1,25(OH)D significantly up-regulates the expression of the NOS2 gene encoding iNOS in bovine submandibular stromal cells compared to EtOH (vehicle) control and this is a maintained response to all three bacterial and viral ligands. We have developed a new in vitro model to allow detailed investigations of mechanisms to enhance oral immunity in cattle. We show that these cells are fibroblast in nature, immunologically competent and vitamin D responsive. Their vitamin D-mediated enhancement of NOS2 expression warrants further investigation in saliva as a potential mechanism to boost oral immunity against infectious agents.


Assuntos
Fibroblastos/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/genética , Vitamina D/análogos & derivados , Adjuvantes Imunológicos/farmacologia , Animais , Bovinos , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Padrões Moleculares Associados a Patógenos/farmacologia , Glândulas Salivares/citologia , Vitamina D/farmacologia
12.
Nat Rev Rheumatol ; 16(6): 316-333, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32393826

RESUMO

Rheumatoid arthritis (RA) is a chronic immune-mediated disease that primarily affects the synovium of diarthrodial joints. During the course of RA, the synovium transforms into a hyperplastic invasive tissue that causes destruction of cartilage and bone. Fibroblast-like synoviocytes (FLS), which form the lining of the joint, are epigenetically imprinted with an aggressive phenotype in RA and have an important role in these pathological processes. In addition to producing the extracellular matrix and joint lubricants, FLS in RA produce pathogenic mediators such as cytokines and proteases that contribute to disease pathogenesis and perpetuation. The development of multi-omics integrative analyses have enabled new ways to dissect the mechanisms that imprint FLS, have helped to identify potential FLS subsets with distinct functions and have identified differences in FLS phenotypes between joints in individual patients. This Review provides an overview of advances in understanding of FLS biology and highlights omics approaches and studies that hold promise for identifying future therapeutic targets.


Assuntos
Artrite Reumatoide/imunologia , Reabsorção Óssea/imunologia , Cartilagem Articular/imunologia , Fibroblastos/imunologia , Membrana Sinovial/imunologia , Sinoviócitos/imunologia , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Linfócitos B/imunologia , Reabsorção Óssea/metabolismo , Caderinas/metabolismo , Cartilagem Articular/metabolismo , Metilação de DNA , Células Endoteliais/metabolismo , Epigênese Genética , Fibroblastos/metabolismo , Humanos , Macrófagos/imunologia , Terapia de Alvo Molecular , Monócitos/imunologia , Neovascularização Patológica/imunologia , Neovascularização Patológica/metabolismo , Osteogênese , Proteínas Tirosina Fosfatases/metabolismo , Membrana Sinovial/citologia , Membrana Sinovial/metabolismo , Sinoviócitos/metabolismo , Linfócitos T/imunologia
13.
Breast Cancer Res ; 22(1): 41, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32370801

RESUMO

BACKGROUND: In utero endocrine disruption is linked to increased risk of breast cancer later in life. Despite numerous studies establishing this linkage, the long-term molecular changes that predispose mammary cells to carcinogenic transformation are unknown. Herein, we investigated how endocrine disrupting compounds (EDCs) drive changes within the stroma that can contribute to breast cancer susceptibility. METHODS: We utilized bisphenol A (BPA) as a model of estrogenic endocrine disruption to analyze the long-term consequences in the stroma. Deregulated genes were identified by RNA-seq transcriptional profiling of adult primary fibroblasts, isolated from female mice exposed to in utero BPA. Collagen staining, collagen imaging techniques, and permeability assays were used to characterize changes to the extracellular matrix. Finally, gland stiffness tests were performed on exposed and control mammary glands. RESULTS: We identified significant transcriptional deregulation of adult fibroblasts exposed to in utero BPA. Deregulated genes were associated with cancer pathways and specifically extracellular matrix composition. Multiple collagen genes were more highly expressed in the BPA-exposed fibroblasts resulting in increased collagen deposition in the adult mammary gland. This transcriptional reprogramming of BPA-exposed fibroblasts generates a less permeable extracellular matrix and a stiffer mammary gland. These phenotypes were only observed in adult 12-week-old, but not 4-week-old, mice. Additionally, diethylstilbestrol, known to increase breast cancer risk in humans, also increases gland stiffness similar to BPA, while bisphenol S does not. CONCLUSIONS: As breast stiffness, extracellular matrix density, and collagen deposition have been directly linked to breast cancer risk, these data mechanistically connect EDC exposures to molecular alterations associated with increased disease susceptibility. These alterations develop over time and thus contribute to cancer risk in adulthood.


Assuntos
Disruptores Endócrinos/toxicidade , Matriz Extracelular/patologia , Glândulas Mamárias Animais/patologia , Efeitos Tardios da Exposição Pré-Natal/patologia , Células Estromais/patologia , Animais , Compostos Benzidrílicos/toxicidade , Estrogênios não Esteroides/farmacologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/imunologia , Feminino , Fibroblastos/imunologia , Fibroblastos/patologia , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/imunologia , Glândulas Mamárias Animais/metabolismo , Camundongos , Fenóis/toxicidade , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Células Estromais/efeitos dos fármacos , Células Estromais/imunologia , Transcriptoma
14.
Immunity ; 52(5): 767-781.e6, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32277911

RESUMO

The enzyme cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA in infected and malignant cells and catalyzes the formation of 2'3'cGMP-AMP (cGAMP), which in turn triggers interferon (IFN) production via the STING pathway. Here, we examined the contribution of anion channels to cGAMP transfer and anti-viral defense. A candidate screen revealed that inhibition of volume-regulated anion channels (VRACs) increased propagation of the DNA virus HSV-1 but not the RNA virus VSV. Chemical blockade or genetic ablation of LRRC8A/SWELL1, a VRAC subunit, resulted in defective IFN responses to HSV-1. Biochemical and electrophysiological analyses revealed that LRRC8A/LRRC8E-containing VRACs transport cGAMP and cyclic dinucleotides across the plasma membrane. Enhancing VRAC activity by hypotonic cell swelling, cisplatin, GTPγS, or the cytokines TNF or interleukin-1 increased STING-dependent IFN response to extracellular but not intracellular cGAMP. Lrrc8e-/- mice exhibited impaired IFN responses and compromised immunity to HSV-1. Our findings suggest that cell-to-cell transmission of cGAMP via LRRC8/VRAC channels is central to effective anti-viral immunity.


Assuntos
Fibroblastos/imunologia , Interferons/imunologia , Proteínas de Membrana/imunologia , Nucleotídeos Cíclicos/imunologia , Canais de Ânion Dependentes de Voltagem/imunologia , Animais , Antivirais/imunologia , Antivirais/metabolismo , Efeito Espectador , Linhagem Celular , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HeLa , Herpes Simples/imunologia , Herpes Simples/virologia , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/fisiologia , Humanos , Interferons/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nucleotídeos Cíclicos/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologia , Nucleotidiltransferases/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo
15.
Cancer Immunol Immunother ; 69(8): 1535-1548, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32300857

RESUMO

With an infection rate of 60-90%, the human cytomegalovirus (HCMV) is very common among adults but normally causes no symptoms. When T cell-mediated immunity is compromised, HCMV reactivation can lead to increased morbidity and mortality. HCMV antigens are processed and presented as peptides on the cell surface via HLA I complexes to the T cell receptor (TCR) of T cells. The generation of antibodies against HCMV peptides presented on HLA complexes (TCR-like antibodies) has been described, but is without therapeutic applications to date due to the polygenic and polymorphic nature of HLA genes. We set out to obtain antibodies specific for HLA/HCMV-peptides, covering the majority of HLA alleles present in European populations. Using phage display technology, we selected 10 Fabs, able to bind to HCMV-peptides presented in the 6 different HLA class I alleles A*0101, A*0201, A*2402, B*0702, B*0801 and B*3501. We demonstrate specific binding of all selected Fabs to HLA-typed lymphoblastoid cell lines (EBV-transformed B cells) and lymphocytes loaded with HCMV-peptides. After infection with HCMV, 4/10 tetramerized Fabs restricted to the alleles HLA-A*0101, HLA-A*0201 and HLA-B*0702 showed binding to infected primary fibroblasts. When linked to the pseudomonas exotoxin A, these Fab antibodies induce highly specific cytotoxicity in HLA matched cell lines loaded with HCMV peptides. TCR-like antibody repertoires therefore represent a promising new treatment modality for viral infections and may also have applications in the treatment of cancers.


Assuntos
Infecções por Citomegalovirus/prevenção & controle , Citomegalovirus/imunologia , Fibroblastos/imunologia , Antígenos HLA/imunologia , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Fragmentos de Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Antígenos Virais/imunologia , Sobrevivência Celular , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/virologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunotoxinas/administração & dosagem , Melanoma/imunologia , Melanoma/metabolismo , Melanoma/patologia , Melanoma/prevenção & controle , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Virais/imunologia
16.
Curr Pharm Biotechnol ; 21(12): 1213-1223, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32297577

RESUMO

BACKGROUND: The first immunosuppressive drug - cyclosporine A (CsA) has many unquestioned merits in maintaining organ transplants in patients, as well as, in the treatment of many inflammatory diseases, also associated with cutaneous manifestations. The main task of this drug is to suppress the inflammatory response at the sites of action, which is not well known. OBJECTIVE: The objective of this study was to evaluate the influence of CsA in therapeutic concentration on the expression of genes associated with the inflammatory response pathway in normal human dermal fibroblasts (NHDF; CC-2511), and this study attempted to determine the mechanism of its action. METHODS: The cytotoxicity MTT test was performed. The expression of the inflammatory response pathway genes was determined using HG-U133A_2.0 oligonucleotide microarrays. Statistical analysis was performed by GeneSpring 13.0 software using the PL-Grid platform. RESULTS: Among the 5,300 mRNA, only 573 were changed significantly in response to CsA compared to the control fibroblasts (P≤0.05). CsA inhibited the expression of most genes associated with the inflammatory response in NHDFs. There were only 19 genes with a fold change (FC) lower than -2.0, among which EGR1, FOS, PBK, CDK1 and TOP2A had the lowest expression, as did CXCL2 which can directly impact inflammation. Furthermore, ZNF451 was strongly induced, and COL1A1, COL3A1, IL33, TNFRSFs were weakly up-regulated (FC lower than 2.0). CONCLUSION: The CsA in therapeutic concentration influences the genes linked to the inflammatory response (in the transcriptional level) in human dermal fibroblasts. The findings suggest that the potential mechanism of CsA action in this concentration and on these genes can be associated with a profibrotic and proapoptotic, and genotoxic effects.


Assuntos
Ciclosporina/farmacologia , Fibroblastos/efeitos dos fármacos , Imunossupressores/farmacologia , Pele/efeitos dos fármacos , Transcriptoma , Técnicas de Cultura de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Relação Dose-Resposta a Droga , Fibroblastos/imunologia , Perfilação da Expressão Gênica , Humanos , Inflamação/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Pele/imunologia , Transcriptoma/efeitos dos fármacos , Transcriptoma/imunologia , Regulação para Cima
17.
Microbiol Immunol ; 64(6): 445-457, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32246487

RESUMO

Chikungunya virus (CHIKV) is a mosquito-borne virus that causes arthralgic fever. Fibroblast-like synoviocytes play a key role in joint damage in inflammatory arthritides and can additionally serve as target cells for CHIKV infection. To gain a better understanding of CHIKV-induced arthralgia, the interaction between CHIKV and synoviocytes was investigated at the protein level. A gel-enhanced liquid chromatography-mass spectrometry (GeLC-MS/MS) approach was used to examine protein expression from primary human fibroblast-like synoviocytes (HFLS) infected with clinical isolates of CHIKV at 12 and 24 hr post infection. Our analysis identified 259 and 241 proteins of known function that were differentially expressed (>1.5 or <-1.5 fold change) following CHIKV infection at 12 and 24 hpi, respectively. These proteins are involved in cellular homeostasis, including cellular trafficking, cytoskeletal organization, immune response, metabolic process, and protein modification. Some of these proteins have previously been reported to participate in arthralgia/arthritis and the death of infected cells. Our results provide information on the CHIKV-induced modulation of cellular proteins of HFLS at an early stage of infection, as well as highlighting biological processes associated with CHIKV infection in the main target cells of the joint.


Assuntos
Febre de Chikungunya , Fibroblastos/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Proteoma/imunologia , Sinoviócitos/imunologia , Células Cultivadas , Febre de Chikungunya/imunologia , Febre de Chikungunya/patologia , Febre de Chikungunya/virologia , Vírus Chikungunya/fisiologia , Fibroblastos/patologia , Humanos , Proteômica/métodos , Sinoviócitos/patologia , Replicação Viral
18.
J Virol ; 94(12)2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32238587

RESUMO

Cyclic GMP-AMP synthase (cGAS) senses double-stranded DNA and synthesizes the second messenger cyclic GMP-AMP (cGAMP), which binds to mediator of IRF3 activation (MITA) and initiates MITA-mediated signaling, leading to induction of type I interferons (IFNs) and other antiviral effectors. Human cytomegalovirus (HCMV), a widespread and opportunistic pathogen, antagonizes the host antiviral immune response to establish latent infection. Here, we identified HCMV tegument protein UL94 as an inhibitor of the cGAS-MITA-mediated antiviral response. Ectopic expression of UL94 impaired cytosolic double-stranded DNA (dsDNA)- and DNA virus-triggered induction of type I IFNs and enhanced viral replication. Conversely, UL94 deficiency potentiated HCMV-induced transcription of type I IFNs and downstream antiviral effectors and impaired viral replication. UL94 interacted with MITA, disrupted the dimerization and translocation of MITA, and impaired the recruitment of TBK1 to the MITA signalsome. These results suggest that UL94 plays an important role in the immune evasion of HCMV.IMPORTANCE Human cytomegalovirus (HCMV), a large double-stranded DNA (dsDNA) virus, encodes more than 200 viral proteins. HCMV infection causes irreversible abnormalities of the central nervous system in newborns and severe syndromes in organ transplantation patients or AIDS patients. It has been demonstrated that HCMV has evolved multiple immune evasion strategies to establish latent infection. Previous studies pay more attention to the mechanism by which HCMV evades immune response in the early phase of infection. In this study, we identified UL94 as a negative regulator of the innate immune response, which functions in the late phase of HCMV infection.


Assuntos
Proteínas do Capsídeo/imunologia , Citomegalovirus/imunologia , Genoma Viral , Evasão da Resposta Imune , Proteínas de Membrana/imunologia , Proteínas Serina-Treonina Quinases/imunologia , RNA Interferente Pequeno/genética , Proteínas do Capsídeo/genética , Núcleo Celular/imunologia , Núcleo Celular/virologia , GMP Cíclico/imunologia , GMP Cíclico/metabolismo , Citomegalovirus/genética , Citomegalovirus/crescimento & desenvolvimento , Citosol/imunologia , Citosol/virologia , DNA/imunologia , DNA/metabolismo , Fibroblastos/imunologia , Fibroblastos/virologia , Regulação da Expressão Gênica , Células HEK293 , Humanos , Imunidade Inata , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Proteínas de Membrana/genética , Cultura Primária de Células , Ligação Proteica , Multimerização Proteica , Transporte Proteico , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/imunologia , Transdução de Sinais , Sequenciamento Completo do Exoma
19.
J Virol ; 94(12)2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32238588

RESUMO

Tetherin/BST-2 is an antiviral protein that blocks the release of enveloped viral particles by linking them to the membrane of producing cells. At first, BST-2 genes were described only in humans and other mammals. Recent work identified BST-2 orthologs in nonmammalian vertebrates, including birds. Here, we identify the BST-2 sequence in domestic chicken (Gallus gallus) for the first time and demonstrate its activity against avian sarcoma and leukosis virus (ASLV). We generated a BST-2 knockout in chicken cells and showed that BST-2 is a major determinant of an interferon-induced block of ASLV release. Ectopic expression of chicken BST-2 blocks the release of ASLV in chicken cells and of human immunodeficiency virus type 1 (HIV-1) in human cells. Using metabolic labeling and pulse-chase analysis of HIV-1 Gag proteins, we verified that chicken BST-2 blocks the virus at the release stage. Furthermore, we describe BST-2 orthologs in multiple avian species from 12 avian orders. Previously, some of these species were reported to lack BST-2, highlighting the difficulty of identifying sequences of this extremely variable gene. We analyzed BST-2 genes in the avian orders Galliformes and Passeriformes and showed that they evolve under positive selection. This indicates that avian BST-2 is involved in host-virus evolutionary arms races and suggests that BST-2 antagonists exist in some avian viruses. In summary, we show that chicken BST-2 has the potential to act as a restriction factor against ASLV. Characterizing the interaction of avian BST-2 with avian viruses is important in understanding innate antiviral defenses in birds.IMPORTANCE Birds are important hosts of viruses that have the potential to cause zoonotic infections in humans. However, only a few antiviral genes (called viral restriction factors) have been described in birds, mostly because birds lack counterparts of highly studied mammalian restriction factors. Tetherin/BST-2 is a restriction factor, originally described in humans, that blocks the release of newly formed virus particles from infected cells. Recent work identified BST-2 in nonmammalian vertebrate species, including birds. Here, we report the BST-2 sequence in domestic chicken and describe its antiviral activity against a prototypical avian retrovirus, avian sarcoma and leukosis virus (ASLV). We also identify BST-2 genes in multiple avian species and show that they evolve rapidly in birds, which is an important indication of their relevance for antiviral defense. Analysis of avian BST-2 genes will shed light on defense mechanisms against avian viral pathogens.


Assuntos
Proteínas Aviárias/imunologia , Vírus do Sarcoma Aviário/imunologia , Antígeno 2 do Estroma da Médula Óssea/imunologia , Evolução Molecular , Galliformes/imunologia , Sarcoma Aviário/imunologia , Sequência de Aminoácidos , Animais , Proteínas Aviárias/genética , Vírus do Sarcoma Aviário/genética , Vírus do Sarcoma Aviário/patogenicidade , Antígeno 2 do Estroma da Médula Óssea/genética , Linhagem Celular , Fibroblastos/imunologia , Fibroblastos/virologia , Galliformes/genética , Galliformes/virologia , Regulação da Expressão Gênica , Células HEK293 , HIV-1/genética , HIV-1/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Passeriformes/genética , Passeriformes/imunologia , Passeriformes/virologia , Sarcoma Aviário/genética , Sarcoma Aviário/virologia , Seleção Genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Liberação de Vírus , Replicação Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia
20.
Mol Immunol ; 121: 136-143, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32200171

RESUMO

The innate immune system is activated upon virus invasion of a host cell by recognizing viral component, such as dsRNA through specific receptors, resulting in the production of type- I IFNs, which confer an antiviral state within the invaded as well as surrounding cells. In the present study, fibroblast, monocyte and macrophage cells derived from water Buffalo (Bubalus bubalis) were exposed to a synthetic dsRNA analogue, poly I:C to mimic viral invasion in each cell type. Recognition of poly I:C through cytosolic helicase receptors RIG-I and MDA5 molecule lead to the activation of the RLR pathway, subsequently activating the MAVS-IRF3/7 cascade and the production of antiviral effector molecule like IFNß and ISGs. Within the different cell types, we identified variability in RLR receptor and IFNß expression after poly I:C administration. Fibroblasts responded quickly and strongly with IFNß production, followed by macrophages and monocytes. Despite absolute expression variability among different cell types the expression trend of RLRs pathway genes were similar. Length of poly I:C molecule also influence IFNß expression in response of RLR pathway. Short (LMW) poly I:C induce stronger IFN-ß expression in myeloid (macrophage and monocyte) cells. In contrast long (HMW) poly I:C preferably elicit higher IFNß expression in non-myeloid (fibroblast) cell. Therefore, MDA5 and RIG-1 plays an indispensable role in eliciting antiviral response in non- immune (fibroblast) host cell. Thus, stimulation of RLR pathway with suitable and potentially cell-type specific agonist molecules successfully elicit antiviral state in the host animal, with fibroblasts conferring a stronger antiviral state compared with the monocytes and macrophages.


Assuntos
Búfalos/imunologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Poli I-C/farmacologia , Animais , Células Cultivadas , Proteína DEAD-box 58/imunologia , Proteína DEAD-box 58/metabolismo , Resistência à Doença/imunologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Fibroblastos/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Interferon beta/imunologia , Interferon beta/metabolismo , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Cultura Primária de Células , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia
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