RESUMO
We present here the data to support the understanding of the implication of Rap2a GTPase in LPS-induced innate immune response and NF-κB activation. The data presented are related to molecular tools that were generated, acquired, optimized or validated to investigate Rap2a expression, activation and its effects in mammalian cells including RAW264.7 macrophages and THP-1 monocytes under inflammatory conditions. These data supplement important technical and biological information on immune function of Rap2a in macrophages activated by LPS, recently reported by us (Carvalho et al., 2019) [1].
RESUMO
Small Ras GTPases are key molecules that regulate a variety of cellular responses in different cell types. Rap1 plays important functions in the regulation of macrophage biology during inflammation triggered by toll-like receptors (TLRs). However, despite sharing a relatively high degree of similarity with Rap1, no studies concerning Rap2 in macrophages and innate immunity have been reported yet. In this work, we show that either way alterations in the levels of Rap2a hampers proper macrophages response to TLR stimulation. Rap2a is activated by LPS in macrophages, and although putative activator TLR-inducible Ras guanine exchange factor RasGEF1b was sufficient to induce, it was not fully required for Rap2a activation. Silencing of Rap2a impaired LPS-induced production of IL-6 cytokine and KC/Cxcl1 chemokine, and also NF-κB activity as measured by reporter gene studies. Surprisingly, overexpression of Rap2a did also lead to marked inhibition of NF-κB activation induced by LPS, Pam3CSK4 and downstream TLR signaling molecules. We also found that Rap2a can inhibit the LPS-induced phosphorylation of the NF-κB subunit p65 at serine 536. Collectively, our data suggest that expression levels of Rap2a in macrophages might be tightly regulated to avoid unbalanced immune response. Our results implicate Rap2a in TLR-mediated responses by contributing to balanced NF-κB activity status in macrophages.
Assuntos
Regulação da Expressão Gênica , Inflamação/genética , Macrófagos/enzimologia , NF-kappa B/metabolismo , Proteínas rap de Ligação ao GTP/metabolismo , Animais , Quimiocina CXCL1/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos , Macrófagos/patologia , Camundongos , Células RAW 264.7 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismo , Proteínas rap de Ligação ao GTP/genética , Fatores ras de Troca de Nucleotídeo GuaninaRESUMO
BACKGROUND: Gastric adenocarcinoma is associated with chronic infection by Helicobacter pylori and with the host inflammatory response triggered by it, with substantial inter-person variation in the immune response profile due to host genetic factors. AIM: To investigate the diversity of the proinflammatory genes IL8, its receptors and PTGS2 in Amerindians; to test whether candidate SNPs in these genes are associated with gastric cancer in an admixed population with high Amerindian ancestry from Lima, Peru; and to assess whether an IL8RB promoter-derived haplotype affects gene expression. METHODS: We performed a Sanger-resequencing population survey, a candidate-gene association study (220 cases, 288 controls) and meta-analyses. We also performed an in vitro validation by a reporter gene assay of IL8RB promoter. RESULTS: The diversity of the promoter of studied genes in Native Americans is similar to Europeans. Although an association between candidate SNPs and gastric cancer was not found in Peruvians, trend in our data is consistent with meta-analyses results that suggest PTGS2-rs689466-A is associated with H. pylori-associated gastric cancer in East Asia. IL8RB promoter-derived haplotype (rs3890158-A/rs4674258-T), common in Peruvians, was up-regulated by TNF-α unlike the ancestral haplotype (rs3890158-G/rs4674258-C). Bioinformatics analysis suggests that this effect stemmed from creation of a binding site for the FOXO3 transcription factor by rs3890158G>A. CONCLUSIONS: Our updated meta-analysis reinforces the role of PTGS2-rs689466-A in gastric cancer in Asians, although more studies that control for ancestry are necessary to clarify its role in Latin Americans. Finally, we suggest that IL8RB-rs3890158G>A is a cis-regulatory SNP.
Assuntos
Adenocarcinoma/etnologia , Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Ciclo-Oxigenase 2/genética , Indígenas Sul-Americanos/genética , Interleucina-8/genética , Polimorfismo de Nucleotídeo Único , Neoplasias Gástricas/etnologia , Neoplasias Gástricas/genética , Adenocarcinoma/metabolismo , Povo Asiático/genética , Sítios de Ligação , População Negra/genética , Estudos de Casos e Controles , Biologia Computacional , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Células HEK293 , Haplótipos , Humanos , Peru/epidemiologia , Fenótipo , Regiões Promotoras Genéticas , Fatores de Risco , Neoplasias Gástricas/metabolismo , Transfecção , População Branca/genéticaRESUMO
In a previous study we had shown that membrane cholesterol removal induced unregulated lysosomal exocytosis events leading to the depletion of lysosomes located at cell periphery. However, the mechanism by which cholesterol triggered these exocytic events had not been uncovered. In this study we investigated the importance of cholesterol in controlling mechanical properties of cells and its connection with lysosomal exocytosis. Tether extraction with optical tweezers and defocusing microscopy were used to assess cell dynamics in mouse fibroblasts. These assays showed that bending modulus and surface tension increased when cholesterol was extracted from fibroblasts plasma membrane upon incubation with MßCD, and that the membrane-cytoskeleton relaxation time increased at the beginning of MßCD treatment and decreased at the end. We also showed for the first time that the amplitude of membrane-cytoskeleton fluctuation decreased during cholesterol sequestration, showing that these cells become stiffer. These changes in membrane dynamics involved not only rearrangement of the actin cytoskeleton, but also de novo actin polymerization and stress fiber formation through Rho activation. We found that these mechanical changes observed after cholesterol sequestration were involved in triggering lysosomal exocytosis. Exocytosis occurred even in the absence of the lysosomal calcium sensor synaptotagmin VII, and was associated with actin polymerization induced by MßCD. Notably, exocytosis triggered by cholesterol removal led to the secretion of a unique population of lysosomes, different from the pool mobilized by actin depolymerizing drugs such as Latrunculin-A. These data support the existence of at least two different pools of lysosomes with different exocytosis dynamics, one of which is directly mobilized for plasma membrane fusion after cholesterol removal.
Assuntos
Membrana Celular/efeitos dos fármacos , Colesterol/química , Fibroblastos/efeitos dos fármacos , Lisossomos/metabolismo , beta-Ciclodextrinas/farmacologia , Actinas/genética , Actinas/metabolismo , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular , Membrana Celular/ultraestrutura , Colesterol/deficiência , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Exocitose/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Lisossomos/classificação , Fluidez de Membrana/efeitos dos fármacos , Camundongos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Sinaptotagminas/antagonistas & inibidores , Sinaptotagminas/genética , Sinaptotagminas/metabolismo , Tiazolidinas/farmacologia , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: Although B cells are important as antigen presenting cells (APC) during the immune response, their role in DNA vaccination models is unknown. METHODS: In this study in vitro and in vivo experiments were performed to evaluate the ability of B cells to protect mice against Mycobacterium tuberculosis challenge. RESULTS: In vitro and in vivo studies showed that B cells efficiently present antigens after naked plasmid pcDNA3 encoding M. leprae 65-kDa heat shock protein (pcDNA3-Hsp65) internalization and protect B knock-out (BKO) mice against Mycobacterium tuberculosis infection. pcDNA3-Hsp65-transfected B cells adoptively transferred into BKO mice rescued the memory phenotypes and reduced the number of CFU compared to wild-type mice. CONCLUSIONS: These data not only suggest that B cells play an important role in the induction of CD8 T cells but also that they improve bacterial clearance in DNA vaccine model.
RESUMO
BACKGROUND: mRNAs are highly versatile, non-toxic molecules that are easy to produce and store, which can allow transient protein expression in all cell types. The safety aspects of mRNA-based treatments in gene therapy make this molecule one of the most promising active components of therapeutic or prophylactic methods. The use of mRNA as strategy for the stimulation of the immune system has been used mainly in current strategies for the cancer treatment but until now no one tested this molecule as vaccine for infectious disease. RESULTS: We produce messenger RNA of Hsp65 protein from Mycobacterium leprae and show that vaccination of mice with a single dose of 10 µg of naked mRNA-Hsp65 through intranasal route was able to induce protection against subsequent challenge with virulent strain of Mycobacterium tuberculosis. Moreover it was shown that this immunization was associated with specific production of IL-10 and TNF-alpha in spleen. In order to determine if antigen presenting cells (APCs) present in the lung are capable of capture the mRNA, labeled mRNA-Hsp65 was administered by intranasal route and lung APCs were analyzed by flow cytometry. These experiments showed that after 30 minutes until 8 hours the populations of CD11c+, CD11b+ and CD19+ cells were able to capture the mRNA. We also demonstrated in vitro that mRNA-Hsp65 leads nitric oxide (NO) production through Toll-like receptor 7 (TLR7). CONCLUSIONS: Taken together, our results showed a novel and efficient strategy to control experimental tuberculosis, besides opening novel perspectives for the use of mRNA in vaccines against infectious diseases and clarifying the mechanisms involved in the disease protection we noticed as well.
Assuntos
Proteínas de Bactérias/administração & dosagem , Chaperonina 60/administração & dosagem , Terapia Genética , RNA Mensageiro/administração & dosagem , Vacinas contra a Tuberculose/administração & dosagem , Tuberculose/prevenção & controle , Administração Intranasal , Animais , Células Apresentadoras de Antígenos/imunologia , Proteínas de Bactérias/imunologia , Linhagem Celular , Chaperonina 60/imunologia , Feminino , Células HEK293 , Humanos , Interleucina-10/imunologia , Pulmão/citologia , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium leprae/imunologia , Mycobacterium tuberculosis/patogenicidade , RNA Mensageiro/imunologia , Tuberculose/imunologia , Vacinas contra a Tuberculose/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
A major malaria vaccine candidate, the circumsporozoite (CS) protein of Plasmodium, is a pre-erythrocytic stage antigen that is attached to the surface of the sporozoites through a glycosylphosphatidylinositol (GPI) anchor. However, here we show that the motif that signals for glycosylphosphatidylinositol anchor addition interferes with the immunogenicity of this protein and reduces protection in mice upon immunization with a recombinant adenovirus. The presence of the glycosylphosphatidylinositol-anchoring motif sequentially affected total circumsporozoite protein production, cellular distribution, antigen processing and secretion, leading to less effective antigen presentation. Consistently, vaccination with an adenovirus recombinant carrying the anchoring motif-disrupted circumsporozoite gene, resulted in significant increase of the number of interferon-gamma (IFN-gamma) producing T cells and specific IgG2a isotype antibodies, ensuing more effective vaccination. Given that the anchoring motif is highly conserved among different species of Plasmodium, anti-malaria subunit vaccines encoded by recombinant vectors that aim at the induction of strong cellular immunity could maximize immunogenicity by removing anchoring motifs.