RESUMO
Neutrophils are increasingly implicated in chronic inflammation and metabolic disorders. Here, we show that visceral adipose tissue (VAT) from individuals with obesity contains more neutrophils than in those without obesity and is associated with a distinct bacterial community. Exploring the mechanism, we gavaged microbiome-depleted mice with stool from patients with and without obesity during high-fat or normal diet administration. Only mice receiving high-fat diet and stool from subjects with obesity show enrichment of VAT neutrophils, suggesting donor microbiome and recipient diet determine VAT neutrophilia. A rise in pro-inflammatory CD4+ Th1 cells and a drop in immunoregulatory T cells in VAT only follows if there is a transient spike in neutrophils. Human VAT neutrophils exhibit a distinct gene expression pattern that is found in different human tissues, including tumors. VAT neutrophils and bacteria may be a novel therapeutic target for treating inflammatory-driven complications of obesity, including insulin resistance and colon cancer.
Assuntos
Dieta Hiperlipídica , Inflamação , Gordura Intra-Abdominal , Neutrófilos , Obesidade , Gordura Intra-Abdominal/imunologia , Gordura Intra-Abdominal/metabolismo , Animais , Obesidade/microbiologia , Obesidade/imunologia , Humanos , Neutrófilos/imunologia , Dieta Hiperlipídica/efeitos adversos , Camundongos , Inflamação/imunologia , Inflamação/microbiologia , Inflamação/patologia , Microbioma Gastrointestinal/imunologia , Masculino , Camundongos Endogâmicos C57BL , Feminino , Fezes/microbiologia , Microbiota/imunologia , Células Th1/imunologia , Infiltração de NeutrófilosRESUMO
In September 2018, India launched Pradhan Mantri Jan Arogya Yojana (PM-JAY), a nationally implemented government-funded health insurance scheme to improve access to quality inpatient care, increase financial protection, and reduce unmet need for the most vulnerable population groups. This protocol describes the methodology adopted to evaluate implementation processes and early effects of PM-JAY in seven Indian states. The study adopts a mixed and multi-methods concurrent triangulation design including three components: 1. demand-side household study, including a structured survey and qualitative elements, to quantify and understand PM-JAY reach and its effect on insurance awareness, health service utilization, and financial protection; 2. supply-side hospital-based survey encompassing both quantitative and qualitative elements to assess the effect of PM-JAY on quality of service delivery and to explore healthcare providers' experiences with scheme implementation; and 3. process documentation to examine implementation processes in selected states transitioning from either no or prior health insurance to PM-JAY. Descriptive statistics and quasi-experimental methods will be used to analyze quantitative data, while thematic analysis will be used to analyze qualitative data. The study design presented represents the first effort to jointly evaluate implementation processes and early effects of the largest government-funded health insurance scheme ever launched in India.
Assuntos
Hospitais , Seguro Saúde , Serviços de Saúde , Acessibilidade aos Serviços de Saúde/organização & administração , Índia , Avaliação de Programas e Projetos de Saúde/métodos , Qualidade da Assistência à SaúdeRESUMO
Dysregulation of monocyte and macrophage responses are often observed in children with systemic juvenile idiopathic arthritis (sJIA) and cytokine storm syndrome (CSS), a potentially fatal complication of chronic rheumatic diseases. Both conditions are associated with activation of TLR signaling in monocyte and macrophage lineage cells, leading to overwhelming inflammatory responses. Despite the importance of TLR engagement in activating proinflammatory macrophages, relatively little is known about activation of intrinsic negative regulatory pathways to attenuate excessive inflammatory responses. In this study, we demonstrate that loss of diacylglycerol (DAG) kinase (Dgk) ζ, an enzyme which converts DAG into phosphatidic acid, limits inflammatory cytokine production in an arthritic mouse model dependent on TLR2 signaling and in a CSS mouse model dependent on TLR9 signaling. In vitro, Dgkζ deficiency results in reduced production of TNF-α, IL-6, and IL-1ß and in limited M1 macrophage polarization. Mechanistically, Dgkζ deficiency decreases STAT1 and STAT3 phosphorylation. Moreover, Dgkζ levels are increased in macrophages derived from mice with CSS or exposed to plasma from sJIA patients with active disease. Our data suggest that Dgkζ induction in arthritic conditions perpetuates systemic inflammatory responses mediated by macrophages and highlight a potential role of Dgkζ-DAG/phosphatidic acid axis as a modulator of inflammatory cytokine production in sJIA and CSS.
Assuntos
Artrite Juvenil/metabolismo , Calcinose/metabolismo , Citocinas/metabolismo , Diacilglicerol Quinase/metabolismo , Modelos Animais de Doenças , Doenças das Valvas Cardíacas/metabolismo , Hipotricose/metabolismo , Macrófagos/metabolismo , Dermatopatias Genéticas/metabolismo , Animais , Artrite Juvenil/imunologia , Artrite Juvenil/patologia , Calcinose/imunologia , Calcinose/patologia , Parede Celular/imunologia , Parede Celular/metabolismo , Células Cultivadas , Citocinas/imunologia , Diacilglicerol Quinase/deficiência , Diacilglicerol Quinase/imunologia , Doenças das Valvas Cardíacas/imunologia , Doenças das Valvas Cardíacas/patologia , Hipotricose/imunologia , Hipotricose/patologia , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Dermatopatias Genéticas/imunologia , Dermatopatias Genéticas/patologiaRESUMO
Chronically activated CD4+ T cells drive uncontrolled inflammation, leading to tissue damage in various autoimmune disorders, such as rheumatoid arthritis (RA). Investigation of the molecular mechanisms involved in RA and recent analysis of transcriptomic profiles has implicated members of the nuclear receptor (NR) superfamily in RA. NRs are required for the development, differentiation, and effector function of CD4+ T cells; therefore, it is thought that NRs are important in shaping the CD4+ T cell repertoire and associated inflammation in RA. Despite their relevance, the full potential of the NR superfamily in RA, either as biomarkers or disease targets, has not been harnessed. To gain insight on the NR members that are closely associated with RA disease activity, we generated an expression atlas for the NR superfamily in CD4+ T cells isolated either in a steady state or over the course of collagen-induced arthritis mouse model of RA. We observed discrete expression patterns among the NR superfamily during the disease stages. NRs that instigate anti-inflammatory programs underwent major downregulation during disease onset; however, during the fully developed disease stage we noticed that NRs that induce proinflammatory programs had reduced transcript levels. These animal findings corroborated well with the expression patterns of NRs in clinical samples obtained from RA patients. Furthermore, we observed that targeting NRs using synthetic ligands alleviates the progression of collagen-induced arthritis. Overall, our data demonstrates the potential of the NR superfamily as novel therapeutic targets for the treatment of autoimmune disorders.
Assuntos
Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Anti-Inflamatórios/uso terapêutico , Anticorpos/imunologia , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/patologia , Colágeno Tipo II/imunologia , Colágeno Tipo II/farmacologia , Citocinas/metabolismo , Feminino , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos DBA , Fenilacetatos/uso terapêutico , Retinoides/uso terapêutico , Líquido Sinovial/metabolismo , Tiazóis/uso terapêutico , Tiossemicarbazonas/uso terapêutico , Transcrição GênicaRESUMO
Store-operated calcium entry (SOCE) modulates cytosolic calcium in multiple cells. Endoplasmic reticulum (ER)-localized STIM1 and plasma membrane (PM)-localized ORAI1 are two main components of SOCE. STIM1:ORAI1 association requires STIM1 oligomerization, its re-distribution to ER-PM junctions, and puncta formation. However, little is known about the negative regulation of these steps to prevent calcium overload. Here, we identified Tmem178 as a negative modulator of STIM1 puncta formation in myeloid cells. Using site-directed mutagenesis, co-immunoprecipitation assays and FRET imaging, we determined that Tmem178:STIM1 association occurs via their transmembrane motifs. Mutants that increase Tmem178:STIM1 association reduce STIM1 puncta formation, SOCE activation, impair inflammatory cytokine production in macrophages and osteoclastogenesis. Mutants that reduce Tmem178:STIM1 association reverse these effects. Furthermore, exposure to plasma from arthritic patients decreases Tmem178 expression, enhances SOCE activation and cytoplasmic calcium. In conclusion, Tmem178 modulates the rate-limiting step of STIM1 puncta formation and therefore controls SOCE in inflammatory conditions.
Assuntos
Cálcio/metabolismo , Proteínas Sensoras de Cálcio Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Células Mieloides/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Células Mieloides/imunologia , Proteínas de Neoplasias/química , Osteogênese/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Molécula 1 de Interação Estromal/químicaRESUMO
Cytokine storm syndrome (CSS) is a life-threatening condition characterized by excessive activation of T cells and uncontrolled inflammation, mostly described in patients with familial hemophagocytic lymphohistiocytosis and certain systemic auto-inflammatory diseases, such as systemic juvenile idiopathic arthritis (sJIA). Defects in T cell cytotoxicity as a mechanism for uncontrolled inflammation following viral infections fail to represent the whole spectrum of CSS. Evidence implicates dysregulated innate immune responses, especially activation of monocytes and macrophages, in patients with CSS. However, the direct contribution of monocytes/macrophages to CSS development and the signaling pathways involved in their activation have not been formally investigated. We find that depletion of monocytes/macrophages during early stages of CSS development, by clodronate-liposomes or neutralizing anti-CSF1 antibody, reduces mortality and inflammatory cytokine levels in two CSS mouse models, one dependent on T cells and the second induced by repeated TLR9 stimulation. We further demonstrate that activation of Plcγ2 in myeloid cells controls CSS development by driving macrophage pro-inflammatory responses. Intriguingly, the Plcγ2 downstream effector Tmem178, a negative modulator of calcium levels, acts in a negative feedback loop to restrain inflammatory cytokine production. Genetic deletion of Tmem178 leads to pro-inflammatory macrophage polarization in vitro and more severe CSS in vivo. Importantly, Tmem178 levels are reduced in macrophages from mice with CSS and after exposure to plasma from sJIA patients with active disease. Our data identify a novel Plcγ2/Tmem178 axis as a modulator of inflammatory cytokine production by monocytes/macrophages. We also find that loss of Tmem178 accentuates the pro-inflammatory responses in CSS.
Assuntos
Síndrome de Ativação Macrofágica/imunologia , Macrófagos/imunologia , Proteínas de Membrana/imunologia , Monócitos/imunologia , Fosfolipase C gama/imunologia , Transdução de Sinais/imunologia , Animais , Humanos , Síndrome de Ativação Macrofágica/genética , Síndrome de Ativação Macrofágica/patologia , Macrófagos/patologia , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Monócitos/patologia , Fosfolipase C gama/genética , Transdução de Sinais/genéticaAssuntos
Ecocardiografia/métodos , Átrios do Coração/diagnóstico por imagem , Comunicação Interatrial/diagnóstico por imagem , Veias Pulmonares/diagnóstico por imagem , Síndrome de Cimitarra/diagnóstico por imagem , Cor , Átrios do Coração/anormalidades , Comunicação Interatrial/fisiopatologia , Humanos , Recém-Nascido , Masculino , Veias Pulmonares/anormalidades , Síndrome de Cimitarra/fisiopatologiaRESUMO
Early stage prostate cancers are dependent on androgens for their growth and survival and androgen withdrawal causes them to regress. Progressive prostate cancers eventually acquire androgen independence rendering anti-androgen therapy ineffective. However, the factors leading to this have not been adequately addressed. This study shows that AIRE finds differential expression in androgen-dependent and -independent prostate cancer cells. AIRE expression is more in androgen-independent cells due to its regulation by transcription factor Elk-1. These enhanced levels of AIRE modulate the prostate tumor microenvironment by transcriptionally activating a malignancy gene IL-6 in androgen-independent cells. Additionally, AIRE prevents the cancer cells from anticancer drug-induced death and enhances their invasiveness. Moreover, AIRE by modulating the cytokine milieu skews the tumor-associated macrophage polarization towards M2 phenotype with increased CD206 and CD163 expression. Subcutaneous mouse model of prostate cancer revealed AIRE+/+ mice forming a palpable tumor and presents lymphadenopathy however, only a small benign tumor is observed in AIRE-/- mice and lymph nodes appear normal in size. In conclusion, our findings suggest AIRE as a probable factor in promoting prostate cancer progression.
RESUMO
Mycobacterium tuberculosis instigates interactions with host factors to promote its survival within the host inimical conditions. Among such factors, nuclear receptors (NRs) seem to be promising candidates owing to their role in bacterial pathogenesis. However, only few members of NR superfamily have been implicated in M. tuberculosis infection and there is a dearth of comprehensive knowledge about expression or function of the entire superfamily. In this study, we performed detailed expression analysis and identified key NRs getting differentially regulated in murine macrophages and dendritic cells (DC) upon infection with H37Rv. The murine macrophages and DCs infected with H37Rv entailed overlapping changes in the expression of certain NRs which reflect upon the possibility that both cells might utilize similar transcriptional programs upon M. tuberculosis infection. We identified Nr4a3 and Rora, which have not been implicated in M. tuberculosis pathogenesis, undergo similar changes in expression in macrophages and DCs upon H37Rv infection. Interestingly, a similar pattern in their expression was also observed in infected human monocyte derived macrophages and the findings corroborated well with PBMCs obtained from TB patients. This all-inclusive analysis provides the basis for a precise approach in identifying NRs that can be targeted therapeutically in intracellular bacterial infections.
Assuntos
Células Dendríticas/microbiologia , Macrófagos/microbiologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Receptores Citoplasmáticos e Nucleares/análise , Animais , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Camundongos Endogâmicos C57BL , Receptores Citoplasmáticos e Nucleares/genéticaRESUMO
Phospholipases Cγ (PLCγ) 1 and 2 are a class of highly homologous enzymes modulating a variety of cellular pathways through production of inositol 1,4,5-trisphosphate and diacylglycerol (DAG). Our previous studies demonstrated the importance of PLCγ2 in osteoclast (OC) differentiation by modulating inositol 1,4,5-trisphosphate-mediated calcium oscillations and the up-regulation of the transcription factor NFATc1. Surprisingly, despite being expressed throughout osteoclastogenesis, PLCγ1 did not compensate for PLCγ2 deficiency. Because both isoforms are activated during osteoclastogenesis, it is plausible that PLCγ1 modulates OC development independently of PLCγ2. Here, we utilized PLCγ1-specific shRNAs to delete PLCγ1 in OC precursors derived from wild type (WT) mice. Differently from PLCγ2, we found that PLCγ1 shRNA significantly suppresses OC differentiation by limiting colony-stimulating factor 1 (CSF-1)-dependent proliferation and ß-catenin/cyclinD1 levels. Confirming the specificity toward CSF-1 signaling, PLCγ1 is recruited to the CSF-1 receptor following exposure to the cytokine. To understand how PLCγ1 controls cell proliferation, we turned to its downstream effector, DAG. By utilizing cells lacking the DAG kinase ζ, which have increased DAG levels, we demonstrate that DAG modulates CSF-1-dependent proliferation and ß-catenin/cyclinD1 levels in OC precursors. Most importantly, the proliferation and osteoclastogenesis defects observed in the absence of PLCγ1 are normalized in PLCγ1/DAG kinase ζ double null cells. Taken together, our study shows that PLCγ1 controls OC numbers via a CSF-1-dependent DAG/ß-catenin/cyclinD1 pathway.
Assuntos
Ciclina D1/metabolismo , Diglicerídeos/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Osteoclastos/metabolismo , Fosfolipase C gama/metabolismo , Transdução de Sinais/fisiologia , beta Catenina/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Ciclina D1/genética , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Diglicerídeos/genética , Técnicas de Silenciamento de Genes , Fator Estimulador de Colônias de Macrófagos/farmacologia , Camundongos , Osteoclastos/citologia , Fosfolipase C gama/genética , Transdução de Sinais/efeitos dos fármacos , beta Catenina/genéticaRESUMO
Mycobacterium tuberculosis can evade host defense processes, thereby ensuring its survival and pathogenesis. In this study, we investigated the role of nuclear receptor, pregnane X receptor (PXR), in M. tuberculosis infection in human monocyte-derived macrophages. In this study, we demonstrate that PXR augments M. tuberculosis survival inside the host macrophages by promoting the foamy macrophage formation and abrogating phagolysosomal fusion, inflammation, and apoptosis. Additionally, M. tuberculosis cell wall lipids, particularly mycolic acids, crosstalk with human PXR (hPXR) by interacting with its promiscuous ligand binding domain. To confirm our in vitro findings and to avoid the reported species barrier in PXR function, we adopted an in vivo mouse model expressing hPXR, wherein expression of hPXR in mice promotes M. tuberculosis survival. Therefore, pharmacological intervention and designing antagonists to hPXR may prove to be a promising adjunct therapy for tuberculosis.
Assuntos
Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia , Receptores de Esteroides/metabolismo , Tuberculose/imunologia , Xenobióticos/metabolismo , Animais , Apoptose , Linhagem Celular , Sobrevivência Celular , Humanos , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fagossomos , Receptor de Pregnano X , Receptores de Esteroides/genética , Transgenes/genéticaRESUMO
The nuclear receptor (NR) superfamily of transcription factors regulates various key aspects of physiological processes; however, their role(s) in immune cells' function are just beginning to unravel. Although few NRs have been shown to be critical for dendritic cell (DC) function, a lack of knowledge about their complete representation in DCs has limited the ability to harness their full potential. Here, we performed a comprehensive NR expression profiling and identified the key members of NR superfamily being expressed in immature, immunogenic, and tolerogenic DCs. Comparative analysis revealed discrete changes in the expression of various NRs among the studied DC subtypes, indicating a likely role in the modulation of DC functionality. Next, we characterized Nr4a2, a member of orphan NR family, and found that it suppresses the activation of bone marrow derived dendritic cells triggered by LPS. Overexpression and knockdown of Nr4a2 demonstrated that Nr4a2 orchestrates the expression of immunoregulatory genes, hence inducing a tolerogenic phenotype in bone marrow derived dendritic cells. Furthermore, we also found that Nr4a2 provides protection from EAE by promoting an increase in Treg cells, while limiting effector T cells. Our findings suggest a previously unidentified role for Nr4a2 as a regulator of DC tolerogenicity and demonstrate its potential as therapeutic target in DC-associated pathophysiologies.
Assuntos
Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental/imunologia , Tolerância Imunológica , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Linfócitos T Reguladores/imunologia , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
Tumor-stroma interactions contribute to tumorigenesis. Tumor cells can educate the stroma at primary and distant sites to facilitate the recruitment of heterogeneous populations of immature myeloid cells, known as myeloid-derived suppressor cells (MDSCs). MDSCs suppress T cell responses and promote tumor proliferation. One outstanding question is how the local and distant stroma modulate MDSCs during tumor progression. Down-regulation of ß-catenin is critical for MDSC accumulation and immune suppressive functions in mice and humans. Here, we demonstrate that stroma-derived Dickkopf-1 (Dkk1) targets ß-catenin in MDSCs, thus exerting immune suppressive effects during tumor progression. Mice bearing extraskeletal tumors show significantly elevated levels of Dkk1 in bone microenvironment relative to tumor site. Strikingly, Dkk1 neutralization decreases tumor growth and MDSC numbers by rescuing ß-catenin in these cells and restores T cell recruitment at the tumor site. Recombinant Dkk1 suppresses ß-catenin target genes in MDSCs from mice and humans and anti-Dkk1 loses its antitumor effects in mice lacking ß-catenin in myeloid cells or after depletion of MDSCs, demonstrating that Dkk1 directly targets MDSCs. Furthermore, we find a correlation between CD15(+) myeloid cells and Dkk1 in pancreatic cancer patients. We establish a novel immunomodulatory role for Dkk1 in regulating tumor-induced immune suppression via targeting ß-catenin in MDSCs.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Células Mieloides/imunologia , Neoplasias Experimentais/imunologia , Neoplasias Pancreáticas/imunologia , Microambiente Tumoral/efeitos dos fármacos , Animais , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Camundongos , Camundongos Knockout , Células Mieloides/patologia , Neoplasias Experimentais/patologia , Neoplasias Pancreáticas/patologia , Linfócitos T/imunologia , Linfócitos T/patologia , Microambiente Tumoral/imunologia , beta Catenina/imunologiaRESUMO
Mycobacterium tuberculosis executes numerous defense strategies for the successful establishment of infection under a diverse array of challenges inside the host. One such strategy that has been delineated in this study is the abrogation of lytic activity of lysozyme by a novel glycosylated and surface-localized lipoprotein, LprI, which is exclusively present in M. tuberculosis complex. The lprI gene co-transcribes with the glbN gene (encoding hemoglobin (HbN)) and both are synchronously up-regulated in M. tuberculosis during macrophage infection. Recombinant LprI, expressed in Escherichia coli, exhibited strong binding (Kd ≤ 2 nm) with lysozyme and abrogated its lytic activity completely, thereby conferring protection to fluorescein-labeled Micrococcus lysodeikticus from lysozyme-mediated hydrolysis. Expression of the lprI gene in Mycobacterium smegmatis (8-10-fold) protected its growth from lysozyme inhibition in vitro and enhanced its phagocytosis and survival during intracellular infection of peritoneal and monocyte-derived macrophages, known to secrete lysozyme, and in the presence of exogenously added lysozyme in secondary cell lines where lysozyme levels are low. In contrast, the presence of HbN enhanced phagocytosis and intracellular survival of M. smegmatis only in the absence of lysozyme but not under lysozyme stress. Interestingly, co-expression of the glbN-lprI gene pair elevated the invasion and survival of M. smegmatis 2-3-fold in secondary cell lines in the presence of lysozyme in comparison with isogenic cells expressing these genes individually. Thus, specific advantage against macrophage-generated lysozyme, conferred by the combination of LprI-HbN during invasion of M. tuberculosis, may have vital implications on the pathogenesis of tuberculosis.
Assuntos
Proteínas de Bactérias/biossíntese , Inibidores Enzimáticos/metabolismo , Lipoproteínas/biossíntese , Macrófagos/microbiologia , Muramidase/antagonistas & inibidores , Mycobacterium tuberculosis/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Linhagem Celular , Inibidores Enzimáticos/química , Lipoproteínas/química , Lipoproteínas/genética , Macrófagos/química , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Macrophages and dendritic cells provide critical effector functions to efficiently resist and promptly eliminate infection. Pattern recognition receptors signaling operative in these cell types is imperative for their innate properties. However, it is now emerging that besides these conventional signaling pathways, nuclear receptors coupled gene regulation and transrepression pathways assemble immune regulatory networks. A couple of these networks associated with members of nuclear receptor superfamily decide heterogeneity in macrophages and dendritic cells population and thereby play decisive role in determining protective immunity against bacteria, viruses, fungi, protozoa and helminths. Pathogens also direct shift in the expression of nuclear receptors and their target genes and this is proclaimed to be a sui generis mechanism whereby microbes disconnect the genomic component from the peripheral immune response. Many endogenous and synthetic nuclear receptor ligands have been tested in various in vitro and in vivo infection models to study their effect on pathogen burden. Here, we discuss current advances in our understanding of the composite interactions between nuclear receptor and pathogens and their implications on the causatum infectious diseases.
Assuntos
Doenças Transmissíveis/imunologia , Células Dendríticas/imunologia , Redes Reguladoras de Genes , Interações Hospedeiro-Patógeno , Macrófagos/imunologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Evasão da Resposta ImuneRESUMO
Orphan nuclear receptors are potential therapeutic targets. The Orphan Nuclear Receptor Ligand Binding Database (ONRLDB) is an interactive, comprehensive and manually curated database of small molecule ligands targeting orphan nuclear receptors. Currently, ONRLDB consists of â¼11,000 ligands, of which â¼6500 are unique. All entries include information for the ligand, such as EC50 and IC50, number of aromatic rings and rotatable bonds, XlogP, hydrogen donor and acceptor count, molecular weight (MW) and structure. ONRLDB is a cross-platform database, where either the cognate small molecule modulators of a receptor or the cognate receptors to a ligand can be searched. The database can be searched using three methods: text search, advanced search or similarity search. Substructure search, cataloguing tools, and clustering tools can be used to perform advanced analysis of the ligand based on chemical similarity fingerprints, hierarchical clustering, binning partition and multidimensional scaling. These tools, together with the Tree function provided, deliver an interactive platform and a comprehensive resource for identification of common and unique scaffolds. As demonstrated, ONRLDB is designed to allow selection of ligands based on various properties and for designing novel ligands or to improve the existing ones. Database URL: http://www.onrldb.org/.
Assuntos
Bases de Dados de Proteínas , Descoberta de Drogas , Receptores Nucleares Órfãos/metabolismo , Análise por Conglomerados , Mineração de Dados , Inflamação/metabolismo , Armazenamento e Recuperação da Informação , Internet , Ligantes , Receptores X do Fígado , PPAR gama/metabolismo , Xenobióticos/química , Xenobióticos/metabolismoRESUMO
The orphan nuclear receptor Nr4a2 is known to modulate both inflammatory and metabolic processes, but the mechanism by which it regulates innate inflammatory homeostasis has not been adequately addressed. This study shows that exposure to ligands for Toll-like receptors (TLRs) robustly induces Nr4a2 and that this induction is tightly regulated by the PI3K-Akt signaling axis. Interestingly, exogenous expression of Nr4a2 in macrophages leads to their alternative phenotype with induction of genes that are prototypical M2 markers. Moreover, Nr4a2 transcriptionally activates arginase 1 expression by directly binding to its promoter. Adoptive transfer experiments revealed that increased survival of animals in endotoxin-induced sepsis is Nr4a2-dependent. Thus our data identify a previously unknown role for Nr4a2 in the regulation of macrophage polarization.
Assuntos
Inflamação/genética , Macrófagos/metabolismo , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Sepse/genética , Animais , Polaridade Celular/genética , Regulação da Expressão Gênica , Humanos , Inflamação/metabolismo , Inflamação/patologia , Ligantes , Lipopolissacarídeos/toxicidade , Macrófagos/patologia , Camundongos , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/biossíntese , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sepse/induzido quimicamente , Sepse/metabolismo , Sepse/patologia , Transdução de Sinais/genética , Receptores Toll-Like/metabolismoRESUMO
BACKGROUND: Complexes of oleic acid (OA) with milk α-lactalbumin, received remarkable attention in view of their selective toxicity towards a spectrum of tumors during the last two decades. OA complexes of some structurally related/unrelated proteins are also tumoricidal. Erythrocytes are among the few differentiated cells that are sensitive and undergo hemolysis when exposed to the complexes. METHODS: The effects of OA complex of bovine α-lactalbumin (Bovine Alpha-lactalbumin Made LEthal to Tumor cells, BAMLET) on human, goat and chicken erythrocytes on calcein leakage, phosphatidylserine exposure, morphological changes and hemolysis were studied by confocal microscopy, FACS analysis, scanning electron microscopy and measuring hemoglobin release. RESULTS: Erythrocytes exposed to BAMLET undergo eryptosis-like alterations as revealed by calcein leakage, surface phosphatidylserine exposure and transformation to echinocytes at low concentrations and hemolysis when the concentration of the complex was raised. Ca(2+) was not essential and restricted the alterations when included in the medium. The BAMLET-induced alterations in human erythrocytes were prevented by the cation channel inhibitors, amiloride and BaCl2 but not by inhibitors of thiol proteases, sphingomyelinase and by the antioxidant N-acetyl cysteine. CONCLUSIONS: The work shows for the first time that low concentrations of BAMLET induces eryptosis in erythrocytes by a novel mechanism not requiring Ca(2+) and hemolysis by detergent-like action by the released OA at higher concentrations. GENERAL SIGNIFICANCE: The study points out to the need for a comprehensive evaluation of the toxicity of OA complexes of α-lactalbumin and other proteins towards erythrocytes and other differentiated cells before being considered for therapy.
Assuntos
Cálcio/farmacologia , Eritrócitos/efeitos dos fármacos , Lactalbumina/farmacologia , Ácidos Oleicos/farmacologia , Amilorida/farmacologia , Animais , Bovinos , Galinhas , Cabras , Hemólise/efeitos dos fármacos , HumanosRESUMO
The ability of the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), to transcriptionally modulate Smads to inhibit Th17 differentiation and experimental autoimmune encephalomyelitis (EAE) has not been adequately studied. This study reports modulation of Smad signaling by the specific binding of the VDR along with its heterodimeric partner RXR to the negative vitamin D response element on the promoter of Smad7, which leads to Smad7 gene repression. The vitamin D receptor-mediated increase in Smad3 expression partially explains the IL10 augmentation seen in Th17 cells. Furthermore, the VDR axis also modulates non-Smad signaling by activating ERK during differentiation of Th17 cells, which inhibits the Th17-specific genes il17a, il17f, il22, and il23r. In vivo EAE experiments revealed that, 1,25(OH)2D3 suppression of EAE correlates with the Smad7 expression in the spleen and lymph nodes. Furthermore, Smad7 expression also correlates well with IL17 and IFNγ expression in CNS infiltered inflammatory T cells. We also observed similar gene repression of Smad7 in in vitro differentiated Th1 cells when cultured in presence of 1,25(OH)2D3. The above canonical and non-canonical pathways in part address the ability of 1,25(OH)2D3-VDR to inhibit EAE.
Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Enzimológica da Expressão Gênica , Proteína Smad7/antagonistas & inibidores , Linfócitos T Auxiliares-Indutores/citologia , Vitamina D/análogos & derivados , Animais , Células COS , Diferenciação Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Chlorocebus aethiops , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Interferon gama/metabolismo , Linfonodos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Fosforilação , Regiões Promotoras Genéticas , Elementos de Resposta , Proteína Smad7/fisiologia , Células Th17 , Transcrição Gênica , Vitamina D/químicaRESUMO
The cell wall of Mycobacterium tuberculosis is configured of bioactive lipid classes that are essential for virulence and potentially involved in the formation of foamy macrophages (FMs) and granulomas. Our recent work established crosstalk between M. tuberculosis cell wall lipids and the host lipid-sensing nuclear receptor TR4. In this study, we have characterized, identified, and adopted a heterologous ligand keto-mycolic acid from among M. tuberculosis lipid repertoire for the host orphan NR TR4. Crosstalk between cell wall lipids and TR4 was analyzed by transactivation and promoter reporter assays. Mycolic acid (MA) was found to transactivate TR4 significantly compared with other cell wall lipids. Among the MA, the oxygenated form, keto-MA, was responsible for transactivation, and the identity was validated by TR4 binding assays followed by TLC and nuclear magnetic resonance. Isothermal titration calorimetry revealed that keto-MA binding to TR4 is energetically favorable. This keto-MA-TR4 axis seems to be essential to this oxygenated MA induction of FMs and granuloma formation as evaluated by in vitro and in vivo model of granuloma formation. TR4 binding with keto-MA features a unique association of host nuclear receptor with a bacterial lipid and adds to the presently known ligand repertoire beyond dietary lipids. Pharmacologic modulation of this heterologous axis may hold promise as an adjunct therapy to frontline tuberculosis drugs.