RESUMO
The cellular cortex provides crucial mechanical support and plays critical roles during cell division and migration. The proteins of the ERM family, comprised of ezrin, radixin, and moesin, are central to these processes by linking the plasma membrane to the actin cytoskeleton. To investigate the contributions of the ERM proteins to leukocyte migration, we generated single and triple ERM knockout macrophages. Surprisingly, we found that even in the absence of ERM proteins, macrophages still form the different actin structures promoting cell migration, such as filopodia, lamellipodia, podosomes, and ruffles. Furthermore, we discovered that, unlike every other cell type previously investigated, the single or triple knockout of ERM proteins does not affect macrophage migration in diverse contexts. Finally, we demonstrated that the loss of ERMs in macrophages does not affect the mechanical properties of their cortex. These findings challenge the notion that ERMs are universally essential for cortex mechanics and cell migration and support the notion that the macrophage cortex may have diverged from that of other cells to allow for their uniquely adaptive cortical plasticity.
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Osteoclasts are multinucleated cells unique in their ability to resorb bone. Osteoclastogenesis involves several steps of actin-driven rearrangements that participate not only in the cell-cell fusion process, but also in the formation of the sealing zone, the adhesive structure determining the resorption area. Despite the importance of these actin cytoskeleton-based processes, their precise mechanisms of regulation are still poorly characterized. Here, we found that moesin, a member of the Ezrin/Radixin/Moesin (ERM) protein family, is activated during osteoclast maturation and plays an instrumental role for both osteoclast fusion and function. In mouse and human osteoclast precursors, moesin is negatively regulated to potentiate their ability to fuse and degrade bone. Accordingly, we demonstrated that moesin depletion decreases membrane-to-cortex attachment and enhances formation of tunneling nanotubes (TNTs), F-actin-containing intercellular bridges that we revealed to trigger osteoclast fusion. In addition, via a ß3-integrin/RhoA/SLK pathway and independently of its role in fusion, moesin regulates the number and organization of sealing zones in mature osteoclast, and thus participates in the control of bone resorption. Supporting these findings, we found that moesin-deficient mice are osteopenic with a reduced density of trabecular bones and increased osteoclast abundance and activity. These findings provide a better understanding of the regulation of osteoclast biology, and open new opportunities to specifically target osteoclast activity in bone disease therapy.
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Macrophages are essential for HIV-1 pathogenesis and represent major viral reservoirs. Therefore, it is critical to understand macrophage infection, especially in tissue macrophages, which are widely infected in vivo, but poorly permissive to cell-free infection. Although cell-to-cell transmission of HIV-1 is a determinant mode of macrophage infection in vivo, how HIV-1 transfers toward macrophages remains elusive. Here, we demonstrate that fusion of infected CD4+ T lymphocytes with human macrophages leads to their efficient and productive infection. Importantly, several tissue macrophage populations undergo this heterotypic cell fusion, including synovial, placental, lung alveolar, and tonsil macrophages. We also find that this mode of infection is modulated by the macrophage polarization state. This fusion process engages a specific short-lived adhesion structure and is controlled by the CD81 tetraspanin, which activates RhoA/ROCK-dependent actomyosin contractility in macrophages. Our study provides important insights into the mechanisms underlying infection of tissue-resident macrophages, and establishment of persistent cellular reservoirs in patients.
Assuntos
Linfócitos T CD4-Positivos , Fusão Celular , Infecções por HIV , Macrófagos , Humanos , Linfócitos T CD4-Positivos/metabolismo , Infecções por HIV/metabolismo , HIV-1/patogenicidade , Macrófagos/metabolismo , Macrófagos/virologia , Actomiosina/metabolismoRESUMO
The lungs harbor multiple resident microbial communities, otherwise known as the microbiota. There is an emerging interest in deciphering whether the pulmonary microbiota modulate local immunity, and whether this knowledge could shed light on mechanisms operating in the response to respiratory pathogens. In this study, we investigate the capacity of a pulmonary Lactobacillus strain to modulate the lung T cell compartment and assess its prophylactic potential upon infection with Mycobacterium tuberculosis, the etiological agent of tuberculosis. In naive mice, we report that a Lactobacillus murinus (Lagilactobacillus murinus) strain (CNCM I-5314) increases the presence of lung Th17 cells and of a regulatory T cell (Treg) subset known as RORγt+ Tregs. In particular, intranasal but not intragastric administration of CNCM I-5314 increases the expansion of these lung leukocytes, suggesting a local rather than systemic effect. Resident Th17 and RORγt+ Tregs display an immunosuppressive phenotype that is accentuated by CNCM I-5314. Despite the well-known ability of M. tuberculosis to modulate lung immunity, the immunomodulatory effect by CNCM I-5314 is dominant, as Th17 and RORγt+ Tregs are still highly increased in the lung at 42-d postinfection. Importantly, CNCM I-5314 administration in M. tuberculosis-infected mice results in reduction of pulmonary inflammation, without increasing M. tuberculosis burden. Collectively, our findings provide evidence for an immunomodulatory capacity of CNCM I-5314 at steady state and in a model of chronic inflammation in which it can display a protective role, suggesting that L. murinus strains found in the lung may shape local T cells in mice and, perhaps, in humans.
Assuntos
Lactobacillus/fisiologia , Pulmão/imunologia , Mycobacterium tuberculosis/fisiologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Tuberculose Pulmonar/imunologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Humanos , Pulmão/microbiologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , PneumoniaRESUMO
Regulated cell necrosis supports immune and anti-infectious strategies of the body; however, dysregulation of these processes drives pathological organ damage. Pseudomonas aeruginosa expresses a phospholipase, ExoU that triggers pathological host cell necrosis through a poorly characterized pathway. Here, we investigated the molecular and cellular mechanisms of ExoU-mediated necrosis. We show that cellular peroxidised phospholipids enhance ExoU phospholipase activity, which drives necrosis of immune and non-immune cells. Conversely, both the endogenous lipid peroxidation regulator GPX4 and the pharmacological inhibition of lipid peroxidation delay ExoU-dependent cell necrosis and improve bacterial elimination in vitro and in vivo. Our findings also pertain to the ExoU-related phospholipase from the bacterial pathogen Burkholderia thailandensis, suggesting that exploitation of peroxidised phospholipids might be a conserved virulence mechanism among various microbial phospholipases. Overall, our results identify an original lipid peroxidation-based virulence mechanism as a strong contributor of microbial phospholipase-driven pathology.
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Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Peroxidação de Lipídeos/fisiologia , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/patogenicidade , Animais , Humanos , Camundongos , Camundongos Knockout , Necrose/metabolismo , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/metabolismo , Virulência/fisiologiaRESUMO
Mycobacterium tuberculosis (Mtb) regulates the macrophage metabolic state to thrive in the host, yet the responsible mechanisms remain elusive. Macrophage activation toward the microbicidal (M1) program depends on the HIF-1α-mediated metabolic shift from oxidative phosphorylation (OXPHOS) toward glycolysis. Here, we ask whether a tuberculosis (TB) microenvironment changes the M1 macrophage metabolic state. We expose M1 macrophages to the acellular fraction of tuberculous pleural effusions (TB-PEs) and find lower glycolytic activity, accompanied by elevated levels of OXPHOS and bacillary load, compared to controls. The eicosanoid fraction of TB-PE drives these metabolic alterations. HIF-1α stabilization reverts the effect of TB-PE by restoring M1 metabolism. Furthermore, Mtb-infected mice with stabilized HIF-1α display lower bacillary loads and a pronounced M1-like metabolic profile in alveolar macrophages (AMs). Collectively, we demonstrate that lipids from a TB-associated microenvironment alter the M1 macrophage metabolic reprogramming by hampering HIF-1α functions, thereby impairing control of Mtb infection.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lipídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Mycobacterium tuberculosis/metabolismo , Tuberculose Pleural/metabolismo , Animais , Carga Bacteriana , Eicosanoides/farmacologia , Feminino , Glicólise/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Humanos , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Derrame Pleural , Tuberculose Pleural/microbiologiaRESUMO
The escape of cancer cells from host immunosurveillance involves a shift in immune responses, including an imbalance in Th1 and Th2 cells. A Th1-dominated immune response predicts positive outcomes in colorectal cancer. The E3 ubiquitin ligase, Asb2α, is expressed in Th2 cells, but its roles in T-cell maturation and cancer are unclear. We provide evidence that the Th2 master regulator, Gata3, induces Asb2 Loss of Asb2 did not affect Th differentiation ex vivo, but reduced IL4 production from Th2 cells. We found that high ASB2 expression was associated with poor outcome in colorectal cancer. Loss of Asb2 from hematopoietic cells promoted a Th1 response and attenuated colitis-associated tumorigenesis in mice. Diminished Th2 function correlated with increased IFNγ production and an enhanced type 1 antitumor immune response in Asb2-deficient mice. Our work suggests that Asb2α promotes a Th2 phenotype in vivo, which in turn is associated with tumor progression in a mouse model of colitis.
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Neoplasias Colorretais/imunologia , Neoplasias Colorretais/metabolismo , Imunomodulação , Células Th2/imunologia , Células Th2/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Sítios de Ligação , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Biologia Computacional/métodos , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunofenotipagem , Camundongos , Ligação Proteica , Recidiva , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
RATIONALE: Heart development involves differentiation of cardiac progenitors and assembly of the contractile sarcomere apparatus of cardiomyocytes. However, little is known about the mechanisms that regulate actin cytoskeleton remodeling during cardiac cell differentiation. OBJECTIVE: The Asb2α (Ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2) CRL5 (cullin 5 RING E3 ubiquitin ligase) triggers polyubiquitylation and subsequent degradation by the proteasome of FLNs (filamins). Here, we investigate the role of Asb2α in heart development and its mechanisms of action. METHODS AND RESULTS: Using Asb2 knockout embryos, we show that Asb2 is an essential gene, critical to heart morphogenesis and function, although its loss does not interfere with the overall patterning of the embryonic heart tube. We show that the Asb2α E3 ubiquitin ligase controls Flna stability in immature cardiomyocytes. Importantly, Asb2α-mediated degradation of the actin-binding protein Flna marks a previously unrecognized intermediate step in cardiac cell differentiation characterized by cell shape changes and actin cytoskeleton remodeling. We further establish that in the absence of Asb2α, myofibrils are disorganized and that heartbeats are inefficient, leading to embryonic lethality in mice. CONCLUSIONS: These findings identify Asb2α as an unsuspected key regulator of cardiac cell differentiation and shed light on the molecular and cellular mechanisms determining the onset of myocardial cell architecture and its link with early cardiac function. Although Flna is known to play roles in cytoskeleton organization and to be required for heart function, this study now reveals that its degradation mediated by Asb2α ensures essential functions in differentiating cardiac progenitors.
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Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Filaminas/metabolismo , Coração/crescimento & desenvolvimento , Miócitos Cardíacos/metabolismo , Ubiquitinação , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Diferenciação Celular , Células Cultivadas , Filaminas/genética , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/citologia , Proteólise , Proteínas Supressoras da Sinalização de CitocinaRESUMO
Conventional dendritic cells (cDCs) comprise distinct populations with specialized immune functions that are mediators of innate and adaptive immune responses. Transcriptomic and proteomic approaches have been used so far to identify transcripts and proteins that are differentially expressed in these subsets to understand the respective functions of cDCs subsets. Here, we showed that the Cullin 5-RING E3 ubiquitin ligase (E3) ASB2α, by driving degradation of filamin A (FLNa) and filamin B (FLNb), is responsible for the difference in FLNa and FLNb abundance in the different spleen cDC subsets. Importantly, the ability of these cDC subsets to migrate correlates with the level of FLNa. Furthermore, our results strongly point to CD4 positive and double negative cDCs as distinct populations. Finally, we develop quantitative global proteomic approaches to identify ASB2α substrates in DCs using ASB2 conditional knockout mice. As component of the ubiquitin-proteasome system (UPS) are amenable to pharmacological manipulation, these approaches aimed to the identification of E3 substrates in physiological relevant settings could potentially lead to novel targets for therapeutic strategies.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Dendríticas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular Tumoral , Filaminas/metabolismo , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteômica/métodos , Proteínas Supressoras da Sinalização de Citocina , Ubiquitina/metabolismoRESUMO
The actin-binding protein filamins (FLNs) are major organizers of the actin cytoskeleton. They control the elasticity and stiffness of the actin network and provide connections with the extracellular microenvironment by anchoring transmembrane receptors to the actin filaments. Although numerous studies have revealed the importance of FLN levels, relatively little is known about the regulation of its stability in physiological relevant settings. Here, we show that the ASB2α cullin 5-ring E3 ubiquitin ligase is highly expressed in immature dendritic cells (DCs) and is down-regulated after DC maturation. We further demonstrate that FLNs are substrates of ASB2α in immature DCs and therefore are not stably expressed in these cells, whereas they exhibit high levels of expression in mature DCs. Using ASB2 conditional knockout mice, we show that ASB2α is a critical regulator of cell spreading and podosome rosette formation in immature DCs. Furthermore, we show that ASB2(-/-) immature DCs exhibit reduced matrix-degrading function leading to defective migration. Altogether, our results point to ASB2α and FLNs as newcomers in DC biology.
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Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Movimento Celular/genética , Células Dendríticas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Contráteis/genética , Proteínas Contráteis/metabolismo , Células Dendríticas/metabolismo , Filaminas , Técnicas de Silenciamento de Genes , Células Progenitoras de Granulócitos e Macrófagos/metabolismo , Células Progenitoras de Granulócitos e Macrófagos/fisiologia , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Células NIH 3T3 , Proteínas Supressoras da Sinalização de Citocina , Transfecção , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/fisiologiaRESUMO
Closely related Escherichia coli B2 strains O1:K1, O2:K1, O18:K1, and O45:K1 constitute a major subgroup causing extraintestinal infections. A DNA pathoarray analysis was used to develop a PCR specific for this subgroup that was included in the multiplex phylogenetic-grouping PCR method. Our PCR may serve to identify this virulent subgroup among different ecological niches.
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Escherichia coli/classificação , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Ribotipagem , VirulênciaRESUMO
Extraintestinal pathogenic Escherichia coli (ExPEC) strains cause a large spectrum of infections. The majority of ExPEC strains are closely related to the B2 or the D phylogenetic group. The aim of our study was to develop a protein-based vaccine against these ExPEC strains. To this end, we identified ExPEC-specific genomic regions, using a comparative genome analysis, between the nonpathogenic E. coli strain K-12 MG1655 and ExPEC strains C5 (meningitis isolate) and CFT073 (urinary tract infection isolate). The analysis of these genomic regions allowed the selection of 40 open reading frames, which are conserved among B2/D clinical isolates and encode proteins with putative outer membrane localization. These genes were cloned, and recombinant proteins were purified and assessed as vaccine candidates. After immunization of BALB/c mice, five proteins induced a significant protective immunity against a lethal challenge with a clinical E. coli strain of the B2 group. In passive immunization assays, antigen-specific antibodies afforded protection to naive mice against a lethal challenge. Three of these antigens were related to iron acquisition metabolism, an important virulence factor of the ExPEC, and two corresponded to new, uncharacterized proteins. Due to the large number of genetic differences that exists between commensal and pathogenic strains of E. coli, our results demonstrate that it is possible to identify targets that elicit protective immune responses specific to those strains. The five protective antigens could constitute the basis for a preventive subunit vaccine against diseases caused by ExPEC strains.
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Infecções por Escherichia coli/prevenção & controle , Proteínas de Escherichia coli/imunologia , Vacinas contra Escherichia coli/imunologia , Escherichia coli/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Transporte Biológico , Modelos Animais de Doenças , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Feminino , Genoma Bacteriano , Humanos , Imunização Passiva , Camundongos , Camundongos Endogâmicos BALB C , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Fatores de Virulência/genética , Fatores de Virulência/imunologiaRESUMO
The biologic role of a majority of the Neisseria meningitidis 2100 predicted coding regions is still to be assigned or experimentally confirmed. Determining the phenotypic effect of gene disruption being a fundamental approach to understanding gene function, we used high-density signature-tagged transposon mutagenesis, followed by a large-scale sequencing of the transposon insertion sites, to construct a genome-wide collection of mutants. The sequencing results for the first half of the 4548 mutants composing the library suggested that we have mutations in 80%-90% of N. meningitidis nonessential genes. This was confirmed by a whole-genome identification of the genes required for resistance to complement-mediated lysis, a key to meningococcal virulence. We show that all the genes we identified, including four previously uncharacterized, were important for the synthesis of the polysialic acid capsule or the lipooligosaccharide (LOS), suggesting that these are likely to be the only meningococcal attributes necessary for serum resistance. Our work provides a valuable and lasting resource that may lead to a global map of gene function in N. meningitidis.