RESUMEN
The spaceflight environment imposes risks for maintaining a healthy skin function as the observed delayed wound healing can contribute to increased risks of infection. To counteract delayed wound healing in space, a better understanding of the fibroblasts' reaction to altered gravity levels is needed. In this paper, we describe experiments that were carried out at the Large Diameter Centrifuge located in ESA-ESTEC as part of the ESA Academy 2021 Spin Your Thesis! Campaign. We exposed dermal fibroblasts to a set of altered gravity levels, including transitions between simulated microgravity and hypergravity. The addition of the stress hormone cortisol to the cell culture medium was done to account for possible interaction effects of gravity and cortisol exposure. Results show a main impact of cortisol on the secretion of pro-inflammatory cytokines as well as extracellular matrix proteins. Altered gravity mostly induced a delay in cellular migration and changes in mechanosensitive cell structures. Furthermore, 20 × g hypergravity transitions induced changes in nuclear morphology. These findings provide insights into the effect of gravity transitions on the fibroblasts' function related to wound healing, which may be useful for the development of countermeasures.
RESUMEN
Antibiotic resistance poses rapidly increasing global problems in combatting multidrug-resistant (MDR) infectious diseases like MDR tuberculosis, prompting for novel approaches including host-directed therapies (HDT). Intracellular pathogens like Salmonellae and Mycobacterium tuberculosis (Mtb) exploit host pathways to survive. Only very few HDT compounds targeting host pathways are currently known. In a library of pharmacologically active compounds (LOPAC)-based drug-repurposing screen, we identify multiple compounds, which target receptor tyrosine kinases (RTKs) and inhibit intracellular Mtb and Salmonellae more potently than currently known HDT compounds. By developing a data-driven in silico model based on confirmed targets from public databases, we successfully predict additional efficacious HDT compounds. These compounds target host RTK signaling and inhibit intracellular (MDR) Mtb. A complementary human kinome siRNA screen independently confirms the role of RTK signaling and kinases (BLK, ABL1, and NTRK1) in host control of Mtb. These approaches validate RTK signaling as a drugable host pathway for HDT against intracellular bacteria.
Asunto(s)
Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Infecciones por Salmonella/enzimología , Salmonella typhimurium/efectos de los fármacos , Tuberculosis/enzimología , Línea Celular , Biología Computacional , Farmacorresistencia Bacteriana , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/fisiología , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Infecciones por Salmonella/genética , Infecciones por Salmonella/microbiología , Salmonella typhimurium/genética , Salmonella typhimurium/fisiología , Transducción de Señal/efectos de los fármacos , Tuberculosis/genética , Tuberculosis/microbiologíaRESUMEN
We have generated unique asymmetric liposomes with phosphatidylserine (PS) distributed at the outer membrane surface to resemble apoptotic bodies and phosphatidic acid (PA) at the inner layer as a strategy to enhance innate antimycobacterial activity in phagocytes while limiting the inflammatory response. Results show that these apoptotic body-like liposomes carrying PA (ABL/PA) (i) are more efficiently internalized by human macrophages than by nonprofessional phagocytes, (ii) induce cytosolic Ca(2+) influx, (iii) promote Ca(2+)-dependent maturation of phagolysosomes containing Mycobacterium tuberculosis (MTB), (iv) induce Ca(2+)-dependent reactive oxygen species (ROS) production, (v) inhibit intracellular mycobacterial growth in differentiated THP-1 cells as well as in type-1 and -2 human macrophages, and (vi) down-regulate tumor necrosis factor (TNF)-α, interleukin (IL)-12, IL-1ß, IL-18, and IL-23 and up-regulate transforming growth factor (TGF)-ß without altering IL-10, IL-27, and IL-6 mRNA expression. Also, ABL/PA promoted intracellular killing of M. tuberculosis in bronchoalveolar lavage cells from patients with active pulmonary tuberculosis. Furthermore, the treatment of MTB-infected mice with ABL/PA, in combination or not with isoniazid (INH), dramatically reduced lung and, to a lesser extent, liver and spleen mycobacterial loads, with a concomitant 10-fold reduction of serum TNF-α, IL-1ß, and IFN-γ compared with that in untreated mice. Altogether, these results suggest that apoptotic body-like liposomes may be used as a Janus-faced immunotherapeutic platform to deliver polar secondary lipid messengers, such as PA, into phagocytes to improve and recover phagolysosome biogenesis and pathogen killing while limiting the inflammatory response.
Asunto(s)
Liposomas/farmacología , Macrófagos/inmunología , Macrófagos/microbiología , Mycobacterium tuberculosis/inmunología , Tuberculosis Pulmonar/tratamiento farmacológico , Tuberculosis Pulmonar/inmunología , Adulto , Animales , Antituberculosos/farmacología , Apoptosis/inmunología , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Calcio/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Inmunidad Innata/inmunología , Isoniazida/farmacología , Leucemia Monocítica Aguda , Liposomas/inmunología , Liposomas/metabolismo , Macrófagos/citología , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Fagocitosis/inmunología , Fosfatidilserinas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
α(1)-Antitrypsin (AAT) acts as an important neutrophil elastase inhibitor in the lung. Although the hepatocyte is considered to be the primary source of AAT, local production by monocytes, macrophages, and epithelial cells may contribute to the formation of an antielastase screen. Because monocytes can differentiate into a heterogeneous population of macrophages with subpopulations ranging from proinflammatory properties (MΦ-1) to antiinflammatory properties (ΜΦ-2) and into dendritic cells (DCs), we studied whether LPS, TNF-α, and oncostatin M (OSM) enhance AAT production differentially in cultured ΜΦ-1, ΜΦ-2, and DCs. Monocytes from healthy blood donors were cultured for 7 days in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor, or GM-CSF with IL-4 to obtain ΜΦ-1, ΜΦ-2, and immature (i)DCs, respectively. Cells were stimulated with LPS, TNF-α, or OSM, and AAT synthesis was assessed by quantitative RT-PCR, immunocytochemistry, and ELISA. Spontaneous release of AAT was higher in ΜΦ-1 than in ΜΦ-2 and iDCs, and only LPS significantly increased AAT production in ΜΦ-1, ΜΦ-2, and DC. TNF-α and OSM did not affect AAT secretion. The secretion levels of the related protease inhibitors α-1 antichymotrypsin and secretory leukocyte proteinase inhibitor were below the limits of detection by ELISA. In contrast to the protein data, analysis by quantitative RT-PCR showed that 24-hour LPS exposure caused a maximal 2.1-fold AAT mRNA increase in ΜΦ-1, a 21-fold increase in ΜΦ-2, and an 11-fold increase in DCs. These data suggest that cellular differentiation is a regulator of local AAT production.
Asunto(s)
Células Dendríticas/metabolismo , Macrófagos/metabolismo , alfa 1-Antitripsina/biosíntesis , Líquido del Lavado Bronquioalveolar , Células Cultivadas , Células Dendríticas/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Inmunohistoquímica , Macrófagos/efectos de los fármacos , Reacción en Cadena de la PolimerasaRESUMEN
During infection of humans and animals, pathogenic mycobacteria manipulate the host cell causing severe diseases such as tuberculosis and leprosy. To understand the basis of mycobacterial pathogenicity, it is crucial to identify the molecular virulence mechanisms. In this study, we address the contribution of ESX-1 and ESX-5--two homologous type VII secretion systems of mycobacteria that secrete distinct sets of immune modulators--during the macrophage infection cycle. Using wild-type, ESX-1- and ESX-5-deficient mycobacterial strains, we demonstrate that these secretion systems differentially affect subcellular localization and macrophage cell responses. We show that in contrast to ESX-1, the effector proteins secreted by ESX-5 are not required for the translocation of Mycobacterium tuberculosis or Mycobacterium marinum to the cytosol of host cells. However, the M. marinum ESX-5 mutant does not induce inflammasome activation and IL-1ß activation. The ESX-5 system also induces a caspase-independent cell death after translocation has taken place. Importantly, by means of inhibitory agents and small interfering RNA experiments, we reveal that cathepsin B is involved in both the induction of cell death and inflammasome activation upon infection with wild-type mycobacteria. These results reveal distinct roles for two different type VII secretion systems during infection and shed light on how virulent mycobacteria manipulate the host cell in various ways to replicate and spread.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Inflamasomas/inmunología , Inflamasomas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Mycobacterium marinum/inmunología , Mycobacterium tuberculosis/inmunología , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Animales , Muerte Celular/inmunología , Línea Celular , Línea Celular Tumoral , Humanos , Inflamación/inmunología , Inflamación/microbiología , Inflamación/patología , Interleucina-1beta/metabolismo , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/microbiología , Ratones , Mycobacterium marinum/patogenicidad , Mycobacterium tuberculosis/patogenicidadRESUMEN
One-third of the world population is infected with Mycobacterium tuberculosis and multi-drug resistant strains are rapidly evolving. The noticeable absence of a whole organism high-throughput screening system for studying the progression of tuberculosis is fast becoming the bottleneck in tuberculosis research. We successfully developed such a system using the zebrafish Mycobacterium marinum infection model, which is a well-characterized model for tuberculosis progression with biomedical significance, mimicking hallmarks of human tuberculosis pathology. Importantly, we demonstrate the suitability of our system to directly study M. tuberculosis, showing for the first time that the human pathogen can propagate in this vertebrate model, resulting in similar early disease symptoms to those observed upon M. marinum infection. Our system is capable of screening for disease progression via robotic yolk injection of early embryos and visual flow screening of late-stage larvae. We also show that this system can reliably recapitulate the standard caudal vein injection method with a throughput level of 2,000 embryos per hour. We additionally demonstrate the possibility of studying signal transduction leading to disease progression using reverse genetics at high-throughput levels. Importantly, we use reference compounds to validate our system in the testing of molecules that prevent tuberculosis progression, making it highly suited for investigating novel anti-tuberculosis compounds in vivo.
Asunto(s)
Biomarcadores/análisis , Ensayos Analíticos de Alto Rendimiento/métodos , Tuberculosis/diagnóstico , Animales , Antituberculosos/aislamiento & purificación , Antituberculosos/uso terapéutico , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Evaluación Preclínica de Medicamentos/métodos , Embrión no Mamífero , Humanos , Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Infecciones por Mycobacterium no Tuberculosas/patología , Mycobacterium marinum/fisiología , Pronóstico , Tuberculosis/tratamiento farmacológico , Tuberculosis/patología , Pez Cebra/embriología , Pez Cebra/crecimiento & desarrollo , Pez Cebra/fisiologíaRESUMEN
Patients with complete IFN-γR deficiency are unable to respond to IFN-γ and have impaired Th1-immunity and recurrent, severe infections with weakly virulent Mycobacteria. Since IFN-α and IFN-γ share signalling pathways, treatment with IFN-α has been proposed in complete IFN-γR deficiency. We stimulated cells from healthy controls and from a patient lacking IFN-γR1 with IFN-α and IFN-γ, to establish whether IFN-α would substitute for IFN-γ effects. IFN-α induced STAT1 phosphorylation in monocytes of the IFN-γR1(-/-) patient, but did not prime for LPS-induced IL-12p70, IL-12p40, IL-23 or TNF production. In control cells, IFN-α inhibited the priming effect of IFN-γ on LPS-induced pro-inflammatory cytokine release. Finally, IFN-γ but not IFN-α induced killing of M. smegmatis in cultured macrophages. In conclusion, no evidence was found to support the use of IFN-α in IFN-γR-deficient patients as intervention against mycobacterial infection; on the contrary, treatment of individuals with IFN-α may even adversely affect host defence against Mycobacteria.
Asunto(s)
Interferón-alfa/uso terapéutico , Interferón gamma/uso terapéutico , Infecciones por Mycobacterium/tratamiento farmacológico , Infecciones por Mycobacterium/inmunología , Receptores de Interferón/genética , Células Cultivadas , Humanos , Interleucinas/biosíntesis , Interleucinas/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/microbiología , Receptores de Lipopolisacáridos/inmunología , Lipopolisacáridos/inmunología , Lipopolisacáridos/metabolismo , Macrófagos/inmunología , Macrófagos/microbiología , Mycobacterium smegmatis/inmunología , Factor de Transcripción STAT1/inmunología , Factor de Transcripción STAT1/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/inmunología , Receptor de Interferón gammaRESUMEN
The phagocyte NAPDH-oxidase complex consists of several phagocyte oxidase (phox) proteins, generating reactive oxygen species (ROS) upon activation. ROS are involved in the defense against microorganisms and also in immune regulation. Defective ROS formation leads to chronic granulomatous disease (CGD) with increased incidence of autoimmunity and disturbed resolution of inflammation. Because regulatory T cells (Tregs) suppress autoimmune T-cell responses and are crucial in down-regulating immune responses, we hypothesized that ROS deficiency may lead to decreased Treg induction. Previously, we showed that in p47(phox)-mutated mice, reconstitution of macrophages (Mph) with ROS-producing capacity was sufficient to protect the mice from arthritis. Now, we present evidence that Mph-derived ROS induce Tregs. In vitro, we showed that Mph ROS-dependently induce Treg, using an NADPH-oxidase inhibitor. This finding was confirmed genetically: rat or human CGD Mph with mutated p47(phox) or gp91(phox) displayed hampered Treg induction and T-cell suppression. However, basal Treg numbers in these subjects were comparable to those in controls, indicating a role for ROS in induction of peripheral Tregs. Induction of allogeneic delayed-type hypersensitivity with p47(phox)-mutated Mph confirmed the importance of Mph-derived ROS in Treg induction in vivo. We conclude that NAPDH oxidase activity in Mph is important for the induction of Tregs to regulate T cell-mediated inflammation.
Asunto(s)
Macrófagos/metabolismo , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Cartilla de ADN/genética , Citometría de Flujo , Enfermedad Granulomatosa Crónica/inmunología , Humanos , Glicoproteínas de Membrana/inmunología , NADPH Oxidasa 2 , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/inmunología , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estadísticas no Paramétricas , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismoRESUMEN
Tuberculosis (TB) is an escalating global health problem and improved vaccines against TB are urgently needed. HLA-E restricted responses may be of interest for vaccine development since HLA-E displays very limited polymorphism (only 2 coding variants exist), and is not down-regulated by HIV-infection. The peptides from Mycobacterium tuberculosis (Mtb) potentially presented by HLA-E molecules, however, are unknown. Here we describe human T-cell responses to Mtb-derived peptides containing predicted HLA-E binding motifs and binding-affinity for HLA-E. We observed CD8(+) T-cell proliferation to the majority of the 69 peptides tested in Mtb responsive adults as well as in BCG-vaccinated infants. CD8(+) T-cells were cytotoxic against target-cells transfected with HLA-E only in the presence of specific peptide. These T cells were also able to lyse M. bovis BCG infected, but not control monocytes, suggesting recognition of antigens during mycobacterial infection. In addition, peptide induced CD8(+) T-cells also displayed regulatory activity, since they inhibited T-cell proliferation. This regulatory activity was cell contact-dependent, and at least partly dependent on membrane-bound TGF-beta. Our results significantly increase our understanding of the human immune response to Mtb by identification of CD8(+) T-cell responses to novel HLA-E binding peptides of Mtb, which have cytotoxic as well as immunoregulatory activity.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos Bacterianos/inmunología , Linfocitos T CD8-positivos/inmunología , Antígenos HLA/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Mycobacterium tuberculosis/inmunología , Subgrupos de Linfocitos T/inmunología , Adulto , Separación Celular , Epítopos de Linfocito T/inmunología , Citometría de Flujo , Humanos , Lactante , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Reguladores/inmunología , Tuberculosis/inmunología , Tuberculosis/prevención & control , Vacunas contra la Tuberculosis/inmunología , Antígenos HLA-ERESUMEN
We have made a detailed inventory of the immune infiltrate of gastrointestinal stromal tumors (GISTs), which originate from mesenchymal cells in the intestinal tract. These sarcomas are heavily infiltrated with macrophages and T cells, while immune cells of other lineages were much less abundant. Dissecting the functional subtypes of T cells with multicolor fluorescent microscopy revealed substantial populations of cytotoxic T cells, helper T cells and FoxP3(+) regulatory T cells. The balance of cytotoxic T cells and FoxP3(+) T cells was toward immune suppression. Analysis of the macrophage population also showed a dominance of anti-inflammatory cells, as the M2 type scavenger receptor CD163 was abundantly present. Other subsets of macrophages (CD14(+)CD163(-)) were occasionally detected. M2 type CD163(+) macrophages were associated with the number of infiltrating FoxP3(+) regulatory T cells and twice as many macrophages were found in metastatic GIST compared to primary lesions. Most metastatic GISTs had been treated with the tyrosine kinase inhibitors imatinib and sunitinib, but the high macrophage infiltrate was not related to this treatment. However, imatinib and sunitinib did induce secretion of anti-inflammatory IL-10 in macrophage cultures, indicating that treatment with these inhibitors might contribute to an immune suppressive microenvironment in GIST. Overall, our data reveal a picture of GIST as an active site of tumor-immune interaction in which suppressive mechanisms overrule potential antitumor responses. Tyrosine kinase inhibitors might promote this negative balance.
Asunto(s)
Tumores del Estroma Gastrointestinal/inmunología , Neoplasias Intestinales/inmunología , Macrófagos/inmunología , Inhibidores de Proteínas Quinasas/uso terapéutico , Linfocitos T Reguladores/inmunología , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Benzamidas , Femenino , Factores de Transcripción Forkhead/metabolismo , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Tumores del Estroma Gastrointestinal/patología , Humanos , Mesilato de Imatinib , Técnicas para Inmunoenzimas , Indoles/uso terapéutico , Interleucina-10/metabolismo , Neoplasias Intestinales/tratamiento farmacológico , Neoplasias Intestinales/patología , Masculino , Piperazinas/uso terapéutico , Pronóstico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/inmunología , Pirimidinas/uso terapéutico , Pirroles/uso terapéutico , Receptores de Superficie Celular/metabolismo , SunitinibRESUMEN
The ESX-5 secretion system of pathogenic mycobacteria is responsible for the secretion of various PPE and PE-PGRS proteins. To better understand the role of ESX-5 effector proteins in virulence, we analyzed the interactions of Mycobacterium marinum ESX-5 mutant with human macrophages (Mphi). Both wild-type bacteria and the ESX-5 mutant were internalized and the ESX-5 mutation did not affect the escape of mycobacteria from phagolysosomes into the cytosol, as was shown by electron microscopy. However, the ESX-5 mutation strongly effected expression of surface Ags and cytokine secretion. Whereas wild-type M. marinum actively suppressed the induction of appreciable levels of IL-12p40, TNF-alpha, and IL-6, infection with the ESX-5 mutant resulted in strongly induced production of these proinflammatory cytokines. By contrast, infection with M. marinum wild-type strain resulted in a significant induction of IL-1beta production as compared with the ESX-5 mutant. These results show that ESX-5 plays an essential role in the modulation of immune cytokine secretion by human Mphi. Subsequently, we show that an intact ESX-5 secretion system actively suppresses TLR signaling-dependent innate immune cytokine secretion. Together, our results show that ESX-5 substrates, directly or indirectly, strongly modulate the human Mphi response at various critical steps.
Asunto(s)
Proteínas Bacterianas/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Infecciones por Mycobacterium no Tuberculosas/inmunología , Mycobacterium marinum/patogenicidad , Factores de Virulencia/inmunología , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/inmunología , Células Cultivadas , Citocinas/biosíntesis , Humanos , Immunoblotting , Factores de Virulencia/metabolismoRESUMEN
CD4(+) T cell differentiation and function are critically dependent on the type of APC and the microenvironment in which Ag presentation occurs. Most studies have documented the effect of dendritic cells on effector and regulatory T cell differentiation; however, macrophages are the most abundant APCs in the periphery and can be found in virtually all organs and tissues. The effect of macrophages, and in particular their subsets, on T cell function has received little attention. Previously, we described distinct subsets of human macrophages (pro- and anti-inflammatory, m phi1 and m phi2, respectively) with highly divergent cell surface Ag expression and cytokine/chemokine production. We reported that human m phi1 promote, whereas m phi2 decrease, Th1 activation. Here, we demonstrate that m phi2, but not m phi1, induce regulatory T cells with a strong suppressive phenotype (T(m phi2)). Their mechanism of suppression is cell-cell contact dependent, mediated by membrane-bound TGFbeta-1 expressed on the regulatory T cell (Treg) population since inhibition of TGFbeta-1 signaling in target cells blocks the regulatory phenotype. T(m phi2), in addition to mediating cell-cell contact-dependent suppression, express typical Treg markers such as CD25, glucocorticoid-induced TNF receptor (GITR), and Foxp3 and are actively induced by m phi2 from CD25-depleted cells. These data identify m phi2 cells as a novel APC subset capable of inducing Tregs. The ability of anti-inflammatory macrophages to induce Tregs in the periphery has important implications for understanding Treg dynamics in pathological conditions where macrophages play a key role in inflammatory disease control and exacerbation.
Asunto(s)
Membrana Celular/metabolismo , Factores de Transcripción Forkhead/metabolismo , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Macrófagos/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Linfocitos T Reguladores/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Comunicación Celular/inmunología , Diferenciación Celular/inmunología , Proliferación Celular , Células Cultivadas , Factores de Transcripción Forkhead/inmunología , Proteína Relacionada con TNFR Inducida por Glucocorticoide , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Subunidad alfa del Receptor de Interleucina-2/inmunología , Isoantígenos/inmunología , Macrófagos/citología , Macrófagos/inmunología , Unión Proteica , Receptores de Factor de Crecimiento Nervioso/inmunología , Receptores del Factor de Necrosis Tumoral/inmunología , Linfocitos T Reguladores/inmunologíaRESUMEN
With the emergence of multidrug resistant (MDR) bacteria, it is imperative to develop new intervention strategies. Current antibiotics typically target pathogen rather than host-specific biochemical pathways. Here we have developed kinase inhibitors that prevent intracellular growth of unrelated pathogens such as Salmonella typhimurium and Mycobacterium tuberculosis. An RNA interference screen of the human kinome using automated microscopy revealed several host kinases capable of inhibiting intracellular growth of S. typhimurium. The kinases identified clustered in one network around AKT1 (also known as PKB). Inhibitors of AKT1 prevent intracellular growth of various bacteria including MDR-M. tuberculosis. AKT1 is activated by the S. typhimurium effector SopB, which promotes intracellular survival by controlling actin dynamics through PAK4, and phagosome-lysosome fusion through the AS160 (also known as TBC1D4)-RAB14 pathway. AKT1 inhibitors counteract the bacterial manipulation of host signalling processes, thus controlling intracellular growth of bacteria. By using a reciprocal chemical genetics approach, we identified kinase inhibitors with antibiotic properties and their host targets, and we determined host signalling networks that are activated by intracellular bacteria for survival.
Asunto(s)
Espacio Intracelular/microbiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Salmonella typhimurium/crecimiento & desarrollo , Animales , Antibacterianos/química , Antibacterianos/farmacología , Línea Celular Tumoral , Humanos , Espacio Intracelular/efectos de los fármacos , Isoquinolinas/química , Isoquinolinas/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Redes y Vías Metabólicas , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Interferencia de ARN , Salmonella typhimurium/efectos de los fármacos , Sulfonamidas/química , Sulfonamidas/farmacologíaRESUMEN
Regulatory T cells (Treg) comprise multiple subsets and are important in controlling immunity and inflammation. However, the induction and mode of action of the various distinct Treg subsets remain ill defined, particularly in humans. Here, we describe a human CD8+ lymphocyte activation gene-3 (LAG-3)+CD25+FoxP3+ Treg subset, which suppresses T cells partly through the secretion of CC chemokine ligand 4 (CCL4), which can inhibit T cell activation by interfering with T cell receptor signaling. CD8+ Tregs are expanded by antigen in in vivo-primed donors, and can be detected in pathogen-infected human tissue. This CD8+LAG-3+CD25+FoxP3+CCL4+ Treg subset thus may play a role in immunoregulation in humans, including infectious diseases.
Asunto(s)
Antígenos CD/análisis , Antígenos CD8/análisis , Quimiocinas CC/fisiología , Factores de Transcripción Forkhead/análisis , Subunidad alfa del Receptor de Interleucina-2/análisis , Linfocitos T Reguladores/inmunología , Animales , Calcio/metabolismo , Células Cultivadas , Quimiocina CCL4 , Quimiocinas CC/análisis , Femenino , Granuloma/inmunología , Humanos , Interleucina-2/farmacología , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
IL-23 is regarded as a major pro-inflammatory mediator in autoimmune disease, a role which until recently was ascribed to its related cytokine IL-12. IL-23, an IL-12p40/p19 heterodimeric protein, binds to IL-12Rbeta1/IL-23R receptor complexes. Mice deficient for p19, p40 or IL-12Rbeta1 are resistant to experimental autoimmune encephalomyelitis or collagen-induced arthritis. Paradoxically, however, IL-12Rbeta2- and IL-12p35-deficient mice show remarkable increases in disease susceptibility, suggesting divergent roles of IL-23 and IL-12 in modulating inflammatory processes. IL-23 induces IL-17, which mediates inflammation and tissue remodeling, but the role of IL-12 in this respect remains unidentified. We investigated the roles of exogenous (recombinant) and endogenous (macrophage-derived) IL-12 and IL-23, on IL-17-induction in human T-cells. IL-23 enhanced IL-17 secretion, as did IL-2, IL-15, IL-18 and IL-21. In contrast, IL-12 mediated specific inhibition of IL-17 production. These data support the role of IL-23 in inflammation through stimulating IL-17 production by T lymphocytes, and importantly indicate a novel regulatory function for IL-12 by specifically suppressing IL-17 secretion. These data therefore extend previous reports that had indicated unique functions for IL-23 and IL-12 due to distinct receptor expression and signal transduction complexes, and provide novel insights into the regulation of immunity, inflammation and immunopathology.
Asunto(s)
Interleucina-12/inmunología , Interleucinas/inmunología , Transducción de Señal/inmunología , Linfocitos T/metabolismo , Animales , Artritis/inducido químicamente , Artritis/inmunología , Células Cultivadas , Colágeno/administración & dosificación , Colágeno/efectos adversos , Colágeno/inmunología , Citocinas/farmacología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Humanos , Inflamación/genética , Inflamación/inmunología , Interleucina-12/deficiencia , Interleucina-12/farmacología , Interleucina-23 , Subunidad p19 de la Interleucina-23 , Interleucinas/deficiencia , Interleucinas/farmacología , Ratones , Ratones Noqueados , Receptores de Interleucina/inmunología , Receptores de Interleucina-12 , Transducción de Señal/efectos de los fármacos , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
The generation of a productive primary immune response is dependent on the ability of naïve T lymphocytes to recirculate through peripheral lymph organs to encounter specific antigen. The process of naïve CD4(+) T cell entry into lymph nodes correlates with cell surface expression of L-selectin (CD62L), which mediates early tethering and rolling events to endothelium prior to entry. Here, we demonstrate that surface expression of CD62L enhances CD4(+) T cell activation in vitro. The synthetic hydroxamate metalloproteinase inhibitor (BB-3103), specifically inhibits activation-induced shedding of CD62L from CD4(+) T cells by TCR cross-linking and lowers proliferation in part by reducing rapid tyrosine phosphorylation of zeta-associated protein 70 kDa (ZAP-70) and by increasing cytosolic free Ca(2+) concentration mobilization. BB-3103 also inhibited the proliferative response of both murine CD4(+) Th1 and Th2 subsets in vitro but the inhibitory effects were sustained only in Th2-type cells. Similarly, BB-3103 mediated prolonged inhibition of allergen-dependent peripheral T cell proliferation in atopic dermatitis patients but not in healthy controls. Analysis of CD62L expression on murine CD4(+) T cell subsets revealed that surface expression was maintained on Th1 cells but not Th2 cells. The differential effects of BB-3103 on primed effector CD4(+) T cells may provide new insights into generating therapeutic agents capable of redressing the Th2/Th1 imbalance in allergic diseases.
Asunto(s)
Linfocitos T CD4-Positivos/fisiología , Ácidos Hidroxámicos/farmacología , Selectina L/fisiología , Metaloendopeptidasas/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T/fisiología , Células TH1/fisiología , Células Th2/fisiología , Animales , Calcio/metabolismo , Citocinas/biosíntesis , Humanos , Selectina L/análisis , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BLRESUMEN
Contact between T cells and APCs results in the orchestrated segregation of molecules at the cell-cell interface and formation of a specialized structure termed the immunological synapse. This model predicts the topological seclusion of large molecules such as CD43 from the site of closest contact between the T cell and APC, allowing for the close apposition of cell membranes and effective TCR engagement. Similarly, during T cell migration segregation of CD43 to the uropod is thought to aid integrin adhesion at the leading edge of the cell by removing steric hindrance. We show in this work that CD43 distribution on T cells is regulated by a membrane proximal ezrin binding site and that failure to displace CD43 from the immunological synapse has no inhibitory effects on primary T cell activation. We also report that CD43 expression at the contact zone between T cells and matrix does not negatively regulate motility but may regulate LFA-1 de-adhesion. These results suggest that the steric barrier model of CD43 is inadequate and that alternative mechanisms account for the negative regulatory properties of CD43.