RESUMEN
BACKGROUND: Cases of the spontaneous regression of multiple pulmonary metastases, after radiofrequency ablation (RFA), of a single lung metastasis, have been documented to be mediated by the immune system. The interaction of immune checkpoints, e.g., PD-1/PD-L1 and CTLA-4/CD80, may explain this phenomenon. The purpose of this study is to identify and quantify immune mechanisms triggered by RFA of pulmonary metastases originating from colorectal cancer. METHODS: We used two-site time-resolved Förster resonance energy transfer as determined by frequency-domain FLIM (iFRET) for the quantification of receptor-ligand interactions. iFRET provides a method by which immune checkpoint interaction states can be quantified in a spatiotemporal manner. The same patient sections were used for assessment of ligand-receptor interaction and intratumoral T-cell labeling. CONCLUSION: The checkpoint interaction states quantified by iFRET did not correlate with ligand expression. We show that immune checkpoint ligand expression as a predictive biomarker may be unsuitable as it does not confirm checkpoint interactions. In pre-RFA-treated metastases, there was a significant and negative correlation between PD-1/PD-L1 interaction state and intratumoral CD3+ and CD8+ density. The negative correlation of CD8+ and interactive states of PD-1/PD-L1 can be used to assess the state of immune suppression in RFA-treated patients.
RESUMEN
Aberrant overactivation of the immune system can give rise to chronic and persistent self-attack, culminating in autoimmune disease. This is currently managed therapeutically using potent immunosuppressive and anti-inflammatory drugs. Class I phosphoinositide-3-kinases (PI3Ks) have been identified as ideal therapeutic targets for autoimmune diseases given their wide-ranging roles in immunological processes. Although progress has been hampered by issues such as poor drug tolerance and drug resistance, several PI3K inhibitors have now received regulatory approval with many others in development, including several intended to suppress the immune response in autoimmune and inflammatory diseases. This chapter reviews the evidence for contribution of aberrant PI3K activity to a range of autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis and type I diabetes) and possible therapeutic application of isoform-specific PI3K inhibitors as immunosuppressive drugs.
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Enfermedades Autoinmunes , Fosfatidilinositol 3-Quinasas , Enfermedades Autoinmunes/tratamiento farmacológico , Humanos , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositoles/uso terapéutico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Isoformas de Proteínas/uso terapéuticoRESUMEN
Innate and adaptive immune systems are built-in homeostatic functions of many multicellular organisms and protect the host against foreign pathogens and infections. Dysregulation of the molecular mechanisms of the immune system can result in autoimmune diseases. The immune system can also be harnessed and manipulated to provide targeted cancer therapies, some of them relying on the blockade of immune-checkpoint receptors. Two prominent immune checkpoints, PD-1/PD-L1 and CTLA-4/CD80, comprise receptor-ligand pairs that prevent the host immune cells from attacking host tissues. However, cancer cells upregulate the respective PD-L1 and CD80 ligands for PD-1 and CTLA-4 and thereby evade the host-immune response. Therapeutic drugs that block PD-1/PD-L1 and CTLA-4/CD80 interactions re-enable the immune system to attack cancer cells, but their prognostic biomarker remains challenging. In this review, we discuss how the use of quantitative molecular imaging can be exploited to predict the response to anti-PD-1/PD-L1 therapies and to identify cancer patients who would benefit from them.
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Antígeno B7-H1 , Inmunoterapia , Neoplasias , Medicina de Precisión , Receptor de Muerte Celular Programada 1 , Proteómica , Humanos , Antígeno B7-H1/genética , Antígeno B7-H1/inmunología , Antígeno CTLA-4/genética , Antígeno CTLA-4/inmunología , Inmunoterapia/métodos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/inmunología , Medicina de Precisión/métodos , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Proteómica/métodosRESUMEN
Implementation of a quantitative molecular imaging method (iFRET), which determines receptor-ligand interactions, has led to the finding that patients with a low extent of PD-1/PD-L1 interaction in metastatic NSCLC, and malignant melanoma, display significantly worsened overall survival compared to those with a high level of interaction.
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Biomarcadores de Tumor/análisis , Imagen Molecular/métodos , Neoplasias/diagnóstico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/fisiología , Biomarcadores de Tumor/aislamiento & purificación , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Humanos , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunohistoquímica/métodos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidad , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/mortalidad , Pronóstico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/fisiología , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
OBJECTIVE: The modified Rodnan skin score (mRSS) remains the preferred method for skin assessment in systemic sclerosis (SSc). There are concerns regarding high interobserver variability of mRSS and negative clinical trials utilizing mRSS as the primary endpoint. High-frequency ultrasound (HFUS) allows objective assessment of cutaneous fibrosis in SSc. We investigated the relationship between HFUS with both mRSS and dermal collagen. METHODS: Skin thickness (ST), echogenicity, and novel shear wave elastography (SWE) were assessed in 53 patients with SSc and 15 healthy controls (HCs) at the finger, hand, forearm, and abdomen. The relationship between HFUS parameters with mRSS (n = 53) and dermal collagen (10 patients with SSc and 10 HCs) was investigated. Intraobserver repeatability of HFUS was calculated using intraclass correlation coefficients (ICCs). RESULTS: HFUS assessment of ST (hand/forearm) and SWE (finger/hand) correlated with local mRSS at some sites. Subclinical abnormalities in ST, echogenicity, and SWE were present in clinically uninvolved SSc skin. Additionally, changes in echogenicity and SWE were sometimes apparent despite objectively normal ST on HFUS. ST, SWE, and local mRSS correlated strongly with collagen quantification (r = 0.697, 0.709, 0.649, respectively). Intraobserver repeatability was high for all HFUS parameters (ICCs for ST = 0.946-0.978; echogenicity = 0.648-0.865; and SWE = 0.953-0.973). CONCLUSION: Our data demonstrate excellent reproducibility and reassuring convergent validity with dermal collagen content. Detection of subclinical abnormalities is an additional benefit of HFUS. The observed correlations with collagen quantification support further investigation of HFUS as an alternative to mRSS in clinical trial settings.
Asunto(s)
Esclerodermia Sistémica , Colágeno , Mano/diagnóstico por imagen , Humanos , Reproducibilidad de los Resultados , Esclerodermia Sistémica/diagnóstico por imagen , Piel/diagnóstico por imagen , UltrasonografíaRESUMEN
Many cancers are termed immunoevasive due to expression of immunomodulatory ligands. Programmed death ligand-1 (PD-L1) and cluster of differentiation 80/86 (CD80/86) interact with their receptors, programmed death receptor-1 (PD-1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4), respectively, on tumor-infiltrating leukocytes eliciting immunosuppression. Immunotherapies aimed at blocking these interactions are revolutionizing cancer treatments, albeit in an inadequately described patient subset. To address the issue of patient stratification for immune checkpoint intervention, we quantitatively imaged PD-1/PD-L1 interactions in tumor samples from patients, employing an assay that readily detects these intercellular protein-protein interactions in the less than or equal to 10 nm range. These analyses across multiple patient cohorts demonstrated the intercancer, interpatient, and intratumoral heterogeneity of interacting immune checkpoints. The PD-1/PD-L1 interaction was not correlated with clinical PD-L1 expression scores in malignant melanoma. Crucially, among anti-PD-1-treated patients with metastatic non-small cell lung cancer, those with lower PD-1/PD-L1 interaction had significantly worsened survival. It is surmised that within tumors selecting for an elevated level of PD-1/PD-L1 interaction, there is a greater dependence on this pathway for immune evasion and hence, they exhibit more impressive patient response to intervention. SIGNIFICANCE: Quantitation of immune checkpoint interaction by direct imaging demonstrates that immunotherapy-treated patients with metastatic NSCLC with a low extent of PD-1/PD-L1 interaction show significantly worse outcome.
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Antígeno B7-H1/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Células Renales/inmunología , Neoplasias Renales/inmunología , Neoplasias Pulmonares/inmunología , Melanoma/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Adulto , Anciano , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/inmunología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Femenino , Transferencia Resonante de Energía de Fluorescencia/métodos , Humanos , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Masculino , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Melanoma/mortalidad , Persona de Mediana Edad , Terapia Molecular Dirigida , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Reproducibilidad de los Resultados , Resultado del TratamientoRESUMEN
Calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 2 (RAMP2) comprise a receptor for adrenomedullin (AM). Although it is known that AM induces internalization of CLRâ¢RAMP2, little is known about the molecular mechanisms that regulate the trafficking of CLRâ¢RAMP2. Using HEK and HMEC-1 cells, we observed that AM-induced activation of CLRâ¢RAMP2 promoted ubiquitination of CLR. A mutant (CLRΔ9KR), lacking all intracellular lysine residues was functional and trafficked similar to the wild-type receptor, but was not ubiquitinated. Degradation of CLRâ¢RAMP2 and CLRΔ9KRâ¢RAMP2 was not dependent on the duration of AM stimulation or ubiquitination and occurred via a mechanism that was partially prevented by peptidase inhibitors. Degradation of CLRâ¢RAMP2 was sensitive to overexpression of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), but not to HRS knockdown, whereas CLRΔ9KRâ¢RAMP2 degradation was unaffected. Overexpression, but not knockdown of HRS, promoted hyperubiquitination of CLR under basal conditions. Thus, we propose a role for ubiquitin and HRS in the regulation of AM-induced degradation of CLRâ¢RAMP2.
Asunto(s)
Adrenomedulina/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Fosfoproteínas/metabolismo , Receptores de Adrenomedulina/metabolismo , Ubiquitinación/fisiología , Proteína Similar al Receptor de Calcitonina/genética , Proteína Similar al Receptor de Calcitonina/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Fosfoproteínas/genética , Transporte de Proteínas , Proteolisis , ARN Interferente Pequeño/metabolismo , Proteína 2 Modificadora de la Actividad de Receptores/genética , Proteína 2 Modificadora de la Actividad de Receptores/metabolismo , Ubiquitina/metabolismoRESUMEN
H2O2 is an early danger cue required for innate immune cell recruitment to wounds. To date, little is known about whether H2O2 is required for the migration of human adaptive immune cells to sites of inflammation. However, oxidative stress is known to impair T cell activity, induce actin stiffness, and inhibit cell polarization. In this study, we show that low oxidative concentrations of H2O2 also impede chemokinesis and chemotaxis of previously activated human T cells to CXCL11, but not CXCL10 or CXCL12. We show that this deficiency in migration is due to a reduction in inflammatory chemokine receptor CXCR3 surface expression and cellular activation of lipid phosphatase SHIP-1. We demonstrate that H2O2 acts through an Src kinase to activate a negative regulator of PI3K signaling, SHIP-1 via phosphorylation, providing a molecular mechanism for H2O2-induced chemotaxis deficiency. We hypothesize that although H2O2 serves as an early recruitment trigger for innate immune cells, it appears to operate as an inhibitor of T lymphocyte immune adaptive responses that are not required until later in the repair process.
Asunto(s)
Movimiento Celular , Quimiocina CXCL11/metabolismo , Peróxido de Hidrógeno/farmacología , Terapia de Inmunosupresión , Linfocitos T/efectos de los fármacos , Actinas/metabolismo , Inmunidad Adaptativa , Adulto , Anciano , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Femenino , Humanos , Inmunidad Innata/efectos de los fármacos , Masculino , Persona de Mediana Edad , Estrés Oxidativo/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/metabolismo , Receptores CXCR3/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Adulto Joven , Familia-src Quinasas/metabolismoRESUMEN
Barrett's metaplasia is the only known morphological precursor to oesophageal adenocarcinoma and is characterized by replacement of stratified squamous epithelium by columnar epithelium. The cell of origin is uncertain and the molecular mechanisms responsible for the change in cellular phenotype are poorly understood. We therefore explored the role of two transcription factors, Cdx2 and HNF4α in the conversion using primary organ cultures. Biopsy samples from cases of human Barrett's metaplasia were analysed for the presence of CDX2 and HNF4α. A new organ culture system for adult murine oesophagus is described. Using this, Cdx2 and HNF4α were ectopically expressed by adenoviral infection. The phenotype following infection was determined by a combination of PCR, immunohistochemical and morphological analyses. We demonstrate the expression of CDX2 and HNF4α in human biopsy samples. Our oesophageal organ culture system expressed markers characteristic of the normal SSQE: p63, K14, K4 and loricrin. Ectopic expression of HNF4α, but not of Cdx2 induced expression of Tff3, villin, K8 and E-cadherin. HNF4α is sufficient to induce a columnar-like phenotype in adult mouse oesophageal epithelium and is present in the human condition. These data suggest that induction of HNF4α is a key early step in the formation of Barrett's metaplasia and are consistent with an origin of Barrett's metaplasia from the oesophageal epithelium.
Asunto(s)
Adenocarcinoma/genética , Esófago de Barrett/genética , Factor de Transcripción CDX2/genética , Neoplasias Esofágicas/genética , Factor Nuclear 4 del Hepatocito/genética , Adenocarcinoma/patología , Adulto , Animales , Esófago de Barrett/patología , Biopsia , Epitelio/patología , Neoplasias Esofágicas/patología , Esófago/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Proteínas de Neoplasias/biosíntesis , Técnicas de Cultivo de ÓrganosRESUMEN
Branching morphogenesis is a critical step in the development of many epithelial organs. The phosphoinositide-3-kinase (PI3K) pathway has been identified as a central component of this process but the precise role has not been fully established. Herein we sought to determine the role of PI3K in murine lung branching using a series of pharmacological inhibitors directed at this pathway. The pan-class I PI3K inhibitor ZSTK474 greatly enhanced the branching potential of whole murine lung explants as measured by an increase in the number of terminal branches compared with controls over 48 hours. This enhancement of branching was also observed following inhibition of the downstream signalling components of PI3K, Akt and mTOR. Isoform selective inhibitors of PI3K identified that the alpha isoform of PI3K is a key driver in branching morphogenesis. To determine if the effect of PI3K inhibition on branching was specific to the lung epithelium or secondary to an effect on the mesenchyme we assessed the impact of PI3K inhibition in cultures of mesenchyme-free lung epithelium. Isolated lung epithelium cultured with FGF7 formed large cyst-like structures, whereas co-culture with FGF7 and ZSTK474 induced the formation of defined branches with an intact lumen. Together these data suggest a novel role for PI3K in the branching program of the murine embryonic lung contradictory to that reported in other branching organs. Our observations also point towards PI3K acting as a morphogenic switch for FGF7 signalling.
Asunto(s)
Factor 7 de Crecimiento de Fibroblastos/metabolismo , Pulmón/crecimiento & desarrollo , Morfogénesis/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Epitelio/efectos de los fármacos , Epitelio/crecimiento & desarrollo , Factor 7 de Crecimiento de Fibroblastos/genética , Humanos , Pulmón/efectos de los fármacos , Pulmón/embriología , Ratones , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositoles/metabolismo , Transducción de Señal/efectos de los fármacos , Triazinas/administración & dosificaciónRESUMEN
Cell penetrating peptides (CPPs) offer the exciting potential of effectively delivering macromolecules to the cytoplasm of a cell that are otherwise impermeable to the plasma membrane. Although the use of these peptides has so far been well tolerated in clinical trials, it is important to remember that some of these CPPs were originally derived from pathogenic material. We therefore sought to determine if three of the most widely studied CPPs; HIV-TAT, Antennapedia and Transportan, initiated an immune response in epithelial cells. Using conditions where these peptides efficiently delivered a rhodamine tagged BSA cargo to the interior of epithelial cells, we failed to observe an effect on cell viability as determined by MTT assay (P>0.05). Further, CPP-mediated delivery of this protein cargo failed to activate NFκB, which would be indicative of toll-like receptor signalling. Finally, no significant increase in the release of the inflammatory cytokines interleukin (IL)-8 and IL-6 was detected in epithelial cells exposed to CPP complexes for 72 h (P>0.05). Together, these results indicate that these commonly used CPPs are passive carriers that do not initiate epithelial cell-associated 'danger signals' during the process of cytoplasmic delivery of a model protein cargo.
Asunto(s)
Células Epiteliales Alveolares/inmunología , Péptidos de Penetración Celular/efectos adversos , Sistemas de Liberación de Medicamentos/efectos adversos , Enterocitos/inmunología , Inmunidad Innata , Queratinocitos/inmunología , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/metabolismo , Proteína con Homeodominio Antennapedia/efectos adversos , Proteína con Homeodominio Antennapedia/química , Transporte Biológico , Línea Celular , Supervivencia Celular , Citocinas/metabolismo , Proteínas de Drosophila/efectos adversos , Proteínas de Drosophila/química , Composición de Medicamentos , Enterocitos/citología , Enterocitos/metabolismo , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Galanina/efectos adversos , Galanina/química , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Oligopéptidos/efectos adversos , Fragmentos de Péptidos/efectos adversos , Proteínas Recombinantes de Fusión/efectos adversos , Proteínas Recombinantes de Fusión/química , Rodaminas/química , Rodaminas/metabolismo , Venenos de Avispas/efectos adversos , Venenos de Avispas/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/efectos adversos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
Migration of naive CD4(+) T lymphocytes into lymphoid tissue is essential for their activation and subsequent roles in adaptive immunity. The adhesion molecule L-selectin (CD62L), critical for this process, is highly expressed on naive CD4(+) T lymphocytes and is downregulated upon T lymphocyte activation. We demonstrate protein expression of P2X7R on naive CD4(+) T lymphocytes and show functional channel activity in whole-cell patch clamp recordings. CD62L downregulation occurs rapidly in response to extracellular ATP, a process that is blocked by selective antagonists of P2X7R. This loss of surface CD62L expression was not associated with externalization of phosphatidylserine. While investigating the mechanisms for this process, we revealed that pharmacological modulation of mitochondrial complex I or III, but not inhibition of NADPH oxidase, enhanced P2X7R-dependent CD62L downregulation by increasing ATP potency. Enhanced superoxide generation in the mitochondria of rotenone- and antimycin A-treated cells was observed and may contribute to the enhanced sensitivity of P2X7R to ATP. P2X7R-dependent exposure of phosphatidylserine was also revealed by preincubation with mitochondrial uncouplers prior to ATP treatment. This may present a novel mechanism whereby P2X7R-dependent phosphatidylserine exposure occurs only when cells have enhanced mitochondrial reactive oxygen species generation. The clearance of apoptotic cells may therefore be enhanced by this mechanism which requires functional P2X7R expression.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular/inmunología , Regulación hacia Abajo , Selectina L , Mitocondrias/metabolismo , Receptores Purinérgicos P2X7/fisiología , Superóxidos/metabolismo , Regulación hacia Arriba/inmunología , Adenosina Trifosfato/fisiología , Apoptosis/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Línea Celular Tumoral , Membrana Celular/inmunología , Membrana Celular/metabolismo , Células Cultivadas , Regulación hacia Abajo/inmunología , Células HEK293 , Humanos , Células Jurkat , Mitocondrias/inmunología , Superóxidos/farmacologíaRESUMEN
The phosphoinositide 3-kinase/mammalian target of rapamycin/protein kinase B (PI3K/mTOR/Akt) signaling pathway is central to a plethora of cellular mechanisms in a wide variety of cells including leukocytes. Perturbation of this signaling cascade is implicated in inflammatory and autoimmune disorders as well as hematological malignancies. Proteins within the PI3K/mTOR/Akt pathway therefore represent attractive targets for therapeutic intervention. There has been a remarkable evolution of PI3K inhibitors in the past 20 years from the early chemical tool compounds to drugs that are showing promise as anticancer agents in clinical trials. The use of animal models and pharmacological tools has expanded our knowledge about the contribution of individual class I PI3K isoforms to immune cell function. In addition, class II and III PI3K isoforms are emerging as nonredundant regulators of immune cell signaling revealing potentially novel targets for disease treatment. Further complexity is added to the PI3K/mTOR/Akt pathway by a number of novel signaling inputs and feedback mechanisms. These can present either caveats or opportunities for novel drug targets. Here, we consider recent advances in 1) our understanding of the contribution of individual PI3K isoforms to immune cell function and their relevance to inflammatory/autoimmune diseases as well as lymphoma and 2) development of small molecules with which to inhibit the PI3K pathway. We also consider whether manipulating other proximal elements of the PI3K signaling cascade (such as class II and III PI3Ks or lipid phosphatases) are likely to be successful in fighting off different immune diseases.
Asunto(s)
Enfermedades Autoinmunes/enzimología , Neoplasias Hematológicas/enzimología , Fosfatidilinositol 3-Quinasas/inmunología , Animales , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/inmunología , Inhibidores Enzimáticos/farmacología , Neoplasias Hematológicas/tratamiento farmacológico , Neoplasias Hematológicas/inmunología , Humanos , Sistema Inmunológico/enzimología , Inflamación/tratamiento farmacológico , Inflamación/enzimología , Inflamación/inmunología , Fosfatidilinositol 3-Quinasas/química , Inhibidores de las Quinasa Fosfoinosítidos-3 , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
The signalling molecule PI3Kγ has been reported to play a key role in the immune system and the inflammatory response. In particular, it facilitates the migration of haemato-poietic cells to the site of inflammation. In this study, we reveal a novel role for PI3Kγ in the regulation of the pro-inflammatory cytokine IL-17. Loss of PI3Kγ or expression of a catalytically inactive mutant of PI3Kγ in mice led to increased IL-17 production both in vitro and in vivo in response to various stimuli. The kinetic profile was unaltered from WT cells, with no effect on proliferation or other cytokines. Elevated levels of IL-17 were not due to an aberrant expansion of IL-17-producing cells. Furthermore, we also identified an increase in IL-17RA expression on PI3Kγ(-/-) CD4(+) T cells, yet these cells exhibited impaired PI3K-dependent signalling in response to IL-17A, and subsequent NF-κB phosphorylation. In vivo, instillation of recombinant IL-17 into the airways of mice lacking PI3Kγ signalling also resulted in reduced phosphorylation of Akt. Cell influx in response to IL-17 was also reduced in PI3Kγ(-/-) lungs. These data demonstrate PI3Kγ-dependent signalling downstream of IL-17RA, which plays a pivotal role in regulating IL-17 production in T cells.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Interleucina-17/inmunología , Fosfatidilinositol 3-Quinasas/inmunología , Receptores de Interleucina-17/inmunología , Transducción de Señal/inmunología , Animales , Linfocitos T CD4-Positivos/metabolismo , Regulación Enzimológica de la Expresión Génica/genética , Regulación Enzimológica de la Expresión Génica/inmunología , Interleucina-17/genética , Interleucina-17/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/genética , Fosforilación/inmunología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismo , Transducción de Señal/genéticaRESUMEN
The predominant expression of the γ and δ isoforms of PI3K in cells of hematopoietic lineage prompted speculation that inhibitors of these isoforms could offer opportunities for selective targeting of PI3K in the immune system in a range of immune-related pathologies. While there has been some success in developing PI3Kδ inhibitors, progress in developing selective inhibitors of PI3Kγ has been rather disappointing. This has prompted the search for alternative targets with which to modulate PI3K signaling specifically in the immune system. One such target is the SH2 domain-containing inositol-5-phosphatase-1 (SHIP-1) which de-phosphorylates PI(3,4,5)P(3) at the D5 position of the inositol ring to create PI(3,4)P(2). In this article, we first describe the current state of PI3K isoform-selective inhibitor development. We then focus on the structure of SHIP-1 and its function in the immune system. Finally, we consider the current state of development of small molecule compounds that potently and selectively modulate SHIP activity and which offer novel opportunities to manipulate PI3K mediated signaling in the immune system.
Asunto(s)
Factores Inmunológicos/uso terapéutico , Antiinflamatorios/uso terapéutico , Enfermedades Autoinmunes/tratamiento farmacológico , Productos Biológicos/uso terapéutico , Humanos , Inflamación/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3RESUMEN
PI3K is critical for the normal function of the immune system, however dysregulated PI3K mediated signaling has been linked to the development of many immune mediated pathologies. This review describes current progress in the development of isoform-specific PI3K inhibitors that hold promise for the treatment of hematopoietic malignancies as well as for inflammatory and autoimmune diseases. A SH2-domain containing inositol-5-phosphatase (SHIP) is a regulator of PI3K signaling, and is also discussed as a potential drug target for immunomodulation and the treatment of leukemia. Recent progress has been made in the development of small molecule compounds that potently and selectively modulate SHIP activity and hence provide a novel mechanism to alter PI3K mediated signaling.
Asunto(s)
Inhibidores de las Quinasa Fosfoinosítidos-3 , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Animales , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Inositol Polifosfato 5-Fosfatasas , Isoenzimas/antagonistas & inhibidores , Isoenzimas/inmunología , Leucemia/tratamiento farmacológico , Leucemia/inmunología , Fosfatidilinositol 3-Quinasas/inmunología , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/inmunologíaRESUMEN
Protein arginine N-methyltransferases (PRMTs) selectively replace N-H for N-CH(3) at substrate protein guanidines, a post-translational modification important for a range of biological processes, such as epigenetic regulation, signal transduction and cancer progression. Selective chemical probes are required to establish the dynamic function of individual PRMTs. Herein, model inhibitors designed to occupy PRMT binding sites for an arginine substrate and S-adenosylmethionine (AdoMet) co-factor are described. Expedient access to such compounds by modular synthesis is detailed. Remarkably, biological evaluation revealed some compounds to be potent inhibitors of PRMT1, but inactive against CARM1. Docking studies show how prototype compounds may occupy the binding sites for a co-factor and arginine substrate. Overlay of PRMT1 and CARM1 binding sites suggest a difference in a single amino acid that may be responsible for the observed selectivity.
Asunto(s)
Arginina/metabolismo , Inhibidores Enzimáticos/síntesis química , Procesamiento Proteico-Postraduccional , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/metabolismo , S-Adenosilmetionina/metabolismo , Arginina/antagonistas & inhibidores , Sitios de Unión , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Epigénesis Genética , Escherichia coli , Humanos , Metilación , Modelos Moleculares , Peso Molecular , Plásmidos , Unión Proteica , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/química , Proteínas Represoras/genética , S-Adenosilmetionina/antagonistas & inhibidores , Especificidad por Sustrato , Transformación BacterianaRESUMEN
XAFS spectroscopy has been used to determine the Ni species in particulate matter collected on quartz thimble filters in the stacks of eight residual (No. 6 fuel) oil-burning electric utility steam-generating units. Proper speciation of nickel in emitted particulate matter is necessary to correctly anticipate potential health risks. Analysis of the spectroscopic data using least-squares linear combination methods and a newly developed method specific for small quantities of Ni sulfide compounds in such emissions show that potentially carcinogenic Ni sulfide compounds are absent within the detection limits of the method (≤ 3% of the total Ni) in the particulate matter samples investigated. In addition to the major nickel sulfate phase (NiSO(4)·6H(2)O), lesser amounts of (Ni,Mg)O and/or NiFe(2)O(4) were also identified in most emission samples. On the basis of the results from these emission characterization studies, the appropriateness of the U.S. Environmental Protection Agency's assumption that the Ni compound mixture emitted from residual oil-fired power plants is 50% as carcinogenic as nickel subsulfide (Ni(3)S(2)) should be re-evaluated.