RESUMEN

Pulmonary infection is a frequent pathology associated with excessive neutrophil infiltration. Ly49Q, an ITIM domain-bearing receptor expressed on different leukocytes, has been recently reported to impact neutrophil migration and polarization. Utilizing a murine model of Klebsiella pneumoniae-induced pulmonary infection in combination with additional in vivo and in vitro assays, we show that Ly49Q is critically involved in different steps of the leukocyte adhesion cascade. Ly49Q deficiency is associated with a reduced rolling velocity, impaired crawling capacity, and diminished transmigration. We show that overactivation of the neutrophil ß2 integrins Mac-1 and LFA-1 is responsible for increased adhesion and reduced neutrophil transmigration, resulting in a strongly impaired immune defense against pulmonary infection. Structure function analysis in vitro and in vivo demonstrated that different domains of Ly49Q are important for its function. In summary, Ly49Q regulates integrin activation and neutrophil recruitment and is required for an adequate immune response in pulmonary infection.

Asunto(s)

Lesión Pulmonar/metabolismo , Pulmón/metabolismo , Subfamilia A de Receptores Similares a Lectina de Células NK/metabolismo , Neutrófilos/metabolismo , Neutrófilos/fisiología , Dominios Proteicos/fisiología , Animales , Antígenos CD18/metabolismo , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Femenino , Infecciones por Klebsiella/metabolismo , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/patogenicidad , Leucocitos/metabolismo , Pulmón/microbiología , Lesión Pulmonar/microbiología , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Antígeno de Macrófago-1/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Infiltración Neutrófila/fisiología

RESUMEN

BACKGROUND AND PURPOSE: Following inflammatory stimuli, neutrophils are recruited to sites of inflammation and exert effector functions that often have deleterious effects on tissue integrity, which can lead to organ failure. Bruton's tyrosine kinase (Btk) is expressed in neutrophils and constitutes a promising pharmacological target for neutrophil-mediated tissue damage. Here, we evaluate a selective reversible inhibitor of Btk, PRN473, for its ability to dampen neutrophil influx via inhibition of adhesion receptor signalling pathways. EXPERIMENTAL APPROACH: In vitro assays were used to assess fMLP receptor 1 (Fpr-1)-mediated binding of ligands to the adhesion receptors macrophage antigen-1 (Mac-1) and lymphocyte function antigen-1. Intravital microscopy of the murine cremaster was used to evaluate post-adhesion strengthening and endoluminal crawling. Finally, neutrophil influx was visualized in a clinically relevant model of sterile liver injury in vivo. Btk knockout animals were used as points of reference for Btk functions. KEY RESULTS: Pharmacological inhibition of Btk by PRN473 reduced fMLP-induced phosphorylation of Btk and Mac-1 activation. Biochemical experiments demonstrated the specificity of the inhibitor. PRN473 (20 mg·kg-1 ) significantly reduced intravascular crawling and neutrophil recruitment into inflamed tissue in a model of sterile liver injury, down to levels seen in Btk-deficient animals. A higher dose did not provide additional reduction of intravascular crawling and neutrophil recruitment. CONCLUSIONS AND IMPLICATIONS: PRN473, a highly selective inhibitor of Btk, potently attenuates sterile liver injury by inhibiting the activation of the ß2 -integrin Mac-1 and subsequently neutrophil recruitment into inflamed tissue.

Asunto(s)

Antígeno de Macrófago-1/metabolismo , Infiltración Neutrófila/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/fisiología , Agammaglobulinemia Tirosina Quinasa , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Relación Dosis-Respuesta a Droga , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infiltración Neutrófila/fisiología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Transducción de Señal/efectos de los fármacos

RESUMEN

Acute kidney injury is a prevalent but underdiagnosed complication that is associated with increased in-hospital morbidity and mortality. The importance of this complication is being increasingly recognized. The lack of timely diagnostic methods and effective preemptive and therapeutic strategies make its perioperative management challenging. To reduce the incidence of acute kidney injury, it is crucial to focus the resources on high-risk patients who can be identified by clinical scoring systems with a high predictive value and newly developed renal biomarkers. The 'Kidney Disease: Improving Global Outcomes' guidelines recommend the implementation of preventive strategies that include a bundle of supportive measures. However, clear evidence to support such an approach is lacking. Previous studies demonstrated improved patients' outcome following remote ischemic preconditioning in high-risk patients. Other studies reached an opposite result. To date, renal replacement therapy is the "gold standard" for the treatment of severe acute kidney injury, although the ideal timing, technique, and application of this therapy remain under debate.

Asunto(s)

Lesión Renal Aguda/prevención & control , Precondicionamiento Isquémico/métodos , Terapia de Reemplazo Renal/métodos , Lesión Renal Aguda/epidemiología , Lesión Renal Aguda/etiología , Animales , Biomarcadores/metabolismo , Humanos , Incidencia , Valor Predictivo de las Pruebas , Factores de Riesgo

RESUMEN

Integrin activation is required for neutrophil functions. Impaired integrin activation on neutrophils is the hallmark of leukocyte adhesion deficiency (LAD) syndrome in humans, characterized by impaired leukocyte recruitment and recurrent infections. The Src kinase-associated phosphoprotein 2 (Skap2) is involved in integrin functions in different leukocyte subtypes. However, the role of Skap2 in ß2 integrin activation and neutrophil recruitment is unknown. In this study, we demonstrate the crucial role of Skap2 in regulating actin polymerization and binding of talin-1 and kindlin-3 to the ß2 integrin cytoplasmic domain, thereby being indispensable for ß2 integrin activation and neutrophil recruitment. The direct interaction of Skap2 with the Wiskott-Aldrich syndrome protein via its SH3 domain is critical for integrin activation and neutrophil recruitment in vivo. Furthermore, Skap2 regulates integrin-mediated outside-in signaling events and neutrophil functions. Thus, Skap2 is essential to activate the ß2 integrins, and loss of Skap2 function is sufficient to cause a LAD-like phenotype in mice.

Asunto(s)

Antígenos CD18/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Infiltración Neutrófila , Neutrófilos/fisiología , Animales , Adhesión Celular , Quimiotaxis de Leucocito , Proteínas del Citoesqueleto/metabolismo , Selectina E/fisiología , Antígeno de Macrófago-1/fisiología , Ratones , Ratones Endogámicos C57BL , Multimerización de Proteína , Talina/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/fisiología , Dominios Homologos src

RESUMEN

Neutrophils are recruited from the blood to sites of sterile inflammation, where they are involved in wound healing but can also cause tissue damage. During sterile inflammation, necrotic cells release pro-inflammatory molecules including formylated peptides. However, the signaling pathway triggered by formylated peptides to integrin activation and leukocyte recruitment is unknown. By using spinning-disk confocal intravital microscopy, we examined the molecular mechanisms of leukocyte recruitment to sites of focal hepatic necrosis in vivo. We demonstrated that the Bruton's tyrosine kinase (Btk) was required for multiple Mac-1 activation events involved in neutrophil recruitment and functions during sterile inflammation triggered by fMLF. The Src family kinase Hck, Wiskott-Aldrich-syndrome protein, and phospholipase Cγ2 were also involved in this pathway required for fMLF-triggered Mac-1 activation and neutrophil recruitment. Thus, we have identified a neutrophil Btk signalosome that is involved in a signaling pathway triggered by formylated peptides leading to the selective activation of Mac-1 and neutrophil recruitment during sterile inflammation.

Asunto(s)

Integrinas/metabolismo , Infiltración Neutrófila/inmunología , Proteínas Tirosina Quinasas/inmunología , Transducción de Señal/inmunología , Agammaglobulinemia Tirosina Quinasa , Animales , Citometría de Flujo , Inflamación , Integrinas/inmunología , Hepatopatías/inmunología , Hepatopatías/metabolismo , Ratones , Microscopía Confocal , N-Formilmetionina Leucil-Fenilalanina/inmunología , Necrosis/inmunología , Proteínas Tirosina Quinasas/metabolismo

RESUMEN

Neutrophil recruitment to the site of inflammation plays a pivotal role in host defense. However, overwhelming activation and accumulation of neutrophils in the tissue may cause tissue damage and autoimmunity due to the release of cytokines, oxidants, and proteases. Neutrophil adhesion in acute inflammation is initiated by activation of αLß2 (LFA-1), which can be induced by rolling on E-selectin (slowly) or by exposure to the chemokine CXCL1 (rapidly). Despite the clinical importance, cell-intrinsic molecular mechanisms of negative regulation of integrin adhesiveness and neutrophil recruitment are poorly understood. Mice deficient in the tyrosine phosphatase Src homology 2 domain-containing protein tyrosine phosphatase 1 (Shp1) show increased leukocyte adhesion, but the interpretation of these data is limited by the severe global phenotype of these mice. In this study, we used mice with global and myeloid-restricted deletion of Shp1 to study neutrophil arrest, adhesion, crawling, and transendothelial migration in vitro and in vivo. Shp1 deficiency results in increased neutrophil adhesion in vivo; however, neutrophil crawling, transmigration, and chemotaxis were reduced in these mice. Mechanistically, Shp1 binds and controls PIPKIγ activity and, thereby, modulates phosphatidylinositol (4,5)-bisphosphate levels and adhesion. Thus, Shp1 is involved in the deactivation of integrins and regulation of neutrophil recruitment into inflamed tissue.

Asunto(s)

Adhesión Celular/inmunología , Infiltración Neutrófila/inmunología , Fosfatidilinositoles/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/inmunología , Animales , Adhesión Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/inmunología , Quimiocina CXCL1/inmunología , Selectina E/inmunología , Activación Enzimática/inmunología , Células HL-60 , Humanos , Inflamación/inmunología , Rodamiento de Leucocito/inmunología , Antígeno-1 Asociado a Función de Linfocito/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infiltración Neutrófila/genética , Neutrófilos/inmunología , Fosfatidilinositoles/biosíntesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo

RESUMEN

Neutrophil recruitment to the site of inflammation plays a pivotal role in host defense. Src family kinases (SFKs) activation is required for integrin and chemokine signaling as well as immune cell function. The receptor-like protein tyrosine phosphatase CD45 positively regulates chemoattractant signaling acting on SFK activity. To further investigate the role of CD45 in neutrophil recruitment and function, we analyzed transgenic mice carrying a single point mutation (CD45E613R), which constitutively activates CD45. By using intravital microscopy experiments, we demonstrated that different steps of the leukocyte recruitment cascade were affected in CD45E613R mutant mice. The rolling velocity of CD45E613R mutant neutrophils was decreased compared with wild-type neutrophils that subsequently resulted in an increased number of adherent cells. The analysis of ß2 integrins LFA-1 and macrophage-1 Ag (Mac-1) showed that in CD45E613R mutant neutrophils LFA-1 adhesiveness was impaired, and avidity was enhanced, whereas Mac-1 adhesiveness was increased. Because of the increased Mac-1 adhesiveness, neutrophil crawling was impaired in CD45E613R mutant compared with wild-type neutrophils. In an Escherichia coli lung infection model, CD45E613R mice displayed a decreased neutrophil recruitment into the alveolar compartment, which resulted in an increased number of CFUs in the lung. Our data demonstrate that the CD45E613R mutation modulates integrin activation and leukocyte recruitment during inflammation.

Asunto(s)

Antígenos Comunes de Leucocito , Rodamiento de Leucocito , Antígeno-1 Asociado a Función de Linfocito , Antígeno de Macrófago-1 , Neutrófilos/inmunología , Mutación Puntual , Sustitución de Aminoácidos , Animales , Escherichia coli/inmunología , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/patología , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/inmunología , Rodamiento de Leucocito/genética , Rodamiento de Leucocito/inmunología , Antígeno-1 Asociado a Función de Linfocito/genética , Antígeno-1 Asociado a Función de Linfocito/inmunología , Antígeno de Macrófago-1/genética , Antígeno de Macrófago-1/inmunología , Ratones , Ratones Mutantes , Neutrófilos/patología , Neumonía Bacteriana/genética , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/patología