RESUMO
Cellular senescence is a hallmark of advanced age and a major instigator of numerous inflammatory pathologies. While endothelial cell (EC) senescence is aligned with defective vascular functionality, its impact on fundamental inflammatory responses in vivo at single-cell level remain unclear. To directly investigate the role of EC senescence on dynamics of neutrophil-venular wall interactions, we applied high resolution confocal intravital microscopy to inflamed tissues of an EC-specific progeroid mouse model, characterized by profound indicators of EC senescence. Progerin-expressing ECs supported prolonged neutrophil adhesion and crawling in a cell autonomous manner that additionally mediated neutrophil-dependent microvascular leakage. Transcriptomic and immunofluorescence analysis of inflamed tissues identified elevated levels of EC CXCL1 on progerin-expressing ECs and functional blockade of CXCL1 suppressed the dysregulated neutrophil responses elicited by senescent ECs. Similarly, cultured progerin-expressing human ECs exhibited a senescent phenotype, were pro-inflammatory and prompted increased neutrophil attachment and activation. Collectively, our findings support the concept that senescent ECs drive excessive inflammation and provide new insights into the mode, dynamics, and mechanisms of this response at single-cell level.
RESUMO
Neutrophil diapedesis is an immediate step following infections and injury and is driven by complex interactions between leukocytes and various components of the blood vessel wall. Here, we show that perivascular mast cells (MC) are key regulators of neutrophil behaviour within the sub-endothelial space of inflamed venules. Using confocal intravital microscopy, we observe directed abluminal neutrophil motility along pericyte processes towards perivascular MCs, a response that created neutrophil extravasation hotspots. Conversely, MC-deficiency and pharmacological or genetic blockade of IL-17A leads to impaired neutrophil sub-endothelial migration and breaching of the pericyte layer. Mechanistically, identifying MCs as a significant cellular source of IL-17A, we establish that MC-derived IL-17A regulates the enrichment of key effector molecules ICAM-1 and CXCL1 in nearby pericytes. Collectively, we identify a novel MC-IL-17A-pericyte axis as modulator of the final steps of neutrophil diapedesis, with potential translational implications for inflammatory disorders driven by increased neutrophil diapedesis.
Assuntos
Neutrófilos , Migração Transendotelial e Transepitelial , Neutrófilos/fisiologia , Pericitos , Interleucina-17 , MastócitosRESUMO
Zika virus (ZIKV) cases continue to be reported, and no vaccine or specific antiviral agent has been approved for the prevention or treatment of infection. Though ZIKV is primarily transmitted by mosquitos, cases of sexual transmission and prolonged viral RNA presence in semen have been reported. In this observational study, we report the mucosal responses to sub-cutaneous and mucosal ZIKV exposure in cynomolgus macaques during acute and late chronic infection. Subcutaneous challenge induced a decrease in the growth factor VEGF in colorectal and cervicovaginal tissues 100 days post-challenge, in contrast to the observed increase in these tissues following vaginal infection. This different pattern was not observed in the uterus, where VEGF was upregulated independently of the challenge route. Vaginal challenge induced a pro-inflammatory profile in all mucosal tissues during late chronic infection. Similar responses were already observed during acute infection in a vaginal tissue explant model of ex vivo challenge. Non-productive and productive infection 100 days post-in vivo vaginal challenge induced distinct proteomic profiles which were characterized by further VEGF increase and IL-10 decrease in non-infected animals. Ex vivo challenge of mucosal explants revealed tissue-specific modulation of cytokine levels during the acute phase of infection. Mucosal cytokine profiles could represent biosignatures of persistent ZIKV infection.
RESUMO
Aging is associated with dysregulated immune functions. Here, we investigated the impact of age on neutrophil diapedesis. Using confocal intravital microscopy, we found that in aged mice, neutrophils adhered to vascular endothelium in inflamed tissues but exhibited a high frequency of reverse transendothelial migration (rTEM). This retrograde breaching of the endothelium by neutrophils was governed by enhanced production of the chemokine CXCL1 from mast cells that localized at endothelial cell (EC) junctions. Increased EC expression of the atypical chemokine receptor 1 (ACKR1) supported this pro-inflammatory milieu in aged venules. Accumulation of CXCL1 caused desensitization of the chemokine receptor CXCR2 on neutrophils and loss of neutrophil directional motility within EC junctions. Fluorescent tracking revealed that in aged mice, neutrophils undergoing rTEM re-entered the circulation and disseminated to the lungs where they caused vascular leakage. Thus, neutrophils stemming from a local inflammatory site contribute to remote organ damage, with implication to the dysregulated systemic inflammation associated with aging.
Assuntos
Envelhecimento/imunologia , Transporte Biológico/imunologia , Inflamação/imunologia , Neutrófilos/imunologia , Animais , Quimiocina CXCL1/imunologia , Células Endoteliais/imunologia , Endotélio Vascular/imunologia , Feminino , Junções Intercelulares/imunologia , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Interleucina-8B/imunologia , Vênulas/imunologiaRESUMO
Increased microvascular permeability to plasma proteins and neutrophil emigration are hallmarks of innate immunity and key features of numerous inflammatory disorders. Although neutrophils can promote microvascular leakage, the impact of vascular permeability on neutrophil trafficking is unknown. Here, through the application of confocal intravital microscopy, we report that vascular permeability-enhancing stimuli caused a significant frequency of neutrophil reverse transendothelial cell migration (rTEM). Furthermore, mice with a selective defect in microvascular permeability enhancement (VEC-Y685F-ki) showed reduced incidence of neutrophil rTEM. Mechanistically, elevated vascular leakage promoted movement of interstitial chemokines into the bloodstream, a response that supported abluminal-to-luminal neutrophil TEM. Through development of an in vivo cell labeling method we provide direct evidence for the systemic dissemination of rTEM neutrophils, and showed them to exhibit an activated phenotype and be capable of trafficking to the lungs where their presence was aligned with regions of vascular injury. Collectively, we demonstrate that increased microvascular leakage reverses the localization of directional cues across venular walls, thus causing neutrophils engaged in diapedesis to reenter the systemic circulation. This cascade of events offers a mechanism to explain how local tissue inflammation and vascular permeability can induce downstream pathological effects in remote organs, most notably in the lungs.
Assuntos
Permeabilidade Capilar/imunologia , Microvasos/imunologia , Ativação de Neutrófilo , Neutrófilos/imunologia , Migração Transendotelial e Transepitelial/imunologia , Animais , Permeabilidade Capilar/genética , Masculino , Camundongos , Camundongos Transgênicos , Microvasos/patologia , Neutrófilos/patologia , Migração Transendotelial e Transepitelial/genéticaRESUMO
The bacterium Escherichia coli contains a single circular chromosome with a defined architecture. DNA replication initiates at a single origin called oriC. Two replication forks are assembled and proceed in opposite directions until they fuse in a specialised zone opposite the origin. This termination area is flanked by polar replication fork pause sites that allow forks to enter, but not to leave. Thus, the chromosome is divided into two replichores, each replicated by a single replication fork. Recently, we analysed the replication parameters in E. coli cells, in which an ectopic origin termed oriZ was integrated in the right-hand replichore. Two major obstacles to replication were identified: (1) head-on replicationâ»transcription conflicts at highly transcribed rrn operons, and (2) the replication fork trap. Here, we describe replication parameters in cells with ectopic origins, termed oriX and oriY, integrated into the left-hand replichore, and a triple origin construct with oriX integrated in the left-hand and oriZ in the right-hand replichore. Our data again highlight both replicationâ»transcription conflicts and the replication fork trap as important obstacles to DNA replication, and we describe a number of spontaneous large genomic rearrangements which successfully alleviate some of the problems arising from having an additional origin in an ectopic location. However, our data reveal additional factors that impact efficient chromosome duplication, highlighting the complexity of chromosomal architecture.
RESUMO
Duplication of bacterial chromosomes is initiated via the assembly of two replication forks at a single defined origin. Forks proceed bi-directionally until they fuse in a specialised termination area opposite the origin. This area is flanked by polar replication fork pause sites that allow forks to enter but not to leave. The precise function of this replication fork trap has remained enigmatic, as no obvious phenotypes have been associated with its inactivation. However, the fork trap becomes a serious problem to cells if the second fork is stalled at an impediment, as replication cannot be completed, suggesting that a significant evolutionary advantage for maintaining this chromosomal arrangement must exist. Recently, we demonstrated that head-on fusion of replication forks can trigger over-replication of the chromosome. This over-replication is normally prevented by a number of proteins including RecG helicase and 3' exonucleases. However, even in the absence of these proteins it can be safely contained within the replication fork trap, highlighting that multiple systems might be involved in coordinating replication fork fusions. Here, we discuss whether considering the problems associated with head-on replication fork fusion events helps us to better understand the important role of the replication fork trap in cellular metabolism.