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ROLE OF THE MUCIN P-SELECTIN GLYCOPROTEIN LIGAND-1 IN THE CONTROL OF IMMUNE SUPPRESSION EXERTED BY REGULATORY T CELLS
No full textLa mucina P-selectin glycoprotein ligand-1 (PSGL-1) lega le selectine L-, E- e P-selectin e controlla il traffico leucocitario in condizioni fisiologiche e patologiche. Il ruolo di PSGL-1 nell’induzione e nello sviluppo di malattie autoimmuni non è chiaro, ma PSGL-1 sembra essere importante nell’omeostasi immunologia dei linfociti T. In questo progetto di Dottorato abbiamo analizzato il ruolo della mucina PSGL-1 nell’induzione dell’encefalomielite sperimentale autoimmune (EAE), una patologia infiammatoria autoimmune a carico del sistema nervoso centrale (SNC), utilizzata come modello sperimentale della sclerosi multipla umana. Partendo dall’osservazione che l’induzione attiva di EAE con il peptide mielinico myelin olygodendrocyte glycoprotein (MOG)35-55 in animali PSGL-1-/- porta ad una malattia più grave rispetto a quella sviluppata da topi WT (wild-type), abbiamo ipotizzato un ruolo del PSGL-1 in meccanismi regolatori coinvolti nello sviluppo della malattia. Questa ipotesi è inoltre accompagnata dal fatto che, in un modello passivo di EAE, cellule T encefalitogeniche MOG35-55-specifiche prodotte da animali PSGL-1-/- inducono una malattia più severa rispetto a cellule encefalitogeniche WT. La nostra attenzione si è rivolta in particolare verso le cellule T regolatorie CD4+CD25+Foxp3+ (Tregs), che regolano l’insorgenza di risposte infiammatorie autoimmuni e sono essenziali per limitare la gravità dell’EAE. In un modello attivo di EAE, abbiamo dimostrato che cellule Tregs deficienti in PSGL-1, iniettate al giorno +7 post-immunizzazione (fase pre-clinica), non sono in grado di sopprimere lo sviluppo dell’EAE, rispetto a Tregs WT. Inoltre, saggi di proliferazione in vitro hanno evidenziato una ridotta capacità delle Tregs deficienti in PSGL-1 di inibire la proliferazione di linfociti T CD4+CD25-. Partendo da tali osservazioni, abbiamo ipotizzato che la mancanza di PSGL-1 sulle cellule Tregs abbia un doppio effetto sulla loro funzionalità, nella fase pre-clinica di malattia:
1. un possibile deficit nel traffico delle Tregs all’interno del SNC infiammato in corso di malattia;
2. un blocco della capacità delle Tregs di sopprimere in vivo l’attivazione dei linfociti T encefalitogenici all’interno dei linfonodi drenanti di topi immunizzati per EAE.
In base a tali ipotesi, abbiamo sviluppato il nostro progetto con due obiettivi:
OBIETTIVO 1: ANALISI DEL TRAFFICO DELLE Tregs NEL SNC IN CORSO DI EAE. Utilizzando tecniche di microscopia intravitale abbiamo dimostrato che cellule Tregs deficienti in PSGL-1 sono sostanzialmente incapaci di interagire con vasi piali cerebrali infiammati. Saggi di migrazione in vivo nel SNC infiammato hanno mostrato che le cellule T regolatorie migrano all’interno del SNC preferenzialmente nella fase pre-clinica di malattia (giorno +7 post-immunizzazione), rispetto al picco di malattia (giorno +14 post-immunizzazione). Inoltre, abbiamo osservato che cellule Tregs PSGL-1-/- sono incapaci di migrare all’interno del SNC infiammato nella fase pre-clinica di malattia (giorno +7 post-immunizzazione), rispetto a cellule Tregs WT. I nostri risultati dimostrano inoltre che la mucina PSGL-1 è funzionalmente attiva sulla superficie di Tregs naïve, al contrario dei linfociti CD4+CD25- naïve, suggerendo un ruolo essenziale per questa molecola nel traffico e nell’omeostasi delle cellule T regolatorie all’interno del sistema immunitario.
IPOTESI 2: ANALISI DELLA AZIONE SOPPRESSORIA DELLE Tregs SULL’ATTIVAZIONE DELLE CELLULE T ANTIGENE-SPECIFICHE NEI LINFONODI DRENANTI DI TOPI CON EAE. Utilizzando la tecnica della microscopia laser multi-fotonica, abbiamo studiato il comportamento di cellule MOG35-55-specifiche all’interno dei linfonodi drenanti di animali immunizzati, in presenza o assenza di Tregs WT o PSGL-1-/- esogene. In particolare, l’analisi è stata suddivisa in due fasi distinte: una fase pre-clinica “precoce” (giorno +1 post-immunizzazione) ed una fase pre-clinica “tardiva” (giorno +7 post-immunizzazione) di malattia. Come precedentemente descritto in letteratura, al giorno +1 post-immunizzazione la presenza di Tregs WT causa un aumento della motilità ed una diminuzione del coefficiente di arresto dei linfociti T MOG35-55-specifici, a causa di una riduzione dei tempi di contatto con le cellule dendritiche presentanti l’antigene. In questa fase precoce, le cellule Tregs PSGL-1-/- sono pienamente efficienti nel modulare la motilità dei linfociti T encefalitogenici. Al contrario, al giorno +7 post-immunizzazione le Tregs deficienti in PSGL-1 non sono in grado di modulare motilità e proliferazione dei linfociti MOG35-55-specifici, mentre le Tregs WT, come al giorno +1, influenzano notevolmente il comportamento dei linfociti T encefalitogenici.
In conclusione, i nostri dati suggeriscono un ruolo chiave per la mucina PSGL-1 nel reclutamento delle cellule Tregs all’interno del SNC infiammato durante la fase pre-clinica dell’EAE; l’espressione di PSGL-1 sulle Tregs sembra inoltre essere essenziale per una efficiente soppressione dell’attivazione dei linfociti T encefalitogenici all’interno dei linfonodi drenanti nella fase pre-clinica “tardiva” di malattia. Globalmente, questi risultati dimostrano un ruolo chiave di PSGL-1 nella funzionalità delle Tregs in un modello di malattia cronica autoimmune.Mucin P-selectin glycoprotein ligand-1 (PSGL-1) binds L-, E- and P-selectin and controls leukocyte trafficking under physiological and pathological conditions. Whereas PSGL-1 role is emerging in T cell homeostasis, its involvement in the induction of autoimmune diseases is not clear. In this PhD project, we studied the role of the mucin PSGL-1 in the induction and development of experimental autoimmune encephalomyelitis (EAE), an autoimmune and inflammatory pathology of the central nervous system (CNS), used as experimental model for human multiple sclerosis. Here we show that active EAE induced with myelin olygodendrocyte glycoprotein (MOG)35-55 peptide was more severe in PSGL-1-/- mice, compared to WT (wild-type) animals. In addition, MOG35-55-specific T cells produced from PSGL-1 deficient mice transferred a significantly more severe disease than WT cells in a passive-transfer model of EAE, suggesting a role for PSGL-1 in regulatory mechanisms during EAE. In particular, we focused our attention on CD4+CD25+Foxp3+ regulatory T (Treg) cells, which are known to regulate autoimmune pathologies development and are crucial in controlling EAE severity. We found that PSGL-1 deficient Tregs, compared to WT Tregs, were not able to suppress EAE when injected in the pre-clinical phase of the disease (day +7 post-immunization). Moreover, in vitro proliferation assays shown that PSGL-1-/- Tregs have a reduced ability to suppress CD4+CD25- T cells proliferation. These results suggested us a dual effect of PSGL-1 deficiency on Treg functionality in the pre-clinical phase of EAE:
1. a possible deficit in Treg trafficking into the inflamed CNS during EAE development;
2. a reduced suppression by Tregs of in vivo encephalitogenic T cells activation in draining lymph nodes of EAE mice.
Based on these hypotheses, we developed our project with two aims:
AIM 1: ANALYSIS OF Treg TRAFFICKING IN THE CNS DURING EAE. Intravital microscopy experiments showed that PSGL-1 deficient Tregs display a dramatic decrease of adhesive interactions in inflamed brain pial vessels, compared to WT Tregs. By performing in vivo migration assays in the inflamed CNS, we observed that regulatory T cells preferentially migrate in the CNS in the pre-clinical phase of the disease (day +7 post-immunization), compared to the disease peak (day +14 post-immunization), and Tregs from PSGL-1-/- mice present a strongly reduced migration capacity into the inflamed CNS in the pre-clinical phase of EAE, when compared to WT Tregs. Our results also demonstrated that PSGL-1 is functionally active on naïve Tregs, compared to naïve CD4+CD25- T cells, suggesting a crucial role for PSGL-1 in Treg homeostasis and trafficking in the immune system.
AIM 2: ANALYSIS OF Treg-MEDIATED SUPPRESSION OF ANTIGEN-SPECIFIC T CELL ACTIVATION IN DRAINING LYMPH NODES OF EAE MICE. By using two-photon laser microscopy, we studied MOG35-55-specific T cell behaviour in draining lymph nodes of EAE mice, in the presence or absence of exogenous WT or PSGL-1-/- Tregs. We analyzed two different time points: an “early” pre-clinical phase of EAE (day +1 post-immunization) and a “late” pre-clinical phase of EAE (day +7 post-immunization). In the early pre-clinical phase of EAE (day +1 post-immunization), as previously shown, we found that MOG35-55-specific T cells showed a significant increase in their motility and decrease of the arrest coefficient in the presence of WT Tregs in draining lymph nodes of EAE mice, due to reduced contact times with antigen-presenting dendritic cells. At this time point, also PSGL-1 deficient Tregs efficiently modulated encephalitogenic T cells behaviour. On the contrary, PSGL-1-/- Tregs failed to modulate the motility behaviour and proliferation of MOG35-55-specific T cells during the late (day +7 post-immunization) pre-clinical phase of the disease, while exogenous WT Tregs still affect MOG35-55-specific T cells motility.
In conclusion, our data demonstrate that PSGL-1 has a key role in CD4+CD25+Foxp3+ Treg migration in the CNS during the pre-clinical phase of EAE; moreover, PSGL-1 expression is necessary for an efficient suppression of late antigen-dependent T cell activation exerted by Tregs in the draining lymph nodes of EAE mice. Overall, our results demonstrate a previously unknown key role for PSGL-1 in Treg functionality in a model of autoimmune disease
Selectins and their ligands as potential immunotherapeutic targets in neurological diseases
No full textSelectins are a family of adhesion receptors that bind to highly glycosylated molecules expressed on the surface of leukocytes and endothelial cells. The interactions between selectins and their ligands control tethering and rolling of leukocytes on the vascular wall during the process of leukocyte migration into the tissues under physiological and pathological conditions. In recent years, it has been shown that leukocyte recruitment in the CNS plays a pivotal role in diseases such as multiple sclerosis, ischemic stroke, epilepsy and traumatic brain injury. In this review, we discuss the role of selectins in leukocyte-endothelial interactions in the pathogenesis of neurological diseases, highlighting new findings suggesting that selectins and their ligands may represent novel potential therapeutic targets for the treatment of CNS diseases
Regulation of T cell trafficking by the T cell immunoglobulin and mucin domain 1 glycoprotein
No full textLeukocyte trafficking is generally considered the initial stage of any immune response, and it involves a multistep intravascular process including capture, rolling, activation, arrest, crawling, and transmigration. Both capture and rolling are predominantly mediated by selectins, which allow circulating leukocytes to sense activating signals on the endothelium and adhere to vessel walls. In this review, we discuss recent data showing that the T cell immunoglobulin and mucin domain 1 (TIM-1) protein is a major ligand for endothelial P-selectin, mediating T helper (Th) cell Th1 and Th17 trafficking in inflamed tissues. We highlight structural and functional features showing that TIM-1 can be included in the restricted group of major adhesion receptors involved in leukocyte trafficking with a pathophysiological role in inflammation and autoimmunity
Selectin-mediated leukocyte trafficking during the development of autoimmune disease
No full textTissue inflammation is a finely regulated process that controls wound healing and allows the clearance of damaged cells, pathogens and irritants. However, excessive or uncontrolled inflammation is detrimental, causing tissue damage and leading to autoimmunity. The recruitment of circulating leukocytes to the target tissue is a key stage in the inflammatory process, and is controlled by a multistep cascade in which adhesive receptors known as selectins mediate initial leukocyte tethering and rolling along vascular surfaces, which is required for their subsequent adhesion and arrest. This review considers the role of selectins and their ligands in the recruitment of circulating leukocytes to peripheral tissues during inflammatory responses that lead to the development of autoimmunity, focusing on data from animal models and clinical trials suggesting that selectins may offer valuable therapeutic targets for the treatment of autoimmune diseases
ROLE OF FUCOSYLTRANSFERASE-VII AND P-SELECTIN GLYCOPROTEIN LIGAND-1 IN THE MIGRATION OF CD4+CD25+ REGULATORY CELLS IN INFLAMED BRAIN
No full textCD4+CD25+ regulatory T (Treg) cells participate in immunologic homeostasis by active suppression of inappropriate immune responses and are able to inhibit a variety of autoimmune and inflammatory diseases. Treg cells inhibit the activation of autoreactive T cells and suppress organ-specific autoimmunity. The mechanisms of Treg cells involved in the regulation of experimental autoimmune encephalomyelitis (EAE) are not well understood. Recent studies have shown a direct involvment of Treg cells in the natural resolution of EAE within the central nervous system (CNS) and a strong correlation between their migration pattern and their ability to control inflammatory responses. However, the molecular mechanisms controlling the migration of Tregs in inflamed brain are not known. P-selectin glycoprotein ligand-1 (PSGL-1) and alpha (1,3) fucosyltransferases (FucT), enzymes that catalyze the glycosylation of PSGL-1 and control its functionality, are molecules involved in the migration of leukocytes in sites of inflammation. The GOAL of this study was to determine the role of PSGL-1 in the migration of Treg cells in mice with EAE.
METHODS: Active and transfer EAE were performed in WT/C57Bl/6, FucT-VII and PSGL-1 deficient (FucT-VII-/- and PSGL-1-/-) mice using MOG35-55 peptide. CD4+CD25+ cells were obtained by magnetic cell sorting. Flow cytometry and ImageStream technology were used to determine the expression and distribution of adhesion molecules and binding capacity to P-selectin and E-selectin chimeras. Migration properties of WT, FucT-VII-/- and PSGL-1-/- Tregs were determined with in vivo migration assays using 3H-glycerol-labeled Tregs. Intravital microscopy experiments were performed in order to determine the ability of WT, FucT-VII-/- and PSGL-1-/- Tregs to interact with inflamed brain endothelium.
RESULTS: Encephalitogenic T cells produced from FucT-VII-/- and PSGL-1-/- mice transferred a significantly more severe disease that WT T cells. We observed no significant differences in the expression of adhesion molecules and IL-4 and IFN-γ production of autoreactive T cells from PSGL-1 and FucT-VII deficient mice. However, co-cultures with CD4+CD25+ Tregs and effector cells showed that deficiency of PSGL-1 and FucT-VII leads to a marked decrease of suppression capacity of Tregs. Interestingly, activated CD4+CD25+ Tregs have increased expression of functional PSGL-1 and a significantly higher suppressor activity in vitro when compared to naïve cells. In addition, activated Tregs display increased migration capacity in inflamed brain in mice with EAE. Both activated and naïve WT Treg cells preferentially migrated into the CNS in the pre-clinical phase of active EAE, than at disease onset. Treg cells from FucT-VII-/- and PSGL-1-/- mice present decreased migration ability to inflamed CNS when compared to WT Tregs. Moreover, intravital microscopy experiments showed a dramatic decrease of adhesive interactions in inflamed brain microcirculation in FucT-VII-/- and PSGL-1-/- Tregs. Finally, Tregs deficient of PSGL-1 and FucT-VII displayed a reduced capacity to suppress active EAE when compared to WT cells.
CONCLUSION: Our data demonstrate that PSGL-1 and the fucosylation of its glycans by FucT-VII are involved in the suppression mediated by CD4+CD25+ Treg cells in MOG-induced EAE. Moreover, in addition to a role in cell-cell contact required for efficient suppression, our results suggest a key role of PSGL-1 and FucT-VII activity in the recruitment of CD4+CD25+ cells into the brain of mice with EAE
Role of fucosyltransferase-VII and P-selectin glycoprotein ligand-1 in the migration of CD4+CD25+ regulatory T cells in inflamed brain
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Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Vascular inflammation in central nervous system diseases: adhesion receptors controlling leukocyte-endothelial interactions
Get PDFLeukocyte trafficking from the blood into the tissues represents a key process during inflammation and requires multiple steps mediated by adhesion molecules and chemoattractants. Inflammation has a detrimental role in several diseases, and in such cases, the molecular mechanisms controlling leukocyte migration are potential therapeutic targets. Over the past 20 years, leukocyte migration in the CNS has been investigated almost exclusively in the context of stroke and MS. Experimental models of ischemic stroke have led to the characterization of adhesion molecules controlling leukocyte migration during acute inflammation, whereas EAE, the animal model of MS, has provided similar data for chronic inflammation. Such experiments have led to clinical trials of antileukocyte adhesion therapy, with consistently positive outcomes in human subjects with MS, showing that interference with leukocyte adhesion can ameliorate chronic inflammatory CNS diseases. This review summarizes our current understanding of the roles of adhesion molecules controlling leukocyte-endothelial interactions in stroke and MS, focusing on recently discovered, novel migration mechanisms. We also discuss the growing evidence suggesting a role for vascular inflammation and leukocyte trafficking in neurodegenerative diseases such as AD. Moreover, we highlight recent findings suggesting a role for leukocyte-endothelial interactions in the pathogenesis of seizures and epilepsy, thus linking endothelial activation and leukocyte trafficking to neuronal electrical hyperactivity. These emerging roles for leukocytes and leukocyte adhesion mechanisms in CNS diseases provide insight into the mechanisms of brain damage and may contribute to the development of novel therapeutic strategies
Editorial: Cell-Cell Interactions Controlling Neuronal Functionality in Health and Disease
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