Role of fucosyltransferase-VII and pselectin glycoprotein ligant-1 in the suppressor activity of CD25+CD4+ regulatory T cells.

Ricerca sulla caratterizzazione dei meccanismi molecolari che controllano la migrazione e la capacità soppressoria dei linfociti CD4+CD25+ (cellule T regolatorie) durante l’encefalomielite sperimentale autoimmune.ope

LFA-1 Controls Th1 and Th17 Motility Behavior in the Inflamed Central Nervous System

Leukocyte trafficking is a key event during autoimmune and inflammatory responses. The subarachnoid space (SAS) and cerebrospinal fluid are major routes for the migration of encephalitogenic T cells into the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis, and are sites of T cell activation before the invasion of CNS parenchyma. In particular, autoreactive Th1 and Th17 cell trafficking and reactivation in the CNS are required for the pathogenesis of EAE. However, the molecular mechanisms controlling T cell dynamics during EAE are unclear. We used two-photon laser microscopy to show that autoreactive Th1 and Th17 cells display distinct motility behavior within the SAS in the spinal cords of mice immunized with the myelin oligodendrocyte glycoprotein peptide MOG(35-55). Th1 cells showed a strong directional bias at the disease peak, moving in a straight line and covering long distances, whereas Th17 cells exhibited more constrained motility. The dynamics of both Th1 and Th17 cells were strongly affected by blocking the integrin LFA-1, which interfered with the deformability and biomechanics of Th1 but not Th17 cells. The intrathecal injection of a blocking anti-LFA-1 antibody at the onset of disease significantly inhibited EAE progression and also strongly reduced neuro-inflammation in the immunized mice. Our results show that LFA-1 plays a pivotal role in T cell motility during EAE and suggest that interfering with the molecular mechanisms controlling T cell motility can help to reduce the pathogenic potential of autoreactive lymphocytes

An unexpected link between fatty acid synthase and cholesterol synthesis in proinflammatory macrophage activation

Different immune activation states require distinct metabolic features and activities in immune cells. For instance, inhibition of fatty acid synthase (FASN), which catalyzes the synthesis of long-chain fatty acids, prevents the proinflammatory response in macrophages; however, the precise role of this enzyme in this response remains poorly defined. Consistent with previous studies, we found here that FASN is essential for lipopolysaccharide-induced, Toll-like receptor (TLR)-mediated macrophage activation. Interestingly, only agents that block FASN upstream of acetoacetyl-CoA synthesis, including the well-characterized FASN inhibitor C75, inhibited TLR4 signaling, while those acting downstream had no effect. We found that acetoacetyl-CoA could overcome C75's inhibitory effect, whereas other FASN metabolites, including palmitate, did not prevent C75-mediated inhibition. This suggested an unexpected role for acetoacetyl-CoA in inflammation that is independent of its role in palmitate synthesis. Our evidence further suggested that acetoacetyl-CoA arising from FASN activity promotes cholesterol production, indicating a surprising link between fatty acid synthesis and cholesterol synthesis. We further demonstrate that this process is required for TLR4 to enter lipid rafts and facilitate TLR4 signaling. In conclusion, we have uncovered an unexpected link between FASN and cholesterol synthesis that appears to be required for TLR signal transduction and proinflammatory macrophage activation

Glutathione Transferase Omega-1 Regulates NLRP3 Inflammasome Activation through NEK7 Deglutathionylation

The NLRP3 inflammasome is a cytosolic complex sensing phagocytosed material and various damage-associated molecular patterns, triggering production of the pro-inflammatory cytokines interleukin-1 beta (IL)-1β and IL-18 and promoting pyroptosis. Here, we characterize glutathione transferase omega 1-1 (GSTO1-1), a constitutive deglutathionylating enzyme, as a regulator of the NLRP3 inflammasome. Using a small molecule inhibitor of GSTO1-1 termed C1-27, endogenous GSTO1-1 knockdown, and GSTO1-1−/− mice, we report that GSTO1-1 is involved in NLRP3 inflammasome activation. Mechanistically, GSTO1-1 deglutathionylates cysteine 253 in NIMA related kinase 7 (NEK7) to promote NLRP3 activation. We therefore identify GSTO1-1 as an NLRP3 inflammasome regulator, which has potential as a drug target to limit NLRP3-mediated inflammation.We would like to acknowledge the following grants: the National Health and Medical Research Council of Australia (NHMRC) is thanked for Project Grant APP1124673 to P.G.B., M.G.C., and L.A.J.O.; Principal Research Fellowship 1117602 to J.B.B.; and NHMRC Project Grant APP1156455 to J.B.B., P.G.B., and M.G.C. The O’Neill laboratory acknowledges the following grant support: European Research Council (ECFP7-ERC-MICROINNATE) and Science Foundation Ireland Investigator Award (SFI 12/IA/1531)

A role for leukocyte-endothelial adhesion mechanisms in epilepsy

The mechanisms involved in the pathogenesis of epilepsy, a chronic neurological disorder that affects approximately 1 percent of the world population, are not well understood1–3. Using a mouse model of epilepsy, we show that seizures induce elevated expression of vascular cell adhesion molecules and enhanced leukocyte rolling and arrest in brain vessels mediated by the leukocyte mucin P-selectin glycoprotein ligand-1 (PSGL-1) and leukocyte integrins α4β1 and αLβ2. Inhibition of leukocyte-vascular interactions either with blocking antibodies, or in mice genetically deficient in functional PSGL-1, dramatically reduced seizures. Treatment with blocking antibodies following acute seizures prevented the development of epilepsy. Neutrophil depletion also inhibited acute seizure induction and chronic spontaneous recurrent seizures. Blood-brain barrier (BBB) leakage, which is known to enhance neuronal excitability, was induced by acute seizure activity but was prevented by blockade of leukocyte-vascular adhesion, suggesting a pathogenetic link between leukocyte-vascular interactions, BBB damage and seizure generation. Consistent with potential leukocyte involvement in the human, leukocytes were more abundant in brains of epileptics than of controls. Our results suggest leukocyte-endothelial interaction as a potential target for the prevention and treatment of epilepsy

ROLE OF THE MUCIN P-SELECTIN GLYCOPROTEIN LIGAND-1 IN THE CONTROL OF IMMUNE SUPPRESSION EXERTED BY REGULATORY T CELLS

La 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

Role of fucosyltransferase-VII and pselectin glycoprotein ligant-1 in the suppressor activity of CD25+CD4+ regulatory T cells.

Ricerca sulla caratterizzazione dei meccanismi molecolari che controllano la migrazione e la capacità soppressoria dei linfociti CD4+CD25+ (cellule T regolatorie) durante l’encefalomielite sperimentale autoimmune

Selectin-mediated leukocyte trafficking during the development of autoimmune disease

Tissue 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

CD4+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-\u3b3 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 na\uefve cells. In addition, activated Tregs display increased migration capacity in inflamed brain in mice with EAE. Both activated and na\uefve 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