Role of integrins in the trafficking of Th1 and Th17 cells in the central nervous system during experimental autoimmune encephalomyelitis

La Sclerosi Multipla (SM) \ue8 una patologia infiammatoria autoimmune cronica, disabilitante e demielinizzante, del sistema nervoso centrale (SNC). La migrazione di cellule T autoreattive e la loro riattivazione attraverso la presentazione antigenica effettuata dalle locali cellule presentanti l\u2019antigene, rappresentano due eventi critici nella patogenesi della SM e nel suo modello animale, l\u2019encefalite autoimmune sperimentale (EAE). Le risposte ai potenziali antigeni all\u2019interno del SNC richiedono una migrazione a lungo raggio, comunicazione a corto raggio e contatti cellula-cellula diretti con le cellule presentanti l\u2019antigene. Il principale obiettivo di questo studio \ue8 indagare il ruolo delle integrine L2 (LFA-1) e 4 (VLA-4 and 47) nella migrazione e nella motilit\ue0 delle cellule autoreattive Th1 e Th17 in corso di EAE all\u2019interno del midollo spinale utilizzando le moderne tecniche di microscopia intravitale. La microscopia intravitale permette di visualizzare questi processi dentro il midollo spinale, il quale rappresenta il principale sito di infiammazione durante l\u2019EAE. Mediante la microscopia intravitale in epifluorescenza abbiamo prima di tutto investigato il ruolo delle integrine 4 e LFA-1 nella migrazione delle cellule autoreattive Th1 e Th17 all\u2019interno dei vasi piali del midollo spinale durante la fase preclinica, l\u2019esordio e la fase cronica di EAE. Abbiamo utilizzato un modello di EAE immunizzando topi C57BL/6 con il peptide MOG35-55. Le cellule Th1 e Th17 MOG-specifiche sono state prodotte in vitro da topi TCR-transgenici 2D2, marcate con label fluorescenti e iniettate in endovena nei topi immunizzati direttamente prima dell\u2019acquisizione. I nostri risultati pongono l\u2019accento su un ruolo selettivo per l\u2019integrina LFA-1 nella migrazione delle cellule Th1 in particolare durante le prime fasi di malattia. Ruolo che invece non \ue8 svolto in fase cronica di malattia. Inoltre, il blocco della subunit\ue0 4, ma non dell\u2019integrina 47 inibisce fortemente sia il rotolamento sia l\u2019adesione stabile delle cellule Th1 all\u2019endotelio infiammato. Questo suggerisce l\u2019integrina VLA-4 come principale mediatrice della migrazione delle Th1 nel SNC infiammato in corso di EAE. Di notevole interesse \ue8 inoltre il selettivo ruolo per l\u2019integrina 47 evidenziato in particolare nell\u2019adesione stabile esclusivamente delle cellule Th17 all\u2019esordio ed in fase cronica di malattia. Successivamente, per studiare la motilit\ue0 intraparenchimale delle cellule Th1 e Th17 nel midollo spinale durante la fase preclinica della patologia, il picco di malattia e la fase cronica abbiamo utilizzato la microsocopia laser a due fotoni. I nostri risultati dimostrano una massiva infiltrazione di Th1 e Th17 nel parenchima del SNC al picco di malattia, mentre la migrazione di queste cellule durante le altre fasi della malattia \ue8 significativamente minore. In seguito, il nostro studio si \ue8 focalizzato sulla fase clinica della malattia e abbiamo osservato che le cellule Th1 e le cellule Th17 mostrano differenze significative nelle componenti direzionali, con le cellule Th1 che si muovono velocemente in direzione rettilinea, coprendo lunghe distanze nel parenchima del midollo spinale, mente le cellule Th17 girano intorno in un volume specifico del tessuto facendo stop and go. In particolare, il blocco dell\u2019integrina LFA-1 influenza drasticamente le dinamiche delle cellule, portando ad una riduzione nella velocit\ue0 e interferendo con il loro pattern di motilit\ue0 rettilinea. Inoltre, in presenza di un anticorpo bloccante anti-LFA-1, le cellule Th17 mostrano una riduzione di velocit\ue0 drastica. Diversamente, l\u2019anticorpo anti-4 non ha nessun effetto sul comportamento motile delle Th1, ma riduce fortemente la velocit\ue0 delle Th17 suggerendo che l\u2019integrina VLA-4 non sia richiesta per la motilit\ue0 intraparenchimale durante l\u2019EAE. Tuttavia, alla luce dei risultati ottenuti in precedenza mediante microscopia intravitale, va preso in considerazione un selettivo ruolo esercitato dall\u2019integrina 47 nella motilit\ue0 intraparenchimale delle Th17. Complessivamente, i nostri risultati suggeriscono che l\u2019LFA-1 sia la principale integrina che controlla la motilit\ue0 delle cellule Th1 e Th17 e che 47 sia invece selettivamente coinvolta nella motilit\ue0 delle cellule Th17 nel midollo spinale in condizioni infiammatorie al picco di malattia. A sostegno di questi risultati abbiamo testato un trattamento terapeutico mediante blocco locale, a livello intratecale, delle integrine LFA-1 e 47 nel nostro modello murino di EAE cronica. Entrambi i trattamenti hanno evidenziato una riduzione nello sviluppo della malattia che suggerisce l\u2019importanza di interferire direttamente con le dinamiche delle cellule pro infiammatorie a livello del SNC. Approfondire dunque la conoscenza dei meccanismi molecolari che controllano la motilit\ue0 intratissutale di cellule T attivate nel SNC potrebbe aiutarci a identificare nuove strategie terapeutiche per le patologie autoimmuni cerebrali croniche.Multiple sclerosis (MS) is a chronic disabling autoimmune inflammatory demyelinating disease of the central nervous system (CNS). The migration of autoreactive T cells from the blood into the CNS and their reactivation through antigen presentation by local antigen presenting cells (APCs) represent critical events in the pathogenesis of MS and its animal model, the experimental autoimmune encephalomyelitis (EAE). The responses to potential antigens inside the CNS require long-range migration of cells, short-range communication and direct cell-cell contact with APCs. The main goal of this study was to investigate the role of L2 (LFA-1) and 4 (VLA-4 and 47) integrins in the migration and motility behavior of Th1 and Th17 cells, which represent key players in the induction of EAE, using intravital microscopy approaches. Intravital microscopy techniques allow the visualization of T cell migration and reactivation in the spinal cord (SC), which represents the main inflammation site during EAE. By using epifluorescence intravital microscopy (IVM) we first studied the roles of 4 and LFA-1 integrins in Th1 and Th17 cell adhesion in the pial vessels of spinal cord (SC) venules in mice immunized with MOG35-55 peptide during the preclinical phase, disease onset and chronic phase of disease. We used an EAE model by immunization of C57BL/6 mice with MOG35-55 peptide. MOG35-55-specific Th1 and Th17 cells were produced in vitro from 2D2 TCR transgenic mice, labeled with fluorescent dyes and intravenously injected in immunized mice before imaging. Our results underlined a selective role for LFA-1 integrin in Th1 cell recruitment in inflamed SC vessels during the early phases of the disease but not during the chronic phase. Moreover, blocking antibodies against the 4\uf020subunit, but not blockade of 47 integrin greatly inhibited rolling and firm adhesion of Th1 cells in the SC venules during all disease phases, suggesting that VLA-4 is the major molecule involved in Th1 cell adhesion in the SC venules during EAE. Interestingly, blockade of 47 integrin led to a significant reduction of firm adhesion in Th17 cells at the onset and chronic phase of EAE indicating a selective role of 47 integrin in the recruitment of Th17 cells in the inflamed CNS. Taking advantage of two-photon laser microscopy (TPLM) approach we next investigated the motility behavior of fluorescently labeled Th1 and Th17 cells within SC parenchyma during different disease phases. Our results showed a massive infiltration of Th1 and Th17 cells in the CNS parenchyma at disease peak, whereas the migration of these cells during other phases of disease was significantly lower. Furthermore, Th1 and Th17 cells displayed significant differences in the directional component, with Th1 cells moving faster in straight directions covering long distances deep in the SC parenchyma, whereas Th17 cells moved around in a specific volume of tissue in a stop and go mode. Notably, the blockade of LFA-1 integrin drastically affected the dynamics of Th1 cells leading to a reduction in velocity and interfering with their straight-line motility pattern. Moreover, Th17 cells displayed a drastic reduction of velocity in the presence of a blocking anti-LFA-1 antibody. The analysis of cellular morphology suggested that LFA-1 is actively involved in the cytoskeleton rearrangements necessary for T cell amoeboid migration inside the CNS, but had no role in the cytoskeleton dynamics in Th17 cells. Notably, 4\uf020integrins had no role in Th1 cells motility, but drastically reduced the dynamics of Th17 cells inside the SC parenchyma. To check the therapeutic relevance of our intravital microscopy findings, we performed intrathecal injection of anti-LFA-1 or anti-47 antibodies at disease onset and observed a significant inhibition of EAE progression in mice immunized with MOG35-55 peptide. Collectively, our data demonstrate that LFA-1 integrin differently controls intraparenchymal Th1 and Th17 cells dynamics, whereas 47 integrin is selectively involved in Th17 cell trafficking in the CNS during EAE. Furthermore, our results suggest that interfering with the molecular mechanisms controlling intraparenchymal dynamics of activated T cells may represent a new therapeutic strategy for CNS autoimmune diseases

"Open Sesame" to the complexity of pattern recognition receptors of myeloid-derived suppressor cells in cancer

Pattern recognition receptors are primitive sensors that arouse a preconfigured immune response to broad stimuli, including nonself pathogen-associated and autologous damage-associated molecular pattern molecules. These receptors are mainly expressed by innate myeloid cells, including granulocytes, monocytes, macrophages, and dendritic cells. Recent investigations have revealed new insights into these receptors as key players not only in triggering inflammation processes against pathogen invasion but also in mediating immune suppression in specific pathological states, including cancer. Myeloid-derived suppressor cells are preferentially expanded in many pathological conditions. This heterogeneous cell population includes immunosuppressive myeloid cells that are thought to be associated with poor prognosis and impaired response to immune therapies in various cancers. Identification of pattern recognition receptors and their ligands increases the understanding of immune-activating and immune-suppressive myeloid cell functions and sheds light on myeloid-derived suppressor cell differences from cognate granulocytes and monocytes in healthy conditions. This review summarizes the different expression, ligand recognition, signaling pathways, and cancer relations and identifies Toll-like receptors as potential new targets on myeloid-derived suppressor cells in cancer, which might help us to decipher the instruction codes for reverting suppressive myeloid cells toward an antitumor phenotype

Pantethine treatment is effective in recovering the disease phenotype induced by ketogenic diet in a pantothenate kinase-associated neurodegeneration mouse model

Pantothenate kinase-associated neurodegeneration, caused by mutations in the PANK2 gene, is an autosomal recessive disorder characterized by dystonia, dysarthria, rigidity, pigmentary retinal degeneration and brain iron accumulation. PANK2 encodes the mitochondrial enzyme pantothenate kinase type 2, responsible for the phosphorylation of pantothenate or vitamin B5 in the biosynthesis of co-enzyme A. A Pank2 knockout (Pank2(−/−)) mouse model did not recapitulate the human disease but showed azoospermia and mitochondrial dysfunctions. We challenged this mouse model with a low glucose and high lipid content diet (ketogenic diet) to stimulate lipid use by mitochondrial beta-oxidation. In the presence of a shortage of co-enzyme A, this diet could evoke a general impairment of bioenergetic metabolism. Only Pank2(−/−) mice fed with a ketogenic diet developed a pantothenate kinase-associated neurodegeneration-like syndrome characterized by severe motor dysfunction, neurodegeneration and severely altered mitochondria in the central and peripheral nervous systems. These mice also showed structural alteration of muscle morphology, which was comparable with that observed in a patient with pantothenate kinase-associated neurodegeneration. We here demonstrate that pantethine administration can prevent the onset of the neuromuscular phenotype in mice suggesting the possibility of experimental treatment in patients with pantothenate kinase-associated neurodegeneration

Extracellular vesicles from adipose mesenchymal stem cells target inflamed lymph nodes in experimental autoimmune encephalomyelitis

Background aims: Adipose mesenchymal stem cells (ASCs) represent a promising therapeutic approach in inflammatory neurological disorders, including multiple sclerosis (MS). Recent lines of evidence indicate that most biological activities of ASCs are mediated by the delivery of soluble factors enclosed in extracellular vesicles (EVs). Indeed, we have previously demonstrated that small EVs derived from ASCs (ASC-EVs) ameliorate experimental autoimmune encephalomyelitis (EAE), a murine model of MS. The precise mechanisms and molecular/cellular target of EVs during EAE are still unknown. Methods: To investigate the homing of ASC-EVs, we intravenously injected small EVs loaded with ultra-small superparamagnetic iron oxide nanoparticles (USPIO) at disease onset in EAE-induced C57Bl/6J mice. Histochemical analysis and transmission electron microscopy were carried out 48 h after EV treatment. Moreover, to assess the cellular target of EVs, flow cytometry on cells extracted ex vivo from EAE mouse lymph nodes was performed. Results: Histochemical and ultrastructural analysis showed the presence of labeled EVs in lymph nodes but not in lungs and spinal cord of EAE injected mice. Moreover, we identified the cellular target of EVs in EAE lymph nodes by flow cytometry: ASC-EVs were preferentially located in macrophages, with a consistent amount also noted in dendritic cells and CD4+ T lymphocytes. Conclusions: This represents the first direct evidence of the privileged localization of ASC-EVs in draining lymph nodes of EAE after systemic injection. These data provide prominent information on the distribution, uptake and retention of ASC-EVs, which may help in the development of EV-based therapy in MS

Blockade of \u3b14 integrins reduces leukocyte-endothelial interactions in cerebral vessels and improves memory in a mouse model of Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive decline associated with the deposition of amyloid-beta (A beta) plaques, hyperphosphorylation of tau protein, and neuronal loss. Vascular inflammation and leukocyte trafficking may contribute to AD pathogenesis, and a better understanding of these inflammation mechanisms could therefore facilitate the development of new AD therapies. Here we show that T cells extravasate in the proximity of cerebral VCAM-1(+) vessels in 3xTg-AD transgenic mice, which develop both A beta and tau pathologies. The counter-ligand of VCAM-1-alpha 4 beta 1 integrin, also known as very late antigen-4 (VLA-4) - was more abundant on circulating CD4(+) T cells and was also expressed by a significant proportion of blood CD8(+) T cells and neutrophils in AD mice. Intravital microscopy of the brain microcirculation revealed that alpha 4 integrins control leukocyte-endothelial interactions in AD mice. Therapeutic targeting of VLA-4 using antibodies that specifically block alpha 4 integrins improved the memory of 3xTg-AD mice compared to an isotype control. These antibodies also reduced neuropathological hallmarks of AD, including microgliosis, A beta load and tau hyperphosphorylation. Our results demonstrate that alpha 4 integrin-dependent leukocyte trafficking promotes cognitive impairment and AD neuropathology, suggesting that the blockade of alpha 4 integrins may offer a new therapeutic strategy in AD

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

Epidemiological study of pathogens isolated from blood in Liguria during 2011

Objectives. An epidemiological study addressed to identify the most represented pathogens isolated from blood and to evaluate their antibiotic susceptibility patterns, was conducted. Methods. Five clinical microbiology laboratories, homogenously distributed in Liguria, were required to collected all consecutive non-duplicates strains isolated from blood cultures during March 2011 to May 2011. the strains were sent to the reference laboratory (Section of Microbiology, DISC, University of Genoa, Italy). Results. A total of 159 microorganisms were enrolled, including 81 Gram positive, 69 Gram negative and 9 fungi.The most represented pathogens were: Escherichia coli (35), Staphylococcus aureus (26), S. epidermidis (20), S. hominis (10). Samples were collected mainly from medicine (59 isolates).Among the staphylococci, the most active molecules were: vancomycin (100% of susceptible strains), teicoplanin (93.4%), trimethoprim-sulfamethoxazole (83.8%) and tobramycin (61.6%). Enterococci showed rates of resistance to vancomycin of 25%. Enterobacteriaceae exhibited resistance to ampicillin (76.9%), ceftriaxone (44.4%), ciprofloxacin (43.3%), trimethoprim-sulfamethoxazole (36.6%) and ceftazidime (32.2%). Conclusions. The data show a higher incidence of Gram positive (51%) in comparison to Gram negative (43.4%). Gram-positive strains showed a high resistance level to fluoroquinolones (92.3%) while Gram-negative resulted resistant to ceftriaxone (44.4%) and fluoroquinolone (43.3%)

Biogenic selenium nanoparticles: characterization, antimicrobial activity and effects on human dendritic cells and fibroblasts

Tailored nanoparticles offer a novel approach to fight antibiotic-resistant microorganisms. We analysed biogenic selenium nanoparticles (SeNPs) of bacterial origin to determine their antimicrobial activity against selected pathogens in their planktonic and biofilm states. SeNPs synthesized by Gram-negative Stenotrophomonas maltophilia [Sm-SeNPs()] and Gram-positive Bacillus mycoides [Bm-SeNPs(+)] were active at low minimum inhibitory concentrations against a number of clinical isolates of Pseudomonas aeruginosa but did not inhibit clinical isolates of the yeast species Candida albicans and C. parapsilosis. However, the SeNPs were able to inhibit biofilm formation and also to disaggregate the mature glycocalyx in both P. aeruginosa and Candida spp. The Sm-SeNPs() and Bm-SeNPs(+) both achieved much stronger antimicrobial effects than synthetic selenium nanoparticles (Ch-SeNPs). Dendritic cells and fibroblasts exposed to Sm-SeNPs(), Bm-SeNPs(+) and Ch-SeNPs did not show any loss of cell viability, any increase in the release of reactive oxygen species or any significant increase in the secretion of pro-inflammatory and immunostimulatory cytokines. Biogenic SeNPs therefore appear to be reliable candidates for safe medical applications, alone or in association with traditional antibiotics, to inhibit the growth of clinical isolates of P. aeruginosa or to facilitate the penetration of P. aeruginosa and Candida spp. biofilms by antimicrobial agents

Epidemiological study of pathogens isolated from blood in Liguria (January-April 2010)

Objectives. An epidemiological study to identify the most represented pathogens isolated from blood and to evaluate their antibiotic susceptibility patterns, was conducted. Methods. Seven clinical microbiology laboratories, homogeneously distributed in the Ligurian area,were required to collected all consecutive non-duplicates strains isolated froom blood cultures during January 2010 to April 2010. The strains were sent to the reference laboratory (Sezione di Microbiologia del DISC, University of Genoa, Italy). Results. A total of 277 microorganisms were enrolled, including 155 Gram positive and 122 Gram negative.The most represented pathogens were: Escherichia coli (68), Staphylococcus aureus (57), Staphylococcus epidermidis (32), Staphylococcus hominis (17), Pseudomonas aeruginosa (15), Klebsiella pneumoniae (15), Enterococcus faecalis (11). Samples were collected mainly from medicine (66, 33.3%, of this number was determined by E. coli), intensive care units (33, 18.2% of this number consisted of S. epidermidis), surgery (24, 33.3% consisted of E. coli) and infectious diseases (20, of which S. aureus, E. coli and S. epidermidis equally represented 20.0%).Among the Staphylococci the most active molecules were: vancomycin and teicoplanin (100% of susceptible strains), chloramphenicol (92.3%) and trimethoprim-sulfamethoxazole (89.8%). Among the OXA-R Staphylococci (81/123, 65.9%) the most active molecules were: vancomycin and teicoplanin (100% of susceptible strains), chloramphenicol (93.8%) and trimethoprim-sulfamethoxazole (84.8%). Enterococci showed rates of resistance to vancomycin of 5.9%. Enterobacteriaceae exhibited resistance to ampicillin (77.5%), trimethoprim-sulfamethoxazole (42.6%), ciprofloxacin (41.2%), ceftriaxone (37.5%), ceftazidime (28.2%), cefepime (26.7%), cefoxitin (22.1%), piperacillintazobactam (20.4%), imipenem (4.7%) and amikacin (2.9%). The Gram negative non-Enterobacteriaceae showed rates of resistance of 100% to ceftriaxone, 81.3% to trimethoprim-sulfamethoxazole, 42.1% to ciprofloxacin and piperacillin-tazobactam, 33.3% to ceftazidime, 31.6% to cefepime, 27.8% to imipenem, 26.3 % to amikacin. Conclusions. The data show a higher incidence of Gram positive (56%) in comparison to Gram negative (44%).This confirms the high incidence of oxacillino-resistance in Staphylococci in our geographic area.Against Enterobacteriaceae rates of resistance were observed in excess of 20% for all drugs tested except imipenem (4.7%) and amikacin (2.9%). The proportion of imipenem-resistant isolates was constituted of strains of K. pneumoniae carbapenemase producers

Epidemiological study of pathogens collected from blood for a period of a year (2008-2009)

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