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Galleria mellonella is an alternative animal model of infection. The use of this species presents a wide range of advantages, as its maintenance and rearing are both easy and inexpensive. Moreover, its use is considered to be more ethically acceptable than other models, it is conveniently sized for manipulation, and its immune system has multiple similarities with mammalian immune systems. Hemocytes are immune cells that help encapsulate and eliminate pathogens and foreign particles. All of these reasons make this insect a promising animal model. However, cultivating G. mellonella hemocytes in vitro is not straightforward and it has many difficult challenges. Here, we present a methodologically optimized protocol to establish and maintain a G. mellonella hemocyte primary culture. These improvements open the door to easily and quickly study the toxicity of nanoparticles and the interactions of particles and materials in an in vitro environment.

Errors in early embryogenesis are a cause of sporadic cell death and developmental failure1,2. Phagocytic activity has a central role in scavenging apoptotic cells in differentiated tissues3-6. However, how apoptotic cells are cleared in the blastula embryo in the absence of specialized immune cells remains unknown. Here we show that the surface epithelium of zebrafish and mouse embryos, which is the first tissue formed during vertebrate development, performs efficient phagocytic clearance of apoptotic cells through phosphatidylserine-mediated target recognition. Quantitative four-dimensional in vivo imaging analyses reveal a collective epithelial clearance mechanism that is based on mechanical cooperation by two types of Rac1-dependent basal epithelial protrusions. The first type of protrusion, phagocytic cups, mediates apoptotic target uptake. The second, a previously undescribed type of fast and extended actin-based protrusion that we call 'epithelial arms', promotes the rapid dispersal of apoptotic targets through Arp2/3-dependent mechanical pushing. On the basis of experimental data and modelling, we show that mechanical load-sharing enables the long-range cooperative uptake of apoptotic cells by multiple epithelial cells. This optimizes the efficiency of tissue clearance by extending the limited spatial exploration range and local uptake capacity of non-motile epithelial cells. Our findings show that epithelial tissue clearance facilitates error correction that is relevant to the developmental robustness and survival of the embryo, revealing the presence of an innate immune function in the earliest stages of embryonic development. Funding: Q.T.-R. acknowledges a grant funded by ‘The Ministerio de Ciencia, Innovación y Universidades and Fondo Social Europeo (FSE)’ (PRE2018-084393). M.I. acknowledges funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (ERC-StG-LS2-63759) and the Spanish Ministry of Economy and Competitiveness (BFU2014-55076-P). S.W. acknowledges support from the Government of Spain (MINECO’s Plan Nacional (PGC2018-098532-A-I00), Severo Ochoa (CEX2019- 000910-S) and Generalitat de Catalunya (CERCA, AGAUR). V.R. acknowledges support from the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa and MINECO’s Plan Nacional (BFU2017-86296-P)

Activating and inhibitory immune receptors play a critical role in regulating systemic and central nervous system (CNS) immune and inflammatory processes. The CD200R1 immunoreceptor induces a restraining signal modulating inflammation and phagocytosis in the CNS under different inflammatory conditions. However, it remains unknown whether CD200R1 has a role in modulating the inflammatory response after a peripheral nerve injury, an essential component of the successful regeneration. Expression of CD200R1 and its ligand CD200 was analyzed during homeostasis and after a sciatic nerve crush injury in C57Bl/6 mice. The role of CD200R1 in Wallerian Degeneration (WD) and nerve regeneration was studied using a specific antibody against CD200R1 injected into the nerve at the time of injury. We found an upregulation of CD200R1 mRNA after injury whereas CD200 was downregulated acutely after nerve injury. Blockade of CD200R1 significantly reduced the acute entrance of both neutrophils and monocytes from blood after nerve injury. When long term regeneration and functional recovery were evaluated, we found that blockade of CD200R1 had a significant effect impairing the spontaneous functional recovery. Taken together, these results show that CD200R1 has a role in mounting a successful acute inflammatory reaction after injury, and contributes to an effective functional recovery.

Phagocytosis is an inherent function of tissue macrophages for the removal of apoptotic cells and cellular debris during acute and chronic injury; however, the dynamics of this event during fibrosis development is unknown. We aim to prove that during the development of kidney fibrosis in the unilateral ureteral obstruction (UUO) model, there are some populations of macrophage with a reduced ability to phagocytose, and whether the infusion of a population of phagocytic macro-phages could reduce fibrosis in the murine model UUO. For this purpose, we have identified the macrophage populations during the development of fibrosis and have characterized their phagocytic ability and their expression of CPT1a. Furthermore, we have evaluated the therapeutic effect of macrophages overexpressing CPT1a with high phagocytic skills. We evidenced that the macro-phage population which exhibits high phagocytic ability (F4/80low-CD11b) in fibrotic animals de-creases during the progression of fibrosis while the macrophage population with lower phagocytic ability (F4/80high-CD11b) in fibrotic conditions, conversely, increases and CPT1a macrophage cell therapy with a strengthening phagocytic ability is associated with a therapeutic effect on kidney fibrosis. We have developed a therapeutic approach to reduce fibrosis in the UUO model by enrichment of the kidney resident macrophage population with a higher proportion of exogenous phagocytic macrophages overexpressing CPT1a., This research was funded by grants from the Ministerio de Economía y Competitividad, reference SAF2015–67770-R, awarded to G.H.; from the Fondo de Investigación Sanitaria (FIS), reference PI20/00900, awarded to G.H and the program Retos Colaboracion, reference: RTC2019- 007197-1, awarded G.H.

Corpora amylacea (CA) in the human brain are polyglucosan bodies that accumulate residual substances originated from aging and both neurodegenerative and infectious processes. These structures, which act as waste containers, are released from the brain to the cerebrospinal fluid, reach the cervical lymph nodes via the meningeal lymphatic system and may be phagocytosed by macrophages. Recent studies indicate that CA present certain neoepitopes (NEs) that can be recognized by natural antibodies of the IgM class, and although evidence of different kinds suggests that these NEs may be formed by carbohydrate structures, their precise nature is unknown. Here, we adapted standard techniques to examine this question. We observed that the preadsorption of IgMs with specific carbohydrates has inhibitory effects on the interaction between IgMs and CA, and found that the digestion of CA proteins had no effect on this interaction. These findings point to the carbohydrate nature of the NEs located in CA. Moreover, the present study indicates that, in vitro, the binding between certain natural IgMs and certain epitopes may be disrupted by certain monosaccharides. We wonder, therefore, whether these inhibitions may also occur in vivo. Further studies should now be carried out to assess the possible in vivo effect of glycemia on the reactivity of natural IgMs and, by extension, on natural immunity.

6 pages, 5 figures, [EN] One of the major transitions in the evolution of life was when one unicellular organism learned to eat another, allowing the establishment of food webs and the formation of complex organisms and ecosystems. Most unicellular eukaryotes known today have maintained this ancestral predation capacity and are very important players in marine food webs, where a large share of primary and secondary production is due to microbes. The smallest predators in marine ecosystems, unicellular colorless flagellated eukaryotes, are the principal agents of prokaryotic mortality and potential prey for upper trophic levels, playing a key role in the remineralization of inorganic nutrients. Here we will review our knowledge of this assemblage of small predators from an ecological and evolutionary perspective, looking at who they are, their numbers and what they do. This essential component of marine systems is beginning to be well characterized in our times, [CAT] Una de les grans transicions en l’evolució de la vida fou quan un organisme unicel·lular va aprendre a menjar-ne un altre, fet que va permetre l’establiment de les xarxes tròfiques i la formació d’organismes i ecosistemes més complexos. La majoria d’eucariotes unicel·lulars actuals han mantingut la capacitat depredadora ancestral i tenen un gran protagonisme en les xarxes tròfiques marines, on bona part de la producció primària i secundària és microbiana. Els depredadors més petits d’aquests ecosistemes, eucariotes unicel ·lulars sense pigments i amb flagel, són els agents principals de la mortalitat de procariotes marins, són preses per a nivells tròfics superiors i tenen un paper clau en la remineralització de nutrients inorgànics. En aquest treball exposarem què sabem d’aquest grup de depredadors des d’un punt de vista ecològic i evolutiu: qui són, quants n’hi ha i què fan. Aquest component essencial dels sistemes marins comença a estar ben caracteritzat, With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

Intensive training and exhausting exercise can disrupt innate and acquired immunity. The flavanone hesperidin has shown immunomodulatory properties in physiological and some pathological conditions, and positive effects on exercise-induced oxidative stress. Nevertheless, it remains uncertain whether it also prevents exhausting exercise-induced immune alterations. The aim of this study was to establish the effect of oral hesperidin supplementation on the systemic immune system in rats following an intensive training and exhausting exercise. For this purpose, female Wistar rats were randomized into an intensive training group or a sedentary group. Intensive training was induced by running in a treadmill 5 days per week (including two exhausting tests) for five weeks. Throughout the training period, 200 mg/kg of hesperidin or vehicle was administered by oral gavage three times per week. At the end, blood, thymus, spleen and macrophages were collected before, immediately after and 24 h after an additional final exhaustion test. Hesperidin supplementation enhanced natural killer cell cytotoxicity and the proportion of phagocytic monocytes, attenuated the secretion of cytokines by stimulated macrophages, prevented the leukocytosis induced by exhaustion and increased the proportion of T helper cells in the thymus, blood and spleen. These results suggest that hesperidin can prevent exhausting exercise-induced immune alterations.

Neonates are born with an immature immune system, which develops during the first stages of life. This early immaturity is more acute in preterm newborns. The aim of the present study was to set up a preterm rat model, in which representative biomarkers of innate and adaptive immunity maturation that could be promoted by certain dietary interventions are established. Throughout the study, the body weight was registered. To evaluate the functionality of the intestinal epithelial barrier, in vivo permeability to dextrans was measured and a histomorphometric study was performed. Furthermore, the blood cell count, phagocytic activity of blood leukocytes and plasmatic immunoglobulins (Ig) were determined. Preterm rats showed lower erythrocyte and platelet concentration but a higher count of leukocytes than the term rats. Although there were no changes in the granulocytes&rsquo, ability to phagocytize, preterm monocytes had lower phagocytic activity. Moreover, lower plasma IgG and IgM concentrations were detected in preterm rats compared to full-term rats, without affecting IgA. Finally, the intestinal study revealed lower permeability in preterm rats and reduced goblet cell size. Here, we characterized a premature rat model, with differential immune system biomarkers, as a useful tool for immunonutritional studies aimed at boosting the development of the immune system.

Significance Aging and neurodegenerative processes induce the formation of waste substances in the brain. Some of these substances accumulate in corpora amylacea (CA). We reveal that CA are released from the brain into the cerebrospinal fluid and are present in the cervical lymph nodes, into which cerebrospinal fluid drains through the meningeal lymphatic system. We also show that CA can be phagocytosed by macrophages. We conclude that CA can act as waste containers and hypothesize that CA are involved in a mechanism that cleans the brain. We also postulate that CA may contain clinical markers of brain disorders and may also play significant roles in some brain autoimmune diseases. These last points merit further study due to their possible clinical implications., Corpora amylacea (CA) in the human brain are granular bodies formed by polyglucosan aggregates that amass waste products of different origins. They are generated by astrocytes, mainly during aging and neurodegenerative conditions, and are located predominantly in periventricular and subpial regions. This study shows that CA are released from these regions to the cerebrospinal fluid and are present in the cervical lymph nodes, into which cerebrospinal fluid drains through the meningeal lymphatic system. We also show that CA can be phagocytosed by macrophages. We conclude that CA can act as containers that remove waste products from the brain and may be involved in a mechanism that cleans the brain. Moreover, we postulate that CA may contribute in some autoimmune brain diseases, exporting brain substances that interact with the immune system, and hypothesize that CA may contain brain markers that may aid in the diagnosis of certain brain diseases.

[Background] Macrophage are specialized cells that contributes to the removal of detrimental contents via phagocytosis. Lipid accumulation in macrophages, whether from phagocytosis of dying cells or from circulating oxidized low-density lipoproteins, alters macrophage biology and functionality. It is known that carnitine palmitoyl transferase 1-a (CPT1a) gene encodes an enzyme involved in fatty acid oxidation and, therefore, lipid content. However, the potential of CPT1a to activate macrophage phagocytic function have not been elucidated., [Methods] Using a murine macrophage cell line, RAW264.7, we determine if intracellular accumulation of 7-ketocholesterol (7-KC) modulates macrophage phagocytic function through CPT1a gene expression. In addition, the effects of CPT1a genetic modification on macrophage phenotype and phagocytosis has been studied., [Results] Our results revealed that CPT1a gene expression decreased by the accumulation of 7-KC at the higher dose of 7-KC. This was concomitant with an impair ability to phagocytize bioparticles and an inflammatory phenotype. GW3965 treatment, which have shown to facilitate the efflux of cholesterol, eliminated the intracellular lipid droplets of 7-KC-laden macrophages, increased the gene expression of CPT1a, diminished the gene expression of the inflammatory marker iNOS and restored macrophage phagocytosis. Furthermore, CPT1a Knockdown per se was detrimental for macrophage phagocytosis whereas transcriptional activation of CPT1a heightened the uptake of bioparticles., [Conclusions] Altogether, our findings indicate that downregulation of CPT1a by lipid content modulates macrophage phagocytosis and inflammatory phenotype., This study was supported by grants from Ministerio de Economía y Competitividad, reference SAF2015–67770-R, awarded to G.H and from Fondo de Investigación Sanitaria (FIS), reference PI17/01411, awarded to A.S.

12 p.-5 fig.-1 tab., Capsular switching allows pre-existing clones of Streptococcus pneumoniae expressing vaccine serotypes to escape the vaccine-induced immunity by acquisition of capsular genes from pneumococci of a non-vaccine serotype. Here, we have analysed the clonal composition of 492 clinical isolates of serotype 11A causing invasive disease in Spain (2000–2012), and their ability to evade the host immune response. Antibiograms, serotyping and molecular typing were performed. The restriction profiles of pbp2x, pbp1a and pbp2b genes were also analysed. Interaction with the complement components C1q, C3b, C4BP, and factor H was explored whereas opsonophagocytosis assays were performed using a human cell line differentiated to neutrophils. Biofilm formation and the polymorphisms of the major autolysin LytA were evaluated. The main genotypes of the 11A pneumococci were: ST62 (447 isolates, 90.6%), followed by ST6521 (35 isolates, 7.3%) and ST838 (10 isolates, 2.1%). Beta lactam resistant serotype 11A variants of genotypes ST838 and ST6521 closely related to the Spain9V-ST156 clone were first detected in 2005. A different pattern of evasion of complement immunity and phagocytosis was observed between genotypes. The emergence of one vaccine escape variant of Spain9V-ST156 (ST652111A), showing a high potential to avoid the host immune response, was observed.In addition, isolates of ST652111A showed higher ability to produce biofilms than ST83811A or ST6211A, which may have contributed to the emergence of this PEN-resistant ST652111A genotype in the last few years. The emergence of penicillin-resistant 11A invasive variants of the highly successful ST156 clonal complex merits close monitoring.

2008/2009 We are exposed daily to a myriad of potential pathogens, mainly through dermal contact, ingestion and inhalation. The innate immune system is of crucial importance in preventing pathogens from colonizing and growing to a point where they can cause life-threatening infections. Different types of cationic antimicrobial peptides (AMP), also known as host defense peptides (HDP), are among the innate immune mechanisms involved in this protective activity. -defensins are an important class of antimicrobial peptides that are present in human beings and have been widely reported to display a direct, salt and medium sensitive bactericidal activity, in vitro, against a broad spectrum of bacteria and fungi. Moreover, there is increasing evidence that they may play a significant role in alerting and enhancing other components of innate and adaptive immunity, thus also playing an indirect role in defense against microbes. Human -defensins are induced in vivo at the sites of microbial invasion, where they are thought to provide a bactericidal barrier, and to form chemotactic gradients that contribute to the recruitment of immune cells to the site of infection. Their interaction with the biological membranes of both microbial and host cells, appears to play a central role in both these types of activities. In this thesis, I was interested in characterizing the role of -defensin hBD2 in modulating activities of important immune host cells, such as monocytes, macrophages and immature dendritic cells, and its ability to bind to and/or be internalized within those cells, in relation to a possible immunomodulatory role. Initially, hBD2 was chosen amongst the human -defensins, and the native peptide synthesized, folded and characterized for antimicrobial activity and cytotoxicity. A fluorescent conjugate, as well as several structural analogues were also designed and prepared, and were respectively used to probe internalization and structure/activity relationships for this defensin. Subsequently, the immunomodulatory potential of hBD2 was probed, considering its possible effects in modulating important processes such as chemotaxis, degranulation, phagocytosis and reactive oxygen species (ROS) production of immune cells it comes into contact with. From this investigation, it emerged that hBD2 displays different effects depending on the cell type which comes into contact with it, and the exposure time. In particular, we confirmed the ability of hBD2 to induced chemotaxis of myeloid derived immature dendritic cells (iDC), contributing to recruitment of these cells to site of infection, although, from our studies, the mechanism of action turned out to be rather more complex than that which had been previously been proposed, and our observations may help reconcile apparently conflicting literature reports. Some novel additional properties of hBD2 were then described. In particular, a short exposure of iDC to hBD2 causes a cell type-dependent degranulation process, which could be involved in the tolerogenic/immunogenic roles of these cells, supporting a further immunomodulatory role for the defensin. A longer term exposure to hBD2 seems to augment the ability iDC to recognize bacteria and interact with them, possibly improving their ability to present antigens and activate adaptive immune responses. An equivalent treatment of macrophages resulted in a significant increase in their phagocytic activity and release of ROS, thus allowing these immune cells to better respond against a bacterial invasion. All these activities were evidenced in vitro at relatively low (micromolar) and sub-cytotoxic concentrations, which are compatible with the likely hBD2 concentration at sites of infection. Moreover, contrary to the direct bactericidal activity, these activities were not found to be salt or medium sensitive, but occur in conditions closely resembling the physiological ones. The activities of hBD2 were also compared to those of the structural analogues to study whether specific structural features of this defensin are required for its activity on immune cells. Results were somewhat puzzling because the evolutionarily conserved defensin scaffold seems to be quite important for exerting some biological function, such as the antimicrobial activity and phagocytosis, but not others, such as chemotaxis. This observation and the time and cell-type dependent activity may point to the concurrent existence of multiple modes of action. In the last part of this thesis, I describe the interaction of fluorescently labelled hBD2 with immune cells, with the aim of determining whether a cellular internalization could play a part for its biological effects. hBD2 was able to interact in a cell specific manner with different types of immune cells, upon short term exposure, leading to differentiated binding and cellular uptake, in a process devoid of cytotoxic or permeabilizing consequences. Macrophages were the most efficient in peptide internalization, whereas iDC seemed to be more avid for peptide surface binding than peptide internalization. The mechanism by which this occurs is as yet unknown, but our preliminary flow-cytometric data indicated that uptake of the defensin was an energy-dependent and temperature-sensitive process, which depends on actin fibers and can reasonably be supposed to involve lipid rafts, and/or clathrin-mediated endocytosis. Moreover, confocal microscopy of macrophages treated with labelled peptide showed that it may interact with specific patches on the membrane and, on binding, the peptide rapidly re-localized in the cytoplasm of cells with a characteristic punctate distribution. Taken together, these data could suggest that the peptide selectively binds to specific sites on membrane, such as lipid rafts, and that endocytosis could be a general mechanism of hBD2 internalization. These observations may thus indicate that hBD2 is capable of modulating activities of host cells not only by interacting with the membrane and surface receptors, but also with cytoplasmic targets. Noi siamo costantemente esposti a una miriade di potenziali patogeni, principalmente attraverso il contatto, l’ingestione e l’inalazione. Il sistema immunitario innato è di importanza cruciale nel prevenire la colonizzazione e la proliferazione dei patogeni che possono causare infezioni pericolose per la sopravvivenza. Tra i diversi meccanismi del sistema immunitario innato coinvolti in questa azione protettiva si contano diversi tipi di peptidi antimicrobici di natura cationica (AMP), anche noti come peptidi di difesa dell’ospite (HDP). Le -defensine sono un’importante classe di peptidi antimicrobici presenti anche nell’uomo che hanno dimostrato di possedere in vitro un’azione battericida diretta sensibile alla concentrazione salina nei confronti di un largo spettro di batteri e funghi. Inoltre è stato dimostrato che esse giocano un ruolo significativo nell’allertare e potenziare altri componenti dell’immunità innata e adattativa, esercitando così anche un’azione indiretta nella difesa contro i patogeni. Le -defensine umane vengono indotte in vivo nei siti dove si verifica un’invasione batterica, formando così una barriera battericida e creando un gradiente chemiotattico che contribuisce a reclutare cellule immunitarie nel sito d’infezione. La loro interazione con le membrane biologiche, sia dei microbi che delle cellule dell’ospite, sembra essere centrale in entrambe le attività. In questa tesi l’interesse principale è caratterizzare il ruolo della -defensina hBD2 nel modulare l’attività di importanti cellule immunitarie, quali monociti, macrofagi e cellule dendritiche immature, e la sua abilità di legarsi a tali cellule e/o di essere internalizzata da esse. Inizialmente, tra le -defensine umane è stata scelta hBD2 e il peptide naturale è stato sintetizzato, “foldato” e caratterizzato per l’attività antimicrobica e la citotossicità. Un coniugato fluorescente e molti analoghi strutturali sono stati inoltre progettati e sintetizzati e sono stati utilizzati, rispettivamente, per indagare l’eventuale internalizzazione e per fare studi di relazione fra struttura e attività. Successivamente è stato analizzato il potenziale immunomodulatorio di hBD2, considerando i suoi possibili effetti nel regolare importanti processi delle cellule immunitarie con cui il peptide viene in contatto, quali ad esempio la chemiotassi, la degranulazione, la fagocitosi e la produzione di specie reattive dell’ossigeno (ROS). Da questa analisi è emerso che hBD2 esercita diverse azioni a seconda del tipo cellulare con cui viene a contatto e del tempo di esposizione delle cellule alla defensina. In particolare, è stata confermata l’abilità di hBD2 a indurre la chemiotassi di cellule dendritiche immature (iDC), contribuendo a reclutare tali cellule al sito d’infezione. Negli studi condotti è però emerso che il meccanismo d’azione potrebbe essere più complesso di quanto proposto fino ad ora; tuttavia le osservazioni proposte in questa tesi appaiono in grado di conciliare i dati contrastanti riportati in letteratura. Sono inoltre emerse alcune nuove proprietà per hBD2. In particolare, un breve tempo di esposizione delle iDC alla defensina causa un processo di degranulazione che risulta dipendente dal tipo cellulare e che potrebbe essere coinvolto nel ruolo immunogenico/tolerogenico di queste cellule, supportando un ruolo immunomodulatorio per hBD2. Un’esposizione per tempi più lunghi a hBD2 sembra incrementare l’abilità delle iDC di riconoscere i batteri e interagire con loro, probabilmente aumentando la loro capacità di presentare gli antigeni e attivare le risposte immunitarie. Un trattamento equivalente sui macrofagi risulta invece in un aumento significativo della loro attività fagocitaria e della produzione di ROS, permettendo così a queste cellule di rispondere meglio ad un’invasione batterica. Tutte queste attività sono state evidenziate in vitro a concentrazioni relativamente basse (micromolari) e sub-citotossiche, compatibili con quelle che hBD2 raggiunge ai siti d’infezione. Inoltre, contrariamente all’attività battericida diretta, l’attività immunomodulatoria non è risultata sensibile alla concentrazione salina, ma si esplica in condizioni simili a quelle fisiologiche. L’attività di hBD2 è stata anche paragonata a quella di analoghi strutturali per studiare quali siano le caratteristiche di questa defensina che sono richieste per le attività sulle cellule immunitarie. I risultati sono per certi versi inspiegabili in quanto la struttura evolutivamente conservata delle defensine sembra essere importante per alcune attività biologiche, come l’attività antimicrobica diretta o la fagocitosi, mentre non lo è in altre, come la chemiotassi. Questa osservazione e la dipendenza dell’attività da tempo di esposizione e dal tipo cellulare considerato potrebbero indicare che esistono diversi meccanismi d’azione. Nell’ultima parte della tesi vengono descritti gli studi effettuati con il coniugato fluorescente di hBD2 con l’obiettivo di determinare se l’internalizzazione del peptide può giocare un ruolo nella sua attività biologica nei confronti delle cellule immunitarie. È emerso che hBD2 è capace di interagire in modo specifico già dopo una breve esposizione con i diversi tipi di cellule immunitarie considerati in questa tesi. Si è evidenziata una diversa capacità di legame e di internalizzazione a seconda del tipo cellulare, ma il processo è risultato sempre non accompagnato da effetti citotossici o permeabilizzazione delle cellule. In particolare, i macrofagi sono risultati i più efficienti nell’internalizzare del peptide, mentre le iDC sembrano più avide nel legare il peptide sulla superficie che nell’internalizzazione. Il meccanismo attraverso cui avvengono legame e internalizzazione è ancora ignoto, ma i nostri dati al citofluorimetro indicano che l’uptake della defensina è un processo che dipendente dall’energia e dalla temperatura, che è legato alla polimerizzazione delle fibre di actina e probabilmente coinvolge un processo di endocitosi mediata dai lipid rafts e/o dalla clatrina. Inoltre, la microscopia confocale di macrofagi trattati con il peptide fluorescente ha mostrato che esso interagisce con specifiche zone della membrana e, dopo il legame, si localizza rapidamente nel citoplasma della cellula con una caratteristica distribuzione puntata. Nell’insieme, questi dati suggeriscono che il peptide si leghi selettivamente a zone specifiche della membrana, come ad esempio i lipid rafts, e che l’endocitosi potrebbe essere un meccanismo generale per l’internalizzazione di hBD2. Tutti queste osservazioni suggeriscono quindi che hBD2 è capace di modulare le attività delle cellule dell’ospite non solo interagendo con le membrane o i recettori di superficie, ma anche con target citoplasmatici. XXI Ciclo 1981

2008/2009 a)STUDIO DELL’INTERAZIONE TRA MASTOCITI, MICRORGANISMI E FIBRE DI AMIANTO INTRODUZIONE: I mastociti (mast cell, MC) sono cellule con un’ampia distribuzione tissutale, che tradizionalmente sono state associate alla risposta allergica, inoltre si pensa che queste cellule siano capaci di regolare il processo infiammatorio. Essi infatti, dopo attivazione, rilasciano istamina, leucotrieni e prostanoidi, come pure proteasi, chemochine e citochine. Questi mediatori possono essere fondamentali nella genesi della risposta infiammatoria. I MC hanno alcune funzioni comuni a quelle dei fagociti professionali. Viste la loro localizzazione e i mediatori prodotti, i mastociti giocano un ruolo attivo in molte patologie, come le allergie, le infezioni, l’asma, l’artrite, i tumori e la fibrosi polmonare. In particolare essendo una fonte di citochine fibrogeniche (TGF-β e PDGF e β-FGF) risultano associati a diverse patologie fibrotiche. Alcuni studi recenti hanno dimostrato che i MC non sono in grado di uccidere microrganismi non opsonizzati a livello intracellulare, una volta che questi sono stati fagocitati. Altri studi hanno dimostrato che i MC sono in grado di uccidere in modo significativo microrganismi opsonizzati. Attualmente non è chiaro si il processo fagocitino dei MC segua o meno le stesse vie e se abbia o meno lo stesso significato di quello dei fagociti professionali. SCOPO: In questo studio abbiamo valutato l’ultrastruttura dei fagosomi che si formano nei MC peritoneali di ratto (RPMC), l’attività di killing contro C. albicans e altri microrganismi. Siamo inoltre a valutare se i MC fossero in grado di interagire direttamente con le fibre di amianto (in particolare Crocidolite) RISULTATI: Abbiamo dimostrato che i RPMC uccidono i microrganismi indipendentemente dalla loro ingestione. L’analisi ultrastrutturale ha dimostrato che i fagosomi dei RPMC contenenti funghi o batteri, hanno una struttura stretta e compatta, e la loro membrana sembra presentare alcune interruzioni. RPMC esprimono NOD1 e NOD2, che appartengono alla famiglia di proteine NLR, il cui ruolo è quello di riconoscere componenti intracellulari di origine sconosciuta. In seguito all’ingestione di C. albicans, entrambe queste molecole vengono traslocate dal citosol, alla zona attorna alla particella ingerita. Queste osservazioni suggeriscono che nei RPMC le particelle ingerite possano entrare in contatto diretto con i NOD, proprio attraverso le interruzioni della membrana del fagosoma, e di conseguenza innescare i meccanismi per la produzione di citochine che vengono innescati dai NOD. Per quanto riguarda l’interazione dei MC con le fibre di amianto, abbiamo osservato che i RPMC, in seguito all’interazione con le fibre, vanno in contro a intensa degranulazione, riversando i granuli sulle fibre stesse, che appaiono ricoperte di granuli. Inoltre durante l’interazione, in presenza di fibre, le RPMC producono anione superossido in quantità maggiore rispetto alle cellule a riposo. Per quanto riguarda la secrezione dell’enzima chimasi, questo sembra calare in maniera significativa nel sopranatante ottenuto dopo centrifugazione del medium di incubazione, mentre per quanto riguarda la Beta-Esosaminidasi (B-Eso), la presenza o meno delle fibre sembra non influenzarne la secrezione. Abbiamo quindi cercato di mettere a punto un modello sperimentale che di permettesse di separare le cellule intere e le fibre con i granuli adesi, dopo l’incubazione. La maggior parte dell’attività enzimatica sembra essere nella nella frazione contenente le cellule residue svuotate e nel pellet, dove si ritrova associata alle fibre. Abbiamo inoltre osservato che le fibre sono in grado di legare l’attività chimasica del lisato di RPMC, mentre sembrano non influenzare l’attività della B-Eso. Tali risultati sembrano suggerire che le fibre sono in grado di legare sia i granuli (e quindi gli enzimi Chimasi e B-Eso), sia la chimasi direttamente. La presenza di enzimi di origine mastocitaria sulle fibre, potrebbe avere un ruolo centrale per permettere il passaggio delle fibre dagli alveoli polmonari alle pleure, dove provocano l’insorgenza di mesotelioma maligno, una neoplasia piuttosto rara, la cui insorgenza è strettamente legata all’esposizione alle fibre di amianto. È inoltre da sviluppare lo studio delle eventuali nuove funzioni che questi enzimi scaricati sulle fibre, conferiscono alle fibre stesse, al fine di capire se le “fibre rivestite” sono in grado di migrare attraverso la matrice extracellulare. Il lavoro sta proseguendo anche al fine di comprendere meglio il tipo di interazione tra MC e le fibre. b)VALUTAZIONE DELLA PRODUZIONE DI CITOCHINE DA PARTE DELL’EPITELIO DELLA MUCOSA ORALE IN UN MODELLO in vitro INTRODUZIONE: La Propoli, un prodotto naturale derivato dall’attività delle api, è nota per una varietà di effetti benefici sfruttati da secoli, tra i quali l’attività antinfiammatoria e quella batterio-statica/battericida sono i principali. I modelli sperimantali per la valutazione dell’attività antinfiammatoria della propoli e dei suoi componenti isolati sono vari, e riguardano per lo più studi in vivo in diversi modelli animali. SCOPO: In questo studio è stata valutata la capacità di un principio attivo costituito da un complesso con propoli e impiegato in alcuni prodotti commerciali per l’igiene orale, di modulare il rilascio spontaneo e indotto da LPS di alcuni mediatori infiammatori, impiegando il modello tridimensionale di epitelio umano orale ricostituito della Ditta Skinethic. La scelta per quanto riguarda i mediatori infiammatori da misurare, è stata fatta in base al loro coinvolgimento in patologie orali quali: periodontite, stomatite aftosa ricorrente e candidosi. Scopo ultimo è quello di mettere a punto un sistema rilevatore semplice e attendibile dell’effetto biologico della propoli e dei suoi componenti su un tessuto umano riprodotto in coltura nelle sue caratteristiche principali. RISULTATI: L’applicazione topica della sola propoli actichelata, induce una riduzione del rilascio basale di IL-6 e IL-8, mentre induce un aumento del rilascio stimolato di entrambe le citochine. Il suo effetto stimolante il rilascio di mediatori infiammatori da parte dell’epitelio orale, soprattutto in condizioni infiammatorie (stimolazione con LPS), meriterebbe ulteriori indagini. Contrariamente, il Tantum verde, e il suo solo principio attivo, inducono un aumento del rilascio basale e stimolato di IL-6 e IL-8 da parte dell’epitelio orale umano. Da quanto osservato risulta quindi che la propoli actichelata, inducendo una riduzione di IL-8 e IL-6, è più efficace nel trattamento di condizioni patologiche che si associano a un’iperattività dei neutrofili come la parodontite, piuttosto che nel trattamento della candidosi orale dove queste cellule svolgono un ruolo fondamentale nella risoluzione. XXII Ciclo