Formyl-peptide Receptor Agonists and Amorphous SiO2-NPs Synergistically and Selectively Increase the Inflammatory Responses of Human Monocytes and PMNs

We tested whether amorphous SiO2-NPs and formyl-peptide receptor (FPRs) agonists synergistically activate human monocytes and neutrophil polymorphonuclear granulocytes (PMNs). Peptide ligands specifically binding to FPR1 (f-MLP) and to FPR2 (MMK-1, WKYMVM and WKYMVm) human isoforms did not modify the association of SiO2-NPs to both cell types or their cytotoxic effects. Similarly, the extent of CD80, CD86, CD83, ICAM-1 and MHCII expression in monocytes treated with SiO2-NPs was not significantly altered by any FPRs agonist. However, FPR1 stimulation with f-MLP strongly increased the secretion of IL-1β, IL-6 and IL-8 by human monocytes, and of IL-8 by PMNs in the presence of SiO2-NPs, due to the synergic stimulation of gene transcription. FPR2 agonists also up-modulated the production of IL-1β induced by monocytes treated with SiO2-NPs. In turn, SiO2-NPs increased the chemotaxis of PMNs toward FPR1-specific ligands, but not toward FPR2-specific ones. Conversely, the chemotaxis of monocytes toward FPR2-specific peptides was inhibited by SiO2-NPs. NADPH-oxidase activation triggered by FPR1- and FPR2-specific ligands in both cell types was not altered by SiO2-NPs. Microbial and tissue danger signals sensed by FPRs selectively amplified the functional responses of monocytes and PMNS to SiO2-NPs, and should be carefully considered in the assessment of the risk associated with nanoparticle exposure

Studier av prionproteinets fysiologiske funksjoner ved bruk av en ny dyremodell

The cellular prion protein (PrPC) has been intensively studied for its role in the pathogenesis of transmissible spongiform encephalopathies, such as Creutzfeldt-Jakob disease in human, bovine spongiform encephalopathy in cattle and scrapie in sheep and goat. Although their causative agent has been identified in the misfolded isoform of the protein, PrPSc, the physiological role of PrPC is still unclear. PrPC is conserved throughout vertebrates, and is expressed in the central nervous system and immune-privileged organs such as ovaries, testes, eye, and placenta. Several lines of Prnp knockout mice have been developed in order to study its functions. These mice develop healthy and are resistant to prion disease. Through their utilization, several roles of PrPC have been proposed, such as cell protection against stressors, metal homeostasis, and regulation of circadian rhythm, among others. We studied the role of PrPC and some phenotypes characterized in mice, with a new animal model, Norwegian dairy goats naturally lacking prion protein due to a stop mutation in the prion protein gene (PRNPTer/Ter). We investigated transcriptomic differences in peripheral blood mononuclear cells (PBMCs) isolated from goats expressing PrPC and in PRNPTer/Ter goats. We identified a primed state of interferonstimulated genes in goats lacking PrPC, suggesting a role for the protein in the dampening of immune responses. We also studied the cytoprotective role of PrPC in goats’ spermatozoa and PBMCs after exposure to stressors. Spermatozoa were exposed to copper ions, and their viability, motility, and adenosine triphosphate (ATP) levels were measured. Moreover, oxidative stress was induced with FeSO4 and H2O2, and the levels of reactive oxygen species (ROS) were measured. PBMCs and a neuroblastoma cell line were also used and the effect of stressors were measured in these cells. We could not detect a stress-protective effect of PrPC in vitro. Our results questioned the in vitro stress-protective role of PrPC . In addition, we carried out morphological analysis of peripheral nerves of goats to investigate a putative function of PrPC in myelin maintenance. We analysed teased fibres, and cross sections of nerves and we identified ultrastructural changes suggesting demyelinating pathology. Moreover, macrophages and T cells infiltrating the nerve were detected. Analysis of peripheral nerve lipid composition revealed significant differences between adult goats with or without PrPC. Such difference was not detected between groups of young goats, indicating a progression of the pathological condition with age. Taken together our results provided evidence of progressive demyelinating pathology in goats lacking prion protein. Altogether, in this thesis we have explored different physiological functions of PrPC. Future research is needed to fully understand PrPC function and the use of a nontransgenic animal model can provide useful advantages to increase the knowledge on this protein.Det cellulære prionproteinet (PrPC) er grundig studert på grunn av dets viktige rolle i utviklingen av overførbare spongiforme encefalopatier, som Creutzfeldt-Jakobs sykdom hos menneske, bovin spongiform encefalopati hos storfe og skrapesyke hos sau og geit. Selv om en feilfoldet variant av proteinet, PrPSc, er årsak til disse sykdommene, er den normale funksjonen til PrPC fortsatt ukjent. PrPC er konservert blant virveldyr og uttrykt i sentralnervesystemet og såkalte immunprivilegerte organer som ovarier, testikler, øye og placenta. Det er utviklet flere linjer av transgene mus hvor genet som koder for PrPC, Prnp er fjernet eller inaktivert (knockout). Gjennom studier av disse muselinjene har man oppdaget mange PrPC-funksjoner for eksempel; beskyttelse mot stress og regulering av metallhomeostase og døgnrytmer. Vi har studert PrPC sine mulige funksjoner i en ny dyremodell, nemlig en linje av Norsk melkegeit som på grunn av en naturlig forekommende «stoppmutasjon» (PRNPTer/Ter) fullstendig mangler PrPC. Vi har studert forskjeller i genuttrykk (transkripsjon) i perifere blod mononukleære blodceller (PBMC) hos geiter med og uten uttrykk av PrPC. Gjennom disse studiene påviste vi at en gruppe interferon-stimulerte gener var mildt oppregulert hos geitene uten PrPC, noe som tyder på at PrPC kan ha en immundempende funksjon. Vi har også studert PrPCs cellebeskyttende funksjoner hos spermier og PBMC’er fra geiter gjennom å utsette celler for ulike former for stressbelastninger. Spermier ble utsatt for kobberioner og cellenes levedyktighet, motilitet (bevegelse) og adenosin trifosfat (ATP) nivåer ble målt. Videre utsatte vi cellene for oksidativt stress gjennom eksponering for jernsulfat (FeSO4) og hydrogenperoksid (H2O2) og vi målte nivåene av reaktive oksygenspecies (ROS). PBMC’er og en human neuroblastom cellelinje ble også benyttet i disse studiene og effektene av stressbelastningene ble sammenlignet hos celler med og uten uttrykk av PrPC. Våre forsøk in vitro (i reagensglass-forsøk) viste ingen forskjell mellom cellenes evne til å tolerere stress om PrPC ble uttrykt eller ikke. Våre resultater stiller spørsmål ved enkelte tidligere publiserte rapporter som har vist en stressbeskyttende funksjon av PrPC i lignende forsøk. I tillegg har vi gjort detaljerte studier av perifere nerver hos geiter med og uten PrPC, for å undersøke om det også hos geit, som hos mus, er tegn til nerveskade (skade av myelinskjedene som omslutter nervene) hos dyr uten PrPC. Vi har analysert isolerte nervefibre med en metode hvor disse trekkes fra hverandre (teasing). Gjennom histologiske snitt av nerver og ved elektronmikroskop har vi kunnet påvise tap av myelinskjeder, en såkalt demyeliniserende nevropati. Videre påviste vi infiltrasjon av immunceller, som makrofager og T celler i nervene. Undersøkelser av fettstoffer (lipidanalyse) fra perifere nerver fra voksne geiter med og uten PrPC viste store forskjeller mellom gruppene, noe som indikerer en gradvis forverrende tilstand hos geitene uten PrPC. Disse funnene underbygger hypotesen om at PrPC har en viktig rolle i vedlikeholdelse av myelinskjedene rundt perifere nerver. Samlet sett har arbeidene i denne avhandlingen belyst ulike mulige fysiologiske roller til PrPC. Flere viktige observasjoner er gjort, men videre undersøkelser er nødvendig for å frembringe detaljert kunnskap om hvordan PrPC utfyller sine fysiologiske funksjoner i kroppen. Våre studier har vist at den ikke-transgene dyremodellene geitene representerer har vært meget nyttig og at potensialet for videre bruke av denne dyremodellen er stort

Loss of prion protein induces a primed state of type I interferon-responsive genes

The cellular prion protein (PrPC) has been extensively studied because of its pivotal role in prion diseases; however, its functions remain incompletely understood. A unique line of goats has been identified that carries a nonsense mutation that abolishes synthesis of PrPC. In these animals, the PrP-encoding mRNA is rapidly degraded. Goats without PrPC are valuable in re-addressing loss-of-function phenotypes observed in Prnp knockout mice. As PrPC has been ascribed various roles in immune cells, we analyzed transcriptomic responses to loss of PrPC in peripheral blood mononuclear cells (PBMCs) from normal goat kids (n = 8, PRNP+/+) and goat kids without PrPC (n = 8, PRNPTer/Ter) by mRNA sequencing. PBMCs normally express moderate levels of PrPC. The vast majority of genes were similarly expressed in the two groups. However, a curated list of 86 differentially expressed genes delineated the two genotypes. About 70% of these were classified as interferon-responsive genes. In goats without PrPC, the majority of type I interferon-responsive genes were in a primed, modestly upregulated state, with fold changes ranging from 1.4 to 3.7. Among these were ISG15, DDX58 (RIG-1), MX1, MX2, OAS1, OAS2 and DRAM1, all of which have important roles in pathogen defense, cell proliferation, apoptosis, immunomodulation and DNA damage response. Our data suggest that PrPC contributes to the fine-tuning of resting state PBMCs expression level of type I interferon-responsive genes. The molecular mechanism by which this is achieved will be an important topic for further research into PrPC physiology

Combined Action of Human Commensal Bacteria and Amorphous Silica Nanoparticles on the Viability and Immune Responses of Dendritic Cells

Dendritic cells (DCs) regulate the host-microbe balance in the gut and skin, tissues likely exposed to nanoparticles (NPs) present in drugs, food, and cosmetics. We analyzed the viability and the activation of DCs incubated with extracellular media (EMs) obtained from cultures of commensal bacteria (Escherichia coli, Staphylococcus epidermidis) or pathogenic bacteria (Pseudomonas aeruginosa, Staphylococcus aureus) in the presence of amorphous silica nanoparticles (SiO2 NPs). EMs and NPs synergistically increased the levels of cytotoxicity and cytokine production, with different nanoparticle dose-response characteristics being found, depending on the bacterial species. E. coli and S. epidermidis EMs plus NPs at nontoxic doses stimulated the secretion of interleukin-1 beta (IL-1 beta), IL-12, IL-10, and IL-6, while E. coli and S. epidermidis EMs plus NPs at toxic doses stimulated the secretion of gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), IL-4, and IL-5. On the contrary, S. aureus and P. aeruginosa EMs induced cytokines only when they were combined with NPs at toxic concentrations. The induction of maturation markers (CD86, CD80, CD83, intercellular adhesion molecule 1, and major histocompatibility complex class II) by commensal bacteria but not by pathogenic ones was improved in the presence of noncytotoxic SiO2 NP doses. DCs consistently supported the proliferation and differentiation of CD4(+) and CD8(+) T cells secreting IFN-gamma and IL-17A. The synergistic induction of CD86 was due to nonprotein molecules present in the EMs from all bacteria tested. At variance with this finding, the synergistic induction of IL-1 beta was prevalently mediated by proteins in the case of E. coli EMs and by nonproteins in the case of S. epidermidis EMs. A bacterial costimulus did not act on DCs after adsorption on SiO2 NPs but rather acted as an independent agonist. The inflammatory and immune actions of DCs stimulated by commensal bacterial agonists might be altered by the simultaneous exposure to engineered or environmental NPs

The peculiar N- and C-termini of trichogin GA IV are needed for membrane interaction and human cell death induction at doses lacking antibiotic activity

none7Peptaibiotics, non-ribosomally synthetized peptides from various ascomycetes, are uniquely characterized by di-alkylated alpha-amino acids, a rigid helical conformation, and membrane permeation properties. Although generally considered as antimicrobial peptides, peptaibiotics may display other toxicological properties, and their function is in many cases unknown. With the goal to define the biological activity and selectivity of the peptaibiotic trichogin GA IV from the human opportunist Trichoderma longibrachiatum we analyzed its membrane interaction, cytotoxic activity and antibacterial effect. Trichogin GA IV effectively killed several types of healthy and neoplastic human cells at doses (EC 50% = 4-6 μM) lacking antibiotic effects on both Gram- and Gram+ bacteria (MIC > 64 μM). The peptaibiotic distinctive C-terminal primary alcohol was found to cooperate with the N-terminal n-octanoyl group to permeate the membrane phospholipid bilayer and to mediate effective binding and active endocytosis of trichogin GA IV in eukaryotic cells, two steps essential for cell death induction. Replacement of one Gly with Lys plus the simultaneous esterification of the C-terminus, strongly increased trichogin GA IV anti-Gram+ activity (MIC 1-4 μM), but further mitigated its cytotoxicity on human cells.restrictedR. Tavano;G. Malachin;M. De Zotti;C. Peggion;B. Biondi;F. Formaggio;E. PapiniTavano, Regina; Malachin, Giulia; DE ZOTTI, Marta; Peggion, Cristina; Biondi, Barbara; Formaggio, Fernando; Papini, Emanuel

Comparison of bactericidal and cytotoxic activities of trichogin analogs

Peptaibiotics are a group of membrane active peptides of fungal origin. They typically contain α-aminoisobutyric acid (Aib; 1-letter code, U) and other non-coded residues (Toniolo and Brückner, 2009; Neumann et al., 2015; Benedett et al., 1982) [1–3] stabilizing their helical structure. Peptaibols are peptaibiotics carrying a 1, 2-aminoalcohol at the C-terminus. When a fatty acid chain (of 8–10 carbon atoms) is present at their N-terminus, they are called lipopeptaibols (Toniolo et al., 2001; Degenkolb et al., 2003) [4,5]. We found (Tavano et al., 2015) [6] that the lipopeptaibol trichogin displays no antibacterial effects up to 64 µM, against both Gram− and Gram+ bacteria, but kills tumor and healthy human cells via a mechanism requiring both the C-terminal primary alcohol group and the N-terminal n-octanoyl moiety, with EC50s around 4–5 µM. However, the substitution of single Gly residues with Lys strongly improves anti-Gram+ activity (Tavano et al., 2015; De Zotti, Biondi, Park et al., 2012; De Zotti, Biondi, Peggion et al., 2012) [6–8]. To further characterize the activity of trichogin analogs as antibiotics and cytotoxic agents, we here manipulated the peptide helix amphipathicity by means of two different substitutions: (i) Aib to Leu (De Zotti et al., 2012) [7] or (ii) multiple Gly to Lys changes (Tavano et al., 2015; De Zotti, Biondi, Park et al., 2012; De Zotti, Biondi, Peggion, Formaggio et al., 2012; De Zotti, Biondi, Peggion, De Poli et al., 2012) [6–9]. The antibacterial activity against four commensal or opportunistic bacterial species and the cytotoxicity against a panel of 9 healthy and tumor-derived eukaryotic cell types (including erythrocytes) are reported as MIC and EC50 (MTS - [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)]-2H-tetrazolium- reduction and LDH - lactate dehydrogenase - release assay). Keywords: Peptaibols, Trichogin, Antibaterial activity, Cytotoxicit

Cellular metabolism dictates T cell effector function in health and disease

In a healthy person, metabolically quiescent T lymphocytes (T cells) circulate between lymph nodes and peripheral tissues in search of antigens. Upon infection, some T cells will encounter cognate antigens followed by proliferation and clonal expansion in a context‐dependent manner, to become effector T cells. These events are accompanied by changes in cellular metabolism, known as metabolic reprogramming. The magnitude and variation of metabolic reprogramming are, in addition to antigens, dependent on factors such as nutrients and oxygen to ensure host survival during various diseases. Herein, we describe how metabolic programmes define T cell subset identity and effector functions. In addition, we will discuss how metabolic programs can be modulated and affect T cell activity in health and disease using cancer and autoimmunity as examples

Goats naturally devoid of PrPC are resistant to scrapie

Prion diseases are progressive and fatal, neurodegenerative disorders described in humans and animals. According to the "protein-only" hypothesis, the normal host-encoded prion protein (PrPC) is converted into a pathological and infectious form (PrPSc) in these diseases. Transgenic knockout models have shown that PrPC is a prerequisite for the development of prion disease. In Norwegian dairy goats, a mutation (Ter) in the prion protein gene (PRNP) effectively blocks PrPC synthesis. We inoculated 12 goats (4 PRNP+/+, 4 PRNP+/Ter, and 4 PRNPTer/Ter) intracerebrally with goat scrapie prions. The mean incubation time until clinical signs of prion disease was 601 days post-inoculation (dpi) in PRNP+/+ goats and 773 dpi in PRNP+/Ter goats. PrPSc and vacuolation were similarly distributed in the central nervous system (CNS) of both groups and observed in all brain regions and segments of the spinal cord. Generally, accumulation of PrPSc was limited in peripheral organs, but all PRNP+/+ goats and 1 of 4 PRNP+/Ter goats were positive in head lymph nodes. The four PRNPTer/Ter goats remained healthy, without clinical signs of prion disease, and were euthanized 1260 dpi. As expected, no accumulation of PrPSc was observed in the CNS or peripheral tissues of this group, as assessed by immunohistochemistry, enzyme immunoassay, and real-time quaking-induced conversion. Our study shows for the first time that animals devoid of PrPC due to a natural mutation do not propagate prions and are resistant to scrapie. Clinical onset of disease is delayed in heterozygous goats expressing about 50% of PrPC levels.publishedVersio

Form Matters: Stable Helical Foldamers Preferentially Target Human Monocytes and Granulocytes

Some hybrid foldamers of various length, all containing the (4R,5S)-4-carboxy-5-methyloxazolidin-2-one (d-Oxd) moiety alternating with an l-amino acid (l-Val, l-Lys, or l-Ala), were prepared in order to study their preferred conformations and to evaluate their biological activity. Surprisingly, only the longer oligomers containing l-Ala fold into well-established helices, whereas all the other oligomers give partially unfolded turn structures. Nevertheless, they all show good biocompatibility, with no detrimental effects up to 64 μm. After equipping some selected foldamers with the fluorescent tag rhodamine B, a quantitative analysis was performed by dose– and time–response fluorescence-activated cell sorting (FACS) assays with human HeLa cells and primary blood lymphocytes, granulocytes, and monocytes. Among the cell types analyzed, the oligomers associated with monocytes and granulocytes with greatest efficacy, still visible after 24 h incubation. This effect is even more pronounced for foldamers that are able to form stable helices

Goats naturally devoid of PrPC are resistant to scrapie

Prion diseases are progressive and fatal, neurodegenerative disorders described in humans and animals. According to the “protein-only” hypothesis, the normal host-encoded prion protein (PrPC) is converted into a pathological and infectious form (PrPSc) in these diseases. Transgenic knockout models have shown that PrPC is a prerequisite for the development of prion disease. In Norwegian dairy goats, a mutation (Ter) in the prion protein gene (PRNP) effectively blocks PrPC synthesis. We inoculated 12 goats (4 PRNP+/+, 4 PRNP+/Ter, and 4 PRNPTer/Ter) intracerebrally with goat scrapie prions. The mean incubation time until clinical signs of prion disease was 601 days post-inoculation (dpi) in PRNP+/+ goats and 773 dpi in PRNP+/Ter goats. PrPSc and vacuolation were similarly distributed in the central nervous system (CNS) of both groups and observed in all brain regions and segments of the spinal cord. Generally, accumulation of PrPSc was limited in peripheral organs, but all PRNP+/+ goats and 1 of 4 PRNP+/Ter goats were positive in head lymph nodes. The four PRNPTer/Ter goats remained healthy, without clinical signs of prion disease, and were euthanized 1260 dpi. As expected, no accumulation of PrPSc was observed in the CNS or peripheral tissues of this group, as assessed by immunohistochemistry, enzyme immunoassay, and real-time quaking-induced conversion. Our study shows for the first time that animals devoid of PrPC due to a natural mutation do not propagate prions and are resistant to scrapie. Clinical onset of disease is delayed in heterozygous goats expressing about 50% of PrPC levels.publishedVersio