Activated, Pro-Inflammatory Th1, Th17, and Memory CD4+ T Cells and B Cells Are Involved in Delayed-Type Hypersensitivity Arthritis (DTHA) Inflammation and Paw Swelling in Mice.

Delayed-type hypersensitivity arthritis (DTHA) is a recently established experimental model of rheumatoid arthritis (RA) in mice with pharmacological values. Despite an indispensable role of CD4+ T cells in inducing DTHA, a potential role for CD4+ T cell subsets is lacking. Here we have quantified CD4+ subsets during DTHA development and found that levels of activated, pro-inflammatory Th1, Th17, and memory CD4+ T cells in draining lymph nodes were increased with differential dynamic patterns after DTHA induction. Moreover, according to B-cell depletion experiments, it has been suggested that this cell type is not involved in DTHA. We show that DTHA is associated with increased levels of B cells in draining lymph nodes accompanied by increased levels of circulating IgG. Finally, using the anti-rheumatoid agents, methotrexate (MTX) and the anti-inflammatory drug dexamethasone (DEX), we show that MTX and DEX differentially suppressed DTHA-induced paw swelling and inflammation. The effects of MTX and DEX coincided with differential regulation of levels of Th1, Th17, and memory T cells as well as B cells. Our results implicate Th1, Th17, and memory T cells, together with activated B cells, to be involved and required for DTHA-induced paw swelling and inflammation.

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.

Demyelinating polyneuropathy in goats lacking prion protein.

Studies in mice with ablation of Prnp, the gene that encodes the cellular prion protein (PrPC ), have led to the hypothesis that PrPC is important for peripheral nerve myelin maintenance. Here, we have used a nontransgenic animal model to put this idea to the test; namely, goats that, due to a naturally occurring nonsense mutation, lack PrPC . Teased nerve fiber preparation revealed a demyelinating pathology in goats without PrPC . Affected nerves were invaded by macrophages and T cells and displayed vacuolated fibers, shrunken axons, and onion bulbs. Peripheral nerve lipid composition was similar in young goats with or without PrPC , but markedly different between corresponding groups of adult goats, reflecting the progressive nature of the neuropathy. This is the first report of a subclinical demyelinating polyneuropathy caused by loss of PrPC function in a nontransgenic mammal.

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.

Stress Resilience of Spermatozoa and Blood Mononuclear Cells without Prion Protein.

The cellular prion protein PrPC is highly expressed in neurons, but also present in non-neuronal tissues, including the testicles and spermatozoa. Most immune cells and their bone marrow precursors also express PrPC. Clearly, this protein operates in highly diverse cellular contexts. Investigations into putative stress-protective roles for PrPC have resulted in an array of functions, such as inhibition of apoptosis, stimulation of anti-oxidant enzymes, scavenging roles, and a role in nuclear DNA repair. We have studied stress resilience of spermatozoa and peripheral blood mononuclear cells (PBMCs) derived from non-transgenic goats that lack PrPC (PRNPTer/Ter) compared with cells from normal (PRNP+/+) goats. Spermatozoa were analyzed for freeze tolerance, DNA integrity, viability, motility, ATP levels, and acrosome intactness at rest and after acute stress, induced by Cu2+ ions, as well as levels of reactive oxygen species (ROS) after exposure to FeSO4 and H2O2. Surprisingly, PrPC-negative spermatozoa reacted similarly to normal spermatozoa in all read-outs. Moreover, in vitro exposure of PBMCs to Doxorubicin, H2O2 and methyl methanesulfonate (MMS), revealed no effect of PrPC on cellular survival or global accumulation of DNA damage. Similar results were obtained with human neuroblastoma (SH-SY5Y) cell lines stably expressing varying levels of PrPC. RNA sequencing of PBMCs (n = 8 of PRNP+/+ and PRNPTer/Ter) showed that basal level expression of genes encoding DNA repair enzymes, ROS scavenging, and antioxidant enzymes were unaffected by the absence of PrPC. Data presented here questions the in vitro cytoprotective roles previously attributed to PrPC, although not excluding such functions in other cell types or tissues during inflammatory stress.

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-1ß (IL-1ß), 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-γ), tumor necrosis factor alpha (TNF-α), 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-γ 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-1ß 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.

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.

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.

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], [2], [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], [7], [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], [7], [8], [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).

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 formylpeptide 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-1ß, 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-1ß 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.

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

Peptaibiotics, non-ribosomally synthetized peptides from various ascomycetes, are uniquely characterized by dialkylated a-amino acids, a rigid heli cal 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 peptaibiotictrichogin GA IV from the human opportunist Trichodenna 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 ~) lacking antibiotic effects on both Gram- and Gram+ bacteria(MIC > 64 ~ ). The peptaibiotic distinctive (-terminal primary alcohol was found to cooperate with theN-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 (-terminus, strongly increased trichogin GA IV anti-Gram+ activity (MIC 1-4 ~ ). but further mitigated its cytotoxicity on human cells.