Emiliana Finamore

Haemophilus influenzae Porin Induces Toll-Like Receptor 2-Mediated Cytokine Production in Human Monocytes and Mouse Macrophages

ABSTRACT The production of proinflammatory cytokines is likely to play a major pathophysiological role in meningitis and other infections caused by Haemophilus influenzae type b (Hib). Previous studies have shown that Hib porin contributes to signaling of the inflammatory cascade. We examined here the role of Toll-like receptors (TLRs) and the TLR-associated adaptor protein MyD88 in Hib porin-induced production of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). Hib porin-induced TNF-α and IL-6 production was virtually eliminated in macrophages from TLR2- or MyD88-deficient mice. In contrast, macrophages from lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice, which are defective in TLR4 function, responded normally to Hib porin. Moreover anti-TLR2 antibodies but not anti-TLR4 antibodies significantly reduced Hib porin-stimulated TNF-α and IL-6 release from the human monocytic cell line THP-1. These data indicate that the TLR2/MyD88 pathway plays an essential role in Hib porin-mediated cytokine production. These findings may be useful in the development of alternative therapies aimed at reducing excessive inflammatory responses during Hib infections.

Dendrimer Functionalization with a Membrane-Interacting Domain of Herpes Simplex Virus Type 1: Towards Intracellular Delivery

A poly(amide)-based dendrimer was synthesized and functionalized with the membrane-interacting peptide gH(625-644) (gH625) derived from the herpes simplex virus type 1 (HSV-1) envelope glycoprotein H, which has previously been shown to assist in delivering large cargoes across the cellular membrane. We demonstrate that the attachment of the gH625 peptide sequence to the termini of a dendrimer allows the conjugate to penetrate into the cellular matrix, whereas the unfunctionalized dendrimer is excluded from translocation. The peptide-functionalized dendrimer is rapidly taken into the cells mainly through a non-active translocation mechanism. Our results suggest that the presented peptidodendrimeric scaffold may be a promising material for efficient drug delivery.

Structural Insights into and Activity Analysis of the Antimicrobial Peptide Myxinidin

ABSTRACT The marine environment has been poorly explored in terms of potential new molecules possessing antibacterial activity. Antimicrobial peptides (AMPs) offer a new potential class of pharmaceuticals; however, further optimization is needed if AMPs are to find broad use as antibiotics. We focused our studies on a peptide derived from the epidermal mucus of hagfish (Myxine glutinosa L.), which was previously characterized and showed high antimicrobial activity against human and fish pathogens. In the present work, the activities of myxinidin peptide analogues were analyzed with the aim of widening the original spectrum of action of myxinidin by suitable changes in the peptide primary structure. The analysis of key residues by alanine scanning allowed for the design of novel peptides with increased activity. We identified the amino acids that are of the utmost importance for the observed antimicrobial activities against a set of pathogens comprising both Gram-negative and Gram-positive bacteria. Overall, optimized bactericidal potency was achieved by adding a tryptophan residue at the N terminus and by the simultaneous substitution of residues present in positions 3, 4, and 11 with arginine. These results indicate that the myxinidin analogues emerge as an attractive alternative for treating drug-resistant infectious diseases and provide key insights into a rational design for novel agents against these pathogens.

Porins from Salmonella enterica serovar Typhimurium induce TNF-α, IL-6 and IL-8 release by CD14-independent and CD11a/CD18-dependent mechanisms

Lipopolysaccharide (LPS) of Gram-negative bacteria and several surface components of Gram-positive bacteria utilize CD14 and CD11a/18 as cellular receptors to induce expression and release of cytokines. Of the surface components of Gram-negative bacteria, porins exhibit a biological activity similar to that of LPS. The results in this paper show that the mechanism of stimulation by porins of THP-1 cells enriched in CD14 receptor after treatment with 1,25-dihydroxyvitamin D(3) (vitamin D(3)) is independent of this receptor, but is partially dependent on CD11a/18 integrins.

Role of mitogen-activated protein kinases in the iNOS production and cytokine secretion by Salmonella enterica serovar Typhimurium porins

The expression of inducible nitric oxide synthase (iNOS) is a critical factor in both physiological and pathological functions. The present study examined the role of mitogen-activated protein kinases (MAPKs) in the regulation of iNOS and proinflammatory cytokine production in RAW 264.7 cells in response to Salmonella enterica serovar Typhimurium porins. By use of Western blotting for iNOS detection and enzyme-linked immunosorbent assay (ELISA) for quantization of cytokine secretion, selective pharmacological inhibitors of MAPK pathways were tested for dissecting the molecular mechanisms underlying the mediation of these signaling in porins-stimulated murine macrophages. S. enterica serovar Typhimurium porins activated iNOS expression, NO production and interleukin (IL)-6, IL-8 and tumor necrosis factor-alpha (TNF-alpha) release. Treatment of cells with SB203580 and SP600125 (inhibitors of p38 and JNK, respectively) significantly affected porin-stimulated iNOS and NO production. Concomitant decrease in the proinflammatory cytokine secretion was detected. These data confirm the importance of the MAPKs cascade in macrophage activation by bacterial product opening up new strategies for therapy of septic shock.

Peptides complementary to the active loop of porin P2 from Haemophilus influenzae modulate its activity

Haemophilus influenzae type b (Hib) is one of the leading causes of invasive bacterial infection in young children. It is characterized by inflammation that is mainly mediated by cytokines and chemokines. One of the most abundant components of the Hib outer membrane is the P2 porin, which has been shown to induce the release of several inflammatory cytokines. A synthetic peptide corresponding to loop L7 of the porin activates JNK and p38 mitogen-activated protein kinase (MAPK) pathways. We report a novel use of the complementary peptide approach to design a peptide that is able to bind selectively to the protein P2, thereby reducing its activity. This work provides insights into essential molecular details of P2 that may affect the pathogenesis of Hib infections where interruption of the signaling cascade could represent an attractive therapeutic strategy.

Evidence for IL-6 promoter nuclear activation in U937 cells stimulated with Salmonella enterica serovar Typhimurium porins

Interleukin-6 (IL-6) is a pleiotropic cytokine and plays an active role in inflammatory and immune responses, contributing to a multitude of physiological and pathophysiological processes. In this study, we address the molecular mechanism of IL-6 transcriptional induction and propose a correlation between activated NF-kappaB localization and IL-6 expression. In particular, we detected, by ChIP assay, that occupation of the IL-6 gene promoter site is dependent on activated NF-kappaB. In fact, after porin stimulation, the NF-kappaB p65 subunit is activated, translocates to the nucleus and binds to the IL-6 promoter sequence.Elucidation of the host signaling pathways and identification of the transcription factors that contribute to IL-6 expression, may aid in the understanding of host susceptibility to gram-negative infections and in identifying new therapeutic strategies in a variety of infectious diseases.

Self-assembled or mixed peptide amphiphile micelles from Herpes simplex virus glycoproteins as potential immunomodulatory treatment.

The use of micelle aggregates formed from peptide amphiphiles (PAs) as potential synthetic self-adjuvant vaccines to treat Herpes simplex virus (HSV) infection are reported here. The PAs were based on epitopes gB409–505 and gD301–309, selected from HSV envelope glycoprotein B (gB) and glycoprotein D (gD), that had their N-terminus modified with hydrophobic moieties containing two C18 hydrocarbon chains. Pure and mixed micelles of gB and/or gD peptide epitopes were easily prepared after starting with the synthesis of corresponding PAs by solid phase methods. Structural characterization of the aggregates confirmed that they were sufficiently stable and compatible with in vivo use: critical micelle concentration values around 4.0 ⋅ 10−7 mol ⋅ Kg−1; hydrodynamic radii (RH) between 50–80 nm, and a zeta potential (ζ) around − 40 mV were found for all aggregates. The in vitro results indicate that both peptide epitopes and micelles, at 10 μM, triggered U937 and RAW 264.7 cells to release appreciable levels of cytokines. In particular, interleukin (IL)-23-, IL-6-, IL-8- or macrophage inflammatory protein (MIP)-2-, and tumor necrosis factor (TNF)-α-release increased considerably when cells were treated with the gB-micelles or gD-micelles compared with the production of the same cytokines when the stimulus was the single gB or gD peptide.

P2 porin and loop L7 from Haemophilus influenzae modulate expression of IL-6 and adhesion molecules in astrocytes

During neuropathological conditions such as infections and degenerative diseases, astrocytes can be activated by infiltrating immune cells. Activated astrocytes can produce chemokines, cytokines and adhesion molecules. In this study, the production of IL‐6 and adhesion molecules intercellular adhesion molecule‐1 (ICAM‐1), vascular cellular adhesion molecule‐1 (VCAM‐1) and E‐selectin by human astroglioma cells stimulated with Gram‐negative surface components was investigated. Haemophilus influenzae type b porin P2 and its selected active peptide, loop L7, were found to induce MEK1‐MEK2/ mitogen‐activated protein kinase phosphorylation in U87‐MG cells as demonstrated by ELISA, and up‐regulate cellular adhesion molecule and interleukin‐6 (IL‐6) production as shown by RT‐PCR and ELISA. Using two potent and selective inhibitors of MEK activation by Raf‐1 (PD‐098059) and p38 (SB‐203580), it was also demonstrated that both ERK1/2 and p38 pathways play key roles in the production of IL‐6 as well as in ICAM‐1, VCAM‐1 and E‐selectin expression by Hib porin.

Viral Fusion Peptides Induce Several Signal Transduction Pathway Activations That Are Essential for Interleukin-10 and Beta-Interferon Production

Objectives: The deciphering of intracellular signaling pathways that are activated by the interaction between viral fusion peptides and cellular membranes are important for the understanding of both viral replication strategies and host defense mechanisms. Methods: Fusion peptides of several enveloped viruses belonging to different virus families were prepared by standard 9-fluorenylmethoxycarbonyl polyamine solid-phase synthesis and used to stimulate U937 cells in vitro to analyze the phosphorylation patterns of the signaling pathways (PKC, Src, Akt, and MAPK pathways). Immunoprecipitation and Western blotting were carried out by using phosphospecific antibodies. All samples were also assayed for the presence of IL-10 and IFN-β by ELISA and activation of nuclear factors (AP-1 and NF-ĸB). Results: We have demonstrated that hydrophobic domains of fusion proteins are able to induce several transduction pathways that lead to cytokine (IFN-β and IL-10) production, an event that appears to be dependent on early activation of AP-1 and NF-ĸB. Conclusions: The results obtained on the signaling activity of fusion peptides from different viruses enabled us to shed some light on the complex mechanism of viral entry and more precisely we focused on the exact signaling event induced by hydrophobic domains characteristic of fusion peptides interacting with the cell membrane.