Sabrina Lisi

Biological Role of the N-Formyl Peptide Receptors

Ligation of N-formyl-methionyl-leucyl-phenylalanine (fMLP) to its specific cell surface receptors triggers different cascades of biochemical events, eventually leading to cellular activation. The formyl peptide receptors (FPRs) are members of the seven-transmembrane, G-protein coupled receptors superfamily, expressed at high levels on polymorphonuclear and mononuclear phagocytes. The main responses elicited upon ligation of formylated peptides, referred to as cellular activation, are those of morphological polarization, locomotion, production of reactive-oxygen species and release of proteolytic enzymes. FPRs have in recent years been shown to be expressed also in several non myelocytic populations, suggesting other unidentified functions for this receptor family, independent of the inflammatory response. Finally, a number of ligands acting as exogenous or host-derived agonists for FPRs, as well as ligands acting as FPRs antagonists, have been described, indicating that these receptors may be differentially modulated by distinct molecules.

Nitric oxide production by macrophages of dogs vaccinated with killed Leishmania infantum promastigotes

Human visceral leishmaniosis is endemic in Southern Italy, where the dog is the main reservoir of viscerotropic strains of Leishmania infantum. The release of nitric oxide (NO) by interferon (IFN)-gamma-activated macrophages is an important leishmanicidal mechanism in several animal species. In this work NO production, phagocytosis and killing capacity of monocyte-derived dog macrophages were evaluated in vitro before and after administration of a vaccine composed of killed Leishmania infantum promastigotes. Moreover, IFN-gamma content was measured in concanavalin A-activated dog peripheral blood mononuclear cell (PBMC) supernatants employed for macrophage stimulation. Phagocytosis, killing capacity and NO production by canine macrophages increased significantly 1 month after vaccine administration, and the increase also persisted 5 months later. In addition, the amount of IFN-gamma in PBMC supernatants was significantly higher after vaccination. Overall, our results suggest the usefulness of evaluating the in vivo protective role of this promastigote preparation in dogs.

Infection with Leishmania infantum Inhibits Actinomycin D‐Induced Apoptosis of Human Monocytic Cell Line U‐937

Abstract. Modulation of host cell apoptosis has been observed in many bacterial, protozoal, and viral infections. The aim of this work was to investigate the effect of viscerotropic Leishmania (L.) infantum infection on actinomycin D‐induced apoptosis of the human monocytic cell line U‐937. Cells were infected with L. infantum promastigotes or treated with the surface molecule lipophosphoglycan (LPG) or with parasite‐free supernatant of Leishmania culture medium and submitted to action of actinomycin D as the apoptosis‐inducing agent. Actinomycin D‐induced apoptosis in U‐937 cells was inhibited in the presence of both viable L. infantum promastigotes and soluble factors contained in Leishmania culture medium or purified LPG. Leishmania infantum affected the survival of U‐937 cells via a mechanism involving inhibition of caspase‐3 activation. Furthermore, protein kinase C δ (PKC δ) cleavage was increased in actinomycin D‐treated U‐937 cells and was inhibited by the addition of LPG. Thus, inhibition of the PKC‐mediated pathways by LPG can be implicated in the enhanced survival of the parasites.

Tumor Necrosis Factor Inhibitors Block Apoptosis of Human Epithelial Cells of the Salivary Glands

Inhibition of tumor necrosis factor‐α (TNF‐α) in organ‐specific autoimmune disease is proving efficacious for a large number of patients. A wide array of biological agents has been designed to inhibit TNF‐α, such as adalimumab (fully humanized) and etanercept (soluble TNF‐α receptor fusion constructs p75 subunit). Recently, we suggested that anti‐Ro and anti‐La autoantibodies (Abs) isolated from patients with Sjögrens syndrome, an autoimmune rheumatic disease, are able to trigger cell death through extrinsic apoptotic mechanisms in human salivary gland epithelial cells (SGEC). We analyzed if primary human SGEC cultures, established from biopsy of labial minor salivary glands, are able to produce TNF‐α, an inductor of the extrinsic apoptotic pathway, when treated with anti‐Ro autoantibodies. A comparative study was performed to test the efficacy of adalimumab and etanercept to block TNF‐α‐mediated apoptosis. ELISA assay and RT‐PCR were employed to visualize TNF‐α production, and apoptosis was evaluated by DNA ladder and flow cytometry. We found that cell treatment with anti‐Ro autoantibodies determines TNF‐α production that reaches a maximum at 16 h and is decreased (P < 0.05) at 24 and 48 h. Adalimumab seems to be more efficacious than etanercept in blocking TNF‐α‐mediated apoptosis. The YOPRO‐1 (+) and propidium iodide (−) method revealed 60% of apoptotic cells after 24 h of incubation with anti‐Ro compared with 15% of apoptotic cells treated with anti‐Ro plus adalimumab and 25% of apoptotic cells treated with anti‐Ro plus etanercept. The antiapoptotic effect of adalimumab and etanercept was supported by inhibition of DNA laddering induced by anti‐Ro Abs. These data validate the therapeutic efficacy of the anti‐TNF reagents in the treatment of autoimmune disorders.

Sjögren's syndrome autoantibodies provoke changes in gene expression profiles of inflammatory cytokines triggering a pathway involving TACE/NF-κB.

We explore the association of the inflammatory gene expression profile observed in the chronic inflammatory autoimmune disorder Sjögrens syndrome (SS) with changes in TNF-α converting enzyme (TACE), tumor necrosis factor (TNF)-α and nuclear factor (NF)-κB levels showing that pathways that include TNF-α signaling converge on NF-κB contributing to exacerbate the diseases. The treatment of human salivary gland epithelial cells (SGECs) with SS anti-Ro/SSA autoantibodies (Abs) result in a progressive increase in NF-κB–DNA binding, that includes a marked enhancement in NF-κB subunit p65 protein–DNA binding. A human cytokine multi-analyte array demonstrated that the NF-κB proinflammatory target genes, increased by anti-Ro/SSA Abs treatment, includes CXC chemokines (CXCL1, CXCL6 and CXCL9), CC chemokines (CCL2, CCL13 and CCL20), interleukins (IL-1α, IL-1β, IL-1F8, IL-6, IL-8, IL-9, IL-13, IL-17 and IL-22) and their receptors (IL-1RN, IL-10Rα, IL-13Rα, CCR1, CCR2, CCR3, CCR4 and CXCR1). Blockade of TACE through the use of the specific inhibitor TAPI-1 regulates proinflammatory cytokines production in SGEC treated with anti-Ro/SSA Abs inhibiting NF-κB nuclear translocation and activation. To further investigate the role of NF-κB on anti-Ro/SSA Abs-determined proinflammatory gene expression, we used the inhibitory protein IκB-α dominant negative super-repressor as inhibitor of NF-κB–DNA binding, demonstrating that transfection with dominant-negative IκB-α in anti-Ro/SSA-treated SGEC determined a marked reduction of proinflammatory cytokines gene expression. Although further studies are needed to clarify the mechanisms underlying SS, our results demonstrate that SS Abs exert their pathogenic effects via triggering the TACE/TNF-α/NF-κB axis.

Pro-inflammatory role of Anti-Ro/SSA autoantibodies through the activation of Furin-TACE-amphiregulin axis.

Prolonged inflammation can be detrimental because it may cause host toxicity and tissue damage. Indeed, excessive production of inflammatory cytokines is often associated with many autoimmune diseases. In this study we demonstrate that the anti-Ro/SSA autoantibodies (Abs) stimulate the production of pro-inflammatory cytokines IL-6 and IL-8 by human healthy salivary gland epithelial cells (healthy SGEC). The secretion of these cytokines is due to amphiregulin (AREG) that is overexpressed in healthy SGEC treated with anti-Ro/SSA Abs and in Sjögrens syndrome. We have discovered that the up-regulation of AREG occurs through TNF-alpha produced following anti-Ro/SSA Abs treatment. The gene silencing technique was used to study the AREG-TNF-alpha-IL-6/IL-8 secretion pathway, demonstrating that: (i) TNF-alpha gene silencing provokes a significant decrease of proinflammatory cytokines production and AREG expression in anti-Ro/SSA Abs-treated healthy SGEC; (ii) AREG gene silencing has a potent inhibitory effect on TNF-alpha-induced IL-6 and IL-8 secretion in healthy SGEC treated with anti-Ro/SSA Abs. These findings indicate that TACE-mediated AREG shedding plays a critical role in TNF-alpha-induced IL-6 and IL-8 secretion by the human healthy salivary gland epithelial cells, suggesting that this may be one of the possible intracellular mechanisms involved in the salivary glands inflammatory response in Sjögrens syndrome.

Sjögren's syndrome pathological neovascularization is regulated by VEGF-A-stimulated TACE-dependent crosstalk between VEGFR2 and NF-κB.

We explore the involvement of tumor necrosis factor α (TNF-α)-converting enzyme (TACE) in vascular endothelial growth factor (VEGF) and its receptor 2 (VEGFR2) (VEGF-A/VEGFR2)-mediated angiogenesis in Sjögren’s syndrome (SS), one of the most common autoimmune rheumatic diseases. To test the hypothesis that SS autoantibodies (Abs) regulate VEGF-A/VEGFR2 expression by a TACE-dependent nuclear factor-κB (NF-κB) activation pathway, their effects on the expression and activation of the VEGF-A/TACE/VEGFR2/NF-κB pathway were determined in human salivary gland epithelial cells (SGEC). An enhanced angiogenesis in SS salivary gland biopsies was observed, associated with an increased VEGF-A expression and activation of VEGF-A/VEGFR2 signaling. Human cytokine array analysis of the pro-inflammatory and pro-angiogenic protein response in SGEC treated with SS Abs revealed an overexpression of multiple pro-angiogenic factors. TACE RNA knockdown, the use of anti-VEGF-A monoclonal antibody and the inhibition of NF-κB activity significantly abrogated the release of pro-angiogenic factors, demonstrating that VEGF-A/TACE/VEGFR2/NF-κB axis dysfunction may be contributory to pathogenesis and exacerbation of this autoimmune condition.

Molecular identification of Mycobacterium avium subspecies paratuberculosis in an Italian patient with Hashimoto's thyroiditis and Melkersson–Rosenthal syndrome

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne’s disease, a chronic granulomatous intestinal condition that affects ruminants, including cattle, sheep, goats and farmed deer (Naser et al., 2004). Water and milk supplies have both been suggested as vehicles of MAP transmission between cattle and humans (Pickup et al., 2006; Groenendaal & Zagmutt, 2008). Milk may be contaminated directly within the udder or indirectly as a result of faecal contamination (Giese & Ahrens, 2000). This bacterium has attracted considerable interest owing to the rapidly growing body of scientific evidence suggesting similarities between Johne’s disease in ruminants and Crohn’s disease in humans, which has led to speculation that MAP might be one of the causative agents in Crohn’s disease (Naser et al., 2004). However, because of the complex nature of human Crohn’s disease, as well as the conflicting experimental evidence, a definitive link between MAP and Crohn’s disease can neither be confirmed nor excluded at present. Some authors believe there is no evidence that a single pathogen, including MAP, is involved in the pathogenesis of inflammatory bowel diseases (Packey & Sartor, 2009), while others have reported that MAP may asymptomatically colonize apparently healthy individuals (Juste et al., 2008). Therefore, no causal link has been scientifically established between MAP and Crohn’s disease, although it is important to note that much evidence supports the possibility of such a relationship. The MAP debate has gathered momentum in recent years because of the more frequent detection of MAP in the blood and tissues of Crohn’s disease patients as compared with controls. The over-representation of MAP DNA in Crohn’s disease patients suggests a probable role for MAP in the aetiology of this pathological condition (Bentley et al., 2008).

ADAM17 at the interface between inflammation and autoimmunity

The discovery of the disintegrin and metalloproteinase 17 (ADAM17), originally identified as tumor necrosis factor-a converting enzyme (TACE) for its ability as sheddase of TNF-α inspired scientists to attempt to elucidate the molecular mechanisms underlying ADAM17 implication in diseased conditions. In recent years, it has become evident that this protease can modify many non matrix substrates, such as cytokines (e.g. TNF-α), cytokine receptors (e.g. IL-6R and TNF-R), ligands of ErbB (e.g. TGF-α and amphiregulin) and adhesion proteins (e.g. Lselectin and ICAM-1). Several recent studies have described experimental model system to better understand the role of specific signaling molecules, the interplay of different signals and tissue interactions in regulating ADAM17-dependent cleavage of most relevant substrates in inflammatory diseases. The central question is whether ADAM17 can influence the outcome of inflammation and if so, how it performs this regulation in autoimmunity, since inflammatory autoimmune diseases are often characterized by deregulated metalloproteinase activities. This review will explore the latest research on the influence of ADAM17 on the progression of inflammatory processes linked to autoimmunity and its role as modulator of inflammation.

Proposing a relationship between Mycobacterium avium subspecies paratuberculosis infection and Hashimoto's thyroiditis

Abstract Humans are widely exposed to Mycobacterium avium subspecies paratuberculosis (MAP), a proven multi-host chronic enteric pathogen that has recently been linked to autoimmune diabetes. In the present study we used a MAP species-specific polymerase chain reaction with the insertion element IS900-specific probe to detect MAP infection in members of the same family suffering from Hashimotos thyroiditis.