Laura Tiberio

HIV‐1 matrix protein p17 binds to monocytes and selectively stimulates MCP‐1 secretion: role of transcriptional factor AP‐1

HIV‐1 matrix protein p17 activates a variety of cell responses which play a critical role in viral replication and infection. Its activity depends on the expression of p17 receptors (p17R) on the surface of target cells. Whether p17 also plays a role in stimulating human monocytes, a major HIV‐1 reservoir, is not known. Here we show that human monocytes constitutively express p17Rs and that p17 selectively triggers these cells to produce MCP‐1. The effect of p17 on MCP‐1 expression was observed at the transcriptional level and was primarily dependent on the activation of the transcription factor AP‐1. p17 increased the binding activity of AP‐1 complexes in a time‐ and dose‐dependent manner. Deletion of the AP‐1 binding sites in the MCP‐1 promoter resulted in the lack of p17‐induced MCP‐1 transcription. In particular, the P3 binding site located between −69 and −63 position seems to be essential to MCP‐1 mRNA induction in p17‐treated monocytes. An ever increasing amount of evidences shows a tight link between biologically dysregulated monocytes, AP‐1 activation, MCP‐1 release and HIV‐1 pathogenesis. Overall our results suggest that p17 may play a critical role in the monocyte‐mediated inflammatory processes, which are suspected to be major precipitating events in AIDS‐defining diseases.

Short- and long- term effects of cigarette smoke exposure on glutathione homeostasis in human bronchial epithelial cells.

Background: Cigarette smoke extract (CSE), a model for studying the effects of tobacco smoke in vivo and in vitro, induces cell oxidative stress and affects the antioxidative glutathione system. We evaluated the impact of CSE on airway epithelial cells and the possible cytoprotective effect of the mucolitic drug S-carboximethilcysteine lysine salt (S-CMC-Lys). Methods: Reduced glutathione (GSH) and reactive oxygen species (ROS) intracellular levels were evaluated by fluorimetry in human bronchial epithelial cells (16-HBE) and the expression and activity of enzymes of the GSH metabolic pathway were investigated by RT-PCR, Western blot and colorimetric assays. Results: CSE significantly increased cell mortality in a time and dose dependent manner, via an apoptosis-independent pathway. Short-term (3 hours) CSE exposure induced an increase in ROS levels and a GSH intracellular concentration drop. In parallel, the expression of glutathione peroxidases 2 and 3, glutathione reductase and glutamate-cysteine-ligase was increased. S-CMC-Lys was effective in counteracting these effects. Conclusion: CSE affects ROS levels, GSH concentration and GSH enzymes pathway. These effects can be to some extent reversed by S-CMC-Lys, that could represent a therapeutic tool to counteract CSE induced oxidative cellular injuries.

Molecular characterization of the new defective Pbrescia alpha1-antitrypsin allele†

Alpha1‐antitrypsin (α1AT) deficiency is a hereditary disorder associated with reduced α1AT serum level, predisposing adults to pulmonary emphysema. Among the known mutations of the α1AT gene (SERPINA1) causing α1AT deficiency, a few alleles, particularly the Z allele, may also predispose adults to liver disease. We have characterized a new defective α1AT allele (c.745G>C) coding for a mutant α1AT (Gly225Arg), named Pbrescia. The Pbrescia α1AT allele was first identified in combination with the rare defective Mwürzburg allele in an 11‐year‐old boy showing significantly reduced serum α1AT level. Subsequently, the Pbrescia allele was found in the heterozygous state with the normal M or the defective Z allele in nine and three adults respectively. In cellular models of the disease, we show that the Pbrescia mutant is retained in the endoplasmic reticulum as ordered polymers and is secreted more slowly than the normal M α1AT. This behaviour recapitulates the abnormal cellular handling and fate of the Z α1AT and suggests that the mutation present in the Pbrescia α1AT causes a conformational change of the protein which, by favouring polymer formation, is etiologic to both severe α1AT deficiency in the plasma and toxic protein‐overload in the liver.

Purification of interleukin-2 antibodies from healthy individuals

By covalent binding of recombinant interleukin-2 (rIL-2) to Sepharose, it was possible to immunopurify specific human anti-IL-2 antibodies from a pool of immunoglobulins obtained from healthy subjects. Since low quantities of the ligand released by the matrix could interfere with the evaluation of the biological activity of anti-IL-2 antibodies, the antibody preparation was subjected to pepsin digestion which is known to destroy the IL-2 molecule. Purified human anti-IL-2 antibodies were found to be mostly IgG1 and able to neutralize IL-2 induced peripheral blood lymphocytes (PBL) proliferation in vitro. The availability of purified anti-IL-2 antibodies, obtained from healthy individuals, able to modulate IL-2 activity, could be important in several therapeutic approaches.

The Decrease of Mineralcorticoid Receptor Drives Angiogenic Pathways in Colorectal Cancer

Angiogenesis plays a crucial role in tumor growth and progression. Low expression of mineralocorticoid receptor (MR) in several malignant tumors correlates with disease recurrence and overall survival. Previous studies have shown that MR expression is decreased in colorectal cancer (CRC). Here we hypothesize that decreased MR expression can contribute to angiogenesis and poor patient survival in colorectal malignancies. In a cohort of CRC patients, we analyzed tumor MR expression, its correlation with tumor microvascular density and its impact on survival. Subsequently, we interrogated the role of MR in angiogenesis in an in vitro model, based on the colon cancer cell line HCT116, ingenierized to re-express a physiologically controlled MR. In CRC, decreased MR expression was associated with increased microvascular density and poor patient survival. In pchMR transfected HCT116, aldosterone or natural serum steroids largely inhibited mRNA expression levels of both VEGFA and its receptor 2/KDR. In CRC, MR activation may significantly decrease angiogenesis by directly inhibiting dysregulated VEGFA and hypoxia-induced VEGFA mRNA expression. In addition, MR activation attenuates the expression of the VEGF receptor 2/KDR, possibly dampening the activation of a VEGFA/KDR dependent signaling pathway important for the survival of tumor cells under hypoxic conditions.

The role of bronchial epithelial cells in the pathogenesis of COPD in Z-alpha-1 antitrypsin deficiency

BackgroundAlpha-1 antitrypsin is the main inhibitor of neutrophil elastase in the lung. Although it is principally synthesized by hepatocytes, alpha-1 antitrypsin is also secreted by bronchial epithelial cells. Gene mutations can lead to alpha-1 antitrypsin deficiency, with the Z variant being the most clinically relevant due to its propensity to polymerize. The ability of bronchial epithelial cells to produce Z-variant protein and its polymers is unknown.We investigated the expression, accumulation, and secretion of Z-alpha-1 antitrypsin and its polymers in cultures of transfected cells and in cells originating from alpha-1 antitrypsin-deficient patients.MethodsExperiments using a conformation-specific antibody were carried out on M- and Z-variant–transfected 16HBE cells and on bronchial biopsies and ex vivo bronchial epithelial cells from Z and M homozygous patients. In addition, the effect of an inflammatory stimulus on Z-variant polymer formation, elicited by Oncostatin M, was investigated. Comparisons of groups were performed using t-test or ANOVA. Non-normally distributed data were assessed by Mann–Whitney U test or the Kruskal-Wallis test, where appropriate. A P value of < 0.05 was considered to be significant.ResultsAlpha-1 antitrypsin polymers were found at a higher concentration in the culture medium of ex vivo bronchial epithelial cells from Z-variant homozygotes, compared with M-variant homozygotes (P < 0.01), and detected in the bronchial epithelial cells and submucosa of patient biopsies. Oncostatin M significantly increased the expression of alpha-1 antitrypsin mRNA and protein (P < 0.05), and the presence of Z-variant polymers in ex vivo cells (P < 0.01).ConclusionsPolymers of Z-alpha-1 antitrypsin form in bronchial epithelial cells, suggesting that these cells may be involved in the pathogenesis of lung emphysema and in bronchial epithelial cell dysfunction.

The atypical receptor CCRL2 is required for CXCR2-dependent neutrophil recruitment and tissue damage

CCRL2 is a 7-transmembrane domain receptor that shares structural and functional similarities with the family of atypical chemokine receptors (ACKRs). CCRL2 is upregulated by inflammatory signals and, unlike other ACKRs, it is not a chemoattractant-scavenging receptor, does not activate β-arrestins, and is widely expressed by many leukocyte subsets. Therefore, the biological role of CCRL2 in immunity is still unclear. We report that CCRL2-deficient mice have a defect in neutrophil recruitment and are protected in 2 models of inflammatory arthritis. In vitro, CCRL2 was found to constitutively form homodimers and heterodimers with CXCR2, a main neutrophil chemotactic receptor. By heterodimerization, CCRL2 could regulate membrane expression and promote CXCR2 functions, including the activation of β2-integrins. Therefore, upregulation of CCRL2 observed under inflammatory conditions is functional to finely tune CXCR2-mediated neutrophil recruitment at sites of inflammation.

Chemokine and chemotactic signals in dendritic cell migration

Dendritic cells (DCs) are professional antigen-presenting cells responsible for the activation of specific T-cell responses and for the development of immune tolerance. Immature DCs reside in peripheral tissues and specialize in antigen capture, whereas mature DCs reside mostly in the secondary lymphoid organs where they act as antigen-presenting cells. The correct localization of DCs is strictly regulated by a large variety of chemotactic and nonchemotactic signals that include bacterial products, DAMPs (danger-associated molecular patterns), complement proteins, lipids, and chemokines. These signals function both individually and in concert, generating a complex regulatory network. This network is regulated at multiple levels through different strategies, such as synergistic interactions, proteolytic processing, and the actions of atypical chemokine receptors. Understanding this complex scenario will help to clarify the role of DCs in different pathological conditions, such as autoimmune diseases and cancers and will uncover new molecular targets for therapeutic interventions.

Leukocyte trafficking in tumor microenvironment

The tumor microenvironment consists of both malignant and non-malignant cells and a plethora of soluble mediators. Different types of tumors have specific tumor microenvironments characterized by distinct chemokines and chemotactic factors that influence leukocyte recruitment. The immune cell infiltrate continuously interacts with stroma cells and influence tumor growth. Emerging evidence suggests that the regulation of the composition and the metabolic state of tumor-associated leukocytes may represent a new promising intervention strategy. Here we summarize the current knowledge on the role of tumor-associated immune cells in tumor growth and dissemination, with a specific focus on the nature of the chemotactic factors responsible for their accumulation and activation in tumors.

The atypical receptor CCRL2 (C-C Chemokine Receptor-Like 2) does not act as a decoy receptor in endothelial cells

C-C chemokine receptor-like 2 (CCRL2) is a non-signaling seven-transmembrane domain (7-TMD) receptor related to the atypical chemokine receptor (ACKR) family. ACKRs bind chemokines but do not activate G protein-dependent signaling or cell functions. ACKRs were shown to regulate immune functions in vivo by their ability to scavenge chemokines from the local environment. This study was performed to investigate whether CCRL2 shares two of the main characteristics of ACKRs, namely the ability to internalize and scavenge the ligands. Cell membrane analysis of CCRL2-transfected cells revealed a weak, constitutive, ligand-independent internalization, and recycling of CCRL2, with a kinetics that was slower than those observed with ACKR3, a prototypic ACKR, or other chemotactic signaling receptors [i.e., chemokine-like receptor 1 and C-X-C motif chemokine receptor 2]. Intracellularly, CCRL2 colocalized with early endosome antigen 1-positive and Rab5-positive vesicles and with recycling compartments mainly characterized by Rab11-positive vesicles. CCRL2-transfected cells and activated mouse blood endothelial cells, that endogenously express CCRL2, were used to investigate the scavenging ability of CCRL2. These experiments confirmed the ability of CCRL2 to bind chemerin, the only recognized ligand, but excluded the ability of CCRL2 to perform scavenging. Collectively, these results identify unique functional properties for this member of the non-signaling 7-TMD receptor family.