Iole Paoletti

Antimicrobial human beta-defensin-2 stimulates migration, proliferation and tube formation of human umbilical vein endothelial cells

Human beta-defensin-2 (hBD-2) is an antimicrobial peptide which is released upon microbial invasion and contributes to mucosal and epithelial defense modulating both innate and adaptive immunity. We found that hBD-2 stimulates chemotaxis of human endothelial cells with an extent similar to that exerted by the vascular endothelial growth factor (VEGF). The hBD-2-dependent chemotaxis is dose-dependent, maximal effect being reached at 500 ng/ml concentration. In the absence of any growth factor, hBD-2 favors wound healing of endothelial cells, causing an about 2-fold increase in the speed of wound closure with respect to the control. Furthermore, hBD-2 promotes endothelial cell proliferation, although at a minor extent as compared to VEGF. When plated on matrigel enriched with angiogenic factors, endothelial cells form a three-dimensional network of tubes that gives rise to capillary-like structures. Similarly to VEGF, hBD-2 promotes capillary-like tube formation of human endothelial cells. Pro-angiogenic effect promoted by hBD-2 is dose-dependent, peaks at a 500 ng/ml hBD-2 concentration and is prevented by blocking anti-alphavbeta3 monoclonal antibody. However, hBD-2-induced pro-angiogenic activity is not due to endogenously produced VEGF because it is not prevented by neutralizing anti-VEGF antibodies. Overall, our findings suggest that hBD-2 could link inflammation and the host defense through its pro-angiogenic activity.

Malassezia furfur induces the expression of β-defensin-2 in human keratinocytes in a protein kinase C-dependent manner

Antimicrobial peptides of the β-defensin family are expressed in all human epithelial tissues tested to date and have recently been the subject of vigorous investigation. Their localization and characteristics support the hypothesis that these peptides play a role in mucosal and skin defense. The lipophilic yeast Malassezia furfur is a saprophyte found in normal human cutaneous flora. Malassezia furfur is not only a saprophyte, but is also associated with several diseases such as Malassezia folliculitis, seborrheic dermatitis and some forms of atopic dermatitis, psoriasis and confluent and reticulate papillomatosis. Little is known about the mechanism by which M. furfur overcomes the natural barrier of the skin. To further define the role of the β-defensins in the innate human skin immune response, we analyzed the mRNA expression of two human β-defensins HBD-1 and HBD-2 in human keratinocytes treated with M. furfur. In addition, we looked into how M. furfur of TGF-β1 and IL-10, cytokines that interfere with the development of protective cell immunity, regulate their expression. Finally, we examined the signal transduction mechanisms involved during M. furfur uptake. Cultured human keratinocytes were treated with M. furfur. The mRNA and protein expression were analyzed, respectively, by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. Our data demonstrate that M. furfur does not modify HBD-1 expression, whereas it up-regulates, via protein kinase C (PKC), the expression of HBD-2, TGFβ-1 and IL-10 48 h after treatment. Our results suggest that β-defensins are integral components of innate host defenses. They play an essential part in the resistance of the human skin surfaces against M. furfur uptake and other microbial invasion.

Possible role of Malassezia furfur in psoriasis: modulation of TGF‐β1, integrin, and HSP70 expression in human keratinocytes and in the skin of psoriasis‐affected patients

Background:  Psoriasis is a disease characterized by an abnormal pattern of keratinocyte growth and differentiation. Malassezia furfur forms part of the normal human skin flora. It may also be involved in the pathogenesis of psoriasis. To define the role of M. furfur in the pathogenesis of psoriasis, we investigated how M. furfur regulates molecules involved in cell migration and proliferation. The experiments were performed using human keratinocytes and skin biopsies from M. furfur‐positive and ‐negative psoriasis‐affected patients. In addition, we examined the signal transduction mechanisms involved.

Malassezia furfur invasiveness in a keratinocyte cell line (HaCat): effects on cytoskeleton and on adhesion molecule and cytokine expression

Abstract The lipophilic yeast Malassezia furfur is a member of the cutaneous microbiota, also associated with several chronic diseases such as pityriasis versicolor, folliculitis, seborrhoeic dermatitis, and some forms of atopic dermatitis, psoriasis and confluent and reticulate papillomatosis. In this study we determined the immunomodulatory and invasive capacity of M. furfur in a human keratinocyte cell culture, HaCat. At a yeast cell to HaCat ratio of 30 : 1, M. furfur penetration was only 30% with poor phagolysosome fusion and with cytoskeleton modification. Transglutaminase I gene expression was also inhibited, supporting the hypothesis that M. furfur causes an initial break in the barrier function of the epidermis. Moreover, we demonstrated that M. furfur modulates proinflammatory and immunomodulatory cytokine synthesis by downregulating IL-1α and by inhibiting IL-6 and TNF-α and by upregulating IL-10 and TGF-β1. The suppressed inflammatory response induced by M. furfur may play a role in chronic disease.

Pemphigus serum and captopril induce heat shock protein 70 and inducible nitric oxide synthase overexpression, triggering apoptosis in human keratinocytes

Background  Captopril is an angiotensin‐converting enzyme inhibitor with sulphydryl groups in its chemical structure. It is commonly used as an antihypertensive drug. The occurrence of pemphigus vulgaris has repeatedly been reported in patients receiving captopril. The capacity of captopril and pemphigus serum to induce acantholysis, in vivo or in vitro, has been demonstrated experimentally.

Bacterial components induce cytokine and intercellular adhesion molecules-1 and activate transcription factors in dermal fibroblasts.

This study investigated the effect of various structural components of Gram-positive (lipotheichoic acid and protein A) and Gram-negative (porins and lipopolysaccharide) bacteria on human dermal fibroblasts. Fibroblasts are important effector cells which have a potential role in augmenting the inflammatory response in various diseases. In this study we present a profile of TNF-alpha, IL-6 and IL-8, the expression of intercellular adhesion molecules (ICAM-1) and the activation of transcriptional nuclear factor NF-kB and AP-1 in human dermal fibroblasts stimulated by bacterial surface components. Compared to the controls, increased ICAM-1, IL-6 and IL-8 gene expression after stimulation of LPS and porins at 2 and 4 h was more evident than that obtained following stimulation of LTA and PA. Gene expression was also associated with the production of cytokine proteins in culture supernatants. TNF-alpha gene expression remained undetectable. Moreover, LPS and porin treatments determined IkBalpha phosphorylation and degradation in human dermal fibroblasts and the subsequent activation of nuclear factors NF-kB and AP-1. These data suggest the importance of such stimuli in the first step of the inflammatory process, as well as the important role played by fibroblasts in skin inflammatory disease.

Anti-inflammatory effects of moxifloxacin and human β-defensin 2 association in human lung epithelial cell line (A549) stimulated with lipopolysaccharide

Epithelia in the human airways, from the nasal aperture to the alveoli, are covered in a protective film of fluid containing a number of antimicrobial proteins. Defensins are single-chain, strongly cationic peptides and are one of the most extensively studied classes of antimicrobial peptides. Moxifloxacin (MXF) is a fluoroquinolone that acts against both Gram positive and Gram negative bacteria. In this study, we evaluated the effects of HBD2, MXF and the association MXF/HBD2 on some cytokines and on the ICAM-1 expression in LPS-stimulated A549 cells. Our results suggest that by lowering the epithelial cell-derived IL-1beta, IL-6, IL-8 and ICAM-1 expression, the MXF/HBD2 association interferes with the multifunctional cytokine network evolving during inflammatory processes of the respiratory tract; this anti-inflammatory potential could be of great value in the treatment of inflammatory disorders.

Ectoine from halophilic microorganisms induces the expression of hsp70 and hsp70B' in human keratinocytes modulating the proinflammatory response.

Abstract The heat shock proteins (Hsps) have an important role in the cytoprotection and repair of cells and tissues. One potential mechanism of protection is the ability of Hsp to inhibit genetic expression of proinflammatory cytokines, the transcription of which is dependent on nuclear factor–kappa B (NF-κB) activation. In this study, we evaluated the ability of ectoine, a novel natural biomolecule produced by halophilic microorganisms, to activate the hsp70 and hsp70B′. By reverse transcriptase–polymerase chain reaction and Western blot analysis, we demonstrated increased hsp70B′ gene expression in human keratinocytes treated with ectoine and heat stressed. In contrast, in the absence of heat shock, ectoine was unable to induce hsp70B′ but had the ability to induce another member of the Hsp family, the hsp70. The latter is not only elevated in response to stress but is also present at basal level in unstressed cells. In addition, ectoine had no effect on proinflammatory cytokines interleukin (IL)-1alpha, IL-6, IL-8, and tumor necrosis factor–alpha and on NF-κB and IκB-α pathway, whereas it downregulated the expression of cited proinflammatory cytokines, in lipopolysaccharides-treated keratinocytes. These results highlighted the ability of ectoine to protect cells from stress conditions and to prevent cell damage by maintaining an elevated level of the Hsp70. Overall, these data might suggest the use of this compatible solute in cosmetic and even pharmaceutical preparations aiming to activate a cytoprotective heat shock response in human cells.

Effect of Temperature on the Shift of Pseudomonas Fluorescens from an Environmental Microorganism to a Potential Human Pathogen

Pseudomonas fluorescens is a Gram-negative bacterium generally considered of scarce clinical significance. However, in the last few years, the isolation of P. fluorescens as causative agent of nosocomial infections has rapidly increased. P. fluorescens is a psychrophile microorganism which grows at an optimal temperature of 25–30°C. In spite of this constraint, it has recently been reported that the human physiological temperature does not appear to be a barrier for this microorganism. In this study we examined the ability of P. fluorescens, grown at 28°C or at 37°C, to adhere to cultured human A549 pulmonary cells and to form biofilm. The ability of P. fluorescens to induce expression of proinflammatory cytokines, beta-defensin 2 and the intercellular adhesion molecule-1 was also investigated. Our results clearly indicate that inflammatory mediators are induced when the microorganism is grown at a lower temperature, while biofilm is formed only at 37°C. The results presented are consistent with previous reports indicating P. fluorescens as an opportunistic pathogen and underscore the urgent need for further studies to better characterize the virulence of this microorganism.

Alpha-MSH reduces the internalization of Staphylococcus aureus and down-regulates HSP 70, integrins and cytokine expression in human keratinocyte cell lines.

Abstract:  α‐melanocyte‐stimulating hormone (α‐MSH) is a neuropeptide predominantly produced by the pituitary gland, but it is also generated by many extra‐pituitary cells including keratinocytes of the skin. This neuropeptide has anti‐inflammatory and antimicrobial effects and probably contributes in innate immunity. Staphylococcus aureus is the aetiological agent of a wide range of infections in humans. Colonization of human skin by S. aureus is a characteristic feature of several skin diseases and is often followed by tissue invasion and severe cell damage. The aim of our study was to detect a possible role of α‐MSH during the early infection stages in the adhesion and penetration of keratinocytes before cell damage. Our data demonstrated that α‐MSH precociously down‐regulates the production of integrins such as β1 and heat shock surface protein 70, essential molecules for the entry of S. aureus. Moreover, in our experimental model, α‐MSH induces the down‐regulation of the pro‐inflammatory cytokine expression and of the adhesion molecules in keratinocytes activated by S. aureus. Our data suggest that α‐MSH plays a protective role in the skin by reducing infection and the inflammatory process.