Angela Catizone

Bovine lactoferrin counteracts Toll-like receptor mediated activation signals in antigen presenting cells.

Lactoferrin (LF), a key element in mammalian immune system, plays pivotal roles in host defence against infection and excessive inflammation. Its protective effects range from direct antimicrobial activities against a large panel of microbes, including bacteria, viruses, fungi and parasites, to antinflammatory and anticancer activities. In this study, we show that monocyte-derived dendritic cells (MD-DCs) generated in the presence of bovine LF (bLF) fail to undergo activation by up-modulating CD83, co-stimulatory and major histocompatibility complex molecules, and cytokine/chemokine secretion. Moreover, these cells are weak activators of T cell proliferation and retain antigen uptake activity. Consistent with an impaired maturation, bLF-MD-DC primed T lymphocytes exhibit a functional unresponsiveness characterized by reduced expression of CD154 and impaired expression of IFN-γ and IL-2. The observed imunosuppressive effects correlate with an increased expression of molecules with negative regulatory functions (i.e. immunoglobulin-like transcript 3 and programmed death ligand 1), indoleamine 2,3-dioxygenase, and suppressor of cytokine signaling-3. Interestingly, bLF-MD-DCs produce IL-6 and exhibit constitutive signal transducer and activator of transcription 3 activation. Conversely, bLF exposure of already differentiated MD-DCs completely fails to induce IL-6, and partially inhibits Toll-like receptor (TLR) agonist-induced activation. Cell-specific differences in bLF internalization likely account for the distinct response elicited by bLF in monocytes versus immature DCs, providing a mechanistic base for its multiple effects. These results indicate that bLF exerts a potent anti-inflammatory activity by skewing monocyte differentiation into DCs with impaired capacity to undergo activation and to promote Th1 responses. Overall, these bLF-mediated effects may represent a strategy to block excessive DC activation upon TLR-induced inflammation, adding further evidence for a critical role of bLF in directing host immune function.

Diabetic rat testes: morphological and functional alterations

Reproductive dysfunction is a consequence of diabetes, but the underlying mechanisms are poorly understood. This study investigated the histological and molecular alterations in the testes of rats injected with streptozotocin at prepuperal (SPI rats) and adult age (SAI rats) to understand whether diabetes affects testicular tissue with different severity depending on the age in which this pathological condition starts. The testes of diabetic animals showed frequent abnormal histology, and seminiferous epithelium cytoarchitecture appeared altered as well as the occludin distribution pattern. The early occurrence of diabetes increased the percentage of animals with high number of damaged tubules. The interstitial compartment of the testes was clearly hypertrophic in several portions of the organs both in SPI and SAI rats. Interestingly, fully developed Leydig cells were present in all the treated animals although abnormally distributed. Besides the above‐described damages, we found a similar decrease in plasma testosterone levels both in SPI and SAI rats. Oxidative stress (OS) is involved in the pathogenesis of various diabetic complications, and in our experimental models we found that manganese superoxide dismutase was reduced in diabetic animals. We conclude that in STZ‐induced diabetes, the altered spermatogenesis, more severe in SPI animals, is possibly due to the effect of OS on Leydig cell function which could cause the testosterone decrease responsible for the alterations found in the seminiferous epithelium of diabetic animals.

Poly(ADP-ribosyl)ation Acts in the DNA Demethylation of Mouse Primordial Germ Cells Also with DNA Damage-Independent Roles

Poly(ADP-ribosyl)ation regulates chromatin structure and transcription driving epigenetic events. In particular, Parp1 is able to directly influence DNA methylation patterns controlling transcription and activity of Dnmt1. Here, we show that ADP-ribose polymer levels and Parp1 expression are noticeably high in mouse primordial germ cells (PGCs) when the bulk of DNA demethylation occurs during germline epigenetic reprogramming in the embryo. Notably, Parp1 activity is stimulated in PGCs even before its participation in the DNA damage response associated with active DNA demethylation. We demonstrate that PARP inhibition impairs both genome-wide and locus-specific DNA methylation erasure in PGCs. Moreover, we evidence that impairment of PARP activity causes a significant reduction of expression of the gene coding for Tet1 hydroxylases involved in active DNA demethylation. Taken together these results demonstrate new and adjuvant roles of poly(ADP-ribosyl)ation during germline DNA demethylation and suggest its possible more general involvement in genome reprogramming.

Pleiotropic activity of hepatocyte growth factor during embryonic mouse testis development

The hepatocyte growth factor (HGF) is a pleiotropic cytokine whose action is mediated by c-met, a glycoproteic receptor with tyrosine kinase activity which transduces its multiple biological activities including cell proliferation, motility and differentiation. During embryonic development HGF acts as a morphogenetic factor as previously demonstrated for metanephric and lung development. Recently, culturing male genital ridges, we demonstrated that HGF is able to support in vitro testicular cord formation. In the present paper we report the expression pattern of the HGF gene during embryonic testis development and the multiple roles exerted by this factor during the morphogenesis of this organ. Northern blot analysis reveals a positive signal in urogenital ridges isolated from 11.5 days post coitum (dpc) embryos and in testes isolated from 13.5 and 15.5 dpc male embryos. On the contrary HGF mRNA is undetectable in ovaries isolated from 13.5 and 15.5 dpc embryos. Moreover, we demonstrate that HGF is synthesized and secreted by the male gonad and is biologically active. These data indicate a male specific biological function of HGF during embryonic gonadal development. This hypothesis is supported by the in vitro demonstration that HGF acts as a migratory factor for male mesonephric cells which is a male specific event. In addition we demonstrate that during testicular development, HGF acts as a morphogenetic factor able to reorganize dissociated testicular cells which, under HGF stimulation, form a tridimensional network of cord-like structures. Finally, we demonstrate that HGF induces testicular cell proliferation in this way being responsible for the size increase of the testis. All together the data presented in this paper demonstrate that HGF is expressed during the embryonic development of the testis and clarify the multiple roles exerted by this factor during the morphogenesis of the male gonad.

Embryonic mouse testis development: Role of platelet derived growth factor (PDGF-BB)

Platelet‐derived growth factors (PDGFs) are paracrine growth factors mediating epithelial–mesenchymal interactions and exerting multiple biological activities which include cell proliferation, motility, and differentiation. As previously demonstrated, PDGFs act during embryonic development and recently, by culturing male genital ridges, we have demonstrated that PDGF‐BB is able to support in vitro testicular cord formation. In the present paper, we report that PDGF‐BB is present during embryonic testis development and, in organ culture, induces cord formation although with reduced diameters compared with the cords formed in the genital ridges cultured in the presence of HGF. Moreover we have analyzed the roles exerted by this growth factor during the morphogenesis of the testis. We demonstrate by immunohistochemical experiments that PDGF‐BB and its receptors are synthesized by the male UGRs isolated from 11.5 and 13.5 dpc embryos and by Western blot that the factor is secreted in a biologically active form by testicular cells isolated from 13.5 dpc embryos. The biological roles of the factor have also been studied and we demonstrate that PDGF‐BB acts as a migratory factor for male mesonephric cells whose migration is a male specific event necessary for a normal testicular morphogenesis. In addition we demonstrate that during testicular development, PDGF‐BB induces testicular cell proliferation being in this way responsible for the increase in size of the testis. Finally we demonstrate that PDGF‐BB is able to reorganize dissociated testicular cells inducing the formation of large cellular aggregates. However the structures formed in vitro under PDGF‐BB stimulation never had a cord‐like morphology similar to the cord‐like structures formed in the presence of HGF (Ricci et al., 2002, Mech Dev 118:19–28), suggesting that this factor does not act as a morphogenetic factor during testicular development. All together the data presented in this paper demonstrate that PDGF‐BB and its receptors (α‐ and β‐subunits) are present during the crucial ages of embryonic mouse testis morphogenesis and indicate the multiple roles exerted by this factor during the development of the male gonad.

Expression of Platelet-Derived Growth Factor (PDGF) in the Epididymis and Analysis of the Epididymal Development in PDGF-A, PDGF-B, and PDGF Receptor β Deficient Mice

Abstract The platelet-derived growth factor (PDGF) family of ligands and receptors play a pivotal role in the development of various organs. The critical importance of the PDGF-mediated signaling during embryonic development and adult physiology of the kidney and the common mesonephric origin of the epididymis and kidney prompted us to investigate the immunohistochemical localization of PDGF A- and B-chain and PDGF receptor (PDGFR) α- and β-subunit in rat and mouse epididymis, the expression profiles of the corresponding mRNAs, and the consequences of a loss-of-function mutation at the PDGF-A, PDGF-B, and PDGFR-β loci on mouse epididymis phenotypic appearance. Prenatally, PDGF-A and PDGFR-α immunohistochemical staining was seen in both species, whereas PDGF-B and PDGFR-β were absent. The cellular localization of PDGF-A within the epithelium and the α-receptor in the mesenchyme in either mouse or rat before birth suggests that the PDGF-A/PDGFR-α system might be involved in the epididymal epithelial-mesenchymal interaction during the fetal period of life. Postnatally, PDGF A- and B-ligand and PDGFR α- and β-subunit were confined in the epithelium. The identity of PDGF and PDGFR proteins were further confirmed by immunoblotting. In line with the immunohistochemical studies, PDGF-A and PDGFR-α mRNAs were seen by reverse transcription–polymerase chain reaction in rat and mouse tissue before birth, whereas PDGF-B and PDGFR-β were almost not detectable. During the first days of life, PDGF-B and PDGFR-β genes started to appear, and the overall trend in mRNA expression throughout postnatal development showed that the transcripts levels for PDGF-A, PDGF-B, PDGFR-β, and PDGFR-α were constant with the only exception of a progressive decrease of PDGFR-α in adult rats. The PDGF-A null mutation strongly influenced the epididymal phenotype starting from puberty; only fetal PDGF-B and PDGFR-β −/− mice were available, and no differences were seen in the epididymis of these animals, compared with wild-type littermates. Taken together, these data indicate that the PDGF system is highly expressed in the epididymis and suggest that PDGF could be involved in the maintenance of morphological structure and functional control of this organ.

Functional and Morphological Improvement of Dystrophic Muscle by Interleukin 6 Receptor Blockade.

The anti-inflammatory agents glucocorticoids (GC) are the only available treatment for Duchenne muscular dystrophy (DMD). However, long-term GC treatment causes muscle atrophy and wasting. Thus, targeting specific mediator of inflammatory response may be more specific, more efficacious, and with fewer side effects. The pro-inflammatory cytokine interleukin (IL) 6 is overproduced in patients with DMD and in the muscle of mdx, the animal model for human DMD. We tested the ability of inhibition of IL6 activity, using an interleukin-6 receptor (Il6r) neutralizing antibody, to ameliorate the dystrophic phenotype. Blockade of endogenous Il6r conferred on dystrophic muscle resistance to degeneration and alleviated both morphological and functional consequences of the primary genetic defect. Pharmacological inhibition of IL6 activity leaded to changes in the dystrophic muscle environment, favoring anti-inflammatory responses and improvement in muscle repair. This resulted in a functional homeostatic maintenance of dystrophic muscle. These data provide an alternative pharmacological strategy for treatment of DMD and circumvent the major problems associated with conventional therapy.

Lactoferrin decreases inflammatory response by cystic fibrosis bronchial cells invaded with Burkholderia cenocepacia iron-modulated biofilm.

In cystic fibrosis (CF) high iron concentration in airway secretion plays a pivotal role in bacterial multiplication and biofilm formation as well as in inflammatory response. Burkholderia cenocepacia, an opportunistic facultative pathogen responsible for chronic lung infections and cepacia syndrome, recurrently infects CF patients. Lactoferrin (Lf), an iron binding multifunctional glycoprotein synthesized by exocrine glands and neutrophils, has been found at higher concentration in the airway secretions of infected CF patients than in healthy subjects. Here the influence of milk derivative bovine lactoferrin (bLf), an emerging important regulator of iron and inflammatory homeostasis, on invasiveness of B. cenocepacia iron-modulated biofilm, as well as on inflammatory response by infected CF bronchial (IB3-1) cells, is reported, bLf did not significantly affect invasion efficacy by biofilm-forming B. cenocepacia clinical strains. Conversely, the addition of bLf to cell monolayers during infection significantly decreased the pro-inflammatory Interleukin (IL)-1β and increased the antiinflammatory IL-11 expression compared to that observed in cells infected in the absence of bLf. The bLf ability to modulate genes expressed following B. cenocepacia infection seems related to its localization to the nucleus of infected IB3-1 cells. These results provide evidence for a role of bLf in the protection of infected CF cells from inflammation-related damage, thus extending the therapeutic potential of this multifunctional natural protein.

Hepatocyte growth factor modulates Sertoli–Sertoli tight junction dynamics

In mammalian testes Sertoli cells form tight junctions whose function is fundamental for the maintenance of a normal spermatogenesis. Hepatocyte growth factor (HGF) is a cytokine influencing the cellular tight junctions either in normal or in tumor cells. We have previously demonstrated that HGF is expressed in the rat testis and influences many functional activities of somatic and germ cells. We now report that HGF decreases the levels of testicular occludin and influences the position of the molecule in the tight junctions as demonstrated by confocal microscopy analysis. In fact in the presence of the factor occludin was mainly localized in the suprabasal region of the tubules whereas in its absence occludin was prevalently localized in the basal region. Occludin production is known to be regulated by different cytokines including TGFβ. We have investigated the role of HGF in the regulation of the levels of TGFβ and we report that HGF significantly increases the amount of the active fraction of the factor without affecting the amount of the total TGFβ. Urokinase type plasminogen activator (uPA) is closely related with the tight junctions and is one of the molecules able to activate the inactive TGF‐β. We found that HGF significantly increases the amount of uPA present in the testis suggesting that HGF regulates the amount of active TGFβ via uPA levels. In conclusion we report that in the testis HGF regulates Sertoli–Sertoli tight junctions inducing a reduction and redistribution of occludin possibly modulating the levels of uPA and active TGFβ. J. Cell. Physiol. 216: 253–260, 2008.

Increased levels of interleukin-6 exacerbate the dystrophic phenotype in mdx mice

Duchenne muscular dystrophy (DMD) is characterized by progressive lethal muscle degeneration and chronic inflammatory response. The mdx mouse strain has served as the animal model for human DMD. However, while DMD patients undergo extensive necrosis, the affected muscles of adult mdx mice rapidly regenerates and regains structural and functional integrity. The basis for the mild effects observed in mice compared with the lethal consequences in humans remains unknown. In this study, we provide evidence that interleukin-6 (IL-6) is causally linked to the pathogenesis of muscular dystrophy. We report that forced expression of IL-6, in the adult mdx mice, recapitulates the severe phenotypic characteristics of DMD in humans. Increased levels of IL-6 exacerbate the dystrophic muscle phenotype, sustaining inflammatory response and repeated cycles of muscle degeneration and regeneration, leading to exhaustion of satellite cells. The mdx/IL6 mouse closely approximates the human disease and more faithfully recapitulates the disease progression in humans. This study promises to significantly advance our understanding of the pathogenic mechanisms that lead to DMD.