Indoleamine 2,3-dioxygenase 1 activation in mature cDC1 promotes tolerogenic education of inflammatory cDC2 via metabolic communication.

Conventional dendritic cells (cDCs), cDC1 and cDC2, act both to initiate immunity and maintain self-tolerance. The tryptophan metabolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is used by cDCs in maintaining tolerance, but its role in different subsets remains unclear. At homeostasis, only mature CCR7+ cDC1 expressed IDO1 that was dependent on IRF8. Lipopolysaccharide treatment induced maturation and IDO1-dependent tolerogenic activity in isolated immature cDC1, but not isolated cDC2. However, both human and mouse cDC2 could induce IDO1 and acquire tolerogenic function when co-cultured with mature cDC1 through the action of cDC1-derived l-kynurenine. Accordingly, cDC1-specific inactivation of IDO1 in vivo exacerbated disease in experimental autoimmune encephalomyelitis. This study identifies a previously unrecognized metabolic communication in which IDO1-expressing cDC1 cells extend their immunoregulatory capacity to the cDC2 subset through their production of tryptophan metabolite l-kynurenine. This metabolic axis represents a potential therapeutic target in treating autoimmune demyelinating diseases.

Indoleamine 2,3-dioxygenase is a signaling protein in long-term tolerance by dendritic cells.

Regulation of tryptophan metabolism by indoleamine 2,3-dioxygenase (IDO) in dendritic cells (DCs) is a highly versatile modulator of immunity. In inflammation, interferon-γ is the main inducer of IDO for the prevention of hyperinflammatory responses, yet IDO is also responsible for self-tolerance effects in the longer term. Here we show that treatment of mouse plasmacytoid DCs (pDCs) with transforming growth factor-ß (TGF-ß) conferred regulatory effects on IDO that were mechanistically separable from its enzymic activity. We found that IDO was involved in intracellular signaling events responsible for the self-amplification and maintenance of a stably regulatory phenotype in pDCs. Thus, IDO has a tonic, nonenzymic function that contributes to TGF-ß-driven tolerance in noninflammatory contexts.

The Polyunsaturated Fatty Acid EPA, but Not DHA, Enhances Neurotrophic Factor Expression through Epigenetic Mechanisms and Protects against Parkinsonian Neuronal Cell Death.

ω-3 Polyunsaturated fatty acids (PUFAs) have been found to exert many actions, including neuroprotective effects. In this regard, the exact molecular mechanisms are not well understood. Parkinson's disease (PD) is the second most common age-related neurodegenerative disease. Emerging evidence supports the hypothesis that PD is the result of complex interactions between genetic abnormalities, environmental toxins, mitochondrial dysfunction, and other cellular processes, such as DNA methylation. In this context, BDNF (brain-derived neurotrophic factor) and GDNF (glial cell line-derived neurotrophic factor) have a pivotal role because they are both involved in neuron differentiation, survival, and synaptogenesis. In this study, we aimed to elucidate the potential role of two PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and their effects on BDNF and GDNF expression in the SH-SY5Y cell line. Cell viability was determined using the MTT assay, and flow cytometry analysis was used to verify the level of apoptosis. Transmission electron microscopy was performed to observe the cell ultrastructure and mitochondria morphology. BDNF and GDNF protein levels and mRNA were assayed by Western blotting and RT-PCR, respectively. Finally, methylated and hydroxymethylated DNA immunoprecipitation were performed in the BDNF and GDNF promoter regions. EPA, but not DHA, is able (i) to reduce the neurotoxic effect of neurotoxin 6-hydroxydopamine (6-OHDA) in vitro, (ii) to re-establish mitochondrial function, and (iii) to increase BNDF and GDNF expression via epigenetic mechanisms.

Testosterone and Follicle Stimulating Hormone-Dependent Glyoxalase 1 Up-Regulation Sustains the Viability of Porcine Sertoli Cells through the Control of Hydroimidazolone- and Argpyrimidine-Mediated NF-κB Pathway.

Because Sertoli cells (SCs) play a central role in germ cell survival, their death may result in marked germ cell loss and infertility. SCs are the only somatic cells within the seminiferous tubules and are essential for regulating spermatogenesis. Factors that enhance or diminish the viability of SCs may have profound effects on spermatogenesis. Yet the mechanisms underlying the maintenance of SC viability remain largely unknown. Glyoxalase 1 (Glo1) detoxifies methylglyoxal (MG), a highly reactive carbonyl species mainly formed during glycolysis, which is a potent precursor of cytotoxic advanced glycation end products (AGEs). Hydroimidazolone (MG-H1) and argpyrimidine (ArgPyr) are AGEs resulting from MG-mediated post-translational modification of arginine residues in various proteins. The role of Glo1 and MG-derived AGEs in regulating the fate of SCs has never been investigated. By using gene silencing and the specific MG scavenger, aminoguanidine, the authors demonstrate that Glo1, under testosterone and follicle-stimulating hormone control, sustains viability of porcine neonatal SCs through a mechanism involving the NF-κB pathway. Glo1 knockdown induces a mitochondrial apoptotic pathway driven by the intracellular accumulation of MG-H1 and ArgPyr that desensitizes NF-κB signaling by modifying the inhibitor of NF-κB kinase, IKKß. This is the first report describing a role for Glo1 and MG-derived AGEs in SC biology, providing valuable new insights into the potential involvement of this metabolic axis into spermatogenesis.

Xenograft of microencapsulated Sertoli cells restores glucose homeostasis in db/db mice with spontaneous diabetes mellitus.

BACKGROUND: Increased abdominal fat and chronic inflammation in the expanded adipose tissue of obesity contribute to the development of insulin resistance and type 2 diabetes mellitus (T2D). The emerging immunoregulatory and anti-inflammatory properties of Sertoli cells have prompted their application to experimental models of autoimmune/inflammatory disorders, including diabetes. The main goal of this work was to verify whether transplantation of microencapsulated prepubertal porcine Sertoli cells (MC-SC) in the subcutaneous abdominal fat depot of spontaneously diabetic and obese db/db mice (homozygous for the diabetes spontaneous mutation [Leprdb ]) would: (i) improve glucose homeostasis and (ii) modulate local and systemic immune response and adipokines profiles. METHODS: Porcine prepubertal Sertoli cells were isolated, according to previously established methods and enveloped in Barium alginate microcapsules by a mono air-jet device. MC-SC were then injected in the subcutaneous abdominal fat depot of db/db mice. RESULTS: We have preliminarily shown that graft of MC-SC restored glucose homeostasis, with normalization of glycated hemoglobin values with improvement of the intraperitoneal glucose tolerance test in 60% of the treated animals. These results were associated with consistent increase, in the adipose tissue, of uncoupling protein 1 expression, regulatory B cells, anti-inflammatory macrophages and a concomitant decrease of proinflammatory macrophages. Furthermore, the treated animals showed a reduction in inducible NOS and proinflammatory molecules and a significant increase in an anti-inflammatory cytokine such as IL-10 along with concomitant rise of circulating adiponectin levels. The anti-hyperglycemic graft effects also emerged from an increased expression of GLUT-4, in conjunction with downregulation of GLUT-2, in skeletal muscle and liver, respectively. CONCLUSIONS: Preliminarily, xenograft of MC-SC holds promises for an effective cell therapy approach for treatment of experimental T2D.

Stem cells from human amniotic fluid exert immunoregulatory function via secreted indoleamine 2,3-dioxygenase1.

Although human amniotic fluid does contain different populations of foetal-derived stem cells, scanty information is available on the stemness and the potential immunomodulatory activity of in vitro expanded, amniotic fluid stem cells. By means of a methodology unrequiring immune selection, we isolated and characterized different stem cell types from second-trimester human amniotic fluid samples (human amniotic fluid stem cells, HASCs). Of those populations, one was characterized by a fast doubling time, and cells were thus designated as fHASCs. Cells maintained their original phenotype under prolonged in vitro passaging, and they were able to originate embryoid bodies. Moreover, fHASCs exhibited regulatory properties when treated with interferon (IFN)-γ, including induction of the immunomodulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1). On coculture with human peripheral blood mononuclear cells, IFN-γ-treated fHASCs caused significantly decreased T-cell proliferation and increased frequency in CD4(+)  CD25(+)  FOXP3(+) regulatory T cells. Both effects required an intact IDO1 function and were cell contact-independent. An unprecedented finding in our study was that purified vesicles from IFN-γ-treated fHASCs abundantly expressed the functional IDO1 protein, and those vesicles were endowed with an fHASC-like regulatory function. In vivo, fHASCs were capable of immunoregulatory function, promoting allograft survival in a mouse model of allogeneic skin transplantation. This was concurrent with the expansion of CD4(+)  CD25(+)  Foxp3(+) T cells in graft-draining lymph nodes from recipient mice. Thus fHASCs, or vesicles thereof, may represent a novel opportunity for immunoregulatory maneuvers both in vitro and in vivo.

In vitro cadmium effects on ECM gene expression in human bronchial epithelial cells.

Occupational and environmental exposure to the heavy metal cadmium (Cd) and its inhalation from cigarette smoke are associated with emphysema. Many growth factors and extracellular matrix (ECM) cell signaling molecules are directly involved in the epithelial bronchial cell pathway. This study investigated the direct effects of Cd on the production of several ECM components in human bronchial epithelial cells (BEAS-2B) that were exposed in vitro for 48 h to sub-toxic and toxic concentrations of Cd. Gene expression of collagens, metalloproteases (MMPs), integrins, tenascin and vitronectin were quantified by RT-PCR. To study apoptosis cascade, annexin assay and cellular cytotoxicity by MTT assay were performed. We also investigated whether an imbalance in the TGFß/TGFß receptor (TGFßR) expression mediated Cd effects. The results showed the sub-toxic Cd dose significantly increased tenascin, vitronectin, ß1 and ß5 integrin gene expression. The toxic Cd dose decreased type IV and V collagen, α1, α2 and ß3 integrins. Both Cd doses down-regulated type I collagen and up-regulated metalloproteases. Each Cd dose caused a different imbalance in the complex pattern of TGFß and its receptors. No alteration in classic apoptotic marker protein expression was observed in presence of the sub-toxic dose of Cd, suggesting this metal alters ECM production without apoptotic activation. In conclusion, all these data show even sub-toxic Cd dose exposure alters the specific gene expression of several ECM components that are crucially implicated in the mechanical properties of lung parenchyma supporting the hypothesis that the mechanism underlying Cd-induced lung disease may involve downstream changes in TGFß/TGFßR signaling.

Long-term stability, functional competence, and safety of microencapsulated specific pathogen-free neonatal porcine Sertoli cells: a potential product for cell transplant therapy.

BACKGROUND: Porcine Sertoli cells (pSCs) have been employed for cell therapy in pre-clinical studies for several chronic/immune diseases as they deliver molecules associated with trophic and anti-inflammatory effects. To be employed for human xenografts, pSCs products need to comply with safety and stability. To fulfill such requirements, we employed a microencapsulation technology to increase pre-transplant storage stability of specific pathogen-free pSCs (SPF-pSCs) and evaluated the in vivo long-term viability and safety of grafts. METHODS: Specific pathogen free neonatal pigs underwent testis excision under sterility. pSCs were isolated, characterized by immunofluorescence (IF) and cytofluorimetric analysis (CA) and examined in terms of viability and function [namely, production of anti-müllerian hormone (AMH), inhibin B, and transforming growth factor beta-1 (TFGß-1)]. After microencapsulation in barium alginate microcapsules (Ba-MC), long-term SPF-pSCs (Ba-MCpSCs) viability and barium concentrations were evaluated at 1, 24 throughout 40 h to establish pre-transplant storage conditions. RESULTS: The purity of isolated pSCs was about 95% with negligible contaminating cells. Cultured pSCs monolayers, both prior to and after microencapsulation, maintained high function and full viability up to 24 h of storage. At 40 h post-encapsulation, pSCs viability decreased to 80%. Barium concentration in Ba-MCpSCs lagged below the normal maximum daily allowance and was stable for 4 months in mice with no evident side effects. CONCLUSIONS: Such results suggest that this protocol for the isolation and microencapsulation of pSCs is compatible with long-haul transportation and that Ba-MCpSCs could be potentially employable for xenotransplantation.

Zinc restores functionality in porcine prepubertal Sertoli cells exposed to subtoxic cadmium concentration via regulating the Nrf2 signaling pathway.

Introduction: Among substances released into the environment by anthropogenic activities, the heavy metal cadmium (Cd) is known to induce severe testicular injury causing male subfertility/infertility. Zinc (Zn) is another heavy metal that, unlike Cd, is physiologically present in the testis, being essential for spermatogenesis. We aimed to examine the possibility that 50 µM ZnCl2 could counteract the toxic effects induced by Cd in an in vitro model of porcine prepubertal Sertoli cells (SCs) exposed to both subtoxic (5 µM) and toxic (10 µM) concentrations of CdCl2 for 48 h. Materials and Methods: Apoptosis, cell cycle, and cell functionality were assessed. The gene expression of Nrf2 and its downstream antioxidant enzymes, ERK1/2, and AKT kinase signaling pathways were evaluated. Materials and Results: We found that Zn, in co-treatment with subtoxic and toxic Cd concentration, increased the number of metabolically active SCs compared to Cd exposure alone but restored SC functionality only in co-treatment with subtoxic Cd concentration with respect to subtoxic Cd alone. Exposure of Cd disrupted cell cycle in SCs, and Zn co-treatment was not able to counteract this effect. Cd alone induced SC death through apoptosis and necrosis in a dose-dependent manner, and co-treatment with Zn increased the pro-apoptotic effect of Cd. Subtoxic and toxic Cd exposures activated the Nrf2 signaling pathway by increasing gene expression of Nrf2 and its downstream genes (SOD, HO-1, and GSHPx). Zn co-treatment with subtoxic Cd attenuated upregulation on the Nrf2 system, while with toxic Cd, the effect was more erratic. Studying ERK1/2 and AKT pathways as a target, we found that the phosphorylation ratio of p-ERK1/2 and p-AKT was upregulated by both subtoxic and toxic Cd exposure alone and in co-treatment with Zn. Discussion: Our results suggest that Zn could counteract Cd effects by increasing the number of metabolically active SCs, fully or partially restoring their functionality by modulating Nrf2, ERK1/2, and AKT pathways. Our SC model could be useful to study the effects of early Cd exposure on immature testis, evaluating the possible protective effects of Zn.

Nickel oxide nanoparticles exposure as a risk factor for male infertility: "In vitro" effects on porcine pre-pubertal Sertoli cells.

Lately, nickel oxide nanoparticles (NiO NPs) have been employed in different industrial and biomedical fields. Several studies have reported that NiO NPs may affect the development of reproductive organs inducing oxidative stress and, resulting in male infertility. We investigated the in vitro effects of NiO NPs on porcine pre-pubertal Sertoli cells (SCs) which undergone acute (24 h) and chronic (from 1 up to 3 weeks) exposure at two subtoxic doses of NiO NPs of 1 µg/ml and 5 µg/ml. After NiO NPs exposure we performed the following analysis: (a) SCs morphological analysis (Light Microscopy); (b) ROS production and oxidative DNA damage, gene expression of antioxidant enzymes (c) SCs functionality (AMH, inhibin B Real-time PCR analysis and ELISA test); (d) apoptosis (WB analysis); (e) pro-inflammatory cytokines (Real-time PCR analysis), and (f) MAPK kinase signaling pathway (WB analysis). We found that the SCs exposed to both subtoxic doses of NiO NPs didn't sustain substantial morphological changes. NiO NPs exposure, at each concentration, reported a marked increase of intracellular ROS at the third week of treatment and DNA damage at all exposure times. We demonstrated, un up-regulation of SOD and HO-1 gene expression, at both concentrations tested. The both subtoxic doses of NiO NPs detected a down-regulation of AMH and inhibin B gene expression and secreted proteins. Only the 5 µg/ml dose induced the activation of caspase-3 at the third week. At the two subtoxic doses of NiO NPs a clear pro-inflammatory response was resulted in an up-regulation of TNF-α and IL-6 in terms of mRNA. Finally, an increased phosphorylation ratio of p-ERK1/2, p-38 and p-AKT was observed up to the third week, at both concentrations. Our results show the negative impact of subtoxic doses NiO NPs chronic exposure on porcine SCs functionality and viability.

Microencapsulated Sertoli cells sustain myoblast proliferation without affecting the myogenic potential. In vitro data.

Sertoli cells (SeC) isolated from porcine testes have shown direct effects on muscle precursor cells sustaining C2C12 myoblasts proliferation and inhibiting oxidative stress and apoptosis in the early phase of the differentiation process, and stimulating myoblast fusion into myotubes and the expression of markers of myogenic differentiation in the late phase. This suggested that the cocktail of factors secreted by SeC stimulates proliferation in myoblasts without weakening their myogenic potential resulting in the formation of the critical myoblast amount necessary to rebuild the required muscle mass upon a damage. Here, we show that co-culturing C2C12 myoblasts with high doses of SeC microencapsulated in clinical grade alginate-based microcapsules (MC-SeC) for three days in differentiation medium (DM) translates into increased cell numbers and almost absence of myotube formation. However, after removal of MC-SeC, an intense fusion activity into myotubes was observed culminating in a fusion index similar to that of control after additional three days of culture in DM. These data definitely demonstrate that SeC-derived factors preserve the myogenic potential while sustaining cell proliferation in C2C12 myoblasts.

Improvements in human sperm quality by long-term in vitro co-culture with isolated porcine Sertoli cells.

BACKGROUND: Spermatogenesis is a complex process where spermatogonial germ cells become spermatozoa with the indispensable support of Sertoli cells (SCs), which provide 'ad hoc' structural and nutritional support. Unfortunately, for most sperm dysfunctions, no therapies are yet available except assisted reproductive technologies (ART) that are based on the use of different culture media to preserve sperm in vitro. However, sperm culture is only possible for short periods of time, since long-term culture would invariably and irreversibly damage the cells with negative impact on their fertilization potential. METHODS: Fresh sperm cells (5 ml of 20 × 10(6)/ml) were co-cultured with SCs layers, derived from prepubertal pig testes or incubated in cell free SC medium or BWW (Biggers, Whitten and Whittingham) medium for 2, 4 or 7 days. Sperm viability, motility, mitochondrial status, DNA fragmentation, chromatin integrity, intracellular calcium and acrosome status were assessed after every co-culture or incubation time, but capacitation and induction of acrosome reaction (AR) with progesterone was only evaluated after 7 days. RESULTS: SCs layers derived from prepubertal pig testes (co-culture of sperm and SC feeder, CCSCF) were able to preserve normal sperm viability, motility and normal mitochondrial function, after 7 days of culture; CCSCF did not induce AR or hyperactivation of spermatozoa, keeping the sperm in a quiescent state for 7 days of culture. Nevertheless, the sperm were readily able to initiate AR after stimulation with progesterone. CONCLUSIONS: CCSCF maintained good sperm viability and motility for 7 days. This approach could improve retention of sperm viability and motility during ART procedures and maintain sperm viability, during transfer between two distant Centres, avoiding the need for cryopreservation.

IDO mediates TLR9-driven protection from experimental autoimmune diabetes.

Originally predicated on the recognition of an increasing prevalence of allergy, the hygiene hypothesis was later found to accommodate the contrasting epidemiologic trends in developed countries for infectious vs autoimmune diseases. Experimentally, reduced exposure to infections will increase the risk of disease in several models of experimental autoimmunity. Although TLRs were initially considered as stimulatory molecules capable of activating early defense mechanisms against invading pathogens, emerging data suggest that they can also exert a regulatory function. In the present study, we evaluated whether TLR3 and TLR9, recognizing microbial dsDNA and CpG-containing DNA sequences, respectively, play a role in the protection from experimental autoimmune diabetes induced in C57BL/6 mice by streptozotocin. In wild-type animals, the disease was accompanied by up-regulation of IDO in pancreatic lymph nodes and would be greatly exacerbated by in vivo administration of an IDO inhibitor. Conversely, administration of a CpG-containing oligodeoxynucleotide greatly attenuated the disease in an IDO-dependent fashion. TLR9-, but not TLR3-deficient mice developed a more robust disease, an event accompanied by lack of IDO induction in pancreatic lymph nodes. Thus, our data suggest that the TLR9-IDO axis may represent a valuable target in the prevention/therapy of type 1 diabetes.

A microbial polysaccharide reduces the severity of rheumatoid arthritis by influencing Th17 differentiation and proinflammatory cytokines production.

Rheumatoid arthritis (RA) is a chronic and debilitating autoimmune disease characterized by chronic joint inflammation with subsequent cartilage and bone destruction. RA is emerging as a model of IL-17-driven autoimmune inflammatory disease. IL-17 is a marker for Th17 cells, with its master regulator being the retinoic acid receptor-related orphan receptor (RORgammat) regulated by STAT3 signaling. Glucuronoxylomannan (GXM), a polysaccharide representing the main component of the capsular material of the opportunistic yeast Cryptococcus neoformans, exhibits potent immunosuppressive properties both in vitro and in vivo. The present study investigates the effects of GXM treatment on the progression of collagen-induced arthritis. GXM suppressed clinical signs of collagen-induced arthritis and blocked joint erosion progression. This effect was mediated by down-regulation of key cytokines involved in the pathogenesis of RA such as TNF-alpha and IL-1beta, and up-regulation of the inhibitory cytokine IL-10. Moreover, a reduction of IL-6 and TGF-beta, which inhibit Th17 differentiation with consequent decreased IL-17 production at the local and systemic level, was observed. The effect of GXM on Th17 differentiation mirrored the reduction in STAT3 activation and inhibition of RORgammat synthesis. Consequently, this work highlights the beneficial properties of an efficacious compound that could eventually be destined to the clinic.

Sertoli Cells Improve Myogenic Differentiation, Reduce Fibrogenic Markers, and Induce Utrophin Expression in Human DMD Myoblasts.

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in DMD gene translating in lack of functional dystrophin and resulting in susceptibility of myofibers to rupture during contraction. Inflammation and fibrosis are critical hallmarks of DMD muscles, which undergo progressive degeneration leading to loss of independent ambulation in childhood and death by early adulthood. We reported that intraperitoneal injection of microencapsulated Sertoli cells (SeC) in dystrophic mice translates into recovery of muscle morphology and performance thanks to anti-inflammatory effects and induction of the dystrophin paralogue, utrophin at the muscle level, opening new avenues in the treatment of DMD. The aim of this study is to obtain information about the direct effects of SeC on myoblasts/myotubes, as a necessary step in view of a translational application of SeC-based approaches to DMD. We show that (i) SeC-derived factors stimulate cell proliferation in the early phase of differentiation in C2C12, and human healthy and DMD myoblasts; (ii) SeC delay the expression of differentiation markers in the early phase nevertheless stimulating terminal differentiation in DMD myoblasts; (iii) SeC restrain the fibrogenic potential of fibroblasts, and inhibit myoblast-myofibroblast transdifferentiation; and, (iv) SeC provide functional replacement of dystrophin in preformed DMD myotubes regardless of the mutation by inducing heregulin ß1/ErbB2/ERK1/2-dependent utrophin expression. Altogether, these results show that SeC are endowed with promyogenic and antifibrotic effects on dystrophic myoblasts, further supporting their potential use in the treatment of DMD patients. Our data also suggest that SeC-based approaches might be useful in improving the early phase of muscle regeneration, during which myoblasts have to adequately proliferate to replace the damaged muscle mass.

Effect of EPA on Neonatal Pig Sertoli Cells "In Vitro": A Possible Treatment to Help Maintain Fertility in Pre-Pubertal Boys Undergoing Treatment With Gonado-Toxic Therapies.

The incidence of cancer in pre-pubertal boys has significantly increased and, it has been recognized that the gonado-toxic effect of the cancer treatments may lead to infertility. Here, we have evaluated the effects on porcine neonatal Sertoli cells (SCs) of three commonly used chemotherapy drugs; cisplatin, 4-Hydroperoxycyclophosphamide and doxorubicin. All three drugs induced a statistical reduction of 5-hydroxymethylcytosine in comparison with the control group, performed by Immunofluorescence Analysis. The gene and protein expression levels of GDNF, were significantly down-regulated after treatment to all three chemotherapy drugs comparison with the control group. Specifically, differences in the mRNA levels of GDNF were: 0,8200 ± 0,0440, 0,6400 ± 0,0140, 0,4400 ± 0,0130 fold change at 0.33, 1.66, and 3.33µM cisplatin concentrations, respectively (**p < 0.01 at 0.33 and 1.66 µM vs SCs and ***p < 0.001 at 3.33µM vs SCs); 0,6000 ± 0,0340, 0,4200 ± 0,0130 fold change at 50 and 100 µM of 4-Hydroperoxycyclophosphamide concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,7000 ± 0,0340, 0,6200 ± 0,0240, 0,4000 ± 0,0230 fold change at 0.1, 0.2 and 1 µM doxorubicin concentrations, respectively (**p < 0.01 at 0.1 and 0.2 µM vs SCs and ***p < 0.001 at 1 µM vs SCs). Differences in the protein expression levels of GDNF were: 0,7400 ± 0,0340, 0,2000 ± 0,0240, 0,0400 ± 0,0230 A.U. at 0.33, 1.66, and 3.33µM cisplatin concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,7300 ± 0,0340, 0,4000 ± 0,0130 A.U. at 50 and 100 µM of 4- Hydroperoxycyclophosphamide concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,6200 ± 0,0340, 0,4000 ± 0,0240, 0,3800 ± 0,0230 A.U. at 0.l, 0.2 and 1 µM doxorubicin concentrations, respectively (**p < 0.01 at 0.1 and 0.2 µM vs SCs and ***p < 0.001 at 1 µM vs SCs). Furthermore, we have demonstrated the protective effect of eicosapentaenoic acid on SCs only at the highest concentration of cisplatin, resulting in an increase in both gene and protein expression levels of GDNF (1,3400 ± 0,0280 fold change; **p < 0.01 vs SCs); and of AMH and inhibin B that were significantly recovered with values comparable to the control group. Results from this study, offers the opportunity to develop future therapeutic strategies for male fertility management, especially in pre-pubertal boys.

Aspergillus fumigatus tryptophan metabolic route differently affects host immunity.

Indoleamine 2,3-dioxygenases (IDOs) degrade l-tryptophan to kynurenines and drive the de novo synthesis of nicotinamide adenine dinucleotide. Unsurprisingly, various invertebrates, vertebrates, and even fungi produce IDO. In mammals, IDO1 also serves as a homeostatic regulator, modulating immune response to infection via local tryptophan deprivation, active catabolite production, and non-enzymatic cell signaling. Whether fungal Idos have pleiotropic functions that impact on host-fungal physiology is unclear. Here, we show that Aspergillus fumigatus possesses three ido genes that are expressed under conditions of hypoxia or tryptophan abundance. Loss of these genes results in increased fungal pathogenicity and inflammation in a mouse model of aspergillosis, driven by an alternative tryptophan degradation pathway to indole derivatives and the host aryl hydrocarbon receptor. Fungal tryptophan metabolic pathways thus cooperate with the host xenobiotic response to shape host-microbe interactions in local tissue microenvironments.

Bioactive long-term release from biodegradable microspheres preserves implanted ALG-PLO-ALG microcapsules from in vivo response to purified alginate.

PURPOSE: To assess whether prevention of unexpected in vivo adverse inflammatory and immune responses to biohybrid organ grafts for the treatment of Type I Diabetes Mellitus (T1DM) is possible by superoxide dismutase and ketoprofen controlled release. METHODS: Superoxide dismutase and ketoprofen-loaded polyester microspheres were prepared by W/O/W and O/W methods, embodied into purified alginate-poly-L-ornithine-alginate microcapsules and intraperitoneally implanted into CD1 mice. The microspheres were characterized for morphology, size, encapsulation efficiency, enzyme activity and in vitro release. Purified alginate contaminants were assayed, and the obtained microcapsules were investigated for size and morphology before and after implantation over 30 days. Cell pericapsular overgrowth and expression were evaluated by optical microscopy and flow cytometry. RESULTS: Superoxide dismutase and ketoprofen sustained release reduced cell pericapsular overgrowth in comparison to the control. Superoxide dismutase release allowed preserving the microcapsules over 30 days. Ketoprofen-loaded microspheres showed some effect in the immediate post-grafting period. A higher macrophage and T-cell expression was observed for the control group. CONCLUSIONS: Microspheres containing superoxide dismutase and ketoprofen may represent novel tools to limit or prevent unpredictable adverse in vivo response to alginate, thus contributing to improve cell transplantation success rates in T1DM treatment.

Artocarpus tonkinensis Extract Inhibits LPS-Triggered Inflammation Markers and Suppresses RANKL-Induced Osteoclastogenesis in RAW264.7.

Artocarpus tonkinensis (At) leaf decoction, a traditional remedy prepared in North Vietnam by the Hmong ethnic group, is a tea extract rich in bioactive compounds that may have therapeutic effects in arthritis and backache. Indeed, it has been demonstrated that At is able to inhibit Th17 lymphocytes development and to protect mice in an experimental model of collagen-induced arthritis. By resorting to macrophage in vitro models of inflammation and osteoclastogenesis, we showed that At extract significantly reduced nitric oxide synthase 2 (NOS2) activity and IL-6 production by RAW 264.7 murine cells. Moreover, At demonstrated an anti-osteoclastogenic effect, as revealed by complete inhibition of TRAP-positive osteoclast formation and decreased expression of key osteoclast-related genes. This At activity likely relies on the inhibition of RANK downstream signaling pathway, as the activation of non-receptor tyrosine kinase Src is reduced upon RANKL-At exposure. Protective effect of At against bone loss was also enlightened in vivo by collagen-induced arthritis (CIA) experiment demonstrating that, although paw edema was only weakly opposed by drinking At decoction, bone and cartilage were well preserved in CIA+At mice and joint tissue expressed decreased levels of osteoclast marker genes respect to CIA control group. Maesopsin 4-O-ß-D-glucoside (i.e., TAT-2, one of the main decoction bioactive components) was capable to contrast NOS2 activity, IL-6 expression and osteoclast formation, too, albeit to a lesser extent when compared to At decoction. Overall, this study enlightens another At cell target, macrophages, beside Th17 lymphocytes, and suggests that the anti-arthritic beneficial effects of At decoction largely derives from its ability to counteract not only inflammation, but also osteoclastogenesis.

Molecular mechanisms underlying eicosapentaenoic acid inhibition of HDAC1 and DNMT expression and activity in carcinoma cells.

DNA methylation and histone acetylation, the most studied epigenetic changes, drive and maintain cancer phenotypes. DNA methyltransferase (DNMT) dysregulation promoted localized hypermethylation in CpG rich regions while upregulated histone deacetylases (HDAC) deacetylated histone tails. Both changes led to close chromatin conformation, suppressing transcription and silencing tumor suppressor genes. Consequently, HDAC and DNMT inhibitors appeared to reprogram the transcriptional circuit and potentiate anti-tumoral activity. Here, we report that eicosapentaenoic acid (EPA), a fatty acid with anti-cancer properties, inhibited HDAC1 and DNMT expression and activity, thus promoting tumor suppressor gene expression. In hepatocarcinoma cells (HCC) EPA bound and activated PPARγ thus downregulating HDAC1 which sequentially reduced expression of DNMT1, 3A and 3B. At the same time, activated PPARγ physically interacted with DNMT1 and HDAC1 in a CpG island on the Hic-1 gene to assemble PPARγ/DNMT1 and PPARγ/HDAC1 protein complexes, which exited from DNA. When EPA and PPARγ were no longer bound, the protein complexes separated into individual proteins. Consequently, DNMT1 and HDAC1 down-regulation and release from DNA inhibited their activities. Overall, EPA-bound PPARγ induced re-expression of the tumor suppressor gene Hic-1. In the present study PPARγ emerged as a master regulator acting synergistically through diverse targets and ways to reveal the epigenetic action of EPA as an HDAC1 and DNMT1 inhibitor.