Functionality of macrophages encapsulated in porcine decellularized adipose matrix hydrogels and interaction with Candida albicans.

Extracellular matrix hydrogels are considered one of the most suitable biomaterials for tissue regeneration due to their similarity with the extracellular microenvironment of the native tissue. Their properties are dependent on their composition, material concentration, fiber density and the fabrication approaches, among other factors. The encapsulation of immune cells in this kind of hydrogels, both in absence or presence of a pathogen, represents a promising strategy for the development of platforms that mimic healthy and infected tissues, respectively. In this work, we have encapsulated macrophages in 3D hydrogels of porcine decellularized adipose matrices (pDAMs) without and with the Candida albicans fungus, as 3D experimental models to study the macrophage immunocompetence in a closer situation to the physiological conditions and to mimic an infection scenario. Our results indicate that encapsulated macrophages preserve their functionality within these pDAM hydrogels and phagocytose live pathogens. In addition, their behavior is influenced by the hydrogel pore size, inversely related to the hydrogel concentration. Thus, larger pore size promotes the polarization of macrophages towards M2 phenotype along the time and enhances their phagocytosis capability. It is important to point out that encapsulated macrophages in absence of pathogen showed an M2 phenotype, but macrophages coencapsulated with C. albicans can switch towards an M1 inflammatory phenotype to resolve the infection, depending on the fungus quantity. The present study reveals that pDAM hydrogels preserve the macrophage plasticity, demonstrating their relevance as new models for macrophage-pathogen interaction studies that mimic an infection scenario with application in regenerative medicine research.

Effects of graphene oxide and reduced graphene oxide nanomaterials on porcine endothelial progenitor cells.

Graphene oxide (GO) and reduced graphene oxide (rGO) have been widely used in the field of tissue regeneration and various biomedical applications. In order to use these nanomaterials in organisms, it is imperative to possess an understanding of their impact on different cell types. Due to the potential of these nanomaterials to enter the bloodstream, interact with the endothelium and accumulate within diverse tissues, it is highly relevant to probe them when in contact with the cellular components of the vascular system. Endothelial progenitor cells (EPCs), involved in blood vessel formation, have great potential for tissue engineering and offer great advantages to study the possible angiogenic effects of biomaterials. Vascular endothelial growth factor (VEGF) induces angiogenesis and regulates vascular permeability, mainly activating VEGFR2 on endothelial cells. The effects of GO and two types of reduced GO, obtained after vacuum-assisted thermal treatment for 15 min (rGO15) and 30 min (rGO30), on porcine endothelial progenitor cells (EPCs) functionality were assessed by analyzing the nanomaterial intracellular uptake, reactive oxygen species (ROS) production and VEGFR2 expression by EPCs. The results evidence that short annealing (15 and 30 minutes) at 200 °C of GO resulted in the mitigation of both the increased ROS production and decline in VEGFR2 expression of EPCs upon GO exposure. Interestingly, after 72 hours of exposure to rGO30, VEGFR2 was higher than in the control culture, suggesting an early angiogenic potential of rGO30. The present work reveals that discrete variations in the reduction of GO may significantly affect the response of porcine endothelial progenitor cells.

Promising biomedical subcutaneous delivery system in opioid disaccustom process: In vitro/in vivo evaluation of naloxone microparticles on antagonist effect of morphine.

The addiction induced by the misuse of opioids, is not only a public health emergency but also a social and economic welfare. The main therapy is based on opioid antagonists. Oral and injectable naltrexone administration is the most widely used, presenting some inconveniences: poor patient adherence to the oral daily dosing schedule, cases of hepatitis and clinically significant liver dysfunction. This study proposes the in vitro e in vivo evaluation of anti-opioid properties of naloxone loaded-poly(lactic-co-glycolic) acid microparticles (NX-MP). In previous studies, NX-MP showed in vitro sustained naloxone release for one week at least. Our results demonstrate the in vitro efficacy of the NX-MP antagonizing for 7 days the morphine effect in SH-SY5Y cells and myenteric plexus-longitudinal muscle preparations isolated from guinea-pig ileum. The in vivo evaluation of the NX-MP was carried out in mice testing the antagonism of the antinociceptive effect of morphine. Results showed that subcutaneous administration of NX-MP blocked the morphine effect. The results of this work suggest that the subcutaneous administration of NX-MP enhances naloxone therapeutic efficacy as non-addictive medication and could be a promising alternative to naltrexone. Furthermore, the dose of NX-MP can be adapted to the patient necessities. It would be an interesting advantage to treat opioid-addiction.

Effects of Graphene Oxide and Reduced Graphene Oxide Nanostructures on CD4+ Th2 Lymphocytes.

The activation of T helper (Th) lymphocytes is necessary for the adaptive immune response as they contribute to the stimulation of B cells (for the secretion of antibodies) and macrophages (for phagocytosis and destruction of pathogens) and are necessary for cytotoxic T-cell activation to kill infected target cells. For these issues, Th lymphocytes must be converted into Th effector cells after their stimulation through their surface receptors TCR/CD3 (by binding to peptide-major histocompatibility complex localized on antigen-presenting cells) and the CD4 co-receptor. After stimulation, Th cells proliferate and differentiate into subpopulations, like Th1, Th2 or Th17, with different functions during the adaptative immune response. Due to the central role of the activation of Th lymphocytes for an accurate adaptative immune response and considering recent preclinical advances in the use of nanomaterials to enhance T-cell therapy, we evaluated in vitro the effects of graphene oxide (GO) and two types of reduced GO (rGO15 and rGO30) nanostructures on the Th2 lymphocyte cell line SR.D10. This cell line offers the possibility of studying their activation threshold by employing soluble antibodies against TCR/CD3 and against CD4, as well as the simultaneous activation of these two receptors. In the present study, the effects of GO, rGO15 and rGO30 on the activation/proliferation rate of these Th2 lymphocytes have been analyzed by studying cell viability, cell cycle phases, intracellular content of reactive oxygen species (ROS) and cytokine secretion. High lymphocyte viability values were obtained after treatment with these nanostructures, as well as increased proliferation in the presence of rGOs. Moreover, rGO15 treatment decreased the intracellular ROS content of Th2 cells in all stimulated conditions. The analysis of these parameters showed that the presence of these GO and rGO nanostructures did not alter the response of Th2 lymphocytes.

Benefits in the Macrophage Response Due to Graphene Oxide Reduction by Thermal Treatment.

Graphene and its derivatives are very promising nanomaterials for biomedical applications and are proving to be very useful for the preparation of scaffolds for tissue repair. The response of immune cells to these graphene-based materials (GBM) appears to be critical in promoting regeneration, thus, the study of this response is essential before they are used to prepare any type of scaffold. Another relevant factor is the variability of the GBM surface chemistry, namely the type and quantity of oxygen functional groups, which may have an important effect on cell behavior. The response of RAW-264.7 macrophages to graphene oxide (GO) and two types of reduced GO, rGO15 and rGO30, obtained after vacuum-assisted thermal treatment of 15 and 30 min, respectively, was evaluated by analyzing the uptake of these nanostructures, the intracellular content of reactive oxygen species, and specific markers of the proinflammatory M1 phenotype, such as CD80 expression and secretion of inflammatory cytokines TNF-α and IL-6. Our results demonstrate that GO reduction resulted in a decrease of both oxidative stress and proinflammatory cytokine secretion, significantly improving its biocompatibility and potential for the preparation of 3D scaffolds able of triggering the appropriate immune response for tissue regeneration.

Effects of Human and Porcine Adipose Extracellular Matrices Decellularized by Enzymatic or Chemical Methods on Macrophage Polarization and Immunocompetence.

The decellularized extracellular matrix (ECM) obtained from human and porcine adipose tissue (AT) is currently used to prepare regenerative medicine bio-scaffolds. However, the influence of these natural biomaterials on host immune response is not yet deeply understood. Since macrophages play a key role in the inflammation/healing processes due to their high functional plasticity between M1 and M2 phenotypes, the evaluation of their response to decellularized ECM is mandatory. It is also necessary to analyze the immunocompetence of macrophages after contact with decellularized ECM materials to assess their functional role in a possible infection scenario. In this work, we studied the effect of four decellularized adipose matrices (DAMs) obtained from human and porcine AT by enzymatic or chemical methods on macrophage phenotypes and fungal phagocytosis. First, a thorough biochemical characterization of these biomaterials by quantification of remnant DNA, lipids, and proteins was performed, thus indicating the efficiency and reliability of both methods. The proteomic analysis evidenced that some proteins are differentially preserved depending on both the AT origin and the decellularization method employed. After exposure to the four DAMs, specific markers of M1 proinflammatory and M2 anti-inflammatory macrophages were analyzed. Porcine DAMs favor the M2 phenotype, independently of the decellularization method employed. Finally, a sensitive fungal phagocytosis assay allowed us to relate the macrophage phagocytosis capability with specific proteins differentially preserved in certain DAMs. The results obtained in this study highlight the close relationship between the ECM biochemical composition and the macrophage's functional role.

Macrophage inflammatory and metabolic responses to graphene-based nanomaterials differing in size and functionalization.

The preparation of graphene-based nanomaterials (GBNs) with appropriate stability and biocompatibility is crucial for their use in biomedical applications. In this work, three GBNs differing in size and/or functionalization have been synthetized and characterized, and their in vitro biological effects were compared. Pegylated graphene oxide (GO-PEG, 200-500 nm) and flavin mononucleotide-stabilized pristine graphene with two different sizes (PG-FMN, 200-400 nm and 100-200 nm) were administered to macrophages, chosen as cellular model due to their key role in the processing of foreign materials and the regulation of inflammatory responses. The results showed that cellular uptake of GBNs was mainly influenced by their lateral size, while the inflammatory potential depended also on the type of functionalization. PG-FMN nanomaterials (both sizes) triggered significantly higher nitric oxide (NO) release, together with some intracellular metabolic changes, similar to those induced by the prototypical inflammatory stimulus LPS. NMR metabolomics revealed that macrophages incubated with smaller PG-FMN displayed increased levels of succinate, itaconate, phosphocholine and phosphocreatine, together with decreased creatine content. The latter two variations were also detected in cells incubated with larger PG-FMN nanosheets. On the other hand, GO-PEG induced a decrease in the inflammatory metabolite succinate and a few other changes distinct from those seen in LPS-stimulated macrophages. Assessment of TNF-α secretion and macrophage surface markers (CD80 and CD206) further corroborated the low inflammatory potential of GO-PEG. Overall, these findings revealed distinct phenotypic and metabolic responses of macrophages to different GBNs, which inform on their immunomodulatory activity and may contribute to guide their therapeutic applications.

Estradiol impairs the Th17 immune response against Candida albicans.

Candida albicans is a commensal opportunistic pathogen that is also a member of gastrointestinal and reproductive tract microbiota. Exogenous factors, such as oral contraceptives, hormone replacement therapy, and estradiol, may affect susceptibility to Candida infection, although the mechanisms involved in this process have not been elucidated. We used a systemic candidiasis model to investigate how estradiol confers susceptibility to infection. We report that estradiol increases mouse susceptibility to systemic candidiasis, as in vivo and ex vivo estradiol-treated DCs were less efficient at up-regulating antigen-presenting machinery, pathogen killing, migration, IL-23 production, and triggering of the Th17 immune response. Based on these results, we propose that estradiol impairs DC function, thus explaining the increased susceptibility to infection during estrus.

The Candida albicans cell wall protein Rhd3/Pga29 is abundant in the yeast form and contributes to virulence.

The glycosylphosphatidylinositol-modified protein Rhd3/Pga29 of the human pathogen Candida albicans belongs to a family of cell wall proteins that are widespread among Candida species but are not found in other fungi. Pga29 is covalently linked to the beta-1,3-glucan framework of the cell wall via beta-1,6-glucan. It is a small and abundant O-glycosylated protein and requires the protein-O-mannosyl transferase Pmt1 for glycosylation. Furthermore, Pga29 is strongly expressed in yeast cells but is downregulated in hyphae. Removal of the PGA29 gene in C. albicans leads to a significant reduction of cell wall mannan; however, Pga29 does not seem to have a major role in maintaining cell wall integrity. In addition, adhesion capacity and hyphae formation appear normal in pga29 deletion mutants. Importantly, the pga29 deletion mutant is less virulent, and infection of reconstituted human epithelium with the pga29 mutant results in a diminished induction of proinflammatory cytokines, such as GM-CSF, TNF, IL-6 and IL-8. We propose that the reduced virulence of the pga29 mutant is a consequence of altered surface properties, resulting in altered fungal recognition.

Candida albicans actively modulates intracellular membrane trafficking in mouse macrophage phagosomes.

The intracellular trafficking/survival strategies of the opportunistic human pathogen Candida albicans are poorly understood. Here we investigated the infection of RAW264.7 macrophages with a virulent wild-type (WT) filamentous C. albicans strain and a hyphal signalling-defective mutant (efg1Delta/cph1Delta). A comparative analysis of the acquisition by phagosomes of actin, and of early/late endocytic organelles markers of the different fungal strains was performed and related to Candida's survival inside macrophages. Our results show that both fungal strains have evolved a similar mechanism to subvert the 'lysosomal' system, as seen by the inhibition of the phagosome fusion with compartments enriched in the lysobisphosphatidic acid and the vATPase, and thereby the acquisition of a low pH from the outset of infection. Besides, the virulent WT strain displayed additional specific survival strategies to prevent its targeting to compartmentsdisplaying late endosomal/lysosomal features, such as induction of active recycling out of phagosomes of the lysosomal membrane protein LAMP-1, the lysosomal protease cathepsin D and preinternalized colloidal gold. Finally, both virulent and efg1Delta/cph1Delta mutant fungal strains actively suppressed the production of macrophage nitric oxide (NO), although their cell wall extracts were potent inducers of NO.

Candida albicans-macrophage interactions: genomic and proteomic insights.

Candida albicans infection is a significant cause of morbidity and mortality in immunocompromised patients. In vivo and in vitro models have been developed to study both the fungal and the mammalian immune responses. Phagocytic cells (i.e., macrophages) play a key role in innate immunity against C. albicans by capturing, killing and processing the pathogen for presentation to T cells. The use of microarray technology to study global fungal transcriptional changes after interaction with different host cells has revealed how C. albicans adapts to its environment. Proteomic tools complement molecular approaches and computational methods enable the formulation of relevant biological hypotheses. Therefore, the combination of genomics, proteomics and bioinformatics tools (i.e., network analyses) is a powerful strategy to better understand the biological situation of the fungus inside macrophages; part of the fungal population is killed while a significantly high percentage survives.

Integrated proteomics and genomics strategies bring new insight into Candida albicans response upon macrophage interaction.

The interaction of Candida albicans with macrophages is considered a crucial step in the development of an adequate immune response in systemic candidiasis. An in vitro model of phagocytosis that includes a differential staining procedure to discriminate between internalized and non-internalized yeast was developed. Upon optimization of a protocol to obtain an enriched population of ingested yeasts, a thorough genomics and proteomics analysis was carried out on these cells. Both proteins and mRNA were obtained from the same sample and analyzed in parallel. The combination of two-dimensional PAGE with MS revealed a total of 132 differentially expressed yeast protein species upon macrophage interaction. Among these species, 67 unique proteins were identified. This is the first time that a proteomics approach has been used to study C. albicans-macrophage interaction. We provide evidence of a rapid protein response of the fungus to adapt to the new environment inside the phagosome by changing the expression of proteins belonging to different pathways. The clear down-regulation of the carbon-compound metabolism, plus the up-regulation of lipid, fatty acid, glyoxylate, and tricarboxylic acid cycles, indicates that yeast shifts to a starvation mode. There is an important activation of the degradation and detoxification protein machinery. The complementary genomics approach led to the detection of specific pathways related to the virulence of Candida. Network analyses allowed us to generate a hypothetical model of Candida cell death after macrophage interaction, highlighting the interconnection between actin cytoskeleton, mitochondria, and autophagy in the regulation of apoptosis. In conclusion, the combination of genomics, proteomics, and network analyses is a powerful strategy to better understand the complex host-pathogen interactions.