Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages.

The fungal pathogen Histoplasma capsulatum (Hc) invades, replicates within, and destroys macrophages. To interrogate the molecular mechanisms underlying this interaction, we conducted a host-directed CRISPR-Cas9 screen and identified 361 genes that modify macrophage susceptibility to Hc infection, greatly expanding our understanding of host gene networks targeted by Hc. We identified pathways that have not been previously implicated in Hc interaction with macrophages, including the ragulator complex (involved in nutrient stress sensing), glycosylation enzymes, protein degradation machinery, mitochondrial respiration genes, solute transporters, and the ER membrane complex (EMC). The highest scoring protective hits included the complement C3a receptor (C3aR), a G-protein coupled receptor (GPCR) that recognizes the complement fragment C3a. Although it is known that complement components react with the fungal surface, leading to opsonization and release of small peptide fragments such as C3a, a role for C3aR in macrophage interactions with fungi has not been elucidated. We demonstrated that whereas C3aR is dispensable for macrophage phagocytosis of bacteria and latex beads, it is critical for optimal macrophage capture of pathogenic fungi, including Hc, the ubiquitous fungal pathogen Candida albicans, and the causative agent of Valley Fever Coccidioides posadasii. We showed that C3aR localizes to the early phagosome during Hc infection where it coordinates the formation of actin-rich membrane protrusions that promote Hc capture. We also showed that the EMC promotes surface expression of C3aR, likely explaining its identification in our screen. Taken together, our results provide new insight into host processes that affect Hc-macrophage interactions and uncover a novel and specific role for C3aR in macrophage recognition of fungi.

Cbp1, a fungal virulence factor under positive selection, forms an effector complex that drives macrophage lysis.

Intracellular pathogens secrete effectors to manipulate their host cells. Histoplasma capsulatum (Hc) is a fungal intracellular pathogen of humans that grows in a yeast form in the host. Hc yeasts are phagocytosed by macrophages, where fungal intracellular replication precedes macrophage lysis. The most abundant virulence factor secreted by Hc yeast cells is Calcium Binding Protein 1 (Cbp1), which is absolutely required for macrophage lysis. Here we take an evolutionary, structural, and cell biological approach to understand Cbp1 function. We find that Cbp1 is present only in the genomes of closely related dimorphic fungal species of the Ajellomycetaceae family that lead primarily intracellular lifestyles in their mammalian hosts (Histoplasma, Paracoccidioides, and Emergomyces), but not conserved in the extracellular fungal pathogen Blastomyces dermatitidis. We observe a high rate of fixation of non-synonymous substitutions in the Cbp1 coding sequences, indicating that Cbp1 is under positive selection. We determine the de novo structures of Hc H88 Cbp1 and the Paracoccidioides americana (Pb03) Cbp1, revealing a novel "binocular" fold consisting of a helical dimer arrangement wherein two helices from each monomer contribute to a four-helix bundle. In contrast to Pb03 Cbp1, we show that Emergomyces Cbp1 orthologs are unable to stimulate macrophage lysis when expressed in the Hc cbp1 mutant. Consistent with this result, we find that wild-type Emergomyces africanus yeast are able to grow within primary macrophages but are incapable of lysing them. Finally, we use subcellular fractionation of infected macrophages and indirect immunofluorescence to show that Cbp1 localizes to the macrophage cytosol during Hc infection, making this the first instance of a phagosomal human fungal pathogen directing an effector into the cytosol of the host cell. We additionally show that Cbp1 forms a complex with Yps-3, another known Hc virulence factor that accesses the cytosol. Taken together, these data imply that Cbp1 is a fungal virulence factor under positive selection that localizes to the cytosol to trigger host cell lysis.

Hypoxia as a Modulator of Inflammation and Immune Response in Cancer.

A clear association between hypoxia and cancer has heretofore been established; however, it has not been completely developed. In this sense, the understanding of the tumoral microenvironment is critical to dissect the complexity of cancer, including the reduction in oxygen distribution inside the tumoral mass, defined as tumoral hypoxia. Moreover, hypoxia not only influences the tumoral cells but also the surrounding cells, including those related to the inflammatory processes. In this review, we analyze the participation of HIF, NF-κB, and STAT signaling pathways as the main components that interconnect hypoxia and immune response and how they modulate tumoral growth. In addition, we closely examine the participation of the immune cells and how they are affected by hypoxia, the effects of the progression of cancer, and some innovative applications that take advantage of this knowledge, to suggest potential therapies. Therefore, we contribute to the understanding of the complexity of cancer to propose innovative therapeutic strategies in the future.

Autophagy as a Potential Therapy for Malignant Glioma.

Glioma is the most frequent and aggressive type of brain neoplasm, being anaplastic astrocytoma (AA) and glioblastoma multiforme (GBM), its most malignant forms. The survival rate in patients with these neoplasms is 15 months after diagnosis, despite a diversity of treatments, including surgery, radiation, chemotherapy, and immunotherapy. The resistance of GBM to various therapies is due to a highly mutated genome; these genetic changes induce a de-regulation of several signaling pathways and result in higher cell proliferation rates, angiogenesis, invasion, and a marked resistance to apoptosis; this latter trait is a hallmark of highly invasive tumor cells, such as glioma cells. Due to a defective apoptosis in gliomas, induced autophagic death can be an alternative to remove tumor cells. Paradoxically, however, autophagy in cancer can promote either a cell death or survival. Modulating the autophagic pathway as a death mechanism for cancer cells has prompted the use of both inhibitors and autophagy inducers. The autophagic process, either as a cancer suppressing or inducing mechanism in high-grade gliomas is discussed in this review, along with therapeutic approaches to inhibit or induce autophagy in pre-clinical and clinical studies, aiming to increase the efficiency of conventional treatments to remove glioma neoplastic cells.

Autophagic and Apoptotic Pathways as Targets for Chemotherapy in Glioblastoma.

Glioblastoma multiforme is the most malignant and aggressive type of brain tumor, with a mean life expectancy of less than 15 months. This is due in part to the high resistance to apoptosis and moderate resistant to autophagic cell death in glioblastoma cells, and to the poor therapeutic response to conventional therapies. Autophagic cell death represents an alternative mechanism to overcome the resistance of glioblastoma to pro-apoptosis-related therapies. Nevertheless, apoptosis induction plays a major conceptual role in several experimental studies to develop novel therapies against brain tumors. In this review, we outline the different components of the apoptotic and autophagic pathways and explore the mechanisms of resistance to these cell death pathways in glioblastoma cells. Finally, we discuss drugs with clinical and preclinical use that interfere with the mechanisms of survival, proliferation, angiogenesis, migration, invasion, and cell death of malignant cells, favoring the induction of apoptosis and autophagy, or the inhibition of the latter leading to cell death, as well as their therapeutic potential in glioma, and examine new perspectives in this promising research field.

Suicide HSVtk gene delivery by neurotensin-polyplex nanoparticles via the bloodstream and GCV Treatment specifically inhibit the growth of human MDA-MB-231 triple negative breast cancer tumors xenografted in athymic mice.

The human breast adenocarcinoma cell line MDA-MB-231 has the triple-negative breast cancer (TNBC) phenotype, which is an aggressive subtype with no specific treatment. MDA-MB-231 cells express neurotensin receptor type 1 (NTSR1), which makes these cells an attractive target of therapeutic genes that are delivered by the neurotensin (NTS)-polyplex nanocarrier via the bloodstream. We addressed the relevance of this strategy for TNBC treatment using NTS-polyplex nanoparticles harboring the herpes simplex virus thymidine kinase (HSVtk) suicide gene and its complementary prodrug ganciclovir (GCV). The reporter gene encoding green fluorescent protein (GFP) was used as a control. NTS-polyplex successfully transfected both genes in cultured MDA-MB-231 cells. The transfection was demonstrated pharmacologically to be dependent on activation of NTSR1. The expression of HSVtk gene decreased cell viability by 49% (P<0.0001) and induced apoptosis in cultured MDA-MB-231 cells after complementary GCV treatment. In the MDA-MB-231 xenograft model, NTS-polyplex nanoparticles carrying either the HSVtk gene or GFP gene were injected into the tumors or via the bloodstream. Both routes of administration allowed the NTS-polyplex nanoparticles to reach and transfect tumorous cells. HSVtk expression and GCV led to apoptosis, as shown by the presence of cleaved caspase-3 and Apostain immunoreactivity, and significantly inhibited the tumor growth (55-60%) (P<0.001). At the end of the experiment, the weight of tumors transfected with the HSVtk gene was 55% less than that of control tumors (P<0.05). The intravenous transfection did not induce apoptosis in peripheral organs. Our results offer a promising gene therapy for TNBC using the NTS-polyplex nanocarrier.

Safety of the intravenous administration of neurotensin-polyplex nanoparticles in BALB/c mice.

Neurotensin (NTS)-polyplex is a gene nanocarrier that has potential nanomedicine-based applications for the treatment of Parkinson's disease and cancers of cells expressing NTS receptor type 1. We assessed the acute inflammatory response to NTS-polyplex carrying a reporter gene in BALB/c mice. The intravenous injection of NTS-polyplex caused the specific expression of the reporter gene in gastrointestinal cells. Six hours after an intravenous injection of propidium iodide labeled-NTS-polyplex, fluorescent spots were located in the cells of the organs with a mononuclear phagocyte system, suggesting NTS-polyplex clearance. In contrast to lipopolysaccharide and carbon tetrachloride, NTS-polyplex did not increase the serum levels of tumor necrosis factor alpha, interleukin (IL)-1ß, IL-6, bilirubin, aspartate transaminase, and alanine transaminase. NTS-polyplex increased the levels of serum amyloid A and alkaline phosphatase, but these levels normalized after 24 h. Compared to carrageenan, the local injection of NTS-polyplex did not produce inflammation. Our results support the safety of NTS-polyplex. FROM THE CLINICAL EDITOR: This study focuses on the safety of neurotensin (NTS)-polyplex, a gene nanocarrier that has potential in the treatment of Parkinson's disease and cancers of cells expressing NTS receptor type 1. NTS polyplex demonstrates a better safety profile compared with carrageenan, lipopolysaccharide, and carbon tetrachloride in a murine model.

Characterization of putrescine uptake in hamster amelanocytic melanoma AMEL-3 cells.

The uptake of putrescine, spermidine and spermine by Fortner's hamster amelanocytic melanoma AMEL-3 cells was observed in this study to be time-dependent, temperature-sensitive, pH-dependent and saturable. Metabolic poisons nullified polyamine uptake, an indication that this is an energy-requiring mechanism. The presence of Na+ ions was found to be requisite to full activity. Valinomycin, gramicidin, monensin and the calcium ionophore calcimycin were also observed to inhibit the process substantially. The transporter active site would seem to contain sulfhydryl groups. Other diamines and polyamine analogues, as well as cationic diamidines, suppressed putrescine uptake. The presence of the ornithine decarboxylase inhibitor DFMO in the culture medium induced putrescine inflows. Putrescine, in turn, induced the negative expression of the carrier, thus suggesting that this influx mechanism is governed by up/down regulation. The cationic diamidine CGP 40215A and its analogue CGP039937A competitively inhibited putrescine transport, with Ki values of 1.9 and 15 microM, respectively. The role of polyamine uptake in these cultures is discussed.

The neurotrophic effect of oleic acid includes dendritic differentiation and the expression of the neuronal basic helix-loop-helix transcription factor NeuroD2.

We have shown recently that the presence of albumin in astrocytes triggers the synthesis and release of oleic acid, which behaves as a neurotrophic factor for neurons. Thus, oleic acid promotes axonal growth together with the expression of the axonal growth-associated protein, GAP-43. Here we attempted to elucidate whether the neurotrophic effect of oleic acid includes dendritic differentiation. Our results indicate that oleic acid induces the expression of microtubule associated protein-2 (MAP-2), a marker of dendritic differentiation. In addition, the presence of oleic acid promotes the translocation of MAP-2 from the soma to the dendrites. The time course of MAP-2 expression during brain development coincides with that of stearoyl-CoA desaturase, the limiting enzyme of oleic acid synthesis, indicating that both phenomena coincide during development. The effect of oleic acid on MAP-2 expression is most probably independent of autocrine factors synthesized by neurons because this effect was also observed at low cellular densities. As oleic acid is an activator of protein kinase C, the possible participation of this transduction pathway was studied. Our results indicate that added oleic acid or oleic acid endogenously synthesized by astrocytes exerts its neurotrophic effect through a protein kinase C-dependent mechanism as the effect was inhibited by sphingosine or two myristoylated peptide inhibitors of protein kinase C. The transduction pathway by which oleic acid induces the expression of genes responsible for neuronal differentiation appears to be mediated by the transcription factor NeuroD2, a regulator of terminal neuronal differentiation.