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Biology subjects

Pineda, E.

Publications and source records attributed to Pineda, E..

9 recordsLinked to original sources

C1q from C1q+ tumor-associated myeloid cells promotes resistance to T-cell engagers and CAR T-cells and is induced by LIF and glucocorticoids

Immunotherapies, particularly T-cell engagers (TCEs) and CAR T-cells, have shown limited efficacy in solid tumors, partly due to an immunosuppressive tumor microenvironment (TME). However, the molecular mechanisms by which the TME impairs immunotherapy remain poorly understood. Here, we found that C1q generated by C1q tumor-associated myeloid cells (TAMs) plays a fundamental role in shaping the immunosuppressive TME in glioblastoma (GBM), one of the most aggressive tumors. C1q suppressed T-cell activation and impaired the activity of T-cell engagers (TCEs) and CAR T-cells. Genetic ablation of C1qa improved anti-tumor responses to TCEs and CAR T-cells. We used innovative patient-derived tumor tissue cultures (PDTTCs), which preserve an intact TME, from 19 GBM patients and identified the LIF cytokine as the main inducer of C1q. Moreover, we discovered that glucocorticoids cooperate with LIF to induce C1q. The identified C1q TAM signature overlapped with an anti-LIF gene signature, was associated with poor prognosis, and was enriched in mesenchymal GBMs with NF1 mutations. The blockade of LIF using an anti-LIF neutralizing antibody decreased the presence of C1q+ TAMs, and we found that the regulation of C1q by anti-LIF is conserved between human and mouse. Using the C1qa-/- GBM mouse model, we showed that C1q mediates the anti-tumor immune response induced by LIF blockade. Our findings identify C1q TAMs as key immunosuppressive players in GBM, impairing CAR T-cells and TCE activity, and position them as therapeutic targets to improve immunotherapy responses in this devastating disease.

cancer biology↗

Defensive lipid droplets are PUFA reservoirs driving bacterial clearance and inflammation

Lipid droplets (LDs) rapidly form in infected cells to participate in the defence against microbes. Here, we investigate the involvement of LD lipids in these immune responses. Comparative shotgun and targeted lipidomics demonstrate that in vivo host LDs accumulate polyunsaturated fatty acids (PUFAs). PUFAs arrive at cells from the bloodstream to be further metabolised into complex PUFAs accrued by LD-triglycerides and -phospholipids. Host lipid metabolism is transcriptionally controlled by rapid, transient, and intricate immune programs initiated by pathogen-associated molecular patterns and relayed by cytokines such as interferons (type I and II), interleukins (IL-1{beta}), and tumour necrosis factor. When this lipid and signalling environment is reproduced in cultured macrophages, newly formed LDs accumulate defensive proteins, coordinate the synthesis of complex PUFAs, and become PUFA reservoirs and suppliers. Among LD-PUFAs, the {omega}-6 arachidonic acid is the most actively metabolised during the initial phases of innate immunity. Released from LDs by adipose triglyceride lipase, arachidonic acid is used by macrophages for prostaglandin synthesis, bacterial phagocytosis, and elimination of microbes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=163 HEIGHT=200 SRC="FIGDIR/small/711356v2_ufig1.gif" ALT="Figure 1"> View larger version (107K): org.highwire.dtl.DTLVardef@14d65eorg.highwire.dtl.DTLVardef@5ecc1org.highwire.dtl.DTLVardef@fa99e5org.highwire.dtl.DTLVardef@8d9632_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

Glycerol metabolism triggers trypanosome differentiation into transmissible forms in mammalian tissue-like conditions

In the mammalian bloodstream, Trypanosoma brucei, the parasite responsible for sleeping sickness, proliferates as slender forms before undergoing quorum-sensing (QS)-mediated differentiation into cell cycle-arrested stumpy forms (stumpy-QS), a transition that regulates parasitaemia and primes parasites for tsetse fly transmission. Beyond the bloodstream, T. brucei also occupies extravascular, adipose-rich tissues such as the skin, a potential reservoir for transmission. Here, we identify an alternative slender-to-stumpy differentiation pathway driven by glycerol, a host metabolite abundant in adipose-rich tissues, that could resolve the long-standing paradox of successful parasite transmission from human hosts during chronic infection despite low parasitaemia. We show that high (10 mM) and non-physiological glycerol concentrations under low glucose conditions (0.5 mM) induce differentiation, generating distinct stumpy-Glyc forms that resemble stumpy-QS parasites but have an extended lifespan. Under conditions mimicking dermal tissue interstitial fluids (4 mM glucose, 0.25 mM glycerol), we show that glycerol promotes the emergence of proliferative intermediate forms that retain transmission potential and can differentiate into fly host-specific procyclic forms in vitro and within tsetse flies. These findings open the door for reevaluation of the model of T. brucei transmission and supports a dominant role for adipocyte-derived glycerol in the skin in sustaining parasite transmission.

microbiology↗

The glycosomal ATP-dependent phosphofructokinase of Trypanosoma brucei operates in the gluconeogenic direction in cellulo under in vivo-like conditions

In the glucose-free environment of the midgut of the tsetse fly vector, the procyclic forms of Trypanosoma brucei primarily consume proline to feed its central carbon and energy metabolism. In this context, the parasite produces through gluconeogenesis glucose 6- phosphate (G6P), the precursor of essential metabolic pathways, from proline catabolism. We showed here that the parasite uses three different enzymes to perform the key gluconeogenic reaction producing fructose 6-phosphate (F6P) from fructose 1,6-bisphosphate, (i) fructose-1,6- bisphosphatase (FBPase), the canonical enzyme performing this reaction, (ii) sedoheptulose- 1,7-bisphosphatase (SBPase) and (iii) more surprisingly ATP-dependent phosphofructokinase (PFK), an enzyme considered to irreversibly catalyse the opposite reaction involved in glycolysis. These three enzymes, as well as six other glycolytic/gluconeogenic enzymes, are located in peroxisome-related organelles, named glycosomes. Incorporation of 13C-enriched glycerol (a more effective alternative to proline for monitoring gluconeogenic activity) into F6P and G6P was more affected in the PFK null mutant than in the FBPase null mutant, suggesting the PFK contributes at least as much as FBPase to gluconeogenesis. We also showed that glucose deprivation did not affect the quantities of PFK substrates and products, whereas a 500-fold increase in the substrate/product ratio was expected for PFK to carry out the gluconeogenic reaction. In conclusion, we showed for the first time that ATP-dependent PFK can function in vivo in the gluconeogenic direction, even in the presence of FBPase activity. This particular feature, which precludes loss of ATP through a futile cycle involving PFK and FBPase working simultaneously in the glycolytic and gluconeogenic directions, respectively, is probably due to the supramolecular organisation of the metabolic pathway within glycosomes to overcome thermodynamic barriers through metabolic channelling.

microbiology↗

Trypanosoma brucei bloodstream form mitochondrion is capable of ATP production by substrate phoshorylation

The bloodstream form Trypanosoma brucei maintains essential mitochondrial membrane potential ({Delta}{Psi}m) through the reverse activity of FoF1-ATP synthase. The ATP that drives this activity is thought to be generated by glycolysis and imported from the cytosol via an ATP/ADP carrier (AAC). We have shown that this carrier is the only carrier that can import ATP into the mitochondrial matrix to power the FoF1-ATPase. Contrary to expectations, its deletion has no effect on parasite growth, virulence and levels of {Delta}{Psi}m, suggesting that ATP is produced intramitochondrially by substrate phosphorylation pathways. Therefore, we knocked out the succinyl-CoA synthetase (SCoAS) gene, a key enzyme that produces ATP through substrate phosphorylation. Its absence resulted in changes in the metabolic landscape of the parasite, lower virulence, and reduced mitochondrial ATP content. This minimal mitochondrial ATP pool was maintained by AAC activity as evidenced by the 25- fold increase in sensitivity of the mutant parasites to AAC inhibitor carboxyatractyloside. Under nutrient-limited conditions, suppression of SCoAS expression by RNA interference negatively affected cell growth and levels of {Delta}{Psi}m. We concluded that the bloodstream mitochondrion is capable of generating ATP via substrate phosphorylation pathways, the importance of which depends on environmental conditions.

microbiology↗

Urbanisation generates multiple trait syndromes for terrestrial taxa worldwide

Cities can host significant biological diversity. Yet, urbanisation leads to the loss of habitats and, potentially, to local extinctions. Understanding how multiple taxa respond to urbanisation globally is essential to promote and conserve biodiversity in cities and surrounding landscapes. Using a dataset with site-level occurrence and trait data of 5302 species from six terrestrial fauna taxonomic groups across 379 cities on 6 continents, we show that urbanisation produces taxon-specific changes in trait composition, with traits related to reproductive strategy consistently showing the strongest response. The effect of urbanisation on community trait composition is strongest at the largest spatial scale considered, and more closely linked to landscape composition (% urban) than arrangement (aggregation), although latitude and climatic variables remain a stronger influence. This study did not find evidence in support of a global urban taxa syndrome, but instead we suggest that there are four general urban trait syndromes, with resources associated with reproduction and diet likely to be driving patterns in traits associated with mobility and body size. Functional diversity measures showed a wide range of responses, leading to a shift in trait space that is most likely driven by the distribution and abundance of critical resources, and the urban trait syndrome displayed by individual species within a community. Further research is required to understand the interactions between the four general urban trait syndromes, resource distribution and abundance and changes in functional diversity of taxa at different spatial and temporal scales. Maximising opportunities to support species within taxa groups with different urban trait syndromes should be pivotal in conservation and management programmes within and among cities. This will reduce the likelihood of biotic homogenisation at the taxa level, and helps ensure that urban environments have the ecological capacity to respond to challenges such as climate change, further habitat fragmentation and loss, and other disruptions. These actions are critical if we are to reframe the role of cities in global biodiversity loss.

ecology↗

Apparent growth paradox of Staphylococcus epidermidis in response to extremely low frequency electromagnetic radiation exposure

Staphylococcus epidermidis is a normal part of the human microbiome; however, it is an opportunistic pathogen and can cause infections when the delicate balance of this microbiome is disrupted. Furthermore, infections caused by this bacterium can be hard to treat as a result of antibiotic resistance and biofilm production. This experiment aimed to determine whether electromagnetic field radiation (ELF-EMF) could be a deterrent of bacterial growth, as an alternative treatment to antibiotics. A non-pathogenic strain of S. epidermidis was used for experimentation, which took place in a school laboratory setting. The experimental group was exposed to ELF-EMF, while the control group did not receive the ELF-EMF treatment. The number of bacterial colonies, represented as colony forming units (CFUs) and area of random colonies were calculated to determine the effect of this treatment. There was no dramatic difference of colony formation on days 0, 1, and 2 of the four day period of ELF-EMF exposure. However, colony formation for days 3 and 4 showed a significant difference between the control and the experimental groups, as the experimental group had a significantly higher CFU count than the control. The average CFU count for day 3 in the control group was 420.6 and 1,097.4 for the experimental group (p<0.0001, t=12.9803). On the final day of the experimentation (Day 4) the average CFU count for the control group was 424.6 and 896.4 for the experimental group (p<0.0001, t=5.8926). The area for five randomly chosen colonies from each petri dish was calculated on the fourth day of experimentation. The area for the experimental group was significantly lower than that control (p<0.0001, with t=6.8659). The average area for the control group was 1.3249 mm2 and a lower average of 0.6375mm2 for the experimental group. These results demonstrate that the ELF-EMF treatment had an inhibitory effect on the area growth of S. epidermidis, but not on the colony-forming ability of S. epidermidis. This suggests that ELF-EMF influences the means by which the bacterium S. epidermidis grows.

microbiology↗

The trypanosome UDP-glucose pyrophosphorylase is imported by piggybacking into glycosomes where unconventional sugar nucleotide synthesis takes place

Glycosomes are peroxisome-related organelles of trypanosomatid parasites containing metabolic pathways usually present in the cytosol of other eukaryotes, such as glycolysis and biosynthesis of sugar nucleotides. UDP-glucose pyrophosphorylase (UGP), the enzyme responsible for the synthesis of the sugar nucleotide UDP-glucose, is localised in the cytosol and glycosomes of the bloodstream and procyclic trypanosomes, despite the absence of any known peroxisomal targeting signal (PTS1 and PTS2). The questions we addressed here are (i) is the unusual glycosomal biosynthetic pathway of sugar nucleotide functional and (ii) how the PTS-free UGP is imported into glycosomes? We showed that UGP is imported into glycosomes by piggybacking on the glycosomal PTS1-containing phosphoenolpyruvate carboxykinase (PEPCK) and identified the domains involved in the UGP/PEPCK interaction. Proximity ligation assays revealed that this interaction occurs in 3-10% of glycosomes, suggesting that these correspond to organelles competent for protein import. We also showed that UGP is essential for growth of trypanosomes and that both the glycosomal and cytosolic metabolic pathways involving UGP are functional, since the lethality of the knock-down UGP mutant cell line (RNAiUGP) was rescued by expressing a recoded UGP in the organelle (RNAiUGP/EXPrUGP-GPDH). Our conclusion was supported by targeted metabolomic analyses (IC-HRMS) showing that UDP-glucose is no longer detectable in the RNAiUGP mutant, while it is still produced in cells expressing UGP exclusively in the cytosol (PEPCK null mutant) or glycosomes (RNAiUGP/EXPrUGP-GPDH). Trypanosomatids are the only known organisms to have selected functional peroxisomal (glycosomal) sugar nucleotide biosynthetic pathways in addition to the canonical cytosolic ones. ImportanceUnusual compartmentalization of metabolic pathways within organelles is one of the most enigmatic features of trypanosomatids. These unicellular eukaryotes are the only organisms that sequestered glycolysis inside peroxisomes (glycosomes), although the selective advantage of this compartmentalization is still not clear. Trypanosomatids are also unique for the glycosomal localisation of enzymes of the sugar nucleotide biosynthetic pathways, which are also present in the cytosol. Here we showed that the cytosolic and glycosomal pathways are functional. Like in all other eukaryotes, the cytosolic pathways feed glycosylation reactions, however the role of the duplicated glycosomal pathways is currently unknown. We also showed that one of these enzymes (UGP) is imported into glycosomes by piggybacking on another glycosomal enzyme (PEPCK), which are not functionally related. The UGP/PEPCK association is unique since all piggybacking examples reported to date involve functionally related interacting partners, which broadens the possible combinations of carrier-cargo proteins being imported as hetero-oligomers.

microbiology↗

Fly stage trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in near physiological conditions

Trypanosoma brucei, a protist responsible for human African trypanosomiasis (sleeping sickness), is transmitted by the tsetse fly, where the procyclic forms of the parasite develop in the proline-rich (1-2 mM) and glucose-depleted digestive tract. Proline is essential for the midgut colonization of the parasite in the insect vector, however other carbon sources could be available and used to feed its central metabolism. Here we show that procyclic trypanosomes can consume and metabolize metabolic intermediates, including those excreted from glucose catabolism (succinate, alanine and pyruvate), with the exception of acetate, which is the ultimate end-product excreted by the parasite. Among the tested metabolites, tricarboxylic acid (TCA) cycle intermediates (succinate, malate and -ketoglutarate) stimulated growth of the parasite in the presence of 2 mM proline. The pathways used for their metabolism were mapped by proton-NMR metabolic profiling and phenotypic analyses of a dozen RNAi and/or null mutants affecting central carbon metabolism. We showed that (i) malate is converted to succinate by both the reducing and oxidative branches of the TCA cycle, which demonstrates that procyclic trypanosomes can use the full TCA cycle, (ii) the enormous rate of -ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and (iii) -ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an -ketoglutarate dehydrogenase null mutant. In addition, epimastigotes produced from procyclics upon overexpression of RBP6, showed a growth defect in the presence of 2 mM proline, which is rescued by -ketoglutarate, suggesting that physiological amounts of proline are not sufficient per se for the development of trypanosomes in the fly. In conclusion, these data show that trypanosomes can metabolize multiple metabolites, in addition to proline, which allows them to confront challenging environments in the fly. Author SummaryIn the midgut of its insect vector, trypanosomes rely on proline to feed their energy metabolism. However, the availability of other potential carbon sources that can be used by the parasite is currently unknown. Here we show that tricarboxylic acid (TCA) cycle intermediates, i.e. succinate, malate and -ketoglutarate, stimulate growth of procyclic trypanosomes incubated in medium containing 2 mM proline, which is in the range of the amounts measured in the midgut of the fly. Some of these additional carbon sources are needed for the development of epimastigotes, which differentiate from procyclics in the midgut of the fly, since their growth defect observed in the presence of 2 mM proline is rescued by addition of -ketoglutarate. In addition, we have implemented new approaches to study a poorly explored branch of the TCA cycle converting malate to -ketoglutarate, which was previously described as non-functional in the parasite, regardless of the glucose levels available. The discovery of this branch reveals that a full TCA cycle can operate in procyclic trypanosomes. Our data broaden the metabolic potential of trypanosomes and pave the way for a better understanding of the parasites metabolism in various organ systems of the tsetse fly, where it evolves.

microbiology↗