Biology subjects

Segovia, M.

Publications and source records attributed to Segovia, M..

5 recordsLinked to original sources

bioRxiv2024

Ribonuclease activity undermines immune sensing of naked extracellular RNA

The plasma membrane and the membrane of endosomal vesicles are considered physical barriers preventing extracellular RNA uptake. While naked RNA can be spontaneously internalized by certain cells types, functional delivery of naked RNA into the cytosol has been rarely observed. Here we show that extracellular ribonucleases, mainly derived from cell culture supplements, have so far hindered the study of extracellular RNA functionality. In the presence of active ribonuclease inhibitors (RI), naked bacterial RNA is pro-inflammatory when spiked in the media of dendritic cells and macrophages. In murine cells, this response mainly depends on the action of endosomal Toll-like receptors. However, we also show that naked RNA can perform endosomal escape and engage with cytosolic RNA sensors and ribosomes. For example, naked mRNAs encoding reporter proteins can be spontaneously internalized and translated by a variety of cell types, in an RI-dependent manner. In vivo, RI co-injection enhances the activation induced by naked extracellular RNA on splenic lymphocytes and myeloid-derived leukocytes. Furthermore, naked extracellular RNA is inherently pro-inflammatory in ribonuclease-poor compartments such as the peritoneal cavity. Overall, these results demonstrate that naked RNA is bioactive and does not need encapsulation inside synthetic or biological lipid vesicles for functional uptake, making a case for nonvesicular extracellular RNA-mediated intercellular communication.

molecular biology↗

bioRxiv2022

Formulating a TMEM176B blocker in nanoparticles uncouples its paradoxical roles in innate and adaptive antitumoral immunity.

The immunoregulatory cation channel TMEM176B plays a dual role in tumor immunity. On one hand, TMEM176B promotes antigen cross-presentation to CD8+ T cells by regulating phagosomal pH in dendritic cells (DCs). On the other hand, TMEM176B inhibits NLRP3 inflammasome activation through ionic mechanisms in DCs, monocytes and macrophages. Moreover, the TMEM176B blocker BayK8644 controls tumor progression through mechanisms involving inflammasome activation in prophylactic but not in therapeutic protocols. We speculated that the limited therapeutic efficacy of the compound may be linked to its potential capacity to inhibit antigen cross-presentation. Here we show that free BayK8644 inhibits antigen cross-presentation by splenic DCs. To prevent such inhibition, we reasoned that formulating BayK8644 in nanoparticles may delay the release of the compound in endosomes. Avoiding TMEM176B inhibition during the first 30 minutes of nanoparticle internalization by DCs may allow efficient cross-presentation to occur during this critical time frame. Indeed, we observed that NP-PEG-BayK8644 did not inhibit antigen cross-presentation, in contrast to the free compound. Moreover, NP-PEG-BayK8644 triggered inflammasome activation in a Tmem176b-dependent manner. We then injected eNP-PEG or NP-PEG-BayK8644 to mice bearing established tumors. NP-PEG-BayK8644 significantly controlled tumor growth and mice survival, as compared to eNP-PEG and free BayK8644, in a Tmem176b-dependent manner in mouse melanoma and lymphoma tumors. Responding animals treated with NP-PEG-BayK8644 showed reinforced tumor infiltration by total and tumor-specific CD8+ T cells. Overall, we rationally developed a formulating method of BayK8644 that improves its anti-tumoral therapeutic efficacy by uncoupling the dual role of TMEM176B on innate and adaptive immunity.

immunology↗

bioRxiv2020

Ivermectin reduces coronavirus infection in vivo: a mouse experimental model

SARS-CoV2 is a single strand RNA virus member of the type 2 coronavirus family, responsible for causing COVID-19 disease in humans. The objective of this study was to test the ivermectin drug in a murine model of coronavirus infection using a type 2 family RNA coronavirus similar to SARS-CoV2, the mouse hepatitis virus (MHV). BALB/cJ female mice were infected with 6,000 PFU of MHV-A59 (Group Infected; n=20) and immediately treated with one single dose of 500 g/kg of ivermectin (Group Infected + IVM; n=20), or were not infected and treated with PBS (Control group; n=16). Five days after infection/treatment, mice were euthanized to obtain different tissues to check general health status and infection levels. Overall results demonstrated that viral infection induces the typical MHV disease in infected animals, with livers showing severe hepatocellular necrosis surrounded by a severe lymphoplasmacytic inflammatory infiltration associated with a high hepatic viral load (52,158 AU), while ivermectin administration showed a better health status with lower viral load (23,192 AU; p<0.05) and few livers with histopathological damage (p<0.05), not showing statistical differences with control mice (P=NS). Furthermore, serum transaminase levels (aspartate aminotransferase and alanine aminotransferase) were significantly lower in treated mice compared to infected animals. In conclusion, ivermectin seems to be effective to diminish MHV viral load and disease in mice, being a useful model for further understanding new therapies against coronavirus diseases.

pathology↗

bioRxiv2020

Genome-wide locus sequence typing (GLST) of eukaryotic pathogens

Analysis of genetic polymorphism is a powerful tool for epidemiological surveillance and research. Powerful inference from pathogen genetic variation, however, is often restrained by limited access to representative target DNA, especially in the study of obligate parasitic species for which ex vivo culture is resource-intensive or bias-prone. Modern sequence capture methods enable pathogen genetic variation to be analyzed directly from vector/host material but are often too complex and expensive for resource-poor settings where infectious diseases prevail. This study proposes a simple, cost-effective genome-wide locus sequence typing (GLST) tool based on massive parallel amplification of information hotspots throughout the target pathogen genome. The multiplexed polymerase chain reaction amplifies hundreds of different, user-defined genetic targets in a single reaction tube, and subsequent agarose gel-based clean-up and barcoding completes library preparation at under 4 USD per sample. Approximately 100 libraries can be sequenced together in one Illumina MiSeq run. Our study generates a flexible GLST primer panel design workflow for Trypanosoma cruzi, the parasitic agent of Chagas disease. We successfully apply our 203-target GLST panel to direct, culture-free metagenomic extracts from triatomine vectors containing a minimum of 3.69 pg/l T. cruzi DNA and further elaborate on method performance by sequencing GLST libraries from T. cruzi reference clones representing discrete typing units (DTUs) TcI, TcIII, TcIV, and TcVI. The 780 SNP sites we identify in the sample set repeatably distinguish parasites infecting sympatric vectors and detect correlations between genetic and geographic distances at regional (< 150 km) as well as continental scales. The markers also clearly separate DTUs. We discuss the advantages, limitations and prospects of our method across a spectrum of epidemiological research.

genetics↗

bioRxiv2020

Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

A major proportion of extracellular RNAs (exRNAs) do not co-isolate with extracellular vesicles (EVs) and remain in ultracentrifugation supernatants of cell-conditioned medium or mammalian blood serum. However, little is known about exRNAs beyond EVs. We have previously shown that the composition of the nonvesicular exRNA fraction is highly biased toward specific tRNA-derived fragments capable of forming RNase-protecting dimers. To solve the problem of stability in exRNA analysis, we developed RI-SEC-seq: a method based on sequencing the size exclusion chromatography (SEC) fractions of nonvesicular extracellular samples treated with RNase inhibitors (RI). This method revealed dramatic compositional changes in exRNA population when enzymatic RNA degradation was inhibited. We demonstrated the presence of ribosomes and full-length tRNAs in cell-conditioned medium of a variety of mammalian cell lines. Their fragmentation generates some small RNAs that are highly resistant to degradation. The extracellular biogenesis of some of the most abundant exRNAs demonstrates that extracellular abundance is not a reliable input to estimate RNA secretion rates. Finally, we showed that chromatographic fractions containing extracellular ribosomes can be sensed by dendritic cells. Extracellular ribosomes and/or tRNAs could therefore be decoded as damage-associated molecular patterns.

cell biology↗