Biology subjects

Prina, E.

Publications and source records attributed to Prina, E..

12 recordsLinked to original sources

bioRxiv2025

Enhanced detection of low-expressed miRNAs in Leishmania-infected macrophages through RNA fractionation and RT-qPCR optimization

MicroRNAs (miRNAs) play critical roles in regulating host responses to Leishmania infections, yet accurate detection of low-abundance miRNAs remains challenging. This study evaluated the impact of RNA fractionation and RT-qPCR protocol optimization on miRNA quantification in Leishmania amazonensis-infected murine macrophages. Using a panel of nine infection-associated miRNAs, we compared small RNA and total RNA fractions for their ability to detect weakly expressed targets. Small RNA consistently provided greater sensitivity and specificity, particularly when combined with a modified RT-qPCR protocol. These findings underscore the importance of RNA preparation methods for studying miRNA dynamics in infectious disease contexts and support improved approaches for detecting biologically relevant, low-expressed miRNAs.

molecular biology↗

bioRxiv2024

MAPPING CHANGES OF MIRNA-MRNA NETWORKS IN LEISHMANIA-INFECTED MACROPHAGES PREDICTS REGULATORY MIRNA-TF LOOPS AS NOVEL TARGETS OF PARASITE IMMUNE SUBVERSION

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level and play a crucial role in numerous disease processes, including infections. Although intracellular microbial pathogens are known to modulate host cell gene expression to establish permissive conditions for infection, the specific role of host-encoded miRNAs underlying such subversion remains poorly understood. In this study, we employed the protozoan parasite Leishmania amazonensis as a model system to investigate how infection of macrophages modifies the host cell miRNA profile to evade antimicrobial functions and to establish permissive conditions for intracellular proliferation. Dual RNA-seq analyses using matched mRNA and miRNA-enriched samples from uninfected and L. amazonensis-infected bone marrow-derived macrophages (BMDMs) revealed 102 differentially expressed miRNAs (padj<0.05), with 18 miRNAs showing reduced and 84 miRNAs showing increased abundance in infected BMDMs. Mapping putative networks of miRNA-mRNA interactions based on the observed expression changes, combined with Gene Ontology enrichment analyses, allowed us to identify potential miRNA target genes involved in key biological processes and metabolic pathways that permit parasite intracellular survival and proliferation. Our analyses predict the existence of a large miRNA-mRNA network affecting the expression level of numerous transcription factors that indicates inhibition of the NF-{kappa}B-dependent inflammatory response or the promotion of cholesterol biosynthesis during infection. In particular, the over 10e3-fold increase in the abundance of mmu-miR-686 in infected BMDMs was correlated with a reduced abundance of putative target transcripts implicated in miRNA biogenesis itself, in RNA binding, and in regulation of apoptosis, such as Caspase 12, the mRNA decay activator protein Zfp36l1 or Leukemia Inhibitory Factor Receptor Alpha. Likewise, the over 200-fold increase in abundance of mmu-miR-6546-3p was associated with a reduced abundance of putative target mRNAs implicated in cytokine-mediated signaling, positive regulation of apoptotic process and regulation of gene expression, affecting, for example, the MADS box transcription enhancer factor 2, the transformation related protein 53 inducible nuclear protein 1, or the G protein-coupled receptor 35. Interestingly, both miRNAs are predicted to simultaneously target 32 mRNAs that showed reduced abundance in infected BMDMs, including Maturin Neural Progenitor Differentiation Regulator (Mturn), a regulator of NF-{kappa}B transcription factor activity. In conclusion, our approach provides novel insight into molecular mechanisms that may govern macrophage subversion and intracellular Leishmania survival. Our results shed new light on the complex relationship among miRNAs, macrophage gene expression and Leishmania infection, proposing regulatory feed-forward loops (FFLs) and feedback loops (FBLs) between miRNAs and TFs as a novel target of Leishmania immune subversion. These findings open exciting new avenues for the development of intervention strategies aimed at disrupting such crucial interactions, for example using an anti-miR (antagomir) approach against mmu-miR-686 and mmu-miR-6546-3p.

microbiology↗

bioRxiv2023

Inhibition of the macrophage demethylase LSD1 reverses Leishmania amazonensis-induced transcriptomic changes and causes a decrease in parasite load

Intracellular pathogens exploit host cell functions to favor their own survival. In recent years, the subversion of epigenetic regulation has emerged as a key microbial strategy to modify host cell gene expression and evade antimicrobial immune responses. Using the protozoan parasite Leishmania as a model system, we have recently demonstrated that infection causes histone H3 hypomethylation, which is associated with the establishment of an anti-inflammatory phenotype, suggesting that host cell demethylases may play a role in the intracellular survival of these parasites. In this study, we combined pharmacological inhibition with RNA sequencing and quantitative immune-precipitation analysis to investigate the role of the macrophage lysine demethylase LSD1 (KDM1a) in Leishmania intracellular infection in vitro. Treatment of infected macrophages with validated, LSD1-specific inhibitors resulted in a significant reduction in parasite burden. We confirmed the impact of these inhibitors on LSD1 activity within macrophage nuclear extracts using an in vitro demethylase assay and established their LSD1 target engagement in situ by cellular thermal shift assay. RNA-seq analysis of infected and inhibitor-treated macrophages linked parasite killing to a partial reversion of infection-dependent expression changes, restoring the macrophage anti-microbial response and limiting cholesterol biosynthesis. While we ruled out any impact of Leishmania on LSD1 expression or localization, we uncovered significant alterations in LSD1 complex formation within infected macrophages, involving unique interactions with host cell regulatory proteins such as Rcor-1. Our study sheds important new light on the epigenetic mechanisms of macrophage immuno-metabolic subversion by intracellular Leishmania and identifies LSD1 as a potential candidate for host-directed, anti-leishmanial therapy.

cell biology↗

bioRxiv2022

Leishmania amazonensis controls macrophage-regulated cell death to establish chronic infection in vitro and in vivo

Pathogenic protists of the genus Leishmania have evolved various strategies to exploit macrophages as host cells and subvert their immuno-metabolic functions to favour intracellular parasite survival. Surprisingly little is known on how Leishmania affects regulated cell death (RCD) pathways of its host cell, even though increased survival of in vitro infected macrophages has been reported, and chronic macrophage infection in vivo causes the devastating immunopathologies of leishmaniasis. To overcome this limitation and gain first systems-level insight into the interaction between intracellular Leishmania and the host cell RCD pathways, including apoptosis, pyroptosis and necroptosis, we applied transcriptomic analyses on L. amazonensis-infected, primary macrophages (termed LIMs) and used YO-PRO-1 to monitor cell death by fluorescent microscopy. RNAseq analyses at day 3 post-infection (PI) revealed dichotomic dysregulation of more than 60% of RCD-related genes in LIMs, characterized by up-regulation of anti-RCD and down-regulation of pro-RCD markers, including key regulators common to the three forms of cell death such as casp8, fadd, tradd, tnfaip3, tax1bp1, birc3, and itch. This profile correlated with expression changes of transcription factors known to regulate RCD, including AP1 and NF-{kappa}B family members, ppar{gamma} and cebp{beta}. Consequently, LIMs showed remarkable longevity in culture for at least 50 days, despite a constant increase of parasite burden to about 100 parasites per cell, while non-infected cells were cleared from the culture in just a few days. Longitudinal expression analysis of LIMs at days 0, 3, 15, and 30 PI by RT-qPCR confirmed stable maintenance of this high longevity profile with the dichotomic decrease and increase of RCD-activators and -inhibitors, respectively. LIMs further showed significant resistance to RCD-inducing signals compared to non-infected cells, including CSF-1 deprivation (intrinsic apoptosis), actinomycin D treatment (extrinsic apoptosis), LPS/ATP stimulation (pyroptosis). Significantly, we extended the anti-RCD expression pattern and RCD resistance phenotype to L. amazonensis-infected macrophages recovered from lesions, thus validating our long-term in vitro infection system as an easily accessible model to study chronic macrophage infection. In conclusion, our analyses firmly document the pan-anti RCD effect of L. amazonensis on its macrophage host cell in vitro and in vivo and shed important new light on mechanisms underlying Leishmania chronic infection.

microbiology↗

bioRxiv2022

Immunometabolic profiling of in vitro and ex vivo Leishmania-infected macrophages (LIMs) reveals unique polarization and bioenergetic signatures

Macrophages are the major host cells of the protozoan parasite Leishmania in mammalian infection. These key innate immune cells display remarkable phenotypic plasticity ranging from pro-inflammatory M1 to anti-inflammatory M2 macrophages that can control infection and tissue homeostasis, respectively. It has been recognized that Leishmania exploits macrophage phenotypic plasticity to establish chronic infection. However, the current notion that these parasites simply trigger an M2-like phenotype seems over-simplified considering the immunopathology observed during leishmaniasis - in particular in response to Leishmania amazonensis - which is often characterized by a mixed Th1/Th2 immune response. Here we combined a series of systems-level analyses to shed new light on the phenotype of Leishmania-infected macrophages (LIMs) during short- and long-term infection, in vitro and in vivo. Immuno-metabolic profiling by RNA-seq, RT-qPCR, cytokine immunoassays, and real-time bioenergetic flux analysis of L. amazonensis-infected bone marrow-derived macrophages (BMDMs) revealed a highly complex and unique phenotypic and bioenergetic signature. In vitro LIMs were characterized by co-expression of both M1 and M2 markers at RNA and protein levels and increased expression of glycolytic genes that matched a progressive metabolic switch from a M2-like respiratory to a M1-like glycolytic energy production observed for both long-term in vitro and in vivo infected macrophages. Unlike in M1 macrophages, glycolytic gene expression did not correlate with increased expression of its key regulatory HIF-1. In contrast, siRNA knock down experiments in primary BMDMs uncovered an essential role of the m6A reader protein IGF2BP2 in stabilizing m6A modified transcripts of the glycolytic pathway, contributing to HIF-1-independent induction of glycolysis. In conclusion, L. amazonensis establishes a complex and unique phenotypic shift in infected macrophages in vitro and in vivo that combines M1-like and M2-like immuno-metabolomic characteristics and implicates differential mRNA stability in induction of aerobic glycolysis. Our data thus uncover epi-transcriptomic regulation as a novel target for Leishmania immune subversion to establish a host cell phenotype beneficial for intracellular parasite development and chronic infection.

immunology↗

bioRxiv2022

Force-biased nuclear import sets nuclear-cytoplasmic volumetric coupling by osmosis

In eukaryotes, cytoplasmic and nuclear volumes are tightly regulated to ensure proper cell homeostasis. However, the detailed mechanisms underlying nucleus-cytoplasm volumetric coupling remain unknown. Recent evidence supports a primary role of osmotic mechanisms in determining a tight link between nuclear and cytoplasmic volume, but this hypothesis remains largely untested in mammalian cells. We approach the question in single cultured adhering human cells, by jointly measuring cytoplasmic and nuclear volumes, in real time and across cell cycles. Surprisingly, we find that cytoplasmic and nuclear volumes follow different average growth laws: while the cytoplasm grows exponentially, the nucleus grows linearly. Moreover, by combining several experimental perturbations and analyzing a mathematical model including osmotic effects and tension, we conclude that the mechanical forces exerted by the cytoskeleton on the nuclear envelope can strongly affect nucleus-cytoplasm volumetric coupling by biasing nuclear import. Our results unveil how osmo-mechanical equilibrium regulates nuclear size in mammalian cells. One-Sentence SummaryCytoskeletal forces exerted on the nuclear envelope impact on nuclear volume through modulation of force-coupled nucleo-cytoplasmic transport, affecting osmosis.

cell biology↗

bioRxiv2020

Inkjet based 3D Printing of bespoke medical devices that resist bacterial biofilm formation

We demonstrate the formulation of advanced functional 3D printing inks that prevent the formation of bacterial biofilms in vivo. Starting from polymer libraries, we show that a biofilm resistant object can be 3D printed with the potential for shape and cell instructive function to be selected independently. When tested in vivo, the candidate materials not only resisted bacterial attachment but drove the recruitment of host defences in order to clear infection. To exemplify our approach, we manufacture a finger prosthetic and demonstrate that it resists biofilm formation – a cell instructive function that can prevent the development of infection during surgical implantation. More widely, cell instructive behaviours can be ‘dialled up’ from available libraries and may include in the future such diverse functions as the modulation of immune response and the direction of stem cell fate.Competing Interest StatementThe authors have declared no competing interest.View Full Text

bioengineering↗

bioRxiv2020

Dynamic High-Content Imaging Reveals Surface Exposure Of Virulent Leishmania Amastigotes In Infected Macrophages Undergoing Pyroptosis

Leishmania spp are obligate intracellular parasites that infect vertebrate phagocytes, notably macrophages. We previously reported that Leishmania amazonensis (L. am) subvert the host cell pro-inflammatory response by dampening the macrophage NLRP3 inflammasome. No information is available on how Leishmania infection affects inflammatory cell death termed pyroptosis, known to limit microbial infection. Here, we provide first evidence that L. amazonensis-infected macrophages can undergo pyroptosis when subjected to pro-inflammatory stimuli. We analyzed the dynamics of the pyroptotic process and the fate of intracellular amastigotes at the single cell level using spinning disk confocal microscopy and high-content, real-time imaging. Bone marrow-derived macrophages (BMDMs) were infected with L. am amastigotes isolated from footpad lesions and sequentially treated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP) for canonical NLRP3 inflammasome priming and activation. Real-time monitoring was performed for 240 min post ATP addition. Longitudinal analyses revealed distinct phases of the pyroptotic process, including rapid decay of the parasitophorous vacuole (PV) as monitored by the pH-sensitive lysotracker fluid phase marker, progressive decrease in macrophage viability as monitored by accumulation of the nuclear dye YO-PRO-1, followed by translocation of the luminal PV membrane to the cell surface observed for 40% of macrophages, resulting in the extracellular exposure of amastigotes that remained anchored to the PV membrane. Scanning and transmission electron microscopy analyses revealed a highly polarized orientation of parasites with exclusive exposure of the anterior pole toward the extracellular milieu, and an attachment site forming a potential biological junction between the parasite posterior pole and the PV membrane. We showed that the exposed parasites are resistant to the cytolytic host cell activities linked to pyroptosis and retain their full infectious potential in reinfection experiments using naive macrophages. Together these data uncover a novel Leishmania immune subversion strategy that may allow stealthy parasite dissemination via the uptake of pyroptotic host debris by uninfected phagocytes.

immunology↗

bioRxiv2019

SILAC-based quantitative proteomics reveals pleiotropic, phenotypic modulation in primary murine macrophages infected with the protozoan pathogen Leishmania donovani

Leishmaniases are major vector-borne tropical diseases responsible for great human morbidity and mortality, caused by protozoan, trypanosomatid parasites of the genus Leishmania. In the mammalian host parasites survive and multiply within mononuclear phagocytes, especially macrophages. However, the underlying mechanisms by which Leishmania spp affect their host, are not fully understood. Herein, proteomic alterations of primary bone marrow-derived, BALB/c macrophages are documented after 72 h of infection with Leishmania donovani insect-stage promastigotes, with the use of a SILAC-based, quantitative proteomics approach. The protocol was optimised by combining strong anion exchange and gel electrophoresis fractionation that displayed similar depth of analysis (>5500 proteins). Our analyses revealed 86 differentially modulated proteins (35 showing increased and 51 decreased abundance) in response to Leishmania donovani infection. The proteomics results were validated by analysing the abundance of selected proteins. Intracellular Leishmania donovani infection led to changes in various host cell biological processes, including primary metabolism and catabolic process, with a significant enrichment in lysosomal organisation. Overall, our analysis allows new technical insight into the challenges of quantitative proteomics applied on primary cells, and establishes the first proteome of bona fide primary macrophages infected ex vivo with Leishmania donovani, revealing new mechanisms acting at the host/pathogen interface.

systems biology↗

bioRxiv2019

The immunomodulatory effect of IrSPI, a tick salivary gland serine protease inhibitor involved in Ixodes ricinus tick feeding

Ticks are strict hematophagous arthropods and are the most important vectors of pathogens affecting both domestic and wild animals worldwide. Moreover, they are second only to mosquitoes as vectors of human pathogens. Hard tick feeding is a slow process--taking up to several days for repletion prior to detachment--and necessitates extended control over the host response. The success of the feeding process depends upon injection of saliva by tick, which not only controls host haemostasis and wound healing, but also subverts the host immune response to avoid tick rejection during this long-lasting process. In turn, the manipulation of the host immune response creates a favourable niche for the survival and propagation of diverse tick-borne pathogens transmitted during feeding. Here, we report on the molecular and biochemical features and functions of IrSPI, an Ixodes ricinus salivary serine protease inhibitor involved in blood meal acquisition. Our results show that IrSPI harbours the typical conformational fold of Kunitz type I serine protease inhibitors and that it functionally inhibits the elastase and, to a lesser extent, chymotrypsin. We also show that IrSPI is injected into the host during feeding. Crucially, we found that IrSPI has no impact on tissue factor pathway-induced coagulation, fibrinolysis, apoptosis, or angiogenesis, but a strong effect on immune cells. IrSPI affects antigen-presenting macrophages by hampering IL-5 production. In addition, IrSPI represses proliferation of mitogen-stimulated CD4+ cells. The inhibition of T cell proliferation was associated with marked reductions in pro-inflammatory cytokine secretion. Our study contributes valuable knowledge to tick-host interactions and provides insights that could be further exploited to design anti-tick vaccines targeting this immunomodulator implicated in successful I. ricinus tick feeding.\n\nAuthor summaryTicks are the most important vector influencing both human and animal health in Europe, where Ixodes ricinus is the most abundant tick species. Ticks feed on animal or human blood for an extended period, during which their saliva allows both feeding and pathogen transmission by interfering with native host responses. A better understanding of tick-host-pathogen interactions is central to the discovery of improved control methods. Within this context, we previously identified IrSPI as an I. ricinus salivary molecule implicated in both tick feeding and bacterial transmission. This serine protease inhibitor was almost characterised as an elastase inhibitor, and here, we show IrSPI overexpression in several tick organs--especially salivary glands--during blood feeding. We demonstrate that IrSPI is injected into the host through saliva, and despite having no impact on endothelial cell angiogenesis or apoptosis during blood feeding, we report an immunomodulatory role, whereby CD4+ T lymphocyte proliferation is repressed and where the cytokine secretion pattern of both splenocytes and macrophages is modified. Our study provides new insights into the complex armament developed by ticks to overcome the host response, and uncovers a potential vaccine target for disruption of feeding processes and pathogen transmission.

animal behavior and cognition↗

bioRxiv2019

Leishmania targets the macrophage epigenome and dampens the NF-κB/NLRP3- mediated inflammatory response

Aberrant macrophage activation during intracellular infection generates important immunopathologies that can cause severe human morbidity. A better understanding of microbial immune subversion strategies and macrophage phenotypic and functional responses is a prerequisite for the design of novel, host-directed intervention strategies. Here, we uncover a fine-tuned transcriptional response induced in primary macrophages infected by the human parasite Leishmania amazonensis that prevents NF-{kappa}B and NLRP3 inflammasome activation. This unusual subversion is characterized by respectively suppression and induction of activating and de-activating components of the NF-{kappa}B and NLRP3 pathways. This dichotomic modulation was associated with histone H3 hypoacetylation at promoters of NF-{kappa}B-related, pro-inflammatory genes. Our results reveal a novel Leishmania immune subversion strategy targeting host cell epigenetic regulation to modulate the macrophage phenotype. Modulation of the macrophage epigenetic landscape establishes conditions beneficial for intracellular parasite survival, and opens interesting new venues for host-directed, anti-microbial drug discovery.

cell biology↗

bioRxiv2018

Geometry alone influences stem cell differentiation in a precision 3D printed stem cell niche

Stem cells within epithelial tissues reside in anatomical structures known as crypts that are known to contribute to the mechanical and chemical milieu important for function. To date, epithelial stem cell therapies have largely ignored the niche and focussed solely on the cell population to be transplanted. Our aim was to recreate the precise geometry of the epithelial stem cell niche using two photon polymerisation and to determine the influence of this structure alone on stem cell phenotype. We were able to recreate crypt structures and following cell seeding, a zonation in cell phenotype along the z-axis emerged. This illustrates that geometry alone, without the use of exogenous signalling molecules, influences cell response. Understanding the role of geometry in the regulation of the stem cell niche will enable significant advances in our ability to influence stem cell behaviour to expedite cellular therapies to the clinic.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC=\"FIGDIR/small/489773v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (30K):\norg.highwire.dtl.DTLVardef@152d03eorg.highwire.dtl.DTLVardef@1e5b6ecorg.highwire.dtl.DTLVardef@4c6c02org.highwire.dtl.DTLVardef@14726a5_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

bioengineering↗