Biology subjects

Martino, N.

Publications and source records attributed to Martino, N..

9 recordsLinked to original sources

bioRxiv2025

Endothelial Cells retain inflammatory memory through chromatin remodeling

Sepsis survivors face a heightened risk of secondary infections and chronic vascular dysfunction despite clinical recovery, yet the underlying mechanisms remain poorly defined. Our study identifies a novel mechanism of endothelial inflammatory memory, wherein transient inflammatory exposure induces durable chromatin remodeling, priming endothelial cells for exaggerated responses to subsequent inflammatory insults. Utilizing a clinically relevant two-hit mouse model, cecal ligation and puncture (CLP) followed by secondary Streptococcus pneumoniae (SP) infection, we reveal persistent transcriptional activation in endothelial cells (ECs), characterized by amplified expression of inflammatory cytokines, adhesion molecules (e.g., ICAM-1), complement factors, and interferon-stimulated genes. Genome-wide ATAC-seq analyses demonstrated stable chromatin accessibility at key inflammatory loci, indicative of epigenetic priming. Mechanistically, we uncovered a critical role for the AP-1 transcription factor JunB in mediating this epigenetic remodeling. JunB knockdown attenuated chromatin accessibility and subsequent transcriptional amplification upon secondary challenge, pinpointing JunB-driven chromatin modifications as central to endothelial reprogramming. Our findings offer mechanistic insights into how transient inflammation creates lasting epigenetic states within the endothelium, highlighting JunB as a potential therapeutic target to mitigate chronic endothelial dysfunction and increased susceptibility to secondary infections post-sepsis.

cell biology↗

bioRxiv2025

LRRC8A-containing anion channels promote glioblastoma proliferation via a WNK1/mTORC2-dependent mechanism

Leucine-rich repeat-containing protein 8A (LRRC8A) is the essential subunit of ubiquitous volume-regulated anion channels (VRACs). LRCC8A is overexpressed in several cancers and promotes negative survival outcomes via a poorly defined mechanism. Here, we explored the role of LRRC8A and VRACs in the progression of glioblastoma (GBM), the most common and deadly primary brain tumor. We found that, as compared to healthy controls, LRRC8A mRNA was strongly upregulated in surgical GBM specimens, patient-derived GBM cell lines, and GBM datasets from The Cancer Genome Atlas (TCGA). Our in-silico analysis indicated that patients belonging to the lowest LRRC8A expression quartile demonstrated a trend for extended life expectancy. In patient-derived GBM cultures, siRNA-driven LRRC8A knockdown reduced cell proliferation and additionally decreased intracellular chloride levels and inhibited activity of mTOR complex 2. The antiproliferative effect of LRRC8A downregulation was recapitulated with a pharmacological inhibitor of VRAC. Our ensuing biochemical and molecular biology analyses established that the LRRC8A-containing VRACs facilitate GBM proliferation via a new mechanism involving non-enzymatic actions of the chloride-sensitive protein kinase WNK1. Accordingly, the chloride-bound WNK1 stimulates mTORC2 and the mTORC2-dependent protein kinases AKT and SGK, which promote proliferation. These findings establish the new mTORC2-centric axis for VRAC dependent regulation of cellular functions and uncover potential targets for GBM intervention. SUBJECT AREASCell biology Molecular biology Cancer

cancer biology↗

bioRxiv2024

Wideband Tuning and Deep-Tissue Spectral Detection of Indium Phosphide Nano-Laser Particles

Laser particles (LPs) emitting narrowband spectra across wide spectral ranges are highly promising for high-multiplex optical barcoding. Here, we present LPs based on indium phosphide (InP) nanodisks, operating in the near-infrared wavelength range of 740-970 nm. Utilizing low-order whispering gallery resonance modes in size-tuned nanodisks, we achieved an ultrawide color palette with 27% bandwidth utilization and nanometer-scale linewidth. The minimum laser size was 430 nm in air and 560 nm within the cytoplasm, operating at mode order 4 or 5. We further demonstrated spectral detection of laser peaks with high signal-to-background ratios in highly-scattering media, including 1-cm-thick chicken breast tissue and blood vessels in live mice.

bioengineering↗

bioRxiv2024

A transient brain endothelial translatome response to endotoxin is associated with mild cognitive changes post-shock in young mice

Sepsis-associated encephalopathy (SAE) is a common manifestation in septic patients that is associated with increased risk of long-term cognitive impairment. SAE is driven, at least in part, by brain endothelial dysfunction in response to systemic cytokine signaling. However, the mechanisms driving SAE and its consequences remain largely unknown. Here, we performed translating ribosome affinity purification and RNA-sequencing (TRAP-seq) from the brain endothelium to determine the transcriptional changes after an acute endotoxemic (LPS) challenge. LPS induced a strong acute transcriptional response in the brain endothelium that partially correlates with the whole brain transcriptional response and suggested an endothelial-specific hypoxia response. Consistent with a crucial role for IL-6, loss of the main regulator of this pathway, SOCS3, leads to a broadening of the population of genes responsive to LPS, suggesting that an overactivation of the IL-6/JAK/STAT3 pathway leads to an increased transcriptional response that could explain our prior findings of severe brain injury in these mice. To identify any potential sequelae of this acute response, we performed brain TRAP-seq following a battery of behavioral tests in mice after apparent recovery. We found that the transcriptional response returns to baseline within days post-challenge. Despite the transient nature of the response, we observed that mice that recovered from the endotoxemic shock showed mild, sex-dependent cognitive impairment, suggesting that the acute brain injury led to sustained, non-transcriptional effects. A better understanding of the transcriptional and non-transcriptional changes in response to shock is needed in order to prevent and/or revert the devastating consequences of septic shock. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/583191v2_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@19515borg.highwire.dtl.DTLVardef@2c307dorg.highwire.dtl.DTLVardef@12393f9org.highwire.dtl.DTLVardef@c7d795_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2022

Regulation of endothelial DNA methylation by the sustained response to IL-6

Endothelial dysfunction is a critical factor in promoting organ failure during septic shock. Organ dysfunction during shock increases the risk of long-term sequelae in survivors through mechanisms that remain unknown. We postulated that vascular dysfunction during shock contributes to the long-term morbidity post shock through transcriptional and epigenetic changes within the endothelium. As we have previously demonstrated that IL-6/JAK/STAT3 signaling in endothelial cells contributes to the inflammatory response following endotoxin, we performed cross-omics analyses on kidney endothelium from acute endotoxin-challenged mice lacking or not the JAK/STAT3 inhibitor SOCS3. This analysis revealed significant DNA methylation changes upon proinflammatory signaling that was significantly associated with transcriptional activity through AP1, STAT, and IRF families, suggesting a mechanism driving transcription-induced gene-specific methylation changes. In vitro, we demonstrated that IL-6 induces similar changes in DNA methylation. Specific genes showed DNA methylation changes in response to an IL-6+R challenge, and consistently, changes in their expression levels by 72 hours of IL-6+R treatment. Further, changes in the endothelial methylome remain in place for prolonged periods in absence of IL-6, suggesting that this cytokine may elicit transcriptional changes long after the resolution of inflammation. Also, demonstrated that DNA methylation changes could directly alter the expression of these genes and that STAT3 activation had a causal role in this transcriptional response. Our findings provide evidence for a critical role for IL-6 signaling in regulating shock-induced epigenetic changes and sustained endothelial activation, offering a new therapeutic target to limit vascular dysfunction and prevent long-term organ damage. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/502384v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@453cbdorg.highwire.dtl.DTLVardef@196b50dorg.highwire.dtl.DTLVardef@1487180org.highwire.dtl.DTLVardef@113a9ee_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Laser particle barcoding for multi-pass high-dimensional flow cytometry

Flow cytometry is a standard technology in life science and clinical laboratories used to characterize the phenotypes and functional status of cells, especially immune cells. Recent advances in immunology and immuno-oncology as well as drug and vaccine discovery have increased the demand to measure more parameters. However, the overlap of fluorophore emission spectra and one-time measurement nature of flow cytometry are major barriers to meeting the need. Here, we present multi-pass flow cytometry, in which cells are tracked and measured repeatedly through barcoding with infrared laser-emitting microparticles. We demonstrate the benefits of this approach on several pertinent assays with human peripheral blood mononuclear cells (PBMCs). First, we demonstrate unprecedented time-resolved flow characterization of T cells before and after stimulation. Second, we show 33-marker deep immunophenotyping of PBMCs, analyzing the same cells in 3 back-to-back cycles. This workflow allowed us to use only 10-13 fluorophores in each cycle, significantly reducing spectral spillover and simplifying panel design. Our results open a new avenue in multi-dimensional single-cell analysis based on optical barcoding of individual cells.

bioengineering↗

bioRxiv2022

SOCS3 limits endotoxin-induced endothelial dysfunction by blocking a required autocrine interleukin 6 signal in human umbilical vein endothelial cells

Increased circulating levels of soluble interleukin (IL)-6 receptor (sIL-6R) are commonly observed during inflammatory responses, allowing for IL-6 signaling to occur in cells that express the ubiquitous receptor subunit gp130 but not IL-6R, such as endothelial cells. Activation of Toll-like receptor (TLR)-4 or the tumor necrosis factor (TNF) receptor leads to NF-{kappa}B-dependent increases in endothelial IL-6 expression. Thus, we hypothesize that danger signals may induce autocrine IL-6 signaling within the endothelium via sIL-6R-mediated trans-signaling. In support of this hypothesis, we recently demonstrated that conditional deletion in the endothelium of the IL-6 signaling inhibitor SOCS3 leads to rapid mortality in mice challenged with the TLR-4 agonist endotoxin through increases in vascular leakage, thrombosis, leukocyte adhesion, and a type I-like interferon response. Here, we sought to directly test a role for sIL-6R in LPS-treated human umbilical vein endothelial cells. We show that cotreatment with sIL-6R dramatically increases the loss of barrier function and the expression of COX2 and tissue factor mRNA levels induced by LPS. This co-treatment led to a strong activation of STAT1 and STAT3 while not affecting LPS-induced activation of p38 and NF-{kappa}B signaling. Similar results were obtained when sIL-6R was added to a TNF challenge. JAK inhibition by pretreatment with ruxolitinib or by SOCS3 overexpression blunted LPS and sIL-6R synergistic effects, while SOCS3 knockdown further increased the response. Together, these findings demonstrate that IL-6 signaling downstream of NF-kB activation leads to a strong endothelial activation and may explain the acute endotheliopathy observed during critical illness.

cell biology↗

bioRxiv2020

Endothelial SOCS3 maintains homeostasis and promotes survival in endotoxemic mice

SOCS3 is the main inhibitor of the JAK/STAT3 pathway. This pathway is activated by interleukin 6 (IL-6), a major mediator of the cytokine storm during shock. To determine its role in the vascular response to shock, we challenged mice lacking SOCS3 in the adult endothelium (SOCS3iEKo) with a non-lethal dose of lipopolysaccharide (LPS). SOCS3iEKo mice died 16-24 hours post-injection after severe kidney failure. Loss of SOCS3 led to an LPS-induced type I interferon-like program, and high expression of pro-thrombotic and pro-adhesive genes. Consistently, we observed intraluminal leukocyte adhesion and NETosis, as well as retinal venular leukoembolization. Notably, heterozygous mice displayed an intermediate phenotype, suggesting a gene dose effect. In vitro studies were performed to study the role of SOCS3 protein levels in the regulation of the inflammatory response. In HUVEC, pulse-chase experiments showed that SOCS3 protein has a half-life below 20 minutes. Inhibition of SOCS3 ubiquitination and proteasomal degradation leads to protein accumulation and a stronger inhibition of IL-6 signaling and barrier function loss. Together, our data demonstrates that the regulation of SOCS3 protein levels is critical to inhibit IL-6-mediated endotheliopathy during shock and provides a promising new therapeutic avenue to prevent MODS though stabilization of endothelial SOCS3. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/424586v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@88eaf4org.highwire.dtl.DTLVardef@f974f1org.highwire.dtl.DTLVardef@16c9fa4org.highwire.dtl.DTLVardef@650229_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2018

Wavelength-encoded laser particles for massively-multiplexed cell tagging

Large-scale single-cell analyses have become increasingly important given the role of cellular heterogeneity in complex biological systems. However, no current techniques enable optical imaging of uniquely-tagged individual cells. Fluorescence-based approaches can only distinguish a handful of distinct cells or cell groups at a time because of spectral crosstalk between conventional fluorophores. Here we show a novel class of imaging probes emitting coherent laser light, called laser particles. Made of silica-coated semiconductor microcavities, these laser particles have single-mode emission over a broad range from 1170 to 1580 nm with sub-nm linewidths, enabling massive spectral multiplexing. We demonstrate the stability and biocompatibility of these probes in vitro and their utility for wavelength-multiplexed cell tagging and imaging. We demonstrate real-time tracking of thousands of individual cells in a 3D tumor model for several days showing different behavioral phenotypes. We expect laser particles will enable new approaches for single-cell analyses.

biophysics↗