Biology subjects

Aguilar, D.

Publications and source records attributed to Aguilar, D..

11 recordsLinked to original sources

bioRxiv2026

Sympathetic neurons control adaptive immunity in response to Streptococcus pneumoniae infection by regulating T cell and B cell effector function

Adaptive immunity in the lung must be rapidly mobilized to control bacterial pathogens, yet the neuroimmune signals that shape local B and T cell responses remain poorly defined. Here we identify a sympathetic-immune axis that regulates lung-resident memory B cells, antigen-specific IgG production, and protection against Streptococcus pneumoniae. Lung-targeted chemical sympathectomy reduced norepinephrine availability, diminished Tbet memory and resident-memory B cell populations, and markedly impaired S. pneumoniae-specific IgG, resulting in increased bacterial burden. Mice lacking Adrb1 & Adrb2-adrenergic receptors phenocopied these defects, and adoptive transfer of Adrb1/Adrb2-/- B cells into Ighm-/- hosts was sufficient to reduce IgG and worsen infection. Combined T cell depletion and B cell-intrinsic adrenergic receptor loss further impaired bacterial control and suppressed IFNgamma, indicating that norepinephrine stimulates T cell IFNgamma release and that norepinephrine and IFNgamma together drive optimal B cell IgG production. In vitro, norepinephrine enhanced T cell IFNgamma secretion, and norepinephrine plus IFNgamma synergistically increased IgG. These findings define norepinephrine as a key regulator of lung-resident humoral immunity and reveal a neuroimmune mechanism that promotes antibody-mediated bacterial clearance at the respiratory mucosa.

immunology↗

bioRxiv2025

A new method to quantify the spatiotemporal localization of SnRK1.1

Maintaining energy homeostasis is a major challenge for plants in the current context of climate change. The Sucrose-non fermenting 1 (SNF1)-related kinase 1 (SnRK1) complex, a member of the SNF1-AMP-activated protein kinase (AMPK)-SnRK1 family of kinase complexes, is a central player in the regulation of cell energy homeostasis. The -subunit of the complex, which possesses kinase activity and is known as SnRK1.1 or KIN10, plays a role in sensing energy status and coordinating metabolic reprogramming to counter any energy imbalance. The discovery of a dual and dynamic intracellular distribution of SnRK1.1 suggests that the activity and function of SnRK1 might be regulated by spatiotemporal changes. To investigate the spatiotemporal distribution of SnRK1.1, we developed a protocol to quantify its intracellular distribution using fluorescence confocal images acquired along the z-axis in plants expressing SnRK1.1-eGFP. Using the open-source software Fiji/ImageJ, we calculated the ratio between nuclear and non-nuclear SnRK1.1 fractions and defined this as the N/ER index. We validated our method by analyzing the response of SnRK1.1 to photosynthesis inhibition by DCMU, including changes in protein levels and phosphorylation status. In addition, comparison with results obtained using a commercial software-based approach confirmed the compatibility of the N/ER index with different segmentation and quantification tools. Originally designed for leaf tissue images, this protocol can be broadly applied to assess the role of intracellular spatiotemporal changes in a wide range of kinases or fluorescently tagged recombinant proteins. Finally, SnRK1.1 intracellular distribution may also serve as a proxy to assess changes in cellular energy status. One sentence summaryNew method to track SnRK1.1 distribution and changes in plant cell energy status

plant biology↗

bioRxiv2024

A chronic Pseudomonas aeruginosa mouse lung infection modeling the pathophysiology and inflammation of human cystic fibrosis

Investigation of chronic cystic fibrosis (CF) lung infections has been limited by a lack of murine models that reproduce obstructive lung pathology, chronicity of bacterial infections, and complex inflammation in human CF lung pathology. Three different approaches have been used separately to address these limitations, including using transgenic Scnn1b-Tg mice overexpressing a lung epithelial sodium channel to mimic the mucus-rich and hyperinflammatory CF lung environment, using synthetic CF sputum medium (SCFM) in an acute infection to induce bacterial phenotypes consistent with human CF, or using agar beads to promote chronic infections. Here, we combine these three models to establish a chronic Pseudomonas aeruginosa lung infection model using SCFM agar beads and Scnn1b-Tg mice (SCFM-Tg-mice) to recapitulate nutrients, mucus, and inflammation characteristic of the human CF lung environment. Like people with CF, SCFM-Tg-mice failed to clear bacterial infections. Lung function measurements showed that infected SCFM-Tg-mice had decreased inspiratory capacity and compliance, elevated airway resistance, and significantly reduced FVC and FEV0.1. Using spectral flow cytometry and multiplex cytokine arrays we show that, like people with CF, SCFM-Tg-mice developed inflammation characterized by eosinophil infiltration and Th2 lymphocytic cytokine responses. Chronically infected SCFM-Tg-mice developed an exacerbated mix of innate and Th1, Th2, and Th17-mediated inflammation, causing higher lung cellular damage, and elevated numbers of unusual Siglec F+ neutrophils. Thus, SCFM-Tg-mice represents a powerful tool to investigate bacterial pathogenesis and potential treatments for chronic CF lung infections and reveal a potential role for Siglec F+ neutrophils in CF inflammation. ImportanceHost-pathogen interaction studies of Pseudomonas aeruginosa cystic fibrosis (CF) lung infections have been hampered by limitations of mouse infection models. Here we combine synthetic CF sputum medium (SCFM) agar beads and Scnn1b-Tg transgenic mice to model the mucus obstructed airways and complex inflammatory characteristic of the human cystic fibrosis lung environment. In this model, which we name SCFM-Tg-mice, we use SCFM to cause changes in bacterial gene expression consistent with sputum collected from people with CF and the Scnn1b-Tg mice produce excessive airway mucus like people with CF. We show that SCMF-Tg-mice infected with P. aeruginosa have defects in lung function and increased inflammation that is consistent with human CF lung infections. This model can be adapted for other bacterial species and can be used to test hypotheses about bacterial pathogenesis and potential treatments in a CF human-like system.

microbiology↗

bioRxiv2024

IspE Kinase as an Anti-infective Target: Role of a Hydrophobic Pocket in Inhibitor Binding

Enzymes of the methylerythritol phosphate (MEP) pathway are potential targets for antimicrobial drug discovery. Here we focus on 4-diphosphocytidyl-2-C-methyl-D-erythritol (IspE) kinase from the MEP pathway. We use biochemical and structural biology methods to investigate homologs from the pathogenic microorganisms; Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii. We determined the X-ray structures of IspE-inhibitor complexes and studied selected inhibitors binding modes targeting the substrate pocket. The experimental results indicate the need for distinct inhibitor strategies due to structural differences among IspE homologs, particularly for A. baumannii IspE, which displays unique inhibitory profile due to a tighter hydrophobic subpocket in the substrate binding site. This study enhances our understanding of the MEP enzymes and sets the stage for structure-based drug design of selective inhibitors to combat pathogenic microorganisms.

biophysics↗

bioRxiv2024

Sensory neurons regulate stimulus-dependent humoral immunity

Sensory neurons sense pathogenic infiltration, informing immune coordination of host defense. However, sensory neuron-immune interactions have been predominantly shown to drive innate immune responses. Humoral memory, whether protective or destructive, is acquired early in life - as demonstrated by both early exposure to streptococci and allergic disease onset. Our study further defines the role of sensory neuron influence on humoral immunity in the lung. Using a murine model of Streptococcus pneumonia pre-exposure and infection and a model of allergic asthma, we show that sensory neurons are required for B-cell and plasma cell recruitment and antibody production. In response to S. pneumoniae, sensory neuron depletion resulted in a larger bacterial burden, reduced B-cell populations, IgG release, and neutrophil stimulation. Conversely, sensory neuron depletion reduced B-cell populations, IgE, and asthmatic characteristics during allergen-induced airway inflammation. The sensory neuron neuropeptide released within each model differed. With bacterial infection, vasoactive intestinal polypeptide (VIP) was preferentially released, whereas substance P was released in response to asthma. Administration of VIP into sensory neuron-depleted mice suppressed bacterial burden and increased IgG levels, while VIP1R deficiency increased susceptibility to bacterial infection. Sensory neuron-depleted mice treated with substance P increased IgE and asthma, while substance P genetic ablation resulted in blunted IgE, similar to sensory neuron-depleted asthmatic mice. These data demonstrate that the immunogen differentially stimulates sensory neurons to release specific neuropeptides that specifically target B-cells. Targeting sensory neurons may provide an alternate treatment pathway for diseases involved with insufficient and/or aggravated humoral immunity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/574231v3_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1a96e0org.highwire.dtl.DTLVardef@1a75c97org.highwire.dtl.DTLVardef@afd6f8org.highwire.dtl.DTLVardef@4a6666_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Circulating tumor extracellular vesicles to monitor metastatic prostate cancer genomics and transcriptomic evolution

Extracellular vesicles (EVs) secreted by tumors are abundant in plasma, but their potential for interrogating the molecular features of tumors through multi-omic profiling remains widely unexplored. Genomic and transcriptomic profiling of circulating EV-DNA and EV-RNA isolated from a range of in-vitro and in-vivo models of metastatic prostate cancer (mPC) revealed a high contribution of tumor material to EV-loaded DNA/RNA. Findings were validated in a cohort of longitudinal plasma samples collected from mPC patients during androgen receptor signaling inhibitor (ARSI) therapy. EV-DNA genomic features recapitulated matched-patient biopsies and associated with clinical progression. We developed a novel approach to enable the transcriptomic profiling of EV-RNA (RExCuE). We report how the transcriptomic profile in mPC EV-RNA is enriched for tumor-associated transcripts when compared to same patient blood RNA and healthy individuals EV-RNA, and reflect early on-therapy tumor adaptation changes. Altogether, we show that EV profiling enables longitudinal transcriptomic and genomic profiling of mPC in liquid biopsy.

cancer biology↗

bioRxiv2022

Macrophage and neutrophil heterogeneity at single-cell spatial resolution in inflammatory bowel disease

Ulcerative colitis (UC) and Crohns disease (CD) are chronic inflammatory intestinal diseases that show a perplexing heterogeneity in manifestations and response to treatment. The molecular basis for this heterogeneity remains uncharacterized. We applied single-cell RNA sequencing and CosMx Spatial Molecular Imaging to human colon and found the highest diversity in cellular composition in the myeloid compartment of UC and CD patients. Besides resident macrophage subsets (M0 and M2), patients showed a variety of activated macrophages including classical (M1 CXCL5 and M1 ACOD1) and new inflammation-dependent alternative (IDA) macrophages. In addition, we captured intestinal neutrophils in three transcriptional states. Subepithelial IDA macrophages expressed NRG1, which promotes epithelial differentiation. In contrast, NRG1low IDA macrophages were expanded within the submucosa and in granulomas, in proximity to abundant inflammatory fibroblasts, which we suggest may promote macrophage activation. We conclude that macrophages sense and respond to unique tissue microenvironments, potentially contributing to patient-to-patient heterogeneity.

immunology↗

bioRxiv2021

Targeting of Protein Kinase CK2 Elicits Antiviral Activity on Bovine Coronavirus Infection

Coronaviruses constitute a global threat to human population since three highly pathogenic coronaviruses (SARS-CoV, MERS-CoV and SARS-CoV-2) have crossed species to cause severe human respiratory disease. Considering the worldwide emergency status due to the current COVID-19 pandemic, effective pan-coronavirus antiviral drugs are required to tackle the ongoing as well as future (re)emerging virus outbreaks. Protein kinase CK2 has been deemed a promising therapeutic target in COVID-19 supported by its in vitro pharmacologic inhibition and molecular studies on SARS-CoV-2 infected cells. CIGB-325 is a first-in-class synthetic peptide impairing the CK2-mediated signaling whose safety and clinical benefit have been evidenced in Covid-19 and cancer patients after intravenous administration. Here, we explored the putative antiviral effect of CIGB-325 over MDBK cells infected by bovine coronavirus (BCoV) Mebus. Importantly, CIGB-325 inhibited both the cytopathic effect and the number of plaques forming units with a half-inhibitory concentrations IC50 = 3.5 M and 17.7 M, respectively. Accordingly, viral protein accumulation at the cytoplasm was clearly reduced by treating BCoV-infected cells with CIGB-325 over time, as determined by immunocytochemistry. Of note, data from pull-down assay followed by western blot and/or mass spectrometry identification revealed physical interaction of CIGB-325 with nucleocapsid (N) protein and a bona fide cellular CK2 substrates. Functional enrichment and network analysis from the CIGB-325 interacting proteins indicated cytoskeleton reorganization and protein folding as the most represented biological processes disturbed by this anti-CK2 peptide. Altogether, our findings not only unveil the direct antiviral activity of CIGB-325 on coronavirus infection but also provide molecular clues underlying such effect. Also, our data reinforce the scientific rationality behind the pharmacologic inhibition of CK2 to treat coronavirus infections.

pharmacology and toxicology↗

bioRxiv2021

Targeting of Protein Kinase CK2 in Acute Myeloid Leukemia Cells Using the Clinical-Grade Synthetic-Peptide CIGB-300

Protein kinase CK2 has emerged as an attractive therapeutic target in acute myeloid leukemia (AML), advent that becomes particularly relevant since the treatment of this hematological neoplasia remains challenging. Here we explored for the first time the effect of the clinical-grade peptide-based CK2 inhibitor CIGB-300 on AML cells proliferation and viability. CIGB-300 internalization and subcellular distribution were also studied, and the role of B23/nucleophosmin 1 (NPM1), a major target for the peptide in solid tumors, was addressed by knock-down in model cell lines. Finally, pull-down experiments and phosphoproteomic analysis were performed to study CIGB-interacting proteins and identify the array of CK2 substrates differentially modulated after treatment with the peptide. Importantly, CIGB-300 elicited a potent anti-proliferative and proapoptotic effect in AML cells, with more than 80% of peptide transduced cells within three minutes. Unlike solid tumor cells, NPM1 did not appear to be a major target for CIGB-300 in AML cells. However, in vivo pull-down experiments and phosphoproteomic analysis evidenced that CIGB-300 targeted the CK2 catalytic subunit, different ribosomal proteins, and inhibited the phosphorylation of a common CK2 substrates array among both AML backgrounds. Remarkably, our results not only provide cellular and molecular insights unveiling the complexity of the CIGB-300 anti-leukemic effect in AML cells, but also reinforce the rationale behind the pharmacologic blockade of protein kinase CK2 for AML targeted therapy.

cancer biology↗

bioRxiv2018

Suppressor of Fused controls the proliferation of postnatal neural stem and precursor cells via a Gli3-dependent mechanism

The ventricular-subventricular zone (V-SVZ) of the forebrain is the source of neurogenic stem/precursor cells for adaptive and homeostatic needs throughout the life of most mammals. Here, we report that Suppressor of Fused (SUFU) plays a critical role in the establishment of the V-SVZ at early neonatal stages by controlling the proliferation of distinct subpopulations of stem/precursor cells. Conditional deletion of Sufu in radial glial progenitor cells (RGCs) at E13.5 resulted in a dramatic increase in the proliferation of Sox2+ Type B cells, as well as Gsx2+ ventral forebrain derived transit amplifying precursor cells (TACs). In contrast, we found a significant decrease in Tbr2+ dorsal forebrain derived TACs indicating that innate differences between dorsal and ventral forebrain derived Type B cells influence Sufu function. However, most precursors failed to survive and accumulated in the dorsal V-SVZ, demonstrating that precursors are unable to transition into functional differentiated progenies. These defects were accompanied by reduced Gli3 expression, yet despite reduced Gli3 levels, activation of Sonic hedgehog (Shh) signaling did not occur implying that the Sufu-Gli3 regulatory axis may influence other signaling pathways in the neonatal dorsal V-SVZ. These data suggest that Sufu plays a critical role in controlling Gli3 function in the establishment and survival of functional stem/precursor cells in the postnatal dorsal V-SVZ.\n\nSUMMARY STATEMENTConditional deletion of Sufu cause dramatic expansion of neural stem/precursor cells in the neonatal ventricular-subventricular (SVZ) zone. This defect occurs through a Gli3-dependent mechanism that results in the downregulation of Shh signaling.

developmental biology↗

bioRxiv2018

Cell adhesion promoted by a unique Shigella IpaA vinculin- and talin-binding site

During Shigella cell invasion, the IpaA effector targets the focal adhesion protein vinculin through three vinculin-binding sites (VBSs). Here, we report that IpaA VBS3 also binds to talin. The 2.5 [A] resolution crystal structure indicates that IpaA VBS3 forms a tightly folded -helical bundle with talin H1-H4, contrasting with bundle unraveling upon vinculin interaction. High-affinity binding of Ipa VBS3 to talin H1-H4 requires a core of hydrophobic residues conserved in vinculin binding and a pair of electrostatic interactions accounting for talin binding specificity. IpaA VBS3 does not bind to talin H1-H5 suggesting the targeting of partially activated stretched talin but not inactive talin. Consistently, IpaA VBS3 labeled filopodial and nascent adhesions and regulated their formation through talin binding. In addition, talin-binding by IpaA VBS3 was required for bacterial capture by filopodia during Shigella invasion and for the stabilization of focal adhesions in infected cells. These findings point to the functional diversity of VBSs and a critical role for talin-binding by Shigella IpaA VBS3 and possibly talin VBSs in the regulation of adhesion structures.

microbiology↗