Biology subjects

Hoffmann, A.

Publications and source records attributed to Hoffmann, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Intubation and mechanical ventilation lead to an extensive expansion of VAP-associated bacterial species in respiratory secretions of ventilated pigs

Ventilator associated pneumonia (VAP) remains one of the most critical nosocomial infections in intensive care medicine. Between 20-50% of ventilated patients develop VAP during treatment, often leading to long term sequelae and death, despite successful therapy of their underlying disease. VAP is typically caused by Gram-negative bacteria that can previously be detected on the mucosal membranes of the oral and upper respiratory tract. To assess and quantify bacterial colonization of the respiratory tract following intubation and ventilation a well-established pig model was used. Methods: After induction of anaesthesia and endotracheal intubation pigs underwent mechanical ventilation continuously for up to three days. To assess bacterial colonization of the upper respiratory tract, samples of respiratory secretions were taken at day 0, 1 and 2 directly above the cuff outside of the endotracheal tube (ETT) and by aspiration of tracheal secretions through the ETT. Quantification of bacteria was carried out by serial dilutions and CFU/ml determinations on sheep blood agar and MacConkey agar plates. Species identification was achieved by 16S-RNA gene sequencing. Results: Respiratory samples of 19 pigs were analysed and showed a sharp increase of predominantly Gram-negative organisms in the respiratory material sampled above the cuff from day 0 to 1. Starting at a median of 7.3 x 103 immediately after intubation numbers increased to a median of 2.0 x 107 Gram-negative bacteria at day 1 and 3.2 x 107 at day 2. Gram-negative species that were detected included Pseudomonas aeruginosa, Acinetobacter baumanii, Enterobacter aerogenes, Klebsiella pneumoniae and other Enterobacteriaceae. Tracheal secretions contained much less bacteria. While no Gram-negative bacteria could be cultured at the beginning of the experiment in 18 of 19 animals, colonization with Gram-negative bacteria gradually increased and was found in 7 of 19 and 9 of 14 animals on day 1 and day 2 respectively. Interestingly, in the vast majority of colonized tracheal secretions the identical Gram-negative species had previously been detected in secretions collected > 24h earlier at the cuff. Conclusion: A massive increase of the concentration of Gram-negative bacteria occurred at 24h of ventilation in mucosal secretions above the cuff. Detected species were the same Gram-negative bacteria that are frequently responsible for human infections, suggesting that especially the accumulation of bacteria in secretions above the cuff may be crucial in the development of VAP.

microbiology↗

bioRxiv2026

Direct 3D isolation in gelatin methacryloyl microbeads versus conventional 2D isolation and culture: a trade-off between mesenchymal stromal cell purity and niche-associated transcriptional profiles

Bone-marrow mesenchymal stromal cell (MSC) isolation typically couples plastic-adherence selection to planar expansion. Therefore, the recovered cells may reflect culture adaptation as much as native stromal biology. Direct isolation within a three-dimensional matrix could reduce planar selection, but its effects on cellular recovery, composition and MSC transcriptional profiles remain insufficiently resolved. We therefore developed a gelatin methacryloyl (GelMA) workflow progressing from bulk hydrogels to medium-surrounded GelMA microbeads (GMBs) and compared 3D-derived cells with conventional 2D cultures. Qualitative live/dead staining with calcein acetoxymethyl ester (CAM) and propidium iodide (PI) and 5-ethynyl-2'-deoxyuridine (EdU) imaging established cellular activity in both GelMA formats. In bulk GelMA cultures from four human donors, CD73-, CD90- and CD105-associated flow-cytometric readouts did not increase from day 7 to day 21. At day 21, the combined marker readout was numerically higher in donor-matched 2D cultures than in bulk 3D cultures, which retained heterogeneous marker-defined populations. GMBs enabled cell recovery and flow-cytometric characterization and motivated an exploratory one-donor single-cell RNA sequencing (scRNA-seq) analysis. The final dataset contained 37,784 cells across four samples and 23 annotated populations. MSC-focused re-embedding resolved 13,175 cells into two 3D and four 2D clusters. A pooled comparison identified 4,840 differentially expressed genes (DEGs, false discovery rate [FDR]<0.01, |log2FC|[≥]1), indicating a broad method-associated expression shift. The 3D C1 cluster combined progenitor-, extracellular-matrix-, adhesion-, cytokine-, hypoxia/glycolysis- and predicted-interaction-associated programmes, whereas 2D clusters showed mitochondrial, cytoskeletal/mechanoadaptive and proliferative programmes. Thus, 2D culture yielded a more strongly represented and compositionally purer MSC compartment, while 3D isolation contained rare MSC clusters with niche-associated transcriptional profiles within a heterogeneous marrow environment. These findings support a testable native-associated tendency for 3D, but donor replication, matched recovery and direct native-reference mapping are required before inferring greater in-vivo similarity or functional superiority.

cell biology↗

bioRxiv2026

AI-enabled Spatial Profiling of Circulating Tumor-Immune Ecosystems Predicts Patient Outcomes Across Cancers

Circulating tumor cells (CTCs) and immune cells form dynamic multicellular ecosystems in blood, but their spatial organization and clinical relevance have not been systematically characterized. We developed the Cell and Cluster Identification Program (CCIP), an artificial intelligence-based framework that analyzes routine multiplex immunofluorescence blood scans to segment cells, identify CTCs and five immune lineages with high accuracy, and quantify multicellular clusters and tumor-immune interactions. Applying CCIP to 2,693 blood scans from 1,399 patients, we profiled over 60 million cells (>7 million multi-cell clusters) and linked imaging-derived features to patient outcomes. Correlated with circulating-tumor DNA mutation burdens, a 14-feature image model predicted overall survival in breast cancer, outperformed clinicopathologic variables and CTC enumeration, and generalized to prostate cancer. Prognostic imaging signatures were also associated with therapy response-related progression-free survival as well as with single-cell RNA sequencing-derived immune suppression states, connecting circulating tumor-immune architecture with systemic immune dysfunction.

cancer biology↗

bioRxiv2026

A conserved transcriptomic model defines metabolic resilience and vulnerability in obesity

BackgroundObesity arises from a complex interplay of genetic and environmental factors, with alterations of transcriptional networks that integrate metabolic, immune, and regulatory pathways. Conventional measures such as body mass index (BMI) quantify body size but fail to capture the molecular heterogeneity underlying divergent metabolic outcomes. We therefore sought to construct a gene expression-based transcriptomic representation of obesity, using BMI as a practical training anchor, and to use this framework to delineate transcriptional programs associated with metabolically healthy and pathogenic obesity, with subsequent projection to mouse transcriptomic data for cross-species validation. MethodsTranscriptome data of human visceral adipose tissue (N= 1,298) were used to derive the transcriptomic BMI model, and genes contributing to the model were functionally annotated by gene set enrichment analysis. The human-trained model was subsequently applied to mouse selection lines (N = 18) with divergent obesity phenotypes. In the human cohort, post hoc stratification into metabolically healthy obesity (MHO) and metabolically unhealthy obesity (MUO) groups was performed using a downstream classification framework incorporating observed BMI together with predicted BMI, to assess whether model-derived predicted BMI reflected obesity-related pathophysiologic status. ResultsModel-selected genes were involved in coordinated regulation of lipid metabolism, immune activation, and growth signaling, extending to mitochondrial and translational pathways. Cross-species analyses uncovered conserved metabolic polarization: DU6 mice exhibited lipid-anabolic and inflammatory remodeling, whereas DU6P mice displayed oxidative, mitochondrial, and GH-axis-enriched transcriptional states. In human cohorts, MHO individuals showed upregulation of mitochondrial energetics and protein synthesis, while MUO individuals were characterized by increased autophagy, lipid catabolism, and stress-adaptive signaling on the transcriptional level. Together, these findings define a conserved molecular continuum linking oxidative efficiency to metabolic health and inflammation to metabolic vulnerability. ConclusionsThis integrative transcriptomic framework bridges human and mouse adipose biology to uncover conserved mechanisms underlying obesity phenotypes. By contrasting mitochondrial and translational programs with inflammatory and catabolic pathways, it provides mechanistic insight into metabolic resilience and a foundation for precision approaches to obesity management.

systems biology↗

bioRxiv2026

Anchored for action: a dual role for integrin β1 in mast cell perivascular positioning and vasoactivity to license leukocyte recruitment

Mast cells (MCs) are tissue-resident sentinels of the innate immune system that play pivotal roles in host defense and inflammation. Perivascular MCs exert a particularly strong influence on the onset and dynamics of inflammation through the rapid, directional release of proinflammatory mediators into the circulation. Yet, the mechanisms governing their attachment to the vessel wall - a prerequisite for intravascular degranulation - remain poorly defined. Using a conditional knockout of integrin {beta}1 (Itgb1) in MCs, we investigated how perivascular positioning, degranulation, and vasoactive function contribute to inflammatory responses. In vivo imaging revealed that Itgb1 is essential for positioning MCs within the perivascular niche, particularly around arterioles. The absence of Itgb1 markedly reduced directional MC degranulation into blood vessels during skin inflammation. In vitro, Itgb1-deficient MCs displayed impaired degranulation kinetics together with altered SHIP1/PI3K-AKT signaling and calcium influx upon P2X7 ligation by ATP. During contact hypersensitivity, mice lacking Itgb1 in MCs exhibited strongly diminished ear swelling and reduced recruitment of multiple leukocyte subsets. Mechanistically, disordered MC positioning and attenuated degranulation impaired endothelial activation, resulting in decreased leukocyte adhesion and extravasation. These findings uncover a dual role for Itgb1 in regulating MC responsiveness and pro-inflammatory vasoactive function, establishing Itgb1-mediated perivascular MC positioning as a key prerequisite for effective leukocyte recruitment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/723399v1_ufig1.gif" ALT="Figure 1"> View larger version (80K): org.highwire.dtl.DTLVardef@2268d8org.highwire.dtl.DTLVardef@1114842org.highwire.dtl.DTLVardef@19efc2borg.highwire.dtl.DTLVardef@bd90cf_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2026

Mapping the MIF-2 Chemokine Interactome Reveals MIF-2-CCL20 Complex Formation in Liver Fibrosis

D-dopachrome tautomerase (D-DT/MIF-2) is an inflammatory cytokine, atypical chemokine (ACK) and member of the macrophage migration-inhibitory factor (MIF) family. While interactions among classical chemokines (CKs) are established, ACK-CK interactions remain underexplored. Here, we screened for MIF-2 binding-partners using a protein array encompassing all CKs and selected ACKs, and validated candidate interactors by surface-plasmon resonance. CCL20/MIP-3 was prioritized based on RNA-sequencing suggesting induction during liver fibrosis. MIF-2/CCL20 complex formation was verified by microscale thermophoresis and interaction interfaces mapped using peptide array and in-silico modeling. The deduced binding-site near the MIF-2 tautomerase pocket was consistent with inhibition of its tautomerase activity by CCL20. We found both proteins abundantly expressed in human liver tissue, with a positive correlation. Pull-down confirmed complex formation and proximity ligation assay demonstrated MIF-2/CCL20 complexes in liver in situ, with higher levels in fibrotic tissue. Functionally, MIF-2/CCL20 complexes suppressed MIF-2-driven CD4 T-cell chemotaxis and fibroblast IL-6 secretion, indicating modulation of immune and stromal responses. This study extends the ACK interactome to MIF-2 and suggests ACK/CK complexes modulate chemokine activities in liver fibrosis.

immunology↗

bioRxiv2026

Kinetics of local C3 production orchestrates neutrophil recruitment in lung injury

Complement component 3 (C3) is crucial for host defense against bacteria. While the liver is the primary source of circulating C3, local C3 production at barrier surfaces such as the lung is key in early responses. Yet, how local complement-mediated responses are initiated at mucosal barriers is unknown. This study investigates the kinetics and necessity of lung-derived C3 during the initial hours of an infection. Using models of bacterial pneumonia in ex vivo-perfused human lungs and mice deficient in liver-derived C3, we demonstrate that intrapulmonary C3 production and activation precedes the accumulation of circulating C3 into the bronchoalveolar space. Utilizing mice deficient in lung-derived C3, we demonstrate that epithelial cell-derived C3 is required for early neutrophil recruitment in pneumonia. Transcriptomic and proteomic analyses reveal that neutrophil chemotactic pathways such as C5a and CXCL2 depend on lung epithelial cell-derived C3. These findings demonstrate how lung epithelial-derived C3 influences early mucosal responses to infection via both canonical (direct) and non-canonical (indirect) pathways. SUMMARYAlburquerque et al show an initial, entirely local phase of complement-mediated mucosal protection before a subsequent, systemic response occurs in the setting of barrier disruption. Their work suggests that complement component C3 derived locally at a barrier from the epithelium influences early responses to infection by recruiting neutrophils via multiple pathways independent of circulating C3.

immunology↗

bioRxiv2025

Animal-Origin-Free Method for Generating Blood Vessel Organoids

BackgroundBlood vessel organoids (BVOs) represent a promising tool for modeling vascular diseases, drug screening, and regenerative therapies. However, current protocols for BVO generation are complex, labor-intensive, and reliant on animal-derived extracellular matrices (ECM) such as Matrigel, limiting reproducibility, scalability, and clinical applicability. MethodsWe developed a simplified, animal-origin-free protocol for BVO generation that addresses current limitations and enables high-throughput automated workflows. The method employs ultra-low attachment 96-well U-bottom plates for standardized aggregation and differentiation of human induced pluripotent stem cells (hiPSCs) in a human derived collagen-based extracellular matrix. Unlike conventional protocols where aggregates are embedded in a two-layer ECM, our approach utilizes a single-layer, which we termed "sitting drop". This innovative approach requires considerably fewer materials and handling steps and is compatible with high-throughput automated machines. ResultsBVO generation utilizing the here described optimized protocol resulted in the formation of BVOs with reproducible morphology and cellular composition. Flow cytometry confirmed the presence of CD31 endothelial cells and PDGFR{beta} pericytes in BVOs, generated in sitting drops, with cell population percentages comparable to those observed in traditional two-layer BVO cultures. In vivo transplantation of mature BVOs in a mouse full-thickness skin wound model demonstrated successful integration of BVO derived cells into host vessels, highlighting their potential in cell-based therapies. ConclusionOur study presents a robust and animal-origin-free method for BVO generation based on single-layer "sitting drop" cultures. This protocol maintains cellular integrity while enhancing reproducibility and automation-readiness, paving the way for high-throughput screening and clinical translation of vascular organoid technology. HighlightsO_LIEntire blood vessel organoid (BVO) workflow performed in a single ultra-low attachment-96 plate C_LIO_LIFully animal-origin-free: no Matrigel or Geltrex required throughout the protocol C_LIO_LIRobust generation of BVOs using human collagen-based ECM C_LIO_LIHigh-throughput compatible and automation-ready "sitting drop" culture system C_LIO_LIIntegration of BVO-derived cells into host vessels in vivo C_LI

bioengineering↗

bioRxiv2025

New genomic resources for an invasive mite pest help guide the development of KASP markers for pesticide resistance screening

The redlegged earth mite, Halotydeus destructor, is an invasive pest of grain and pasture crops in Australia. Australian populations are increasingly developing resistance to broad-spectrum pesticides. Here, we assembled a chromosome-level genome for H. destructor, comprising nine chromosomes and totalling approximately 46 Mb. We also generated a genomic dataset from 190 populations collected across thousands of kilometres of its invasive Australian range and across four years (2018, 2021, 2022, and 2023), and used this dataset to develop KASP (Kompetitive allele-specific PCR) markers for identifying resistance mutations. We combined pooled whole-genome sequencing with resistance testing for organophosphate pesticides, chlorpyrifos and omethoate, and the pyrethroid pesticide bifenthrin. Target-site resistance to chlorpyrifos was strongly associated with the common ace G119S mutation. A second mutation, ace A201S, was also common, although its association with chlorpyrifos or omethoate resistance was less clear. Target-site resistance to bifenthrin was strongly associated with the common para L1024F mutation. We also detected several rare target-site mutations that likely contribute to resistance in specific populations, including F331Y in ace, and M918T, M918V, L925I, F1020S, and F1538I in para. Copy number variation in ace showed mixed associations with resistance, potentially reflecting the co-occurrence of variable gene copies and target-site mutations segregating within and among populations. Finally, we observed putative compositional and structural differences in genomic regions containing carboxylesterases with high ace-like homology, a group of genes that might be involved in organophosphate metabolism. This study advances the genomic and molecular resources available for H. destructor, and represents the most comprehensive investigation to date of resistance mechanisms and their spatiotemporal distribution in this invasive pest. Our data showed that pesticide resistance in H. destructor likely involves many genes. Nevertheless, target-site mutations are likely to remain key markers for rapid resistance diagnostics, for which our KASP markers will provide valuable tools.

genomics↗

bioRxiv2025

A data-driven model of macrophage polarization states reveals an IFN macrophage signature in active Crohn's disease

Macrophages are key innate immune cells responsible for initiating and coordinating immune responses. A major determinant of macrophage function is their tissue-context-dependent polarization state, regulated by the cytokines present in the tissue microenvironment. Yet, the capability of characterizing macrophage polarization states in clinical studies remains limited. Here, we used a defined set of cytokines to polarize human PBMC derived macrophages and determine their transcriptional signatures and stimulus responsiveness. The resultant atlas of transcriptional signatures for human macrophage polarization states was applied to a dataset of intestinal biopsies from Crohns disease patients and healthy controls. Our analysis identified a dominant population of IFN{gamma}-polarized macrophages in areas of active Crohns intestinal inflammation and a loss of wound healing IL-4-, IL-10- and IL-13-polarized macrophages. This study demonstrates that in vitro datasets of macrophages in defined conditions can be leveraged to interpret the functionality of cells transcriptional profiled in clinical studies.

immunology↗

bioRxiv2025

SARS-CoV-2 nucleocapsid induces hyperinflammation and vascular leakage through the Toll-like receptor signaling axis in macrophages

Tens of thousands of severe COVID-19 cases are hospitalized weekly in the U.S., often driven by an imbalance between antiviral responses and inflammatory signaling, leading to uncontrolled cytokine secretion. The SARS-CoV-2 nucleocapsid (N) protein is a known immune antagonist, but its role in macrophage-driven cytokine storms is unclear. We demonstrate that N functions in a pathway-specific manner, specifically amplifying nuclear factor {kappa}B-related transcripts upon Toll-like receptor 7/8 stimulation. Moreover, we show that this is a conserved feature of pathogenic coronaviruses, with the delta variant N being the most pro-inflammatory. Our interaction networks suggest the delta variant N drives inflammation through interactions with several stress granule-related proteins. Profiling of secreted cytokines revealed that supernatants from the delta variant N-expressing macrophages disrupt brain and heart endothelial barriers, implicating N in COVID-19-associated cognitive and cardiac complications. Our findings highlight N-mediated immune imbalance as a driver of severe COVID-19 and identify N as a promising therapeutic target to mitigate hyperinflammation.

microbiology↗

bioRxiv2025

Diet and temperature interactively impact brown adipose tissue gene regulation controlled by DNA methylation

Controlling brown adipose tissue (BAT) plasticity in adulthood holds promising potential for effective new obesity therapies by targeting the mechanisms of adaptive thermogenesis. Recent studies have shown that BAT development and function are under epigenetic control, with DNA methylation linked to the regulation of key thermogenic and metabolic genes. Here we sought to understand how diet and cold exposure interactively shape BAT gene regulation controlled by DNA methylation. Mice (N = 8 per group) were housed under cold exposure (8{degrees}C) or thermoneutrality (30{degrees}C) and fed either chow or high-fat diet (HFD). BAT was isolated for transcriptome (RNAseq) and methylome (RRBS) analyses. We identified differentially methylated and expressed genes (DMEGs) by comparing the effects of cold exposure under chow and high-fat diet, as well as by analyzing the interaction between temperature and diet. Functional pathway enrichment and EpiFactors Database screening were used to assess epigenetic regulators, and candidate gene expression was validated by modulating DNA-methylation in vitro. We identified [~]1,360 differentially expressed genes (DEGs) uniquely affected by the diet-temperature interaction with most downregulated in HFD-fed mice, indicating that obesity limits the transcriptional response of BAT to cold. 65 DMEGs (4% of DEGs) were largely diet-specific in response to cold exposure, suggesting that DNA methylation contributes to a selective layer of gene regulation during BAT adaptation to distinct metabolic states. In HFD-fed mice, DMEGs were enriched in pathways related to mitochondrial dysfunction, altered lipid metabolism, neuroendocrine signaling, and compensatory stress responses, contrasting the adaptive thermogenic profile observed in chow-fed controls. Differentially expressed genes of epigenetic regulators such as Tet2, Dnmt3a and Apobec1 showed diet- and cold-dependent regulation, indicating impaired methylation flexibility under obesogenic conditions. Using cell culture experiments, we confirmed the regulation of gene expression of candidate genes, validating the functional link between DNA methylation and thermogenic gene regulation. This is the first study to demonstrate an epigenetic response to cold exposure that differs by dietary condition, particularly in obesity. Our findings highlight a coordinated transcriptional and epigenetic remodeling of BAT, shaped by both environmental and metabolic signals. These insights may inform targeted epigenetic or nutritional strategies to restore BAT function and would further strengthen our understanding of how these might be used as therapeutic basis to improve metabolic health in obesity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/672110v2_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1f99b80org.highwire.dtl.DTLVardef@502d02org.highwire.dtl.DTLVardef@1bb0a93org.highwire.dtl.DTLVardef@b34e4c_HPS_FORMAT_FIGEXP M_FIG C_FIG Mice were housed under cold exposure or thermoneutrality and fed either chow or high-fat diet (HFD). RNA sequencing (RNAseq) and reduced representation bisulfite sequencing (RRBS) was performed on brown adipose tissue (BAT) which provide the fundament for the identification of differentially methylated positions (DMP) and regions (DMR) as well as differentially expressed genes (DEG) in three models: COLDchow, comparing 8{degrees} vs 30{degrees}C mice on chow diet; COLDHFD, comparing 8{degrees} vs 30{degrees}C mice on HFD diet and{Delta} COLDdiet, comparing COLDchow vs COLDHFD. Differentially methylated and expressed genes (DMEGs; circos plot shows a representative example) were identified for all comparisons based on those DMPs which correlated significantly with differentially expressed genes (DEGs; the heatmap shows a representative example). DMEGs were taken forward for pathways enrichment analyses and to identify differentially regulated epigenetic regulators using the EpiFactors Database. The figure was created with BioRender.

genomics↗

bioRxiv2025

Phased epigenetic fragility and stability accelerate the genetic evolution of B-cells

Non-genetic heterogeneity can provide population robustness when responding to threats or making developmental decisions, but when the biological process rests on specific individuals (tissue-resident macrophages or genetic evolution), non-genetic heterogeneity degrades performance. Vaccine responses depend on the Darwinian evolution of B-cells to generate high-affinity, genetically-encoded antibodies, yet B-cell decision-making is non-genetically variable. In fact, B-cell epigenetic states are fragile during selection but stable during the proliferation burst. Here we report that the iterative dynamics of epigenetic fragility and stability of B-cell fate decisions do not impair but accelerate affinity maturation, by modulating the generation and removal of high-affinity outliers on a fitness landscape that is non-monotonic defined not only by survival/proliferation but plasma cell differentiation. These insights reconcile classical B-cell clonal selection theory with experimentally observed dynamics of epigenetic variability. The resulting model correctly predicts emergent vaccine response properties in alternate mouse strains and may contribute to personalized vaccination strategies. HIGHLIGHTSO_LIB-cell epigenetic states are fragile during antigen selection but stable within proliferative bursts C_LIO_LIEpigenetic state fragility allows high affinity cells to escape plasma cell differentiation C_LIO_LIEpigenetic state stability within proliferative bursts maximizes progeny of fittest cells C_LIO_LIPhased epigenetic dynamics of B-cells predicts emergent vaccine response properties C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/661408v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1c2af9forg.highwire.dtl.DTLVardef@980003org.highwire.dtl.DTLVardef@128d8c0org.highwire.dtl.DTLVardef@ae48b8_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2025

IFNγ-induced memory in human macrophages is not sustained by epigenetic changes but the durability of the cytokine itself

Macrophages, as key sentinel cells of the innate immune system, can retain memory of prior stimulus exposure. Interferon gamma (IFN{gamma}) plays a central role in maintaining trained immunity in vivo and can induce potent memory in macrophages. Such memory is associated with the formation of de novo enhancers that alter gene expression responses to subsequent stimuli. However, how such enhancers are maintained after cytokine exposure remains unclear. We report that durable IFN{gamma}-induced enhancers can last for days after cytokine washout, yet the underlying persistence mechanism is not cell-intrinsic. IFN{gamma}-treated macrophages continue to exhibit JAK/STAT signaling days after cytokine removal. Blocking IFN{gamma} signaling with a JAK inhibitor or anti-IFN{gamma} neutralizing antibodies after cytokine removal is sufficient to reverse IFN{gamma}-induced enhancers and erase the potentiated state of the treated macrophages. Our findings suggest that epigenetic changes in macrophages do not inherently encode innate immune memory or a "potentiated" macrophage state, but in fact are themselves dependent on ongoing cytokine signaling. These findings suggest new possibilities for pharmacologic interventions to reverse aberrantly trained immune states associated with pathology.

immunology↗

bioRxiv2025

EGFR activation correlates with intracranial pressure and outcome in a mixed intracranial bleeding porcine model

Intracranial hypertension is a major driver of secondary injury after acute subdural hematoma (ASDH), yet how mechanical stress is translated into neuroinflammatory signaling remains poorly understood. Here, we identify a mechanosensitive astrocyte signaling pathway that links elevated intracranial pressure (ICP) to inflammatory amplification in the injured brain. Using a clinically relevant porcine ASDH model combined with mechanistic studies in human iPSC-derived astrocytes, we demonstrate that sustained ICP elevation induces bilateral neuroinflammation together with coordinated upregulation of mechanosensitive ion channels and receptor tyrosine kinase (RTK) pathways. Integrative analysis of molecular and physiological datasets identified astrocytes as the principal cellular responders to ICP and revealed epidermal growth factor receptor (EGFR) as the astrocyte-associated RTK most strongly correlated with ICP dynamics, inflammatory chemokine expression, and survival. Pharmacological activation of the mechanosensitive channel Piezo1 in human astrocytes was sufficient to trigger EGFR internalization, site-specific phosphorylation, and ERK signaling, promoting structural remodeling and robust induction of pro-inflammatory mediators including CCL2, IL-6, and IL-8. Conversely, EGFR inhibition attenuated inflammatory signaling while enhancing astrocytic programs associated with water handling and edema containment. In vivo, increased expression of EGFR ligands together with elevated EGFR phosphorylation supported sustained pathway engagement following ASDH, and correlation analyses linked Piezo1 expression and EGFR activation with ICP severity and adverse outcome. Together, these findings define a mechanotransduction axis in which astrocytic Piezo1 signaling integrates mechanical stress with EGFR-dependent neuroimmune responses, positioning EGFR as a translationally accessible target to modulate inflammation-driven secondary brain injury.

neuroscience↗

bioRxiv2025

A computational workflow for assessing drug effects on temporal signaling dynamics reveals robustness in stimulus-specific NFκB signaling

Single-cell studies of signal transduction have revealed complex temporal dynamics that determine downstream biological function. For example, the stimulus-specific dynamics of the transcription factor NF{kappa}B specify stimulus-specific gene expression programs, and loss of specificity leads to disease. Thus, it is intriguing to consider drugs that may restore signaling specificity in disease contexts, or reduce activity but maintain signaling specificity to avoid unwanted side effects. However, while steady-state dose-response relationships have been the focus of pharmacological studies, there are no established methods for quantifying drug impact on stimulus-response signaling dynamics. Here we evaluated how drug treatments affect the stimulus-specificity of NF{kappa}B activation dynamics and its ability to accurately code ligand identity and dose. Specifically, we simulated the dynamic NF{kappa}B trajectories in response to 15 stimuli representing various immune threats under treatment of 10 representative drugs across 20 dosage levels. To quantify the effects on coding capacity, we introduced a Stimulus Response Specificity (SRS) score and a stimulus confusion score. We constructed stimulus confusion maps by employing epsilon network clustering in the trajectory space and in various dimensionally reduced spaces: canonical polyadic decomposition (CPD), functional principal component analysis (fPCA), and NF{kappa}B signaling codons (i.e. established, informative dynamic features). Our results indicated that the SRS score and the stimulus confusion map based on signaling codons are best-suited to quantify stimulus-specific NF{kappa}B dynamics confusion under pharmacological perturbations. Using these tools we found that temporal coding capacity of the NF{kappa}B signaling network is generally robust to a variety of pharmacological perturbations, thereby enabling the targeting of stimulus-specific dynamics without causing broad side-effects.

pharmacology and toxicology↗

bioRxiv2025

Macrophages maintain signaling fidelity in response to ligand mixtures

As immune sentinel cells, macrophages are required to respond specifically to diverse immune threats and initiate appropriate immune responses. This stimulus-response specificity (SRS) is in part encoded in the signaling dynamics of the NF{kappa}B transcription factor. While experimental stimulus-response studies have typically focused on single defined ligands, in physiological contexts cells are exposed to multi-ligand mixtures. It remains unclear how macrophages combine multi-ligand information and whether they are able to maintain SRS in such complex exposure conditions. Here, we leveraged an established mathematical model that captures the heterogeneous single-cell NF{kappa}B responses of macrophage populations to extend experimental studies with systematic simulations of complex mixtures containing up to five ligands. Live-cell microscopy experiments for some conditions validated model predictions but revealed a discrepancy when TLR3 and TLR9 are stimulated. Refining the model suggested that the observed but unexpected ligand antagonism arises from a limited capacity for endosomal transport which is required for responses to CpG and pIC. With the updated model, we systematically analyzed SRS across all combinatorial-ligand conditions and employed three ways of quantifying SRS involving trajectory decomposition into informative trajectory features or machine learning. Our findings show that macrophages most effectively distinguish single-ligand stimuli, and distinguishability declines as more ligands are combined. However, even in complex combinatorial conditions, macrophages still maintain statistically significant distinguishability. These results indicate a robustness of innate immune response specificity: even in the context of complex exposure conditions, the NF{kappa}B temporal signaling code of macrophages can still classify immune threats to direct an appropriate response. Significance ([≤]120)Macrophages sense diverse pathogens within complex environments and respond appropriately. Experimental studies have found that the NF{kappa}B pathway responds with stimulus-specific dynamics when macrophages are exposed to single ligand stimuli. However, it remains unclear complex contexts might erode this stimulus-specificity. Here we systematically studies NF{kappa}B responses using a mathematical model that provides simulations of the heterogeneous population of single cell responses. We show that although the model is parameterized to single ligand data it can predict the responses to multi-ligand mixtures. Indeed, model validation uncovered signaling antagonism between two ligands and the underlying mechanism. Importantly, we found that NF{kappa}B signaling dynamics distinguish ligands within multi-ligand mixtures indicating a robustness of the NF{kappa}B temporal code that was not previously appreciated.

systems biology↗

bioRxiv2025

Temporal dose inversion properties of adaptive biomolecular circuits

Cells have the capacity to encode and decode information in the temporal features of molecular signals. Many pathways, for example, generate either sustained or pulsatile responses depending on the context, and such diverse temporal behaviors have a profound impact on cell fate. Here we focus on how molecular pathways can convert the temporal features of dynamic signals, in particular how they can convert transient signals into persistent downstream events and vice versa. We refer to this type of behavior as temporal dose inversion, and we demonstrate that it can be achieved through adaptive molecular circuits. Using a cell-free synthetic gene circuit implementing an incoherent feedforward loop (IFFL), we experimentally demonstrate the temporal dose inversion. To understand the design principles of the temporal dose inversion, we analyze adaptive circuit motifs including incoherent feedforward loops (IFFLs) and negative feedback loops (NFLs). Through numerical simulations with expensive parameter exploration, we identify parametric regimes in which these circuits exhibit temporal dose inversion. We further examine more detailed biological models of the IFFL and NFL circuits, including enzymatic signaling models and gene regulatory network models, showing that the IFFL circuits is more likely to exhibit temporal dose inversion compared with the NFL circuits. Finally, we analyze a generalized IFFL topology, and we find that both the time delay in the inhibition pathway and the relative signal intensities of the activation and inhibition signals are key determinants for temporal dose inversion. Together, our results establish a design principle of temporal dose inversion on the adaptive biomolecular circuits and provide mechanistic insight into how molecular networks process temporal information in dynamic signals.

systems biology↗