Biology subjects

Dropmann, A.

Publications and source records attributed to Dropmann, A..

3 recordsLinked to original sources

bioRxiv2025

Immunoglobulin G binding as a quantitative marker of hepatocellular death across acute and chronic liver injury

Background & AimsAccurate detection of hepatocellular death is fundamental for understanding liver injury, intoxication, regeneration, and fibrosis. Conventional markers, such as serum transaminases and histopathological scoring, suffer from limited temporal resolution, high variability, and observer dependence. We evaluated immunoglobulin G (IgG) binding as a quantitative and spatially resolved marker of hepatocyte death in acute and chronic liver injury models. MethodsMale C57BL/6 mice were subjected to acute carbon tetrachloride (CCl) intoxication (1600 mg/kg, single dose), dose-escalation (0-800 mg/kg), and chronic injury paradigms including Western diet (WD), WD+CCl, and Mdr2-/- mice with or without a single CCl challenge. The serum ALT and AST levels were measured. Liver sections were stained with IgG, Hematoxylin and Eosin (H&E), bromodeoxyuridine (BrdU), glutamine synthetase (GS), CD26, and alpha-smooth muscle actin (Acta2). Spatial and integrative transcriptomic analyses were performed to characterize the IgG hepatocyte dead regions. ResultsHepatocellular IgG labeling emerged as early as 6h post-CCl, peaked at 72-96h, and declined during regeneration. IgG-positive areas correlated strongly with Ishak necroinflammatory score (r=0.70) and serum transaminase levels (p=0.74-0.85), surpassing both in Receiver Operating Characteristic (ROC) analyses (AUC=0.92-0.95). IgG bound to both apoptotic (TUNEL) and necrotic (TUNEL-) hepatocytes. In chronic liver injury models, IgG deposition was localized to the injury zones and correlated with ALT/AST, irrespective of etiology. Multiplex imaging revealed IgG-positive necrotic cores surrounded by proliferating hepatocytes and Acta2+ myofibroblasts. Spatial transcriptomics identified immune cell enrichment, Fc{gamma}R-mediated signaling, phagocytosis, and vascular remodeling within the IgG-marked regions. ConclusionsIgG immunostaining provides a robust, quantitative, and pathologist-independent readout of hepatocellular death, which scales with injury severity, delineates necrotic zones, and reveals immune-active microenvironments. These findings establish IgG-based detection as a versatile, high-resolution tool for assessing liver injury, regeneration, and fibrosis. Conflict of Interest declarationThe authors declare that they have no affiliations with or involvement in any organization or entity with any financial interest in the subject matter or materials discussed in this manuscript. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/690787v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1d9b05eorg.highwire.dtl.DTLVardef@ca2ad8org.highwire.dtl.DTLVardef@c2d384org.highwire.dtl.DTLVardef@cca46d_HPS_FORMAT_FIGEXP M_FIG C_FIG Liver injury induced by toxins, dietary stress, or genetic knockout provokes chemokine release (i.e. CXCL1, CXCL2, and CCL3) and recruitment of immune cells, including T cells, NK cells (NKs), and dendritic cells (DCs). Activated immune cells secrete IgG, which binds to damaged hepatocytes, leading to IgG deposition and opsonization. Opsonized hepatocytes expose "eat me" signals, promoting their phagocytic clearance and contributing to the resolution of liver injury and restoration of hepatic homeostasis.

cell biology↗

bioRxiv2025

CD271 sorting for improved liver cell isolation: Semiautomated and simultaneous preparation of parenchymal and non-parenchymal cells from mouse and human livers

BackgroundA detailed understanding of the dynamic fate changes of hepatocytes, hepatic stellate cells (HSC), Kupffer cells (KC), and liver sinusoidal endothelial cells (LSEC) is critical for studying liver (patho)physiology during disease progression. Current isolation methods often focus on single cell types, limiting utility in comprehensive research. AimTo develop a novel, semi-automated protocol for the simultaneous isolation of hepatocytes and non-parenchymal cells (NPCs), including HSC, KC, and LSEC, from mouse and human, with high yield, purity, and viability from healthy and diseased livers. MethodThe protocol employs a two-step EGTA and collagenase II perfusion for tissue digestion. Hepatocytes were isolated by low-speed centrifugation and a Percoll gradient. Subsequently, magnetic-activated cell separation, using CD271 as a selective surface marker for HSC, CD11b for KC and CD146 for LSEC) was performed. Validation was achieved with immunofluorescence staining, flow cytometry, RT-PCR, and UV fluorescence, whereby yield, purity, and viability were assessed. ResultsWith our method, yield of hepatocytes, HSC, KC, and LSEC, is 33.4{+/-}5.5x10, 5.2{+/-}6.3x10, 12.4{+/-}4.8x10 and 18.2{+/-}8.9x10 cells per healthy mouse liver, respectively, with cell viabilities exceeding 89%, and purity surpassing 90%. CD271 was validated as an effective marker for purifying HSC in healthy and diseased human (n=4-6) and mouse livers. Compared to microfluidic and organ-on-a-chip approaches, with our protocol, we achieve higher yield and purity values while enabling the simultaneous isolation of multiple cell types from a single sample. ConclusionOur semi-automated protocol offers a scalable, reliable, and versatile solution for isolating main liver cell types with high yield, purity, and viability from both healthy and diseased tissues, advancing liver research and facilitating downstream investigations. Impact and implicationsO_LIBroad applicability: The CD271-based method efficiently isolates key liver cell types (Hepatocyte, HSC, KC, LSEC) simultaneously. C_LIO_LIVersatility in disease models: Effective for studying healthy, fibrotic, and damaged liver tissues. C_LIO_LIRobust across variability: Works reliably across different mouse strains, age groups, and conditions. C_LIO_LIHuman research potential: Scalable for high-purity isolation from human liver tissue, enabling translational studies. C_LIO_LIHigh-quality results: Ensures >85% viability and >90% purity, supporting reproducible liver research applications. C_LI

cell biology↗

bioRxiv2023

TGF-β1 inhibits cholesterol metabolism in hepatocytes to facilitate cell death, EMT and signals for HSC activation.

Background and Aims: Transforming growth factor-{beta}1 (TGF-{beta}1) plays important roles in chronic liver diseases, including metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD involves various biological processes including dysfunctional cholesterol metabolism and contributes to progression to metabolic dysfunction-associated steatohepatitis (MASH) and hepatocellular carcinoma (HCC). However, the reciprocal regulation of TGF-{beta}1 signaling and cholesterol metabolism in MASLD is yet unknown. Methods: Changes in transcription of genes associated with cholesterol metabolism were assessed by RNA-Seq of murine hepatocyte cell line (AML12) and mouse primary hepatocytes (MPH) treated with TGF-{beta}1. Functional assays were performed on AML12 cells (untreated, TGF-{beta}1 treated, or subjected to cholesterol enrichment (CE) or depletion (CD)), and on mice injected with adeno-associated virus 8 (AAV8)-Control/TGF-{beta}1. Results: TGF-{beta}1 inhibited mRNA expression of several cholesterol metabolism regulatory genes, including rate-limiting enzymes of cholesterol biosynthesis in AML12 cells, MPHs, and AAV8-TGF-{beta}1-treated mice. Total cholesterol levels and lipid droplet accumulation in AML12 cells and liver tissue were also reduced upon TGF-{beta}1 treatment. Smad2/3 phosphorylation following 2 h TGF-{beta}1 treatment persisted after CE or CD and was mildly increased following CD, while TGF-{beta}1-mediated AKT phosphorylation (30 min) was inhibited by CE. Furthermore, CE protected AML12 cells from several effects mediated by 72 h incubation with TGF-{beta}1, including EMT, actin polymerization, and apoptosis. CD mimicked the outcome of long term TGF- {beta}1 administration, an effect that was blocked by an inhibitor of the type I TGF-{beta} receptor. Additionally, the supernatant of CE- or CD-treated AML12 cells inhibited or promoted, respectively, the activation of LX-2 hepatic stellate cells. Conclusions: TGF-{beta}1 inhibits cholesterol metabolism while cholesterol attenuates TGF-{beta}1 downstream effects in hepatocytes.

cell biology↗