a-multimodal-assay-to-infer-body-temperature-set-point-shifts-freely

A Multimodal Assay to Infer Body Temperature Set-Point Shifts in Freely Moving Mice

Homeothermic animals maintain a stable core body temperature (Tc) at a set-point around 37 &#176;C. Thermoregulatory homeostasis maintains Tc around the ...

Chinese Academy of Medical Sciences & Peking Union Medical College

Chinese Institute for Brain Research (CIBR)

Beijing Key Laboratory of Brain Science and Brain-Machine Interface

Hepatic fibrosis is characterized by the activation of hepatic stellate cells (HSCs). Migration of the activated HSCs to the site of injury is one of the key characteristics during the wound healing process. We have previously demonstrated that 14 kDa phosphohistidine phosphatase (PHP14) is involved in migration and lamellipodia formation of HSCs. However, the role of PHP14 in liver fibrosis remains unknown. In this study, we first assessed PHP14 expression and distribution in liver fibrotic tissues using western blot, immunohistochemistry, and double immunofluorescence staining. Next, we investigated the role of PHP14 in liver fibrosis and, more specifically, the migration of HSCs by Transwell assay and 3D collagen matrices assay. Finally, we explored the possible molecular mechanisms of the effects of PHP14 on these processes. Our results show that the PHP14 expression is up-regulated in fibrotic liver and mainly in HSCs. Importantly, TGF-β1 can induce PHP14 expression in HSCs accompanied with the activation of HSCs. Consistent with the previous study, PHP14 promotes HSCs migration, especially, promotes 3D floating collagen matrices contraction but inhibits stressed-released matrices contraction. Mechanistically, the PI3Kγ/AKT/Rac1 pathway is involved in migration regulated by PHP14. Moreover, PHP14 specifically mediates the TGF-β1 signaling to PI3Kγ/AKT pathway and regulates HSC migration, and thus participates in liver fibrosis. Our study identified the role of PHP14 in liver fibrosis, particularly HSC migration, and suggested a novel mediator of transducting TGF-β1 signaling to PI3Kγ/AKT/Rac1 pathway.

PHP14 regulates hepatic stellate cells migration in liver fibrosis via mediating TGF-β1 signaling to PI3Kγ/AKT/Rac1 pathway.

The actin cytoskeleton of hepatic stellate cells (HSCs) is reorganized when they are cultured in 3D collagen matrices. Here, we investigated the molecular mechanism of actin cytoskeleton reorganization in HSCs cultured in 3D floating collagen matrices (FCM) compared to those on 2D polystyrene surfaces (PS). First, we found that the generation of dendritic cellular processes was controlled by Rac1. Next, we examined the differential gene expression of HSCs cultured on 2D PS and in 3D FCM by RNA-Seq and focused on the changes of actin cytoskeleton reorganization-related molecular components and guanine nucleotide exchange factors (GEFs). The results showed that the expression of genes associated with actin cytoskeleton reorganization-related cellular components, filopodia and lamellipodia, were significantly decreased, but podosome-related genes was significantly increased in 3D FCM. Furthermore, we found that a Rac1-specific GEF, ARHGEF4, played roles in morphological changes, migration and podosome-related gene expression in HSCs cultured in 3D FCM. Abbreviations: 2D PS: 2-dimensional polystyrene surface; 3D FCM: 3-dimensional floating collagen matrices; ARHGEF4: Rho guanine nucleotide exchange factor 4; ARHGEF6: Rho guanine nucleotide exchange factor 6; GEF: guanine nucleotide exchange factor; HSC: hepatic stellate cell.

ARHGEF4-mediates the actin cytoskeleton reorganization of hepatic stellate cells in 3-dimensional collagen matrices.

To investigate CD4+CD25+FoxP3+ T regulatory cells (Tregs) in the peripheral blood of patients with atopic dermatitis (AD) and its correlation with disease severity.

Correlation of Blood FoxP3+ Regulatory T Cells and Disease Activity of Atopic Dermatitis.

Krüppel-like factor 5 (KLF5) can both promote and suppress cell migration, but the underlying mechanisms have not been elucidated. In this study, we show that the function of KLF5 in epithelial-mesenchymal transition (EMT) and migration of liver cancer cells depends on the status of the cellular tumor antigen p53 (p53). Furthermore, zinc finger E-box-binding homeobox 2 (ZEB2) is the main regulator of KLF5 in EMT in liver cancer cells in the context of p53 loss. Most importantly, the regulation of ZEB2 by p53 and KLF5 is indirect and that miR-192 mediates this regulation. Finally, we find that in invasive liver cancer, KLF5 is absent in the context of p53 loss or mutation.

KLF5 regulates epithelial-mesenchymal transition of liver cancer cells in the context of p53 loss through miR-192 targeting of ZEB2.

Liver fibrosis, a major cause of morbidity and mortality worldwide, leads to liver damage, seriously threatening human health. In our previous study, we demonstrated that 14 kDa phosphohistidine phosphatase (PHP14) was upregulated in fibrotic liver tissue and involved in the migration and lamellipodia formation of hepatic stellate cells (HSCs). In this study, we evaluated PHP14 as a therapeutic target for liver fibrosis and investigated the mechanism by which it mediates liver fibrosis. AAV-sh administration significantly attenuates CCl-induced liver fibrosis in mice. In particular, fibrosis-associated inflammatory infiltration was significantly suppressed after PHP14 knockdown. Mechanistically, PHP14 regulated macrophage recruitment, infiltration, and migration by affecting podosome formation of macrophages. Inhibition of PHP14 decreased the expression of the fibrogenic signature at the early stage of liver fibrogenesis and the activation of HSCs in vivo. Thus, PHP14 can be considered a potential therapeutic target for liver fibrosis. PHP14 inhibition via adeno-associated virus (AAV)-mediated gene silencing could potently attenuate carbon tetrachloride (CCl)-induced liver fibrosis. PHP14 could regulate the migration of macrophages to the site of injury in vivo. PHP14 knockdown in vivo influenced the environment of fibrogenesis and relevant signaling pathways, subsequently affecting myofibroblast activation.

14-kDa phosphohistidine phosphatase is a potential therapeutic target for liver fibrosis.

Adipose-derived stem cells (ADSCs) are able to modulate the immune response and are used for treating ulcerative colitis (UC). However, it is possible that ADSCs from patients with inflammatory or autoimmune disorders may show defective immunosuppression. We investigated the use of ADSCs from UC patients for autologous cell treatment, specifically, ADSCs from healthy donors (H-ADSCs) and UC patients (P-ADSCs) in terms of various functions, including differentiation, proliferation, secretion, and immunosuppression. The efficacy of P-ADSCs for treating UC was examined in mouse models of acute or chronic colitis. Both H-ADSCs and P-ADSCs were similar in cell morphology, size, adipogenic differentiation capabilities, and cell surface markers. We found that P-ADSCs had lower proliferative capacity, cloning ability, and osteogenic and chondrogenic differentiation potential than H-ADSCs. P-ADSCs exhibited a diminished capacity to inhibit peripheral blood mononuclear cell proliferation, suppress CD25 and CD69 marker expression, decrease the production of inflammation-associated cytokines interferon-γ and tumor necrosis factor-α, and reduce their cytotoxic effect on A549 cells. When primed with inflammatory cytokines, P-ADSCs secreted lower levels of prostaglandin E, indoleamine 2, 3-dioxygenase, and tumor necrosis factor-α-induced protein 6, which mediated their reduced immunopotency. Moreover, P-ADSCs exhibited weaker therapeutic effects than H-ADSCs, determined by disease activity, histology, myeloperoxidase activity, and body weight. These findings indicate that the immunosuppressive properties of ASCs are affected by donor metabolic characteristics. This study shows, for the first time, the presence of defective ADSC immunosuppression in UC, indicating that autologous transplantation of ADSCs may be inappropriate for patients with UC.

Adipose-Derived Stem Cells From Patients With Ulcerative Colitis Exhibit Impaired Immunosuppressive Function.

To analyze the predictive value of the Th17/Treg ratio for renal injury in sepsis patients, a prospective observational study was conducted. Adult patients with sepsis were enrolled and divided into a sepsis-induced acute kidney injury (SAKI) group and a sepsis-without-AKI group. Logistic regression was used to analyze the independent predictors of SAKI, and the ROC curve was plotted to evaluate the predictive value of the Th17/Treg ratio for renal injury in patients with sepsis. A total of 124 patients were enrolled in this study, including 60 cases (48.39%) of SAKI. Patients who developed sepsis-induced acute kidney injury had a higher Th17/Treg ratio level compared to patients without it (0.11 [0.07, 0.28] versus 0.06 [0.05, 0.16], p < 0.05, respectively. The area under the receiver operating characteristic curve of the Th17/Treg ratio to predict sepsis-induced acute kidney injury was 0.669 (95% CI 0.574−0.763, p < 0.05). The Th17/Treg ratio was associated with SAKI (OR 1.15, 95%CI [1.06−1.24], p < 0.05, non-adjusted and R 1.12, 95%CI [1.00−1.25], p < 0.05, adjusted). The use of the Th17/Treg ratio improved the prediction performance of the prediction model of NAGL. The median Th17/Treg ratio significantly increased with the stratified KDIGO stage (p < 0.05). Th17/Treg imbalance was associated with occurrence of acute kidney injury and AKI severity in patients with sepsis. The Th17/Treg ratio could be a potential predictive marker of sepsis-induced acute kidney injury.

Th17/Regulatory T-Cell Imbalance and Acute Kidney Injury in Patients with Sepsis.

Ferroptosis is a recently identified iron-dependent form of nonapoptotic cell death characterized by reactive oxygen species (ROS) generation and lipid peroxidation. Here, we report a novel iron-dependent form of ferroptosis induced by labile iron and investigate the mechanism underlying this process. We find that labile iron-induced ferroptosis is distinct from canonical ferroptosis and is linked to the mitochondrial pathway. Specifically, the mitochondrial calcium uniporter mediates the ferroptosis induced by labile iron. Interestingly, cells undergoing labile iron-induced ferroptosis exhibit cytoplasmic features of oncosis and nuclear features of apoptosis. Furthermore, labile iron-induced ferroptosis involves a unique set of genes. Finally, labile iron-induced ferroptosis was observed in liver subjected to acute iron overload in vivo. Our study reveals a novel form of ferroptosis that may be implicated in diseases caused by acute injury.

An iron-dependent form of non-canonical ferroptosis induced by labile iron.

Microglia and astrocytes are key players in neuroinflammation and ischemic stroke. A1 astrocytes are a subtype of astrocytes that are extremely neurotoxic and quickly kill neurons. Although the detrimental A1 astrocytes are present in many neurodegenerative diseases and are considered to accelerate neurodegeneration, their role in the pathophysiology of ischemic stroke is poorly understood. Here, we combined RNA-seq, molecular and immunological techniques, and behavioral tests to investigate the role of A1 astrocytes in the pathophysiology of ischemic stroke. We found that astrocyte phenotypes change from a beneficial A2 type in the acute phase to a detrimental A1 type in the chronic phase following ischemic stroke. The activated microglial IL1α, TNF, and C1q prompt commitment of A1 astrocytes. Inhibition of A1 astrocytes induction attenuates reactive gliosis and ameliorates morphological and functional defects following ischemic stroke. The crosstalk between astrocytic C3 and microglial C3aR contributes to the formation of A1 astrocytes and morphological and functional defects. In addition, NF-κB is activated following ischemic stroke and governs the formation of A1 astrocytes via direct targeting of inflammatory cytokines and chemokines. Taken together, we discovered that A2 astrocytes and A1 astrocytes are enriched in the acute and chronic phases of ischemic stroke respectively, and that the C3/C3aR/NF-κB signaling leads to A1 astrocytes induction. Therefore, the C3/C3aR/NF-κB signaling is a novel therapeutic target for ischemic stroke treatment.

Transcriptome Analysis Reveals Dynamic Microglial-Induced A1 Astrocyte Reactivity via C3/C3aR/NF-κB Signaling After Ischemic Stroke.

Stroke is a major global cause of death and disability, with ischemic stroke being the most common type. The disruption of the blood-brain barrier (BBB) is a key factor in the pathophysiology of ischemic stroke, allowing immune cells to infiltrate and worsening neuroinflammation. This study uses single-cell RNA sequencing (scRNA-seq) to examine the transcriptional changes in neutrophils, endothelial cells, and T cells during ischemic stroke. Our findings indicate a significant increase in neutrophil and lymphocyte infiltration, along with a notable decrease in endothelial cell populations, demonstrating severe BBB disruption. Differential gene expression analysis shows that endothelial cells lose important characteristics post-stroke, while lymphocytes activate cytotoxic pathways that may lead to neuronal damage. Additionally, we reveal the contrasting roles of CXCR2 and CXCR4 in neutrophil movement and identify neutrophil-derived damage-associated molecular patterns (DAMPs) and matrix metalloproteinases (MMPs) as key drivers of endothelial cell apoptosis. Notably, the S100A8/A9 inhibitor paquinimod significantly protects neurons and reduces lymphocyte infiltration, suggesting that targeting S100A8/A9 could be a promising therapeutic strategy for reducing neurological deficits after ischemic stroke. Overall, these results enhance our understanding of the complex interactions between immune cells and the BBB in ischemic stroke, paving the way for innovative therapeutic approaches aimed at maintaining brain integrity and improving patient outcomes.

Single-cell RNA sequencing reveals S100A8/A9 neutrophils-induced endothelial cell death and lymphocyte infiltration after ischemic stroke.

Stroke is a worldwide leading cause of death and long-term disability, with ischemic strokes making up approximately 85% of all cases. There is a significant need for an ideal animal model that accurately replicates the disease's pathology to study the molecular mechanisms of brain injury. Various experimental models have been created to induce middle cerebral artery occlusion (MCAO), including intraluminal MCAO, photothrombotic models, endothelin-1 injections, and electrocoagulation. However, these often result in large infarct or lesion volumes accompanied by considerable variability. In this study, we present a ministroke model that specifically targets the mouse barrel cortex, making it suitable for investigating the mechanisms of minor strokes and stroke recurrence. In our model, the distal branch of the right middle cerebral artery (MCA), which supplies the sensorimotor cortex, is permanently ligated using 10-0 sutures. This is followed by a 7-min occlusion of the bilateral common carotid arteries (CCAs) and subsequent reperfusion. This approach produces a mild stroke characterized by small and consistent lesion volumes and very low mortality rates. A well-trained experimenter can achieve nearly zero mortality with this technique. Furthermore, this model of localized ischemia induces lesions in the functionally defined barrel cortex, allowing the use of the vibrissae-evoked forelimb placing test to assess functional outcomes. Key features • Introduces a novel ministroke model targeting the mouse barrel cortex, specifically designed for studying minor strokes. • Achieves consistently small infarct sizes and low mortality rates, allowing for long-term assessments of functional outcomes. • Requires specialized surgical skills for the permanent ligation of the right middle cerebral artery and temporary occlusion of common carotid arteries. • The adhesive removal test and the vibrissae-evoked forelimb placing test exhibit remarkable sensitivity in this model.

Article	Year
PHP14 regulates hepatic stellate cells migration in liver fibrosis via mediating TGF-β1 signaling to PI3Kγ/AKT/Rac1 pathway. Journal of molecular medicine (Berlin, Germany)\| PubMed ID: 29098317	2018
ARHGEF4-mediates the actin cytoskeleton reorganization of hepatic stellate cells in 3-dimensional collagen matrices. Cell adhesion & migration\| PubMed ID: 30871422	2019
Correlation of Blood FoxP3+ Regulatory T Cells and Disease Activity of Atopic Dermatitis. Journal of immunology research\| PubMed ID: 31637264	2019
KLF5 regulates epithelial-mesenchymal transition of liver cancer cells in the context of p53 loss through miR-192 targeting of ZEB2. Cell adhesion & migration\| PubMed ID: 32965165	2020
14-kDa phosphohistidine phosphatase is a potential therapeutic target for liver fibrosis. American journal of physiology. Gastrointestinal and liver physiology\| PubMed ID: 33406007	2021
Adipose-Derived Stem Cells From Patients With Ulcerative Colitis Exhibit Impaired Immunosuppressive Function. Frontiers in cell and developmental biology\| PubMed ID: 35252190	2022
Th17/Regulatory T-Cell Imbalance and Acute Kidney Injury in Patients with Sepsis. Journal of clinical medicine\| PubMed ID: 35887790	2022
An iron-dependent form of non-canonical ferroptosis induced by labile iron. Science China. Life sciences\| PubMed ID: 36515861	2023
Transcriptome Analysis Reveals Dynamic Microglial-Induced A1 Astrocyte Reactivity via C3/C3aR/NF-κB Signaling After Ischemic Stroke. Molecular neurobiology\| PubMed ID: 38713438	2024
Single-cell RNA sequencing reveals S100A8/A9 neutrophils-induced endothelial cell death and lymphocyte infiltration after ischemic stroke. Biochemical and biophysical research communications\| PubMed ID: 39579533	2024
Developing a Ministroke Model in Mouse Barrel Cortex. Bio-protocol\| PubMed ID: 40395851	2025

Jingyi Yao

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