Cell Infiltration Research Articles

Enhanced cancer cell proliferation and aggressive phenotype counterbalance in breast cancer with high BRCA1 gene expression.

While comprehensive research exists on the mutation of the DNA repair gene BRCA1, limited information is available regarding the clinical significance of BRCA1 gene expression. Given that cancer cell proliferation is aggrevated by DNA repair, we hypothesized that high BRCA1 gene expression breast cancer (BC) might be linked with aggressive tumor biology and poor clinical outcomes. The cohorts: The Cancer Genome Atlas (TCGA, n = 1069), METABRIC (n = 1903), and SCAN-B (n = 3273) were utilzed to obtain data of 6245 BC patients. BC patients without BRCA1 mutation exhibited higher BRCA1 expression, which was associated with DNA repair functionality. However, no such correlation was observed with BRCA2 expression. The association of high BRCA1 expression with cancer cell proliferation was evidenced by significant enrichment of cell proliferation-related gene sets, higher histological grade, and proliferation score. Furthermore, increased levels of homologous recombination deficiency, intratumoral heterogeneity, and altered fractions were associated with high BRCA1 expression. Moreover, BC with high BRCA1 expression exhibited reduced infiltration of dendritic cells and CD8 T-cells, while showing increased infiltration of Th1 cells. Surprisingly, BRCA1 expression was not associated with the survival of BC irrespective of the subtypes. Conversely, BC with low BRCA1 expression enriched cancer aggravating pathway gene sets, such as Cancer Stem Cell-related signaling (NOTCH and HEDGEHOG), Angiogenesis, Epithelial-Mesenchymal Transition, Inflammatory Response, and TGF-beta signaling. Despite being linked to heightened proliferation of cancer cells and unassertive phenotype, BRCA1 expression did not show any association with survival in BC.

A Novel Gene Signature Based on Immune Cell Infiltration Landscape Predicts Prognosis in Lung Adenocarcinoma Patients.

The tumor microenvironment (TME) is created by the tumor and dominated by tumor-induced interactions. Long-term survival of lung adenocarcinoma (LUAD) patients is strongly influenced by immune cell infiltration in TME. The current article intends to construct a gene signature from LUAD ICI for predicting patient outcomes. For the initial phase of the study, the TCGA-LUAD dataset was chosen as the training group for dataset selection. We found two datasets named GSE72094 and GSE68465 in the Gene Expression Omnibus (GEO) database for model validation. Unsupervised clustering was performed on the training cohort patients using the ICI profiles. We employed Kaplan-Meier estimators and univariate Cox proportional-hazard models to identify prognostic differentially expressed genes in immune cell infiltration (ICI) clusters. These prognostic genes are then used to develop a LASSO Cox model that generates a prognostic gene signature. Validation was performed using Kaplan-Meier estimation, Cox, and ROC analysis. Our signature and vital immune-relevant signatures were analyzed. Finally, we performed gene set enrichment analysis (GSEA) and immune infiltration analysis on our finding gene signature to further examine the functional mechanisms and immune cellular interactions. Our study found a sixteen-gene signature (EREG, HPGDS, TSPAN32, ACSM5, SFTPD, SCN7A, CCR2, S100P, KLK12, MS4A1, INHA, HOXB9, CYP4B1, SPOCK1, STAP1, and ACAP1) to be prognostic based on data from the training cohort. This prognostic signature was certified by Kaplan-Meier, Cox proportional-hazards, and ROC curves. 11/15 immune-relevant signatures were related to our signature. The GSEA results indicated our gene signature strongly correlates with immune-related pathways. Based on the immune infiltration analysis findings, it can be deduced that a significant portion of the prognostic significance of the signature can be attributed to resting mast cells. We used bioinformatics to determine a new, robust sixteen-gene signature. We also found that this signature's prognostic ability was closely related to the resting mast cell infiltration of LUAD patients.

Subcutaneously transplanted xenogeneic protein recruits treg cells and M2 macrophages to induce browning of inguinal white adipose tissue.

To study whether subcutaneously embedding xenogeneic protein threads or synthetic polymer absorbable threads can improve obesity phenotypes and metabolic conditions, and to further explore its underlying mechanism. Thirty-six 8-week-old ob/ob mice were randomly allocated to three groups, respectively, receiving catgut embedding, PGA thread embedding or sham treatment bilaterally to the groin. Individual parameters including weight, food intake, and core temperature are recorded and metabolism assessment, energy expenditure analysis, and PET/CT scanning are also performed at fixed timepoints. After surgical incision, the inguinal white adipose tissue was histologically examined and its expression profile was tested and compared among groups 4 weeks and 12 weeks after operation. Catgut embedding reduced weight gain and improved metabolic status in ob/ob mice. Browning of bilateral inguinal WAT (white adipose tissue) was induced after catgut embedding, with massive infiltration of Treg cells and M2 macrophages in the tissue slices of fat pads. IL-10 and TGF-β released by Treg cells targeted macrophages and the induced M2 macrophages increased the expression of thermogenic and anti-inflammatory genes in fat. The secretion of catecholamines by polarized M2 macrophages led to the activation of β3-AR-related pathways in adipocytes and the browning of adipose tissue. Abdominal subcutaneous catgut embedding has the potential to combat obesity through the induction of WAT browning mediated by infiltrated Treg cells and macrophages.

Evaluation of the Safety and Efficacy of Curcumin-Synthesized Silver Nanoparticles in Rats Exposed to Chlorpyrifos During Puberty Development.

Silver nanoparticles (Ag-NPs) have garnered significant attention in recent years due to their therapeutic effects. Curcumin (CUR) has been utilized as a coating agent for synthesizing Ag-NPs, intended to act as a potential drug. This study was designed to evaluate the safety and efficacy of curcuminsynthesized silver nanoparticles on rats exposed to chlorpyrifos (CPF) during their pubertal development. Forty-two male Wistar rats, 23 days old, were selected and randomly divided into 7 groups (n=6) as follows: positive control, negative control, CPF (5 mg/kg), silver nanoparticles synthesized using curcumin at 40 μg/kg (CUR-Ag-NPs 40), CUR-Ag-NPs 80, CPF+ CUR-Ag-NPs 40, CPF+ CUR-AgNPs 80. All treatments were administered via gavage for 30 days. At the end of the study, rats were anesthetized using ketamine (50 mg/kg), and xylazine, (10 mg/kg) and blood was collected from the heart for serum analysis of liver enzymes, urea, and creatinine. Liver and kidney tissues were isolated for histopathological analysis. No significant differences were observed in serum levels of AST, ALT, and ALP enzymes as well as urea and creatinine levels among the different groups. Light microscopy observation revealed multifocal inflammatory mononuclear cell subsets in liver tissue associated with mild inflammatory mononuclear cell infiltration in the portal region in CPF, CUR-Ag-NPs 40, CUR-Ag-NPs 80, CPF+CUR-Ag-NPs 40, and CPF+CUR-Ag- NPs 80 groups. Histological examination of kidney tissue showed degenerative changes in the tubular epithelium, congestion, and mild infiltration of mononuclear inflammatory cells in the renal interstitial tissue in the CPF group, CUR-Ag-NPs 40, CUR-Ag-NPs 80, CPF+CUR-Ag-NPs 40 and CPF+CUR-Ag-NPs 80 groups. This study failed to establish the safety and efficacy of CUR-Ag-NP at 40 and 80 μg/kg in prepubertal rats exposed to CPF. However, further studies should be conducted to thoroughly characterize the efficacy of CUR-Ag-NP in developmental animal models.

Berberine attenuates ECM accumulation and the progression of acute liver failure through inhibition of NLRP3 inflammasome signalling

Acute liver failure (ALF) is a life-threatening disease, characterized by upregulated extracellular matrix deposition and inflammatory signalling, with no effective treatment options and targets. The present study was designed to investigate the preventive and therapeutic effects of berberine (BBR) and its underlying mechanism in thioacetamide (TAA)-induced ALF. Male SD rats were administered with TAA 300 mg/kg, i.p., thrice to induce ALF and pre- or post-treated with BBR. To decipher the effects of BBR LFT markers, histopathological analysis of key fibrotic and inflammatory proteins was performed. In addition, the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were assessed by ELISA. Our work showed TAA-induced ALF animals were associated with increased ALT, AST, bilirubin (LFT markers) and histopathological alterations with profuse infiltration of inflammatory cells in the liver tissue. Treatment with BBR has significantly inhibited LFT markers and histological alterations triggered by TAA. In addition, TAA animals demonstrated increased collagen accumulation and upregulated expression of TGF-β1, vimentin, and α-SMA compared to control. The excessive accumulation of collagen, TGF-β1, vimentin, and α-SMA were significantly modulated with BBR treatment. Further, the fluorescence intensity of ROS an activator of NLRP3 including the NLRP3 inflammasome, and its downstream signalling ASC, cleaved IL-1β, and other pro-inflammatory cytokines like TNF-α and IL-6 stimulated by TAA were attenuated by BBR treatment. The current work indicated that BBR significantly ameliorated TAA-induced ALF by inhibiting the extracellular matrix accumulation associated with the NLRP3/IL-1β signalling pathway and could be a viable therapeutic option to treat ALF and other fibroinflammatory diseases.

Establishment of human acute monocytic leukemia model with systemic infiltration in NPG mice.

A model construction of systemic acute leukemia is challenging. Herein, we established a systemic leukemia mouse model using highly immunodeficient NPG mice without any immunosuppressive treatments. NPG mice received tail intravenous injection of SHI-1 cells at the concentration of 1×107 cells (group A) or 5×107 cells (group B) and randomly sacrificed each seven days post-inoculation. Tumor development was monitored using nested-PCR, peripheral blood-smear analysis, flow cytometry, pathological examinations, and immunohistochemistry. The median survival of mice in groups A and B were 33.0 and 30.0 days, respectively. Blast cells in peripheral blood appeared on day 14 in group B, and on day 21 in group A. In addition, SHI-1 cell specific MLL-AF6 mRNA was detected in both spleen and bone marrow on day 14 post-inoculation. 21 days after inoculation, we observed human CD45+CD33+ cells with an SH-1-immunophenotype in the peripheral blood, spleen, and bone marrow, as well as solid neoplasms in multiple organs. Moreover, the histologically infiltrated leukemic cells expressed CD45. In conclusion, the current study demonstrated the normal growth of SHI-1 cells in the NPG mice without immunosuppression, which caused systemic leukemia similar to that observed in acute leukemia patients. We developed an efficient and reproducible model to study leukemia pathogenesis and progression.

NCAPD3 is a prognostic biomarker and is correlated with immune infiltrates in glioma.

Non-SMC Condensin II Complex Subunit D3 (NCAPD3) has been linked with the genesis and progression of multiple human cancers. Nevertheless, the scientific value and molecular process of NCAPD3 in glioma remain unclear. We explored the level of NCAPD3 expression in pan-cancer by multiple online databases. And we focused on the level and prognostic value of NCAPD3 expression in glioma by immunohistochemistry (IHC) and survival analysis. Meanwhile, we verified the relationship between NCAPD3, biological function and immune infiltration in glioma by Linkedomics and SangerBox databases. The expression of NCAPD3 was increased in a variety of cancers, including glioma. Its high expression was strongly related to WHO grade (P=0.002) and programmed cell death ligand 1 (PD-L1) expression of glioma (P=0.001). Patients with a high level of NCAPD3 expression had a lower overall survival (OS) in glioma than patients with a low level of NCAPD3 expression. Multivariate statistical analyses showed NCAPD3 expression (P=0.040), WHO grade (P<0.001), 1p/19q codeletion (P<0.001), recurrence (P<0.001), age (P=0.023), and chemotherapy status (P=0.001) were meaningful independent prognostic factors in patients with glioma. Furthermore, bioinformatics analysis proved that NCAPD3 has been linked to immune infiltration in glioma. High level of NCAPD3 expression may serve as a promising prognostic biomarker and correlate with dendritic cell infiltration, representing a potential immunotherapy target in glioma.

Co-mutation of TP53 and TTN is Correlated with the Efficacy of Immunotherapy in Lung Squamous Cell Carcinoma.

Immunotherapy has been a promising treatment in advanced lung cancer. However, only a few patients could benefit from it. Herein, we aimed to explore mutationrelated predictive biomarkers in lung squamous cell carcinoma (LUSC), which could help develop clinical immunotherapy strategies and screen beneficial populations. Co-occurrence and mutually exclusive analysis was conducted on the TCGA-LUSC cohort. Correlations between the gene mutation status and tumor mutation burden (TMB) levels, and neo-antigen levels were analyzed by Wilcoxon test. Kaplan-Meier method was employed to analyze the progression-free survival (PFS) of lung cancer patients with immunotherapy. Gene set enrichment analysis (GSEA) was used to investigate the functional changes affected by TP53mut/TTNmut. The immune cell infiltration landscape in co-mutation subgroups was analyzed using CIBERSORT. 1) TP53, TTN, CSMD3, MUC16, RYR2, LRP1B, USH2A, SYNE1, ZFHX4, FAM135B, KMT2D, and NAV3 were frequently mutated in LUSC patients. 2) TMB levels in highly mutated groups were higher than that in wild type groups. 3) There were higher neoantigen levels in mutation group compared to the wild-type group, and LUSC patients in mutation group had longer PFS. 4) TP53mut/TTNmut co-mutation group exhibited higher TMB levels and better response to immunotherapy. 5) A host of immune-related signaling pathways was inhibited in TP53mut/TTNmut subgroup. 6) There were more T follicular helper cells and NK cells were in TP53mut/TTNmut subgroup than in the WT subgroup. The LUSC patients with TP53 and TTN co-mutation had higher TMB levels and better response to immunotherapy. The TP53 and TTN co-mutation is a promising novel biomarker to assist LUSC immunotherapy evaluation.

Plastic bronchitis associated with human bocavirus 1 infection in children.

Plastic bronchitis (PB) is a clinical-pathological syndrome characterized by the abnormal accumulation of endogenous substances in the bronchial airways, causing partial or complete obstruction and resulting in impaired lung ventilation. In this retrospective analysis, we aim to summarize the clinical manifestations, imaging characteristics, diagnostic methods, and treatment approaches to enhance clinicians' ability to detect children who are infected withhuman bocavirus 1(hBoV 1) and develop PB. In the period from January 2021 to January 2024, a total of six hBoV 1 infection children were diagnosed with PB through bronchoscopy. The onset of the condition was mainly concentrated between June and December. The detection methods used included metagenomic next-generation sequencing for pathogen identification (three cases) and respiratory pathogen nucleic acid 13-plex detection (oropharyngeal swab) (three cases), both of which confirmed the presence of hBoV 1. Out of the six children with PB, two were girls and four were boys. Their ages ranged from 10 months to 4 years old. Common symptoms reported by all patients included fever, cough,and wheezing. Chest high-resolution computed tomography scans revealed atelectasis in six cases, in addition to pneumonia. After the removal of the plastic bronchi via bronchoscopy, the airway obstruction symptoms in the children were relieved, and no recurrence was observed during the follow-up period. Pathological findings indicated cellulose exudation and inflammatory cell infiltration, consistent with nonlymphatic PB. When children infected with hBoV 1 exhibit persistent or worsening symptoms such as cough, fever, and wheezing despite treatment, clinicians should remain highly vigilant for the potential occurrence of PB. Bronchoscopy plays a crucial role not only in diagnosing the presence of a plastic bronchus but also in effectively treating PB.

SIRT5 participates in the suppressive tumor immune microenvironment of EGFR-mutant LUAD by regulating the succinylation of ACAT1

Epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma (LUAD) exhibits a poor response to immune checkpoint inhibitors (ICIs) by shaping a suppressive tumor immune microenvironment (TIME), which characters as lacking immune cell infiltration; however, the underlying mechanism remains to be elucidated. Here, we demonstrated that Sirtuin 5 (SIRT5), a member of the deacetylase SIRT family, functions as a desuccinylase of acetyl-CoA acetyltransferase 1 (ACAT1) and enhances the enzymatic activity of ACAT1 to activate the NRF2 pathway, inhibiting the secretion of the chemokines CCL5 and CXCL10, which are important for recruiting CD8+ T cells, thereby participating in the formation of an inhibitory TIME in EGFR-mutant LUAD. In conclusion, we propose that the combination of a SIRT5 inhibitor with ICIs therapy may be a promising therapeutic approach for patients with EGFR-mutant LUAD.

TRPV1 alleviates APOE4-dependent microglial antigen presentation and T cell infiltration in Alzheimer's disease

BackgroundPersistent innate and adaptive immune responses in the brain contribute to the progression of Alzheimer’s disease (AD). APOE4, the most important genetic risk factor for sporadic AD, encodes apolipoprotein E4, which by itself is a potent modulator of immune response. However, little is known about the immune hub that governs the crosstalk between the nervous and the adaptive immune systems. Transient receptor potential vanilloid type 1 (TRPV1) channel is a ligand-gated, nonselective cation channel with Ca2+ permeability, which has been proposed as a neuroprotective target in AD.MethodsUsing Ca2+-sensitive dyes, dynamic changes of Ca2+ in microglia were measured, including exogenous Ca2+ uptake and endoplasmic reticulum Ca2+ release. The mRFP-GFP-tagged LC3 plasmid was expressed in microglia to characterize the role of TRPV1 in the autophagic flux. Transcriptomic analyses and flow cytometry were performed to investigate the effects of APOE4 on brain microglia and T cells from APOE-targeted replacement mice with microglia-specific TRPV1 gene deficiency.ResultsBoth APOE4 microglia derived from induced pluripotent stem cells of AD patients and APOE4-related tauopathy mouse model showed significantly increased cholesterol biosynthesis and accumulation compared to their APOE3 counterparts. Further, cholesterol dysregulation was associated with persistent activation of microglia and elevation of major histocompatibility complex II-dependent antigen presentation in microglia, subsequently accompanied by T cell infiltration. In addition, TRPV1-mediated transient Ca2+ influx mitigated cholesterol biosynthesis in microglia by suppressing the transcriptional activation of sterol regulatory element-binding protein 2, promoted autophagic activity and reduced lysosomal cholesterol accumulation, which were sufficient to resolve excessive immune response and neurodegeneration in APOE4-related tauopathy mouse model. Moreover, microglia-specific deficiency of TRPV1 gene accelerated glial inflammation, T cell response and associated neurodegeneration in an APOE4-related tauopathy mouse model.ConclusionsThe findings provide new perspectives for the treatment of APOE4-dependent neurodegeneration including AD.

Novel TBC1D8B variant causes neonatal nephrotic syndrome combined with acute kidney injury

BackgroundNephrotic syndrome (NPHS), characterized by proteinuria, hypoalbuminemia, and edema, can be caused by genetic variations. TBC1D8B was recently discovered as a novel disease-causing gene for X-linked NPHS. With only a few reported cases, the clinical manifestations associated with variants of this gene need to be further examined.MethodsWe recruited a newborn with NPHS complicated by acute kidney injury (AKI) and his parents and tested the potential genetic cause of the disease through trio-whole exome sequencing and Sanger sequencing. Western blotting (WB) was performed using a mutant plasmid to evaluate mutant protein expression levels. Since the TBC1D8B protein interacts with RAB proteins to catalyze the GTPase hydrolysis process, immunofluorescence (IF) can be used to verify the interaction between the TBC1D8B mutant protein and RAB11A/RAB11B, and thus to confirm its effect on the endocytosis and vesicle recycling functions of RAB proteins within the cell.ResultsThe child, at 1 month, showed severe edema and proteinuria and unexplained coma with epilepsy. Ultrasound examination revealed multiple organ enlargement, and MRI showed nonspecific high diffusion-weighted imaging signal characteristics in the splenium of the corpus callosum. Hematoxylin and eosin staining showed diffuse inflammatory cell infiltration in the renal interstitium and multifocal renal tubule lumen expansion. Diffuse fusion of podocyte foot processes was observed under electron microscopy, indicating glomerular podocyte lesions. Genetic testing revealed a maternally inherited novel hemizygous variant, NM_017752: c.628 A > T, p.Lys210Ter, in TBC1D8B. In vitro functional experiments showed that this variant may lead to TBC1D8B protein degradation. IF results showed disrupted interaction with RAB11A/RAB11B, that then affects the biological function of RAB proteins in the process of cell intimal vesicle formation and intracellular transport.ConclusionThis study will enrich the mutational and phenotypic spectra of TBC1D8B and demonstrate the potential of this gene variants to cause early-onset NPHS leading to severe kidney disease.

Disrupting Intracellular Homeostasis by Copper-Based Nanoinducer with Multiple Enzyme-Mimicking Activities to Induce Disulfidptosis-Enhanced Pyroptosis for Tumor Immunotherapy.

Given the crucial role of abnormal homeostasis in tumor cells for maintaining their growth, it may be more efficient with less effort to develop anti-tumor strategies that target multiple combined mechanisms by disrupting intracellular homeostasis. Here, a copper-based nanoinducer (CGBH NNs) with multiple enzyme-like activities is designed and constructed to induce disulfidptosis-enhanced pyroptosis through disrupting multiple intracellular homeostasis for effective tumor immunotherapy. Within the tumor microenvironment (TME), CGBH NNs can disrupt intracellular glucose homeostasis and inhibit NADPH production, leading to accumulation of cystine, which further blocked the substrate and key enzyme for synthesizing glutathione. Subsequently, through cascade catalytic reactions involving enzyme activities (glutathione peroxidase-like, glucose oxidase and peroxidase-like activities), CGBH NNs can produce massive reactive oxygen species(ROS) and further disrupt intracellular redox homeostasis, resulting in the disulfidptosis-enhanced pyroptosis. The tumor cells undergoing immunogenic pyroptosis can release various cytosolic contents and inflammatory factors, eliciting robust immune responses by facilitating immune cell infiltration, and reprogramming the immunosuppressive TME. After the combination with immune checkpoint blockade therapy, CGBH NNs can effectively suppress the tumor growth and prolong the survival time of tumor-bearing mice. This work presents a novel paradigm to trigger disulfidptosis-enhanced pyroptosis by destroying intracellular homeostasis for anti-tumor immunotherapy.

Etomidate Inhibits Hepatic Ischemia-Reperfusion Injury Depending on the Activation of Nrf2-HO-1 Signaling Pathway.

Hepatic ischemia-reperfusion (I/R) injury (HIRI) is recognized as a local aseptic inflammatory response driven by innate immunity and is considered a leading cause of early organ dysfunction and failure following liver transplantation. Etomidate (Eto), an anesthetic drug known for its ability to inhibit inflammatory response and apoptosis, was the focus of our investigation. In this study, we conducted hepatic I/R surgery invivo on C57 mice, analyzing liver damage through histopathology. Additionally, primary hepatocytes isolated from mice were cultured and subjected to hypoxia/reoxygenation (H/R) insult in vitro, with cell activity assessed using the CCK8 assay and immunofluorescence staining employed to analyze liver inflammatory cell infiltration and apoptosis. Results showed that Eto effectively inhibited liver injury, inflammatory response, and apoptosis induced by HIRI surgery, with the greatest effect observed at an Eto concentration of 10 mg/kg. Furthermore, Eto also showed the ability to inhibit H/R-induced cell damage, inflammatory activation, and apoptosis in primary hepatocytes. Further mechanistic studies revealed that Eto could promote the activation of the Nrf2-HO-1 signaling pathway, and the protective effect of Eto on HIRI was nullified when the Nrf2 inhibitor ML385 was utilized. This study highlights the potential of Eto to protect against HIRI by promoting the Nrf2-HO-1 signaling axis.

Role of inflammation and macrophages in the formation of the atrial cardiomyopathy associated with obesity

Abstract Introduction The atrial fibrillation (AF), the most frequent cardiac arrhythmia in clinical practice is often associated with an atrial cardiomyopathy (ACM) which is favoured by several clinical conditions including metabolic diseases such as obesity. Recently, the epicardial adipose tissue (EAT) has emerged as an important component of ACM notably in the context of metabolic diseases. Epicardial progenitor cells (EPC) are the main source of EAT. The replacement of EAT by fibrosis is arrhythmogenic and involved inflammation but not yet characterized. The hypothesis tested here is that macrophages are central players for both the recruitment of EPC and replacement of EAT by fibrosis. Purpose-Aims First to characterize subpopulation of macrophages involves in the progression of ACM. Second, to study how the inflammatory environment regulates EPC differentiation. Method A spatial gene expression was used to analyze human samples (n=4) (Agreement, CODECOH, Declaration or authorization of activities involving the preservation and preparation of human body parts for scientific purposes DC-2020-4183). A well-characterized mouse model of atrial cardiomyopathy associated with obesity induced by high fat diet (HFD) was used to characterize macrophages associated with progression of ACM by using immunolabeling together with single nuclei RNA sequencing approaches. ACM was characterized by using echocardiography and atrial burst pacing to evaluate determine AF vulnerability. Transgenic RGB-MAC mice with macrophages tagged with McApple-Csf1R and ECGFP-CX3CR1 or with a knock-out for CCR2 were used to follow infiltration of myeloid cells in the atrial at the different stages of the ACM. Bi-photon microscopy was used to visualize cell subpopulations in the distinct area of the atrial wall. In some experiments EPC and macrophages were cocultured with transwell. Results Macrophages were detected in subepicardium of human atrial samples in area of fibro-fatty infiltrates. After 2 months of HFD, monocytes were infiltrated atria and macrophages were overexpressed in atria from 1 to 4 months of HFD. Using Bi-photon microscopy it was possible to visualise resident ECGFP-CX3CR1+ macrophages at the contact of EAT whereas McApple-Csfr1r+ macrophages were at the junction with fibrosis (figure 1). Preliminary data generated by single nuclei RNA sequencing showed clusters of different subpopulations of macrophages at different time point of the ACM (figure 2). We will present results on the effects of suppression of the CCR2 subpopulation of macrophages on characteristics of the ACM. Conclusion Distinct populations of macrophages i.e resident and monocyte-derived macrophages, participate to the distinct stages of the ACM caused by obesity in mice.Myeloid cells infiltration in epicardiumCCR2+cells infiltration in EAT-fibrosis

Unraveling immune checkpoint inhibitors effects on atherosclerosis: mechanisms and preventive approaches

Abstract Background Immune checkpoint inhibitors (ICIs) represent a novel and rapidly evolving field in cancer treatment. Despite increasing reports of accelerated atherosclerosis and ICI-related atherosclerotic cardiovascular (CV) events in clinical studies and case reports, there is a notable lack of data concerning the underlying mechanisms, appropriate monitoring, and potential interventions. Objectives To explore the role of ICIs in triggering and/or accelerating the atherosclerotic process, with a specific focus on the involvement of CD8+ T-cells. Additionally, we aimed to evaluate and compare the potential beneficial effects of statins and Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) inhibitors on atherosclerosis under ICIs treatment. Methods Thirty 8-week-old C57BL mice were divided into six groups as follows: (1) Control, normal diet; (2) Control, atherogenic diet; (3) Atherogenic diet + anti PD-1; (4) Atherogenic diet + statins + anti-PD1; (5) Atherogenic diet + PCSK9-i + anti PD-1; (6) Atherogenic diet + PCSK9 inhibitors. At 16 weeks, the carotid arteries and heart were removed for histological examination, and serum was withdrawn for blood work and further analysis. Results Anti-PD1 treatment resulted in extensive lymphocytic infiltration of the carotid intima, primarily constituted of CD8+ T cells. This infiltration was significantly attenuated when statins or PCSK-9 inhibitors were used in conjunction with anti-PD1 treatment. Serum analysis for CD4+ and CD8+ T cells by ELISA revealed no difference in CD4+ abundance between the groups. However, plasma CD8+ T cells were significantly increased with anti-PD1 treatment, and furthermore, when PCSK9 inhibitors were used in combination; but not when statins were used. Conclusion The induction of CD8+ T cell infiltration in the arterial carotid walls by anti-PD-1 was prevented with the use of either statins or PCSK9 inhibitors. In the plasma, while statins reduced the hyper-expression of CD8+ T cells observed under PD-1 treatment, PCSK9 inhibitors enhanced this expression. This may provide a first indication of the potential benefit of PCSK9 inhibitor treatment for both preventing arterial inflammation and enhancing the desired anti-tumor immune response under ICIs treatment. Figure legend: 1A. A cross-section of the carotid artery after 16 weeks of treatment, stained with H&amp;E. Magnification is X100. 1B. A cross-section of the carotid artery after 16 weeks of treatment, stained with anti-CD8 Ab. Magnification is X100. 1C. quantitative CD8+ T cells staining in the carotid arteries. 1D. quantitative results of ELISA for plasma CD8+ T cells.

Sinomenine hydrochloride alleviates autoimmune myocarditis via suppressing Th1 cell induced-M1 macrophage pyroptosis

Abstract Background Myocarditis is typical of the complex inflammatory response in the heart with high morbidity and mortality, accompanied by tissue damage and cardiac fibrosis. Previous studies have demonstrated that CD4+ T cells are crucial to the cardiac autoimmunity of myocarditis, but the role of different CD4+ T cell subsets remains unclear. Recently, sinomenine hydrochloride, a natural compound from the traditional Chinese herb Sinomenium acutum, is reported to inhibit T cell proliferation in some systemic autoimmune diseases such as rheumatoid arthritis. Given that autoimmune myocarditis is an organ-specific autoimmune disease, the effect of sinomenine hydrochloride on autoimmune myocarditis demands further study. Purpose This study aims to elucidate the role of CD4+ T-helper 1 (Th1) cells in regulating inflammatory cell death in autoimmune myocarditis and investigate the pharmacological effect of sinomenine hydrochloride on CD4+ Th1 cells and autoimmune myocarditis. Methods Male Balb/c mice were immunized with myosin heavy chain-α peptides to establish the experimental autoimmune myocarditis (EAM) model, followed by the treatments with sinomenine hydrochloride by gavage. Tohoku Hospital Pediatrics-1 (THP-1) cell line and murine T cells were used for in vitro experiments. Results In the acute phase of EAM, we observed plenty of CD4+ T cells infiltrating the heart tissue. RNA sequencing revealed that among the genes encoding typical transcription factors of CD4+ T cell subset, only Tbx21 and Stat4, both representing Th1 cells, were upregulated, which consisted with immunofluorescence staining. Further studies indicated that Th1 cells not only mediated classical activated macrophage (M1 macrophage) polarization by secreting interferon-γ (IFN-γ) but also associated with pro-inflammatory macrophage pyroptosis in the EAM, with the cleavage of gasdermin D (GSDMD), caspase-1 and IL-1β. Subsequent in vitro experiments confirmed that IFN-γ could dramatically promote macrophage pyroptosis combined with ATP, leading to the release of IL-1β and IL-18(Figure 1). To find an effective method for Th1 cell suppression and myocarditis remission, we identified that sinomenine hydrochloride impeded Th1 cell activation, differentiation and proliferation in vitro. Furthermore, sinomenine hydrochloride inhibited Th1 cell infiltration and M1 macrophage pyroptosis in the EAM heart, In the chronic phase, cardiac remodeling and fibrosis were suppressed, and cardiac systolic function was significantly improved in the treatment group (Figure 2). Conclusion Our study demonstrates that Th1 cells-induced M1 macrophage pyroptosis is an essential pathogenic mechanism of EAM. Sinomenine hydrochloride can alleviate the severity and progression of EAM, which may become a novel therapeutic strategy for treating myocarditis.Figure 1Figure 2

IL-33 Increases the Magnitude of the Tissue-Resident Memory T Cell Response in Intestinal Tissues during Local Infection.

IL-33 plays an important role in the early programming of CD8 T cells; however, its contribution to the differentiation of tissue-resident memory T cells invivo remains poorly defined. After infection of mice with Yersinia pseudotuberculosis, IL-33 expression was increased in the intestinal tissue, and this coincided with the expression of ST2 on T cells infiltrating the intestinal epithelium and lamina propria. Blocking IL-33 signaling after T cell infiltration of the intestinal tissue did not significantly impact the number or phenotype of tissue-resident memory T cells generated. However, overexpression of ST2 on T cells was able to increase expression of TCF1 and T cell number in the intestine compared with the lymphoid organs during infection. We also observed that enhanced accumulation and maintenance of ST2-overexpressing cells in the intestine postinfection were resolved. This points to a role for IL-33 in increasing the number of T cells that commit to intestinal tissue residency invivo.

Recruitment of CCR5-positive T cells in pulmonary vascular lesions in idiopathic- and connective tissue disease associated- pulmonary arterial hypertension

Abstract Background C-C chemokine receptor type 5 (CCR5) is a chemokine receptor predominantly expressed on leukocytes including T cells, macrophages, and dendritic cells, and is involved in the process by which T cells are attracted to specific tissues and organs. Although CCR5 has been shown to be expressed on macrophages and pulmonary artery smooth muscle cells in lung samples from human idiopathic pulmonary arterial hypertension (PAH) and some animal models, its expression on T cells in human PAH lungs has not been investigated. Purpose To investigate the immunohistochemical expression of CCR5 on T cells infiltrating in pulmonary vascular lesions of human PAH tissues, particularly from patients with idiopathic PAH and PAH associated with connective tissue disease (CTD-PAH), where inflammation has been implicated in the pathogenesis. Methods Subjects included 25 postmortem cases of idiopathic PAH (14 cases, a mean age of 34 ± 10 years, 8 female cases) and CTD-PAH (11 cases, 48 ± 13 years, all female). We performed immunohistochemistry using antibodies against CCR5 and CD3 (for T cells) on formalin-fixed paraffin-embedded sections. To semi-quantify, we selected the three small muscular pulmonary arteries (80-150 µm in external diameter) with the strongest CCR5-positive cell infiltration in each case, counted the number of positive cells, and the average were shown as the number of cells per vessel. Results In all cases, regardless of whether IPAH or CTD-PAH, a subset of infiltrating inflammatory cells into or accumulating around vessels was CCR5-positive. Specifically, CCR5-positive inflammatory cells were found in remodelled pulmonary arteries, such as plexiform lesions and constrictive lesions with intimal thickening and/or medial hypertrophy, as well as perivascular space. The average number of CCR5-positive cells per small muscular pulmonary artery was 8.1/vessel. Where pulmonary venous occlusive lesions were seen, CCR5-positive cells were also present in and around them. The majority of CCR5-positive inflammatory cells appeared to be T cells, based on morphology and CD3 immunopositivity in the serial sections. Conclusion(s) Activated T cells showing CCR5 immunopositivity were recruited to remodelled pulmonary vascular lesions in both idiopathic PAH and CTD-PAH. This implicates CCR5-positive T cells as a common key player and that CCR5 signalling is involved in the pathogenesis of PAH in concert with other inflammatory cells, vascular endothelial cells, and pulmonary artery smooth muscle cells.

A Novel Mouse Model for Cerebral Inflammatory Demyelination in X-Linked Adrenoleukodystrophy: Insights into Pathogenesis and Potential Therapeutic Targets.

X-linked adrenoleukodystrophy (ALD) is caused by mutations in ABCD1, a peroxisomal gene. More than half of males with an ABCD1 mutation develop inflammatory cerebral demyelination (cALD), but underlying mechanisms remain unknown and therapies are limited. We sought to develop and characterize a mouse model of cALD to facilitate study of disease mechanisms and therapy development. We used immunoassays and immunohistochemistry to assess novel (interleukin 18 [IL-18]) and established molecular markers in cerebrospinal fluid (CSF) and postmortem brain tissue from cALD patients. We generated a cALD phenotype in Abcd1-knockout mice using a 2-hit method that combines cuprizone and experimental autoimmune encephalomyelitis models. We then used magnetic resonance imaging (MRI) and immunohistochemistry to assess the fidelity of cALD molecular markers in the mice. Human and mouse cALD lesions shared histologic features of myelin phagocytosis, myelin loss, abundant microglial activation, T and B-cell infiltration, and astrogliosis. Compared to wild-type controls, Abcd1-knockout mice displayed more cerebral demyelination, blood-brain barrier disruption, and perivascular immune cell infiltration. This enhanced inflammatory response was associated with higher levels of fibrin deposition, oxidative stress, demyelination, and axonal injury. IL-18 immunoreactivity co-localized with perivascular monocytes/macrophages in both human and mouse brain tissue. In cALD patients, CSF IL-18 levels correlated with MRI lesion severity. Our results suggest loss of Abcd1 function in mice predisposes to more severe blood-brain barrier disruption, cerebral inflammation driven by the infiltration of peripheral immune cells, demyelination, and axonal damage, replicating human cALD features. This novel mouse model could shed light on cALD mechanisms and accelerate cALD therapy development. ANN NEUROL 2024.