Leukocyte Migration Research Articles

DKK1+ tumor cells inhibited the infiltration of CCL19+ fibroblasts and plasma cells contributing to worse immunotherapy response in hepatocellular carcinoma

Intra-tumor immune infiltration plays a pivotal role in the interaction with tumor cells in hepatocellular carcinoma (HCC). However, its phenotype and related spatial structure remained elusive. To address these limitations, we conducted a comprehensive study combining spatial data (38,191 spots from eight samples) and single-cell data (56,022 cells from 20 samples). Our analysis revealed two distinct infiltration patterns: immune exclusion and immune activation. Plasma cells emerged as the primary cell type within intra-tumor immune clusters. Notably, we observed the co-location of CCL19+ fibroblasts with plasma cells, which secrete chemokines and promote T-cell activation and leukocyte migration. Conversely, in immune-exclusion samples, this co-location was primarily observed in the adjacent normal area. This co-localization correlated with T cell infiltration and the formation of tertiary lymphoid structures, validated by multiplex immunofluorescence conducted on twenty HCC samples. Both CCL19+ fibroblasts and plasma cells were associated with favorable survival outcomes. In an immunotherapy cohort, HCC patients who responded favorably exhibited higher infiltration of CCL19+ fibroblasts and plasma cells. Additionally, we observed the accumulation of DKK1+ tumor cells within the tumor area in immune-exclusion samples, particularly at the tumor boundary, which inhibited the infiltration of CCL19+ fibroblasts and plasma cells into the tumor area. Furthermore, in immune-exclusion samples, the SPP1 signaling pathway demonstrated the highest activity in communication between tumor and immune clusters, and CCL19-CCR7 played a pivotal role in the self-communication of immune clusters. This study elucidates immune exclusion and immune activation patterns in HCC and identifies relevant factors contributing to immune resistance.

Luteolin alleviates cerebral ischemia/reperfusion injury by regulating cell pyroptosis.

This study aimed to clarify the roles and underlying mechanisms of luteolin in the progression of cerebral ischemia/reperfusion injury (CIRI). A mouse model of CIRI was established using the middle cerebral artery occlusion (MCAO) method, after which luteolin was administered. Subsequently, neuronal apoptosis and pyroptosis were measured and the brain tissues of each group were subjected to RNA sequencing. Luteolin alleviated MCAO-induced brain infarction, apoptosis, and pyroptosis. RNA sequencing identified 3,379, 2,777, and 3,933 differentially expressed genes (DEGs) in the MCAO vs sham, MCAO vs MCAO + luteolin, and MCAO + luteolin vs sham groups, respectively. The identified DEGs showed enrichment in multiple processes, including pattern specification, forebrain development, anion transport, leukocyte migration, regulation of cell-cell adhesion, and positive regulation of the response to external stimuli, as well as the calcium, PI3K-AKT, JAK-STAT, NF-kappa B, IL-17, cAMP, cGMP-PKG, and Wnt signaling pathways. In addition, Ccl2 and Angpt2 interacted more with the other top 30 DEGs with high interaction weights. Finally, RT-qPCR results showed that MCAO induction significantly up-regulated the expression of Stoml3, Eomes, and Ms4a15 and down-regulated Nms, Ttr, and Avpr1a; however, luteolin could partially reverse the expression caused by MCAO. Luteolin can alleviate brain infarction, apoptosis, and pyroptosis in CIRI, and may improve MCAO-induced CIRI by targeting the identified DEGs and their enriched pathways.

Cysteine Leukotriene Receptor Antagonist-Montelukast Effects on Diabetic Retinal Microvascular Endothelial Cells Curtail Autophagy.

Diabetic macular edema (DME) is the primary cause of vision impairment in diabetic retinopathy (DR) patients. A previous study has shown the efficacy of montelukast, a cysteinyl leukotriene receptor (CysLTR)1 antagonist, in a diabetic mouse model. This study aims to understand the CysLTR1 signaling in retinal endothelial cells and the impact of montelukast. Primary human retinal microvascular endothelial cells (HRECs) challenged with 20 ng/mL TNF-α and 30mM D-glucose (D-glu) for six to 24 hours served as a model of endothelial activation. HRECs were incubated with L-glucose (L-glu) as an osmotic control. CysLTR1 knockdown and montelukast pretreatment assessed CysLTR1 antagonism. Gene expression, protein expression, and cell-permeable dyes were utilized to measure autophagy and inflammation. Transendothelial electrical resistance (TER) and transendothelial migration of mononuclear leukocytes across HRECs monolayer were measured as a functional assessment of vascular permeability. Endothelial activation induced by hyperglycemia and inflammation increased CysLTR1 expression, triggering autophagy within two to six hours, IL-1β production, loss of junction integrity, decreased TER, and increased leukocyte migration within six to 24 hours. Pretreatment with montelukast effectively alleviated these effects, demonstrating its dependence on CysLTR1. Dysfunctional retinal endothelium initiates a self-reinforcing loop of inflammation, autophagy, and compromised integrity associated with heightened CysLTR1 levels. The antagonistic effect of montelukast against CysLTR1 effectively mitigates these detrimental changes. This study reveals CysLTR1 as a potential therapeutic target in treating DME and offers a novel strategy to mitigate detrimental changes in DR.

Characterization of Immune-Related circRNAs and mRNAs in Human Chronic Atrophic Gastritis.

Chronic atrophic gastritis (CAG) is a severe condition characterized by inflammation and loss of appropriate mucosal glands in the stomach. The underlying mechanisms of CAG development remain unclear. Exploring immune-related circular RNAs (circRNAs) could provide insights for potential diagnostic and therapeutic strategies. Samples from 40 patients with CAG and non-CAG (CNAG) underwent high-throughput sequencing, and EdgeR analysis identified differentially expressed circRNAs and mRNAs. Gene Ontology (GO) analysis elucidated biological functions, while Immune Cell Abundance Identifier (ImmuCellAI) estimated immune cell abundance. Flow cytometry analyzed immune cell infiltration. Weighted gene co-expression network analysis (WGCNA) identified hub genes related to the immune response in CAG. CircRNA-mRNA networks were constructed, and qRT-PCR validated findings. A total of 163 differentially expressed immune-related genes (DEIRGs) were identified between CAG and CNAG. The upregulated immune-related mRNAs in CAG were significantly enriched in antimicrobial humoral response, viral entry into host cells, neutrophil activation, and leukocyte migration. Conversely, downregulated immune-related mRNAs were linked to regulation of natural killer cell-mediated cytotoxicity, positive regulation of adaptive immune response, antigen receptor-mediated signaling pathway, and B cell activation. Immune Cell Abundance Identifier (ImmuCellAI) and flow cytometry confirmed increased neutrophil infiltration in CAG compared to CNAG. WGCNA identified 56hub immune-related genes. Additionally, circRNA expression profiles in CNAG and CAG were explored, with 19 upregulated and 23 downregulated circRNAs identified in CAG. The upregulated circRNAs were associated with biological processes like carnitine metabolic process and regulation of B cell receptor signaling pathway. A circRNA-mRNA co-expression network was constructed based on five circRNAs highly related to hub immune-related genes. Furthermore, the expression of eight immune-related mRNAs and five circRNAs were validated in CAG. This study is the first systematic analysis of circRNA profiles in CAG and provide important insights for potential immunotherapeutic strategies and early diagnostic biomarkers in CAG treatment.

Sex-Specific Transcriptomic Effects of Low-Dose Inorganic Arsenic Exposure on Bone Marrow-Derived Macrophages.

Both tissue-resident macrophages and monocytes recruited from the bone marrow that transform into tissue-resident cells play critical roles in mediating homeostasis as well as in the pathology of inflammatory diseases. Inorganic arsenic (iAs) is the most common drinking water contaminant worldwide and represents a major public health concern. There are numerous diseases caused by iAs exposure in which macrophages are involved, including cardiovascular disease, cancer, and increased risk of (respiratory) infectious diseases. Notably, prenatal iAs exposure is also associated with negative birth outcomes and developmental immunotoxicity (DIT) contributing to long-term adverse outcomes of these immune-related diseases. Therefore, understanding the effects of iAs exposure on macrophages, particularly during immune development or tissue injury and inflammation, can help us better grasp the full range of arsenic immunotoxicity and better design therapeutic targets for iAs-induced diseases particularly in exposed populations. In contrast to prior published studies which often only focused on the effect of iAs on mature macrophages after development, in this study, we analyzed the transcriptome of M0-, M1- and M2-polarized male and female murine bone marrow-derived macrophages (BMDMs) which were exposed to iAs during the differentiation phase, as a model to study iAs (developmental) immunotoxicity. We identified differentially expressed genes by iAs in a sex- and stimulation-dependent manner and used bioinformatics tools to predict protein-protein interactions, transcriptional regulatory networks, and associated biological processes. Overall, our data suggest that M1-stimulated, especially female-derived, BMDMs are most susceptible to iAs exposure during differentiation. Most notably, we observed significant downregulation of major proinflammatory transcription factors, like IRF8, and its downstream targets, as well as genes encoding proteins involved in pattern recognition and antigen presentation, such as TLR7, TLR8, and H2-D1, potentially providing causal insight regarding the role of (early-life) arsenic exposure in perturbing immune responses to infectious diseases. We also observed significant downregulation of genes involved in processes crucial to coordinating a proinflammatory response including leukocyte migration, differentiation, and cytokine and chemokine production and response. Finally, we discovered that 24 X-linked genes were dysregulated in iAs-exposed female stimulation groups compared to only 3 across the iAs-exposed male stimulation groups. These findings elucidate the potential mechanisms underlying the sex-differential iAs-associated immune-related disease risk.

Discovery of PANoptosis-related signatures correlates with immune cell infiltration in psoriasis.

Psoriasis is an inflammatory skin disease that relapses frequently. Keratinocyte apoptosis dysregulation plays a crucial role in the pathological mechanisms of psoriasis. PANoptosis is a process with intermolecular interaction among pyroptosis, apoptosis, and necroptosis. The mechanism of PANoptosis in the occurrence and development of psoriasis is still unclear. Here we present a novel approach by identifying PANoptosis-related signatures (PANoptosis-sig) from skin tissue of psoriasis patients and healthy controls on transcriptional and protein levels. Five PANoptosis-sig (TYMP, S100A8, S100A9, NAMPT, LCN2) were identified. Enrichment analysis showed they were mainly enriched in response to leukocyte aggregation, leukocyte migration, chronic inflammatory response and IL-17 signaling pathway. Single cell transcriptome analysis showed TYMP and NAMPT were expressed in almost all cell populations, while LCN2, S100A8 and S100A9 were significantly highly expressed in keratinocyte. We then constructed predictive and diagnostic models with the PANoptosis-sig and evaluated their performance. Finally, unsupervised consensus clustering analysis was conducted to ascertain psoriasis molecular subtypes by the PANoptosis-sig. The psoriasis cohort was divided into two distinct subtypes. Immune landscape showed that the stromal score of cluster 1 was significantly higher than cluster 2, while the immune and estimate scores of cluster 2 were expressively higher than cluster 1. Cluster 1 exhibited high expression of Plasma cells, Tregs and Mast cells resting, while cluster 2 showed high expression of T cells, Macrophages M1, Dendritic cells activated, and Neutrophils in immune infiltration analysis. And cluster 2 was more sensitive to immune checkpoints. In conclusion, our findings revealed potential biomarkers and therapeutic targets for the prevention, diagnosis, and treatment of psoriasis, enhancing our understanding of the molecular mechanisms underlying PANoptosis.

Comparative analysis of human and mouse transcriptomes during skin wound healing.

Skin wound healing is a complex process which involves multiple molecular events and the underlying mechanism is not fully understood. We presented a comparative transcriptomic analysis of skin wound healing in humans and mice to identify shared molecular mechanisms across species. We analyzed transcriptomes from three distinct stages of the healing process and constructed protein-protein interaction networks to elucidate commonalities in the healing process. A substantial number of differentially expressed genes (DEGs) were identified in human transcriptomes, particularly upregulated genes before and after wound injury, and enriched in processes related to extracellular matrix organization and leukocyte migration. Similarly, the mouse transcriptome revealed thousands of DEGs, with shared biological processes and enriched KEGG pathways, highlighting a conserved molecular signature in skin wound healing. A total of 21 common DEGs were found across human comparisons, and 591 in mouse comparisons, with four genes (KRT2, MARCKSL1, MMP1, and TNC) consistently differentially expressed in both species, suggesting critical roles in mammalian skin wound healing. The expression trends of these genes were consistent, indicating their potential as therapeutic targets. The molecular network analysis identified five subnetworks associated with collagen synthesis, immunity, cell-cell adhesion, and extracellular matrix, with hub genes such as COL4A1, TLR7, TJP3, MMP13, and HIF1A exhibited significant expression changes before and after wound injury in humans and mice. In conclusion, our study provided a detailed molecular network for understanding the healing process in humans and mice, revealing conserved mechanisms that could help the development of targeted therapies across species.

Role of Galectin-3 in resident macrophages and effects on atherosclerosis

Abstract Background Arterial macrophages play an important role in atherosclerosis, exhibiting heterogeneity in their origins as either resident or inflammatory monocyte-derived macrophages. Resident macrophages originate predominantly from the yolk sac, and they are endowed with homeostatic functions and can have anti-inflammatory properties under inflammation [1]. Resident macrophages pre-existing within the tissue differentiate into foamy macrophages before monocytes appear in early atherosclerotic lesions [2]. Galectin-3 (Gal-3) encoded by the gene Lgals3, is a crucial effector molecule that is prominently expressed by macrophages [3]. Studies have provided conflicting evidence regarding the role of Gal-3 in atherosclerosis by affecting macrophage functions and monocyte recruitment [4,5]. Purpose This study aims to investigate the role of Gal-3 in arterial resident macrophages for atherosclerosis and reveal the underlying mechanisms. Methods General Lgals3 knockout (Lgals3-/-) and resident macrophage specific knockout (Lgals3-/-Rank-Cretg) mice on the Apoe-deficient background were generated and atherosclerotic lesion development (8 weeks of western diet) was compared. Results Our study revealed that Lgals3-/- mice exhibited increased numbers of monocytes and neutrophils in the blood, which was associated with the development of larger atherosclerotic plaques. Plasma Gal-3 concentration from Lgals3-/-Rank-Cretg mice was decreased by 60%, indicating that the majority of circulating Gal-3 is derived from resident macrophages. Surprisingly, Lgals3-/-Rank-Cretg mice did not show the observed increase in blood monocytes and neutrophils, suggesting that this effect was independent of circulating Gal-3. Consistent with a causal role of monocytosis and leukocytosis in atherogenesis, plaque formation in Lgals3-/-Rank-Cretg was not significantly affected. Additionally, Gal-3 deficiency promoted M1 polarization and reduced efferocytosis, phagocytosis and chemokine receptor expression in BMDMs, and the addition of exogenous Gal-3 restored the effect on phagocytosis and blocked CXCL12 and CCL5-induced leukocyte migration. Conclusion This study reveals a prominent protective role of Gal-3, primarily through inflammatory macrophages rather than resident macrophages for the development of atherosclerotic lesions. This protective effect is attributed to its homeostatic effects on monocyte and neutrophil numbers, as well as its influence on macrophage functions.

Global deficiency in the inflammatory chemokine receptors 1,2,3 and 5 prevents vascular dysfunction in angiotensin II-induced hypertension and selectively modulates aortic myeloid cell phenotype

Abstract Background Leukocyte migration to the vasculature and the heart plays a critical role in hypertensive immune responses and is a carefully orchestrated process, regulated by the interactions of inflammatory chemokines with their receptors, notably CCR1, CCR2, CCR3, and CCR5 (collectively termed inflammatory chemokine receptors (iCCRs)). However, the inflammatory chemokine/receptor system is characterised by redundancy, ligand sharing and overlapping expression patterns. Consequently, our understanding of the specific and combinatory roles of chemokine receptors in cardiovascular inflammation remains incomplete, thus hindering the development of targeted therapies. To address this challenge, we have utilised two novel mouse models: iREP mice, carrying fluorescent reporters of inflammatory chemokine receptor expression, and TACKO mice, in which the entire iCcr locus containing Ccr1, Ccr2, Ccr3, and Ccr5 has been deleted. Purpose To evaluate the expression pattern of iCCRs in experimental hypertension and understand how the global deletion of iCCRs impacts disease progression. Methods We induced hypertension in mice by subcutaneously implanting osmotic minipumps administering angiotensin II (Ang II) for a duration of 14 days. Single-cell RNA sequencing was utilised for investigating iCCR expression in aortas during hypertension. Additionally, we employed flow cytometry and confocal microscopy to track iCCR expression and localization in aortas and hearts obtained from iREP mice. TACKO mice were utilised to assess the effect of global iCCr deficiency on hypertensive phenotypes. Blood pressure was measured via telemetry and vascular function was assessed using myography. Vascular and heart remodelling were evaluated through histology. Molecular mechanisms were analysed using Western Blotting and RT-qPCR, while immune cell changes in the aorta were evaluated through cytometry of time-of-flight (CyTOF). Results Ang II infusion induced higher iCCRs expression levels in both the aorta and the heart in comparison with sham treatment, particularly in fibrotic areas. After Ang II-treatment, global deletion of iCCRs in TACKO mice resulted in the prevention of endothelial dysfunction (n=5/group, P<0.05) and perivascular fibrosis (n=6-9, P<0.01), and a reduction in cardiomyocyte size (n=12-16, P<0.01) compared to wild type mice. However, no significant improvements were observed in other hypertensive phenotypes, including blood pressure, heart fibrosis, vascular NO synthase, and oxidative stress. CyTOF analysis revealed reductions in inflammatory monocytes and conventional type 2 dendritic cells in TACKO aorta, accompanied by a phenotypic switch in macrophages towards a less inflammatory phenotype. Conclusion Deficiency in iCCRs confers protective effects against vascular dysfunction in experimental hypertension, primarily by selectively reducing inflammatory myeloid cell homing to the aorta.

Role of C-C chemokine receptor type 5 in pathogenesis of malaria and its severe forms.

Malaria is a mosquito-borne disease caused by Plasmodium parasites, responsible for a significant impact on public health in several tropical and sub-tropical countries. The majority of infection cases are classified as uncomplicated malaria, causing mild symptoms such as fever and headache. However, the disease may progress to severe malaria and death if the infection is not properly treated. Furthermore, malaria poses a major concern for children, pregnant women and immunosuppressed individuals. Exacerbated inflammation is characteristic of severe malaria cases. The C-C chemokine receptor type 5 (CCR5) is an important molecule for leukocyte migration and regulation of inflammation. Although widely known as an HIV-1 co-receptor, CCR5 also affects the susceptibility and progression of autoimmune and inflammatory diseases. There is evidence supporting the participation of CCR5 in malaria manifestations, with the evaluation of CCR5 gene expression levels suggested as a marker to monitor malaria severity. Certain genetic variants in the CCR5 gene affect CCR5 expression, potentially altering CCR5-mediated inflammatory responses during malaria infection. However, the complex influences of CCR5 on malaria remain underexplored. Therefore, this review examines and updates the role of CCR5 in various contexts of malaria infection, including uncomplicated malaria, Plasmodium/HIV co-infection, pregnancy and severe (cerebral) malaria.

Leukocyte infiltration and cross-talk with cardiomyocytes exploit intracellular stress pathways in dilated cardiomyopathy of idiopathic origin.

Dilated cardiomyopathy (DCM) is a prevalent form of heart failure results in dilation and disruption of heart. Most strikingly a majority of the DCM cases do not have any identified etiology, hence known as idiopathic DCM (IDCM). Our study aimed to investigate the cross-talk between leukocytes and cardiomyocytes in terms of cardiac inflammation and stress response in IDCM. 60 IDCM patients and 60 age and sex matched healthy volunteers were recruited in this study based on the New York Heart Association (NYHA) guidelines. Their echocardiographic and biochemical markers were assessed and PBMCs were analyzed for leukocyte migration and inflammation. Also C2C12 myocyte cells were cultured with LPS-activated RAW264.7 monocytes to investigate the cross-talk between them. Left ventricular (LV) dysfunction was evident in the IDCM patients which were correlated with their physical discomfort level according to NYHA classification. Their serum levels of IL-1β and TNF-α (≈ 20 pg/ml) were found to be very high along with hs-CRP and IL-2. Elevated levels of ROCK, SMA and ICAM-1 proteins indicated activation and migration of the leukocytes. During monocyte-myocyte co-culture, robust diapedesis was observed in the cultured macrophage cells towards myocytes through the transwell pores (8 µM) in presence of IL-1β and TNF-α causing ER stress and cell death in the myocytes. Inhibition of this migration or by alleviating ER stress inhibits leukocyte recruitment and ensures protection to the myocytes. The present study showed that alleviating cellular stress and managing leukocyte migration promotes protection to the heart.

Protective Role of High-Density Lipoprotein in Multiple Sclerosis

Multiple sclerosis (MS) is a chronic, progressive demyelinating disease with a most likely autoimmune background and a neurodegenerative component. Besides the demyelinating process caused by autoreactive antibodies, an increased permeability in the blood–brain barrier (BBB) also plays a key role. Recently, there has been growing interest in assessing lipid profile alterations in patients with MS. As a result of myelin destruction, there is an increase in the level of cholesterol released from cells, which in turn causes disruptions in lipid metabolism homeostasis both in the central nervous system (CNS) and peripheral tissues. Currently, there is a growing body of evidence suggesting a protective role of HDL in MS through its effect on the BBB by decreasing its permeability. This follows from the impact of HDL on the endothelium and its anti-inflammatory effect, mostly by interacting with adhesion molecules like vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and E-selectin. HDL, through its action via sphingosine-1-phosphate, exerts an inhibitory effect on leukocyte migration, and its antioxidant properties contribute to the improvement of the BBB function. In this review, we want to summarize these studies and focus on HDL as a mediator of the anti-inflammatory response in MS.

Leukocyte kinetics and bacterial clearance during S. pneumoniae pneumonia and contributions of ICAM-1.

Streptococcus pneumoniae is a leading cause of community-acquired pneumonia. Intercellular adhesion molecule-1 (ICAM-1) is an adhesion molecule that is highly expressed on the pulmonary capillary endothelium, alveolar epithelium and other cell types within the lung. ICAM-1 plays important roles in leukocyte adhesion, migration, and motility. To determine the contributions of ICAM-1 to bacterial clearance and leukocyte kinetics during pneumonia, mice were inoculated with S. pneumoniae and evaluated 1, 4 and 7 days later. Our results show that Icam1-/-mice have a greater number of viable bacteria within the lung at each time point. The impaired clearance observed in Icam1-/- mice was not due to an impediment in leukocyte recruitment. In fact, Icam1-/- mice had a greater number of neutrophils and recruited inflammatory macrophages in the lung tissue and the alveoli/airways on day 7. In contrast, fewer alveolar macrophages were present in the BAL of Icam1-/-mice. The loss of body weight and the concentrations of inflammatory mediators in the BAL were also significantly greater in Icam1-/- mice. Mechanistic studies to understand the defect in clearance show that neutrophils and macrophage subpopulations had no defect in phagocytosis or acidification of phagosomes. RNA sequencing reveals many differences in gene expression, but no suggestion of a defect in phagocytosis or killing. Thus, that ICAM-1 is necessary for the clearance of S. pneumoniae and for the resolution of pneumonia, but is not required for the recruitment of neutrophils or inflammatory macrophages into the pneumonic lung parenchyma or the alveoli/airways during S. pneumoniae-induced pneumonia.

Multiple Machine Learning Models, Molecular Subtyping and Singlecell Analysis Identify PANoptosis-related Core Genes and their Association with Subtypes in Crohn's Disease.

PANoptosis plays an important role in many inflammatory diseases. However, there are no reports on the association between PANoptosis and CD. This study used five machine learning algorithms - least absolute shrinkage and selection operator, support vector machine, random forest, decision tree and Gaussian mixture models - to construct CD's PANoptosis signature. Unsupervised hierarchical clustering analysis was used to identify PANoptosis-associated subgroups of CD. Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA) were conducted to compare the PANoptosis-associated subgroups of CD among the potential biological mechanisms. Single sample GSEA was used to assess immune microenvironmental differences among the subgroups. The potential role of PANoptosis in CD was further explored using single-cell RNA-Seq (scRNA-Seq) for PANoptosis scoring, differential analysis, pseudotime analysis, cellular communication analysis and weighted gene co-expression network analysis (WGCNA) analysis. CD's PANoptosis signature consisted of seven genes: CEACAM6, CHP2, PIK3R1, CASP10, PSMB1, PSMB8 and UBC. The PANoptosis signature in multiple cohorts had a strong ability to recognise CD. The levels of immune cell infiltration and the vigour of the immune responses significantly varied between the two subpopulations of CD associated with PANoptosis. Multiple lines of evidence from the GO, KEGG, GSEA, GSVA, scRNA-Seq and WGCNA analyses suggested that I-kappaB kinase/NF- kappaB signalling, mitogen-activated protein kinase (MAPK), leukocyte activation and leukocyte migration were mechanisms closely associated with PANoptosis in CD. This study is the first to construct a PANoptosis signature with excellent efficacy in recognising CD. PANoptosis may mediate the process of CD through inflammatory and immune mechanisms, such as NF- kappaB, MAPK and leukocyte migration.

Evaluation of Bioactive Compounds, Antioxidant Capacity, and Anti-Inflammatory Effects of Lipophilic and Hydrophilic Extracts of the Pericarp of Passiflora tripartita var. mollissima at Two Stages of Ripening.

Chronic disease inflammation requires safe complementary treatments. The pericarp of Passiflora tripartita var. mollissima (PTM) contains potential anti-inflammatory metabolites. This study aimed to evaluate the bioactive components, antioxidant capacity, and anti-inflammatory effects of PTM extracts at two ripening stages. The bioactive compounds in the hydrophilic and lipophilic extracts of mature and green pericarps were identified by GC-MS and UV-VIS, while the antioxidant capacity was measured by free radical reduction. Anti-inflammatory effects were tested using a rat paw edema model with carrageenan-induced edema, indomethacin, or PTM extracts (100, 250, and 500 mg/kg). The effect of mature hydrophilic extract was further evaluated in an air pouch model, where rats received the placebo, carrageenan, indomethacin, or the extract (500 and 1000 mg/kg). Leukocytes, cytokines, and markers of oxidative stress were evaluated. The results showed the presence of organic compounds, total phenols, and flavonoids. The mature hydrophilic extract exhibited the highest antioxidant activity. At 500 mg/kg, it reduced edema, leukocyte migration, and levels of IL-1β, IL-6, and TNF-α while managing oxidative stress and preventing histological damage. In conclusion, PTM contains bioactive compounds with potential pharmacological properties. The hydrophilic extract of the mature pericarp, at a dose of 500 mg/kg, exhibits an enhanced antioxidant and anti-inflammatory effect.

BRAF Modulates the Interplay Between Cell-Cell and Cell-Extracellular Matrix Adhesions in PECAM-1-Mediated Mechanotransduction.

Mechanotransduction, the process of how cells sense and convert mechanical stimuli into biochemical response, is crucial in the migration of leukocytes or cancer cells through the endothelium during inflammation or metastasis. Migrating cells exert forces on the endothelium through cell surface adhesion molecules, such as platelet endothelial adhesion molecule PECAM-1, and this is essential for a successful transmigration. To study PECAM-1-mediated mechanotransduction, we applied PECAM-1-antibody-coated magnetic beads and exerted about 40 pN force on the endothelial monolayer. We show that force increases cell-ECM adhesion in the cell center and is accompanied by the opening of cell-cell junctions. Upon depletion of the MEK/ERK kinase, BRAF force increases cell-ECM adhesion both at the cell periphery and in the cell center, but this does not result in the opening of cell-cell junctions. Decreasing cell-ECM adhesion in BRAF-depleted cells through FAK inhibition results in the remodeling of cell-cell junctions. Force-induced increase in cell-ECM adhesion in the cell center correlates with the activation of the transcriptional cofactor Yes-associated protein (YAP). Furthermore, the induced activation of YAP through LATS inhibition prevents junctional remodeling in control cells. Thus, the activation of YAP might determine the strength of cell-cell junctions during PECAM-1-mediated mechanotransduction.

Intravital Microscopy With an Airy Beam Light Sheet Microscope Improves Temporal Resolution and Reduces Surgical Trauma.

Intravital microscopy has emerged as a powerful imaging tool, which allows the visualization and precise understanding of rapid physiological processes at sites of inflammation in vivo, such as vascular permeability and leukocyte migration. Leukocyte interactions with the vascular endothelium can be characterized in the living organism in the murine cremaster muscle. Here, we present a microscopy technique using an Airy Beam Light Sheet microscope that has significant advantages over our previously used confocal microscopy systems. In comparison, the light sheet microscope offers near isotropic optical resolution and faster acquisition speed, while imaging a larger field of view. With less invasive surgery we can significantly reduce side effects such as bleeding, muscle twitching, and surgical inflammation. However, the increased acquisition speed requires exceptional tissue stability to avoid imaging artefacts. Since respiratory motion is transmitted to the tissue under investigation, we have developed a relocation algorithm that removes motion artefacts from our intravital microscopy images. Using these techniques, we are now able to obtain more detailed 3D time-lapse images of the cremaster vascular microcirculation, which allow us to observe the process of leukocyte emigration into the surrounding tissue with increased temporal resolution in comparison to our previous confocal approach.

Cyperus articulatus: Anti-inflammatory and antinociceptive activity of a medicinal plant from the Amazon

Ethnopharmacological relevanceCyperus articulatus L., popularly known as priprioca, is a plant used in the Amazon for perfumed baths and homemade perfumes. In traditional medicine, its rhizomes are used to treat diseases related to inflammatory processes. Aim of the studyDue to its promising bioactive properties, this study sought to investigate its phytochemistry and the anti-inflammatory and antinociceptive activity of the essential oil obtained from C. articulatus (CAEO) in in vitro and in vivo tests. Material and methodsThe essential oil was obtained from the rhizomes of C. articulatus and extraction was carried out via hydrodistillation. Then, the oil was analyzed by GC-MS analyses. Initially, culture of RAW 264.7 macrophages stimulated by lipopolysaccharide (LPS) was used to evaluate cytotoxicity and interference in the production of mediators of the inflammatory process (nitrite, IL-1β, TNF-α and PGE2) after exposure to CAEO. The acute toxicity of CAEO was evaluated and the results were used to define doses of 10, 100 and 400 mg kg−1 for evaluation of CAEO in in vivo tests using mice. The carrageenan-induced air pouch models and the Evans test were used to evaluate the anti-inflammatory activity by measuring the number of total leukocytes and vascular permeability. Antinociceptive activity was evaluated via tests of contortions induced by acetic acid, hot plate, and formalin. ResultsTreatment with CAEO reduced the levels of nitrite IL-1β, TNF-α and PGE2 in the macrophage culture, revealing its anti-inflammatory potential. CAEO decreased carrageenan-induced leukocyte migration and vascular permeability, which are important events related to the acute inflammatory response. Nociceptive activity was significantly inhibited by CAEO in the acetic acid-induced contortions model, hot plate, and in both phases of the formalin test. The treatment with naloxane reversed the antinociceptive effect observed in the formalin test, suggesting the participation of opioid receptors in the mechanism of action of CAEO. ConclusionThe observed results reveal the anti-inflammatory and antinocipeptive activity of C. articulatus essential oil in vivo and support the traditional use of this plant in the treatment of different diseases involving inflammation and pain.

Integrated machine learning and Mendelian randomization reveal PALMD as a prognostic biomarker for nonspecific orbital inflammation

Background: Nonspecific Orbital Inflammation (NSOI) remains a perplexing enigma among proliferative inflammatory disorders. Its etiology is idiopathic, characterized by distinctive and polymorphous lymphoid infiltration within the orbital region. Preliminary investigations suggest that PALMD localizes within the cytosol, potentially playing a crucial role in cellular processes, including plasma membrane dynamics and myogenic differentiation. The potential of PALMD as a biomarker for NSOI warrants meticulous exploration. Methods: PALMD was identified through the intersection analysis of common DEGs from datasets GSE58331 and GSE105149 from the GEO database, alongside immune-related gene lists from the ImmPort database, using Lasso regression and SVM-RFE analysis. GSEA and GSVA were conducted with gene sets co-expressed with PALMD. To further investigate the correlation between PALMD and immune-related biological processes, the CIBERSORT algorithm and ESTIMATE method were employed to evaluate immune microenvironment characteristics of each sample. The expression levels of PALMD were subsequently validated using GSE105149. Results: Among the 314 DEGs identified, several showed significant differences. Lasso and SVM-RFE algorithms pinpointed 15 hub genes. Functional analysis of PALMD emphasized its involvement in cell-cell adhesion, leukocyte migration, and leukocyte-mediated immunity. Enrichment analysis revealed that gene sets positively correlated with PALMD were enriched in immune-related pathways. Immune infiltration analysis indicated that resting dendritic cells, resting mast cells, activated NK cells, and plasma cells positively associate with PALMD expression. Conversely, naive B cells, activated dendritic cells, M0 and M1 macrophages, activated mast cells, activated CD4 memory T cells, and naive CD4 T cells showed a negative correlation with PALMD expression. PALMD demonstrated significant diagnostic potential in differentiating NSOI. Conclusions: This study identifies PALMD as a potential biomarker linked to NSOI, providing insights into its pathogenesis and offering new avenues for tracking disease progression.

Extracellular vesicles in sepsis plasma mediate neuronal inflammation in the brain through miRNAs and innate immune signaling

BackgroundNeuroinflammation reportedly plays a critical role in the pathogenesis of sepsis-associated encephalopathy (SAE). We previously reported that circulating plasma extracellular vesicles (EVs) from septic mice are proinflammatory. In the current study, we tested the role of sepsis plasma EVs in neuroinflammation.MethodsTo track EVs in cells and tissues, HEK293T cell-derived EVs were labeled with the fluorescent dye PKH26. Cecal ligation and puncture (CLP) was conducted to model polymicrobial sepsis in mice. Plasma EVs were isolated by ultracentrifugation and their role in promoting neuronal inflammation was tested following intracerebroventricular (ICV) injection. miRNA inhibitors (anti-miR-146a, -122, -34a, and -145a) were applied to determine the effects of EV cargo miRNAs in the brain. A cytokine array was performed to profile microglia-released protein mediators. TLR7- or MyD88-knockout (KO) mice were utilized to determine the underlying mechanism of EVs-mediated neuroinflammation.ResultsWe observed the uptake of fluorescent PKH26-EVs inside the cell bodies of both microglia and neurons. Sepsis plasma EVs led to a dose-dependent cytokine release in cultured microglia, which was partially attenuated by miRNA inhibitors against the target miRNAs and in TLR7-KO cells. When administered via the ICV, sepsis plasma EVs resulted in a marked increase in the accumulation of innate immune cells, including monocyte and neutrophil and cytokine gene expression, in the brain. Although sepsis plasma EVs had no direct effect on cytokine production or neuronal injury in vitro, the conditioned media (CM) of microglia treated with sepsis plasma EVs induced neuronal cell death as evidenced by increased caspase-3 cleavage and Annexin-V staining. Cytokine arrays and bioinformatics analysis of the microglial CM revealed multiple cytokines/chemokines and other factors functionally linked to leukocyte chemotaxis and migration, TLR signaling, and neuronal death. Moreover, sepsis plasma EV-induced brain inflammation in vivo was significantly dependent on MyD88.ConclusionsCirculating plasma EVs in septic mice cause a microglial proinflammatory response in vitro and a brain innate immune response in vivo, some of which are in part mediated by TLR7 in vitro and MyD88 signaling in vivo. These findings highlight the importance of circulating EVs in brain inflammation during sepsis.