Immune Cell Migration Research Articles

Exosomes-Mediated Signaling Pathway: A New Direction for Treatment of Organ Ischemia-Reperfusion Injury.

Ischemia reperfusion (I/R) is a common pathological process which occurs mostly in organs like the heart, brain, kidney, and lung. The injury caused by I/R gradually becomes one of the main causes of fatal diseases, which is an urgent clinical problem to be solved. Although great progress has been made in therapeutic methods, including surgical, drug, gene therapy, and transplant therapy for I/R injury, the development of effective methods to cure the injury remains a worldwide challenge. In recent years, exosomes have attracted much attention for their important roles in immune response, antigen presentation, cell migration, cell differentiation, and tumor invasion. Meanwhile, exosomes have been shown to have great potential in the treatment of I/R injury in organs. The study of the exosome-mediated signaling pathway can not only help to reveal the mechanism behind exosomes promoting reperfusion injury recovery, but also provide a theoretical basis for the clinical application of exosomes. Here, we review the research progress in utilizing various exosomes from different cell types to promote the healing of I/R injury, focusing on the classical signaling pathways such as PI3K/Akt, NF-κB, Nrf2, PTEN, Wnt, MAPK, toll-like receptor, and AMPK. The results suggest that exosomes regulate these signaling pathways to reduce oxidative stress, regulate immune responses, decrease the expression of inflammatory cytokines, and promote tissue repair, making exosomes a competitive emerging vector for treating I/R damage in organs.

Abstract TP286: Spatial Transcriptome Profile of Stem Cell-Mediated Recovery in Stroke-Injured Rat Brains

Introduction: Intraparenchymal stem cell transplantation shows therapeutic promise for chronic ischemic stroke patients. Our previous phase 1/2a trial revealed a transient T2-FLAIR (fluid-attenuated inversion recovery) MRI signal in the premotor cortex following intraparenchymal stem cell transplantation, the size of which positively correlated with the extent of clinical recovery. We sought to elucidate the underlying molecular mechanisms in this FLAIR region that may facilitate post-stroke recovery using spatial transcriptomic profiling in stroke-injured rat brains. Methods: Adult male SD rats underwent 30 min transient middle cerebral artery occlusion followed at 1 month by transplantation of human ES-derived neural stem cells (NR1) or buffer into the ipsilesional striatum. Whisker paw test and various MRI sequences (T2, FLAIR, DWI) were performed at multiple time points. The Visium platform (10X Genomics) was used for unbiased spatial transcriptome analysis in two stem-cell and two vehicle-treated brains at the peak of FLAIR. Results: We successfully replicated transplant-induced FLAIR after chronic stroke in rats and found a positive correlation between FLAIR and improved neurological outcomes. Visium identified specific gene clusters for the stroke injury, transplant/needle-track, stem cell graft, and the cortical ‘FLAIR’ region. The latter consisted of a central core containing the gene signature of the needle track and blood, and a wider region around this core which encompassed the FLAIR lesion seen by MRI. This wider FLAIR region displayed a predominantly microglia/inflammation-associated signature and was best characterized by the upregulation of apoptosis-associated genes. Pathway analysis of the differentially-expressed genes in the FLAIR cluster revealed a significant representation of immune cell responses (e.g. phagocytosis, immune cell migration, cytokine signaling), as well as ER stress mechanisms. Conclusion: This pilot study provides initial evidence that altered immune responses in the stem cell transplant-induced FLAIR region may promote recovery after chronic stroke. Further research on the role of microglia/neuroinflammation is needed to understand the mechanisms of stem cell-mediated brain repair.

Abstract B028: Investigating immune activity caused by a novel anti-cancer cell penetrating peptide

Abstract Activating the immune system against cancerous cells represents a promising therapy against multiple cancer types, particularly breast cancer. The epidermal growth factor receptor (EGFR) represents an emerging target in immunotherapy. A novel anti-cancer cell penetrating peptide was recently discovered to affect immune recruitment and potentially function in a murine model of breast cancer. This peptide, termed cSNX1.3, is a targeted therapy to block the nuclear localization of the epidermal growth factor receptor (EGFR), a phenomenon thought to occur primarily in cancerous cells. This suggests that nuclear localized EGFR may play a role in suppressing an anti-cancer immune response. To gain a better understanding of this in vivo data, we used in vitro immune cell co-culture techniques, migration assays, and other techniques to investigate immune recruitment and function after cancer cells are treated with this novel peptide therapy. In addition, we are investigating the mechanism of cSNX1.3 mediated cell death to determine if it can induce immunogenic cell death. To determine if nuclear EGFR plays a role in immune cell suppression, we are using a CRISPR/Cas9 strategy to mutate the nuclear localization sequence. This will provide a valuable tool to better understand the seemingly cancer-specific trafficking phenomenon. Together, this work will expand our understanding of therapy induced immune cell recruitment and trafficking. Citation Format: Ryan Hecksel, Angelica Escoto, Joyce Schroeder. Investigating immune activity caused by a novel anti-cancer cell penetrating peptide [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B028.

Abstract WMP119: CD13 Facilitates Immune Cell Migration and Aggravates Acute Injury but Promotes Chronic Post-Stroke Recovery

Introduction: Acute stroke leads to the activation of myeloid cells. Activated myeloid cells express CD13, which facilitates their migration into the site of injury. Chronically after stroke, repair processes, including angiogenesis, are activated and enhance post-stroke recovery. However, angiogenic blood vessels which play a role in recovery also express CD13. Overall, the specific contribution of CD13 to acute and chronic stroke outcomes is unknown. Methods: CD13 expression was estimated in mice and humans after the ischemic stroke. Young (8-12 weeks) male wild-type (WT) and global CD13 knockout (KO) mice were used for this study. Mice underwent 60 minutes of middle cerebral artery occlusion (MCAO) followed by reperfusion. For acute studies, the mice were euthanized at either 24- or 72 hours post-stroke. For chronic studies, the Y maze, Barnes maze, and the open field were performed on day 7 and day 28 post-stroke. Mice were euthanized at day 30 post-stroke and the brains were collected for assessment of inflammation, white matter injury, tissue loss, and angiogenesis. Flow cytometry was performed on days 3 and 7 post-stroke to quantify infiltrated monocytes and neutrophils and CXCL12/CXCR4 signaling. Results: Brain CD13 expression and infiltrated CD13 + monocytes and neutrophils increased acutely after the stroke. The brain CD13 + lectin + blood vessels increased on day 15 after the stroke. Similarly, an increase in the percentage area CD13 was observed in human stroke patients at the subacute time after stroke. Deletion of CD13 resulted in reduced infarct volume and improved neurological recovery after acute stroke. However, CD13KO mice had significantly worse memory deficits, amplified gliosis, and white matter damage compared to WT animals at chronic time points. CD13KO mice had an increased percentage of CXCL12 + cells but a reduced percentage of CXCR4 + cells and decreased angiogenesis at day 30 post-stroke. Conclusions: CD13 is involved in the transmigration of monocytes and neutrophils after stroke, and acutely, led to decreased infarct size and improved behavioral outcomes. However, loss of CD13 led to reductions in post-stroke angiogenesis by reducing CXCL12/CXCR4 signaling.

The Role of Chemokines in Orchestrating the Immune Response to Pancreatic Ductal Adenocarcinoma

Chemokines are small molecules that function as chemotactic factors which regulate the migration, infiltration, and accumulation of immune cells. Here, we comprehensively assess the structural and functional role of chemokines, examine the effects of chemokines that are present in the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME), specifically those produced by cancer cells and stromal components, and evaluate their impact on immune cell trafficking, both in promoting and suppressing anti-tumor responses. We further explore the impact of chemokines on patient outcomes in PDAC and their role in the context of immunotherapy treatments, and review clinical trials that have targeted chemokine receptors and ligands in the treatment of PDAC. Lastly, we highlight potential strategies that can be utilized to harness chemokines in order to increase cytotoxic immune cell infiltration and the anti-tumor effects of immunotherapy.

P172 Spatial Transcriptomics Characterization of S1P Pathway Genes in Ulcerative Colitis and Crohn’s Disease Colon Tissues

Abstract Background Inflammatory bowel diseases (IBD), such as ulcerative colitis (UC) and Crohn’s disease (CD) are chronic inflammatory diseases of the gastrointestinal tract, related to the confluence of environmental and immunological factors in genetically predisposed individuals. Activated T and B cells migrate from the lymphoid tissue into the circulation through the interaction of Sphingosine-1-phosphate (S1P) and the S1P1 receptor (S1P1), which can perpetuate inflammation and lead to tissue damage. Targeting of cellular migratory pathways has emerged as a novel therapeutic paradigm in IBD. The aim of this study is to characterize the S1P pathway-related genes and signaling pathways driving the pathogenesis of UC and CD. Methods Spatial transcriptomics analysis was performed in health volunteer (HV, n=2), UC (n=4) and CD (n=2) FFPE colon tissues using the GeoMX platform and NanoString whole transcriptome atlas (WTA) assay. The WTA panel was run on 8 samples stained with antibodies to S1P1, pan Cytokeratin, and CD45. Six regions of interests (ROIs) were selected in each sample based on the staining and each ROI were split into two areas (CD45+ or CD45-). Differential gene expression and enriched pathways analysis including S1P pathway genes were performed in CD45+ and CD45- segments in HV, UC and CD colon tissues. Results A total of 6085 and 6054 differentially expressed genes were identified from the CD45+ segments in UC and CD respectively, compared to HV colon (P=0.05), including the genes that are involved in T cell activation (e.g., HLA-DPA1, CCDC13), immune cell migration (e.g., ADAM15, CCL5) and tissue damage (e.g., FABP6, PDK4). Interestingly, significant differences were identified in UC and CD in S1P pathway gene expression. Specifically, SMPD1, a gene involved in generating ceramide and regulating inflammatory responses, was the most significantly up-regulated gene from both CD45+ and CD45- compartments in UC compared to HV colon. While CERS2 and CERS6, genes related to S1P metabolism, were significantly down-regulated genes in CD45+ and CD45- compartment in UC respectively, as compared to HV colon. Additionally, RTN4 (participates in the intestinal epithelial barrier function) and PLPP3 (dephosphorylates extracellular S1P to sphingosine) were most significantly down-regulated genes from CD45+ segments in both UC and CD compared to HV colon. Conclusion In summary, our study successfully validated the GeoMx transcriptome analysis approach in FFPE colon tissue and assessed the S1P pathway gene signature difference in HV, UC and CD colon tissues supporting the pathway’s role in IBD. Results could also facilitate the identification of potential S1P pathway UC and CD disease biomarkers.

OP30 Endothelial cell-mediated smooth muscle hyperplasia in Crohn’s disease intestinal strictures: Caveolin 1 as a potential therapeutic target

Abstract Background Intestinal stricture is a severe and common complication of Crohn’s disease (CD). Current therapies offer limited success and surgery is often needed. Smooth muscle hyperplasia and hypertrophy play a key role in CD stricture development. This study explores the transcriptomic features of fibrostenotic CD, focusing on the endothelial contribution to smooth muscle changes within intestinal strictures. Methods 9 CD patients undergoing surgery for fibrotic strictures were prospectively enrolled. Dissected samples from the stricture and the adjacent proximal and distal non-strictured areas were obtained from fresh specimens collected directly from the operating theatre. RNA was extracted from all samples for bulk-transcriptomic analysis. Differentially expressed genes (adj. p-value <0.05) were identified and pathway enrichment analysis pinpointed relevant pathways. Subsequently, single-cell RNA sequencing (scRNAseq) analysis was performed using the 10x Chromium Controller® platform. A total of 31.195 cells were sequenced and bioinformatic analysis revealed 13 distinct clusters based on the expression of key lineage target genes. To better characterise the nature of clusters expressing stromal like markers and positioned closely in the UMAP plot, a further subclustering was performed. This resulted in 5 groups, including plasma cells, endothelial cells, fibroblasts, epithelial cells and dendritic like cells. Results Bulk-transcriptomic analysis identified 81 genes differentially expressed in strictures compared to both proximal and distal regions. Notably, genes associated with leukocyte and macrophage recruitment and chemotaxis, namely Platelet and Endothelial Cell Adhesion Molecule 1 (PECAM1), C-C motif Chemokine Ligand 2 (CCL2), and Endothelin Receptor Type B (EDNRB), resulted overexpressed in strictures. scRNAseq identified a distinct subset of endothelial cells (CD34+, PECAM1+). Caveolin 1 (CAV1), a gene encoding for a structural protein predominantly expressed in smooth muscle cell plasma membrane invaginations, was exclusively up-regulated in endothelial cells of ileal stricture samples. Previous research linked CAV1 to smooth muscle proliferation and fibrosis in various organs. Conclusion This study highlights the pivotal role of endothelial cells in CD strictures. The transcriptomic analysis shows the involvement of inflammatory targets featured genes involved in transendothelial immune cell migration. Furthermore, the up-regulation of CAV1 in endothelial cells within stricture segments suggests a mechanistic connection between endothelial cells and smooth muscle hyperplasia in intestinal strictures. CAV1 holds promise as a potential therapeutic target in fibrostenotic CD.

HCMV IE1/IE1mut Therapeutic Vaccine Induces Tumor Regression via Intratumoral Tertiary Lymphoid Structure Formation and Peripheral Immunity Activation in Glioblastoma Multiforme

Glioblastoma multiforme (GBM), a highly malignant invasive brain tumor, is associated with poor prognosis and survival and lacks an effective cure. High expression of the human cytomegalovirus (HCMV) immediate early protein 1 (IE1) in GBM tissues is strongly associated with their malignant progression, presenting a novel target for therapeutic strategies. Here, the bioluminescence imaging technology revealed remarkable tumor shrinkage and improved survival rates in a mouse glioma model treated with HCMV IE1/IE1mut vaccine. In addition, immunofluorescence data demonstrated that the treated group exhibited significantly more and larger tertiary lymphoid structures (TLSs) than the untreated group. The presence of TLS was associated with enhanced T cell infiltration, and a large number of proliferating T cells were found in the treated group. Furthermore, the flow cytometry results showed that in the treatment group, cytotoxic T lymphocytes exhibited partial polarization toward effector memory T cells and were activated to play a lethal role in the peripheral immunological organs. Furthermore, a substantial proportion of B cells in the draining lymph nodes expressed CD40 and CD86. Surprisingly, quantitative polymerase chain reaction indicated that a high expression of cytokines, including chemokines in brain tumors and immune tissues, induced the differentiation, development, and chemokine migration of immune cells in the treated group. Our study data demonstrate that IE1 or IE1mut vaccination has a favorable effect in glioma mice models. This study holds substantial implications for identifying new and effective therapeutic targets within GBM.

Unbalancing cAMP and Ras/MAPK pathways as a therapeutic strategy for cutaneous neurofibromas.

Cutaneous neurofibromas (cNFs) are benign Schwann cell (SC) tumors arising from subepidermal glia. Individuals with neurofibromatosis type 1 (NF1) may develop thousands of cNFs, which greatly affect their quality of life. cNF growth is driven by the proliferation of NF1-/- SCs and their interaction with the NF1+/- microenvironment. We analyzed the crosstalk between human cNF-derived SCs and fibroblasts (FBs), identifying an expression signature specific to the SC-FB interaction. We validated the secretion of proteins involved in immune cell migration, suggesting a role of SC-FB crosstalk in immune cell recruitment. The signature also captured components of developmental signaling pathways, including the cAMP elevator G protein-coupled receptor 68 (GPR68). Activation of Gpr68 by ogerin in combination with the MEK inhibitor (MEKi) selumetinib reduced viability and induced differentiation and death of human cNF-derived primary SCs, a result corroborated using an induced pluripotent stem cell-derived 3D neurofibromasphere model. Similar results were obtained using other Gpr68 activators or cAMP analogs/adenylyl cyclase activators in combination with selumetinib. Interestingly, whereas primary SC cultures restarted their proliferation after treatment with selumetinib alone was stopped, the combination of ogerin-selumetinib elicited a permanent halt on SC expansion that persisted after drug removal. These results indicate that unbalancing the Ras and cAMP pathways by combining MEKi and cAMP elevators could be used as a potential treatment for cNFs.

Methazolamide Reduces the AQP5 mRNA Expression and Immune Cell Migration-A New Potential Drug in Sepsis Therapy?

Sepsis is a life-threatening condition caused by the dysregulated host response to infection. Novel therapeutic options are urgently needed and aquaporin inhibitors could suffice as aquaporin 5 (Aqp5) knockdown provided enhanced sepsis survival in a murine sepsis model. Potential AQP5 inhibitors provide sulfonamides and their derivatives. In this study, we tested the hypothesis that sulfonamides reduce AQP5 expression in different conditions. The impact of sulfonamides on AQP5 expression and immune cell migration was examined in cell lines REH and RAW 264.7 by qPCR, Western blot and migration assay. Subsequently, whether furosemide and methazolamide are capable of reducing AQP5 expression after LPS incubation was investigated in whole blood samples of healthy volunteers. Incubation with methazolamide (10-5 M) and furosemide (10-6 M) reduced AQP5 mRNA and protein expression by about 30% in REH cells. Pre-incubation of the cells with methazolamide reduced cell migration towards SDF1-α compared to non-preincubated cells to control level. Pre-incubation with methazolamide in PBMCs led to a reduction in LPS-induced AQP5 expression compared to control levels, while furosemide failed to reduce it. Methazolamide appears to reduce AQP5 expression and migration of immune cells. However, after LPS administration, the reduction in AQP5 expression by methazolamide is no longer possible. Hence, our study indicates that methazolamide is capable of reducing AQP5 expression and has the potential to be used in sepsis prophylaxis.

Histological examination of the young Oncorhynchus mykiss intestines using the feed with chelated and probiotic supplements.

Macronutrient and micronutrient deficiencies in the fish diet can affect fish growth rate, ability to resist disease, and fillet quality. Determination of effective dose of developed feed additives consisting of chelate compounds of biogenic elements and probiotic preparation based on Bacillus subtilis. Young rainbow trout were divided into four groups consisting of 100 fish each. The control group received standard food; the experimental groups received food with chelates and probiotics in different concentrations. At the end of the experiment (30 days), the middle intestine histology and morphometric parameters were studied. The use of chelated compounds alone affected the state of the intestinal villi and the infiltration of the epithelium by lymphocytes. The addition of a probiotic had a positive effect on the migration of immune cells in the intestinal villi; however, the probiotic in high concentrations contributed to a decrease in the number and area of goblet cells. Synergistic effects of chelated compounds and probiotic preparations on the morphometric parameters of the medium intestine were observed. In the proper doses, the studied feed additives might be recommended for rainbow trout farming under industrial aquaculture conditions.

Contractive function of mesenteric lymph nodes in obese rats

Over the past 50 years, the prevalence of obesity around the world has increased several times and has become a pandemic. The effect of obesity on the lymphatic system, which plays a key role in the regulation of fluid homeostasis, immune cell migration, antigen presentation, and resolution of inflammatory responses, is poorly understood, and there is no data on the contractile activity of the lymph nodes in obesity. The purpose of the research was to investigate the parameters and mechanisms of dysfunction of the contractile function of the mesenteric lymph nodes of rats in obesity caused by the feeding with the high-fat diet (HFD). Material and methods. The study was conducted on 50 male Sprague-Dawley rats. Rats aged 6 weeks were randomly divided into groups: a control group (n=10) fed a standard diet and a group of rats (n=40) kept on HFD (60% fat content by calorie value). Rats received food and water ad libitum for 16 weeks. Before the end of the experiment, four groups of HFD rats were formed: obesity resistant animals (HFD-OR, n=11), without additional interventions (HFD, n=10), rats which were administered dexamethasone three days before the study (HFD+Dexa, n=9), HFD followed by 8-week diet restriction (HFD+DR, n=9). At the end of the experiment, mesenteric lymph nodes (LNs) were taken from rats under anesthesia and their contractile function was studied in a myograph using 1400W, dynastat and Tempol. Results. LNs of control rats had a high level of tone and generated spontaneous high-amplitude phasic contractions. The LNs of HFD rats had a low initial tone, and rare low-amplitude phasic contractions were recorded in them. The parameters of contractile activity of the LNs of rats in HFD-OR and HFD+Dexa groups differed slightly from the corresponding parameters of the LNs of rats in the control group. Calorie restriction for 8 weeks in obese rats (HFD+DR) resulted in an increase in tone, frequency and amplitude of phasic contractions of the LNs compared to those in HFD rats. iNOS inhibition caused a significant increase in the tone, amplitude and frequency of phasic contractions of the LNs in the HFD group. An increase in the frequency of phasic contractions was observed only in the LNs of HFD+Dex and HFD+DR rats. Inhibition of cyclooxygenase 2 did not affect the contractile function of the LNs of rats of all groups, with the exception of animals from the HFD group (increase in the amplitude and frequency of phasic contractions). Tempol significantly increased the tone, frequency and amplitude of phasic contractions of the LNs in rats of the HFD group and increased the frequency of phasic contractions of the LNs of the HFD+DR rats. Conclusion. A high-fat diet leads to impaired contractile function of rat LNs and can create additional obstacles to the movement of lymph, promoting its leakage into surrounding tissues. Obesity is accompanied by the development of inflammation in the LNs and perinodal adipose tissue, which induces the expression of inducible NO synthase, cyclooxygenase-2 and the accumulation of reactive oxygen species (ROS). NO, prostaglandins and ROS have an inhibitory effect on the SMC capsules of the LNs, leading to a decrease in tonic tension and a weakening of spontaneous phasic contractions. The reason for inhibition of LN contractile function is obesity, but not consumption of food high in fat. Transferring obese rats to a calorie-restricted diet results in a decrease in body weight and visceral fat mass and an improvement in LN contractile function.

Beet supplementation mitigates post-exercise inflammation.

This study investigated the efficacy of a mixed beet-based supplement (BEET) versus placebo (PL) in countering inflammation during recovery from 2.25 h of intensive cycling in 20 male and female cyclists. A multi-omics approach was used that included untargeted proteomics and a targeted oxylipin panel. A randomized, placebo-controlled, double-blind, crossover design was used with two 2-week supplementation periods and a 2-week washout period. Supplementation periods were followed by a 2.25 h cycling bout at close to 70%VO2max. The BEET supplement provided 212 mg of nitrates per day, 200 mg caffeine from green tea extract, 44 mg vitamin C from Camu Camu berry, B-vitamins from quinoa sprouts (40% Daily Value for thiamin, riboflavin, niacin, and vitamin B6), and 2.5 g of a mushroom blend containing Cordyceps sinensis and Inonotus obliquus. Six blood samples were collected before and after supplementation (overnight fasted state), immediately post-exercise, and at 1.5 h-, 3 h-, and 24 h-post-exercise. The 2.25 h cycling bout increased plasma levels of 41 of 67 oxylipins detected. BEET supplementation significantly increased plasma nitrate (NO3 -) and nitrite (NO2 -) (sum, NO3 - + NO2 -) concentrations (interaction effect, p < 0.001) and two anti-inflammatory oxylipins [18-hydroxyeicosapentaenoic acid (18-HEPE) and 4-hydroxy-docosahexanoic acid (4-HDoHE)]. The untargeted proteomics analysis identified 616 proteins (458 across all times points), and 2-way ANOVA revealed a cluster of 45 proteins that were decreased and a cluster of 21 that were increased in the BEET versus PL trials. Functional enrichment supported significant BEET-related reductions in inflammation-related proteins including several proteins related to complement activation, the acute phase response, and immune cell adhesion, migration, and differentiation. Intake of a BEET-based supplement during a 2-week period was linked to higher plasma levels of NO3 - + NO2 -, elevated post-exercise levels of two anti-inflammatory oxylipins, and a significant decrease in a cluster of proteins involved in complement activation and inflammation. These data support that 2-weeks intake of nitrate from a mixed beet-based supplement moderated protein biomarkers of exercise-induced inflammation in athletes.

Lipocalin-2 drives neuropsychiatric and cutaneous disease in MRL/lpr mice.

Approximately 20-40% of patients with systemic lupus erythematosus (SLE) experience neuropsychiatric SLE (NPSLE), which often manifests as cognitive dysfunction and depression. Currently, there are no approved treatments for NPSLE because its underlying mechanisms are unclear. Identifying relevant mediators and understanding their contribution to pathogenesis are crucial for developing targeted treatment options. Lipocalin 2 (LCN2) is a multifunctional acute-phase protein that plays important roles in immune cell differentiation, migration, and function. LCN2 has been implicated in models of neuroinflammatory disease. We generated an LCN2-deficient MRL/lpr mouse to evaluate the effects of LCN2 on this classic NPSLE model. To evaluate the effects of LCN2 deficiency on behavior, the mice underwent a battery of behavioral tests evaluating depression, memory, and anxiety. Flow cytometry was used to quantify immune cell populations in the brain, blood, and secondary lymphoid organs. Cutaneous disease was quantified by scoring lesional skin, and skin infiltrates were quantified through immunofluorescent staining. Systemic disease was evaluated through measuring anti-nuclear antibodies by ELISA. In this study, we found that LCN2 deficiency significantly attenuates neuropsychiatric and cutaneous disease in MRL/lpr lupus prone mice, likely by decreasing local infiltration of immune cells into the brain and skin and reducing astrocyte activation in the hippocampus. Anti-nuclear antibodies and kidney disease were not affected by LCN2. As there was no effect on systemic disease, our results suggest that the inflammatory effects of LCN2 were localized to the skin and brain in this model. This study further establishes LCN2 as a potential target to ameliorate organ injury in SLE, including neuropsychiatric and cutaneous disease.

Rapid Preparation of Living Drosophila Pupal Macrophages for Ex Vivo Imaging.

The fruit fly Drosophila is a well-established invertebrate model that enables in vivo imaging of innate immune cell (e.g., macrophage) migration and signaling at high spatiotemporal resolution within the intact, living animal. While optimized methods already exist to enable flow cytometry-based macrophage isolation from Drosophila at various stages of development, there remains a need for more rapid and gentle methods to isolate living macrophages for downstream ex vivo applications. Here, we describe techniques for rapid and direct isolation of living macrophages from mature Drosophila pupae and their downstream ex vivo preparation for live imaging and immunostaining. This strategy enables straightforward access to physiologically relevant innate immune cells, both circulating and tissue-resident populations, for subsequent imaging of signal transduction.

Chemotaxis Assay of Bone Marrow-Derived Macrophages.

Immune responses rely on efficient and coordinated migration of immune cells to the site of infection or injury. To reach the site of immunological threat often requires long-range navigation of immune cells through complex tissue and vascular networks. Chemotaxis, cell migration steered by gradients of cell-attractive chemicals that bind sensory receptors, is central to this response. Chemoattractant receptors mostly belong to the G-protein-coupled receptor (GPCR) family, but the way attractant-receptor signaling directs cell migration is not fully understood. Direct-viewing chemotaxis chambers combined with time-lapse microscopy give a powerful tool to study the dynamic details of cells' responses to different attractant landscapes. Here, we describe the application of one such chamber (the Dunn chamber) to study bone marrow-derived macrophage chemotaxis to gradients of complement C5a.

Emerging Trends in the Application of Extracellular Vesicles as Novel Oral Delivery Vehicles for Therapeutics in Inflammatory Diseases.

Inflammation involves complex immune responses where cytokines such as TNF-α, IL-1, and IL-6 promote vasodilation and increased vascular permeability to facilitate immune cell migration to inflammation sites. Persistent inflammation is linked to diseases like cancer, arthritis, and neurodegenerative disorders. Although oral anti-inflammatory drugs are favored for their non-invasiveness and cost-effectiveness, their efficacy is often compromised due to gastrointestinal degradation and limited bioavailability. Recent advancements highlight the potential of extracellular vesicles (EVs) as nanocarriers that enhance drug delivery by encapsulating therapeutic agents, ensuring targeted release and reduced toxicity. These EVs, derived from dietary sources and cell cultures, exhibit excellent biocompatibility and stability, presenting a novel approach in anti-inflammatory therapies. This review discusses the classification and advantages of orally administered EVs (O-EVs), their mechanism of action, and their emerging role in treating inflammatory conditions, positioning them as promising vectors in the development of innovative anti-inflammatory drug delivery systems.

Adipose Tissue Hypoxia in Obesity: Clinical Reappraisal of Hypoxia Hypothesis.

Obese subjects exhibit lower adipose tissue oxygen consumption in accordance with the lower adipose tissue blood flow. Thereby, compared to lean subjects, obese individuals have almost half lower capillary density and more than half lower vascular endothelial growth factor (VEGF). The VEGF expression together with hypoxia-inducible transcription factor-1 alpha (HIF-1α) activity also requires phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR)-mediated signaling. Especially HIF-1α is an important signaling molecule for hypoxia to induce the inflammatory responses. Hypoxia contributes to several biological functions, such as angiogenesis, cell proliferation, apoptosis, inflammation, and insulin resistance (IR). Pathogenesis of obesity-related comorbidities is attributed to intermittent hypoxia (IH), which is mostly observed in visceral obesity. Proinflammatory phenotype of the adipose tissue is a crucial link between IH and the development of IR. Inhibition of adaptive unfolded protein response (UPR) in hypoxia increases β cell death. Moreover, deletion of HIF-1α worsens β cell function. Oxidative stress, as well as the release of proinflammatory cytokines/adipokines in obesity, is proportional to the severity of IH. Reactive oxygen species (ROS) generation at mitochondria is responsible for propagation of the hypoxic signal; however, mitochondrial ROS production is required for hypoxic HIF-1α protein stabilization. Alterations in oxygen availability of adipose tissue directly affect the macrophage polarization and are responsible for the dysregulated adipocytokines production in obesity. Hypoxia both inhibits adipocyte differentiation from preadipocytes and macrophage migration from the hypoxic adipose tissue. Upon reaching a hypertrophic threshold beyond the adipocyte fat loading capacity, excess extracellular matrix (ECM) components are deposited, causing fibrosis. HIF-1α initiates the whole pathological process of fibrosis and inflammation in the obese adipose tissue. In addition to stressed adipocytes, hypoxia contributes to immune cell migration and activation which further aggravates adipose tissue fibrosis. Therefore, targeting HIF-1α might be an efficient way to suppress hypoxia-induced pathological changes in the ECM. The fibrosis score of adipose tissue correlates negatively with the body mass index and metabolic parameters. Inducers of browning/beiging adipocytes and adipokines, as well as modulations of matrix remodeling enzyme inhibitors, and associated gene regulators, are potential pharmacological targets for treating obesity.

GLIS1, Correlated with Immune Infiltrates, Is a Potential Prognostic Biomarker in Prostate Cancer.

Prostate cancer (PCa) is a prevalent malignant disease and the primary reason for cancer-related mortality among men globally. GLIS1 (GLIS family zinc finger 1) is a key regulator in various pathologies. However, the expression pattern, clinical relevance, and immunomodulatory function of GLIS1 in PCa remain unclear. In this study, GLIS1 was discovered to serve as a key gene in PCa by integrating mRNA and miRNA expression profiles from GEO database. We systematically explored the expression and prognostic values of GLIS1 in cancers using multiple databases. Additionally, we examined the functions of GLIS1 and the relationship between GLIS1 expression levels and immune infiltration in PCa. Results showed that GLIS1 was differentially expressed between normal and tumor tissues in various cancer types and was significantly low-expressed in PCa. Low GLIS1 expression was associated with poor PCa prognosis. GLIS1 was also involved in the activation, proliferation, differentiation, and migration of immune cells, and its expression showed a positive correlation with the infiltration of various immune cells. Moreover, GLIS1 expression was positively associated with various chemokines/chemokine receptors, indicating the involvement in regulating immune cell migration. In summary, GLIS1 is a potential prognostic biomarker and a therapeutic target to modulate anti-tumor immune response in PCa.

Anti-Inflammatory Activity of Pequi Oil (Caryocar brasiliense): A Systematic Review.

Disorders in the inflammatory process underlie the pathogenesis of numerous diseases. The utilization of natural products as anti-inflammatory agents is a well-established approach in both traditional medicine and scientific research, with studies consistently demonstrating their efficacy in managing inflammatory conditions. Pequi oil, derived from Caryocar brasiliense, is a rich source of bioactive compounds including fatty acids and carotenoids, which exhibit immunomodulatory potential. This systematic review aims to comprehensively summarize the scientific evidence regarding the anti-inflammatory activity of pequi oil. Extensive literature searches were conducted across prominent databases (Scopus, BVS, CINAHL, Cochrane, LILACS, Embase, MEDLINE, ProQuest, PubMed, FSTA, ScienceDirect, and Web of Science). Studies evaluating the immunomodulatory activity of crude pequi oil using in vitro, in vivo models, or clinical trials were included. Out of the 438 articles identified, 10 met the stringent inclusion criteria. These studies collectively elucidate the potential of pequi oil to modulate gene expression, regulate circulating levels of pro- and anti-inflammatory mediators, and mitigate oxidative stress, immune cell migration, and cardinal signs of inflammation. Moreover, negligible to no toxicity of pequi oil was observed across the diverse evaluated models. Notably, variations in the chemical profile of the oil were noted, depending on the extraction methodology and geographical origin. This systematic review strongly supports the utility of pequi oil in controlling the inflammatory process. However, further comparative studies involving oils obtained via different methods and sourced from various regions are warranted to reinforce our understanding of its effectiveness and safety.