Changes In Immune Cell Populations Research Articles

P0050 4-Octyl Itaconate Protects Against Colitis by Modulating Neutrophil Function and Enhancing Intestinal Epithelial Barrier Integrity

Abstract Background Inflammatory bowel disease (IBD) is a complex gastrointestinal condition marked by immune system dysfunction and intestinal barrier damage. The pathogenesis involves a mix of genetic, environmental, and immunological factors, with the potential for itaconic acid derivatives like 4-Octyl itaconate (4-OI) to modulate immune responses and offer therapeutic benefits. Methods The study utilized a mouse model of acute colitis induced by dextran sulfate sodium (DSS) to evaluate the effects of 4-OI. C57BL/6J mice were divided into control and treatment groups, with interventions including 4-OI administration. Lamina Propria Mononuclear Cells (LPMC) isolation, flow cytometry, neutrophil studies, Western blot, qRT-PCR, and RNA-seq profiling were conducted to assess changes in immune cell populations, protein and gene expression, and global transcriptional responses. Results 4-OI treatment resulted in the amelioration of DSS-induced colitis, evidenced by improved colonic epithelial barrier function, reduced secretion of pro-inflammatory factors, and dampened inflammatory signaling pathways. Notably, 4-OI reduced neutrophil infiltration and activity, including the formation of neutrophil extracellular traps (NETs), with minimal impact on T cells and macrophages. Moreover, we discovered that 4-OI primarily targeted the acute phase of the disease, with less satisfactory effects on alleviating chronic colitis models. Conclusion 4-OI demonstrates potential as a therapeutic agent in acute colitis by preserving the intestinal epithelial barrier and modulating neutrophil activity. While the study provides evidence of 4-OI's effects on neutrophils and epithelial cells, further research is necessary to understand its mechanisms, particularly regarding chronic colitis and potential clinical applications.

Efekti adjuvantne terapije kolhicinom na kontrolu bolesti, serum NALP3, sICAM-1, MMP-9 i MMP-13 kod pacijenata sa koronarnom bolešću srca i akutnim napadom gihta

Background: To investigate the impact of colchicine adjuvant therapy on disease control and serum levels of nucleotide-binding oligomerization domain-like receptor (NALP) 3, soluble intercellular adhesion molecule (sICAM)-1, matrix metalloproteinase (MMP)-9, and MMP13 in patients with coronary heart disease (CHD) complicated by acute gout attacks. Methods: Ninety-two patients with CHD and acute gout attacks admitted to our hospital from October 2021 to January 2023 were randomly divided into an observation group and a control group, with 46 patients in each group. The control group received conventional treatment, while the observation group received colchicine adjuvant therapy on top of the control group's treatment for 7 days. Clinical efficacy in both groups was assessed. Before and after treatment, cardiac function indicators (left ventricular ejection fraction (LVEF), left ventricular end-diastolic diameter (LVEDD), left ventricular posterior wall thickness (LVPWT)), vascular endothelial function indicators (sICAM-1, endothelin-1 (ET-1), and vascular endothelial growth factor (VEGF)), inflammatory factors (NALP3, MMP-9, MMP-13) levels, changes in immune cell populations (CD3+ lymphocytes, CD3+CD4+ lymphocytes, CD3+CD8+ lymphocytes ratio, and CD3+CD4+/CD3+CD8+ ratio) were compared, and the incidence of adverse reactions was recorded. Three months after treatment, the occurrence of major adverse cardiovascular events was also recorded. Results: The total effective rate in the observation group was significantly higher than that in the control group (93.48% vs 79.07%) (P<0.05). After treatment, the levels of NALP3, MMP-9, and MMP-13 in both groups decreased, with the observation group being lower than the control group (P<0.05). After treatment, LVPWT and LVEDD levels in the observation group were lower than those in the control group, and LVEF was higher (P<0.05). After treatment, the levels of ET-1 and sICAM-1 in the observation group were lower than those in the control group, and VEGF levels were higher (P<0.05). After treatment, the proportions of CD3+ lymphocytes, CD3+CD4+ lymphocytes, and CD3+CD4+/CD3+CD8+ ratio were significantly higher in the observation group than in the control group (P<0.05). There was no significant difference in the incidence of adverse reactions between the two groups (P>0.05). The occurrence of major adverse cardiovascular events in the observation group was lower than that in the control group (2.17% vs 13.04%). Conclusions: Colchicine adjuvant therapy improves the efficacy of CHD patients with acute gout attacks, helps improve cardiac function and vascular endothelial function, reduces serum levels of NALP3, sICAM-1, MMP-9, and MMP-13, enhances patient immunity, and controls disease progression.

Single-cell RNA sequencing of the spleen reveals differences in Salmonella typhimurium infection mechanisms between different chicken breeds.

Bacterial infections remain an important cause of morbidity in poultry production. The molecular characteristics and dynamic changes in immune cell populations after bacterial infection have yet to be fully understood. Beijing-You chicken and Cobb broiler, two broiler breeds with different disease resistance, were infected with Salmonella typhimurium, and inflammation models were constructed. Compared to Beijing-You, Cobb showed higher survival rates, lower liver load, and milder spleen damage after Salmonella infection. We characterized chicken spleen CD45+ immune cells by single-cell RNA sequencing and identified 9 distinct cell types among 54,487 cells. In Beijing-You, mono-macrophages expressed higher levels of pro-inflammatory factors, including IL1B, IL6, and M-CSF, after bacterial infection. In Cobb, Tregs exhibited intense inflammatory inhibition and highly expressed CTLA4, LAG3 and other immunosuppressive regulators. In addition, we found complex macrophage phenotypes during bacterial infection, with a tendency in macrophages from pro-inflammatory phenotypes (Mac-IL1B) to anti-inflammatory phenotypes (Mac-C1QC/Mac-MARCO). This study represents the first single-cell transcriptomic analysis of chicken spleen and compares the immune responses of Beijing-You and Cobb after bacterial infection. These findings provide insight into the mechanism of inflammation regulation in different broiler breeds.

Abstract C008: New insights from a novel mouse model of synovial sarcoma

Abstract Mouse models are the basis of preclinical and translational research in solid tumors. Multiple methods exist to induce tumor formation in mice, including genetically engineered mouse models, chemotoxic agents, injection of tumor cells, and xenograft approaches. Synovial sarcoma, a rare highly malignant soft-tissue tumor, poses a serious threat to patients due to its aggressive nature and associated high rates of mortality and morbidity. This disease's underlying cause and development remain poorly comprehended, and existing treatment approaches have yielded unsatisfactory outcomes. In the last two decades, numerous effective animal models of soft tissue sarcoma (STS) have been created, primarily utilizing genetically engineered mice and zebrafish, however long latency of tumor development for several months in these models creates a barrier for practical in-vivo experiments for drug testing or manipulating the cellular composition of the tumor microenvironment. In studying cancer immunotherapy, it is important to consider aspects of antitumor immune responses and to produce a model that mimics the complexity of the immune system We have generated a new mouse model of synovial sarcoma of exceptionally fast tumor development. Intramuscular delivery of Cre recombinase protein in mice which drives conditional knockdown in PTEN, stabilization of β-catenin, and formation of fusion oncogenes of SS18-SSX2 was sufficient to initiate high-grade synovial sarcomas with myofibroblastic differentiation in 3 weeks. As immunotherapy is increasingly applied to sarcomas, our mouse model will serve as a great tool for preclinical data. Flow cytometry-based analysis of peripheral blood shows significant changes in immune cell populations as early as five days after Tat-Cre injection and continues throughout the development of synovial sarcoma. These results suggest a significant systemic phenotypic alteration of circulating immune cells, which may impact other major organs in this genetic mouse model of synovial sarcoma. Single- cell RNA sequencing of the CD45+ leukocytes in the tumor microenvironment reveals subsets that may promote tumor progression. Our work promises to significantly enhance our understanding of the pathogenesis of this disease and establish a reliable preclinical platform for devising and assessing therapeutic interventions. Citation Format: Hesham Mohei, Donn Van Deren, Irene S. Molina, Malay Haldar. New insights from a novel mouse model of synovial sarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C008.

Blood integrin- and cytokine-producing T cells are associated with stage and genetic risk score in age-related macular degeneration

Age-related macular degeneration (AMD) remains a leading cause of vision loss in the geriatric population. There are age-related changes in peripheral blood leukocyte composition, but their significance for AMD remains unclear. We aimed to determine changes in immune cell populations in the blood of AMD patients. A standardized 31-parameter flow cytometry analysis was conducted on peripheral blood mononuclear cells from 59 patients with early and advanced AMD and 39 controls without AMD, all older than 65 years. Fundus photography and optical coherence tomography were used to classify disease stages and a custom genotype array was used to compute an AMD genetic risk score based on 52 AMD disease risk variants (GRS-52). A generalized linear regression model corrected for age, sex, and smoking status revealed that AMD patients showed decreased frequencies of CD4+ T helper cell population expressing Integrin Alpha E (CD103) (Padj = 0.019). We further noted that early AMD was characterized by increased interleukin-4 (IL-4)-producing CD4+ T helper cells (Padj = 0.013; <0.001), as well as IL-4-producing cytotoxic CD8+ T cells (Padj = 0.016; <0.001). Reclassification of samples based on the GRS-52 revealed that IL-17-producing T cells decreased incrementally across GRS-52 categories. In AMD, alterations in peripheral blood leukocyte populations are associated with genetic risk score and disease stage and include specifically IL-4 and IL-17A cytokine-producing and CD103 integrin-expressing T cell populations.

Changes in immune cell populations during acclimatization to high altitude.

The immune response to acute hypoxemia may play a critical role in high-altitude acclimatization and adaptation. However, if not properly controlled, hypoxemia-induced inflammation may exacerbate high-altitude pathologies, such as acute mountain sickness (AMS), or other hypoxia-related clinical conditions. Several studies report changes in immune cell subsets at high altitude. However, the mechanisms underlying these changes, and if these alterations are beneficial or maladaptive, remains unknown. To address this, we performed multiparameter flow cytometry on peripheral blood mononuclear cells (PBMCs) collected throughout 3 days of high-altitude acclimatization in healthy sea-level residents (n = 20). Additionally, we conducted invitro stimulation assays to test if high-altitude hypoxia exposure influences responses of immune cells to subsequent inflammatory stimuli. We found several immune populations were altered at high altitude, including monocytes, T cells, and B cells. Some changes in immune cell populations are potentially correlated with AMS incidence and severity. Invitro high-altitude PBMC cultures stimulated with lipopolysaccharide (LPS) showed no changes in pro-inflammatory cytokine production after 1 day at high-altitude. However, by day three pro-inflammatory cytokine production in response to LPS decreased significantly. These results indicate that high-altitude exposure may initiate an inflammatory response that encompasses innate immune sensitization, with adaptive immune suppression following acclimatization.

Early-Life Respiratory Syncytial Virus (RSV) Infection Triggers Immunological Changes in Gut-Associated Lymphoid Tissues in a Sex-Dependent Manner in Adulthood.

Severe respiratory syncytial virus (RSV) infection during early life has been linked to gut dysbiosis, which correlates with increased disease severity and a higher risk of developing asthma later in life. However, the impact of such early-life RSV infections on intestinal immunity in adulthood remains unclear. Herein, we show that RSV infection in 3-week-old mice induced persistent differential natural killer (NK) and T cell profiles within the lungs and gastrointestinal (GI) lymphoid tissues (GALT) in adulthood. Notably, male mice exhibited more pronounced RSV-induced changes in immune cell populations in both the lungs and GALT, while female mice displayed greater resilience. Importantly, early-life RSV infection was associated with the chronic downregulation of CD69-expressing T lymphocytes, particularly T regulatory cells in Peyer's patches, which could have a significant impact on T cell functionality and immune tolerance. We propose that RSV infection in early life is a trigger for the breakdown in immune tolerance at mucosal surfaces, with potential implications for airways allergic disease, food allergies, and other GI inflammatory diseases.

Bioinformatics analysis of therapeutic targets for idiopathic pulmonary fibrosis and exploration of immune cell infiltration patterns

Idiopathic pulmonary fibrosis (IPF) is a severe progressive lung disease characterized by fibrotic changes in lung tissue, with limited treatment options. This study analyzed gene expression data from three gene expression omnibus datasets (GSE2052, GSE53845, and GSE110147) using R and LIMMA to identify differentially expressed genes (DEGs) in IPF samples. We identified 215 DEGs, comprising 106 upregulated and 109 downregulated genes. Weighted gene coexpression network analysis revealed five gene modules, with the module eigengene yellow showing the strongest correlation with IPF. Functional enrichment analysis of 40 consensus genes in this module indicated their significant involvement in extracellular matrix (ECM) organization. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed pathways related to protein digestion, cell adhesion molecules, and the advanced glycation end product&amp;ndash;receptor for advanced glycation end product signaling pathway. Based on protein&amp;ndash;protein interaction network analysis, collagen type XV alpha 1 chain (COL15A1) and collagen type VI alpha 3 chain (COL6A3) were identified as upregulated hub genes in IPF, which were regulated by microRNAs and transcription factors. Immune cell infiltration analysis showed significant changes in immune cell populations in IPF samples, with increases in memory B cells, plasma cells, and M0 macrophages and decreases in CD8 T cells, and resting natural killer cells. Potential drugs targeting COL15A1 and COL6A3 were predicted using multiple databases, revealing compounds such as (+)-JQ1, aristolochic acid I, and dexamethasone with promising binding potential. These findings suggest that COL15A1 and COL6A3 are central hub genes in IPF, are associated with ECM organization and immune response, and serve as therapeutic targets for IPF.

Extracorporeal photopheresis vs. systemic immunosuppression for immune-related adverse events: Interim analysis of a prospective two-arm study

BackgroundCheckpoint inhibitor-induced steroid-refractory (sr) and steroid-dependent (sd) immune-related adverse events (irAE) account for about 11 % of irAE. Although these patients face worse outcomes due to irAE mortality and/or sustained immunosuppression, which impairs anti-tumor response, there is no established second-line treatment based on prospective trial data. MethodsThis prospective comparative study investigates outcomes of extracorporeal photopheresis (ECP), an immunomodulating therapy, versus second-line immunosuppressants (SLI) in sr/sd-irAE. The primary endpoint was longitudinal change in immunophenotype; secondary endpoints were outcome of irAE and tumor response. Patient demographics, quality of life (EORTC QLQ-C30; global health status (GHS/QoL)) and longitudinal blood samples were analyzed at baseline; in weeks 1, 4, 8, and 12. ResultsAt interim analysis, 21 patients (11 ECP, 10 SLI) with 7 different sr/sd-irAE were included. Compared with the SLI group, the ECP group demonstrated a higher clinical response rate of irAE (93 % vs. 80 %; 95 % CI 0.83–1.92; P = 0.54) and a better GHS/QoL score throughout all follow-up visits. ECP patients showed a numerically higher overall survival (23 vs. 12 months; 95 % CI 0.02–3.02; P = 0.27) and lower cancer progression rates (33 % vs. 67 %; 95 % CI 0.09–1.60; P = 0.52). Immunophenotyping revealed changes in immune cell populations and the regulation of immune checkpoints. There were no significant safety issues in either treatment group. ConclusionThis prospective comparative study supports the clinical efficacy of ECP in the treatment of sr/sd-irAE in comparison to the SLI cohort. Thus, ECP represents a potential treatment option for this indication, given its good safety profile while maintaining anti-tumor response. Trial RegistrationClinicalTrials.gov, NCT05700565, https://classic.clinicaltrials.gov/ct2/show/NCT05700565.

Cigarette smoking prolongs inflammation associated with influenza infection and delays its clearance in mice.

Epidemiological studies have shown that smoking is associated with increased incidence of severe viral infections leading to hospitalization. Moreover, studies in experimental models have identified impaired antiviral responses and altered inflammatory responses, yet it is unclear how the effects of smoke exposure and influenza A infection interact and how this varies over the course of infection. We hypothesized that smoking would exacerbate innate immune responses against influenza. To test this, female BALB/c mice were exposed to cigarette smoke or air twice a day for 24-28 days and (mock) infected with H3N2 influenza A on day 21 while smoking continued. Three and seven days after infection, changes in immune cell populations, the transcriptome, and viral clearance in lung tissue were analyzed. After influenza A infection, smoke-exposed mice lost significantly more weight than air-exposed controls, indicating that smoking resulted in more severe disease. Immune cell and lung tissue transcriptome analysis revealed that neutrophil infiltration was prolonged and macrophage activation dysregulated after infection of smoke-exposed mice compared to air-exposed controls. Expression of genes in IL-6 and interferon pathways was similarly longer active. In parallel, we observed lower clearance of influenza virus in smoke-exposed mice after infection compared to air-exposed controls, indicating ineffective antiviral responses. Altogether, the data from our mouse model indicate that cigarette smoke exposure prolongs innate immune responses against influenza A. The results from this study help to explain the susceptibility of current smokers to severe influenza A disease.

Bone Marrow and Peripheral Blood Mononuclear Cell Phenotype Changes after Cultivation and Autologous Infusion in Patients with Primary Biliary Cholangitis.

Primary biliary cholangitis (PBC) is a condition affecting the liver and immune system. In this study, the impact of autologous bone marrow-derived mononuclear cell (BM-MNC) transplantation on PBC patients was investigated. Sixteen eligible PBC patients participated at the National Scientific Medical Center in Astana, Kazakhstan, between 2017 and 2022, and BM-MNCs were harvested from their anterior iliac crest. After isolating and cultivating the BM-MNCs, they were infused back into the patient's peripheral veins. Changes in BM-MNC and peripheral blood mononuclear cell (PB-MNC) phenotypes were assessed before and after a 24-hour cultivation period and 72 hours post-transplantation. We monitored liver function parameters over 6-month intervals and conducted flow cytometry analysis to assess CD markers on BM-MNCs before and after cultivation and PB-MNCs before and after transplantation. Statistical analysis included the Friedman test for liver parameters and the Wilcoxon signed-rank test for BM-MNC and PB-MNC comparisons. Our findings revealed significant reductions in liver function tests after multiple transplantations. Flow cytometry analysis before and after a 24-hour culture and autologous BM-MNC infusion revealed the expansion of specific cell populations, with significant increases in CD3+, CD4+, CD16+, CD20+, CD25+, CD34+, CD105+, CD73+, СD117+, and CD34+populations, while CD4+25+, CD34+105+, and CD4+FOXP3+ populations decreased. Interestingly, a contradictory finding was observed with a decrease in bone marrow CD34+105+ cell lines (P=0.03) alongside an increase in peripheral CD34+105+ population (P=0.03). In summary, our study shows that BM-MNC transplantation in PBC patients leads to changes in immune cell populations and liver function. These findings suggest potential therapeutic applications of BM-MNC transplantation in managing PBC and offer insights into the dynamics of immune cells associated with this treatment approach.

Time to resolution of airway inflammation caused by bronchoalveolar lavage in healthy horses.

Bronchoalveolar lavage (BAL) is a common procedure for evaluation of the equine lower airways. Time to resolution of post-BAL inflammation has not been clearly defined. Residual inflammation, evident by changes in immune cell populations and inflammatory cytokines, will resolve by 72 hours after BAL. Six adult, healthy, institution-owned horses. Randomized, complete cross-over design. Each horse underwent 3 paired BALs, including a baseline and then 48, 72, and 96 hours later, with a 7-day washout between paired BALs. Each sample underwent cytological evaluation and cytokine concentrations were determined by a commercially available multiplex bead immunoassay. Statistical analysis was performed by multilevel mixed-effects Poisson regression analysis. Data are reported as marginal means and 95% confidence interval (CI). Neutrophil, eosinophil and mast cell percentages were not significantly different at any time points. Macrophage percentages were higher at 72 hours (45.0 [95% CI, 41.6-48.4]%) and 96 hours (45.3 [95% CI, 42.9-47.7]%) vs baseline (37.4 [95% CI, 33.5-41.4]%; P < .001 and P = .01, respectively), and at 72 hours and 96 hours vs 48 hours (31.9 [95% CI, 28.1-35.6]%; P < .001). Neutrophil percentage was not significantly increased at 48 hours (P = .11). Interleukin (IL)-6 concentration was increased at 72 hours (5.22 [95% CI, 3.44-6.99] pg/mL) vs 48 hours (4.38 [95% CI, 2.99-5.78] pg/mL; P < .001). Significant lung inflammation was not detected at 72 and 96 hours, suggesting that repeating BAL at 72 hours or more can be done without concern of residual inflammation.

Isolating Immune Cells from Mouse Brain and Skull.

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only in the brain, but also in the skull. A key step toward analyzing changes in immune cell populations in both the brain and skull bone marrow after brain damage (e.g., stroke) is to obtain sufficient numbers of high-quality immune cells for downstream analyses. Here, two optimized protocols are provided for isolating immune cells from the brain and skull bone marrow. The advantages of both protocols are reflected in their simplicity, speed, and efficacy in yielding a large quantity of viable immune cells. These cells may be suitable for a range of downstream applications, such as cell sorting, flow cytometry, and transcriptomic analysis. To demonstrate the effectiveness of the protocols, immunophenotyping experiments were performed on stroke brains and normal brain skull bone marrow using flow cytometry analysis, and the results aligned with findings from published studies.

Endoplasmic reticulum stress-related gene expression patterns in glioblastoma models identified by single-cell RNA sequencing: Implications for prognosis and treatment

Objective: Endoplasmic reticulum (ER) stress has therapeutic potential for a variety of malignancies, including glioma. In this study, bioinformatics was used to analyze ER stress-related genes (ERGs) in glioblastoma (GBM), explore their functions and pathways, construct prognostic models, and explore new treatment strategies. Methods: Various bioinformatics algorithms were utilized to screen for ERGs and construct a risk model. According to the expression of ERGs, different subtypes were classified using the consensus clustering method. Gene Set Variation Analysis (GSVA) and Gene Set Enrichment Analysis (GSEA) were performed on the subtypes. Based on screened risk genes, GBM patients were divided into Train and Test groups in a 1:1 ratio. The prognostic model was validated through Receiver Operating Characteristic (ROC) curve analysis and independent prognostic analysis. The model was further validated by comparing the risk scores between high-risk and low-risk groups, and comparisons were made in terms of survival time, immune microenvironment, and pathway regulation. Drug sensitivity was used to screen drugs for low- and high- risk group, and single-cell RNA sequencing (scRNA-seq) analysis were utilized to explore the expression distribution of risk genes in GBM. Results: According to the ERGs, GBM samples can be divided into two groups with significant differences. Cluster A showed better survival rates compared to Cluster B. GSVA and GSEA analysis revealed that Cluster A was mainly enriched in glutamate receptor signaling pathway, synaptic transmission between neurons, postsynaptic density membrane, postsynaptic membrane, and synaptic vesicle membrane functions. It is worth noting that 8 ERGs were screened as model genes, which can effectively and independently predicate the survival risk of GBM patients with high accuracy and discrimination ability. Subsequently, changes in immune cell populations were observed in high-risk and low-risk groups, with differences in memory B cells and resting CD4 memory T cells between the high-risk and low-risk groups. The high-risk group had higher levels of memory B cells, while the low-risk group had higher levels of resting CD4 memory T cells. Furthermore, potential therapeutic strategies were identified, with BI-2536, Daporinad, SB505124, UMI-77, and Vorinostat identified for the low-risk group, while AZD8055, Camptothecin, Gemcitabine, PD0325901, and Topotecan identified for the high-risk group. scRNA-seq identified Ribosomal Protein L10 (RPL10) as one of the eight ER stress-related genes, primarily expressed in malignant cells of various tumors. Conclusion: This study identified eight ERGs and constructed a risk model based on bioinformatics analysis, which can be used for prognosis prediction and drug screening.

Dynamic changes in immune cell populations by AXL kinase targeting diminish liver inflammation and fibrosis in experimental MASH.

Metabolic dysfunction-associated steatohepatitis (MASH) is a significant health concern with limited treatment options. AXL, a receptor tyrosine kinase activated by the GAS6 ligand, promotes MASH through activation of hepatic stellate cells and inflammatory macrophages. This study identified cell subsets affected by MASH progression and the effect of AXL inhibition. Mice were fed chow or different fat-enriched diets to induce MASH, and small molecule AXL kinase inhibition with bemcentinib was evaluated. Gene expression was measured by qPCR. Time-of-flight mass cytometry (CyTOF) used single cells from dissociated livers, acquired on the Fluidigm Helios, and cell populations were studied using machine learning. In mice fed different fat-enriched diets, liver steatosis alone was insufficient to elevate plasma soluble AXL (sAXL) levels. However, in conjunction with inflammation, sAXL increases, serving as an early indicator of steatohepatitis progression. Bemcentinib, an AXL inhibitor, effectively reduced proinflammatory responses in MASH models, even before fibrosis appearance. Utilizing CyTOF analysis, we detected a decreased population of Kupffer cells during MASH while promoting infiltration of monocytes/macrophages and CD8+ T cells. Bemcentinib partially restored Kupffer cells, reduced pDCs and GzmB- NK cells, and increased GzmB+CD8+ T cells and LSECs. Additionally, AXL inhibition enhanced a subtype of GzmB+CD8+ tissue-resident memory T cells characterized by CX3CR1 expression. Furthermore, bemcentinib altered the transcriptomic landscape associated with MASH progression, particularly in TLR signaling and inflammatory response, exhibiting differential cytokine expression in the plasma, consistent with liver repair and decreased inflammation. Our findings highlight sAXL as a biomarker for monitoring MASH progression and demonstrate that AXL targeting shifted liver macrophages and CD8+ T-cell subsets away from an inflammatory phenotype toward fibrotic resolution and organ healing, presenting a promising strategy for MASH treatment.

Immune Activation Promotes Cardiac Remodeling in a Mouse Model of Takotsubo Cardiomyopathy

Takotsubo Cardiomyopathy (TCM) is an acute, life-threatening condition characterized by a drop in ejection fraction (EF) and apical ballooning of the left ventricle (LV). TCM is most frequently observed in women and is associated with an emotional or physical stressor. Importantly, with medical intervention, cardiac dysfunction is transient, and EF is often restored over weeks to months. Despite this, patients who experience TCM are at an increased risk of additional cardiovascular events and premature mortality. The precise etiology of TCM is unknown but a wave of excess circulating catecholamines is thought to be a trigger. Furthermore, the cause for increased cardiovascular risk post-TCM is unknown. The goal of this study is to understand how the immune system may contribute to TCM and the associated cardiovascular event risk. To induce TCM, mice were given a single dose of 200mg/kg isoproterenol (ISO), i.p. and cardiac function was assessed by conscious echocardiography (ECG). An initial study compared the effect of ISO between sexes (n=4-6). Though ISO induced a drop in EF in both sexes after 24 hours (p&lt;0.001 compared to vehicle (Veh)), females exhibited lower EF (46.64±3.11%) than males (68.36±2.62%, p&lt;0.0001). This mirrors the increased prevalence of clinically defined TCM in females in human populations. For this reason, female mice were used for the following experiments. A time course study demonstrated that EF begins to normalize by Day 3 (78.78±2.70 v 90.80±1.00%, p&lt;0.001) and is restored to baseline levels by Day 7 (90.65±1.17%). Flow cytometry was used to identify changes in immune cell populations in the heart on Days 0, 1, 3 and 7 (n=4-5). Neutrophils and inflammatory monocytes increased on Day 1, returning to baseline by Day 3. Dendritic cells and macrophages were elevated on Day 3 post-ISO, with a specific increase in CCR2+ TIM4- recruited inflammatory macrophages (p&lt;0.0001). Across all subsets, the number of immune cells returned to baseline levels by Day 7. Due to the robust immune response observed, we tested the specific hypothesis that transfer of immune cells from mice who have experienced TCM would induce cardiac dysfunction in naïve recipient mice. Donor mice were treated with either Veh or ISO and 1 day later splenocyte isolation and transfer was performed. Importantly, transfer of splenocytes from mice who had TCM induced a significant drop in EF the following day in the recipient mice (86.30±1.08%, p=0.001, n=10) concurrent with an increase in LV inner diameter and volume during systole. A final study tested the hypothesis that a history of TCM would increase risk for future cardiovascular disease. Veh or ISO-treated mice were allowed to recover for 10 days. Tail-cuff plethysmography showed no significant difference in baseline blood pressure and minipumps were implanted to deliver a constant infusion of Angiotensin II (Ang II, 140ng/kg/min). Mice with a history of TCM exhibited higher blood pressures compared to controls (135.11±3.32 v 116.9±6.41mmhg, n=6/9, p&lt;0.05) by week 2 of Ang II infusion. These results together demonstrated that TCM is associated with a robust immune response and points to a possible causal contribution of immune activation to the cardiac dysfunction observed in TCM. Furthermore, a history of TCM promotes a hypertensive phenotype, but additional studies are needed to test whether this is through immunological mechanisms. AHAPOST837441 DJF, 19EIA34480023-MSM, and 1R01 HL161212-MSM. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Predictive biomarkers and initial analysis of maternal immune alterations in postpartum preeclampsia reveal an immune-driven pathology.

Postpartum preeclampsia (PPPE) is an under-diagnosed condition, developing within 48 hours to 6 weeks following an uncomplicated pregnancy. The etiology of PPPE is still unknown, leaving patients vulnerable and making the identification and treatment of patients requiring postpartum care an unmet need. We aimed to understand the immune contribution to PPPE at the time of diagnosis, as well as uncover the predictive potential of perinatal biomarkers for the early postnatal identification of high-risk patients. Placentas were collected at delivery from uncomplicated pregnancies (CTL) and PPPE patients for immunohistochemistry analysis. In this initial study, blood samples in PPPE patients were collected at the time of PPPE diagnosis (48h-25 days postpartum; mean 7.4 days) and compared to CTL blood samples taken 24h after delivery. Single-cell transcriptomics, flow cytometry, intracellular cytokine staining, and the circulating levels of inflammatory mediators were evaluated in the blood. Placental CD163+ cells and 1st trimester blood pressures can be valuable non-invasive and predictive biomarkers of PPPE with strong clinical application prospects. Furthermore, changes in immune cell populations, as well as cytokine production by CD14+, CD4+, and CD8+ cells, suggested a dampened response with an exhausted phenotype including decreased IL1β, IL12, and IFNγ as well as elevated IL10. Understanding maternal immune changes at the time of diagnosis and prenatally within the placenta in our sizable cohort will serve as groundwork for pre-clinical and clinical research, as well as guiding clinical practice for example in the development of immune-targeted therapies, and early postnatal identification of patients who would benefit from more thorough follow-ups and risk education in the weeks following an uncomplicated pregnancy.

Cerebral and Peripheral Immune Cell Changes following Rodent Juvenile Traumatic Brain Injury.

Traumatic brain injury (TBI) is one of the leading causes of death and disability. TBI is associated with neuroinflammation, but temporal changes in immune and inflammatory signaling following TBI have not been fully elucidated. Furthermore, there have been no previous studies on changes in immune cell populations following TBI via the Closed Head Injury Model of Engineered Rotational Acceleration (CHIMERA). The current study aimed to determine the time course changes to inflammatory marker mRNA expression in the acute period following TBI in juvenile rats and to determine acute changes to brain and circulating immune cell populations. For this study, post-natal day (PND)-30 male Long Evans rats sustained a TBI or Sham TBI and were euthanized at 0, 3, 6, 12, 24, or 96 h post-injury. Prefrontal cortex and hippocampus samples were used to determine mRNA expression changes of inflammatory factors. The mRNA expression of the pro-inflammatory cytokine TNF-α was significantly elevated at 6 h post-injury in both regions evaluated. To evaluate immune cell populations, male Long Evans rats were euthanized at 48 h post-injury, and brain and blood samples were used for cell sorting by marker-specific antibodies. In the peripheral blood, there was an elevation in CD3+ total T cells, CD45R+ total B cells, and CD3+CD4+ helper T cells in the TBI subjects. However, there were no changes to natural killer cells or CD3+CD8+ cytotoxic T cell populations. In the brain, there was a reduction in CD11b/c+ monocytes/macrophages, but no changes in other immune cell populations. At 48 h post-injury, the TBI subjects also demonstrated expansion of the thymic medulla. These changes in the cerebral and blood immune cell populations and thymic medulla expansion may implicate the subacute recovery timeframe as a vulnerable window for the immune system in the pediatric population.

(9) - High-Dimensional Lung Tissue Imaging Reveals Temporal Changes in Immune Cell Populations and Cell Interactions During Progression of Chronic Lung Allograft Dysfunction (CLAD)

(9) - High-Dimensional Lung Tissue Imaging Reveals Temporal Changes in Immune Cell Populations and Cell Interactions During Progression of Chronic Lung Allograft Dysfunction (CLAD)

Abstract 3388: Targeting liver specific βII-Spectrin to prevent the development of metabolic dysfunction associated steatohepatitis (MASH) through CEACAM1 axis

Abstract Background: Obesity and pro-inflammatory alteration of the gut microbiome are risk factors for gastrointestinal (GI) cancers, including liver cancer (HCC). Dysregulated TGF-β signaling and loss of CEACAM1 activity are implicated in fatty liver disease. Clinically, 40% of human liver and GI cancers have genetic alterations in the TGF-β pathway members. CEACAM1 expression is significantly decreased in the liver of patients with Metabolic dysfunction-associated steatohepatitis (MASH). In contrast, in hepatocellular carcinoma (HCC), high CEACAM1 is associated with invasion, metastasis and poor prognosis. We hypothesized that TGF-β signaling and βII-Spectrin modulate MASH and HCC through CEACAM1. Methods: Wild type and the liver-specific βII-Spectrin (Sptbn1) knockout (LSKO) mice fed a high-fat diet (HFD) or Western diet (WD) and analyzed for Ceacam1 expression, changes in immune cell population, liver inflammation, and cancer. We used Alphafold and molecular modeling to identify the CEACAM1 and SPTBN1 interaction sites. Results: Single cell sequencing analysis of MASH mice liver revealed that Ceacam1 expression is significantly decreased in hepatocytes. Interestingly, we found that CEACAM1 levels are restored to normal in tissues from liver-specific βII-Spectrin knockout (LSKO) mice. Transcriptomic and multiplex imaging analysis suggest that proinflammatory TGF-β signaling is activated and infiltration of CD11b+ cells is increased in MASH liver. These phenotypes are blocked in LSKO mice. Furthermore, siRNA-knockdown of βII-Spectrin blocks diet-induced obesity, fibrosis, lipid accumulation, tissue damage, and cancer susceptibility. We have also found that CEACAM1 interacts with the Smad3 adaptor, βII-Spectrin. Furthermore, structural analysis of CECAM1 and βII-Spectrin revealed critical residues involved in the interaction. Conclusion: These data suggest that βII-Spectrin regulates CEACAM1 expression in the liver and is a potential target for therapeutic intervention to treat MASH and prevent progression to HCC. Citation Format: Krishanu Bhowmick, Kazufumi Ohshiro, Xiyan Xiang, Sahara John, Xiaochun Yang, Nicole Beauchemin, Mohammad I. Hassan, Taj Mohammad, Lopa Mishra. Targeting liver specific βII-Spectrin to prevent the development of metabolic dysfunction associated steatohepatitis (MASH) through CEACAM1 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3388.