Proportion Of Dendritic Cells Research Articles

A β-Carboline Derivate PAD4 Inhibitor Reshapes Neutrophil Phenotype and Improves the Tumor Immune Microenvironment against Triple-Negative Breast Cancer.

Triple-negative breast cancer is a highly aggressive and heterogeneous breast cancer subtype characterized by early metastasis, poor prognosis, and high recurrence. Targeting histone citrullination-mediated chromatin dysregulation to induce epigenetic alterations shows great promise in TNBC therapy. We report the synthesis, optimization, and evaluation of a novel series of β-carboline-derived peptidyl arginine deiminase 4 inhibitors that exhibited potent inhibition of TNBC cell proliferation. The most outstanding PAD4 inhibitor, compound 28, hindered the PAD4-H3cit-NET signaling pathway and inhibited the growth of solid tumors and pulmonary metastatic nodules in the 4T1 in situ mouse model. Furthermore, 28 improved the tumor immune microenvironment by reshaping neutrophil phenotype, upregulating the proportions of dendritic cells and M1 macrophages, and reducing the amount of myeloid-derived suppressor cells. In conclusion, our work offered 28 as an efficacious PAD4 inhibitor that exerts a combination of conventional chemotherapy and immune-boosting effects, which represents a potential therapy strategy for TNBC.

Rho-kinase inhibitor alleviates CD4+T cell mediated corneal graft rejection by modulating its STAT3 and STAT5 activation

Penetrating keratoplasty remains the most common treatment to restore vision for corneal diseases. Immune rejection after corneal transplantation is one of the major causes of graft failure. In recent years, Rho-associated protein kinase (ROCK) inhibitors have been found to be associated with the activation of the STATs pathway and are widely studied in autoimmune diseases. Therefore, it may be possible that the ROCK inhibitors also participate in the local and systemic immune regulation in corneal transplantation through activation of the STATs pathway and affect the CD4+ T cell differentiation. This study aimed to explore the role of ROCK-STATs pathway in the occurrence of immune rejection in corneal transplantation by applying Y27632, a ROCK inhibitor, to the recipient mice and peripheral CD4+ T cells. We found that Y27632 significantly up-regulated the phosphorylation level of STAT5 in both spleen and lymph nodes, down-regulated the phosphorylation level of STAT3 in the CD4+ T cells in the spleen. It also increased the proportion of CD4+CD25+Foxp3+Helios+ Tregs while decreased CD4+IL17A+ -Th17 cells. Moreover, Y27632 also reduced the proportion of dendritic cells in both spleen and lymph nodes, as well as the expression level of CD86 on their surfaces in the spleen, while the proportion of macrophages was not affected. The expression levels of ROCK1, ROCK2, CD11c and IL-17A mRNA were also found to be low in the graft tissue while the expression of Helios was upregulated. Rho-kinase inhibitor can modulate the balance of Tregs/Th17 by regulating the phosphorylation levels of both STAT3 and STAT5, thereby inhibiting the occurrence of immune rejection in allogeneic corneal transplantation.

In vivo evaluations of Lactobacillus-fermented Eucheuma spinosum polysaccharides on alleviating food allergy activity.

In order to explore the in vivo anti-food allergy activity of Lactobacillus sakei subsp. sakei-fermented Eucheuma spinosum polysaccharides F1-ESP-3, an ovalbumin (OVA)-induced food allergy mouse model was established by ascites immunization and gavage. The weight, temperature, incidence of diarrhea, levels of allergic mediators and inflammatory factors in the serum of mice were analyzed. We analyzed the differentiation of mouse spleen lymphocytes and the proportion of sensitized mast cells by flow cytometry. The intestinal barrier status of mice was analyzed by intestinal pathological tissue sections and microbiota sequencing. The results showed that F1-ESP-3 could alleviate the food allergy symptoms of mice, such as hypothermia and loose stool; levels of OVA-specific immunoglobulin E, mast cell protease and histamine in the serum of sensitized mice and the proportion of dendritic cells and mast cells in mouse spleen were significantly reduced; in addition, F1-ESP-3 may protect the intestinal barrier and further improve the intestinal microenvironment of food-allergic mice by regulating the abundance of Bacteroidetes and Firmicutes. F1-ESP-3 can further improve the intestinal microenvironment of food-allergic mice by upregulating the levels of Lachnospiraceae, and may affect the signal pathways such as NOD-like receptor, MAPK, I kappa B and antigen processing and presentation.

Calcium electroporation of esophageal cancer induces gene expression changes: a sub-study of a phase I clinical trial.

In this study, we aim to investigate gene expression changes in tumor samples obtained from patients with esophageal cancer treated with calcium electroporation. Previously, local treatment with calcium electroporation has been shown to induce gene expression alterations, potentially contributing to a more tumor-hostile microenvironment. In this sub-study of a phase I clinical trial, we included five patients with esophageal cancer treated with calcium electroporation. We compared cancer-associated gene expression patterns in tumor samples before and after treatment. Furthermore, we used linear support vector regression to predict the cellular composition of tumor samples. Using differential expression analysis, we identified the downregulation of CXCL14 and upregulation of CCL21, ANGPTL4, and CRABP2 genes. We also found a decreased predicted proportion of dendritic cells while the proportion of neutrophils was increased. This study provides evidence that calcium electroporation for esophageal cancer induces local transcriptional changes and possibly alters the cellular composition of the tumor microenvironment. The results are explorative, larger studies are needed to confirm and further correlate our findings with clinical outcomes.

Synchronous targeted delivery of TGF-β siRNA to stromal and tumor cells elicits robust antitumor immunity against triple-negative breast cancer by comprehensively remodeling the tumor microenvironment

The poor permeability of therapeutic drugs, limited T-cell infiltration, and strong immunosuppressive tumor microenvironment of triple-negative breast cancer (TNBC) acts as a prominent barrier to the delivery of drugs and immunotherapy including programmed cell death ligand-1 antibody (anti-PD-L1). Transforming growth factor (TGF)-β, an important cytokine produced by cancer-associated fibroblasts (CAFs) and tumor cells contributes to the pathological vasculature, dense tumor stroma and strong immunosuppressive tumor microenvironment (TME). Herein, a nanomedicine platform (HA-LSL/siTGF-β) employing dual-targeting, alongside hyaluronidase (HAase) and glutathione (GSH) triggered release was elaborately constructed to efficiently deliver TGF-β small interference RNA (siTGF-β). It was determined that this system was able to improve the efficacy of anti-PD-L1. The siTGF-β nanosystem efficiently silenced TGF-β-related signaling pathways in both activated NIH 3T3 cells and 4T1 cells in vitro and in vivo. This occurred firstly, through CD44-mediated uptake, followed by rapid escape mediated by HAase in endo/lysosomes and release of siRNA mediated by high GSH concentrations in the cytoplasm. By simultaneous silencing of TGF-β in stromal and tumor cells, HA-LSL/siTGF-β dramatically reduced stroma deposition, promoted the penetration of nanomedicines for deep remodeling of the TME, improved oxygenation, T cells infiltration and subsequent anti-PD-L1 deep penetration. The double suppression of TGF-β has been demonstrated to promote blood vessel normalization, inhibit an epithelial-to-mesenchymal transition (EMT), and further modify the immunosuppressive TME, which was supported by an overall increase in the proportion of dendritic cells and cytotoxic T cells. Further, a reduction in the proportion of immunosuppression cells such as regulatory T cells and myeloid-derived suppressor cells was also observed in the TME. Based on the comprehensive remodeling of the tumor microenvironment by this nanosystem, subsequent anti-PD-L1 therapy elicited robust antitumor immunity. Specifically, this system was able to suppress the growth of both primary and distant tumor while preventing tumor metastasis to the lung. Therefore, the combination of the dual-targeted siTGF-β nanosystem, alongside anti-PD-L1 may serve as a novel method to enhance antitumor immunotherapy against stroma-rich TNBC.

Abstract 6675: Integration of associations of immune profiles in peripheral blood and tumor microenvironment with bladder cancer outcomes

Abstract Immune cell profiles in peripheral blood have been associated with bladder cancer outcomes, however, their association with response to immunotherapy and the tumor microenvironment is a major unresolved issue. Although tumor growth can be attenuated via the activation of tumor-infiltrating effector T cells, the relationship between tumor infiltration and immune activation remains unclear. This study explored the interaction between bladder cancer outcomes and immune profiles within peripheral blood and a tumor microenvironment (TME) based on DNA methylation profiles. Peripheral blood and the matched tumor FFPE DNA methylation profiles of 60 non-muscle-invasive bladder cancer (NMIBC) and 12 muscle-invasive bladder cancer (MIBC) patients. Cell-type deconvolution approaches were applied to estimate 12 peripheral immune cell-type proportions and 17 cell-type proportions within TME. We found a positive correlation between dendritic cell proportions in the TME with peripheral CD8T memory cell proportions (r = 0.35, P = 0.003) and a negative correlation between dendritic cell proportions in the TME with peripheral regulatory T cell proportions (r = -0.28, P = 0.021). In addition, monocyte cell proportions in TME had a positive correlation with peripheral B memory (r = 0.37, P = 0.002) and CD8T memory cell proportions (r = 0.43, P = 0.0002). To investigate associations of bladder cancer outcomes with immune cell profiles, using Cox proportional hazard models, we observed an association between the fraction of dendritic cells and the hazard of death (HR = 1.27, 95% CI = 1.06-1.53). Further, a high endothelial cell proportion was significantly associated with an increased hazard of death and tumor recurrence (HR = 1.06, 95% CI = 1.01-1.13) in TME. In addition, the peripheral neutrophil-to-lymphocyte ratio (HR = 1.49, 95% CI = 1.01-2.22), monocyte (HR = 1.17, 95% CI = 1.05-1.31), neutrophil (HR = 1.04, 95% CI = 1.01-1.07), and basophil (HR = 1.35, 95% CI = 1.01-1.81) cell proportions were associated with an increased hazard of death and tumor recurrence. Our results integrated the information on bladder cancer outcomes and cell profiles in TME and peripheral blood, providing biomarkers for estimating bladder cancer prognosis using genome-scale DNA methylation measures. Citation Format: Ji-Qing Chen, Lucas A. Salas, John K. Wiencke, Devin C. Koestler, Annette M. Molinaro, Angeline S. Andrew, John D. Seigne, Margaret R. Karagas, Karl T. Kelsey, Brock C. Christensen. Integration of associations of immune profiles in peripheral blood and tumor microenvironment with bladder cancer outcomes. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6675.

Time-Course Transcriptomic Analysis Reveals the Crucial Roles of PANoptosis in Fungal Keratitis.

Fungal keratitis (FK) is a serious corneal infection with high morbidity. Host immune responses function as a double-edged sword by eradicating fungal pathogens while also causing corneal damage, dictating the severity, progression, and outcome of FK. However, the underlying immunopathogenesis remains elusive. Time-course transcriptome was performed to illustrate the dynamic immune landscape in a mouse model of FK. Integrated bioinformatic analyses included identification of differentially expressed genes, time series clustering, Gene Ontology enrichment, and inference of infiltrating immune cells. Gene expression was verified by quantitative polymerase chain reaction (qPCR), Western blot, or immunohistochemistry. FK mice exhibited dynamic immune responses with concerted trends with clinical score, transcriptional alteration, and immune cell infiltration score peaking at 3 days post infection (dpi). Disrupted substrate metabolism, broad immune activation, and corneal wound healing occurred sequentially in early, middle, and late stages of FK. Meanwhile, dynamics of infiltrating innate and adaptive immune cells displayed distinct characteristics. Proportions of dendritic cells showed overall decreasing trend with fungal infection, whereas that of macrophages, monocytes, and neutrophils rose sharply in early stage and then gradually decreased as inflammation resolved. Activation of adaptive immune cells was also observed in late stage of infection. Furthermore, shared immune responses and activation of AIM2-, pyrin-, and ZBP1-mediated PANoptosis were revealed across different time points. Our study profiles the dynamic immune landscape and highlights the crucial roles of PANoptosis in FK pathogenesis. These findings provide novel insights into host responses to fungi and contribute to the development of PANoptosis-targeted therapeutics for patients with FK.

Identify Key Genes Correlated to Ischemia-Reperfusion Injury in Aging Livers.

With the intensification of population aging, the proportion of aging livers in the donor pool is increasing rapidly. Compared with young livers, aging livers are more susceptible to ischemia-reperfusion injury (IRI) during liver transplantation, which greatly affects the utilization rate of aging livers. The potential risk factors associated with IRI in aging livers have not been fully elucidated. In this work, five human liver tissue expression profiling datasets (GSE61260, GSE107037, GSE89632, GSE133815, and GSE151648) and a total of 28 young and aging liver tissues of human (N = 20) and mouse (N = 8) were used to screen and verify the potential risk factors associated with aging livers being more prone to IRI. DrugBank Online was used to screen drugs with potential to alleviate IRI in aging livers. The gene expression profile and immune cell composition between young and aging livers had significant differences. Among the differentially expressed genes, aryl hydrocarbon receptor nuclear translocator-like (ARNTL), BTG antiproliferation factor 2 (BTG2), C-X-C motif chemokine ligand 10 (CXCL10), chitinase 3-like 1 (CHI3L1), immediate early response 3 (IER3), Fos proto-oncogene, AP-1 transcription factor subunit (FOS), and peroxisome proliferative activated receptor, gamma, coactivator 1 alpha (PPARGC1A), mainly involved in the regulation of cell proliferation, metabolism, and inflammation, were also dysregulated in liver tissues suffered from IRI and could form a FOS-centered interaction network. Nadroparin was screened out with the potential to target FOS in DrugBank Online. In addition, the proportion of dendritic cells (DCs) was significantly upregulated in aging livers. We combined the expression profiling datasets of liver tissues and samples collected in our hospital for the first time to reveal that the changes in the expression of ARNTL, BTG2, CXCL10, CHI3L1, IER3, FOS, and PPARGC1A and the proportion of dendritic cells may be associated with aging livers being more prone to IRI. Nadroparin may be used to mitigate IRI in aging livers by targeting FOS, and regulation of DC activity may also reduce IRI.

Probiotic supplementation has sex-dependent effects on immune responses in association with the gut microbiota in community-dwelling older adults: a randomized, double-blind, placebo-controlled, multicenter trial.

Probiotics have been suggested as potent modulators of age-related disorders in immunological functions, yet little is known about sex-dependent effects of probiotic supplements. Therefore, we aimed to investigate sex-dependent effects of probiotics on profiles of the gut microbiota and peripheral immune cells in healthy older adults. In a randomized, double-blind, placebo-controlled, multicenter trial, healthy elderly individuals ≥ 65 yrs old were administered probiotic capsules (or placebo) for 12 wk. Gut microbiota was analyzed using 16S rRNA gene sequencing and bioinformatic analyses. Peripheral immune cells were profiled using flow cytometry for lymphocytes (natural killer, B, CD4+ T, and CD8+ T cells), dendritic cells, monocytes, and their subpopulations. Compared with placebo, phylum Firmicutes was significantly reduced in the probiotic group in women, but not in men. At the genus level, sex-specific responses included reductions in the relative abundances of pro-inflammatory gut microbes, including Catabacter and unclassified_Coriobacteriales, and Burkholderia and unclassified Enterobacteriaceae, in men and women, respectively. Peripheral immune cell profiling analysis revealed that in men, probiotics significantly reduced the proportions of dendritic cells and CD14+ CD16- monocytes; however, these effects were not observed in women. In contrast, the proportion of total CD4+ T cells was significantly reduced in women in the probiotic group. Additionally, serum lipopolysaccharide-binding protein levels showed a decreasing tendency that were positively associated with changes in gut bacteria, including Catabacter (ρ = 0.678, P < 0.05) and Burkholderia (ρ = 0.673, P < 0.05) in men and women, respectively. These results suggest that probiotic supplementation may reduce the incidence of inflammation-related diseases by regulating the profiles of the gut microbiota and peripheral immune cells in healthy elders in a sex-specific manner.

Innate Immune Microenvironment of Indolent B Cell Splenic Lymphomas: Role in Diagnosis and Treatment

Indolent splenic B-cell lymphomas are rare diseases (incidence <1:100,000) that include splenic marginal zone lymphoma (SMZL), hairy cell leukemia (cHCL), hairy cell leukaemia variant (HCLv), and splenic diffuse red pulp small B-cell lymphoma (SDRPL). These splenic lymphomas can have overlapping clinical presentations with only subtle differences in their morphology and phenotypes. However, there are considerable differences in prognosis, response to treatment, and outcomes. Better understanding of the tumor microenvironment (TME) is essential to design expedient and accurate diagnostic methods and precision drug treatments for each type of splenic B-cell lymphoma. We hypothesized each splenic lymphoma would have a distinct TME with some overlap for the red pulp lymphomas (cHCL, HCLv, and SDRPL). Human specimen collection and usage was approved by the Institutional Research Subjects Review Board. We studied cryo-preserved bulk splenic tumor suspension cell isolates from fresh (<4 hour) splenectomy tissue and intact formalin-fixed paraffin embedded (FFPE) tissue sections from 17 splenic lymphoma patients and 4 trauma patients (controls). FFPE tissue sections were studied by immunohistochemical (IHC) staining and splenic suspension cells analyzed by high-parameter fluorescence flow cytometry with a 29-color myeloid panel using a Cytek Aurora spectral flow cytometer. Initial IHC characterization of the splenic TME illustrated a markedly different distribution of intratumoral T cells (CD3) and macrophages (Mθ) (CD163). In cHCL, residual T cell zones appear retained, albeit diminished, while HCLv and SDRPL have markedly reduced T cells throughout with non-distinct zonation, and SMZL has prominent peritumoral collections of T cells at the interface of the neoplastic white pulp. Tissue architecture, as evidenced by splenic macrophage distribution, was markedly different. cHCL and HCLv showed diffuse but distorted networks of interdigitating macrophages, while SDRPL retained a nearly normal sinusoidal distribution of abundant macrophages, and SMZL showed total exclusion of CD163-positive Mθ from areas of confluent neoplastic white pulp. Analysis of splenic suspension cells showed an average of 72% B cells in the lymphoma specimens, twice that of trauma spleens. T cells made up the greatest fraction of non-B cells (~63%) for both the diseased and control samples. We analyzed the non-T and non-B cells to examine the innate immune cells. Granulocytes comprised on average 4% with no difference between the splenic lymphomas or trauma (Fig.1A). Trauma spleen had 86% macrophage/monocytes (M/Mθ)s, with similar values for HCLv (80%) and cHCL (74%). The percentage of M/Mθ was lower in SDRPL (55%) and SMZL (48%). We then analyzed the percentage of M/Mθ subtypes. As shown in (Fig.1B), CD14+CD16- was the major subtype for trauma (58%) and HCLv (50%). Conversely for cHCL, CD14loCD16+ was predominant (60%) subtype. SDRPL and SMZL shared similar subtype profiles with equal proportions of CD14+CD16- and CD14loCD16+ (40%). We then measured the dendritic cells of the innate immune population. SDRPL (41%) and SMZL (48%) had higher percentages than trauma (12%), HCLv (16%), or cHCL (20%). Conventional dendritic cell 2 (cDC2) was the predominant subtype comprising 50-74% of the DCs. Average conventional dendritic cell 1 (cDC1) and plasmacytoid dendritic cells (pDCs) were 30% and 9%, respectively. pDC was lower in cHCL than the other lymphomas (1%). Our data show a similar M/Mθ component of the innate immune TME in the red pulp lymphoma SDRPL and white pulp lymphoma SMZL. SDRPL and SMZL also had higher proportions of dendritic cells compared to cHCL, HCLv, or trauma spleens. cDC2 was the predominant DC type in the spleen regardless of disease state. We are currently analyzing flow cytometry panels to access natural killer cell distribution and T cell functionality. We aim to better understand immune cell interactions in the TME and discover variations between the splenic B-cell lymphomas, resulting in improved diagnosis and therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Midazolam exhibits antitumour and enhances the efficiency of Anti-PD-1 immunotherapy in hepatocellular carcinoma

BackgroundMidazolam (MDZ) is an anaesthetic that is widely used for anxiolysis and sedation. More recently, MDZ has also been described to be related to the outcome of various types of carcinomas. However, how MDZ influences the progression of hepatocellular carcinoma (HCC) and its effects on the biological function and tumour immune microenvironment of this type of tumour remain unknown.MethodsThe effects of MDZ on the proliferation, invasion, and migration of HCC cell lines were examined in vitro using the Cell Counting Kit 8 (CCK8), 5-ethynyl-2ʹ-deoxyuridine (EdU), Transwell, and wound healing assays. Additionally, western blotting was employed to confirm that PD-L1 was expressed. Chromatin immunoprecipitation-seq (ChIP-seq) analysis was used to pinpoint the transcriptional regulation regions of NF-κB and programmed death-ligand 1 (PD-L1). A C57BL/6 mouse model was used to produce subcutaneous HCC tumors in order to evaluate the in vivo performance of MDZ. Mass spectrometry was also used to assess changes in the tumour immunological microenvironment following MDZ injection.ResultsThe HCC-LM3 and Hep-3B cell lines’ proliferation, invasion, and migration were controlled by MDZ, according to the results of the CCK8, EdU, Transwell, and wound healing assays. PD-L1 expression was shown by ChIP-seq analysis to be boosted by NF-κB, and by Western blotting analysis, it was shown that MDZ downregulated the expression of NF-κB. Additionally, in vivo tests revealed that intraperitoneal MDZ injections reduced HCC tumor development and enhanced the effectiveness of anti-PD-1 therapy. The CD45+ immune cell proportions were higher in the MDZ group than in the PBS group, according to the mass spectrometry results. Injection of MDZ resulted in a decrease in the proportions of CD4+ T cells, CD8+ T cells, natural killer (NK) cells, monocytes, Tregs, and M2 macrophages and a rise in the proportion of dendritic cells. Additionally, the concentrations of the cytokines IFN-g and TNF-a were noticeably raised whereas the concentrations of the CD8+ T-cell fatigue markers ICOS, TIGIT, and TIM3 were noticeably lowered.ConclusionAccording to this study, MDZ inhibited the progression of HCC by inhibiting the NF-κB pathway and reducing the exhaustion of CD8+ T cells. In clinical practice, MDZ combined with anti-PD-1 therapy might contribute to synergistically improving the antitumor efficacy of HCC treatment.

Absence of NC14A Domain of COLXVII/BP180 in Mice Results in IL-17‒Associated Skin Inflammation

The deletion of exon 18 from Col17a1 in transgenic ΔNC14A mice results in the absence of the NC14Adomain. NC14A corresponds to the human NC16A domain, the immunodominant epitope in bullous pemphigoid. Before the age of 1 year, 84% of ΔNC14A mice have developed severe itch and skin erosion. Further characterization of mice with mutated CoLXVII (Bp180) revealed acanthosis; subepidermal blistering; and inflammatory cell infiltrates, especially neutrophils, eosinophils, and mast cells in the lesional skin. Direct immunofluorescence analysis detected linear complement C3, IgG, and/or IgA deposition in the dermo‒epidermal junction of symptomatic ΔNC14A mice. Elevated gene expression of IL-17‒associated cytokines was detected in the lesional skin. An increased proportion of dendritic cells, myeloid-derived suppressor cells, and NK cells and a decrease of T cells were found in both the spleen and lymph nodes of symptomatic ΔNC14A mice. The proportions of B cells and regulatory T cells were increased in lymph nodes. An 8-week treatment with an anti‒IL-17A decreased the expression of Il6, Il23a, and Cxcl1 in the nonlesional skin. Our results suggest that the absence of the NC14A domain of CoLXVII in mice causes an autoimmune response against the cutaneous basement membrane and manifests as an IL-17‒associated inflammation in the skin.

Expression Profile of Inflammation Response Genes and Potential Regulatory Mechanisms in Dilated Cardiomyopathy.

Background The inflammatory response is important in dilated cardiomyopathy (DCM). However, the expression of inflammatory response genes (IRGs) and regulatory mechanisms in DCM has not been well characterized. Methods We analyzed 27,665 cells of single-cell RNA sequencing dataset of four DCM samples and two healthy controls (HC). IRGs among differentially expressed genes (DEGs) of active cell clusters were screened from the Molecular Signatures Database (MSigDB). The bulk sequencing dataset of 166 DCM patients and 166 HC was analyzed to explore the common IRGs. The biological functions of the IRGs were analyzed according to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. IRG-related transcription factors (TFs) were determined using the TRRUST database. The protein–protein interaction (PPI) network was constructed using the STRING database. Then, we established the noncoding RNA (ncRNA) regulatory network based on the StarBase database. Finally, the potential drugs that target IRGs were explored using the Drug Gene Interaction Database (DGIdb). Results The proportions of dendritic cells (DCs), B cells, NK cells, and T cells were increased in DCM patients, whereas monocytes were decreased. DCs expressed more IRGs in DCM. The GO and KEGG analyses indicated that the functional characteristics of active cells mainly focused on the immune response. Thirty-nine IRGs were commonly expressed among active cell cluster DEGs, bulk RNA DEGs, and inflammatory response-related genes. ETS1 plays an important role in regulation of IRG expression. The competing endogenous RNA regulatory network showed the relationship between ncRNA and IRGs. Sankey diagram showed that arachidonate 5-lipoxygenase (ALOX5) played a major role in regulation between TFs and potential drugs. Conclusion DCs infiltrate into the myocardium and contribute to the immune response in DCM. The transcription factor ETS1 plays an important role in regulation of IRGs. Moreover, ALOX5 may be a potential therapeutic target for DCM.

The Crosstalk Between Immune Infiltration, Circulating Tumor Cells, and Metastasis in Pancreatic Cancer: Identification of HMGB3 From a Multiple Omics Analysis.

Metastasis is the major cause of death in patients with pancreatic ductal adenocarcinoma (PDAC), and circulating tumor cells (CTCs) play an important role in the development of metastasis. However, few studies have uncovered the metastasis mechanism of PDAC based on CTCs. In this study, the existing bulk RNA-sequencing (bulk RNA-seq) and single-cell sequencing (scRNA-seq) data for CTCs in pancreatic cancer were obtained from the Gene Expression Omnibus (GEO) database. Analysis of tumor-infiltrating immune cells (TIICs) by CIBERSORT showed that the CTCs enriched from the peripheral blood of metastatic PDAC were found to contain a high proportion of T cell regulators (Tregs) and macrophages, while the proportion of dendritic cells (DCs) was lower than that enriched from localized PDAC. Through weighted gene co-expression network analysis (WGCNA) and the result of scRNA-seq, we identified the hub module (265 genes) and 87 marker genes, respectively, which were highly associated with metastasis. The results of functional enrichment analysis indicated that the two gene sets mentioned above are mainly involved in cell adhesion and cytoskeleton and epithelial–mesenchymal transition (EMT). Finally, we found that HMGB3 was the hub gene according to the Venn diagram. The expression of HMGB3 in PDAC was significantly higher than that in normal tissues (protein and mRNA levels). HMGB3 expression was significantly positively correlated with both EMT-related molecules and CTC cluster–related markers. Furthermore, it was also found that HMGB3 mutations were favorably related to tumor-associated immune cells through the TIMER2.0 online tool. We further demonstrated that PDAC patients with higher HMGB3 expression had significantly worse overall survival (OS) in multiple datasets. In summary, our study suggests that HMGB3 is a hub gene associated with EMT in CTCs, the formation of CTC clusters, and infiltration patterns of immune cells favorable for tumor progression and metastasis to distant organs.

The Analysis of Phagocytic Myeloid Cells in Low and High Fiber Fed Mice after Three Weeks of Hypoxia

Pulmonary hypertension (PH) is a devastating disease that is characterized by increased blood pressure (BP) in the pulmonary vasculature. Immune cells participate in adverse cardiovascular remodelling of pulmonary vessels and could represent targets for PH treatment. It has been shown that patients with PH have increased numbers of interstitial macrophages and dendritic cells in the lungs that originate from circulating immune cells. These myeloid immune cells migrate to the vessel wall and promote vascular hypertrophy. Attenuation of immune cell invasion into the vessel wall could potentially slow the progression of PH. One of the possible ways to reduce inflammation in the cardiovascular system is dietary modification with increased content of soluble fiber. Soluble fiber increases the level of short chain fatty acids (SCFAs) in the plasma which demonstrate anti‐inflammatory properties in systemic hypertension and cardiac overload. In different models of cardiovascular diseases high fiber (HFi) diet and SCFAs improve cardiovascular functions reducing the level of immune cells at the site of injury. The effects of HFi diet on the pulmonary circulation are unknown. This study establishes the effects of low (LFi) and HFi diet on macrophage and dendritic cell composition in the lungs in the model of hypoxia‐induced PH.C57bl/6j mice were fed with LFi and HFi diet five weeks before and three weeks during hypoxic exposure (10% O2). After that hematocrit, cecum, spleen and body weight were measured. Flow cytometry was used to identify the proportion of alveolar and interstitial macrophages and dendritic cells in the lungs.Hypoxic animals had increased hematocrit level compared to normoxic controls. Hematocrit did not differ between LFi fed animals and HFi fed animals in hypoxic conditions but in normoxia hematocrit was higher in LFi diet group. Cecum weight was significantly higher in HFi fed mice than in LFi fed mice whereas total body weight and spleen weight was not affected by diet composition. The percentage of alveolar macrophages (CD11b+CD11c+CD64+) did not differ between groups. Hypoxia increased the proportion of dendritic cells (CD11b+MHCll+CD24+) and reduced the proportion of interstitial macrophages (CD11b+MHCll+CD64+) among CD11b+MHC ll+ cells. Dietary fiber did not affect the proportion of dendritic cells and interstitial macrophages in normoxic and hypoxic animals.This data demonstrates that three weeks of hypoxia changes the proportion of CD11b+ dendritic cells and interstitial macrophages in the mouse lungs however soluble dietary fiber did not affect it.

Comparative study of doxycycline, sancycline, and 4-dedimethylamino sancycline (CMT-3) on epidermal melanogenesis.

Melanogenesis is regulated by melanocytes, which synthesize the pigment melanin inside melanosomes; these melanosomes are exported through dendritic extensions to adjacent keratinocytes and result in skin coloration. Chemically modified tetracyclines (CMTs) are nonantimicrobial tetracyclines that retain the capacity to inhibit matrix metalloproteinases (MMPs) and have shown several biological benefits; in particular, CMT-3 [(4-dedimethylamino sancycline (SAN)] has emerged as a candidate for therapeutic benefits in our previous studies. However, to date, studies of the effects of CMT-3 or SAN on melanogenesis are lacking. We have previously reported the anti-melanogenic activity of CMT-308 (the 9-amino derivative of CMT-3). Herein, we have compared the three tetracycline analogs, doxycycline (DOX), SAN, and CMT-3, for their effects on melanogenesis using B16F10 mouse melanoma cells and have validated results in primary human melanocytes (HEMn-DP). DOX did not show any significant effects on intracellular melanin or melanosome export in DP cells while SAN was cytotoxic at high doses but without effects on melanogenesis at lower doses. However, CMT-3 showed a robust suppression of dendricity parameters (dendrite number, dendrite length, and proportion of dendritic cells) in DP cells which was associated, at least in part, with a significant reduction of intracellular tyrosinase activity. In spite of its inhibition of tyrosinase activity, CMT-3 had no significant effects on intracellular melanin levels, suggesting that it selectively targets melanosome export. Our results demonstrate a unique structure-activity relationship (SAR) for the effects of these compounds on melanogenesis and support the conclusion that removal of the 4-dimethylamino moiety confers the selective capacity to suppress melanosome export. Collectively, these results indicate that CMT-3 might be a candidate for diminishing hyperpigmentation skin disorders.

The Upregulation of PLXDC2 Correlates with Immune Microenvironment Characteristics and Predicts Prognosis in Gastric Cancer.

Tumor microenvironment (TME) has been demonstrated to exhibit a regulatory effect on the progressions of gastric cancer (GC). However, the related functions of stromal and immune components (TME-associated genes) in TME remain largely unclear. From the TCGA dataset, we downloaded the clinical data of 375 GC cases and then estimated the percentage of tumor-infiltrating immunocytes (TICs) and the levels of immune and stromal constituents by the use of CIBERSORT and ESTIMATE tolls. Univariate assays were applied to study the differentially expressed genes. The associations between the clinical information of GC patients and the expressions of the specific genes were analyzed based on the TCGA datasets. The effect of Plexin domain containing 2 (PLXDC2) expression on TICs was conducted. We observed that PLXDC2 expression was distinctly upregulated in GC specimens compared with nontumor gastric specimens. Its upregulation was associated with advanced clinical stages and predicted a shorter overall survival of GC patients. The genes in the group of higher expressing PLXDC2 were primarily enriched in immunity-associated events. By the use of CIBERSORT, we observed that PLXDC2 expressions were related to the proportion of dendritic cells resting, T cell CD4 memory resting, eosinophils, mastocyte resting, mononuclear cells, plasma cells, T cell follicle helper, macrophage M2, and dendritic cells activated. Overall, our discoveries revealed that the expression of PLXDC2 was remarkable in GC, might be a possible biomarker for GC, and provided novel contents regarding immune infiltrates, offering novel insight for treatments of GC.

Peripheral Immune Cells Exhaustion and Functional Impairment in Patients With Chronic Hepatitis B.

After infection of hepatitis B virus (HBV), the virus induces a variety of immune disorders in the host, leading to immune escape and, finally, the chronicity of the disease. This study investigated immune cell defects and functional impairment in patients with chronic hepatitis B (CHB). We analyzed the percentage, function, and phenotypes of various immune cell subpopulations in the peripheral blood along with the concentrations of cytokines in the plasma. We compared the results between patients with CHB and healthy individuals. It was found that in patients with CHB, the cell function was impaired and, there was increased expression of inhibitory receptors, such as NKG2A and PD-1 in both NK and T cells. The impairment of function was mainly in cytokine secretion, and the cytotoxicity was not significantly diminished. We also found that the proportion of dendritic cells (DC) decreased and regulatory B cells (Breg) increased in CHB. In addition, the Breg cells were negatively correlated with T cell cytokine and positively correlated with ALT and HBV viral load. Taken together, various disorders and functional impairments were found in the immune cells of peripheral blood in CHB patients, especially NK and T cells. These cells showed exhaustion and the increase of regulatory B cells may be one of the reasons for this phenomenon.

Celiac Disease Defined by Over-Sensitivity to Gliadin Activation and Superior Antigen Presentation of Dendritic Cells

The autoimmune condition, Celiac Disease (CeD), displays broad clinical symptoms due to gluten exposure. Its genetic association with DQ variants in the human leukocyte antigen (HLA) system has been recognised. Monocyte-derived mature dendritic cells (MoDCs) present gluten peptides through HLA-DQ and co-stimulatory molecules to T lymphocytes, eliciting a cytokine-rich microenvironment. Having access to CeD associated families prevalent in the Czech Republic, this study utilised an in vitro model to investigate their differential monocyte profile. The higher monocyte yields isolated from PBMCs of CeD patients versus control individuals also reflected the greater proportion of dendritic cells derived from these sources following lipopolysaccharide (LPS)/ peptic-tryptic-gliadin (PTG) fragment stimulation. Cell surface markers of CeD monocytes and MoDCs were subsequently profiled. This foremost study identified a novel bio-profile characterised by elevated CD64 and reduced CD33 levels, unique to CD14++ monocytes of CeD patients. Normalisation to LPS stimulation revealed the increased sensitivity of CeD-MoDCs to PTG, as shown by CD86 and HLA-DQ flow cytometric readouts. Enhanced CD86 and HLA-DQ expression in CeD-MoDCs were revealed by confocal microscopy. Analysis highlighted their dominance at the CeD-MoDC membrane in comparison to controls, reflective of superior antigen presentation ability. In conclusion, this investigative study deciphered the monocytes and MoDCs of CeD patients with the identification of a novel bio-profile marker of potential diagnostic value for clinical interpretation. Herein, the characterisation of CD86 and HLA-DQ as activators to stimulants, along with robust membrane assembly reflective of efficient antigen presentation, offers CeD targeted therapeutic avenues worth further exploration.

A Diet High in Lipid Soluble Forms of Choline Modulates Gut-Associated Immune Function in Sprague-Dawley Dams

ObjectivesOral tolerance is the state of unresponsiveness to dietary antigens that are not harmful to the host, a process that begins in the gut and influences both local and peripheral immune responses. Lipid soluble forms of choline have been shown to beneficial affect peripheral immune responses during pregnancy and lactation, yet little is known about the impact of the different choline forms on the gut-associate lymphoid tissue (GALT). Therefore, the objective of this study was to determine the effect of feeding buttermilk-derived choline forms on the GALT in lactating dams. MethodsSprague-Dawley dams were randomized to consume one of three diets, all containing 1.9 g/kg of total choline: 1-Control (100% free choline (FC)), 2-Buttermilk (37% phosphatidylcholine (PC), 34% sphingomyelin, 17% glycerophosphocholine (GPC), 7% FC, 5% phosphocholine) and 3-Placebo (50% PC, 25% FC, 25% GPC). Immune cell phenotypes of mesenteric lymph nodes (MLN) and Peyer’s patches (PP) were measured by flow cytometry. Ex vivo cytokine production by immune cells isolated from MLN stimulated with Concanavalin A (ConA) and Ovalbumin (OVA) was measured by ELISA. ResultsIn MLN, dams from the buttermilk and placebo groups had a higher production of IL-10 after both ConA and OVA stimulation and a higher proportion of helper T cells expressing co-inhibitory marker (CD4+CD152+) when compared to the control diet (both P < 0.05). No changes were found in IL-2, TNF-α, or IL-6 production and the proportion of regulatory T cells (CD3+CD4+CD25+FoxP3+) among groups. In PP, dams from the buttermilk group had a higher proportion of CD45RA + cells (B cells) compared to both the placebo and control diets and dams from the placebo diet had a higher proportion of dendritic cells (DC, OX62+OX6+) compared to both the buttermilk and control diets (both P < 0.05). ConclusionsCompared to the control diet (only FC), the buttermilk and placebo diets appear to beneficially modulate oral tolerance by increasing the production of IL-10 (an important mediator) and could be partially explain by the higher proportion of CD4+CD152+cells in MLN. In PP, the higher proportion of B cells in the buttermilk and DC in the placebo groups, suggests that dams from these groups may have better antigen presentation function, which may further assist the induction of oral tolerance. Funding SourcesDairy Farmers of Canada, NSERC, CONACYT.