Inflammatory Chemokines Research Articles

CDC7 inhibition induces replication stress-mediated aneuploid cells with an inflammatory phenotype sensitizing tumors to immune checkpoint blockade

Serine/threonine kinase, cell division cycle 7 (CDC7) is critical for initiating DNA replication. TAK-931 is a specific CDC7 inhibitor, which is a next-generation replication stress (RS) inducer. This study preclinically investigates TAK-931 antitumor efficacy and immunity regulation. TAK-931 induce RS, generating senescence-like aneuploid cells, which highly expressed inflammatory cytokines and chemokines (senescence-associated secretory phenotype, SASP). In vivo multilayer-omics analyses in gene expression panel, immune panel, immunohistochemistry, RNA sequencing, and single-cell RNA sequencing reveal that the RS-mediated aneuploid cells generated by TAK-931 intensively activate inflammatory-related and senescence-associated pathways, resulting in accumulation of tumor-infiltrating immune cells and potent antitumor immunity and efficacy. Finally, the combination of TAK-931 and immune checkpoint inhibitors profoundly enhance antiproliferative activities. These findings suggest that TAK-931 has therapeutic antitumor properties and improved clinical benefits in combination with conventional immunotherapy.

Inflammatory and Metabolic Signaling Interfaces of the Hypertrophic and Senescent Chondrocyte Phenotypes Associated with Osteoarthritis.

Osteoarthritis (OA) is a complex disease of whole joints with progressive cartilage matrix degradation and chondrocyte transformation. The inflammatory features of OA are reflected in increased synovial levels of IL-1β, IL-6 and VEGF, higher levels of TLR-4 binding plasma proteins and increased expression of IL-15, IL-18, IL-10 and Cox2, in cartilage. Chondrocytes in OA undergo hypertrophic and senescent transition; in these states, the expression of Sox-9, Acan and Col2a1 is suppressed, whereas the expression of RunX2, HIF-2α and MMP-13 is significantly increased. NF-kB, which triggers many pro-inflammatory cytokines, works with BMP, Wnt and HIF-2α to link hypertrophy and inflammation. Altered carbohydrate metabolism and the upregulation of GLUT-1 contribute to the formation of end-glycation products that trigger inflammation via the RAGE pathway. In addition, a glycolytic shift, increased rates of oxidative phosphorylation and mitochondrial dysfunction generate reactive oxygen species with deleterious effects. An important surveyor mechanism, the YAP/TAZ signaling system, controls chondrocyte differentiation, inhibits ageing by protecting the nuclear envelope and suppressing NF-kB, MMP-13 and aggrecanases. The inflammatory microenvironment and synthesis of key matrix components are also controlled by SIRT1 and mTORc. Senescent chondrocytes represent the functional end stage of hypertrophic differentiation and characteristically upregulate p16 and p21, but also a variety of inflammatory cytokines, chemokines and metalloproteinases, developing the senescence-associated secretory phenotype. Senolysis with dendrobin, miR29b-5p and other agents has been shown to be efficient under experimental conditions, and appears to be a promising tool for the treatment of OA, as it restores COL2A1 and aggrecan synthesis, suppressing NF-kB and destructive metalloproteinases.

Vitronectin and Fetuin-A: Newfangled Glycoproteins in Periodontits and Coronary Artery Diseases: A Narrative Review

Periodontitis, an intricate inflammatory condition impacting oral support structures, is increasingly recognized as a potential contributor to systemic ailments, notably cardiovascular diseases. Its pathophysiology involves the release of inflammatory molecules like cytokines and chemokines, exacerbating conditions like atherosclerosis. Within this complex web of inflammation, glycoproteins like vitronectin and fetuin-A have emerged as pivotal players, with vitronectin's multifunctional roles in cellular processes and fetuin-A's influence on metabolic pathways. Understanding their involvement in periodontitis and cardiovascular disorders offers promise in leveraging them as diagnostic biomarkers, potentially enhancing disease management and prevention.

TMIC-78. ENDOTHELIAL REGULATION OF THE IMMUNOSUPPRESSIVE PHENOTYPE OF TUMOR-ASSOCIATED MICROGLIA IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is considered a non-immunogenic “cold tumor” with an immunosuppressive microenvironment that blocks T-cell infiltration and function, limiting the efficacy of targeted immunotherapy. Tumor-associated microglia and macrophages (TAM) constitute approximately 30-50% of the non-neoplastic immune cells, and they are closely associated with blood vessels in GBM. However, the precise mechanism by which TAM acquire the immunosuppressive phenotype remains poorly understood. In this study, we show that tumor endothelial cells (TEC) from primary and recurrent GBM express inflammatory cytokines and chemokines that influence the function and polarization of microglia. We demonstrate that conditioned media (CM) of TEC promotes the immunosuppressive phenotype of normal microglia and patient-derived TAM. RNA-sequencing of TAM treated with TEC-CM shows upregulation of immunosuppressive cytokines, as well as T-cell co-inhibitory molecules such as PD-L1. Culturing CD8 T-cells in CM of TAM-polarized by TEC inhibits their cytotoxic function and cytokine production, and induces expression of PD-1 and CTLA-4. Furthermore, gene set enrichment analysis uncovers alterations in calcium signaling-related pathways in TAM polarized by TEC-CM. Blocking calcium signaling with small molecule inhibitors in microglia reverses the immunosuppressive phenotype induced by TEC. To determine whether the interactions between GBM, endothelial cells and TAM promotes immunosuppression in vivo, we utilize the humanized immune system mouse model (huNOG-EXL) that harbor human lymphocytes and monocyte populations. In preliminary studies, we found that co-transplantation of GBM tumor cells with TAM and TEC significantly promotes tumor growth compared with GBM cells alone. Single-cell mass cytometry analysis of peripheral blood mononuclear cells (PBMC) pre- and post-transplant, and scRNA-sequencing and spatial-transcriptomic studies of these co-transplanted mice will potentially reveal key insights into local- and peripheral immune responses regulated by tumor endothelial-microglia interactions. Together, our findings highlight a key role for endothelial-mediated calcium signaling in regulation of the immunosuppressive phenotype of microglia in GBM.

Global deficiency in the inflammatory chemokine receptors 1,2,3 and 5 ameliorates atherosclerosis and selectively modulates aortic myeloid cell phenotype

Abstract Background Atherosclerosis is a chronic inflammatory disease in which leukocytes traffic to the vessel wall, a process in part mediated by the inflammatory chemokine receptors (iCCRs): Ccr1, Ccr2, Ccr3 and Ccr5. ICCRs display significant ligand promiscuity and redundancy, which have hampered the definitive deduction of the isolated and collective roles of these receptors. Purpose To assess iCCR expression in human and mouse atherosclerosis and determine the contribution of global iCCR expression to atherosclerosis formation in mice. Methods We derived data from the human carotid endarterectomy biobanks, Athero-Express and Biobank of Karolinska Endarterectomy (BiKE) to investigate iCCR expression. Next, we employed fluorescent reporter mice (iREP) to track iCCR expression via flow cytometry and confocal microscopy. To ascertain the contribution of iCCRs to atherosclerosis, we utilised mice in which the complete iCcr locus incorporating Ccr1, Ccr2, Ccr3 and Ccr5 has been deleted (TACKO mice). We induced atherosclerosis using tail vein injection of AAV8- proprotein convertase subtilisin/kexin type 9 (PCSK9) in combination with 12- or 20-weeks Western diet (WD). Plaque size and composition was assessed via histology while cytometry of time-of-flight (CyTOF) and single-cell RNA-sequencing (scRNA-seq) were used to assess cellular changes. Results By analysing scRNA-seq data from Athero-Express, CCR1 emerged as the most strongly expressed iCCR and was highly localised to macrophages. Next, by utilizing BiKE data, we discovered that CCR1, 2 and 5 expression increased in plaques compared to healthy vessels, with CCR1 and CCR5 more strongly associated with symptomatic patients compared with asymptomatic patients. In iREP mice, CCR1, 2 and 5+ cell numbers were significantly increased in the atherosclerotic aorta compared with healthy mice. Cellular expression levels were variable with CCR2 being dominant in the plaque and increasing markedly on macrophages. Following 12 weeks WD, there was a reduction in mean plaque area within the aortic sinus of TACKO mice (WT 0.20 mm² vs. TACKO 0.10 mm², n=19-20, P<0.0001). Following 20 weeks WD, mean plaque area was also reduced in TACKO mice (WT 0.41 mm² vs. TACKO 0.25 mm², n=9-12, P<0.01) and this was associated with a significant reduction in necrotic core area and a reduction in calcification, reflecting a shift towards a more stable plaque phenotype, as classified according to the Virmani grading system. CyTOF analysis revealed reductions in inflammatory monocytes and conventional type 2 dendritic cells in TACKO aorta accompanied by a phenotypic switch in macrophages towards a less inflammatory phenotype. Finally, scRNA-seq analysis revealed phenotypic differences in non-immune vascular cells between WT and TACKO mice. Conclusion Our results support an athero-protective, plaque-stabilizing effect of iCcr deletion with a selective reduction in inflammatory myeloid cell aortic homing.

Abstract 11795: CXCL5 Inhibition Recovers Neovascularization in Diabetes Mellitus

Introduction: Diabetes mellitus (DM) is a chronic inflammatory disease accompanied by dysfunction of vascular endothelial cells. Vascular complications are the main causes of death in patients with DM. CXCL5 is an inflammatory chemokine that upregulated in patients with type 2 DM. However, its role in diabetic vasculopathy was not clarified. This study aimed to explore the impacts and mechanistic insights of CXCL5 in neovasculogenesis in DM. Methods: Endothelial progenitor cells (EPCs) and human aortic endothelial cells (HAECs) were used in vitro. Streptozotocin-induced diabetic mice and dbdb mice were used as type 1 and type 2 DM models. CXCL5 knockout mice were also used to generate the diabetic animal model. The hindlimb ischemia surgery, matrigel plug assay, and aortic ring assay were conducted. Flow cytometry were used to evaluate the circulating number of EPCs. Results: CXCL5 concentrations were increased in plasma and EPCs culture medium from type 2 DM patients. CXCL5 inhibition by neutralizing antibody upregulated VEGF/SDF-1 expression and improved migration and tube formation abilities in EPCs from type 2 DM patients and high glucose-treated EPCs from non-diabetic subjects as well as HAECs. Intraperitoneal injection of CXCL5 neutralizing antibody increased circulating EPC number, enhanced capillary density, and recovered the blood flow after hindlimb ischemia surgery in both type 1 and type 2 diabetic animals. Protein levels of VEGF and SDF-1 in ischemic muscle were upregulated in the CXCL5 inhibition group. Additionally, diabetic mice injected with CXCL5 neutralizing antibody showed higher hemoglobin contents and neovascular branches in the matrigel plug assay and aortic ring assay, respectively. Importantly, CXCL5 knockout diabetic mice showed improved ischemia-induced angiogenesis and higher expression of angiogenic proteins in ischemic muscle compared with wild-type diabetic mice. Conclusion: CXCL5 inhibition can improve neovasculogenesis via protecting vascular endothelial function and upregulating angiogenic protein expressions in both type 1 and type 2 DM. Further clinical studies are needed to verify that CXCL5 may be a potential therapeutic target for DM vascular complications.

Abstract 18007: Role of Interferon Lambda in Influenza-Induced Exacerbation of Atherosclerosis

Introduction and Hypothesis: Influenza infection is a significant public health and economic threat around the world. Although pneumonia is the most common complication associated with influenza, there are several clinical reports demonstrating increased risk for cardiovascular disease. In influenza infection type I, type II and type III IFNs play an indispensable role in controlling the virus. In atherosclerosis, type I and type II IFNs promote atherosclerosis by inducing chemokines from macrophages and vascular endothelial cells. Studies have shown that the influenza infection exacerbates atherosclerosis. However, there are no studies determining the role of type III IFNs on influenza-induced exacerbation of atherosclerosis. In this study, we hypothesize that the type III IFN (IFNL3) may play a protective role in atherosclerosis. Methods: ApoE -/- mice were fed with a high-fat diet (HFD) containing 42% Kcal fat (Teklad) for 11 weeks. Mice were then infected with influenza A/PR/8/34 (H1N1) (100 pfu) and treated with recombinant IFNL3 by oropharyngeal (O/P) aspiration or intraperitoneal (I/P) injection, and weight loss, and gene expression of inflammatory cytokines and chemokines were measured. In another study, HFD-fed ApoE -/- and ApoE -/- IFNLR1 -/- mice were infected with influenza, and the disease outcomes were measured as described above. Results and Conclusions: IFNL3 treatment decreased influenza-induced weight loss in ApoE -/- mice during the early phase (4-6 days) of influenza infection. Further, IFNL3 treatment decreased gene expression levels of IL-6 and CCL2 in the aorta in influenza-infected ApoE -/- mice. Based on these findings, it seems likely that INFL3 plays a protective role in acute influenza-induced exacerbation of atherosclerosis. However, ApoE -/- IFNLR1 -/- mice recovered body weight faster than wild-type mice suggesting a detrimental role for IFNL after acute infection. These data show that IFNLR plays a protective role during the early phase of influenza infection and potentially a pathological role during the recovery phase of influenza infection.

EZH2 inhibition stimulates repetitive element expression and viral mimicry in resting splenic B cells.

Mammalian cells repress expression of repetitive genomic sequences by forming heterochromatin. However, the consequences of ectopic repeat expression remain unclear. Here we demonstrate that inhibitors of EZH2, the catalytic subunit of the Polycomb repressive complex 2 (PRC2), stimulate repeat misexpression and cell death in resting splenic B cells. B cells are uniquely sensitive to these agents because they exhibit high levels of histone H3 lysine 27 trimethylation (H3K27me3) and correspondingly low DNA methylation at repeat elements. We generated a pattern recognition receptor loss-of-function mouse model, called RIC, with mutations in Rigi (encoding for RIG-I), Ifih1 (MDA5), and Cgas. In both wildtype and RIC mutant B cells, EZH2 inhibition caused loss of H3K27me3 at repetitive elements and upregulated their expression. However, NF-κB-dependent expression of inflammatory chemokines and subsequent cell death was suppressed by the RIC mutations. We further show that inhibition of EZH2 in cancer cells requires the same pattern recognition receptors to activate an interferon response. Together, the results reveal chemokine expression induced by EZH2 inhibitors in B cells as a novel inflammatory response to genomic repeat expression. Given the overlap of genes induced by EZH2 inhibitors and Epstein-Barr virus infection, this response can be described as a form of viral mimicry.

Abstract 14108: Eicosapentaenoic Acid and Docosahexaenoic Acid and Their Association With Oral Inflammatory Markers in Subjects With Clinical Coronary Artery Disease

Background: Our group previously reported that subjects with coronary artery disease (CAD) randomized to the omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), had lower levels of blood inflammatory markers and prevention of progression of non-calcified coronary plaque. Macrophage inhibitory protein (MIP)-1β is an inflammatory chemokine expressed by T cells in human plaques; circulating levels are increased in patients with atherosclerosis. The relation between EPA+DHA supplementation and oral inflammatory markers is unclear. Aim: To determine if EPA+DHA supplementation lowers levels of oral inflammatory markers. Methods: Subjects with clinical CAD were randomized to EPA+DHA, 3.36 g daily, or none for 30 months; 199 underwent a dental exam. Gingival crevicular fluid was collected at baseline and 30 months and analyzed for inflammatory markers. Results: MIP-1-β level was significantly lower in the EPA+DHA group compared to control at 30 months in the left upper quadrant of the mouth but not in the right upper quadrant (Table). This difference could potentially be explained by handedness. For example, Right-handed people tend to brush the left side of the mouth better whereas left-handed individuals and ambidextrous individuals have better brushing skills on the right side of the mouth. Other inflammatory markers (MCP, TNF-a, IL-1 and IL-6) were not different between groups on either side. Conclusions: Subjects with CAD randomized to EPA+DHA had reduced levels of the oral inflammatory marker, MIP-1β, at 30 months, suggesting that EPA+DHA can confer oral health benefits in addition to lowering systemic inflammation. For greatest benefit, brushing equally on both sides of the mouth should be encouraged, for which a powered toothbrush can help as it doesn’t rely on manual dexterity and handedness. Future research should examine whether the benefit of omega-3 fatty acids on oral health increases the benefit on coronary plaque.

Abstract 18831: The Protective Effect of Duffy Antigen Receptor for Chemokines Gene Deletion on Abdominal Aortic Aneurysm Formation: Role of Lysyl Oxidase

Introduction: Epidemiologic studies indicate that Blacks are protected against abdominal aortic aneurysm (AAA), despite similar risk factors as Caucasians. Approximately 70% of Blacks harbor a variant in the Duffy Antigen Receptor for Chemokines (DARC), a non-signaling receptor expressed primarily on erythrocytes which binds and regulates inflammatory chemokines. Since chemokines play a critical role in AAA, we tested the hypothesis that lack of DARC expression on erythrocytes protects against AAA, and we investigated the mechanisms. Methods: Mice lacking DARC on hematopoietic cells were created by mating DARC flox/flox mice with Vav1-Cre mice. At 10-14 weeks of age, both Vav1-Cre positive (DARC HKO ) and Vav1-Cre - mice [wild type (WT) littermate controls] received PCSK9 adeno-associated virus injection and were started on high fat diet. After 1 week, mice were infused with angiotensin II (AngII, 1,000 ng/kg/min) via osmotic minipump for 26 days to induce AAA. Results: Compared to WT, there was no difference in blood pressure or cholesterol levels in AngII-infused DARC HKO mice, which were confirmed to lack DARC on erythrocytes by flow cytometry. Aortic diameter and AAA incidence were significantly decreased in DARC HKO compared to WT mice (n=16-20, p=0.0257), in conjunction with reduced elastin degradation and macrophage infiltration. We did not find differences in levels of DARC-bound chemokines that could account for the protection against AAA. Interestingly, we found that the level and activity of lysyl oxidase (LOX), a cytokine-like enzyme that crosslinks extracellular matrix proteins and protects against AAA, were higher in DARC HKO mice compared to WT mice. Conclusions: Loss of DARC on hematopoietic cells protects against AAA formation, likely by upregulating aortic LOX. These findings provide novel insight into ethnic differences in susceptibility to AAA.

Cytokine and Chemokine Production in Mice Inoculated with NVX-CoV2373 (Nuvaxovid®) in Comparison with Omicron BA.4/5 Bivalent BNT162b2 (Comirnaty®).

A recombinant SARS-CoV-2 spike protein vaccine (NVX-CoV2373) has been licensed and has a lesser incidence of adverse events. To know the immunological mechanisms of adverse events, the production of cytokines and chemokines was investigated in mice inoculated with NVX-CoV2373. Serum IL-6 was detected on Day 1 of the first and second doses and the IFN-γ, IL-4, IL-10, TNF-α, and IL-6 levels increased on Day 1 of the second dose at the inoculation site. The enhanced production of the inflammatory chemokines (CCL2), homeostatic chemokine (CXCL13), and Th2 chemokine (CCL17) was observed at the inoculation site on Day 1 of the second dose. These findings were compared with data obtained following inoculation with BNT162b2 bivalent vaccine containing omicron BA.4/5. Significantly lower levels of inflammatory chemokines were detected on Day 1 after the first dose of NVX-CoV2373 in sera and inoculation site than those following inoculation with bivalent BNT162b2 (p < 0.01), reflecting a lower incidence of adverse events after immunization with NVX-CoV2373 in humans. NVX-CoV2373 induced significantly higher concentrations of IFN-γ, TNF-α, and IL-10 at the inoculation site obtained on Day 1 of the second dose (p < 0.05). Significant higher levels of Th2 chemokines, CCL11 and CCL17, were induced at the inoculation site on Day 1 of the second dose (p < 0.01) and they explain the booster IgG EIA antibody response after the second dose of NVX-CoV2373.

The Sesquiterpene, Selin-11-en-4α-ol, from Artemisia vulgaris Inhibits Lipopolysaccharide-Induced Inflammatory Responses in RAW264.7 Cells via MAPK and NF-κB Signaling Pathways

Objective Our study aimed to investigate the underlying molecular mechanisms of selin-11-en-4α-ol purified from Artemisia vulgaris against anti-inflammation through network pharmacology, molecular docking, and in vitro experiments. Methods Potential targets for selin-11-en-4α-ol and inflammation were obtained using Swiss Target Prediction, Pharm Mapper, and GeneCards databases. Venn diagrams were used to obtain the targets of selin-11-en-4α-ol for the treatment of inflammation, and the intersecting targets were uploaded to the STRING database to construct a protein–protein interaction (PPI) network of selin-11-en-4α-ol anti-inflammatory target proteins for further screening of the core targets. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using the DAVID database. In addition, molecular docking validation of core target binding to selin-11-en-4α-ol was performed. The anti-inflammatory effects were verified by in vitro experiments of lipopolysaccharide-induced RAW264.7 cells. Results PPI network analysis showed that mitogen-activated protein kinase 14 (MAPK14), SRC, and HSP90AA1 were predicted as potential anti-inflammatory targets, and KEGG enrichment analysis revealed that the MAPK signaling pathway presented the highest gene enrichment counts. Molecular docking and western blot results suggested that selin-11-en-4α-ol regulated MAPK and nuclear transcription factor-κB (NF-κB) signaling pathways to alleviate inflammation. In addition, selin-11-en-4α-ol inhibited the production of inflammatory factors, chemokines, and reactive oxygen species and exhibited anti-inflammatory and antioxidant effects. Conclusion Selin-11-en-4α-ol treats inflammation by targeting the MAPK and NF-κB signaling pathway, demonstrating that network pharmacology and molecular docking are effective tools for studying traditional Chinese medicine and providing a theoretical basis for developing anti-inflammatory drugs.

ABX464 (obefazimod) for patients with COVID-19 at risk for severe disease: miR-AGE, a randomized, double-blind placebo-controlled trial.

ABX464 (obefazimod) is a small molecule that upregulates a single microRNA (miR-124) in immune cells and reduces the production of various inflammatory cytokines and chemokines. We assessed the efficacy and safety of the standard of care (SoC) plus oral obefazimod (SoC plus ABX464), 50 mg once daily, versus the SoC plus placebo for prevention of severe acute respiratory syndrome in patients with coronavirus disease 2019 (COVID-19) who are at risk for severe disease. Eligible patients for this phase 2/3 double-blind, placebo-controlled miR-AGE study were randomized (2:1) into 2 groups: SoC-ABX464 (n= 339) and SoC-placebo (n= 170). The primary end point was the percentage of patients who did not require use of high-flow oxygen or invasive or noninvasive mechanical ventilation within 28 days. The safety analyses included patients who had been randomly assigned and had received at least 1 dose of the study treatment. At the time of the interim analysis, obefazimod showed no benefit over placebo when added to the SoC; the study enrollment was stopped for futility. The evaluation of the safety of obefazimod in 505 patients showed significantly more treatment-emergent adverse events in the SoC-ABX464 group than in the SoC-placebo group (P= .007). Frequently reported AEs in the SoC-ABX464 group included headache (14.6%), abdominal pain (9.6%), diarrhea (9.0%), back pain (6.9%), and nausea (6.0%). No treatment-related changes in laboratory parameters were reported. For patients who have severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and are at risk for severe COVID-19, obefazimod, 50 mg, provided no benefit over placebo when added to the SoC, although it did have a good safety profile (comparable to that reported in many therapeutic areas).

Anti-Atopic Dermatitis Effect of TPS240, a Novel Therapeutic Peptide, via Suppression of NF-κB and STAT3 Activation

Atopic dermatitis (AD) is a relapsing skin disease with persistent inflammation as a causal factor for symptoms and disease progression. Current therapies provide only temporary relief and require long-term usage accompanied by side effects due to persistent relapses. A short peptide, TPS240, has been tested for its potential to subside AD. In this study, we confirmed the anti-atopic effect of TPS240 in vivo and in vitro using a DNCB-induced AD mouse model and TNF-α/IFN-γ-stimulated HaCaT cells. In the AD mouse model, topical treatment with TPS240 diminished AD-like skin lesions and symptoms such as epidermal thickening and mast cell infiltration induced by DNCB, similar to the existing treatment, dexamethasone (Dex). Furthermore, skin atrophy, weight loss, and abnormal organ weight changes observed in the Dex-treated group were not detected in the TPS240-treated group. In TNF-α/IFN-γ-stimulated HaCaT cells, TPS240 reduced the expression of the inflammatory chemokines CCL17 and CCL22 and the pruritic cytokines TSLP and IL-31 by inhibiting NF-κB and STAT3 activation. These results suggest that TPS240 has an anti-atopic effect through immunomodulation of AD-specific cytokines and chemokines and can be used as a candidate drug for the prevention and treatment of AD that can solve the safety problems of existing treatments.

Expression and metabolism profiles of CVT associated with inflammatory responses and oxygen carrier ability in the brain.

As the main type of stroke, the incidence of cerebral venous thrombosis (CVT) has been rising. However, the comprehensive mechanisms behind it remain unclear. Thus, the multi-omics study is required to investigate the mechanism after CVT and elucidate the characteristic pathology of venous stroke and arterial stroke. Adult rats were subjected to CVT and MCAO models. Whole-transcriptome sequencing (RNA-seq) and untargeted metabolomics analysis were performed to construct the transcriptome and metabolism profiles of rat brains after CVT and also MCAO. The difference analysis, functional annotation, and enrichment analysis were also performed. Through RNA-seq analysis, differentially expressed genes (DEGs) were screened. 174 CVT specific genes including Il1a, Ccl9, Cxxl6, Tnfrsf14, etc., were detected. The hemoglobin genes, including both Hba and Hbb, were significantly downregulated after CVT, compared both to the MCAO and Sham groups. Metabolism analysis showed that CVT had higher heterogeneity of metabolism compared to MCAO. Metabolites including N-stearoyltyrosine, 5-methoxy-3-indoleaceate, Afegostat, pipecolic acid, etc. were specially regulated in CVT. Through the immune infiltration analysis, it was found that CVT had a higher immune response, with the abundance of certain types of immune cells increased, especially T helper cells. It was important to find the prevalence of the activation of inflammatory chemokine, cytokine, NOD-like pathway, and neutrophil extracellular trap. We explored and analyzed the gene expression and metabolomic characteristics of CVT, revealed the specific inflammatory reaction mechanism of CVT and found the markers in transcriptome and metabolism levels. It points out the direction for CVT early diagnosis and treatment.

43 Impact of Pregnancy Status on the Transcriptome of Peripheral White Blood Cells

Abstract Most embryo losses occur in the first trimester of pregnancy in cows, resulting in negative economic impact for dairy production systems. The current scientific paradigm implies the conceptus evades rejection by the maternal immune system. However, the cellular and molecular mechanisms behind the maternal immune response to the growing embryo have not been fully characterized. Hence, this study examined gene expression profiles of peripheral white blood cells (PWBC) from pregnant cows at 21 days after an embryo was transferred, (Bos taurus taurus), and cows that were treated equally but lost the embryo. We investigated the effect of pregnancy status on differential gene expression in PBMCs. We obtained and compared the total transcripts of PWBC from heifers that became pregnant at day 21 (N = 5) or failed to become pregnant after the embryo transfer (N = 5t). The total RNA transcripts, where an average of 96 % of the total reads were successfully mapped to the bovine reference genome. Each sample generates between 32 to 43 million reads. Sequencing data can be accessed by GEO with the accession number GSE210665. There was no need to remove samples in this study. Based on the length and abundance of the reads that cover the bovine genomic, we conclude that deep sequencing is suitable for the experiment outcome. A total of 13.167 genes were tested for differential expression by controlling the false discovery rate (FDR). A positive fold change denotes an overexpression and a negative fold change value a downregulation gene between treatments. After statistical analysis, 682 of these genes present a P-value smaller than 0.01, a total of 302 genes were upregulated, and 382 downregulated. The top 7 expression fold change top upregulated (logFC &amp;gt; 2) genes were COL1A2, LOC100337213, H2AC18, HTRA1, MMP14, CD5L, and LOC100297044. The top 4 downregulated (logFC&amp;lt; − 2) were ADAMDEC1, MYO1A, RPL39, and Histone H4. These data show differentially expressed genes associated with pregnancy status were both upregulated and downregulated as a response to early pregnancy, or a failure to become pregnant after embryo transfer. Our results show that these are key maternal genes for promoting tolerance allowing developing of the embryo. In summary, this study presented the genes related to positive regulation of inflammatory chemokine activity and immune defense response in the maternal PWBC.

Tunable Sulfated Alginate-based Hydrogel Platform with enhanced anti-inflammatory and antioxidant capacity for promoting burn wound repair

Amidst progressive advancements in tissue engineering, there has been a significant enhancement in the efficacy of anti-inflammatory hydrogel dressings, addressing a myriad of clinical challenges on wound healing. A frequent complication during the initial stages of deep second-degree burn wound healing is the onset of an inflammatory storm, typically occurring without effective intervention. This event disrupts normal biological healing sequences, leading to undesirable regression. In response, we have customized a tunable, multidimensional anti-inflammatory hydrogel platform based on sulfated alginates (Algs), loaded with Prussian blue (PB) nanozymes. This platform competently eliminates surplus reactive oxygen species (ROS) present in the wound bed. Algs, functioning as a mimic of sulfated glycosaminoglycans (including heparin, heparan sulfate, and chondroitin sulfate) in the extracellular matrices (ECM), demonstrate a high affinity towards inflammatory chemokines such as interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1). This affinity effectively impedes the infiltration of inflammatory cells into the wound. Concurrently, Algs markedly modulate the macrophage phenotype transition from M1 to M2. Ultimately, our potent anti-inflammatory hydrogels, which strategically target inflammatory chemokines, M1 macrophages, and ROS, successfully attenuate dysregulated hyperinflammation in wound sites. Precise immunomodulation administered to deep second-degree burn wounds in mice has demonstrated promotion of neovascular maturation, granulation tissue formation, collagen deposition, and wound closure. Our biomimetic hydrogels, therefore, represent a significant expansion in the repertoire of anti-inflammatory strategies available for clinical practice.

Weizmannia coagulans Extracellular Proteins Reduce Skin Acne by Inhibiting Pathogenic Bacteria and Regulating TLR2/TRAF6-Mediated NF-κB and MAPKs Signaling Pathways.

As a probiotic, Weizmannia coagulans (W. coagulans) is often used in food and medicine to regulate intestinal flora and exert anti-inflammatory effects. In this study, the anti-acne efficacy and mechanism of extracellular proteins (YTCY-EPs) from W. coagulans YTCY strain are analyzed. The main components of YTCY-EPs, extracted and separated from the fermentation broth, are peptides ranging from 1.51 to 11.44kDa, accounting for about 80%. Among the peptides identified by LC/MS-MS, YTCY_A-F possess the properties of antimicrobial peptides, while YTCY_1-4 possess antioxidative properties. These peptides have a strong effect on Cutibacterium acnes (C. acnes) and significantly inhibit Staphylococcus aureus. The inhibition rate of biofilm adhesion of YT-EPs to C. acnes reached 50% under the MIC. It was found that YTCY-EPs possess strong antioxidant and anti-inflammatory properties. It can effectively reduce active oxygen nearly 3 times and can reduce the downstream TLR2/NF-κB and MAPKs/AP-1 pathways by regulating the nuclear translocation of NF-κB and AP-1 in vitro. The transcriptional expression of inflammatory cytokines, inflammatory chemokines, and matrix metalloproteinase genes is also regulated, thereby slowing the recruitment of inflammatory cells and the development of inflammation, and increasing keratinocyte mobility. In addition, the expression levels of inflammatory factors and matrix metalloproteinases in the rabbit ears with acne problems that were tested with YTCY-EPs were significantly reduced, and it was obviously observed that the rabbit ear inflammation, acne, and keratinization problems were repaired. The results of this study prove that YTCY-EPs can be used as a potential anti-acne raw material in cosmetics.

Development of a novel human triculture model of non-alcoholic fatty liver disease and identification of berberine as ameliorating steatosis, oxidative stress and fibrosis.

Objectives: Non-alcoholic fatty liver disease (NAFLD) and its progression to non-alcoholic steatohepatitis (NASH) and hepatocarcinoma is a serious and growing problem. However, the development of new therapies is severely hindered by a lack of high-throughput assays for drug testing. Methods: We have developed a simple transwell assay comprised of HepG2 hepatocytes, hepatic LX-2 stellate cells, and differentiated THP-1 cells. The cells were incubated with an activating mixture containing the NASH-associated risk factors, glucose, insulin, free fatty acids (FFAs), and lipopolysaccharide (LPS) for 72h. We compared different combinations of culture conditions to obtain a model system that recapitulates the main features of NAFLD/NASH, i.e., increased steatosis, reactive oxygen species (ROS), secretion of pro-inflammatory cytokines/chemokines, and presence of fibrosis. To confirm the usefulness of the optimized model system, we screened for compounds that inhibit steatosis in the hepatocytes and evaluated the most effective compound in the triculture model system. Results: The activating mixture stimulated HepG2 cells in this triculture to accumulate more fat and produce higher levels of reactive oxygen species (ROS) than HepG2 cells in monocultures. As well, higher levels of inflammatory cytokines and chemokines (IL-8, IL-6, MIP-1α, etc.) were produced in this triculture compared to monocultures. In addition, in all LX-2 monocultures and cocultures, exposure to the activating mixture increased markers of fibrosis. A major strength of our triculture system is that it makes possible the simultaneous monitoring of 4 main features of NASH, i.e., steatosis, oxidative stress, inflammation and fibrosis. Screening potential modulators that may reduce steatosis in HepG2 cells revealed the protective effects of the isoalkaloid, berberine. Tested using this novel triculture assay, treatment with 5µM berberine decreased steatosis and ROS in HepG2 hepatocytes, reduced inflammatory cytokine production and inhibited collagen production from LX-2 cells. Conclusion: This simple triculture model recapitulates the main features of NAFLD/NASH and should be useful for high-throughput preclinical drug discovery. In this model, berberine showed promising results in decreasing steatosis and ROS and protection against fibrosis.

Cellular Heterogeneity of Activated Primary Human Macrophages and Associated Drug-Gene Networks: From Biology to Precision Therapeutics.

Interferon-γ (IFNγ) signaling plays a complex role in atherogenesis. IFNγ stimulation of macrophages permits in vitro exploration of proinflammatory mechanisms and the development of novel immune therapies. We hypothesized that the study of macrophage subpopulations could lead to anti-inflammatory interventions. Primary human macrophages activated by IFNγ (M(IFNγ)) underwent analyses by single-cell RNA sequencing, time-course cell-cluster proteomics, metabolite consumption, immunoassays, and functional tests (phagocytic, efferocytotic, and chemotactic). RNA-sequencing data were analyzed in LINCS (Library of Integrated Network-Based Cellular Signatures) to identify compounds targeting M(IFNγ) subpopulations. The effect of compound BI-2536 was tested in human macrophages in vitro and in a murine model of atherosclerosis. Single-cell RNA sequencing identified 2 major clusters in M(IFNγ): inflammatory (M(IFNγ)i) and phagocytic (M(IFNγ)p). M(IFNγ)i had elevated expression of inflammatory chemokines and higher amino acid consumption compared with M(IFNγ)p. M(IFNγ)p were more phagocytotic and chemotactic with higher Krebs cycle activity and less glycolysis than M(IFNγ)i. Human carotid atherosclerotic plaques contained 2 such macrophage clusters. Bioinformatic LINCS analysis using our RNA-sequencing data identified BI-2536 as a potential compound to decrease the M(IFNγ)i subpopulation. BI-2536 in vitro decreased inflammatory chemokine expression and secretion in M(IFNγ) by shrinking the M(IFNγ)i subpopulation while expanding the M(IFNγ)p subpopulation. BI-2536 in vivo shifted the phenotype of macrophages, modulated inflammation, and decreased atherosclerosis and calcification. We characterized 2 clusters of macrophages in atherosclerosis and combined our cellular data with a cell-signature drug library to identify a novel compound that targets a subset of macrophages in atherosclerosis. Our approach is a precision medicine strategy to identify new drugs that target atherosclerosis and other inflammatory diseases.