Upregulation Of Inflammatory Genes Research Articles

Early-life thymectomy upregulates proinflammatory chemokine signaling in visceral adipose tissue

We have previously reported that early-life thymectomy (thymex) induces an aged T cell phenotype (i.e. shift from naïve to memory) and subsequent glucose intolerance at 9mo compared to control mice. To determine potential mechanisms of inflammation and glucose intolerance in these mice, we preformed RNA sequencing (RNAseq) on the perigonadal white adipose tissue (pWAT) from thymex and control mice. We hypothesized that thymex mice would exhibit an upregulation of inflammatory genes compared to controls. This study was approved by the IACUC at the University of Texas Arlington. Male C57BL/6 mice underwent thymectomy (n=4-10) at 3wks of age or were left with their thymus intact (control n=4-10). At 9mo of age, mice were euthanized and both pWAT fat pads were dissected. One fat pad was used to determine T cell phenotype via flow cytometry, RNA was extracted from the other fat pat for RNAseq. Group differences were assessed by a students t test; RNAseq p values were adjusted for false discovery rate. RNAseq data is reported as a fold change (normalized to control group gene expression); T cell data reported as mean ± SEM (control vs thymex). There were no statistical differences in the total weight of the pWAT nor the total number of T cells in the fat pads. Gene ontology and KEGG analysis revealed that the adaptive immune response, chemokine signaling, chemokine binding and chemokine receptor binding pathways, all markers of inflammation, were upregulated in the pWAT of thymex mice compared to controls. Further analysis of these pathways revealed that gene expression for the chemokines ccl2 (MCP-1) (3.2 times greater than control; p=<0.01) and ccl5 (RANTES)(2.3 times greater than control; p=<0.01) were significantly upregulated in thymex mice compared to controls. In addition, gene expression for the receptors of these chemokines, ccr2 (1.7 times greater than control; p=<0.01) and ccr5 (4.9 times greater than control; p<0.01) were up-regulated in the pWAT of thymex mice. To determine if these chemokines were actively recruiting T cells to the pWAT, we assessed CCR2 and CCR5 expression on CD4+ (Helper) and CD8+ (Cytotoxic) T cells in the pWAT using flow cytometery. Thymex mice had significantly greater proportions of CD4+ CCR2+ (54.4±4.7%, 72.2±4.3%; p=<0.01) and CCR5+ (57.4±5.1%, 75.4±5.0%; p=0.01) cells compared to controls. Similarly, thymex mice had significantly greater proportions of CD8+ CCR2+ (23.1±4.3%, 43.3±5.4%; p=<0.01) and CCR5+(22.7±5.1%, 41.8±7.1%; p=0.02) cells. These results indicate that early life thymectomy induces upregulation of proinflammatory chemokine signaling in the pWAT. These chemokines appear to recruit T cells and potentially other immune cells to the pWAT, contributing to inflammation and potentially metabolic dysfunction. Funding: AHA 940023, NIH R01AG060395, K01AG061271. 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.

Epigenetic control of inflammation by histone methylation in cardiometabolic heart failure with preserved ejection fraction

Abstract Background Obesity represents one of the most common comorbidities in patients with heart failure with preserved ejection fraction (HFpEF). Histone post-translational modifications by chromatin modifying enzymes (CMEs) are emerging as pivotal regulators of gene transcription in the heart. Purpose To investigate the role of chromatin remodelling in obese HFpEF (obHFpEF). Methods Unbiased gene expression profiling of CMEs (PCR array) was performed in left ventricular (LV) myocardial specimens from obHFpEF patients and age-matched control donors (n=8/group). Among myocardial CMEs, the methyltransferase SETD7 showed the highest variation in gene expression. Hence, we investigated the role of SETD7 and its chromatin mark H3K4me1 in a murine model of obHFpEF. Mice with cardiomyocyte-specific deletion of SETD7 (c-SETD7-/-) and control littermates (SETD7fl/fl) were generated and subjected to high fat diet feeding and L-NAME treatment for 15 weeks to induce obHFpEF. Echocardiography and Treadmill exhaustion test were performed. ChIP-Seq datasets were employed to determine the biological pathways regulated by SETD7, whereas chromatin immunoprecipitation assays (ChIP) were performed to investigate SETD7/H3k4me1 enrichment on target gene promoters. SETD7 gain- and loss-of-function experiments were performed in cultured neonatal rat ventricular myocytes (NRVMs) exposed to palmitic acid (200µM) for 48h. Selective inhibition of SETD7 by (R)-PFI-2 was performed in skinned cardiomyocytes isolated from left ventricular specimens of obHFpEF patients. Passive stiffness, a main feature of HFpEF, was assessed before and after RPFI-2 treatment. Results CMEs profiling showed SETD7 as the top-ranking transcript (fold change, 7.36, P < 0.01) in myocardial specimens from obHFpEF patients as compared to controls. ChIP-Seq in CMs showed a strong enrichment of SETD7 and H3k4me1 on the promoter of NF-kB p65 gene, a master regulator of inflammation. SETD7 and H3k4me1 were upregulated in HFpEF vs. control mouse hearts, showed enrichment on NF-kB p65 promoter and were associated with IL-1β and IL-6 upregulation. In HFpEF mice, cardiomyocyte-specific deletion of SETD7 protected against LV hypertrophy, diastolic dysfunction (assessed by E/E’ ratio) and lung congestion while improving exercise tolerance. At the molecular level, SETD7 deletion blunted H3K4me1 enrichment on p65 promoter thus preventing the upregulation of inflammatory genes and myocardial apoptosis. In cultured CMs exposed to PA, SETD7 inhibition by (R)-PFI-2 prevented H3k4me1-driven p65 upregulation, whereas SETD7 overexpression mimicked HFpEF features. Moreover, knockdown of NF-kB p65 prevented IL-1β/IL-6 transcription in SETD7-overexpressing CMs. Of clinical relevance, (R)-PFI-2 reduced passive stiffness in skinned CMs isolated from obHFpEF patients. Conclusions Pharmacological targeting of SETD7 may represent a new strategy to prevent myocardial inflammation in cardiometabolic HFpEF.

Therapeutic S100A8/A9 blockade inhibits myocardial and systemic inflammation and mitigates sepsis-induced myocardial dysfunction

Background and AimsThe triggering factors of sepsis-induced myocardial dysfunction (SIMD) are poorly understood and are not addressed by current treatments. S100A8/A9 is a pro-inflammatory alarmin abundantly secreted by activated neutrophils during infection and inflammation. We investigated the efficacy of S100A8/A9 blockade as a potential new treatment in SIMD.MethodsThe relationship between plasma S100A8/A9 and cardiac dysfunction was assessed in a cohort of 62 patients with severe sepsis admitted to the intensive care unit of Linköping University Hospital, Sweden. We used S100A8/A9 blockade with the small-molecule inhibitor ABR-238901 and S100A9−/− mice for therapeutic and mechanistic studies on endotoxemia-induced cardiac dysfunction in mice.ResultsIn sepsis patients, elevated plasma S100A8/A9 was associated with left-ventricular (LV) systolic dysfunction and increased SOFA score. In wild-type mice, 5 mg/kg of bacterial lipopolysaccharide (LPS) induced rapid plasma S100A8/A9 increase and acute LV dysfunction. Two ABR-238901 doses (30 mg/kg) administered intraperitoneally with a 6 h interval, starting directly after LPS or at a later time-point when LV dysfunction is fully established, efficiently prevented and reversed the phenotype, respectively. In contrast, dexamethasone did not improve cardiac function compared to PBS-treated endotoxemic controls. S100A8/A9 inhibition potently reduced systemic levels of inflammatory mediators, prevented upregulation of inflammatory genes and restored mitochondrial function in the myocardium. The S100A9−/− mice were protected against LPS-induced LV dysfunction to an extent comparable with pharmacologic S100A8/A9 blockade. The ABR-238901 treatment did not induce an additional improvement of LV function in the S100A9−/− mice, confirming target specificity.ConclusionElevated S100A8/A9 is associated with the development of LV dysfunction in severe sepsis patients and in a mouse model of endotoxemia. Pharmacological blockade of S100A8/A9 with ABR-238901 has potent anti-inflammatory effects, mitigates myocardial dysfunction and might represent a novel therapeutic strategy for patients with severe sepsis.Graphical

Tumor microenvironment modifications induced by afatinib in squamous cell carcinoma of the head and neck - a window-of-opportunity study (EORTC90111-24111).

The EORTC-90111-24111 phase II window study evaluated afatinib versus no preoperative treatment in patients with primary squamous cell carcinoma of the head and neck (HNSCC). We investigated afatinib-induced tumor and microenvironment modifications by comparing pre- and post-treatment tumor biopsies. Thirty treatment-naïve patients with primary HNSCC were randomized. Twenty-five patients received afatinib for 14 days before surgery (40mg 1x/day) and five patients were attributed to the control arm. Biopsies were taken at work-up and during surgery. Good quality RNA samples were used for omics analyses. The control arm was enlarged by samples coming from our previous similar window study (Schmitz et al. 2013). Immunohistochemistry analyses of afatinib-treated tumor biopsies showed a decrease in pEGFR (P≤.05) and pERK (P≤.05); an increase in CD3+ (P≤.01) and CD8+ (P≤.01) T-cell infiltration, and in CD3+ (P≤.05) T-cell density. RNA sequencing analyses of afatinib-treated tumor samples showed upregulation of inflammatory genes and increased expression scores of signatures predictive of response to PD-1 blockade (P≤.05). In post-treatment biopsies of afatinib-treated patients, two clusters were observed. Cluster 1 showed a higher expression of markers and gene sets implicated in epithelial-to-mesenchymal transition (EMT) and activation of cancer-associated fibroblasts (CAFs) compared to cluster 2 and controls. Short-term treatment with afatinib in primary HNSCC induces CD3+ and CD8+ tumor infiltration and, in some patients, EMT and CAFs activation. These results open perspectives to overcome resistance mechanisms to anti-HER therapy and to potentiate the activity of immune checkpoint inhibitors.

ETV6 represses inflammatory response genes and regulates HSPC function during stress hematopoiesis in mice

AbstractETS variant 6 (ETV6) encodes a transcriptional repressor expressed in hematopoietic stem and progenitor cells (HSPCs), where it is required for adult hematopoiesis. Heterozygous pathogenic germline ETV6 variants are associated with thrombocytopenia 5 (T5), a poorly understood genetic condition resulting in thrombocytopenia and predisposition to hematologic malignancies. To elucidate how germline ETV6 variants affect HSPCs and contribute to disease, we generated a mouse model harboring an Etv6R355X loss-of-function variant, equivalent to the T5-associated variant ETV6R359X. Under homeostatic conditions, all HSPC subpopulations are present in the bone marrow (BM) of Etv6R355X/+ mice; however, these animals display shifts in the proportions and/or numbers of progenitor subtypes. To examine whether the Etv6R355X/+ mutation affects HSPC function, we performed serial competitive transplantation and observed that Etv6R355X/+ lineage−sca1+cKit+ (LSK) cells exhibit impaired reconstitution, with near complete failure to repopulate irradiated recipients by the tertiary transplant. Mechanistic studies incorporating cleavage under target and release under nuclease assay, assay for transposase accessible chromatin sequencing, and high-throughput chromosome conformation capture identify ETV6 binding at inflammatory gene loci, including multiple genes within the tumor necrosis factor (TNF) signaling pathway in ETV6-sufficient mouse and human HSPCs. Furthermore, single-cell RNA sequencing of BM cells isolated after transplantation reveals upregulation of inflammatory genes in Etv6R355X/+ progenitors when compared to Etv6+/+ counterparts. Corroborating these findings, Etv6R355X/+ HSPCs produce significantly more TNF than Etv6+/+ cells post-transplantation. We conclude that ETV6 is required to repress inflammatory gene expression in HSPCs under conditions of hematopoietic stress, and this mechanism may be critical to sustain HSPC function.

Therapeutic role of mesenchymal stem cell-derived extracellular vesicles in neuroinflammation and cognitive dysfunctions induced by binge-like ethanol treatment in adolescent mice.

Extracellular vesicles (EVs) are heterogeneous membrane vesicles secreted by cells in extracellular spaces that play an important role in intercellular communication under both normal and pathological conditions. Mesenchymal stem cells (MSC) are anti-inflammatory and immunoregulatory cells capable of secreting EVs, which are considered promising molecules for treating immune, inflammatory, and degenerative diseases. Our previous studies demonstrate that, by activating innate immune receptors TLR4 (Toll-like receptor 4), binge-like ethanol exposure in adolescence causes neuroinflammation and neural damage. To evaluate whether the intravenous administration of MSC-derived EVs is capable of reducing neuroinflammation, myelin and synaptic alterations, and the cognitive dysfunction induced by binge-like ethanol treatment in adolescent mice. MSC-derived EVs obtained from adipose tissue were administered in the tail vein (50 microg/dose, one weekly dose) to female WT adolescent mice treated intermittently with ethanol (3.0 g/kg) during two weeks. MSC-derived EVs from adipose tissue ameliorate ethanol-induced up-regulation of inflammatory genes (e.g., COX-2, iNOS, MIP-1α, NF-κB, CX3CL1, and MCP-1) in the prefrontal cortex of adolescent mice. Notably, MSC-derived EVs also restore the myelin and synaptic derangements, and the memory and learning impairments, induced by ethanol treatment. Using cortical astroglial cells in culture, our results further confirm that MSC-derived EVs decrease inflammatory genes in ethanol-treated astroglial cells. This, in turn, confirms in vivo findings. Taken together, these results provide the first evidence for the therapeutic potential of the MSC-derived EVs in the neuroimmune response and cognitive dysfunction induced by binge alcohol drinking in adolescence.

1684-P: Transgene Integration-Induced Genomic Rearrangement in a Noncoding Region of Chromosome 1 Leads to Spontaneous Obesity and Type 2 Diabetes in Mice

Obesity is a critical risk factor for type 2 diabetes (T2D) and has become a global health issue. We found that a mouse line (C57/B6J) of Floxed-stop over expression of IF1 (ATPase Inhibitory Factor 1) (hereon called FEI) develops spontaneous obesity without induction of transgenic expression. Targeted-Locus-Amplification (TLA) analyses revealed a rearrangement in upstream sequences of the transgene integration site, and a duplication downstream in a non-coding region of chromosome 1. Male and female FEI mice show early dyslipidemia before weight gain at two months, reaching 50-60 grams at 6-8 months. Body composition revealed FEI mice have markedly increased fat mass and modestly increased lean mass, consistent with morphometric findings of substantially increased peripheral fat pads. FEI mice were hyperphagic. When subjected to pair-feeding, they reverted to comparable WT body weight levels, then gained weight again when fed ad libidum. Adult FEI mice maintain comparable physical activity levels to their WT counterparts, but indirect metabolic levels among FEI mice declined. These mice exhibited glucose and insulin tolerance at six weeks of age, as well as hyperglycemia, hyper-insulinemia, and hyper-leptinemia. Histology H&E staining revealed markedly enlarged adipocytes with crown-like structures on visceral white adipose tissue sections, and increased fat deposition in liver sections. RNAseq analyses from subcutaneous white adipose tissues of 5-month-old FEI mice demonstrate upregulation of adipogenic and inflammatory genes. Isolated FEI preadipocytes exhibited increased proliferation but impaired lipid deposition. RT-PCR revealed upregulation of inflammatory genes (IFI202b, TNF-α, and IL6). Together, we show FEI mice are a novel obese mouse model. Preclinical studies using this model may help yield insights into novel pathogenic mechanisms and effective treatments for obesity. Disclosure D.White: None. S.Alahari: None. E.D.Lazartigues: None. Q.Yang: None. J.Wu: None. S.Kang: None. L.Lauterboeck: None. P.Mobasheran: None. F.Paliarin: None. T.Nguyen: None. M.Elgazzaz: None. R.Maiya: None. Funding American Diabetes Association (1-17-IBS-184 to Q.Y.)

RELA governs a network of islet-specific metabolic genes necessary for beta cell function

Aims/hypothesisNF-κB activation unites metabolic and inflammatory responses in many diseases yet less is known about the role that NF-κB plays in normal metabolism. In this study we investigated how RELA impacts the beta cell transcriptional landscape and provides network control over glucoregulation.MethodsWe generated novel mouse lines harbouring beta cell-specific deletion of either the Rela gene, encoding the canonical NF-κB transcription factor p65 (βp65KO mice), or the Ikbkg gene, encoding the NF-κB essential modulator NEMO (βNEMOKO mice), as well as βA20Tg mice that carry beta cell-specific and forced transgenic expression of the NF-κB-negative regulator gene Tnfaip3, which encodes the A20 protein. Mouse studies were complemented by bioinformatics analysis of human islet chromatin accessibility (assay for transposase-accessible chromatin with sequencing [ATAC-seq]), promoter capture Hi-C (pcHi-C) and p65 binding (chromatin immunoprecipitation–sequencing [ChIP-seq]) data to investigate genome-wide control of the human beta cell metabolic programme.ResultsRela deficiency resulted in complete loss of stimulus-dependent inflammatory gene upregulation, consistent with its known role in governing inflammation. However, Rela deletion also rendered mice glucose intolerant because of functional loss of insulin secretion. Glucose intolerance was intrinsic to beta cells as βp65KO islets failed to secrete insulin ex vivo in response to a glucose challenge and were unable to restore metabolic control when transplanted into secondary chemical-induced hyperglycaemic recipients. Maintenance of glucose tolerance required Rela but was independent of classical NF-κB inflammatory cascades, as blocking NF-κB signalling in vivo by beta cell knockout of Ikbkg (NEMO), or beta cell overexpression of Tnfaip3 (A20), did not cause severe glucose intolerance. Thus, basal p65 activity has an essential and islet-intrinsic role in maintaining normal glucose homeostasis. Genome-wide bioinformatic mapping revealed the presence of p65 binding sites in the promoter regions of specific metabolic genes and in the majority of islet enhancer hubs (~70% of ~1300 hubs), which are responsible for shaping beta cell type-specific gene expression programmes. Indeed, the islet-specific metabolic genes Slc2a2, Capn9 and Pfkm identified within the large network of islet enhancer hub genes showed dysregulated expression in βp65KO islets.Conclusions/interpretationThese data demonstrate an unappreciated role for RELA as a regulator of islet-specific transcriptional programmes necessary for the maintenance of healthy glucose metabolism. These findings have clinical implications for the use of anti-inflammatories, which influence NF-κB activation and are associated with diabetes.Graphical

Abstract 4833: Inhibiting the mitochondrial RNA degradosome complex SUV3 and PNPase increases dsRNA in the cytoplasm, triggers a viral mimicry response and kills AML cells and progenitors

Abstract Eukaryotic cells have two separate genomes; nuclear chromosomal DNA and circular mitochondrial DNA. Mitochondrial DNA lacks introns, and encodes 2 rRNAs, 22 t-RNAs and 13 of the 90 proteins that constitute the mitochondrial respiratory chain. To maintain homeostasis, mitochondrial RNA degradation machinery regulates RNA turnover. ATP-dependent helicase, SUV3 (gene SUPV3L1), and exonuclease PNPase (gene PNPT1) function as a complex to degrade mitochondrial dsRNA. By immunoblotting, PNPase and SUV3 proteins were increased in 7/7 AML patient samples and 13/13 of AML cell lines, compared to the normal hematopoietic cells. Analysis of the TARGET AML dataset revealed AML patients with increased expression of SUPV3L1 (p = 0.051, p= 0.045) and PNPT1 (p = 0.0013, p = 0.018) had decreased overall and event free survival. Genetic knockdown or knockout using shRNA or sgRNA against PNPT1 or SUPV3L1 decreased growth and viability of OCI-AML2, TEX, K562, U937, NB4 and OCI-AML 8227 cells. Furthermore, SUPV3L1 & PNPT1 ranked in the top 5.2% and 7.4% of essential genes in 26 leukemia cell lines in CRISPR screens and 2.7% and 4.9% in RNAi screens (depmap.org). Knockdown of PNPT1 & SUPV3L1 also reduced the clonogenic growth of OCI-AML2, TEX and U937 cells and significantly reduced engraftment of TEX cells into the marrow of immune deficient mice, demonstrating the functional importance on leukemia initiating cells in vivo. SUPV3L1 knockdown in primary AML cells reduced engraftment in marrow of immune deficient mice. Bioinformatics analysis to detect processes associated with PNPT1 and SUPV3L1, we identified associations with Response to exogenous dsRNA, Response to virus, and RNA catabolic process ontologies. Consistent with this, we observed knockdown of PNPT1 or SUPV3L1 increased expression of genes (INFgR1, ICAM, IRF7 & JAK/STAT) suggesting an interferon response. As PNPT1 and SUPV3L1 degrade mitochondrial dsRNA, we measured levels of dsRNA after knockdown of these genes. Knockdown of PNPT1 and SUPV3L1 in OCI-AML2 cells increased levels of cytoplasmic/mitochondrial dsRNA 3-4 fold compared to control. Knockdown of PNPT1 and SUPV3L1 also increased dsRNA in 143B cells, but not Rho (0) 143B cells that lack mitochondrial DNA. Upregulation of inflammatory genes leads to viral mimicry and can increase sensitivity to immune mediated killing. We observed enhanced sensitivity to Double Negative T (DNT) cells mediated killing in PNPT1 and SUPV3L1 knockdown OCI-AML2 cells compared to control cells. In summary, RNA degradosome complex proteins SUPV3L1 and PNPT1 are overexpressed in AML, and are essential for the survival of AML cells and AML stem/progenitors. These enzymes regulate levels of mitochondrial dsRNA, and their inhibition leads to increased cytoplasmic dsRNA triggering a viral mimicry response and enhanced sensitivity to immune-mediated killing. Citation Format: Geethu Emily Thomas, Kazem Nouri, Jong Bok Lee, Rose Hurren, Yongran Yan, Neil MacLean, Yulia Jitkova, Li Ma, Xiao Ming Wang, Chaitra Sarathy, Andrea Arruda, Mark D. Minden, Li Zhang, Vito Spadavecchio, Aaron Schimmer. Inhibiting the mitochondrial RNA degradosome complex SUV3 and PNPase increases dsRNA in the cytoplasm, triggers a viral mimicry response and kills AML cells and progenitors. [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 4833.

Lack of STAT1 co-operative DNA binding protects against adverse cardiac remodelling in acute myocardial infarction.

In this study, we addressed the functional significance of co-operative DNA binding of the cytokine-driven transcription factor STAT1 (signal transducer and activator of transcription 1) in an experimental murine model of acute myocardial infarction (MI). STAT1 knock-in mice expressing a phenylalanine-to-alanine substitution at position 77 in the STAT1 amino-terminal domain were examined for the early clinical effects produced by ligation of the left anterior descending coronary artery (LAD), an established model for MI. The F77A mutation has been previously reported to disrupt amino-terminal interactions between adjacent STAT1 dimers resulting in impaired tetramerization and defective co-operative binding on DNA, while leaving other protein functions unaffected. Our results demonstrate that a loss of STAT1 tetramer stabilization improves survival of adult male mice and ameliorates left ventricular dysfunction in female mice, as determined echocardiographically by an increased ejection fraction and a reduced left intra-ventricular diameter. We found that the ratio of STAT3 to STAT1 protein level was higher in the infarcted tissue in knock-in mice as compared to wild-type (WT) mice, which was accompanied by an enhanced infiltration of immune cells in the infarcted area, as determined by histology. Additionally, RNA sequencing of the infarcted tissue 24 h after LAD ligation revealed an upregulation of inflammatory genes in the knock-in mice, as compared to their WT littermates. Concomitantly, genes involved in oxidative phosphorylation and other metabolic pathways showed a significantly more pronounced downregulation in the infarcted tissue from STAT1F77A/F77A mice than in WT animals. Based on these results, we propose that dysfunctional STAT1 signalling owing to a lack of oligomerisation results in a compensatory increase in STAT3 expression and promotes early infiltration of immune cells in the infarcted area, which has beneficial effects on left ventricular remodelling in early MI following LAD ligation.

Microglia Exhibit Distinct Heterogeneity Rather than M1/M2 Polarization within the Early Stage of Acute Ischemic Stroke.

The classification of microglial M1/M2 polarization in the acute phase of ischemic stroke remains controversial, which has limited further advances in neuroprotective strategy. To thoroughly assess the microglial phenotypes, we made the middle cerebral artery occlusion model in mice to simulate the acute pathological processes of ischemic stroke from normal conditions to acute cerebral ischemia and then to the early reperfusion period. The temporal changes in gene profiles, cell subtypes, and microglial function were comprehensively analyzed using single-cell RNA sequencing. We identified 37,614 microglial cells and divided them into eight distinct subpopulations. Mic_home, Mic_pre1, and Mic_pre2 subpopulations were three clusters mainly composed of cells from the control samples, in which Mic_home was a homeostatic subpopulation characterized by high expression of Hpgd and Tagap, and Mic_pre1 and Mic_pre2 were two clusters with preliminary inflammatory activation characteristics marked by P2ry13 and Wsb1 respectively. Mic_M1L1 and Mic_M1L2 subpopulations exhibited M1-like polarization manifested by the upregulation of inflammatory genes after ischemic stroke, while the intrinsic heterogeneity on the level of inflammatory responses and neurotrophic support properties was observed. Moreover, we identified three unique clusters of cells with low inflammation levels. Mic_np1, Mic_np2, and Mic_np3 were characterized by high expression of Arhgap45, Rgs10, and Pkm respectively. However, these cells did not show significant M2-like characteristics and their classic microglia function was also attenuated. These subpopulations exhibited higher activation of neuropeptide functional pathways. At last, we performed cell-cell communication analysis and identified major couplings contributing to the interaction between microglia and other cell populations. In summary, our study elucidated the temporal heterogeneity of microglia in the acute phase of ischemic stroke, which may facilitate the identification of effective neuroprotective targets to curb ischemic damage at an early stage.

Aggregated alpha-synuclein transcriptionally activates pro-inflammatory canonical and non-canonical NF-κB signaling pathways in peripheral monocytic cells

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by chronic neuroinflammation, loss of dopaminergic neurons in the substantia nigra, and in several cases accumulation of alpha-synuclein fibril (α-syn) containing Lewy-bodies (LBs). Peripheral inflammation may play a causal role in inducing and perpetuating neuroinflammation in PD and accumulation of fibrillar α-syn has been reported at several peripheral sites including the gut and liver. Peripheral fibrillar α-syn may induce activation of monocytes via recognition by toll-like receptors (TLRs) and stimulation of downstream NF-κB signaling; however, the specific mechanism by which this occurs is not defined. In this study we utilized the THP-1 monocytic cell line to model the peripheral transcriptional response to preformed fibrillar (PFF) α-syn. Compared to monomeric α-syn, PFF α-syn displays overt inflammatory gene upregulation and pathway activation including broad pan-TLR signaling pathway activation and increases in TNF and IL1B gene expression. Notably, the non-canonical NF-κB signaling pathway gene and PD genome wide association study (GWAS) candidate NFKB2 was upregulated. Additionally, non-canonical NF-κB activation-associated RANK and CD40 pathways were also upregulated. Transcriptional-phenotype analysis suggests PFFs induce transcriptional programs associated with differentiation of monocytes towards macrophages and osteoclasts via non-canonical NF-κB signaling as a potential mechanism in which myeloid/monocyte cells may contribute to peripheral inflammation and pathogenesis in PD.

Evaluation of cytotoxicity, analysis of metals and cumulative risk assessment in microalgae

Microalgae are one promising source for the production of bioactive compounds. However, microalgae can accumulate harmful substances. So, our objectives were (i) to evaluate cell viability after Phaeodactylum triconutum (0% and 65% cell disruption, DR) and Tetraselmis chuii (0% and 67% DR) freeze-dried exposure in HepG2 cells by MTT assay; (ii) to evaluate cell viability after P. triconutum and T. chuii extract exposure; (iii) to assess the effect in cell viability when they were simultaneously exposed to T-2 toxin and, (iv) to evaluate if inflammatory response is related to the mechanism of toxicity of these microalgae by qPCR assays. Results demonstrated that cell viability did not increase after freeze-dried microalgae exposure in HepG2 cells. And, no IC50 values were observed. However, an increase in HepG2 cell viability after exposure of T. chuii 0% DR extract at 5, 25 and 100 µg/mL was observed. Additionally, 1:64 diluted T. chuii 0% DR with IC50/4 T-2 and with IC50/2 T-2 and 1:32 diluted T. chuii 0% DR with IC50/4 T-2 showed an increase in cell viability. Both microalgae increased the relative TNF-α, IL-1β and IL-6 mRNA expression. Concluding, no cytotoxic effect was evidenced but, it was noted up-regulation of inflammatory genes after T. chuii exposure in HepG2 cell. Thus, more studies related the mechanistic toxicity of microalgae are needed to evaluate the potential toxicological risk of inflammation of these novel foods. .

Influenza A Virus Exacerbates Group A Streptococcus Infection and Thwarts Anti-Bacterial Inflammatory Responses in Murine Macrophages.

Seasonal influenza epidemics pose a considerable hazard for global health. In the past decades, accumulating evidence revealed that influenza A virus (IAV) renders the host vulnerable to bacterial superinfections which in turn are a major cause for morbidity and mortality. However, whether the impact of influenza on anti-bacterial innate immunity is restricted to the vicinity of the lung or systemically extends to remote sites is underexplored. We therefore sought to investigate intranasal infection of adult C57BL/6J mice with IAV H1N1 in combination with bacteremia elicited by intravenous application of Group A Streptococcus (GAS). Co-infection in vivo was supplemented in vitro by challenging murine bone marrow derived macrophages and exploring gene expression and cytokine secretion. Our results show that viral infection of mice caused mild disease and induced the depletion of CCL2 in the periphery. Influenza preceding GAS infection promoted the occurrence of paw edemas and was accompanied by exacerbated disease scores. In vitro co-infection of macrophages led to significantly elevated expression of TLR2 and CD80 compared to bacterial mono-infection, whereas CD163 and CD206 were downregulated. The GAS-inducible upregulation of inflammatory genes, such as Nos2, as well as the secretion of TNFα and IL-1β were notably reduced or even abrogated following co-infection. Our results indicate that IAV primes an innate immune layout that is inadequately equipped for bacterial clearance.

Supraphysiologic Vaginal Estrogen Therapy in Aged Mice Mitigates Age-Associated Bladder Inflammatory Response to Urinary Tract Infections.

Bladder diseases characterized by chronic inflammation are highly prevalent in older women, as are recurrent urinary tract infections (rUTIs). Recurrent urinary tract infections lead to chronic inflammation of the bladder mucosa and cause lower urinary tract symptoms that persist even after the infection is cleared. Vaginal estrogen therapy (VET) has long been used for the treatment of rUTIs; however, its mechanism of action remains unclear. The objective of this study was to examine the mechanism(s) by which VET affects bladder inflammation and response to rUTIs. Here, we induced surgical menopause in aged (18 months old) mice followed by VET. Mice were then infected with uropathogenic Escherichia coli , and course of infection was investigated. Inflammatory cytokine response was assessed before and during infection using enzyme-linked immunosorbent assay. RNA sequencing analysis was used to compare the inflammatory status of the young versus aged bladder and principal changes confirmed via quantitative reverse transcriptase-polymerase chain reaction to determine the effects of VET on bladder inflammation. Impact on age-associated bladder tertiary lymphoid tissue formation was evaluated histologically. In the ovariectomized aged model, VET not only mitigated uterine atrophy but was also associated with reduced rUTIs, number of bacterial reservoirs, dampened immune response, and promotion of terminal differentiation of urothelial cells. Bladder tertiary lymphoid tissue lesions were also reduced with VET, with an associated decrease in signals important for bladder tertiary lymphoid tissue formation. Finally, we determined that VET reverses age-associated upregulation of inflammatory genes and pathways. Our data suggest that VET is effective by reducing age-associated hyperinflammatory conditions in bladder mucosa and in enhancing the host response to infection.

A chromatin mark by SETD7 regulates myocardial inflammation in obesity-related heart failure with preserved ejection fraction

Abstract Background Obesity is a major risk factor for heart failure with preserved ejection fraction (HFpEF). Post-translational modification of histones by chromatin modifying enzymes (CMEs) are emerging as pivotal regulators of gene transcription in cardiovascular disease. Purpose To investigate the role of chromatin remodelling in obese HFpEF (obHFpEF). Methods Gene expression profiling of CMEs (PCR array) was performed in left ventricular (LV) myocardial specimens from obHFpEF patients and age-matched control donors (n=8/group). Among myocardial CMEs, the methyltransferase SETD7 showed the highest variation in gene expression. Hence, we investigated the role of SETD7 and its chromatin mark H3K4me1 in a murine model of obHFpEF. Mice with cardiomyocyte-specific deletion of SETD7 (c-SETD7−/−) and control littermates (SETD7fl/fl) were subjected to high fat diet feeding and L-NAME treatment for 15 weeks to induce obHFpEF. Echocardiography and Treadmill exhaustion test were performed. ChIP-Seq datasets were employed to determine the biological pathways regulated by SETD7, whereas chromatin immunoprecipitation assays (ChIP) were performed to investigate SETD7/H3k4me1 enrichment on target gene promoters. SETD7 gain- and loss-of-function experiments were performed in cultured neonatal rat ventricular myocytes (NRVMs) exposed to palmitic acid (200μM) for 48h. Selective inhibition of SETD7 by (R)-PFI-2 was performed in skinned cardiomyocytes isolated from left ventricular specimens of obHFpEF patients. Passive stiffness, a main feature of HFpEF, was assessed before and after (R)-PFI-2 treatment. Results CMEs profiling showed SETD7 as the top-ranking transcript (fold change, 7.36, P<0.01) in myocardial specimens from obHFpEF patients as compared to controls. ChIP-Seq in CMs showed a strong enrichment of SETD7 and H3k4me1 on the promoter of NF-kB p65 gene, a master regulator of inflammation. SETD7 and H3k4me1 were upregulated in HFpEF vs. control mouse hearts, showed enrichment on NF-kB p65 promoter and were associated with IL-1β and IL-6 upregulation. In HFpEF mice, cardiomyocyte-specific deletion of SETD7 protected against LV hypertrophy, diastolic dysfunction (assessed by E/E' ratio) and lung congestion while improving exercise tolerance. At the molecular level, SETD7 deletion blunted H3K4me1 enrichment on p65 promoter thus preventing the upregulation of inflammatory genes and myocardial apoptosis. In cultured CMs exposed to PA, SETD7 inhibition by (R)-PFI-2 prevented H3k4me1-driven p65 upregulation, whereas SETD7 overexpression mimicked HFpEF features. Moreover, knockdown of NF-kB p65 prevented IL-1β/IL-6 transcription in SETD7-overexpressing CMs. Of clinical relevance, (R)-PFI-2 reduced passive stiffness in skinned CMs isolated from obHFpEF patients. Conclusions Our results unveil a new epigenetic mechanism underpinning inflammation in obHFpEF. Targeting SETD7 may represent a novel therapeutic approach to prevent HFpEF in obesity. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Swiss National Science Foundation

Effect of triadimefon on rat placental morphology, function, and gene expression

Triadimefon is a fungicide that is broadly used to treat fungal diseases of plants. It causes developmental toxicity in the animal model. Whether triadimefon disrupts the placental function and the underlying mechanism remains unclear. Thirty-six female pregnant Sprague-Dawley rats were randomly assigned into four groups and were orally administered via gavage of triadimefon (0, 25, 50, and 100 mg/kg/day) for 10 days from gestational day (GD) 12–21. Triadimefon disrupted the structure of the placenta, leading to hypertrophy, abnormal hemodynamics, including fibrin exudation, edema, hemorrhage, infarction, and inflammation. RNA-seq analysis showed that triadimefon down-regulated the expression of developmental and metabolic genes, while up-regulating the immune/inflammatory genes. The qPCR showed that triadimefon markedly down-regulated the expression of Cpt1c, Scd2, Ldlr, Dvl1, Flt4, and Vwf and their proteins, while up-regulating the expression of Cyp1a1, Star, Ccl5, and Cx3cr1 and their proteins at 25–100 mg/kg. Western blot showed that triadimefon reduced the level of STAT3 at doses of 50 and 100 mg/kg and the phosphorylation of AMPK at 100 mg/kg. In conclusion, triadimefon severely damages the structure and function of the placenta, leading to placental hypertrophy, local blood circulation disorders, and inflammation and this may be associated with its down-regulation of genes related to metabolism and nutrient transport and the up-regulation of inflammatory genes via STAT3 and AMPK signals.

Inflammatory stimuli alter bone marrow composition and compromise bone health in the malnourished host.

Inflammation has a role in the pathogenesis of childhood malnutrition. We investigated the effect of malnutrition and inflammatory challenge on bone marrow composition and bone health. We studied an established murine model of moderate acute malnutrition at baseline and after acute inflammatory challenge with bacterial lipopolysaccharide (LPS), a surrogate of Gram-negative bacterial sepsis, or Leishmania donovani, the cause of visceral leishmaniasis. Both of these infections cause significant morbidity and mortality in malnourished children. Of the 2 stimuli, LPS caused more pronounced bone marrow changes that were amplified in malnourished mice. LPS challenge led to increased inflammatory cytokine expression (Il1b, Il6, and Tnf), inflammasome activation, and inflammatory monocyte accumulation in the bone marrow of malnourished mice. Depletion of inflammatory monocytes in Csfr1-LysMcre-DT malnourished mice significantly reduced the inflammasome activation and IL1-ß production after LPS challenge. The inflammatory challenge also led to increased expansion of mesenchymal stem cells (MSCs), bone marrow adiposity, and expression of genes (Pparg, Adipoq, and Srbp1) associated with adipogenesis in malnourished mice. This suggests that inflammatory challenge promotes differentiation of BM MSCs toward the adipocyte lineage rather than toward bone-forming osteoblasts in the malnourished host. Concurrent with this reduced osteoblastic potential there was an increase in bone-resorbing osteoclasts, enhanced osteoclast activity, upregulation of inflammatory genes, and IL-1B involved in osteoclast differentiation and activation. The resulting weakened bone formation and increased bone resorption would contribute to the bone fragility associated with malnutrition. Lastly, we evaluated the effect of replacing lipid rich in omega-6 fatty acids (corn oil) with lipid-rich in omega-3 fatty acids (fish oil) in the nutrient-deficient diet. LPS-challenged malnourished mice that received dietary fish oil showed decreased expression of inflammatory cytokines and Rankl and reduced osteoclast differentiation and activation in the bone marrow. This work demonstrates that the negative effect of inflammatory challenge on bone marrow is amplified in the malnourished host. Increasing dietary intake of omega-3 fatty acids may be a means to reduce inflammation and improve bone health in malnourished children.

Abstract 124: PRDX4 links ER-specific redox vulnerability to innate immunity in pancreatic cancer

Abstract The prognosis of pancreatic ductal carcinoma (PDAC) remains dismal, with an overall 5-year survival rate of 7%. Immunotherapy (IT) has provided new hope for other hard-to-treat cancers, but PDAC has shown striking resistance to IT by orchestrating mechanisms of immune escape. This spurs an interest to identify novel strategies to improve PDAC sensitivity to IT. We recently unveiled that perturbating redox homeostasis by targeting the antioxidant protein peroxiredoxin 4 (PRDX4) render pancreatic cancer cells highly vulnerable. PRDX4 supports redox homeostasis by metabolizing H2O2 in the endoplasmic reticulum (ER), and its loss leads to oxidative stress and toxicity. Interestingly, cell death induced by PRDX4 depletion is accompanied by DNA damage and accumulation of cytosolic DNA, a potent primer for immune therapy. In line with that, PRDX4 knockdown results in the transcriptional upregulation of inflammatory genes, including type I interferons (IFNs), primary cytokines and interferon-stimulated genes (ISGs). Importantly, our data indicate that this induction is dependent on the critical regulator of the innate immune responses, STING, as treatment with a STING inhibitor, or a STING knockdown, substantially blunts their upregulation. It is known that following DNA damage detection, the ER-localized STING translocates to the Golgi, where it associates with the kinase TBK1 and mediates the induction of inflammatory target genes, depending on IRF3 and NF-κB transcription factors. We observe an activation of downstream STING effectors upon PRDX4 loss, and co-depletion with NFκB significantly rescues the upregulation of those genes. As such, we propose that PRDX4 links ER-specific redox vulnerability to innate immunity signaling in PDAC. Since PRDX4 loss causes increased DNA damage and inflammatory signaling, our preliminary data suggest that PRDX4 could be a novel therapeutic target that may act synergistically with immune checkpoint inhibitors. Citation Format: Lucie Malbeteau, Pallavi Jain, Shane Harding, Bradley Wouters, Marianne Koritzinsky. PRDX4 links ER-specific redox vulnerability to innate immunity in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 124.

Aggregated Alpha‐Synuclein Activates Pro‐Inflammatory NFKB Signaling Pathways Through TLR‐Dependent and Independent Mechanisms in Peripheral Monocytic Cells

Toll‐like receptors (TLRs) are pattern recognition molecules that induce inflammatory signaling cascades upon recognition of pathogen or damage associated molecules. In addition to peripheral immune cells including monocytes, TLRs are broadly expressed on cells of the central nervous system (CNS) including microglia, astrocytes, and neurons and their activation induces production of pro‐inflammatory mediators inducing neuroinflammation and neurodegeneration in the chronic state. Prolonged neuroinflammation in the substantia nigra (SN) leading to accumulation of alpha‐synuclein fibril (α‐syn) containing Lewy‐bodies (LBs) and ultimately death of dopaminergic neurons is a pathological hallmark of Parkinson’s disease (PD) and is thought to be a major mechanism driving disease progression. TLRs are an integral component of the feed‐forward neuroinflammatory loop where damage from neurodegeneration generates TLR ligands that further amplify the inflammatory circuit. Importantly, aggregate fibrillar α‐syn can interact with TLRs on microglia in the brain, of which TLR2 and 4 are best characterized; however, the extent to which fibrillar α‐syn interacts with peripheral immune cells including monocytes is poorly understood. Peripheral inflammation may play a causal role in inducing and perpetuating neuroinflammation and accumulation of fibrillar α‐syn has been reported at several peripheral sites including the gut and liver in PD. To model the peripheral immune response to fibrillar α‐syn we exposed THP‐1 monocytic cells in vitro to determine inflammatory signaling pathways altered by α‐syn. Compared to monomeric α‐syn, preformed‐fibrillar (PFF) α‐syn displays overt inflammatory gene upregulation and pathway activation as assessed by RNA‐Seq. Both MyD88 and TRIF mediated TLR signaling pathways were broadly overrepresented in the PFF condition as well as RNAK/RANKL signaling supported by large increases in NFKB signaling and pro‐inflammatory cytokine production including TNF and IL‐1B. These results indicate that α‐syn may act broadly across all TLRs and other pro‐inflammatory receptors such as RANK.