Veranstaltungsprogramm

Background:

Obesity is a global health burden and a chronic life-threatening disease associated with low-grade inflammation and metabolic dysfunction. The intestinal immune system has emerged as a central regulator of systemic inflammation and glucose homeostasis. Inhibition of gut immune cell trafficking may therefore represent a novel mechanism linking intestinal immunity to metabolic dysfunction. The mucosal adhesion molecule MAdCAM-1 mediates the recruitment of α4β7 expressing lymphocytes to the intestinal mucosa, but its contribution to obesity-associated inflammation and metabolic dysfunction remains unknown. The aim of this study was to investigate the effect of MAdCAM-1-deficiency on metabolism and obesity.

Methods:

Male MAdCAM-1–/– mice (n=13) and wild type mice (n=10) were fed a high-fat diet (HFD) and compared to control groups (MAdCAM-1–/–, n=5, MAdCAM-1 WT, n=6) fed chow diet for 20 weeks. Body weight, fasted blood glucose levels, and insulin tolerance (ITT) were assessed. Systemic immune cell profiling was performed by multicolor Aurora flow cytometry (FACS) of blood, white adipose tissue (WAT), liver, kidney, spleen, and bone marrow. In addition, intestinal immune cell subsets were analyzed in the lamina propria (LP) and intraepithelial lymphocytes (IEL) of the small intestine.

Results:

Despite similar body weight gain under HFD, MAdCAM-1–/– mice showed a non- significant trend to reduced fasting blood glucose levels (10.48 ± 1.35 vs. 7.98 ± 0.41 mmol/l in WT vs. MAdCAM-1–/– mice, p=0.08). Furthermore, mice lacking MAdCAM-1 had improved insulin tolerance compared to WT mice (ITT AUC of blood glucose levels over time: 742.3 ± 0.48 vs. 985.4 ± 0.37, p=0.02). FACS analysis revealed a reduction of Ly6Chigh monocytes in the blood (33%), WAT (49%) and in the kidney (28%) in MAdCAM-1–/– mice vs. WT mice. In WT mice, obesity induced by HFD led to a distinct redistribution of intestinal immune cells, characterized by a 11-fold increase in lamina propria (LP) leukocytes and a reduction in intraepithelial lymphocytes (IEL) compared to control mice without obesity (chow diet), indicating obesity-associated intestinal immune cell activation.

Conclusion:

These data suggest that HFD-induced obesity shifts intestinal immune cells from the epithelial layer to the lamina propria, which might indicate gut immune cell activation. Despite similar body weight under HFD, MAdCAM-1 deficiency protected against obesity- induced metabolic dysfunction by improving insulin sensitivity and limiting inflammatory monocyte expansion. MAdCAM-1 associated intestinal inflammation might be a novel potential therapeutic target in obesity and metabolic dysfunction.

The CRISPR/dCas9 system, which facilitates control of gene expression without DNA editing, has generated important attention for therapeutic applications due to its high efficiency and specificity. Enhancing the quantity and quality of anti-atherogenic high-density lipoproteins (HDL), primarily produced in the liver, represents a promising therapeutic target in atherosclerosis. The present study aimed to use CRISPR/dCas9 activation technology to stimulate the transcription of key HDL-associated apolipoprotein A1 (ApoA1) and paraoxonase 1 (PON1) in hepatocytes (Hep). Hep belonging to the Huh7 human-derived hepatocyte cell line were transfected with CRISPR/dCas9 activation plasmids targeting ApoA1 or PON1. Following antibiotics selection homogenous cell cultures stably overexpressing the targeted proteins were obtained. ApoA1 and PON1 were secreted in the culture media, which were collected and serve as conditioned media (CM). The CM were used to investigate the functional activities of the secreted ApoA1 and PON1 by determining the effect on oxidative stress (total reactive oxygen species, ROS), inflammatory stress (vascular cell adhesion protein 1, VCAM-1 and monocyte chemoattractant protein 1, MCP-1) and endoplasmic reticulum stress markers (glucose-regulated protein 78, GRP78, spliced X-box binding protein 1, sXBP1, activating transcription factor 4, ATF4) in TNFα-treated endothelial cells (EA.hy926, EC) compared to Control EC. Bulk long-RNA sequencing (RNAseq) analysis was done on total RNA isolated from selected transfected Hep. Bioinformatic analysis of transcriptomic data was performed using the principal component analysis (PCA) and the functional enrichment analysis of differential expressed genes (DEG) using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome databases. Results indicate that: (i) the CRISPR/dCas9 activation system generated an elevated transcription of ApoAI and PON1, as demonstrated by the increased mRNA expression 10- and 18-fold respectively, whereas protein levels in the Hep were augmented 5-fold for both ApoAI and PON1. Concomitantly, protein content in the CM increased 22-fold for ApoA1 and 6-fold for PON1; (ii) CM from transfected Hep added to the EA.hy926 cells reduced significantly the TNFα-induced oxidative stress by decreasing ROS production by 20% for ApoA1 and 10% for PON1. Gene expression for VCAM-1 was reduced by 40% for ApoA1 and 70% for PON1, whereas for MCP-1 was lowered by 20% for both ApoA1 and PON1). The treatment of TNFα-stimulated EC with the CM also decreased significantly the gene expression of endoplasmic reticulum stress sensors : GRP78 (30% for ApoA1 and 80% for PON1), ATF4 (30% for ApoA1 and 60% for PON1 and sXBP1 (20% for ApoA1 and 70% for PON1); (iii) bioinformatic analysis of RNAseq data showed that CRISPR/dCas9-mediated activation of ApoA1 and PON1 induced significant changes in the metabolic profiles of the transfected hepatocytes by modulating the lipid metabolic pathways (lipid transport, fatty acid metabolism, plasma lipoprotein assembly, regulation by nuclear receptors, bile secretion) and insulin signaling and resistance in hepatocytes. Conclusion: Long-term upregulation of ApoA1 or PON1 has been achieved in human Huh7 hepatocytes using CRISPR/dCas9 activation system. The robust overexpression of these major HDL proteins obtained by CRISPR/dCas9 technology offers a promising strategy to enhance their hepatic biosynthesis and secretion, which can be used to alleviate the inflammatory stress induced EC dysfunction and to impede vascular atherosclerosis.

Funded by Romanian Academy, Romania’s National Recovery and Resilience Plan (PNRR) Program Grant CF197-2022/PNRR-III-C9-2022-I8 (contract no. 760059/23.05.2023).

Background

Antimicrobial peptides can kill or inhibit pathogenic bacteria and thus play an important role in the innate immune defence. Cathelicidin antimicrobial peptide (CAMP, mouse homologue = CRAMP) is one of these peptides; it binds lipopolysaccharide (LPS) and thereby neutralises the pathogenic effects of the Toll-like receptor 4 (TLR) ligand LPS, e.g. in sepsis-induced myocardial dysfunction (cardiomyopathy). Here, we investigate LPS-dependent and -independent effects of CRAMP on NF-κB activation, cytokine expression, endothelial adhesion and transmigration of monocytes.

Methods and Results

As expected, CRAMP almost completely blocked NF-κB activation in THP-1 reporter cells by LPS (p<0.001), but not by the TLR2/6 agonist Pam2 or the TLR 2/1 agonist Pam3. In endothelial cells (MyEND) and monocytes (J774A.1), real-time PCR analysis showed that Cramp expression was strongly upregulated by LPS (>250-fold, p<0.001) and remained significantly upregulated by Pam2 and Pam3 (2-50-fold, p<0.01-p<0.001). The LPS-dependent upregulation of the proinflammatory cytokines Il-1β, Il-6, Tnf-α, Ccl-2 in both MyEND and J774A.1 cells was markedly attenuated by the addition of CRAMP (real-time PCR, p<0.001). In MyEND cells, the LPS-induced expression of the adhesion molecules Vcam-1, Icam-1, E-selectin, P-selectin was likewise inhibited by CRAMP (real-time PCR, p<0.001), and subsequently, the adhesion of J774A.1 cells to a MyEND cell monolayer (425 vs. 505 cells/hpf, p<0.01).

In addition, we also observed LPS-independent effects of CRAMP. Thus, CRAMP led to a moderate induction of inflammatory genes in MyEND and J774A.1 cells and E-selectin and P-selectin in MyEND cells (real-time PCR, p<0.01-p<0.001). Accordingly, we observed a slightly increased adherence of J774A.1 cells to a MyEND cell monolayer with CRAMP and in transmigration assays with J774A.1 cells through a MyEND cell monolayer, CRAMP was even more potent than LPS as a chemoattractant.

Conclusion

CRAMP is a potent inhibitor of LPS-dependent cytokine induction in monocytes and endothelial cells and of adhesion of monocytes to the endothelium, with potential benefits in curbing the cytokine storm and blood monocyte activity in sepsis, including sepsis-induced cardiomyopathy.

Aim:

Vascular smooth muscle cells (VSMCs) are involved in all stages of atherosclerosis,

from early lesions to advanced plaques. Thinning of the fibrous cap of the plaque,

caused by the death of VSMCs and the degradation of collagen and extracellular matrix

(ECM), increases the risk of plaque rupture. Previous studies have shown that PACAP

(pituitary adenylate cyclase-activating polypeptide) deficiency in ApoE-/- mice fed a

standard diet has proatherogenic effects. The cellular mechanisms of these effects are

still unclear. Therefore, the aim of this study is to investigate the effects of PACAP on

viability, migratory ability, and mitochondrial dynamics/dysfunction in human coronary

artery smooth muscle cells (HCASMC).

Methods and Results:

To investigate the effects of PACAP deficiency in atherosclerosis development under

standard chow (SC), PACAP−/− mice were crossbred with ApoE−/− mice to generate

PACAP−/−

/ApoE−/− mice. Plaque areas in the aorta were analyzed using ORO staining

and ImageJ (Fiji). In vitro, HCASMC were treated with 25 µg/ml oxLDL with/without

PACAP38. Viability was analyzed with PrestoblueTM, and oxLDL uptake and

accumulation were analyzed with BodipyTM, measured by ELISA. Cell migration was

assessed using the scratch assay and the MRI wound healing tool in ImageJ (Fiji).

Mitochondrial morphology was examined by immunofluorescence staining with

MitoTrackerTM and antibodies against cytochrome c. Mitochondrial morphology was

analyzed using the MiNA tool in ImageJ (Fiji).

In vivo data show that PACAP deficiency increased the plaque area in the aorta in

ApoE−/− mice after 30 weeks SC. In vitro data reveal that PACAP38 had no effect on

lipid accumulation, but increased viability in oxLDL-treated HCASMC. Both, oxLDL and

PACAP38 slowed cell migration after 5 hours compared to negative control. Analyses

of mitochondrial morphology indicate that oxLDL and PACAP38 increase individual

(puncta, rods) and network structure of mitochondria with a reduction in the mean

number of branches per network.

Conclusion:

Our data suggest that PACAP plays an important regulatory role in HCASMC migration

and improves mitochondrial health by promoting fission and fusion processes

independent of the lipid content. Thus, PACAP appears to have a protective function

in HCASMC.

Macrophages play a pivotal role in inflammation and metabolic dysfunction. In obesity, adipose tissue macrophages (ATMs) expand in number and contribute to chronic low-grade inflammation. Recent studies have revealed that ATMs are heterogeneous, comprising distinct subsets with unique signaling pathways and functions. However, the specific ATM subsets responsible for obesity-induced inflammation remain unclear. Notably, olfactory receptor 2 (Olfr2), an odorant receptor, has recently been implicated as an inflammatory signaling mediator in macrophages. Here, we identified Olfr2-expressing ATMs in the epididymal adipose tissue (EAT) of diet-induced obese mice. Genetic deletion of Olfr2 protected against obesity and metabolic dysfunction by attenuating inflammation in EAT. Among ATM subtypes, lipid-associated macrophages displayed the highest expression of Olfr2 and enriched fatty acid metabolism signatures, which were diminished in Olfr2-deficient mice. Loss of Olfr2 did not affect the respiratory exchange ratio or energy expenditure. Together, our findings identify an inflammatory ATM subset, Olfr2 macrophages, that contributes to obesity-induced inflammation, implicating Olfr2 as a potential therapeutic target.

Introduction:

CHIP (clonal hematopoiesis of indeterminate potential) is an acquired, genetic risk factor of cardiovascular diseases. The most common CHIP-driver gene is DNMT3A, an epigenetic regulator. Studies in human and mice suggest that inflammatory myeloid cells mediate CHIP-associated cardiovascular risks. We aim to establish an in vitro system that models myelopoiesis in a milieu with DNMT3A-mutated cells, using human induced pluripotent stem cells (hiPSC).

Methods:

We generated DNMT3A-mutated hiPSC lines using CRISPR/Cas9-mediated gene editing techniques to eliminate the enzymatic domain of the DNMT3A gene (exon 21-23). Next, we differentiated hiPSC into hematopoietic stem cells (HSC) and further into monocytes with a newly established extrinsic factor-guided differentiation protocol. We collected the mutated and non-mutated hiPSC-derived HSC and monocytes using fluorescence-activated cell sorting. Genomic DNA isolated from the sorted cells was subjected to bisulfite sequencing for the examination of genome-wide DNA methylation. RNA sequencing was performed to profile transcriptome. We plan to co-culture mutated and non-mutated hiPSC-derived HSC during monocyte differentiation for modelling the clonal chimerism in vivo.

Results:

Our CRISPR/Cas9 system effectively edited 30% of hiPSC, from which we produced 24 single cell-derived hiPSC clones. Applying multiple quality control measures, we identified two pure hiPSC clones, one had truncated DNMT3A (the mutated) while the other had intact DNMT3A (the non-mutated). Bisulfite sequencing of these two hiPSC lines and the derived HSC and monocytes revealed a distinctive genome-wide DNA methylation between the mutated and non-mutated cells. DNMT3A-mutated cells are significantly hypomethylated compared to non-mutated cells. Hypomethylated promoters or enhancers of genes, such as IL6 and TNF receptors, however, may indicate a higher expression of proinflammatory cytokines in the mutated cells. Transcriptomes showed a more inflammatory and proliferative phenotype in mutated cells, and thus correlated with the results of the methylation analyses.

Conclusion:

Our in vitro model demonstrated a genome-wide hypomethylation during DNMT3A-deficient myelopoiesis, leading to a more inflammatory and proliferative phenotype. This model serves as a foundation for studying hematopoiesis and will support deciphering how CHIP mutations lead to cell function changes, on the basis of which therapeutic strategies can be developed against CHIP-aggravated cardiovascular diseases.

Background: Periodontitis is a highly prevalent chronic inflammatory disease of the gum tissue and has been epidemiologically linked to heightened cardiovascular risk, even after clinically successful treatment. Recent experimental evidence indicates that periodontal inflammation can epigenetically reprogram hematopoietic stem cells, generating myeloid progeny with exaggerated inflammatory potential. However, whether such inflammatory memory influences the development of future cardiovascular disease remains unknown.

Aims: To investigate the impact of antecedent periodontitis on abdominal aortic aneurysm (AAA) formation in mice.

Methods: Juvenile male C57BL/6J mice (mean age: 10 +/-1.5 weeks) underwent bilateral ligature-induced periodontitis (LIP) or sham surgery (n=8-10/group). Ligature placement elicited consistent periodontal disease by day 21, after which ligatures were removed and a 2-week healing period was allowed. Mice were then subjected to porcine pancreatic elastase–induced AAA formation via transient intraaortic infusion. Longitudinal blood sampling and high-resolution ultrasonography were used to assess leukocyte dynamics and aneurysm progression.

Results: At day 3 following elastase infusion, mice with prior LIP exhibited significantly elevated circulating leukocyte counts (10x10^x/uL +/- 2.34 SD) compared to sham controls (7.83x10^3/uL +/- 1,45 SD; p=0.03), driven predominantly by neutrophilia and monocytosis. By day 28, mice previously exposed to LIP developed greater aortic dilation (204% ± 21.19 fold change compared to baseline) than sham-treated animals (180.5% ± 23.62; p = 0.044), despite normalization of peripheral leukocyte numbers.

Conclusion: These findings indicate that previous periodontal inflammation can influence subsequent aortic aneurysm development in this murine model. Ongoing work aims to clarify the underlying mechanisms and to determine whether similar processes contribute to cardiovascular risk in humans.