Daphne Dekker

CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL

The low-density lipoprotein receptor (LDLR) plays a pivotal role in clearing atherogenic circulating low-density lipoprotein (LDL) cholesterol. Here we show that the COMMD/CCDC22/CCDC93 (CCC) and the Wiskott–Aldrich syndrome protein and SCAR homologue (WASH) complexes are both crucial for endosomal sorting of LDLR and for its function. We find that patients with X-linked intellectual disability caused by mutations in CCDC22 are hypercholesterolaemic, and that COMMD1-deficient dogs and liver-specific Commd1 knockout mice have elevated plasma LDL cholesterol levels. Furthermore, Commd1 depletion results in mislocalization of LDLR, accompanied by decreased LDL uptake. Increased total plasma cholesterol levels are also seen in hepatic COMMD9-deficient mice. Inactivation of the CCC-associated WASH complex causes LDLR mislocalization, increased lysosomal degradation of LDLR and impaired LDL uptake. Furthermore, a mutation in the WASH component KIAA0196 (strumpellin) is associated with hypercholesterolaemia in humans. Altogether, this study provides valuable insights into the mechanisms regulating cholesterol homeostasis and LDLR trafficking.

Porphyromonas Gingivalis and E-coli induce different cytokine production patterns in pregnant women

Objective Pregnant individuals of many species, including humans, are more sensitive to various bacteria or their products as compared with non-pregnant individuals. Pregnant individuals also respond differently to different bacteria or their products. Therefore, in the present study, we evaluated whether the increased sensitivity of pregnant women to bacterial products and their heterogeneous response to different bacteria was associated with differences in whole blood cytokine production upon stimulation with bacteria or their products. Methods Blood samples were taken from healthy pregnant and age-matched non-pregnant women and ex vivo stimulated with bacteria or LPS from Porphyromonas Gingivalis (Pg) or E-coli for 24 hrs. TNFα, IL-1ß, IL-6, IL-12 and IL-10 were measured using a multiplex Luminex system. Results We observed a generally lower cytokine production after stimulation with Pg bacteria or it’s LPS as compared with E-coli bacteria. However, there was also an effect of pregnancy upon cytokine production: in pregnant women the production of IL-6 upon Pg stimulation was decreased as compared with non-pregnant women. After stimulation with E-coli, the production of IL-12 and TNFα was decreased in pregnant women as compared with non-pregnant women. Conclusion Our results showed that cytokine production upon bacterial stimulation of whole blood differed between pregnant and non-pregnant women, showing that the increased sensitivity of pregnant women may be due to differences in cytokine production. Moreover, pregnancy also affected whole blood cytokine production upon Pg or E-coli stimulation differently. Thus, the different responses of pregnant women to different bacteria or their products may result from variations in cytokine production.

A cell-type-specific role for murine Commd1 in liver inflammation

The transcription factor NF-κB plays a critical role in the inflammatory response and it has been implicated in various diseases, including non-alcoholic fatty liver disease (NAFLD). Although transient NF-κB activation may protect tissues from stress, a prolonged NF-κB activation can have a detrimental effect on tissue homeostasis and therefore accurate termination is crucial. Copper Metabolism MURR1 Domain-containing 1 (COMMD1), a protein with functions in multiple pathways, has been shown to suppress NF-κB activity. However, its action in controlling liver inflammation has not yet been investigated. To determine the cell-type-specific contribution of Commd1 to liver inflammation, we used hepatocyte and myeloid-specific Commd1-deficient mice. We also used a mouse model of NAFLD to study low-grade chronic liver inflammation: we fed the mice a high fat, high cholesterol (HFC) diet, which results in hepatic lipid accumulation accompanied by liver inflammation. Depletion of hepatocyte Commd1 resulted in elevated levels of the NF-κB transactivation subunit p65 (RelA) but, surprisingly, the level of liver inflammation was not aggravated. In contrast, deficiency of myeloid Commd1 exacerbated diet-induced liver inflammation. Unexpectedly we observed that hepatic and myeloid Commd1 deficiency in the mice both augmented hepatic lipid accumulation. The elevated levels of proinflammatory cytokines in myeloid Commd1-deficient mice might be responsible for the increased level of steatosis. This increase was not seen in hepatocyte Commd1-deficient mice, in which increased lipid accumulation appeared to be independent of inflammation. Our mouse models demonstrate a cell-type-specific role for Commd1 in suppressing liver inflammation and in the progression of NAFLD.

Cytokine production induced by non-encapsulated and encapsulated Porphyromonas gingivalis strains

OBJECTIVE Although the exact reason is not known, encapsulated gram-negative Porphyromonas gingivalis strains are more virulent than non-encapsulated strains. Since difference in virulence properties may be due to difference in cytokine production following recognition of the bacteria or their products by the host inflammatory cells, we compared cytokine production following stimulation with bacteria or lipopolysaccharides (LPS) of a non-encapsulated and an encapsulated P. gingivalis strain (K(-) and K1). DESIGN Tumour necrosis factor-alpha (TNF-α) production following stimulation of the cell-line Mono Mac 6 with bacteria or LPS of both P. gingivalis strains was determined using flow cytometry. Furthermore, we investigated the effects of the two P. gingivalis strains or their LPS on TNF-α and Interleukin (IL-1β, IL-6, IL-12 and IL-10) production in whole blood using Luminex. In both experiments, Escherichia coli bacteria and LPS were used as a reference. RESULTS Both P. gingivalis strains induced lower cytokine production than E. coli with the exception of IL-6. P. gingivalis K1 bacteria elicited a higher overall cytokine production than P. gingivalis K(-). In contrast, P. gingivalis K1 LPS stimulation induced a lower cytokine production than P. gingivalis K(-) LPS. CONCLUSIONS Our findings suggest that the encapsulated P. gingivalis K1 bacteria induce higher cytokine production than the non-encapsulated P. gingivalis K(-). This was not due to its LPS. The stronger induction of cytokines may contribute to the higher virulence of P. gingivalis K1.

COMMD Family Regulates Plasma LDL Levels and Attenuates Atherosclerosis Through Stabilizing the CCC Complex in Endosomal LDLR Trafficking

Rationale: COMMD (copper metabolism MURR1 domain)-containing proteins are a part of the CCC (COMMD–CCDC22 [coiled-coil domain containing 22]–CCDC93 [coiled-coil domain containing 93]) complex facilitating endosomal trafficking of cell surface receptors. Hepatic COMMD1 inactivation decreases CCDC22 and CCDC93 protein levels, impairs the recycling of the LDLR (low-density lipoprotein receptor), and increases plasma low-density lipoprotein cholesterol levels in mice. However, whether any of the other COMMD members function similarly as COMMD1 and whether perturbation in the CCC complex promotes atherogenesis remain unclear. Objective: The main aim of this study is to unravel the contribution of evolutionarily conserved COMMD proteins to plasma lipoprotein levels and atherogenesis. Methods and Results: Using liver-specific Commd1, Commd6, or Commd9 knockout mice, we investigated the relation between the COMMD proteins in the regulation of plasma cholesterol levels. Combining biochemical and quantitative targeted proteomic approaches, we found that hepatic COMMD1, COMMD6, or COMMD9 deficiency resulted in massive reduction in the protein levels of all 10 COMMDs. This decrease in COMMD protein levels coincided with destabilizing of the core (CCDC22, CCDC93, and chromosome 16 open reading frame 62 [C16orf62]) of the CCC complex, reduced cell surface levels of LDLR and LRP1 (LDLR-related protein 1), followed by increased plasma low-density lipoprotein cholesterol levels. To assess the direct contribution of the CCC core in the regulation of plasma cholesterol levels, Ccdc22 was deleted in mouse livers via CRISPR/Cas9-mediated somatic gene editing. CCDC22 deficiency also destabilized the complete CCC complex and resulted in elevated plasma low-density lipoprotein cholesterol levels. Finally, we found that hepatic disruption of the CCC complex exacerbates dyslipidemia and atherosclerosis in ApoE3*Leiden mice. Conclusions: Collectively, these findings demonstrate a strong interrelationship between COMMD proteins and the core of the CCC complex in endosomal LDLR trafficking. Hepatic disruption of either of these CCC components causes hypercholesterolemia and exacerbates atherosclerosis. Our results indicate that not only COMMD1 but all other COMMDs and CCC components may be potential targets for modulating plasma lipid levels in humans.

NF-κB p65 serine 467 phosphorylation sensitizes mice to weight gain and TNFα-or diet-induced inflammation

The NF-κB family of transcription factors is essential for an effective immune response, but also controls cell metabolism, proliferation and apoptosis. Its broad relevance and the high connectivity to diverse signaling pathways require a tight control of NF-κB activity. To investigate the control of NF-κB activity by phosphorylation of the NF-κB p65 subunit, we generated a knock-in mouse model in which serine 467 (the mouse homolog of human p65 serine 468) was replaced with a non-phosphorylatable alanine (S467A). This substitution caused reduced p65 protein synthesis and diminished TNFα-induced expression of a selected group of NF-κB-dependent genes. Intriguingly, high-fat fed S467A mice displayed increased locomotor activity and energy expenditure, which coincided with a reduced body weight gain. Although glucose metabolism or insulin sensitivity was not improved, diet-induced liver inflammation was diminished in S467A mice. Altogether, this study demonstrates that phosphorylation of p65 serine 467 augment NF-κB activity and exacerbates various deleterious effects of overnutrition in mice.

Gut microbiota dysbiosis augments atherosclerosis in LDLR-/- mice

Aim: In recent years the gut microbiome has been recognized as an important participant in the etiology of obesity and -associated comorbidities. Moreover, recent studies have implicated an involvement of the gut microbiota in both cardiovascular health and disease. Although the gut microbiome has been suggested as a contributor to atherosclerosis, firm evidence to support a causal role for the gut microbiota in atherosclerosis is limited. Methods: To investigate the effect of the gut microbiota in atherosclerosis, we performed fecal microbiota transplantation to transplant the gut microbiota of caspase1-/- mice, an established model for dysbiosis, into LDLR-/- mice (LDLR-/-[casp1-/-]). Fecal microbiota transplantation of the gut microbiota of LDLR-/- mice into LDLR-/- mice (LDLR-/-[LDLR-/-]) served as control. Mice were fed a chow or high-fat cholesterol diet (HFC, 0,21% cholesterol) for 13 weeks and fecal samples were collected to determine microbiota composition by 16S rRNA sequencing. Plaque size was analyzed in the aortic root and immune cell subsets were analyzed by flow cytometry. Results: 16S rRNA sequencing of fecal samples confirmed the induction of dysbiosis in LDLR-/-[casp1-/-] compared to LDLR-/-[LDLR-/-] mice. Body weight, plasma triglyceride and cholesterol levels were significantly increased by HFC-feeding in LDLR-/-[casp1-/-] and LDLR-/-[LDLR-/-] mice. However, dysbiosis did not affect these parameters. In contrast, plaque size was significantly increased in HFC-fed LDLR-/-[casp1-/-] compared to HFC-fed (LDLR-/-[LDLR-/-] mice. Furthermore, dysbiosis in LDLR-/- mice was associated with a moderate increase in circulatory levels of Ly6C-high monocytes. Conclusions: Our data shows that gut microbiota dysbiosis augments atherosclerosis, possibly by exacerbating low-grade systemic inflammation.