Reduced levels of SCD1 accentuate palmitate-induced stress in insulin-producing β-cells

<p>Abstract</p> <p>Background</p> <p>Stearoyl-CoA desaturase 1 (SCD1) is an ER resident enzyme introducing a double-bond in saturated fatty acids. Global knockout of SCD1 in mouse increases fatty acid oxidation and insulin sensitivity which makes the animal resistant to diet-induced obesity. Inhibition of SCD1 has therefore been proposed as a potential therapy of the metabolic syndrome. Much of the work has focused on insulin target tissue and very little is known about how reduced levels of SCD1 would affect the insulin-producing β-cell, however. The aim of the present study was therefore to investigate how reduced levels of SCD1 affect the β-cell.</p> <p>Results</p> <p>Insulin-secreting MIN6 cells with reduced levels of SCD1 were established by siRNA mediated knockdown. When fatty acid oxidation was measured, no difference between cells with reduced levels of SCD1 and mock-transfected cells were found. Also, reducing levels of SCD1 did not affect insulin secretion in response to glucose. To investigate how SCD1 knockdown affected cellular mechanisms, differentially regulated proteins were identified by a proteomic approach. Cells with reduced levels of SCD1 had higher levels of ER chaperones and components of the proteasome. The higher amounts did not protect the β-cell from palmitate-induced ER stress and apoptosis. Instead, rise in levels of p-eIF2α and CHOP after palmitate exposure was 2-fold higher in cells with reduced levels of SCD1 compared to mock-transfected cells. Accordingly, apoptosis rose to higher levels after exposure to palmitate in cells with reduced levels of SCD1 compared to mock-transfected cells.</p> <p>Conclusions</p> <p>In conclusion, reduced levels of SCD1 augment palmitate-induced ER stress and apoptosis in the β-cell, which is an important caveat when considering targeting this enzyme as a treatment of the metabolic syndrome.</p

Genetic variations in A20 DUB domain provide a genetic link to citrullination and neutrophil extracellular traps in systemic lupus erythematosus

Objectives: Genetic variations in TNFAIP3 (A20) de-ubiquitinase (DUB) domain increase the risk of systemic lupus erythematosus (SLE) and rheumatoid arthritis. A20 is a negative regulator of NF-κB but the role of its DUB domain and related genetic variants remain unclear. We aimed to study the functional effects of A20 DUB-domain alterations in immune cells and understand its link to SLE pathogenesis. Methods: CRISPR/Cas9 was used to generate human U937 monocytes with A20 DUB-inactivating C103A knock-in (KI) mutation. Whole genome RNA-sequencing was used to identify differentially expressed genes between WT and C103A KI cells. Functional studies were performed in A20 C103A U937 cells and in immune cells from A20 C103A mice and genotyped healthy individuals with A20 DUB polymorphism rs2230926. Neutrophil extracellular trap (NET) formation was addressed ex vivo in neutrophils from A20 C103A mice and SLE-patients with rs2230926. Results: Genetic disruption of A20 DUB domain in human and murine myeloid cells did not give rise to enhanced NF-κB signalling. Instead, cells with C103A mutation or rs2230926 polymorphism presented an upregulated expression of PADI4, an enzyme regulating protein citrullination and NET formation, two key mechanisms in autoimmune pathology. A20 C103A cells exhibited enhanced protein citrullination and extracellular trap formation, which could be suppressed by selective PAD4 inhibition. Moreover, SLE-patients with rs2230926 showed increased NETs and increased frequency of autoantibodies to citrullinated epitopes. Conclusions: We propose that genetic alterations disrupting the A20 DUB domain mediate increased susceptibility to SLE through the upregulation of PADI4 with resultant protein citrullination and extracellular trap formation

Epithelial IL-6 trans-signaling defines a new asthma phenotype with increased airway inflammation

Background: Although several studies link high levels of IL-6 and soluble IL-6 receptor (sIL-6R) to asthma severity and decreased lung function, the role of IL-6 trans-signaling (IL-6TS) in asthmatic patients is unclear. Objective: We sought to explore the association between epithelial IL-6TS pathway activation and molecular and clinical phenotypes in asthmatic patients. Methods: An IL-6TS gene signature obtained from air-liquid interface cultures of human bronchial epithelial cells stimulated with IL-6 and sIL-6R was used to stratify lung epithelial transcriptomic data (Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes [U-BIOPRED] cohorts) by means of hierarchical clustering. IL-6TS-specific protein markers were used to stratify sputum biomarker data (Wessex cohort). Molecular phenotyping was based on transcriptional profiling of epithelial brushings, pathway analysis, and immunohistochemical analysis of bronchial biopsy specimens. Results: Activation of IL-6TS in air-liquid interface cultures reduced epithelial integrity and induced a specific gene signature enriched in genes associated with airway remodeling. The IL-6TS signature identified a subset of patients with IL-6TS-high asthma with increased epithelial expression of IL-6TS-inducible genes in the absence of systemic inflammation. The IL-6TS-high subset had an overrepresentation of frequent exacerbators, blood eosinophilia, and submucosal infiltration of T cells and macrophages. In bronchial brushings Toll-like receptor pathway genes were upregulated, whereas expression of cell junction genes was reduced. Sputum sIL-6R and IL-6 levels correlated with sputum markers of remodeling and innate immune activation, in particular YKL-40, matrix metalloproteinase 3, macrophage inflammatory protein 1 beta, IL-8, and IL-1 beta. Conclusions: Local lung epithelial IL-6TS activation in the absence of type 2 airway inflammation defines a novel subset of asthmatic patients and might drive airway inflammation and epithelial dysfunction in these patients.Peer reviewe

Epithelial IL-6 trans-signaling defines a new asthma phenotype with increased airway inflammation

Background: Although several studies link high levels of IL-6 and soluble IL-6 receptor (sIL-6R) with asthma severity and decreased lung function, the role of IL-6 trans-signaling (IL-6TS) in asthma is unclear. Objective: To explore the association between epithelial IL-6TS pathway activation and molecular and clinical phenotypes in asthma. Methods: An IL-6TS gene signature, obtained from air-liquid interface (ALI) cultures of human bronchial epithelial cells stimulated with IL-6 and sIL-6R, was used to stratify lung epithelium transcriptomic data (U-BIOPRED cohorts) by hierarchical clustering. IL-6TS-specific protein markers were used to stratify sputum biomarker data (Wessex cohort). Molecular phenotyping was based on transcriptional profiling of epithelial brushings, pathway analysis and immunohistochemical analysis of bronchial biopsies. Results: Activation of IL-6TS in ALI cultures reduced epithelial integrity and induced a specific gene signature enriched in genes associated with airway remodeling. The IL-6TS signature identified a subset of IL-6TS. High asthma patients with increased epithelial expression of IL-6TS inducible genes in absence of systemic inflammation. The IL-6TS High subset had an overrepresentation of frequent exacerbators, blood eosinophilia, and submucosal infiltration of T cells and macrophages. In bronchial brushings, TLR pathway genes were up-regulated while the expression of tight junction genes was reduced. Sputum sIL-6R and IL-6 levels correlated with sputum markers of remodeling and innate immune activation, in particular YKL-40, MMP3, MIP-1β, IL-8 and IL-1β. Conclusions: Local lung epithelial IL-6TS activation in absence of type 2 airway inflammation defines a novel subset of asthmatics and may drive airway inflammation and epithelial dysfunction in these patients

Palmitate-induced Apoptosis in Insulin-producing β-cells

Type 2 diabetes is a disease characterized by the inability of pancreatic β-cells to secrete sufficient amounts of insulin to maintain normoglycemia. Increased levels of saturated fatty acids such as palmitate are believed to contribute to β-cell failure and the development of the disease. In the present thesis, mechanisms behind palmitate-induced β-cell apoptosis were explored. Palmitate augmented insulin secretion after short exposure to the fatty acid, but attenuated the secretory response after longer exposure. Elevated levels of palmitate increased endoplasmic reticulum (ER) stress and induced apoptosis. When insulin secretion was inhibited by diazoxide, palmitate-induced ER stress and apoptosis were reduced. In comparison to palmitate, the mono-unsaturated fatty acid oleate increased neither ER stress nor apoptosis. Furthermore, shuttling of fatty acids into triglycerides and β-oxidation was favored in cells exposed to oleate compared to palmitate. When the levels of stearoyl-CoA desaturase 1 (SCD1), the enzyme responsible for conversion of saturated to mono-unsaturated fatty acids, were reduced, up-regulation of ER chaperones and components of the proteasome was observed. Cells with reduced levels of SCD1 showed increased sensitivity to palmitate, as exposure to the fatty acid increased levels of ER stress and apoptosis. Palmitate-induced apoptosis of the β-cell has been linked to alterations in sphingolipid metabolism. In cells with reduced levels of sphingosine kinase (SphK) 2, palmitate failed to induce apoptosis, and ER stress was reduced. Furthermore, SphK2 was required for the palmitate-induced activation of c-Jun N-terminal kinase (JNK). In contrast, knockdown of SphK1 sensitized the cell to palmitate-induced apoptosis independently of ER stress. In summary, palmitate induces β-cell apoptosis, which is partly dependent on the induction of ER stress. The mechanisms investigated support the notion that increased protein load on the ER, low degree of triglyceride formation and β-oxidation, and perturbations in sphingolipid metabolism contribute to palmitate-induced apoptosis in insulin-producing β-cells

Palmitate-induced Apoptosis in Insulin-producing β-cells

Type 2 diabetes is a disease characterized by the inability of pancreatic β-cells to secrete sufficient amounts of insulin to maintain normoglycemia. Increased levels of saturated fatty acids such as palmitate are believed to contribute to β-cell failure and the development of the disease. In the present thesis, mechanisms behind palmitate-induced β-cell apoptosis were explored. Palmitate augmented insulin secretion after short exposure to the fatty acid, but attenuated the secretory response after longer exposure. Elevated levels of palmitate increased endoplasmic reticulum (ER) stress and induced apoptosis. When insulin secretion was inhibited by diazoxide, palmitate-induced ER stress and apoptosis were reduced. In comparison to palmitate, the mono-unsaturated fatty acid oleate increased neither ER stress nor apoptosis. Furthermore, shuttling of fatty acids into triglycerides and β-oxidation was favored in cells exposed to oleate compared to palmitate. When the levels of stearoyl-CoA desaturase 1 (SCD1), the enzyme responsible for conversion of saturated to mono-unsaturated fatty acids, were reduced, up-regulation of ER chaperones and components of the proteasome was observed. Cells with reduced levels of SCD1 showed increased sensitivity to palmitate, as exposure to the fatty acid increased levels of ER stress and apoptosis. Palmitate-induced apoptosis of the β-cell has been linked to alterations in sphingolipid metabolism. In cells with reduced levels of sphingosine kinase (SphK) 2, palmitate failed to induce apoptosis, and ER stress was reduced. Furthermore, SphK2 was required for the palmitate-induced activation of c-Jun N-terminal kinase (JNK). In contrast, knockdown of SphK1 sensitized the cell to palmitate-induced apoptosis independently of ER stress. In summary, palmitate induces β-cell apoptosis, which is partly dependent on the induction of ER stress. The mechanisms investigated support the notion that increased protein load on the ER, low degree of triglyceride formation and β-oxidation, and perturbations in sphingolipid metabolism contribute to palmitate-induced apoptosis in insulin-producing β-cells

Palmitate-induced Apoptosis in Insulin-producing β-cells

Type 2 diabetes is a disease characterized by the inability of pancreatic β-cells to secrete sufficient amounts of insulin to maintain normoglycemia. Increased levels of saturated fatty acids such as palmitate are believed to contribute to β-cell failure and the development of the disease. In the present thesis, mechanisms behind palmitate-induced β-cell apoptosis were explored. Palmitate augmented insulin secretion after short exposure to the fatty acid, but attenuated the secretory response after longer exposure. Elevated levels of palmitate increased endoplasmic reticulum (ER) stress and induced apoptosis. When insulin secretion was inhibited by diazoxide, palmitate-induced ER stress and apoptosis were reduced. In comparison to palmitate, the mono-unsaturated fatty acid oleate increased neither ER stress nor apoptosis. Furthermore, shuttling of fatty acids into triglycerides and β-oxidation was favored in cells exposed to oleate compared to palmitate. When the levels of stearoyl-CoA desaturase 1 (SCD1), the enzyme responsible for conversion of saturated to mono-unsaturated fatty acids, were reduced, up-regulation of ER chaperones and components of the proteasome was observed. Cells with reduced levels of SCD1 showed increased sensitivity to palmitate, as exposure to the fatty acid increased levels of ER stress and apoptosis. Palmitate-induced apoptosis of the β-cell has been linked to alterations in sphingolipid metabolism. In cells with reduced levels of sphingosine kinase (SphK) 2, palmitate failed to induce apoptosis, and ER stress was reduced. Furthermore, SphK2 was required for the palmitate-induced activation of c-Jun N-terminal kinase (JNK). In contrast, knockdown of SphK1 sensitized the cell to palmitate-induced apoptosis independently of ER stress. In summary, palmitate induces β-cell apoptosis, which is partly dependent on the induction of ER stress. The mechanisms investigated support the notion that increased protein load on the ER, low degree of triglyceride formation and β-oxidation, and perturbations in sphingolipid metabolism contribute to palmitate-induced apoptosis in insulin-producing β-cells

Optimised generation of iPSC-derived macrophages and dendritic cells that are functionally and transcriptionally similar to their primary counterparts.

Induced pluripotent stem cells (iPSC) offer the possibility to generate diverse disease-relevant cell types, from any genetic background with the use of cellular reprogramming and directed differentiation. This provides a powerful platform for disease modeling, drug screening and cell therapeutics. The critical question is how the differentiated iPSC-derived cells translate to their primary counterparts. Our refinement of a published differentiation protocol produces a CD14+ monocytic lineage at a higher yield, in a smaller format and at a lower cost. These iPSC-derived monocytes can be further differentiated into macrophages or dendritic cells (DC), both with similar morphological and functional profiles as compared to their primary counterparts. Transcriptomic analysis of iPSC-derived cells at different stages of differentiation as well as comparison to their blood-derived counterparts demonstrates a complete switch of iPSCs to cells expressing a monocyte, macrophage or DC specific gene profile. iPSC-derived macrophages respond to LPS treatment by inducing expression of classic macrophage pro-inflammatory response markers. Interestingly, though iPSC-derived DC show similarities to monocyte derived DC, they are more similar transcriptionally to a newly described subpopulation of AXL+ DC. Thus, our study provides a detailed and accurate profile of iPSC-derived monocytic lineage cells

Cytokine production by activated plasmacytoid dendritic cells and natural killer cells is suppressed by an IRAK4 inhibitor

Background: In systemic lupus erythematosus (SLE), immune complexes (ICs) containing self-derived nucleic acids trigger the synthesis of proinflammatory cytokines by immune cells. We asked how an interleukin (IL)-1 receptor-associated kinase 4 small molecule inhibitor (IRAK4i) affects RNA-IC-induced cytokine production compared with hydroxychloroquine (HCQ). Methods: Plasmacytoid dendritic cells (pDCs) and natural killer (NK) cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy individuals. PBMCs from SLE patients and healthy individuals were depleted of monocytes. Cells were stimulated with RNA-containing IC (RNA-IC) in the presence or absence of IRAK4i I92 or HCQ, and cytokines were measured by immunoassay or flow cytometry. Transcriptome sequencing was performed on RNA-IC-stimulated pDCs from healthy individuals to assess the effect of IRAK4i and HCQ. Results: In healthy individuals, RNA-IC induced interferon (IFN)-α, tumor necrosis factor (TNF)-α, IL-6, IL-8, IFN-γ, macrophage inflammatory protein (MIP)1-α, and MIP1-β production in pDC and NK cell cocultures. IFN-α production was selective for pDCs, whereas both pDCs and NK cells produced TNF-α. IRAK4i reduced the pDC and NK cell-derived cytokine production by 74–95%. HCQ interfered with cytokine production in pDCs but not in NK cells. In monocyte-depleted PBMCs, IRAK4i blocked cytokine production more efficiently than HCQ. Following RNA-IC activation of pDCs, 975 differentially expressed genes were observed (false discovery rate (FDR) &lt; 0.05), with many connected to cytokine pathways, cell regulation, and apoptosis. IRAK4i altered the expression of a larger number of RNA-IC-induced genes than did HCQ (492 versus 65 genes). Conclusions: The IRAK4i I92 exhibits a broader inhibitory effect than HCQ on proinflammatory pathways triggered by RNA-IC, suggesting IRAK4 inhibition as a therapeutic option in SLE