Proinflammatory cytokines induce rapid, NO-independent apoptosis, expression of chemotactic mediators and interleukin-32 secretion in human pluripotent stem cell-derived beta cells

Abstract

Aims/hypothesis The aim of this study was to examine the effects of proinflammatory cytokines on cells of different developmental stages during the generation of stem cell-derived beta cells (SC-beta cells) from human pluripotent stem cells (hPSCs). We wanted to find out to what extent human SC-beta cells are suitable as an experimental cellular model and, with regard to a possible therapeutic use, whether SC-beta cells have a comparable vulnerability to cytokines as bona fide beta cells. Methods hPSCs were differentiated towards pancreatic organoids (SC-organoids) using a 3D production protocol. SC-beta cells and non-insulin-producing cells were separated by FACS and differential gene expression profiles of purified human SC-beta cells, progenitor stages and the human beta cell line EndoC-βH1, as a reference, were determined after 24 h incubation with the proinflammatory cytokines IL-1β, TNF-α and IFN-γ via a transcriptome microarray. Furthermore, we investigated apoptosis based on caspase cleavage, the generation of reactive oxygen species and activation of mitogen-activated protein-kinase (MAPK) stress-signalling pathways. Results A 24 h exposure of SC-beta cells to proinflammatory cytokines resulted in significant activation of caspase 3/7 and apoptosis via the extrinsic and intrinsic apoptosis signalling pathways. At this time point, SC-beta cells showed a markedly higher sensitivity towards proinflammatory cytokines than non-insulin-producing cells and EndoC-βH1 cells. Furthermore, we were able to demonstrate the generation of reactive oxygen species and rule out the involvement of NO-mediated stress. A transient activation of stress-signalling pathways p38 mitogen-activated protein kinases (p38) and c-Jun N-terminal kinase (JNK) was already observed after 10 min of cytokine exposure. The transcriptome analysis revealed that the cellular response to proinflammatory cytokines increased with the degree of differentiation of the cells. Cytokines induced the expression of multiple inflammatory mediators including IL-32, CXCL9 and CXCL10 in SC-beta cells and in non-insulin-producing cells. Conclusions/interpretation Our results indicate that human SC-beta cells respond to proinflammatory cytokines very similarly to human islets. Due to the fast and fulminant cellular response of SC-beta cells, we conclude that SC-beta cells represent a suitable model for diabetes research. In light of the immaturity of SC-beta cells, they may be an attractive model for developmentally young beta cells as they are, for example, present in patients with early-onset type 1 diabetes. The secretion of chemotactic signals may promote communication between SC-beta cells and immune cells, and non-insulin-producing cells possibly participate in the overall immune response and are thus capable of amplifying the immune response and further stimulating inflammation. We demonstrated that cytokine-treated SC-organoids secrete IL-32, which is considered a promising candidate for type 1 diabetes onset. This underlines the need to ensure the survival of SC-beta cells in an autoimmune environment such as that found in type 1 diabetes. Graphical abstract

Transcription and splicing regulation by NLRC5 shape the interferon response in human pancreatic β cells

“…As expected, PDX1 expression was higher in -like cells, while Aristaless Related Homeobox (ARX), a transcription factor specifically expressed in glucagon-producing  cells, was much higher in -like cells. -Like cells had higher expression of the proapoptotic ER stress marker DDIT3 (also known as CHOP) [log 2 fold change (log 2 FC) = 2.20, adjusted P << 0.0001] and also XBP1 (log 2 FC = 0.70, adjusted P << 0.0001) [in line with (32)], while -like cells showed higher expression of the protective chaperone HSPA5 (also known as BiP) (log 2 FC = 0.13, adjusted P << 0.0001). Compared to -like cells, -like cells also had a higher expression of the antiapoptotic family member BCL2L1 (also known as Bcl-XL) (log 2 FC = 0.13, adjusted P << 0.0001), of the viral sensor IFIH1 (MDA5) (log 2 FC = 0.97, adjusted P << 0.0001), and of the inhibitory variant HLA-E (log 2 FC = 0.76, adjusted P << 0.0001).…”

Resistance of Human Liver Mesenchymal Stem Cells to FAS-Induced Cell Death

“…For some cell types, pro-inflammatory cytokines such as IL-1β in combination with IFN-γ, as well as TNF-α have been shown to induce cytokine-dependent apoptosis [ 41 , 42 ]. These cytokines also increase the sensitivity of hepatocytes to Fas-induced cell death [ 43 , 44 ].…”

Fas Ligand-Modified Scaffolds Protect Stem Cell Derived β-Cells by Modulating Immune Cell Numbers and Polarization

“…The increase in apoptosis and decline in expression of maturation markers for the SA-engineered scaffold condition is consistent with the findings by others for β-cells and hPSC derived β-cells. 27,50,61 Moreover, SA-FasL-engineered scaffolds-maintained differentiation state and reduced caspase-dependent apoptosis. An analysis of cytokine expression within the conditioned media did not provide a simple mechanistic explanation for the decreased apoptosis with SA-FasL conditioned media compared to the control conditioned Our studies identified increased expression of one pro-inflammatory factor, one anti-inflammatory factor, and undetectable levels of several other cytokines that are pro-or anti-inflammatory.…”

“…Cytokines such as TNF-a, IFN, IL-6, and CXCL8 38 directly act on islet derived β-cells to induce apoptosis, 51,55−57 with TNF-α being one of the primary factors that are detrimental to β-cells. Apoptosis occurs through activated caspase-dependent apoptosis via the extrinsic and intrinsic apoptosis signaling pathways, 50,58 with activation of stress-induced kinases IKKβ and JNK, and initiation of a signaling cascade involving NFκB and NLRP3 in β-cells. 51,55−57 Inflammation and the associated proinflammatory factors within the in vivo microenvironment is expected to reduce insulin secretion and delay maturation of the hPSC derived β-cells.…”