Abstract
In this study, we took advantage of human-induced pluripotent stem cells (hiPSC) and CRISPR/Cas9 technology to investigate the potential roles of RIPK1 in regulating hematopoiesis and macrophage differentiation, proinflammatory activation, and cell death pathways. Knock-out of RIPK1 in hiPSCs demonstrated that this protein is not required for erythro-myeloid differentiation. Using a well-established macrophage differentiation protocol, knock-out of RIPK1 did not block the differentiation of iPSC-derived macrophages, which displayed a similar phenotype to WT hiPSC-derived macrophages. However, knock-out of RIPK1 leads to a TNFα-dependent apoptotic death of differentiated hiPSC-derived macrophages (iPS-MΦ) and progressive loss of iPS-MΦ production irrespective of external pro-inflammatory stimuli. Live video analysis demonstrated that TLR3/4 activation of RIPK1 KO hiPSC-derived macrophages triggered TRIF and RIPK3-dependent necroptosis irrespective of caspase-8 activation. In contrast, TLR3/4 activation of WT macrophages-induced necroptosis only when caspases were inhibited, confirming the modulating effect of RIPK1 on RIPK3-mediated necroptosis through the FADD, Caspase-8 pathway. Activation of these inflammatory pathways required RIPK3 kinase activity while RIPK1 was dispensable. However, loss of RIPK1 sensitizes macrophages to activate RIPK3 in response to inflammatory stimuli, thereby exacerbating a potentially pathological inflammatory response. Taken together, these results reveal that RIPK1 has an important role in regulating the potent inflammatory pathways in authentic human macrophages that are poised to respond to external stimuli. Consequently, RIPK1 activity might be a valid target in the development of novel therapies for chronic inflammatory diseases.
Highlights
Macrophages are key cells of the innate immune system.They are distributed throughout the tissues of the body, and play a key role in host defense, tissue homeostasis, Official journal of the Cell Death Differentiation AssociationBuchrieser et al Cell Death and Disease (2018)9:973 receptor-interacting serine/threonine-protein kinase 1 (RIPK1) plays an essential role[5,6,7,8]
All human-induced pluripotent stem cells (hiPSC) lines were capable of forming embryoid bodies (EBs), with no significant differences in size and morphology between EBs from both WT and RIPK1 KO clones (Supplementary Figure 3)
In order to investigate the relative contribution of RIPK1 to inflammatory and cell death pathways in normal human macrophage development and function, in this study we have generated KO of RIPK1 in human iPS cells, and used a well-established model of iPS-MΦ differentiation
Summary
Macrophages are key cells of the innate immune system.They are distributed throughout the tissues of the body, and play a key role in host defense, tissue homeostasis, Official journal of the Cell Death Differentiation AssociationBuchrieser et al Cell Death and Disease (2018)9:973 receptor-interacting serine/threonine-protein kinase 1 (RIPK1) plays an essential role[5,6,7,8]. Macrophages are key cells of the innate immune system. They are distributed throughout the tissues of the body, and play a key role in host defense, tissue homeostasis, Official journal of the Cell Death Differentiation Association. RIPK1 has been reported to shift the balance between cell survival, apoptosis, and necroptosis upon TNFα stimulation. It was reported to act as a kinase in the formation of the “necrosome” and triggering of RIPK3-dependent necroptosis[9,10]. A kinase-independent role for RIPK1 was later described, which suggests a scaffolding role for RIPK1 to inhibit caspase-8-dependent apoptosis and, paradoxically, necroptosis[11,12]. RIPK1 has been shown to play a role in the induction of proinflammatory gene expression independently of cell death[5,6,14]
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