Necroptotic Death Of RIPK1-Deficient HSC Compromises Hematopoiesis
Authors: Croker Ben A , Joanne O'Donnell A , Metcalf Donald , James Rickard , Evans Joseph , Masters Seth L. , Roberts Andrew W. , Gerlic Motti , Silke John 
Published: October 2019

Abstract

Introduction
Cell death can be triggered by many stimuli leading to apoptosis, pyroptosis (Caspase-1-dependent cell death) or necroptosis (Receptor-interacting serine/threonine-protein kinase (RIPK)-1/RIPK3-dependent cell death). RIPK1 is engaged by TNFR or Fas/CD95 ligation, and can induce NF-κB activation and cell death. FADD and Caspase-8 modulate RIPK1 and RIPK3 activity to prevent inappropriate induction of necroptosis (Oberst et al., Nature 2011, 471: 363-367; Zhang et al., Nature 2011, 471: 373-376). Modulation of necroptosis by small molecule inhibitors of RIPK1 has emerged as an exciting approach to intervene in inflammatory disease, ischemia reperfusion injury, pancreatitis and in mouse models of sepsis (He et al., Cell 2009, 137: 1100-1111; McNeal et al., Shock 2011, 35: 499-505; Oerlemans et al., Basic Res Cardiol 2012, 107: 270; Lukens et al., Nature 2013, 498: 224-227). However, RIPK1-deficient neonates die at birth and exhibit inflammatory disease and anemia, suggesting that inhibitors of RIPK1 may alter hematopoiesis. We have therefore investigated the hematological consequences of RIPK1 deficiency.

Methods
Fetal liver chimeras and competitive transplants were generated using E13.5 Ripk1-/-, Ripk3-/-and Ripk1-/-Ripk3-/- fetal liver cells. Serial transplants were established using 106 fetal liver cells for primary transplants and 0.2-5 x 106 bone marrow cells for secondary transplants. The survival of recipient mice and frequency of donor, competitor and recipient cells was assessed by flow cytometry up to 6 months post transplantation. The frequency of hematopoietic progenitor cells was assessed using in vitro clonal culture assays of E13.5-E18.5 fetal liver cells stimulated with SCF+IL-3+Epo in the presence or absence of TNFα or FasL. The contribution of TNFα and FasL to hematopoiesis was examined using TNFα neutralizing antibody in lethally-irradiated recipients of Ripk1-/- cells or by engrafting Ripk1-/- cells into lethally-irradiated Tnfa-/-Faslgld/gldrecipient mice.

Results
Ripk1 -/- fetal liver cells fail to engraft in lethally-irradiated recipients, with defects evident in lymphoid and myeloid lineages in the bone marrow, peripheral blood and spleen between 4 and 26 weeks post-transplant. In competitive fetal liver transplant experiments, Ripk1-/- hematopoietic stem and progenitor cells failed to compete with wild-type counterparts, indicating a cell-intrinsic defect in hematopoietic progenitor cells that cannot be attributed to the inflammatory disease evident in Ripk1-/- embryos. Ripk1-/- myeloid progenitor cells were sensitive to death induced by TNFα or FasL stimulation. Only minor abnormalities in hematopoiesis were detected when Ripk1-/- fetal liver cells were transplanted into lethally-irradiated Tnfa-/-Faslgld/gld recipient mice, or when lethally-irradiated wild-type recipient mice receiving Ripk1-/- fetal liver cells were treated with a TNFα neutralizing antibody, indicating key roles for TNFα and FasL during engraftment. A compound deficiency in RIPK3 rescued the reconstitution defects seen in Ripk1-/- cells suggesting that RIPK1-deficient hematopoietic cells undergo RIPK3-dependent necroptotic death. A residual defect in Ripk1-/-Ripk3-/-T lymphopoiesis suggests that RIPK1 deficiency induces other forms of cell death or that RIPK1 is required for other essential signaling pathways such as NF-κB signaling.

Conclusion
These data demonstrate essential roles for RIPK1 in hematopoiesis at steady state. Our results indicate that small molecule RIPK1 inhibitors should be used with caution in the clinic to avoid activation of RIPK3-dependent cell death pathways leading to cytopenia, immunosuppression and bone marrow failure. Finally, this work highlights that studies using RIPK1-deficient cells to study the roles for RIPK1 in inflammatory disease must draw conclusions with care considering the critical role of RIPK1 in hematopoiesis.

Disclosures:
No relevant conflicts of interest to declare.