Matija Zelic

RIPK1 maintains epithelial homeostasis by inhibiting apoptosis and necroptosis

Necroptosis has emerged as an important pathway of programmed cell death in embryonic development, tissue homeostasis, immunity and inflammation. RIPK1 is implicated in inflammatory and cell death signalling and its kinase activity is believed to drive RIPK3-mediated necroptosis. Here we show that kinase-independent scaffolding RIPK1 functions regulate homeostasis and prevent inflammation in barrier tissues by inhibiting epithelial cell apoptosis and necroptosis. Intestinal epithelial cell (IEC)-specific RIPK1 knockout caused IEC apoptosis, villus atrophy, loss of goblet and Paneth cells and premature death in mice. This pathology developed independently of the microbiota and of MyD88 signalling but was partly rescued by TNFR1 (also known as TNFRSF1A) deficiency. Epithelial FADD ablation inhibited IEC apoptosis and prevented the premature death of mice with IEC-specific RIPK1 knockout. However, mice lacking both RIPK1 and FADD in IECs displayed RIPK3-dependent IEC necroptosis, Paneth cell loss and focal erosive inflammatory lesions in the colon. Moreover, a RIPK1 kinase inactive knock-in delayed but did not prevent inflammation caused by FADD deficiency in IECs or keratinocytes, showing that RIPK3-dependent necroptosis of FADD-deficient epithelial cells only partly requires RIPK1 kinase activity. Epidermis-specific RIPK1 knockout triggered keratinocyte apoptosis and necroptosis and caused severe skin inflammation that was prevented by RIPK3 but not FADD deficiency. These findings revealed that RIPK1 inhibits RIPK3-mediated necroptosis in keratinocytes in vivo and identified necroptosis as a more potent trigger of inflammation compared with apoptosis. Therefore, RIPK1 is a master regulator of epithelial cell survival, homeostasis and inflammation in the intestine and the skin.

Cutting Edge: RIPK1 Kinase Inactive Mice Are Viable and Protected from TNF-Induced Necroptosis In Vivo

The serine/threonine kinase RIPK1 is recruited to TNFR1 to mediate proinflammatory signaling and to regulate TNF-induced cell death. A RIPK1 deficiency results in perinatal lethality, impaired NFκB and MAPK signaling, and sensitivity to TNF-induced apoptosis. Chemical inhibitor and in vitro–reconstitution studies suggested that RIPK1 displays distinct kinase activity–dependent and –independent functions. To determine the contribution of RIPK1 kinase to inflammation in vivo, we generated knock-in mice endogenously expressing catalytically inactive RIPK1 D138N. Unlike Ripk1−/− mice, which die shortly after birth, Ripk1D138N/D138N mice are viable. Cells expressing RIPK1 D138N are resistant to TNF- and polyinosinic-polycytidylic acid–induced necroptosis in vitro, and Ripk1D138N/D138N mice are protected from TNF-induced shock in vivo. Moreover, Ripk1D138N/D138N mice fail to control vaccinia virus replication in vivo. This study provides genetic evidence that the kinase activity of RIPK1 is not required for survival but is essential for TNF-, TRIF-, and viral-initiated necroptosis.

Hematopoietic RIPK1 deficiency results in bone marrow failure caused by apoptosis and RIPK3-mediated necroptosis

Significance Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is involved in TNF signaling and interacts with the related RIPK3 to regulate cell death and inflammation. RIPK1 has kinase-independent prosurvival and kinase-dependent prodeath functions. To identify the lineages that depend on RIPK1 for survival, we generated conditional Ripk1 mice. Acute Ripk1 deletion results in rapid death of the animal caused by extensive cell death in the intestinal and hematopoietic lineages. A hematopoietic RIPK1 deficiency stimulates proinflammatory cytokine/chemokine production and cell death, resulting in bone marrow failure. Hematopoietic failure is partially rescued by a RIPK3 deficiency, indicating that RIPK1-deficient hematopoietic cells undergo RIPK3-mediated necroptosis. These findings show that in the hematopoietic lineage RIPK1 suppresses RIPK3 activity and suggest that RIPK-dependent necroptosis may contribute to human bone marrow failure syndromes. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is recruited to the TNF receptor 1 to mediate proinflammatory signaling and to regulate TNF-induced cell death. RIPK1 deficiency results in postnatal lethality, but precisely why Ripk1−/− mice die remains unclear. To identify the lineages and cell types that depend on RIPK1 for survival, we generated conditional Ripk1 mice. Tamoxifen administration to adult RosaCreERT2Ripk1fl/fl mice results in lethality caused by cell death in the intestinal and hematopoietic lineages. Similarly, Ripk1 deletion in cells of the hematopoietic lineage stimulates proinflammatory cytokine and chemokine production and hematopoietic cell death, resulting in bone marrow failure. The cell death reflected cell-intrinsic survival roles for RIPK1 in hematopoietic stem and progenitor cells, because Vav-iCre Ripk1fl/fl fetal liver cells failed to reconstitute hematopoiesis in lethally irradiated recipients. We demonstrate that RIPK3 deficiency partially rescues hematopoiesis in Vav-iCre Ripk1fl/fl mice, showing that RIPK1-deficient hematopoietic cells undergo RIPK3-mediated necroptosis. However, the Vav-iCre Ripk1fl/fl Ripk3−/− progenitors remain TNF sensitive in vitro and fail to repopulate irradiated mice. These genetic studies reveal that hematopoietic RIPK1 deficiency triggers both apoptotic and necroptotic death that is partially prevented by RIPK3 deficiency. Therefore, RIPK1 regulates hematopoiesis and prevents inflammation by suppressing RIPK3 activation.

CYLD proteolysis protects macrophages from TNF-mediated auto-necroptosis induced by LPS and licensed by type I IFN

Tumor necrosis factor (TNF) induces necroptosis, a RIPK3/MLKL-dependent form of inflammatory cell death. In response to infection by Gram-negative bacteria, multiple receptors on macrophages, including TLR4, TNF, and type I IFN receptors, are concurrently activated, but it is unclear how they crosstalk to regulate necroptosis. We report that TLR4 activates CASPASE-8 to cleave and remove the deubiquitinase cylindromatosis (CYLD) in a TRIF- and RIPK1-dependent manner to disable necroptosis in macrophages. Inhibiting CASPASE-8 leads to CYLD-dependent necroptosis caused by the TNF produced in response to TLR4 ligation. While lipopolysaccharides (LPS)-induced necroptosis was abrogated in Tnf(-/-) macrophages, a soluble TNF antagonist was not able to do so in Tnf(+/+) macrophages, indicating that necroptosis occurs in a cell-autonomous manner. Surprisingly, TNF-mediated auto-necroptosis of macrophages requires type I IFN, which primes the expression of key necroptosis-signaling molecules, including TNFR2 and MLKL. Thus, the TNF necroptosis pathway is regulated by both negative and positive crosstalk.

Kinase Activities of RIPK1 and RIPK3 Can Direct IFN-β Synthesis Induced by Lipopolysaccharide

The innate immune response is a central element of the initial defense against bacterial and viral pathogens. Macrophages are key innate immune cells that upon encountering pathogen-associated molecular patterns respond by producing cytokines, including IFN-β. In this study, we identify a novel role for RIPK1 and RIPK3, a pair of homologous serine/threonine kinases previously implicated in the regulation of necroptosis and pathologic tissue injury, in directing IFN-β production in macrophages. Using genetic and pharmacologic tools, we show that catalytic activity of RIPK1 directs IFN-β synthesis induced by LPS in mice. Additionally, we report that RIPK1 kinase–dependent IFN-β production may be elicited in an analogous fashion using LPS in bone marrow–derived macrophages upon inhibition of caspases. Notably, this regulation requires kinase activities of both RIPK1 and RIPK3, but not the necroptosis effector protein, MLKL. Mechanistically, we provide evidence that necrosome-like RIPK1 and RIPK3 aggregates facilitate canonical TRIF–dependent IFN-β production downstream of the LPS receptor TLR4. Intriguingly, we also show that RIPK1 and RIPK3 kinase–dependent synthesis of IFN-β is markedly induced by avirulent strains of Gram-negative bacteria, Yersinia and Klebsiella, and less so by their wild-type counterparts. Overall, these observations identify unexpected roles for RIPK1 and RIPK3 kinases in the production of IFN-β during the host inflammatory responses to bacterial infection and suggest that the axis in which these kinases operate may represent a target for bacterial virulence factors.

Dendritic Cell RIPK1 Maintains Immune Homeostasis by Preventing Inflammation and Autoimmunity

Necroptosis is a form of cell death associated with inflammation; however, the biological consequences of chronic necroptosis are unknown. Necroptosis is mediated by RIPK1, RIPK3, and MLKL kinases but in hematopoietic cells RIPK1 has anti-inflammatory roles and functions to prevent necroptosis. Here we interrogate the consequences of chronic necroptosis on immune homeostasis by deleting Ripk1 in mouse dendritic cells. We demonstrate that deregulated necroptosis results in systemic inflammation, tissue fibrosis, and autoimmunity. We show that inflammation and autoimmunity are prevented upon expression of kinase inactive RIPK1 or deletion of RIPK3 or MLKL. We provide evidence that the inflammation is not driven by microbial ligands, but depends on the release of danger-associated molecular patterns and MyD88-dependent signaling. Importantly, although the inflammation is independent of type I IFN and the nucleic acid sensing TLRs, blocking these pathways rescues the autoimmunity. These mouse genetic studies reveal that chronic necroptosis may underlie human fibrotic and autoimmune disorders.

RIP kinase 1–dependent endothelial necroptosis underlies systemic inflammatory response syndrome

Receptor interacting protein kinase 1 (RIPK1) has important kinase-dependent and kinase-independent scaffolding functions that activate or prevent apoptosis or necroptosis in a cell context–dependent manner. The kinase activity of RIPK1 mediates hypothermia and lethality in a mouse model of TNF-induced shock, reflecting the hyperinflammatory state of systemic inflammatory response syndrome (SIRS), where the proinflammatory “cytokine storm” has long been viewed as detrimental. Here, we demonstrate that cytokine and chemokine levels did not predict survival and, importantly, that kinase-inactive Ripk1D138N/D138N hematopoietic cells afforded little protection from TNF- or TNF/zVAD-induced shock in reconstituted mice. Unexpectedly, RIPK1 kinase–inactive mice transplanted with WT hematopoietic cells remained resistant to TNF-induced shock, revealing that a nonhematopoietic lineage mediated protection. TNF-treated Ripk1D138N/D138N mice exhibited no significant increases in intestinal or vascular permeability, nor did they activate the clotting cascade. We show that TNF administration damaged the liver vascular endothelium and induced phosphorylated mixed lineage kinase domain-like (phospho-MLKL) reactivity in endothelial cells isolated from TNF/zVAD-treated WT, but not Ripk1D138N/D138N, mice. These data reveal that the tissue damage present in this SIRS model is reflected, in part, by breaks in the vasculature due to endothelial cell necroptosis and thereby predict that RIPK1 kinase inhibitors may provide clinical benefit to shock and/or sepsis patients.

Analyzing Necroptosis Using an RIPK1 Kinase Inactive Mouse Model of TNF Shock

The serine/threonine kinase RIPK1 has numerous biological and pathological functions, mediating prosurvival as well as prodeath apoptotic and necroptotic signaling pathways downstream of various receptors, including death receptors and Toll-like receptors (TLRs). RIPK1 has been implicated in various diseases, including ischemia-reperfusion injury and inflammatory bowel disease (IBD). The recent generation of RIPK1 kinase inactive mice has enabled us to genetically interrogate the role of RIPK1 kinase-mediated necroptosis in disease models. Here, we describe procedures utilizing kinase inactive Ripk1D138N/D138N mice to analyze necroptosis induction in vitro in bone-marrow derived macrophages (BMDMs) and in vivo in a murine model of TNF-induced shock.

Correction: Dendritic Cell RIPK1 Maintains Immune Homeostasis by Preventing Inflammation and Autoimmunity

O’Donnell, J. A., J. Lehman, J. E. Roderick, D. Martinez-Marin, M. Zelic, C. Doran, N. Hermance, S. Lyle, M. Pasparakis, K. A. Fitzgerald, A. Marshak-Rothstein, and M. A. Kelliher. 2018. Dendritic cell RIPK1 maintains immune homeostasis by preventing inflammation and autoimmunity. J. Immunol. 200

Abstract B14: Hematopoietic RIPK1 deficiency results in bone marrow failure due to apoptosis and RIPK3-mediated necroptosis.

The serine/threonine kinase RIPK1 is recruited to the TNF receptor 1 to mediate pro-inflammatory signaling and to regulate TNF-induced apoptosis and necroptosis. The kinase activity of RIPK1 is required for RIPK3 activation and induction of necroptosis, an inflammatory form of cell death. Although Ripk3-/- mice are viable, a RIPK1 deficiency results in postnatal lethality. To identify the lineages and cell types that depend on RIPK1 for survival, we generated conditional Ripk1 mice. Tamoxifen administration to adult RosaCreERT2Ripk1fl/fl mice results in lethality due to cell death in the intestinal and hematopoietic lineages. Similarly, Ripk1 deletion in cells of the hematopoietic lineage stimulates pro-inflammatory cytokine and chemokine production and hematopoietic cell death, resulting in bone marrow failure. The cell death reflects cell intrinsic survival roles for RIPK1 in hematopoietic stem and progenitor cells, as Vav-iCre Ripk1fl/fl fetal liver cells failed to reconstitute hematopoiesis in lethally irradiated recipients. We demonstrate that a RIPK3 deficiency partially rescues hematopoiesis in Vav-iCre Ripk1fl/fl mice, revealing that RIPK1 mediates survival by preventing RIPK3-mediated necroptosis. However, Vav-iCre Ripk1fl/flRipk3-/- progenitors remain TNF sensitive in vitro and fail to repopulate irradiated mice. These genetic studies reveal that a hematopoietic RIPK1 deficiency triggers both apoptotic and necroptotic death that is partially prevented by a RIPK3 deficiency. Thus, in contrast to in vitro studies, RIPK1 functions in hematopoietic cells to negatively regulate RIPK3 and prevent inflammation. Citation Format: Justine R. Roderick, Nicole C. Hermance, Matija Zelic, Apostolos Polykratis, Manolis Pasparakis, Michelle A. Kelliher. Hematopoietic RIPK1 deficiency results in bone marrow failure due to apoptosis and RIPK3-mediated necroptosis. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B14.