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Summary A short form of cellular FLICE-inhibitory protein encoded by CFLARs promotes necroptosis. Although necroptosis is involved in various pathological conditions, the detailed mechanisms are not fully understood. Here we generated transgenic mice wherein CFLARs was integrated onto the X chromosome. All male CFLARs Tg mice died perinatally due to severe ileitis. Although necroptosis was observed in various tissues of CFLARs Tg mice, large numbers of intestinal epithelial cells (IECs) died by apoptosis. Deletion of Ripk3 or Mlkl, essential genes of necroptosis, prevented both necroptosis and apoptosis, and rescued lethality of CFLARs Tg mice. Type 3 innate lymphoid cells (ILC3s) were activated and recruited to the small intestine along with upregulation of interleukin-22 (Il22) in CFLARs Tg mice. Deletion of ILC3s or Il22 rescued lethality of CFLARs Tg mice by preventing apoptosis, but not necroptosis of IECs. Together, necroptosis-dependent activation of ILC3s induces lethal ileitis in an IL-22-dependent manner., Graphical Abstract, Highlights • CFLARs Tg mice develop severe ileitis in utero • Intestinal epithelial cells die by apoptosis and necroptosis in CFLARs Tg mice • Blockade of necroptosis rescues lethality of CFLARs Tg mice • Necroptosis activates type 3 innate lymphoid cells, resulting in severe ileitis, Immunology; Immune Response; Cell Biology; Functional Aspects of Cell Biology

Regenerating islet-derived protein (Reg)3β belongs to a member of the Reg family of proteins and has pleiotropic functions, including antimicrobial activity and tissue repair. However, whether Reg3β plays a protective role in the development of colitis and ileitis has not been fully investigated. We generated transgenic mice expressing a short form of cellular FLICE-inhibitory protein (cFLIPs) that promotes necroptosis, a regulated form of cell death. cFLIPs transgenic (CFLARs Tg) mice develop severe ileitis in utero. Although Reg3β is undetectable in the small intestine of wild-type embryos, its expression is aberrantly elevated in the small intestine of CFLARs Tg embryos. To test whether elevated Reg3β attenuates or exacerbates ileitis in CFLARs Tg mice, we generated a Reg3b−/− strain. Reg3b−/− mice grew to adulthood without apparent abnormalities. Deletion of Reg3b in CFLARs Tg mice exacerbated the embryonic lethality of CFLARs Tg mice. Dextran sulfate sodium-induced colitis, characterized by body weight loss and infiltration of neutrophils, was exacerbated in Reg3b−/− compared to wild-type mice. Moreover, the expression of Interleukin 6, an inflammatory cytokine and Chitinase-like 3, a marker for tissue repair macrophages was elevated in the colon of Reg3b−/− mice compared to wild-type mice after DSS treatment. Together, these results suggest that attenuation of colitis and ileitis is a result of Reg3β′s real function., Graphical abstract Image 1, Highlights • The expression of Reg3β is elevated in the embryonic small intestine of CFLARs Tg mice. • Reg3b−/− mice grow to adulthood without apparent abnormalities. • Dextran sulfate sodium-induced colitis is exacerbated in Reg3b−/− mice. • Deletion of Reg3b exacerbates ileitis in CFLARs Tg mice.

Apoptosis is a prototype of regulated cell death and plays a crucial role in the development of various organs and maintaining tissue homeostasis. Recent studies have revealed that new types of regulated cell death, including necroptosis, ferroptosis, and pyroptosis are molecularly identified. In this review, we discuss the molecular mechanisms and the functions of new types of regulated cell death.

Necroptosis is a regulated form of necrosis that depends on receptor-interacting protein kinase (RIPK)3 and mixed lineage kinase domain-like (MLKL). While danger-associated molecular pattern (DAMP)s are involved in various pathological conditions and released from dead cells, the underlying mechanisms are not fully understood. Here we develop a fluorescence resonance energy transfer (FRET) biosensor, termed SMART (a sensor for MLKL activation by RIPK3 based on FRET). SMART is composed of a fragment of MLKL and monitors necroptosis, but not apoptosis or necrosis. Mechanistically, SMART monitors plasma membrane translocation of oligomerized MLKL, which is induced by RIPK3 or mutational activation. SMART in combination with imaging of the release of nuclear DAMPs and Live-Cell Imaging for Secretion activity (LCI-S) reveals two different modes of the release of High Mobility Group Box 1 from necroptotic cells. Thus, SMART and LCI-S uncover novel regulation of the release of DAMPs during necroptosis., Necroptotic cells activate MLKL and release inflammatory DAMPs, although the underlying regulatory mechanisms of this process are poorly understood. Here, Murai et al. develop a necroptosis-specific FRET sensor (SMART) that monitors MLKL membrane translocation to identify two modes of DAMP release.

Cellular FLICE-inhibitory protein (cFLIP) is a catalytically inactive homolog of the initiator caspase, caspase 8 and blocks apoptosis through binding to caspase 8. Human CFLAR gene encodes two proteins, a long form cFLIP (cFLIPL) and a short form cFLIP (cFLIPs) due to an alternative splicing. Recent studies have shown that expression of cFLIPs, but not cFLIPL promotes programmed necrosis (also referred to as necroptosis) in an immortalized human keratinocyte cell line, HaCaT. Here, we found that expression of cFLIPs similarly promoted necroptosis in immortalized fibroblasts. To further expand this observation and exclude the possibility that immortalization process of keratinocytes or fibroblasts might affect the phenotype induced by cFLIPs expression, we generated human CFLARs transgenic (Tg) mice. Primary fibroblasts derived from CFLARs Tg mice were increased in susceptibility to TNFα-induced necroptosis, but not apoptosis compared to wild-type (WT) fibroblasts. Moreover, hallmarks of necroptosis, such as phosphorylation of receptor-interacting protein kinase (RIPK)1 and RIPK3, and oligomer formation of mixed lineage kinase domain-like (MLKL) were robustly induced in CFLARs Tg fibroblasts compared to wild-type fibroblasts following TNFα stimulation. Thus, cFLIPs-dependent promotion of necroptosis is not unique to immortalized keratinocytes or fibroblasts, but also to generalized to primary fibroblasts.

Background A delicate balance between cell death and keratinocyte proliferation is crucial for normal skin development. Previous studies have reported that cellular FLICE (FADD-like ICE)-inhibitory protein plays a crucial role in prevention of keratinocytes from TNF-α–dependent apoptosis and blocking of dermatitis. However, a role for cellular FLICE-inhibitory protein in TNF-α–independent cell death remains unclear. Objective We investigated contribution of TNF-α–dependent and TNF-α–independent signals to the development of dermatitis in epidermis-specific Cflar -deficient (Cflar E-KO ) mice. Methods We examined the histology and expression of epidermal differentiation markers and inflammatory cytokines in the skin of Cflar E-KO ; Tnfrsf1a +/− and Cflar E-KO ; Tnfrsf1a −/− mice. Mice were treated with neutralizing antibodies against Fas ligand and TNF-related apoptosis-inducing ligand to block TNF-α–independent cell death of Cflar E-KO ; Tnfrsf1a −/− mice. Results Cflar E-KO ; Tnfrsf1a −/− mice were born but experienced severe dermatitis and succumbed soon after birth. Cflar E-KO ;Tnfrsf1a +/− mice exhibited embryonic lethality caused by massive keratinocyte apoptosis. Although keratinocytes from Cflar E-KO ; Tnfrsf1a −/− mice still died of apoptosis, neutralizing antibodies against Fas ligand and TNF-related apoptosis-inducing ligand substantially prolonged survival of Cflar E-KO ; Tnfrsf1a −/− mice. Expression of inflammatory cytokines, such as Il6 and Il17a was increased; conversely, expression of epidermal differentiation markers was severely downregulated in the skin of Cflar E-KO ; Tnfrsf1a −/− mice. Treatment of primary keratinocytes with IL-6 and, to a lesser extent, IL-17A suppressed expression of epidermal differentiation markers. Conclusion TNF receptor superfamily 1 (TNFR1)–dependent or TNFR1-independent apoptosis of keratinocytes promotes inflammatory cytokine production, which subsequently blocks epidermal differentiation. Thus blockade of both TNFR1-dependent and TNFR1-independent cell death might be an alternative strategy to treat skin diseases when treatment with anti–TNF-α antibody alone is not sufficient.

While apoptosis has been considered to be identical to programmed cell death, necroptosis, which is morphologically related to necrosis, has emerged as a novel type of programmed cell death. Necroptosis depends on two structurally related kinases, receptor-interacting serine-threonine kinase (RIPK)1 and RIPK3. RIPK1 is activated through oligomerization of upstream adaptor molecules such as Fas-associated protein with death domain (FADD) and TNF receptor-associated death domain (TRADD) that are triggered by TNFα or Fas ligand. Activated RIPK1 subsequently interacts with and activates RIPK3, resulting in necroptosis. However, contribution of oxidative stress to execution of necroptosis is still controversial. We found that a selective inhibitor for RIPK1, necrostatin-1 (Nec-1) significantly blocked TNFα-induced cell death and ROS accumulation in NF-κB activation-deficient cells. This suggests that these cells mostly died by necroptosis upon TNFα stimulation. Intriguingly, an antioxidant, butylated hydroxyanisole (BHA) blocked TNFα-induced necroptosis and ROS accumulation in NF-κB activation-deficient cells. However, Nec-1, but not BHA, inhibited TNFα-induced phosphorylation of RIPK1 in these cells, suggesting that ROS play a crucial role in execution of necroptosis downstream of RIPK1 activation. Structural and functional analyses using BHA related compounds revealed that both tert-butyl and hydroxy groups of BHA are crucial for its anti-necroptotic function. Together, these results suggest that TNFα-induced necroptosis is tightly associated with oxidative stress, and oxidative stress is induced downstream of RIPK1 activation.

Cellular FLICE-inhibitory protein (cFLIP) is structurally related to caspase-8, but lacks its protease activity. Cflip gene encodes several splicing variants including short form (cFLIPs) and long form (cFLIP

Cellular FLICE-inhibitory protein (cFLIP) is structurally related to caspase-8, but lacks its protease activity. Cflip gene encodes several splicing variants including short form (cFLIPs) and long form (cFLIPL). cFLIPL is composed of two death effector domains at the N terminus and a C-terminal caspase-like domain, and cFLIPs lacks the caspase-like domain. Our studies reveal that cFLIP plays a central role in NF-κB-dependent survival signals that control apoptosis and programmed necrosis. Germline deletion of Cflip results in embryonic lethality due to enhanced apoptosis and programmed necrosis; however, the combined deletion of the death-signaling regulators, Fadd and Ripk3, prevents embryonic lethality in Cflip-deficient mice. Moreover, tissue-specific deletion of Cflip reveals cFLIP as a crucial regulator that maintains tissue homeostasis of immune cells, hepatocytes, intestinal epithelial cells, and epidermal cells by preventing apoptosis and programmed necrosis.