RIP3-deficient Mice Research Articles

RIP3 deficiency alleviates liver fibrosis by inhibiting ROCK1-TLR4-NF-κB pathway in macrophages.

Liver fibrosis is an important pathologic process in injured liver tissues. A protein kinase, receptor-interacting protein (RIP)3, plays a crucial role in mediating different diseases. However, the role of RIP3 in macrophages in liver fibrosis has not yet been studied. In our study, we found that RIP3 expression was up-regulated in liver tissues and macrophages of humans and mice with liver fibrosis. Absence of RIP3 in macrophages could alleviate inflammation and macrophage or neutrophil accumulation in mice after carbon tetrachloride (CCl4) or bile duct ligation (BDL) treatment. Importantly, RIP3 deficiency in macrophages could decrease CCl4-induced and BDL-induced liver fibrosis in mice. Moreover, RIP3 deficiency could inhibit the TLR4-NF-κB pathway through suppressing Rho-associated coiled-coil containing protein kinase (ROCK)1 in macrophages. To explore the connection of ROCK1 and RIP3 in macrophages of mice with liver fibrosis in vivo, ROCK1-overexpressed macrophages were infused to RIP3-deficient mice, which resulted in increased inflammation and liver fibrosis. In conclusion, our findings suggest that RIP3 plays a crucial proinflammatory role in liver fibrosis by regulating the ROCK1-TLR4-NF-κB signaling pathway in macrophages and therefore may be a potential therapeutic target for immune-mediated liver fibrosis.-Wei, S., Zhou, H., Wang, Q., Zhou, S., Li, C., Liu, R., Qiu, J., Shi, C., Lu, L. RIP3 deficiency alleviates liver fibrosis by inhibiting ROCK1-TLR4-NF-κB pathway in macrophages.

RIP3 promotes colitis-associated colorectal cancer by controlling tumor cell proliferation and CXCL1-induced immune suppression.

Rationale: Necroptosis is a programmed form of non-apoptotic cell death that requires receptor-interacting protein 3 (RIP3). RIP3 has been shown to be relevant in multiple tumor types and has differential impact on tumor progression. We investigated whether RIP3 is involved in the progression of colitis-associated cancer (CAC) in mice.Methods: Tissues from colorectal cancer patients were examined for RIP3 expression. CAC was induced using azoxymethane (AOM) injection followed by dextran sodium sulfate (DSS) treatment in RIP3-deficient or wild-type mice. Colon tissues were collected and analyzed by Western blotting and gene expression profile analyses. Immune cell infiltration and CXCL1 expression were examined by flow cytometry and Real-time PCR, respectively.Results: RIP3 expression was upregulated in mouse CAC and human colon cancer. RIP3-deficient mice showed significantly attenuated colitis-associated tumorigenesis. Bone marrow transplantation experiments suggested that RIP3's function in hematopoietic cells primarily contributes to the phenotype. RIP3 supported epithelial proliferation and tumor growth via JNK signaling but had no effect on apoptosis. RIP3 deletion increased T cell accumulation and reduced infiltration by immunosuppressive subsets of myeloid cells during acute colitis and CAC. The immune-suppressive tumor microenvironment was dependent on RIP3-induced expression of the chemokine attractant CXCL1, and administration of recombinant CXCL1 during CAC restored tumorigenesis in Rip3-/- mice.Conclusion: Our results reveal an unexpected function of RIP3 in enhancing the proliferation of premalignant intestinal epithelial cells (IECs) and promoting myeloid cell-induced adaptive immune suppression. These two distinct mechanisms of RIP3-induced JNK and CXCL1 signalling contribute to CAC progression.

Mito-tempo protects against acute liver injury but induces limited secondary apoptosis during the late phase of acetaminophen hepatotoxicity.

We previously reported that delayed treatment with Mito-tempo (MT), a mitochondria-targeted superoxide dismutase mimetic, protects against the early phase of acetaminophen (APAP) hepatotoxicity by inhibiting peroxynitrite formation. However, whether this protection is sustained to the late phase of toxicity is unknown. To investigate the late protection, C57Bl/6J mice were treated with 300mg/kg APAP followed by 20mg/kg MT 1.5h or 3h later. We found that both MT treatments protected against the late phase of APAP hepatotoxicity at 12 and 24h. Surprisingly, MT-treated mice demonstrated a significant increase in apoptotic hepatocytes, while the necrotic phenotype was observed almost exclusively in mice treated with APAP alone. In addition, there was a significant increase in caspase-3 activity and cleavage in the livers of MT-treated mice. Immunostaining for active caspase-3 revealed that the positively stained hepatocytes were exclusively in centrilobular areas. Treatment with the pan-caspase inhibitor ZVD-fmk (10mg/kg) 2h post-APAP neutralized this caspase activation and provided additional protection against APAP hepatotoxicity. Treatment with N-acetylcysteine, the current standard of care for APAP poisoning, protected but did not induce this apoptotic phenotype. Mechanistically, MT treatment inhibited APAP-induced RIP3 kinase expression, and RIP3-deficient mice showed caspase activation and apoptotic morphology in hepatocytes analogous to MT treatment. These data suggest that while necrosis is the primary cause of cell death after APAP hepatotoxicity, treatment with the antioxidant MT may switch the mode of cell death to secondary apoptosis in some cells. Modulation of mitochondrial oxidative stress and RIP3 kinase expression play critical roles in this switch.

RIP3 deficiency protects against traumatic brain injury (TBI) through suppressing oxidative stress, inflammation and apoptosis: Dependent on AMPK pathway

Traumatic brain injury (TBI) is a leading cause of disability and mortality in young adults worldwide. The pathophysiology is not fully understood. Programmed necrosis (necroptosis) is a newly identified mechanism of cell death combining features of both apoptosis and necrosis. Receptor-interacting protein 3 (RIP3) plays an important role in programmed necrosis. However, the effect of RIP3-related pathway in TBI is little to be known. We attempted to explore the significance of RIP3 in regulating TBI in vivo. Significantly, TBI induced over-expression of RIP3 in the hippocampus of mice, as well as RIP1 and phosphorylated mixed lineage kinase domain-like protein (MLKL). Mice after TBI exhibited cognitive dysfunction and activation of glia cells, which were significantly attenuated by RIP3-knockout (KO). Moreover, inflammation and oxidative stress in hippocampus were markedly induced by TBI in wild type (WT) mice. Of note, the reduction of pro-inflammatory cytokines and oxidants was observed in RIP3-deficient mice, which was linked to the blockage of NLR pyrin domain containing 3 (NLRP3)/apoptosis-associated speck-like protein containing a CARD (ASC)/Caspase-1 and kelch-like ECH-associated protein 1 (Keap 1) pathways. Further, TBI induced hippocampus apoptosis, evidenced by the increase of cleaved Caspase-8/-3 and poly (ADP)-ribose polymerase (PARP) in WT mice, whereas being decreased by RIP3-knockout. In addition, RIP3 knockout led to phosphorylation of AMP-activated protein kinase α (AMPKα) in hippocampus of mice after TBI. And of note, the in vitro findings indicated that RIP3-ablation attenuated oxidative stress, inflammation and apoptosis in astrocytes, which was dependent on AMPKα activation. Together, suppressing RIP3 might be served as a therapeutic target against brain injury through inhibiting inflammation, oxidative stress and apoptosis.

Environmental cues in Casp8-deficient mice enhance virus-specific T cell expansion and terminal differentiation

Abstract Cell autonomous death is balanced against survival signaling to establish T cell levels in the course of primary as well as secondary responses to infection. It is known that mitochondrial (intrinsic) cell death contributes to T cell contraction; however, the question remains about the role of extrinsic cell death in the T cell response. Caspase8 (Casp8) initiates apoptosis downstream of death receptor activation while also suppresses RIP3-mediated necroptosis. We have employed wild type (WT), Rip3−/− and Casp8−/− Rip3−/− (DKO) mice to address the contribution of Casp8-dependent apoptosis as well as RIP3-dependent necroptosis in the antiviral T cell response. Although both Casp8 and RIP3 are dispensable for the T cell response to murine cytomegalovirus (MCMV) infection, we found that DKO mice exhibit differences in T cell characteristics compared to WT or RIP3-deficient mice. Interestingly, the T cell populations in naïve DKO mice show increased activation markers (CD44LhiKLRG1hiCD127lo). This pre-activated status results in more abundant virus-specific effector T cells in DKO mice following expansion at 7 dpi. Importantly, DKO T cells respond the same as WT T cells when adoptively transferred into WT environment; however, adoptive transfer of DKO splenocytes into WT recipients results in greater expansion and terminal differentiation of the recipient (WT) T cell population during infection. Altogether, even though Casp8 and RIP3 are dispensable for T cell expansion and contraction when examined directly, Casp8-dependent environmental cues influence T cell expansion during infection and even drive WT T cells to behave like Casp8-deficient T cells when present together during virus infection.

Type I interferon and TNF signaling plays key synergistic role in programmed necrosis

Abstract Pathogen detection and downstream signaling events are important for protection against disease and clearance of infection. Upon sensing pathogen-associated molecular patterns (PAMPs), cells often produce interferons (IFNs), which drive multiple transcriptional changes, allowing a cell to enter an antiviral state. Intriguingly, the IFN-α/β receptor (IFNAR) has recently been shown to be important in triggering the cell death program necroptosis (or programmed necrosis), although the mechanism is unclear. Unlike apoptosis (which relies on caspase activation), necroptosis requires the activation of receptor interacting kinase 3 (RIP3) and leads to the swelling and bursting of the cell. Death receptor or Toll-like receptor signaling can activate RIP3 in certain cellular contexts, leading to cellular destruction by necroptosis. Furthermore, RIP3-deficient mice are highly susceptible to multiple viral infections, highlighting RIP3’s antiviral role. How IFN signaling interfaces with programmed necrosis remains to be discovered. My studies have illuminated the requirement of TNFα production downstream of IFN signaling to trigger necroptosis. We believe promoting necroptosis through the production of TNFα may be an underappreciated role of the IFN-driven antiviral response.

Role of RIP1 in physiological enterocyte turnover in mouse small intestine via nonapoptotic death.

Enterocyte shedding in the small intestine is often referred as an example of programmed cell death. However, little is known about the underlying mechanisms, although both apoptotic and nonapoptotic cell death have been suggested to play an important role. Here, we show by electron microscope that the majority of cells dying in the mouse small intestine do not display apoptotic characteristics. Chemical biological approach in vivo and in an organ culture showed that necrostatin-1 (Nec-1), an inhibitor of receptor-interacting protein 1 (RIP1, also called RIPK1), inhibited the shedding/nonapoptotic death of enterocyte, resulting in suppression of physiological enterocyte turnover. Moreover, RIP1 knockdown in vivo and RIP1 haploinsufficiency significantly suppressed physiological enterocyte turnover. Unlike Nec-1-sensitive (RIP1-dependent) cell death, so called necroptosis, which is also dependent on RIP3, physiological enterocyte turnover in RIP3-deficient mice was executed normally and still inhibited by Nec-1. As inhibition of the shedding/nonapoptotic death of enterocyte by Nec-1 resulted in suppression of crypt cell proliferation, the shedding process plays a dominant role over cell proliferation in maintaining homeostasis of enterocyte turnover. These results indicate that RIP1 plays a major role in physiological enterocyte turnover through a RIP3-independent nonapoptotic death mechanism in the mouse small intestine.

Receptor interacting protein kinase 3 is a critical early mediator of acetaminophen-induced hepatocyte necrosis in mice

Acetaminophen (APAP) overdose is a major cause of hepatotoxicity and acute liver failure in the U.S., but the pathophysiology is incompletely understood. Despite evidence for apoptotic signaling, hepatic cell death after APAP is generally considered necrotic in mice and in humans. Recent findings suggest that the receptor interacting protein kinase 3 (RIP3) acts as a switch from apoptosis to necrosis (programmed necrosis). Thus, the aim of the current investigation was to determine if RIP3 is involved in APAP-induced liver cell death. APAP (200-300 mg/kg) caused glutathione depletion and protein adduct formation, oxidant stress, mitochondrial release of apoptosis inducing factor, and nuclear DNA fragmentation resulting in centrilobular necrosis in C57Bl/6J mice. Inhibiting RIP3 protein induction with antisense morpholinos in wild-type animals or using RIP3-deficient mice had no effect on protein adduct formation but attenuated all other parameters, including necrotic cell death, at 6 hours after APAP. In addition, cultured hepatocytes from RIP3-deficient mice showed reduced injury compared to wild-type cells after 24 hours. Interestingly, APAP-induced mitochondrial translocation of dynamin-related protein 1 (Drp1), the initiator of mitochondrial fission, was inhibited by reduced RIP3 protein expression and the Drp1 inhibitor MDIVI reduced APAP-induced cell death at 24 hours. All of these protective effects were lost after 24 hours in vivo or 48 hours in vitro. RIP3 is an early mediator of APAP hepatotoxicity, involving modulation of mitochondrial dysfunction and oxidant stress. Controlling RIP3 expression could be a promising new approach to reduce APAP-induced liver injury, but requires complementary strategies to control mitochondrial dysfunction for long-term protection.

Dichotomy between RIP1- and RIP3-Mediated Necroptosis in Tumor Necrosis Factor-α-Induced Shock

Tumor necrosis factor receptor (TNFR) signaling may result in survival, apoptosis or programmed necrosis. The latter is called necroptosis if the receptor-interacting protein 1 (RIP1) inhibitor necrostatin-1 (Nec-1) or genetic knockout of RIP3 prevents it. In the lethal mouse model of TNFα-mediated shock, addition of the pan-caspase inhibitor zVAD-fmk (zVAD) accelerates time to death. Here, we demonstrate that RIP3-deficient mice are protected markedly from TNFα-mediated shock in the presence and absence of caspase inhibition. We further show that the fusion protein TAT-crmA, previously demonstrated to inhibit apoptosis, also prevents necroptosis in L929, HT29 and FADD-deficient Jurkat cells. In contrast to RIP3-deficient mice, blocking necroptosis by Nec-1 or TAT-crmA did not protect from TNFα/zVAD-mediated shock, but further accelerated time to death. Even in the absence of caspase inhibition, Nec-1 application led to similar kinetics. Depletion of macrophages, natural killer (NK) cells, granulocytes or genetic deficiency for T lymphocytes did not influence this model. Because RIP3-deficient mice are known to be protected from cerulein-induced pancreatitis (CIP), we applied Nec-1 and TAT-crmA in this model and demonstrated the deterioration of pancreatic damage upon addition of these substances. These data highlight the importance of separating genetic RIP3 deficiency from RIP1 inhibition by Nec-1 application in vivo and challenge the current definition of necroptosis.

Virus Inhibition of RIP3-Dependent Necrosis

Viral infection activates cytokine expression and triggers cell death, the modulation of which is important for successful pathogenesis. Necroptosis is a form of programmed necrosis dependent on two related RIP homotypic interaction motif (RHIM)-containing signaling adaptors, receptor-interacting protein kinases (RIP) 1 and 3. We find that murine cytomegalovirus infection induces RIP3-dependent necrosis. Whereas RIP3 kinase activity and RHIM-dependent interactions control virus-associated necrosis, virus-induced death proceeds independently of RIP1 and is therefore distinct from TNFalpha-dependent necroptosis. Viral M45-encoded inhibitor of RIP activation (vIRA) targets RIP3 during infection and disrupts RIP3-RIP1 interactions characteristic of TNFalpha-induced necroptosis, thereby suppressing both death pathways. Importantly, attenuation of vIRA mutant virus in wild-type mice is normalized in RIP3-deficient mice. Thus, vIRA function validates necrosis as central to host defense against viral infections and highlights the benefit of multiple virus-encoded cell-death suppressors that inhibit not only apoptotic, but also necrotic mechanisms of virus clearance.

A Role for RIP1/RIP3-Dependent Programmed Necrosis in Innate Immune Responses Against Vaccinia Virus Infections (135.44)

Abstract Cell death by programmed necrosis (also known as caspase-independent cell death or necroptosis) is characterized by rapid loss of plasma membrane integrity prior to the exposure of phagocytic signal. The release of endogenous "danger signals" from necrotic cells trigger inflammation and may have important physiological and pathological consequences in immune responses, inflammatory diseases and cancer growth. However, the role of programmed necrosis in immune responses and inflammation has not been adequately tested, in part because the molecular pathway that regulates programmed necrosis was ill-defined. The serine/threonine kinase RIP1 plays crucial role in programmed necrosis induced by TNF and other stimuli. We performed a RNA interference (RNAi) screen to identify downstream regulators of RIP1-dependent programmed and identified RIP3 to be another kinase critically required for TNF-induced programmed necrosis. In order to test the role of programmed necrosis in inflammation and anti-viral immune responses, we infected RIP3-deficient mice with vaccinia virus and found that the RIP3-deficient mice were highly sensitive to vaccinia virus infections. The details of these studies and the physiological implications of programmed necrosis will be discussed.

Kinase RIP3 is dispensable for normal NF-kappa Bs, signaling by the B-cell and T-cell receptors, tumor necrosis factor receptor 1, and Toll-like receptors 2 and 4.

RIP3 is a member of the RIP kinase family. It is expressed in the embryo and in multiple adult tissues, including most hemopoietic cell lineages. Several studies have implicated RIP3 in the regulation of apoptosis and NF-kappa B signaling, but whether RIP3 promotes or attenuates activation of the NF-kappa B family of transcription factors has been controversial. We have generated RIP3-deficient mice by gene targeting and find RIP3 to be dispensable for normal mouse development. RIP3-deficient cells showed normal sensitivity to a variety of apoptotic stimuli and were indistinguishable from wild-type cells in their ability to activate NF-kappa B signaling in response to the following: human tumor necrosis factor (TNF), which selectively engages mouse TNF receptor 1; cross-linking of the B- or T-cell antigen receptors; peptidoglycan, which activates Toll-like receptor 2; and lipopolysaccharide (LPS), which stimulates Toll-like receptor 4. Consistent with these observations, RIP3-deficient mice exhibited normal antibody production after immunization with a T-dependent antigen and normal interleukin-1 beta (IL-1 beta), IL-6, and TNF production after LPS treatment. Thus, we can exclude RIP3 as an essential modulator of NF-kappa B signaling downstream of several receptor systems.