New insights into the regulation of apoptosis, necroptosis, and pyroptosis by receptor interacting protein kinase 1 and caspase-8

Necroptosis and pyroptosis are inflammatory forms of regulated necrotic cell death as opposed to apoptosis that is generally considered immunologically silent. Recent studies revealed unexpected links in the pathways regulating and executing cell death in response to activation of signaling cascades inducing apoptosis, necroptosis, and pyroptosis. Emerging evidence suggests that receptor interacting protein kinase 1 and caspase-8 control the cross-talk between apoptosis, necroptosis, and pyroptosis and determine the type of cell death induced in response to activation of cell death signaling

Complementation of Lymphotoxin α Knockout Mice with Tumor Necrosis Factor–expressing Transgenes Rectifies Defective Splenic Structure and Function

Lymphotoxin (LT)α knockout mice, as well as double LTα/tumor necrosis factor (TNF) knockout mice, show a severe splenic disorganization with nonsegregating T/B cell zones and complete absence of primary B cell follicles, follicular dendritic cell (FDC) networks, and germinal centers. In contrast, as shown previously and confirmed in this study, LTβ-deficient mice show much more conserved T/B cell areas and a reduced but preserved capacity to form germinal centers and FDC networks. We show here that similar to the splenic phenotype of LTβ-deficient mice, complementation of LTα knockout mice with TNF-expressing transgenes leads to a p55 TNF receptor–dependent restoration of B/T cell zone segregation and a partial preservation of primary B cell follicles, FDC networks, and germinal centers. Notably, upon lipopolysaccharide challenge, LTα knockout mice fail to produce physiological levels of TNF both in peritoneal macrophage supernatants and in their serum, indicating a coinciding deficiency in TNF expression. These findings suggest that defective TNF expression contributes to the complex phenotype of the LTα knockout mice, and uncover a predominant role for TNF and its p55 TNF receptor in supporting, even in the absence of LTα, the development and maintenance of splenic B cell follicles, FDC networks, and germinal centers

Endothelial and Macrophage-Specific Deficiency of P38α MAPK Does Not Affect the Pathogenesis of Atherosclerosis in ApoE−/− Mice

BACKGROUND: The p38α Mitogen-Activated Protein Kinase (MAPK) regulates stress- and inflammation-induced cellular responses. Factors implicated in the development of atherosclerosis including modified low-density lipoprotein (LDL), cytokines and even shear stress induce p38 activation in endothelial cells and macrophages, which may be important for plaque formation. This study investigates the effects of endothelial- and macrophage-specific deficiency of p38α in atherosclerosis development, in Apolipoprotein E deficient (ApoE(-/-)) mice. METHODOLOGY/PRINCIPAL FINDINGS: ApoE(-/-) mice with macrophage or endothelial cell-specific p38α deficiency were fed a high cholesterol diet (HCD) for 10 weeks and atherosclerosis development was assessed by histological and molecular methods. Surprisingly, although p38α-deficiency strongly attenuated oxidized LDL-induced expression of molecules responsible for monocyte recruitment in endothelial cell cultures in vitro, endothelial-specific p38α ablation in vivo did not affect atherosclerosis development. Similarly, macrophage specific deletion of p38α did not affect atherosclerotic plaque development in ApoE(-/-) mice. CONCLUSIONS: Although previous studies implicated p38α signaling in atherosclerosis, our in vivo experiments suggest that p38α function in endothelial cells and macrophages does not play an important role in atherosclerotic plaque formation in ApoE deficient mice

IκB Kinase Signaling Is Essential for Maintenance of Mature B Cells

Nuclear factor (NF)-κB proteins play crucial roles in immune responses and cellular survival. Activation of NF-κB is mediated by the IκB kinase (IKK) complex, which is composed of two kinases, IKK1 and IKK2, and a regulatory subunit termed NF-κB essential modulator (NEMO). IKK2- and NEMO-deficient mice die at early embryonic stages. We therefore used conditional gene targeting to evaluate the role of these proteins in B cells in adult mice. B lineage–specific disruption of either IKK signaling by deletion of NEMO, or of IKK2-specific signals by ablation of IKK2 activity leads to the disappearance of mature B lymphocytes. We conclude that maintenance of mature B cells depends on IKK-mediated activation of NF-κB

The role of OTULIN in skin inflammation and cell death

The skin epithelium provides a physical and immunologically active multilayered barrier that protects the body from environmental factors and mediates cellular immune responses. Especially mechanisms regulating the cell survival and cell death of epidermal keratinocytes play a crucial role in maintaining tissue homeostasis. Inflammatory and cell death signalling is tightly controlled by linear ubiquitination, mediated via the E3 ligase Linear Ubiquitin Assembly Complex (LUBAC) to initiate innate immune responses. LUBAC itself is controlled by OTU deubiquitinase with linear specificity (OTULIN), a deubiquitinating enzyme that specifically degrades linear ubiquitin chains. Mouse model studies suggested that OTULIN deficiency results in loss of LUBAC components and cell death-mediated inflammation. Interestingly, mutations in the gene encoding OTULIN cause the OTULIN-related autoinflammatory syndrome (ORAS), a human genetic disease presented with complex pathological features and systemic inflammation affecting multiple organs, including the skin. The role of OTULIN in regulating skin homeostasis, inflammation and cell death is poorly understood and remains to be elucidated. The aim of this study was to investigate the biological function of OTULIN in the skin and to unravel the underlying cellular processes. The results acquired in this thesis reveal that OTULIN prevents skin inflammation by inhibiting keratinocyte necroptosis. In detail, deleting OTULIN in the mouse epidermis causes skin lesions that develop in different parts of the skin, including the tail. Those lesions resemble papilloma-like structures and are displayed with an inflammatory gene expression profile. Our genetic studies demonstrate that this skin pathology is driven by TNFR1 mediated RIPK1 kinase-dependent cell death. Furthermore, RIPK3 and MLKL deficiency strongly protect mice lacking OTULIN in keratinocytes from skin inflammation, thereby identifying necroptosis as a key driver, whereas FADD-dependent apoptosis plays a minor role in the skin disease development. Additionally, MyD88 deficiency strongly delayed and ameliorated skin pathology, implying that microbiota-dependent stimuli contribute to the pathogenesis in OTULINE-KO mice. Taken together, the data presented in this study uncover a key role for OTULIN in the epidermis in preventing cell death and inflammation to maintain skin homeostasis

FADD and Caspase-8 Regulate Gut Homeostasis and Inflammation by Controlling MLKL- and GSDMD-Mediated Death of Intestinal Epithelial Cells

Pathways controlling intestinal epithelial cell (IEC) death regulate gut immune homeostasis and contribute to the pathogenesis of inflammatory bowel diseases. Here we show that caspase-8 and its adapter FADD act in IECs to regulate intestinal inflammation downstream of Z-DNA binding protein 1 (ZBP1)- and tumor necrosis factor receptor-1 (TNFR1)-mediated receptor interacting protein kinase 1 (RIPK1) and RIPK3 signaling. Mice with IEC-specific FADD or caspase-8 deficiency developed colitis dependent on mixed lineage kinase-like (MLKL)-mediated epithelial cell necroptosis. However, MLKL deficiency fully prevented ileitis caused by epithelial caspase-8 ablation, but only partially ameliorated ileitis in mice lacking FADD in IECs. Our genetic studies revealed that caspase-8 and gasdermin-D (GSDMD) were both required for the development of MLKL-independent ileitis in mice with epithelial FADD deficiency. Therefore, FADD prevents intestinal inflammation downstream of ZBP1 and TNFR1 by inhibiting both MLKL-induced necroptosis and caspase-8-GSDMD-dependent pyroptosis-like death of epithelial cells

Hepatocyte IKK2 Protects Mdr2−/− Mice from Chronic Liver Failure

Mice lacking the Abc4 protein encoded by the multidrug resistance-2 gene (Mdr2−/−) develop chronic periductular inflammation and cholestatic liver disease resulting in the development of hepatocellular carcinoma (HCC). Inhibition of NF-κB by expression of an IκBα super-repressor (IκBαSR) transgene in hepatocytes was shown to prevent HCC development in Mdr2−/− mice, suggesting that NF-κB acts as a tumour promoter in this model of inflammation-associated carcinogenesis. On the other hand, inhibition of NF-κB by hepatocyte specific ablation of IKK2 resulted in increased liver tumour development induced by the chemical carcinogen DEN. To address the role of IKK2-mediated NF-κB activation in hepatocytes in the pathogenesis of liver disease and HCC in Mdr2−/− mice, we generated Mdr2-deficient animals lacking IKK2 specifically in hepatocytes using the Cre-loxP system. Mdr2−/− mice lacking IKK2 in hepatocytes developed spontaneously a severe liver disease characterized by cholestasis, major hyperbilirubinemia and severe to end-stage fibrosis, which caused muscle wasting, loss of body weight, lethargy and early spontaneous death. Cell culture experiments showed that primary hepatocytes lacking IKK2 were more sensitive to bile acid induced death, suggesting that hepatocyte-specific IKK2 deficiency sensitized hepatocytes to the toxicity of bile acids under conditions of cholestasis resulting in greatly exacerbated liver damage. Mdr2−/−IKK2Hep-KO mice remarkably recapitulate chronic liver failure in humans and might be of special importance for the study of the mechanisms contributing to the pathogenesis of end-stage chronic liver disease or its implications on other organs. Conclusion: IKK2-mediated signaling in hepatocytes protects the liver from damage under conditions of chronic inflammatory cholestasis and prevents the development of severe fibrosis and liver failure

Autophosphorylation at serine 166 regulates RIP kinase 1-mediated cell death and inflammation

Receptor interacting protein kinase 1 (RIPK1) regulates cell death and inflammatory responses downstream of TNFR1 and other receptors, and has been implicated in the pathogenesis of inflammatory and degenerative diseases. RIPK1 kinase activity induces apoptosis and necroptosis, however the mechanisms and phosphorylation events regulating RIPK1-dependent cell death signaling remain poorly understood. Here we show that RIPK1 autophosphorylation at serine 166 plays a critical role for the activation of RIPK1 kinase-dependent apoptosis and necroptosis. Moreover, we show that S166 phosphorylation is required for RIPK1 kinase-dependent pathogenesis of inflammatory pathologies in vivo in four relevant mouse models. Mechanistically, we provide evidence that trans autophosphorylation at S166 modulates RIPK1 kinase activation but is not by itself sufficient to induce cell death. These results show that S166 autophosphorylation licenses RIPK1 kinase activity to induce downstream cell death signaling and inflammation, suggesting that S166 phosphorylation can serve as a reliable biomarker for RIPK1 kinase-dependent pathologies

Generalized immune activation as a direct result of activated CD4+ T cell killing

Background: In addition to progressive CD4+ T cell immune deficiency, HIV infection is characterized by generalized immune activation, thought to arise from increased microbial exposure resulting from diminishing immunity. Results: Here we report that, in a virus-free mouse model, conditional ablation of activated CD4+ T cells, the targets of immunodeficiency viruses, accelerates their turnover and produces CD4+ T cell immune deficiency. More importantly, activated CD4+ T cell killing also results in generalized immune activation, which is attributable to regulatory CD4+ T cell insufficiency and preventable by regulatory CD4+ T cell reconstitution. Immune activation in this model develops independently of microbial exposure. Furthermore, microbial translocation in mice with conditional disruption of intestinal epithelial integrity affects myeloid but not T cell homeostasis. Conclusions: Although neither ablation of activated CD4+ T cells nor disruption of intestinal epithelial integrity in mice fully reproduces every aspect of HIV-associated immune dysfunction in humans, ablation of activated CD4+ T cells, but not disruption of intestinal epithelial integrity, approximates the two key immune alterations in HIV infection: CD4+ T cell immune deficiency and generalized immune activation. We therefore propose activated CD4+ T cell killing as a common etiology for both immune deficiency and activation in HIV infection

Tumor Necrosis Factor (TNF) Receptor Shedding Controls Thresholds of Innate Immune Activation That Balance Opposing TNF Functions in Infectious and Inflammatory Diseases

Tumor necrosis factor (TNF) is a potent cytokine exerting critical functions in the activation and regulation of immune and inflammatory responses. Due to its pleiotropic activities, the amplitude and duration of TNF function must be tightly regulated. One of the mechanisms that may have evolved to modulate TNF function is the proteolytic cleavage of its cell surface receptors. In humans, mutations affecting shedding of the p55TNF receptor (R) have been linked with the development of the TNFR-associated periodic syndromes, disorders characterized by recurrent fever attacks and localized inflammation. Here we show that knock-in mice expressing a mutated nonsheddable p55TNFR develop Toll-like receptor–dependent innate immune hyperreactivity, which renders their immune system more efficient at controlling intracellular bacterial infections. Notably, gain of function for antibacterial host defenses ensues at the cost of disbalanced inflammatory reactions that lead to pathology. Mutant mice exhibit spontaneous hepatitis, enhanced susceptibility to endotoxic shock, exacerbated TNF-dependent arthritis, and experimental autoimmune encephalomyelitis. These results introduce a new concept for receptor shedding as a mechanism setting up thresholds of cytokine function to balance resistance and susceptibility to disease. Assessment of p55TNFR shedding may thus be of prognostic value in infectious, inflammatory, and autoimmune diseases