Receptor Interacting Protein Kinase Pathways Regulate Innate B Cell Developmental Checkpoints But Not Effector Function in Mice.

Mutations in the scaffolding domain of Receptor Interacting Protein kinases (RIP) underlie the recently described human autoimmune syndrome, CRIA, characterized by lymphadenopathy, splenomegaly, and autoantibody production. While disease mechanisms for CRIA remain undescribed, RIP kinases work together with caspase-8 to regulate cell death, which is critical for normal differentiation of many cell types. Here, we describe a key role for RIP1 in facilitating innate B cell differentiation and subsequent activation. By comparing RIP1, RIP3, and caspase-8 triple deficient and RIP3, caspase-8 double deficient mice, we identified selective contributions of RIP1 to an accumulation of murine splenic Marginal Zone (MZ) B cells and B1-b cells. We used mixed bone-marrow chimeras to determine that innate B cell commitment required B cell-intrinsic RIP1, RIP3, and caspase-8 sufficiency. RIP1 regulated MZ B cell development rather than differentiation and RIP1 mediates its innate immune effects independent of the RIP1 kinase domain. NP-KLH/alum and NP-Ficoll vaccination of mice doubly deficient in both caspase-8 and RIP3 or deficient in all three proteins (RIP3, caspase-8, and RIP1) revealed uniquely delayed T-dependent and T-independent IgG responses, abnormal splenic germinal center architecture, and reduced extrafollicular plasmablast formation compared to WT mice. Thus, RIP kinases and caspase-8 jointly orchestrate B cell fate and delayed effector function through a B cell-intrinsic mechanism.

Cutting Edge: Caspase-8 Is a Linchpin in Caspase-3 and Gasdermin D Activation to Control Cell Death, Cytokine Release, and Host Defense during Influenza A Virus Infection.

Programmed cell death (PCD) is essential for the innate immune response, which serves as the first line of defense against pathogens. Caspases regulate PCD, immune responses, and homeostasis. Caspase-8 specifically plays multifaceted roles in PCD pathways including pyroptosis, apoptosis, and necroptosis. However, because caspase-8-deficient mice are embryonically lethal, little is known about how caspase-8 coordinates different PCD pathways under physiological conditions. Here, we report an anti-inflammatory role of caspase-8 during influenza A virus infection. We generated viable mice carrying an uncleavable version of caspase-8 (Casp8 DA/DA). We demonstrated that caspase-8 autoprocessing was responsible for activating caspase-3, thereby suppressing gasdermin D-mediated pyroptosis and inflammatory cytokine release. We also found that apoptotic and pyroptotic pathways were activated at the same time during influenza A virus infection, which enabled the cell-intrinsic anti-inflammatory function of the caspase-8-caspase-3 axis. Our findings provide new insight into the immunological consequences of caspase-8-coordinated PCD cross-talk under physiological conditions.

Environmental allergens trigger type 2 inflammation through ripoptosome activation.

Environmental allergens, including fungi, insects and mites, trigger type 2 immunity; however, the innate sensing mechanisms and initial signaling events remain unclear. Herein, we demonstrate that allergens trigger RIPK1-caspase 8 ripoptosome activation in epithelial cells. The active caspase 8 subsequently engages caspases 3 and 7, which directly mediate intracellular maturation and release of IL-33, a pro-atopy, innate immunity, alarmin cytokine. Mature IL-33 maintained functional interaction with the cognate ST2 receptor and elicited potent pro-atopy inflammatory activity in vitro and in vivo. Inhibiting caspase 8 pharmacologically and deleting murine Il33 and Casp8 each attenuated allergic inflammation in vivo. Clinical data substantiated ripoptosome activation and IL-33 maturation as likely contributors to human allergic inflammation. Our findings reveal an epithelial barrier, allergen-sensing mechanism that converges on the ripoptosome as an intracellular molecular signaling platform, triggering type 2 innate immune responses. These findings have significant implications for understanding and treating human allergic diseases.

Multiple roles of caspase-8 in cell death, inflammation, and innate immunity.

Caspase-8 is an apical caspase involved in the programmed form of cell death called apoptosis that is critically important for mammalian development and immunity. Apoptosis was historically described as immunologically silent in contrast to other types of programmed cell death such as necroptosis or pyroptosis. Recent reports suggest considerable crosstalk between these different forms of cell death. It is becoming increasingly clear that caspase-8 has many non-apoptotic roles, participating in multiple processes including regulation of necroptosis (mediated by receptor-interacting serine/threonine kinases, RIPK1-RIPK3), inflammatory cytokine expression, inflammasome activation, and cleavage of IL-1ß and gasdermin D, and protection against shock and microbial infection. In this review, we discuss the involvement of caspase-8 in cell death and inflammation and highlight its role in innate immune responses and in the relationship between different forms of cell death. Caspase-8 is one of the central components in this type of crosstalk.

CD8dimCD3+ lymphocytes in fever patients might be biomarkers of active EBV infection and exclusion indicator of T-LGLL.

Background: Massive monoclonal or oligoclonal expansion of CD8+ T cells is a notable feature of primary infections of the Epstein-Barr virus (EBV). However, the clinical significance of this expansion is not clear. Results: An increase in the CD8dimCD3+ lymphocyte subset in patients with active EBV infection was due to caspase-8-dependent apoptosis was found using flow cytometry in this study. The number of these cells was associated with the illness severity. Pan-T-cell antigen and receptor analyses were also compared in patients with active EBV infections and T-cell large granular lymphocytic leukemia to provide additional diagnostic information. Conclusion: The increase in CD8dimCD3+ cells could be a biomarker of active EBV infection and an exclusion indicator of T-cell large granular lymphocytic leukemia with flow cytometric analysis.

SARS-CoV-2 triggers inflammatory responses and cell death through caspase-8 activation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can lead to respiratory illness and multi-organ failure in critically ill patients. Although the virus-induced lung damage and inflammatory cytokine storm are believed to be directly associated with coronavirus disease 2019 (COVID-19) clinical manifestations, the underlying mechanisms of virus-triggered inflammatory responses are currently unknown. Here we report that SARS-CoV-2 infection activates caspase-8 to trigger cell apoptosis and inflammatory cytokine processing in the lung epithelial cells. The processed inflammatory cytokines are released through the virus-induced necroptosis pathway. Virus-induced apoptosis, necroptosis, and inflammation activation were also observed in the lung sections of SARS-CoV-2-infected HFH4-hACE2 transgenic mouse model, a valid model for studying SARS-CoV-2 pathogenesis. Furthermore, analysis of the postmortem lung sections of fatal COVID-19 patients revealed not only apoptosis and necroptosis but also massive inflammatory cell infiltration, necrotic cell debris, and pulmonary interstitial fibrosis, typical of immune pathogenesis in the lung. The SARS-CoV-2 infection triggered a dual mode of cell death pathways and caspase-8-dependent inflammatory responses may lead to the lung damage in the COVID-19 patients. These discoveries might assist the development of therapeutic strategies to treat COVID-19.

NLRC4 biology in immunity and inflammation.

Inflammasomes are cytosolic multiprotein complexes that sense microbial infections or host cell damage, triggering cytokine production and a proinflammatory form of cell death, called pyroptosis. Whereas pyroptosis and cytokine production may often promote host resistance to infections, uncontrolled inflammasome activation leads to autoinflammatory diseases in humans. Among the multiple inflammasomes described, the neuronal apoptosis inhibitory protein/nucleotide-binding domain leucine-rich repeat-containing protein family caspase activation and recruitment domain-containing protein 4 (NLRC4) inflammasome emerged as a critical component for the restriction of bacterial infections. Accordingly, our understanding of this inflammasome advanced remarkably over the last 10 yr, expanding our knowledge about ligand-receptor interaction; cryo-EM structure; and downstream effectors and substrates, such as gasdermin-D, caspase-1, caspase-8, and caspase-7. In this review, we discuss recent advances on the biology of the NLRC4 inflammasome, in terms of structure and activation mechanisms, importance in bacterial and nonbacterial diseases, and the identification of NLRC4 gain-of-function mutations leading to NLRC4-associated autoinflammatory diseases in humans.

Ptpn6 inhibits caspase-8- and Ripk3/Mlkl-dependent inflammation.

Ptpn6 is a cytoplasmic phosphatase that functions to prevent autoimmune and interleukin-1 (IL-1) receptor-dependent, caspase-1-independent inflammatory disease. Conditional deletion of Ptpn6 in neutrophils (Ptpn6∆PMN) is sufficient to initiate IL-1 receptor-dependent cutaneous inflammatory disease, but the source of IL-1 and the mechanisms behind IL-1 release remain unclear. Here, we investigate the mechanisms controlling IL-1α/ß release from neutrophils by inhibiting caspase-8-dependent apoptosis and Ripk1-Ripk3-Mlkl-regulated necroptosis. Loss of Ripk1 accelerated disease onset, whereas combined deletion of caspase-8 and either Ripk3 or Mlkl strongly protected Ptpn6∆PMN mice. Ptpn6∆PMN neutrophils displayed increased p38 mitogen-activated protein kinase-dependent Ripk1-independent IL-1 and tumor necrosis factor production, and were prone to cell death. Together, these data emphasize dual functions for Ptpn6 in the negative regulation of p38 mitogen-activated protein kinase activation to control tumor necrosis factor and IL-1α/ß expression, and in maintaining Ripk1 function to prevent caspase-8- and Ripk3-Mlkl-dependent cell death and concomitant IL-1α/ß release.

Innate immune priming in the absence of TAK1 drives RIPK1 kinase activity-independent pyroptosis, apoptosis, necroptosis, and inflammatory disease.

RIPK1 kinase activity has been shown to be essential to driving pyroptosis, apoptosis, and necroptosis. However, here we show a kinase activity-independent role for RIPK1 in these processes using a model of TLR priming in a TAK1-deficient setting to mimic pathogen-induced priming and inhibition. TLR priming of TAK1-deficient macrophages triggered inflammasome activation, including the activation of caspase-8 and gasdermin D, and the recruitment of NLRP3 and ASC into a novel RIPK1 kinase activity-independent cell death complex to drive pyroptosis and apoptosis. Furthermore, we found fully functional RIPK1 kinase activity-independent necroptosis driven by the RIPK3-MLKL pathway in TAK1-deficient macrophages. In vivo, TAK1 inactivation resulted in RIPK3-caspase-8 signaling axis-driven myeloid proliferation and a severe sepsis-like syndrome. Overall, our study highlights a previously unknown mechanism for RIPK1 kinase activity-independent inflammasome activation and pyroptosis, apoptosis, and necroptosis (PANoptosis) that could be targeted for treatment of TAK1-associated myeloid proliferation and sepsis.

Caspase-8 restricts antiviral CD8 T cell hyperaccumulation.

The magnitude of CD8 T cell responses against viruses is checked by the balance of proliferation and death. Caspase-8 (CASP8) has the potential to influence response characteristics through initiation of apoptosis, suppression of necroptosis, and modulation of cell death-independent signal transduction. Mice deficient in CASP8 and RIPK3 (Casp8-/-Ripk3-/- ) mount enhanced peak CD8 T cell levels against the natural mouse pathogen murine cytomegalovirus (MCMV) or the human pathogen herpes simplex virus-1 compared with littermate control RIPK3-deficient or WT C57BL/6 mice, suggesting an impact of CASP8 on the magnitude of antiviral CD8 T cell expansion and not on contraction. The higher peak response to MCMV in Casp8-/-Ripk3-/- mice resulted from accumulation of greater numbers of terminally differentiated KLRG1hi effector CD8 T cell subsets. Antiviral Casp8-/-Ripk3-/- T cells exhibited enhanced proliferation when splenocytes were transferred into WT recipient mice. Thus, cell-autonomous CASP8 normally restricts CD8 T cell proliferation following T cell receptor activation in response to foreign antigen. Memory inflation is a hallmark quality of the T cell response to cytomegalovirus infection. Surprisingly, MCMV-specific memory inflation was not sustained long-term in Casp8-/-Ripk3-/- mice even though these mice retained immunity to secondary challenge. In addition, the accumulation of abnormal B220+CD3+ T cells in these viable CASP8-deficient mice was reduced by chronic MCMV infection. Combined, these data brings to light the cell death-independent role of CASP8 during CD8 T cell expansion in mice lacking the confounding impact of RIPK3-mediated necroptosis.

Gasdermin-D and Caspase-7 are the key Caspase-1/8 substrates downstream of the NAIP5/NLRC4 inflammasome required for restriction of Legionella pneumophila.

Inflammasomes are cytosolic multi-protein complexes that detect infection or cellular damage and activate the Caspase-1 (CASP1) protease. The NAIP5/NLRC4 inflammasome detects bacterial flagellin and is essential for resistance to the flagellated intracellular bacterium Legionella pneumophila. The effectors required downstream of NAIP5/NLRC4 to restrict bacterial replication remain unclear. Upon NAIP5/NLRC4 activation, CASP1 cleaves and activates the pore-forming protein Gasdermin-D (GSDMD) and the effector caspase-7 (CASP7). However, Casp1-/- (and Casp1/11-/-) mice are only partially susceptible to L. pneumophila and do not phenocopy Nlrc4-/-mice, because NAIP5/NLRC4 also activates CASP8 for restriction of L. pneumophila infection. Here we show that CASP8 promotes the activation of CASP7 and that Casp7/1/11-/- and Casp8/1/11-/- mice recapitulate the full susceptibility of Nlrc4-/- mice. Gsdmd-/- mice exhibit only mild susceptibility to L. pneumophila, but Gsdmd-/-Casp7-/- mice are as susceptible as the Nlrc4-/- mice. These results demonstrate that GSDMD and CASP7 are the key substrates downstream of NAIP5/NLRC4/CASP1/8 required for resistance to L. pneumophila.

Dysregulation of Inflammasome Priming and Activation by MicroRNAs in Human Immune-Mediated Diseases.

Inflammasomes are protein complexes that respond to a wide range of pathogens and cellular damage signals. Their activation prompts the caspase-1-mediated cleavage of the proinflammatory cytokines IL-1ß and IL-18. Inflammasome dysregulation has been demonstrated to play a role in a range of diseases involving the adaptive immune system like multiple sclerosis, rheumatic diseases, and type 1 diabetes. Priming and activation of inflammasomes can be modulated by microRNAs (miRNAs), small noncoding RNAs that regulate gene expression posttranscriptionally. miRNAs, such as miR-223-3p, have been demonstrated to directly target the inflammasome components NLRP3, caspase-1, and caspase-8. Other miRNAs like miR-155-5p modulate TLR-, IL-1R-, TNFR-, and IFNAR-mediated signaling pathways upstream of the inflammasomes. In this study, we discuss how a more detailed elucidation of miRNA-driven inflammasome regulation helps in understanding the molecular processes underlying immune-mediated human diseases, holds potential for the identification of biomarkers and may offer novel targets for the development of future therapeutics.

Molecular characterization of caspase-8-like and its expression induced by microcystin-LR in grass carp (Ctenopharygodon idella).

Caspase-8, an initiator caspase, plays a vital role in apoptosis. In this study, caspase-8-like (named as Cicaspase-8-like), a homologue of caspase-8, was identified in grass carp (Ctenopharygodon idella). The full-length cDNA sequence of CiCaspase-8-like was 1409 bp and contained a 162 bp 5'-UTR, a 239 bp 3'-UTR and a 1008 bp coding sequence. The putative amino acids sequence was 335 residues long, including a large subunit (P20) and a small subunit (P10), but lacking conserved death effector domains. A histidine active site DHSQMDAFVCCVLSHG and a cysteine active-site motif KPKLFFIQACQG were found in P20. Phylogenetic analysis showed that Cicaspase-8-like clustered with the caspase-8 and caspase-8-like of other fish and grouped closely with Carassius auratus caspase-8-like. Quantitative real-time PCR revealed that the Cicaspase-8-like mRNA were expressed constitutively in all tested tissues from healthy grass carp, with high expression level in the blood, spleen, liver and gill, indicating its role in immune reaction. The expression of Cicaspase-8-like mRNA was decreased significantly in the liver because of the stress caused by microcystin-LR (MC-LR) (75 and 100 µg MC-LR/kg BW) at 24 h and 96 h post injection (P < 0.05), but it was increased significantly in grass carp treated with 25 µg MC-LR/kg BW at 24 h (P < 0.05) post injection. Cleaved fragments of Cicaspase-8-like were observed using western blot analysis, and the expression of Cicaspase-8-like protein was increased after MC-LR treatments. Moreover, the expression of both caspase-9 and caspase-3 mRNA increased significantly after treatment with the three doses of MC-LR. TUNEL assay results showed remarkable changes in apoptosis after the MC-LR treatment. These results suggest that Cicaspase-8-like is an important caspase and plays an essential role in MC-LR-induced apoptosis.

Betaine Inhibits Interleukin-1ß Production and Release: Potential Mechanisms.

Betaine is a critical nutrient for mammal health, and has been found to alleviate inflammation by lowering interleukin (IL)-1ß secretion; however, the underlying mechanisms by which betaine inhibits IL-1ß secretion remain to be uncovered. In this review, we summarize the current understanding about the mechanisms of betaine in IL-1ß production and release. For IL-1ß production, betaine affects canonical and non-canonical inflammasome-mediated processing of IL-1ß through signaling pathways, such as NF-κB, NLRP3 and caspase-8/11. For IL-1ß release, betaine inhibits IL-1ß release through blocking the exocytosis of IL-1ß-containing secretory lysosomes, reducing the shedding of IL-1ß-containing plasma membrane microvesicles, suppressing the exocytosis of IL-1ß-containing exosomes, and attenuating the passive efflux of IL-1ß across hyperpermeable plasma membrane during pyroptotic cell death, which are associated with ERK1/2/PLA2 and caspase-8/A-SMase signaling pathways. Collectively, this review highlights the anti-inflammatory property of betaine by inhibiting the production and release of IL-1ß, and indicates the potential application of betaine supplementation as an adjuvant therapy in various inflammatory diseases associating with IL-1ß secretion.

p21-activated kinase 4 as a switch between caspase-8 apoptosis and NF-κB survival signals in response to TNF-α in hepatocarcinoma cells.

PAK4 is overexpressed in a variety of human cancers and considered a promising candidate for therapeutic target. However, its functions remain poorly understood, especially in liver carcinogenesis which could be triggered by inflammation. In the present study, endogenous PAK4 was knockdown using siRNA in HepG2 and SK-Hep1 cells. The two cell lines performed reduced cell viability, altered cell cycle composed of decreased S and arrest in G2, and apoptosis. Meanwhile, expression of NF-κB p65 in the nuclei and caspase-8 activity did not show significant differences from control. However, after treating cells with TNF-α, an inflammatory cytokine, we investigated repressed nuclear expression and localization of NF-κB p65, and induced apoptosis with increased caspase-8 activity in PAK4-knockdown cells. The findings revealed that ablation of PAK4 inhibited cell viability via blocking cell cycle and progressing apoptosis. The apoptosis was partially dependent upon caspase-8 concomitant with attenuated NF-κB survival signal due to stimulus of TNF-α. It suggests that PAK4 as target is a switch between caspase-8 apoptosis and NF-κB survival signals induced by TNF-α in hepatocarcinoma cells.

SIRT1 inhibits rheumatoid arthritis fibroblast-like synoviocyte aggressiveness and inflammatory response via suppressing NF-κB pathway.

Rheumatoid arthritis (RA) is an autoimmune disease of the joints characterized by synovial hyperplasia and chronic inflammation. Fibroblast-like synoviocytes (FLS) play a central role in RA initiation, progression, and perpetuation. Prior studies showed that sirtuin 1 (SIRT1), a deacetylase participating in a broad range of transcriptional and metabolic regulations, may impact cell proliferation and inflammatory responses. However, the role of SIRT1 in RA-FLS was unclear. Here, we explored the effects of SIRT1 on the aggressiveness and inflammatory responses of cultured RA-FLS. SIRT1 expression was significantly lower in synovial tissues and FLS from RA patients than from healthy controls. Overexpression of SIRT1 significantly inhibited RA-FLS proliferation, migration, and invasion. SIRT1 overexpression also significantly increased RA-FLS apoptosis and caspase-3 and -8 activity. Focusing on inflammatory phenotypes, we found SIRT1 significantly reduced RA-FLS secretion of TNF-α, IL-6, IL-8, and IL-1ß. Mechanistic studies further revealed SIRT1 suppressed NF-κB pathway by reducing p65 protein expression, phosphorylation, and acetylation in RA-FLS. Our results suggest SIRT1 is a key regulator in RA pathogenesis by suppressing aggressive phenotypes and inflammatory response of FLS. Enhancing SIRT1 expression or function in FLS could be therapeutic beneficial for RA by inhibiting synovial hyperplasia and inflammation.

Staphylococcus aureus triggers a shift from influenza virus-induced apoptosis to necrotic cell death.

Superinfections with Staphylococcus aureus are a major complication of influenza disease, causing excessive inflammation and tissue damage. This enhanced cell-damaging effect is also observed in superinfected tissue cultures, leading to a strong decrease in overall cell viability. In our analysis of the underlying molecular mechanisms, we observed that, despite enhanced cell damage in superinfection, S. aureus did not increase but rather inhibited influenza virus (IV)-induced apoptosis in cells on the level of procaspase-8 activation. This apparent contradiction was solved when we observed that S. aureus mediated a switch from apoptosis to necrotic cell death of IV-infected cells, a mechanism that was dependent on the bacterial accessory gene regulator ( agr) locus that promotes bacterial survival and spread. This so far unknown action may be a bacterial strategy to enhance dissemination of intracellular S. aureus and may thereby contribute to increased tissue damage and severity of disease.-Van Krüchten, A., Wilden, J. J., Niemann, S., Peters, G., Löffler, B., Ludwig, S., Ehrhardt, C. Staphylococcus aureus triggers a shift from influenza virus-induced apoptosis to necrotic cell death.

The Effects of Dendritic Cell Hypersensitivity on Persistent Viral Infection.

Caspase-8 (CASP8) is known as an executioner of apoptosis, but more recent studies have shown that it participates in the regulation of necroptosis and innate immunity. In this study, we show that CASP8 negatively regulates retinoic acid-inducible gene I (RIG-I) signaling such that, in its absence, stimulation of the RIG-I pathway in dendritic cells (DCs) produced modestly enhanced activation of IFN regulatory factor 3 with correspondingly greater amounts of proinflammatory cytokines. In addition, mice lacking DC-specific CASP8 (dcCasp8-/- mice) develop age-dependent symptoms of autoimmune disease characterized by hyperactive DCs and T cells, spleen and liver immunopathology, and the appearance of Th1-polarized CD4+ T cells. Such mice infected with chronic lymphocytic choriomeningitis virus, an RNA virus detected by RIG-I, mounted an enhanced lymphocytic choriomeningitis virus-specific immune response as measured by increased proportions of Ag-specific CD4+ T cells and multicytokine-producing CD4+ and CD8+ T cells. These results show that CASP8 subtly modulates DC maturation, which controls the spontaneous appearance of autoimmune T cells while simultaneously attenuating the acquired immune system and its potential to control a persistent viral infection.

Caspase-1 Is an Apical Caspase Leading to Caspase-3 Cleavage in the AIM2 Inflammasome Response, Independent of Caspase-8.

Canonical inflammasomes are multiprotein complexes that can activate both caspase-1 and caspase-8. Caspase-1 drives rapid lysis of cells by pyroptosis and maturation of interleukin (IL)-1ß and IL-18. In caspase-1-deficient cells, inflammasome formation still leads to caspase-3 activation and slower apoptotic death, dependent on caspase-8 as an apical caspase. A role for caspase-8 directly upstream of caspase-1 has also been suggested, but here we show that caspase-8-deficient macrophages have no defect in AIM2 inflammasome-mediated caspase-1 activation, pyroptosis, and IL-1ß cleavage. In investigating the inflammasome-induced apoptotic pathway, we previously demonstrated that activated caspase-8 is essential for caspase-3 cleavage and apoptosis in caspase-1-deficient cells. However, here we found that AIM2 inflammasome-initiated caspase-3 cleavage was maintained in Ripk3-/-Casp8-/- macrophages. Gene knockdown showed that caspase-1 was required for the caspase-3 cleavage. Thus inflammasomes activate a network of caspases that can promote both pyroptotic and apoptotic cell death. In cells where rapid pyroptosis is blocked, delayed inflammasome-dependent cell death could still occur due to both caspase-1- and caspase-8-dependent apoptosis. Initiation of redundant cell death pathways is likely to be a strategy for coping with pathogen interference in death processes.