Caspase-6 Is a Key Regulator of Innate Immunity, Inflammasome Activation, and Host Defense.

Caspases regulate cell death, immune responses, and homeostasis. Caspase-6 is categorized as an executioner caspase but shows key differences from the other executioners. Overall, little is known about the functions of caspase-6 in biological processes apart from apoptosis. Here, we show that caspase-6 mediates innate immunity and inflammasome activation. Furthermore, we demonstrate that caspase-6 promotes the activation of programmed cell death pathways including pyroptosis, apoptosis, and necroptosis (PANoptosis) and plays an essential role in host defense against influenza A virus (IAV) infection. In addition, caspase-6 promoted the differentiation of alternatively activated macrophages (AAMs). Caspase-6 facilitated the RIP homotypic interaction motif (RHIM)-dependent binding of RIPK3 to ZBP1 via its interaction with RIPK3. Altogether, our findings reveal a vital role for caspase-6 in facilitating ZBP1-mediated inflammasome activation, cell death, and host defense during IAV infection, opening additional avenues for treatment of infectious and autoinflammatory diseases and cancer.

A novel caspase-6 from sea cucumber Holothuria leucospilota: Molecular characterization, expression analysis and apoptosis detection.

In this study, a novel caspase-6 named HLcaspase-6 was identified from sea cucumber Holothuria leucospilota. The full-length cDNA of HLcaspase-6 is 2195 bp in size, containing a 126 bp 5'-untranslated region (UTR), a 1043 bp 3'-UTR and a 1026 bp open reading frame (ORF) encoding a protein of 341 amino acids with a deduced molecular weight of 38.57 kDa. HLcaspase-6 contains the common signatures of the caspase family, including the conserved pentapeptide motif QACRG, as well as the P20 and P10 domains. In addition, HLcaspase-6 contains a short pro-domain. HLcaspase-6 mRNA is ubiquitously expressed in all tissues examined, with the highest transcript level in the intestine, followed by coelomocytes. In in vitro experiments, the expression of HLcaspase-6 mRNA in coelomocytes was significantly up-regulated by lipopolysaccharides (LPS) or polyriboinosinic-polyribocytidylic acid [poly (I:C)] challenge, suggesting that HLcaspase-6 might play important roles in the innate immune defense of sea cucumber against bacterial and viral infections. Moreover, we further confirmed that overexpression of HLcaspase-6 could induce apoptosis and activate the p53 signal pathway.

Caspase 3, 6, 8, and 9 Gene Expression in Peripheral Blood Leukocytes and Plasma Concentrations of IL-6 and TNF-α in Carriers of Different Polymorphic Marker -174G>C Genotypes of IL6 Gene Associated with the Risk of Nonalcoholic Steatohepatitis.

We revealed an association of IL6 gene -174G>C polymorphism with the development of nonalcoholic steatohepatitis in the Russian population. The risk is significantly higher in carriers of C allele: OR=1.77 (1.04; 3.02). The effects of -174G>C substitution in IL6 gene involving caspase 9 gene transcripts in peripheral blood leukocytes and on blood content of TNF-α in healthy individuals without clinical manifestations of nonalcoholic steatohepatitis were detected. The content of caspase 9 gene transcripts in peripheral blood leukocytes and plasma level of TNF-α were significantly higher in healthy subjects carrying C allele than in carriers of GG genotype. The levels of caspases 3, 6, 8, and 9 gene transcripts in peripheral blood leukocytes and plasma concentrations of TNF-α in patients with nonalcoholic steatohepatitis did not depend on IL6 genotype by -174G

Identification of Caspase-6 as a New Regulator of Alternatively Activated Macrophages.

Alternatively activated macrophages (AAMs) play essential roles in the promotion of tissue remodeling, vasculogenesis, and tumor progression; however, the detailed mechanisms underlying the activation of AAMs remain largely unknown. Here, by using quantitative proteomic analysis, we identified 62 proteins that were up-regulated in IL-4-induced macrophages. Among these, Caspase-6 was increased significantly. Caspase-6 is important in the apoptotic signaling pathway; however, its role in non-apoptosis is also reported. Here, we first examined the non-apoptotic role of Caspase-6 in the alternative activation of macrophages after administration of IL-4, 4T1 tumor conditional medium, or co-culture with 4T1 cells. Both treatments promoted alternative activation of RAW264.7 cells and primary macrophages, whereas disruption of caspase-6 expression and activity could markedly suppress the biomarker levels of AAMs. Overexpression of Caspase-6 could significantly promote the activation of AAMs. Importantly, we further present evidence that caspase-6 could regulate breast cancer cell invasion by modulating MMP-2 and MMP-9 expression in 4T1 tumor-associated macrophages, as ablation of protein levels or activity of caspase-6 suppressed tumor cell invasion in vitro In conclusion, the observed results markedly expanded our views of the dynamic changes in protein composition during alternative activation of macrophages, and they revealed a critical new role of caspase-6 in regulating this cellular biological process, which suggested that caspase-6 might be a key nod molecule to regulate immunological steady-state and be a therapeutic candidate for tumor immunotherapy.

A pathogenic role for ER stress-induced autophagy and ER chaperone GRP78/BiP in T lymphocyte systemic lupus erythematosus.

Abnormal regulation of ER stress and apoptosis has been implicated in autoimmune disorders. Particularly, ER stress-induced autophagy and the role of GRP78, or BiP in T lymphocyte survival and death in SLE are poorly understood. This study investigated the pathogenic roles of ER stress-induced autophagy and GRP78/BiP in apoptosis of T lymphocytes. We compared spontaneous and induced autophagy and apoptosis of T lymphocytes in healthy donors and patients with SLE. The molecular mechanism of altered autophagy and apoptosis was investigated in T lymphocytes transfected with siRNA for beclin 1 and CHOP and T lymphocytes overexpressing GRP78. Decreased autophagy and increased apoptosis in response to TG-induced ER stress were observed in lupus T lymphocytes. GRP78 and ER stress-signaling molecules, such as PERK, p-eIF2α, IRE1, and ATF6 decreased, whereas CHOP levels increased in lupus T cells in response to TG. The levels antiapoptotic molecules, Bcl-2 and Bcl-XL decreased, whereas the proapoptotic molecules, Bax and caspase 6, increased in lupus T cells. The TG-induced ER stress altered autophagy and apoptosis, which in turn, led to abnormal T cell homeostasis with increased apoptotic T cell death. We hypothesize that aberrant autophagy of T lymphocytes as a result of ER stress and decreased GRP78 expression is involved in the pathogenesis of SLE and might serve as important therapeutic targets.

Involvement of glycoreceptors in galactoxylomannan-induced T cell death.

The major virulence factor of Cryptococcus neoformans is its capsular polysaccharide, which is also released into tissues. The shed polysaccharide is composed of glucuronoxylomannan, galactoxylomannan (GalXM), and mannoproteins. In a previous study, we demonstrated a direct interaction of purified soluble GalXM with T cells that induced their apoptosis. In this study, we focus on the mechanisms involved in the apoptotic effect of GalXM. In our experimental system, we analyzed the effect of GalXM on purified human T cells and Jurkat cells, a T cell line routinely used for apoptotic studies. Our results reveal that GalXM activates the extrinsic and intrinsic apoptotic pathways through the cleavage and recruitment of caspase-8. Caspase-8 elicits the downstream executioner caspase-3, caspase-6, and caspase-7 both directly and indirectly, via Bid cleavage and caspase-9 activation. These effects appeared to be primarily mediated by the interaction of GalXM with the glycoreceptors, which differed in human T and Jurkat cells. CD45 was primarily involved in Jurkat cells apoptosis while CD7 and CD43 mediated human T cell apoptosis. Our results highlight a new mechanism by which a microbial product can contribute to virulence through direct interaction with T cell glycoreceptors, thereby triggering lymphocyte apoptosis.

c-Maf increases apoptosis in peripheral CD8 cells by transactivating Caspase 6.

In addition to transactivation of interleukin-4 (IL-4), cellular muscular aponeurotic fibrosarcoma (c-Maf) enhances CD4 cell apoptosis by limiting Bcl-2 expression. The CD8 cells also express c-Maf and peripheral CD8 cell numbers are reduced in c-Maf transgenic mice, suggesting that c-Maf may influence CD8 cell survival in a manner similar to CD4 cells. Here we confirm that, similar to CD4 cells, c-Maf enhances CD8 cell susceptibility to apoptosis induced by multiple stimuli, independent of IL-4. However, unlike CD4 cells, c-Maf enhancement of apoptosis is independent of Bcl-2, suggesting that c-Maf uses other mechanisms to regulate CD8 cell apoptosis. Real-time reverse transcription-polymerase chain reaction reveals that the pro-apoptotic gene Caspase 6 is upregulated in c-Maf transgenic CD8 cells, suggesting that Caspase 6 is a novel c-Maf target gene. Luciferase reporter assays and site-directed mutagenesis reveal a functional c-Maf recognition element (MARE) within the first intron of Caspase 6. Binding of c-Maf to the MARE site is detectable by chromatin immunoprecipitation using non-transgenic T-cell lysates, so c-Maf can interact with the Caspase 6 MARE site in normal T cells. Furthermore, caspase 6 activity is increased among CD8 cells from c-Maf transgenic mice following T-cell receptor engagement. As expected, activity of the downstream caspases 3 and 7 is also increased. Consistent with the ability of caspase 6 to participate in positive feedback loops, cytochrome c release and caspase 8 activation are also increased. Together these results indicated that c-Maf increases CD8 cell sensitivity to apoptotic stimuli, at least in part, by direct transactivation of Caspase 6, providing increased substrate for Caspase 6-dependent apoptosis pathways.

Caspase 6 regulates B cell activation and differentiation into plasma cells.

Caspase (Casp) family proteases regulate not only lymphocyte apoptosis but also lymphocyte activation and development. In this study, we show that Casp6 regulates B cell activation and differentiation into plasma cells by modifying cell cycle entry. B cells from Casp6 knockout (Casp6 KO) mice examined ex vivo have more cells in G(1) than wild-type B cells, and mitogen-induced G(1) entry of Casp6 KO B cells is much faster than that of wild-type B cells. Even so, S phase entry and proliferation are not increased in Casp6 KO B cells. Rather than proliferating, activated Casp6 KO B cells preferentially differentiate into syndecan-1(+) plasma cells and produce Abs. In Casp6 KO mice compared with WT mice, serum levels of IgG1, IgG2a, and IgG2b are increased and Ag-specific Ab responses are also enhanced along with increased percentages of syndecan-1(+) plasma cells. Casp6 may regulate both B cell activation and differentiation by modifying requirements for G(0) B cells to enter G(1).