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Interferon-induced with helicase C domain 1 (IFIH1) is a cytosolic sensor of dsRNA that induces an anti-viral Type I interferon (IFN) state. A gain-of-function mutation in IFIH1 can cause increased Type I IFN activity and is clinically associated with Aicardi-Goutieres syndrome (AGS). AGS is a multisystem disease, characterized as an early-onset progressive encephalopathy with basal ganglia calcification and systemic lupus erythematosus-like features. Gastrointestinal manifestation is rare in AGS patients. We described a 10-year-old female patient with a heterozygous IFIH1 gene mutation who presented with gastrointestinal colitis, cystitis and very severe diarrhea as initial major manifestations of AGS. Proteinuria with high titer of antinuclear antibody and anti-double-stranded DNA was found in this patient. She also had growth retardation and a history of seizures (about two episodes each year) but without attacks until 7 years old. Serum cytokines detected by flow cytometry indicated extremely high level of interleukin 6 (1970.1 pg/ml) and IFN-α (204.1 pg/ml). A contrast-enhanced CT scan of the whole abdomen and an intestinal hydro-MRI indicated that the walls of her stomach, small bowel, colon, and bladder were in various degrees of edema and thickened states. Whole exome sequencing analysis indicated that she harbors an IFIH1 heterozygous mutation (c.2336G > A (p.R779H)) in both blood and intestinal samples. Abundant inflammatory cells infiltration into the intestinal epithelium was observed by immunohistochemical staining. Positive staining of caspase 4 and caspase 5 suggested that the signaling pathway of pyroptosis was involved in the mechanism of intestinal inflammation in AGS. Diarrhea was significantly improved after steroids and intravenous immunoglobulin treatments. Gastrointestinal colitis and cystitis can be rare manifestations of AGS with IFIH1 mutation. Caspase and its related inflammasome pathway may involve in the pathogenesis of AGS.

Activation of the NLRP3 inflammasome has been shown to play a major role in the neuroinflammation that accompanies Alzheimer's disease (AD); interventions that down regulate the NLRP3 inflammasome could thus be beneficial in AD. Parasite infections were recently shown to be associated with improved cognitive functions in Apolipoprotein E4 (ApoE4)-expressing members of an Amazonian tribe. We verified in an in vitro model whether Leishmania infantum infection could reduce NLRP3. Results obtained in an initial experimental model in which PBMC were LPS primed and nigericin-stimulated showed that L. infantum infection significantly reduced ASC-speck formation (i.e. intracellular inflammasome proteins assembly), as well as the production of activated caspase 5 and IL-1β, but increased that of activated caspase 1 and IL-18. Moreover, L. infantum infection induced the generation of an anti-inflammatory milieu by suppressing the production of TNFα and increasing that of IL-10. These results were replicated when cells that had been LPS-primed were stimulated with Aβ42 and infected with L. infantum. Results herein indicate that Leishmania infection favors an anti-inflammatory milieu, which includes the down-regulation of NLRP3 inflammasome activation, possibly to facilitate its survival inside host cells. A side effect of Leishmaniasis would be the hampering of neuroinflammation; this could play a protective role against AD development.

Pigs are especially useful large animal models, however, limited availability of commercially available antibodies for immunoblotting presents a significant obstacle facing preclinical xenotransplantation research. Major histocompatibility complex class I (MHC-I) molecule expression enhancement by nucleotide-binding oligomerization domain (NOD)-like receptor family with a caspase recruitment domain (CARD) containing caspase 5 (NLRC5) is fundamental to understanding porcine xenoantigen presentation. Swine Leukocyte Antigens (SLAs) are the porcine MHC homologs for human leukocyte antigens. SLA-I is a known xenoantigen that causes T cell activation. NLRC5, SLA-I, and B2M are all targets of immune modulation in genetically engineered pigs in xenotransplantation research with the goal to reduce SLA-I expression. In the present study, the human anti-NLRC5 (ab105411), anti-NLRC5 (ab117624), anti-NLRC5 N-terminal (ab178767), anti-HLA E (ab203082), anti-HLA E (ab135826), anti-HLA E (ab2216) and anti-β2 M (ab75853) antibodies were examined using immunoblots for porcine cross-reactivity. The anti-human antibodies ab117624, ab105411, ab178767, ab2216, and ab75853 cross reacted with cognate proteins in porcine cell lysates. Antibody reagents from this study will allow for validation of NLRC5, B2M, MHC-I expression in future research studies. In addition, following the methodology described in this study for other xenotransplantation targets may provide an alternative to custom antibody development.

Altered intestinal microbial composition promotes intestinal barrier dysfunction and triggers the initiation and recurrence of inflammatory bowel disease (IBD). Current treatments for IBD are focused on control of inflammation rather than on maintaining intestinal epithelial barrier function. Here, we show that the internalization of Gram-negative bacterial outer membrane vesicles (OMVs) in human intestinal epithelial cells promotes recruitment of caspase-5 and PIKfyve to early endosomal membranes via sorting nexin 10 (SNX10), resulting in LPS release from OMVs into the cytosol. Caspase-5 activated by cytosolic LPS leads to Lyn phosphorylation, which in turn promotes nuclear translocalization of Snail/Slug, downregulation of E-cadherin expression, and intestinal barrier dysfunction. SNX10 deletion or treatment with DC-SX029, a novel SNX10 inhibitor, rescues OMV-induced intestinal barrier dysfunction and ameliorates colitis in mice by blocking cytosolic LPS release, caspase-5 activation, and downstream signaling. Our results show that targeting SNX10 may be a new therapeutic approach for restoring intestinal epithelial barrier function and promising strategy for IBD treatment.

Altered intestinal microbial composition promotes intestinal barrier dysfunction and triggers the initiation and recurrence of inflammatory bowel disease (IBD). Current treatments for IBD are focused on control of inflammation rather than on maintaining intestinal epithelial barrier function. Here, we show that the internalization of Gram-negative bacterial outer membrane vesicles (OMVs) in human intestinal epithelial cells promotes recruitment of caspase-5 and PIKfyve to early endosomal membranes via sorting nexin 10 (SNX10), resulting in LPS release from OMVs into the cytosol. Caspase-5 activated by cytosolic LPS leads to Lyn phosphorylation, which in turn promotes nuclear translocalization of Snail/Slug, downregulation of E-cadherin expression, and intestinal barrier dysfunction. SNX10 deletion or treatment with DC-SX029, a novel SNX10 inhibitor, rescues OMV-induced intestinal barrier dysfunction and ameliorates colitis in mice by blocking cytosolic LPS release, caspase-5 activation, and downstream signaling. Our results show that targeting SNX10 may be a new therapeutic approach for restoring intestinal epithelial barrier function and promising strategy for IBD treatment.

Pattern Recognition Receptors (PRRs, [104]) (nomenclature as agreed by NC-IUPHAR sub-committee on Pattern Recognition Receptors, [18]) participate in the innate immune response to microbial agents, the stimulation of which leads to activation of intracellular enzymes and regulation of gene transcription. PRRs express multiple leucine-rich regions to bind a range of microbially-derived ligands, termed PAMPs or pathogen-associated molecular patterns or endogenous ligands, termed DAMPS or damage-associated molecular patterns. These include peptides, carbohydrates, peptidoglycans, lipoproteins, lipopolysaccharides, and nucleic acids. PRRs include both cell-surface and intracellular proteins. PRRs may be divided into signalling-associated members, identified here, and endocytic members, the function of which appears to be to recognise particular microbial motifs for subsequent cell attachment, internalisation and destruction. Some are involved in inflammasome formation, and modulation of IL-1β cleavage and secretion, and others in the initiation of the type I interferon response. PRRs included in the Guide To PHARMACOLOGY are:Catalytic PRRs (see links below this overview)Toll-like receptors (TLRs)Nucleotide-binding oligomerization domain, leucine-rich repeat containing receptors (NLRs, also known as NOD (Nucleotide oligomerisation domain)-like receptors)RIG-I-like receptors (RLRs)Caspase 4 and caspase 5 Non-catalytic PRRsAbsent in melanoma (AIM)-like receptors (ALRs)C-type lectin-like receptors (CLRs)Other pattern recognition receptorsAdvanced glycosylation end-product specific receptor (RAGE)

Excessive release of heme from red blood cells is a key pathophysiological feature of several disease states including bacterial sepsis, malaria, and sickle cell disease. This hemolysis results in an increased level of free heme that has been implicated in the inflammatory activation of monocytes, macrophages, and endothelium. Here, we show that extracellular heme engages the human inflammatory caspases, caspase-1, caspase-4, and caspase-5, resulting in the release of IL-1β. Heme-induced IL-1β release was further increased in macrophages from patients with sickle cell disease. In human primary macrophages, heme activated caspase-1 in an inflammasome-dependent manner, but heme-induced activation of caspase-4 and caspase-5 was independent of canonical inflammasomes. Furthermore, we show that both caspase-4, and caspase-5 are essential for heme-induced IL-1β release, while caspase-4 is the primary contributor to heme-induced cell death. Together, we have identified that extracellular heme acts as a damage associated molecular pattern (DAMP) that can engage canonical and non-canonical inflammasome activation as a key mediator of inflammation in macrophages.FootnotesFunding for the project includes NIH/NIDDK T32DK060445 (BEB, BAR), NIH/NIDDK F32DK121479 (BEB), NIH/NIGMS R01GM121389 (LBH), NIH/NHLBI R01HL114567 (JDB, GMV), NIH/NHLBI R01-HL136415 (JMF), and CPRIT-RP180672, NIHCA125123 and NIHRR024574 (Cytometry and Cell Sorting Core at Baylor College of Medicine)Key PointsHeme induces oligomerization of caspase-1, caspase-4, and caspase-5.Heme-induced IL-1β release requires both caspase-4 and caspase-5.Caspase-4 alone contributes to heme-induced cell death.

Excessive release of heme from RBCs is a key pathophysiological feature of several disease states, including bacterial sepsis, malaria, and sickle cell disease. This hemolysis results in an increased level of free heme that has been implicated in the inflammatory activation of monocytes, macrophages, and the endothelium. In this study, we show that extracellular heme engages the human inflammatory caspases, caspase-1, caspase-4, and caspase-5, resulting in the release of IL-1β. Heme-induced IL-1β release was further increased in macrophages from patients with sickle cell disease. In human primary macrophages, heme activated caspase-1 in an inflammasome-dependent manner, but heme-induced activation of caspase-4 and caspase-5 was independent of canonical inflammasomes. Furthermore, we show that both caspase-4 and caspase-5 are essential for heme-induced IL-1β release, whereas caspase-4 is the primary contributor to heme-induced cell death. Together, we have identified that extracellular heme is a damage-associated molecular pattern that can engage canonical and noncanonical inflammasome activation as a key mediator of inflammation in macrophages.

Pattern Recognition Receptors (PRRs, [83]) (nomenclature as agreed by NC-IUPHAR sub-committee on Pattern Recognition Receptors, [15]) participate in the innate immune response to microbial agents, the stimulation of which leads to activation of intracellular enzymes and regulation of gene transcription. PRRs express multiple leucine-rich regions to bind a range of microbially-derived ligands, termed PAMPs or pathogen-associated molecular patterns or endogenous ligands, termed DAMPS or damage-associated molecular patterns. These include peptides, carbohydrates, peptidoglycans, lipoproteins, lipopolysaccharides, and nucleic acids. PRRs include both cell-surface and intracellular proteins. PRRs may be divided into signalling-associated members, identified here, and endocytic members, the function of which appears to be to recognise particular microbial motifs for subsequent cell attachment, internalisation and destruction. Some are involved in inflammasome formation, and modulation of IL-1β cleavage and secretion, and others in the initiation of the type I interferon response. PRRs included in the Guide To PHARMACOLOGY are:Catalytic PRRs (see links below this overview)Toll-like receptors (TLRs)Nucleotide-binding oligomerization domain, leucine-rich repeat containing receptors (NLRs, also known as NOD (Nucleotide oligomerisation domain)-like receptors)RIG-I-like receptors (RLRs)Caspase 4 and caspase 5 Non-catalytic PRRsAbsent in melanoma (AIM)-like receptors (ALRs)C-type lectin-like receptors (CLRs)Other pattern recognition receptorsAdvanced glycosylation end-product specific receptor (RAGE)

Inflammasomes are multiprotein innate immune complexes that regulate caspase-dependent inflammation and cell death. Pattern recognition receptors, such as nucleotide-binding oligomerization domain (NOD)-like receptors and absent in melanoma 2 (AIM2)-like receptors, sense danger signals or cellular events to activate canonical inflammasomes, resulting in caspase 1 activation, pyroptosis and the secretion of IL-1β and IL-18. Non-canonical inflammasomes can be activated by intracellular lipopolysaccharides, toxins and some cell signalling pathways. These inflammasomes regulate the activation of alternative caspases (caspase 4, caspase 5, caspase 11 and caspase 8) that lead to pyroptosis, apoptosis and the regulation of other cellular pathways. Many inflammasome-related genes and proteins have been implicated in animal models of kidney disease. In particular, the NLRP3 (NOD-, LRR- and pyrin domain-containing 3) inflammasome has been shown to contribute to a wide range of acute and chronic microbial and non-microbial kidney diseases via canonical and non-canonical mechanisms that regulate inflammation, pyroptosis, apoptosis and fibrosis. In patients with chronic kidney disease, immunomodulation therapies targeting IL-1β such as canakinumab have been shown to prevent cardiovascular events. Moreover, findings in experimental models of kidney disease suggest that small-molecule inhibitors targeting NLRP3 and other inflammasome components are promising therapeutic agents. In this Review, the authors discuss the biology of canonical and non-canonical inflammasomes and inflammasome-forming genes in the kidney, including their different functions in the various kidney compartments and their potential as therapeutic targets.

In our previous studies, programmed cell death (PCD) was induced in human periodontal ligament (PDL) cells, through activation of caspase-3 and upregulation of CASP5 gene (encoding caspase-5 protein), in response to mechanical stretch loading. The aim of this study is to explore the relationship between the inflammatory caspase, caspase-5, and the apoptotic executioner protein, caspase-3, in human PDL cells. Here, we found that cyclic stretching upregulated the activity and the protein expression level of caspase-3 and -5 and the addition of the caspase-3 inhibitor or caspase-5 inhibitor significantly inhibited the stretch-induced PCD. Meanwhile, the inhibition of caspase-5 inhibited the activation of caspase-3 and vice versa. The result of coimmunoprecipitation also demonstrated that the expression of caspase-3 was immunoprecipitated with caspase-5. Thus, our study revealed that the in vitro application of cyclic stretching induced PCD by activation of caspase-3 and -5 in human PDL cells, and these two caspases could interact with each other after mechanical stretch loading. The study may facilitate further studies on the mechanism of stretch-induced PCD and help us understand the force-related periodontal homeostasis and remodeling better.

Humans encode two inflammatory caspases that detect cytoplasmic LPS, caspase-4 and caspase-5. When activated, these trigger pyroptotic cell death and caspase-1-dependent IL-1β production; however the mechanism underlying this process is not yet confirmed. We now show that a specific NLRP3 inhibitor, MCC950, prevents caspase-4/5-dependent IL-1β production elicited by transfected LPS. Given that both caspase-4 and caspase-5 can detect cytoplasmic LPS, it is possible that these proteins exhibit some degree of redundancy. Therefore, we generated human monocytic cell lines in which caspase-4 and caspase-5 were genetically deleted either individually or together. We found that the deletion of caspase-4 suppressed cell death and IL-1β production following transfection of LPS into the cytoplasm, or in response to infection with Salmonella typhimurium. Although deletion of caspase-5 did not confer protection against transfected LPS, cell death and IL-1β production were reduced after infection with Salmonella. Furthermore, double deletion of caspase-4 and caspase-5 had a synergistic effect in the context of Salmonella infection. Our results identify the NLRP3 inflammasome as the specific platform for IL-1β maturation, downstream of cytoplasmic LPS detection by caspase-4/5. We also show that both caspase-4 and caspase-5 are functionally important for appropriate responses to intracellular Gram-negative bacteria.

Purpose Microsatellite instability (MSI) resulting from loss of functional DNA mismatch repair was recently found in various haematological disorders. In coding sequences, MSI leads to frameshift mutations (FSMs) and the production of C-terminally altered proteins which are foreign to the immune system. Here, we wondered whether these frame-shifted peptide (FSP) sequences represent tumour-specific antigens also for MSI + leukaemia and lymphomas (L/L). Material and methods A total of 33 coding region microsatellites were examined in MSI + L/L cell lines for the presence of FSMs. Thereafter, recognition of MSI + cells by established FSP-specific CD8 + T cell lines was quantified using interferon (IFN)-γ enzyme-linked immunospot (ELISpot) assays. In each experiment, MSI + L/L cell lines and T2 targets exogenously loaded with the cognate peptide (=internal control) were employed. Supplementary, lytic activity towards tumour cells was analysed by standard chromium release assay ( 51 Cr). Results Mutational profiling of 33 coding microsatellite loci in nine MSI + L/L cell lines revealed instability in at least nine microsatellites. In each cell line, a distinct mutational profile was observed. Only three of the 33 loci were stable. FSP-specific and human leukocyte antigen-A2 (HLA-A2)-restricted T cells specifically recognised MSI + L/L cells endogenously expressing TGFβRII (-1), Caspase 5 (-1) and MSH3 (-1) in ELISpot assays. Moreover, specific killing of Caspase 5 (-1) and MSH3 (-1) expressing L/L cell lines was achieved in functional cytotoxicity assays. Conclusion Data presented here expand the importance of FSPs as shared and general tumour-specific antigens. Consequently, they open new avenues for specific immunotherapies not only for solid but also for MSI + haematological malignancies.

The cytosolic protease caspase 11 senses and is activated by the microbial product lipopolysaccharide (LPS), which leads to cell death by pyroptosis and the activation of caspase 1, which is required for secretion of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. Uncontrolled cell death and inflammatory cytokine production in infected patients result in sepsis, which is often fatal. Napier et al . performed a CRISPR/Cas9-based screen in a mouse macrophage cell line and found that loss of the gene encoding the complement-related peptidase carboxypeptidase B1 (Cpb1) reduced caspase 11–dependent death in cells exposed to cytosolic LPS. Cpb1 processes a cleavage product of the complement factor C3a to generate a ligand for the G protein–coupled receptor C3aR. Knockout of Cpb1 in a mouse macrophage cell line also decreased activation of caspase 11 and reduced expression of the gene encoding caspase 11 in cells exposed to LPS. Incubation of Cbp1-deficient macrophages with conditioned medium from LPS-treated wild-type or C3aR-deficient macrophages (both of which contained functional Cbp1) led to the expression of genes encoding inflammatory cytokines; however, conditioned medium from LPS-treated Cbp1-deficient macrophages had no such effect, suggesting that the Cbp1-C3-C3aR pathway acts in both a cell-autonomous and non–cell-autonomous manner. Macrophages deficient in Cpb1, C3, or C3aR were as protected from death induced by bacterial infection as were macrophages deficient in caspase 11. Compared with wild-type mice, C3aR-deficient mice had decreased serum concentrations of inflammatory cytokines and increased survival in response to injection with LPS. Furthermore, treatment of wild-type mice with a pharmacological inhibitor of C3aR protected them from the effects of LPS. Exposure of human macrophages to cytosolic LPS led to increased expression of the genes encoding caspases 4 and 5, orthologs of mouse caspase 11, which was blocked by the C3aR inhibitor. Last, compared with healthy controls, human sepsis patients showed increased expression of the genes encoding caspase 5 and C3aR. Together, these data suggest that the Cbp1-C3-C3aR axis enhances caspase 11–dependent inflammation and sepsis, providing a potentially useful therapeutic target for treating sepsis. B. A. Napier, S. W. Brubaker, T. E. Sweeney, P. Monette, G. H. Rothmeier, N. A. Gertsvolf, A. Puschnik, J. E. Carette, P. Khatri, D. M. Monack, Complement pathway amplifies caspase-11–dependent cell death and endotoxin-induced sepsis severity. J . Exp . Med . 213 , 2365–2382 (2016). [[PubMed]][1] [1]: https://www.ncbi.nlm.nih.gov/pubmed/27697835

The inflammatory caspases (caspase-1, -4 and -5) are potential therapeutic targets for autoimmune and inflammatory diseases due to their involvement in the immune response upon inflammasome formation. A series of small molecules based on the 4-(piperazin-1-yl)-2,6-di(pyrrolidin-1-yl)pyrimidine scaffold were synthesized with varying substituents on the piperazine ring. Several compounds were pan-selective inhibitors of the inflammatory caspases, caspase-1, -4 and -5, with the ethylbenzene derivative CK-1-41 displaying low nanomolar Ki values across this family of caspases. Three analogs were nearly 10 fold selective for caspase-5 over caspase-1 and -4. The compounds display non-competitive, time dependent inhibition profiles. To our knowledge, this series is the first example of small molecule inhibitors of all three inflammatory caspases.

We previously reported that ductal infiltrating carcinomas (d.i.c.) of the human breast display profound modifications of the stromal architecture, associated with anomalous collagen composition. Among the major alterations observed in the interstitial collagen, the relative increase of type V collagen content was detected. When type V collagen was used as an "in vitro" substrate for 8701-BC d.i.c. cells, it appeared able to restrain cell growth, inhibit cell motility and invasion "in vitro", and modify the expression levels of genes coding for apoptosis factors, caspases and stress response proteins. In the present paper we demonstrate that type V collagen induces the down-regulation of protein kinase C η, an event that may be, at least in part, responsible of the previously-reported modifications of cell morphology and growth rate, and that appears to be involved in the already-observed changes of expression levels of genes encoding for anti- (Bcl-2) and pro-apoptotic factors (Bad, Dapk, Bcl-Xs) and enzymes (caspase 5 and 8).

The development of highly selective small molecule inhibitors for individual caspases, a class of cysteine-dependent aspartate-specific proteases, has been challenging due to conservation of the active site. Previously, we discovered an allosteric site at the dimer interface of caspases-3, -7, and -1 using disulfide trapping. Here, we show this approach can generate selective tethered ligands and inhibitors for caspase-5, which is remarkable considering its high sequence similarity to caspase-1. Among the 62 hits of a screen of ∼15 000 thiol-containing fragments, a naphthyl–thiazole-containing molecule was identified that selectively inhibited and labeled the allosteric cysteine in the p10 subunit of caspase-5, but caused very little inhibition or labeling of caspase-1. Interestingly, some of allosteric tethered compounds to caspase-5 did not inhibit its enzymatic activity, suggesting that thiol-labeling itself is not sufficient to drive inhibition. These studies validate an allosteric site on caspase-5 and provide a useful starting point to develop selective compounds to probe the role of caspase-5 separate from caspase-1 in the innate immune response.

The generation of peptides presented by MHC class I molecules requires the proteolytic activity of the proteasome and/or other peptidases. The processing of a short vaccinia virus-encoded antigen can take place by a proteasome-independent pathway involving initiator caspase-5 and -10, which generate antigenic peptides recognized by CD8(+) T lymphocytes. In the present study, comparing single versus double enzyme digestions by mass spectrometry analysis, both qualitative and quantitative differences in the products obtained were identified. These in vitro data suggest that each enzyme can use the degradation products of the other as substrate for new cleavages, indicating concerted endoproteolytic activity of caspase-5 and -10., This work was supported by grants provided by the European Union, Ministerio de Ciencia e Innovación, Comunidad de Madrid, and Instituto de Salud Carlos III to M. D. V., and by Programa Ramón y Cajal and FIPSE Foundation to D. L.

The inflammasome is a cytosolic multiprotein complex with two major functions: recognizing pathogen-associated molecular patterns and reacting to these through activation of the proinflammatory cytokines IL-1β and IL-18. In this study, we characterized the expression of inflammasome components in psoriatic skin and other common inflammatory skin diseases. Human skin biopsy specimens, cultured primary human keratinocytes, and peripheral blood mononuclear cells (PBMCs) were analyzed using quantitative reverse transcriptase-PCR (RT-PCR) and semiquantitative western blotting. mRNA expression of the inflammasome components NALP1, NALP3, ASC, caspase-1, caspase-4, and caspase-5 was detected in psoriatic skin. Interestingly, we found an extensive, 20-fold upregulation (P0.01) of caspase-5 mRNA in lesional compared with nonlesional psoriatic skin, whereas caspase-1, caspase-4, and ASC (apoptosis-associated speck-like protein with CARD domain) mRNAs were upregulated by only 1.5- to 2.6-fold (P0.01). Caspase-5 mRNA was not increased in biopsies from other inflammatory skin diseases, suggesting that this finding could be psoriasis specific. In vitro experiments revealed that caspase-5 mRNA was induced in primary keratinocytes as well as PBMCs stimulated with IFN-γ. Inhibition studies suggested that caspase-5 mRNA upregulation was mediated through the NF-κB pathway. Our findings suggest that caspase-5 and the inflammasome may have an important role in the inflammatory response in psoriasis.

Mounting evidence indicates that deregulation of apoptosis is involved in the mechanisms of cancer development. Mutations of genes encoding caspases, the executioners of apoptosis, have been detected in human cancers, indicating inactivation of apoptosis by the mutations of caspase is an important mechanism in cancer development. The aim of this study was to see whether genes encoding human caspases 1, 4, and 5 are mutated in human cancers. We analyzed the entire coding region and all splice sites of human caspase 1, 4, and 5 genes for the detection of somatic mutations in 337 human cancers, including 103 colorectal, 54 gastric, 60 breast, 60 hepatocellular, and 60 lung carcinomas by a single-strand conformation polymorphism assay. We detected 2 (0.6%) caspase-1, 2 (0.6%) caspase-4, and 15 (4.4%) caspase-5 mutations in the 343 cancers. The mutations were detected in 11 gastric carcinomas (2 caspase-1 and 9 caspase-5 mutations), 6 colorectal carcinomas (2 caspase-4 and 4 caspase-5 mutations), 1 breast carcinoma (1 caspase-5 mutation), and 1 lung carcinoma (1 caspase-5 mutation). The mutations consisted of 11 mutations in exons and 8 mutations in noncoding sequences. The 11 mutations in the exons consisted of 3 missense, 1 silent, and 7 frameshift mutation(s). Of note, most (6/9) of the caspase-5 mutations in the coding sequences were detected in microsatellite instability (MSI)-positive cancers. These data indicate that somatic mutations of caspase-1 and caspase-4 genes are rare in common solid cancers. In addition, the data indicate that caspase-5 gene is commonly mutated in the MSI-positive cancers, and suggest that inactivation of caspase-5 may play a role in the tumorigenesis of MSI-positive cancers.

Several studies have implicated a remarkable dysfunctional apoptotic state and/or response in ESRD patients. Previously published studies are controversial with respect to acute effects of haemodialysis (HD) treatment on up- or downregulation of apoptotic genes. Twenty-eight chronic HD patients were haemodialysed for 4 hours with a 4008 dialyser using high-flux membranes. For subgroup analysis, patients were separated into a low (up to 0.5 mg/dl) and a high (0.5 to 5.0 mg/dl) CRP group. Blood was drawn prior to HD and 240 min after initiation of HD. Acute changes of transcript levels encoding pro- or anti-apoptotic genes were analyzed in RNA immediately isolated from blood leukocytes using quantitative real-time PCR. In the present study, we detected a significant elevation of the death receptor CD95/Fas (induction factor (IF) 1.55 +/- 0.16), the death receptor 5 (DR5) (IF 1.17 +/- 0.08), and caspase 8 (IF 1.37 +/- 0.14) gene expression during HD. mRNA levels of the respective ligands (CD95L, TRAIL), of the caspase 5 and anti-apoptotic Bcl-2 family members such as Bcl-2 and Bcl2l2 were slightly, but not significantly, increased after HD treatment. An additional anti-apoptotic molecule, BAG3, was found to be slightly, but significantly, induced after HD (IF 1.16 +/- 0.07). In addition to being an activator of immune cells, CD40L has been shown to be strongly induced after HD treatment (IF 1.70 +/- 0.20). Subgroup analysis revealed no significant differences between low vs. high CRP patient groups or diabetic vs. non-diabetic patients. These results indicate a marked influence of routine haemodialysis treatment on the transcription of pro- and anti-apoptotic molecules and the involvement of the extrinsic pathway for apoptosis through the activation of death receptors and the initiator caspase 8. Furthermore, following dialysis, lymphocytes seem to be activated by CD40L, which represents an early T-cell activation marker.

Background and objective As caspase-5 plays a role in apoptosis, the present study aimed to identify the expression and activation of caspase-5 in human periodontal ligament cells in response to cyclic stretch and the role of caspase-5 in stretch-induced apoptosis. Methods Human PDL cells were exposed to 10% or 20% stretch strain for 6 or 24 h, and the mRNA and protein expressions of caspase-5 were analyzed with real-time PCR and Western blot, respectively. The caspase-5 activation was detected by colorimetric assay. Then the influence of the inhibition of caspase-5 on the stretch-induced apoptosis and caspase-3 activation were analyzed with flow cytometry and colorimetric assay, respectively. Results Both 6 and 24 h stretches increased mRNA, protein expression and activation of caspase-5 in human PDL cells. Inhibition of caspase-5 inhibited the stretch-induced apoptosis and caspase-3 activation in human PDL cells. Conclusion This study for the first time identified the expression and activation of caspase-5 in cyclic stretched human PDL cells and found that the stretch-induced apoptosis and caspase-3 activation were caspase-5 dependent.

Monocytes promote the early host response to infection releasing key pro-inflammatory cytokines, such as IL-1β. The biologically inactive IL-1β precursor is processed to active form by inflammasomes, multi-protein complexes activating caspase-1. Human monocytes exhibit an unconventional one-step pathway of inflammasome activation in response to lipopolysaccharide (LPS) alone. Although this lineage-restricted mechanism is likely to contribute to the pathology of endotoxin shock, signalling pathways regulating this mechanism are currently unknown. Here we report that caspase-4 and caspase-5 mediate IL-1α and IL-1β release from human monocytes after LPS stimulation. Although caspase-4 remains uncleaved, caspase-5 undergoes rapid processing upon LPS treatment. We also identify an additional caspase-5 cleavage product in LPS-stimulated monocytes, which correlates with IL-1 secretion. This one-step pathway requires Syk activity and Ca2+ flux instigated by CD14/TLR4-mediated LPS internalization. Identification of caspase-4/5 as the key determinants of one-step inflammasome activation in human monocytes provides potential targets for therapeutic intervention in endotoxin shock., Human monocytes exhibit an unconventional one-step pathway of inflammasome activation and IL-1 release in response to LPS. Here the authors show that it is mediated by caspases 4 and 5, and characterize caspase 5 cleavage, Syk and calcium signalling as key mediators of this pathway.

IL-1β is a potent player in cutaneous inflammation and central for the development of a Th17 micro-milieu in autoinflammatory diseases including psoriasis. Its production is controlled at the transcriptional level and by subsequent posttranslational processing via inflammatory caspases. In this study, we detected inflammatory caspase-5 active in epidermal keratinocytes and in psoriatic skin lesions. Further, interferon-γ and interleukin-17A synergistically induced caspase-5 expression in cultured keratinocytes, which was dependent on the antimicrobial peptide psoriasin (S100A7). However, diseases-relevant triggers for caspase-5 activity and IL-1β production remain unknown. Recently, extranuclear DNA has been identified as danger-signals abundant in the psoriatic epidermis. Here, we could demonstrate that cytosolic double-stranded (ds) DNA transfected into keratinocytes triggered the activation of caspase-5 and the release of IL-1β. Further, interleukin-17A promoted caspase-5 function via facilitation of the NLRP1-inflammasome. Anti-inflammatory vitamin D interfered with the IL-1β release and suppressed caspase-5 in keratinocytes and in psoriatic skin lesions. Our data link the disease-intrinsic danger signals psoriasin (S100A7) and dsDNA for NLPR1-dependent caspase-5 activity in psoriasis providing potential therapeutic targets in Th17-mediated skin autoinflammation.

Bisphosphonates (BPs) like zoledronic acid (ZOL) are widely used for the treatment of different diseases such as osteoporosis, metastatic bone diseases and hypercalcaemia. However, the effects of BPs on apoptosis of the liver and kidney after treatment are unclear. Furthermore, basic fibroblast growth factor (bFGF) is an angiogenic molecule, which plays an important role in angiogenesis and tissue repair. The present study investigated the expression of caspase-3, -5, -7 and apoptotic protease-activating factor-1 (APAF-1) in the liver and kidney of rats treated with ZOL and bFGF.The present study investigated the expression of caspase-3, -5, -7 and apoptotic protease-activating factor-1 (APAF-1) in the liver and kidney of rats treated with ZOL and bFGF.An animal model with 32 male Sprague Dawley rats was used. The effects of ZOL and bFGF on liver and kidney with the expressions of different apoptosis markers were studied histopathologically and immunohistochemically. Data were analyzed using Cronbach's alpha, Kruskal-Wallis and Bonnferroni-Dunn tests.The main microscopic findings were mononuclear cell infiltrations around the bile ducts, binuclear and markedly enlarged hepatocytes (cytomegaly) and mitotic figures in the liver of rats treated with ZOL only. Immunohistochemically, both APAF-1 and caspase-3, -5 and -7 expressions were found elevated significantly (P0.05) in the liver and kidney of these rats.Our findings showed that ZOL treatment increased while bFGF treatment decreased apoptosis significantly in the liver and kidney of Sprague Dawley rats.The addition of bFGF to ZOL treatment of various diseases might reduce the ZOL effects.

SummaryBackground Psoriasis involves a multifaceted interplay of keratinocytes, blood vessels, immune system mediators and expressed genes. Aim To determine whether genetic polymorphisms in the angiopoietin-1 (ANGPT1), angiopoietin-2 (ANGPT2) and caspase-5 (CASP5) genes are associated with an increased risk of developing psoriasis vulgaris (PV) in a Han population in northeastern China. Methods We analysed single nucleotide polymorphisms of ANGPT1 (rs2507800, rs1954727 and rs1010824), ANGPT2 (rs3739390, rs2442598 and rs1868554) and caspase-5 (rs507879, rs518604 and rs523104) in 343 patients with PV and 347 healthy controls (HCs) by the SNaPshot method, and evaluated the risk for psoriasis conferred by the individual polymorphisms and haplotypes. Results The rs2442598 polymorphism of ANGPT2 was significantly associated with PV. The CT+TT genotype was present at an increased frequency in patients with PV (adjusted OR = 1.45, 95% CI 1.04–2.03, P = 0.03). However, the distribution of the other genotypes (rs2507800, rs1954727 and rs1010824 of ANGPT1; rs3739390 and rs1868554 of ANGPT2; and rs507879, rs518604 and rs523104 of caspase-5) was not significantly different between patients with PV and the HCs. Furthermore, none of the genes in the haplotype-based analysis showed any association with psoriasis risk. Conclusions Our data suggest that ANGPT2 variants may be involved in PV risk in a Han population in northeastern China.

Based on high sequence homology, there are six members in the caspase-1 subfamily: caspases 1, 4, 5, and 13 in humans and caspases 1, 11, and 12 in mice. Only caspase-1 is known to activate interleukin-1beta and interleukin-18, and caspase-11 activates pro-caspase-1 in vivo. Almost nothing is known about caspases 4, 5, and 13. Here we report a sensitive and specific polymerase chain reaction system to analyze closely related genes. We employed this system to analyze the gene expression and regulation of human caspases 1, 4, 5, and 13, demonstrating that they have different expression patterns in normal tissues and cell lines. Interferon-gamma strongly induced CASP1 and CASP5 but not CASP4 or CASP13 gene expression in HT-29 colon carcinoma cells. In contrast to the mRNA, interferon-gamma up-regulated caspase-1 but not caspase-5 protein. In the monocytic cell line THP-1, CASP1 mRNA and caspase-1 protein are expressed constitutively, and their levels were not increased by lipopolysaccharide, whereas both CASP5 mRNA and caspase-5 protein were induced by lipopolysaccharide. Caspase-1 subfamily members displayed different in vitro activities toward pro-caspases 1 and 3 and pro-interleukin-1beta. Our results demonstrate that caspase-1 and caspase-5 levels are modulated by interferon-gamma and lipopolysaccharide, respectively, and suggest that caspase-1 subfamily members are differentially regulated and may have distinct functions.

Proteases of the caspase family are implicated in mammalian apoptosis and constitute a protease cascade. We characterized caspase-4 (TX/ICH-2/ICErelII) and caspase-5 (ICErelIII/TY), which are most closely related to caspase-1 (ICE) among the caspase family. Although overexpression of caspase-4 and caspase-5 induced apoptosis, confirming previous observations, this apoptosis was not inhibited by a caspase-1-specific tetrapeptide inhibitor (Ac-YVAD-CHO), suggesting that caspase-4 and caspase-5 have different substrate specificities from caspase-1 and also that caspase-4- and caspase-5-induced apoptosis is not mediated by caspase-1. CrmA, a cowpox virus-derived caspase-1 inhibitor that prevents apoptosis induced by various stimuli, was cleaved by caspase-4 and caspase-5, and inhibited their proteolytic activity as assessed by cleavage of pro-caspase-3 (pro-CPP32/Yama/apopain). Thus, caspase-4 and caspase-5 are CrmA-inhibitable proteases like caspase-1 and might be involved in apoptosis.

Cysteine proteases related to mammalian interleukin-1β-converting enzyme (ICE) and the nematode cell death abnormal ced-3 gene product have been implicated in the effector mechanism of apoptotic cell death. Two novel members of this new family of ICE/CED-3-related proteases, designated ICErel-II and ICErel-III, were cloned from human monocytic cells. Both were highly homologous to human ICE (52% identical) and CED-3 (25% identical) and both contained the absolutely conserved pentapeptide sequence Gln-Ala-Cys-Arg-Asp containing the catalytic cysteine residue. Other structural motifs that were comparable with ICE suggest that ICErel-II and ICErel-III are also synthesized as larger proenzymes which are proteolytically processed to form heterodimeric active enzymes. Pro-interleukin-1β processing activity could not be detected in cells transfected with ICErel-II or ICErel-III, but pro-domain-less truncated forms of ICErel-II and ICErel-III were capable of effectively inducing fibroblast apoptosis. ICErel-II and ICErel-III may, therefore, participate in proteolytic events culminating in the apoptotic death of human cells.

A previous data showed that the hypoxia mimetic compound CoCl(2) induced cleavage of HuR and subsequent apoptosis in human oral cancer cells. We also previously demonstrated that exposure of NT-2 human neuronal precursor cells to hypoxia resulted in changes in sphingolipid levels and apoptosis. Since it is known that CoCl(2) induces cleavage of HuR, we investigated whether there is a link between HuR cleavage and the observed sphingolipid changes in cells exposed to hypoxia, and whether this link is associated with the induction of apoptosis. Exposure of hepatocytes to direct hypoxia by means of a hypoxic chamber resulted in acid sphingomyelinase activation and ceramide elevation. The elevation in ceramide levels was associated with activation of caspase 5 and the subsequent cleavage of HuR and apoptotic cell death. These data raise the possibility that acid sphingomyelinase and caspase 5 are each potential targets for treating hypoxia (ischemia)-induced liver injury.

Caspase-5 is a caspase-1-like protease with pro-apoptotic and pro-inflammatory activities. Here we have identified a novel exon at the 5'-end of the human caspase-5 gene. This novel exon was present in six alternatively spliced caspase-5 mRNA variants expressed in human peripheral blood mononuclear cells (PBMC) and encoded the previously unknown amino-terminus of caspase-5. The genomic region upstream of this exon contained sequence elements homologous to those of the caspase-11 promoter in the mouse, and transcription of caspase-5 was upregulated by lipopolysaccharide (LPS) in a caspase-11-like manner in human PBMC in vivo. Taken together, our findings call for a revision of the structure of caspase-5 at the genomic level as well as at the mRNA and protein levels.

Microsatellite instability (MSI) in tumors is diagnostic for inactive DNA mismatch repair. It is widespread among some tumor types, such as colorectal or endometrial carcinoma, but is rarely found in leukemia. Therapy-related acute myeloid leukemia/myelodysplastic syndrome (tAML/MDS) is an exception, and MSI is frequent in tAML/MDS following cancer chemotherapy or organ transplantation. The development of MSI+ tumors is associated with an accumulation of insertion/deletion mutations in repetitive sequences. These events can cause inactivating frameshifts or loss of expression of key growth control proteins. We examined established MSI+ cell lines and tAML/MDS cases for frameshift-like mutations of repetitive sequences in several genes that have known, or suspected, relevance to leukemia. CASPASE-5, an acknowledged frameshift target in MSI+ gastrointestinal tract tumors, was frequently mutated in MSI+ cell lines (67%) and in tAML/MDS (29%). Frameshift-like mutations were also observed in the NF1 and FANCD2 genes that are associated with genetic conditions conferring a predisposition to leukemia. Both genes were frequent targets for mutation in MSI+ cell lines and colorectal carcinomas. FANCD2 mutations were also common in MSI+ tAML/MDS, although NF1 mutations were not observed. A novel FANCD2 polymorphism was also identified.

Methyltransferase inhibitors commonly used in clinical trials promote tumor cell death, but their detailed cytotoxic action is not yet fully understood. A deeper knowledge about their apotosis-inducing mechanisms and their interaction with DNA methyltransferases (DNMTs) DNMT1, DNMT3a, and DNMT3b might allow the design of more effective drugs with lower cytotoxicity. 5-aza-cytidine (5-aza-CR), a potent inhibitor of DNMT1, is known to induce demethylation and reactivation of silenced genes. In this study, we investigated the p53 dependence of apoptotic, cell cycle, and growth inhibitory effects of 5-aza-CR, as well as the influence on the expression level of DNMT1, DNMT3a, and DNMT3b in the colon cancer cell line HCT-116. Exposure to 5-aza-CR induced the up-regulation of genes promoting cell cycle arrest and DNA repair (p21(WAF1) and GADD45) or apoptosis (p53, RIPK2, Bak1, caspase 5, and caspase 6). In parallel, there was a down-regulation of antiapoptotic Bcl2 protein and the G(2)/M-mediator cyclin B1. Co-incubation with pifithrin-alpha (PFT-alpha), a selective p53 inhibitor, restored GADD45, Bcl2, cyclin B1, and p21(WAF1) expression levels and almost completely reversed the growth inhibitory, cell cycle, and apoptotic effects of 5-aza-CR. 5-aza-CR treatment caused global demethylation and reactivation of p16(INK4) expression. There was a marked decrease in DNMT1 and DNMT3a mRNA expression, with PFT-alpha reversing these effects. However, 5-aza-CR treatment did not modulate DNMT3b expression. Our data demonstrate that 5-aza-CR action in HCT-116 is mediated by p53 and its downstream effectors p21(WAF1) and GADD45. This is the first report to show a link between p53 and regulation of DNMT1 and de novo methyltransferase DNMT3a.

The process of apoptosis, or programmed cell death, is fundamental during normal development and homeostasis and aberrant apoptosis has been implicated in a number of human diseases. The cellular machinery involved in the execution of apoptosis includes a family of cysteine proteases termed caspases. Caspases exhibit the rare substrate preference of cleavage C-terminal to aspartate residues, a property shared only by the cytotoxic lymphocyte serine protease, granzyme B. Experimental evidence demonstrates a vital role for caspase activation in the apoptotic pathway, and, as such, caspases are a target for the development of agents that can modulate their activity. This article reviews the members of the caspase family and the role that each contributes to the execution of cell death induced by apoptotic stimuli.

Background Caspases form an evolutionarily conserved family of cytosolic, aspartate-specific, cysteine proteases. Beyond its role in apoptosis, certain caspases (caspase-1, -4, -5, -11 and -12) exert non-apoptotic functions, including cytokine maturation during innate immunity, cell differentiation, proliferation, and NF-κB activation. Objectives This study investigated expression of caspase-5 and effect of Z-WEHD-fmk, a selective caspase-5 inhibitor, on pro-inflammatory gene expressions and cellular proliferation in fibroblast-like synoviocyte (FLS) in patients with rheumatoid arthritis (RA). Methods Synovial tissues were obtained from patients with RA during total knee replacement surgery then FLS were isolated and cultured. Expression of caspase-5 in rheumatoid FLS were measured after stimulation either without or with pro-inflammatory agents IL-1β (2 ng/mL), TNF-α (20 ng/mL), lipopolysaccharide (1000 ng/mL) by real-time quantitative RT-PCR. The involvement of caspase-5 in inflammation and cellular proliferaion was further examined by treating the cells with caspase-5 inhibitor Z-WEHD-fmk. The mRNA expressions of IL-1β, IL-6, IL-18, COX-2, MMP-9 and CCL-2 after caspase-5 inhibitor Z-WEHD-fmk treatment were determined by real-time quantitative RT-PCR, and FLS proliferation in response to caspase-5 inhibitor Z-WEHD-fmk treatment was measured by MTT assay. Results Caspase-5 mRNA expression in RA FLS was increased by all the pro-inflammatory agents compared to control. Caspase-5 inhibitor Z-WEHD-fmk treatment induced a reduction in mRNA expressions of IL-1β, IL-6, IL-18, COX-2, MMP-9 and CCL-2 from RA FLS. But, cellular proliferation of rheumatoid FLS was not suppressed by caspase-5 inhibitor Z-WEHD-fmk. Conclusions Caspase-5 inhibitor treatment inhibited suppressed various pro-inflammatory gene expressions. These results suggest that inhibition of Caspase-5 can be a novel therapeutic approach in human RA. References Martinon F, Tschopp J. Inflammatory caspases and inflammasomes: master switches of inflammation. Cell Death Differ. 2007;14:10 –22. Scott AM, Saleh M. The inflammatory caspases: guardians against infections and sepsis. Cell Death Differ. 2007;14:23–31. Lamkanfi M, Kalai M, Vandenabeele P. Caspase-12: an overview. Cell Death Differ. 2004;11:365–368. Schwerk C, Schulze-Osthoff K. Non-apoptotic functions of caspases in cellular proliferation and differentiation. Biochem Pharmacol 2003;66:1453-8. Disclosure of Interest None Declared

Background The inflammasome is a multi-protein complex which regulates the activation of caspase-1. This activation results in the cleavage and secretion of the IL-1β super family cytokines, IL-1β, IL-18, and IL-33. NLR family-pyrin domain containing- 3 (NLRP3) is a nucleotide binding domain-leucine rich repeat (NLR) family protein responsible for sensitization and oligomerization of the NLRP3 inflammasome complex. Although various damage and pathogen associated patterns have been implicated as stimuli, the exact mechanism of activation has yet to be elucidated. Capase-5, an inflammatory caspase with similar homology to caspase-1, is a key molecule activation of the NLRP1 inflammasome. Caspase-4, an evolutionary duplicate in humans to murine caspase 12 along with caspase 5, is important in IL-1β processing; its involvement with the NLRP3 inflammasome is unknown. We therefore investigated whether caspase-4 plays a role in the activation of the NLRP3 inflammasome. Methods Inflammasomes in THP-1 macrophages were activated using Nigericin (10 μg/mL), a bacterial pore causing toxin and NLRP3 inflammasome activator, in the presence or absence of various concentrations (0.1 μM, 1 μM, and 10 μM) of caspase-4 inhibitor, Z-YVAD-FMK. We analyzed the inflammasome activation, caspase-1 cleavage, and IL-1β release by western blot and ELISA analysis. Results Our results indicate that inhibition of caspase-4 leads to a dose dependant decrease in IL-1β secretion. In addition, our results show that caspase-4 contributes to IL-1β and caspase-1 cleavage, both of which are hall marks of inflammasome activation. Conclusions These findings suggest that caspase-4 is important to the activation of the NLRP3 inflammasome. In modulating the inflammasome, caspase-4 appears to be a druggable target for treatment of chronic inflammatory pulmonary conditions such as allergy and asthma.

To investigate the expression, activation, and functional involvement of caspase-5 in human retinal pigment epithelial (hRPE) cells.Expression and activation of caspase-5 in primary cultured hRPE cells, telomerase-immortalized hTERT-RPE1 cells (hTERT-RPE1), or both, were measured after stimulation with proinflammatory agents IL-1β, TNF-α, lipopolysaccharide (LPS), interferon-γ, monocyte coculture, adenosine triphosphate (ATP), or endoplasmic reticulum (ER) stress inducers. Immunomodulating agents dexamethasone (Dex), IL-10, and triamcinolone acetonide (TA) were used to antagonize proinflammatory stimulation. Cell death ELISA and TUNEL staining assays were used to assess apoptosis.Caspase-5 mRNA expression and protein activation were induced by LPS and monocyte-hRPE coculture. Caspase-5 activation appeared as early as 2 hours after challenge by LPS and consistently increased to 24 hours. Meanwhile, caspase-1 expression and protein activation were induced by LPS. Activation of caspase-5 was blocked or reduced by Dex, IL-10, and TA. Activation of caspase-5 and -1 was also enhanced by ATP and ER stress inducers. Expression and activation of caspase-5 were inhibited by a caspase-1-specific inhibitor. Caspase-5 knockdown reduced caspase-1 protein expression and activation and inhibited TNF-α-induced IL-8 and MCP-1. In contrast to caspase-4, the contribution of caspase-5 to stress-induced apoptosis was moderate.Caspase-5 mRNA synthesis, protein expression, and catalytic activation were highly regulated in response to various proinflammatory stimuli, ATP, and ER stress inducers. Mutual activation between caspase-5 and -1 suggests caspase-5 may work predominantly in concert with caspase-1 in modulating hRPE inflammatory responses.

Human guanylate binding protein-1 (GBP-1) belongs to the family of large GTPases. The expression of GBP-1 is inducible by inflammatory cytokines, and the protein is involved in inflammatory processes and host defence against cellular pathogens. GBP-1 is the first GTPase which was described to be secreted by eukaryotic cells. Here, we report that precipitation of GBP-1 with GMP-agarose from cell culture supernatants co-purified a 47-kD fragment of GBP-1 (p47-GBP-1) in addition to the 67-kD full-length form. MALDI-TOF sequencing revealed that p47-GBP-1 corresponds to the C-terminal helical part of GBP-1 and lacks most of the globular GTPase domain. In silico analyses of protease target sites, together with cleavage experiments in vitro and in vivo, showed that p67-GBP-1 is cleaved by the inflammatory caspases 1 and 5, leading to the formation of p47-GBP-1. Furthermore, the secretion of p47-GBP-1 was found to occur via a non-classical secretion pathway and to be dependent on caspase-1 activity but independent of inflammasome activation. Finally, we showed that p47-GBP-1 represents the predominant form of secreted GBP-1, both in cell culture supernatants and, in vivo, in the cerebrospinal fluid of patients with bacterial meningitis, indicating that it may represent the biologically active form of extracellular GBP-1. These findings confirm the involvement of caspase-1 in non-classical secretion mechanisms and open novel perspectives for the extracellular function of secreted GBP-1.