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Current Protocols in Immunology Dec 2020This article presents assays that allow induction and measurement of activation of different inflammasomes in mouse macrophages, human peripheral blood mononuclear cell...
This article presents assays that allow induction and measurement of activation of different inflammasomes in mouse macrophages, human peripheral blood mononuclear cell (PBMC) cultures, and mouse peritonitis and endotoxic shock models. Basic Protocol 1 describes how to prime the inflammasome in mouse macrophages with different Toll-like receptor agonists and TNF-α; how to induce NLRP1, NLRP3, NLRC4, and AIM2 inflammasome activation by their corresponding stimuli; and how to measure inflammasome activation-mediated maturation of interleukin (IL)-1β and IL-18 and pyroptosis. Since the well-established agonists for NLRP1 are inconsistent between mice and humans, Basic Protocol 2 describes how to activate the NLRP1 inflammasome in human PBMCs. Basic Protocol 3 describes how to purify, crosslink, and detect the apoptosis-associated speck-like protein containing a CARD (ASC) pyroptosome. Formation of the ASC pyroptosome is a signature of inflammasome activation. A limitation of ASC pyroptosome detection is the requirement of a relatively large cell number. Alternate Protocol 1 is provided to stain ASC pyroptosomes using an anti-ASC antibody and to measure ASC specks by fluorescence microscopy in a single cell. Intraperitoneal injection of lipopolysaccharides (LPS) and inflammasome agonists will induce peritonitis, which is seen as an elevation of IL-1β and other proinflammatory cytokines and an infiltration of neutrophils and inflammatory monocytes. Basic Protocol 4 describes how to induce NLRP3 inflammasome activation and peritonitis by priming mice with LPS and subsequently challenging them with monosodium urate (MSU). The method for measuring cytokines in serum and through peritoneal lavage is also described. Finally, Alternate Protocol 2 describes how to induce noncanonical NLRP3 inflammasome activation by high-dose LPS challenge in a sepsis model. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Priming and activation of inflammasomes in mouse macrophages Basic Protocol 2: Activation of human NLRP1 inflammasome by DPP8/9 inhibitor talabostat Basic Protocol 3: Purification and detection of ASC pyroptosome Alternate Protocol 1: Detection of ASC speck by immunofluorescence staining Basic Protocol 4: Activation of canonical NLRP3 inflammasome in mice by intraperitoneal delivery of MSU crystals Alternate Protocol 2: Activation of noncanonical NLRP3 inflammasome in mice by intraperitoneal delivery of LPS.
Topics: Animals; Cell Culture Techniques; Disease Models, Animal; Humans; Immunoassay; Inflammasomes; Leukocytes, Mononuclear; Macrophages; Mice; Peritonitis; Pyroptosis; Shock, Septic
PubMed: 33017103
DOI: 10.1002/cpim.107 -
International Journal of Molecular... Jul 2020Inflammasomes represent a group of protein complexes that contribute to host defense against pathogens and repair processes upon the induction of inflammation. However,... (Review)
Review
Inflammasomes represent a group of protein complexes that contribute to host defense against pathogens and repair processes upon the induction of inflammation. However, aberrant and chronic inflammasome activation underlies the pathology of numerous common inflammatory diseases. Inflammasome assembly causes activation of the protease caspase-1 which in turn activates proinflammatory cytokines and induces a lytic type of cell death termed pyroptosis. Although NLRP1 (NACHT, leucine-rich repeat and pyrin domain containing 1) was the first inflammasome sensor, described almost 20 years ago, the molecular mechanisms underlying its activation and the resulting downstream events are incompletely understood. This is partially a consequence of the poor conservation of the NLRP1 pathway between human and mice. Moreover, recent evidence demonstrates a complex and multi-stage mechanism of NLRP1 inflammasome activation. In contrast to other inflammasome sensors, NLRP1 possesses protease activity required for proteolytic self-cleavage and activation mediated by the function-to-find domain (FIIND). CARD8 is a second FIIND protein and is expressed in humans but not in mice. In immune cells and AML (acute myeloid leukemia) cells, the anti-cancer drug talabostat induces CARD8 activation and causes caspase-1-dependent pyroptosis. In contrast, in human keratinocytes talabostat induces NLRP1 activation and massive proinflammatory cytokine activation. NLRP1 is regarded as the principal inflammasome sensor in human keratinocytes and UVB radiation induces its activation, which is believed to underlie the induction of sunburn. Moreover, gain-of-function mutations of cause inflammatory skin syndromes and a predisposition for the development of skin cancer. SNPs (single nucleotide polymorphisms) of are associated with several (auto)inflammatory diseases with a major skin phenotype, such as psoriasis or vitiligo. Here, we summarize knowledge about NLRP1 with emphasis on its role in human keratinocytes and skin. Due to its accessibility, pharmacological targeting of NLRP1 activation in epidermal keratinocytes represents a promising strategy for the treatment of the numerous patients suffering from NLRP1-dependent inflammatory skin conditions and cancer.
Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis Regulatory Proteins; CARD Signaling Adaptor Proteins; Humans; Inflammasomes; Inflammation; Keratinocytes; NLR Proteins; Neoplasm Proteins; Skin; Skin Neoplasms
PubMed: 32640751
DOI: 10.3390/ijms21134788 -
Role of Fibroblast Activation Protein Alpha in Fibroblast-like Synoviocytes of Rheumatoid Arthritis.Iranian Journal of Allergy, Asthma, and... Jun 2021Fibroblast-like synoviocytes (FLSs) have been introduced in recent years as a key player in the pathogenesis of rheumatoid arthritis (RA), but the exact mechanisms of...
Fibroblast-like synoviocytes (FLSs) have been introduced in recent years as a key player in the pathogenesis of rheumatoid arthritis (RA), but the exact mechanisms of their transformation and intracellular pathways have not yet been determined. This study aimed to investigate the role of fibroblast activation protein-alpha (FAP-α) in the regulation of genes involved in the transformation and pathogenic activity of RA FLSs. Synovial FLSs were isolated from RA patients and non-arthritic individuals (n=10 in both groups) and characterized; using immunocytochemistry and flow cytometry analysis. FLSs were divided into un-treated and Talabostat-treated groups to evaluate the FAP-α effect on the selected genes involved in cell cycle regulation (p21, p53, CCND1), apoptosis (Bcl-2, PUMA), and inflammatory and destructive behavior of FLSs (IL-6, TGF-β1, MMP-2, MMP-9, P2RX7). Gene expression analysis was performed by quantitative real-time polymerase chain reaction (qRT-PCR), and immunoblotting was carried out to evaluate FAP-α protein levels. The basal level of FAP-α protein in RA patients was significantly higher than non-arthritic control individuals. However, no differences were observed between RA and non-arthritic FLSs, at the baseline mRNA levels of all the genes. Talabostat treatment significantly reduced FAP-α protein levels in both RA and non-arthritic FLSs, however, had no effect on mRNA expressions except an upregulated TGF-β1 expression in non-arthritic FLSs. A significantly higher protein level of FAP-α in FLSs of RA patients compared with that of healthy individuals may point to the pathogenic role of this protein in RA FLSs. However, more investigations are necessary to address the mechanisms mediating the FAP-α pathogenic role in RA FLSs.
Topics: Adult; Aged; Apoptosis Regulatory Proteins; Arthritis, Rheumatoid; Case-Control Studies; Cell Cycle Proteins; Cells, Cultured; Endopeptidases; Female; Fibroblasts; Gene Expression Regulation; Humans; Inflammation Mediators; Male; Membrane Proteins; Middle Aged; Signal Transduction; Synoviocytes
PubMed: 34134455
DOI: 10.18502/ijaai.v20i3.6335 -
Cell Chemical Biology Mar 2018Val-boroPro (PT-100, Talabostat) induces powerful anti-tumor immune responses in syngeneic cancer models, but its mechanism of action has not yet been established....
Val-boroPro (PT-100, Talabostat) induces powerful anti-tumor immune responses in syngeneic cancer models, but its mechanism of action has not yet been established. Val-boroPro is a non-selective inhibitor of post-proline-cleaving serine proteases, and the inhibition of the highly related cytosolic serine proteases Dpp8 and Dpp9 (Dpp8/9) by Val-boroPro was recently demonstrated to trigger an immunostimulatory form of programmed cell death known as pyroptosis selectively in monocytes and macrophages. Here we show that Dpp8/9 inhibition activates the inflammasome sensor protein Nlrp1b, which in turn activates pro-caspase-1 to mediate pyroptosis. This work reveals a previously unrecognized mechanism for activating an innate immune pattern recognition receptor and suggests that Dpp8/9 serve as an intracellular checkpoint to restrain Nlrp1b and the innate immune system.
Topics: Animals; Apoptosis Regulatory Proteins; Boronic Acids; Caspase 1; Dipeptidases; Dipeptides; Female; HEK293 Cells; Humans; Immunity, Innate; Inflammasomes; Macrophages; Male; Mice; Mice, Inbred C57BL; Proteasome Endopeptidase Complex; Proteolysis; Pyroptosis; RAW 264.7 Cells
PubMed: 29396289
DOI: 10.1016/j.chembiol.2017.12.013 -
BMC Cancer Jul 2009Metastatic melanoma is an incurable disease with an average survival of less than one year. Talabostat is a novel dipeptidyl peptidase inhibitor with immunostimulatory...
BACKGROUND
Metastatic melanoma is an incurable disease with an average survival of less than one year. Talabostat is a novel dipeptidyl peptidase inhibitor with immunostimulatory properties.
METHODS
This phase II, open label, single arm study was conducted to evaluate the safety and efficacy of 75-100 mg/m2 cisplatin combined with 300-400 mcg talabostat bid for 6, 21-day cycles. The primary endpoint was overall response. The rate of complete responses, duration of overall objective response, progression-free survival (PFS), and overall survival were the secondary endpoints.
RESULTS
Six objective partial responses were recorded in the 74 patients (8.1%) in the intention-to-treat population. Five of these responses involved the 40 evaluable patients (12.5%). Thirty-one percent of patients reported SAEs to the combination of talabostat and cisplatin.
CONCLUSION
Acceptable tolerability was observed in the intention-to-treat population and antitumor activity was observed in 12.5% of evaluable patients, which is not greater than historical expectation with cisplatin alone.
Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Boronic Acids; Cisplatin; Dipeptides; Disease Progression; Disease-Free Survival; Female; Humans; Male; Melanoma; Middle Aged; Prognosis; Skin Neoplasms; Treatment Outcome
PubMed: 19643020
DOI: 10.1186/1471-2407-9-263 -
Theranostics 2023Chemoimmunotherapy is a promising approach in cancer immunotherapy. However, its therapeutic efficacy is restricted by high reactive oxygen species (ROS) levels, an...
Chemoimmunotherapy is a promising approach in cancer immunotherapy. However, its therapeutic efficacy is restricted by high reactive oxygen species (ROS) levels, an abundance of cancer-associated fibroblasts (CAFs) in tumor microenvironment (TME) as well as immune checkpoints for escaping immunosurveillance. Herein, a new type of TME and reduction dual-responsive polymersomal prodrug (TRPP) nanoplatform was constructed when the D-peptide antagonist (PPA-1) of programmed death ligand-1 was conjugated onto the surface, and talabostat mesylate (Tab, a fibroblast activation protein inhibitor) was encapsulated in the watery core (PPA-TRPP/Tab). Doxorubicin (DOX) conjugation in the chain served as an immunogenic cell death (ICD) inducer and hydrophobic part. PPA-TRPP/Tab reassembled into a micellar structure with TME modulation by Tab, ROS consumption by 2, 2'-diselanediylbis(ethan-1-ol), immune checkpoint blockade by PPA-1 and ICD generation by DOX. This resolved the dilemma between a hydrophilic Tab release in the TME for CAF inhibition and intracellular hydrophobic DOX release for ICD via re-assembly in weakly acidic TME with polymersome-micelle transformation. results indicated that PPA-TRPP/Tab could improve tumor accumulation, suppress CAF formation, downregulate regulatory T cells and promote T lymphocyte infiltration. In mice, it gave a 60% complete tumor regression ratio and a long-term immune memory response. The study offers potential in tumor eradication via exploiting an "all-in-one" smart polymeric nanoplatform.
Topics: Animals; Mice; Prodrugs; Immune Checkpoint Inhibitors; Tumor Microenvironment; Reactive Oxygen Species; Immunogenic Cell Death; Antineoplastic Agents; Immunotherapy; Doxorubicin; Neoplasms; Micelles; Cell Line, Tumor
PubMed: 37064869
DOI: 10.7150/thno.83912 -
Molecular Metabolism Jan 2019Fibroblast Activation Protein (FAP), an enzyme structurally related to dipeptidyl peptidase-4 (DPP-4), has garnered interest as a potential metabolic drug target due to...
OBJECTIVE
Fibroblast Activation Protein (FAP), an enzyme structurally related to dipeptidyl peptidase-4 (DPP-4), has garnered interest as a potential metabolic drug target due to its ability to cleave and inactivate FGF-21 as well as other peptide substrates. Here we investigated the metabolic importance of FAP for control of body weight and glucose homeostasis in regular chow-fed and high fat diet-fed mice.
METHODS
FAP enzyme activity was transiently attenuated using a highly-specific inhibitor CPD60 and permanently ablated by genetic inactivation of the mouse Fap gene. We also assessed the FAP-dependence of CPD60 and talabostat (Val-boroPro), a chemical inhibitor reportedly targeting both FAP and dipeptidyl peptidase-4 RESULTS: CPD60 robustly inhibited plasma FAP activity with no effect on DPP-4 activity. Fap gene disruption was confirmed by assessment of genomic DNA, and loss of FAP enzyme activity in plasma and tissues. CPD60 did not improve lipid tolerance but modestly improved acute oral and intraperitoneal glucose tolerance in a FAP-dependent manner. Genetic inactivation of Fap did not improve glucose or lipid tolerance nor confer resistance to weight gain in male or female Fap mice fed regular chow or high-fat diets. Moreover, talabostat markedly improved glucose homeostasis in a FAP- and FGF-21-independent, DPP-4 dependent manner.
CONCLUSION
Although pharmacological FAP inhibition improves glucose tolerance, the absence of a metabolic phenotype in Fapmice suggest that endogenous FAP is dispensable for the regulation of murine glucose homeostasis and body weight. These findings highlight the importance of characterizing the specificity and actions of FAP inhibitors in different species and raise important questions about the feasibility of mouse models for targeting FAP as a treatment for diabetes and related metabolic disorders.
Topics: Animals; Blood Glucose; Body Weight; Diabetes Mellitus; Diet, High-Fat; Dipeptidyl Peptidase 4; Dipeptidyl-Peptidase IV Inhibitors; Endopeptidases; Female; Fibroblast Growth Factors; Gelatinases; Glucagon-Like Peptide 1; Glucose; Homeostasis; Insulin; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Serine Endopeptidases; Weight Gain
PubMed: 30477988
DOI: 10.1016/j.molmet.2018.10.011 -
Advanced Science (Weinheim,... Jan 2022Diabetic ulcers, a difficult problem faced by clinicians, are strongly associated with an increase in cellular senescence. Few empirical studies have focused on...
Diabetic ulcers, a difficult problem faced by clinicians, are strongly associated with an increase in cellular senescence. Few empirical studies have focused on exploring a targeted strategy to cure diabetic wounds by eliminating senescent fibroblasts (SFs) and reducing side effects. In this study, poly-l-lysine/sodium alginate (PLS) is modified with talabostat (PT100) and encapsulates a PARP1 plasmid (PARP1@PLS-PT100) for delivery to target the dipeptidyl peptidase 4 (DPP4) receptor and eliminate SFs. PARP1@PLS-PT100 releases encapsulated plasmids, displaying high selectivity for SFs over normal fibroblasts by targeting the DPP4 receptor, decreasing senescence-associated secretory phenotypes (SASPs), and stimulating the secretion of anti-inflammatory factors. Furthermore, the increased apoptosis of SFs and the disappearance of cellular senescence alleviates SASPs, accelerates re-epithelialization and collagen deposition, and significantly induces macrophage M2 polarization, which mediates tissue repair and the inflammatory response. This innovative strategy has revealed the previously undefined role of PARP1@PLS-PT100 in promoting diabetic wound healing, suggesting its therapeutic potential in refractory wound repair.
Topics: Alginates; Animals; Cells, Cultured; Cellular Senescence; Diabetes Mellitus, Experimental; Dipeptidyl Peptidase 4; Disease Models, Animal; Nanospheres; Poly (ADP-Ribose) Polymerase-1; Polylysine; Rats; Rats, Sprague-Dawley; Wound Healing
PubMed: 34738744
DOI: 10.1002/advs.202104128 -
Nature Chemical Biology Jan 2017Val-boroPro (Talabostat, PT-100), a nonselective inhibitor of post-proline cleaving serine proteases, stimulates mammalian immune systems through an unknown mechanism of...
Val-boroPro (Talabostat, PT-100), a nonselective inhibitor of post-proline cleaving serine proteases, stimulates mammalian immune systems through an unknown mechanism of action. Despite this lack of mechanistic understanding, Val-boroPro has attracted substantial interest as a potential anticancer agent, reaching phase 3 trials in humans. Here we show that Val-boroPro stimulates the immune system by triggering a proinflammatory form of cell death in monocytes and macrophages known as pyroptosis. We demonstrate that the inhibition of two serine proteases, DPP8 and DPP9, activates the pro-protein form of caspase-1 independent of the inflammasome adaptor ASC. Activated pro-caspase-1 does not efficiently process itself or IL-1β but does cleave and activate gasdermin D to induce pyroptosis. Mice lacking caspase-1 do not show immune stimulation after treatment with Val-boroPro. Our data identify what is to our knowledge the first small molecule that induces pyroptosis and reveals a new checkpoint that controls the activation of the innate immune system.
Topics: Animals; Boronic Acids; Caspase 1; Cell Line; Dipeptidases; Dipeptides; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Dose-Response Relationship, Drug; Humans; Leukocytes, Mononuclear; Macrophages; Mice; Molecular Conformation; Pyroptosis; Serine Proteinase Inhibitors; Structure-Activity Relationship
PubMed: 27820798
DOI: 10.1038/nchembio.2229 -
Cell Reports Oct 2020Several cytosolic pattern-recognition receptors (PRRs) form multiprotein complexes called canonical inflammasomes in response to intracellular danger signals. Canonical...
Several cytosolic pattern-recognition receptors (PRRs) form multiprotein complexes called canonical inflammasomes in response to intracellular danger signals. Canonical inflammasomes recruit and activate caspase-1 (CASP1), which in turn cleaves and activates inflammatory cytokines and gasdermin D (GSDMD), inducing pyroptotic cell death. Inhibitors of the dipeptidyl peptidases DPP8 and DPP9 (DPP8/9) activate both the human NLRP1 and CARD8 inflammasomes. NLRP1 and CARD8 have different N-terminal regions but have similar C-terminal regions that undergo autoproteolysis to generate two non-covalently associated fragments. Here, we show that DPP8/9 inhibition activates a proteasomal degradation pathway that targets disordered and misfolded proteins for destruction. CARD8's N terminus contains a disordered region of ∼160 amino acids that is recognized and destroyed by this degradation pathway, thereby freeing its C-terminal fragment to activate CASP1 and induce pyroptosis. Thus, CARD8 serves as an alarm to signal the activation of a degradation pathway for disordered and misfolded proteins.
Topics: Animals; Boronic Acids; CARD Signaling Adaptor Proteins; Dipeptides; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; HEK293 Cells; Humans; Inflammasomes; Intrinsically Disordered Proteins; Lysine; Mice; Neoplasm Proteins; Proteolysis; Proteostasis; RAW 264.7 Cells; THP-1 Cells
PubMed: 33053349
DOI: 10.1016/j.celrep.2020.108264