O reconhecimento da flagelina é compartilhado pelo receptor transmembrânico TLR5 e citosólico NAIP5/NLRC4. Entretanto, pouco se sabe sobre os mecanismos efetores individuais induzidos a partir do reconhecimento extra e intracelular da flagelina. Aqui, nós demonstramos que macrófagos estimulados com a flagelina citosólica (FLA-BSDot) induziu a expressão de iNOS, enzima responsável pela produção do óxido nítrico (NO). A expressão de iNOS foi dependente do eixo NAIP5/NLRC4/caspase-1 e independente de IL-1β, IL-18 e MyD88, descartando a via de ativação dos TLRs. Ainda, esta via não requer a ativação do fator de transcrição IRF-1, mas envolve a ativação do NF-kB, assim como a clivagem da enzima PARP-1 (poly(ADP-ribose)polymerase-1). Por fim, avaliamos a relevância biológica desta via no controle das infecções por L. pneumophila e S. Typhimurium, dados que definem um mecanismo efetor adicional no controle de patógenos. / Recognition of flagellin is shared by transmembranic TLR5 and cytosolic NAIP5/NLRC4. However, little is known about the individual effector mechanisms induced by extra and intracellular flagellin. Here, we have demonstrated that cytosolic flagellin-stimulated macrophages (FLA-BSDot) induced iNOS expression, an enzyme responsible for the production of nitric oxide (NO). iNOS expression was dependent of the NAIP5/NLRC4/caspase-1 axis and independent of IL-1β, IL-18 and MyD88, discarding TLRs signaling pathway. Still, this pathway do not require the activation of IRF-1 transcriptional factor, but involves NF-kB activation as well as the cleavage of the enzyme, PARP-1 (poly(ADP-ribose)polymerase-1). Finally, we have evaluated the biological relevance of this pathway in the control of the infections by L. pneumophila e S. Typhimurium, which define an additional effector mechanism to the control of pathogens.

Acute myocarditis is an acute inflammatory syndrome characterized by myocardial damage and dysfunction often due to a viral infection followed by a variable development over time. There are currently no specific treatments and standard treatments for heart failure are generally applied. The inflammasome is a recently identified macromolecular structure that occupies a central role in the amplification of the inflammatory response and promotion of cell death during acute and chronic infections. We hypothesized the formation of the inflammasome in acute myocarditis. To investigate, samples of patients were collected from the Cardiomyopathy Registry in Trieste, with 12 cases of biopsy-proven myocarditis and 11 cases of autopsy-proven myocarditis stained for major components of the inflammasome through immunofluorescence; 10 of the 12 (83.3%) biopsy cases and 8 of the 11 (72.7%) autopsy cases presented formation of the inflammasome in a variety of cells including resident cells (i.e. cardiomyocytes, endothelial cells, fibroblasts) and infiltrating cells (i.e. leukocytes) while varying in intensity and distribution. Control samples of 5 subjects not presenting with any acute cardiac events showed no formation of the inflammasome. While further studies should look to elucidate the correlation of inflammasome-formation and progression of disease, this finding paves the way for further insight into the pathophysiology of acute myocarditis.

Celiac disease is an autoimmune disease which occurs in susceptible individuals after ingestion of food containing gluten. Gluten and its monomeric fraction gliadin induce inflammatory damage of the small intestine by activating the immune cells that react strongly to gluten peptides. Gluten peptides have the ability to activate cells of adaptive as well as innate immune system. This work is focused on the production of interleukin (IL)-1 in antigen presenting cells stimulated with peptic gliadin digest. We found that monocytes and peripheral blood mononuclear cells (PBMC) isolated from blood of celiac patients secrete significantly more IL-1α and IL-1β than cells of healthy donors after stimulation with gliadin digest. The gliadin-induced IL-1β expression is controlled by a signaling cascade that includes MAPK kinase family molecules and transcription factor NF-κB. Moreover, we found that the adaptor proteins MyD88 and TRIF as well as Toll-like receptor (TLR) 2 and 4 play a role in the signaling cascade underlying gliadin-induced IL-1β expression by using murine bone marrow derived dendritic cells (BMDC). The precursor form of IL-1β in gliadin- stimulated PBMC and murine BMDC is maturated by caspase-1. In celiac PBMC the gliadin- induced maturation and secretion of IL-1β depends on the potassium...

Pattern recognition receptors coordinate innate immune responses by sensing infection or injury. Nucleotide-binding, leucine rich repeat, and pyrin domain-containing protein 3 (NLRP3) is a cytosolic PRR which perceives diverse pathogenic and sterile insults. NLRP3 orchestrates inflammatory signaling responses by forming inflammasomes with the adaptor protein apoptosis-associated speck like protein with a caspase recruitment domain (ASC) and the cysteine protease caspase-1. Assembly of the intracellular macromolecular inflammasome complex culminates in proximity-induced autocatalysis of caspase-1. Caspase-1 activation promotes cell death by pyroptosis and activation and secretion of proinflammatory cytokines interleukin (IL)-1β and IL-18. While NLRP3-mediated inflammation protects against bacterial, fungal, viral, and parasitic infections, aberrant NLRP3 activation is implicated in numerous inflammatory diseases and heritable syndromes. Mechanistically, inflammasome activation requires a preliminary NF-κB-activating priming step (signal 1) and a subsequent NLRP3-specific stimulus (signal 2). While there is enormous molecular diversity among NLRP3-specific agonists, this second signal appears to engage a common pathway involving cation flux. Furthermore, NLRP3 associates with mitochondria and mitochondrial damage is implicated in NLRP3 activation, although the precise role for mitochondria in inflammasome assembly remains controversial. We previously demonstrated that the mitochondrial phospholipid cardiolipin binds to NLRP3 and is critical for NLRP3 inflammasome activation. Here, we further investigated how mitochondria contribute to NLRP3 activation. We found that liposomes containing molar concentrations of cardiolipin that resemble mitochondrial cardiolipin levels can induce NLRP3-dependent caspase-1 autoactivation. Unexpectedly, we discovered that caspase-1 binds directly to cardiolipin, causing inflammasome-independent caspase-1 complex formation and autocatalysis at higher cardiolipin densities. Finding that caspase-1 and NLRP3 are independently capable of binding to cardiolipin, we more thoroughly examined the association of inflammasome components with mitochondria. Normally confined within mitochondrial inner membranes, cardiolipin relocates to outer membranes of stressed mitochondria. We found that reactive oxygen species (ROS) produced in response to signal 1 facilitate cardiolipin externalization to the outer membrane during priming. We also determined that this coincides with ROS-dependent recruitment of NLRP3 and caspase-1 to the outer membrane of mitochondria at priming. In contrast, we found that NLRP3 activation by the signal 2 agonist nigericin induces calcium-dependent recruitment of the adaptor ASC to mitochondria and caspase-1 activation. Finally, to determine what type of mitochondrial damage was necessary to promote NLRP3 inflammasome activation, we examined how different NLRP3 agonists affect mitochondria. We found substantial variability in the extent of mitochondrial damage induced among different NLRP3 agonists. Collectively, our findings illustrate that mitochondria serve as innate immune signaling platforms through multiple stages of NLRP3 inflammasome activation. Further, paralleling lipid A interactions with caspase-11, we have demonstrated that caspase-1 is capable of binding to the phospholipid cardiolipin.

Pseudomonas aeruginosa-induced pneumonia is serious problem that results in severe inflammation response and high mortality in the host. Interleukin-1£] (IL-1£]) is one of the major extracellular proinflammatory cytokines thought to be involved in many acute and chronic lung diseases. To investigate the role of caspase-1, IL-1£] and macrophage inflammatory protein-2 (MIP-2) in P. aeruginosa pneumonia induced lung damage, C57BL/6 (WT) and CASP-1-/- mice were subjected to pneumonia induced by intratracheal injection of P. aeruginosa. The lung permeability, bacterial content in blood and lung, lung myeloperoxidase (MPO) activity, total cell counts and protein in bronchoalveolar lavage fluid (BALF), NF-£eB activation as well as expression of IL-1£] and MIP-2 were assayed at 8 hr after P. aeruginosa injection. The IL-1£] inhibitor, anakinra, was also used to evaluate the role of IL-1£]. P. aeruginosa injection increased the lung permeability, lung MPO activity, bacterial counts in blood, total cell counts and protein in BALF, NF-£eB activation and expression of IL-1£] and MIP-2 in WT mice; and these increases were all decreased by administration of anakinra in WT mice or in CASP-1-/- mice. Furthermore, the lung MPO activity, total protein in BALF and expression of IL-1£] and MIP-2 were decreased in CASP-1-/- ¡÷ WT but not in WT ¡÷ CASP-1-/- chimeric mice, suggesting that pneumonia induced lung damage and IL-1£] and MIP-2 expressions depend on caspase-1 signaling of the resident cells.

Thesis (Ph. D.)--Case Western Reserve University, 2004. / [School of Medicine] Department of Pathology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Note: Portions of this abstract have been previously published in the journal Blood, Basiorka et al. Blood. 2016 Oct 13, and has been reproduced in this manuscript with permission from the publisher. Myelodysplastic syndromes (MDS) are genetically diverse hematopoietic stem cell malignancies that share a common phenotype of cytological dysplasia, ineffective hematopoiesis and aberrant myeloid lineage maturation. Apoptotic cell death potentiated by inflammatory cytokines has been considered a fundamental feature of MDS for over two decades. However, this non-inflammatory form of cell death cannot account for the inflammatory nature of these disorders. We report that a hallmark of lower-risk (LR) MDS is activation of the NLRP3 inflammasome, which drives clonal expansion and pyroptosis, a caspase-1-dependent programmed cell death induced by danger-associated molecular pattern (DAMP) signals. Independent of genotype, MDS hematopoietic stem and progenitor cells (HSPC) overexpress pyroptosis-related transcripts, inflammasome proteins and manifest activated NLRP3 inflammasome complexes that direct caspase-1 activation, IL-1β and IL-18 maturation and pyroptotic cell death. Using the S100A9 transgenic (S100A9Tg) mouse model that phenocopies human MDS, we demonstrated that forced expression of S100A9 was sufficient to drive pyroptosis in vivo, implicating pyroptosis as the principal mechanism of HSPC cell death in S100A9Tg mice. The lytic cell death releases intraceullar contents that include alarmins and catalytically active ASC specks, which can propagate bystander inflammation. Notably, MDS mesenchymal stromal cells (MSC) and stromal-derived linages were found to predominantly undergo pyroptosis, with marked activation of caspase-1 and NLRP3 inflammasome complexes. These findings may account for the clusters of both HSPC and stromal cell death previously described in the bone marrows of patients with MDS. Mechanistically, pyroptosis is triggered by the alarmin S100A9 that is found in excess in MDS HSPC and bone marrow (BM) plasma. Further, both somatic gene mutations and S100A9-induced signaling activate NADPH oxidase (NOX), generating reactive oxygen species (ROS) that initiate cation influx, cell swelling and β-catenin activation. Accordingly, ROS and active β-catenin were significantly increased in MDS BM mononuclear cells (BM-MNC) and S100A9Tg mice compared to normal controls, as well as in human cell lines harboring gene mutations and in murine models of gene mutation knock-in or gene loss. ROS and β-catenin nuclear translocation were significantly reduced by NLRP3 or NOX inhibition, indicating that S100A9 and somatic gene mutations prime cells to undergo NOX1/4-dependent NLRP3 inflammasome assembly, pyroptosis and β-catenin activation. Together, these data explain the concurrent proliferation and inflammatory cell death characteristic of LR-MDS. Given that loss of a gene-rich area in del(5q) disease results in derepression of innate immune signaling, we hypothesized that this genetic deficit would trigger assembly of the NLRP3 inflammasome complex, akin to the pathobiological mechanism characteristic of non-del(5q) MDS. To this end, we utilized two distinct murine models of del(5q) disease, namely in the context of Rps14 haploinsufficiency and concurrent loss of mDia1 and microRNA (miR)-146a. In both models, pyroptosis was not evident in the HSPC compartment; however, early erythroid progenitors displayed high fractions of pyroptotic cells. This was associated with significant increases in caspase-1 and NLRP3 inflammasome activation, ROS and nuclear localization of β-catenin, which was extinguished by inflammasome or NOX complex inhibition. These data suggest that early activation of the inflammasome drives cell death and prevents terminal maturation of erythroid precursors, accounting for the progressive anemia characteristic of del(5q) disease, whereby hematopoietic defects are primarily restricted to the erythroid compartment. Importantly, these data implicate a similar pathobiological mechanism in del(5q) MDS as is observed in non-del(5q) patients. The identification of the NLRP3 inflammasome as a pathobiological driver of the LR non-del(5q) and del(5q) MDS phenotype allows for novel therapeutic agent development. Notably, knockdown of NLRP3 or caspase-1, neutralization of S100A9, and pharmacologic inhibition of NLRP3 or NOX suppresses pyroptosis, ROS generation and nuclear β-catenin in MDS, and are sufficient to restore effective hematopoiesis. In del(5q) murine models, inhibition of the NLRP3 inflammasome significantly improved erythroid colony forming capacity by a mechanism distinct from that of lenalidomide, highlighting the translational potential for targeting this innate immune complex in this subset of MDS. Thus, alarmins and founder gene mutations in MDS license a common redox-sensitive inflammasome circuit, which suggests new avenues for therapeutic intervention. Furthermore, aggregated clusters of the NLRP3 adaptor protein ASC [apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD)] are referred to as ASC specks. During pyroptosis, ASC specks are released from dying cells and function as DAMP signals that propagate inflammation. In this way, specks are a surrogate marker for NLRP3 inflammasome activation and pyroptotic cell death. Given that pyroptosis is the predominant mechanism of cell death in MDS and ASC specks are readily quantified by flow cytometry, we hypothesized that BM or peripheral blood (PB) plasma-derived ASC specks may be a biologically rational biomarker for the diagnosis of MDS. The percentage of ASC specks were significantly increased in MDS BM plasma compared to normal, healthy donors, which was validated by confocal microscopy. PB plasma-derived ASC specks were significantly greater in LR- versus HR-MDS, consistent with the greater extent of cell death and myeloid-derived suppressor cell (MDSC) expansion in LR disease. As hyperglycemia induces NLRP3 inflammasome activation, plasma glucose levels were measured to adjust for this confounding variable. Subsequently, the percentage of glucose-adjusted, PB plasma-derived ASC specks was measured in a panel of specimens of varied hematologic malignancies. The corrected percentage of ASC specks was significantly increased in MDS compared to normal donors and to each other malignancy investigated, including other myeloid and lymphoid leukemias, myeloproliferative neoplasms and overlap syndromes, like chronic myelomonocytic leukemia (CMML). These data indicate that the glucose-adjusted ASC speck percentage is MDS-specific and may be a valuable diagnostic biomarker. At a cutoff of 0.039, the biomarker minimizes misclassification error and achieves 95% sensitivity and 82% specificity in classifying MDS from normal donors, other hematologic malignancies and T2D. Lastly, the biomarker declined with treatment response to lenalidomide in LR-MDS patients, but not to erythropoietin stimulating agent (ESA) or hypomethylating agent (HMA) therapy. As such, the percentage of ASC specks represents the first biologically rational, diagnostic biomarker for MDS that can be implemented with current diagnostic practices to reduce diagnostic error.

Vascular access dysfunction is a cause of morbidity and mortality in chronic kidney disease (CKD) patients that require hemodialysis. The major cause of vascular access failure is venous stenosis due to neointimal hyperplasia (NH). Vascular smooth muscle cells (VSMC) are critical for the development of NH lesions, as they have the ability to modulate their phenotype from a "contractile" to a "synthetic" phenotype in the presence of uremia, through the regulation of sensor genes for uremia danger signals and VSMC-specific differentiation genes. Recent research indicates that Caspase-1 (casp-1) activation plays an essential role in sensing metabolic danger signal-associated molecular patterns and initiating vascular inflammation. Carbamylated LDL, a uremic toxin that has been shown to be found in higher levels in patients with CKD and in CKD murine models when compared to controls, and could play a role in casp-1 activation. Therefore, the goal of this project is to examine the role of cLDL/CKD-driven casp-1 activation in VSMC and CKD-related NH. We have established a CKD mouse model and published on CKD-associated vascular remodeling. We exposed wild type and caspase-1 knockout mice to our CKD model, analyzed and quantified the NH lesion formed. We also examined in vitro and ex-vivo changes in VSMC-specific differentiation genes when exposed to uremic serum and cLDL, in the presence or absence of caspase-1 inhibitor. We found that CKD serum induces with casp-1 activation and phenotypic changes in VSMCs from a "contractile" to a "synthetic" phenotype, which are reversed with casp-1 inhibition. In an ex-vivo model using relative quantification we found that VSMC contractile markers α -Actin, Calponin, SM-22, and Smoothelin gene expression of CKD mouse carotid VSMC were higher in casp-1 knockout mice when compared to wild-type (1.40, 1.28, 1.22, 1.41 respectively). Also using an in-vivo model, relative quantification of α-actin decreased from 1.0 to 0.329 when VSMCs were exposed to uremic serum and but increased back to 0.588 when Caspase-1 inhibitor is added. The relative quantification of Calponin also decreased from 1.0 to 0.394 when exposed to uremic serum and increased back to 0.601 with caspase-1 inhibitor. We also found that caspase-1 deficiency significantly reversed CKD-related vascular remodeling in casp-1 knockout mice and reduced NH volume by 50% from 1,440,023in wild-type mice to 71,069 µm2 in casp-1 knockouts (p-value 0.002). This evidence provides evidence that casp-1 plays a critical role in NH formation. Furthermore our results provide a novel insight over the therapeutic potential of casp-1 inhibitors for CKD induced NH and other inflammation induced vascular remodeling. / Public Health

It is well established that cytosolic caspase-1 activation, mediated by inflammasome after pathogens-associated molecular patterns (PAMP) and metabolic danger-associated molecular patterns (DAMPs), mediates the initiation of inflammation in endothelial cells by its downstream targets such as Interleukin-1β (IL-1β), Interleukin-18 (IL-18), and Sirtuin-1. However, it remains unknown whether proatherogenic lipids lysophosphatidylcholine (LPC) and reactive oxygen species (ROS) can promote nuclear localization of caspase-1. Using biochemical, bioinformatic, and immunologic approaches, we made the following findings: (1) DNA damage was found in atherosclerotic mice. (2) A nuclear exportation signal was mapped in the CARD domain of pro-caspase-1. LPC promotes nuclear localization of pro-caspase-1 in human aortic endothelial cells (HAECs), which may interrupt DNA damage and repair pathways. (3) Blockage of caspase-1 nuclear cytosol trafficking in HAEC activated by LPC may mediate inflammation and interrupt cell cycle regulation. (4) Pro-caspase-1 in the nucleus inhibits inflammation but promotes interferon pathways. Activation of caspase-1 in the nucleus promotes aging- and fos-related antigen 2 (FRA2) mediated DNA damage and apoptosis. (5) Inhibition of SUMOylation decreases pro-caspase-1 translocation into the cytosol from the nucleus. (6) Blockage of caspase-1 cytosol nuclear trafficking in HAEC activated by H2O2 may decrease caspase-1 activity and increase cell viability. Our results demonstrate, for the first time, that caspase-1 patrols in the cell, senses danger signals and interrupts the balance between DNA damage and DNA repair pathways. It is a novel insight that not only should we suppress the inflammation in the cytosol but also in the nucleus, which is important for the future development of therapeutics for cardiovascular diseases and other inflammatory diseases. / Biomedical Sciences

L'inflammasome est une voie de signalisation du système immunitaire inné impliquée dans la lutte contre les pathogènes et notamment dans la réponse aux infections bactérienne. L'activation de l'inflammasome entraine la sécrétion de cytokines pro-inflammatoires et une mort cellulaire caspase-1 dépendante. Des dérégulations de l'inflammasome conduisent aussi à des syndromes auto-inflammatoires graves ; il est donc essentiel de mieux comprendre sa régulation. Francisella tularensis est une bactérie intracellulaire facultative responsable de la tularémie. Son pouvoir pathogène est lié à sa capacité à s'échapper rapidement de son phagosome. Le système de surveillance du macrophage détecte la présence de F. tularensis via l'inflammasome AIM2. La détection de l'ADN bactérien induit la formation d'un large complexe composé de AIM2, le récepteur, d'ASC, l'adaptateur et de caspase-1, l'effecteur ; ce complexe forme un speck visible dans la cellule. Nous avons utilisé l'infection par F. tularensis de macrophages primaires murins pour étudier la régulation de l'inflammasome AIM2 dans un contexte physiologique. Nous avons ainsi identifié une boucle de rétrocontrôle, médiée par la caspase-1, qui régule négativement la formation/stabilité des specks AIM2. Nous avons étudié le rôle de facteurs vacuolaires et des espèces réactives de l'oxygène et de l'azote dans l'activation de l'inflammasome AIM2 lors de l'infection par Francisella. Nous avons ainsi mis en évidence le rôle clef des péroxynitrites dans cette activation. Nos résultats suggèrent que des décomposeurs catalytiques des péroxynitrites pourraient avoir un rôle thérapeutique dans les maladies liées à l'inflammasome / The inflammasome is an innate immune signaling pathway involved in the fight against pathogens. This pathway can also be activated by danger signals. Inflammasome activation induces the release of the pro-inflammatory cytokines IL-1b and IL-18 and cell death in a caspase-1 dependent manner. The inflammasome pathway is a key antibacterial pathway. Deregulation of the inflammasome pathway can lead to serious auto-inflammatory syndromes ; it is therefore critical to better understand inflammasome regulation. Francisella tularensis is a facultative intracellular bacterium responsible for tularemia. Its ability to cause disease is linked to its ability to rapidly escape from the phagosome into the host cytosol where it replicates. The macrophage surveillance system can detect F. tularensis presence in the cytosol through the AIM2 inflammasome. Recognition of DNA induces the formation of a large complex consisting of AIM2, the receptor; ASC, the adaptor and caspase-1, the effector; this complex is visible as a speck within the cell. We used F. tularensis infection of bone marrow derived macrophages to study the activation of the AIM2 inflammasome in a physiological context. We have identified a feedback loop, dependent on caspase-1, negatively regulating speck formation/stability. Then, we studied the role of vacuolar factors and reactive oxygen and nitrogen species in the AIM2 inflammasome activation during Francisella infection. We also described a key role for peroxynitrite in this activation. Our results suggest that catalytic decomposer of peroxynitrite may have a therapeutic potential in diseases linked to inflammasome