Defect of suppression of inflammasome-independent interleukin-8 secretion from SW982 synovial sarcoma cells by familial Mediterranean fever-derived pyrin mutations

Familial Mediterranean fever (FMF) is a recessive inherited autoinflammatory syndrome. Patients with FMF have symptoms such as recurrent fever and abdominal pain, sometimes accompanied by arthralgia. Biopsy specimens have revealed substantial neutrophil infiltration into synovia. FMF patients have a mutation in the Mediterranean fever gene, encoding pyrin, which is known to regulate the inflammasome, a platform for processing interleukin (IL)-1β. FMF patients heterozygous for E148Q mutation, heterozygous for M694I mutation, or combined heterozygous for E148Q and M694I mutations, which were found to be major mutations in an FMF study group in Japan, suffer from arthritis, the severity of which is likely to be lower than in FMF patients with M694V mutations. Expression plasmids of wild-type (WT) pyrin and mutated pyrin, such as E148Q, M694I, M694V, and E148Q+M694I, were constructed, and SW982 synovial sarcoma cells were transfected with these expression plasmids. IL-8 and IL-6 were spontaneously secreted from the culture supernatant of SW982 cells without any stimulation, whereas IL-1β and TNF-α could not be detected even when stimulated with lipopolysaccharide. Notably, two inflammasome components, ASC and caspase-1, could not be detected in SW982 cells by Western blotting. IL-8 but not IL-6 secretion from SW982 cells was largely suppressed by WT pyrin, but less suppressed by mutated pyrin, which appeared to become weaker in the order of E148Q, M694I, E148Q+M694I, and M694V mutations. As for IL-8 and IL-6, similar results were obtained using stable THP-1 cells expressing the WT pyrin or mutated pyrins, such as M694V or E148Q, when stimulated by LPS. In addition, IL-8 secretion from mononuclear cells of FMF patients was significantly higher than that of healthy volunteers when incubated on a culture plate. Thus, our results suggest that IL-8 secretion from SW982 synovial sarcoma cells suppressed by pyrin independently of inflammasome is affected by pyrin mutations, which may reflect the activity in FMF arthritis.     

Critical roles of ASC inflammasomes in caspase-1 activation and host innate resistance to Streptococcus pneumoniae infection

Streptococcus pneumoniae is a Gram-positive, extracellular bacterium that is responsible for significant mortality and morbidity worldwide. Pneumolysin (PLY), a cytolysin produced by all clinical isolates of the pneumococcus, is one of the most important virulence factors of this pathogen. We have previously reported that PLY is an essential factor for activation of caspase-1 and consequent secretion of IL-1β and IL-18 in macrophages infected with S. pneumoniae. However, the host molecular factors involved in caspase-1 activation are still unclear. To further elucidate the mechanism of caspase-1 activation in macrophages infected with S. pneumoniae, we examined the involvement of inflammasomes in inducing this cellular response. Our study revealed that apoptosis-associated specklike protein containing a caspase recruitment domain (ASC), an adaptor protein for inflammasome receptors such as nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) and absent in melanoma 2 (AIM2), is essentially required for the induction of caspase-1 activation by S. pneumoniae. Caspase-1 activation was partially impaired in NLRP3(-/-) macrophages, whereas knockdown and knockout of AIM2 resulted in a clear decrease in caspase-1 activation in response to S. pneumoniae. These results suggest that ASC inflammasomes, including AIM2 and NLRP3, are critical for caspase-1 activation induced by S. pneumoniae. Furthermore, ASC(-/-) mice were more susceptible than wild-type mice to S. pneumoniae, with impaired secretion of IL-1β and IL-18 into the bronchoalveolar lavage after intranasal infection, suggesting that ASC inflammasomes contribute to the protection of host from infection with PLY-producing S. pneumoniae.     

ASC,which is composed of a PYD and a CARD,is up-regulated by inflammation and apoptosis in human neutrophils

ASC is an adaptor protein that is composed of two protein-protein interaction domains, a PYRIN domain (PYD), and a caspase-recruitment domain (CARD). Recently, ASC was identified as a binding partner of pyrin, which is the product of MEFV, a gene causing familial Mediterranean fever (FMF). Mutations in MEFV result in defects in control of neutrophil-mediated inflammation. Thus we focused on the expression of ASC in neutrophils. Immunohistochemical study showed that ASC is increased in neutrophils in severe inflammatory sites of gangrenous appendicitis. We, then, tested whether proinflammatory mediators induce ASC using peripheral blood neutrophils in vitro. ASC expression was transiently up-regulated by IL-1alpha, IL-1beta, IFN-alpha, IFN-gamma, TNFalpha, and LPS. ASC was also increased by incubation with either anti-Fas antibody or recombinant soluble Fas ligand. The Fas-mediated induction of ASC was inhibited by a general caspase inhibitor, z-VAD-fmk, and an immunocytochemical study showed that ASC was increased in neutrophils exhibiting characteristic phenotypes for apoptosis. These findings suggest that up-regulation of ASC is closely associated with inflammation and apoptosis in neutrophils.     

Significant role of IL-1 signaling, but limited role of inflammasome activation, in oviduct pathology during Chlamydia muridarum genital infection

IL-1β has been implicated in the development of oviduct pathology during Chlamydia muridarum genital infection in the mouse model. The goal of this study was to characterize the role of IL-1 signaling and the inflammasome-activation pathways during genital chlamydial infection. Compared with control mice, IL-1R-deficient mice displayed delayed clearance and increased chlamydial colonization. Consistent with the role for IL-1 signaling in infection clearance, mice deficient for the IL-1R antagonist cleared infection at a faster rate. Despite increased infection, IL-1R-deficient mice had significantly reduced oviduct pathology, which was associated with decreased numbers of neutrophils, but more macrophages, in the genital tract. IL-1β secretion is dependent on caspase-1 and apoptosis-associated speck-like protein containing caspase recruitment domain (ASC) inflammasome during in vitro infection of primed macrophages with C. muridarum. To investigate the role of inflammasome components during in vivo genital infection, mice lacking NLRP3, NLRC4, and ASC were tested and found to display no reduction in oviduct pathology compared with control mice. Mice deficient for ASC displayed a prolonged course of infection, which was associated with reduced T cell recruitment and proliferation. Further, ASC-deficient mice displayed normal levels of IL-1β in genital secretions. However, a significant decrease in caspase-1-dependent IL-18 was observed in both ASC- and NLRP3-deficient mice. These data demonstrate a major role for IL-1 signaling, but a limited role for the inflammasome pathway, in IL-1β secretion and development of oviduct pathology during genital chlamydial infection. The data also suggest an IL-1-independent role for ASC in adaptive immunity during genital chlamydial infection.     

Pyrin, product of the MEFV locus, interacts with the proapoptotic protein, Siva

Mutations in pyrin cause the autoinflammatory disorder familial Mediterranean fever (FMF), a syndrome characterized by sporadic and unpredictable attacks of fever and localized severe pain. Currently, it is not clear how attacks are triggered, nor why they spontaneously resolve after 2 or 3 days. In fact, the cellular function of the pyrin protein and the molecular underpinnings of its malfunction in FMF have so far eluded clear definition. The identification of pyrin-interacting proteins has the potential to increase our understanding of the cellular networks in which pyrin functions. Previous reports have established that pyrin interacts with the apoptotic protein ASC, the cytoskeletal adaptor protein PSTPIP1, the inflammatory caspase, Caspase-1 and certain forms of the cytosolic anchoring protein 14-3-3. Here, we report that pyrin also interacts with Siva, a pro-apoptotic protein first identified for its interaction with the cytosolic tail of CD27, a TNF family receptor. The interaction between pyrin and Siva involves the C-terminal B30.2/rfp/SRPY domain of pyrin and exon 1 of Siva. We show that Siva and pyrin are indeed co-expressed in human neutrophils, monocytes, and synovial cells. Furthermore, using a novel protein/protein interaction assay, we demonstrate that pyrin can recruit Siva to ASC specks, establishing a potential platform for intersection of ASC and Siva function. Finally, we show that pyrin modulates the apoptotic response to oxidative stress mediated by Siva. Thus, the Siva-pyrin interaction may be a potential target for future therapeutic strategies.     

The SPRY domain of Pyrin, mutated in familial Mediterranean fever patients, interacts with inflammasome components and inhibits proIL-1beta processing

The autoinflammatory disorders Muckle-Wells syndrome, familial cold urtecaria and chronic infantile neurological cutaneous and articular syndrome are associated with mutations in the NALP3 (Cryopyrin) gene, which is the central platform of the proinflammatory caspase-1 activating complex, named the inflammasome. In patients with another autoinflammatory disorder, familial Mediterranean fever (FMF), mutations in the SPRY domain of the Pyrin protein are frequently found. Recent evidence suggests that Pyrin associates with ASC, an inflammasome component, via its Pyrin domain, thereby halting the inflammatory response. This interaction, however, does not explain the effects of mutations of the SPRY domain found in FMF patients. Here we show that the Pyrin SPRY domain not only interacts with NALP3, but also with caspase-1 and its substrate pro-interleukin(IL)-1beta. Whereas a Pyrin knockdown results in increased caspase-1 activation and IL-1beta secretion, overexpression of the SPRY domain alone blocks these processes. Thus Pyrin binds to several inflammasome components thereby modulating their activity.     

Pyrin levels in human monocytes and monocyte-derived macrophages regulate IL-1beta processing and release

Macrophages and their precursors, monocytes, are key cells involved in the innate immune response. Although both monocytes and macrophages produce caspase-1, the key enzyme responsible for pro-IL-1beta processing; macrophages are limited in their ability to activate the enzyme and release functional IL-1beta. In this context, because mutations in the pyrin gene (MEFV) cause the inflammatory disorder familial Mediterranean fever, pyrin is believed to regulate IL-1beta processing. To determine whether variations in pyrin expression explain the difference between monocytes and macrophages in IL-1beta processing and release, pyrin was studied in human monocytes and monocyte-derived macrophages. Although monocytes express pyrin mRNA and protein, which is readily inducible by endotoxin, monocyte-derived macrophages express significantly less pyrin mRNA and protein. Pyrin levels directly correlated with IL-1beta processing in monocytes and macrophages; therefore, we asked whether pyrin might promote IL-1beta processing and release. HEK293 cells were transfected with pyrin, caspase-1, apoptotic speck protein with a caspase recruitment domain, and IL-1beta. Pyrin induced IL-1beta processing and release in a dose-dependent manner. Conversely, pyrin small interference RNA suppressed pro-IL-1beta processing in both THP-1 cells and fresh human monocytes. In summary, both pyrin expression and IL-1beta processing and release are diminished upon the maturation of monocytes to macrophages. When pyrin is ectopically expressed or silenced, IL-1beta processing and release parallels the level of pyrin. In conclusion, in the context of endotoxin-induced activation of mononuclear phagocytes, pyrin augments IL-1beta processing and release.     

Activation of the pyrin inflammasome by intracellular Burkholderia cenocepacia

Burkholderia cenocepacia is an opportunistic pathogen that causes chronic infection and induces progressive respiratory inflammation in cystic fibrosis patients. Recognition of bacteria by mononuclear cells generally results in the activation of caspase-1 and processing of IL-1β, a major proinflammatory cytokine. In this study, we report that human pyrin is required to detect intracellular B. cenocepacia leading to IL-1β processing and release. This inflammatory response involves the host adapter molecule ASC and the bacterial type VI secretion system (T6SS). Human monocytes and THP-1 cells stably expressing either small interfering RNA against pyrin or YFP-pyrin and ASC (YFP-ASC) were infected with B. cenocepacia and analyzed for inflammasome activation. B. cenocepacia efficiently activates the inflammasome and IL-1β release in monocytes and THP-1. Suppression of pyrin levels in monocytes and THP-1 cells reduced caspase-1 activation and IL-1β release in response to B. cenocepacia challenge. In contrast, overexpression of pyrin or ASC induced a robust IL-1β response to B. cenocepacia, which correlated with enhanced host cell death. Inflammasome activation was significantly reduced in cells infected with T6SS-defective mutants of B. cenocepacia, suggesting that the inflammatory reaction is likely induced by an as yet uncharacterized effector(s) of the T6SS. Together, we show for the first time, to our knowledge, that in human mononuclear cells infected with B. cenocepacia, pyrin associates with caspase-1 and ASC forming an inflammasome that upregulates mononuclear cell IL-1β processing and release.     

Expression of ASC in renal tissues of familial mediterranean fever patients with amyloidosis: postulating a role for ASC in AA type amyloid deposition

Familial Mediterranean fever (FMF) is characterized by recurrent attacks of fever and serositis; in some cases, ensuing amyloidosis results in kidney damage. Treatment with colchicine reduces the frequency and severity of FMF attacks and prevents amyloidosis, although the mechanisms behind these effects are unknown. Pyrin, the protein product of the MEFV gene, interacts with ASC, a key molecule in apoptotic and inflammatory processes. ASC forms intracellular speck-like aggregates that presage cell death. Here we show that cell death after ASC speck formation is much slower in nonmyeloid cells than in myeloid cells. Additionally, we demonstrate that colchicine prevents speck formation and show that specks can survive in the extracellular space after cell death. Because we also found that ASC is expressed in renal glomeruli of patients with FMF but not in those of control patients, we posit that high local ASC expression may result in speck formation and that specks from dying cells may persist in the extracellular space where they have the potential (perhaps in association with pyrin) to nucleate amyloid. The fact that speck formation requires an intact microtubule network as shown here could potentially account for the ability of prophylactic colchicine to prevent or reverse amyloidosis in patients with FMF.     

Differential regulation of caspase-1 activation, pyroptosis, and autophagy via Ipaf and ASC in Shigella-infected macrophages

Shigella infection, the cause of bacillary dysentery, induces caspase-1 activation and cell death in macrophages, but the precise mechanisms of this activation remain poorly understood. We demonstrate here that caspase-1 activation and IL-1beta processing induced by Shigella are mediated through Ipaf, a cytosolic pattern-recognition receptor of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, and the adaptor protein apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). We also show that Ipaf was critical for pyroptosis, a specialized form of caspase-1-dependent cell death induced in macrophages by bacterial infection, whereas ASC was dispensable. Unlike that observed in Salmonella and Legionella, caspase-1 activation induced by Shigella infection was independent of flagellin. Notably, infection of macrophages with Shigella induced autophagy, which was dramatically increased by the absence of caspase-1 or Ipaf, but not ASC. Autophagy induced by Shigella required an intact bacterial type III secretion system but not VirG protein, a bacterial factor required for autophagy in epithelial-infected cells. Treatment of macrophages with 3-methyladenine, an inhibitor of autophagy, enhanced pyroptosis induced by Shigella infection, suggesting that autophagy protects infected macrophages from pyroptosis. Thus, Ipaf plays a critical role in caspase-1 activation induced by Shigella independently of flagellin. Furthermore, the absence of Ipaf or caspase-1, but not ASC, regulates pyroptosis and the induction of autophagy in Shigella-infected macrophages, providing a novel function for NLR proteins in bacterial-host interactions.     

Cryopyrin and pyrin activate caspase-1, but not NF-kappaB, via ASC oligomerization

Mutations in cryopyrin and pyrin proteins are responsible for several autoinflammatory disorders in humans, suggesting that these proteins play important roles in regulating inflammation. Using a HEK293 cell-based reconstitution system that stably expresses ASC and procaspase-1 we demonstrated that neither cryopyrin nor pyrin or their corresponding disease-associated mutants could significantly activate NF-kappaB in this system. However, both cryopyrin and two disease-associated cryopyrin mutants induced ASC oligomerization and ASC-dependent caspase-1 activation, with the disease-associated mutants being more potent than the wild-type (WT) cryopyrin, because of increased self-oligomerization. Contrary to the proposed anti-inflammatory activity of WT pyrin, our results demonstrated that pyrin, like cryopyrin, can also assemble an inflammasome complex with ASC and procaspase-1 leading to ASC oligomerization, caspase-1 activation and interleukin-1beta processing. Thus, we propose that pyrin could function as a proinflammatory molecule.     

Distinct roles of TLR2 and the adaptor ASC in IL-1beta/IL-18 secretion in response to Listeria monocytogenes

Apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC) is an adaptor molecule that has recently been implicated in the activation of caspase-1. We have studied the role of ASC in the host defense against the intracellular pathogen Listeria monocytogenes. ASC was found to be essential for the secretion of IL-1beta/IL-18, but dispensable for IL-6, TNF-alpha, and IFN-beta production, in macrophages infected with Listeria. Activation of caspase-1 was abolished in ASC-deficient macrophages, whereas activation of NF-kappaB and p38 was unaffected. In contrast, secretion of IL-1beta, IL-6, and TNF-alpha was reduced in TLR2-deficient macrophages infected with Listeria; this was associated with impaired activation of NF-kappaB and p38, but normal caspase-1 processing. Analysis of Listeria mutants revealed that cytosolic invasion was required for ASC-dependent IL-1beta secretion, consistent with a critical role for cytosolic signaling in the activation of caspase-1. Secretion of IL-1beta in response to lipopeptide, a TLR2 agonist, was greatly reduced in ASC-null macrophages and was abolished in TLR2-deficient macrophages. These results demonstrate that TLR2 and ASC regulate the secretion of IL-1beta via distinct mechanisms in response to Listeria. ASC, but not TLR2, is required for caspase-1 activation independent of NF-kappaB in Listeria-infected macrophages.     

Interaction between pyrin and the apoptotic speck protein (ASC) modulates ASC-induced apoptosis

Patients with familial Mediterranean fever suffer sporadic inflammatory attacks characterized by fever and intense pain (in joints, abdomen, or chest). Pyrin, the product of the MEFV locus, is a cytosolic protein whose function is unknown. Using pyrin as a "bait" to probe a yeast two-hybrid library made from neutrophil cDNA, we isolated apoptotic speck protein containing a caspase recruitment domain (CARD) (ASC), a proapoptotic protein that induces the formation of large cytosolic "specks" in transfected cells. We found that when HeLa cells are transfected with ASC, specks are formed. After co-transfection of cells with ASC plus wild type pyrin, an increase in speck-positive cells is found, and speck-positive cells show increased survival. Immunofluorescence studies show that pyrin co-localizes with ASC in specks. Speck localization requires exon 1 of pyrin, but exon 1 alone of pyrin does not result in an increase in the number of specks. Exon 1 of pyrin and exon 1 of ASC show 42% sequence similarity and resemble death domain-related structures in modeling studies. These findings link pyrin to apoptosis pathways and suggest that the modulation of cell survival may be a component of the pathophysiology of familial Mediterranean fever.     

The role of the pro-apoptotic protein Siva in the pathogenesis of Familial Mediterranean fever: A structural and functional analysis

Familial Mediterranean fever (FMF) is an autosomal, recessive disease, attributed to mutations in MEFV gene encoding pyrin, which is characterized by recurrent, acute and self-limiting attacks of fever as well as an increased neutrophil and monocyte apoptosis. Most disease-associated mutations in MEFV gene reside on the C-terminal PRYSPRY (B30.2) domain of pyrin, an area found to interact with the pro-apoptotic protein Siva. Because apoptotic events may be contributing to endogenous inflammation we hypothesized that mutations in pyrin may affect Siva-mediated apoptosis. The confirmation of this hypothesis would be of a great biological significance since it would be demonstrated a connection between apoptosis and inflammation. We used homology modeling to construct a 3-D model of Siva protein and the constructed model of Siva defined structural elements with potential of binding other proteins to induce apoptosis. Given that Siva protein binds pyrin as shown by transfection and immunoprecipitation experiments, apoptosis was assessed by FACS and Western blotting. No differences in rates of apoptosis in myeloid cells (THP-1) upon transfection with either wt pyrin or mutant forms of pyrin were found. Patients with FMF did not display any mutations in the Siva-1 (full length) gene. Siva-1 was not linked to pyrin in the major predicted FMF gene network constructed using a literature-curated gene signature for FMF. These results suggest that Siva-mediated unprovoked apoptosis is not likely to be involved in the pathogenesis of FMF.     

Apoptosis-associated speck-like protein containing a caspase recruitment domain is a regulator of procaspase-1 activation

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/target of methylation-induced silencing/PYCARD represents one of only two proteins encoded in the human genome that contains a caspase recruitment domain (CARD) together with a pyrin, AIM, ASC, and death domain-like (PAAD)/PYRIN/DAPIN domain. CARDs regulate caspase family proteases. We show here that ASC binds by its CARD to procaspase-1 and to adapter proteins involved in caspase-1 activation, thereby regulating cytokine pro-IL-1beta activation by this protease in THP-1 monocytes. ASC enhances IL-1beta secretion into the cell culture supernatants, at low concentrations, while suppressing at high concentrations. When expressed in HEK293 cells, ASC interferes with Cardiak/Rip2/Rick-mediated oligomerization of procaspase-1 and suppresses activation this protease, as measured by protease activity assays. Moreover, ASC also recruits procaspase-1 into ASC-formed cytosolic specks, separating it from Cardiak. We also show that expression of the PAAD/PYRIN family proteins pyrin or cryopyrin/PYPAF1/NALP3 individually inhibits IL-1beta secretion but that coexpression of ASC with these proteins results in enhanced IL-1beta secretion. However, expression of ASC uniformly interferes with caspase-1 activation and IL-1beta secretion induced by proinflammatory stimuli such as LPS and TNF, suggesting pathway competition. Moreover, LPS and TNF induce increases in ASC mRNA and protein expression in cells of myeloid/monocytic origin, revealing another level of cross-talk of cytokine-signaling pathways with the ASC-controlled pathway. Thus, our results suggest a complex interplay of the bipartite adapter protein ASC with PAAD/PYRIN family proteins, LPS (Toll family receptors), and TNF in the regulation of procaspase-1 activation, cytokine production, and control of inflammatory responses.     

Immunological function of familial Mediterranean fever disease protein Pyrin

Pyrin,encoded by MEFV gene,is conserved in humans and mice.Mutations in the MEFV gene are associated with the human autoinflammatory disease familial Mediterranean fever(FMF).Pyrin can interact with the inflammasome adaptor ASC and induce inflammatory caspase-1 activation in monocytic cells,but the physiological function of Pyrin has been unknown for many years.Here we summarize previous studies of Pyrin function under the context of FMF and immunity,and discuss a recent study demonstrating that Pyrin forms an inflammasome complex for caspase-1 activation in innate immunity.Pyrin inflammasome detects inactivating modifications of host Rho GTPases by diverse bacterial toxins and infections,including Clostridium difficile glucosylating cytotoxin Tcd B,FIC-domain adenylyltransferase effectors from Vibrio parahaemolyticus and Histophilus somni,ADP-ribosylating Clostridium botulinum C3 toxin as well as Burkholderia cenocepacia infection.The mode of Pyrin action,i.e.,sensing pathogen virulence activity rather than directly recognizing a microbial molecule,represents a new paradigm in innate immunity.     

RAC1 expression and role in IL-1β production and oxidative stress generation in familial Mediterranean fever (FMF) patients

Objectives

Familial Mediterranean fever (FMF) is a recessively inherited autoinflammatory disorder. The caspase-1-dependent cytokine, IL-1β, plays an important role in FMF pathogenesis, and RAC1 protein has been recently involved in IL-1β secretion. This study aims to investigate RAC1 expression and role in IL-1β and caspase-1 production and oxidative stress generation in FMF.

Materials and methods

The study included 25 FMF patients (nine during attack and remission, and 16 during remission only), and 25 controls. RAC1 expression levels were analyzed by real-time PCR. Ex vivo production of caspase-1, IL-1β, IL-6 and markers of oxidative stress (malondialdehyde, catalase, and glutathione system) were evaluated respectively in supernatants of patients’ and controls’ PBMC and PMN cultures, in the presence and absence of RAC1 inhibitor.

Results

RAC1 gene expression and IL-1β levels were increased in patients in crises compared to those in remission or controls. RAC1 expression levels were correlated with MEFV genotypes, patients carrying the M694V/M694V genotype having a two-fold increase in the expression levels compared to those carrying other genotypes. Caspase-1 levels were higher in LPS-induced PBMC of patients in remission than controls. Spontaneous and LPS-induced IL-1β production were comparable in patients in remission and controls, whereas LPS-induced IL-6 production was enhanced in patients, compared to controls. RAC1 inhibition resulted in a decrease in caspase-1 and IL-1β, but not IL-6, levels. Malondialdehyde levels produced by LPS-stimulated PMNs were increased in patients in remission compared to those in controls, but decreased following RAC1 inhibition. Catalase and GSH activities were reduced in unstimulated PMN culture supernatants of patients in remission compared to controls and were increased in the presence of RAC1 inhibitor.

Conclusion

These results show the involvement of RAC1 in the inflammatory process of FMF by enhancing IL-1β production, through caspase-1 activation, and generating oxidative stress, even during asymptomatic periods.

     

ASC/PYCARD and caspase-1 regulate the IL-18/IFN-gamma axis during Anaplasma phagocytophilum infection

Anaplasma phagocytophilum is an obligate intracellular pathogen that resides within neutrophils and can cause fever, pancytopenia, or death. IFN-gamma plays a critical role in the control of A. phagocytophilum; however, the mechanisms that regulate IFN-gamma production remain unclear. In this study, we demonstrate that apoptotic specklike protein with a caspase-activating recruiting domain (ASC)/PYCARD, a central adaptor molecule in the Nod-like receptor (NLR) pathway, regulates the IL-18/IFN-gamma axis during A. phagocytophilum infection through its effect on caspase-1. Caspase-1- and asc-null mice were more susceptible than control animals to A. phagocytophilum infection due to the absence of IL-18 secretion and reduced IFN-gamma levels in the peripheral blood. Moreover, caspase-1 and ASC deficiency reduced CD4+ T cell-mediated IFN-gamma after in vitro restimulation with A. phagocytophilum. The NLR family member IPAF/NLRC4, but not NALP3/NLRP3, was partially required for IFN-gamma production in response to A. phagocytophilum. Taken together, our data demonstrate that ASC and caspase-1 are critical for IFN-gamma-mediated control of A. phagocytophilum infection.     

A role for Nod-like receptors in autophagy induced by Shigella infection

Shigella infection, the cause of bacillary dysentery, induces caspase-1 activation and cell death in macrophages, but the precise mechanisms remain poorly understood. In our recent study, we presented evidence that caspase-1 activation and IL-1beta processing induced by Shigella are mediated through Ipaf, a cytosolic pattern-recognition receptor of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family and the adaptor protein apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). We also show that Ipaf and caspase-1 were critical for pyroptosis, a specialized form of caspase-1-dependent cell death induced in macrophages by bacterial infection, whereas ASC is dispensable. Notably, infection of macrophages with Shigella induced autophagy, which was dramatically increased by the absence of caspase-1 or Ipaf, but not ASC. Furthermore, autophagy induction was associated with transient resistance to pyroptosis. These results indicate that autophagy in macrophages is regulated by the Ipaf inflammasome, providing a novel function for NLR proteins in bacterial-host interactions.     

The effect of colchicine on pyrin and pyrin interacting proteins

MEFV which encodes pyrin, cause familial Mediterranean fever (FMF), the most common auto‐inflammatory disease. Pyrin is believed to be a regulator of inflammation, though the nature of this regulatory activity remains to be identified. Prophylactic treatment with colchicine, a microtubule toxin, has had a remarkable effect on disease progression and outcome. It has been thought that, inhibition of microtubule polymerization is the main mechanism of action of colchicine. But, the exact cellular mechanism explaining the efficacy of colchicine in suppressing FMF attacks is still unclear. Given the ability of colchicine treatment to be considered as a differential diagnosis criteria of FMF, we hypothesized that colchicine may have a specific effect on pyrin and pyrin interacting proteins. This study showed that colchicine prevents reticulated fibrils formed by PSTPIP1 filaments and reduces ASC speck rates in transfected cells. We further noted that, colchicine down‐regulates MEFV expression in THP‐1 cells. We also observed that colchicine causes re‐organization of actin cytoskeleton in THP‐1 cells. Pyrin is an actin‐binding protein that specifically localizes with polymerizing actin filaments. Thus, MEFV expression might be affected by re‐organization of actin cytoskeleton. The data presented here reveal an important connection between colchicine and pyrin which might explain the remarkable efficacy of colchicine in preventing FMF attacks. J. Cell. Biochem. 113: 3536–3546, 2012. © 2012 Wiley Periodicals, Inc.