Modulation of Inflammasome Activity by miR-197-3p in Familial Mediterranean Fever Mouse Macrophages.

Autophagy is a key regulator of cellular homeostasis that can be activated by pathogen-associated molecules and recently has been shown to influence IL-1β secretion by macrophages. However, the mechanisms behind this are unclear. Here, we describe a novel role for autophagy in regulating the production of IL-1β in antigen-presenting cells. After treatment of macrophages with Toll-like receptor ligands, pro-IL-1β was specifically sequestered into autophagosomes, whereas further activation of autophagy with rapamycin induced the degradation of pro-IL-1β and blocked secretion of the mature cytokine. Inhibition of autophagy promoted the processing and secretion of IL-1β by antigen-presenting cells in an NLRP3- and TRIF-dependent manner. This effect was reduced by inhibition of reactive oxygen species but was independent of NOX2. Induction of autophagy in mice in vivo with rapamycin reduced serum levels of IL-1β in response to challenge with LPS. These data demonstrate that autophagy controls the production of IL-1β through at least two separate mechanisms: by targeting pro-IL-1β for lysosomal degradation and by regulating activation of the NLRP3 inflammasome.

Renal amyloidosis in familial Mediterranean fever

NOD-Like Receptor Protein 3 Inflammasome Priming and Activation in Barrett’s Epithelial Cells

Syndrome of undifferentiated recurrent fever (SURF) is characterized by recurrent fevers, a lack of confirmed molecular diagnosis, and a complete or partial response to colchicine. Despite the clinical similarities to familial Mediterranean fever (FMF), the underlying inflammatory mechanisms of SURF are not yet understood. We here analyzed the in vitro activation of the pyrin inflammasome in a cohort of SURF patients compared to FMF and PFAPA patients. Peripheral blood mononuclear cells (PBMC) were collected from SURF (both colchicine-treated and untreated), FMF, PFAPA patients, and healthy donors. PBMC were stimulated ex vivo with Clostridium difficile toxin A (TcdA) and a PKC inhibitor (UCN-01), in the presence or absence of colchicine. The assembly of the pyrin inflammasome was evaluated by measuring the presence of apoptosis-associated Speck-like protein containing caspase recruitment domain (ASC) specks in monocytes using flow cytometry. IL-1β secretion was quantified using an ELISA assay. No differences in TcdA-induced activation of pyrin inflammasome were observed among FMF, PFAPA, and healthy donors. Untreated SURF patients showed a reduced response to TcdA, which was normalized after colchicine treatment. In contrast to FMF, SURF patients, similar to PFAPA patients and healthy donors, did not exhibit pyrin inflammasome activation in response to UCN-01-mediated pyrin dephosphorylation. These data demonstrate that in vitro functional analysis of pyrin inflammasome activation can differentiate SURF from FMF and PFAPA patients, suggesting the involvement of the pyrin inflammasome in the pathophysiology of SURF.

Familial Mediterranean fever (FMF) is an autoinflammatory disease. Its major clinical manifestations are recurrent paroxysmal fever, serositis and arthritis. Activated neutrophils are involved in the pathogenesis of FMF and abundant neutrophils are found in the affected regions of individuals with FMF.1 Although myalgia is well documented in FMF patients, the histological manifestations of the affected muscles remain unclear. We herein describe the first report of a patient with FMF in whom the infiltration of neutrophil was histologically confirmed, and we describe how treatment with tocilizumab (TCZ) significantly improved C-reactive protein (CRP) levels and the symptoms.2 The patient was a 64-year-old Japanese woman who had been treated with prednisolone (PSL; 10–20 mg/day) for the prior 7 years for undiagnosed recurrent fever and myalgia. PSL was not considered to be effective since her intermittent fever and myalgia continued. Her case did not fulfill the classification criteria of dermatomyositis or polymyositis developed by Bohan and Peter.2 She had no family history of recurrent fever or myalgia. On admission to our hospital in September 2013, her body temperature was 39.2°C. Her blood pressure, pulse rate and respiratory rate were normal. Physical examination revealed marked swelling and muscle grasp pain on the left thigh. The initial laboratory studies revealed: white blood cell count 9.2 109/L (87.8% neutrophils, 4.2% lymphocytes), hemoglobin 133 g/L, platelet count 188 109/L, and CRP 22.8 mg/L. The aspartate aminotransferase (AST) level was 147 IU/L, alanine aminotransferase (ALT) was 71 IU/L, lactate dehydrogenase was 286 IU/L and creatine phosphokinase was 154 IU/L. Serum immunoglobulin levels, complements and ferritin were normal. Rheumatoid factor, anti-nuclear antibodies, anti-DNA antibodies, anti-ribonucleoprotein antibodies, anti-Sjögren syndrome-A antibodies, anticyclic citrullinated peptide antibodies, anti-jo-1 antibodies and anti-aminoacyl tRNA synthetase antibodies were negative. Magnetic resonance imaging (MRI) of the lower limbs showed high intensity in femoral muscle and fascia on short TI inversion recovery (STIR) images. Figure 1 shows the MRI of the patient's lower limbs at the time of relapse of myalgia over the period 2006–2014, including this admission. The high intensity of muscle and fascia had appeared in different sites in each attack. A muscle biopsy of left quadriceps revealed the infiltration of neutrophils into the fascia as well as vascular endothelium of fascia (Fig. 2). A gene analysis revealed the E148Q hetero variant of the Mediterranean fever (MEFV) gene. We treated the patient with colchicine, which resulted in a rapid improvement of her fever, myalgia and inflammatory reaction symptoms. We thus made the diagnosis of FMF based on Tel-Hashomer criteria.3 Although colchicine treatment showed a remarkable effect for several months, an FMF attack occurred again 5 months after the initiation of colchicine treatment. Therefore, we added TCZ at the dose of 8 mg/kg every 4 weeks by intravenous administration. Since the initiation of this TCZ treatment, the FMF attacks of fever and myalgia have been completely inhibited for 9 months. Myalgia has been reported in many patients with FMF. The incidence of myalgia in Japanese FMF patients was reported to be 11.9%.4 The myalgia in FMF patients shows the following three clinical patterns: the spontaneous pattern, the exertion-induced pattern and protracted febrile myalgia syndrome (PFMS) pattern, seen in 8%, 81% and 11% of 65 patients, respectively.5 PFMS is characterized by the presence of persistent febrile paralyzing myalgia that may last as long as 1 month.6 PFMS is resistant to colchicine, but responds well to steroid treatment. We concluded that the present case corresponds to the spontaneous pattern. To the best of our knowledge, only two cases of histological examinations of myositis in patients with FMF have been reported. Gdynia et al.7 described the case of a 40-year-old Turkish man with FMF presenting myalgia. He had an M694I mutation in the MEFV gene. The muscle biopsy showed no inflammatory infiltrates, but an immunohistochemical analysis revealed a high deposition of immunoglobulin G (IgG) and major histocompatibility complex 1 (MHC1) with sarcolemmal staining, which are signs of an inflammatory component with single muscle fiber degeneration. The other case was described by Estublier et al.8: a 39-year-old Jewish man with FMF presented with myalgia with the M694I homozygous mutation in the MEFV gene. Similar to the former case, a muscle biopsy showed only nonspecific inflammation of the muscle. On the other hand, our patient's histological findings showed an abundant infiltration of neutrophils into the fascia. Some of the neutrophils also showed rolling in the vascular endothelium of fascia. The protein pyrin, encoded by MEFV gene, is considered to be responsible for the pathogenesis of FMF. Pyrin is highly expressed in neutrophils, and it regulates processes of neutrophil activation such as adhesion and migration at the level of the cytoskeleton.9 An FMF attack is characterized by the massive infiltration of neutrophils into the site of inflammation.1 The present case report is the first in which the infiltration of neutrophils was histologically proven at the site of a myalgia attack of FMF. The positive effect of TCZ therapy was demonstrated in the present case. Although the efficacy of biologic agents against colchicine-resistant FMF is limited and needs to be discussed, Bilgen et al.10 reported that tumor necrosis factor (TNF) inhibitors have been used successfully as treatment for colchicine-resistant FMF patients. In their report, etanercept, infliximab and adalimumab were administered to a total of 10 patients. After the introduction of TNF inhibitors, the FMF attacks disappeared in seven patients, and the frequency of attacks decreased in the other three patients. The anakinra, an interleukin (IL)-1 receptor antagonist, was also reported to be effective for 28 colchicine-resistant FMF patients; complete remission was achieved by 21 patients and partial remission was observed in the other seven patients;8 however, anakinra is not yet approved in Japan. We recently treated another FMF patient successfully with TCZ.11 FMF is thought to be an essentially IL-1-mediated disease, and IL-1 induces IL-6 transcript; a significant increase in serum IL-6 levels is observed in FMF patients.12 In addition, Hamanoue et al.13 reported successful treatment with TCZ in a case of amyloid A (AA) amyloidosis complicated by FMF possessing E148Q/M694I variants. Therefore, we selected TCZ for the present patient, and her clinical course showed the remarkable effect of TCZ not only for fever but also for myalgia. The level of CRP was also completely suppressed to the normal range. In conclusion, in a patient with FMF, we first detected neutrophil-dependent inflammation in the muscle tissues histologically. TCZ can be a good treatment option for colchicine-resistant FMF patients. We thank the late Toshiro Yoshimura, Nagasaki University School of Health Sciences, for the muscle biopsy. None.

Familial Mediterranean Fever (FMF) is the most common monogenic autoinflammatory disorder. FMF is caused by mutations in the MEFV gene, encoding pyrin, an inflammasome sensor. The best characterized pathogenic mutations associated with FMF cluster in exon 10. Yet, mutations have been described along the whole MEFV coding sequence. Exon 10 encodes the B30.2 domain of the pyrin protein, but the function of this human-specific domain remains unclear. Pyrin is an inflammasome sensor detecting RhoA GTPase inhibition following exposure to bacterial toxins such as TcdA. Here, we demonstrate that the B30.2 domain is dispensable for pyrin inflammasome activation in response to this toxin. Deletion of the B30.2 domain mimics the most typical FMF-associated mutation and confers spontaneous inflammasome activation in response to pyrin dephosphorylation. Our results indicate that the B30.2 domain is a negative regulator of the pyrin inflammasome that acts independently from and downstream of pyrin dephosphorylation. In addition, we identify the central helical scaffold (CHS) domain of pyrin, which lies immediately upstream of the B30.2 domain as a second regulatory domain. Mutations affecting the CHS domain mimic pathogenic mutations in the B30.2 domain and render the pyrin inflammasome activation under the sole control of the dephosphorylation. In addition, specific mutations in the CHS domain strongly increase the cell susceptibility to steroid catabolites, recently described to activate pyrin, in both a cell line model and in monocytes from genotype-selected FMF patients. Taken together, our work reveals the existence of two distinct regulatory regions at the C-terminus of the pyrin protein, that act in a distinct manner to regulate positively or negatively inflammasome activation. Furthermore, our results indicate that different mutations in pyrin regulatory domains have different functional impacts on the pyrin inflammasome which could contribute to the diversity of pyrin-associated autoinflammatory diseases.

Enterococcus Faecalis activates NLRP3 inflammasomes leading to increased interleukin-1 beta secretion and pyroptosis of THP-1 macrophages

Aberrant pyrin inflammasome activity triggers familial Mediterranean fever (FMF) pathogenesis, but the exact mechanism remains elusive and an obstacle to efficient treatment. We undertook this study to identify pyrin inflammasome-specific mechanisms to improve FMF treatment and diagnostics in the future. Pyrin-specific protein secretion was assessed by proteome analysis in U937-derived macrophages, and specific findings were confirmed in pyrin inflammasome-activated monocytes from healthy blood donors and patients with FMF, stratified according to MEFV genotype categories corresponding to a suspected increase in FMF disease severity. Proteome data revealed a differential secretion pattern of interleukin-1 receptor antagonist (IL-1Ra) from pyrin- and NLRP3-activated U937-derived macrophages, which was verified by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. Moreover, pyrin activation significantly reduced IL1RN messenger RNA expression (P < 0.001) and IL-1Ra secretion (P < 0.01) in healthy donor and FMF monocytes, respectively. Independent of MEFV genotype, unstimulated FMF monocytes from colchicine-treated patients secreted lower amounts of IL-1Ra compared to healthy donors (P < 0.05) and displayed decreased ratios of IL-1Ra:IL-1β (P < 0.05), suggesting a reduced antiinflammatory capacity. Our data show an inherent lack of IL-1Ra expression specific to pyrin inflammasome activation, suggesting a new mechanism underlying FMF pathogenesis. The reduced IL-1Ra levels in FMF monocytes suggest a diminished antiinflammatory capacity that potentially leaves FMF patients sensitive to proinflammatory stimuli, regardless of receiving colchicine therapy. Thus, considering the potential clinical consequence of reduced monocyte IL-1Ra secretion in FMF patients, we suggest further investigation into IL-1Ra dynamics and its potential implications for FMF treatment in the future.

Protective effect of gedunin on TLR-mediated inflammation by modulation of inflammasome activation and cytokine production: Evidence of a multitarget compound

Familial Mediterranean fever (FMF) is a monogenic autoinflammatory disorder with recurrent fever, abdominal pain, serositis, articular manifestations, erysipelas-like erythema, and renal complications as its main features. Caused by the mutations in the MEditerranean FeVer (MEFV) gene, it mainly affects people of Mediterranean descent with a higher incidence in the Turkish, Jewish, Arabic, and Armenian populations. As our understanding of FMF improves, it becomes clearer that we are facing with a more complex picture of FMF with respect to its pathogenesis, penetrance, variant type (gain-of-function vs. loss-of-function), and inheritance. In this study, MEFV gene analysis results and clinical findings of 27,504 patients from 35 universities and institutions in Turkey and Northern Cyprus are combined in an effort to provide a better insight into the genotype-phenotype correlation and how a specific variant contributes to certain clinical findings in FMF patients. Our results may help better understand this complex disease and how the genotype may sometimes contribute to phenotype. Unlike many studies in the literature, our study investigated a broader symptomatic spectrum and the relationship between the genotype and phenotype data. In this sense, we aimed to guide all clinicians and academicians who work in this field to better establish a comprehensive data set for the patients. One of the biggest messages of our study is that lack of uniformity in some clinical and demographic data of participants may become an obstacle in approaching FMF patients and understanding this complex disease.

Atheroprone flow promotes inflammation in endothelial cells, and this process is critical for pathogenesis of many chronic inflammatory conditions such as coronary and carotid artery atherosclerosis, as well as abdominal aortic aneurysm. Signal mediators activated by atheroprone (disturbed) flow that have been described include nuclear factor κB and protein kinase ζ, which is very different from atheroprotective (steady laminar) flow.1 In this issue of Circulation , an article from Xiao et al2 shows the critical role of sterol regulatory element binding protein 2 (SREBP2) on atheroprone flow–mediated Nod-like receptor protein 3 (NLRP3) inflammasome activation. In particular, they showed that atheroprone flow induced both mature form of SREBP2 (SREBP2-N) and SREBP2 mRNA induction, which transcriptionally increase NADPH oxidase 2 (Nox2) and NLRP3 expression, thereby leading to interleukin 1β (IL-1β) expression and endothelial inflammation (Figure 1). In this editorial, we briefly review the NLRP3 inflammasome and SREBP activation system, which play a key role in modulating atheroprone flow–mediated endothelial cell inflammation. We also discuss the following important considerations for the future: the role of local NLRP3 and IL-1β expression, mechanisms for two different types of flow (atheroprone flow versus atheroprotective flow) on SREBP2 activation, and other NLRP3 activators including thioredoxin-interacting protein (TXNIP). Figure 1. Scheme for sterol regulatory element binding protein 2 (SREBP2)-mediated Nod-like receptor protein 3 (NLRP3) inflammasome activation. IL indicates interleukin; Nox, NADPH oxidase; ROS, reactive oxygen species; and TXNIP, thioredoxin-interacting protein. Article see p 632 The inflammasome is a protein complex that serves as a platform for the maturation of caspase-1 subsequent activation, leading to proteolytic maturation and secretion of IL-1β and IL-18 (Figure 8 in Xiao et al2). Three essential components of inflammasome are a sensor protein, the adapter protein ASC, and the inflammatory protease caspase-1. As a sensor protein, Nod-like receptor (NLR) family (NLRP1, NLRP3, and …

Prevalence of the MEFV Gene Mutations in Childhood Polyarteritis Nodosa

Initial periodontal treatment affects nucleotide-binding domain leucine-rich repeat-containing protein 3 inflammasome priming in peripheral blood mononuclear cells

Pyrin is an inflammasome sensor that promotes caspase-1-mediated pyroptotic cell death and maturation of proinflammatory cytokines IL-1β and IL-18. Familial Mediterranean fever (FMF), an autoinflammatory disorder, is associated with mutations in the gene encoding pyrin (MEFV). FMF-knockin (FMF-KI) mice that express chimeric pyrin protein with FMF mutation (MefvV726A/V726A) exhibit an autoinflammatory disorder mediated by autoactivation of the pyrin inflammasome. Increase in the levels of TNF are observed in FMF-KI mice, and many features of FMF overlap with the autoinflammatory disorder associated with TNF receptor signaling. In this study, we assessed the contribution of TNF signaling to pyrin inflammasome activation and its consequent role in distinct FMF pathologies. TNF signaling promoted the expression of pyrin in response to multiple stimuli and was required for inflammasome activation in response to canonical pyrin stimuli and in myeloid cells from FMF-KI mice. TNF signaling promoted systemic wasting, anemia, and neutrophilia in the FMF-KI mice. Further, TNF-induced pathology was induced specifically through the TNFR1 receptor, while TNFR2-mediated signaling was distinctly protective in colitis and ankle joint inflammation. Overall, our data show that TNF is a critical modulator of pyrin expression, inflammasome activation, and pyrin-inflammasomopathy. Further, specific blockade of TNFR1 or activation of TNFR2 could provide substantial protection against FMF pathologies.

Familial Mediterranean fever (FMF) is the most frequent hereditary systemic autoinflammatory syndrome. FMF is usually caused by biallelic mutations in the MEFV gene, encoding Pyrin. Conclusive genetic evidence lacks for about 30% of patients diagnosed with clinical FMF. Pyrin is an inflammasome sensor maintained inactive by two kinases (PKN1/2). The consequences of MEFV mutations on inflammasome activation are still poorly understood. Here, we demonstrate that PKC superfamily inhibitors trigger inflammasome activation in monocytes from FMF patients while they trigger a delayed apoptosis in monocytes from healthy donors. The expression of the pathogenic p.M694V MEFV allele is necessary and sufficient for PKC inhibitors (or mutations precluding Pyrin phosphorylation) to trigger caspase‐1‐ and gasdermin D‐mediated pyroptosis. In line with colchicine efficacy in patients, colchicine fully blocks this response in FMF patients’ monocytes. These results indicate that Pyrin inflammasome activation is solely controlled by Pyrin (de)phosphorylation in FMF patients while a second control mechanism restricts its activation in healthy donors/non‐FMF patients. This study paves the way toward a functional characterization of MEFV variants and a functional test to diagnose FMF.