Pyrin Inflammasome Research Articles

A type VI secretion system in Burkholderia species cenocepacia and orbicola triggers distinct macrophage death pathways independent of the pyrin inflammasome.

The Burkholderia cepacia complex contains opportunistic pathogens that cause chronic infections and inflammation in the lungs of people with cystic fibrosis. Two closely related species within this complex are Burkholderia cenocepacia and the recently classified Burkholderia orbicola. B. cenocepacia and B. orbicola encode a type VI secretion system and the effector TecA, which is detected by the pyrin/caspase-1 inflammasome, and triggers macrophage inflammatory death. We previously showed that the pyrin inflammasome was dispensable for lung inflammation in mice infected with B. orbicola AU1054, indicating this species activates an alternative pathway of macrophage inflammatory death. Notably, B. cenocepacia strains J2315 and K56-2 can damage macrophage phagosomes, and K56-2 triggers activation of the caspase-11 inflammasome, which detects cytosolic lipopolysaccharide. Here, we investigated inflammatory cell death in pyrin- (Mefv-/-) or caspase-1/caspase-11- (Casp1/11-/-) deficient mouse macrophages infected with B. cenocepacia J2315 or K56-2 or B. orbicola AU1054 or PC184. Macrophage inflammatory death was measured by cleavage of gasdermin D protein, the release of cytokines IL-1α and IL-1β, and plasma membrane rupture. We found that J2315 and K56-2 are detected by the caspase-11 inflammasome in Mefv-/- macrophages, resulting in IL-1β release. By contrast, inflammasome activation was not detected in Mefv-/- macrophages infected with AU1054 or PC184. Instead, AU1054 triggered an alternative macrophage inflammatory death pathway that required TecA and resulted in plasma membrane rupture and IL-1α release. Structural modeling of TecA orthologs in B. cenocepacia and B. orbicola suggested that amino acid changes in the latter may underlie its ability to trigger a non-inflammasome macrophage death pathway.

The Potential Role of Cell-Death Mechanisms in the Pathogenesis of Familial Mediterranean Fever Attacks: Granzyme A and Beyond.

FMF is the most common autoinflammatory disease. The activation of the pyrin inflammasome is the mainstay of the pathogenesis, which might lead to a specific cell-death mechanism, pyroptosis. Pyroptosis is a programmed inflammatory cell death mediated by gasdermin proteins, featuring cell swelling, membrane rupture, and release of inflammatory contents Aim: In this study we aimed to analyze the cell-death mechanisms in the pathogenesis of FMF attacks. Twenty-five FMF patients were included, and PFAPA patients (n = 10) and healthy controls (HC, n = 10) served as controls. We collected plasma samples from FMF and PFAPA patients during the attack and the attack-free period. We measured the soluble plasma levels of sFas, sFasL, granzyme A, granzyme B, perforin, granulysin, IL-2, IL-4, IL-10, IL-6, IL-17A, TNF-α, and IFN-γ by commercial pre-defined cytometric bead array kits. There was no significant difference between groups in terms of sex and age between FMF patients and HCs, but PFAPA patients were younger than other groups due to the nature of the disease. We then analyzed the components of apoptosis and pyroptosis. The levels of sFasL (p = 0.035) and granzyme A (p = 0.038) in FMF patients were significantly increased during the attack period and decreased to levels comparable to HCs during the attack-free period. This increase was not seen in the PFAPA patients, with comparable levels with the HC group both during attack period and attack-free period. During the attack period of FMF patients, granzyme B (p = 0.145) and perforin (p = 0.203) levels were also increased; however, the differences were not statistically significant. The levels of sFasL, granzyme A, granzyme B, and perforin were closely correlated with each other during the attack period of FMF patients. Our study on death pathways during an FMF attack, suggests an upregulation in both pyroptosis through the granzyme-gasdermin pathway and apoptosis with the increased FasL and perforin levels, which was different from PFAPA patients. These findings might shed light on the reason for the nature of self-limited attacks, but further studies are needed to prove this hypothesis.

CircCDC42-encoded CDC42-165aa regulates macrophage pyroptosis in Klebsiella pneumoniae infection through Pyrin inflammasome activation

The circular RNA (circRNA) family is a group of endogenous non-coding RNAs (ncRNAs) that have critical functions in multiple physiological and pathological processes, including inflammation, cancer, and cardiovascular diseases. However, their roles in regulating innate immune responses remain unclear. Here, we define Cell division cycle 42 (CDC42)−165aa, a protein encoded by circRNA circCDC42, which is overexpressed in Klebsiella pneumoniae (KP)-infected alveolar macrophages. High levels of CDC42-165aa induces the hyperactivation of Pyrin inflammasomes and aggravates alveolar macrophage pyroptosis, while the inhibition of CDC42-165aa reduces lung injury in mice after KP infection by inhibiting Pyrin inflammasome-mediated pyroptosis. Overall, these results demonstrate that CDC42-165aa stimulates Pyrin inflammasome by inhibiting CDC42 GTPase activation and provides a potential clinical target for pathogenic bacterial infection in clinical practice.

Exploring S100A8/A9, neopterin, and MMP3 in familial Mediterranean fever.

Familial Mediterranean fever (FMF) is characterized by inflammatory attacks due to overactivation of pyrin inflammasome. This study aimed to investigate the reliability of S100A8/A9, neopterin, and matrix metalloproteinase 3 (MMP3) at monitoring subclinical inflammation and disease activity, and at differentiating FMF attacks from appendicitis, the most common misdiagnosis among FMF patients. Blood samples (n = 75), comprising from FMF patients during an attack (n = 20), the same FMF patients during the attack-free period (n = 14), patients with appendicitis (n = 24), and healthy volunteers (n = 17) were obtained. Duplicate determinations of S100A8/A9, neopterin, and MMP-3 levels were conducted using the enzyme-linked immunosorbent assay (ELISA). FMF patients with and without attack and patients with appendicitis had significantly elevated S100A8/A9 levels compared to healthy volunteers (P-values: < 0.001, 0.036, 0.002, respectively). Patients with appendicitis and FMF patients with and without attack had significantly increased serum neopterin levels compared to healthy volunteers (P-value: < 0.001). MMP3 levels were significantly higher among patients with appendicitis and FMF patients during attack compared to healthy controls (P-values: < 0.001, 0.001). Serum levels of S100A8/A9, neopterin, and MMP3 were increased significantly during attacks compared to attack-free periods among FMF patients (P-values: 0.03, 0.047, 0.007). S100A8/A9 emerges as a valuable marker for monitoring disease activity. Neopterin and S100A8/A9 might help physicians to monitor subclinical inflammation during the attack-free periods of FMF patients. MMP3 might aid in diagnosing FMF attacks when distinguishing between attack and attack-free periods is challenging.

Interrupting an IFN-γ-dependent feedback loop in the syndrome of pyogenic arthritis with pyoderma gangrenosum and acne

ObjectivesTo study the molecular pathogenesis of PAPA (pyogenic arthritis, pyoderma gangrenosum and acne) syndrome, a debilitating hereditary autoinflammatory disease caused by dominant mutation in PSTPIP1.MethodsGene knock-out and knock-in mice were...

The inflammasome pathway is activated by dengue virus non-structural protein 1 and is protective during dengue virus infection.

Dengue virus (DENV) is a medically important flavivirus causing an estimated 50-100 million dengue cases annually, some of whom progress to severe disease. DENV non-structural protein 1 (NS1) is secreted from infected cells and has been implicated as a major driver of dengue pathogenesis by inducing endothelial barrier dysfunction. However, less is known about how DENV NS1 interacts with immune cells and what role these interactions play. Here we report that DENV NS1 can trigger activation of inflammasomes, a family of cytosolic innate immune sensors that respond to infectious and noxious stimuli, in mouse and human macrophages. DENV NS1 induces the release of IL-1β in a caspase-1 dependent manner. Additionally, we find that DENV NS1-induced inflammasome activation is independent of the NLRP3, Pyrin, and AIM2 inflammasome pathways, but requires CD14. Intriguingly, DENV NS1-induced inflammasome activation does not induce pyroptosis and rapid cell death; instead, macrophages maintain cellular viability while releasing IL-1β. Lastly, we show that caspase-1/11-deficient, but not NLRP3-deficient, mice are more susceptible to lethal DENV infection. Together, these results indicate that the inflammasome pathway acts as a sensor of DENV NS1 and plays a protective role during infection.

Pyrin Inflammasome Activation Defines Colchicine-Responsive SURF Patients from FMF and Other Recurrent Fevers.

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.

The pyrin inflammasome, a leading actor in pediatric autoinflammatory diseases.

The activation of the pyrin inflammasome represents a highly intriguing mechanism employed by the innate immune system to effectively counteract pathogenic agents. Despite its key role in innate immunity, pyrin has also garnered significant attention due to its association with a range of autoinflammatory diseases (AIDs) including familial Mediterranean fever caused by disruption of the MEFV gene, or in other genes involved in its complex regulation mechanisms. Pyrin activation is strictly dependent on homeostasis-altering molecular processes, mostly consisting of the disruption of the small Ras Homolog Family Member A (RhoA) GTPases by pathogen toxins. The downstream pathways are regulated by the phosphorylation of specific pyrin residues by the kinases PKN1/2 and the binding of the chaperone 14-3-3. Furthermore, a key role in pyrin activation is played by the cytoskeleton and gasdermin D, which is responsible for membrane pores in the context of pyroptosis. In addition, recent evidence has highlighted the role of steroid hormone catabolites and alarmins S100A8/A9 and S100A12 in pyrin-dependent inflammation. The aim of this article is to offer a comprehensive overview of the most recent evidence on the pyrin inflammasome and its molecular pathways to better understand the pathogenesis behind the significant group of pyrin-related AIDs.

Recent Insights in Pyrin Inflammasome Activation: Identifying Potential Novel Therapeutic Approaches in Pyrin-Associated Autoinflammatory Syndromes.

Pyrin is a cytosolic protein encoded by the MEFV gene, predominantly expressed in innate immune cells. Upon activation, it forms an inflammasome, a multimolecular complex that enables the activation and secretion of IL-1β and IL-18. In addition, the Pyrin inflammasome activates Gasdermin D leading to pyroptosis, a highly pro-inflammatory cell death. Four autoinflammatory syndromes are associated with Pyrin inflammasome dysregulation: familial Mediterranean fever, hyper IgD syndrome/mevalonate kinase deficiency, pyrin-associated autoinflammation with neutrophilic dermatosis, and pyogenic arthritis, pyoderma gangrenosum, and acne syndrome. In this review, we discuss recent advances in understanding the molecular mechanisms regulating the two-step model of Pyrin inflammasome activation. Based on these insights, we discuss current pharmacological options and identify a series of existing molecules with therapeutic potential for the treatment of pyrin-associated autoinflammatory syndromes.

Phagocytic cell death leads to enhanced release of pro-inflammatory S100A12 in familial Mediterranean fever

BackgroundFamilial Mediterranean fever (FMF) is a prototypical autoinflammatory syndrome associated with phagocytic cell activation. Pyrin mutations are the genetic basis of this disease, and its expression has been shown in monocytes, granulocytes, dendritic cells, and synovial fibroblasts. Pyrin functions as a cytosolic pattern recognition receptor and forms a distinct pyrin inflammasome. The phagocyte-specific protein S100A12 is predominantly expressed in granulocytes and belongs to the group of damage associated molecular patterns (DAMP). S100A12 can be detected at massively elevated levels in the serum of FMF patients, even in clinically inactive disease. Whether this is crucial for FMF pathogenesis is as yet unknown, and we therefore investigated the mechanisms of S100A12 release from granulocytes of FMF patients presenting clinically inactive.ResultsWe demonstrate that FMF neutrophils from patients in clinical inactive disease possess an intrinsic activity leading to cell death even in exogenously unstimulated neutrophils. Cell death resembles NETosis and is dependent on ROS and pore forming protein gasdermin D (GSDMD), as inhibitors for both are capable of completely block cell death and S100A12 release. When pyrin-activator TcdA (Clostridium difficile toxin A) is used to stimulate, neutrophilic cell death and S100A12 release are significantly enhanced in neutrophils from FMF patients compared to neutrophils from HC.ConclusionsWe are able to demonstrate that activation threshold of neutrophils from inactive FMF patients is decreased, most likely by pre-activated pyrin. FMF neutrophils present with intrinsically higher ROS production, when cultured ex vivo. This higher baseline ROS activity leads to increased GSDMD cleavage and subsequent release of, e.g., S100A12, and to increased cell death with features of NETosis and pyroptosis. We show for the first time that cell death pathways in neutrophils of inactive FMF patients are easily triggered and lead to ROS- and GSDMD-dependent activation mechanisms and possibly pathology. This could be therapeutically addressed by blocking ROS or GSDMD cleavage to decrease inflammatory outbreaks when becoming highly active.

A mathematical model of Familial Mediterranean Fever predicts mechanisms controlling inflammation

BackgroundFamilial Mediterranean Fever (FMF) is a monogenic disease caused by gain-of-function mutations in the MEditerranean FeVer (MEFV) gene. The molecular dysregulations induced by these mutations and the associated causal mechanisms are complex and intricate. ObjectiveWe sought to provide a computational model capturing the mechanistic details of biological pathways involved in FMF physiopathology and enabling the study of the patient's immune cell dynamics. MethodsWe carried out a literature survey to identify experimental studies published from January 2000 to December 2020, and integrated its results into a molecular map and a mathematical model. Then, we studied the network of molecular interactions and the dynamic of monocytes to identify key players for inflammation phenotype in FMF patients. ResultsWe built a molecular map of FMF integrating in a structured manner the current knowledge regarding pathophysiological processes participating in the triggering and perpetuation of the disease flares. The mathematical model derived from the map reproduced patient's monocyte behavior, in particular its proinflammatory role via the Pyrin inflammasome activation. Network analysis and in silico experiments identified NF-κB and JAK1/TYK2 as critical to modulate IL-1β- and IL-18-mediated inflammation. ConclusionThe in silico model of FMF monocyte proved its ability to reproduce in vitro observations. Considering the difficulties related to experimental settings and financial investments to test combinations of stimuli/perturbation in vitro, this model could be used to test complex hypotheses in silico, thus narrowing down the number of in vitro and ex vivo experiments to perform.

A dominant pathogenic MEFV mutation causes atypical pyrin-associated periodic syndromes.

Pyrin, a protein encoded by the MEFV gene, plays a vital role in innate immunity by sensing modifications in Rho GTPase and assembling the pyrin inflammasome, which in turn activates downstream immune responses. We identified a novel and de novo MEFV p.E583A dominant variant in 3 patients from the same family; the variant was distinct from the previously reported S242 and E244 sites. These patients exhibited a phenotype that diverged from those resulting from classical MEFV gene mutations, characterized by the absence of recurrent fever but the presence of recurrent chest and abdominal pain. Colchicine effectively controlled the phenotype, and the mutation was found to induce pyrin inflammasome assembly and activation in patients' peripheral blood mononuclear cells (PBMCs) and cell lines. Mechanistically, truncation experiments revealed that the E583A variant affected the autoinhibitory structure of pyrin. Our study offers insights into the mechanisms underlying pyrin inflammasome activation.

Phosphoprotein phosphatase activity positively regulates oligomeric pyrin to trigger inflammasome assembly in phagocytes.

Pyrin, a unique cytosolic receptor, initiates inflammatory responses against RhoA-inactivating bacterial toxins and effectors like Yersinia's YopE and YopT. Understanding pyrin regulation is crucial due to its association with dysregulated inflammatory responses, including Familial Mediterranean Fever (FMF), linked to pyrin gene mutations. FMF mutations historically acted as a defense mechanism against plague. Negative regulation of pyrin through PKN phosphorylation is well established, with Yersinia using the YopM effector to promote pyrin phosphorylation and counteract its activity. This study highlights the importance of phosphoprotein phosphatase activity in positively regulating pyrin inflammasome assembly in phagocytic cells of humans and mice. Oligomeric murine pyrin has S205 phosphorylated before inflammasome assembly, and this study implicates the dephosphorylation of murine pyrin S205 by two catalytic subunits of PP2A in macrophages. These findings offer insights for investigating the regulation of oligomeric pyrin and the balance of kinase and phosphatase activity in pyrin-associated infectious and autoinflammatory diseases.

C-terminal variants in CDC42 drive type I interferon-dependent autoinflammation in NOCARH syndrome reversible by ruxolitinib

C-terminal variants in CDC42 encoding cell division control protein 42 homolog underlie neonatal-onset cytopenia, autoinflammation, rash, and hemophagocytic lymphohistiocytosis (NOCARH). Pyrin inflammasome hyperactivation has been shown to contribute to disease pathophysiology. However, mortality of NOCARH patients remains high despite inflammasome-focused treatments. Here, we demonstrate in four NOCARH patients from three families that cell-intrinsic activation of type I interferon (IFN) is a previously unrecognized driver of autoinflammation in NOCARH. Our data show that aberrant innate immune activation is caused by sensing of cytosolic nucleic acids released from mitochondria, which exhibit disturbances in integrity and dynamics due to CDC42 dysfunction. In one of our patients, treatment with the Janus kinase inhibitor ruxolitinib led to complete remission, indicating that inhibition of type I IFN signaling may have an important role in the management of autoinflammation in patients with NOCARH.

Hyperinflammatory Immune Response in COVID-19: Host Genetic Factors in Pyrin Inflammasome and Immunity to Virus in a Spanish Population from Majorca Island.

The hyperinflammatory response caused by SARS-CoV-2 infection contributes to its severity, and many critically ill patients show features of cytokine storm (CS) syndrome. We investigated, by next-generation sequencing, 24 causative genes of primary immunodeficiencies whose defect predisposes to CS. We studied two cohorts with extreme phenotypes of SARS-CoV-2 infection: critical/severe hyperinflammatory patients (H-P) and asymptomatic patients (AM-risk-P) with a high risk (older age) to severe COVID-19. To explore inborn errors of the immunity, we investigated the presence of pathogenic or rare variants, and to identify COVID-19 severity-associated markers, we compared the allele frequencies of common genetic polymorphisms between our two cohorts. We found: 1 H-P carries the likely pathogenic variant c.887-2 A>C in the IRF7 gene and 5 H-P carries variants in the MEFV gene, whose role in the pathogenicity of the familial Mediterranean fever (FMF) disease is controversial. The common polymorphism analysis showed three potential risk biomarkers for developing the hyperinflammatory response: the homozygous haplotype rs1231123A/A-rs1231122A/A in MEFV gene, the IFNAR2 p.Phe8Ser variant, and the CARMIL2 p.Val181Met variant. The combined analysis showed an increased risk of developing severe COVID-19 in patients that had at least one of our genetic risk markers (odds ratio (OR) = 6.2 (95% CI) (2.430-16.20)).

Alpha-linolenic acid pretreatment alleviates NETs-induced alveolar macrophage pyroptosis by inhibiting pyrin inflammasome activation in a mouse model of sepsis-induced ALI/ARDS.

Neutrophil extracellular traps (NETs) can cause acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) by inducing macrophage pyroptosis. The purpose of this study was to find out whether pretreatment of alpha-linolenic acid (ALA) could inhibit NETs-induced macrophage pyroptosis in sepsis-induced ALI/ARDS, as well as to identify which inflammasome is involved in this process. LPS was instilled into the trachea to establish sepsis-induced ALI/ARDS in a mouse model. ​Lung injury was assessed by microscopic examination of lung tissue after hematoxylin and eosin staining, pathology score, and bronchoalveolar lavage fluid (BALF) total protein concentration. The level of NETs in lung tissue was detected by MPO-DNA ELISA. Purified NETs, extracted from peritoneal neutrophils, induced macrophage pyroptosis in vitro. Expression of pyroptosis-related proteins (Cl-caspase-1, Cl-GSDMD, ASC) and IL-1β in the lung tissue and bone marrow-derived macrophages (BMDMs) were determined by western blotting or ELISA. Specks of Pyrin/ASC were examined by confocal immunofluorescence microscopy. Mefv (Pyrin)-/- mice were used to study the role of Pyrin in the process of sepsis-induced ALI/ARDS. ALA alleviated LPS-induced lung injury. ALA reduced the level of NETs, pyroptosis-related proteins (Cl-caspase-1, Cl-GSDMD, ASC), and IL-1β in the lung tissue of sepsis mice. In vitro, NETs increased the expression of pyroptosis-related proteins (Cl-caspase-1, Cl-GSDMD, ASC) and IL-1β significantly in BMDMs. Pyrin protein was found to be higher and form the inflammasome with ASC in NETs challenged-BMDMs. Knockout of Mefv (Pyrin) gene fully restored the increased expression of pyroptosis-related proteins (Cl-caspase-1, Cl-GSDMD, ASC) and IL-1β in vitro and in vivo. Lung injury was alleviated significantly in Mefv (Pyrin)-/- mice as well.​ ALA suppresses all the NETs-induced changes as mentioned above. Our study is the first to demonstrate Pyrin inflammasome driving NETs-induced macrophage pyroptosis, and ALA may reduce ALI/ARDS by inhibiting the activation of the Pyrin inflammasome-driven macrophage pyroptosis.

Mutations in the B30.2 and the central helical scaffold domains of pyrin differentially affect inflammasome activation

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.

The Clinical Journey of a Patient with Familial Mediterranean Fever: One Gene, Three Diseases.

Familial Mediterranean fever (FMF) is the most common monogenic autoinflammatory disease characterized by recurrent self-limiting attacks of fever and serositis (Ben-Chetrit E and Levy M, Lancet 1998; 351: 659–664). The gain-of-function mutations in the responsible gene encoding pyrin, known as the MEditerranean FeVer (MEFV) gene, cause a reduction in the activation threshold of pyrin inflammasome triggering recurrent clinical attacks, subclinical inflammation, and a tendency for other inflammatory diseases (Jamilloux Y et al., Rheumatology (Oxford) 2018; 57: 100–111). Familial Mediterranean fever is more prevalently seen in the Middle Eastern communities. A relatively severe disease phenotype together with several diseases associated with FMF was frequently reported around this part of the world (Ben-Chetrit E and Yazici H, Clinical and Experimental Rheumatology 2019; 37 (Suppl 121): 18–22).

Detection of a rare variant in PSTPIP1 through three generations in a family with an initial diagnosis of FMF/MKD-overlapping phenotype.

The presence of FMF cases without MEFV (MEFV innate immunity regulator, pyrin) pathogenic variants led us to search for other genes' involvement in the disease development. Here, we describe the presence of genetic heterogeneity in a three-generation family with an FMF/mevalonate kinase deficiency (MKD)-overlapping phenotype without MEFV/MVK (mevalonate kinase) pathogenic variants. Targeted sequencing revealed a rare, fully penetrant variant in PSTPIP1 (p.Arg228Cys, rs781341816). Computational stability analyses of PSTPIP1 protein were performed. PSTPIP1-pyrin protein interaction was examined by immunoprecipitation and immunoblotting in peripheral blood mononuclear cells (PBMCs) of patients and healthy controls. PBMCs were cultured, and inflammation was induced by LPS+ATP treatment, followed by protein level measurements of caspase-1, IL1ß, pyrin and PSTPIP1 in cell lysates and mature caspase-1 and mature IL1ß in supernatants. The conserved, rare (GnomAD, 0.000028) PSTPIP1 p.Arg228Cys variant, previously reported in ClinVar as a variant with uncertain significance, showed complete penetrance in the family presenting an autosomal dominant pattern. Computational analyses showed a potentially destabilizing effect of the variant on PSTPIP1 protein. Accordingly, PSTPIP1-pyrin interaction was increased in patients harboring the variant, which resulted in elevated levels of mature caspase-1 and IL1ß in the inflammation-induced patient samples. Unlike previously described cases with pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA)-associated PSTPIP1 variants, our patients with the p.Arg228Cys variant presented with an FMF/MKD-overlapping phenotype. As additional data on the genetic heterogeneity in the variable clinical spectrum of autoinflammatory syndromes, we suggest that the p.Arg228Cys variant in PSTPIP1 is related to inflammation responses through strong PSTPIP1-pyrin interaction and pyrin inflammasome activation.

Inducing Pyroptosis Via the Pyrin Inflammasome.

The pyrin inflammasome detects bacterial toxins and effectors that inhibit RhoA GTPases and triggers inflammatory cytokine release and a fast cell death termed pyroptosis. In addition, various endogenous molecules, drugs, synthetic molecules, or mutations can trigger pyrin inflammasome activation. The pyrin protein differs between humans and mice, and the repertoire of pyrin activators is also species-specific. Here, we present the various pyrin inflammasome activators, inhibitors, the kinetics of pyrin activation in response to the various activators, and their species specificity. In addition, we present different methods to monitor pyrin-mediated pyroptosis.