The NACHT-, leucine-rich-repeat-, and pyrin domain-containing protein 3 (NLRP3) is a critical intracellular sensor of the innate immune system that detects various pathogen- and danger-associated molecular patterns, leading to the assembly of the NLRP3 inflammasome and release of interleukin (IL) 1β and IL-18. However, the abnormal activation of the NLRP3 inflammasome has been implicated in the pathogenesis of autoinflammatory diseases such as cryopyrin-associated autoinflammatory syndromes (CAPS) and common diseases such as Alzheimer\'s disease and asthma. Recent studies have revealed that pyrin functions as an indirect sensor, similar to the plant guard system, and is regulated by binding to inhibitory 14-3-3 proteins. Upon activation, pyrin transitions to its active form. NLRP3 is predicted to follow a similar regulatory mechanism and maintain its inactive form in the cage model, as it also acts as an indirect sensor. Additionally, newly developed NLRP3 inhibitors have been found to inhibit NLRP3 activity by stabilizing its inactive form. Most studies and reviews on NLRP3 have focused on the activation of the NLRP3 inflammasome. This review highlights the molecular mechanisms that regulate NLRP3 in its resting state, and discusses how targeting this inhibitory mechanism can lead to novel therapeutic strategies for NLRP3-related diseases.

Cholera toxin (CT), a bacterial exotoxin composed of one A subunit (CTA) and five B subunits (CTB), functions as an immune adjuvant. CTB can induce production of interleukin-1β (IL-1β), a proinflammatory cytokine, in synergy with a lipopolysaccharide (LPS), from resident peritoneal macrophages (RPMs) through the pyrin and NLRP3 inflammasomes. However, how CTB or CT activates these inflammasomes in the macrophages has been unclear. Here, we clarify the roles of inositol-requiring enzyme 1 alpha (IRE1α), an endoplasmic reticulum (ER) stress sensor, in CT-induced IL-1β production in RPMs. In RPMs, CTB is incorporated into the ER and induces ER stress responses, depending on GM1, a cell membrane ganglioside. IRE1α-deficient RPMs show a significant impairment of CT- or CTB-induced IL-1β production, indicating that IRE1α is required for CT- or CTB-induced IL-1β production in RPMs. This study demonstrates the critical roles of IRE1α in activation of both NLRP3 and pyrin inflammasomes in tissue-resident macrophages.

PSTPIP1 (proline-serine-threonine phosphatase-interactive protein 1)-associated myeloid-related proteinemia inflammatory (PAMI) syndrome is a rare autoinflammatory disease caused by heterozygous gain-of-function mutation in PSTPIP1. As one of the PSTPIP1-associated inflammatory diseases (PAIDs), neutropenia is a distinct manifestation to separate PAMI syndrome from other PAIDs. This study aimed to investigate the potential role of neutrophils and inflammatory signatures in the pathogenesis of PAMI. PAMI neutrophils displayed markedly increased production of interleukin-1β (IL-1β) and IL-18 by enzyme linked immunosorbent assay (ELISA) assay and intracellular cytokine staining. ASC speck formation and lactic dehydrogenase (LDH) release are also increased in patient neutrophils suggesting elevated pyrin inflammasome activation followed by upregulated cell death in PAMI neutrophils. RNA sequencing result showed strong inflammatory signals in both nuclear-factor kappa B (NF-κB) pathway and interferon (IFN) pathway in patient neutrophils. This study highlighted that elevated proinflammatory cytokines IL-1β and IL-18, increased pyrin inflammasome activation, and upregulation of NF-κB and IFN signaling pathways in neutrophils play important roles in pathogenicity of PAMI syndrome.

Pathogenic MEFV variants cause pyrin-associated autoinflammatory diseases (PAADs), which include familial Mediterranean fever (FMF), FMF-like disease, and pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). The diagnosis of PAADs is established by clinical phenotypic and genetic analyses. However, the pathogenicity of most MEFV variants remains controversial, as they have not been functionally evaluated. This study aimed to establish and validate a new functional assay to evaluate the pathogenicity of MEFV variants.
We transfected THP-1 monocytes with 32 MEFV variants and analyzed their effects on cell death with or without stimulation with Clostridium difficile toxin A (TcdA) or UCN-01. These variants were classified using hierarchical cluster analysis. Macrophages were obtained from three healthy controls and two patients with a novel homozygous MEFVP257L variant, for comparison of IL-1β secretion using a cell-based assay and a novel THP-1-based assay.
Disease-associated MEFV variants induced variable degrees of spontaneous or TcdA/UCN-01-induced cell death in THP-1. Cell death was caspase-1 dependent and was accompanied by ASC speck formation and IL-1β secretion, indicating that pathogenic MEFV variants induced abnormal pyrin inflammasome activation and subsequent pyroptotic cell deaths in this assay. The MEFV variants (n = 32) exhibiting distinct response signatures were classified into 6 clusters, which showed a good correlation with the clinical phenotypes. Regarding the pathogenicity of MEFVP257L variants, the results were consistent between the cell-based assay and the THP-1-based assay.
Our assay facilitates a rapid and comprehensive assessment of the pathogenicity of MEFV variants and contributes to a refined definition of PAAD subtypes.

The pyrin inflammasome has evolved as an innate immune sensor to detect bacterial toxin-induced Rho guanosine triphosphatase (Rho GTPase)-inactivation, a process that is similar to the \"guard\" mechanism in plants. Rho GTPases act as molecular switches to regulate a variety of signal transduction pathways including cytoskeletal organization. Pathogens can modulate Rho GTPase activity to suppress host immune responses such as phagocytosis. Pyrin is encoded by MEFV, the gene that is mutated in patients with familial Mediterranean fever (FMF). FMF is the prototypic autoinflammatory disease characterized by recurring short episodes of systemic inflammation and is a common disorder in many populations in the Mediterranean basin. Pyrin specifically senses modifications in the activity of the small GTPase RhoA, which binds to many effector proteins including the serine/threonine-protein kinases PKN1 and PKN2 and actin-binding proteins. RhoA activation leads to PKN-mediated phosphorylation-dependent pyrin inhibition. Conversely, pathogen virulence factors downregulate RhoA activity in a variety of ways, and these changes are detected by the pyrin inflammasome irrespective of the type of modifications. MEFV pathogenic variants favor the active state of pyrin and elicit proinflammatory cytokine release and pyroptosis. They can be inherited either as a dominant or recessive trait depending on the variant\'s location and effect on the protein function. Mutations in the C-terminal B30.2 domain are usually considered recessive, although heterozygotes may manifest a biochemical or even a clinical phenotype. These variants are hypomorphic in regard to their effect on intramolecular interactions, but ultimately accentuate pyrin activity. Heterozygous mutations in other domains of pyrin affect residues critical for inhibition or protein oligomerization, and lead to constitutively active inflammasome. In healthy carriers of FMF mutations who have the subclinical inflammatory phenotype, the increased activity of pyrin might have been protective against endemic infections over human history. This finding is supported by the observation of high carrier frequencies of FMF-mutations in multiple populations. The pyrin inflammasome also plays a role in mediating inflammation in other autoinflammatory diseases linked to dysregulation in the actin polymerization pathway. Therefore, the assembly of the pyrin inflammasome is initiated in response to fluctuations in cytoplasmic homeostasis and perturbations in cytoskeletal dynamics.

Cholera toxin B (CTB) is a subunit of cholera toxin, a bacterial enterotoxin secreted by Vibrio cholerae and also functions as an immune adjuvant. However, it remains unclear how CTB activates immune cells. We here evaluated whether or how CTB induces production of a pro-inflammatory cytokine, interleukin-1β (IL-1β). CTB induced IL-1β production not only from bone marrow-derived macrophages (BMMs) but also from resident peritoneal macrophages in synergy with O111:B4-derived lipopolysaccharide (LPS O111:B4) that can bind to CTB. Meanwhile, when prestimulated with O55:B5-derived LPS (LPS O55:B5) that fails to bind to CTB, resident peritoneal macrophages, but not BMMs, produced IL-1β in response to CTB. The CTB-induced IL-1β production in synergy with LPS in both peritoneal macrophages and BMMs was dependent on ganglioside GM1, which is required for internalization of CTB. Notably, not only the NLRP3 inflammasome but also the pyrin inflammasome were involved in CTB-induced IL-1β production from resident peritoneal macrophages, while only the NLRP3 inflammasome was involved in that from BMMs. In response to CTB, a Rho family small GTPase, RhoA, which activates pyrin inflammasome upon various kinds of biochemical modification, increased its phosphorylation at serine-188 in a GM1-dependent manner. This phosphorylation as well as CTB-induced IL-1β productions were dependent on protein kinase A (PKA), indicating critical involvement of PKA-dependent RhoA phosphorylation in CTB-induced IL-1β production. Taken together, these results suggest that CTB, incorporated through GM1, can activate resident peritoneal macrophages to produce IL-1β in synergy with LPS through novel mechanisms in which pyrin as well as NLRP3 inflammasomes are involved.

The monogenic autoinflammatory diseases are a group of illnesses with prominent rheumatic manifestations that are characterized by genetically determined recurrent sterile inflammation and are thus inborn errors of innate immunity. Molecular targeted therapies against inflammatory cytokines, such as interleukin 1 and tumor necrosis factor, and intracellular cytokine signaling pathways have proved effective in many cases. Emerging next-generation sequencing technologies have accelerated the identification of previously unreported genes causing autoinflammatory diseases. This review covers several of the prominent recent advances in the field of autoinflammatory diseases, including gene discoveries, the elucidation of new pathogenic mechanisms, and the development of effective targeted therapies.