Flora Magnotti

Variants of uncertain significance (VUS) are a major obstacle in genetic diagnosis, particularly when involving gain-of-function (GoF) mutations that are poorly predicted in silico. MEFV, which encodes the inflammasome sensor pyrin, is mutated in two autoinflammatory diseases, familial Mediterranean fever (FMF) and pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). Here, we developed SpeckSeq, a method that combines DNA bar-coding, ASC speck–based single-cell sorting and next-generation sequencing to systematically identify hypermorphic MEFV variants in response to different stimuli. SpeckSeq identified 49 GoF mutations separated into two distinct groups containing either PAAND variants or FMF variants. SpeckSeq was validated using patients’ cells and supported a reclassification of MEFV variant pathogenicity, leading to novel diagnoses. As a large-scale mutagenesis approach, using human genetics as a guide, SpeckSeq revealed structural and functional pyrin features, including a putative ligand-accommodating cavity in the B30.2 domain. Altogether, SpeckSeq classifies VUS to refine molecular diagnostics and improve our knowledge on the pyrin inflammasome.

Juvenile idiopathic arthritis is the most common chronic rheumatic disease in children, and its etiology remains poorly understood. Here, we explored four families with early-onset arthritis carrying homozygous loss-of-expression mutations in LACC1. To understand the link between LACC1 and inflammation, we performed a functional study of LACC1 in human immune cells. We showed that LACC1 was primarily expressed in macrophages upon mTOR signaling. We found that LACC1 deficiency had no obvious impact on inflammasome activation, type I interferon response, or NF-κB regulation. Using bimolecular fluorescence complementation and biochemical assays, we showed that autophagy-inducing proteins, RACK1 and AMPK, interacted with LACC1. Autophagy blockade in macrophages was associated with LACC1 cleavage and degradation. Moreover, LACC1 deficiency reduced autophagy flux in primary macrophages. This was associated with a defect in the accumulation of lipid droplets and mitochondrial respiration, suggesting that LACC1-dependent autophagy fuels macrophage bioenergetics metabolism. Altogether, LACC1 deficiency defines a novel form of genetically inherited juvenile arthritis associated with impaired autophagy in macrophages.

Numerous pathogens including Clostridium difficile and Yersinia pestis have evolved toxins or effectors targeting GTPases from the RhoA subfamily (RhoA/B/C) to inhibit or hijack the host cytoskeleton dynamics. The resulting impairment of RhoA GTPases activity is sensed by the host via an innate immune complex termed the pyrin inflammasome in which caspase-1 is activated. The cascade leading to activation of the pyrin inflammasome has been recently uncovered. In this review, following a brief presentation of RhoA GTPases-modulating toxins, we present the pyrin inflammasome and its regulatory mechanisms. Furthermore, we discuss how some pathogens have developed strategies to escape detection by the pyrin inflammasome. Finally, we present five monogenic autoinflammatory diseases associated with pyrin inflammasome deregulation. The molecular insights provided by the study of these diseases and the corresponding mutations on pyrin inflammasome regulation and activation are presented.

Caspase-4/5 in humans and caspase-11 in mice bind hexa-acylated lipid A, the lipid moeity of lipopolysaccharide (LPS), to induce the activation of non-canonical inflammasome. Pathogens such as Francisella novicida express an under-acylated lipid A and escape caspase-11 recognition in mice. Here, we show that caspase-4 drives inflammasome responses to F. novicida infection in human macrophages. Caspase-4 triggers F. novicida-mediated, gasdermin D-dependent pyroptosis and activates the NLRP3 inflammasome. Inflammasome activation could be recapitulated by transfection of under-acylated LPS from different bacterial species or synthetic tetra-acylated lipid A into cytosol of human macrophage. Our results indicate functional differences between human caspase-4 and murine caspase-11. We further establish that human Guanylate-binding proteins promote inflammasome responses to under-acylated LPS. Altogether, our data demonstrate a broader reactivity of caspase-4 to under-acylated LPS than caspase-11, which may have important clinical implications for management of sepsis.

FMF is the most frequent autoinflammatory disease and is associated in most patients with bi-allelic MEFV mutations. MEFV encodes Pyrin, an inflammasome sensor activated following RhoGTPase inhibition. The functional consequences of MEFV mutations on the ability of Pyrin variants to act as inflammasome sensors are largely unknown. The aim of this study was to assess whether MEFV mutations affect the ability of Pyrin to detect RhoGTPase inhibition and other inflammasome stimuli. IL-1β and IL-18 released by monocytes from healthy donors (HDs) and FMF patients were measured upon specific engagement of the Pyrin, NLRP3 and NLRC4 inflammasomes. Cell death kinetics following Pyrin activation was monitored in real time. Monocytes from FMF patients secreted significantly more IL-1β and IL-18 and died significantly faster than HD monocytes in response to low concentrations of Clostridium difficile toxin B (TcdB), a Pyrin-activating stimulus. Monocytes from patients bearing two MEFV exon 10 pathogenic variants displayed an increased Pyrin inflammasome response compared with monocytes from patients with a single exon 10 pathogenic variant indicating a gene-dosage effect. Using a short priming step, the response of monocytes from FMF patients to NLRP3- and NLRC4-activating stimuli was normal indicating that MEFV mutations trigger a specific hypersensitivity of monocytes to low doses of a Pyrin-engaging stimulus. Contrary to the NLRP3 mutations described in cryopyrin-associated periodic syndrome, FMF-associated MEFV mutations do not lead to a constitutive activation of Pyrin. Rather, FMF-associated mutations are hypermorphic mutations that specifically decrease the activation threshold of the Pyrin inflammasome without affecting other canonical inflammasomes.

The knowledge on systemic autoinflammatory disorders (SAID) is expanding rapidly and new signalling pathways are being decrypted. The concept of autoinflammation has been proposed since 1999, to define a group of diseases with abnormal innate immunity activation. Since then, more than 30 monogenic SAID have been described. In this review, we first describe inflammasomopathies and SAID related to the interleukin-1 pathway. Recent insights into the pathogenesis of familial Mediterranean fever and the function of Pyrin are detailed. In addition, complex or polygenic SAID, such as Still’s disease or PFAPA syndrome, are also discussed. Then, major players driving autoinflammation, such as type-1 interferonopathies (including the recently described haploinsuffiency in A20 and otulipenia), TNF-associated periodic syndromes, defects in ubiquitination, and SAID with overlapping features of autoimmunity or immunodeficiency. Discoveries of the pathogenic role of mosaicism, intronic defects coupled to the likelihood to identify digenic or polygenic diseases are providing new challenges for physicians and geneticists. This comprehensive review depicts the various SAID, presenting them according to their predominant pathophysiological mechanism, with a particular emphasis on recent findings. Epidemiologic data are also presented. Finally, we propose a practical diagnostic approach to the most common monogenic SAID, based on the most characteristic clinical presentation of these disorders.

Behcet's disease (BD) is a systemic inflammatory disease with a still unclear pathogenesis. Although several inflammatory molecules have been studied, current biomarkers are largely insensitive in BD and unable to predict disease progression and response to treatment. Our primary aim was to explore serum levels of soluble CD40 L (sCD40L), soluble intracellular adhesion molecule (sICAM‐1), monocyte chemoattractant protein‐1 (MCP‐1), myeloperoxidase (MPO), leptin, resistin, osteoprotegerin (OPG), soluble type 1 tumour necrosis factor receptor (sTNFR), interleukin (IL)−6 and serum amyloid A (SAA) serum concentration in a cohort of 27 BD patients. The secondary aim was to evaluate potential correlations between the putative circulating biomarkers, demographic profile of patients, the status of disease activity, the specific organ involvement at the time of sample collection and different therapeutic regimens. Serum concentrations of sTNFR (P = 0·008), leptin (P = 0·0011), sCD40L (P < 0·0001) and IL‐6 (P = 0·0154) were significantly higher in BD patients than in HC, while no difference was found in MCP‐1, MPO and resistin serum levels. Moreover, we observed significantly higher sTNFR serum concentrations in BD patients presenting inactive disease than HC (P = 0·0108). A correlation between sTNFR and age was also found, with higher levels in patients over 40 years than HC (P = 0·0329). Although further research is warranted to elucidate the role of circulating biomarkers, some of that may contribute to the understanding of the physiopathology processes underlying BD activity and damage as well as to provide useful tools for prognostic purposes and a personalized treatment approach.