Sepsis is characterized by a systemic inflammatory response followed by immunosuppression of the host. Metabolic defects and mitochondrial failure are common in immunocompromised patients with sepsis. The NLRP3 inflammasome is important for establishing an inflammatory response after activation by the purinergic P2X7 receptor. Here, we study a cohort of individuals with intra-abdominal origin sepsis and show that patient monocytes have impaired NLRP3 activation by the P2X7 receptor. Furthermore, most sepsis-related deaths are among patients whose NLRP3 activation is profoundly altered. In monocytes from sepsis patients, the P2X7 receptor is associated with mitochondrial dysfunction. Furthermore, activation of the P2X7 receptor results in mitochondrial damage, which in turn inhibits NLRP3 activation by HIF-1α. We show that mortality increases in a mouse model of sepsis when the P2X7 receptor is activated in vivo. These data reveal a molecular mechanism initiated by the P2X7 receptor that contributes to NLRP3 impairment during infection.

Objective Pyrin-Associated Autoinflammation with Neutrophilic Dermatosis (PAAND) is a recently described monogenic autoinflammatory disease. The causal p.S242R MEFV mutation disrupts a binding motif of the regulatory 14-3-3 proteins within pyrin. Here, we investigate a family with clinical features consistent with PAAND in whom the novel p.E244K MEFV mutation, located in the +2 site of the 14-3-3 binding motif in pyrin, has been found. Methods Multiplex cytokine analyses were performed on p.E244K patient and control serum. Peripheral blood mononuclear cells were stimulated ex vivo with lipopolysaccharide (LPS). In vitro, inflammasome complex formation was evaluated by flow cytometry of Apoptosis-associated Speck-like protein containing a Caspase recruitment domain (ASC) specks. Interleukin-1β (IL-1β) and IL-18 production was quantified by ELISA. The ability of the p.E244K pyrin mutation to interact with 14-3-3 was assessed by immunoprecipitation. Results PAAND p.E244K patient serum displayed a different cytokine profile compared with patients with Familial Mediterranean Fever (FMF). In overexpression models, p.E244K pyrin was associated with decreased 14-3-3 binding and increased ASC speck formation. THP-1 monocytes expressing PAAND pyrin mutations demonstrated spontaneous caspase-1-dependent IL-1β and IL-18 secretion, as well as cell death, which were significantly greater than those of wild-type and the FMF-associated mutation p.M694V. Conclusion In PAAND, disruption of the +2 position of a 14-3-3 binding motif in pyrin results in its constitutive activation, with spontaneous production of IL-1β and IL-18, associated with inflammatory cell death. The altered serum cytokine profile may explain the different clinical features exhibited by PAAND patients compared with those with FMF.

Objective: Gain‐of‐function NLRP3 mutations cause cryopyrin‐associated periodic syndrome (CAPS), with gene mosaicism playing a relevant role in the pathogenesis. This study was undertaken to characterize the genetic cause underlying late‐onset but otherwise typical CAPS.Methods: We studied a 64‐year‐old patient who presented with recurrent episodes of urticaria‐like rash, fever, conjunctivitis, and oligoarthritis at age 56 years. DNA was extracted from both unfractionated blood and isolated leukocyte and CD34+ subpopulations. Genetic studies were performed using both the Sanger method of DNA sequencing and next‐generation sequencing (NGS) methods. In vitro and ex vivo analyses were performed to determine the consequences that the presence of the variant have in the normal structure or function of the protein of the detected variant.Results: NGS analyses revealed the novel p.Gln636Glu NLRP3 variant in unfractionated blood, with an allele frequency (18.4%) compatible with gene mosaicism. Sanger sequence chromatograms revealed a small peak corresponding to the variant allele. Amplicon‐based deep sequencing revealed somatic NLRP3 mosaicism restricted to myeloid cells (31.8% in monocytes, 24.6% in neutrophils, and 11.2% in circulating CD34+ common myeloid progenitor cells) and its complete absence in lymphoid cells. Functional analyses confirmed the gain‐of‐function behavior of the gene variant and hyperactivity of the NLRP3 inflammasome in the patient. Treatment with anakinra resulted in good control of the disease.Conclusion: We identified the novel gain‐of‐function p.Gln636Glu NLRP3 mutation, which was detected as a somatic mutation restricted to myeloid cells, as the cause of late‐onset but otherwise typical CAPS. Our results expand the diversity of CAPS toward milder phenotypes than previously reported, including those starting during adulthood.

Autoinflammatory diseases (AIDs) were first described as clinical disorders characterized by recurrent episodes of seemingly unprovoked sterile inflammation. In the past few years, the identification of novel AIDs expanded their phenotypes toward more complex clinical pictures associating vasculopathy, autoimmunity, or immunodeficiency. Herein, we describe two unrelated patients suffering since the neonatal period from a complex disease mainly characterized by severe sterile inflammation, recurrent bacterial infections, and marked humoral immunodeficiency. Whole-exome sequencing detected a novel, de novo heterozygous PLCG2 variant in each patient (p.Ala708Pro and p.Leu845_Leu848del). A clear enhanced PLCγ2 activity for both variants was demonstrated by both ex vivo calcium responses of the patient’s B cells to IgM stimulation and in vitro assessment of PLC activity. These data supported the autoinflammation and PLCγ2-associated antibody deficiency and immune dysregulation (APLAID) diagnosis in both patients. Immunological evaluation revealed a severe decrease of immunoglobulins and B cells, especially class-switched memory B cells, with normal T and NK cell counts. Analysis of bone marrow of one patient revealed a reduced immature B cell fraction compared with controls. Additional investigations showed that both PLCG2 variants activate the NLRP3-inflammasome through the alternative pathway instead of the canonical pathway. Collectively, the evidences here shown expand APLAID diversity toward more severe phenotypes than previously reported including dominantly inherited agammaglobulinemia, add novel data about its genetic basis, and implicate the alternative NLRP3-inflammasome activation pathway in the basis of sterile inflammation.

The nucleotide-binding domain and leucine-rich repeat-containing receptor with a pyrin domain 3 (NLRP3) inflammasome is a sensor for different types of infections and alterations of homeostatic parameters, including abnormally high levels of the extracellular nucleotide ATP or crystallization of different metabolites. All NLRP3 activators trigger a similar intracellular pathway, where a decrease in intracellular K⁺ concentration and permeabilization of plasma membrane are key steps. Cationic amphipathic antimicrobial peptides and peptide toxins permeabilize the plasma membrane. In fact, some of them have been described to activate the NLRP3 inflammasome. Among them, the bee venom antimicrobial toxin peptide melittin is known to elicit an inflammatory reaction via the NLRP3 inflammasome in response to bee venom. Our study found that melittin induces canonical NLRP3 inflammasome activation by plasma membrane permeabilization and a reduction in the intracellular K⁺ concentration. Following melittin treatment, the apoptosis-associated speck-like protein, an adaptor protein with a caspase recruitment domain (ASC), was necessary to activate caspase-1 and induce IL-1β release. However, cell death induced by melittin prevented the formation of large ASC aggregates, amplification of caspase-1 activation, IL-18 release and execution of pyroptosis. Therefore, melittin-induced activation of the NLRP3 inflammasome results in an attenuated inflammasome response that does not result in caspase-1 dependent cell death.

Steps of the NLRP3 inflammasome activation pathway where some selected polyphenols have shown a positive modulatory effect on different cardiometabolic alterations.