Inhibition of SlSKOR by SlCIPK23-SlCBL1/9 uncovers CIPK-CBL-target network rewiring in land plants.

Transport of K+ to the xylem is a key process in the mineral nutrition of the shoots. Although CIPK-CBL complexes have been widely shown to regulate K+ uptake transport systems, no information is available about the xylem ones. Here, we studied the physiological roles of the voltage-gated K+ channel SlSKOR and its regulation by the SlCIPK23-SlCBL1/9 complexes in tomato plants. We phenotyped gene-edited slskor and slcipk23 tomato knockout mutants and carried out two-electrode voltage-clamp (TEVC) and BiFC assays in Xenopus oocytes as key approaches. SlSKOR was preferentially expressed in the root stele and was important not only for K+ transport to shoots but also, indirectly, for that of Ca2+ , Mg2+ , Na+ , NO3 - , and Cl- . Surprisingly, the SlCIPK23-SlCBL1/9 complexes turned out to be negative regulators of SlSKOR. Inhibition of SlSKOR by SlCIPK23-SlCBL1/9 was observed in Xenopus oocytes and tomato plants. Regulation of SKOR-like channels by CIPK23-CBL1 complexes was also present in Medicago, grapevine, and lettuce but not in Arabidopsis and saltwater cress. Our results provide a molecular framework for coordinating root K+ uptake and its translocation to the shoot by SlCIPK23-SlCBL1/9 in tomato plants. Moreover, they evidenced that CIPK-CBL-target networks have evolved differently in land plants.

Early-onset recurrent panniculitis as a phenotype of NLRC4-associated autoinflammatory syndrome: Characterization of pathogenicity of the p.Ser445Pro NLRC4 variant.

Monoallelic NLRC4 gain-of-function variants cause an inflammasomopathy with diverse clinical forms including infantile enterocolitis, recurrent macrophage activation syndrome, cold-induced urticaria-like lesions (or familial-cold autoinflammatory syndrome, FCAS4), and painful subcutaneous nodules. Here, we identified a large family with six consecutive generations affected. Genetic analyses detected the heterozygous p.Ser445Pro NLRC4 variant in three patients, which has been previously reported in a Dutch family with FCAS4. We aimed to describe the clinicopathological features and the functional consequences of the detected NLRC4 variant. Patients presented an early-onset (3 months-6 years) inflammatory disease characterized by recurrent panniculitis, fever and arthralgia. Histopathological examination showed perivascular and interstitial lymphohistiocytic infiltrates in the dermis and mixed panniculitis. Functional analysis supported the conclusion that the p.Ser445Pro NLRC4 variant leads to a constitutive activation of NLRC4-inflammasome and increased plasma levels of IL-18. Prompt recognition of early-onset panniculitis through clinicopathological examination and laboratory biomarkers may allow targeted therapies.

Evolutionary analyses of the gasdermin family suggest conserved roles in infection response despite loss of pore-forming functionality.

BACKGROUND: Gasdermins are ancient (>500million-years-ago) proteins, constituting a family of pore-forming proteins that allow the release of intracellular content including proinflammatory cytokines. Despite their importance in the immune response, and although gasdermin and gasdermin-like genes have been identified across a wide range of animal and non-animal species, there is limited information about the evolutionary history of the gasdermin family, and their functional roles after infection. In this study, we assess the lytic functions of different gasdermins across Metazoa species, and use a mouse model of sepsis to evaluate the expression of the different gasdermins during infection. RESULTS: We show that the majority of gasdermin family members from distantly related animal clades are pore-forming, in line with the function of the ancestral proto-gasdermin and gasdermin-like proteins of Bacteria. We demonstrate the first expansion of this family occurred through a duplication of the ancestral gasdermin gene which formed gasdermin E and pejvakin prior to the divergence of cartilaginous fish and bony fish ~475 mya. We show that pejvakin from cartilaginous fish and mammals lost the pore-forming functionality and thus its role in cell lysis. We describe that the pore-forming gasdermin A formed ~320 mya as a duplication of gasdermin E prior to the divergence of the Sauropsida clade (the ancestral lineage of reptiles, turtles, and birds) and the Synapsid clade (the ancestral lineage of mammals). We then demonstrate that the gasdermin A gene duplicated to form the rest of the gasdermin family including gasdermins B, C, and D: pore-forming proteins that present a high variation of the exons in the linker sequence, which in turn allows for diverse activation pathways. Finally, we describe expression of murine gasdermin family members in different tissues in a mouse sepsis model, indicating function during infection response. CONCLUSIONS: In this study we explored the evolutionary history of the gasdermin proteins in animals and demonstrated that the pore-formation functionality has been conserved from the ancient proto-gasdermin protein. We also showed that one gasdermin family member, pejvakin, lost its pore-forming functionality, but that all gasdermin family members, including pejvakin, likely retained a role in inflammation and the physiological response to infection.

Techniques to Study Inflammasome Activation and Inhibition by Small Molecules.

Inflammasomes are immune cytosolic oligomers involved in the initiation and progression of multiple pathologies and diseases. The tight regulation of these immune sensors is necessary to control an optimal inflammatory response and recover organism homeostasis. Prolonged activation of inflammasomes result in the development of chronic inflammatory diseases, and the use of small drug-like inhibitory molecules are emerging as promising anti-inflammatory therapies. Different aspects have to be taken in consideration when designing inflammasome inhibitors. This review summarizes the different techniques that can be used to study the mechanism of action of potential inflammasome inhibitory molecules.

Pathogenic NLRP3 mutants form constitutively active inflammasomes resulting in immune-metabolic limitation of IL-1ß production.

Cryopyrin-associated periodic syndrome (CAPS) is an autoinflammatory condition resulting from monoallelic NLRP3 variants that facilitate IL-1ß production. Although these are gain-of-function variants characterized by hypersensitivity to cell priming, patients with CAPS and animal models of the disease may present inflammatory flares without identifiable external triggers. Here we find that CAPS-associated NLRP3 variants are forming constitutively active inflammasome, which induce increased basal cleavage of gasdermin D, IL-18 release and pyroptosis, with a concurrent basal pro-inflammatory gene expression signature, including the induction of nuclear receptors 4 A. The constitutively active NLRP3-inflammasome of CAPS is responsive to the selective NLRP3 inhibitor MCC950 and its activation is regulated by deubiquitination. Despite their preactivated state, the CAPS inflammasomes are responsive to activation of the NF-κB pathway. NLRP3-inflammasomes with CAPS-associated variants affect the immunometabolism of the myeloid compartment, leading to disruptions in lipids and amino acid pathways and impaired glycolysis, limiting IL-1ß production. In summary, NLRP3 variants causing CAPS form a constitutively active inflammasome inducing pyroptosis and IL-18 release without cell priming, which enables the host's innate defence against pathogens while also limiting IL-1ß-dependent inflammatory episodes through immunometabolism modulation.

Increased NLRP3 Inflammasome Activation and Pyroptosis in Patients With Multiple Sclerosis With Fingolimod Treatment Failure.

BACKGROUND AND OBJECTIVES: Inflammasomes are involved in the pathogenesis of different neuroimmune and neurodegenerative diseases, including multiple sclerosis (MS). In a previous study by our group, the nucleotide-binding oligomerization domain, leucine-rich repeat receptor and pyrin-domain-containing 3 (NLRP3) inflammasome was reported to be associated with the response to interferon-beta in MS. Based on recent data showing the potential for the oral therapy fingolimod to inhibit NLRP3 inflammasome activation, here we investigated whether fingolimod could also be implicated in the response to this therapy in patients with MS. METHODS: NLRP3 gene expression levels were measured by real-time PCR in peripheral blood mononuclear cells at baseline and after 3, 6, and 12 months in a cohort of patients with MS treated with fingolimod (N = 23), dimethyl fumarate (N = 21), and teriflunomide (N = 21) and classified into responders and nonresponders to the treatment according to clinical and radiologic criteria. In a subgroup of fingolimod responders and nonresponders, the percentage of monocytes with an oligomer of ASC was determined by flow cytometry, and the levels of interleukin (IL)-1ß, IL-18, IL-6, tumor necrosis factor (TNF)α, and galectin-3 were quantified by ELISA. RESULTS: NLPR3 expression levels were significantly increased in fingolimod nonresponders after 3 (p = 0.03) and 6 months (p = 0.008) of treatment compared with the baseline but remained similar in responders at all time points. These changes were not observed in nonresponders to the other oral therapies tested. The formation of an oligomer of ASC in monocytes after lipopolysaccharide and adenosine 5'-triphosphate stimulation was significantly decreased in responders (p = 0.006) but increased in nonresponders (p = 0.0003) after 6 months of fingolimod treatment compared with the baseline. Proinflammatory cytokine release from stimulated peripheral blood mononuclear cells was comparable between responders and nonresponders, but galectin-3 levels on cell supernatants, as a marker of cell damage, were significantly increased in fingolimod nonresponders (p = 0.02). DISCUSSION: The differential effect of fingolimod on the formation of an inflammasome-triggered ASC oligomer in monocytes between responders and nonresponders could be used as a response biomarker after 6 months of fingolimod treatment and suggests that fingolimod may exert their beneficial effects by reducing inflammasome signaling in a subset of patients with MS.

NLRP3 inflammasome activation and symptom burden in KRAS-mutated CMML patients is reverted by IL-1 blocking therapy.

Chronic myelomonocytic leukemia (CMML) is frequently associated with mutations in the rat sarcoma gene (RAS), leading to worse prognosis. RAS mutations result in active RAS-GTP proteins, favoring myeloid cell proliferation and survival and inducing the NLRP3 inflammasome together with the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which promote caspase-1 activation and interleukin (IL)-1ß release. Here, we report, in a cohort of CMML patients with mutations in KRAS, a constitutive activation of the NLRP3 inflammasome in monocytes, evidenced by ASC oligomerization and IL-1ß release, as well as a specific inflammatory cytokine signature. Treatment of a CMML patient with a KRASG12D mutation using the IL-1 receptor blocker anakinra inhibits NLRP3 inflammasome activation, reduces monocyte count, and improves the patient's clinical status, enabling a stem cell transplant. This reveals a basal inflammasome activation in RAS-mutated CMML patients and suggests potential therapeutic applications of NLRP3 and IL-1 blockers.

Characterization of P2X7 Receptors in Human Blood Cells.

P2X7 receptors are expressed in different circulating immune cells. Depending on the cell in which they are expressed, their function could vary from activation of the leucine-rich repeat receptor pyrin domain containing 3 (NLRP3) inflammasome or the shedding of different surface receptors. The P2X7 receptor has been involved in the evolution of several inflammatory diseases, and thus its characterization in blood samples is important to investigate its role in human diseases. In this chapter, we present different methods to detect the presence and the activation of P2X7 receptors in whole blood samples. These methods could be used to evaluate the degree of P2X7 receptor activation in blood samples from patients suffering different chronic inflammatory diseases.

Assessment of ASC Oligomerization by Flow Cytometry.

Inflammasomes are multiprotein complexes that critically control different aspects of innate and adaptive immunity. Upon activation, inflammasome proteins oligomerize forming scaffolds to nucleate the apoptosis-associated speck-like protein containing a CARD (ASC) in filaments that will finally result in large ASC oligomers that are commonly named as ASC specks. In this chapter, we present a method to monitor NLRP3 or pyrin inflammasome activation in human monocytes upon extracellular ATP or Clostridium difficile toxin B treatment, respectively, by detecting intracellular oligomers of ASC by flow cytometry. This method could be used to evaluate the degree of inflammasome activation in blood samples from patients suffering from different chronic inflammatory diseases.

NLRP3 Inflammasome and Pyroptosis in Liver Pathophysiology: The Emerging Relevance of Nrf2 Inducers.

Inflammasomes, particularly the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing 3 (NLRP3) inflammasome, apparently serve as crucial regulators of the inflammatory response through the activation of Caspase-1 and induction of pro-inflammatory cytokines and pyroptotic cell death. Pyroptosis is a type of programmed cell death mediated by Caspase-1 cleavage of Gasdermin D and the insertion of its N-terminal fragment into the plasma membrane, where it forms pores, enabling the release of different pro-inflammatory mediators. Pyroptosis is considered not only a pro-inflammatory pathway involved in liver pathophysiology but also an important pro-fibrotic mediator. Diverse molecular mechanisms linking oxidative stress, inflammasome activation, pyroptosis, and the progression of liver pathologies have been documented. Numerous studies have indicated the protective effects of several antioxidants, with the ability to induce nuclear factor erythroid 2-related factor 2 (Nrf2) activity on liver inflammation and fibrosis. In this review, we have summarised recent studies addressing the role of the NLRP3 inflammasome and pyroptosis in the pathogenesis of various hepatic diseases, highlighting the potential application of Nrf2 inducers in the prevention of pyroptosis as liver protective compounds.

Soluble P2X7 Receptor Is Elevated in the Plasma of COVID-19 Patients and Correlates With Disease Severity.

Inflammation is a tightly coordinated response against bacterial and viral infections, triggered by the production of pro-inflammatory cytokines. SARS-CoV-2 infection induces COVID-19 disease, characterized by an inflammatory response mediated through the activation of the NLRP3 inflammasome, which results in the production of IL-1ß and IL-18 along with pyroptotic cell death. The NLRP3 inflammasome could be also activated by sterile danger signals such as extracellular ATP triggering the purinergic P2X7 receptor. Severe inflammation in the lungs of SARS-CoV-2-infected individuals is associated with pneumonia, hypoxia and acute respiratory distress syndrome, these being the causes of death associated with COVID-19. Both the P2X7 receptor and NLRP3 have been considered as potential pharmacological targets for treating inflammation in COVID-19. However, there is no experimental evidence of the involvement of the P2X7 receptor during COVID-19 disease. In the present study, we determined the concentration of different cytokines and the P2X7 receptor in the plasma of COVID-19 patients and found that along with the increase in IL-6, IL-18 and the IL-1 receptor antagonist in the plasma of COVID-19 patients, there was also an increase in the purinergic P2X7 receptor. The increase in COVID-19 severity and C-reactive protein concentration positively correlated with increased concentration of the P2X7 receptor in the plasma, but not with the IL-18 cytokine. The P2X7 receptor was found in the supernatant of human peripheral blood mononuclear cells after inflammasome activation. Therefore, our data suggest that determining the levels of the P2X7 receptor in the plasma could be a novel biomarker of COVID-19 severity.

First Description of Late-Onset Autoinflammatory Disease Due to Somatic NLRC4 Mosaicism.

OBJECTIVE: Autoinflammatory diseases are inherited disorders of innate immunity that usually start during childhood. However, several recent reports have described an increasing number of patients with autoinflammatory disease starting in adulthood. This study was undertaken to investigate the underlying cause of a case of late-onset uncharacterized autoinflammatory disease. METHODS: Genetics studies were performed using Sanger sequencing and next-generation sequencing (NGS) methods. In silico, in vitro, and ex vivo analyses were performed to determine the functional consequences of the detected variant. RESULTS: We studied a 57-year-old woman who at the age of 47 years began to have recurrent episodes of fever, myalgias, arthralgias, diffuse abdominal pain, diarrhea, adenopathies, and systemic inflammation, which were relatively well controlled with anti-interleukin-1 (anti-IL-1) drugs. NGS analyses did not detect germline variants in any of the known autoinflammatory disease-associated genes, but they identified the p.Ser171Phe NLRC4 variant in unfractionated blood, with an allele fraction (2-4%) compatible with gene mosaicism. Structural modeling analyses suggested that this missense variant might favor the open, active conformation of the NLRC4 protein, and in vitro and ex vivo analyses confirmed its propensity to oligomerize and activate the NLRC4 inflammasome, with subsequent overproduction of IL-18. CONCLUSION: Our findings indicate that the postzygotic p.Ser171Phe NLRC4 variant is a plausible cause of the disease in the enrolled patient. Functional and structural studies clearly support, for the first time, its gain-of-function behavior, consistent with previously reported NLRC4 pathogenic variants. These novel findings should be considered in the diagnostic evaluation of patients with adult-onset uncharacterized autoinflammatory disease.