The inflammasome adaptor protein ASC promotes amyloid deposition in Cryopyrin-associated Periodic Syndromes

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Mateo, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5416087/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Amyloid A (AA)-type amyloidosis is a complication associated with various autoinflammatory diseases, including cryopyrin-associated periodic syndromes (CAPS). The activation of the NLRP3-inflammasome in CAPS leads to the release of inflammasome oligomers, which are primarily composed of oligomeric filaments of the protein ASC. Recent studies have demonstrated that extracellular deposition of circulating ASC oligomers in tissues can serve as seeds for amyloid deposition, potentially culminating in clinical AA-type amyloidosis. In this study, we found that extracellular ASC in a late-onset, myeloid-restricted CAPS is associated with AA-type amyloidosis. Furthermore, intraperitoneally injected ASC oligomers produced from NLRP3 CAPS variants resulted in increased amyloid deposition. Overall, extracellular ASC oligomers present during inflammatory flares in CAPS promote amyloid deposition in tissues. Targeting extracellular ASC may represent a potential therapeutic strategy to prevent the development of AA-type amyloidosis in autoinflammatory diseases. Immunology Amyloidosis autoinflammatory diseases inflammasome NLRP3 IL-1 serum amyloid A Figures Figure 1 INTRODUCTION Amyloid A (AA)-type amyloidosis is a severe complication associated with different monogenic autoinflammatory diseases (Lachmann et al , 2007). We present a patient diagnosed with late-onset cryopyrin-associated periodic syndromes (CAPS), who started with recurrent inflammatory manifestation at 51 years including fever, chills, urticaria-like rash, fatigue, myalgias, and nephrotic-range proteinuria (>12 mg/day). The patient was found to carry the myeloid-restricted c.924A>T: p.Q308H (RefSeq NM_001243133.1) variant in the gene nucleotide oligomerization-domain, leucine-rich repeats, and pyrin domain-containing 3 ( NLRP3 ), with a variant allele frequency (VAF) in peripheral blood of 5.1%, compatible with gene mosaicism (Mensa-Vilaró et al , 2019). Clinical manifestations were effectively managed with anti-IL-1 therapy (Figure 1A). Additionally, the patient experienced fecal incontinence due to intestinal AA-type amyloidosis (Figure 1B) and had elevated levels of serum amyloid A protein (SAA), which were normalized during anti-IL-1 treatment (Figure 1C). Since the p.Q308H NLRP3 variant has been reported in a limited number of patients as a “likely pathogenic” variant (Mensa-Vilaró et al , 2019), with no functional studies performed, herein we aimed to characterize its consequences on the NLRP3-inflammasome activity. For this proposal, we ectopically expressed this variant in HEK293T cells and observed a significant increased puncta distribution of mutant NLRP3 compared to wild-type NLRP3 expression (Figure 1D and Figure EV1A). This puncta distribution have been previously found for other CAPS-associated NLRP3 variants and during NLRP3-inflammasome activation (Baroja-Mazo et al , 2014). Additionally, co-expression of the inflammasome adaptor protein apoptosis-associated speck-like protein containing a caspase activation domain (ASC) with mutant p.Q308H NLRP3 resulted in a higher number of cells with ASC oligomers (Figure 1E and Figure EV1B). These evidences confirm that the p.Q308H NLRP3 variant promotes the formation of active NLRP3-inflammasomes and support for a gain-of-function behaviour. This aligns with CAPS pathophysiology, which is characterized by constitutive activation of the NLRP3-inflammasome as a consequence of gain-of-function NLRP3 variants (Molina-López et al , 2024). The activation of the NLRP3-inflammasome in CAPS also leads to the release of large inflammasome oligomers, primarily composed of ASC oligomeric filaments, which have been identified extracellularly during acute inflammatory flares in CAPS patients (Baroja-Mazo et al , 2014). We have previously found ASC oligomers in the plasma of different late-onset CAPS with myeloid-restricted NLRP3 variants and developing AA-type amyloidosis (Rowczenio et al , 2017). Additionally, mouse models of CAPS exhibit tissue amyloid deposition (Bertoni et al , 2020), and the macrophages are responsible for the inflammatory phenotype (Frising et al , 2022). Recent studies have demonstrated that extracellular deposition of circulating ASC oligomers in tissues can act as seeds for amyloid deposition, which may finally lead to clinical AA-type amyloidosis (Losa et al , 2024; Venegas et al , 2017). We observed the presence of ASC alongside amyloid foci in the intestine of the late-onset CAPS patient here described (Figure 1B), similar to the observation that ASC expression correlated with amyloidosis in familial Mediterranean fever patients (Balci-Peynircioglu et al , 2008). Since the late-onset CAPS patient here reported carried a myeloid-restricted NLRP3 gain-of-function variant, it is tentative to hypothesize that ASC oligomers could be released from myeloid cells in CAPS. To confirm this hypothesis, we analyzed ASC oligomerization in different leukocyte subpopulations from CAPS patients carrying different germline NLRP3 gain-of-function variants. After 4 hours of in vitro culture we observed that monocytes, but not neutrophils or lymphocytes, were producing ASC oligomerization (Figure 1F), albeit neutrophils were expressing ASC at higher levels than lymphocytes, but less than monocytes (Figure EV1C), indicating that monocytes will be the source of extracellular ASC oligomers in CAPS. To confirm a role of ASC in AA-type amyloid deposition, we used a mouse model of AA-type amyloidosis by administering casein subcutaneously daily over 25 days. This treatment led to amyloid deposition in the liver, kidney, and spleen (Figure 1G and Figure EV2), along with a corresponding increase in serum SAA concentration (Figure 1H). ASC-deficient ( Pycard –/– ) mice showed a reduction in amyloid deposition in tissues (Figure 1G and Figure EV2), despite serum SAA concentration remained elevated (Figure 1H). Notably, when comparing serum SAA concentrations in the serum of casein-treated wild-type mice to Pycard –/– mice after 25 days, SAA concentration was significantly lower in Pycard –/– (Figure 1H). As expected, Pycard –/– mice exhibited decreased production of IL-1β but maintained normal levels of IL-6 following casein administration (Figure 1I). In wild-type mice, casein induces steatohepatitis and significant inflammatory infiltration in the liver, which was absent in Pycard –/– mice (Figure 1G and Figure EV2). To confirm that extracellular ASC oligomeric particles promote amyloid deposition in CAPS, we intraperitoneally injected ASC oligomers produced with p.D305N NLRP3 seeds into Pycard –/– mice. ASC oligomers administration resulted in increased SAA concentrations in the serum (Figure 1J). These mice also showed presence of amyloid deposition in the kidneys and lungs (Figure 1K and Figure EV3). Overall, NLRP3-inflammasome activation in CAPS is associated with inflammation, elevated serum SAA concentration, and increase of monocyte-dependent extracellular ASC oligomers that promote amyloid deposition in tissues, leading to AA-type amyloidosis. Recently, various nanobodies have been developed to target extracellular ASC (Bertheloot et al , 2022; Losa et al , 2024), offering a potential therapy to prevent AA-type amyloidosis development in CAPS and potentially in other chronic inflammatory diseases. METHODS Preparation of experimental models and subject details CAPS patient Written informed consent was obtained from the human volunteers involved in this study to use their biological samples and clinical data for this study. Human samples were used following standard operating procedures with appropriate approval of the Ethical Committee of the Clinical University Hospital Virgen de la Arrixaca (Murcia, Spain) and the principles of the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report. We describe a patient in their 60s (2017) of Spanish descent, born from non-consanguineous parents, with no family history of CAPS. The medical history of the patient includes uterine cancer with hysterectomy in 1995, bilateral neurosensorial hearing loss and papilledema diagnosed in 2010, and ankylosing spondylitis diagnosed in 2014. In 2015, she was diagnosed with faecal incontinence without identifiable triggers. The symptoms included low-grade fever, conjunctivitis, polyarthralgia, and oligoarthritis affecting the knees, wrists, elbows, and ankles. Elevated serum amyloid protein levels were detected (Figure 1C), and AA-type amyloidosis were found in the duodenum (Figure 1B) and rectum, but not in the oesophagus or ileum. Initial treatment began in 2014 with various anti-TNF biologics (Adalimumab, Golimumab, and Certolizumab), with the latter showing limited improvement in dermatological lesions and alleviating inflammatory back pain. Anti-TNF treatment was discontinued in 2017, and Anakinra was initiated, resulting in a partial response. In 2018, Anakinra was replaced with Canakinumab (300 mg every 15 days), which led to a dramatic positive response and good control of clinical symptoms. A myeloid–restricted NLRP3 variant in exon 3 (c.924A>T; p.Q308H; RefSeq NM_001243133.1) was identified in mosaicism (Mensa-Vilaró et al , 2019), leading to a diagnosis of late–onset Muckle-Wells syndrome, the intermediate severity among CAPS phenotypes. Treatment with anti-IL-1 drugs was maintained every 15 days. However, during the COVID-19 pandemic, the anti-IL-1 treatment was interrupted, and the patient developed progressive systemic AA-type amyloidosis, severe diarrhoea, and renal failure, which led to a kidney transplantation. Post-transplantation, anti-IL-1 treatment was resumed, but the clinical symptoms did not improve. Switching to Tocilizumab also failed to alleviate the symptoms. Canakinumab was then administered every 28 days, which improved the diarrhoea and skin urticarial-like rash. In 2022, the patient suffered a stroke due to hypertension, resulting in cognitive impairment. In June 2024, the patient passed away due to sepsis and multiorgan failure. In this study we also analysed blood samples collected in EDTA anticoagulated tubes from healthy donors ( n = 5) and CAPS (Muckle-Wells syndrome) patients who carried the following germline NLRP3 gain-of-function variants: p.R262W ( n = 1), p.D305N ( n = 1), p.T350M ( n = 1) and p.A441T ( n = 2). By the time the blood was extracted, all CAPS patients were under IL-1 blocking therapy with inactive disease. Cell culture and transfections Human blood (50 μl) was cultured in polystyrene flow cytometry tubes (Falcon) with RPMI 1640 medium (Sigma) containing 10% FCS and 2 mM Glutamax (Thermo Fisher) for 4 hours at 37ºC in a humidified 5% CO 2 incubator. HEK293T cells (CRL-11268, American Type Culture Collection) were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/ F-12 (1:1) (Biowest) supplemented with 10% fetal calf serum (FCS) (Biowest) and 2 mM L-glutamin (Gibco). Cells were maintained at 37 ºC in a humidified 5% CO 2 incubator. Cell line was not authenticated but was free of mycoplasma by routinely testing with the MycoProbe Mycoplasma Detection Kit following manufacturer instructions (R&D Systems). Lipofectamine 2000 (Invitrogen) was used according to the manufacturer's instructions for the transfection of HEK293T cells using 0.1 to 1 μg of NLRP3 wild-type or p.Q308H construct, and with 0.1 μg of ASC tagged with Red Fluorescent Protein (RFP). Microscopy assays were performed after 24h of transfection. Animals All experimental protocols for animal handling were refined and approved by the ethics committee of the University of Murcia (reference CEEA 554/2019), the Biosecurity committee of the University of Murcia (reference CBE217/2019) and the Animal Health Service of the General Directorate of Fishing and Farming of the Council of Murcia ( Servicio de Sanidad Animal, Dirección General de Ganadería y Pesca, Consejería de Agricultura y Agua Región de Murcia , reference A13211201). C57BL/6 mice (WT, wild-type, RRID: IMSR_JAX:000664) were obtained from the Jackson Laboratories, and ASC deficient mice in C57BL/6 background ( Pycard –/– ) (Baroja-Mazo et al , 2014) was a generous gift from I. Couillin (University of Orleans and CNRS, Orleans, France). For all experiments, male and female mice between 8-10 weeks of age were used, no differences were observed in SAA concentrations among males and females. Mice were bred in specific pathogen-free conditions with a 12:12 h light-dark cycle and used in accordance with the Spanish national (Royal Decree 1201/2005 and Law 32/2007) and EU (86/609/EEC and 2010/63/EU) legislation. Sample size was calculated estimating an alpha of 0.05 and a statistical power of 94%. Animals were randomized before starting procedures and initial weight of animals were similar among randomized groups. Subcutaneous daily injection of 500 ml of saline sterile solution or 10% vitamin-free casein (Merck) in 0.05M of NaHCO 3 for 5 or 28 days. In another set of experiments, recombinant ASC oligomers were intraperitoneal injected at a dose of 4x10 6 for mice. Mice were sacrifice at days 7, 10, 14 and 18 after injection. After procedures the animals were euthanized by CO 2 inhalation and peritoneal lavages and blood and tissue samples were collected. Blood samples were obtained by thoracic aorta and were centrifuged at 12,500 g for 10 min. The recovered serum was stored at -80ºC until further use. For tissue harvesting the abdominal wall was exposed, the organs were removed using scissors and forceps and were fixed and paraffin-embedded or stored at -80°C for future analysis. Plasmid constructs The mutation of human NLRP3 (UniProt #Q96P20) was generated by overlapping polymerase chain reaction (PCR) to introduce a point mutation (p.Q308H) and cloned into pcDNA3.1/V5-His Topo (Life Technologies). Sequencing of the construct was performed to confirm correct modification and the absence of unwanted mutations. The construct was double tagged using overlapping PCR in the N terminus to YFP. On the other hand, human ASC (Uniprot #Q9ULZ3) expression vector tagged with RFP at the C terminus was kindly provided by E. Latz (Institute of Innate Immunity, University Hospital Bonn, Bonn, Germany). ASC oligomer production We followed the protocol registered at doi: 10.21769/BioProtoc.1480 with modifications. In particular, HEK293T cells stably expressing human NLRP3 p.D305N were transiently transfected with an expression vector coding for human ASC-YFP tagged at C-terminal using Lipofectamine TM 2000 according to the manufacturer instructions. After 48 h of transfection, ASC oligomerization was confirmed by fluorescence microscope (Nikon Eclipse Ti) and cells were resuspended on 600mL Buffer A composed of 20 mM HEPES (Sigma Aldrich), 300 mM Sucrose (Sigma Aldrich), 10 mM KCl (Panreac), 1.5 mM MgCl 2 (Sigma Aldrich), 1 mM EDTA (Sigma Aldrich), 1 mM EGTA (Sigma Aldrich). Cell suspension was mechanically lysed by pressuring 10 times with 1ml syringe and 20G needle followed by pressuring 20 times with 1ml syringe and 25G needle. Mechanical cell lysis was accompanied of a final heat shock cell lysis with fast freezing on liquid N 2 , followed by 37ºC water bath unfreezing. Lysates were centrifuged at 400 g for 8 min at 4ºC. Resulting supernatants were mixed with 2 volumes of 2x CHAPS buffer composed of Buffer A with 0.2% CHAPS (Calbiochem), and then centrifuged at 2000 g for 10 min at 4ºC. Filtered volume was mixed 1:1 with 2x CHAPS buffer and centrifuged at 2,300 g for 8 min at 4ºC. Pellet was washed by resuspending it in 1mL 1x CHAPS buffer followed by centrifugation at 5,000 g for 8 min at 4ºC. This step was repeated 3 times. Pellet was added into a 40% Percoll (Merck Millipore) diluted in 1x CHAPS, and centrifuged at 16,000 g for 10 min at 4ºC. After centrifugation, the intermedial phase was collected and washed with 1mL 1x CHAPS buffer and centrifuged at 16,000 g for 10 min at 4ºC. Pellet was resuspended in 100 mL 1x CHAPS buffer and obtained ASC oligomers were counted by fluorescence microscope (Nikon Eclipse Ti). ASC oligomers were diluted into 1x PBS at a concentration of 4x10 6 oligomers per 100ml, ready to use for in vivo experiments. Fluorescence microscopy To analyse wild-type or p.Q308H NLRP3 puncta distribution, HEK293T cells were transfected as described before. Images were acquired with a Nikon Eclipse Ti microscope equipped with a 20× S Plan Fluor objective (numerical aperture, 0.45) and a digital Sight DS-QiMc camera (Nikon) and 472 nm/520 nm and 543 nm/593 nm filter sets (Semrock), and the NIS-Elements AR software (Nikon). An example of puncta distribution in NLRP3 expressing cells is shown in Figure EV1A. Images were analysed with ImageJ (US National Institutes of Health). Flow cytometry Intracellular ASC-RFP-speck formation in HEK293T cells was evaluated after 24 h post-transfection by Time of Flight for Inflammasome Evaluation (TOFIE) in different gates with increasing mean fluorescence intensity for NLRP3-YFP. Samples were analysed by flow cytometry using LSRFortessa (BD Biosciences) and FlowJo™ v10.8 Software (BD Life Sciences). Intracellular ASC oligomerization in blood human leukocytes was evaluated by TOFIE flow cytometry technique. Cultured human whole blood cells were stained using the phycoerythrin (PE) conjugated mouse monoclonal anti-ASC antibody (clone HASC-71, catalogue 653903, Biolegend, 1:500). Monocytes were stained and gated with fluorescein (FITC)-conjugated mouse anti-CD14 monoclonal antibody (clone M5E2; catalogue #557153; BD Biosciences, 1:10) and neutrophils were gated in a forward scatter/side scatter (FSC/SSC) dot plot and gated with allophycocyanin (APC)-conjugated mouse anti-CD15 monoclonal antibody (clone HI98, catalogue 551376, BD Biosciences, 1:10). Lymphocytes were gated in a FSC/SSC dot plot. Human blood samples were analysed by flow cytometry using FACS Canto (BD Biosciences) and the FlowJo™ v10.8 Software (BD Life Sciences). Histology To determine presence of ASC expression associated with amyloid deposits within the affected tissue, samples from human intestine (duodenum) were obtained by endoscopic biopsy, formalin-fixed and paraffin embedded. 4 μm-thick sections stained with a standard haematoxylin and eosin (H&E) technique. Briefly, after deparaffination and rehydration, sections were stained with Mayer’s haematoxylin (Thermo Scientific) and alcoholic eosin Y (Thermo Scientific). To study co-localization of ASC and amyloid deposits, a combined indirect immunohistochemical procedure and a Congo red staining were performed in 10 μm-thick sections. For immunohistochemistry, after deparaffination and rehydration, a heat-induced antigen demasking procedure was carried out using a commercial kit (High pH target retrieval solution, Agilent-Dako). After the endogenous peroxidase inactivation and background blockage, the sections were incubated at 4 ºC overnight with the primary anti-ASC antibody (Biolegend mouse anti-ASC clone HASC-71; ref: 653902; dilution 1:250). Sections were then incubated with the secondary-HRP labelled polymer (Vector ImmPress anti-mouse, Vector Laboratories, Burlingame, USA) for 30 minutes at 37ºC. Immunoreaction was finally revealed with 3-3´ diaminobencidine (DAB) for 5 min at room temperature by using a commercial kit (DAB chromogen kit, Agilent-Dako). Positive immunoreaction was identified as a dark-brown precipitated. After the immunohistochemical procedure, a Congo red stain was performed on the sections by using a commercial kit (Congo red stain, Abcam) following the manufacturer instructions. The sections were finally contrasted with Mayer’s haematoxylin, dehydrated, and mounted in permanent media. Sections from human kidney inflammatory amyloidosis were complementary used as a positive control. Immunohistochemical staining was evaluated by using a brightfield standard microscope (Zeiss Axio Scope A10, Karl Zeiss). To evaluate the presence of amyloid deposits the sections were visualized under polarized light by using a polarization module on the same microscope. The amyloid protein was identified as light-green extracellular deposits. Representative images were obtained with a high-resolution digital camera (AxioCam 506 color, Karl Zeiss), by using a specialized software (Zeiss Zen Ver. 3.0 Blue Edition). Pathologist was blinded to sample identity. ELISA Mouse serum was used with the following ELISA kits according to manufacturer instructions: mouse IL-1b (RRID: AB_2574946, Thermo Fisher Scientific), mouse IL-6 (RRID: AB_2877063, R&D System), and mouse SAA (ORB170167, Biorbyt). ELISA was read in a Synergy Mx (BioTek) plate reader at 450 nm and corrected at 540 nm. Statistical analysis Statistical analyses were performed using GraphPad Prism version 9 (GraphPad Software Inc). Normality of the samples was determined with D’Angostino and Pearson omnibus K2 normality test. Outliers from data sets were identified by the ROUT method with Q = 1% and were eliminated from the analysis and representation. All data are shown as mean values and errors bars represent standard error (SEM). Unpaired t -test, two tails, was used to compare two groups, for comparison of multiple groups a two-way ANOVA was used. Significance p values are either indicated by exact number in the figure legend and using the following symbols in the figure: **** p <0.0001; ** p <0.01. Declarations Data availability This study includes no data deposited in external repositories. Disclosure Statement & Competing Interests PP and JJMG declares that they are inventors in a patent filled on March 2020 by the Fundación para la Formación e Investigación Sanitaria de la Región de Murcia (PCT/EP2020/056729) for a method to identify NLRP3-immunocompromised sepsis patients. PP, LH-N, JJMG and ABM are co-founders of Viva in vitro diagnostics SL, but declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The remaining authors declare no competing interests. Acknowledgments We thank E. Latz (Institute of Innate Immunity, University Hospital Bonn, Bonn, Germany) for the human ASC expression vector tagged with RFP and I. Couillin (University of Orleans and CNRS, Orleans, France) for ASC deficient mice. We thank the pathology service or the Biomedical Research Institute of Murcia (Spain), the central core facility of cell culture of the University of Murcia (Spain) for flow cytometry facilities, and animal facilities at University of Murcia and the Biomedical Research Institute of Murcia (Spain). This work was supported by grants from MCIN/AEI/10.13039/501100011033 (grants PID2020-116709RB-I00, CNS2022-135101 and RED2022-134511-T to PP), FEDER/Ministerio de Ciencia, Innovación y Universidades – Agencia Estatal de Investigación (grant SAF2017‐88276‐R to PP), Fundación Séneca (grant 21897/PI/22 to PP, grant 21967/JLI/22 to JJMG), and the Instituto Salud Carlos III (grant AC22/00009 to PP; PI20/00185 to ABM). LH-N was supported by the fellowship 21214/FPI/19 (Fundación Séneca, Región de Murcia, Spain). CM-L was funded by the fellowship PRE2018-086824 (Ministerio economía y competitividad). SVM was supported by a fellowship from Instituto Salud Carlos III (FI21/00073). JJGM. was supported by a Maria Zambrano fellowship at University of Murcia. References Balci-Peynircioglu B, Waite AL, Schaner P, Taskiran ZE, Taskiran ZE, Richards N, Orhan D, Orhan D, Gucer S, Gucer S, et al (2008) Expression of ASC in renal tissues of familial mediterranean fever patients with amyloidosis: postulating a role for ASC in AA type amyloid deposition. Experimental Biology and Medicine 233: 1324–1333 Baroja-Mazo A, Martín-Sánchez F, Gomez AI, Martínez CM, Amores-Iniesta J, Compan V, Barberà-Cremades M, Yagüe J, Ruiz-Ortiz E, Antón J, et al (2014) The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response. Nature Immunology 15: 738–748 Bertheloot D, Wanderley CW, Schneider AH, Schiffelers LD, Wuerth JD, Tödtmann JM, Maasewerd S, Hawwari I, Duthie F, Rohland C, et al (2022) Nanobodies dismantle post‐pyroptotic ASC specks and counteract inflammation in vivo. EMBO Molecular Medicine 14: e15415 Bertoni A, Carta S, Baldovini C, Penco F, Balza E, Borghini S, Di Duca M, Ognio E, Signori A, Nozza P, et al (2020) A novel knock-in mouse model of cryopyrin-associated periodic syndromes with development of amyloidosis: Therapeutic efficacy of proton pump inhibitors. The Journal of Allergy and Clinical Immunology 145: 368-378.e13 Franklin BS, Bossaller L, De Nardo D, Ratter JM, Stutz A, Engels G, Brenker C, Nordhoff M, Mirandola SR, Al-Amoudi A, et al (2014) The adaptor ASC has extracellular and ‘prionoid’ activities that propagate inflammation. Nature Immunology 15: 727–737 Frising UC, Ribo S, Doglio MG, Malissen B, van Loo G & Wullaert A (2022) Nlrp3 inflammasome activation in macrophages suffices for inducing autoinflammation in mice. EMBO Reports 23: e54339 Lachmann HJ, Goodman HJB, Gilbertson JA, Gallimore JR, Sabin CA, Gillmore JD & Hawkins PN (2007) Natural history and outcome in systemic AA amyloidosis. New England Journal of Medicine 356: 2361–2371 Losa M, Emmenegger M, De Rossi P, Schürch PM, Serdiuk T, Pengo N, Capron D, Bieli D, Bargenda N, Rupp NJ, et al (2024) The ASC inflammasome adapter governs SAA-derived protein aggregation in inflammatory amyloidosis. EMBO Molecular Medicine 16: 2024–2042 Mariathasan S, Newton K, Monack DM, Vucic D, French DM, Lee WP, Roose-Girma M, Erickson S & Dixit VM (2004) Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature 430: 213–218 Mensa-Vilaró A, García-Morato MB, de la Calle-Martin O, Franco-Jarava C, Martínez-Saavedra MT, González-Granado LI, González-Roca E, Fuster JL, Alsina L, Mutchinick OM, et al (2019) Unexpected Relevant Role of Gene Mosaicism in Primary Immunodeficiency Diseases. Journal of Allergy and Clinical Immunology 143: 359–368 Molina-López C, Hurtado-Navarro L, García CJ, Angosto-Bazarra D, Vallejo F, Tapia-Abellán A, Marques-Soares JR, Vargas C, Bujan-Rivas S, Tomás-Barberán FA, et al (2024) Pathogenic NLRP3 mutants form constitutively active inflammasomes resulting in immune-metabolic limitation of IL-1β production. Nature Communications 15: 1096 Rowczenio DMDM, Gomes SMSM, Aróstegui JIJI, Mensa-Vilaro A, Omoyinmi E, Trojer H, Baginska A, Baroja-Mazo A, Pelegrin P, Savic S, et al (2017) Late-Onset Cryopyrin-Associated Periodic Syndromes Caused by Somatic NLRP3 Mosaicism—UK Single Center Experience. Frontiers in Immunology 8: 1410 Venegas C, Kumar S, Franklin BS, Dierkes T, Brinkschulte R, Tejera D, Vieira-Saecker A, Schwartz S, Santarelli F, Kummer MP, et al (2017) Microglia-derived ASC specks cross-seed amyloid-β in Alzheimer’s disease. Nature 552: 355–361 Additional Declarations The authors declare potential competing interests as follows: PP and JJMG declares that they are inventors in a patent filled on March 2020 by the Fundación para la Formación e Investigación Sanitaria de la Región de Murcia (PCT/EP2020/056729) for a method to identify NLRP3-immunocompromised sepsis patients. PP, LH-N, JJMG and ABM are co-founders of Viva in vitro diagnostics SL, but declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The remaining authors declare no competing interests. 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Gómez","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"I.","lastName":"Gómez","suffix":""},{"id":375722526,"identity":"c03ffa22-80b5-4c72-9792-6681178c62c2","order_by":8,"name":"Javier Gómez-Román","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Javier","middleName":"","lastName":"Gómez-Román","suffix":""},{"id":375722527,"identity":"9408626e-e8f9-4599-ac8f-91911ed25a0a","order_by":9,"name":"Alberto Baroja-Mazo","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Alberto","middleName":"","lastName":"Baroja-Mazo","suffix":""},{"id":375722528,"identity":"7e3de1a5-7573-448b-8d77-9ef06431ed91","order_by":10,"name":"Juan I. Arostegui","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"I.","lastName":"Arostegui","suffix":""},{"id":375722529,"identity":"7b313384-cf73-45c6-986b-934433e4caff","order_by":11,"name":"Natalia Palmou-Fontana","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Natalia","middleName":"","lastName":"Palmou-Fontana","suffix":""},{"id":375722530,"identity":"7e168319-10ea-4755-8f07-4babe5bf5b23","order_by":12,"name":"Juan J. Martínez-García","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABE0lEQVRIiWNgGAWjYBACxgYgIQHnGgAxe2PjA+K1HABp4Tl82IB4Ow+ACIm0NAl8iphnJD97YFFRJ8cgdvjZ5w8Fd+TkG3LMqnlzGIAMHA6bkWZuIHHmsDGDdJrxjAMGz4wZG86Y3ebdxmBscACXlgQzCcm2A4kN0gnGQL8cTmxm7AFrSdyAw2GMM9K/AbXU1TdIp38GaalvY+YxKwZqqZ+P02E5IFuYExikc8C2JPCwsaUxA7UkMOByWM+bMgmgXwzbpHOKGc4YHDacwcN8WHLuNgnDDTi0GLanb5OWqKiT55dO38xQ8eewvPz8h40f3m6zkccVYoZAcWZQNLChSeCOGnmQ4z7glB4Fo2AUjIJRAAQARP9WEQ8h0kYAAAAASUVORK5CYII=","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Juan","middleName":"J.","lastName":"Martínez-García","suffix":""},{"id":375722531,"identity":"1e785cd8-7b5e-47e6-adc0-73929cc4cf6e","order_by":13,"name":"Pablo Pelegrín","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsElEQVRIiWNgGAWjYFACHsYDQFKGnxQtDCAtPJINJGsxOECsBnP2swcOfNxhw2N8IzuB4cMfIrRY9uQlHJx5Jo3H7EbuBsaZbURoMTiQY3CYt+0wWAszbwMxWs6/AWn5z2M8A6jlDzEOM7gBtuUAj4EEUAsDGxFaLGe8A/klmUfizNsNB3uJ8Ys5f+7BBx932Mnxt+dufPCDKIeBCMYGCOcAERrQtIyCUTAKRsEowAoAdRQ78RHLb5AAAAAASUVORK5CYII=","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Pablo","middleName":"","lastName":"Pelegrín","suffix":""}],"badges":[],"createdAt":"2024-11-08 11:14:53","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":true,"conflictsOfInterestStatement":true,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":true,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-5416087/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5416087/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68779824,"identity":"0aaff61d-4a8d-4354-a1c4-8b277116781f","added_by":"auto","created_at":"2024-11-12 02:26:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":488279,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAmyloid deposition correlates with extracellular ASC in CAPS. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Cutaneous manifestations of a patient with late–onset Cryopyrin–Associated Periodic Syndrome (CAPS) with myeloid–restricted gain-of-function \u003cem\u003eNLRP3\u003c/em\u003e somatic mutation p.Q308H before and after Anakinra treatment. (\u003cstrong\u003eB\u003c/strong\u003e) Congo-red and ASC-stained duodenum from the CAPS patient with amyloidosis. (\u003cstrong\u003eC\u003c/strong\u003e) Serum amyloid A (SAA) concentration in the plasma of the CAPS patient before and after IL-1 blocking therapy, red dotted line denotes 6 mg/l which are the reference value for a healthy adult. (\u003cstrong\u003eD\u003c/strong\u003e) Percentage of HEK293T cells with puncta distribution of NLRP3 wild type (WT) or p.Q308H; Centre values represent the mean (\u003cem\u003en\u003c/em\u003e= 5 independent experiments) and error bars represent s.e.m. Unpaired \u003cem\u003et\u003c/em\u003e-test, two tails (****\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001). (\u003cstrong\u003eE\u003c/strong\u003e) Percentage of HEK293T cells with ASC oligomers after expression of different amounts of NLRP3 WT (\u003cem\u003en\u003c/em\u003e= 5 independent experiments) or p.Q308H (\u003cem\u003en\u003c/em\u003e= 5 independent experiments); Centre values represent the mean and error bars represent s.e.m. Two-way ANOVA (****\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001). (\u003cstrong\u003eF\u003c/strong\u003e) Percentage of ASC specking cells in whole blood cultured for 4 hours gating on lymphocytes (white bar), neutrophils (grey bar) and monocytes (blue bar) from healthy donors and germline CAPS patients with the NLRP3 variants p.R262W, p.D305N, p.T350M and p.A441T; Centre values represent the mean (\u003cem\u003en\u003c/em\u003e= 5 healthy, \u003cem\u003en\u003c/em\u003e= 5 patients) and error bars represent s.e.m; one-way ANOVA (*\u003cem\u003ep\u003c/em\u003e= 0.0270; **\u003cem\u003ep\u003c/em\u003e= 0.0061; \u003cem\u003ens\u003c/em\u003e: not significant \u003cem\u003ep\u003c/em\u003e\u0026gt;0.05). (\u003cstrong\u003eG\u003c/strong\u003e) Congo-red stained in liver, kidney and spleen sections from C57BL/6 and \u003cem\u003ePycard\u003c/em\u003e\u003csup\u003e–/–\u003c/sup\u003e mice after 25 days of casein administration. (\u003cstrong\u003eH,I\u003c/strong\u003e) Concentration of SAA (H), IL-1b and IL-6 (I) in the serum of mice treated as in F after 5 and 25 days (H) or 25 days (I); for H, Two-way ANOVA (**\u003cem\u003ep\u003c/em\u003e= 0.0069; ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001) with saline: \u003cem\u003en\u003c/em\u003e= 2 mice day 2, \u003cem\u003en\u003c/em\u003e= 4 mice day 25; casein: \u003cem\u003en\u003c/em\u003e= 6 mice day 5, \u003cem\u003en\u003c/em\u003e= 9 mice WT day 25, \u003cem\u003en\u003c/em\u003e= 10 mice \u003cem\u003ePycard\u003c/em\u003e\u003csup\u003e–/–\u003c/sup\u003e day 25; for I, IL-6, saline: \u003cem\u003en\u003c/em\u003e= 4 mice, casein: \u003cem\u003en\u003c/em\u003e= 9 mice WT, \u003cem\u003en\u003c/em\u003e= 10 mice \u003cem\u003ePycard\u003c/em\u003e\u003csup\u003e–/–\u003c/sup\u003e; for I, IL-1b \u003cem\u003en\u003c/em\u003e= 3 mice; Centre values represent the mean and error bars represent s.e.m. (\u003cstrong\u003eJ\u003c/strong\u003e) Concentration of SAA in serum of \u003cem\u003ePycard\u003c/em\u003e\u003csup\u003e–/–\u003c/sup\u003e mice after 7, 10, 14 or 18 days of intraperitoneal injection of ASC oligomers; \u003cem\u003en\u003c/em\u003e= 2 mice for control, \u003cem\u003en\u003c/em\u003e= 3 mice for the different days; Centre values represent the mean and error bars represent s.e.m. (\u003cstrong\u003eK\u003c/strong\u003e) Congo-red stained in kidney and lung sections from \u003cem\u003ePycard\u003c/em\u003e\u003csup\u003e–/–\u003c/sup\u003e mice after 10 and 18 days of intraperitoneal ASC oligomers administration (as control, kidney sections from the casein model presented in G is shown). Arrowheads denotes areas of amyloid deposition. Scale bars: 50 mm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5416087/v1/4dfb8c27559e01c9168ea1c1.png"},{"id":68779826,"identity":"3ae96799-e4c8-4d5a-8b47-3f02e95cd910","added_by":"auto","created_at":"2024-11-12 02:26:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":917189,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5416087/v1/77df4cd5-2cd3-43b2-a039-bf49d74da26a.pdf"},{"id":68779825,"identity":"95619b47-4c5c-47e1-85ed-31e0ba7df86e","added_by":"auto","created_at":"2024-11-12 02:26:34","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":562787693,"visible":true,"origin":"","legend":"","description":"","filename":"FigureEV.docx","url":"https://assets-eu.researchsquare.com/files/rs-5416087/v1/641b3983392fc743c01c3c6a.docx"}],"financialInterests":"The authors declare potential competing interests as follows: PP and JJMG declares that they are inventors in a patent filled on March 2020 by the Fundación para la Formación e Investigación Sanitaria de la Región de Murcia (PCT/EP2020/056729) for a method to identify NLRP3-immunocompromised sepsis patients. PP, LH-N, JJMG and ABM are co-founders of Viva in vitro diagnostics SL, but declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The remaining authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eThe inflammasome adaptor protein ASC promotes amyloid deposition in Cryopyrin-associated Periodic Syndromes\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAmyloid A (AA)-type amyloidosis is a severe complication associated with different monogenic autoinflammatory diseases\u0026nbsp;(Lachmann \u003cem\u003eet al\u003c/em\u003e, 2007). We present a patient diagnosed with late-onset cryopyrin-associated periodic syndromes (CAPS), who started with recurrent inflammatory manifestation at 51 years including fever, chills, urticaria-like rash, fatigue, myalgias, and nephrotic-range proteinuria (\u0026gt;12 mg/day). The patient was found to carry the myeloid-restricted c.924A\u0026gt;T: p.Q308H (RefSeq NM_001243133.1) variant in the gene nucleotide oligomerization-domain, leucine-rich repeats, and pyrin domain-containing 3 (\u003cem\u003eNLRP3\u003c/em\u003e), with a variant allele frequency (VAF) in peripheral blood of 5.1%, compatible with gene mosaicism\u0026nbsp;(Mensa-Vilar\u0026oacute; \u003cem\u003eet al\u003c/em\u003e, 2019). Clinical manifestations were effectively managed with anti-IL-1 therapy (Figure 1A). Additionally, the patient experienced fecal incontinence due to intestinal AA-type amyloidosis (Figure 1B) and had elevated levels of serum amyloid A protein (SAA), which were normalized during anti-IL-1 treatment (Figure 1C). Since the p.Q308H NLRP3 variant has been reported \u0026nbsp;in a limited number of patients as a \u0026ldquo;likely pathogenic\u0026rdquo; variant\u0026nbsp;(Mensa-Vilar\u0026oacute; \u003cem\u003eet al\u003c/em\u003e, 2019),\u0026nbsp;with no functional studies performed, herein we aimed to characterize its consequences on the NLRP3-inflammasome activity. For this proposal, we ectopically expressed this variant in HEK293T cells and observed a significant increased puncta distribution of mutant NLRP3 compared to wild-type NLRP3 expression (Figure 1D and Figure EV1A). This puncta distribution have been previously found for other CAPS-associated NLRP3 variants and during NLRP3-inflammasome activation\u0026nbsp;(Baroja-Mazo \u003cem\u003eet al\u003c/em\u003e, 2014). Additionally, co-expression of the inflammasome adaptor protein apoptosis-associated speck-like protein containing a caspase activation domain (ASC) with mutant p.Q308H NLRP3 resulted in a higher number of cells with ASC oligomers (Figure 1E and Figure EV1B). These evidences confirm that the p.Q308H NLRP3 variant promotes the formation of active NLRP3-inflammasomes and support for a gain-of-function behaviour. This aligns with CAPS pathophysiology, which is characterized by constitutive activation of the NLRP3-inflammasome as a consequence of gain-of-function \u003cem\u003eNLRP3\u003c/em\u003e variants\u0026nbsp;(Molina-L\u0026oacute;pez \u003cem\u003eet al\u003c/em\u003e, 2024). The activation of the NLRP3-inflammasome in CAPS also leads to the release of large inflammasome oligomers, primarily composed of ASC oligomeric filaments, which have been identified extracellularly during acute inflammatory flares in CAPS patients\u0026nbsp;(Baroja-Mazo \u003cem\u003eet al\u003c/em\u003e, 2014). We have previously found ASC oligomers in the plasma of different late-onset CAPS with myeloid-restricted NLRP3 variants and developing AA-type amyloidosis\u0026nbsp;(Rowczenio \u003cem\u003eet al\u003c/em\u003e, 2017). Additionally, mouse models of CAPS exhibit tissue amyloid deposition\u0026nbsp;(Bertoni \u003cem\u003eet al\u003c/em\u003e, 2020), and the macrophages are responsible for the inflammatory phenotype\u0026nbsp;(Frising \u003cem\u003eet al\u003c/em\u003e, 2022). Recent studies have demonstrated that extracellular deposition of circulating ASC oligomers in tissues can act as seeds for amyloid deposition, which may finally lead to clinical AA-type amyloidosis\u0026nbsp;(Losa \u003cem\u003eet al\u003c/em\u003e, 2024; Venegas \u003cem\u003eet al\u003c/em\u003e, 2017).\u0026nbsp;We observed the presence of ASC alongside amyloid foci in the intestine of the late-onset CAPS patient here described (Figure 1B), similar to the observation that\u0026nbsp;ASC expression correlated with amyloidosis\u0026nbsp;in\u0026nbsp;familial Mediterranean fever patients\u0026nbsp;(Balci-Peynircioglu \u003cem\u003eet al\u003c/em\u003e, 2008). Since the late-onset CAPS patient here reported carried a myeloid-restricted \u003cem\u003eNLRP3\u003c/em\u003e gain-of-function variant, it is tentative to hypothesize that ASC oligomers could be released from myeloid cells in CAPS. To confirm this hypothesis, we analyzed ASC oligomerization in different leukocyte subpopulations from CAPS patients carrying different germline \u003cem\u003eNLRP3\u0026nbsp;\u003c/em\u003egain-of-function variants. After 4 hours of \u003cem\u003ein vitro\u003c/em\u003e culture we observed that monocytes, but not neutrophils or lymphocytes, were producing ASC oligomerization (Figure 1F), albeit neutrophils were expressing ASC at higher levels than lymphocytes, but less than monocytes (Figure EV1C), indicating that monocytes will be the source of extracellular ASC oligomers in CAPS.\u0026nbsp;To confirm a role of ASC in AA-type amyloid deposition, we used a mouse model of AA-type amyloidosis by administering casein subcutaneously daily over 25 days. This treatment led to amyloid deposition in the liver, kidney, and spleen (Figure 1G and Figure EV2), along with a corresponding increase in serum SAA concentration (Figure 1H). ASC-deficient (\u003cem\u003ePycard\u003c/em\u003e\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e) mice showed a reduction in amyloid deposition in tissues (Figure 1G and Figure EV2), despite serum SAA concentration remained elevated (Figure 1H). Notably, when comparing serum SAA concentrations in the serum of casein-treated wild-type mice to \u003cem\u003ePycard\u003c/em\u003e\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice after 25 days, SAA concentration was significantly lower in \u003cem\u003ePycard\u003c/em\u003e\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e (Figure 1H). As expected, \u003cem\u003ePycard\u003c/em\u003e\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice exhibited decreased production of IL-1\u0026beta; but maintained normal levels of IL-6 following casein administration (Figure 1I). In wild-type mice, casein induces steatohepatitis and significant inflammatory infiltration in the liver, which was absent in \u003cem\u003ePycard\u003c/em\u003e\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice (Figure 1G and Figure EV2). To confirm that extracellular ASC oligomeric particles promote amyloid deposition in CAPS, we intraperitoneally injected ASC oligomers produced with p.D305N NLRP3 seeds into \u003cem\u003ePycard\u003c/em\u003e\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice. ASC oligomers administration resulted in increased SAA concentrations in the serum (Figure 1J). These mice also showed presence of amyloid deposition in the kidneys and lungs (Figure 1K and Figure EV3). Overall, NLRP3-inflammasome activation in CAPS is associated with inflammation, elevated serum SAA concentration, and increase of monocyte-dependent extracellular ASC oligomers that promote amyloid deposition in tissues, leading to AA-type amyloidosis. Recently, various nanobodies have been developed to target extracellular ASC\u0026nbsp;(Bertheloot \u003cem\u003eet al\u003c/em\u003e, 2022; Losa \u003cem\u003eet al\u003c/em\u003e, 2024), offering a potential therapy to prevent AA-type amyloidosis development in CAPS and potentially in other chronic inflammatory diseases.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cstrong\u003ePreparation of experimental models and subject details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCAPS patient\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from the human volunteers involved in this study to use their biological samples and clinical data for this study. Human samples were used following standard operating procedures with appropriate approval of the Ethical Committee of the Clinical University Hospital Virgen de la Arrixaca (Murcia, Spain) and the principles of the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.\u003c/p\u003e\n\u003cp\u003eWe describe a patient in their 60s (2017) of Spanish descent, born from non-consanguineous parents, with no family history of CAPS. The medical history of the patient includes uterine cancer with hysterectomy in 1995, bilateral neurosensorial hearing loss and papilledema diagnosed in 2010, and ankylosing spondylitis diagnosed in 2014. In 2015, she was diagnosed with faecal incontinence without identifiable triggers. The symptoms included low-grade fever, conjunctivitis, polyarthralgia, and oligoarthritis affecting the knees, wrists, elbows, and ankles. Elevated serum amyloid protein levels were detected (Figure 1C), and AA-type amyloidosis were found in the duodenum (Figure 1B) and rectum, but not in the oesophagus or ileum. Initial treatment began in 2014 with various anti-TNF biologics (Adalimumab, Golimumab, and Certolizumab), with the latter showing limited improvement in dermatological lesions and alleviating inflammatory back pain. Anti-TNF treatment was discontinued in 2017, and Anakinra was initiated, resulting in a partial response. In 2018, Anakinra was replaced with Canakinumab (300 mg every 15 days), which led to a dramatic positive response and good control of clinical symptoms. A myeloid\u0026ndash;restricted \u003cem\u003eNLRP3\u003c/em\u003e variant in exon 3 (c.924A\u0026gt;T; p.Q308H; RefSeq NM_001243133.1) was identified in mosaicism\u0026nbsp;(Mensa-Vilar\u0026oacute; \u003cem\u003eet al\u003c/em\u003e, 2019),\u0026nbsp;leading to a diagnosis of late\u0026ndash;onset Muckle-Wells syndrome, the intermediate severity among CAPS phenotypes. Treatment with anti-IL-1 drugs was maintained every 15 days. However, during the COVID-19 pandemic, the anti-IL-1 treatment was interrupted, and the patient developed progressive systemic AA-type amyloidosis, severe diarrhoea, and renal failure, which led to a kidney transplantation. Post-transplantation, anti-IL-1 treatment was resumed, but the clinical symptoms did not improve. Switching to Tocilizumab also failed to alleviate the symptoms. Canakinumab was then administered every 28 days, which improved the diarrhoea and skin urticarial-like rash. In 2022, the patient suffered a stroke due to hypertension, resulting in cognitive impairment. In June 2024, the patient passed away due to sepsis and multiorgan failure.\u003c/p\u003e\n\u003cp\u003eIn this study we also analysed blood samples collected in EDTA anticoagulated tubes from healthy donors (\u003cem\u003en\u003c/em\u003e= 5) and CAPS (Muckle-Wells syndrome) patients who carried the following germline NLRP3 gain-of-function variants: p.R262W (\u003cem\u003en\u003c/em\u003e= 1), p.D305N (\u003cem\u003en\u003c/em\u003e= 1), p.T350M (\u003cem\u003en\u003c/em\u003e= 1) and p.A441T (\u003cem\u003en\u003c/em\u003e= 2). By the time the blood was extracted, all CAPS patients were under IL-1 blocking therapy with inactive disease.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCell culture and transfections\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman blood (50 \u0026mu;l) was cultured in polystyrene flow cytometry tubes (Falcon) with RPMI 1640 medium (Sigma) containing 10% FCS and 2 mM Glutamax (Thermo Fisher) for 4 hours at 37\u0026ordm;C in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e incubator.\u003c/p\u003e\n\u003cp\u003eHEK293T cells (CRL-11268, American Type Culture Collection) were maintained in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM)/ F-12 (1:1) (Biowest) supplemented with 10% fetal calf serum (FCS) (Biowest) and 2 mM L-glutamin (Gibco). Cells were maintained at 37 \u0026ordm;C in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. Cell line was not authenticated but was free of mycoplasma by routinely testing with the MycoProbe Mycoplasma Detection Kit following manufacturer instructions (R\u0026amp;D Systems). Lipofectamine 2000 (Invitrogen) was used according to the manufacturer\u0026apos;s instructions for the transfection of HEK293T cells using 0.1 to 1 \u0026mu;g of NLRP3 wild-type or p.Q308H construct, and with 0.1 \u0026mu;g of ASC tagged with Red Fluorescent Protein (RFP). Microscopy assays were performed after 24h of transfection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnimals\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental protocols for animal handling were refined and approved by the ethics committee of the University of Murcia (reference CEEA 554/2019), the Biosecurity committee of the University of Murcia (reference CBE217/2019) and the Animal Health Service of the General Directorate of Fishing and Farming of the Council of Murcia (\u003cem\u003eServicio de Sanidad Animal, Direcci\u0026oacute;n General de Ganader\u0026iacute;a y Pesca, Consejer\u0026iacute;a de Agricultura y Agua Regi\u0026oacute;n de Murcia\u003c/em\u003e, reference A13211201). C57BL/6 mice (WT, wild-type, RRID: IMSR_JAX:000664) were obtained from the Jackson Laboratories, and ASC deficient mice in C57BL/6 background (\u003cem\u003ePycard\u003c/em\u003e\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e)\u0026nbsp;(Baroja-Mazo \u003cem\u003eet al\u003c/em\u003e, 2014)\u0026nbsp;was a generous gift from I. Couillin (University of Orleans and CNRS, Orleans, France). For all experiments, male and female mice between 8-10 weeks of age were used, no differences were observed in SAA concentrations among males and females. Mice were bred in specific pathogen-free conditions with a 12:12 h light-dark cycle and used in accordance with the Spanish national (Royal Decree 1201/2005 and Law 32/2007) and EU (86/609/EEC and 2010/63/EU) legislation. Sample size was calculated estimating an alpha of 0.05 and a statistical power of 94%. Animals were randomized before starting procedures and initial weight of animals were similar among randomized groups. Subcutaneous daily injection of 500\u0026nbsp;ml of saline sterile solution or 10% vitamin-free casein (Merck) in 0.05M of NaHCO\u003csub\u003e3\u003c/sub\u003e for 5 or 28 days. In another set of experiments, recombinant ASC oligomers were intraperitoneal injected at a dose of 4x10\u003csup\u003e6\u003c/sup\u003e for mice. Mice were sacrifice at days 7, 10, 14 and 18 after injection.\u0026nbsp;After procedures the animals were euthanized by CO\u003csub\u003e2\u003c/sub\u003e inhalation and peritoneal lavages and blood and tissue samples were collected. Blood samples were obtained by thoracic aorta and were centrifuged at 12,500 \u003cem\u003eg\u0026nbsp;\u003c/em\u003efor 10 min. The recovered serum was stored at -80\u0026ordm;C until further use. For tissue harvesting the abdominal wall was exposed, the organs were removed using scissors and forceps and were fixed and paraffin-embedded or stored at -80\u0026deg;C for future analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePlasmid constructs\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mutation of human NLRP3 (UniProt #Q96P20) was generated by overlapping polymerase chain reaction (PCR) to introduce a point mutation (p.Q308H) and cloned into pcDNA3.1/V5-His Topo (Life Technologies). Sequencing of the construct was performed to confirm correct modification and the absence of unwanted mutations. The construct was double tagged using overlapping PCR in the N terminus to YFP. On the other hand, human ASC (Uniprot #Q9ULZ3) expression vector tagged with RFP at the C terminus was kindly provided by E. Latz (Institute of Innate Immunity, University Hospital Bonn, Bonn, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eASC oligomer production\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe followed the protocol registered at doi: 10.21769/BioProtoc.1480 with modifications. In particular, HEK293T cells stably expressing human NLRP3 p.D305N were transiently transfected with an expression vector coding for human ASC-YFP tagged at C-terminal using Lipofectamine\u003csup\u003eTM\u003c/sup\u003e 2000 according to the manufacturer instructions. After 48 h of transfection, ASC oligomerization was confirmed by fluorescence microscope (Nikon Eclipse Ti) and cells were resuspended on 600mL Buffer A composed of 20 mM HEPES (Sigma Aldrich), 300 mM Sucrose (Sigma Aldrich), 10 mM KCl (Panreac), 1.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e (Sigma Aldrich), 1 mM EDTA (Sigma Aldrich), 1 mM EGTA (Sigma Aldrich). Cell suspension was mechanically lysed by pressuring 10 times with 1ml syringe and 20G needle followed by pressuring 20 times with 1ml syringe and 25G needle. Mechanical cell lysis was accompanied of a final heat shock cell lysis with fast freezing on liquid N\u003csub\u003e2\u003c/sub\u003e, followed by 37\u0026ordm;C water bath unfreezing. Lysates were centrifuged at 400 \u003cem\u003eg\u003c/em\u003e for 8 min at 4\u0026ordm;C. Resulting supernatants were mixed with 2 volumes of 2x CHAPS buffer composed of Buffer A with 0.2% CHAPS (Calbiochem), and then centrifuged at 2000 \u003cem\u003eg\u003c/em\u003e for 10 min at 4\u0026ordm;C. Filtered volume was mixed 1:1 with 2x CHAPS buffer and centrifuged at 2,300 \u003cem\u003eg\u003c/em\u003e for 8 min at 4\u0026ordm;C. Pellet was washed by resuspending it in 1mL 1x CHAPS buffer followed by centrifugation at 5,000 \u003cem\u003eg\u003c/em\u003e for 8 min at 4\u0026ordm;C. This step was repeated 3 times. Pellet was added into a 40% Percoll (Merck Millipore) diluted in 1x CHAPS, and centrifuged at 16,000 \u003cem\u003eg\u003c/em\u003e for 10 min at 4\u0026ordm;C. After centrifugation, the intermedial phase was collected and washed with 1mL 1x CHAPS buffer and centrifuged at 16,000 \u003cem\u003eg\u003c/em\u003e for 10 min at 4\u0026ordm;C. Pellet was resuspended in 100 mL 1x CHAPS buffer and obtained ASC oligomers were counted by fluorescence microscope (Nikon Eclipse Ti). ASC oligomers were diluted into 1x PBS at a concentration of 4x10\u003csup\u003e6\u003c/sup\u003e oligomers per 100ml, ready to use for \u003cem\u003ein vivo\u003c/em\u003e experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFluorescence microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo analyse wild-type or p.Q308H NLRP3 puncta distribution, HEK293T cells were transfected as described before. Images were acquired with a Nikon Eclipse Ti microscope equipped with a 20\u0026times; S Plan Fluor objective (numerical aperture, 0.45) and a digital Sight DS-QiMc camera (Nikon) and 472 nm/520 nm and 543 nm/593 nm filter sets (Semrock), and the NIS-Elements AR software (Nikon). An example of puncta distribution in NLRP3 expressing cells is shown in Figure EV1A. Images were analysed with ImageJ (US National Institutes of Health).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntracellular ASC-RFP-speck formation in HEK293T cells was evaluated after 24\u0026thinsp;h post-transfection by Time of Flight for Inflammasome Evaluation (TOFIE) in different gates with increasing mean fluorescence intensity for NLRP3-YFP. Samples were analysed by flow cytometry using LSRFortessa (BD Biosciences) and FlowJo\u0026trade; v10.8 Software (BD Life Sciences).\u003c/p\u003e\n\u003cp\u003eIntracellular ASC oligomerization in blood human leukocytes was evaluated by TOFIE flow cytometry technique. Cultured human whole blood cells were stained using the phycoerythrin (PE) conjugated mouse monoclonal anti-ASC antibody (clone HASC-71, catalogue 653903, Biolegend, 1:500). Monocytes were stained and gated with fluorescein (FITC)-conjugated mouse anti-CD14 monoclonal antibody (clone M5E2; catalogue #557153; BD Biosciences, 1:10) and neutrophils were gated in a forward scatter/side scatter (FSC/SSC) dot plot and gated with allophycocyanin (APC)-conjugated mouse anti-CD15 monoclonal antibody (clone HI98, catalogue 551376,\u0026nbsp;BD Biosciences, 1:10). Lymphocytes were gated in a FSC/SSC dot plot. Human blood samples were analysed by flow cytometry using FACS Canto (BD Biosciences) and the FlowJo\u0026trade; v10.8 Software (BD Life Sciences).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine presence of ASC expression associated with amyloid deposits within the affected tissue, samples from human intestine (duodenum) were obtained by endoscopic biopsy, formalin-fixed and paraffin embedded. 4 \u0026mu;m-thick sections stained with a standard haematoxylin and eosin (H\u0026amp;E) technique. Briefly, after deparaffination and rehydration, sections were stained with Mayer\u0026rsquo;s haematoxylin (Thermo Scientific) and alcoholic eosin Y (Thermo Scientific). To study co-localization of ASC and amyloid deposits, a combined indirect immunohistochemical procedure and a Congo red staining were performed in 10 \u0026mu;m-thick sections. For immunohistochemistry, after deparaffination and rehydration, a heat-induced antigen demasking procedure was carried out using a commercial kit (High pH target retrieval solution, Agilent-Dako). After the endogenous peroxidase inactivation and background blockage, the sections were incubated at 4 \u0026ordm;C overnight with the primary anti-ASC antibody (Biolegend mouse anti-ASC clone HASC-71; ref: 653902; dilution 1:250). Sections were then incubated with the secondary-HRP labelled polymer (Vector ImmPress anti-mouse, Vector Laboratories, Burlingame, USA) for 30 minutes at 37\u0026ordm;C. Immunoreaction was finally revealed with 3-3\u0026acute; diaminobencidine (DAB) for 5 min at room temperature by using a commercial kit (DAB chromogen kit, Agilent-Dako). Positive immunoreaction was identified as a dark-brown precipitated. After the immunohistochemical procedure, a Congo red stain was performed on the sections by using a commercial kit (Congo red stain, Abcam) following the manufacturer instructions. The sections were finally contrasted with Mayer\u0026rsquo;s haematoxylin, dehydrated, and mounted in permanent media. Sections from human kidney inflammatory amyloidosis were complementary used as a positive control. Immunohistochemical staining was evaluated by using a brightfield standard microscope (Zeiss Axio Scope A10, Karl Zeiss). To evaluate the presence of amyloid deposits the sections were visualized under polarized light by using a polarization module on the same microscope. The amyloid protein was identified as light-green extracellular deposits. Representative images were obtained with a high-resolution digital camera (AxioCam 506 color, Karl Zeiss), by using a specialized software (Zeiss Zen Ver. 3.0 Blue Edition). Pathologist was blinded to sample identity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse serum was used with the following ELISA kits according to manufacturer instructions: mouse IL-1b\u0026nbsp;(RRID: AB_2574946, Thermo Fisher Scientific), mouse IL-6 (RRID: AB_2877063, R\u0026amp;D System), and mouse SAA (ORB170167, Biorbyt). ELISA was read in a Synergy Mx (BioTek) plate reader at 450 nm and corrected at 540 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using GraphPad Prism version 9 (GraphPad Software Inc). Normality of the samples was determined with D\u0026rsquo;Angostino and Pearson omnibus K2 normality test. Outliers from data sets were identified by the ROUT method with \u003cem\u003eQ\u003c/em\u003e= 1% and were eliminated from the analysis and representation. All data are shown as mean values and errors bars represent standard error (SEM). Unpaired \u003cem\u003et\u003c/em\u003e-test, two tails, was used to compare two groups, for comparison of multiple groups a two-way ANOVA was used. Significance \u003cem\u003ep\u003c/em\u003e values are either indicated by exact number in the figure legend and using the following symbols in the figure: ****\u003cem\u003ep\u003c/em\u003e \u0026lt;0.0001; **\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study includes no data deposited in external repositories.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure Statement \u0026amp; Competing Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePP and JJMG declares that they are inventors in a patent filled on March 2020 by the \u003cem\u003eFundaci\u0026oacute;n para la Formaci\u0026oacute;n e Investigaci\u0026oacute;n Sanitaria de la Regi\u0026oacute;n de Murcia\u003c/em\u003e (PCT/EP2020/056729) for a method to identify NLRP3-immunocompromised sepsis patients. PP, LH-N, JJMG and ABM are co-founders of Viva in vitro diagnostics SL,\u0026nbsp;but declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The remaining authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank E. Latz (Institute of Innate Immunity, University Hospital Bonn, Bonn, Germany) for the human ASC expression vector tagged with RFP and I. Couillin (University of Orleans and CNRS, Orleans, France) for ASC deficient mice. We thank the pathology service or the Biomedical Research Institute of Murcia (Spain), the central core facility of cell culture of the University of Murcia (Spain) for flow cytometry facilities, and animal facilities at University of Murcia and the Biomedical Research Institute of Murcia (Spain). This work was supported by grants from MCIN/AEI/10.13039/501100011033 (grants PID2020-116709RB-I00, CNS2022-135101 and RED2022-134511-T to PP), FEDER/Ministerio de Ciencia, Innovaci\u0026oacute;n y Universidades \u0026ndash; Agencia Estatal de Investigaci\u0026oacute;n (grant SAF2017‐88276‐R to PP), Fundaci\u0026oacute;n S\u0026eacute;neca (grant 21897/PI/22 to PP, grant 21967/JLI/22 to JJMG), and the Instituto Salud Carlos III (grant AC22/00009 to PP; PI20/00185 to ABM). LH-N was supported by the fellowship 21214/FPI/19 (Fundaci\u0026oacute;n S\u0026eacute;neca, Regi\u0026oacute;n de Murcia, Spain). CM-L was funded by the fellowship PRE2018-086824 (Ministerio econom\u0026iacute;a y competitividad). SVM was supported by a fellowship from Instituto Salud Carlos III (FI21/00073). JJGM. was supported by a Maria Zambrano fellowship at University of Murcia.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBalci-Peynircioglu B, Waite AL, Schaner P, Taskiran ZE, Taskiran ZE, Richards N, Orhan D, Orhan D, Gucer S, Gucer S, \u003cem\u003eet al\u003c/em\u003e (2008) Expression of ASC in renal tissues of familial mediterranean fever patients with amyloidosis: postulating a role for ASC in AA type amyloid deposition. \u003cem\u003eExperimental Biology and Medicine\u003c/em\u003e 233: 1324\u0026ndash;1333\u003c/li\u003e\n \u003cli\u003eBaroja-Mazo A, Mart\u0026iacute;n-S\u0026aacute;nchez F, Gomez AI, Mart\u0026iacute;nez CM, Amores-Iniesta J, Compan V, Barber\u0026agrave;-Cremades M, Yag\u0026uuml;e J, Ruiz-Ortiz E, Ant\u0026oacute;n J, \u003cem\u003eet al\u003c/em\u003e (2014) The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response. \u003cem\u003eNature Immunology\u003c/em\u003e 15: 738\u0026ndash;748\u003c/li\u003e\n \u003cli\u003eBertheloot D, Wanderley CW, Schneider AH, Schiffelers LD, Wuerth JD, T\u0026ouml;dtmann JM, Maasewerd S, Hawwari I, Duthie F, Rohland C, \u003cem\u003eet al\u003c/em\u003e (2022) Nanobodies dismantle post‐pyroptotic ASC specks and counteract inflammation in vivo. \u003cem\u003eEMBO Molecular Medicine\u003c/em\u003e 14: e15415\u003c/li\u003e\n \u003cli\u003eBertoni A, Carta S, Baldovini C, Penco F, Balza E, Borghini S, Di Duca M, Ognio E, Signori A, Nozza P, \u003cem\u003eet al\u003c/em\u003e (2020) A novel knock-in mouse model of cryopyrin-associated periodic syndromes with development of amyloidosis: Therapeutic efficacy of proton pump inhibitors. \u003cem\u003eThe Journal of Allergy and Clinical Immunology\u003c/em\u003e 145: 368-378.e13\u003c/li\u003e\n \u003cli\u003eFranklin BS, Bossaller L, De Nardo D, Ratter JM, Stutz A, Engels G, Brenker C, Nordhoff M, Mirandola SR, Al-Amoudi A, \u003cem\u003eet al\u003c/em\u003e (2014) The adaptor ASC has extracellular and \u0026lsquo;prionoid\u0026rsquo; activities that propagate inflammation. \u003cem\u003eNature Immunology\u003c/em\u003e 15: 727\u0026ndash;737\u003c/li\u003e\n \u003cli\u003eFrising UC, Ribo S, Doglio MG, Malissen B, van Loo G \u0026amp; Wullaert A (2022) Nlrp3 inflammasome activation in macrophages suffices for inducing autoinflammation in mice. \u003cem\u003eEMBO Reports\u003c/em\u003e 23: e54339\u003c/li\u003e\n \u003cli\u003eLachmann HJ, Goodman HJB, Gilbertson JA, Gallimore JR, Sabin CA, Gillmore JD \u0026amp; Hawkins PN (2007) Natural history and outcome in systemic AA amyloidosis. \u003cem\u003eNew England Journal of Medicine\u003c/em\u003e 356: 2361\u0026ndash;2371\u003c/li\u003e\n \u003cli\u003eLosa M, Emmenegger M, De Rossi P, Sch\u0026uuml;rch PM, Serdiuk T, Pengo N, Capron D, Bieli D, Bargenda N, Rupp NJ, \u003cem\u003eet al\u003c/em\u003e (2024) The ASC inflammasome adapter governs SAA-derived protein aggregation in inflammatory amyloidosis. \u003cem\u003eEMBO Molecular Medicine\u003c/em\u003e 16: 2024\u0026ndash;2042\u003c/li\u003e\n \u003cli\u003eMariathasan S, Newton K, Monack DM, Vucic D, French DM, Lee WP, Roose-Girma M, Erickson S \u0026amp; Dixit VM (2004) Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf.\u0026nbsp;\u003cem\u003eNature\u003c/em\u003e 430: 213\u0026ndash;218\u003c/li\u003e\n \u003cli\u003eMensa-Vilar\u0026oacute; A, Garc\u0026iacute;a-Morato MB, de la Calle-Martin O, Franco-Jarava C, Mart\u0026iacute;nez-Saavedra MT, Gonz\u0026aacute;lez-Granado LI, Gonz\u0026aacute;lez-Roca E, Fuster JL, Alsina L, Mutchinick OM, \u003cem\u003eet al\u003c/em\u003e (2019) Unexpected Relevant Role of Gene Mosaicism in Primary Immunodeficiency Diseases.\u0026nbsp;\u003cem\u003eJournal of Allergy and Clinical Immunology\u003c/em\u003e 143: 359\u0026ndash;368\u003c/li\u003e\n \u003cli\u003eMolina-L\u0026oacute;pez C, Hurtado-Navarro L, Garc\u0026iacute;a CJ, Angosto-Bazarra D, Vallejo F, Tapia-Abell\u0026aacute;n A, Marques-Soares JR, Vargas C, Bujan-Rivas S, Tom\u0026aacute;s-Barber\u0026aacute;n FA, \u003cem\u003eet al\u003c/em\u003e (2024) Pathogenic NLRP3 mutants form constitutively active inflammasomes resulting in immune-metabolic limitation of IL-1\u0026beta; production. \u003cem\u003eNature Communications\u003c/em\u003e 15: 1096\u003c/li\u003e\n \u003cli\u003eRowczenio DMDM, Gomes SMSM, Ar\u0026oacute;stegui JIJI, Mensa-Vilaro A, Omoyinmi E, Trojer H, Baginska A, Baroja-Mazo A, Pelegrin P, Savic S, \u003cem\u003eet al\u003c/em\u003e (2017) Late-Onset Cryopyrin-Associated Periodic Syndromes Caused by Somatic NLRP3 Mosaicism\u0026mdash;UK Single Center Experience. \u003cem\u003eFrontiers in Immunology\u003c/em\u003e 8: 1410\u003c/li\u003e\n \u003cli\u003eVenegas C, Kumar S, Franklin BS, Dierkes T, Brinkschulte R, Tejera D, Vieira-Saecker A, Schwartz S, Santarelli F, Kummer MP, \u003cem\u003eet al\u003c/em\u003e (2017) Microglia-derived ASC specks cross-seed amyloid-\u0026beta; in Alzheimer\u0026rsquo;s disease. \u003cem\u003eNature\u003c/em\u003e 552: 355\u0026ndash;361\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"77e5cf91-5a2a-4b76-a672-2a65aa443a6f","identifier":"10.13039/501100004837","name":"Ministerio de Ciencia e Innovación","awardNumber":"CNS2022-135101","order_by":0},{"identity":"182b3f03-6ed1-4ebc-84a9-a0844d77a273","identifier":"10.13039/501100004837","name":"Ministerio de Ciencia e Innovación","awardNumber":"PID2020-116709RB-I00","order_by":1},{"identity":"4b6c1432-42d5-4cf5-8753-417baee4bcee","identifier":"10.13039/100007801","name":"Fundación Séneca","awardNumber":"21897/PI/22 ","order_by":2},{"identity":"14222599-f86a-4b7a-8a2c-d772fcc1454d","identifier":"10.13039/100007801","name":"Fundación Séneca","awardNumber":"21967/JLI/22","order_by":3}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Instituto Murciano de Investigación Biosanitaria","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Amyloidosis, autoinflammatory diseases, inflammasome, NLRP3, IL-1, serum amyloid A","lastPublishedDoi":"10.21203/rs.3.rs-5416087/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5416087/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAmyloid A (AA)-type amyloidosis is a complication associated with various autoinflammatory diseases, including cryopyrin-associated periodic syndromes (CAPS). The activation of the NLRP3-inflammasome in CAPS leads to the release of inflammasome oligomers, which are primarily composed of oligomeric filaments of the protein ASC. Recent studies have demonstrated that extracellular deposition of circulating ASC oligomers in tissues can serve as seeds for amyloid deposition, potentially culminating in clinical AA-type amyloidosis. In this study, we found that extracellular ASC in a late-onset, myeloid-restricted CAPS is associated with AA-type amyloidosis. Furthermore, intraperitoneally injected ASC oligomers produced from NLRP3 CAPS variants resulted in increased amyloid deposition. Overall, extracellular ASC oligomers present during inflammatory flares in CAPS promote amyloid deposition in tissues. Targeting extracellular ASC may represent a potential therapeutic strategy to prevent the development of AA-type amyloidosis in autoinflammatory diseases.\u003c/p\u003e","manuscriptTitle":"The inflammasome adaptor protein ASC promotes amyloid deposition in Cryopyrin-associated Periodic Syndromes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-12 02:26:21","doi":"10.21203/rs.3.rs-5416087/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d0e76b3a-762a-4e21-94fb-f8a50a1bd5f4","owner":[],"postedDate":"November 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":40002034,"name":"Immunology"}],"tags":[],"updatedAt":"2024-11-12T02:26:21+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-12 02:26:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5416087","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5416087","identity":"rs-5416087","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}