The inflammasome adaptor protein ASC promotes amyloid deposition in cryopyrin-associated periodic syndromes

Abstract

In this Correspondence, P. Pelegrin and colleagues found that the deposition of amyloid in tissues in Cryopyrin-Associated Periodic Syndrome were promoted by the extracellular presence of the inflammasome adaptor protein ASC, opening exciting new directions in clinical practice to obtain a novel therapy towards secondary amyloidosis in inflammasomopathies.

Figure 1. Amyloid deposition correlates with extracellular ASC in CAPS.

(A) Cutaneous manifestations of a patient with late-onset cryopyrin-associated periodic syndrome (CAPS) with myeloid-restricted gain-of-function NLRP3 somatic mutation p.Q308H before and after Anakinra treatment. (B) Congo-red and ASC-stained duodenum from the CAPS patient with amyloidosis. (C) 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. (D) Percentage of HEK293T cells with puncta distribution of NLRP3 wild type (WT) or p.Q308H; Centre values represent the mean (n = 5 independent experiments) and error bars represent s.e.m. Unpaired t-test, two tails (****p < 0.0001). (E) Percentage of HEK293T cells with ASC oligomers after expression of different amounts of NLRP3 WT (n = 5 independent experiments) or p.Q308H (n = 5 independent experiments); Centre values represent the mean and error bars represent s.e.m. Two-way ANOVA (****p < 0.0001). (F) Percentage of ASC specking cells in whole blood cultured for 4 h 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 (n = 5 healthy, n = 5 patients) and error bars represent s.e.m.; one-way ANOVA (*p = 0.0270; **p = 0.0061; ns: not significant p > 0.05). (G) Congo-red stained in liver, kidney and spleen sections from C57BL/6 and Pycard^–/– mice after 25 days of casein administration. (H, I) Concentration of SAA (H), IL-1β 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 (**p = 0.0069; ****p < 0.0001) with saline: n = 2 mice day 2, n = 4 mice day 25; casein: n = 6 mice day 5, n = 9 mice WT day 25, n = 10 mice Pycard^–/– day 25; for (I), IL-6, saline: n = 4 mice, casein: n = 9 mice WT, n = 10 mice Pycard^–/–; for (I), IL-1β n = 3 mice; Centre values represent the mean and error bars represent s.e.m. (J) Concentration of SAA in serum of Pycard^–/– mice after 7, 10, 14, or 18 days of intraperitoneal injection of ASC oligomers; n = 2 mice for control, n = 3 mice for the different days; Centre values represent the mean and error bars represent s.e.m. (K) Congo-red stained in kidney and lung sections from Pycard^–/– 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 denote areas of amyloid deposition. Scale bars: 50 μm. Source data are available online for this figure.

Figure EV1. NLRP3 p.Q308H resulted in puncta distribution and ASC oligomerization.

(A) Representative images of HEK293T cells transfected with NLRP3 wild type (WT) or p.Q308H tagged with YFP; Arrowheads denote cells with a puncta distribution of NLRP3. Scale bars: 10 μm. (B) Gating strategy to analyse the percentage of ASC specking cells in five different gates with low constant ASC-RFP expression and increased expression of NLRP3-YFP wild type (top) or p.Q308H (bottom) calculated as mean fluorescence intensity (MFI). NLRP3-YFP MFI corresponding to the five gates are: 1: 300.25; 2: 564; 3: 1063.25; 4: 2023.75; 5: 3693. As example, in the right panels, the percentage of ASC specking cells is shown in the gate number three for NLRP3 expression. (C) ASC MFI in human blood lymphocytes (white bar), neutrophils (grey bar) and monocytes (blue bar) cultured for 4 h, from healthy donors and germline CAPS patients whole blood with the NLRP3 variants p.R262W, p.D305N, p.T350M and p.A441T; Centre values represent the mean (n = 5 healthy, n = 5 patients) and error bars represent s.e.m; one-way ANOVA (^##p = 0.0019; **p = 0.0080; ⁺⁺p = 0.0040; ns: not significant p > 0.05).

Figure EV2. Amyloidosis is reduced in ASC-deficient mice.

Congo-red and Haematoxylin and eosin stained in liver, kidney and spleen sections from C57BL/6 and Pycard^–/– mice after 25 days of casein administration. Please note that part of these images are also presented in Fig. 1F. Scale bars: 100 μm.

Figure EV3. Administration of ASC oligomers induce amyloidosis in vivo.

Congo-red stained in kidney and lung sections from Pycard^–/– mice after 10, 14 and 18 days of intraperitoneal ASC oligomers administration. Pictures of brightfield and polarized light are shown. As control, kidney, spleen and liver sections from C57BL/6 mice after 18 days of casein administration were stained with Congo-red and visualized under polarized light. Arrowheads denote areas of amyloid deposition. Please note that part of these images are also presented in Fig. 1J. Scale bars: 50 μm.