Fig 2: Anakinra treatment reduces NLRP3 inflammasome activation(A and B) Release of IL-1β from peripheral blood mononuclear cells (A) and percentage of ASC-specking monocytes (B), at baseline or after canonical activation of NLRP3 (LPS+ATP) and pyrin (LPS+TcdB) inflammasome, in the absence/presence of MCC950 from healthy donors (n = 2–3, each dot represents an individual donor, white bars) and the index patient with the KRASG12D mutation before anakinra administration (gray bars).(C) Percentage of monocytes with an ASC oligomer from the index patient over the time points examined before and after anakinra treatment.(D) Percentage of monocytes with an ASC oligomer from the index patient after canonical NLRP3 inflammasome activation with LPS+ATP (green) or pyrin inflammasome activation with LPS+TcdB (dark blue) over the time points examined before and after anakinra treatment.(E and F) Release of IL-1β (E) and IL-18 (F) from peripheral blood mononuclear cells, at baseline or treated, as indicated for NLRP3 (LPS+ATP) or pyrin (LPS+TcdB) inflammasome canonical activation, in healthy donors (n = 3, each dot represents an individual donor, white bars) and the index patient during anakinra treatment (green bars).(G) Plasma levels of ASC in the index patient along the time points examined before and after anakinra treatment.(H) Concentration of IL-1 family cytokines in the plasma of the CMML KRASG12D index patient in response to anakinra.For (A)–(F), are represented as mean ± SEM, and each dot represents an individual donor.

Fig 3: Monocytes from CMML KRASmut patients present a constitutive inflammasome activation(A) Percentage of ASC-specking monocytes in healthy controls (white bars), CMML patients without KRAS mutation (KRASwt, blue bars), and CMML patients with a KRAS mutation (KRASmut, orange bars) at baseline or treated as indicated for NLRP3 (LPS+ATP) or pyrin (LPS+TcdB) inflammasome activation.(B and C) Release of IL-1β from peripheral blood mononuclear cells (PBMCs) (B) and the formation of ASC specks in monocytes (C) in CMML KRASmut patients at baseline or after the indicated stimulation or treatment (LPS+ATP and LPS+TcdB for NLRP3 or pyrin inflammasome, respectively), in the absence/presence of MCC950. (B) Fold increase was calculated to control non-stimulated conditions, where the average of the higher value used to calculate fold increase is 1,066.58 pg/mL.(D) Percentage of extracellular LDH from untreated PBMCs from healthy donors (white bar), KRASwt patients (blue bar), and KRASmut patients (orange bar). Data are normalized to the percentage of monocytes.(E) Plasma concentration of HMGB1 and P2X7 receptor from healthy donors (white bar), KRASwt patients (blue bar), and KRASmut patients (orange bar).For (A)–(E), data are represented as mean ± SEM; each dot represents an individual patient; ordinary one-way ANOVA test (two-tailed) was used for (A) (∗ compares CMML KRASmut vs. healthy controls; † compares CMML KRASmut vs. CMML KRASwt), and two-tailed t test in (B)–(E). Note that ∗ or †p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗ or ††††p < 0.0001; ns, no significant difference (p > 0.05).

Fig 4: Levels of acute phase reactants and proteins related to pyroptosis and neutrophil activity in the different study groups, following the grade 2–3 double contour-tophi definition for deposits in asymptomatic hyperuricaemia. Data shown as medians and 95% confidence intervals. P-values in the boxes at the bottom right corner of each figure correspond to the comparisons of all four groups by the Kruskal–Wallis test, while P-values for the AH group comparisons are based on the Mann–Whitney U-test. AH: asymptomatic hyperuricaemia; ASC: apoptosis-associated Speck-like protein containing a caspase activation and recruitment domain; H3Cit: citrullinated histone H3; hsCRP: high-sensitivity CRP; MSU: monosodium urate; NLR: neutrophil to lymphocyte ratio; NU: normouricaemia; SAA: serum amyloid-A