Figure 1(A, Neutrophils isolated from tamoxifen-treated Nlrp3A350V/wt CreT+ and Nlrp3A350V/wt CreT- mice were incubated with LPS (3 hr) before culture media were analysed for secreted IL-1β (A) Cytokine values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA and P-values were corrected for multiple comparisons (Bonferroni). **, p ≤ 0.01.B). Neutrophils isolated from tamoxifen-treated Nlrp3A350V/wt CreT+ and Nlrp3A350V/wt CreT- mice were incubated with LPS and cell lysates and culture media were immunoblotted for β-actin, caspase-1 and IL-1β (B).(C) Representative pictures of Nlrp3A350V/wt MRP8-CreTg and Nlrp3A350V/wt MRP8-Cre- mice on days 4 and 8 after birth. Left side in each picture: Nlrp3A350V/wt MRP8-Cre- mice; right side in each picture: Nlrp3A350V/wt MRP8-CreTg. Abscesses (day 4) and scaling erythema (day 8) are observed in Nlrp3A350V/wt MRP8-CreTg mice.(D) Survival curves of Nlrp3A350V/wt MRP8-CreTg (n=22) and littermate Nlrp3A350V/wt MRP8-Cre- (n=21) mice. Statistical significance was analysed by the log-rank Mantel-Cox test. ****, p ≤0.0001.(E) Representative H&E-stained sections of spleen (top, scale bar: 100 µM) and liver (bottom, scale bar: 200 µM) of Nlrp3A350V/wt MRP8-CreTg and littermate Nlrp3A350V/wt MRP8-Cre- mice. Spleens of Nlrp3A350V/wt MRP8-Cre- control mice (top left) display characteristic white pulp (WP) regions (example shown surrounded by dashed line) composed of lymphoid follicles and periarteriolar lymphoid sheaths (PALS), which at this magnification are recognized by dark-purple staining, large aggregates of lymphocytes surrounding the central artery (CA) and their distinctive separation from the red-staining red pulp (RP). WP was largely absent in spleens of Nlrp3A350V/wt MRP8-CreTg (top middle). Megakaryocytes (MK) are indicated for easier recognition of areas of extramedullary hematopoiesis (EMH). Most of the spleen parenchyma of Nlrp3A350V/wt MRP8-CreTg animals was replaced by EMH of mostly myeloid lineage, which can be recognized by the medium-sized cells with more cytoplasm, giving a lighter appearance (inset). In both the overview picture (top middle picture) and inset (top right) of Nlrp3A350V/wt MRP8-CreTg spleen, a small amount of EMH of the erythroid lineage is present that can be recognized by small aggregates of darker-stained, smaller cells (asterix,*). Liver sections of both Nlrp3A350V/wtMRP8-Cre- control mice and diseased Nlrp3A350V/wt MRP8-CreTg pups displayed comparable EMH of the erythroid lineage (bottom left picture, indicated with yellow arrows). Liver sections of Nlrp3A350V/wt MRP8-CreTg pups also showed a modest increase in EMH of the myeloid lineage (bottom middle picture and inset (scale bar: 100 µM) in bottom right, indicated with yellow arrowheads), as well as hepatocyte enlargement. (F-I) H&E-stained histological sections of spleen (F, G) or liver (H, I) of Nlrp3A350V/wt MRP8-Cre- (Cre-, n=8) and Nlrp3A350V/wt MRP8-CreTg (CreTg, n=6) mice were assigned a score of 0-5 for lymphoid depletion (F) or EMH (G, H) or hepatocyte enlargement (I) by a board-certified pathologist. Values represent mean ± SEM of n=6-8 biological repeats. Statistical significance was analysed by the Mann-Whitney U-test. ***, p ≤0.001.(J-L) Cytokine secretion analysis (Luminex) of serum samples obtained at day 7 from Nlrp3A350V/wt MRP8-Cre- (Cre-, n=8) and Nlrp3A350V/wt MRP8-CreTg (CreTg, n=6) pups. Values represent mean ± SEM of n=6-8 biological repeats. Statistical analysis of serum cytokine levels was performed by the Welch's t-test. *, p ≤0.05; **, p ≤0.01.