Figure 4. NLRP1a activation influences myelopoiesis.

(A) Transfer of Nlrp1a^Q593P/Q593P bone marrow to lethally irradiated wild-type recipients results in neutrophilia and B and T lymphopenia. The transfer of wild-type bone marrow to lethally-irradiated Nlrp1a^Q593P/Q593P mice is sufficient to restore normal hematopoiesis. (B,C) Reduced reconstitution efficiency of Nlrp1a^Q593P/Q593P bone marrow in (B) the bone marrow and (C) peripheral blood of irradiated recipients 8 weeks post transplant. *p<0.05 by ANOVA and SNK. (D) Expression of Nlrp1a, Nlrp3, Asc and Actin in purified populations of hematopoietic stem cells (HSC), lin⁻c-kit⁺Sca1⁺ (LSK), common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP) and common lymphoid progenitors (CLP). (E) Decrease of lin⁻c-kit⁺ hematopoietic progenitor cells in the bone marrow of Nlrp1a^Q593P/Q593P and Il1r^−/−Nlrp1a^Q593P/Q593P mice. (F) Contribution of wild-type and Nlrp1a^Q593P/Q593P bone marrow to lethally-irradiated recipients, 8 weeks post-transplant. Equal numbers of wild-type and Nlrp1a^Q593P/Q593P bone marrow cells were initially transplanted. (G,H) The differentiation of progenitor cells in blast colonies is altered by the Nlrp1a^Q593P mutation through Caspase-1 activity, and occurs independently of IL-1β. Individual blast colonies from whole bone marrow cultures stimulated with SCF+IL-6 for 7 days were resuspended in agar and incubated for a further 7 days in the presence of M-CSF, GM-CSF or SCF+IL-3+Epo before colony types were enumerated. G: granulocyte; GM: granulocyte-macrophage; M:macrophage. (G) *p<0.05, mean ± SEM, Il1r^−/−Nlrp1a^Q593P/Q593P vs. Il1r^−/−, n=14–16 independent recloned colonies from 2 independent experiments. (H) *p<0.05, mean ± SEM, n=19–20 independent recloned colonies from 2 independent experiments. (I) Enumeration of lymphocytes, neutrophils, platelets, monocytes, eosinophils and red blood cells in Il1r^−/−Nlrp1a^Q593P/Q593P mice. *p<0.05. (J) Caspase-1 activity in lin⁻c-kit⁺ bone marrow progenitor cells using FAM-YVAD-FMK Caspase-1 substrate. MFI: Mean fluorescence intensity. (K) IL-1R-independent but Caspase-1-dependent reduction in viability of Nlrp1a^Q593P/Q593P lin⁻c-kit⁺ hematopoietic progenitor cells.