NLRP1 inflammasome activation induces pyroptosis of hematopoietic progenitor cells

Abstract

Cytopenias are key prognostic indicators of life-threatening infection, contributing to immunosuppression and mortality. Here we define a role for Caspase-1-dependent death, known as pyroptosis, in infection-induced cytopenias by studying inflammasome activation in hematopoietic progenitor cells. The NLRP1a inflammasome is expressed in hematopoietic progenitor cells and its activation triggers their pyroptotic death. Active NLRP1a induced a lethal systemic inflammatory disease that was driven by Caspase-1 and IL-1β but was independent of apoptosis-associated speck-like protein containing a CARD (ASC) and ameliorated by IL-18. Surprisingly, in the absence of IL-1β-driven inflammation, active NLRP1a triggered pyroptosis of hematopoietic progenitor cells resulting in leukopenia at steady state. During periods of hematopoietic stress induced by chemotherapy or lymphocytic choriomeningitis virus (LCMV) infection, active NLRP1a caused prolonged cytopenia, bone marrow hypoplasia, and immunosuppression. Conversely, NLRP1-deficient mice showed enhanced recovery from chemotherapy and LCMV infection, demonstrating that NLRP1 acts as a cellular sentinel to alert Caspase-1 to hematopoietic and infectious stress.

Figure 1. Inflammatory disease induced by the NLRP1a^Q593P mutation

Hematoxylin and eosin-stained tissues show pneumonitis (A), meningitis (B) and hepatitis (C).

Figure 2. Caspase-1 and IL-1β are major contributors to NLRP1a^Q593P–induced disease

(A) Effects of heterozygous and homozygous Nlrp1a^Q593P mutations on peripheral blood neutrophil numbers in mice on a BALB/c background. (B) Nlrp1a^Q593P/Q593P macrophages were treated with 2 ng/mL LPS for 4h before the inflammasome was activated with 5 mM ATP for 30 min or MDP complexed with TiO₂, 100 µg/mL uric acid crystals (MSU), 7 µg/mL poly(dA:dT) (dsDNA) or alum for 15 h. IL-1β secretion was assessed by immunoblot using antibodies specific for the 35 kDa pro-IL-1β and the processed, bioactive 17 kDa IL-1β. (C) Cytokine levels were measured in the serum of wild-type and Nlrp1a^Q593P/Q593P mice. *p<0.05, mean ± SD, n=3 independent samples. (D) Disease-free survival in Nlrp1a^Q593P/Q593P mice deficient in the IL-1R. (E–J) Neutrophil numbers and disease-free survival in Nlrp1a^Q593P/Q593P mice deficient in IL-1α, Caspase-1 or ASC. Neutrophil numbers in the peripheral blood were analysed at 7 weeks age. A significant change (p<0.05) in disease-free survival is evident between Nlrp1a^Q593P/Q593P Il1a^−/− and Nlrp1a^Q593P/Q593P mice, as well as between Nlrp1a^Q593P/Q593P Casp1^−/− and Nlrp1a^Q593P/Q593P mice. (K) Enumeration of neutrophils, lymphocytes, monocytes and eosinophils in Casp11^−/− Nlrp1a^Q593P/Q593P mice. *p<0.05.

Figure 3. IL-18 inhibits the lethal multiorgan inflammatory disease in Nlrp1a^Q593P/Q593P mice

(A, B) Serum IL-18 was measured by ELISA in wild-type, Casp1^−/−, Nlrp1a^Q593P/Q593P, Nlrp1a^Q593P/Q593P Casp1^−/−, Il1r^−/− and Nlrp1a^Q593P/Q593P Il1r^−/− mice. (C) IL-18 production by Nlrp1a^Q593P/Q593P macrophages following stimulation with 2 ng/mL LPS and 5mM ATP. (D) Serum IL-18 levels in lethally-irradiated mice after reconstitution with Nlrp1a^Q593P/Q593P or wild-type bone marrow cells. (E–L) Neutrophil numbers in the peripheral blood of Nlrp1a^Q593P/Q593P mice lacking IL-18 (E), IFNγ (G), T cells (I) or B cells (K). Survival of Nlrp1a^Q593P/Q593P mice lacking IL-18 (F), IFNγ (H), T cells (J) or B cells (L). A significant change (p<0.05) in disease-free survival is evident between Nlrp1a^Q593P/Q593P Il18^−/− and Nlrp1a^Q593P/Q593P mice, as well as between Nlrp1a^Q593P/Q593P Cd3^−/− and Nlrp1a^Q593P/Q593P mice. (M) Neutrophil numbers in the peripheral blood of germ-free Nlrp1a^Q593P/Q593P Il18^−/− and Nlrp1a^Q593P/Q593P mice. *p<0.05.

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.

Figure 5. NLRP1a activation prevents recovery from hematopoietic stress

(A) Leukopenia induced by 150 mg/kg 5-fluorouracil in Il1r^−/−Nlrp1a^Q593P/Q593P mice at Day 12 but not Il1r^−/− littermate controls. *p<0.05, mean ± SEM, n=3–4 mice. (B) Reticulocyte numbers 12 days following challenge with 5-fluorouracil. *p<0.05, mean ± SEM, n=4 mice. (C) Hematoxylin and eosin staining of bone marrow of Il1r^−/−Nlrp1a^Q593P/Q593P and Il1r^−/− mice 12 days after treatment with 5-FU. (D) The composition of blood following injection with 150 mg/kg 5-fluorouracil. *p<0.05, mean ± SEM, n=8–18 mice. (E) Reticulocyte numbers 12 days following challenge with 5-fluorouracil. *p<0.05, mean ± SEM, n=8–18 mice. (F) Spleen weight at steady state or 12 days post-injection with 5-FU. *p<0.05, mean ± SEM, n=3–18 mice.

Figure 6. LCMV infection induces prolonged cytopenia in NLRP1a mutant mice

(A) Enumeration and analysis of red blood cells (RBC), reticulocytes, platelets (PLT), neutrophils, lymphocytes and monocytes in the blood of mice infected with LCMV docile. *p<0.05, mean ± SEM, n=4–15. (B) Nucleated bone marrow and spleen cells 16 days after infection. *p<0.05, mean ± SEM, n=4–8. (C) Spleen weight of infected mice 16 days after infection. *p<0.05, mean ± SEM, n=4–8. (D) Disease-free survival of Il1r^−/−Nlrp1a^Q593P/Q593P mice and littermate controls injected with 2 × 10⁶ PFU LCMV. *p<0.05, n=5–17. (E) Viral titers in the lung, kidney, brain and spleen calculated by plaque assay. (F) Tetramer-positive, CD8+ T cells 16 days after infection. *p<0.05, Mean ± SEM, n=4–8. (G) Hematoxylin and eosin staining of bone marrow from mice 8 days after infection with LCMV. (H) Infection of purified lin⁻c-kit⁺ hematopoietic progenitor cells cultured in SCF+IL-3+Epo for 3 days. Viability was assessed using LIVE/DEAD yellow stain and virus was detected by intracellular staining for LCMV nucleoprotein. Representative data from triplicate wells of 5 independent experiments.

Figure 7. NLRP1 deficiency enhances hematopoietic responses to LCMV

(A) Peripheral blood parameters of LCMV-infected mice at steady state (day 0) and day 4 and 11 post-infection with LCMV (clone 13). *p<0.05, mean ± SEM, n=4–7. (B) Weight of mice after infection with LCMV. *p<0.05, mean ± SEM, n=6. (C) Spleen weight 12 days after LCMV infection. *p<0.05, mean ± SEM, n=6. (D) Total nucleated cells in the bone marrow and spleen 12 days after LCMV infection. *p<0.05, mean ± SEM, n=6. (E) Tetramer-positive (LCMV-specific) CD8⁺ T cells in the spleen 12 days post LCMV-infection. *p<0.05, mean ± SEM, n=6. (F) Tetramer-positive (LCMV-specific) CD8⁺ T cells in the spleen of bone marrow chimeras 12 days post LCMV-infection. Chimeric mice were generated by transplanting lethally-irradiated wild-type recipients with equal numbers of wild-type and Nlrp1^−/− bone marrow cells. *p<0.05, Mean ± SEM, n=4.