Divergence of IL-1, IL-18, and cell death in NLRP3 inflammasomopathies

Abstract

The inflammasome is a cytoplasmic multiprotein complex that promotes proinflammatory cytokine maturation in response to host- and pathogen-derived signals. Missense mutations in cryopyrin (NLRP3) result in a hyperactive inflammasome that drives overproduction of the proinflammatory cytokines IL-1β and IL-18, leading to the cryopyrin-associated periodic syndromes (CAPS) disease spectrum. Mouse lines harboring CAPS-associated mutations in Nlrp3 have elevated levels of IL-1β and IL-18 and closely mimic human disease. To examine the role of inflammasome-driven IL-18 in murine CAPS, we bred Nlrp3 mutations onto an Il18r-null background. Deletion of Il18r resulted in partial phenotypic rescue that abolished skin and visceral disease in young mice and normalized serum cytokines to a greater extent than breeding to Il1r-null mice. Significant systemic inflammation developed in aging Nlrp3 mutant Il18r-null mice, indicating that IL-1 and IL-18 drive pathology at different stages of the disease process. Ongoing inflammation in double-cytokine knockout CAPS mice implicated a role for caspase-1-mediated pyroptosis and confirmed that CAPS is inflammasome dependent. Our results have important implications for patients with CAPS and residual disease, emphasizing the need to explore other NLRP3-mediated pathways and the potential for inflammasome-targeted therapy.

Figure 1. Myeloid cells expressing mutant NLRP3 proteins secrete IL-18.

Tamoxifen-treated MWS CreT, FCAS CreT, and littermate WT BMDCs were incubated with pure LPS, with and without ATP at (A) 37°C or (B) 32°C. IL-18 in the supernatants was measured by ELISA (n = 4, mean and SEM). Tamoxifen-treated MWS CreT and FCAS CreT peritoneal macrophages (PM) were incubated at 32°C, and (C) IL-1β and (D) IL-18 were measured in the supernatants by ELISA (n = 2, mean and SEM). (E) Adherent monocytes from patients with FCAS were incubated at 37°C or 32°C, and IL-18 was measured in the culture supernatants by ELISA (n = 10). *P < 0.05, **P < 0.005, by Student’s t test.

Figure 2. IL-18R deficiency ameliorates inflammation in murine CAPS.

(A) Survival and (B) growth of FCAS Il18r^–/–, Il18r^–/–, FCAS ll1r^–/–, and Il1r^–/– mice (n = 16–30 for survival and 1–29 for growth). (C) Survival and (D) growth of MWS, MWS Il18r^–/–, MWS Il1r^–/–, and WT littermates (n = 4–33 for survival and 1–27 for growth). Statistical significance was tested by log-rank (Mantel-Cox) comparison. Error bars shown for mean daily weights on growth curves are SEM.

Figure 3. IL-18R signaling drives skin and liver pathology in murine CAPS.

(A) H&E-stained tissue sections from skin, liver, and bone marrow from 9-day-old littermate WT, MWS, MWS Il18r^–/–, and MWS Il1r^–/– mice (original magnification, ×20). (B) Immunohistochemistry on skin and liver from WT and MWS pups for IL-18 followed by hematoxylin staining (original magnification, ×10; ×40 [insets]). Sections are representative of at least 3 mice per strain.

Figure 4. Disruption of IL-18 signaling normalizes key CAPS serum cytokine and SAA levels.

Multiplex cytokine and ELISA analysis of serum obtained at days 6–8 from WT, MWS, MWS Il1r^–/–, and MWS Il18r^–/– pups (n ≥ 5 mice); each graph point represents 1 mouse, with mean identified by horizontal bar. *P < 0.05, **P < 0.005 by Student’s t test.

Figure 5. Aging mutant CAPS mice on IL-18R knockout background develop systemic inflammation.

(A) Extended survival and (B) growth of MWS Il18r^–/–, Il18r^–/–, MWS Il1r^–/–, and Il1r^–/– mice (n = 7–46 for survival and 1–38 for growth, mean ± SEM). Statistical significance was tested by log-rank (Mantel-Cox) comparison. (C) Complete peripheral blood counts for MWS Il1r^–/– and Il18r^–/– mice at 16 weeks of age (n ≥ 3 mice; each graph point represents 1 mouse, with mean identified by horizontal bar). (D) Spleens from MWS Il18r^–/–, Il18r^–/–, MWS Il1r^–/–, Il11r^–/–, and WT littermates were weighed at 8 or 15 weeks (n = 3–12 mice; each graph point represents 1 mouse, with mean identified by horizontal bar). *P < 0.05, **P < 0.005, by Student’s t test. WBC, white blood cells; RBC, red blood cells; ANC, absolute neutrophil count.

Figure 6. Inducible and chimeric mutant CAPS models confirm age-related cytokine dependence.

(A) Survival of FCAS CreT, FCAS Il18r^–/–, or FCAS Il1r^–/– mice, displayed as days after initiation of tamoxifen treatment (n = 4–14 mice). (B) Schematic of bone marrow transplants performed. Survival of transplanted mice, displayed as days after initiation of tamoxifen treatment (n = 5–14 mice). P values by log-rank (Mantel-Cox) comparison.

Figure 7. Caspase-1 induces inflammation in CAPS via cytokine mediators and pyroptosis.

(A) Survival and (B) growth of MWS ll1r^–/–Il18^–/–, Il1r^–/–Il18^–/–, MWS Il18^–/–, and MWS Il1r^–/– mice (n = 7–32 for survival and 1–29 for growth). (C) Survival and (D) growth of FCAS Casp1^–/–, FCAS Il1r^–/–Il18^–/–, Il1r^–/–Il18^–/–, FCAS Il18^–/–, and FCAS Il1r^–/– mice (n = 7–51 for survival and 1–40 for growth). Weights are shown as mean ± SEM. Bone marrow cells were isolated from 16-week-old FCAS Casp1^–/–, FCAS Il1r^–/–Il18^–/–, and WT mice and evaluated for (E) membrane asymmetry and (F) dye exclusion by flow cytometry. Data are representative of 3 independent experiments (mean ± SEM shown). *P < 0.05, by Student’s t test.