Differential roles of caspase-1 and caspase-11 in infection and inflammation

Abstract

Caspase-1, also known as interleukin-1β (IL-1β)-converting enzyme (ICE), regulates antimicrobial host defense, tissue repair, tumorigenesis, metabolism and membrane biogenesis. On activation within an inflammasome complex, caspase-1 induces pyroptosis and converts pro-IL-1β and pro-IL-18 into their biologically active forms. "ICE^-/-" or "Casp1^-/-" mice generated using 129 embryonic stem cells carry a 129-associated inactivating passenger mutation on the caspase-11 locus, essentially making them deficient in both caspase-1 and caspase-11. The overlapping and unique functions of caspase-1 and caspase-11 are difficult to unravel without additional genetic tools. Here, we generated caspase-1-deficient mouse (Casp1^Null) on the C57BL/6 J background that expressed caspase-11. Casp1^Null cells did not release IL-1β and IL-18 in response to NLRC4 activators Salmonella Typhimurium and flagellin, canonical or non-canonical NLRP3 activators LPS and ATP, Escherichia coli, Citrobacter rodentium and transfection of LPS, AIM2 activators Francisella novicida, mouse cytomegalovirus and DNA, and the infectious agents Listeria monocytogenes and Aspergillus fumigatus. We further demonstrated that caspase-1 and caspase-11 differentially contributed to the host defense against A. fumigatus infection and to endotoxemia.

Figure 1. Caspase-11 is expressed in Casp1^Null bone marrow-derived macrophages.

Immunoblot analysis of caspase-1, caspase-11 and GAPDH (loading control) in unprimed WT or mutant BMDMs at various times (above lane) after stimulation with IFN-β (250U/ml), IFN-γ (100 ng/ml) or LPS (100 ng/ml). Data are representative of two independent experiments.

Figure 2. Responses of Casp1^Null bone marrow-derived macrophages to the activation of canonical inflammasomes.

(A) Top, immunoblot analysis of pro-caspase-1 (Pro-Casp-1) and the caspase-1 subunit p20 (Casp-1 p20) in unprimed WT or mutant BMDMs left untreated (medium alone [Med]) or assessed 4 h after infection with Salmonella Typhimurium (MOI, 1; left) or 4 h after transfection of S Typhimurium flagellin (4 μg/ml; middle) or in LPS-primed BMDMs left untreated (Med) or assessed 30 min after stimulation with 5 mM ATP (LPS + ATP, right). Bottom, immunoblot analysis of pro-caspase-1 (Pro-Casp-1) and the caspase-1 subunit p20 (Casp-1 p20) in unprimed WT or mutant BMDMs left uninfected (medium alone [Med]) or assessed 20 h after infection with F. novicida (MOI, 100; left) or 5 h after transfection with poly(dA:dT) (5 μg/ml; middle) or 10 h after infection with mouse cytomegalovirus (MCMV, MOI, 10; right). (B–D) Release of IL-1β, IL-18, death of BMDMs, and release of TNF after treatment as in (A). Cell death indicates % of LDH release relative to total lysis, set at 100% (B–D). Data in (A–D) are representative of three independent experiments (mean and s.e.m. of values from three independent experiments in B–D).

Figure 3. Responses of Casp1^Null bone marrow-derived macrophages and mice to activation of the non-canonical inflammasome.

(A) Immunoblot analysis of pro-caspase-1 (Pro-Casp-1) and the caspase-1 subunit p20 (Casp-1 p20) in unprimed WT or mutant BMDMs left untreated (medium alone [Med]) or assessed 20 h after infection with C. rodentium (MOI, 20, left), E. coli (MOI, 20, middle), or 10 h after LPS transfection (4 μg/ml, right). (B) Release of IL-1β, IL-18, death of BMDMs, and release of TNF after treatment as in (A). (C) Survival of 8-week-old WT and mutant mice injected intraperitoneally with 54 mg LPS per kg body weight. NS, not statistically significant, **P < 0.01 and ****P < 0.0001 (log-rank test). Data are representative of two (C) or three independent experiments (A and B; mean and s.e.m. are representative of values from three independent experiments in B).

Figure 4. Differential roles of caspase-1 and caspase-11 in response to infection with Aspergillus fumigatus.

(A) Immunoblot analysis of pro-caspase-1 (Pro-Casp-1) and the caspase-1 subunit p20 (Casp-1 p20) and GAPDH (loading control) in unprimed WT or mutant bone marrow-derived dendritic cells left untreated (medium alone [Med]) or assessed 20 h after infection with A. fumigatus (MOI, 10). (B) Release of IL-1β and IL-18 after treatment as in (A). (C) Survival of 8-week-old WT and mutant mice infected with 5 × 10⁵ A. fumigatus conidia after immunosuppression with cyclophosphamide and cortisone acetate. *P < 0.05, ****P < 0.0001 (log-rank test). Data are representative of two (C) or three independent experiments (A and B; mean and s.e.m. are representative of values from three independent experiments in B).