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

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 (Casp1Null) on the C57BL/6 J background that expressed caspase-11. Casp1Null 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.

contributed to lethality during LPS-induced endotoxemia. This caspase-1-deficient mouse strain provides the scientific community with an exciting opportunity to refine the roles of caspase-1 and caspase-11 in health and disease.
Inflammasome activities are impaired in Casp1 Null BMDMs in response to canonical inflammasome activation. Caspase-1 is activated within an inflammasome following engagement of the inflammasome-initiating sensors NAIP-NLRC4, NLRP3 and AIM2 13 . To systematically validate the Casp1 Null line, we measured inflammasome responses from primary BMDMs generated from this line following stimulation with known inflammasome triggers. The NAIP-NLRC4 inflammasome can be activated by Salmonella enterica serovar Typhimurium (S. Typhimurium) which had been grown to a log-phase or through transfection of bacterial flagellin into the host cytoplasm [17][18][19][20][21][22] . Activation of the NLRC4 inflammasome using S. Typhimurium or transfection of flagellin from S. Typhimurium led to robust activation of caspase-1, release of IL-1β and/or IL-18, and cell death in WT and Casp11 −/− BMDMs, but not in Casp1 Null and Casp1 −/− Casp11 −/− BMDMs ( Fig. 2A and  B).

Secretion of IL-1β and IL-18, but not pyroptosis, is impaired in Casp1 Null BMDMs in response to non-canonical inflammasome activation. Gram-negative bacteria, including Escherichia coli and
Citrobacter rodentium, introduce LPS into the host cytoplasm during infection and engage non-canonical activation of the NLRP3 inflammasome via caspase-11 16 . Casp1 Null BMDMs did not secrete IL-1β and IL-18 in response to infection by E. coli and C. rodentium or transfection of LPS ( Fig. 3A and B). However, cell death was observed in Casp1 Null BMDMs in response to infection by E. coli and C. rodentium or transfection of LPS (Fig. 3B), which is consistent with the model that, in response to non-canonical activation of the NLRP3 inflammasome, pyroptosis is driven by caspase-11 rather than caspase-1 16,42,44,50 .
Differential roles of caspase-1 and caspase-11 in response to infection with the fungal pathogen Aspergillus fumigatus. In addition to its function in the recognition of bacteria and viruses, inflammasomes have a central role in the control of fungal pathogens, including Aspergillus fumigatus 54 . We found that A. fumigatus failed to induce the release of IL-1β and IL-18 in Casp1 Null and Casp1 −/− Casp11 −/− bone marrow-derived dendritic cells (BMDCs), whereas maturation of caspase-1 and the release of IL-1β and IL-18 in WT and Casp11 −/− BMDCs were observed ( Fig. 4A and B). This finding supported our previous observations showing that caspase-11 is dispensable for activation of the inflammasome induced by A. fumigatus infection 55 . Similar to BMDMs, Casp1 Null BMDCs retained the ability to express the caspase-11 protein (Fig. 4A).
We have previously found that Casp1 −/− Casp11 −/− mice were extremely sensitive to infection by A. fumigatus compared with WT mice 55 . However, whether caspase-1 or caspase-11 contributed to the host defense against A. fumigatus infection in vivo has remained unclear. To investigate this, we immunocompromised WT, Casp1 Null , Casp1 −/− Casp11 −/− and Casp11 −/− mice with cyclophosphamide and cortisone acetate and intranasally infected these mice with A. fumigatus conidia. Immunosuppression procedures were used because immunocompetent WT mice and mice lacking components of the inflammasome do not succumb to infection with A. fumigatus 55   which is in line with the observation that only immunocompromised individuals in large are susceptible to invasive pulmonary aspergillosis 56 .
Following intranasal infection with A. fumigatus conidia, Casp1 Null mice were substantially more susceptible to A. fumigatus-induced mortality compared with WT mice (Fig. 4C). The hypersusceptibility of Casp1 Null mice to A. fumigatus was phenocopied by Casp1 −/− Casp11 −/− mice (Fig. 4C). In addition, we found that Casp11 −/− mice were also more susceptible to infection with A. fumigatus conidia compared with WT mice (Fig. 4C). However, Casp11 −/− mice succumbed to infection with a delayed kinetic compared with Casp1 Null mice or Casp1 −/− Casp11 −/− mice. Although caspase-11 had no role in the activation of the inflammasome in BMDCs in response to A. fumigatus, caspase-11 contributed to the host defense against A. fumigatus infection in vivo. It is possible that activation of caspase-11 might induce pyroptosis and/or actin-mediated phagosomal killing in a cell-type-specific manner in order to control A. fumigatus dissemination in vivo [57][58][59] . Indeed, the release of IL-18 via A. fumigatus-sensing NLRP3 and AIM2 inflammasomes induces production of IFN-γ , which might provide a priming signal for caspase-11 to clear A. fumigatus in vivo 55 . This IL-18-IFN-γ -Caspase-11 signaling pathway and defense strategy has been reported in the host clearance of the cytosolic bacterium Burkholderia thailandensis 60 . Overall, our study has generated and validated a valuable genetic tool to enable us to refine the differential contribution of caspase-1 and capsase-11 in health and disease in future studies.

Discussion
Inflammatory caspases are multi-functional proteins which mediate host defense to infectious diseases and regulate tumor development, metabolic syndromes, autoinflammatory disease, tissue repair, and cell survival 1,2 . Previously generated caspase-1-deficient mouse strains using embryonic stem cells of the 129 background lack caspase-11 expression, essentially rendering them deficient in both caspase-1 and caspase-11 16 . Therefore, the biological insights of caspase-1 gained from using Casp1 −/− Casp11 −/− mice should be re-examined. We generated caspase-1-deficient mouse strain on the C57BL/6 J background to provide the scientific community a genetic tool to revisit the biological functions of caspase-1 and caspase-11.
Studies in mouse models have revealed differential contributions between caspase-1 and caspase-11 in infection and cancer. Casp1 −/− Casp11 −/− mice are hypersusceptible to infection by S. Typhimurium 18,65,85,86 . A further study has found that the caspase-11-expressing mouse strain Casp1 −/− Casp11 Tg (generated via microinjection of a bacterial artificial chromosome transgene encoding caspase-11 into Casp1 −/− Casp11 −/− mouse embryos) revealed that they were more susceptible to infection with S. Typhimurium than Casp1 −/− Casp11 −/− mice 65 . However, this study reported that Casp11 −/− mice were not more susceptible compared with wild-type mice 65 . These data would suggest that caspase-11 is detrimental to the host only in the absence of caspase-1. However, others have reported a protective role for caspase-11 during salmonellosis exclusively in the intestine 63,87 . In mouse macrophages or embryonic fibroblasts, both caspase-1 and caspase-11 contribute to cell-autonomous control of intracellular replication of S. Typhimurium and other bacteria 21,58,88-90 . In the context of intestinal inflammation, Casp1 −/− Casp11 −/− mice are susceptible to colitis induced by the colitogen dextran sulfate sodium (DSS) [91][92][93] . More recent studies have now revealed that mice lacking caspase-11 alone are sensitive to DSS-induced colitis [94][95][96] . The susceptibility observed in Casp11 −/− mice largely phenocopies that of Casp1 −/− Casp11 −/− mice 94 , suggesting that caspase-11 could be a dominant inflammatory caspase that drives a protective response in the intestine. This protective response in the intestine might be attributed to the ability of caspase-11 to mediate secretion of IL-18 95,96 , a cytokine generally considered to be protective in colitis. Caspase-1 could have non-redundant functions with caspase-11 in the intestine and that further studies are required to investigate their relative effect during intestinal inflammation.
The caspase-11 homologs in humans, caspase-4 and caspase-5, also recognize LPS and activate caspase-1, mediate cleavage of gasdermin D, and drive pyroptosis 43,69,70 . However, subtle differences have been reported between human caspase-4 and human caspase-5. Caspase-4, but not caspase-5, is required for cell death and IL-1β production in the human monocytic THP-1 cell line following transfection of LPS 97,98 . Caspase-4 also mediates secretion of IL-1β and IL-18 in LPS-stimulated mouse BMDMs engineered to express human caspase-4 99 . However, other studies have suggested that both caspase-4 and caspase-5 are required for IL-1β release in human monocytes stimulated with LPS or in the THP-1 cell line infected with S. Typhimurium 97,100 , suggesting non-redundant activities between these caspases. A further study has demonstrated that caspase-4 is not necessary for IL-1β release in primary human macrophages infected with S. Typhimurium, L. pneumophila and Y. pseudotuberculosis 101 . The function of these inflammatory caspases is likely to be cell-type-and species-specific and influenced by the type of activators delivered into the cell.
Overall, our study has generated and validated a mouse strain for use in unraveling the specific function of caspase-1 without the confounding absence of caspase-11. We also showed the unique and overlapping functions of caspase-1 and caspase-11 during infection and inflammation. Two recent studies have now reported caspase-1-deficient mice on the C57BL/6 background 102,103 . Together with our study, the availability of caspase-1-deficient mice provides a valuable tool for the scientific community to propel investigations that aim to refine the differential functions of caspase-1 and caspase-11 in health and disease.

Methods
Mice. Casp1 −/− Casp11 −/− (also known as Casp1 −/− Casp11 129mt/129mt ) and Casp11 −/− mice have been described previously 16 (Fig. S1), and were subsequently surgically transplanted into the oviducts of pseudo pregnant CD1 females. Newborn mice bearing a null allele of Casp1 (Casp1 Null ) were identified by amplification of a 716 bp fragment using primers flanking the 2 break sites [Casp1-F51 and Casp1-R32 (Tables S1 and S2)]. Sanger sequencing of the 716 bp amplicon confirmed proper deletion of the ~3.8 kb fragment containing exons 2-4. sgRNAs were designed and generated as described previously 104 . The Cas9 mRNA transcript was generated as described previously 104 . Potential off-target sites were identified using Cas-OFFinder and each locus was PCR-amplified and sequenced (Table S3) 105 . No off-target site cleavage was observed.
The following conditions were used to stimulate BMDMs: Immunoblotting analysis. Cells and supernatant were lysed in RIPA buffer and sample loading buffer containing SDS and 100 mM DTT. Proteins were separated on 8-12% polyacrylamide gels. Following electrophoretic transfer of protein onto PVDF membranes (IPVH00010, Millipore), membranes were blocked in 5% skim milk and incubated with primary antibodies against caspase-1 (1:3,000 dilution, AG-20B-0042, Adipogen), caspase-11 (1:1,000 dilution, NB120-10454, Novus) and GAPDH (1:10,000 dilution, #5174, Cell Signaling Technologies). Membranes were then incubated with HRP-conjugated secondary antibody for 1 h and proteins were visualized using Super Signal Femto substrate (34096, ThermoFisher Scientific). Statistical analysis. GraphPad Prism 6.0 software was used for data analysis. Data are shown as mean ± s.e.m. Statistical significance was determined by a log-rank test. P < 0.05 was considered statistically significant.