Recent study of the CAPS disease spectrum has led to significant advances in our understanding of the NLRP3 inflammasome and IL-1β–mediated inflammation. While translational studies have resulted in vital therapies for patients with CAPS, many questions about the inflammasome and other caspase-1–dependent pathways remain. Inflammasome-mediated IL-18 has largely been overlooked in the context of human disease. In addition, recent studies have demonstrated caspase-1 and inflammasome functions extending beyond cytokine maturation to cellular death pathways, but whether these processes have any bearing on CAPS remains to be seen. Here, we take advantage of mutant NLRP3 knockin mouse lines to investigate these questions.
Our studies demonstrate that dysregulated IL-18 secretion occurs from both patient and Nlrp3 mutant mouse cells in a manner similar to IL-1β. In vitro, a hallmark of CAPS is lack of reliance on the 2-signal paradigm generally required for secretion of active IL-1β. We used this mechanism of inflammasome activation to evaluate IL-18 secretion. Both patient and mouse CAPS cells require some stimulation to transcribe immature cytokine proforms; however, the threshold for mature protein production appears much lower and can be reached with normally inconsequential stimuli such as cold temperature. IL-1β and IL-18 are secreted at similar levels in response to cold by FCAS mutant murine BMDCs, peritoneal macrophages, and FCAS patient peripheral blood monocytes, yet LPS treatment results in nearly 100-fold excess of IL-1β compared with IL-18. These differences suggest that pathogen-associated molecular patterns and cold are sensed differently by the inflammasome and that IL-1β and IL-18 secretion may be differentially regulated.
In vivo, we demonstrated increased IL-18 in regions of inflamed tissue and increased transcription of some IL-18 targets. Initially, elimination of IL-18R signaling appeared to provide improved phenotypic rescue compared with blockade of IL-1 in young mice, a surprising finding given the profound effect of IL-1 blockade in patients with CAPS. MWS Il18r–/– mice are often indistinguishable from their WT littermates at 6 weeks of age. However, upon extended observation, aging MWS Il18r–/– mice demonstrated evidence of systemic inflammation in the absence of cutaneous pathology sometimes observed in MWS Il1r–/– mice. It is therefore likely that the two inflammasome-dependent cytokines have different temporal impacts during the natural progression of disease.
Further dissection of cytokine signaling also revealed spatial differences in the influence of each cytokine. During bone marrow transplant studies, Il18r–/–
recipients transplanted with FCAS CreT
bone marrow succumbed early, whereas Il1r–/–
recipients survived, indicating a critical role for IL-1R signaling, but a more limited role for IL-18R, in somatic tissues. Like IL-1β, monocytes and dendritic cells are the primary sources of active IL-18, which has a variety of effects. Depending on context, IL-18 stimulates expression of adhesion molecules; induces production of GM-CSF, TNF-α, IL-6, and IL-8; enhances neutrophil, NK, and CD8+
T cell cytotoxicity; and stimulates Th1, Th2, or Th17 responses (18
), all hematopoietic targets that would be unaffected in the transplant model. However, unlike IL-1β, which requires a stimulus for transcription, pro–IL-18 is constitutively expressed in most tissues by epithelial cells, keratinocytes, and synovial fibroblasts. These steady-state pools of inactive cytokine may then be released under conditions of stress and cleaved exogenously by proteinase-3 (19
). Thus, cells that lack all the components for inflammasome formation may still contribute to IL-18–driven inflammation. Therefore, only complete elimination of IL-18R signaling in both the hematopoietic and somatic compartments would lead to a measurable difference in disease activity.
We have extended our previous findings that CAPS is inflammasome dependent (9
) by demonstrating that intact caspase-1 is required for disease. Clearly other inflammasome-dependent disease mechanisms are at play, as 50% of FCAS Il1r–/–Il18–/–
mice still succumbed to disease before reaching adulthood. It is possible that the residual inflammation seen on the Il1r–/–Il18–/–
background is due to increased pyroptosis. In support of this hypothesis, FCAS Il1r–/–Il18–/–
mouse bone marrow cells demonstrated increased caspase-1–dependent, pyroptotic cell death in the bone marrow. Pyroptosis is associated with cleavage and release of IL-18, IL-1β, and other inflammatory cellular contents, resulting in activation of nearby cells and potentiating the inflammatory response (21
). The relative contribution of this pathway to disease pathogenesis remains to be determined but may be a potential target for therapeutic development.
Our study is limited by the breeding schemes undertaken. By breeding mutant Nlrp3
mice to Il1r–/–
mice, we have eliminated specific receptor signaling but cannot account for signaling via alternate receptors or pleiotropic effects due to loss of signaling by alternate ligands. A previous study demonstrated a discrepancy in the susceptibility of Il18r–/–
mice in a model of experimental autoimmune encephalitis, suggesting that other ligand(s) for the IL-18R could exist (22
). Therefore, the rescue seen on the Il18r–/–
background could be due to loss of signaling via an unknown ligand independent of IL-18. This scenario is unlikely, since breeding the CAPS mutation onto an Il18–/–
background resulted in an inflammatory phenotype identical to that seen in mutant Il18r–/–
mice (our unpublished results). In addition, the caspase-1 knockout mice used in our study were recently found to be deficient in caspase-11 also. However, studies suggest that caspase-11 is activated by an inflammasome distinct from NLRP3, making its absence unlikely to significantly influence our results (23
IL-18 signaling appears to be vital to the initial development of inflammation in murine CAPS. Since only modest improvement is observed when IL-1 signaling is disrupted (9
), it is surprising that anti–IL-1 therapies are so effective in patients with CAPS (8
). The improvement in inflammation we observed in young mice in the absence of IL-18 signaling suggests that in pups disease is largely driven by IL-18, which then influences IL-1β production. This notion is supported by multiplex results demonstrating that both IL-1β and IL-18 normalize in MWS Il18r–/–
serum, while remaining elevated in MWS Il1r–/–
serum. The question therefore remains as to why there are not more anti–IL-1 treatment failures, especially in young patients with CAPS. The differential development of mice in utero compared with human fetuses may provide some insight into this difference. Hematopoietic stem cell development in humans occurs relatively earlier compared with that in mice, at 70 days (first trimester) versus 15.5 days gestation (third trimester) (24
), respectively. It is possible that, in humans, the proinflammatory effects of IL-18 occur mainly in utero and IL-1β then drives the pathology observed in young patients with CAPS. To date, neither increased rate of miscarriage, nor extreme prematurity have been described in patients with CAPS. Prematurity has been reported in the related autoinflammatory disease deficiency of the IL-1R antagonist (25
), although the role of other cytokines was not investigated.
Despite these questions, our results have important implications for patients with CAPS with residual disease and for patients with conditions involving other NLRP3 and IL-1β–mediated pathways (26
). For patients with other inherited autoinflammatory syndromes (28
), or more common inflammatory diseases such as heart disease and rheumatoid arthritis, IL-1 blockade has been a successful alternative to traditional therapy, even in patients previously receiving biologics (36
). However, reports of patients with IL-1β–driven diseases that have failed IL-1–targeted therapy are emerging (39
), suggesting that therapeutics aimed either at IL-18 or upstream at the level of the inflammasome may be valuable. IL-18 binding protein (IL-18BP), a naturally occurring, specific inhibitor that blocks the binding of mature IL-18 to its receptor (41
), has been used in mouse models of metastatic melanoma and myocardial ischemia (42
). The elevated expression of IL-18BP in sepsis (45
) and macrophage activation syndrome (46
) suggests that blockade of IL-18 may be useful in cases refractory to therapy by IL-1 inhibition. Whether similar increases in IL-18BP exist in patients with NLRP3-driven autoinflammatory syndromes remains to be determined.
Recently, small molecule inhibitors have been developed that have the potential for inflammasome-targeted therapy. In vitro experiments with PBMCs from patients with FCAS have demonstrated effectiveness of the orally active ICE/caspase-1 inhibitor, VX-765 (47
). Specific caspase-1 or other inflammasome-targeted therapies would affect not only IL-1β and IL-18 secretion but other caspase-1 substrates and pyroptotic cell death (48
). These benefits have been demonstrated in studies of myocardial ischemia, in which IL-1 and IL-18 acted in synergy to cause postischemic dysfunction, which could be prevented, with preservation of cell viability, using caspase 1 inhibitors (49
). Investigation into these alternative pathways may be instrumental in our knowledge of the autoinflammatory disease spectrum and function of the NLRP3 inflammasome.