Interleukins and Atherosclerosis: a Dysfunctional Family Grows

Abstract

Atherosclerosis is driven by the release of cytokines from macrophages, and the β isoform of interleukin 1 (IL-1β) is a prime suspect in disease progression. Freigang et al. (2013) now suggest that IL-1α, a close relative, is selectively induced by fatty acids independent of the inflammasome to promote vascular inflammation.

Cytokines evolved as part of the immune response to protect mammals from an environment filled with threats, but those threats now include an overeating-induced excess of lipids that prompts cytokine responses more appropriate for pathogens. That cytokines mediate disease is now well established in metabolic and vascular medicine, but the precise cytokines involved in atherosclerosis and their relative importance are still under debate. A large body of evidence suggests that interleukin-1 (IL-1) signaling is proatherogenic. Two prominent members of the IL-1 family are the isoforms IL-1α and IL-1β, which use a shared receptor, the interleukin 1 receptor type I (IL-1R1), to promote downstream signaling pathways. Of the two isoforms, IL-1β is by far the more extensively studied. In fact, the ongoing Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS) is evaluating the efficacy of IL-1β inhibition in reducing cardiovascular events in one of the first true tests of the inflammation hypothesis of atherosclerosis in humans (Ridker et al., 2011). However, the biology of IL-1 signaling is complex and the role played by IL-1β may be less than explicit. Although mice deficient in IL-1β (Kirii et al., 2003) or injected with anti-IL-1β neutralizing antibody (Bhaskar et al., 2011) have reduced plaque formation, deficiency of IL-1α has been reported to afford more protection from atherosclerosis (Kamari et al., 2007). Mice deficient inIL-1R1 have a complex phenotype that includes features suggestive of plaque instability (Alexander et al., 2012). Both IL-1α and IL-1β lack a signal sequence required for conventional secretory pathways, and they appear to utilize different mechanisms for secretion. The NLRP3 inflammasome is essential for IL-1β secretion, whereas IL-1α secretion can be induced by inflammasome-independent mechanisms that involve calcium flux (Gross et al., 2012).

A recent study (Freigang et al., 2013) sheds light on the roles of macrophage IL-1α and IL-1β in the context of atherosclerosis. Freigang et al. transplanted bone marrow from mice lacking either IL-1α or IL-1β into animals that are prone to develop diet-induced atherosclerosis, LDL receptor-deficient mice. This manipulation resulted in atherosclerosis-susceptible mice with a deficiency in IL-1α or IL-1β limited to the hematopoietic compartment. After 4 months of an atherogenic diet containing 0.5% cholesterol, mice lacking bone marrow-derived IL-1α had fewer atherosclerotic lesions than controls, and the effect was more pronounced than in mice lacking bone marrow-derived IL-1β, confirming previous results (Kamari et al., 2007). In a different atherosclerosis-prone mouse model with normal expression of IL-1 isoforms - theapoE-deficient mouse - lipid mass spectrometry analysis of lesions after three months of atherogenic feeding showed a predominance of the saturated fatty acids palmitate (16:0) and stearate (18:0). Three months later, after 6 months of atherogenic feeding, oleic acid (18:1) was most abundant and there were increases in other unsaturated fatty acids such as linoleate (18:2).

When wild-type macrophages were first activated by lipopoly saccharide, treatment with oleic acid induced IL-1α but not IL-1β secretion. Some other unsaturated fatty acids had the same effect. Saturated fatty acids did not stimulate the release of either isoform. Oleic acid-induced IL-1α secretion was inflammasome-independent. Mice fed an oleic acid-enriched diet for 12 weeks had more atherosclerosis than mice fed a chow diet. Additional data in cultured bone marrow-derived macrophages suggested that the promotion of IL-1α secretion to the exclusion of IL-1β secretion was mediated by oleic acid-induced mitochondrial respiratory uncoupling, leading to increased calcium flux. Vascular respiratory uncoupling is known to increase atherosclerosis (Bernal-Mizrachi et al., 2005).

Although oleic acid induces IL-1α secretion in primed macrophages, it is not clear that this process is entirely independent of IL-1β. Freigang et al. also injected oleic acid to induce peritonitis. Neutrophil migration was blunted in mice transplanted with bone marrow deficient in either IL-1α or IL-1β. However, only antibodies to IL-1α but not to IL-1β protected against oleic acid-induced neutrophil migration, suggesting an intracellular requirement of IL-1β for IL-1α secretion. Since neutrophils are not a major contributor to diet-induced atherosclerosis, it would be of interest to know if mice deficient in IL-1α or IL-1β are protected from oleic acid-induced atherosclerosis.

The discovery of selective induction of secretion of macrophage IL-1α by oleic acid and other unsaturated fatty acids is intriguing (Figure 1), but a looming issue is the overall significance of this pathway to atherogenesis. It is curious that lipid analysis of lesions closest to the time of a demonstrated protective effect of IL-1α deficiency showed a predominance of saturated fatty acids, which are apparently incapable of stimulating IL-1α secretion. Lipid analysis at 6 months showed enrichment with oleic acid, reflecting a phenomenon first described in de novo lipogenesis studies more than 40 years ago: as cholesterol-induced atherosclerotic lesions progress, oleic acid becomes more abundant in lesions as it is preferentially used to form cholesteryl esters (St Clair et al., 1968). The authors studied responses of cells to free (unesterified) fatty acids, but fatty acids in cells are rapidly modified and incorporated into cholesteryl esters (comprising more than 80% of the lipid mass of fatty streaks), phospholipids, triglycerides, and other forms. It is unclear how free oleate would be trafficked from one of these sites to the mitochondrial membrane to induce respiratory uncoupling. It is also difficult to reconcile this mouse work with a large body of human data indicating that oleic acid-enriched diets decrease clinical atherosclerosis. In one striking example, a randomized trial recently showed that humans assigned to a Mediterranean diet supplemented with extra virgin olive oil (consisting mostly of oleic acid with some of the other unsaturated fatty acids shown to increase IL-1α secretion in the Freigang study) had fewer major cardiovascular events (Estruch et al., 2013).

Figure 1. Inflammatory cytokines promote atherosclerosis.

Macrophages secrete IL-1β (bottom), which is thought to be important in this process, but recent evidence implicates IL-1α (top) through a mechanism involving fatty acid-induced respiratory uncoupling and alterations of calcium flux.

Freigang and colleagues present important findings that expand our understanding of macrophage biology. Their work demonstrates that IL-1α can be selectively induced without increasing IL-1β secretion and implicate unsaturated fatty acids in this effect. The idea that IL-1β is the dominant family member promoting vascular wall inflammatory dysfunction may be naive. The poet and satirist Alexander Pope is credited with the observation that a family is but too often a commonwealth of malignants. In atherosclerosis, the interleukin family is dysfunctional, but additional studies will be required to clarify which member, IL-α or IL-β, is most malignant in mediating progression of vascular disease in a world intent on promoting inflammation by eating too much.