Sustained low-grade inflammation mediated by non-resolving inflammatory monocytes has long been suspected in the pathogenesis of atherosclerosis; however, the molecular mechanisms responsible for the sustainment of non-resolving inflammatory monocytes during atherosclerosis are poorly understood. Here we observe that subclinical endotoxemia, often seen in humans with chronic inflammation, aggravates murine atherosclerosis through programming monocytes into a non-resolving inflammatory state with elevated Ly6C, CCR5, MCP-1 and reduced SR-B1. The sustainment of inflammatory monocytes is due to the disruption of homeostatic tolerance through the elevation of miR-24 and reduction of the key negative-feedback regulator IRAK-M. miR-24 reduces the levels of Smad4 required for the expression of IRAK-M and also downregulates key lipid-processing molecule SR-B1. IRAK-M deficiency in turn leads to elevated miR-24 levels, sustains disruption of monocyte homeostasis and aggravates atherosclerosis. Our data define an integrated feedback circuit in monocytes and its disruption may lead to non-resolving low-grade inflammation conducive to atherosclerosis.

Low-dose endotoxemia is prevalent in humans with adverse health conditions, and it correlates with the pathogenesis of chronic inflammatory diseases such as atherosclerosis, diabetes, and neurologic inflammation. However, the underlying molecular mechanisms are poorly understood. In this study, we demonstrate that subclinical low-dose LPS skews macrophages into a mild proinflammatory state, through cell surface TLR4, IL-1R–associated kinase-1, and the Toll-interacting protein. Unlike high-dose LPS, low-dose LPS does not induce robust activation of NF-κB, MAPKs, PI3K, or anti-inflammatory mediators. Instead, low-dose LPS induces activating transcription factor 2 through Toll-interacting protein–mediated generation of mitochondrial reactive oxygen species, allowing mild induction of proinflammatory mediators. Low-dose LPS also suppresses PI3K and related negative regulators of inflammatory genes. Our data reveal novel mechanisms responsible for skewed and persistent low-grade inflammation, a cardinal feature of chronic inflammatory diseases.

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Inflammatory stimulants such as bacterial endotoxin (lipopolysaccharide (LPS)) are known to induce tissue damage and injury partly through the induction of reactive oxygen species (ROS). Although it is recognized that the induction of ROS in macrophages byReactive oxygen species (ROS) 2 play a critical role in the regulation of inflammatory processes causing the oxidation of lipids and proteins and eventually leading to tissue damage and organ failure. The generation of ROS is modulated by two families of opposing enzymes, oxidative enzymes such as NADPH oxidase and antioxidative enzymes, including glutathione peroxidase, catalase, and superoxide dismutase. Bacterial products such as lipopolysaccharide (LPS) selectively induce the expression and activation of oxidative enzymes, while decreasing the expression of antioxidative enzymes (1, 2). Taken together, LPS challenge significantly contributes to the production of ROS and the pathogenesis of diverse inflammatory diseases.Most of the published studies regarding NADPH oxidase have been specifically focused on the regulation and activation of NOX-2, the enzymatic NADPH oxidase component primarily expressed in neutrophils (3, 4). NOX-2 protein is constitutively expressed and is not regulated transcriptionally (3). LPS challenge causes rapid translocation of the functional NOX-2 containing NADPH oxidase to the membrane complex, leading to its activation (3). In contrast, NOX-1, the primary NADPH oxidase in macrophages, can be both transcriptionally induced and post-transcriptionally activated by LPS. However, the molecular mechanism for LPS-induced expression and activation of NOX-1 is poorly defined. Based on studies done in other cell types (5, 6), it is conceivable that LPS may contribute to the activation of NOX-1 containing NADPH oxidase via the small GTPase Rac1 in macrophages (7). However, the detailed molecular mechanism underlying LPS-mediated activation of Rac1 in macrophages is not known.On the other hand, LPS treatment decreases the levels of nuclear receptor family transcription factors such as PPAR␣ and PGC-1, which are responsible for the sustained expression of antioxidative enzymes, including glutathione peroxidase and catalase (8 -11). Collectively, the LPS-triggered up-regulation of oxidative enzymes and concurrent down-regulation of antioxidases leads to the generation and accumulation of ROS and tissue damage.IRAK-1 is one of many intracellular signaling components downstream of the LPS receptor (TLR4) (12)(13)(14). A series of studies have revealed that IRAK-1 positively contributes to the activation of NFB, STAT1/3, and IRF5/7, while negatively regulating the activities of nuclear factor of activated T-cells and nuclear receptors (15)(16)(17)(18)(19)(20). Despite the prominent role that IRAK-1 plays within the TLR4 signaling pathway, its involve-

Subclinical levels of circulating endotoxin are associated with the pathogenesis of diverse human inflammatory diseases, by mildly inducing the expression of proinflammatory mediators. In this study, we examined the molecular mechanism responsible for the effect of low-dose LPS in macrophages. In contrast to high-dose LPS, which activates NF-κB and induces the robust expression of proinflammatory mediators, we observed that low-dose LPS failed to activate NF-κB. Instead, it selectively activated C/EBPδ and removed nuclear repressors, including peroxisome proliferator-activated receptor α and retinoic acid receptor α, enabling a mild and leaky expression of proinflammatory mediators. The effect of low-dose LPS required IRAK-1, which interacts with and acts upstream of IκB kinase ε to contribute to LPS-mediated induction of C/EBPδ and proinflammatory mediators. Additionally, mice fed a high-fat diet acquired elevated levels of endotoxin and proinflammatory mediators in an IRAK-1–dependent fashion. Taken together, these data reveal a distinct pathway preferentially used by low-dose endotoxin in initiating low-grade inflammation.