Urmila Maitra

IRAK-1 contributes to lipopolysaccharide-induced reactive oxygen species generation in macrophages by inducing NOX-1 transcription and Rac1 activation and suppressing the expression of antioxidative enzymes.

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 by LPS depends upon the expression and activation of NADPH oxidase, as well as the suppression of antioxidative enzymes involved in ROS clearance, the underlying molecular mechanisms are poorly defined. In this study, we examined the contribution of the interleukin-1 receptor-associated kinase 1 (IRAK-1) to LPS-induced generation of ROS. We observed that LPS induced significantly less ROS in IRAK-1−/− macrophages, indicating that IRAK-1 is critically involved in the induction of ROS. Mechanistically, we observed that IRAK-1 is required for LPS-induced expression of NOX-1, a key component of NADPH oxidase, via multiple transcription factors, including p65/RelA, C/EBPβ, and C/EBPδ. On the other hand, we demonstrated that IRAK-1 associated with and activated small GTPase Rac1, a known activator of NOX-1 oxidase enzymatic activity. IRAK-1 forms a close complex with Rac1 via a novel LWPPPP motif within the variable region of IRAK-1. On the other hand, we also observed that IRAK-1 is required for LPS-mediated suppression of peroxisome proliferator-activated receptor α and PGC-1α, nuclear factors essential for the expression of antioxidative enzymes such as GPX3 and catalase. Consequently, injection of LPS causes significantly less plasma lipid peroxidation in IRAK-1−/− mice compared with wild type mice. Taken together, our study reveals IRAK-1 as a novel component involved in the generation of ROS induced by LPS.

Molecular Mechanisms Responsible for the Selective and Low-Grade Induction of Proinflammatory Mediators in Murine Macrophages by Lipopolysaccharide

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.

Differential Regulation of Foxp3 and IL-17 Expression in CD4 T Helper Cells by IRAK-1

Host immune responses are finely regulated by the opposing effects of Th17 and T regulatory (Treg) cells. Treg cells help to dampen inflammatory processes and Th17 cells facilitate various aspects of immune activation. The differentiation of Th cells depends on a unique combination of stimulants and subsequent activation of diverse transcription factors. In particular, cooperative activation of NFAT and Smad3 leads to the induction of Treg cells, and cooperation among STAT3 and Smad3 switches to the induction of Th17 cells. We have previously shown that the IL-1 receptor associated kinase 1 (IRAK-1) selectively activates STAT3 and inactivates NFAT. Physiological studies have shown that IRAK-1−/− mice are protected from developing various inflammatory diseases, including experimental autoimmune encephalomyelitis and atherosclerosis with unknown mechanism. In this study, we demonstrate that IRAK-1 plays a critical modulatory role in the differentiation of Th17 and Treg cells. Following stimulation with TCR agonists and TGFβ, IRAK-1−/− CD4 Th cells display elevated nuclear NFATc2 levels and increased interaction of NFATc2 and Smad3, resulting in increased expression of Foxp3, a key marker for Treg cells. IRAK-1−/− mice have constitutively higher populations of Treg cells. In contrast, when stimulated with TCR agonists together with IL-6 and TGF-β, IRAK-1−/− CD4 Th cells exhibit attenuated STAT3 Ser727 phosphorylation and reduced expression of IL-17 and RORγt compared with wild-type cells. Correspondingly, IRAK-1 deletion results in decreased IL-17 expression and dampened inflammatory responses in acute and chronic inflammatory mice models. Our data provides mechanistic explanation for the anti-inflammatory phenotypes of IRAK-1−/− mice.

Low-Dose Endotoxin Induces Inflammation by Selectively Removing Nuclear Receptors and Activating CCAAT/Enhancer-Binding Protein δ

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.

Molecular mechanism responsible for the priming of macrophage activation

Background: Macrophages can adopt primed or tolerance state, depending upon the history of prior challenges. Results: IRAK-1 and Tollip facilitate priming of macrophages by super low dose endotoxin. Conclusion: Super low dose endotoxin primes macrophages by inactivating molecular suppressors, including RelB. Significance: Revealing mechanisms of macrophage priming is crucial for understanding the pathogenesis of inflammatory diseases. Host macrophages can be preprogrammed into opposing primed or tolerant states depending upon the nature and quantities of external stimulants. The paradigm of priming and tolerance has significant implications in the pathogenesis and resolution of both acute and chronic inflammatory diseases. However, the responsible mechanisms are not well understood. Here, we report that super low dose bacterial endotoxin lipopolysaccharide (LPS), as low as 5 pg/ml, primes the expression of proinflammatory mediators in macrophages upon a second high dose LPS challenge (100 ng/ml), although 5 pg/ml LPS itself does not trigger noticeable macrophage activation. Mice primed with super low dose LPS (0.5 μg/kg body weight) in vivo experience significantly elevated mortality following a second hit of high dose LPS as compared with saline-primed control mice. Mechanistically, we demonstrate that LPS primes macrophages by removing transcriptional suppressive RelB through interleukin receptor-associated kinase 1 and Tollip (Toll-interacting protein)-dependent mechanisms. This is in sharp contrast to the well documented RelB stabilization and induction by high dose LPS, potentially through the phosphoinositide 3-kinase (PI3K) pathway. Super low dose and high dose LPS cause opposing modulation of interleukin receptor-associated kinase 1 and PI3K pathways and lead to opposing regulation of RelB. The pathway switching induced by super low versus high dose LPS underscores the importance of competing intracellular circuitry during the establishment of macrophage priming and tolerance.

An Innate Immunity Signaling Process Suppresses Macrophage ABCA1 Expression through IRAK-1-Mediated Downregulation of Retinoic Acid Receptor α and NFATc2

ABSTRACT ATP-binding cassette transporter A1 (ABCA1) plays a central role in promoting cholesterol efflux from macrophages, thereby reducing the risk of foam cell formation and atherosclerosis. The expression of ABCA1 is induced by members of the nuclear receptor family of transcription factors, including retinoic acid receptors (RARs). A key innate immunity signaling kinase, IRAK-1, has been associated with an increased risk of atherosclerosis in humans and mice. This prompted us to investigate the potential connection between IRAK-1 and the expression of ABCA1. Here, we demonstrate that nuclear RARα levels are dramatically elevated in IRAK-1−/− macrophages. Correspondingly, IRAK-1−/− macrophages exhibit increased expression of ABCA1 mRNA and protein, as well as elevated cholesterol efflux in response to the RAR ligand ATRA. Analysis of the ABCA1 proximal promoter revealed binding sites for both RAR and NFAT. Chromatin immunoprecipitation assays demonstrated increased binding of RARα and NFATc2 to the ABCA1 promoter in IRAK-1−/− macrophages compared to wild-type macrophages. Additionally, lipopolysaccharide pretreatment reduced the nuclear levels of RARα and decreased ABCA1 expression and cholesterol efflux in wild-type but not in IRAK-1−/− cells. In summary, this study reveals a novel connection between innate immunity signaling processes and the regulation of ABCA1 expression in macrophages and defines a potential therapeutic target for treating atherosclerosis.

Molecular mechanism underlying the suppression of lipid oxidation during endotoxemia.

Although both inflammatory and metabolic complications occur during sepsis and endotoxemia, relatively few studies have examined the molecular mechanism underlying LPS-modulated metabolic changes during sepsis. In this report, we have demonstrated that LPS suppresses free fatty acid (FFA) oxidation, and consequently contributes to elevated plasma levels of FFA and triglyceride (TG). Furthermore, this process depends upon the interleukin-1 receptor associated kinase 1 (IRAK-1), one of the key TLR4 intracellular signaling kinases. IRAK-1(-/-) mice fail to exhibit the dramatic rise in plasma FFA and TG levels compared to wild-type (WT) mice following lethal LPS injection. Mechanistically, we demonstrated that LPS suppresses FFA oxidation through decreasing the expression levels of key FFA oxidative genes including CPT-1 and MCAD in both liver and kidney tissues of WT but not IRAK-1(-/-) mice. The expression of CPT-1 and MCAD is controlled by nuclear receptors and co-receptors including PPARalpha and PGC-1alpha. We observed that LPS selectively suppresses the levels of PPARalpha and PGC-1alpha in tissues from WT, but not IRAK-1(-/-) mice. Consequently, IRAK-1(-/-) mice have a higher survival rate following a lethal dose of LPS. Our current study reveals a novel role for IRAK-1 in the metabolic alterations induced by LPS.

Molecular Mechanisms Responsible for the Reduced Expression of Cholesterol Transporters From Macrophages by Low-Dose Endotoxin

Objective—Atherosclerosis is characterized as a chronic inflammatory condition that involves cholesterol deposition in arteries. Together with scavenger receptor B1 (SR-B1), the ATP-binding cassette transporters ABCA1 and ABCG1 are the major components of macrophage cholesterol efflux. Recent studies have shown that low-grade inflammation plays a distinct regulatory role in the expression of SR-B1 and ABCA1/ABCG1. However, the mechanisms linking low-grade inflammation and cholesterol accumulation are poorly understood. Methods and Results—Using primary bone-marrow-derived macrophages, we demonstrate that subclinical low-dose lipopolysaccharide potently reduces the expression of SR-B1 and ABCA1/ABCG1, as well as cholesterol efflux from macrophages through interleukin-1 receptor-associated kinase 1 and Toll-interacting-protein. Low-dose lipopolysaccharide downregulates the nuclear levels of retinoic acid receptor-&agr;, leading to their reduced binding to the promoters of SR-B1 and ABCA1/ABCG1. We observe that glycogen synthase kinase 3&bgr; activation by low-dose lipopolysaccharide through interleukin-1 receptor-associated kinase 1 and Toll-interacting-protein is responsible for reduced levels of retinoic acid receptor-&agr;, and reduced expression of SR-B1 and ABCA1/ABCG1. Interleukin-1 receptor-associated kinase M, however, counteracts the function of interleukin-1 receptor associated kinase 1. Conclusion—Collectively, our data reveal a novel intracellular network regulated by low-dose endotoxemia that disrupts cholesterol efflux from macrophages and leads to the pathogenesis of atherosclerosis.

Causes and consequences of low grade endotoxemia and inflammatory diseases

Increasing clinical observations reveal that persistent low-grade inflammation is associated with the pathogenesis of severe chronic diseases such as atherosclerosis, diabetes, and aging-related neurological diseases. Intriguingly, low levels of circulating Gram-negative bacterial endotoxin lipopolysaccharide (LPS) appear to be one of the key culprits in provoking a non-resolving low-grade inflammation. Adverse life styles, chronic infection, and aging can all contribute to the rise of circulating endotoxin levels and lead to low-grade endotoxemia. As a consequence, low-grade endotoxemia may skew host immune environment into a mild non-resolving pro-inflammatory state, which eventually leads to the pathogenesis and progression of inflammatory diseases. This review aims to highlight the recent progress in the causes and consequences of low-grade endotoxemia, as well as the emerging molecular mechanisms responsible.

The homeodomain protein CDP regulates mammary-specific gene transcription and tumorigenesis.

ABSTRACT The CCAAT-displacement protein (CDP) has been implicated in developmental and cell-type-specific regulation of many cellular and viral genes. We previously have shown that CDP represses mouse mammary tumor virus (MMTV) transcription in tissue culture cells. Since CDP-binding activity for the MMTV long terminal repeat declines during mammary development, we tested whether binding mutations could alter viral expression. Infection of mice with MMTV proviruses containing CDP binding site mutations elevated viral RNA levels in virgin mammary glands and shortened mammary tumor latency. To determine if CDP has direct effects on MMTV transcription rather than viral spread, virgin mammary glands of homozygous CDP-mutant mice lacking one of three Cut repeat DNA-binding domains (ΔCR1) were examined by reverse transcription-PCR. RNA levels of endogenous MMTV as well as α-lactalbumin and whey acidic protein (WAP) were elevated. Heterozygous mice with a different CDP mutation that eliminated the entire C terminus and the homeodomain (ΔC mice) showed increased levels of MMTV, β-casein, WAP, and α-lactalbumin RNA in virgin mammary glands compared to those from wild-type animals. No differences in amounts of WDNM1, ε-casein, or glyceraldehyde-3-phosphate dehydrogenase RNA were observed between the undifferentiated mammary tissues from wild-type and mutant mice, indicating the specificity of this effect. These data show independent contributions of different CDP domains to negative regulation of differentiation-specific genes in the mammary gland.