Interleukin-1 receptor-associated kinase-3 is a key inhibitor of inflammation in obesity and metabolic syndrome

Abstract

Background: Visceral obesity is associated with the rising incidence of type 2 diabetes and metabolic syndrome. Low-grade chronic inflammation and oxidative stress synergize in obesity and obesity-induced disorders.

Objective: We searched a cluster of molecules that support interactions between these stress conditions in monocytes.

Methods: RNA expressions in blood monocytes of two independent cohorts comprising 21 and 102 obese persons and 46 age-matched controls were determined by microarray and independently validated by quantitative RT-PCR analysis. The effect of three-month weight loss after bariatric surgery was determined. The effect of RNA silencing on inflammation and oxidative stress was studied in human monocytic THP-1 cells.

Results: Interleukin-1 receptor-associated kinase-3 (IRAK3), key inhibitor of IRAK/NFκB-mediated chronic inflammation, is downregulated in monocytes of obese persons. Low IRAK3 was associated with high superoxide dismutase-2 (SOD2), a marker of mitochondrial oxidative stress. A comparable expression profile was also detected in visceral adipose tissue of the same obese subjects. Low IRAK3 and high SOD2 was associated with a high prevalence of metabolic syndrome (odds ratio: 9.3; sensitivity: 91%; specificity: 77%). By comparison, the odds ratio of high-sensitivity C-reactive protein, a widely used marker of systemic inflammation, was 4.3 (sensitivity: 69%; specificity: 66%). Weight loss was associated with an increase in IRAK3 and a decrease in SOD2, in association with a lowering of systemic inflammation and a decreasing number of metabolic syndrome components. We identified the increase in reactive oxygen species in combination with obesity-associated low adiponectin and high glucose and interleukin-6 as cause of the decrease in IRAK3 in THP-1 cells in vitro.

Conclusion: IRAK3 is a key inhibitor of inflammation in association with obesity and metabolic syndrome. Our data warrant further evaluation of IRAK3 as a diagnostic and prognostic marker, and as a target for intervention.

Figure 1. IRAK3 is a key inhibitor in monocyte-related mechanisms underlying inflammation and oxidative stress during obesity.

(A) A structural model containing TLR2 as cell surface marker, NFκB as transcription factor, TNFα as inflammatory output, SOD2 as oxidative stress marker, and IRAK3 and TNFAIP3 as putative inhibitors was determined by promoter and gene annotation analysis of deregulated genes. Flow of the pathway at the protein interaction level is indicated by black arrows. Blunted arrows indicate inhibition. Phosphorylation is indicated by ???. Note that NFκB is constitutively bound to IκB molecules, which confine its localization to the cytosol. IKK complex phosphorylation of IκB promotes its degradation, thereby freeing NFκB to enter the nucleus and activate transcription of target genes. Gene expression of key molecules in the TLR2/NFκB inflammatory pathway was measured by qRT-PCR in blood monocytes of (B) the first cohort comprising 14 lean controls and 21 obese patients, and (C) the second cohort comprising 25 lean controls and 102 obese patients. Data are expressed as means. ^** P<0.01 and ^*** P<0.001 obese persons compared with lean controls.

Figure 2. Gene expressions of the IRAK3-related pathway and adipocyte differentiation markers in visceral adipose tissue.

(A) Gene expression in visceral adipose tissue was analyzed by measuring relative RNA levels using qRT-PCR for key molecules in the TLR2/NFκB inflammatory pathway. The adipose tissue specific antioxidant gene SOD3 instead of SOD2 was used as oxidative stress marker in visceral adipose tissue. (B) Relative RNA levels of markers of adipocyte differentiation (PPARs and ADIPOQ), insulin signaling (INSR) and glucose uptake (GLUT4) in visceral adipose tissue as determined by qRT-PCR. Data shown are means. ^* P<0.05 and ^** P<0.01 obese persons compared with lean controls; lean controls (n = 7), obese patients (n = 21).

Figure 3. Regulation of IRAK3 expression in THP-1 monocytes.

(A) Gene expression was analyzed by measuring relative RNA levels using qRT-PCR, protein expression and ROS production were determined by flow cytometry in THP-1 cells exposed to 1 or 10 µg/ml gADIPOQ (n = 6) or in IRAK3-depleted THP-1 cells exposed to 10 µg/ml gADIPOQ (n = 4) for 6 h and 24 h. Data shown are means ± SEM of 24 h exposed cells normalized to 6 h exposed cells. ^* P<0.05, ^** P<0.01 and ^*** P<0.001 compared with THP-1 cells exposed to high gADIPOQ; ^$$ P<0.01 and ^$$$ P<0.001 compared with THP-1 cells exposed to low gADIPOQ. (B) Gene/protein expression in THP-1 cells exposed to 10 µg/ml gADIPOQ and 10 µg/ml ox-LDL (n = 6), 1 µg/ml gADIPOQ and 25 µg/ml ox-LDL (n = 6) or 10 µg/ml gADIPOQ and 25 µg/ml ox-LDL (n = 6). Data are expressed as means ± SEM. ^** P<0.01 compared with THP-1 cells exposed to 10 µg/ml gADIPOQ and 10 µg/ml ox-LDL; ^$ P<0.05 and ^$$ P<0.01 compared with THP-1 cells exposed to 1 µg/ml gADIPOQ and 25 µg/ml ox-LDL. Abbreviations: gADIPOQ, globular adiponectin, iROS, intracellular ROS; mROS, mitochondrial ROS; ox-LDL, oxidized LDL; ROS, reactive oxygen species.

Figure 4. Exposure of IRAK3-depleted THP-1 cells to additional stress results in more inflammation and ROS.

Gene expression was analyzed using qRT-PCR and mROS production was determined by flow cytometry in THP-1 cells exposed to (A) 5.5 mM D-glucose and 9.5 mM D-mannitol (osmotic control) or 15 mM D-glucose (n = 6), and (B) 100 ng/ml IL-6 (n = 6). Data shown are means ± SEM. ^* P<0.05, ^** P<0.01 and ^*** P<0.001 compared with THP-1 cells exposed to 5.5 mM D-glucose or PBS vehicle. (C) Gene/protein expression and ROS production in THP-1 cells transiently transfected with siRNA targeting IRAK3 (n = 10) or in THP-1 cells exposed to 100 mU/ml glucose oxidase with (n = 4) or without (n = 5) silencing of IRAK3. Data shown are means ± SEM. ^* P<0.05, ^** P<0.01 and ^*** P<0.001 compared with THP-1 control cells or THP-1 cells transfected with negative control siRNA; ^$$ P<0.01 and ^$$$ P<0.001 compared with THP-1 cells transfected with IRAK3 siRNA; ^## P<0.01 and ^### P<0.001 compared with THP-1 cells exposed to glucose oxidase. Abbreviations: iROS, intracellular ROS; mROS, mitochondrial ROS; ROS, reactive oxygen species.