The D299G/T399I Toll-Like Receptor 4 Variant Associates with Body and Liver Fat: Results from the TULIP and METSIM Studies

BACKGROUND: Toll-like-receptor 4 (TLR) is discussed to provide a molecular link between obesity, inflammation and insulin resistance. Genetic studies with replications in non-diabetic individuals in regard to their fat distribution or insulin resistance according to their carrier status of a common toll-like receptor 4 (TLR4) variant (TLR4(D299G/T399I)) are still lacking. METHODOLOGY/PRINCIPAL FINDINGS: We performed a cross-sectional analysis in individuals phenotyped for prediabetic traits as body fat composition (including magnetic resonance imaging), blood glucose levels and insulin resistance (oral glucose tolerance testing, euglycemic hyperinsulinemic clamp), according to TLR4 genotype determined by candidate SNP analyses (rs4986790). We analyzed N = 1482 non-diabetic individuals from the TÜF/TULIP cohort (South Germany, aged 39±13 y, BMI 28.5±7.9, mean±SD) and N = 5327 non-diabetic participants of the METSIM study (Finland, males aged 58±6 y, BMI 26.8±3.8) for replication purposes. German TLR4(D299G/T399I) carriers had a significantly increased body fat (XG in rs4986790: +6.98%, p = 0.03, dominant model, adjusted for age, gender) and decreased insulin sensitivity (XG: -15.3%, Matsuda model, p = 0.04; XG: -20.6%, p = 0.016, clamp; both dominant models adjusted for age, gender, body fat). In addition, both liver fat (AG: +49.7%; p = 0.002) and visceral adipose tissue (AG: +8.2%; p = 0.047, both adjusted for age, gender, body fat) were significantly increased in rs4986790 minor allele carriers, and the effect on liver fat remained significant also after additional adjustment for visceral fat (p = 0.014). The analysis in METSIM confirmed increased body fat content in association with the rare G allele in rs4986790 (AG: +1.26%, GG: +11.0%; p = 0.010, additive model, adjusted for age) and showed a non-significant trend towards decreased insulin sensitivity (AG: -0.99%, GG: -10.62%). CONCLUSIONS/SIGNIFICANCE: TLR4(D299G/T399I) associates with increased total body fat, visceral fat, liver fat and decreased insulin sensitivity in non-diabetic Caucasians and may contribute to diabetes risk. This finding supports the role of TLR4 as a molecular link between obesity and insulin resistance

Effects of functional Toll-like receptor-4 mutations on the immune response to human and experimental sepsis.

Genetically determined responsiveness to microbial stimuli such as lipopolysaccharide (LPS) may affect the pathophysiology of human sepsis. The D299G mutation in human Toll-like receptor-4 (TLR4) impairs LPS signalling in homozygous and heterozygous individuals. To investigate whether the presence of the TLR4(D299G) mutation may correlate with the development or outcome of sepsis following major visceral surgery the presence of TLR4(D299G) mutation was analysed in 307 Caucasian patients (154 without and 153 with sepsis). Sepsis was caused in 84% of patients by polymicrobial infection. The presence of the mutant TLR4 did not significantly correlate with development or outcome of sepsis. Serum levels of tumour necrosis factor, interleukin (IL)-10, and IL-6 at sepsis onset did not significantly differ between patients carrying wild-type and mutant TLR4. Moreover, studies in a murine model of polymicrobial septic peritonitis demonstrated that TLR4-deficiency did neither influence the systemic cytokine response nor the development of organ injury. The results suggest that the signalling capacity of TLR4 as affected by loss-of-function mutations does not influence human or experimental sepsis caused by polymicrobial infection. Thus, in polymicrobial infection, other innate immune receptors may compensate for TLR4 defects

Attenuated pathogenesis of polymicrobial peritonitis in mice after TLR2 agonist pre-treatment involves ST2 up-regulation.

The innate immune system uses Toll-like receptors (TLRs) to activate and instruct immune responses against microbial pathogens. Administration of TLR agonists to mice induces a state of hyporesponsiveness, or tolerance, characterized by reduced cytokine production upon subsequent second challenge. The present study examined the effects of pre-treatment of mice with TLR2-dependent stimuli on the host defense against acute polymicrobial infection. Immune priming of mice with macrophage-activating lipopeptide-2 (MALP-2) 4 days prior to infection greatly improves survival and bacterial clearance in a model of polymicrobial septic peritonitis which is associated with enhanced accumulation of effector neutrophils in the peritoneal cavity. Further, the systemic and local production of both myeloid differentiation factor 88 (MyD88)-dependently and MyD88-independently produced cytokines was substantially diminished, but not completely absent, in TLR2-treated mice. While pre-treatment with MALP-2 does not involve differential expression of TLR and IL-1R-associated kinase proteins, ST2, a negative regulator of TLR signaling, was up-regulated after treatment of mice with either MALP-2 or N-alpha-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-L-cysteine. Therefore, ST2 may be a mechanism, among others, to attenuate the sepsis-induced cytokine burst. Thus, these results suggest that immune protection in mice after pre-treatment with TLR2-dependent stimuli involves the induction of enhanced pathogen defense by neutrophils. In addition, up-regulation of ST2 could contribute to the diminished cytokine production

CC chemokine receptor 2 regulates leukocyte recruitment and IL-10 production during acute polymicrobial sepsis.

Chemokine receptors are important for recruiting leukocytes to sites of infection and may contribute to immune cell activation. The present study investigated the role of the chemokine receptor CCR2 in polymicrobial septic peritonitis. The results showed that peritoneal production of the CCR2 ligands CCL2 and CCL12 in septic mice was largely independent of the common Toll-like receptor signaling adaptor MyD88. Antibody blockade of CCR2 reduced the recruitment of macrophages and neutrophils to the infected peritoneal cavities of both wild-type and MyD88-deficient mice, suggesting that CCR2 engagement contributes to the MyD88-independent cellular response against polymicrobial septic peritonitis. Notably, administration of blocking CCR2 antibodies markedly increased local and systemic IL-10 levels in septic wild-type mice, whereas IL-10 was not detected in MyD88-deficient mice irrespective of whether CCR2 was blocked or not. Inhibition of CCR2 directly augmented Toll-like receptor-induced IL-10, but not TNF and IL-6, production of macrophages in vitro. Concomitant with enhanced IL-10 production, CCR2 blockade caused impaired bacterial clearance and aggravated kidney injury in wild-type, but not MyD88-null mice. These results indicate that CCR2 engagement modulates the innate immune response to polymicrobial septic peritonitis by both MyD88-dependent and -independent processes and suggest that a major function of CCR2 in sepsis is to attenuate IL-10 production and IL-10-mediated suppression of host defense