In this study of obese subjects, we demonstrated both qualitative and quantitative evidence of differential TLR activity in human adipose tissue, extending knowledge from previous animal studies. Under basal conditions, we demonstrated greatest expression of the TLR4 subtype using two separate complementary methods that included enhanced immunofluorescence and gene expression, and confirmed TLR colocalization with adiponectin in adipocytes. Additionally, we demonstrated that TLRs are functionally inducible and associated with downstream NF-κB activation and proinflammatory cytokine production. These current findings provide evidence that adipose stores play a dynamic role in mechanisms of innate or adaptive immune responses in human subjects, and provide additional data to our growing understanding of the active inflammatory pathways in human adipose tissue.
TLRs represent a family of receptors that are critical to the innate immune response against foreign pathogens and microorganisms (6
). The TLR family has significant homology in its cytoplasmic domain to the IL-1 receptor, and recognizes both endogenous and exogenous ligands. Currently, up to 10 mammalian TLRs have been identified, primarily in vascular and immune cells, myocytes, and platelets, with receptors functioning both within the plasma membrane and intracellular space. Adipose tissue has also been shown to express TLR in experimental studies (5
). TLR2 has been cloned and characterized in mouse adipocytes, and their inflammatory stimulation induces changes in TLR2 expression and secretory cytokines, such as IL-6. In agreement with our findings, LPS has been shown to induce proinflammatory chemokine gene expression in differentiated human adipocytes through TLR and NF-κB action (14
). In this study, we additionally demonstrated that human adipocytes respond robustly to other agonists such as Pam3CSK4 as manifest by significant upregulation of TLR2. We also provide novel data by demonstrating NF-κB activation and nuclear translocation in human adipocytes using confocal immunofluorescence imaging techniques which had been previously reported in vascular endothelial cells (16
). These findings lend support to the presence of a functionally intact and diverse pathway of innate immunity in fat that is subject to activation and immunomodulation.
The functional significance of differential TLR expression patterns in human adipose tissue remains unknown. Although TLR expression in immune cells appears teleologically adaptive about host defense, its functional presence in fat cells raises the question of whether adipocytes are not only intricately involved in homeostatic immune regulation but may also play a functional role in disease states pathophysiologically linked to low-grade systemic inflammation. Stimulation of TLRs initiates intracellular signaling cascades resulting in downstream NF-κB and mitogen-activated protein kinase activation and production of proinflammatory chemokines associated with mechanisms of metabolic dysfunction and cardiovascular disease progression. Chronic inflammation owing to excessively efficient or hyperactive innate immunity, while protective against pathogens, may contribute to mechanisms of systemic disease. This observation is best clinically supported by a study demonstrating that the TLR4 polymorphism is associated with attenuated receptor signaling, reduced levels of circulating proinflammatory mediators, and decreased carotid atherosclerosis, while conferring increased susceptibility to bacterial infection (8
A salient finding in our study was evidence for greatest expression of TLR4 in fat tissue among the TLR family, raising the possibility that its activity may play a prominent role in obesity-associated inflammation and cardiometabolic risk. In support of this, both animal and human studies have supported a mechanistic role for TLR4 in systemic disease. TLR4-deficient mice crossed with ApoE−/−
mice develop smaller and fewer atherosclerotic lesions than ApoE−/−
mice alone (17
). TLR4 has been shown to recognize endogenous ligands such as oxidized-low-density lipoprotein and fibrinogen that may have relevance to atherothrombotic events. TLR4 expression is enhanced in both murine atherosclerotic lesions and lipid-rich, macrophage-infiltrated human coronary plaques, particularly in shoulder regions prone to rupture (10
). Elevated fatty acids levels associated with obesity activate TLR4 signaling in fat cells and macrophages, and induce insulin resistance in murine models (20
). Genotypic variations in TLR4 have been associated with carotid intimal thickening and increased risk of myocardial infarction (8
). In addition, cardiac myocytes constitutively express TLR4 that is upregulated in failing hearts (22
). Other members of the TLR family have also been implicated in atherogenesis. TLR1–6 are differentially expressed in human atherosclerotic plaques, although TLR2 and TLR4 appear to be expressed more widely (23
). TLR2 in mice is linked to increased atherosclerotic lesion formation, and TLR2 genetic variants have been associated with diabetes mellitus (24
). Meanwhile, adiponectin suppresses NF-κB activation in vascular cells, macrophages, and adipocytes (25
). Collectively, the findings lend support to a functional link between TLR signaling and obesity-related inflammation and cardiometabolic dysfunction.
This study has several limitations. First, the study was largely exploratory with the primary objective of examining different TLR subtype expression patterns in human adipose tissue under basal conditions. Our functional studies however were limited to TLR2 and TLR4 immunomodulation owing to clinical relevance based on previous studies. Second, we examined TLRs exclusively in obese patients as securing fat tissue was technically safer and more feasible in these subjects. Additionally, a recent report had previously examined this issue in adipose tissue from lean subjects (26
). Finally, we did not find significant correlation between TLR4 expression and cardiovascular risk factors, which is likely a limitation of the small sample size. The finding of a relationship between tissue TLR8 expression and circulating CRP levels supports a parallel link in local and systemic inflammatory activation in obesity and warrants further investigation.
In summary, this study demonstrated that human adipose tissue expresses TLRs with greatest expression of TLR4. TLR expression is inducible in adipose tissue and linked with downstream NF-κB activation and upregulated cytokine release. Enhanced TLR-dependent inflammatory activation owing to excess fat mass may represent a mechanistic link between obesity and cardiometabolic risk.