Adipose tissue plays a significant role in the response to inflammatory stimuli. This role is conserved from drosophila to mammals, and because of their strategic location around organs, adipocytes are able to participate in the recognition and neutralization of multiple pathogens. Thus adipocytes facilitate a robust innate immune defense system [1
]. The innate immune response mediated by adipocytes is primarily mediated by adipokines released in response to inflammatory stimuli [26
]. Although the expression of TLR2 in adipocytes suggests a capability to respond to TLR2 ligands, [5
], little is known regarding the activation of inflammatory response by peptidoglycan in adipocytes. We have provided evidence herein that adipocytes respond directly to TLR2 activation with the peptidoglycan component of gram positive bacteria. The ability of adipocytes to recognize gram positive bacteria component fills a critical gap regarding the capability of adipocytes to neutralize both classes of bacteria pathogens and demonstrates the versatility of immune reaction mediated by the adipocytes. The recent evidence that fatty acids act as endogenous ligands for TLR2 in hypertrophic adipose tissue leading to the activation of a subset of adipose tissue macrophages [28
] supports a relevant role for TLR2 in vivo. Since fatty acid concentrations are elevated in obesity, fatty acids could be the major endogenous ligands for TLR2 in adipose tissue. Whereas TLR4 activation induces a rapid acute response, the inflammatory response to TLR2, demonstrated by the induction of IL6 in our case, is slower, but surprisingly prolonged. Therefore, although activation of both receptors elicits an inflammatory response, TLR2 induces a chronic inflammatory state. Although peptidoglycan recognition protein (PGRP) and NOD have been shown to mediate peptidoglycan response in some cell types [29
], their role in mediating the response obtained in this experiment is limited because immunoneutralization with a TLR2 specific antibody totally ablates response to peptidoglycan, and a TLR2 specific siRNA drastically (approx. 80%) reduces IL6 induction by peptidoglycan. Obesity and type 2 diabetes are associated with a chronic low grade systemic and adipose tissue inflammation [30
]. Interestingly, obesity and type 2 diabetes are also associated with increased expression of TLR2, and obesity induces the expression of a subset of adipocytes that over express both TLR2 and TNFα [8
]. The reduction in the expression of adiponectin receptors in response to TLR2 activation with peptidoglycan also agrees with the finding that these receptors are downregulated in adipose tissue of obese and insulin resistant mice [22
]. This raises the possibility that activation of TLR2 in obesity may contribute to a state of adiponectin resistance in obesity. Because adiponectin exerts anti-inflammatory effects [16
], a reduction in the expression of its receptors could attenuate this critical role of adiponectin. Therefore, TLR2 may be an important player in the perpetuation of inflammation that characterizes obesity.
Our work and that of others have shown that multiple signaling pathways mediate LPS induction of IL6 [1
]. These pathways include NFκB, c-JNK, ERK, inhibitory G protein and PKC mediated processes. Toll like receptors activate similar but distinct signaling pathways due to their ability to recruit different adapter proteins. TLR2 is able to recruit TIRAP/Mal, and this allows it to regulate the expression of a distinct set of inflammatory genes such as IL6, TNFα and IL12 [33
]. Our work has shown that inhibition of NFκB failed to suppress induction of IL6 by peptidoglycan and also had no effect on TLR2 mRNA expression. It is unknown whether this observation is unique to the 3T3-L1 adipocytes or related to our experimental conditions, but parallels the inability of NFκB inhibition to suppress IL6 induction by LPS in myocytes [23
]. However, additional studies will be needed to fully characterize the uniqueness of the signaling characteristics of TLR2 in adipocytes in comparison with other toll receptors.
The strong induction of TLR2 mRNA by both LPS and peptidoglycan that was observed in this study clearly supports a role for TLR2 as a strong marker of inflammation in adipocytes. This corroborates the induction of TLR2, but not TLR4, by LPS in mouse splenic macrophages [35
]. Since obesity is an inflammatory condition that is also associated with elevated TLR2 expression in adipose tissue, adipocyte TLR2 may indeed mediate part of the inflammatory environment that characterizes obesity [8
]. Therefore, targeting TLR2 may contribute to prevention of obesity-induced inflammation. Since obesity is also associated with elevated fatty acid levels, the induction of TLR2 expression by both DHA and EPA individually and additively with peptidoglycan and the additive induction of TLR2 by both linoleic acid and peptidoglycan suggests that fatty acids may be partly responsible for the upregulation of TLR2 in obesity. This also suggests that regulation of TLR2 mediated cellular responses may be fatty acid specific. Elevated fatty acid concentrations in obesity may amplify the inflammatory cascade that is induced by yet unidentified endogenous ligands for TLR2. Although omega-3 fatty acids, EPA and DHA at moderate levels are known to exert anti-inflammatory effects, elevated levels of these fatty acids in circulation has been demonstrated to cause increased inflammation characterized by increased macrophage infiltration into adipose tissue [36
]. Therefore, the levels of these fatty acids as utilized in this experiment mimic more closely the hyperlipidemic condition that is characterized by elevated fatty acid concentrations. The additivity of effects of fatty acids and peptidoglycan on the induction of TLR expression suggests that under the hyperlipidemic conditions of obesity fatty acids and ligands of TLRs may co-operate to amplify the inflammatory state by further increasing the expression of TLRs. This may be a mechanism to prevent desensitization to the effects of TLR ligands in obesity.
Interestingly, whereas inhibition of p44/42 MAPK and c-JNK suppressed peptidoglycan induction of IL6, it amplifies the induction of TLR2 mRNA by peptidoglycan. This observation agrees with the upreguation of TLR2 mRNA by p44/42 MAPK inhibition with PD 98059 in RAW 264.7 macrophages [35
]. Furthermore, it indicates that these kinase pathways, although they mediate positively the induction of IL6 by peptidoglycan, they may also be involved in a negative feedback mechanism to prevent an upregulation of TLR2 during inflammation, perhaps to prevent on overzealous inflammatory reaction.
The downregulation of expression of both adiponectin receptors by peptidoglycan parallels the reduction of soluble adiponectin receptor expression after the administration of LPS to human subjects [37
] and suggests that TLR2 activation in obesity may partly be responsible for the downregulation of adiponectin receptor expression in adipose tissue in obesity [22
]. Therefore, this may implicate TLR2 in phenomenon of obesity-induced adiponectin resistance. Because reduced expression of adiponectin receptors correlates with reduced AMPK activation, TLR2 activation may play an active role in the worsening insulin resistance in obesity.
In conclusion, we have provided evidence that adipocytes are able to respond to gram positive bacteria component in a TLR2 dependent manner and the response to TLR2 stimulation appears to perpetuate a chronic immune response. Since TLR2 expression is induced in obesity and type 2 diabetes, TLR2 may play a prominent role in the initiation of obesity induced inflammation. The use of TLR2 knockout animal models will provide further insight into this mechanism. An understanding of the role of TLR2 and its interactions with other receptors in the adipocyte may yield significant insights into the regulation of inflammation in obesity and may help in the search for new therapeutic targets against obesity-induced impairment of insulin signaling.