The primary findings of the current study are that global TLR4 deficiency reduces AT inflammation concomitant with a shift in ATM polarization toward an alternatively activated state. To our knowledge, this is the first direct experimental evidence for a role of TLR4 in ATM polarization, which is further supported by the finding that hematopoietic cell TLR4 deficiency is sufficient to elicit the alternative activation of ATMs irrespective of recipient genotype.
Despite using a dietary strategy aimed at maximizing our ability to detect TLR4-dependent effects of dietary SFA content, we failed to observe a robust difference in the metabolic or inflammatory response of TLR4−/−
mice to the HFMUFA
diets. Results of previous studies suggesting that SFAs directly activate TLR4 (4
) have been questioned. A recent report by Erridge and Samani (27
) demonstrates that lipopolysaccharide and lipopeptide contamination of BSA accounts for the TLR4 activation commonly attributed to SFA treatment. We recognize that the current study does not directly address this issue; however, our findings do not generally support the notion that dietary SFAs directly induce TLR4 signaling. Irrespective of whether SFAs act as a TLR4 ligand, several lines of evidence suggest a much more complex and multifaceted immunomodulatory role for dietary fatty acids, such as inducing a shift to gram-negative intestinal microbiota and increasing gut permeability (28
The finding of reduced weight gain in TLR4−/−
mice after HF feeding is in line with some (5
)—but not all—previous investigations (6
). Careful examination of the available literature highlights the difficulty in drawing direct comparisons between previous studies because important differences exist with respect to 1
) sex and age of the mice used; 2
) percentage of dietary fat (ranging from 42–60% kcal from fat); 3
) carbohydrate source (e.g., sucrose or corn starch); 4
) TLR4-deficient model (i.e., C57BL/10ScN, C3H/HeJ, or TLR4−/−
mice on a BL/6 or BL/10 background); and 5
) study duration. Unfortunately, no discernible patterns exist that might explain the various weight gain results. Taken in context with the current literature, our study suggests that any protection TLR4 deficiency may confer with respect to weight gain is likely very modest and of questionable physiologic relevance.
Our results are in agreement with the general consensus of previous studies demonstrating a reduction in hepatic lipid accumulation in TLR4−/−
mice after HF feeding (5
). Perhaps surprising was the lack of effect noted in our BMT model with respect to the influence of parenchymal or hematopoietic cell TLR4 deficiency on hepatic steatosis. Whereas we observed similar levels of hepatic TG accumulation in WT and TLR4−/−
recipients, regardless of hematopoietic cell TLR4 expression, Saberi et al. (32
) recently reported a dramatic reduction in hepatic TG concentrations after HF feeding in WT mice reconstituted with TLR4−/−
BM. The reasons for this inconsistency are unclear but likely include differences in diet and the duration of HF feeding.
Similar to previous investigations (10
), we found that global as well as hematopoietic cell TLR4 deficiency produces modest reductions in AT inflammation despite comparable increases in ATM accumulation after HF feeding, suggesting that the absence of TLR4 signaling may influence ATM phenotype. Support for this possibility comes from the finding that the expression of various markers of M2 macrophage polarization are elevated in the AT of TLR4−/−
mice. Likewise, the proportion of M2 ATMs (i.e., F4/80+
) is significantly greater in HFSFA
mice than in WT counterparts. Interestingly, our BMT model points to a direct influence of TLR4 signaling on ATM polarization, as shown by the elevation of M2 marker expression in the SVF of mice reconstituted with TLR4−/−
BM, regardless of recipient genotype (i.e., WTTLR4−/−BM
). Additional support for a direct role of TLR4 in macrophage polarization comes from the finding that TLR4−/−
PMs are skewed toward an alternatively activated expression profile. In addition to the potential for TLR4 deficiency to directly modulate ATM polarization, the possibility that the alternative activation observed in TLR4-deficient ATMs may actually represent a relative resistance to the shift in ATM polarization toward an M1 phenotype in response to elevated endotoxin concentrations during HF feeding should not be discounted.
Despite displaying reductions in body weight, hepatic steatosis, and AT inflammation, we failed to detect any beneficial impact of TLR4 deficiency on systemic IR. Although previous studies have reported a lack of TLR4 signaling (via knockout or loss-of-function mutation) attenuates lipid infusion–induced IR (5
), the effect of TLR4 deficiency on insulin sensitivity in the setting of DIO remains unclear. Regarding these inconsistencies, it is interesting to note that those studies reporting an attenuation of IR after an HFD used diets providing 55–60% kcal from fat (7
), whereas studies (including the present) using diets consisting of 42–45% kcal from fat consistently fail to show an effect of TLR4 deficiency on IR (5
). This raises the possibility that our ability to detect the influence of TLR4 on systemic insulin sensitivity was compromised by the use of 45% fat diets.
Similar to models of global TLR4 deficiency, the role of hematopoietic cell TLR4 signaling in HFD-induced IR is uncertain. Olefsky and colleagues (32
) recently reported that hematopoietic cell TLR4 deficiency, as well as lentiviral mediated knockdown of TLR4 in hematopoietic cells, attenuates IR after HF feeding. In contrast, the current study and a previous report by our laboratory (13
) both failed to detect an improvement in systemic insulin sensitivity in mice lacking hematopoietic TLR4 signaling. Notably, TLR4−/−
recipients (i.e., TLR4−/−WTBM
) were significantly more glucose- and insulin-intolerant than WT recipients (i.e., WTWTBM
), regardless of hematopoietic cell TLR4 expression. Because previous studies have only used WT recipients, we are unable to reconcile this unexpected finding. Nonetheless, this does not impact our finding that hematopoietic cell TLR4 deficiency does not influence glucose or insulin tolerance.
In summary, the current study demonstrates that TLR4 deficiency promotes the alternative activation of ATMs. Furthermore, the alternative activation of ATMs in hematopoietic cell TLR4–deficient chimeras suggests that TLR4 signaling plays a direct role in mediating ATM phenotype in DIO. Lastly, it should be emphasized that, despite the observed influence on ATM phenotype and AT inflammation, in our hands, global and hematopoietic TLR4 deficiency produces a modest phenotype that does not manifest in improved systemic glucose homeostasis.