Discussion
Despite the fact that CD40L has been shown to play a pivotal role in the pathogenesis of DIO, our findings indicate that T cell-specific CD40L is not a main driver of obesity-induced metabolic dysfunction. Previous studies report that total-body CD40L deficient mice subjected to DIO exhibit a strong decrease in adipose tissue inflammation, increased insulin sensitivity, and decreased hepatosteatosis.14 19 23 However, our data reveal that T cell CD40L deficiency only results in minor improvements in insulin sensitivity and hepatic inflammation. These results indicate that other CD40L expressing cell types, such as platelets or ECs, may play a more prominent role in obesity-associated adipose tissue inflammation and metabolic dysfunction than T cell CD40L.
Indeed, there are indications that other CD40L expressing cells play an important role in DIO. Recently, it was reported that ECs experience increased oxidative stress, as observed in db/db mice, as a consequence of increased CD40L expression during obesity.19 Additionally, EC oxidative stress caused by activated CD40L expressing platelets can enhance expression of endothelial-CD40L, while expression of soluble and cell surface chemoattractant CCL2 (MCP-1) was also increased.30 In accordance, data obtained from obese full-body CD40L deficient mice showed that adipose tissue exhibited decreased expression of CCL2, which was associated with a decrease in immune cell infiltration of the adipose tissue.14 19 23 As our study found no differences in CCL2 expression and adipose tissue inflammation, other CD40L expressing cells such as ECs, VSMCs, and platelets may mediate CD40L-driven immune cell infiltration in adipose tissue.
The role of platelet CD40L in DIO is emphasized by several clinical studies correlating sCD40L with insulin resistance. Subjects with a BMI >30 kg/m2 have increased plasma levels of sCD40L, which is mainly platelet derived.17–21 Increased plasma sCD40L is correlates with fasting glucose, insulin resistance and leukocyte counts in human cross-sectional studies.17–20 Furthermore, when patients with T2D were compared with age-matched controls, plasma sCD40L was significantly higher, independent of cholesterol, waist circumference, BMI or sex.17 This suggests that platelet CD40L may play a more important role in metabolic dysfunction than T cell CD40L. Combined, these data show the crucial roles of induction and maintenance of inflammation in DIO by CD40L expressing cells other than T cells.
In our mouse model, we found that 78% of T cell CD40L was depleted, which resulted in major changes in T cell subsets and their expression profiles. Although we cannot rule out residual T cell CD40L activity, the fact that T effector cell numbers have decreased substantially suggests that the remaining CD40L+ T cell population plays a minor role. Our results show that T cell CD40L is pivotal for the generation of CD4+ effector T cells and CD4+ Tregs, cell types that are considered major regulators of obesity-induced adipose tissue inflammation and insulin resistance.31 32 Adipose tissue T cell quantity and T cell subtype content correlates with BMI and waist circumference in patients with obesity and T2D. During obesity, the number of Th2 cells and anti-inflammatory Tregs decreases in adipose tissue and are replaced with a gross increase in pro-inflammatory Th1 cells.31 32 Correlation between increased Th1 cells and altered glucose metabolism in DIO was found especially for patients with T2D, whereas in patients with obesity this correlation is less evident.33 34 During DIO, the fraction of CD4+ effector T cells increases significantly, causing local inflammation by secretion of inflammatory cytokines in the adipose tissue. However, these pro-inflammatory effector T cells also migrate to other peripheral sites, such as the liver, further inducing inflammation.35 36 The increase in effector T cells is mediated by lymphocyte central memory cells. Central memory cells respond to inflammation and obesity-induced metabolic stress by proliferating and attaining an effector memory profile,37 contributing in part to the increase in T cells in adipose tissue during DIO.7 38 39 In our CD4CreCD40Lfl/fl mice, we observe a decrease in effector T cells in spleen and lymphoid organs, which is accompanied by a decrease in the expression of the pro-migratory chemokine receptor CXCR3. This suggests that absence of T cell CD40L affects migration of effector T cells into the periphery.40 However, in the adipose tissue the amount of effector T cells was unaffected when CD40L was absent on T cells. T lymphocyte central memory cells were also unaffected in the adipose tissue of CD4CreCD40Lfl/fl mice and these cells could thereby maintain the local effector memory populations. The decrease in CXCR3 expression does hamper effector T cells migratory ability, decreasing circulating effector cells and protecting peripheral organs such as the liver from these pro-inflammatory cells. As such, our data suggest that T cell CD40L deficiency does not limit the local expansion of the effector T cell, a culprit behind increased adipose tissue inflammation.
To maintain adipose tissue homeostasis Tregs are essential.31 32 These adipose tissue Tregs are predominantly induced Tregs (iTregs), which develop from naïve T cells, whereas natural Tregs (nTregs) develop in the thymus and largely depend on IL2 production.41 42 The CD4CreCD40Lfl/fl mice had decreased Treg numbers in blood, LNs, and spleen, but Treg numbers were maintained in the adipose tissue. This is probably due to the difference in development between the nTreg and iTreg. Blocking CD40L on lymphocytes leads to decreased IL2 production, as well as impaired de novo production of nTregs.29 The CD4CreCD40Lfl/fl mice seem to have a decrease in nTreg development due to T cell CD40L deficiency and reduction in IL2 expression online supplementary figure S1D). Adipose tissue iTregs are largely dependent on the B7 co-stimulatory pathway and peroxisome proliferator-activated receptor-γ (PPARγ).41 42 The B7 co-stimulatory molecules (CD80/CD86) on APCs play a critical role in iTreg differentiation, expansion, and function in adipose tissue, partly via induction of PPARγ in the differentiating naïve T cell.41 42 It is conceivable that these adipose tissue iTregs balance the local inflammation in our CD4CreCD40Lfl/fl mice. However, the amount of iTreg cells significantly decreases in DIO, and cross-sectional studies in humans have shown Treg decline to coincide with a decrease in B7 expression during obesity.9 41 This indicates the dependence of iTregs on the B7 proteins, rather than on CD40L, which is increased in DIO. We assume that in the T cell CD40L deficient mice, nTreg development was negatively affected, while iTreg development in adipose tissue was unaffected by CD40L deficiency, but iTreg cell numbers still decreased due to DIO.
Although the fraction of T cells seems to decrease in the CD4CreCD40Lfl/fl mice, we found an increase in the B cell fraction. On the contrary, total plasma IgG immunoglobulins were significantly decreased (online supplementary figure S7). This resembles findings from CD40L knock-out mice, where it was found that pro-inflammatory anti-oxidized lipid IgGs were reduced, correlating to an ameliorated metabolic phenotype in DIO.23 However, as we find limited changes in the metabolic syndrome of our T cell CD40L deficient mice, the increase in B cells and decrease in plasma IgGs do not seem to indicate biological relevance in our DIO model.
Apart from the shifts observed in immune cells, deficiency of T cell CD40L caused a more favorable metabolic and inflammatory profile in livers of obese mice, with a reduction in genes involved in lipid synthesis, a slight decrease in hepatosteatosis, a decrease in plasma insulin levels, and an altered immune cell composition. Gene expression levels of IL2 and IL12 were reduced in the liver, indicating reduced T cell proliferation.35 This was corroborated by a decrease in CD3+ expression, indicative of the decrease in effector T cells and Tregs. On the other hand, gene expression of pro-inflammatory IL6 was increased in the liver. However, data on the hepatocyte-specific deletion of the IL6 receptor in mice have shown to induce early onset insulin resistance and hepatosteatosis in DIO,43 demonstrating the protective function of IL6 in liver metabolism.
It seems that T cell CD40L orchestrates inflammation and metabolism via multiple mechanisms, although it does appear that the net result may be tissue dependent, of which the consequence seems to indicate a limited influence of T cell CD40L in diet-induced metabolic dysfunction on a whole. This reiterates the complicated role of the CD40-CD40L inflammatory dyad in DIO, where it has presented itself as a protective as well as detrimental mediator of obesity-associated metabolic syndromes.13–16 19 The most plausible explanation for the opposing phenotypes caused by CD40 and CD40L deficiency during DIO, is the presence of CD40 and CD40L on an abundance of cell types, all playing different roles in obesity. This has already become evident in previous studies. Although we and others found that total body CD40 deficiency was detrimental in DIO,13–16 we recently found that macrophage-specific CD40 deletion (LysMCreCD40fl/fl mice) had no effect on obesity-induced inflammation or metabolic dysregulation.24
Moreover, besides cell type specific effects, CD40 can also use different signaling routes via its TNF receptor-associated factors (TRAF) adaptor molecules to exert its effects. Obese mice deficient in CD40-TRAF2/3/5 signaling had aggravated adipose tissue inflammation and increased hepatosteatosis, whereas CD40-TRAF6 deficient obese mice had a decrease in adipose tissue inflammation and hepatosteatosis.13 Similar disparate effects are seen for the T cell co-stimulatory molecules CD137-CD137L, which are also expressed on an abundance of different cell types. Both inhibition of CD137 as well as antibody stimulation of CD137 gave similar results in DIO.44 45 These, sometimes contradicting results demonstrate the disparate effects co-stimulatory molecules may have in DIO, while highlighting the importance of understanding the effects of the co-stimulatory molecules on different cell types.
To conclude, T cell-specific CD40L is essential for effector T cell and Treg development, which are both important mediators in regulating obesity-associated inflammation. However, due to the opposing nature of these cells, T cell CD40L has limited effects on diet-induced metabolic dysregulation. Overall, T cell-specific CD40L deficiency only resulted in a slight improvement in insulin resistance, hepatic lipid uptake and hepatic inflammation, and therefore does not effectively reduce metabolic dysfunction and inflammation in obesity. As blocking CD40L in general constitutes a potent therapeutic strategy to reduce obesity-induced inflammation and its associated metabolic dysfunction,14 further research into the function of other CD40L expressing cell types, including the endothelial cell and the platelet, in DIO is warranted.