Periodic Reporting for period 4 - FAT NKT (Targeting iNKT cell and adipocyte crosstalk for control of metabolism and body weight)
Período documentado: 2021-03-01 hasta 2022-08-31
Obesity is one of the greatest global issues we face today. The biggest health and economic burden of obesity is obesity-asscoaited diseases. Obesity is far more complex than previously appreciated, and therapies for obesity are limited by an incomplete understanding of how body weight is controlled. In the last 20 years, it has become clear that the immune system is involved in regulating body weight and metabolism, and the adipose tissue has its own immune system. In obesity, inflammation occurs in adipose tissue and interferes with insulin signaling causing metabolic disease and worsening obesity. Inflammation also underlies other obesity-related diseases including liver disease, atherosclerosis, and certain cancers. It is therefore crucial that we gain greater understanding of the interactions between the immune and metabolic systems. A picture has emerged in which a battle ensues between inflammatory cells and regulatory protective immune cells including invariant natural killer T (iNKT) cells and T regulatory cells (Tregs) that can reverse these inflammation and correct the metabolic disorder. Thus, the involvement of the immune system in metabolism and weight control represents an entirely new therapeutic direction for treating obesity and type 2 diabetes. However, in order to target immunometabolic pathways in obesity and diabetes, it is critical that we understand the key players and molecules that regulate them. Less is known about iNKT cells than other T cells but iNKT cells are the innate lipid-sensing arm of the immune system. This means that unlike adaptive T cells that recognize peptide through MHC molecules, iNKT cells recognize lipid antigens presented by CD1d. This project is built on the central hypothesis that resident adipose iNKT cells play a critical role in keeping local inflammation at bay, and protecting against metabolic disorder in obesity. The ability to activate adipose iNKT cells will provide entirely new therapeutic directions for treating obesity and diabetes.
Objective 1:
iNKT cells are activated by lipid antigens presented by CD1d molecules on antigen presenting cells (APCs), and this lipid-CD1d complex is recognized by the invariant TCR. Surprisingly adipose tissue expresses CD1d but the significance of this is yet to be discovered. We investigated what happened if only adipocytes didn’t express CD1d (Adipo1-CD1d-/-). Adipo1-CD1d-/- were significantly heavier than littermate controls. We found that iNKT cells in adipose tissue are uniquely anti-inflammatory and regulatory, unlike iNKT cells elsewhere, and that the transcription factor E4BP4 controls their regulatory IL-10 production. Adipose iNKT cells also express Nur77, indicative of chronic stimulation through their T cell receptor. In the absence of CD1d on adipocytes, iNKT cells expressed less Nur77, although adipocyte expression of CD1d is not required for iNKT cell recruitment or survival in adipose tissue but is important for their Nur77 expression. Macrophage specific CD1d ko mice and it did not effect Nur77 expression.
Objective 2: What drives adipose iNKT cell phenotype
While one recent study demonstrated that mutation of the iNKT TCR resulted in a relative accumulation of adipose iNKT cells (Vieth et al., 2016), single-cell RNA sequencing of the mouse thymus did not identify an
E4BP4-positive, IL-10 producing iNKT cell population (Engel et al., 2016). Therefore, we hypothesized that the regulatory phenotype of adipose iNKT cells is instructed signals in the adipose microenvironment. In line with
this hypothesis, the project has evolved to answer the following two questions:
1) What factor(s) and pathway(s) drive E4BP4 expression and IL-10 production in adipose iNKT cells?
2) Is there functional heterogeneity within the adipose iNKT cell compartment?
Our current model is that iNKT cells arrive at the adipose tissue, encounter FFAs, and experience ER stress. This leads to the activation of the IRE1α/XBP1s arm of the UPR, the upregulation of E4BP4, and
consequentially the competence to produce IL-10. Within the adipose iNKT cell compartment there are at least two populations of iNKT cells, distinguished by the expression of NK1.1. NK1.1POS iNKT cells are cytotoxic,
produce IFNγ, and drive inflammation and metabolic dysfunction in adipose tissue. NK1.1NEG iNKT cells are anti-inflammatory, expand M2 macrophages, and promote glucose tolerance (Cell Metabolism, 2019).
Further, we discovered additional insight into iNKT cell biology. While the transcriptional profiles of naive, effector, and memory adaptive T cells have been well studied, less is known about the transcriptional regulation of different iNKT cell activation states. We used single-cell RNA-sequencing, to perform longitudinal profiling of activated iNKT cells in adipose tissue compared to spleen, generating a transcriptomic atlas of iNKT cell activation states. We found that transcriptional signatures of activation are highly conserved among heterogeneous iNKT cell populations, including NKT1, NKT2, and NKT17 subsets, and human iNKT cells. Strikingly, we found that regulatory iNKT cells, such as adipose iNKT cells, undergo blunted activation and display constitutive enrichment of memory-like cMAF+ and KLRG1+ populations. Moreover, we identify a conserved cMAF-associated transcriptional network among NKT10 cells, providing novel insights into the biology of regulatory and antigen-experienced iNKT cells. These regulatory adipose iNKt cells have all the hallmarks of antigen experiences iNKT cells suggesting they are in contact with lipid antigens presented in adipose tissue (ELife 2022)
Objective 3: Identify soluble ‘weight-loss/ thermogenic’ factor produced by adipose iNKT cells.
Adipose iNKT cells can regulate body weight, although the mechanism is not fully understood. In the absence of iNKT cells (CD1dko mice), obesity is more severe, and when iNKT cells are activated, mice lose weight through thermogenesis. Using ERC funded metabolic cages, we completed whole metabolic analysis of CD1dko mice in vivo. Despite increased obesity, CD1dko mice ate less and had the same activity as WT controls. However, CD1dko mice had a lower metabolic rate, highlighting that the obese diabetic phenotype was due to decreased energy expenditure. ne major goal was to publish the data on iNKT cell induced weight loss. We identified that iNKT cells induced weight loss through browning of white fat, which turned on b-oxidation and generated heat. We identified FGF21 as a critical step in this process and found that in the absence of FGF21, iNKT cell induced weight loss was significantly less (Cell Metabolism 2016, and selected for BioCentury Innovations magazine).
iNKT cells are activated by lipid antigens presented by CD1d molecules on antigen presenting cells (APCs), and this lipid-CD1d complex is recognized by the invariant TCR. Surprisingly adipose tissue expresses CD1d but the significance of this is yet to be discovered. We investigated what happened if only adipocytes didn’t express CD1d (Adipo1-CD1d-/-). Adipo1-CD1d-/- were significantly heavier than littermate controls. We found that iNKT cells in adipose tissue are uniquely anti-inflammatory and regulatory, unlike iNKT cells elsewhere, and that the transcription factor E4BP4 controls their regulatory IL-10 production. Adipose iNKT cells also express Nur77, indicative of chronic stimulation through their T cell receptor. In the absence of CD1d on adipocytes, iNKT cells expressed less Nur77, although adipocyte expression of CD1d is not required for iNKT cell recruitment or survival in adipose tissue but is important for their Nur77 expression. Macrophage specific CD1d ko mice and it did not effect Nur77 expression.
Objective 2: What drives adipose iNKT cell phenotype
While one recent study demonstrated that mutation of the iNKT TCR resulted in a relative accumulation of adipose iNKT cells (Vieth et al., 2016), single-cell RNA sequencing of the mouse thymus did not identify an
E4BP4-positive, IL-10 producing iNKT cell population (Engel et al., 2016). Therefore, we hypothesized that the regulatory phenotype of adipose iNKT cells is instructed signals in the adipose microenvironment. In line with
this hypothesis, the project has evolved to answer the following two questions:
1) What factor(s) and pathway(s) drive E4BP4 expression and IL-10 production in adipose iNKT cells?
2) Is there functional heterogeneity within the adipose iNKT cell compartment?
Our current model is that iNKT cells arrive at the adipose tissue, encounter FFAs, and experience ER stress. This leads to the activation of the IRE1α/XBP1s arm of the UPR, the upregulation of E4BP4, and
consequentially the competence to produce IL-10. Within the adipose iNKT cell compartment there are at least two populations of iNKT cells, distinguished by the expression of NK1.1. NK1.1POS iNKT cells are cytotoxic,
produce IFNγ, and drive inflammation and metabolic dysfunction in adipose tissue. NK1.1NEG iNKT cells are anti-inflammatory, expand M2 macrophages, and promote glucose tolerance (Cell Metabolism, 2019).
Further, we discovered additional insight into iNKT cell biology. While the transcriptional profiles of naive, effector, and memory adaptive T cells have been well studied, less is known about the transcriptional regulation of different iNKT cell activation states. We used single-cell RNA-sequencing, to perform longitudinal profiling of activated iNKT cells in adipose tissue compared to spleen, generating a transcriptomic atlas of iNKT cell activation states. We found that transcriptional signatures of activation are highly conserved among heterogeneous iNKT cell populations, including NKT1, NKT2, and NKT17 subsets, and human iNKT cells. Strikingly, we found that regulatory iNKT cells, such as adipose iNKT cells, undergo blunted activation and display constitutive enrichment of memory-like cMAF+ and KLRG1+ populations. Moreover, we identify a conserved cMAF-associated transcriptional network among NKT10 cells, providing novel insights into the biology of regulatory and antigen-experienced iNKT cells. These regulatory adipose iNKt cells have all the hallmarks of antigen experiences iNKT cells suggesting they are in contact with lipid antigens presented in adipose tissue (ELife 2022)
Objective 3: Identify soluble ‘weight-loss/ thermogenic’ factor produced by adipose iNKT cells.
Adipose iNKT cells can regulate body weight, although the mechanism is not fully understood. In the absence of iNKT cells (CD1dko mice), obesity is more severe, and when iNKT cells are activated, mice lose weight through thermogenesis. Using ERC funded metabolic cages, we completed whole metabolic analysis of CD1dko mice in vivo. Despite increased obesity, CD1dko mice ate less and had the same activity as WT controls. However, CD1dko mice had a lower metabolic rate, highlighting that the obese diabetic phenotype was due to decreased energy expenditure. ne major goal was to publish the data on iNKT cell induced weight loss. We identified that iNKT cells induced weight loss through browning of white fat, which turned on b-oxidation and generated heat. We identified FGF21 as a critical step in this process and found that in the absence of FGF21, iNKT cell induced weight loss was significantly less (Cell Metabolism 2016, and selected for BioCentury Innovations magazine).
We established the first metabolic core in Ireland. We performed state of the art single cell sequencing on adipose iNKT cells for the first time. We have also performed this in response to these perturbations, which we predict will highlight key roles of adipose resident iNKT cells in different physiological but non pathological settings.