ATP Adenosiediphosphate AUC Area Under the Curve BAT Brown Adipose Tissue BCR B Sel Reseptor BMI Body Mass Index. UMAP Uniform Manifold Approximation and Projection VANTAGE VANderbilt Technologies for Advanced GEnomics BTW Visceral Adipose Tissue.
The obesity crisis
Weight cycling: The yo-yo diet effect
Consequences of weight cycling
Diabetes: types 1 and 2
Type 1 Diabetes Mellitus
Type 2 Diabetes Mellitus
Systemic regulation of blood glucose
Insulin production and secretion
ATP-dependent potassium channels close in response to increasing ratio of ATP to adenosine diphosphate (ADP), causing the cell membrane to depolarize. Changes in membrane voltage cause the opening of voltage-gated calcium channels and subsequent Ca2+ influx into the cell.
Insulin delivery
Immunofluorescence imaging of insulin (green), glucagon (red) and somatostatin (blue) in mouse (A; left) and human (B; right) islets. Importantly, changes in insulin delivery are challenging to measure and are therefore rarely quantified during assessments of insulin resistance.
Insulin action
Therefore, changes in blood flow through capillaries, capillary permeability, and capillary structure can all disrupt insulin release at the site of insulin action [64–66]. Chronic hyperinsulinemia, as a result of compensatory increases in insulin secretion and decreases in insulin extraction, can also increase insulin receptor endocytosis, reducing surface insulin receptors and exacerbating insulin resistance [76].
Adipose tissue homeostasis
During obesity, systemic leptin levels are elevated (i.e. hyperleptinemia) and leptin resistance (i.e. impaired leptin responsiveness) is observed. In rodent models of diet-induced obesity, neurons in the arcuate nucleus of the hypothalamus, important for the regulation of feeding behavior, have reduced projections [92].
Innate immune cells
Both cDCs subsets found in AT are typically found in a relatively immature state expressing low levels of CD80 and CD86 [119]. In addition to the cells of the myeloid and granulocyte developmental pathways, a small population of innate lymphoid cells (ILCs) can be found in AT.
Adaptive immune cells
CD8+ T cells are present in lean AT but are much more abundant in AT during weight gain. Typically, CD8+ T cells are associated with cytolytic activity and are often referred to as cytotoxic T lymphocytes (CTLs).
Adipose tissue immune cells
Reproduced with permission from Villarroyaet al., 2018. IL-10, IL-13 and IL-33, respectively), preserve WAT function and engage transcriptional activation patterns for wound repair genes and repression of genes encoding inflammatory cytokines. Palmitate and other saturated fatty acids interact with many surface receptors, including toll-like receptors (TLRs) found on innate immune cells (eg TLR-4), inducing activation and production of cytokines in one loop forward [180. 181].
CD8 + T cells in systemic immunity
- TCR-dependent signal transduction in CD8 + T cells
- Clonal expansion
- Long-lived memory T cells
- Age-associated T cells
- T cell exhaustion
The linear model of development (A) suggests that memory T cells differentiate from residual effector T cells in the absence of further antigen stimulation. The differentiation potential model (B) suggests that memory T cells are formed from cells that exit the effector differentiation pathway early.
Adipose tissue as a T cell reservoire
Mouse models deficient in PD-1 develop immunopathology either due to an impaired T cell response against pathogens or dysregulated autoimmune activity of effector T cells [ 221 , 222 ]. Interestingly, while obese individuals with cancer have faster tumor progression and associated immune dysfunction, in some cases checkpoint blockade (eg, PD-1 immunotherapy) has improved efficacy and overall survival during treatment [225].
Summary
While this finding requires further experimentation, it raises an interesting question: Could AT act as a reservoir for T cells that contribute to disease pathology, partially explaining the increased risk of disease progression in many obesity-related comorbidities.
Animal models
Diet-induced obesity
Between weeks 7 and 8, mice were treated every other day (3 times) with 100 µg of anti-CD8 monoclonal antibody (BioLegend Cat#114120) or IgG-matched control antibody (BioLegend Cat#400566) by intraperitoneal delivery. Between weeks 8 and 12, mice were treated every other day (3 times) with 200 µg of anti-PD-1 monoclonal antibody (BioXCell Cat#BE0146) or IgG matched control (BioXCell Cat#BE0089) antibody by intraperitoneal delivery.
Immune cell isolation and flow cytometry
Tissue dissociation
Remaining cells were resuspended in 400 µL cold PBS with 1% FBS solution and passed through a 35 µm filter for downstream processing step. Remaining cells were resuspended in 1 ml of cold PBS with 1% FBS solution and passed through a 35 µm filter for downstream processing steps.
Flow cytometry
Cell sorting
For magnetic cell sorting, samples were prepared and a surface marker was immunolabeled with an antibody conjugated to magnetic beads.
Ex vivo cell stimulation
RNA isolation
RNA sequencing
Bulk RNA-seq
Poly-A selection for mRNA enrichment was performed using poly-A magnetic beads (New England Biolabs #E7490L) and cDNA library preparation was performed using the NEBNext Ultra kit (New England Biolabs #7760L). Quantification of sequences was performed using the Salmon version by aligning to the C57BL/6J GRCm39 reference assembly.
Single cell RNA-seq and CITE-seq
Differential expression was performed using the Wilcoxon Ranking-Sums test in Seurat V4 for single cells. Metaclusters identified by MiloR were further tested with permutation testing using the scProportionTest R package V0.0.9000 (https://github.com/rpolicastro/scProportionTest).
Single cell V(D)J sequencing
Sample code to reproduce our processed and integrated Seurat v4 object used in Chapter III is available at: https://github.com/HastyLab/Multiomics-WeightCycling-Vignettes. All the code needed to reproduce the figures for Chapter III is available on GitHub at: https://github.com/HastyLab/Multiomics- WeightCycling-Figures .
Immunoblotting
After incubation of primary antibodies, membranes were washed and probed with species-appropriate HRP-conjugated secondary antibodies in 5% skim milk powder for 2 h at room temperature. For quantification of phosphorylated proteins, band intensities were expressed as a ratio to the parent protein (e.g., pAKT to total AKT).
Tissue imaging
- Tissue preparation
- Histology
- Immunofluorescence imaging
- Electron Microscopy
Immunolabeling with primary antibody was performed overnight at 4°C and with secondary at room temperature for 2 hours. Segmentation of individual insulin granules was performed using semi-supervised machine learning segmentation tools and thresholding.
Glucose tolerance test
Briefly, mature insulin granules in random quadrants (between 256 and 512 pixels in XY) of EM micrographs of large islets were manually segmented using Labkit to create binary masks that could serve as ground truth (GT) images. Individual beads were approximated using a binary flow area and the “measure” function was used to determine the area of the insulin beads.
Mixed-meal tolerance test
Plasma Biochemistry
Islet perifusion
Hyperinsulinemic euglycemic clamp
Hyperglycemic clamp
Statistics
Introduction
Multiomics reveals the persistence of obesity-associated immune cell phenotypes in adipose tissue during weight loss and weight gain in mice as LAMs, which are unique from traditional macrophage polarization states. scRNA-seq enables the interpretation of changes in immune populations and unbiased gene expression across the entire immune landscape simultaneously. To date, no studies have thoroughly characterized AT immune populations after WC using a comprehensive technique such as scRNA-seq, and many published scRNA-seq datasets are difficult to further explore without bioinformatics expertise.
Results
- Diet induced WC exacerbates glucose intolerance in male mice
- Multimodal single-cell sequencing highlights the diversity of adi-
- Obesity-associated immune cell phenotypes are confirmed by sin-
- Obesity-associated T cell exhaustion persists after WL
- Monocytes are abundant in AT, but do not have an altered tran-
- DCs shift towards an activated transcriptional signature with obe-
- Macrophage populations are highly adaptable to change in dietary
- Alterations in macrophage phenotype remain unresolved with WL 62
However, WL rats had reduced eAT mass compared to lean rats, and WC rats had increased eAT mass compared to obese rats (Fig. 3.3a). TRMs were enriched for expression of Klf4, Cbr2, andStab1[276], while LAMs expressed many different genes associated with lipid interactions (Trem2,Cd9,Lpl) [116], and cycling macrophages expressed cycle genes cellular (Stmn1, Pclaf). 261] (Fig. 3.13c).
Discussion
In support of this, AT DCs switch to a mature, activated state with the onset of obesity and persist during WL and WC. However, expression of Cd163, another M2-like marker, is lost with the onset of obesity, and the loss continues during WL and WC.
Preface
Introduction
Obesity-induced acceleration of CD8+T cell senescence in adipose tissue that persists during WL and weight gain. In this chapter, CD8+ T cells in AT during WL and WC are examined using common surrogate surface markers and functional assessments of T cell cytokine production.
Results
- Effector memory T cells persist in AT after WL
- Depletion of CD8 + T cells during weight regain does not protect
- Anti-PD-1 immunotherapy does not reduce obesity-associated im-
- AT CD8 + T EM acquire an age-associated phenotype during obe-
- AT CD8 + T cells are clonally enriched during WL and WC
The number of CD8+T cells per gram of tissue was increased in obese and WC eAT compared to lean eAT and increased even further in WL eAT (Fig. 4.2a). The percentage of CD8+ T cells was increased in obese and WC eAT compared to lean eAT and even further increased in WL eAT.
Discussion
However, WC mice do not undergo T1D-associated β-cell destruction as we discuss in the next chapter. At least in the AT where we measured these clonal types, the clonal insulin-responsive T cells do not express GZMB or perforin.
Future Directions
Therefore, subsequent adoptive cell transfer studies are likely essential to determine whether AT is a specific reservoir for GZMK+ T cells in the context of obesity. It is already known that obese individuals have an improved response to immunotherapy in the context of many cancers.
Introduction
WC impairs pancreatic insulin secretion but does not interfere with whole-body insulin action in diet-induced obese mice. Instead, we report that WC-induced glucose intolerance is not explained by impaired whole-body insulin action, but rather is attributable to insufficient insulin secretion directly linked to loss of β-cell identity and adaptive plasticity.
Results
- WC-induced glucose intolerance is not attributable to impaired in-
- WC impairs in vivo glucose stimulated insulin secretion indepen-
- Insulin secretion is suppressed in WC islets
- Components of β -cell transcriptional identity are downregulated
Muscle (gastrocnemius, vastus lateralis and soleus) and eAT Rg, were reduced in WC mice (Fig. 5.1k). Multiple drivers of β-cell identity and function, Nkx6.1, Mafa, Pdx1, Ucn3 and Slc2a2, were also downregulated in WC islets compared to obese islets (Fig. 5.8a).
Discussion
However, few cells stained positive for Ki-67 within the islets, indicating that β-cell proliferation is low in obese and WC mice after 27 weeks of diet. Specifically, the decrease in transcript and protein levels of the β-cell transcription factors NKX6.1 and MAFA in WC mice indicates a loss of appropriate β-cell compensation normally seen only in obesity.
Progress in understanding obesity and T2D
As discussed in Chapter I and shown in Chapter III, obesity-associated changes in AT immune cell populations include both loss of immunoregulatory cells and enrichment for pro-inflammatory populations. Importantly, our lack of understanding of AT immune cells and mechanisms driving their recruitment, activation, and function likely limits our ability to effectively target obesity-associated inflammation.
WL and WC in basic research
Therefore, the potential for managing obesity-related inflammation to reduce the risk of comorbidities, such as T2D, is great.
Caveats of WL and WC mouse models
We have taken female mice through our WC protocol previously (Fig. 3.4) and observed a non-significant defect in glucose tolerance. During the second HFD period, when the mice are much older, female mice gain weight at a much greater rate.
WL, AT remodeling, and weight regain
Taken together, these data suggest that immune cells persisting in AT after WL are likely to contribute to the rate of weight gain and the metabolic consequences of WC. For example, it is unknown whether therapies that promote macrophage ovulation of AT will support weight maintenance and AT remodeling.
The complex role of T cells in AT
First, how do CD8+ T cells survive in the AT and does the AT act as a unique reservoir for these cells. We observed reduced Gzmb expression and decreased cytokine production, but are there different roles for these cells in AT inflammation (eg GZMK production).
WL and WC in the pancreatic islet
Furthermore, whether CD8+ T cells can migrate from the AT and infiltrate other tissues such as the pancreas or liver. For example, we identified T1D-associated TCRs and began to generate retrogenic models to determine whether TCR signaling during obesity drives these cells in the AT and pancreas.
Future Directions
For example, we still have little insight into the mechanisms that signal and promote the clearance of dying adipocytes by macrophages (i.e., in CLS) and what, if any, effects rapid lipid uptake has on macrophage phenotype. Furthermore, whether T cells can directly migrate from AT to tumors (eg, in breast cancer) remains unclear, but an expanded pool of PD-1+ T cells found in obese, WL, and WC AT may be therapeutically valuable be in the context of anti -PD-1 immunotherapy.
Summary
Kratz, M. et al. Metabolic dysfunction induces a mechanistically distinct proinflammatory phenotype in adipose tissue macrophages. Vatarescu, M. et al. Adipose tissue supports normalization of liver macrophage and lipid handling in obesity reversal.
Systemic regulation of blood glucose
Species differences in islet architecture
Electron microscopy of white and brown adipocytes
Adipose tissue depots in humans and mice
The fat mouse grows up
Crown-like structures
V(D)J recombination
Immune cells in lean and obese adipose tissue
Models of memory T cell differentiation
Mouse models of lean, obese, weight loss (WL) and weight cycling
Interim body mass and ipGTT for male mice
Tissue mass and adipocyte size for male mice
Weight cycling in female C57BL/6J mice
Validation of CITE-seq antibodies
Biological replicates identified by Hashtag demultiplexing are well
Adipose tissue immune cell populations observed by CITE-seq
CITE-seq recapitulates obesity-associated immune cell changes in
Obesity-induced changes in adipose tissue T cells are not reversed
Adipose tissue T cells are retained and express markers of exhaus-
Monocytes upregulate features of lipid handling during obesity, weight
Dendritic cells shift towards a mature, activated status in mice that
Diet-induced obesity causes persistent changes in adipose tissue macrophages,
Surface T cell markers are sensitive to collagenase digestion
AT CD4 + and CD8 + are enriched after WL
CD8 + T cells recover within 3 weeks of antibody-mediated CD8 + T
CD8 + T cell depletion prior to weight regain does not protect glucose
PD-1 expression in AT T cells during WL and WC
PD-1 neutralizing antibodies do not affect weight gain or protect
PD-1 neutralizing antibodies do not induce leukocyte infiltration
CD8 + T cells have impaired cytokine response to stimulation follow-
Lean adipose is enriched for clonal T regs while obese, weight loss,
T1D-associated T cell clonotypes found in WL and WC express Gzmk. 82
WC-induced glucose intolerance is not attributable to impaired in-
Obese and WC sAT and skeletal muscle have similar response to ex
Differences in diet composition does not alter body composition or
WC impairs in vivo glucose stimulated insulin secretion indepen-
WC decreases nutrient clearance in response to a mixed meal
Ex vivo insulin secretion is reduced in WC mice
WC islets are transcriptionally distinct from obese islets
Expression of β –cell transcription factors and glucose transporter 2
Insulin granule loading is impaired by WC
