Adipose tissue & Immunity: The basics 2

Lymphoid structures in adipose tissue

Leukocytes present in the omentum are organized in clusters called "milky spots" (MS), which composition varies between species and is determined by antigenic exposure. The omentum has immunological and wound-healing properties, probably due to its angiogenic potential, large surface area and immunological activity (1). MS are composed primarily of macrophages and B1 cells. B1 cells are a subset of B cells that are different from conventional B cells (B2) in that they express different markers and antigen receptors that can bind common bacterial epitopes. These cells are also able to produce natural antibodies that provide a first protection to bacterial infections (2). The importance of MS as secondary lymphoid organs was highlighted by the work of Rangel-Moreno, et al. (3). They showed that SLP mice (spleen lymph node and Peyer's patch deficient) are able to generate B-cell responses to peritoneal antigens and present MS, which develop in the absence of LTi (lymphoid tissue inducer) cells. The local role for MS seems to be presenting  antigens derived from the peritoneal cavity to recirculating T and B lymphocytes, which enter MS from the blood. MS could also support somatic hypermutation and affinity maturation of B cells, and proliferation of T cells. Lymphocytes activated elsewhere were also found in MS, supporting the view of MS as an important part of the immune system. 

In the human mesentery, there is the presence of lymphoid clusters similar to MS called FALCs (fat-associated lymphoid clusters), in that both contain lymphocytes framed by adipose tissue in the peritoneal cavity. However, FALCs contain a great number (20-40% of total lymphocytes) of Lin- c-Kit+ Sca-1+ cells (4)*, which also express ILR7a. This suggests a progenitor potential. Moreover, these cells are capable of producing large amounts of IL2, IL4, IL5, IL6, GM-CSF and a moderate amount of IFN-gamma. Because of its properties (Th2-type lymphocytes with innate like characteristics), these FALC cells were termed as "natural helper cells" by the authors.

B cells

B1 cells, as has been mentioned, provides a first-line defense against bacterial and some viral infections. They have a less-diverse antibody repertoire, but respond to pathogen-associated molecules more rapidly than B2 cells.  B1 cells have been related to some metabolic diseases, due to the fact that they are responsive to LPS and cytokines (5). Importantly, these cells also produce IL-10 (6), which can regulate the activity of regulatory B cells (Bregs) (7). The implications of obesity in the function of B1 cells has yet to be discovered, but given that endotoxemia (8) and increased pro-inflammatory cytokine secretion are associated with obesity, overactivation of B1 cells could lead to the production of autoantibodies, promoting autoimmunity (9). Autoantibody production by B1 cells can also be promoted by endocrine disruptors (10). Finally, in contrast to their interaction with Bregs, B1 cells are unable to promote the conversion of naive Foxp3-CD4+ T cells into Foxp3+Treg cells (T-regulatory cells) compared to B2 cells, and promote Th1 and Th17 cell differentiation (see below) (11). 

The role of B2 cells in the pathogenesis of obesity has not been studied much, although there is an increase in total B cell count in adipose tissue from mice fed a high fat diet (HFD) (12). B cells might have a role mediating glucose intolerance in the obese, probably through IgG (12). Of note, some authors have suggested that B2 cells are atherogenic, while B1 cells are atheroprotective because BAFF-R deficient ApoE -/- mice (which are B2 depleted) show reduced inflammation and atherosclerosis (13, 14). 

T-cells

Infiltration of T lymphocytes to adipose tissue occurs rapidly in mice fed a HFD and correlates with the impairment in insulin sensitivity and glucose metabolism (15). Inducing obesity in mice produces an increase in CD8+ T cell levels in adipose tissue (epididymal fat), concomitantly with a reduction in CD4+ and Treg levels; accumulation which precedes macrophage infiltration (16). Furthermore, in vitro experiments have demonstrated that obese epididymal fat induces T cell proliferation and co-culturing obese adipose tissue with CD8+ T cells and monocytes induces the differentiation of macrophages to an inflammatory phenotype (16). Wu, et al. (17) found that obesity increases the level of regulated on activation, normal T cell expressed and secreted (RANTES) and CCR5 (one of RANTES receptors) in adipose tissue. This increases the migration of T-cells, as RANTES is chemotactic for these cells. The number of adipose tissue lymphocytes, as well as the expression of CCL20, increases with the degree of adiposity (18). Demonstrating a tight relationship between inflammation in adipose tissue and metabolic dysregulation, conditioned media from human adipose tissue lymphocytes is able to inhibit insulin-mediated FAS and LPL upregulation in mature adipocytes and to downregulate PIK3R1, effects probably mediated by IFN-gamma (18). In an elegant study, Winer, et al. (19) found that inducing obesity in mice produced the following changes in T-cell subsets in adipose tissue:

• IFN-gamma secreting Th1 lymphocytes were higher in visceral adipose tissue (VAT) compared to subcutaneous adipose tissue (SAT).
• The proportion of CD4+Foxp3+ T cells (Tregs) was 70% lower in VAT from obese mice, which produced an increase in the Th1:Treg ratio from 1.5:1 in lean mice to 6.5:1. 
• In absolute numbers, almost three times more Th1 cells accumulated per gram of fat in obese mice.
• In VAT from obese humans, the ratio of Th1:Treg was 12:1, compared to 6:1 in lean subjects. 
• T-cell expansion in VAT seems to be antigen-driven, given that OVA-specific T cells in OT2 TCR transgenic mice (20) showed secondary TCR-alpha rearrangements (thus, suggesting a positive selection towards an antigen present in VAT). These T-cells undergoing secondary TCR-alpha rearrangements in VAT showed a very narrow TCR-alpha diversity, implying a strong positive selection. Moreover, a negative pressure against selection of most of systemic TCR V-alpha (variable region in the alpha chain) was seen, given to the loss of some of these receptors**. 
• To characterize the role of T-cells in obesity, the authors used Rag1-null mice, which lack lymphocytes. These mice, when fed a HFD, gain more weight and visceral fat than HFD fed wild type mice. These mice also developed glucose intolerance, showing hyperglycemia, hyperinsulinemia and low insulin sensitivity. These mice also displayed a significant elevation of blood glucose when fed a normal diet, suggesting a physiological role of lymphocytes on metabolism.
• Reconstitution of these mice with CD4+ (but not CD8+) improved the metabolic abnormalities. It also induced Treg repopulation in VAT, albeit slowly. The effect of CD4+ cells was not mediated through differentiation into Tregs, nor by increasing IL-10 levels but by differentiation into Th2 cells. 
• Antibody treatment with anti-CD3 or F(ab')2 increased Treg levels in VAT and improved metabolic abnormalities. Additionally, F(ab')2 treatment increased the numbers of macrophage mannose receptor (MMR) positive cells (alternatively activated macrophages), increasing to almost 300% the production of IL-10.

The role of other inflammatory type of T-cell, Th17 cells, has been associated with obesity and metabolic complications. Diet-induced obese mice show an increased proliferation and differentiation towards Th17 cells, via an IL-6 dependent pathway (21). This increase in Th17 potentiated the severity of EAE and experimental colitis, reflecting the relationship between obesity and autoimmune risk. 

T-regulatory cells (Treg)

Treg cells have gotten much attention because they can regulate almost all immune responses. Further from being equal, different Treg populations display gene-expression differences (22), and subphenotype differences in Treg cells from different organs suggest that these tissue-specific Tregs modulate the immune response in a given organ. Of relevance to the topic, adipose-tissue-resident Tregs have been characterized (23). These fat Tregs had a different pattern of gene expression than their counterparts from other lymphoid organs: they over-represented genes coding for molecules involved in leukocyte migration and extravasation (CCR1, CCR2, CCR9, CXCL10, etc) and under-represented CCL5 and CXCR3. Fat Treg cells also seem to produce (and respond to, judging by expression of genes downstream of the IL-10 receptor) large amounts of IL-10, 136-fold higher than lymph node Tregs. The TCR repertoire of fat Treg cells was markedly different than that of adipose tissue T-cells and from other organs, suggesting that it is unlikely that these cells were the result of conversion of local T-cells. Finally, inducing T-cell conversion into Treg cells seem to ameliorate some metabolic abnormalities in rodent models (23). 

Adipose tissue effects on adaptive immune cells. Increases in adipose tissue mass  promotes changes in adaptive immune cell subtypes, such as T and B cells. The production of pro-inflammatory and chemotactic molecules (RANTES, IL-6, TNF-alpha, IFN-gamma, CCR1, CCR2, etc.) induces Th1 and Th17 cell polarization, as well as secretion of IgG by B-cells. 

Metabolic and immune integration

The function, differentiation and proliferation of several immune cell types is influenced by molecules that also play a key role in regulating metabolic homeostasis. Undoubtedly, leptin has gotten the most interest in recent years. Leptin is a master modulator of T-cell functioning: it promotes proliferation of peripheral blood lymphocytes and CD4+ T cells and induces the secretion of Th1 cytokines, such as IFN-gamma, while supressing the production of Th2 cytokines (24, 25). Leptin also modulates apoptosis of Th1 cells, promoting their survival and it is necessary for a Th2 response in vivo (26, 27). How does leptin modulates polarization and proliferation of lymphocytes? Compared to wild type mice and reconstituted ob/ob mice, autoreactive CD4+ T-cells from leptin-deficient mice have lower levels of Bcl-2 and phospho-ERK1/2, correlated with a reduction in the degradation of p27Kip1, a cell cycle inhibitor (28). Treatment with rapamycin mimics the effects of leptin deficiency (28), suggesting that leptin acts through the mTOR pathway. The effects of leptin in cells from the innate and adaptive immune system are shown in the following table (adapted from Procaccini, et al., 2012):

Cell type	Effect
Monocytes/macrophages	↑Phagocytosis ↑TNF-α, IL-6, IL-12 ↑Activation markers ↑Chemotaxis
Dendritic cells (DC)	↑TGFβ ↑Th1 priming ↑Survival ↑Immature DC migration ↑Stimulation of allogenic T cells
Neutrophils	↑Chemotaxis ↑ROS
NK cells	↑Activation marker ↑Cytotoxic activity ↑Perforin production
B cells	↑Lymphopoiesis and maturation ↑IL-6, IL-10, TNF-α ↑IgG2a
T cells	↑Activation markers ↑Proliferation ↑Th1 response ↑Adhesion molecules
Treg	↓Proliferation ↓Activity

mTOR

The mTOR pathway plays a very important role regulating immune function. Because of its role as a cellular energy sensor, it integrates metabolism and immunity, and provides a link for regulation of both by different molecules, such as leptin. 

Besides commonly known upstream activators of mTOR (for example, growth factors like insulin), immune-associated molecules are also able to induce mTOR activity. CD28, a T-cell costimulatory receptor, is a potent activator of mTOR through the activation of PI3K (29). Cytokines, such as IL-2, IL-4, IL-12, leptin, IFN-gamma and IL-1 are also able to activate mTOR (30). Conversely, PD-1 ligand 1 inhibits mTOR activity (31). Proteins normally thought to function only as metabolic sensors can also regulate mTOR activity, providing the first mechanism by which metabolism and immunity are related. For example, AMPK phoshporylates TSC, promoting the inhibition of mTOR (32, 33). The importance of AMPK for regulating the immune response is highlighted by the findings that loss of AMPK aggraviates EAE in mice (34). Aminoacids are potent activators of mTOR (35) and Treg induction from naïve T-cells is dependent on amino acid availability (36).

mTOR controls T-cell activation and differentiation 

mTOR plays a very important role integrating environmental cues for T-cell differentiation and activation. For a T-cell to become activated, two signals must be present. Signal 1 involves the recognition of antigens by TCR. On the other hand, Signal 2 has been defined as an species-specific accesory signal delivered by the stimulatory cell to the T-cell (37).  This means that for full T-cell activation, the two signals must be present. Signal 2, far from implicating only one type of interaction, is the result of the integration of multiple signals, which seem to be under the control of mTOR. For example, in Th1 cells, activation of mTOR is necessary for full T-cell activation and anergy reversal (37). Supporting the model of mTOR as Signal 2, experiments done on CD4+ T cells show that depending on the microenvironment (levels of specific cytokines), mTOR is necessary to differentiate T-cells into different subsets. Delgoffe, et al. (38) deleted mTOR specifically in T-cells. These mutant cells proliferated normally (albeit slower than wild type), but werent able to differentiate into Th1, Th2 or Th17 after skewing conditions. This correlated with decreased STAT activation (STAT4 for Th1, STAT6 for Th2, STAT3 for Th17) and lack of upregulation of T-bet, GATA-3 and ROR-gammat expression (specific transcription factors for Th1, Th2 and Th17 cells, respectively). Surprisingly, TCR engagement in the absence of mTOR induced the differentiation of Foxp3+ cells. 

mTOR also influences the activation of CD8+ cells. These cells are maintained in a quiescent state by several transcription factors, such as ELF4 and KLF4. mTOR controls CD8+ cell activation by inhibiting the expression of these proteins, reversing the quiescent state (39, 40). CD8+ cells lacking mTORC1 fail to become effector cells, and transition to memory cells seems to be also under control of mTOR (30). Besides controlling activation, the PI3K-mTOR axis regulates T-lymphocyte trafficking (41). 

mTOR effects on T-cell subsets. mTORC1 is activated by growth factors (insulin, IGF-1, EGF, etc.), aminoacids (BCAAs), co-stimulatory molecules (CD-28) and several cytokines. Upon activation, mTORC1 promotes Th1 differentiation of CD4+ T-cells by activating STAT4, which in turns promotes transcription of T-bet; and Th17 by activating STAT3, which increases the expression of ROR-gammat. This effect is mediated by inhibition of SOCS3. Additionally, mTORC1 leads to activation of CD8+ T-cells by inhibiting the expression of ELF4 and KLF4, which maintains CD8+T-cells in a quiescent state. Upstream activation of mTORC2 is poorly understood, but growth factors and, recently, ribosomes have shown to activate this complex. mTORC2 promotes CD4+T-cell differentiation into Th2 cells, by inhibiting SOCS5, enhancing STAT6 phosphorylation. This leads to increased expression of GATA-3 transcription factor. Activation of mTORC2 also inhibits FOXO proteins. Both complexes inhibit Foxp3, thereby reducing Treg differentiation.

mTOR promotes survival and maturation of B-cells

The direct effect of mTOR signaling in B-cell function has not been addressed. However, conditional deletion of TSC1 (mTORC1 inhibitor) in murine B-cells caused an impairment on B-cell maturation and response to T-cell dependent and independent antigens (42). Moreover, survival and proliferation of activated B-cells requires PI3K and mTOR activation (43).

mTOR controls dendritic cell (DC) activation/maturation

Treatment of DC with rapamycin (mTOR inhibitor) inhibits maturation by IL-4, GM-CSF and IL-1b (37). These DC are poor stimulators of T-cells but promote the induction of T-cell tolerance by Treg induction (44, 45). Rapamycin also produces a decrease in the expression of receptors involved in antigen presentation and uptake (46) and synthesis of some cytokines, like IL-18 (47). In LPS-stimulated DC, rapamycin enhances IL-12 production  and supresses IL-10 expression (48). This suggests that mTOR TLR-induced activation promotes the secretion of IL-10 while inhibiting the production of IL-12. Finally, mTOR is critical for monocyte-derived DC survival and immunostimulatory potential (49).

mTOR effects on immune cells is related to their metabolism

Every cell needs ATP for performing their specific functions. Unlike most differentiated cells (which utilize the citric acid cycle and mitochondrial respiration in the presence of oxygen), lymphocytes show a similar metabolic phenotype as cancer cells: they utilize oxidative glycolysis (Warburg effect) (30). In the resting state, lymphocytes are in a catabolic state, and molecules required for protein synthesis and energy are provided by autophagy. Upon activation, when energy requirements increase, there is an upregulation in protein, nucleotide and lipid biosynthesis. This anabolic switch is mediated by immunostimulatory molecules. For instance, CD28 activates PI3K, which activates Akt, which in turn promotes membrane translocation of glucose transporters (principally GLUT1 in lymphocytes). This results in increased glucose uptake and glycolysis, in excess of what is required for sustaining an adequate cellular level of ATP or macromolecular synthesis (50, 51). CTLA-4, an inhibitory receptor, inhibits CD28-induced upregulation of glucose metabolism (50). Activation of T-cells also leads to an increase in other glucose utilization pathways, such as the pentose phosphate pathway, which provides substrates for nucleic acid synthesis and NADPH (52). 

The importance of these metabolic changes on T-cell functioning is shown by experiments in which blocking the PI3K-Akt-mTOR pathway inhibit T-cell activation. AICAR, which activates AMPK (and thereby inhibiting mTORC1) inhibits IL-2 synthesis and promotes T-cell anergy (53). NALA, a leucine antagonist, and 2-deoxyglucose inhibit IL-2 and IFN-gamma synthesis, as well as proliferation of Th1 cells (54). This effect was observed even in the presence of co-stimulation. 

Treg cells display a different metabolic phenotype than effector CD4+ T cells. Whereas the latter exhibit a glycolytic dependent metabolism and high surface expression of GLUT1, Treg cells are dependent on lipid metabolism (55). Michalek et al. found that glucose deficiency inhibits Th17 polarization, without affecting Treg differentiation. Moreover, blocking CPT-I supressed Treg generation and the addition of exogenous fatty acids (oleate/palmitate mixture) inhibited the production of Th1, Th2 and Th17 cytokines (specifically, Th1 differentiation was reduced) but enhanced Treg expression of Foxp3. Finally, rapamycin and metformin increased lipid oxidation in CD4+ cells and Treg generation, while decreasing GLUT1 expression in CD4+ T-cells in a murine model of allergic asthma. 

Summary

• Obesity alterates the immune balance in all immune cell types, by increasing the secretion of several cytokines and chemotactic molecules. Specifically, obesity is associated with Th1 and Th17 skewing, CD8+ T-cell activation and proliferation and reduction in Treg cell numbers. It also promotes IgG production by B-cells, which seems to be important for the metabolic alterations observed.  
• Leptin, the most famous adipocytokine, is central to the relationship between immunity and metabolism. 
• Leptin effects are mediated by mTOR, which in turn, is the link between metabolism and immune function. mTOR is now being proposed as Signal 2, which is required for full T-cell activation.
• mTOR controls T-cell activation, proliferation and differentiation. Activation of mTOR promotes Th1, Th17 and Th2 differentiation of CD4+ T-cells and inhibits Treg generation. It also controls survival, activation and maturation of B-cells and dendritic cells. 
• Lymphocytes display a glycolytic metabolism, oxidizing glucose preferentially via aerobic glycolysis. Upon activation, mTOR and Akt increase the surface expression of GLUT1 and several enzymes involved in glucose uptake and oxidation. Conversely, Treg cells display a lipolytic metabolism, which is essential for their proliferation, differentiation and activity. 
• The function, differentiation and activity of the different subsets of lymphocytes is dependent on nutrient and energy availability. 

The results discussed above provide the basis for the potential of nutrition as a therapeutic tool in inflammatory and autoimmune conditions. In the next posts, I will expose my nutritional recommendations for treating these disorders, offering an up-to-date and detailed science-based nutritional immunotherapy.


* For flow cytometric analysis, specific cell surface markers are used for identifying distinct cell populations. Usually, a cell positive for a given marker is denoted by the marker and a plus sign (+), and a negative cell is written as the marker and a negative sign (-). So, for example, these cells present in FALCs express c-Kit and Sca-1, but not Lin markers.  
** For understanding the importance of these results, I strongly suggest some basic reading on TCR and V(D)J recombination. 

Addendum

In the first part of these series, I ommitted the evidence linking mast cells in the development of obesity and diabetes. I find utterly surprising that common mast cell stabilizers are able to improve these conditions. 

Powell JD, Pollizzi KN, Heikamp EB, & Horton MR (2011). Regulation of Immune Responses by mTOR. Annual review of immunology PMID: 22136167

Winer S, Chan Y, Paltser G, Truong D, Tsui H, Bahrami J, Dorfman R, Wang Y, Zielenski J, Mastronardi F, Maezawa Y, Drucker DJ, Engleman E, Winer D, & Dosch HM (2009).Normalization of obesity-associated insulin resistance through immunotherapy. Nature medicine, 15 (8), 921-9 PMID: 19633657

Procaccini C, Jirillo E, & Matarese G (2012). Leptin as an immunomodulator. Molecular aspects of medicine, 33 (1), 35-45 PMID: 22040697