Data-dependent and -independent acquisition lipidomics analysis reveals the tissue-dependent effect of metformin on lipid metabolism.

INTRODUCTION: Despite the well-recognized health benefits, the mechanisms and site of action of metformin remains elusive. Metformin-induced global lipidomic changes in plasma of animal models and human subjects have been reported. However, there is a lack of systemic evaluation of metformin-induced lipidomic changes in different tissues. Metformin uptake requires active transporters such as organic cation transporters (OCTs), and hence, it is anticipated that metformin actions are tissue-dependent. In this study, we aim to characterize metformin effects in non-diabetic male mice with a special focus on lipidomics analysis. The findings from this study will help us to better understand the cell-autonomous (direct actions in target cells) or non-cell-autonomous (indirect actions in target cells) mechanisms of metformin and provide insights into the development of more potent yet safe drugs targeting a particular organ instead of systemic metabolism for metabolic regulations without major side effects. OBJECTIVES: To characterize metformin-induced lipidomic alterations in different tissues of non-diabetic male mice and further identify lipids affected by metformin through cell-autonomous or systemic mechanisms based on the correlation between lipid alterations in tissues and the corresponding in-tissue metformin concentrations. METHODS: A dual extraction method involving 80% methanol followed by MTBE (methyl tert-butyl ether) extraction enables the analysis of free fatty acids, polar metabolites, and lipids. Extracts from tissues and plasma of male mice treated with or without metformin in drinking water for 12 days were analyzed using HILIC chromatography coupled to Q Exactive Plus mass spectrometer or reversed-phase liquid chromatography coupled to MS/MS scan workflow (hybrid mode) on LC-Orbitrap Exploris 480 mass spectrometer using biologically relevant lipids-containing inclusion list for data-independent acquisition (DIA), named as BRI-DIA workflow followed by data-dependent acquisition (DDA), to maximum the coverage of lipids and minimize the negative effect of stochasticity of precursor selection on experimental consistency and reproducibility. RESULTS: Lipidomics analysis of 6 mouse tissues and plasma allowed a systemic evaluation of lipidomic changes induced by metformin in different tissues. We observed that (1) the degrees of lipidomic changes induced by metformin treatment overly correlated with tissue concentrations of metformin; (2) the impact on lysophosphatidylcholine (lysoPC) and cardiolipins was positively correlated with tissue concentrations of metformin, while neutral lipids such as triglycerides did not correlate with the corresponding tissue metformin concentrations; (3) increase of intestinal tricarboxylic acid (TCA) cycle intermediates after metformin treatment. CONCLUSION: The data collected in this study from non-diabetic mice with 12-day metformin treatment suggest that the overall metabolic effect of metformin is positively correlated with tissue concentrations and the effect on individual lipid subclass is via both cell-autonomous mechanisms (cardiolipins and lysoPC) and non-cell-autonomous mechanisms (triglycerides).

Fructose regulates the pentose phosphate pathway and induces an inflammatory and resolution phenotype in Kupffer cells.

Over-consumption of fructose in adults and children has been linked to increased risk of non-alcoholic fatty liver disease (NAFLD). Recent studies have highlighted the effect of fructose on liver inflammation, fibrosis, and immune cell activation. However, little work summarizes the direct impact of fructose on macrophage infiltration, phenotype, and function within the liver. We demonstrate that chronic fructose diet decreased Kupffer cell populations while increasing transitioning monocytes. In addition, fructose increased fibrotic gene expression of collagen 1 alpha 1 (Col1a1) and tissue metallopeptidase inhibitor 1 (Timp1) as well as inflammatory gene expression of tumor necrosis factor alpha (Tnfa) and expression of transmembrane glycoprotein NMB (Gpnmb) in liver tissue compared to glucose and control diets. Single cell RNA sequencing (scRNAseq) revealed fructose elevated expression of matrix metallopeptidase 12 (Mmp12), interleukin 1 receptor antagonist (Il1rn), and radical S-adenosyl methionine domain (Rsad2) in liver and hepatic macrophages. In vitro studies using IMKC and J774.1 cells demonstrated decreased viability when exposed to fructose. Additionally, fructose increased Gpnmb, Tnfa, Mmp12, Il1rn, and Rsad2 in unpolarized IMKC. By mass spectrometry, C13 fructose tracing detected fructose metabolites in glycolysis and the pentose phosphate pathway (PPP). Inhibition of the PPP further increased fructose induced Il6, Gpnmb, Mmp12, Il1rn, and Rsad2 in nonpolarized IMKC. Taken together, fructose decreases cell viability while upregulating resolution and anti-inflammatory associated genes in Kupffer cells.

Data-dependent and -independent acquisition lipidomics analysis reveals the tissue-dependent effect of metformin on lipid metabolism.

Introduction: Despite the well-recognized health benefits, the mechanisms and site of action of metformin remains elusive. Metformin-induced global lipidomic changes in plasma of animal models and human subjects have been reported. However, there is a lack of systemic evaluation of metformin-induced lipidomic changes in different tissues. Metformin uptake requires active transporters such as organic cation transporters (OCTs), and hence, it is anticipated that metformin actions are tissue-dependent. In this study, we aim to characterize metformin effects in non-diabetic male mice with a special focus on lipidomics analysis. The findings from this study will help us to better understand the cell-autonomous (direct actions in target cells) or non-cell-autonomous (indirect actions in target cells) mechanisms of metformin and provide insights into the development of more potent yet safe drugs targeting a particular organ instead of systemic metabolism for metabolic regulations without major side effects. Objectives: To characterize metformin-induced lipidomic alterations in different tissues of non-diabetic male mice and further identify lipids affected by metformin through cell-autonomous or systemic mechanisms based on the correlation between lipid alterations in tissues and the corresponding in-tissue metformin concentrations. Methods: Lipids were extracted from tissues and plasma of male mice treated with or without metformin in drinking water for 12 days and analyzed using MS/MS scan workflow (hybrid mode) on LC-Orbitrap Exploris 480 mass spectrometer using biologically relevant lipids-containing inclusion list for data-independent acquisition (DIA), named as BRI-DIA workflow followed by data-dependent acquisition (DDA), to maximum the coverage of lipids and minimize the negative effect of stochasticity of precursor selection on experimental consistency and reproducibility. Results: Lipidomics analysis of 6 mouse tissues and plasma using MS/MS combining BRI-DIA and DDA allowed a systemic evaluation of lipidomic changes induced by metformin in different tissues. We observed that 1) the degrees of lipidomic changes induced by metformin treatment overly correlated with tissue concentrations of metformin; 2) the impact on lysophosphorylcholine and cardiolipins was positively correlated with tissue concentrations of metformin, while neutral lipids such as triglycerides did not correlate with the corresponding tissue metformin concentrations. Conclusion: The data collected in this study from non-diabetic mice with 12-day metformin treatment suggest that the overall metabolic effect of metformin is positively correlated with tissue concentrations and the effect on individual lipid subclass is via both cell-autonomous mechanisms (cardiolipins and lysoPC) and non-cell-autonomous mechanisms (triglycerides).

Myeloid cell MHC I expression drives CD8+ T cell activation in nonalcoholic steatohepatitis.

Background & aims: Activated CD8+ T cells are elevated in Nonalcoholic steatohepatitis (NASH) and are important for driving fibrosis and inflammation. Despite this, mechanisms of CD8+ T cell activation in NASH are largely limited. Specific CD8+ T cell subsets may become activated through metabolic signals or cytokines. However, studies in NASH have not evaluated the impact of antigen presentation or the involvement of specific antigens. Therefore, we determined if activated CD8+ T cells are dependent on MHC class I expression in NASH to regulate fibrosis and inflammation. Methods: We used H2Kb and H2Db deficient (MHC I KO), Kb transgenic mice, and myeloid cell Kb deficient mice (LysM Kb KO) to investigate how MHC class I impacts CD8+ T cell function and NASH. Flow cytometry, gene expression, and histology were used to examine hepatic inflammation and fibrosis. The hepatic class I immunopeptidome was evaluated by mass spectrometry. Results: In NASH, MHC class I isoform H2Kb was upregulated in myeloid cells. MHC I KO demonstrated protective effects against NASH-induced inflammation and fibrosis. Kb mice exhibited increased fibrosis in the absence of H2Db while LysM Kb KO mice showed protection against fibrosis but not inflammation. H2Kb restricted peptides identified a unique NASH peptide Ncf2 capable of CD8+ T cell activation in vitro. The Ncf2 peptide was not detected during fibrosis resolution. Conclusion: These results suggest that activated hepatic CD8+ T cells are dependent on myeloid cell MHC class I expression in diet induced NASH to promote inflammation and fibrosis. Additionally, our studies suggest a role of NADPH oxidase in the production of Ncf2 peptide generation.

Prioritize biologically relevant ions for data-independent acquisition (BRI-DIA) in LC-MS/MS-based lipidomics analysis.

INTRODUCTION: Data-dependent acquisition (DDA) is the most commonly used MS/MS scan method for lipidomics analysis on orbitrap-based instrument. However, MS instrument associated software decide the top N precursors for fragmentation, resulting in stochasticity of precursor selection and compromised consistency and reproducibility. We introduce a novel workflow using biologically relevant lipids to construct inclusion list for data-independent acquisition (DIA), named as BRI-DIA workflow. OBJECTIVES: To ensure consistent coverage of biologically relevant lipids in LC-MS/MS-based lipidomics analysis. METHODS: Biologically relevant ion list was constructed based on LIPID MAPS and lipidome atlas in MS-DIAL 4. Lipids were extracted from mouse tissues and used to assess different MS/MS scan workflow (DDA, BRI-DIA, and hybrid mode) on LC-Orbitrap Exploris 480 mass spectrometer. RESULTS: DDA resulted in more MS/MS events, but the total number of unique lipids identified by three methods (DDA, BRI-DIA, and hybrid MS/MS scan mode) is comparable (580 unique lipids across 44 lipid subclasses in mouse liver). Major cardiolipin molecular species were identified by data generated using BRI-DIA and hybrid methods and allowed calculation of cardiolipin compositions, while identification of the most abundant cardiolipin CL72:8 was missing in data generated using DDA method, leading to wrong calculation of cardiolipin composition. CONCLUSION: The method of using inclusion list comprised of biologically relevant lipids in DIA MS/MS scan is as efficient as traditional DDA method in profiling lipids, but offers better consistency of lipid identification, compared to DDA method. This study was performed using Orbitrap Exploris 480, and we will further evaluate this workflow on other platforms, and if verified by future work, this biologically relevant ion fragmentation workflow could be routinely used in many studies to improve MS/MS identification capacities.

Beneficial effects of eicosapentaenoic acid on the metabolic profile of obese female mice entails upregulation of HEPEs and increased abundance of enteric Akkermansia muciniphila.

Eicosapentaenoic acid (EPA) ethyl esters are of interest given their clinical approval for lowering circulating triglycerides and cardiometabolic disease risk. EPA ethyl esters prevent metabolic complications driven by a high fat diet in male mice; however, their impact on female mice is less studied. Herein, we first investigated how EPA influences the metabolic profile of female C57BL/6J mice consuming a high fat diet. EPA lowered murine fat mass accumulation, potentially through increased biosynthesis of 8-hydroxyeicosapentaenoic acid (HEPE), as revealed by mass spectrometry and cell culture studies. EPA also reversed the effects of a high fat diet on circulating levels of insulin, glucose, and select inflammatory/metabolic markers. Next, we studied if the improved metabolic profile of obese mice consuming EPA was associated with a reduction in the abundance of key gut Gram-negative bacteria that contribute toward impaired glucose metabolism. Using fecal 16S-ribosomal RNA gene sequencing, we found EPA restructured the gut microbiota in a time-dependent manner but did not lower the levels of key Gram-negative bacteria. Interestingly, EPA robustly increased the abundance of the Gram-negative Akkermansia muciniphila, which controls glucose homeostasis. Finally, predictive functional profiling of microbial communities revealed EPA-mediated reversal of high fat diet-associated changes in a wide range of genes related to pathways such as Th-17 cell differentiation and PI3K-Akt signaling. Collectively, these results show that EPA ethyl esters prevent some of the deleterious effects of a high fat diet in female mice, which may be mediated mechanistically through 8-HEPE and the upregulation of intestinal Akkermansia muciniphila.

Vitamin E does not prevent Western diet-induced NASH progression and increases metabolic flux dysregulation in mice.

Fatty liver involves ectopic lipid accumulation and dysregulated hepatic oxidative metabolism, which can progress to a state of elevated inflammation and fibrosis referred to as nonalcoholic steatohepatitis (NASH). The factors that control progression from simple steatosis to NASH are not fully known. Here, we tested the hypothesis that dietary vitamin E (VitE) supplementation would prevent NASH progression and associated metabolic alterations induced by a Western diet (WD). Hyperphagic melanocortin-4 receptor-deficient (MC4R-/-) mice were fed chow, chow+VitE, WD, or WD+VitE starting at 8 or 20 weeks of age. All groups exhibited extensive hepatic steatosis by the end of the study (28 weeks of age). WD feeding exacerbated liver disease severity without inducing proportional changes in liver triglycerides. Eight weeks of WD accelerated liver pyruvate cycling, and 20 weeks of WD extensively upregulated liver glucose and oxidative metabolism assessed by 2H/13C flux analysis. VitE supplementation failed to reduce the histological features of NASH. Rather, WD+VitE increased the abundance and saturation of liver ceramides and accelerated metabolic flux dysregulation compared with 8 weeks of WD alone. In summary, VitE did not limit NASH pathogenesis in genetically obese mice, but instead increased some indicators of metabolic dysfunction.

Lipid Droplet Accumulation in Human Pancreatic Islets Is Dependent On Both Donor Age and Health.

Human but not mouse islets transplanted into immunodeficient NSG mice effectively accumulate lipid droplets (LDs). Because chronic lipid exposure is associated with islet ß-cell dysfunction, we investigated LD accumulation in the intact human and mouse pancreas over a range of ages and states of diabetes. Very few LDs were found in normal human juvenile pancreatic acinar and islet cells, with numbers subsequently increasing throughout adulthood. While accumulation appeared evenly distributed in postjuvenile acinar and islet cells in donors without diabetes, LDs were enriched in islet α- and ß-cells from donors with type 2 diabetes (T2D). LDs were also found in the islet ß-like cells produced from human embryonic cell-derived ß-cell clusters. In contrast, LD accumulation was nearly undetectable in the adult rodent pancreas, even in hyperglycemic and hyperlipidemic models or 1.5-year-old mice. Taken together, there appear to be significant differences in pancreas islet cell lipid handling between species, and the human juvenile and adult cell populations. Moreover, our results suggest that LD enrichment could be impactful to T2D islet cell function.

CD8+ T cells regulate liver injury in obesity-related nonalcoholic fatty liver disease.

Nonalcoholic steatohepatitis (NASH) has increased in Western countries due to the prevalence of obesity. Current interests are aimed at identifying the type and function of immune cells that infiltrate the liver and key factors responsible for mediating their recruitment and activation in NASH. We investigated the function and phenotype of CD8+ T cells under obese and nonobese NASH conditions. We found an elevation in CD8 staining in livers from obese human subjects with NASH and cirrhosis that positively correlated with α-smooth muscle actin, a marker of hepatic stellate cell (HSC) activation. CD8+ T cells were elevated 3.5-fold in the livers of obese and hyperlipidemic NASH mice compared with obese hepatic steatosis mice. Isolated hepatic CD8+ T cells from these mice expressed a cytotoxic IL-10-expressing phenotype, and depletion of CD8+ T cells led to significant reductions in hepatic inflammation, HSC activation, and macrophage accumulation. Furthermore, hepatic CD8+ T cells from obese and hyperlipidemic NASH mice activated HSCs in vitro and in vivo. Interestingly, in the lean NASH mouse model, depletion and knockdown of CD8+ T cells did not impact liver inflammation or HSC activation. We demonstrated that under obese/hyperlipidemia conditions, CD8+ T cell are key regulators of the progression of NASH, while under nonobese conditions they play a minimal role in driving the disease. Thus, therapies targeting CD8+ T cells may be a novel approach for treatment of obesity-associated NASH.NEW & NOTEWORTHY Our study demonstrates that CD8+ T cells are the primary hepatic T cell population, are elevated in obese models of NASH, and directly activate hepatic stellate cells. In contrast, we find CD8+ T cells from lean NASH models do not regulate NASH-associated inflammation or stellate cell activation. Thus, for the first time to our knowledge, we demonstrate that hepatic CD8+ T cells are key players in obesity-associated NASH.

Obesity-induced reduction of adipose eosinophils is reversed with low-calorie dietary intervention.

While many studies have characterized the inflammatory disposition of adipose tissue (AT) during obesity, far fewer have dissected how such inflammation resolves during the process of physiological weight loss. In addition, new immune cells, such as the eosinophil, have been discovered as part of the AT immune cell repertoire. We have therefore characterized how AT eosinophils, associated eosinophilic inflammation, and remodeling processes, fluctuate during a dietary intervention in obese mice. Similar to previous reports, we found that obesity induced by high-fat diet feeding reduced the AT eosinophil content. However, upon switching obese mice to a low fat diet, AT eosinophils were restored to lean levels as mice reached the body weight of controls. The rise in AT eosinophils during dietary weight loss was accompanied by reduced macrophage content and inflammatory expression, upregulated tissue remodeling factors, and a more uniformly distributed AT vascular network. Additionally, we show that eosinophils of another metabolically relevant tissue, the liver, did not oscillate with either dietary weight gain or weight loss. This study shows that eosinophil content is differentially regulated among tissues during the onset and resolution of obesity. Furthermore, AT eosinophils correlated with AT remodeling processes during weight loss and thus may play a role in reestablishing AT homeostasis.

High CD8 T-Cell Receptor Clonality and Altered CDR3 Properties Are Associated With Elevated Isolevuglandins in Adipose Tissue During Diet-Induced Obesity.

Adipose tissue (AT) CD4+ and CD8+ T cells contribute to obesity-associated insulin resistance. Prior studies identified conserved T-cell receptor (TCR) chain families in obese AT, but the presence and clonal expansion of specific TCR sequences in obesity has not been assessed. We characterized AT and liver CD8+ and CD4+ TCR repertoires of mice fed a low-fat diet (LFD) and high-fat diet (HFD) using deep sequencing of the TCRß chain to quantify clonal expansion, gene usage, and CDR3 sequence. In AT CD8+ T cells, HFD reduced TCR diversity, increased the prevalence of public TCR clonotypes, and selected for TCR CDR3 regions enriched in positively charged and less polarized amino acids. Although TCR repertoire alone could distinguish between LFD- and HFD-fed mice, these properties of the CDR3 region of AT CD8+ T cells from HFD-fed mice led us to examine the role of negatively charged and nonpolar isolevuglandin (isoLG) adduct-containing antigen-presenting cells within AT. IsoLG-adducted protein species were significantly higher in AT macrophages of HFD-fed mice; isoLGs were elevated in M2-polarized macrophages, promoting CD8+ T-cell activation. Our findings demonstrate that clonal TCR expansion that favors positively charged CDR3s accompanies HFD-induced obesity, which may be an antigen-driven response to isoLG accumulation in macrophages.

MFehi adipose tissue macrophages compensate for tissue iron perturbations in mice.

Resident adipose tissue macrophages (ATMs) play multiple roles to maintain tissue homeostasis, such as removing excess free fatty acids and regulation of the extracellular matrix. The phagocytic nature and oxidative resiliency of macrophages not only allows them to function as innate immune cells but also to respond to specific tissue needs, such as iron homeostasis. MFehi ATMs are a subtype of resident ATMs that we recently identified to have twice the intracellular iron content as other ATMs and elevated expression of iron-handling genes. Although studies have demonstrated that iron homeostasis is important for adipocyte health, little is known about how MFehi ATMs may respond to and influence adipose tissue iron availability. Two methodologies were used to address this question: dietary iron supplementation and intraperitoneal iron injection. Upon exposure to high dietary iron, MFehi ATMs accumulated excess iron, whereas the iron content of MFelo ATMs and adipocytes remained unchanged. In this model of chronic iron excess, MFehi ATMs exhibited increased expression of genes involved in iron storage. In the injection model, MFehi ATMs incorporated high levels of iron, and adipocytes were spared iron overload. This acute model of iron overload was associated with increased numbers of MFehi ATMs; 17% could be attributed to monocyte recruitment and 83% to MFelo ATM incorporation into the MFehi pool. The MFehi ATM population maintained its low inflammatory profile and iron-cycling expression profile. These studies expand the field's understanding of ATMs and confirm that they can respond as a tissue iron sink in models of iron overload.

Macrophage-Targeted Therapeutics for Metabolic Disease.

Macrophages are cells of the innate immune system that are resident in all tissues, including metabolic organs such as the liver and adipose tissue (AT). Because of their phenotypic flexibility, they play beneficial roles in tissue homeostasis, but they also contribute to the progression of metabolic disease. Thus, they are ideal therapeutic targets for diseases such as insulin resistance (IR), nonalcoholic fatty liver disease (NAFLD), and atherosclerosis. Recently, discoveries in the area of drug delivery have facilitated phenotype-specific targeting of macrophages. In this review we discuss advances in potential therapeutics for metabolic diseases via macrophage-specific delivery. We highlight micro- and nanoparticles, liposomes, and oligopeptide complexes, and how they can be used to alter macrophage phenotype for a more metabolically favorable tissue environment.

Elevating adipose eosinophils in obese mice to physiologically normal levels does not rescue metabolic impairments.

OBJECTIVE: Obesity is a metabolic disorder that has reached epidemic proportions worldwide and leads to increased risk for diabetes, cardiovascular disease, asthma, certain cancers, and various other diseases. Obesity and its comorbidities are associated with impaired adipose tissue (AT) function. In the last decade, eosinophils have been identified as regulators of proper AT function. Our study aimed to determine whether normalizing the number of AT eosinophils in obese mice, to those of lean healthy mice, would reduce obesity and/or improve metabolic fitness. METHODS: C57BL/6J mice fed a high fat diet (HFD) were simultaneously given recombinant interleukin-5 (rIL5) for 8 weeks to increase AT eosinophils. Metabolic fitness was tested by evaluating weight gain, AT inflammation, glucose, lipid, and mixed-meal tolerance, AT insulin signaling, energy substrate utilization, energy expenditure, and white AT beiging capacity. RESULTS: Eosinophils were increased ∼3-fold in AT of obese HFD-fed mice treated with rIL5, and thus were restored to levels observed in lean healthy mice. However, there were no significant differences in rIL5-treated mice among the above listed comprehensive set of metabolic assays, despite the increased AT eosinophils. CONCLUSIONS: We have shown that restoring obese AT eosinophils to lean healthy levels is not sufficient to allow for improvement in any of a range of metabolic features otherwise impaired in obesity. Thus, the mechanisms that identified eosinophils as positive regulators of AT function, and therefore systemic health, are more complex than initially understood and will require further study to fully elucidate.

Adipose Tissue is Enriched for Activated and Late-Differentiated CD8+ T Cells and Shows Distinct CD8+ Receptor Usage, Compared With Blood in HIV-Infected Persons.

BACKGROUND: Adverse viral and medication effects on adipose tissue contribute to the development of metabolic disease in HIV-infected persons, but T cells also have a central role modulating local inflammation and adipocyte function. We sought to characterize potentially proinflammatory T-cell populations in adipose tissue among persons on long-term antiretroviral therapy and assess whether adipose tissue CD8 T cells represent an expanded, oligoclonal population. METHODS: We recruited 10 HIV-infected, non-diabetic, overweight or obese adults on efavirenz, tenofovir, and emtricitabine for >4 years with consistent viral suppression. We collected fasting blood and subcutaneous abdominal adipose tissue to measure the percentage of CD4 and CD8 T cells expressing activation, exhaustion, late differentiation/senescence, and memory surface markers. We performed T-cell receptor (TCR) sequencing on sorted CD8 cells. We compared the proportion of each T-cell subset and the TCR repertoire diversity, in blood versus adipose tissue. RESULTS: Adipose tissue had a higher percentage of CD3CD8 T cells compared with blood (61.0% vs. 51.7%, P < 0.01) and was enriched for both activated CD8HLA-DR T cells (5.5% vs. 0.9%, P < 0.01) and late-differentiated CD8CD57 T cells (37.4% vs. 22.7%, P < 0.01). Adipose tissue CD8 T cells displayed distinct TCRß V and J gene usage, and the Shannon Entropy index, a measure of overall TCRß repertoire diversity, was lower compared with blood (4.39 vs. 4.46; P = 0.05). CONCLUSIONS: Adipose tissue is enriched for activated and late-differentiated CD8 T cells with distinct TCR usage. These cells may contribute to tissue inflammation and impaired adipocyte fitness in HIV-infected persons.

CC-chemokine receptor 7 (CCR7) deficiency alters adipose tissue leukocyte populations in mice.

The mechanism by which macrophages and other immune cells accumulate in adipose tissue (AT) has been an area of intense investigation over the past decade. Several different chemokines and their cognate receptors have been studied for their role as chemoattractants in promoting recruitment of immune cells to AT However, it is also possible that chemoattractants known to promote clearance of immune cells from tissues to regional lymph nodes might be a critical component to overall AT immune homeostasis. In this study, we evaluated whether CCR7 influences AT macrophage (ATM) or T-cell (ATT) accumulation. CCR7-/- and littermate wild-type (WT) mice were placed on low-fat diet (LFD) or high-fat diet (HFD) for 16 weeks. CCR7 deficiency did not impact HFD-induced weight gain, hepatic steatosis, or glucose intolerance. Although lean CCR7-/- mice had an increased proportion of alternatively activated ATMs, there were no differences in ATM accumulation or polarization between HFD-fed CCR7-/- mice and their WT counterparts. However, CCR7 deficiency did lead to the preferential accumulation of CD8+ ATT cells, which was further exacerbated by HFD feeding. Finally, expression of inflammatory cytokines/chemokines, such as Tnf, Il6, Il1ß, Ccl2, and Ccl3, was equally elevated in AT by HFD feeding in CCR7-/- and WT mice, while Ifng and Il18 were elevated by HFD feeding in CCR7-/- but not in WT mice. Together, these data suggest that CCR7 plays a role in CD8+ATT cell egress, but does not influence ATM accumulation or the metabolic impact of diet-induced obesity.

Role of lipids in the metabolism and activation of immune cells.

Immune cell plasticity has extensive implications in the pathogenesis and resolution of metabolic disorders, cancers, autoimmune diseases and chronic inflammatory disorders. Over the past decade, nutritional status has been discovered to influence the immune response. In metabolic disorders such as obesity, immune cells interact with various classes of lipids, which are capable of controlling the plasticity of macrophages and T lymphocytes. The purpose of this review is to discuss lipids and their impact on innate and adaptive immune responses, focusing on two areas: (1) the impact of altering lipid metabolism on immune cell activation, differentiation and function and (2) the mechanism by which lipids such as cholesterol and fatty acids regulate immune cell plasticity.

CCR2 deficiency leads to increased eosinophils, alternative macrophage activation, and type 2 cytokine expression in adipose tissue.

Adipose tissue (AT) inflammation during obesity is mediated by immune cells and closely correlates with systemic insulin resistance. In lean AT, eosinophils are present in low but significant numbers and capable of promoting alternative macrophage activation in an IL-4/IL-13-dependent manner. In WT mice, obesity causes the proportion of AT eosinophils to decline, concomitant with inflammation and classical activation of AT macrophages. In this study, we show that CCR2 deficiency leads to increased eosinophil accumulation in AT. Furthermore, in contrast to WT mice, the increase in eosinophils in CCR2(-/-) AT is sustained and even amplified during obesity. Interestingly, a significant portion of eosinophils is found in CLSs in AT of obese CCR2(-/-) mice, which is the first time eosinophils have been shown to localize to these inflammatory hot spots. CCR2(-/-) bone marrow precursors displayed increased expression of various key eosinophil genes during in vitro differentiation to eosinophils, suggesting a potentially altered eosinophil phenotype in the absence of CCR2. In addition, the proportion of eosinophils in AT positively correlated with local expression of Il5, a potent eosinophil stimulator. The increase in eosinophils in CCR2(-/-) mice was detected in all white fat pads analyzed and in the peritoneal cavity but not in bone marrow, blood, spleen, or liver. In AT of CCR2(-/-) mice, an increased eosinophil number positively correlated with M2-like macrophages, expression of the Treg marker Foxp3, and type 2 cytokines, Il4, Il5, and Il13. This is the first study to link CCR2 function with regulation of AT eosinophil accumulation.

Activation of NF-κB drives the enhanced survival of adipose tissue macrophages in an obesogenic environment.

OBJECTIVE: Macrophage accumulation in adipose tissue (AT) during obesity contributes to inflammation and insulin resistance. Recruitment of monocytes to obese AT has been the most studied mechanism explaining this accumulation. However, recent evidence suggests that recruitment-independent mechanisms may also regulate pro-inflammatory AT macrophage (ATM) numbers. The role of increased ATM survival during obesity has yet to be explored. RESULTS: We demonstrate that activation of apoptotic pathways is significantly reduced in ATMs from diet-induced and genetically obese mice. Concurrently, pro-survival Bcl-2 family member protein levels and localization to the mitochondria is elevated in ATMs from obese mice. This increased pro-survival signaling was associated with elevated activation of the transcription factor, NF-κB, and increased expression of its pro-survival target genes. Finally, an obesogenic milieu increased ATM viability only when NF-κB signaling pathways were functional. CONCLUSIONS: Our data demonstrate that obesity promotes survival of inflammatory ATMs, possibly through an NF-κB-regulated mechanism.

Obesity alters adipose tissue macrophage iron content and tissue iron distribution.

Adipose tissue (AT) expansion is accompanied by the infiltration and accumulation of AT macrophages (ATMs), as well as a shift in ATM polarization. Several studies have implicated recruited M1 ATMs in the metabolic consequences of obesity; however, little is known regarding the role of alternatively activated resident M2 ATMs in AT homeostasis or how their function is altered in obesity. Herein, we report the discovery of a population of alternatively activated ATMs with elevated cellular iron content and an iron-recycling gene expression profile. These iron-rich ATMs are referred to as MFe(hi), and the remaining ATMs are referred to as MFe(lo). In lean mice, ~25% of the ATMs are MFe(hi); this percentage decreases in obesity owing to the recruitment of MFe(lo) macrophages. Similar to MFe(lo) cells, MFe(hi) ATMs undergo an inflammatory shift in obesity. In vivo, obesity reduces the iron content of MFe(hi) ATMs and the gene expression of iron importers as well as the iron exporter, ferroportin, suggesting an impaired ability to handle iron. In vitro, exposure of primary peritoneal macrophages to saturated fatty acids also alters iron metabolism gene expression. Finally, the impaired MFe(hi) iron handling coincides with adipocyte iron overload in obese mice. In conclusion, in obesity, iron distribution is altered both at the cellular and tissue levels, with AT playing a predominant role in this change. An increased availability of fatty acids during obesity may contribute to the observed changes in MFe(hi) ATM phenotype and their reduced capacity to handle iron.