Metabolic dysfunction mediated by HIF-1α contributes to epithelial differentiation defects in eosinophilic esophagitis.

BACKGROUND: Investigating the contributory role that epithelial cell metabolism plays in allergic inflammation is a key factor to understanding what influences dysfunction and the pathogenesis of the allergic disease eosinophilic esophagitis (EoE). We previously highlighted that the absence of hypoxia signaling through hypoxia-inducible factor (HIF)-1α in EoE contributes to esophageal epithelial dysfunction. However, metabolic regulation by HIF-1α has not been explored in esophageal allergy. OBJECTIVES: We sought to define the role of HIF-1α-mediated metabolic dysfunction in esophageal epithelial differentiation processes and barrier function in EoE. METHODS: In RNA sequencing of EoE patient biopsy samples, we observed the expression pattern of key genes involved in mitochondrial metabolism/oxidative phosphorylation (OXPHOS) and glycolysis. Seahorse bioenergetics analysis was performed on EPC2-hTERT cells to decipher the metabolic processes involved in epithelial differentiation processes. In addition, air-liquid interface cultures were used to delineate metabolic dependency mechanisms required for epithelial differentiation. RESULTS: Transcriptomic analysis identified an increase in genes associated with OXPHOS in patients with EoE. Epithelial origin of this signature was confirmed by complex V immunofluorescence of patient biopsy samples. Bioenergetic analysis in vitro revealed that differentiated epithelium was less reliant on OXPHOS compared with undifferentiated epithelium. Increased OXPHOS potential and reduced glycolytic capacity was mirrored in HIF1A-knockdown EPC2-hTERT cells that exhibited a significant absence of terminal markers of epithelial differentiation, including involucrin. Pharmacologic glucose transport inhibition phenocopied this, while rescue of the HIF-1α-deficient phenotype using the pan-prolyl hydroxylase inhibitor dimethyloxalylglycine resulted in restored expression of epithelial differentiation markers. CONCLUSIONS: An OXPHOS-dominated metabolic pattern in EoE patients, brought about largely by the absence of HIF-1α-mediated glycolysis, is linked with the deficit in esophageal epithelial differentiation.

Iron Is Critical for Mucosal-Associated Invariant T Cell Metabolism and Effector Functions.

Mucosal-Associated Invariant T (MAIT) cells are a population of innate T cells that play a critical role in host protection against bacterial and viral pathogens. Upon activation, MAIT cells can rapidly respond via both TCR-dependent and -independent mechanisms, resulting in robust cytokine production. The metabolic and nutritional requirements for optimal MAIT cell effector responses are still emerging. Iron is an important micronutrient and is essential for cellular fitness, in particular cellular metabolism. Iron is also critical for many pathogenic microbes, including those that activate MAIT cells. However, iron has not been investigated with respect to MAIT cell metabolic or functional responses. In this study, we show that human MAIT cells require exogenous iron, transported via CD71 for optimal metabolic activity in MAIT cells, including their production of ATP. We demonstrate that restricting iron availability by either chelating environmental iron or blocking CD71 on MAIT cells results in impaired cytokine production and proliferation. These data collectively highlight the importance of a CD71-iron axis for human MAIT cell metabolism and functionality, an axis that may have implications in conditions where iron availability is limited.

NK cells vs. obesity: A tale of dysfunction & redemption.

Natural killer (NK) cells are critical in protecting the body against infection and cancer. NK cells can rapidly respond to these threats by directly targeting the infected or transformed cell using their cytotoxic machinery or by initiating and amplifying the immune response via their production of cytokines. Additionally, NK cells are resident across many tissues including adipose, were their role extends from host protection to tissue homeostasis. Adipose resident NK cells can control macrophage polarization via cytokine production, whilst also regulating stressed adipocyte fate using their cytotoxic machinery. Obesity is strongly associated with increased rates of cancer and a heightened susceptibility to severe infections. This is in part due to significant obesity-related immune dysregulation, including defects in both peripheral and adipose tissue NK cells. In this review, we detail the literature to date on NK cells in the setting of obesity - outlining the consequences, mechanisms and therapeutic interventions.

Glycogen-fuelled metabolism supports rapid mucosal-associated invariant T cell responses.

Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells which recognize a limited repertoire of ligands presented by the MHC class-I like molecule MR1. In addition to their key role in host protection against bacterial and viral pathogens, MAIT cells are emerging as potent anti-cancer effectors. With their abundance in human, unrestricted properties, and rapid effector functions MAIT cells are emerging as attractive candidates for immunotherapy. In the current study, we demonstrate that MAIT cells are potent cytotoxic cells, rapidly degranulating and inducing target cell death. Previous work from our group and others has highlighted glucose metabolism as a critical process for MAIT cell cytokine responses at 18 h. However, the metabolic processes supporting rapid MAIT cell cytotoxic responses are currently unknown. Here, we show that glucose metabolism is dispensable for both MAIT cell cytotoxicity and early (<3 h) cytokine production, as is oxidative phosphorylation. We show that MAIT cells have the machinery required to make (GYS-1) and metabolize (PYGB) glycogen and further demonstrate that that MAIT cell cytotoxicity and rapid cytokine responses are dependent on glycogen metabolism. In summary, we show that glycogen-fueled metabolism supports rapid MAIT cell effector functions (cytotoxicity and cytokine production) which may have implications for their use as an immunotherapeutic agent.

Glucagon-like peptide-1 therapy in people with obesity restores natural killer cell metabolism and effector function.

OBJECTIVE: People with obesity (PWO) have functionally defective natural killer (NK) cells, with a decreased capacity to produce cytokines and kill target cells, underpinned by defective cellular metabolism. It is plausible that the changes in peripheral NK cell activity are contributing to the multimorbidity in PWO, which includes an increased risk of cancer. This study investigated whether therapy with long-acting glucagon-like peptide-1 (GLP-1) analogues, which are an effective treatment for obesity, could restore NK cell functionality in PWO. METHODS: In a cohort of 20 PWO, this study investigated whether 6 months of once weekly GLP-1 therapy (semaglutide) could restore human NK cell function and metabolism using multicolor flow cytometry, enzyme-linked immunosorbent assays, and cytotoxicity assays. RESULTS: These data demonstrate that PWO who received GLP-1 therapy have improved NK cell function, as measured by cytotoxicity and interferon-γ/granzyme B production. In addition, the study demonstrates increases in a CD98-mTOR-glycolysis metabolic axis, which is critical for NK cell cytokine production. Finally, it shows that the reported improvements in NK cell function appear to be independent of weight loss. CONCLUSIONS: The restoration, by GLP-1 therapy, of NK cell functionality in PWO may be contributing to the overall benefits being seen with this class of medication.

Antigen specific T cells in people with obesity at five months following ChAdOx1 COVID-19 vaccination.

BACKGROUND/OBJECTIVES: People with obesity (PWO) face an increased risk of severe outcomes from COVID-19, including hospitalisation, ICU admission and death. Obesity has been seen to impair immune memory following vaccination against influenza, hepatitis B, tetanus, and rabies. Little is known regarding immune memory in PWO following COVID-19 adenovirus vector vaccination. SUBJECTS/METHODS: We investigated SARS-CoV-2 specific T cell responses in 50 subjects, five months following a two-dose primary course of ChAdOx1 nCoV-19 (AZD1222) vaccination. We further divided our cohort into PWO (n = 30) and matched controls (n = 20). T cell (CD4+, CD8+) cytokine responses (IFNγ, TNFα) to SARS-CoV-2 spike peptide pools were determined using multicolour flow cytometry. RESULTS: Circulating T cells specific for SARS-CoV-2 were readily detected across our cohort, with robust responses to spike peptide stimulation across both T cell lines. PWO and controls had comparable levels of both CD4+ and CD8+ SARS-CoV-2 spike specific T cells. Polyfunctional T cells - associated with enhanced protection against viral infection - were detected at similar frequencies in both PWO and controls. CONCLUSIONS: These data indicate that PWO who have completed a primary course of ChAdOx1 COVID-19 vaccination have robust, durable, and functional antigen specific T cell immunity that is comparable to that seen in people without obesity.

Translating MSC Therapy in the Age of Obesity.

Mesenchymal stromal cell (MSC) therapy has seen increased attention as a possible option to treat a number of inflammatory conditions including COVID-19 acute respiratory distress syndrome (ARDS). As rates of obesity and metabolic disease continue to rise worldwide, increasing proportions of patients treated with MSC therapy will be living with obesity. The obese environment poses critical challenges for immunomodulatory therapies that should be accounted for during development and testing of MSCs. In this review, we look to cancer immunotherapy as a model for the challenges MSCs may face in obese environments. We then outline current evidence that obesity alters MSC immunomodulatory function, drastically modifies the host immune system, and therefore reshapes interactions between MSCs and immune cells. Finally, we argue that obese environments may alter essential features of allogeneic MSCs and offer potential strategies for licensing of MSCs to enhance their efficacy in the obese microenvironment. Our aim is to combine insights from basic research in MSC biology and clinical trials to inform new strategies to ensure MSC therapy is effective for a broad range of patients.

Individuals with obesity who survive SARS-CoV-2 infection have preserved antigen-specific T cell frequencies.

OBJECTIVE: Obesity is a major risk factor for severe disease in COVID-19, with increased hospitalization, intensive care unit admission, and mortality. This increased impact of COVID-19 in people with obesity (PWO) is likely driven, in part, by the well-described obesity-induced immune dysregulation. Obesity has also been associated with impaired immune memory in many settings, including weakened responses to hepatitis B, tetanus, rabies, and influenza vaccination. Recently, it was reported that PWO who have COVID-19 have reduced IgG antibody titers with defective neutralizing capabilities. However, it remains unknown whether PWO generate durable T cell immunity to SARS-CoV-2. METHODS: This study investigated SARS-CoV-2-specific T cell responses in a cohort of 40 patients (n = 20 PWO and n = 20 matched control individuals) who had recovered from COVID-19. T cell (CD4+ , CD8+ ) cytokine responses (IFNγ, TNFα) to SARS-CoV-2 peptide pools (spike, membrane) were determined using multicolor flow cytometry. RESULTS: Circulating T cells specific for SARS-CoV-2 were readily detected in the total cohort. PWO displayed comparable levels of SARS-CoV-2 spike- and membrane-specific T cells, with both T cell subsets responding. CONCLUSIONS: These data indicate that PWO who survive COVID-19 generate robust and durable SARS-CoV-2-specific T cell immunity that is equivalent to that seen in those without obesity.

Mucosal-associated invariant T cells are associated with insulin resistance in childhood obesity, and disrupt insulin signalling via IL-17.

AIMS/HYPOTHESIS: Mucosal-associated invariant T cells (MAIT cells) are an abundant population of innate T cells. When activated, MAIT cells rapidly produce a range of cytokines, including IFNγ, TNF-α and IL-17. Several studies have implicated MAIT cells in the development of metabolic dysfunction, but the mechanisms through which this occurs are not fully understood. We hypothesised that MAIT cells are associated with insulin resistance in children with obesity, and affect insulin signalling through their production of IL-17. METHODS: In a cross-sectional observational study, we investigated MAIT cell cytokine profiles in a cohort of 30 children with obesity and 30 healthy control participants, of similar age, using flow cytometry. We then used a cell-based model to determine the direct effect of MAIT cells and IL-17 on insulin signalling and glucose uptake. RESULTS: Children with obesity display increased MAIT cell frequencies (2.2% vs 2.8%, p=0.047), and, once activated, these produced elevated levels of both TNF-α (39% vs 28%, p=0.03) and IL-17 (1.25% vs 0.5%, p=0.008). The IL-17-producing MAIT cells were associated with an elevated HOMA-IR (r=0.65, p=0.001). The MAIT cell secretome from adults with obesity resulted in reduced glucose uptake when compared with the secretome from healthy adult control (1.31 vs 0.96, p=0.0002), a defect that could be blocked by neutralising IL-17. Finally, we demonstrated that recombinant IL-17 blocked insulin-mediated glucose uptake via inhibition of phosphorylated Akt and extracellular signal-regulated kinase. CONCLUSIONS/INTERPRETATIONS: Collectively, these studies provide further support for the role of MAIT cells in the development of metabolic dysfunction, and suggest that an IL-17-mediated effect on intracellular insulin signalling is responsible.

Cytokine-induced natural killer cell training is dependent on cellular metabolism and is defective in obesity.

Natural killer (NK) cells are a population of innate immune cells that can rapidly kill cancer cells and produce cytokines such as interferon-γ. A key feature of NK cells is their ability to respond without prior sensitization; however, it is now well established that NK cells can possess memory-like features. After activation with cytokines, NK cells demonstrate enhanced effector functions upon restimulation days or weeks later. This demonstrates that NK cells may be trained to be more effective killers and harnessed as more potent cancer immunotherapy agents. We have previously demonstrated that cellular metabolism is essential for NK cell responses, with NK cells upregulating both glycolysis and oxidative phosphorylation upon cytokine stimulation. Limiting NK cell metabolism results in reduced cytotoxicity and cytokine production. We have also demonstrated that defective NK cell responses in obesity are linked to defective cellular metabolism. In the current study, we investigated if cellular metabolism is required during the initial period of NK cell cytokine training and if NK cells from people with obesity (PWO) can be effectively trained. We show that increased flux through glycolysis and oxidative phosphorylation during the initial cytokine activation period is essential for NK cell training, as is the metabolic signaling factor Srebp. We show that NK cells from PWO, which are metabolically defective, display impaired NK cell training, which may have implications for immunotherapy in this particularly vulnerable group.

Mucosal Associated Invariant T Cells in Cancer-Friend or Foe?

Mucosal associated invariant T (MAIT) cells are a population of unconventional T cells which can bridge the innate and adaptive immune systems. Well-described roles for MAIT cells include host protection against invading bacteria, fungi and viruses. Upon activation, MAIT cells become prolific effector cells, capable of producing a range of cytokines and lytic molecules. In addition to their anti-microbial role, MAIT cells have been implicated in immune responses to cancer, with opposing beneficial and pathogenic roles reported. On the one hand, MAIT cells can home to the site of the tumour in many human cancers and can produce anti-tumour molecules. On the other, MAIT cells can display defective phenotypes in certain cancers and produce pro-tumour molecules. In this review, we discuss the current literature on the diverse roles for MAIT cells in cancer, outlining their frequencies, functions and associations with N staging and prognosis. We also discuss potential mechanisms underpinning cancer-related alterations in MAIT cells and highlight therapeutic approaches to harness or target MAIT cells in cancer.

Role for Retinoic Acid-Related Orphan Receptor Alpha (RORα) Expressing Macrophages in Diet-Induced Obesity.

The transcription factor RORα plays an important role in regulating circadian rhythm, inflammation, metabolism, and cellular development. Herein we show a role for RORα-expressing macrophages in the adipose tissue in altering the metabolic state of mice on a high-fat diet. The expression of Rora and RORA is elevated in white adipose tissue from obese mice and humans when compared to lean counterparts. When fed a high-fat diet Rora reporter mice revealed increased expression of Rora-YFP in macrophages in white adipose tissue deposits. To further define the potential role for Rora-expressing macrophages in the generation of an aberrant metabolic state Rorafl/flLysMCre/+ mice, which do not express Rora in myeloid cells, were maintained on a high-fat diet, and metabolic parameters assessed. These mice had significantly impaired weight gain and improved metabolic parameters in comparison to Rorafl/fl control mice. Further analysis of the immune cell populations within white adipose tissue deposits demonstrates a decrease in inflammatory adipose tissue macrophages (ATM). In obese reporter mouse there was increased in Rora-YFP expressing ATM in adipose tissue. Analysis of peritoneal macrophage populations demonstrates that within the peritoneal cavity Rora-expression is limited to myeloid-derived macrophages, suggesting a novel role for RORα in macrophage development and activation, which can impact on metabolism, and inflammation.

Interleukin-17 producing mucosal associated invariant T cells - emerging players in chronic inflammatory diseases?

Mucosal associated invariant T (MAIT) cells are a population of evolutionarily conserved T cells, which express an invariant T cell receptor (TCR) and represent a significant subset of innate-like T cells in humans, yet their role in immunity is still emerging. Unlike conventional αß T cells, MAIT cells are not restricted by MHC molecules, but instead uniquely recognize microbially derived vitamin metabolites presented by the MHC-I like molecule MR1. MAIT cells are enriched in mucosal sites and tissues including liver and adipose tissue where they are thought to play an important role in immunosurveillance and immunity against microbial infection. In addition to their putative role in antimicrobial immunity, recent research on MAIT cells, in particular IL-17 producing MAIT cells, has demonstrated their involvement in numerous chronic inflammatory conditions. In this review, we give an overview of the work to date on the function and subsets of MAIT cells. We also examine the role of IL-17 producing MAIT cells in chronic inflammatory diseases ranging from autoimmune conditions, metabolic diseases to cancer. Furthermore, we discuss the most recent findings from the clinic that might help deepen our understanding about the biology of MAIT cells.

Mucosal-Associated Invariant T Cells Display Diminished Effector Capacity in Oesophageal Adenocarcinoma.

Oesophageal adenocarcinoma (OAC) is an aggressive malignancy with poor prognosis, and incidence is increasing rapidly in the Western world. Mucosal-associated invariant T (MAIT) cells recognize bacterial metabolites and kill infected cells, yet their role in OAC is unknown. We aimed to elucidate the role of MAIT cells during cancer development by characterizing the frequency, phenotype, and function of MAIT cells in human blood and tissues, from OAC and its pre-malignant inflammatory condition Barrett's oesophagus (BO). Blood and tissues were phenotyped by flow cytometry and conditioned media from explanted tissue was used to model the effects of the tumor microenvironment on MAIT cell function. Associations were assessed between MAIT cell frequency, circulating inflammatory markers, and clinical parameters to elucidate the role of MAIT cells in inflammation driven cancer. MAIT cells were decreased in BO and OAC blood compared to healthy controls, but were increased in oesophageal tissues, compared to BO-adjacent tissue, and remained detectable after neo-adjuvant treatment. MAIT cells in tumors expressed CD8, PD-1, and NKG2A but lower NKG2D than BO cohorts. MAIT cells produced less IFN-γ and TNF-α in the presence of tumor-conditioned media. OAC cell line viability was reduced upon exposure to expanded MAIT cells. Serum levels of chemokine IP-10 were inversely correlated with MAIT cell frequency in both tumors and blood. MAIT cells were higher in the tumors of node-negative patients, but were not significantly associated with other clinical parameters. This study demonstrates that OAC tumors are infiltrated by MAIT cells, a type of CD8 T cell featuring immune checkpoint expression and cytotoxic potential. These findings may have implications for immunotherapy and immune scoring approaches.

Dysregulation of Natural Killer Cells in Obesity.

Natural killer (NK) cells are a population of lymphocytes which classically form part of the innate immune system. They are defined as innate lymphocytes, due to their ability to kill infected or transformed cells without prior activation. In addition to their cytotoxic abilities, NK cells are also rapid producers of inflammatory cytokines such as interferon gamma (IFN-γ) and are therefore a critical component of early immune responses. Due to these unique abilities, NK cells are a very important component of host protection, especially anti-tumour and anti-viral immunity. Obesity is a worldwide epidemic, with over 600 million adults and 124 million children now classified as obese. It is well established that individuals who are obese are at a higher risk of many acute and chronic conditions, including cancer and viral infections. Over the past 10 years, many studies have investigated the impact of obesity on NK cell biology, detailing systemic dysregulation of NK cell functions. More recently, several studies have investigated the role of NK cells in the homeostasis of adipose tissue and the pathophysiology of obesity. In this review, we will discuss in detail these studies and focus on emerging data detailing the metabolic mechanisms altering NK cells in obesity.

Nucleic acid cytokine responses in obese children and infants of obese mothers.

Almost a third of Irish children are now overweight and the country ranks 58th out of 200 countries for its proportion of overweight youths. With the rising obesity epidemic, and the impaired immune responses of this population, it is vital to understand the effects that obesity has on the immune system and to design future therapeutics, adjuvants and vaccines with overweight and obese populations in mind. Many current vaccines use adjuvants that have been found to be less effective at stimulating the immune response in children compared with adults and there is now substantial effort to design paediatric-focused adjuvants. Additionally, vaccine responses have been shown to be less effective in obese populations indicating that this is a particularly vulnerable population. We have recently identified cytosolic nucleic acids (CNAs), as novel candidate adjuvants for childhood vaccines. Here we investigated whether immune responses to these candidate adjuvants were adversely affected in infants born to overweight or obese mothers, and in overweight and obese children. Type I Interferon (IFN) and proinflammatory cytokines such as Tumor Necrosis Factor α (TNFα) are vital for driving innate and adaptive immune responses. We found that childhood obesity conferred no significant adverse effect on CNA-induced Type I IFN responses when compared with lean children. Similarly, Type I IFN responses were intact in the cord blood of babies delivered from overweight and obese mothers, when compared with lean mothers. There was also no significant impact of obesity on CNA-induced TNFα responses in children or from cord blood of infants born to overweight/obese mothers. In all cases, there was a tendency towards decreased production of innate cytokine Type I Interferon and TNFα, however there was no significant negative correlation. Interestingly, high maternal BMI showed weak and moderate positive correlation with IL-12p70 and IFNγ, respectively, in response to CNA stimulation. This study demonstrates that future adjuvants can be tailored for these populations through the use of activators of CNA sensors.

NK cells in childhood obesity are activated, metabolically stressed, and functionally deficient.

Childhood obesity is a major global concern, with over 50 million children now classified as obese. Obesity has been linked to the development of numerous chronic inflammatory diseases, including type 2 diabetes and multiple cancers. NK cells are a subset of innate effector cells, which play an important role in the regulation of adipose tissue and antitumor immunity. NK cells can spontaneously kill transformed cells and coordinate subsequent immune responses through their production of cytokines. We investigated the effect of obesity on NK cells in a cohort of obese children, compared to children with a healthy weight. We demonstrated a reduction in peripheral NK cell frequencies in childhood obesity and inverse correlations with body mass index and insulin resistance. Compared with NK cells from children with normal weight, we show increased NK cell activation and metabolism in obese children (PD-1, mTOR activation, ECAR, and mitochondrial ROS), along with a reduced capacity to respond to stimulus, ultimately leading to loss of function (proliferation and tumor lysis). Collectively we show that NK cells from obese children are activated, metabolically stressed, and losing the ability to perform their basic duties. Paired with the reduction in NK cell frequencies in childhood obesity, this suggests that the negative effect on antitumor immunity is present early in the life course of obesity and certainly many years before the development of overt malignancies.

Adipose Type One Innate Lymphoid Cells Regulate Macrophage Homeostasis through Targeted Cytotoxicity.

Adipose tissue has a dynamic immune system that adapts to changes in diet and maintains homeostatic tissue remodeling. Adipose type 1 innate lymphoid cells (AT1-ILCs) promote pro-inflammatory macrophages in obesity, but little is known about their functions at steady state. Here we found that human and murine adipose tissue harbor heterogeneous populations of AT1-ILCs. Experiments using parabiotic mice fed a high-fat diet (HFD) showed differential trafficking of AT1-ILCs, particularly in response to short- and long-term HFD and diet restriction. At steady state, AT1-ILCs displayed cytotoxic activity toward adipose tissue macrophages (ATMs). Depletion of AT1-ILCs and perforin deficiency resulted in alterations in the ratio of inflammatory to anti-inflammatory ATMs, and adoptive transfer of AT1-ILCs exacerbated metabolic disorder. Diet-induced obesity impaired AT1-ILC killing ability. Our findings reveal a role for AT1-ILCs in regulating ATM homeostasis through cytotoxicity and suggest that this function is relevant in both homeostasis and metabolic disease.

The helminth T2 RNase ω1 promotes metabolic homeostasis in an IL-33- and group 2 innate lymphoid cell-dependent mechanism.

Induction of a type 2 cellular response in the white adipose tissue leads to weight loss and improves glucose homeostasis in obese animals. Injection of obese mice with recombinant helminth-derived Schistosoma mansoni egg-derived ω1 (ω1), a potent inducer of type 2 activation, improves metabolic status involving a mechanism reliant upon release of the type 2 initiator cytokine IL-33. IL-33 initiates the accumulation of group 2 innate lymphoid cells (ILC2s), eosinophils, and alternatively activated macrophages in the adipose tissue. IL-33 release from cells in the adipose tissue is mediated by the RNase activity of ω1; however, the ability of ω1 to improve metabolic status is reliant upon effective binding of ω1 to CD206. We demonstrate a novel mechanism for RNase-mediated release of IL-33 inducing ILC2-dependent improvements in the metabolic status of obese animals.