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.

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.

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.

Serum IL-36ß levels are associated with Insulin sensitivity in paediatric patients with obesity.

Although the orchestrating role of Interleukin-36 cytokines in regulating inflammation at barrier tissue sites, is well established, whether they play a significant role in the settings of metabolic health and disease, has yet to be fully established. Several recent studies have demonstrated that IL-36 cytokine expression is elevated among adult patients with obesity, and can play roles in regulating both insulin sensitivity and driving inflammation. In this report, we have extended these analyses to paediatric patients and identified an association between elevated serum levels of expression of the specific Interleukin-36 subfamily member, IL-36ß, among children with obesity displaying insulin sensitivity, compared to children with obesity who are insulin resistant. While these data further indicate a possible protective role for IL-36 in metabolic health, they also differ with previous findings from an adult patient cohort, where elevated levels of the related cytokine, IL-36γ, were found to occur in association with improved metabolic health. While highlighting important differences between paediatric and adult patient cohorts in the context of metabolic disease associated with obesity, these data underscore the need for a deeper mechanistic analysis of the role of IL-36 cytokines in disease.

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.

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.

The E3 ubiquitin ligase Pellino3 protects against obesity-induced inflammation and insulin resistance.

Diet-induced obesity can induce low-level inflammation and insulin resistance. Interleukin-1ß (IL-1ß) is one of the key proinflammatory cytokines that contributes to the generation of insulin resistance and diabetes, but the mechanisms that regulate obesity-driven inflammation are ill defined. Here we found reduced expression of the E3 ubiquitin ligase Pellino3 in human abdominal adipose tissue from obese subjects and in adipose tissue of mice fed a high-fat diet and showing signs of insulin resistance. Pellino3-deficient mice demonstrated exacerbated high-fat-diet-induced inflammation, IL-1ß expression, and insulin resistance. Mechanistically, Pellino3 negatively regulated TNF receptor associated 6 (TRAF6)-mediated ubiquitination and stabilization of hypoxia-inducible factor 1α (HIF1α), resulting in reduced HIF1α-induced expression of IL-1ß. Our studies identify a regulatory mechanism controlling diet-induced insulin resistance by highlighting a critical role for Pellino3 in regulating IL-1ß expression with implications for diseases like type 2 diabetes.

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.

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.

Adipose tissue invariant NKT cells protect against diet-induced obesity and metabolic disorder through regulatory cytokine production.

Invariant natural killer T (iNKT) cells are evolutionarily conserved innate T cells that influence inflammatory responses. We have shown that iNKT cells, previously thought to be rare in humans, were highly enriched in human and murine adipose tissue, and that as adipose tissue expanded in obesity, iNKT cells were depleted, correlating with proinflammatory macrophage infiltration. iNKT cell numbers were restored in mice and humans after weight loss. Mice lacking iNKT cells had enhanced weight gain, larger adipocytes, fatty livers, and insulin resistance on a high-fat diet. Adoptive transfer of iNKT cells into obese mice or in vivo activation of iNKT cells via their lipid ligand, alpha-galactocylceramide, decreased body fat, triglyceride levels, leptin, and fatty liver and improved insulin sensitivity through anti-inflammatory cytokine production by adipose-derived iNKT cells. This finding highlights the potential of iNKT cell-targeted therapies, previously proven to be safe in humans, in the management of obesity and its consequences.

Obesity Reduces mTORC1 Activity in Mucosal-Associated Invariant T Cells, Driving Defective Metabolic and Functional Responses.

Obesity underpins the development of numerous chronic diseases, such as type II diabetes mellitus. It is well established that obesity negatively alters immune cell frequencies and functions. Mucosal-associated invariant T (MAIT) cells are a population of innate T cells, which we have previously reported are dysregulated in obesity, with altered circulating and adipose tissue frequencies and a reduction in their IFN-γ production, which is a critical effector function of MAIT cells in host defense. Hence, there is increased urgency to characterize the key molecular mechanisms that drive MAIT cell effector functions and to identify those which are impaired in the obesity setting. In this study, we found that MAIT cells significantly upregulate their rates of glycolysis upon activation in an mTORC1-dependent manner, and this is essential for MAIT cell IFN-γ production. Furthermore, we show that mTORC1 activation is dependent on amino acid transport via SLC7A5. In obese patients, using RNA sequencing, Seahorse analysis, and a series of in vitro experiments, we demonstrate that MAIT cells isolated from obese adults display defective glycolytic metabolism, mTORC1 signaling, and SLC7A5 aa transport. Collectively, our data detail the intrinsic metabolic pathways controlling MAIT cell cytokine production and highlight mTORC1 as an important metabolic regulator that is impaired in obesity, leading to altered MAIT cell responses.

Type 1 IFN Induction by Cytosolic Nucleic Acid Is Intact in Neonatal Mononuclear Cells, Contrasting Starkly with Neonatal Hyporesponsiveness to TLR Ligation Due to Independence from Endosome-Mediated IRF3 Activation.

Two million infants die each year from infectious diseases before they reach 12 mo; many of these diseases are vaccine preventable in older populations. Pattern recognition receptors represent the critical front-line defense against pathogens. Evidence suggests that the innate immune system does not fully develop until puberty, contributing to impaired response to infection and impaired vaccine responses in neonates, infants, and children. The activity of the pattern recognition receptor family of cytosolic nucleic acid (CNA) sensors in this pediatric population has not been reported. We show that in direct contrast to weak TLR-induced type I IFN in human cord blood mononuclear cells, cord blood mononuclear cells are capable of initiating a potent response to CNA, inducing both antiviral type I IFN and, unexpectedly, proinflammatory TNF-α. A deficiency in Rab11-GTPase endosome formation and consequent lack of IRF3 activation in neonatal monocytes is at least in part responsible for the marked disparity in TLR-induced IFN production between neonatal and adult monocytes. CNA receptors do not rely on endosome formation, and therefore, these responses remain intact in neonates. Heightened neonatal responses to CNA challenge are maintained in children up to 2 y of age and, in marked contrast to TLR4/9 agonists, result in IL-12p70 and IFN-γ generation. CNA sensors induce robust antiviral and proinflammatory pathways in neonates and children and possess great potential for use as immunostimulants or vaccine adjuvants for targeted neonatal and pediatric populations to promote cell-mediated immunity against invasive infectious disease.

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.

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.

Altered distribution and increased IL-17 production by mucosal-associated invariant T cells in adult and childhood obesity.

Mucosal-associated invariant T (MAIT) cells are innate MHC-unrestricted cells that regulate inflammatory responses through the rapid production of cytokines. In this article, we show that circulating MAIT cells are depleted in obese adults, and depletion is associated with diabetic status. Circulating MAIT cells more frequently produced IL-17 upon stimulation ex vivo, a cytokine implicated in insulin resistance. MAIT cells were enriched in adipose tissue (AT) compared with blood. AT MAIT cells, but not circulating MAIT cells, were capable of producing IL-10. In AT from obese subjects, MAIT cells were depleted, were less likely to produce IL-10, and more frequently produced IL-17. Finally, we show that IL-17(+) MAIT cells are also increased in childhood obesity, and altered MAIT cell frequencies in obese children are positively associated with insulin resistance. These data indicate that MAIT cells are enriched in human AT and display an IL-17(+) phenotype in both obese adults and children, correlating with levels of insulin resistance. The alterations in MAIT cells may be contributing to obesity-related sterile inflammation and insulin resistance.

Human invariant NKT cell subsets differentially promote differentiation, antibody production, and T cell stimulation by B cells in vitro.

Invariant NK T (iNKT) cells can provide help for B cell activation and Ab production. Because B cells are also capable of cytokine production, Ag presentation, and T cell activation, we hypothesized that iNKT cells will also influence these activities. Furthermore, subsets of iNKT cells based on CD4 and CD8 expression that have distinct functional activities may differentially affect B cell functions. We investigated the effects of coculturing expanded human CD4(+), CD8α(+), and CD4(-)CD8α(-) double-negative (DN) iNKT cells with autologous peripheral B cells in vitro. All iNKT cell subsets induced IgM, IgA, and IgG release by B cells without needing the iNKT cell agonist ligand α-galactosylceramide. Additionally, CD4(+) iNKT cells induced expansions of cells with phenotypes of regulatory B cells. When cocultured with α-galactosylceramide-pulsed B cells, CD4(+) and DN iNKT cells secreted Th1 and Th2 cytokines but at 10-1000-fold lower levels than when cultured with dendritic cells. CD4(+) iNKT cells reciprocally induced IL-4 and IL-10 production by B cells. DN iNKT cells expressed the cytotoxic degranulation marker CD107a upon exposure to B cells. Remarkably, whereas iNKT cell subsets could induce CD40 and CD86 expression by B cells, iNKT cell-matured B cells were unable to drive proliferation of autologous and alloreactive conventional T cells, as seen with B cells cultured in the absence of iNKT cells. Therefore, human CD4(+), CD8α(+), and DN iNKT cells can differentially promote and regulate the induction of Ab and T cell responses by B cells.

Cutting edge: CD1d restriction and Th1/Th2/Th17 cytokine secretion by human Vδ3 T cells.

Human γδ T cells expressing the Vδ3 TCR make up a minor lymphocyte subset in blood but are enriched in liver and in patients with some chronic viral infections and leukemias. We analyzed the frequencies, phenotypes, restriction elements, and functions of fresh and expanded peripheral blood Vδ3 T cells. Vδ3 T cells accounted for ~0.2% of circulating T cells, included CD4(+), CD8(+), and CD4(-)CD8(-) subsets, and variably expressed CD56, CD161, HLA-DR, and NKG2D but neither NKG2A nor NKG2C. Vδ3 T cells were sorted and expanded by mitogen stimulation in the presence of IL-2. Expanded Vδ3 T cells recognized CD1d but not CD1a, CD1b, or CD1c. Upon activation, they killed CD1d(+) target cells, released Th1, Th2, and Th17 cytokines, and induced maturation of dendritic cells into APCs. Thus, Vδ3 T cells are glycolipid-reactive T cells with distinct Ag specificities but functional similarities to NKT cells.

Glucagon-like peptide 1 analogue therapy directly modulates innate immune-mediated inflammation in individuals with type 2 diabetes mellitus.

AIMS/HYPOTHESIS: Glucagon-like peptide 1 (GLP-1) is a gut hormone used in the treatment of type 2 diabetes mellitus. There is emerging evidence that GLP-1 has anti-inflammatory activity in humans, with murine studies suggesting an effect on macrophage polarisation. We hypothesised that GLP-1 analogue therapy in individuals with type 2 diabetes mellitus would affect the inflammatory macrophage molecule soluble CD163 (sCD163) and adipocytokine profile. METHODS: We studied ten obese type 2 diabetes mellitus patients starting GLP-1 analogue therapy at a hospital-based diabetes service. We investigated levels of sCD163, TNF-α, IL-1ß, IL-6, adiponectin and leptin by ELISA, before and after 8 weeks of GLP-1 analogue therapy. RESULTS: GLP-1 analogue therapy reduced levels of the inflammatory macrophage activation molecule sCD163 (220 ng/ml vs 171 ng/ml, p < 0.001). This occurred independent of changes in body weight, fructosamine and HbA1c. GLP-1 analogue therapy was associated with a decrease in levels of the inflammatory cytokines TNF-α (264 vs 149 pg/ml, p < 0.05), IL-1ß (2,919 vs 748 pg/ml, p < 0.05) and IL-6 (1,379 vs 461 pg/ml p < 0.05) and an increase in levels of the anti-inflammatory adipokine adiponectin (4,480 vs 6,290 pg/ml, p < 0.002). CONCLUSIONS/INTERPRETATION: In individuals with type 2 diabetes mellitus, GLP-1 analogue therapy reduces the frequency of inflammatory macrophages. This effect is not dependent on the glycaemic or body weight effects of GLP-1.

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.