The glucose transporter 2 regulates CD8+ T cell function via environment sensing.

T cell activation is associated with a profound and rapid metabolic response to meet increased energy demands for cell division, differentiation and development of effector function. Glucose uptake and engagement of the glycolytic pathway are major checkpoints for this event. Here we show that the low-affinity, concentration-dependent glucose transporter 2 (Glut2) regulates the development of CD8+ T cell effector responses in mice by promoting glucose uptake, glycolysis and glucose storage. Expression of Glut2 is modulated by environmental factors including glucose and oxygen availability and extracellular acidification. Glut2 is highly expressed by circulating, recently primed T cells, allowing efficient glucose uptake and storage. In glucose-deprived inflammatory environments, Glut2 becomes downregulated, thus preventing passive loss of intracellular glucose. Mechanistically, Glut2 expression is regulated by a combination of molecular interactions involving hypoxia-inducible factor-1 alpha, galectin-9 and stomatin. Finally, we show that human T cells also rely on this glucose transporter, thus providing a potential target for therapeutic immunomodulation.

Circulating c-Met-Expressing Memory T Cells Define Cardiac Autoimmunity.

BACKGROUND: Autoimmunity is increasingly recognized as a key contributing factor in heart muscle diseases. The functional features of cardiac autoimmunity in humans remain undefined because of the challenge of studying immune responses in situ. We previously described a subset of c-mesenchymal epithelial transition factor (c-Met)-expressing (c-Met+) memory T lymphocytes that preferentially migrate to cardiac tissue in mice and humans. METHODS: In-depth phenotyping of peripheral blood T cells, including c-Met+ T cells, was undertaken in groups of patients with inflammatory and noninflammatory cardiomyopathies, patients with noncardiac autoimmunity, and healthy controls. Validation studies were carried out using human cardiac tissue and in an experimental model of cardiac inflammation. RESULTS: We show that c-Met+ T cells are selectively increased in the circulation and in the myocardium of patients with inflammatory cardiomyopathies. The phenotype and function of c-Met+ T cells are distinct from those of c-Met-negative (c-Met-) T cells, including preferential proliferation to cardiac myosin and coproduction of multiple cytokines (interleukin-4, interleukin-17, and interleukin-22). Furthermore, circulating c-Met+ T cell subpopulations in different heart muscle diseases identify distinct and overlapping mechanisms of heart inflammation. In experimental autoimmune myocarditis, elevations in autoantigen-specific c-Met+ T cells in peripheral blood mark the loss of immune tolerance to the heart. Disease development can be halted by pharmacologic c-Met inhibition, indicating a causative role for c-Met+ T cells. CONCLUSIONS: Our study demonstrates that the detection of circulating c-Met+ T cells may have use in the diagnosis and monitoring of adaptive cardiac inflammation and definition of new targets for therapeutic intervention when cardiac autoimmunity causes or contributes to progressive cardiac injury.

Obesity and diabetes are major risk factors for epicardial adipose tissue inflammation.

BACKGROUNDEpicardial adipose tissue (EAT) directly overlies the myocardium, with changes in its morphology and volume associated with myriad cardiovascular and metabolic diseases. However, EAT's immune structure and cellular characterization remain incompletely described. We aimed to define the immune phenotype of EAT in humans and compare such profiles across lean, obese, and diabetic patients.METHODSWe recruited 152 patients undergoing open-chest coronary artery bypass grafting (CABG), valve repair/replacement (VR) surgery, or combined CABG/VR. Patients' clinical and biochemical data and EAT, subcutaneous adipose tissue (SAT), and preoperative blood samples were collected. Immune cell profiling was evaluated by flow cytometry and complemented by gene expression studies of immune mediators. Bulk RNA-Seq was performed in EAT across metabolic profiles to assess whole-transcriptome changes observed in lean, obese, and diabetic groups.RESULTSFlow cytometry analysis demonstrated EAT was highly enriched in adaptive immune (T and B) cells. Although overweight/obese and diabetic patients had similar EAT cellular profiles to lean control patients, the EAT exhibited significantly (P ≤ 0.01) raised expression of immune mediators, including IL-1, IL-6, TNF-α, and IFN-γ. These changes were not observed in SAT or blood. Neither underlying coronary artery disease nor the presence of hypertension significantly altered the immune profiles observed. Bulk RNA-Seq demonstrated significant alterations in metabolic and inflammatory pathways in the EAT of overweight/obese patients compared with lean controls.CONCLUSIONAdaptive immune cells are the predominant immune cell constituent in human EAT and SAT. The presence of underlying cardiometabolic conditions, specifically obesity and diabetes, rather than cardiac disease phenotype appears to alter the inflammatory profile of EAT. Obese states markedly alter EAT metabolic and inflammatory signaling genes, underlining the impact of obesity on the EAT transcriptome profile.FUNDINGBarts Charity MGU0413, Abbott, Medical Research Council MR/T008059/1, and British Heart Foundation FS/13/49/30421 and PG/16/79/32419.

Isolation of stromal vascular fraction cell suspensions from mouse and human adipose tissues for downstream applications.

This protocol outlines a reliable and versatile approach to isolate stromal vascular fraction cells from different adipose tissues across human and mouse species. A number of downstream applications can then be performed to gain an appreciation of the functional activity of unique adipose tissue-resident cell populations. For complete details on the use and execution of this protocol, please refer to Macdougall et al. (2018).

HIF1α activation in dendritic cells under sterile conditions promotes an anti-inflammatory phenotype through accumulation of intracellular lipids.

Obesity is among the leading causes of elevated cardiovascular disease mortality and morbidity. Adipose tissue dysfunction, insulin resistance and inflammation are recognized as important risk factors for the development of cardiovascular disorders in obesity. Hypoxia appears to be a key factor in adipose tissue dysfunction affecting not only adipocytes but also immune cell function. Here we examined the effect of hypoxia-induced transcription factor HIF1α activation on classical dendritic cell (cDCs) function during obesity. We found that deletion of Hif1α on cDCs results in enhanced adipose-tissue inflammation and atherosclerotic plaque formation in a mouse model of obesity. This effect is mediated by HIF1α-mediated increased lipid synthesis, accumulation of lipid droplets and alter synthesis of lipid mediators. Our findings demonstrate that HIF1α activation in cDCs is necessary to control vessel wall inflammation.

Inflammation and adiposity: new frontiers in atrial fibrillation.

The aetiology of atrial fibrillation (AF) remains poorly understood, despite its growing prevalence and associated morbidity, mortality, and healthcare costs. Obesity is implicated in myriad different disease processes and is now recognized a major risk factor in the pathogenesis of AF. Moreover, the role of distinct adipose tissue depots is a matter of intense scientific interest with the depot directly surrounding the heart-epicardial adipose tissue (EAT) appearing to have the greatest correlation with AF presence and severity. Similarly, inflammation is implicated in the pathophysiology of AF with EAT thought to act as a local depot of inflammatory mediators. These can easily diffuse into atrial tissue with the potential to alter its structural and electrical properties. Various meta-analyses have indicated that EAT size is an independent risk factor for AF with adipose tissue expansion being inevitably associated with a local inflammatory process. Here, we first briefly review adipose tissue anatomy and physiology then move on to the epidemiological data correlating EAT, inflammation, and AF. We focus particularly on discussing the mechanistic basis of how EAT inflammation may precipitate and maintain AF. Finally, we review how EAT can be utilized to help in the clinical management of AF patients and discuss future avenues for research.

Constitutive Activation of ß-Catenin in Conventional Dendritic Cells Increases the Insulin Reserve to Ameliorate the Development of Type 2 Diabetes in Mice.

ß-Cell failure is central to the development of type 2 diabetes mellitus (T2DM). Dysregulation of metabolic and inflammatory processes during obesity contributes to the loss of islet function and impaired ß-cell insulin secretion. Modulating the immune system, therefore, has the potential to ameliorate diseases. We report that inducing sustained expression of ß-catenin in conventional dendritic cells (cDCs) provides a novel mechanism to enhance ß-cell insulin secretion. Intriguingly, cDCs with constitutively activated ß-catenin induced islet expansion by increasing ß-cell proliferation in a model of diet-induced obesity. We further found that inflammation in these islets was reduced. Combined, these effects improved ß-cell insulin secretion, suggesting a unique compensatory mechanism driven by cDCs to generate a greater insulin reserve in response to obesity-induced insulin resistance. Our findings highlight the potential of immune modulation to improve ß-cell mass and function in T2DM.

Adipose tissue dendritic cells in steady-state.

Healthy white adipose tissue (WAT) participates in regulating systemic metabolism, whereas dysfunctional WAT plays a prominent role in the development of obesity-associated co-morbidities. Tissue-resident immune cells are important for maintaining WAT homeostasis, including conventional dendritic cells (cDCs) which are critical in the initiation and regulation of adaptive immune responses. Due to phenotypic overlap with other myeloid cells, the distinct contribution of WAT cDCs has been poorly understood. This review will discuss the contribution of cDCs in the maintenance of WAT homeostasis. In particular, the review will focus on the metabolic cross-talk between cDCs and adipocytes that regulates local immune responses during physiological conditions.

Correction: Signatures of inflammation and impending multiple organ dysfunction in the hyperacute phase of trauma: A prospective cohort study.

[This corrects the article DOI: 10.1371/journal.pmed.1002352.].

Visceral Adipose Tissue Immune Homeostasis Is Regulated by the Crosstalk between Adipocytes and Dendritic Cell Subsets.

Visceral adipose tissue (VAT) has multiple roles in orchestrating whole-body energy homeostasis. In addition, VAT is now considered an immune site harboring an array of innate and adaptive immune cells with a direct role in immune surveillance and host defense. We report that conventional dendritic cells (cDCs) in VAT acquire a tolerogenic phenotype through upregulation of pathways involved in adipocyte differentiation. While activation of the Wnt/ß-catenin pathway in cDC1 DCs induces IL-10 production, upregulation of the PPARγ pathway in cDC2 DCs directly suppresses their activation. Combined, they promote an anti-inflammatory milieu in vivo delaying the onset of obesity-induced chronic inflammation and insulin resistance. Under long-term over-nutrition, changes in adipocyte biology curtail ß-catenin and PPARγ activation, contributing to VAT inflammation.

Signatures of inflammation and impending multiple organ dysfunction in the hyperacute phase of trauma: A prospective cohort study.

BACKGROUND: Severe trauma induces a widespread response of the immune system. This "genomic storm" can lead to poor outcomes, including Multiple Organ Dysfunction Syndrome (MODS). MODS carries a high mortality and morbidity rate and adversely affects long-term health outcomes. Contemporary management of MODS is entirely supportive, and no specific therapeutics have been shown to be effective in reducing incidence or severity. The pathogenesis of MODS remains unclear, and several models are proposed, such as excessive inflammation, a second-hit insult, or an imbalance between pro- and anti-inflammatory pathways. We postulated that the hyperacute window after trauma may hold the key to understanding how the genomic storm is initiated and may lead to a new understanding of the pathogenesis of MODS. METHODS AND FINDINGS: We performed whole blood transcriptome and flow cytometry analyses on a total of 70 critically injured patients (Injury Severity Score [ISS] ≥ 25) at The Royal London Hospital in the hyperacute time period within 2 hours of injury. We compared transcriptome findings in 36 critically injured patients with those of 6 patients with minor injuries (ISS ≤ 4). We then performed flow cytometry analyses in 34 critically injured patients and compared findings with those of 9 healthy volunteers. Immediately after injury, only 1,239 gene transcripts (4%) were differentially expressed in critically injured patients. By 24 hours after injury, 6,294 transcripts (21%) were differentially expressed compared to the hyperacute window. Only 202 (16%) genes differentially expressed in the hyperacute window were still expressed in the same direction at 24 hours postinjury. Pathway analysis showed principally up-regulation of pattern recognition and innate inflammatory pathways, with down-regulation of adaptive responses. Immune deconvolution, flow cytometry, and modular analysis suggested a central role for neutrophils and Natural Killer (NK) cells, with underexpression of T- and B cell responses. In the transcriptome cohort, 20 critically injured patients later developed MODS. Compared with the 16 patients who did not develop MODS (NoMODS), maximal differential expression was seen within the hyperacute window. In MODS versus NoMODS, 363 genes were differentially expressed on admission, compared to only 33 at 24 hours postinjury. MODS transcripts differentially expressed in the hyperacute window showed enrichment among diseases and biological functions associated with cell survival and organismal death rather than inflammatory pathways. There was differential up-regulation of NK cell signalling pathways and markers in patients who would later develop MODS, with down-regulation of neutrophil deconvolution markers. This study is limited by its sample size, precluding more detailed analyses of drivers of the hyperacute response and different MODS phenotypes, and requires validation in other critically injured cohorts. CONCLUSIONS: In this study, we showed how the hyperacute postinjury time window contained a focused, specific signature of the response to critical injury that led to widespread genomic activation. A transcriptomic signature for later development of MODS was present in this hyperacute window; it showed a strong signal for cell death and survival pathways and implicated NK cells and neutrophil populations in this differential response.

The Heterogeneity of Ly6Chi Monocytes Controls Their Differentiation into iNOS+ Macrophages or Monocyte-Derived Dendritic Cells.

Inflammation triggers the differentiation of Ly6Chi monocytes into microbicidal macrophages or monocyte-derived dendritic cells (moDCs). Yet, it is unclear whether environmental inflammatory cues control the polarization of monocytes toward each of these fates or whether specialized monocyte progenitor subsets exist before inflammation. Here, we have shown that naive monocytes are phenotypically heterogeneous and contain an NR4A1- and Flt3L-independent, CCR2-dependent, Flt3+CD11c-MHCII+PU.1hi subset. This subset acted as a precursor for FcγRIII+PD-L2+CD209a+, GM-CSF-dependent moDCs but was distal from the DC lineage, as shown by fate-mapping experiments using Zbtb46. By contrast, Flt3-CD11c-MHCII-PU.1lo monocytes differentiated into FcγRIII+PD-L2-CD209a-iNOS+ macrophages upon microbial stimulation. Importantly, Sfpi1 haploinsufficiency genetically distinguished the precursor activities of monocytes toward moDCs or microbicidal macrophages. Indeed, Sfpi1+/- mice had reduced Flt3+CD11c-MHCII+ monocytes and GM-CSF-dependent FcγRIII+PD-L2+CD209a+ moDCs but generated iNOS+ macrophages more efficiently. Therefore, intercellular disparities of PU.1 expression within naive monocytes segregate progenitor activity for inflammatory iNOS+ macrophages or moDCs.

Direct type I IFN but not MDA5/TLR3 activation of dendritic cells is required for maturation and metabolic shift to glycolysis after poly IC stimulation.

Type I interferons (IFNs) play an important role in direct antiviral defense as well as linking the innate and adaptive immune responses. On dendritic cells (DCs), IFNs facilitate their activation and contribute to CD8(+) and CD4(+) T cell priming. However, the precise molecular mechanism by which IFNs regulate maturation and immunogenicity of DCs in vivo has not been studied in depth. Here we show that, after in vivo stimulation with the TLR ligand poly IC, IFNs dominate transcriptional changes in DCs. In contrast to direct TLR3/mda5 signaling, IFNs are required for upregulation of all pathways associated with DC immunogenicity. In addition, metabolic pathways, particularly the switch from oxidative phosphorylation to glycolysis, are also regulated by IFNs and required for DC maturation. These data provide evidence for a metabolic reprogramming concomitant with DC maturation and offer a novel mechanism by which IFNs modulate DC maturation.

A new synthetic TLR4 agonist, GLA, allows dendritic cells targeted with antigen to elicit Th1 T-cell immunity in vivo.

Protein-based vaccines offer safety and cost advantages but require adjuvants to induce immunity. Here we examined the adjuvant capacity of glucopyranosyl lipid A (GLA), a new synthetic non-toxic analogue of lipopolysaccharide. In mice, in comparison with non-formulated LPS and monophosphoryl lipid A, formulated GLA induced higher antibody titers and generated Type 1 T-cell responses to HIV gag-p24 protein in spleen and lymph nodes, which was dependent on TLR4 expression. Immunization was greatly improved by targeting HIV gag p24 to DCs with an antibody to DEC-205, a DC receptor for antigen uptake and processing. Subcutaneous immunization induced antigen-specific T-cell responses in the intestinal lamina propria. Immunity did not develop in mice transiently depleted of DCs. To understand how GLA works, we studied DCs directly from vaccinated mice. Within 4 h, GLA caused DCs to upregulate CD86 and CD40 and produce cytokines including IL-12p70 in vivo. Importantly, DCs removed from mice 4 h after vaccination became immunogenic, capable of inducing T-cell immunity upon injection into naïve mice. These data indicate that a synthetic and clinically feasible TLR4 agonist rapidly stimulates full maturation of DCs in vivo, allowing for adaptive immunity to develop many weeks to months later.

Microbial stimulation fully differentiates monocytes to DC-SIGN/CD209(+) dendritic cells for immune T cell areas.

Dendritic cells (DCs), critical antigen-presenting cells for immune control, normally derive from bone marrow precursors distinct from monocytes. It is not yet established if the large reservoir of monocytes can develop into cells with critical features of DCs in vivo. We now show that fully differentiated monocyte-derived DCs (Mo-DCs) develop in mice and DC-SIGN/CD209a marks the cells. Mo-DCs are recruited from blood monocytes into lymph nodes by lipopolysaccharide and live or dead gram-negative bacteria. Mobilization requires TLR4 and its CD14 coreceptor and Trif. When tested for antigen-presenting function, Mo-DCs are as active as classical DCs, including cross-presentation of proteins and live gram-negative bacteria on MHC I in vivo. Fully differentiated Mo-DCs acquire DC morphology and localize to T cell areas via L-selectin and CCR7. Thus the blood monocyte reservoir becomes the dominant presenting cell in response to select microbes, yielding DC-SIGN(+) cells with critical functions of DCs.

Dendritic cells require a systemic type I interferon response to mature and induce CD4+ Th1 immunity with poly IC as adjuvant.

Relative to several other toll-like receptor (TLR) agonists, we found polyinosinic:polycytidylic acid (poly IC) to be the most effective adjuvant for Th1 CD4(+) T cell responses to a dendritic cell (DC)-targeted HIV gag protein vaccine in mice. To identify mechanisms for adjuvant action in the intact animal and the polyclonal T cell repertoire, we found poly IC to be the most effective inducer of type I interferon (IFN), which was produced by DEC-205(+) DCs, monocytes, and stromal cells. Antibody blocking or deletion of type I IFN receptor showed that IFN was essential for DC maturation and development of CD4(+) immunity. The IFN-AR receptor was directly required for DCs to respond to poly IC. STAT 1 was also essential, in keeping with the type I IFN requirement, but not type II IFN or IL-12 p40. Induction of type I IFN was mda5 dependent, but DCs additionally used TLR3. In bone marrow chimeras, radioresistant and, likely, nonhematopoietic cells were the main source of IFN, but mda5 was required in both marrow-derived and radioresistant host cells for adaptive responses. Therefore, the adjuvant action of poly IC requires a widespread innate type I IFN response that directly links antigen presentation by DCs to adaptive immunity.

CD59 blockade enhances antigen-specific CD4+ T cell responses in humans: a new target for cancer immunotherapy?

CD59, a broadly expressed GPI-anchored molecule, regulates formation of the membrane attack complex of the complement cascade. We previously demonstrated that mouse CD59 also down-modulates CD4(+) T cell activity in vivo. In this study, we explored the role of CD59 on human CD4(+) T cells. Our data demonstrate that CD59 is up-regulated on activated CD4(+) T cells and serves to down-modulate their activity in response to polyclonal and Ag-specific stimulation. The therapeutic potential of this finding was explored using T cells isolated from colorectal cancer patients. The findings were striking and indicated that blockade of CD59 significantly enhanced the CD4(+) T cell response to two different tumor Ags. These data highlight the potential for manipulating CD59 expression on T cells for boosting weak immune responses, such as those found in individuals with cancer.

CD62L (L-selectin) down-regulation does not affect memory T cell distribution but failure to shed compromises anti-viral immunity.

The down-regulation of CD62L that accompanies T lymphocyte activation is thought to redirect cells away from lymph nodes to sites of infection. In this study, CD62L was maintained on Ag-activated T cells and their distribution, and ability to clear pathogen from peripheral sites determined. CD62L was down-regulated on Ag-specific CD8 T cells in lungs of C57BL/6 mice but maintained in CD62L transgenic mice at day 8 after influenza infection. However, the numbers of influenza-specific CD8 T cells recruited were similar in CD62L transgenic and C57BL/6 mice. Memory CD8 T cell numbers in the lungs and noninvolved organs 100 days after primary infection were similar in CD62L transgenic and C57BL/6 mice, despite differing CD62L expression. Transgenic mice expressing wild-type CD62L cleared a recombinant vaccinia virus expressing an influenza-derived CD8 T cell epitope as efficiently as C57BL/6 mice. However, transgenic mice expressing a protease resistant mutant of CD62L showed significantly delayed viral clearance, despite normal CTL generation and the presence of CD107a and IFN-gamma expressing influenza-specific CD8 T cells. These results demonstrate that CD62L down-regulation is not required for CD8 memory cells to home to sites of infection. However, their ability to clear virus is significantly compromised if CD62L shedding is abrogated.

CD59a deficiency exacerbates influenza-induced lung inflammation through complement-dependent and -independent mechanisms.

Influenza-specific immune activity not only promotes virus clearance but also causes immunopathology, thereby underlining the importance of mounting a measured anti-viral immune response. Since complement bridges both the innate and adaptive immune systems and has been implicated in defence against influenza, the role of the complement regulator CD59a in modulating the response to influenza was explored. For this purpose, immune responses to influenza virus, strain E61-13-H17, in mice deficient in the complement regulator protein CD59a (Cd59a(-/-) mice) were compared to those in wild-type mice. The severity of lung inflammation was significantly enhanced in the lungs of Cd59a(-/-) mice with increased numbers of infiltrating neutrophils and CD4(+) T cells. When complement was inhibited using soluble complement receptor 1, the frequency of lung-infiltrating neutrophils in influenza-infected Cd59a(-/-) mice was much reduced whilst numbers of CD4(+) T cells remained unchanged. These results demonstrate that CD59a, previously defined as a complement regulator, modulates both the innate and adaptive immune response to influenza virus by both complement-dependent and -independent mechanisms.

CD59a deficient mice display reduced B cell activity and antibody production in response to T-dependent antigens.

CD59a is the primary regulator of membrane attack complex in mice. Recently, we have shown that CD59a-deficient (Cd59a-/-) mice exhibit enhanced CD4+ T cell responses. Here, we explored the effects of CD59a on B cell function and antibody production. Contrary to our expectations, Cd59a-/- mice showed a decreased humoral immune response to a T cell dependent antigen, sheep red blood cells. We found that the decreased humoral immune response was associated with a reduction in plasma cell number in vivo and reduced ability to respond to stimuli during in vitro culture experiments. Using MLR studies in which purified wild type or Cd59a-/- CD4+ T cells were mixed with purified B cells from each source, we found that the reduced B cell activation was largely due to the absence of CD59a on CD4+ T cells. Furthermore, a CD59a fusion protein bound specifically to mouse B cells, and enhanced B cell proliferation in a MLR, demonstrating that B cells express an as yet unidentified ligand for CD59a that aids in B cell activation.