Metabolic reprogramming of tumor-associated macrophages using glutamine antagonist JHU083 drives tumor immunity in myeloid-rich prostate and bladder cancer tumors.

Glutamine metabolism in tumor microenvironments critically regulates anti-tumor immunity. Using glutamine-antagonist prodrug JHU083, we report potent tumor growth inhibition in urologic tumors by JHU083-reprogrammed tumor-associated macrophages (TAMs) and tumor-infiltrating monocytes (TIMs). We show JHU083-mediated glutamine antagonism in tumor microenvironments induces TNF, pro-inflammatory, and mTORC1 signaling in intratumoral TAM clusters. JHU083-reprogrammed TAMs also exhibit increased tumor cell phagocytosis and diminished pro-angiogenic capacities. In vivo inhibition of TAM glutamine consumption resulted in increased glycolysis, a broken TCA cycle, and purine metabolism disruption. Although the anti-tumor effect of glutamine antagonism on tumor-infiltrating T cells was moderate, JHU083 promoted a stem cell-like phenotype in CD8+ T cells and decreased Treg abundance. Finally, JHU083 caused a ubiquitous shutdown in glutamine utilizing metabolic pathways in tumor cells, leading to reduced HIF-1alpha, c-MYC phosphorylation, and induction of tumor cell apoptosis, all key anti-tumor features.

Prostaglandin E2 controls the metabolic adaptation of T cells to the intestinal microenvironment.

Immune cells must adapt to different environments during the course of an immune response. Here we study the adaptation of CD8+ T cells to the intestinal microenvironment and how this process shapes the establishment of the CD8+ T cell pool. CD8+ T cells progressively remodel their transcriptome and surface phenotype as they enter the gut wall, and downregulate expression of mitochondrial genes. Human and mouse intestinal CD8+ T cells have reduced mitochondrial mass, but maintain a viable energy balance to sustain their function. We find that the intestinal microenvironment is rich in prostaglandin E2 (PGE2), which drives mitochondrial depolarization in CD8+ T cells. Consequently, these cells engage autophagy to clear depolarized mitochondria, and enhance glutathione synthesis to scavenge reactive oxygen species (ROS) that result from mitochondrial depolarization. Impairing PGE2 sensing promotes CD8+ T cell accumulation in the gut, while tampering with autophagy and glutathione negatively impacts the T cell pool. Thus, a PGE2-autophagy-glutathione axis defines the metabolic adaptation of CD8+ T cells to the intestinal microenvironment, to ultimately influence the T cell pool.

A Delphi approach to establishing consensus on best practice for the diagnosis and treatment of Demodex blepharitis.

Demodex blepharitis does not have agreed standardized guidelines. The aim of this study was to classify signs and symptoms and to develop appropriate management strategies for Demodex blepharitis from a consensus of expert advice. METHODS: A total of 11 anterior segment experts (ophthalmologists, optometrists and a contact lens optician) working in the United Kingdom participated in a modified 2-round Delphi panel. A mixed-methods approach was adopted and a survey questionnaire for round 1 was formulated, constructed from information in the available literature. Based on panel responses from round 1, feedback was provided and a round 2 questionnaire was formulated. More than two-thirds majority (72%) was used for consensus building. RESULTS: Based on the clinical presentation of signs and symptoms along with associated conditions and risk factors, a diagnostic algorithm was proposed for the clinical investigation of Demodex blepharitis. A treatment algorithm was also proposed with first-line and second-line treatment recommendations for Demodex blepharitis. CONCLUSION: The recommendation from this study provides the first effort in formulating clinical diagnostic algorithm and management guidelines for Demodex blepharitis. The guidelines include appropriate magnification on the slit lamp, associated signs, symptoms, risk factors and suggested management options. These guidelines can be used in a routine eyecare setting to encourage eyecare practitioners in tailoring the investigation and management of Demodex blepharitis.

Cooperation of ILC2s and TH2 cells in the expulsion of intestinal helminth parasites.

Type 2 immune responses form a critical defence against enteric worm infections. In recent years, mouse models have revealed shared and unique functions for group 2 innate lymphoid cells and T helper 2 cells in type 2 immune response to intestinal helminths. Both cell types use similar innate effector functions at the site of infection, whereas each population has distinct roles during different stages of infection. In this Perspective, we review the underlying mechanisms used by group 2 innate lymphoid cells and T helper 2 cells to cooperate with each other and suggest an overarching model of the interplay between these cell types over the course of a helminth infection.

Metabolic and functional remodeling of colonic macrophages in response to high-fat diet-induced obesity.

Little is known about the effects of high-fat diet (HFD)-induced obesity on resident colonic lamina propria (LP) macrophages (LPMs) function and metabolism. Here, we report that obesity and diabetes resulted in increased macrophage infiltration in the colon. These macrophages exhibited the residency phenotype CX3CR1hiMHCIIhi and were CD4-TIM4-. During HFD, resident colonic LPM exhibited a lipid metabolism gene expression signature that overlapped that used to define lipid-associated macrophages (LAMs). Via single-cell RNA sequencing, we identified a sub-cluster of macrophages, increased in HFD, that were responsible for the LAM signature. Compared to other macrophages in the colon, these cells were characterized by elevated glycolysis, phagocytosis, and efferocytosis signatures. CX3CR1hiMHCIIhi colonic resident LPMs had fewer lipid droplets (LDs) and decreased triacylglycerol (TG) content compared to equivalent cells in lean mice and exhibited increased phagocytic capacity, suggesting that HFD induces adaptive responses in LPMs to limit bacterial translocation.

Metabolic rewiring tunes dermal macrophages in staphylococcal skin infection.

The skin needs to balance tolerance of colonizing microflora with rapid detection of potential pathogens. Flexible response mechanisms would seem most suitable to accommodate the dynamic challenges of effective antimicrobial defense and restoration of tissue homeostasis. Here, we dissected macrophage-intrinsic mechanisms and microenvironmental cues that tune macrophage signaling in localized skin infection with the colonizing and opportunistic pathogen Staphylococcus aureus. Early in skin infection, the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by γδ T cells and hypoxic conditions within the dermal microenvironment diverted macrophages away from a homeostatic M-CSF- and hypoxia-inducible factor 1α (HIF-1α)-dependent program. This allowed macrophages to be metabolically rewired for maximal inflammatory activity, which requires expression of Irg1 and generation of itaconate, but not HIF-1α. This multifactorial macrophage rewiring program was required for both the timely clearance of bacteria and for the provision of local immune memory. These findings indicate that immunometabolic conditioning allows dermal macrophages to cycle between antimicrobial activity and protection against secondary infections.

Demodex Blepharitis: A Survey-Based Approach to Investigate Knowledge, Attitudes, and Practices Among Optometrists in India.

Purpose: To investigate the knowledge, attitudes, and practice towards Demodex blepharitis among optometrists in India. Methods: The study was conducted in the form of an online survey using Research Electronic Data Capture (REDCap). The survey link was distributed via direct e-mail and social media platforms, and it was comprised of 20 questions divided into two sections. The first section focused on the practitioners' demographics and their views on the general health of the eyelid. The second section of the survey was specific and aimed at obtaining information on identifying and treating Demodex blepharitis, and was only completed by those respondents who looked for Demodex mites. Results: The survey was completed by 174 optometrists. The prevalence of blepharitis in the general population was judged by the respondents to be 40%, whereas the prevalence of Demodex mites was estimated to be 29%. Interestingly, the prevalence of Demodex mites in people with blepharitis was estimated to be 30%. This estimated prevalence was substantially lower than that reported in the literature on the subject. 66% of participants believed Demodex mites to be a significant cause of ocular discomfort, whereas only 30% of participants would intervene to diagnose and manage Demodex blepharitis in their patients. Optometrists differed in their preferred method of diagnosis and management of Demodex infestation in eyelids. Conclusion: The result of this survey suggests that Demodex blepharitis is a highly under-diagnosed condition in India, with nearly 30% of surveyed optometrists managing this condition. The study also observed a lack of awareness and consensus among surveyed optometrists with regards to diagnosis and appropriate treatment methods to control Demodex infestation in eyelids.

Metabolism in type 2 immune responses.

The immune response is tailored to the environment in which it takes place. Immune cells sense and adapt to changes in their surroundings, and it is now appreciated that in addition to cytokines made by stromal and epithelial cells, metabolic cues provide key adaptation signals. Changes in immune cell activation states are linked to changes in cellular metabolism that support function. Furthermore, metabolites themselves can signal between as well as within cells. Here, we discuss recent progress in our understanding of how metabolic regulation relates to type 2 immunity firstly by considering specifics of metabolism within type 2 immune cells and secondly by stressing how type 2 immune cells are integrated more broadly into the metabolism of the organism as a whole.

Prostaglandin E 2 controls the metabolic adaptation of T cells to the intestinal microenvironment.

Immune cells must adapt to different environments during the course of an immune response. We studied the adaptation of CD8 + T cells to the intestinal microenvironment and how this process shapes their residency in the gut. CD8 + T cells progressively remodel their transcriptome and surface phenotype as they acquire gut residency, and downregulate expression of mitochondrial genes. Human and mouse gut-resident CD8 + T cells have reduced mitochondrial mass, but maintain a viable energy balance to sustain their function. We found that the intestinal microenvironment is rich in prostaglandin E 2 (PGE 2 ), which drives mitochondrial depolarization in CD8 + T cells. Consequently, these cells engage autophagy to clear depolarized mitochondria, and enhance glutathione synthesis to scavenge reactive oxygen species (ROS) that result from mitochondrial depolarization. Impairing PGE 2 sensing promotes CD8 + T cell accumulation in the gut, while tampering with autophagy and glutathione negatively impacts the T cell population. Thus, a PGE 2 -autophagy-glutathione axis defines the metabolic adaptation of CD8 + T cells to the intestinal microenvironment, to ultimately influence the T cell pool.

Helminth egg derivatives as proregenerative immunotherapies.

The immune system is increasingly recognized as an important regulator of tissue repair. We developed a regenerative immunotherapy from the helminth Schistosoma mansoni soluble egg antigen (SEA) to stimulate production of interleukin (IL)-4 and other type 2-associated cytokines without negative infection-related sequelae. The regenerative SEA (rSEA) applied to a murine muscle injury induced accumulation of IL-4-expressing T helper cells, eosinophils, and regulatory T cells and decreased expression of IL-17A in gamma delta (γδ) T cells, resulting in improved repair and decreased fibrosis. Encapsulation and controlled release of rSEA in a hydrogel further enhanced type 2 immunity and larger volumes of tissue repair. The broad regenerative capacity of rSEA was validated in articular joint and corneal injury models. These results introduce a regenerative immunotherapy approach using natural helminth derivatives.

Phosphoinositide acyl chain saturation drives CD8+ effector T cell signaling and function.

How lipidome changes support CD8+ effector T (Teff) cell differentiation is not well understood. Here we show that, although naive T cells are rich in polyunsaturated phosphoinositides (PIPn with 3-4 double bonds), Teff cells have unique PIPn marked by saturated fatty acyl chains (0-2 double bonds). PIPn are precursors for second messengers. Polyunsaturated phosphatidylinositol bisphosphate (PIP2) exclusively supported signaling immediately upon T cell antigen receptor activation. In late Teff cells, activity of phospholipase C-γ1, the enzyme that cleaves PIP2 into downstream mediators, waned, and saturated PIPn became essential for sustained signaling. Saturated PIP was more rapidly converted to PIP2 with subsequent recruitment of phospholipase C-γ1, and loss of saturated PIPn impaired Teff cell fitness and function, even in cells with abundant polyunsaturated PIPn. Glucose was the substrate for de novo PIPn synthesis, and was rapidly utilized for saturated PIP2 generation. Thus, separate PIPn pools with distinct acyl chain compositions and metabolic dependencies drive important signaling events to initiate and then sustain effector function during CD8+ T cell differentiation.

Resident TH2 cells orchestrate adipose tissue remodeling at a site adjacent to infection.

Type 2 immunity is associated with adipose tissue (AT) homeostasis and infection with parasitic helminths, but whether AT participates in immunity to these parasites is unknown. We found that the fat content of mesenteric AT (mAT) declined in mice during infection with a gut-restricted helminth. This was associated with the accumulation of metabolically activated, interleukin-33 (IL-33), thymic stromal lymphopoietin (TSLP), and extracellular matrix (ECM)-producing stromal cells. These cells shared transcriptional features, including the expression of Dpp4 and Pi16, with multipotent progenitor cells (MPC) that have been identified in numerous tissues and are reported to be capable of differentiating into fibroblasts and adipocytes. Concomitantly, mAT became infiltrated with resident T helper 2 (TH2) cells that responded to TSLP and IL-33 by producing stromal cell-stimulating cytokines, including transforming growth factor ß1 (TGFß1) and amphiregulin. These TH2 cells expressed genes previously associated with type 2 innate lymphoid cells (ILC2), including Nmur1, Calca, Klrg1, and Arg1, and persisted in mAT for at least 11 months after anthelmintic drug-mediated clearance of infection. We found that MPC and TH2 cells localized to ECM-rich interstitial spaces that appeared shared between mesenteric lymph node, mAT, and intestine. Stromal cell expression of epidermal growth factor receptor (EGFR), the receptor for amphiregulin, was required for immunity to infection. Our findings point to the importance of MPC and TH2 cell interactions within the interstitium in orchestrating AT remodeling and immunity to an intestinal infection.

An LKB1-mitochondria axis controls TH17 effector function.

CD4+ T cell differentiation requires metabolic reprogramming to fulfil the bioenergetic demands of proliferation and effector function, and enforce specific transcriptional programmes1-3. Mitochondrial membrane dynamics sustains mitochondrial processes4, including respiration and tricarboxylic acid (TCA) cycle metabolism5, but whether mitochondrial membrane remodelling orchestrates CD4+ T cell differentiation remains unclear. Here we show that unlike other CD4+ T cell subsets, T helper 17 (TH17) cells have fused mitochondria with tight cristae. T cell-specific deletion of optic atrophy 1 (OPA1), which regulates inner mitochondrial membrane fusion and cristae morphology6, revealed that TH17 cells require OPA1 for its control of the TCA cycle, rather than respiration. OPA1 deletion amplifies glutamine oxidation, leading to impaired NADH/NAD+ balance and accumulation of TCA cycle metabolites and 2-hydroxyglutarate-a metabolite that influences the epigenetic landscape5,7. Our multi-omics approach revealed that the serine/threonine kinase liver-associated kinase B1 (LKB1) couples mitochondrial function to cytokine expression in TH17 cells by regulating TCA cycle metabolism and transcriptional remodelling. Mitochondrial membrane disruption activates LKB1, which restrains IL-17 expression. LKB1 deletion restores IL-17 expression in TH17 cells with disrupted mitochondrial membranes, rectifying aberrant TCA cycle glutamine flux, balancing NADH/NAD+ and preventing 2-hydroxyglutarate production from the promiscuous activity of the serine biosynthesis enzyme phosphoglycerate dehydrogenase (PHGDH). These findings identify OPA1 as a major determinant of TH17 cell function, and uncover LKB1 as a sensor linking mitochondrial cues to effector programmes in TH17 cells.

Trained immunity of alveolar macrophages requires metabolic rewiring and type 1 interferon signaling.

Environmental microbial triggers shape the development and functionality of the immune system. Alveolar macrophages (AMs), tissue-resident macrophages of the lungs, are in constant and direct contact with inhaled particles and microbes. Such exposures likely impact AM reactivity to subsequent challenges by immunological imprinting mechanisms referred to as trained immunity. Here, we investigated whether a ubiquitous microbial compound has the potential to induce AM training in vivo. We discovered that intranasal exposure to ambient amounts of lipopolysaccharide (LPS) induced a pronounced AM memory response, characterized by enhanced reactivity upon pneumococcal challenge. Exploring the mechanistic basis of AM training, we identified a critical role of type 1 interferon signaling and found that inhibition of fatty acid oxidation and glutaminolysis significantly attenuated the training effect. Notably, adoptive transfer of trained AMs resulted in increased bacterial loads and tissue damage upon subsequent pneumococcal infection. In contrast, intranasal pre-exposure to LPS promoted bacterial clearance, highlighting the complexity of stimulus-induced immune responses, which likely involve multiple cell types and may depend on the local immunological and metabolic environment. Collectively, our findings demonstrate the profound impact of ambient microbial exposure on pulmonary immune memory and reveal tissue-specific features of trained immunity.

Intracellular infection and immune system cues rewire adipocytes to acquire immune function.

Adipose tissue (AT) plays a central role in systemic metabolic homeostasis, but its function during bacterial infection remains unclear. Following subcutaneous bacterial infection, adipocytes surrounding draining lymph nodes initiated a transcriptional response indicative of stimulation with IFN-γ and a shift away from lipid metabolism toward an immunologic function. Natural killer (NK) and invariant NK T (iNKT) cells were identified as sources of infection-induced IFN-γ in perinodal AT (PAT). IFN-γ induced Nos2 expression in adipocytes through a process dependent on nuclear-binding oligomerization domain 1 (NOD1) sensing of live intracellular bacteria. iNOS expression was coupled to metabolic rewiring, inducing increased diversion of extracellular L-arginine through the arginosuccinate shunt and urea cycle to produce nitric oxide (NO), directly mediating bacterial clearance. In vivo, control of infection in adipocytes was dependent on adipocyte-intrinsic sensing of IFN-γ and expression of iNOS. Thus, adipocytes are licensed by innate lymphocytes to acquire anti-bacterial functions during infection.

Leptin Signaling Suppression in Macrophages Improves Immunometabolic Outcomes in Obesity.

Obesity is a major concern for global health care systems. Systemic low-grade inflammation in obesity is a major risk factor for insulin resistance. Leptin is an adipokine secreted by the adipose tissue that functions by controlling food intake, leading to satiety. Leptin levels are increased in obesity. Here, we show that leptin enhances the effects of LPS in macrophages, intensifying the production of cytokines, glycolytic rates, and morphological and functional changes in the mitochondria through an mTORC2-dependent, mTORC1-independent mechanism. Leptin also boosts the effects of IL-4 in macrophages, leading to increased oxygen consumption, expression of macrophage markers associated with a tissue repair phenotype, and wound healing. In vivo, hyperleptinemia caused by diet-induced obesity increases the inflammatory response by macrophages. Deletion of leptin receptor and subsequently of leptin signaling in myeloid cells (ObR-/-) is sufficient to improve insulin resistance in obese mice and decrease systemic inflammation. Our results indicate that leptin acts as a systemic nutritional checkpoint to regulate macrophage fitness and contributes to obesity-induced inflammation and insulin resistance. Thus, specific interventions aimed at downstream modulators of leptin signaling may represent new therapeutic targets to treat obesity-induced systemic inflammation.

A common framework of monocyte-derived macrophage activation.

Macrophages populate every organ during homeostasis and disease, displaying features of tissue imprinting and heterogeneous activation. The disconnected picture of macrophage biology that has emerged from these observations is a barrier for integration across models or with in vitro macrophage activation paradigms. We set out to contextualize macrophage heterogeneity across mouse tissues and inflammatory conditions, specifically aiming to define a common framework of macrophage activation. We built a predictive model with which we mapped the activation of macrophages across 12 tissues and 25 biological conditions, finding a notable commonality and finite number of transcriptional profiles, in particular among infiltrating macrophages, which we modeled as defined stages along four conserved activation paths. These activation paths include a "phagocytic" regulatory path, an "inflammatory" cytokine-producing path, an "oxidative stress" antimicrobial path, or a "remodeling" extracellular matrix deposition path. We verified this model with adoptive cell transfer experiments and identified transient RELMɑ expression as a feature of monocyte-derived macrophage tissue engraftment. We propose that this integrative approach of macrophage classification allows the establishment of a common predictive framework of monocyte-derived macrophage activation in inflammation and homeostasis.

Gene-selective transcription promotes the inhibition of tissue reparative macrophages by TNF.

Anti-TNF therapies are a core anti-inflammatory approach for chronic diseases such as rheumatoid arthritis and Crohn's Disease. Previously, we and others found that TNF blocks the emergence and function of alternative-activated or M2 macrophages involved in wound healing and tissue-reparative functions. Conceivably, anti-TNF drugs could mediate their protective effects in part by an altered balance of macrophage activity. To understand the mechanistic basis of how TNF regulates tissue-reparative macrophages, we used RNAseq, scRNAseq, ATACseq, time-resolved phospho-proteomics, gene-specific approaches, metabolic analysis, and signaling pathway deconvolution. We found that TNF controls tissue-reparative macrophage gene expression in a highly gene-specific way, dependent on JNK signaling via the type 1 TNF receptor on specific populations of alternative-activated macrophages. We further determined that JNK signaling has a profound and broad effect on activated macrophage gene expression. Our findings suggest that TNF's anti-M2 effects evolved to specifically modulate components of tissue and reparative M2 macrophages and TNF is therefore a context-specific modulator of M2 macrophages rather than a pan-M2 inhibitor.

Macrophages acquire a TNF-dependent inflammatory memory in allergic asthma.

BACKGROUND: Infectious agents can reprogram or "train" macrophages and their progenitors to respond more readily to subsequent insults. However, whether such an inflammatory memory exists in type 2 inflammatory conditions such as allergic asthma was not known. OBJECTIVE: We sought to decipher macrophage-trained immunity in allergic asthma. METHODS: We used a combination of clinical sampling of house dust mite (HDM)-allergic patients, HDM-induced allergic airway inflammation in mice, and an in vitro training setup to analyze persistent changes in macrophage eicosanoid, cytokine, and chemokine production as well as the underlying metabolic and epigenetic mechanisms. Transcriptional and metabolic profiles of patient-derived and in vitro trained macrophages were assessed by RNA sequencing or metabolic flux analysis and liquid chromatography-tandem mass spectrometry analysis, respectively. RESULTS: We found that macrophages differentiated from bone marrow or blood monocyte progenitors of HDM-allergic mice or asthma patients show inflammatory transcriptional reprogramming and excessive mediator (TNF-α, CCL17, leukotriene, PGE2, IL-6) responses upon stimulation. Macrophages from HDM-allergic mice initially exhibited a type 2 imprint, which shifted toward a classical inflammatory training over time. HDM-induced allergic airway inflammation elicited a metabolically activated macrophage phenotype, producing high amounts of 2-hydroxyglutarate (2-HG). HDM-induced macrophage training in vitro was mediated by a formyl peptide receptor 2-TNF-2-HG-PGE2/PGE2 receptor 2 axis, resulting in an M2-like macrophage phenotype with high CCL17 production. TNF blockade by etanercept or genetic ablation of Tnf in myeloid cells prevented the inflammatory imprinting of bone marrow-derived macrophages from HDM-allergic mice. CONCLUSION: Allergen-triggered inflammation drives a TNF-dependent innate memory, which may perpetuate and exacerbate chronic type 2 airway inflammation and thus represents a target for asthma therapy.