Targeted removal of macrophage-secreted interleukin-1 receptor antagonist protects against lethal Candida albicans sepsis.

Invasive fungal infections are associated with high mortality rates, and the lack of efficient treatment options emphasizes an urgency to identify underlying disease mechanisms. We report that disseminated Candida albicans infection is facilitated by interleukin-1 receptor antagonist (IL-1Ra) secreted from macrophages in two temporally and spatially distinct waves. Splenic CD169+ macrophages release IL-1Ra into the bloodstream, impeding early neutrophil recruitment. IL-1Ra secreted by monocyte-derived tissue macrophages further impairs pathogen containment. Therapeutic IL-1Ra neutralization restored the functional competence of neutrophils, corrected maladapted hyper-inflammation, and eradicated the otherwise lethal infection. Conversely, augmentation of macrophage-secreted IL-1Ra by type I interferon severely aggravated disease mortality. Our study uncovers how a fundamental immunoregulatory mechanism mediates the high disease susceptibility to invasive candidiasis. Furthermore, interferon-stimulated IL-1Ra secretion may exacerbate fungal dissemination in human patients with secondary candidemia. Macrophage-secreted IL-1Ra should be considered as an additional biomarker and potential therapeutic target in severe systemic candidiasis.

PPAR-γ regulates the effector function of human T helper 9 cells by promoting glycolysis.

T helper 9 (TH9) cells promote allergic tissue inflammation and express the type 2 cytokines, IL-9 and IL-13, as well as the transcription factor, PPAR-γ. However, the functional role of PPAR-γ in human TH9 cells remains unknown. Here, we demonstrate that PPAR-γ drives activation-induced glycolysis, which, in turn, promotes the expression of IL-9, but not IL-13, in an mTORC1-dependent manner. In vitro and ex vivo experiments show that the PPAR-γ-mTORC1-IL-9 pathway is active in TH9 cells in human skin inflammation. Additionally, we find dynamic regulation of tissue glucose levels in acute allergic skin inflammation, suggesting that in situ glucose availability is linked to distinct immunological functions in vivo. Furthermore, paracrine IL-9 induces expression of the lactate transporter, MCT1, in TH cells and promotes their aerobic glycolysis and proliferative capacity. Altogether, our findings uncover a hitherto unknown relationship between PPAR-γ-dependent glucose metabolism and pathogenic effector functions in human TH9 cells.

Differential sensitivity of inflammatory macrophages and alternatively activated macrophages to ferroptosis.

Acumulation of oxidized membrane lipids ultimately results in ferroptotic cell death, which can be prevented by the selenoenzyme glutathione peroxidase 4 (Gpx4). In vivo conditions promoting ferroptosis and susceptible cell types are still poorly defined. In this study, we analyzed the conditional deletion of Gpx4 in mice specifically in the myeloid cell lineages. Surprisingly, development and maintenance of LysM+ macrophages and neutrophils, as well as CD11c+ monocyte-derived macrophages and dendritic cells were unaffected in the absence of Gpx4. Gpx4-deficient macrophages mounted an unaltered proinflammatory cytokine response including IL-1ß production following stimulation with TLR ligands and activation of several inflammasomes. Accordingly, Gpx4fl/fl LysM-cre mice were protected from bacterial and protozoan infections. Despite having the capacity to differentiate to alternatively activated macrophages (AAM), these cells lacking Gpx4 triggered ferroptosis both in vitro and in vivo following IL-4 overexpression and nematode infection. Exposure to nitric oxide restored viability of Gpx4-deficient AAM, while inhibition of iNOS in proinflammatory macrophages had no effect. These data together suggest that activation cues of tissue macrophages determine sensitivity to lipid peroxidation and ferroptotic cell death.

Serpinb1a Is Dispensable for the Development and Cytokine Response of Invariant Natural Killer T Cell Subsets.

Invariant natural killer T (iNKT) cells are innate-like T lymphocytes. They quickly respond to antigenic stimulation by producing copious amounts of cytokines and chemokines. iNKT precursors differentiate into three subsets iNKT1, iNKT2, and iNKT17 with specific cytokine production signatures. While key transcription factors drive subset differentiation, factors that regulate iNKT subset homeostasis remain incompletely defined. Transcriptomic analyses of thymic iNKT subsets indicate that Serpinb1a is one of the most specific transcripts for iNKT17 cells suggesting that iNKT cell maintenance and function may be regulated by Serpinb1a. Serpinb1a is a major survival factor in neutrophils and prevents cell death in a cell-autonomous manner. It also controls inflammation in models of bacterial and viral infection as well as in LPS-driven inflammation. Here, we examined the iNKT subsets in neutropenic Serpinb1a-/- mice as well as in Serpinb1a-/- mice with normal neutrophil counts due to transgenic re-expression of SERPINB1 in neutrophils. In steady state, we found no significant effect of Serpinb1a-deficiency on the proliferation and numbers of iNKT subsets in thymus, lymph nodes, lung, liver and spleen. Following systemic activation with α-galactosylceramide, the prototypic glycolipid agonist of iNKT cells, we observed similar serum levels of IFN-γ and IL-4 between genotypes. Moreover, splenic dendritic cells showed normal upregulation of maturation markers following iNKT cell activation with α-galactosylceramide. Finally, lung instillation of α-galactosylceramide induced a similar recruitment of neutrophils and production of iNKT-derived cytokines IL-17, IFN-γ, and IL-4 in wild-type and Serpinb1a-/- mice. Taken together, our results indicate that Serpinb1a, while dominantly expressed in iNKT17 cells, is not essential for iNKT cell homeostasis, subset differentiation and cytokine release.

PLCγ1 suppression promotes the adaptation of KRAS-mutant lung adenocarcinomas to hypoxia.

Mutant KRAS modulates the metabolic plasticity of cancer cells to confer a growth advantage during hypoxia, but the molecular underpinnings are largely unknown. Using a lipidomic screen, we found that PLCγ1 is suppressed during hypoxia in KRAS-mutant human lung adenocarcinoma cancer cell lines. Suppression of PLCγ1 in hypoxia promotes a less oxidative cancer cell metabolism state, reduces the formation of mitochondrial reactive oxygen species and switches tumour bioenergetics towards glycolysis by impairing Ca2+ entry into the mitochondria. This event prevents lipid peroxidation, antagonizes apoptosis and increases cancer cell proliferation. Accordingly, loss of function of Plcg1 in a mouse model of KrasG12D-driven lung adenocarcinoma increased the expression of glycolytic genes, boosted tumour growth and reduced survival. In patients with KRAS-mutant lung adenocarcinomas, low PLCγ1 expression correlates with increased expression of hypoxia markers and predicts poor patient survival. Thus, our work reveals a mechanism of cancer cell adaptation to hypoxia with potential therapeutic value.

TNIK signaling imprints CD8+ T cell memory formation early after priming.

Co-stimulatory signals, cytokines and transcription factors regulate the balance between effector and memory cell differentiation during T cell activation. Here, we analyse the role of the TRAF2-/NCK-interacting kinase (TNIK), a signaling molecule downstream of the tumor necrosis factor superfamily receptors such as CD27, in the regulation of CD8+ T cell fate during acute infection with lymphocytic choriomeningitis virus. Priming of CD8+ T cells induces a TNIK-dependent nuclear translocation of ß-catenin with consecutive Wnt pathway activation. TNIK-deficiency during T cell activation results in enhanced differentiation towards effector cells, glycolysis and apoptosis. TNIK signaling enriches for memory precursors by favouring symmetric over asymmetric cell division. This enlarges the pool of memory CD8+ T cells and increases their capacity to expand after re-infection in serial re-transplantation experiments. These findings reveal that TNIK is an important regulator of effector and memory T cell differentiation and induces a population of stem cell-like memory T cells.

Reciprocal Crosstalk between Dendritic Cells and Natural Killer T Cells: Mechanisms and Therapeutic Potential.

Natural killer T cells carrying a highly conserved, semi-invariant T cell receptor (TCR) [invariant natural killer T (iNKT) cells] are a subset of unconventional T lymphocytes that recognize glycolipids presented by CD1d molecules. Although CD1d is expressed on a variety of hematopoietic and non-hematopoietic cells, dendritic cells (DCs) are key presenters of glycolipid antigen in vivo. When stimulated through their TCR, iNKT cells rapidly secrete copious amounts of cytokines and induce maturation of DCs, thereby facilitating coordinated stimulation of innate and adaptive immune responses. The bidirectional crosstalk between DCs and iNKT cells determines the functional outcome of iNKT cell-targeted responses and iNKT cell agonists are used and currently being evaluated as adjuvants to enhance the efficacy of antitumor immunotherapy. This review illustrates mechanistic underpinnings of reciprocal DCs and iNKT cell interactions and discusses how those can be harnessed for cancer therapy.

The autophagy machinery restrains iNKT cell activation through CD1D1 internalization.

Invariant natural killer T (iNKT) cells are innate T cells with powerful immune regulatory functions that recognize glycolipid antigens presented by the CD1D protein. While iNKT cell-activating glycolipids are currently being explored for their efficacy to improve immunotherapy against infectious diseases and cancer, little is known about the mechanisms that control CD1D antigen presentation and iNKT cell activation in vivo. CD1D molecules survey endocytic pathways to bind lipid antigens in MHC class II-containing compartments (MIICs) before recycling to the plasma membrane. Autophagosomes intersect with MIICs and autophagy-related proteins are known to support antigen loading for increased CD4+ T cell immunity. Here, we report that mice with dendritic cell (DC)-specific deletion of the essential autophagy gene Atg5 showed better CD1D1-restricted glycolipid presentation in vivo. These effects led to enhanced iNKT cell cytokine production upon antigen recognition and lower bacterial loads during Sphingomonas paucimobilis infection. Enhanced iNKT cell activation was independent of receptor-mediated glycolipid uptake or costimulatory signals. Instead, loss of Atg5 in DCs impaired clathrin-dependent internalization of CD1D1 molecules via the adaptor protein complex 2 (AP2) and, thus, increased surface expression of stimulatory CD1D1-glycolipid complexes. These findings indicate that the autophagic machinery assists in the recruitment of AP2 to CD1D1 molecules resulting in attenuated iNKT cell activation, in contrast to the supporting role of macroautophagy in CD4+ T cell stimulation.

Cyclopentenone-containing oxidized phospholipids and their isoprostanes as pro-resolving mediators of inflammation.

Inflammation represents a powerful innate immune response that defends tissue homeostasis. However, the appropriate termination of inflammatory processes is essential to prevent the development of chronic inflammatory disorders. The resolution of inflammation is actively induced by specialized pro-resolving lipid mediators, which include eicosanoids, resolvins, protectins and maresins. The responsible pro-resolution pathways have emerged as promising targets for anti-inflammatory therapies since they mitigate excessive inflammation without compromising the anti-microbial defenses of the host. We have recently shown that the lipid peroxidation of membrane phospholipids, which is associated with inflammatory conditions, generates oxidized phospholipid (OxPL) species with potent pro-resolving activities. These pro-resolving OxPLs contain a cyclopentenone as their common determinant, and are structurally and functionally related to endogenous pro-resolving prostaglandins. Here, we review the regulation of inflammatory responses by OxPLs with particular focus on the bioactivities and structural characteristics of cyclopentenone-OxPLs, and discuss the impact of the responsible signaling pathways on inflammatory diseases. This article is part of a Special Issue entitled: Lipid modification and lipid peroxidation products in innate immunity and inflammation edited by Christoph J. Binder.

TREM-1 links dyslipidemia to inflammation and lipid deposition in atherosclerosis.

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a potent amplifier of pro-inflammatory innate immune responses, but its significance in non-infectious diseases remains unclear. Here, we demonstrate that TREM-1 promotes cardiovascular disease by exacerbating atherosclerosis. TREM-1 is expressed in advanced human atheromas and is highly upregulated under dyslipidemic conditions on circulating and on lesion-infiltrating myeloid cells in the Apoe-/- mouse model. TREM-1 strongly contributes to high-fat, high-cholesterol diet (HFCD)-induced monocytosis and synergizes with HFCD serum-derived factors to promote pro-inflammatory cytokine responses and foam cell formation of human monocyte/macrophages. Trem1-/-Apoe-/- mice exhibit substantially attenuated diet-induced atherogenesis. In particular, our results identify skewed monocyte differentiation and enhanced lipid accumulation as novel mechanisms through which TREM-1 can promote atherosclerosis. Collectively, our findings illustrate that dyslipidemia induces TREM-1 surface expression on myeloid cells and subsequently synergizes with TREM-1 to enhance monopoiesis, pro-atherogenic cytokine production and foam cell formation.

The regulation of inflammation by oxidized phospholipids.

During inflammation or under conditions of oxidative stress, the polyunsaturated fatty acid side chains of phospholipids in cellular membranes or lipoproteins can be oxidatively modified. This process generates a complex mixture of structurally diverse oxidized phospholipid (OxPL) species, each of which may exert distinct biological effects. The presence of OxPLs has been documented in acute and chronic microbial infections, metabolic disorders, and degenerative diseases. It is now well recognized that OxPLs actively influence biological processes and contribute to the induction and resolution of inflammation. While many pro- and anti-inflammatory effects have been documented for bulk OxPL preparations, we are only beginning to understand the exact molecular mechanisms and signaling events that mediate the individual proinflammatory or anti-inflammatory bioactivities of discrete isolated OxPL species. Here, we review the current knowledge on the regulation of inflammation by OxPLs and summarize recent studies that establish cyclopentenone-containing OxPLs as a category of potent anti-inflammatory lipid mediators.

Total Synthesis of Prostaglandin 15d-PGJ(2) and Investigation of its Effect on the Secretion of IL-6 and IL-12.

An efficient synthesis of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2, 1) is reported. The route described allows for diversification of the parent structure to prepare seven analogues of 1 in which the positioning of electrophilic sites is varied. These analogues were tested in SAR studies for their ability to reduce the secretion of proinflammatory cytokines. It was shown that the endocyclic enone is crucial for the bioactivity investigated and that the conjugated ω-side chain serves in a reinforcing manner.

T cell lipid peroxidation induces ferroptosis and prevents immunity to infection.

The selenoenzyme glutathione peroxidase 4 (Gpx4) is a major scavenger of phospholipid hydroperoxides. Although Gpx4 represents a key component of the reactive oxygen species-scavenging network, its relevance in the immune system is yet to be defined. Here, we investigated the importance of Gpx4 for physiological T cell responses by using T cell-specific Gpx4-deficient mice. Our results revealed that, despite normal thymic T cell development, CD8(+) T cells from T(ΔGpx4/ΔGpx4) mice had an intrinsic defect in maintaining homeostatic balance in the periphery. Moreover, both antigen-specific CD8(+) and CD4(+) T cells lacking Gpx4 failed to expand and to protect from acute lymphocytic choriomeningitis virus and Leishmania major parasite infections, which were rescued with diet supplementation of high dosage of vitamin E. Notably, depletion of the Gpx4 gene in the memory phase of viral infection did not affect T cell recall responses upon secondary infection. Ex vivo, Gpx4-deficient T cells rapidly accumulated membrane lipid peroxides and concomitantly underwent cell death driven by ferroptosis but not necroptosis. These studies unveil an essential role of Gpx4 for T cell immunity.

Phospholipid oxidation generates potent anti-inflammatory lipid mediators that mimic structurally related pro-resolving eicosanoids by activating Nrf2.

Exposure of biological membranes to reactive oxygen species creates a complex mixture of distinct oxidized phospholipid (OxPL) species, which contribute to the development of chronic inflammatory diseases and metabolic disorders. While the ability of OxPL to modulate biological processes is increasingly recognized, the nature of the biologically active OxPL species and the molecular mechanisms underlying their signaling remain largely unknown. We have employed a combination of mass spectrometry, synthetic chemistry, and immunobiology approaches to characterize the OxPL generated from the abundant phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and investigated their bioactivities and signaling pathways in vitro and in vivo. Our study defines epoxycyclopentenones as potent anti-inflammatory lipid mediators that mimic the signaling of endogenous, pro-resolving prostanoids by activating the transcription factor nuclear factor E2-related factor 2 (Nrf2). Using a library of OxPL variants, we identified a synthetic OxPL derivative, which alleviated endotoxin-induced lung injury and inhibited development of pro-inflammatory T helper (Th) 1 cells. These findings provide a molecular basis for the negative regulation of inflammation by lipid peroxidation products and propose a novel class of highly bioactive compounds for the treatment of inflammatory diseases.

Discovery of a highly potent anti-inflammatory epoxyisoprostane-derived lactone.

Epoxyisoprostanes EI (1) and EC (2) are effective inhibitors of the secretion of proinflammatory cytokines IL-6 and IL-12. In detailed studies toward the investigation of the molecular mode of action of these structures, a highly potent lactone (3) derived from 1 was identified. The known isoprostanoids 1 and 2 are most likely precursors of 3, the product of facile intramolecular reaction between the epoxide with the carboxylic acid in 2.

Fatty acid-induced mitochondrial uncoupling elicits inflammasome-independent IL-1α and sterile vascular inflammation in atherosclerosis.

Chronic inflammation is a fundamental aspect of metabolic disorders such as obesity, diabetes and cardiovascular disease. Cholesterol crystals are metabolic signals that trigger sterile inflammation in atherosclerosis, presumably by activating inflammasomes for IL-1ß production. We found here that atherogenesis was mediated by IL-1α and we identified fatty acids as potent inducers of IL-1α-driven vascular inflammation. Fatty acids selectively stimulated the release of IL-1α but not of IL-1ß by uncoupling mitochondrial respiration. Fatty acid-induced mitochondrial uncoupling abrogated IL-1ß secretion, which deviated the cholesterol crystal-elicited response toward selective production of IL-1α. Our findings delineate a previously unknown pathway for vascular immunopathology that links the cellular response to metabolic stress with innate inflammation, and suggest that IL-1α, not IL-1ß, should be targeted in patients with cardiovascular disease.

IL-21 restricts virus-driven Treg cell expansion in chronic LCMV infection.

Foxp3+ regulatory T (Treg) cells are essential for the maintenance of immune homeostasis and tolerance. During viral infections, Treg cells can limit the immunopathology resulting from excessive inflammation, yet potentially inhibit effective antiviral T cell responses and promote virus persistence. We report here that the fast-replicating LCMV strain Docile triggers a massive expansion of the Treg population that directly correlates with the size of the virus inoculum and its tendency to establish a chronic, persistent infection. This Treg cell proliferation was greatly enhanced in IL-21R-/- mice and depletion of Treg cells partially rescued defective CD8+ T cell cytokine responses and improved viral clearance in some but not all organs. Notably, IL-21 inhibited Treg cell expansion in a cell intrinsic manner. Moreover, experimental augmentation of Treg cells driven by injection of IL-2/anti-IL-2 immune complexes drastically impaired the functionality of the antiviral T cell response and impeded virus clearance. As a consequence, mice became highly susceptible to chronic infection following exposure to low virus doses. These findings reveal virus-driven Treg cell proliferation as potential evasion strategy that facilitates T cell exhaustion and virus persistence. Furthermore, they suggest that besides its primary function as a direct survival signal for antiviral CD8+ T cells during chronic infections, IL-21 may also indirectly promote CD8+ T cell poly-functionality by restricting the suppressive activity of infection-induced Treg cells.

Transport and uptake of immunogenic lipids.

An increasing number of lipid mediators have been identified as key modulators of immunity. Among these is a family of glycolipids capable of cellular uptake, loading onto the MHC-like molecule CD1d and stimulation of NKT cells. NKT cells are particularly interesting because they bridge innate and adaptive immunity by coordinating the early events of dendritic cell maturation, recruitment of NK cells, CD4 and CD8 T cells, and B cells at the site of microbial injury. As such, their therapeutic manipulation could be of the greatest interest in vaccine design or active immunotherapy. However, the use of NKT cells as cellular adjuvant of immunity in the clinic will require a better knowledge of the pharmacology of lipid agonists in order to optimize their action and avoid potential unseen off-target effects. We have been studying extracellular transport and cellular uptake of NKT agonists for the past few years. This field is confronted to a very limited prior knowledge and a small set of usable tools. New technology must be put in place and adapted to answering basic immunology questions related to NKT cells. The intimate link between the pharmacology of glycolipids and lipid metabolism makes us believe that great variations of bioactivity could be seen in the general population when NKT agonists are used therapeutically.

Scavenger receptors target glycolipids for natural killer T cell activation.

NKT cells are innate-like T cells with powerful regulatory functions that are a promising target for immunotherapy. The efficacy of glycolipids, such as the prototypic NKT cell antagonist α-galactosylceramide (αGalCer), is currently being evaluated in clinical trials, but little is known about factors that target lipid antigens for CD1d presentation and NKT cell activation in vivo. Lipid uptake via the LDL receptor (LDLR) has been shown for digalactosylceramide; however, whether this pathway contributes to CD1d presentation of other important NKT cell agonists remains unclear. We therefore investigated receptor-mediated uptake pathways for CD1d presentation using a panel of structurally diverse lipid antigens. We found that uptake via scavenger receptors was essential for the CD1d presentation of αGalCer and Sphingomonas glycolipids. Moreover, in vivo NKT cell responses, i.e., cytokine production, proliferation, and NKT cell help for adaptive CD4+ and CD8+ T cells, required the uptake of αGalCer via scavenger receptor A. Importantly, our data indicate that structural characteristics of glycolipids determine their receptor binding and direct individual lipids toward different uptake pathways. These results reveal an important contribution of scavenger receptors in the selection of lipids for CD1d presentation and identify structural motifs that may prove useful for therapeutic NKT cell vaccination.