Key Points: A new bone marrow DC culture method with FLT3L and GM-CSF efficiently generates functional Batf3-dependent CD103+ DCs. GM-CSF promotes DC precursor polarization and CD103+ DC proliferation.

The hypoxic environment of cystic fibrosis airways allows the persistence of facultative anaerobic bacteria, which can produce short-chain fatty acids (SCFAs) through fermentation. However, the relevance of SCFAs in cystic fibrosis lung disease is unknown. We show that SCFAs are present in sputum samples from cystic fibrosis patients in millimolar concentrations (mean±sem1.99±0.36 mM).SCFAs positively correlated with sputum neutrophil count and higher SCFAs were predictive for impaired nitric oxide production. We studied the effects of the SCFAs acetate, propionate and butyrate on airway inflammatory responses using epithelial cell lines and primary cell cultures. SCFAs in concentrations present in cystic fibrosis airways (0.5–2.5 mM) affected the release of granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor and interleukin (IL)-6. SCFAs also resulted in higher IL-8 release from stimulated cystic fibrosis transmembrane conductance regulator (CFTR) F508del-mutant compared to wild-type CFTR-corrected bronchial epithelial cells. At 25 mM propionate reduced IL-8 release in control but not primary cystic fibrosis epithelial cells. Low (0.5–2.5 mM) SCFA concentrations increased, while high (25–50 mM) concentrations decreased inducible nitric oxide synthase expression. In addition, SCFAs affected the growth ofPseudomonas aeruginosain a concentration- and pH-dependent manner.Thus, our data suggest that SCFAs contribute to cystic fibrosis-specific alterations of responses to airway infection and inflammation.

Choline is an essential nutrient that is required for synthesis of the main eukaryote phospholipid, phosphatidylcholine (PC). Macrophages are innate immune cells that survey and respond to danger and damage signals. Although it is well known that energy metabolism can dictate macrophage function, little is known as to the importance of choline homeostasis in macrophage biology. We hypothesized that the uptake and metabolism of choline is important for macrophage inflammation. Polarization of primary bone marrow macrophages with LPS resulted in an increased rate of choline uptake and higher levels of PC synthesis. This was attributed to a substantial increase in the transcript and protein expression of the choline transporter-like protein-1 (CTL1) in polarized cells. We next sought to determine the importance of choline uptake and CTL1 for macrophage immune responsiveness. Chronic pharmacological or CTL1 antibody-mediated inhibition of choline uptake resulted in altered cytokine secretion in response to LPS, which was associated with increased the levels of diacylglycerol (DAG) and activation of protein kinase C (PKC). These experiments establish a previously unappreciated link between choline phospholipid metabolism and macrophage immune responsiveness, highlighting a critical and regulatory role for macrophage choline uptake via the CTL1 transporter.

Upon inflammation, NK cells undergo metabolic changes to support their high energy demand for effector function and proliferation. The metabolic changes are usually accompanied by an increase in the expression of nutrient transporters leading to increased nutrient uptake. Among various cytokines inducing NK cell proliferation, the mechanisms underlying the effect of IL-18 in promoting NK cell proliferation is not completely understood. Here, we demonstrate that IL-18 is a potent cytokine that can enhance the expression of the nutrient transporter CD98/LAT1 for amino acids independently of mTORC1 pathway and thereby induce a dramatic metabolic change, associated with an increased proliferation of NK cells. Notably, treatment of IL-18-stimulated NK cells with leucine activates the metabolic sensor mTORC1, indicating that the high expression of amino acid transporters induces amino acid-driven mTORC1 activation. Inhibition of the amino acid transporter CD98/LAT1 abrogated the leucine-driven mTORC1 activation and reduced NK cell effector function. Taken together, our study identified a novel role of IL-18 in upregulating nutrient transporters on NK cells and thereby inducing metabolic changes including the mTORC1 activation by amino acids.

Foxp3 specifies the Treg cell lineage and is indispensable for immune tolerance. Accordingly, rare Foxp3 mutations cause lethal autoimmunity. The mechanisms precipitating more prevalent human autoimmune diseases are poorly understood, but involve a combination of genetic and environmental factors. Many autoimmune diseases associate with a partial Treg‐cell dysfunction, yet mouse models reflecting such complex pathophysiological processes are rare. Around 95% of Foxp3⁺ Treg cells can be specifically depleted in bacterial artifical chromosome (BAC)‐transgenic Depletion of REGulatory T cells (DEREG) mice through diphtheria toxin (DT) treatment. However, Treg‐cell depletion fails to cause autoimmunity in adult DEREG mice for unclear reasons. By crossing Foxp3^GFP knock‐in mice to DEREG mice, we introduced additional genetic susceptibility that does not affect untreated mice. Strikingly, DT treatment of DEREG × Foxp3^GFP mice rapidly causes autoimmunity characterized by blepharitis, tissue damage, and autoantibody production. This inflammatory disease is associated with augmented T‐cell activation, increased Th2 cytokine production and myeloproliferation, and is caused by defective Treg‐cell homeostasis, preventing few DT‐insensitive Treg cells from repopulating the niche after Treg‐cell depletion. Our study provides important insights into self‐tolerance. We further highlight DEREG × Foxp3^GFP mice as a model to investigate the role of environmental factors in precipitating autoimmunity. This may help to better understand and treat human autoimmunity.

Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, is able to efficiently manipulate the host immune system establishing chronic infection, yet the underlying mechanisms of immune evasion are not fully understood. Evidence suggests that this pathogen interferes with host cell lipid metabolism to ensure its persistence. Fatty acid metabolism is regulated by acetyl-CoA carboxylase (ACC) 1 and 2; both isoforms catalyze the conversion of acetyl-CoA into malonyl-CoA, but have distinct roles. ACC1 is located in the cytosol, where it regulates de novo fatty acid synthesis (FAS), while ACC2 is associated with the outer mitochondrial membrane, regulating fatty acid oxidation (FAO). In macrophages, mycobacteria induce metabolic changes that lead to the cytosolic accumulation of lipids. This reprogramming impairs macrophage activation and contributes to chronic infection. In dendritic cells (DCs), FAS has been suggested to underlie optimal cytokine production and antigen presentation, but little is known about the metabolic changes occurring in DCs upon mycobacterial infection and how they affect the outcome of the immune response. We therefore determined the role of fatty acid metabolism in myeloid cells and T cells during Mycobacterium bovis BCG or Mtb infection, using novel genetic mouse models that allow cell-specific deletion of ACC1 and ACC2 in DCs, macrophages, or T cells. Our results demonstrate that de novo FAS is induced in DCs and macrophages upon M. bovis BCG infection. However, ACC1 expression in DCs and macrophages is not required to control mycobacteria. Similarly, absence of ACC2 did not influence the ability of DCs and macrophages to cope with infection. Furthermore, deletion of ACC1 in DCs or macrophages had no effect on systemic pro-inflammatory cytokine production or T cell priming, suggesting that FAS is dispensable for an intact innate response against mycobacteria. In contrast, mice with a deletion of ACC1 specifically in T cells fail to generate efficient T helper 1 responses and succumb early to Mtb infection. In summary, our results reveal ACC1-dependent FAS as a crucial mechanism in T cells, but not DCs or macrophages, to fight against mycobacterial infection.