The evolutionary tug-of-war of macrophage metabolism during bacterial infection.

The function and phenotype of macrophages are intimately linked with pathogen detection. On sensing pathogen-derived signals and molecules, macrophages undergo a carefully orchestrated process of polarization to acquire pathogen-clearing properties. This phenotypic change must be adequately supported by metabolic reprogramming that is now known to support the acquisition of effector function, but also generates secondary metabolites with direct microbicidal activity. At the same time, bacteria themselves have adapted to both manipulate and take advantage of macrophage-specific metabolic adaptations. Here, we summarize the current knowledge on macrophage metabolism during infection, with a particular focus on understanding the 'arms race' between host immune cells and bacteria during immune responses.

Inhibition of Mitochondrial Translation Ameliorates Imiquimod-Induced Psoriasis-Like Skin Inflammation by Targeting Vγ4+ γδ T Cells.

Psoriasis is an inflammatory skin disorder that is characterized by keratinocyte hyperproliferation in response to immune cell infiltration and cytokine secretion in the dermis. γδ T cells expressing the Vγ4 TCR chain are among the highest contributors of IL-17A, which is a major cytokine that drives a psoriasis flare, making Vγ4+ γδ T cells a suitable target to restrict psoriasis progression. In this study, we demonstrate that mitochondrial translation inhibition within Vγ4+ γδ T cells effectively reduced erythema, scaling, and skin thickening in a murine model of psoriatic disease. The antibiotic linezolid, which blocks mitochondrial translation, inhibited the production of mitochondrial-encoded protein cytochrome c oxidase in Vγ4+ γδ T cells and systemically reduced the frequencies of IL-17A+ Vγ4+ γδ T cells, effectively resolving IL-17A-dependent inflammation. Inhibiting mitochondrial translation could be a novel metabolic approach to interrupt IL-17A signaling in Vγ4+ T cells and reduce psoriasis-like skin pathophysiology.

Tregs protect against invariant NKT cell-mediated autoimmune colitis and hepatitis.

Immunomodulatory T cells play a pivotal role in protection against (auto)immune-mediated diseases that open perspectives for therapeutic modulation. However, how immune regulatory networks operate in vivo is less understood. To this end, we focused on FOXP3+CD4+CD25+ regulatory T cells (Tregs) and invariant natural killer T (iNKT) cells, two lymphocyte populations that independently regulate adaptive and innate immune responses. In vitro, a functional interplay between Tregs and iNKT cells has been described, but whether Tregs modulate the function and phenotype of iNKT cell subsets in vivo and whether this controls iNKT-mediated autoimmunity is unclear. Taking advantage of the conditional depletion of Tregs, we examined the in vivo interplay between iNKT and Treg cells in steady state and in preclinical models of liver and gut autoimmunity. Under non-inflamed conditions, Treg depletion enhanced glycolipid-mediated iNKT cell responses, with a general impact on Type 1, 2 and 17 iNKT subsets. Moreover, in vivo iNKT activation in the absence of Tregs suppressed the induction of iNKT anergy, consistent with a reduction in programmed cell death receptor 1 (PD-1) expression. Importantly, we unveiled a clear role for an in vivo Treg-iNKT crosstalk both in concanavalin A-induced acute hepatitis and oxazolone-induced colitis. Here, the absence of Tregs led to a markedly enhanced liver and gut pathology, which was not observed in iNKT-deficient mice. Taken together, these results provide evidence for a functional interplay between regulatory T cell subsets critical in controlling the onset of autoimmune disease.

A clinically-relevant STING agonist restrains human TH17 cell inflammatory profile.

The STING signaling pathway has gained attention over the last few years due to its ability to incite antimicrobial and antitumoral immunity. Conversely, in mouse models of autoimmunity such as colitis and multiple sclerosis, where TH17 cells are implicated in tissue inflammation, STING activation has been associated with the attenuation of immunogenic responses. In this line, STING was found to limit murine TH17 pro-inflammatory program in vitro. Here we demonstrate that 2'3'-c-di-AM(PS)2(Rp,Rp), a STING agonist that has been undergoing clinical trials for antitumor immunotherapy, activates the STING signalosome in differentiating human TH17 cells. Of particular interest, 2'3'-c-di-AM(PS)2(Rp,Rp) reduces IL-17A production and IL23R expression by human TH17 cells while it favors the generation of regulatory T (Treg) cells. These findings suggest that STING agonists may be promising approaches for treating human TH17-mediated chronic inflammation.

Targeting ACC1 in T cells ameliorates psoriatic skin inflammation.

Psoriasis is a chronic inflammatory skin disease driven by the IL-23/IL-17 axis. It results from excessive activation of effector T cells, including T helper (Th) and cytotoxic T (Tc) cells, and is associated with dysfunctional regulatory T cells (Tregs). Acetyl-CoA carboxylase 1 (ACC1), a rate-limiting enzyme of fatty acid synthesis (FAS), directs cell fate decisions between Th17 and Tregs and thus could be a promising therapeutic target for psoriasis treatment. Here, we demonstrate that targeting ACC1 in T cells by genetic ablation ameliorates skin inflammation in an experimental model of psoriasis by limiting Th17, Tc17, Th1, and Tc1 cells in skin lesions and increasing the frequency of effector Tregs in skin-draining lymph nodes (LNs). KEY MESSAGES : ACC1 deficiency in T cells ameliorates psoriatic skin inflammation in mice. ACC1 deficiency in T cells reduces IL-17A-producing Th17/Tc17/dysfunctional Treg populations in psoriatic lesions. ACC1 deficiency in T cells restrains IFN-γ-producing Th1/Tc1 populations in psoriatic skin lesions and skin-draining LNs. ACC1 deficiency promotes activated CD44+CD25+ Tregs and effector CD62L-CD44+ Tregs under homeostasis and psoriatic conditions.

Distinct immune modulatory roles of regulatory T cells in gut versus joint inflammation in TNF-driven spondyloarthritis.

OBJECTIVES: Gut and joint inflammation commonly co-occur in spondyloarthritis (SpA) which strongly restricts therapeutic modalities. The immunobiology underlying differences between gut and joint immune regulation, however, is poorly understood. We therefore assessed the immunoregulatory role of CD4+FOXP3+ regulatory T (Treg) cells in a model of Crohn's-like ileitis and concomitant arthritis. METHODS: RNA-sequencing and flow cytometry was performed on inflamed gut and joint samples and tissue-derived Tregs from tumour necrosis factor (TNF)∆ARE mice. In situ hybridisation of TNF and its receptors (TNFR) was applied to human SpA gut biopsies. Soluble TNFR (sTNFR) levels were measured in serum of mice and patients with SpA and controls. Treg function was explored by in vitro cocultures and in vivo by conditional Treg depletion. RESULTS: Chronic TNF exposure induced several TNF superfamily (TNFSF) members (4-1BBL, TWEAK and TRAIL) in synovium and ileum in a site-specific manner. Elevated TNFR2 messenger RNA levels were noted in TNF∆ARE/+ mice leading to increased sTNFR2 release. Likewise, sTNFR2 levels were higher in patients with SpA with gut inflammation and distinct from inflammatory and healthy controls. Tregs accumulated at both gut and joints of TNF∆ARE mice, yet their TNFR2 expression and suppressive function was significantly lower in synovium versus ileum. In line herewith, synovial and intestinal Tregs displayed a distinct transcriptional profile with tissue-restricted TNFSF receptor and p38MAPK gene expression. CONCLUSIONS: These data point to profound differences in immune-regulation between Crohn's ileitis and peripheral arthritis. Whereas Tregs control ileitis they fail to dampen joint inflammation. Synovial resident Tregs are particularly maladapted to chronic TNF exposure.

Antibodies to variable surface antigens induce antigenic variation in the intestinal parasite Giardia lamblia.

The genomes of most protozoa encode families of variant surface antigens. In some parasitic microorganisms, it has been demonstrated that mutually exclusive changes in the expression of these antigens allow parasites to evade the host's immune response. It is widely assumed that antigenic variation in protozoan parasites is accomplished by the spontaneous appearance within the population of cells expressing antigenic variants that escape antibody-mediated cytotoxicity. Here we show, both in vitro and in animal infections, that antibodies to Variant-specific Surface Proteins (VSPs) of the intestinal parasite Giardia lamblia are not cytotoxic, inducing instead VSP clustering into liquid-ordered phase membrane microdomains that trigger a massive release of microvesicles carrying the original VSP and switch in expression to different VSPs by a calcium-dependent mechanism. This novel mechanism of surface antigen clearance throughout its release into microvesicles coupled to the stochastic induction of new phenotypic variants not only changes current paradigms of antigenic switching but also provides a new framework for understanding the course of protozoan infections as a host/parasite adaptive process.

Regulation of DC metabolism by nitric oxide in murine GM-CSF cultures.

The CD11c+ MHCII+ compartment within GM-CSF cultures consists of a MHCIIlow CD11bhigh population (GM-Macs) and a MHCIIhigh CD11bint population (GM-DCs), with different metabolic profiles. GM-Macs upregulate iNOS and produce nitric oxide (NO) upon TLR activation inhibiting mitochondrial respiration (OXPHOS) while promoting glycolytic metabolism in GM-DCs, which naturally do not express iNOS.

Guidelines for mouse and human DC generation.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. This article provides protocols with top ticks and pitfalls for preparation and successful generation of mouse and human DC from different cellular sources, such as murine BM and HoxB8 cells, as well as human CD34+ cells from cord blood, BM, and peripheral blood or peripheral blood monocytes. We describe murine cDC1, cDC2, and pDC generation with Flt3L and the generation of BM-derived DC with GM-CSF. Protocols for human DC generation focus on CD34+ cell culture on OP9 cell layers for cDC1, cDC2, cDC3, and pDC subset generation and DC generation from peripheral blood monocytes (MoDC). Additional protocols include enrichment of murine DC subsets, CRISPR/Cas9 editing, and clinical grade human DC generation. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

Guidelines for mouse and human DC functional assays.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Recent studies have provided evidence for an increasing number of phenotypically distinct conventional DC (cDC) subsets that on one hand exhibit a certain functional plasticity, but on the other hand are characterized by their tissue- and context-dependent functional specialization. Here, we describe a selection of assays for the functional characterization of mouse and human cDC. The first two protocols illustrate analysis of cDC endocytosis and metabolism, followed by guidelines for transcriptomic and proteomic characterization of cDC populations. Then, a larger group of assays describes the characterization of cDC migration in vitro, ex vivo, and in vivo. The final guidelines measure cDC inflammasome and antigen (cross)-presentation activity. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

γδ T cells license immature B cells to produce a broad range of polyreactive antibodies.

Immature autoreactive B cells are present in all healthy individuals, but it is unclear which signals are required for their maturation into antibody-producing cells. Inducible depletion of γδ T cells show that direct interaction between γδ T cells and immature B cells in the spleen support an "innate" transition to mature B cells with a broad range of antigen specificities. IL-4 production of γδ T cells and cell-to-cell contact via CD30L support B cell maturation and induce genes of the unfolded protein response and mTORC1 signaling. Eight days after in vivo depletion of γδ T cells, increased numbers of B cells are already stuck in the transitional phase and express increased levels of IgD and CD21. Absence of γδ T cells leads also to reduced levels of serum anti-nuclear autoantibodies, making γδ T cells an attractive target to treat autoimmunity.

STING is an intrinsic checkpoint inhibitor that restrains the TH17 cell pathogenic program.

External and intrinsic factors regulate the transcriptional profile of T helper 17 (TH17) cells, thereby affecting their pathogenic potential and revealing their context-dependent plasticity. The stimulator of interferon genes (STING), a component of the intracellular DNA-sensing pathway, triggers immune responses but remains largely unexplored in T cells. Here, we describe an intrinsic role of STING in limiting the TH17 cell pathogenic program. We demonstrate that non-pathogenic TH17 cells express higher levels of STING than those activated under pathogenic conditions. Activation of STING induces interleukin-10 (IL-10) production in TH17 cells, decreasing IL-17A and IL-23R expression in a type I interferon (IFN)-independent manner. Mechanistically, STING-induced IL-10 production partially requires aryl hydrocarbon receptor (AhR) signaling, while the decrease of IL-17A expression occurs due to a reduction of Rorγt transcriptional activity. Our findings reveal a regulatory function of STING in the TH17 cell activation program, proposing it as a valuable target to limit TH17-cell-mediated inflammation.

Correction: TGFß-activation by dendritic cells drives Th17 induction and intestinal contractility and augments the expulsion of the parasite Trichinella spiralis in mice.

[This corrects the article DOI: 10.1371/journal.ppat.1007657.].

Transient Depletion of Foxp3+ Regulatory T Cells Selectively Promotes Aggressive ß Cell Autoimmunity in Genetically Susceptible DEREG Mice.

Type 1 diabetes (T1D) represents a hallmark of the fatal multiorgan autoimmune syndrome affecting humans with abrogated Foxp3+ regulatory T (Treg) cell function due to Foxp3 gene mutations, but whether the loss of Foxp3+ Treg cell activity is indeed sufficient to promote ß cell autoimmunity requires further scrutiny. As opposed to human Treg cell deficiency, ß cell autoimmunity has not been observed in non-autoimmune-prone mice with constitutive Foxp3 deficiency or after diphtheria toxin receptor (DTR)-mediated ablation of Foxp3+ Treg cells. In the spontaneous nonobese diabetic (NOD) mouse model of T1D, constitutive Foxp3 deficiency did not result in invasive insulitis and hyperglycemia, and previous studies on Foxp3+ Treg cell ablation focused on Foxp3DTR NOD mice, in which expression of a transgenic BDC2.5 T cell receptor (TCR) restricted the CD4+ TCR repertoire to a single diabetogenic specificity. Here we revisited the effect of acute Foxp3+ Treg cell ablation on ß cell autoimmunity in NOD mice in the context of a polyclonal TCR repertoire. For this, we took advantage of the well-established DTR/GFP transgene of DEREG mice, which allows for specific ablation of Foxp3+ Treg cells without promoting catastrophic autoimmune diseases. We show that the transient loss of Foxp3+ Treg cells in prediabetic NOD.DEREG mice is sufficient to precipitate severe insulitis and persistent hyperglycemia within 5 days after DT administration. Importantly, DT-treated NOD.DEREG mice preserved many clinical features of spontaneous diabetes progression in the NOD model, including a prominent role of diabetogenic CD8+ T cells in terminal ß cell destruction. Despite the severity of destructive ß cell autoimmunity, anti-CD3 mAb therapy of DT-treated mice interfered with the progression to overt diabetes, indicating that the novel NOD.DEREG model can be exploited for preclinical studies on T1D under experimental conditions of synchronized, advanced ß cell autoimmunity. Overall, our studies highlight the continuous requirement of Foxp3+ Treg cell activity for the control of genetically pre-installed autoimmune diabetes.

CD4+ T-cell differentiation and function: Unifying glycolysis, fatty acid oxidation, polyamines NAD mitochondria.

The progression through different steps of T-cell development, activation, and effector function is tightly bound to specific cellular metabolic processes. Previous studies established that T-effector cells have a metabolic bias toward aerobic glycolysis, whereas naive and regulatory T cells mainly rely on oxidative phosphorylation. More recently, the field of immunometabolism has drifted away from the notion that mitochondrial metabolism holds little importance in T-cell activation and function. Of note, T cells possess metabolic promiscuity, which allows them to adapt their nutritional requirements according to the tissue environment. Altogether, the integration of these metabolic pathways culminates in the generation of not only energy but also intermediates, which can regulate epigenetic programs, leading to changes in T-cell fate. In this review, we discuss the recent literature on how glycolysis, amino acid catabolism, and fatty acid oxidation work together with the tricarboxylic acid cycle in the mitochondrion. We also emphasize the importance of the electron transport chain for T-cell immunity. We also discuss novel findings highlighting the role of key enzymes, accessory pathways, and posttranslational protein modifications that distinctively regulate T-cell function and might represent prominent candidates for therapeutic purposes.

Structure-Activity Relationship and Mode-Of-Action Studies Highlight 1-(4-Biphenylylmethyl)-1H-imidazole-Derived Small Molecules as Potent CYP121 Inhibitors.

CYP121 of Mycobacterium tuberculosis (Mtb) is an essential target for the development of novel potent drugs against tuberculosis (TB). Besides known antifungal azoles, further compounds of the azole class were recently identified as CYP121 inhibitors with antimycobacterial activity. Herein, we report the screening of a similarity-oriented library based on the former hit compound, the evaluation of affinity toward CYP121, and activity against M. bovis BCG. The results enabled a comprehensive SAR study, which was extended through the synthesis of promising compounds and led to the identification of favorable features for affinity and/or activity and hit compounds with 2.7-fold improved potency. Mode of action studies show that the hit compounds inhibit substrate conversion and highlighted CYP121 as the main antimycobacterial target of our compounds. Exemplified complex crystal structures of CYP121 with three inhibitors reveal a common binding site. Engaging in both hydrophobic interactions as well as hydrogen bonding to the sixth iron ligand, our compounds block a solvent channel leading to the active site heme. Additionally, we report the first CYP inhibitors that are able to reduce the intracellular replication of M. bovis BCG in macrophages, emphasizing their potential as future drug candidates against TB.

Clarithromycin impairs tissue-resident memory and Th17 responses to macrolide-resistant Streptococcus pneumoniae infections.

The increasing prevalence of antimicrobial resistance in pathogens is a growing public health concern, with the potential to compromise the success of infectious disease treatments in the future. Particularly, the number of infections by macrolide antibiotics-resistant Streptococcus pneumoniae is increasing. We show here that Clarithromycin impairs both the frequencies and number of interleukin (IL)-17 producing T helper (Th) 17 cells within the lungs of mice infected with a macrolide-resistant S. pneumoniae serotype 15A strain. Subsequently, the tissue-resident memory CD4+ T cell (Trm) response to a consecutive S. pneumoniae infection was impaired. The number of lung resident IL-17+ CD69+ Trm was diminished upon Clarithromycin treatment during reinfection. Mechanistically, Clarithromycin attenuated phosphorylation of the p90-S6-kinase as part of the ERK pathway in Th17 cells. Moreover, a strong increase in the mitochondrial-mediated maximal respiratory capacity was observed, while mitochondrial protein translation and mTOR sisgnaling were unimpaired. Therefore, treatment with macrolide antibiotics may favor the spread of antimicrobial-resistant pathogens not only by applying a selection pressure but also by decreasing the natural T cell immune response. Clinical administration of macrolide antibiotics as standard therapy procedure during initial hospitalization should be reconsidered accordingly and possibly be withheld until microbial resistance is determined. KEY MESSAGES: • Macrolide-resistant S. pneumoniae infection undergoes immunomodulation by Clarithromycin • Clarithromycin treatment hinders Th17 and tissue-resident memory responses • Macrolide antibiotics impair Th17 differentiation in vitro by ERK-pathway inhibition.

Inflammation in the Human Periodontium Induces Downregulation of the α1- and ß1-Subunits of the sGC in Cementoclasts.

Nitric oxide (NO) binds to soluble guanylyl cyclase (sGC), activates it in a reduced oxidized heme iron state, and generates cyclic Guanosine Monophosphate (cGMP), which results in vasodilatation and inhibition of osteoclast activity. In inflammation, sGC is oxidized and becomes insensitive to NO. NO- and heme-independent activation of sGC requires protein expression of the α1- and ß1-subunits. Inflammation of the periodontium induces the resorption of cementum by cementoclasts and the resorption of the alveolar bone by osteoclasts, which can lead to tooth loss. As the presence of sGC in cementoclasts is unknown, we investigated the α1- and ß1-subunits of sGC in cementoclasts of healthy and inflamed human periodontium using double immunostaining for CD68 and cathepsin K and compared the findings with those of osteoclasts from the same sections. In comparison to cementoclasts in the healthy periodontium, cementoclasts under inflammatory conditions showed a decreased staining intensity for both α1- and ß1-subunits of sGC, indicating reduced protein expression of these subunits. Therefore, pharmacological activation of sGC in inflamed periodontal tissues in an NO- and heme-independent manner could be considered as a new treatment strategy to inhibit cementum resorption.

Dendritic cell metabolism: moving beyond in vitro-culture-generated paradigms.

Dendritic cells (DCs) are key orchestrators of immunity and tolerance. It has become evident that DC function can be influenced by cellular metabolic programs. However, conclusions from early metabolic studies using in vitro GM-CSF DC cultures fail to correlate with bona fide DC populations. Here, we discuss the existing paradigms in the DC metabolism field, focusing on the limitations of the models utilized. Furthermore, we introduce alternative models to generate DCs in vitro that better emulate DCs found in vivo. Finally, we highlight new techniques to evaluate DC metabolism at the single-cell level. The combination of these two strategies could help advance the DC metabolism field towards a more physiological understanding, which is crucial for the development of effective DC-based therapies.

Targeting cellular fatty acid synthesis limits T helper and innate lymphoid cell function during intestinal inflammation and infection.

CD4+ T cells contribute critically to a protective immune response during intestinal infections, but have also been implicated in the aggravation of intestinal inflammatory pathology. Previous studies suggested that T helper type (Th)1 and Th17 cells depend on de novo fatty acid (FA) synthesis for their development and effector function. Here, we report that T-cell-specific targeting of the enzyme acetyl-CoA carboxylase 1 (ACC1), a major checkpoint controlling FA synthesis, impaired intestinal Th1 and Th17 responses by limiting CD4+ T-cell expansion and infiltration into the lamina propria in murine models of colitis and infection-associated intestinal inflammation. Importantly, pharmacological inhibition of ACC1 by the natural compound soraphen A mirrored the anti-inflammatory effects of T-cell-specific targeting, but also enhanced susceptibility toward infection with C. rodentium. Further analysis revealed that deletion of ACC1 in RORγt+ innate lymphoid cells (ILC), but not dendritic cells or macrophages, decreased resistance to infection by interfering with IL-22 production and intestinal barrier function. Together, our study suggests pharmacological targeting of ACC1 as an effective approach for metabolic immune modulation of T-cell-driven intestinal inflammatory responses, but also reveals an important role of ACC1-mediated lipogenesis for the function of RORγt+ ILC.