Itaconate is a metabolic regulator of bone formation in homeostasis and arthritis.

OBJECTIVES: Bone remodelling is a highly dynamic process dependent on the precise coordination of osteoblasts and haematopoietic-cell derived osteoclasts. Changes in core metabolic pathways during osteoclastogenesis, however, are largely unexplored and it is unknown whether and how these processes are involved in bone homeostasis. METHODS: We metabolically and transcriptionally profiled cells during osteoclast and osteoblast generation. Individual gene expression was characterised by quantitative PCR and western blot. Osteoblast function was assessed by Alizarin red staining. immunoresponsive gene 1 (Irg1)-deficient mice were used in various inflammatory or non-inflammatory models of bone loss. Tissue gene expression was analysed by RNA in situ hybridisation. RESULTS: We show that during differentiation preosteoclasts rearrange their tricarboxylic acid cycle, a process crucially depending on both glucose and glutamine. This rearrangement is characterised by the induction of Irg1 and production of itaconate, which accumulates intracellularly and extracellularly. While the IRG1-itaconate axis is dispensable for osteoclast generation in vitro and in vivo, we demonstrate that itaconate stimulates osteoblasts by accelerating osteogenic differentiation in both human and murine cells. This enhanced osteogenic differentiation is accompanied by reduced proliferation and altered metabolism. Additionally, supplementation of itaconate increases bone formation by boosting osteoblast activity in mice. Conversely, Irg1-deficient mice exhibit decreased bone mass and have reduced osteoproliferative lesions in experimental arthritis. CONCLUSION: In summary, we identify itaconate, generated as a result of the metabolic rewiring during osteoclast differentiation, as a previously unrecognised regulator of osteoblasts.

Protocol to assess the tolerogenic properties of adoptively transferred dendritic cells during murine experimental autoimmune encephalomyelitis.

By their capacity to induce peripheral T cell tolerance, dendritic cells (DCs) present a promising target cell and therapeutic strategy for treatment of several autoimmune diseases including multiple sclerosis (MS). This protocol describes how to determine the tolerogenic capacities of DCs in the context of the murine MS model, experimental autoimmune encephalomyelitis (EAE). We provide a step-by-step instruction for EAE induction, antigen-loaded bone-marrow-derived-DC (BM-DC) generation, adoptive cell transfer, and analysis of DC-mediated changes in regulatory T cell populations. For complete details on the use and execution of this protocol, please refer to Vogel et al. (2022).

PI3K Signaling in Dendritic Cells Aggravates DSS-Induced Colitis.

Aberrant innate immune responses to the gut microbiota are causally involved in the pathogenesis of inflammatory bowel diseases (IBD). The exact triggers and main signaling pathways activating innate immune cells and how they modulate adaptive immunity in IBD is still not completely understood. Here, we report that the PI3K/PTEN signaling pathway in dendritic cells enhances IL-6 production in a model of DSS-induced colitis. This results in exacerbated Th1 cell responses and increased mortality in DC-specific PTEN knockout (PTENΔDC) animals. Depletion of the gut microbiota using antibiotics as well as blocking IL-6R signaling rescued mortality in PTENΔDC mice, whereas adoptive transfer of Flt3L-derived PTEN-/- DCs into WT recipients exacerbated DSS-induced colitis and increased mortality. Taken together, we show that the PI3K signaling pathway in dendritic cells contributes to disease pathology by promoting IL-6 mediated Th1 responses.

JAK1 signaling in dendritic cells promotes peripheral tolerance in autoimmunity through PD-L1-mediated regulatory T cell induction.

Dendritic cells (DCs) induce peripheral T cell tolerance, but cell-intrinsic signaling cascades governing their stable tolerogenesis remain poorly defined. Janus Kinase 1 (JAK1) transduces cytokine-receptor signaling, and JAK inhibitors (Jakinibs), including JAK1-specific filgotinib, break inflammatory cycles in autoimmunity. Here, we report in heterogeneous DC populations of multiple secondary lymphoid organs that JAK1 promotes peripheral T cell tolerance during experimental autoimmune encephalomyelitis (EAE). Mice harboring DC-specific JAK1 deletion exhibit elevated peripheral CD4+ T cell expansion, less regulatory T cells (Tregs), and worse EAE outcomes, whereas adoptive DC transfer ameliorates EAE pathogenesis by inducing peripheral Tregs, programmed cell death ligand 1 (PD-L1) dependently. This tolerogenic program is substantially reduced upon the transfer of JAK1-deficient DCs. DC-intrinsic IFN-γ-JAK1-STAT1 signaling induces PD-L1, which is required for DCs to convert CD4+ T cells into Tregs in vitro and attenuated upon JAK1 deficiency and filgotinib treatment. Thus, DC-intrinsic JAK1 promotes peripheral tolerance, suggesting potential unwarranted DC-mediated effects of Jakinibs in autoimmune diseases.

Itaconate confers tolerance to late NLRP3 inflammasome activation.

Itaconate is a unique regulatory metabolite that is induced upon Toll-like receptor (TLR) stimulation in myeloid cells. Here, we demonstrate major inflammatory tolerance and cell death phenotypes associated with itaconate production in activated macrophages. We show that endogenous itaconate is a key regulator of the signal 2 of NLR family pyrin domain containing 3 (NLRP3) inflammasome activation after long lipopolysaccharide (LPS) priming, which establishes tolerance to late NLRP3 inflammasome activation. We show that itaconate acts synergistically with inducible nitric oxide synthase (iNOS) and that the ability of various TLR ligands to establish NLRP3 inflammasome tolerance depends on the pattern of co-expression of IRG1 and iNOS. Mechanistically, itaconate accumulation upon prolonged inflammatory stimulation prevents full caspase-1 activation and processing of gasdermin D, which we demonstrate to be post-translationally modified by endogenous itaconate. Altogether, our data demonstrate that metabolic rewiring in inflammatory macrophages establishes tolerance to NLRP3 inflammasome activation that, if uncontrolled, can result in pyroptotic cell death and tissue damage.

Comprehensive Profiling of an Aging Immune System Reveals Clonal GZMK+ CD8+ T Cells as Conserved Hallmark of Inflammaging.

Systematic understanding of immune aging on a whole-body scale is currently lacking. We characterized age-associated alterations in immune cells across multiple mouse organs using single-cell RNA and antigen receptor sequencing and flow cytometry-based validation. We defined organ-specific and common immune alterations and identified a subpopulation of age-associated granzyme K (GZMK)-expressing CD8+ T (Taa) cells that are distinct from T effector memory (Tem) cells. Taa cells were highly clonal, had specific epigenetic and transcriptional signatures, developed in response to an aged host environment, and expressed markers of exhaustion and tissue homing. Activated Taa cells were the primary source of GZMK, which enhanced inflammatory functions of non-immune cells. In humans, proportions of the circulating GZMK+CD8+ T cell population that shares transcriptional and epigenetic signatures with mouse Taa cells increased during healthy aging. These results identify GZMK+ Taa cells as a potential target to address age-associated dysfunctions of the immune system.

The PI3K pathway preserves metabolic health through MARCO-dependent lipid uptake by adipose tissue macrophages.

Adipose tissue macrophages (ATMs) display tremendous heterogeneity depending on signals in their local microenvironment and contribute to the pathogenesis of obesity. The phosphoinositide 3-kinase (PI3K) signalling pathway, antagonized by the phosphatase and tensin homologue (PTEN), is important for metabolic responses to obesity. We hypothesized that fluctuations in macrophage-intrinsic PI3K activity via PTEN could alter the trajectory of metabolic disease by driving distinct ATM populations. Using mice harbouring macrophage-specific PTEN deletion or bone marrow chimeras carrying additional PTEN copies, we demonstrate that sustained PI3K activity in macrophages preserves metabolic health in obesity by preventing lipotoxicity. Myeloid PI3K signalling promotes a beneficial ATM population characterized by lipid uptake, catabolism and high expression of the scavenger macrophage receptor with collagenous structure (MARCO). Dual MARCO and myeloid PTEN deficiencies prevent the generation of lipid-buffering ATMs, reversing the beneficial actions of elevated myeloid PI3K activity in metabolic disease. Thus, macrophage-intrinsic PI3K signalling boosts metabolic health by driving ATM programmes associated with MARCO-dependent lipid uptake.

Environmental arginine controls multinuclear giant cell metabolism and formation.

Multinucleated giant cells (MGCs) are implicated in many diseases including schistosomiasis, sarcoidosis and arthritis. MGC generation is energy intensive to enforce membrane fusion and cytoplasmic expansion. Using receptor activator of nuclear factor kappa-Β ligand (RANKL) induced osteoclastogenesis to model MGC formation, here we report RANKL cellular programming requires extracellular arginine. Systemic arginine restriction improves outcome in multiple murine arthritis models and its removal induces preosteoclast metabolic quiescence, associated with impaired tricarboxylic acid (TCA) cycle function and metabolite induction. Effects of arginine deprivation on osteoclastogenesis are independent of mTORC1 activity or global transcriptional and translational inhibition. Arginine scarcity also dampens generation of IL-4 induced MGCs. Strikingly, in extracellular arginine absence, both cell types display flexibility as their formation can be restored with select arginine precursors. These data establish how environmental amino acids control the metabolic fate of polykaryons and suggest metabolic ways to manipulate MGC-associated pathologies and bone remodelling.

PI3K activity in dendritic cells exerts paradoxical effects during autoimmune inflammation.

The peripheral activation of autoreactive T cells and subsequent central nervous system (CNS) immune cell infiltration are key events relevant for experimental autoimmune encephalomyelitis (EAE), a commonly employed multiple sclerosis (MS) model, influenced by TH1 and TH17 mediated immunity. The phosphoinositide-3-kinase (PI3K)-AKT kinase pathway modulates outcome during EAE, with direct actions of PI3K on adaptive immunity implicated in deleterious and effects on antigen presenting cells involved in beneficial responses during EAE. Here, by genetically deleting the regulatory subunit of Class Ia PI3K, p85α, in selective myeloid cells, we aimed to resolve the impact of PI3K in EAE. While genetically deleting PI3K in LysM expressing cells exerted unremarkable effects, attenuating PI3K function in CD11c+ dendritic cells (DCs), promoted secretion of pathogenic EAE promoting cytokines, particularly skewing TH1 and TH17 immunity, while notably, improving health in EAE. Neutralizing IFN-γ activity using blocking antibodies revealed a prolonged TH1 response was critical for the decreased disease of these animals. Thus, PI3K-AKT signaling in DCs acts in a paradoxical manner. While attenuating EAE associated TH1 and TH17 responses, it impairs health during autoimmune inflammation.

UDP-sulfoquinovose formation by Sulfolobus acidocaldarius.

The UDP-sulfoquinovose synthase Agl3 from Sulfolobus acidocaldarius converts UDP-D-glucose and sulfite to UDP-sulfoquinovose, the activated form of sulfoquinovose required for its incorporation into glycoconjugates. Based on the amino acid sequence, Agl3 belongs to the short-chain dehydrogenase/reductase enzyme superfamily, together with SQD1 from Arabidopsis thaliana, the only UDP-sulfoquinovose synthase with known crystal structure. By comparison of sequence and structure of Agl3 and SQD1, putative catalytic amino acids of Agl3 were selected for mutational analysis. The obtained data suggest for Agl3 a modified dehydratase reaction mechanism. We propose that in vitro biosynthesis of UDP-sulfoquinovose occurs through an NAD(+)-dependent oxidation/dehydration/enolization/sulfite addition process. In the absence of a sulfur donor, UDP-D-glucose is converted via UDP-4-keto-D-glucose to UDP-D-glucose-5,6-ene, the structure of which was determined by (1)H and (13)C-NMR spectroscopy. During the redox reaction the cofactor remains tightly bound to Agl3 and participates in the reaction in a concentration-dependent manner. For the first time, the rapid initial electron transfer between UDP-D-glucose and NAD(+) could be monitored in a UDP-sulfoquinovose synthase. Deuterium labeling confirmed that dehydration of UDP-D-glucose occurs only from the enol form of UDP-4-keto-glucose. The obtained functional data are compared with those from other UDP-sulfoquinovose synthases. A divergent evolution of Agl3 from S. acidocaldarius is suggested.