Metabolic profiling of single cells by exploiting NADH and FAD fluorescence via flow cytometry.

The metabolism of different cells within the same microenvironment can differ and dictate physiological or pathological adaptions. Current single-cell analysis methods of metabolism are not label-free. The study introduces a label-free, live-cell analysis method assessing endogenous fluorescence of NAD(P)H and FAD in surface-stained cells by flow cytometry. OxPhos inhibition, mitochondrial uncoupling, glucose exposure, genetic inactivation of glucose uptake and mitochondrial respiration alter the optical redox ratios of FAD and NAD(P)H as measured by flow cytometry. Those alterations correlate strongly with measurements obtained by extracellular flux analysis. Consequently, metabolically distinct live B-cell populations can be resolved, showing that human memory B-cells from peripheral blood exhibit a higher glycolytic flexibility than naïve B cells. Moreover, the comparison of blood-derived B- and T-lymphocytes from healthy donors and rheumatoid arthritis patients unleashes rheumatoid arthritis-associated metabolic traits in human naïve and memory B-lymphocytes. Taken together, these data show that the optical redox ratio can depict metabolic differences in distinct cell populations by flow cytometry.

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.

Caspase-8 promotes scramblase-mediated phosphatidylserine exposure and fusion of osteoclast precursors.

Efficient cellular fusion of mononuclear precursors is the prerequisite for the generation of fully functional multinucleated bone-resorbing osteoclasts. However, the exact molecular factors and mechanisms controlling osteoclast fusion remain incompletely understood. Here we identify RANKL-mediated activation of caspase-8 as early key event during osteoclast fusion. Single cell RNA sequencing-based analyses suggested that activation of parts of the apoptotic machinery accompanied the differentiation of osteoclast precursors into mature multinucleated osteoclasts. A subsequent characterization of osteoclast precursors confirmed that RANKL-mediated activation of caspase-8 promoted the non-apoptotic cleavage and activation of downstream effector caspases that translocated to the plasma membrane where they triggered activation of the phospholipid scramblase Xkr8. Xkr8-mediated exposure of phosphatidylserine, in turn, aided cellular fusion of osteoclast precursors and thereby allowed generation of functional multinucleated osteoclast syncytia and initiation of bone resorption. Pharmacological blockage or genetic deletion of caspase-8 accordingly interfered with fusion of osteoclasts and bone resorption resulting in increased bone mass in mice carrying a conditional deletion of caspase-8 in mononuclear osteoclast precursors. These data identify a novel pathway controlling osteoclast biology and bone turnover with the potential to serve as target for therapeutic intervention during diseases characterized by pathologic osteoclast-mediated bone loss. Proposed model of osteoclast fusion regulated by caspase-8 activation and PS exposure. RANK/RANK-L interaction. Activation of procaspase-8 into caspase-8. Caspase-8 activates caspase-3. Active capase-3 cleaves Xkr8. Local PS exposure is induced. Exposed PS is recognized by the fusion partner. FUSION. PS is re-internalized.

B-cell depletion in autoimmune diseases.

B cells have a pivotal function in the pathogenesis of autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis and systemic lupus erythematosus. In autoimmune disease, B cells orchestrate antigen presentation, cytokine production and autoantibody production, the latter via their differentiation into antibody-secreting plasmablasts and plasma cells. This article addresses the current therapeutic strategies to deplete B cells in order to ameliorate or potentially even cure autoimmune disease. It addresses the main target antigens in the B-cell lineage that are used for therapeutic approaches. Furthermore, it summarises the current evidence for successful treatment of autoimmune disease with monoclonal antibodies targeting B cells and the limitations and challenges of these approaches. Finally, the concept of deep B-cell depletion and immunological reset by chimeric antigen receptor T cells is discussed, as well as the lessons from this approach for better understanding the role of B cells in autoimmune disease.

Selective CAR T cell-mediated B cell depletion suppresses IFN signature in SLE.

Applying advanced molecular profiling together with highly specific targeted therapies offers the possibility to better dissect the mechanisms underlying immune-mediated inflammatory diseases such as systemic lupus erythematosus (SLE) in humans. Here we apply a combination of single-cell RNA-Seq and T/B cell repertoire analysis to perform an in-depth characterization of molecular changes in the immune-signature upon CD19 CAR T cell-mediated depletion of B cells in patients with SLE. The resulting data sets not only confirm a selective CAR T cell-mediated reset of the B cell response but simultaneously reveal consequent changes in the transcriptional signature of monocyte and T cell subsets that respond with a profound reduction in type I IFN signaling. Our current data, thus, provide evidence for a causal relationship between the B cell response and the increased IFN signature observed in SLE and additionally demonstrate the usefulness of combining targeted therapies and analytic approaches to decipher molecular mechanisms of immune-mediated inflammatory diseases in humans.

Discrepant Phenotyping of Monocytes Based on CX3CR1 and CCR2 Using Fluorescent Reporters and Antibodies.

Monocytes, as well as downstream macrophages and dendritic cells, are essential players in the immune system, fulfilling key roles in homeostasis as well as in inflammatory conditions. Conventionally, driven by studies on reporter models, mouse monocytes are categorized into a classical and a non-classical subset based on their inversely correlated surface expression of Ly6C/CCR2 and CX3CR1. Here, we aimed to challenge this concept by antibody staining and reporter mouse models. Therefore, we took advantage of Cx3cr1GFP and Ccr2RFP reporter mice, in which the respective gene was replaced by a fluorescent reporter protein gene. We analyzed the expression of CX3CR1 and CCR2 by flow cytometry using several validated fluorochrome-coupled antibodies and compared them with the reporter gene signal in these reporter mouse strains. Although we were able to validate the specificity of the fluorochrome-coupled flow cytometry antibodies, mouse Ly6Chigh classical and Ly6Clow non-classical monocytes showed no differences in CX3CR1 expression levels in the peripheral blood and spleen when stained with these antibodies. On the contrary, in Cx3cr1GFP reporter mice, we were able to reproduce the inverse correlation of the CX3CR1 reporter gene signal and Ly6C surface expression. Furthermore, differential CCR2 surface expression correlating with the expression of Ly6C was observed by antibody staining, but not in Ccr2RFP reporter mice. In conclusion, our data suggest that phenotyping strategies for mouse monocyte subsets should be carefully selected. In accordance with the literature, the suitability of CX3CR1 antibody staining is limited, whereas for CCR2, caution should be applied when using reporter mice.

Inflammatory tissue priming: novel insights and therapeutic opportunities for inflammatory rheumatic diseases.

Due to optimised treatment strategies and the availability of new therapies during the last decades, formerly devastating chronic inflammatory diseases such as rheumatoid arthritis or systemic sclerosis (SSc) have become less menacing. However, in many patients, even state-of-the-art treatment cannot induce remission. Moreover, the risk for flares strongly increases once anti-inflammatory therapy is tapered or withdrawn, suggesting that underlying pathological processes remain active even in the absence of overt inflammation. It has become evident that tissues have the ability to remember past encounters with pathogens, wounds and other irritants, and to react more strongly and/or persistently to the next occurrence. This priming of the tissue bears a paramount role in defence from microbes, but on the other hand drives inflammatory pathologies (the Dr Jekyll and Mr Hyde aspect of tissue adaptation). Emerging evidence suggests that long-lived tissue-resident cells, such as fibroblasts, macrophages, long-lived plasma cells and tissue-resident memory T cells, determine inflammatory tissue priming in an interplay with infiltrating immune cells of lymphoid and myeloid origin, and with systemically acting factors such as cytokines, extracellular vesicles and antibodies. Here, we review the current state of science on inflammatory tissue priming, focusing on tissue-resident and tissue-occupying cells in arthritis and SSc, and reflect on the most promising treatment options targeting the maladapted tissue response during these diseases.

Bispecific T cell engager therapy for refractory rheumatoid arthritis.

Bispecific T cell engagers (BiTEs) kill B cells by engaging T cells. BiTEs are highly effective in acute lymphoblastic leukemia. Here we treated six patients with multidrug-resistant rheumatoid arthritis (RA) with the CD19xCD3 BiTE blinatumomab under compassionate use. Low doses of blinatumomab led to B cell depletion and concomitant decrease of T cells, documenting their engager function. Treatment was safe, with brief increase in body temperature and acute phase proteins during first infusion but no signs of clinically relevant cytokine-release syndrome. Blinatumomab led to a rapid decline in RA clinical disease activity in all patients, improved synovitis in ultrasound and FAPI-PET-CT and reduced autoantibodies. High-dimensional flow cytometry analysis of B cells documented an immune reset with depletion of activated memory B cells, which were replaced by nonclass-switched IgD-positive naïve B cells. Together, these data suggest the feasibility and potential for BiTEs to treat RA. This approach warrants further exploration on other B-cell-mediated autoimmune diseases.

Metabolic rewiring promotes anti-inflammatory effects of glucocorticoids.

Glucocorticoids represent the mainstay of therapy for a broad spectrum of immune-mediated inflammatory diseases. However, the molecular mechanisms underlying their anti-inflammatory mode of action have remained incompletely understood1. Here we show that the anti-inflammatory properties of glucocorticoids involve reprogramming of the mitochondrial metabolism of macrophages, resulting in increased and sustained production of the anti-inflammatory metabolite itaconate and consequent inhibition of the inflammatory response. The glucocorticoid receptor interacts with parts of the pyruvate dehydrogenase complex whereby glucocorticoids provoke an increase in activity and enable an accelerated and paradoxical flux of the tricarboxylic acid (TCA) cycle in otherwise pro-inflammatory macrophages. This glucocorticoid-mediated rewiring of mitochondrial metabolism potentiates TCA-cycle-dependent production of itaconate throughout the inflammatory response, thereby interfering with the production of pro-inflammatory cytokines. By contrast, artificial blocking of the TCA cycle or genetic deficiency in aconitate decarboxylase 1, the rate-limiting enzyme of itaconate synthesis, interferes with the anti-inflammatory effects of glucocorticoids and, accordingly, abrogates their beneficial effects during a diverse range of preclinical models of immune-mediated inflammatory diseases. Our findings provide important insights into the anti-inflammatory properties of glucocorticoids and have substantial implications for the design of new classes of anti-inflammatory drugs.

CD19 CAR T-Cell Therapy in Autoimmune Disease - A Case Series with Follow-up.

BACKGROUND: Treatment for autoimmune diseases such as systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, and systemic sclerosis often involves long-term immune suppression. Resetting aberrant autoimmunity in these diseases through deep depletion of B cells is a potential strategy for achieving sustained drug-free remission. METHODS: We evaluated 15 patients with severe SLE (8 patients), idiopathic inflammatory myositis (3 patients), or systemic sclerosis (4 patients) who received a single infusion of CD19 chimeric antigen receptor (CAR) T cells after preconditioning with fludarabine and cyclophosphamide. Efficacy up to 2 years after CAR T-cell infusion was assessed by means of Definition of Remission in SLE (DORIS) remission criteria, American College of Rheumatology-European League against Rheumatism (ACR-EULAR) major clinical response, and the score on the European Scleroderma Trials and Research Group (EUSTAR) activity index (with higher scores indicating greater disease activity), among others. Safety variables, including cytokine release syndrome and infections, were recorded. RESULTS: The median follow-up was 15 months (range, 4 to 29). The mean (±SD) duration of B-cell aplasia was 112±47 days. All the patients with SLE had DORIS remission, all the patients with idiopathic inflammatory myositis had an ACR-EULAR major clinical response, and all the patients with systemic sclerosis had a decrease in the score on the EUSTAR activity index. Immunosuppressive therapy was completely stopped in all the patients. Grade 1 cytokine release syndrome occurred in 10 patients. One patient each had grade 2 cytokine release syndrome, grade 1 immune effector cell-associated neurotoxicity syndrome, and pneumonia that resulted in hospitalization. CONCLUSIONS: In this case series, CD19 CAR T-cell transfer appeared to be feasible, safe, and efficacious in three different autoimmune diseases, providing rationale for further controlled clinical trials. (Funded by Deutsche Forschungsgemeinschaft and others.).

Eosinophils preserve bone homeostasis by inhibiting excessive osteoclast formation and activity via eosinophil peroxidase.

Eosinophils are involved in tissue homeostasis. Herein, we unveiled eosinophils as important regulators of bone homeostasis. Eosinophils are localized in proximity to bone-resorbing osteoclasts in the bone marrow. The absence of eosinophils in ΔdblGATA mice results in lower bone mass under steady-state conditions and amplified bone loss upon sex hormone deprivation and inflammatory arthritis. Conversely, increased numbers of eosinophils in IL-5 transgenic mice enhance bone mass under steady-state conditions and protect from hormone- and inflammation- mediated bone loss. Eosinophils strongly inhibit the differentiation and demineralization activity of osteoclasts and lead to profound changes in the transcriptional profile of osteoclasts. This osteoclast-suppressive effect of eosinophils is based on the release of eosinophil peroxidase causing impaired reactive oxygen species and mitogen-activated protein kinase induction in osteoclast precursors. In humans, the number and the activity of eosinophils correlates with bone mass in healthy participants and rheumatoid arthritis patients. Taken together, experimental and human data indicate a regulatory function of eosinophils on bone.

Abatacept inhibits inflammation and onset of rheumatoid arthritis in individuals at high risk (ARIAA): a randomised, international, multicentre, double-blind, placebo-controlled trial.

BACKGROUND: Individuals with anti-citrullinated protein antibodies (ACPAs) and subclinical inflammatory changes in joints are at high risk of developing rheumatoid arthritis. Treatment strategies to intercept this pre-stage clinical disease remain to be developed. We aimed to assess whether 6-month treatment with abatacept improves inflammation in preclinical rheumatoid arthritis. METHODS: The abatacept reversing subclinical inflammation as measured by MRI in ACPA positive arthralgia (ARIAA) study is a randomised, international, multicentre, double-blind, placebo-controlled trial done in 14 hospitals and community centres across Europe (11 in Germany, two in Spain, and one in the Czech Republic). Adults (aged ≥18 years) with ACPA positivity, joint pain (but no swelling), and signs of osteitis, synovitis, or tenosynovitis in hand MRI were randomly assigned (1:1) to weekly subcutaneous abatacept 125 mg or placebo for 6 months followed by a double-blind, drug-free, observation phase for 12 months. The primary outcome was the proportion of participants with any reduction in inflammatory MRI lesions at 6 months. The primary efficacy analysis was done in the modified intention-to-treat population, which included participants who were randomly assigned and received study medication. Safety analyses were conducted in participants who received the study medication and had at least one post-baseline observation. The study was registered with the EUDRA-CT (2014-000555-93). FINDINGS: Between Nov 6, 2014, and June 15, 2021, 139 participants were screened. Of 100 participants, 50 were randomly assigned to abatacept 125 mg and 50 to placebo. Two participants (one from each group) were excluded due to administration failure or refusing treatment; thus, 98 were included in the modified intention-to-treat population. 70 (71%) of 98 participants were female and 28 (29%) of 98 were male. At 6 months, 28 (57%) of 49 participants in the abatacept group and 15 (31%) of 49 participants in the placebo group showed improvement in MRI subclinical inflammation (absolute difference 26·5%, 95% CI 5·9-45·6; p=0·014). Four (8%) of 49 participants in the abatacept group and 17 (35%) of 49 participants in the placebo group developed rheumatoid arthritis (hazard ratio [HR] 0·14 [0·04-0·47]; p=0·0016). Improvement of MRI inflammation (25 [51%] of 49 participants in the abatacept group, 12 [24%] of 49 in the placebo group; p=0·012) and progression to rheumatoid arthritis (17 [35%] of 49, 28 [57%] of 49; HR 0·14 [0·04-0·47]; p=0·018) remained significantly different between the two groups after 18 months, 12 months after the end of the intervention. There were 12 serious adverse events in 11 participants (four [8%] of 48 in the abatacept group and 7 [14%] of 49 in the placebo group). No deaths occurred during the study. INTERPRETATION: 6-month treatment with abatacept decreases MRI inflammation, clinical symptoms, and risk of rheumatoid arthritis development in participants at high risk. The effects of the intervention persist through a 1-year drug-free observation phase. FUNDING: Innovative Medicine Initiative.

Bona fide dendritic cells are pivotal precursors for osteoclasts.

OBJECTIVES: Osteoclasts (OCs) are myeloid-derived multinucleated cells uniquely able to degrade bone. However, the exact nature of their myeloid precursors is not yet defined. METHODS: CD11c-diphtheria toxin receptor (CD11cDTR) transgenic mice were treated with diphtheria toxin (DT) or phosphate buffered saline (PBS) during serum transfer arthritis (STA) and human tumour necrosis factor transgenic (hTNFtg) arthritis and scored clinically and histologically. We measured cytokines in synovitis by quantitative polymerase chain reaction (qPCR). We performed ovariectomy in CD11cDTR mice treated with PBS or DT. We analysed CD11cDTR, CD11c-Cre/CX3CR1-STOP-DTR and Zbtb46-DTR-treated mice with DT using histomorphometry and OC of CD11c and Zbtb46 fate reporter mice by fluorescent imaging. We sorted murine and human OC precursors and stimulated them with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL) to generate OCs. RESULTS: Targeting CD11c+ cells in vivo in models of inflammatory arthritis (STA and hTNFtg) ameliorates arthritis by reducing inflammatory bone destruction and OC generation. Targeting CD11c-expressing cells in unchallenged mice removes all OCs in their long bones. OCs do not seem to be derived from CD11c+ cells expressing CX3CR1+, but from Zbtb46+conventional dendritic cells (cDCs) as all OCs in Zbtb46-Tomato fate reporter mice are Tomato+. In line, administration of DT in Zbtb46-DTR mice depletes all OCs in long bones. Finally, human CD1c-expressing cDCs readily differentiated into bone resorbing OCs. CONCLUSION: Taken together, we identify DCs as important OC precursors in bone homeostasis and inflammation, which might open new avenues for therapeutic interventions in OC-mediated diseases.

Baricitinib Improves Bone Properties and Biomechanics in Patients With Rheumatoid Arthritis: Results of the Prospective Interventional BARE BONE Trial.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by erosive joint damage, deterioration of bone mass, and biomechanics. Preclinical evidence suggests a beneficial effect of Janus kinase inhibition (JAKi) on bone properties, but clinical data are scarce to date. In this study, we evaluated the effect of JAKi through baricitinib (BARI) on 1) volumetric bone mineral density (vBMD), bone microstructure, biomechanics, and erosion repair and 2) synovial inflammation in RA patients. METHODS: Prospective, single-arm, interventional, open-label, single-center phase 4 study in RA patients with pathological bone status and clinical indication of JAKi (BARE BONE trial). Participants received BARI (4 mg/day) over 52 weeks. To assess bone properties and synovial inflammation, high-resolution computed tomography scans and magnetic resonance imaging were performed at baseline (BL), week 24, and week 52. Clinical response and safety were monitored. RESULTS: Thirty RA patients were included. BARI significantly improved disease activity (Disease Activity Score in 28 joints using the erythrocyte sedimentation rate: 4.82 ± 0.90 to 2.71 ± 0.83) and synovial inflammation (RAMRIS synovitis score: 5.3 [4.2] to 2.7 [3.5]). We observed a significant improvement in trabecular vBMD with a mean change of 6.11 mgHA/mm3 (95% confidence interval [95% CI] 0.01-12.26). Biomechanical properties also improved with mean change from baseline in estimated stiffness of 2.28 kN/mm (95% CI 0.30-4.25) and estimated failure load of 98.8 N (95% CI 15.9-181.7). The number and size of erosions in the metacarpal joints remained stable. No new safety signals with BARI treatment were observed. CONCLUSION: Bones of RA patients improve with BARI therapy, as shown by an increase in trabecular bone mass and an improvement of biomechanical properties.

Stunning of neutrophils accounts for the anti-inflammatory effects of clodronate liposomes.

Clodronate liposomes (Clo-Lip) have been widely used to deplete mononuclear phagocytes (MoPh) to study the function of these cells in vivo. Here, we revisited the effects of Clo-Lip together with genetic models of MoPh deficiency, revealing that Clo-Lip exert their anti-inflammatory effects independent of MoPh. Notably, not only MoPh but also polymorphonuclear neutrophils (PMN) ingested Clo-Lip in vivo, which resulted in their functional arrest. Adoptive transfer of PMN, but not of MoPh, reversed the anti-inflammatory effects of Clo-Lip treatment, indicating that stunning of PMN rather than depletion of MoPh accounts for the anti-inflammatory effects of Clo-Lip in vivo. Our data highlight the need for a critical revision of the current literature on the role of MoPh in inflammation.

Non-invasive metabolic profiling of inflammation in joints and entheses by multispectral optoacoustic tomography.

OBJECTIVE: To explore the metabolic characteristics of arthritis and enthesitis using multispectral opto-acoustic tomography (MSOT), a technology using near-infrared multispectral laser to stimulate tissues and detect the emitted acoustic energy, enabling non-invasive quantification of tissue components in vivo based on differential absorbance at multiple wavelengths. METHODS: We performed a cross-sectional study in patients with RA or PsA and healthy controls (HCs). Participants underwent clinical, ultrasonographic and MSOT examination of MCP and wrist joints as well as the entheses of the common extensor tendon at the lateral humeral epicondyles and of the patellar, quadriceps and Achilles tendon. MSOT-measured haemoglobin (Hb), oxygen saturation, collagen and lipid levels were quantified and scaled mean differences between affected and unaffected joints and entheses were calculated as defined by clinical examination or ultrasonography using linear mixed effects models. RESULTS: We obtained 1535 MSOT and 982 ultrasonography scans from 87 participants (34 PsA, 17 RA, 36 HCs). Entheseal tenderness was not associated with significant metabolic changes, whereas enthesitis-related sonographic changes were associated with increased total Hb, oxygen saturation and collagen content. In contrast, the presence of arthritis-related clinical and sonographic findings showed increased Hb levels, reduced oxygen saturation and reduced collagen content. Synovial hypertrophy was associated with increased lipid content in the joints. CONCLUSION: MSOT allows determination of distinct metabolic differences between arthritis and enthesitis in a non-invasive setting in humans in vivo.

Macrophages inhibit Coxiella burnetii by the ACOD1-itaconate pathway for containment of Q fever.

Infection with the intracellular bacterium Coxiella (C.) burnetii can cause chronic Q fever with severe complications and limited treatment options. Here, we identify the enzyme cis-aconitate decarboxylase 1 (ACOD1 or IRG1) and its product itaconate as protective host immune pathway in Q fever. Infection of mice with C. burnetii induced expression of several anti-microbial candidate genes, including Acod1. In macrophages, Acod1 was essential for restricting C. burnetii replication, while other antimicrobial pathways were dispensable. Intratracheal or intraperitoneal infection of Acod1-/- mice caused increased C. burnetii burden, weight loss and stronger inflammatory gene expression. Exogenously added itaconate restored pathogen control in Acod1-/- mouse macrophages and blocked replication in human macrophages. In axenic cultures, itaconate directly inhibited growth of C. burnetii. Finally, treatment of infected Acod1-/- mice with itaconate efficiently reduced the tissue pathogen load. Thus, ACOD1-derived itaconate is a key factor in the macrophage-mediated defense against C. burnetii and may be exploited for novel therapeutic approaches in chronic Q fever.