Exploration of ACOD1-itaconate in controlling Coxiella burnetii infection in mice

Background and objectives: The zoonotic disease Q fever is caused by the obligate intracellular gram-negative bacterium Coxiella (C.) burnetii. Humans are infected by inhalation of infectious material from the reservoir animals (sheep, goats, cattle). Q fever is usually a self-limiting asymptomatic or mild flu-like respiratory infection but can become chronic and cause endocarditis in some patients. Despite extended treatment courses antibiotics are frequently inefficient in chronic Q fever. In activated macrophages Acod1 expression is upregulated and ITA exerts anti-microbial and immunoregulatory effects. MES and CTC have different but overlapping effects on the expression of inflammatory genes and macrophage metabolism. Here we investigated how C. burnetii replication is regulated by the mitochondrial enzyme cis-aconitate decarboxylase 1 (ACOD1 or IRG1) and its product itaconate (ITA). We also investigated the effect of ITA isomers mesaconate (MES) and citraconate (CTC) on C. burnetii. Methods: The attenuated Nine Mile phase II (NMII) strain of C. burnetii was used for infection of bone-marrow derived macrophages (BMM) and of mice. Following infection, BMM were treated with ITA and its isomers (MES and CTC). MTT assay was used to determine cellular toxicity of the supplemented isomers. The intracellular concentration of the isomers was measured by gas chromatography mass spectrometry analysis (GC-MS). Cytokine proteins were analyzed by ELISA and by multiplex bead-based analysis (LEGENDplex assay). The impact of Acod1 knock-out and of ITA supplementation on the control of C. burnetii replication was determined by different readouts like immunofluorescent microscopy, FACS analysis, qPCR and immunohistochemistry from infected BMM and mouse organ samples. For in vivo experiments, mice were infected via the intraperitoneal and intratracheal routes following ITA treatment (i.p). In adoptive cell transfer experiments BMM were transferred to the mice via i.p. route the day before and after C. burnetii infection (i.p.). Expression analysis of different genes of interest was performed by qRT-PCR form infected BMM and mice organs. Finally, the direct effect of ITA and its isomers on C. burnetii was determined by OD measurement and CFU count. Results: Expression of Acod1 mRNA was upregulated after C. burnetii infection and led to ITA production in BMM and in organs of infected mice, which was essential for restricting C. burnetii replication. BMM from Acod1-/- mice was unable to control C. burnetii growth. In vivo, an increased bacterial burden along with weight loss and stronger inflammatory gene expression were noticed in Acod1-/- compared to control mice after intraperitoneal and intratracheal infection. Exogenously supplemented ITA and its isomers were easily taken up by BMM, where ITA but not its isomers restricted C. burnetii replication in Acod1-/- BMM. Following infection, enhanced uptake of ITA and MES but not CTC was noticed in BMM where MES but not CTC competed with ITA for uptake. In contrast, all three isomers directly inhibited C. burnetii growth in axenic culture in a dose-dependent manner, and ITA was found more potent than MES and CTC. Finally, exogenous ITA treatment efficiently reduced the tissue pathogen load in Acod1-/- mice. However, treatment with ITA was not effective in other C. burnetiid-susceptible mice deficient in TLR-signaling (Myd88-/-), production of IFNγ (Ifnγ-/-) or lymphoid immune cells (Rag2-/-γChain-/-). On the other hand, adoptive transfer of Acod1+/+ BMM the day before infection promoted control of bacterial replication in Acod1-/- as well as Myd88-/- mice, indicating a synergistic effect of ITA and other mechanisms in pathogen control. Conclusions and discussion: Being a key factor in macrophage-mediated control of C. burnetii, ACOD1-derived ITA needs further investigation to develop new treatment strategies for chronic Q fever. A special focus should be on the yet unknown mechanistic details of C. burnetii control in BMM and in mice. In addition, the divergent effect of ITA isomers in controlling C. burnetii in BMM and in axenic culture suggests differences in their uptake and intracellular transport mechanisms, which may have important consequences for employing ITA and its isomers for treatment of bacterial infections. Furthermore, investigation of ITA isomers’ effect on C. burnetii should be extended to our in vivo mouse model of Q fever.