Highlights d Hypoxia curtails C. burnetii replication in macrophages without reducing bacterial viability d Hypoxia induces robust HIF1a accumulation in infected macrophages d HIF1a reduces STAT3 activation and citrate availability in infected hypoxic macrophages d Low levels of TCA metabolites impede C. burnetii replication in macrophages

The intracellular pathogen Coxiella (C.) burnetii causes Q fever, a usually self-limiting respiratory infection that becomes chronic and severe in some patients. Innate immune recognition of C. burnetii and its role in the decision between resolution and chronicity is not understood well. However, TLR2 is important for the response to C. burnetii in mice, and genetic polymorphisms in Myd88 have been associated with chronic Q fever in humans. Here, we have employed MyD88-deficient mice in infection models with the attenuated C. burnetii Nine Mile phase II strain (NMII). Myd88−/− macrophages failed to restrict the growth of NMII in vitro, and to upregulate production of the cytokines TNF, IL-6, and IL-10. Following intraperitoneal infection, NMII bacterial burden was significantly higher on day 5 and 20 in organs of Myd88−/− mice. After infection via the natural route by intratracheal injection, a higher bacterial load in the lung and increased dissemination of NMII to other organs was observed in MyD88-deficient mice. While wild-type mice essentially cleared NMII on day 27 after intratracheal infection, it was still readily detectable on day 42 in multiple organs in the absence of MyD88. Despite the elevated bacterial load, Myd88−/− mice had less granulomatous inflammation and expressed significantly lower levels of chemoattractants, inflammatory cytokines, and of several IFNγ-induced genes relevant for control of intracellular pathogens. Together, our results show that MyD88-dependent signaling is essential for early control of C. burnetii replication and to prevent systemic spreading. The continued presence of NMII in the organs of Myd88−/− mice constitutes a new mouse model to study determinants of chronicity and resolution in Q fever.

Various factors of the tissue microenvironment such as the oxygen concentration influence the host–pathogen interaction. During the past decade, hypoxia-driven signaling via hypoxia-inducible factors (HIF) has emerged as an important factor that affects both the pathogen and the host. In this chapter, we will review the current knowledge of this complex interplay, with a particular emphasis given to the impact of hypoxia and HIF on the inflammatory and antimicrobial activity of myeloid cells, the bacterial responses to hypoxia and the containment of bacterial infections under oxygen-limited conditions. We will also summarize how low oxygen concentrations influence the metabolism of neutrophils, macrophages and dendritic cells. Finally, we will discuss the consequences of hypoxia and HIFα activation for the invading pathogen, with a focus on Pseudomonas aeruginosa, Mycobacterium tuberculosis, Coxiella burnetii, Salmonella enterica and Staphylococcus aureus. This includes a description of the mechanisms and microbial factors, which the pathogens use to sense and react to hypoxic conditions.

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.

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BackgroundCoxiella burnetii is a zoonotic pathogen, infecting humans, livestock, pets, birds and ticks. Domestic ruminants such as cattle, sheep, and goats are the main reservoir and major cause of human infection. Infected ruminants are usually asymptomatic, while in humans infection can cause significant disease. Human and bovine macrophages differ in their permissiveness for C. burnetii strains from different host species and of various genotypes and their subsequent host cell response, but the underlying mechanism(s) at the cellular level are unknown.MethodsC. burnetii infected primary human and bovine macrophages under normoxic and hypoxic conditions were analyzed for (i) bacterial replication by CFU counts and immunofluorescence; (ii) immune regulators by westernblot and qRT-PCR; cytokines by ELISA; and metabolites by gas chromatography-mass spectrometry (GC-MS).ResultsHere, we confirmed that peripheral blood-derived human macrophages prevent C. burnetii replication under oxygen-limiting conditions. In contrast, oxygen content had no influence on C. burnetii replication in bovine peripheral blood-derived macrophages. In hypoxic infected bovine macrophages, STAT3 is activated, even though HIF1α is stabilized, which otherwise prevents STAT3 activation in human macrophages. In addition, the TNFα mRNA level is higher in hypoxic than normoxic human macrophages, which correlates with increased secretion of TNFα and control of C. burnetii replication. In contrast, oxygen limitation does not impact TNFα mRNA levels in C. burnetii-infected bovine macrophages and secretion of TNFα is blocked. As TNFα is also involved in the control of C. burnetii replication in bovine macrophages, this cytokine is important for cell autonomous control and its absence is partially responsible for the ability of C. burnetii to replicate in hypoxic bovine macrophages. Further unveiling the molecular basis of macrophage-mediated control of C. burnetii replication might be the first step towards the development of host directed intervention measures to mitigate the health burden of this zoonotic agent.

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, significant 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.

HIF1α is an important transcription factor regulating not only cellular responses to hypoxia, but also anti-infective defense responses. We recently showed that HIF1α hampers replication of the obligate intracellular pathogen Coxiella burnetii which causes the zoonotic disease Q fever. Prior to development of chronic Q fever, it is assumed that the bacteria enter a persistent state. As HIF1α and/or hypoxia might be involved in the induction of C. burnetii persistence, we analyzed the role of HIF1α and hypoxia in the interaction of macrophages with C. burnetii to understand how the bacteria manipulate HIF1α stability and activity. We demonstrate that a C. burnetii-infection initially induces HIF1α stabilization, which decreases then over the course of an infection. This reduction depends on bacterial viability and a functional type IV secretion system (T4SS). While neither the responsible T4SS effector protein(s) nor the molecular mechanism leading to this partial HIF1α destabilization have been identified, our results demonstrate that C. burnetii influences the expression of HIF1α target genes in multiple ways. Therefore, a C. burnetii infection promotes HIF1α-mediated upregulation of several metabolic target genes; affects apoptosis-regulators towards a more pro-apoptotic signature; and under hypoxic conditions, shifts the ratio of the inflammatory genes analyzed towards a pro-inflammatory profile. Taken together, C. burnetii modulates HIF1α in a still elusive manner and alters the expression of multiple HIF1α target genes.