Die HIF1α-induzierte Citratverminderung inhibiert die Replikation von Coxiella burnetii in Makrophagen

Coxiella burnetii is a Gram-negative obligate intracellular bacterium and the causative agent of the zoonotic disease Q fever. Humans are infected through the inhalation of contaminated aerosols. Once inhaled, alveolar macrophages are the first line of defense against inhaled C. burnetii. Infected individuals can be asymptomatic or can develop a flu-like illness, atypical pneumonia or even hepatitis. Apart from acute Q fever, around 2-5% of C. burnetii infected humans will develop chronic Q fever, which mainly manifests as endocarditis years after exposure to the pathogen. Treatments of chronic Q fever are inefficient and harsh, as a combination of doxycycline and hydroxychloroquine is applied for 12 months. Therefore, the establishment of new therapies is inevitable, presumably therapies that prevent persistence of the pathogen in the first place. Yet, details about how C. burnetii escapes the immune system and persists for years inside the host are not established. Here, we examined the role of oxygen in C. burnetii replication and persistence. Indeed, C. burnetii replication is not possible in hypoxic macrophages, while C. burnetii replicates in normoxic macrophages. Of note, C. burnetii stays viable under hypoxia. The reason behind the impairment of replication is not lack of maturation of the Coxiella-containing vacuole (CCV) or changes in phagocytic uptake between normoxia and hypoxia. However, hypoxia-induced HIF1α stabilization contributes to the replication defect in hypoxic macrophages. Thus, HIF1α stabilization leads to reduced Stat3 phosphorylation and translocation into the nucleus. As a result, impairment of C. burnetii replication is caused by HIF1α-induced Stat3 inhibition. Moreover, HIF1α and Stat3 are involved in the regulation of the TCA cycle, including citrate. Therefore, the importance of citrate was analyzed for intracellular C. burnetii. Indeed, intracellular citrate levels are lower in hypoxic macrophages as compared to normoxic macrophages. This is regulated by the HIF1α-Stat3 axis. In fact, HIF1α reduces citrate levels, while Stat3 enhances it. Therefore, HIF1α-induced Stat3 inhibition results in less citrate levels in the cell. Importantly, C. burnetii is affected by this decrease in citrate levels. Inhibiting the mitochondrial citrate transporter protein leads to an impairment of C. burnetii replication in normoxic macrophages. On the contrary, supplementing hypoxic macrophages with exogenous citrate results in boosting replication of C. burnetii enormously. This shows the importance of citrate in C. burnetii replication although it is still unclear how citrate is utilized 2 by the pathogen. Moreover, C. burnetii reacts to the reduced host citrate by adjusting the transcription of its genes involved in citrate metabolism. Furthermore, C. burnetii attempts to counteract the effects of hypoxia by modulating HIF1α stabilization through secreting the type IV effector protein NopA. NopA destabilizes HIF1α, but how this destabilization is mediated requires further investigation. In summary, our data suggest that regulation of citrate levels by HIF1α represents a novel principle of nutritional pathogen-containment of C. burnetii, which might lead to a state of persistence and thus, to chronic Q fever.Bei Coxiella burnetii handelt es sich um ein Gram-negatives, obligat intrazelluläres Bakterium, welches die Zoonose Q-Fieber auslöst. Menschen werden überwiegend durch das Einatmen bakteriell kontaminierter Aerosole infiziert. Nach dem Einatmen treten eingeatmete C. burnetii zunächst mit Alveolarmakrophagen in Wechselwirkung. Infizierte Personen können asymptomatisch sein oder aber eine grippeähnliche Krankheit, eine atypische Lungenentzündung oder sogar eine Hepatitis entwickeln. Abgesehen von diesen Formen des akuten Q-Fiebers entwickeln etwa 2-5% der mit C. burnetii infizierten Menschen chronisches Q-Fieber, das sich Jahre nach Exposition gegenüber dem Erreger hauptsächlich als potentiell letale Endokarditis manifestiert. Die Behandlung von chronischem Q-Fieber ist ineffizient und schwierig, da eine Kombination aus Doxycyclin und Hydroxychloroquin über 12 Monate angewendet werden muss. Daher ist die Einführung neuer Therapien dringend notwendig. Diese Therapien könnten in erster Linie auf die Persistenz des Erregers abzielen. Wie C. burnetii dem Immunsystem ausweicht und jahrelang im Wirt überlebt, ist bislang nicht erforscht. In der vorliegenden Studie wird die Rolle von Sauerstoff bei der Replikation und Persistenz von C. burnetii untersucht. In der Tat ist eine Replikation von C. burnetii in hypoxischen Makrophagen nicht möglich, während C. burnetii in normoxischen Makrophagen normal repliziert. Bemerkenswerterweise bleibt C. burnetii unter Hypoxie allerdings lebensfähig. Der Grund für die Beeinträchtigung der Replikation ist nicht die mangelnde Reifung der Coxiella-haltigen Vakuole (CCV) oder Veränderungen der phagozytischen Aufnahme zwischen Normoxie und Hypoxie. Die Hypoxie-induzierte HIF1α-Stabilisierung trägt zum Replikationsdefekt in hypoxischen Makrophagen bei, indem sie zu einer verringerten Stat3-Phosphorylierung und -Translokation in den Kern führt. Infolgedessen wird eine Beeinträchtigung der C. burnetii-Replikation durch eine HIF1α-induzierte Stat3-Inhibierung verursacht. Darüber hinaus sind HIF1α und Stat3 an der Regulation des TCA-Zyklus einschließlich Citrat beteiligt. Daher wurde die Bedeutung von Citrat für intrazelluläre C. burnetii analysiert. Der intrazelluläre Citratspiegel ist bei hypoxischen Makrophagen im Vergleich zu normoxischen Makrophagen verringert, was durch die HIF1α-Stat3-Achse reguliert wird. Tatsächlich reduziert HIF1α den Citratspiegel, während Stat3 ihn erhöht. Daher führt die HIF1α-induzierte 4 Stat3-Hemmung zu niedrigeren Citratspiegeln in der Zelle. C. burnetii ist von dieser Abnahme des Citratspiegels direkt betroffen. Die Hemmung des mitochondrialen Citrattransporterproteins führt zu einer Beeinträchtigung der C. burnetii-Replikation in normoxischen Makrophagen. Im Gegensatz dazu führt die Ergänzung hypoxischer Makrophagen-Kulturen mit exogenem Citrat zu einer enormen Steigerung der Replikation von C. burnetii. Dies zeigt die Bedeutung von Citrat bei der Replikation von C. burnetii, wobei noch unklar ist, welche Rolle Citrat im Metabolismus des Pathogens spielt. Darüber hinaus reagiert C. burnetii auf das reduzierte Wirtscitrat, indem es die Transkription seiner am Citrat Stoffwechsel beteiligten Gene anpasst. C. burnetii versucht den Auswirkungen der Hypoxie entgegenzuwirken, indem es die HIF1α-Stabilisierung durch Sekretion des Typ IV Effektorproteins NopA moduliert. NopA destabilisiert HIF1α, wobei der zu Grunde liegende Mechanismus noch unklar ist. Zusammenfassend legen unsere Daten nahe, dass die Regulierung des Citratspiegels durch HIF1α ein neues Prinzip der Pathogen-Eindämmung basierend auf Nährstoffverminderung für C. burnetii darstellt, das zu einem Zustand der Persistenz und damit zu chronischem Q-Fieber führen kann

Coxiella burnetii Affects HIF1α Accumulation and HIF1α Target Gene Expression

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

Mechanisms controlling bacterial infection in myeloid cells under hypoxic conditions

Abstract 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

MyD88 Is Required for Efficient Control of Coxiella burnetii Infection and Dissemination

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

Bovine blood derived macrophages are unable to control Coxiella burnetii replication under hypoxic conditions

Background Coxiella 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. Methods C. 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). Results Here, 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

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

Bovine blood derived macrophages are unable to control replication under hypoxic conditions

Background: Coxiella 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. Methods: C. 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). Results: Here, 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

Limitation of TCA Cycle Intermediates Represents an Oxygen-Independent Nutritional Antibacterial Effector Mechanism of Macrophages

Summary: In hypoxic and inflamed tissues, oxygen (O2)-dependent antimicrobial defenses are impaired due to a shortage of O2. To gain insight into the mechanisms that control bacterial infection under hypoxic conditions, we infected macrophages with the obligate intracellular pathogen Coxiella burnetii, the causative agent of Q fever. Our experiments revealed that hypoxia impeded C. burnetii replication in a hypoxia-inducible factor (HIF) 1α-dependent manner. Mechanistically, under hypoxia, HIF1α impaired the activity of STAT3, which in turn reduced the intracellular level of TCA cycle intermediates, including citrate, and impeded C. burnetii replication in macrophages. However, bacterial viability was maintained, allowing the persistence of C. burnetii, which is a prerequisite for the development of chronic Q fever. This knowledge will open future research avenues on the pathogenesis of chronic Q fever. In addition, the regulation of TCA cycle metabolites by HIF1α represents a previously unappreciated mechanism of host defense against intracellular pathogens. : The mechanisms that control bacterial infection under hypoxic conditions are only partially understood. Hayek et al. show that hypoxia-mediated stabilization of HIF1α results in the inhibition of STAT3 activation and the reduction of TCA metabolite levels, including citrate, in macrophages. This prevents C. burnetii replication without reducing bacterial viability. Keywords: Coxiella burnetii, HIF1α, STAT3, citrate, macrophag