Primary Role for Toll-Like Receptor-Driven Tumor Necrosis Factor Rather than Cytosolic Immune Detection in Restricting Coxiella burnetii Phase II Replication within Mouse Macrophages

Abstract

dCoxiella burnetii replicates within permissive host cells by employing a Dot/Icm type IV secretion system (T4SS) to translocate effector proteins that direct the formation of a parasitophorous vacuole. C57BL/6 mouse macrophages restrict the intracellular replication of the C. burnetii Nine Mile phase II (NMII) strain. However, eliminating Toll-like receptor 2 (TLR2) permits bacterial replication, indicating that the restriction of bacterial replication is immune mediated. Here, we examined whether additional innate immune pathways are employed by C57BL/6 macrophages to sense and restrict NMII replication. In addition to the known role of TLR2 in detecting and restricting NMII infection, we found that TLR4 also contributes to cytokine responses but is not required to restrict bacterial replication. Furthermore, the TLR signaling adaptors MyD88 and Trif are required for cytokine responses and restricting bacterial replication. The C. burnetii NMII T4SS translocates bacterial products into C57BL/6 macrophages. However, there was little evidence of cytosolic immune sensing of NMII, as there was a lack of inflammasome activation, T4SS-dependent cytokine responses, and robust type I interferon (IFN) production, and these pathways were not required to restrict bacterial replication. Instead, endogenous tumor necrosis factor (TNF) produced upon TLR sensing of C. burnetii NMII was required to control bacterial replication. Therefore, our findings indicate a primary role for TNF produced upon immune detection of C. burnetii NMII by TLRs, rather than cytosolic PRRs, in enabling C57BL/6 macrophages to restrict bacterial replication.T o initiate innate immune defense against bacterial pathogens, infected host cells utilize pattern recognition receptors (PRRs) to detect pathogen-associated molecular patterns (PAMPs) (1-3). Toll-like receptors (TLRs) located at the cell surface and within endosomes detect extracellular PAMPs such as bacterial lipoproteins and lipopolysaccharide (LPS) (4). Downstream of TLRs, the adaptor proteins MyD88 and Trif activate several signaling pathways, including NF-B, mitogen-activated protein kinases (MAPKs), and interferon (IFN) regulatory factor 3 (IRF3), which direct the expression of proinflammatory cytokines and other antimicrobial effectors (4). For intracellular bacterial pathogens, cytosolic PRRs, such as those of the nucleotide binding domain/ leucine-rich repeat (NLR) and RIG-I-like receptor (RLR) families, often are critical for host defense as they respond to PAMPs introduced into the host cell cytosol by bacterial pore-forming toxins or specialized secretion systems (5-8). In addition, cytosolic sensing can lead to the assembly of a multiprotein complex termed the inflammasome, which activates the host proteases caspase-1 and caspase-11, resulting in the release of IL-1 family cytokines and a form of cell death known as pyroptosis (9-16). These innate immune pathways collaborate to restrict intracellular bacterial infection through both cell-intrinsic and -extrinsic mechanisms (17)(18...

Host and Bacterial Factors Control Susceptibility of Drosophila melanogaster to Coxiella burnetii Infection

“…Interestingly, no difference in Coxiella growth was observed in Eiger mutant flies compared to control w 1118 flies, indicating dissociation between mortality and bacterial growth in these animals. Two recent studies have shown that in mouse bone marrow-derived macrophages there is production of TNF (66,67). Additionally, cells lacking TLR2 or its downstream signaling components exhibited reduced TNF production and increased levels of Coxiella (66).…”

Right on Q: Genetics begin to Unravel Coxiella Burnetii host cell Interactions

“…Essential for inflammasome activity is PAMP recognition by cytosolic Nod-like receptors. Coxiella infection of primary mouse macrophages fails to induce caspase-1 and capase-11 activation and the subsequent release of IL-1 cytokines [37,98]. Furthermore, Coxiella inhibits induction of caspase-11-mediated, noncanonical activation of the inflammasome by L. pneumophila in macrophage coinfection experiments [98].…”

“…Although potentiated innate immune signaling also occurs during NMII interactions ex vivo with primary human macrophages and dendritic cells [32,40], no growth defects relative to NMI are observed. However, the resulting robust immune response elicited by NMII during primary infection of mammals is thought to render the strain avirulent [37,40].…”

“…Increased surface hydrophobicity and protein exposure due to missing O-antigen are thought to facilitate NMII-host plasma membrane interactions [34,35]. Greater exposure of lipoprotein, a potent TLR-2 ligand, may also explain the unusual growth restriction of NMII, but not NMI, by primary mouse macrophages [36,37]. Ligation of TLR-2 triggers synthesis of the future science group www.futuremedicine.com proinflammatory cytokine tumor necrosis factor, which in turn elicits production of the antibacterial effector nitric oxide that inhibits NMII replication [37][38][39].…”

“…Greater exposure of lipoprotein, a potent TLR-2 ligand, may also explain the unusual growth restriction of NMII, but not NMI, by primary mouse macrophages [36,37]. Ligation of TLR-2 triggers synthesis of the future science group www.futuremedicine.com proinflammatory cytokine tumor necrosis factor, which in turn elicits production of the antibacterial effector nitric oxide that inhibits NMII replication [37][38][39]. Although potentiated innate immune signaling also occurs during NMII interactions ex vivo with primary human macrophages and dendritic cells [32,40], no growth defects relative to NMI are observed.…”