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 - PubMed (original) (raw)

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

William P Bradley et al. Infect Immun. 2016.

Abstract

Coxiella 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.

PubMed Disclaimer

Figures

FIG 1

TLR2 and TLR4 mediate immune responses to Coxiella burnetii Nine Mile II in C57BL/6 macrophages. (A and B) C57BL/6, _Tlr2_−/−, _Tlr4_−/−, and _Tlr2_−/− _Tlr4_−/− bone marrow-derived macrophages (BMDMs) were infected with WT C. burnetii NMII at an MOI of 5 or 50 for 24 h. Levels of TNF and IL-6 in the supernatants were measured by ELISA. Graphs show the means ± standard errors of the means (SEM) from triplicate wells. Results are representative of three independent experiments. (C) C57BL/6, _Tlr2_−/−, _Tlr4_−/−, and _Tlr2_−/− _Tlr4_−/− BMDMs were infected with WT C. burnetii NMII at an MOI of 100. At days 1 and 7 postinfection, bacterial uptake and replication were measured as genomic equivalents (GEs) by qPCR. Graphs show the fold change in GEs on day 7 relative to GEs on day 1 ± SEM from triplicate wells. (D) Seven days postinfection, BMDMs of the indicated genotypes infected with mCherry-expressing WT C. burnetii NMII at an MOI of 100 were fixed, stained with DAPI, and examined by fluorescence microscopy. The number of large mCherry-expressing NMII-containing vacuoles greater than 5 μm in size was determined and calculated as a percentage of the total cell number. Graphs show the mean percentage of cells containing large NMII vacuoles ± SEM from triplicate coverslips. At least 300 cells were counted per coverslip. Results are representative of two independent experiments. (E) Representative fluorescence micrographs of mouse BMDMs of the indicated genotypes infected with mCherry-expressing C. burnetii (Cb) NMII at an MOI of 100 and fixed and stained with DAPI on day 7 postinfection. Images were taken at ×40 magnification. Scale bars represent 25 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significance.

FIG 2

Myd88 and Trif are required for cytokine production and the restriction of intracellular Coxiella burnetii Nine Mile II replication in C57BL/6 macrophages. (A and B) C57BL/6, _Trif_−/−, _Myd88_−/−, and _Myd88_−/− _Trif_−/− BMDMs were infected with WT C. burnetii NMII at MOIs of 5 and 50 for 24 h. Levels of TNF and IL-6 in the supernatants were measured by ELISA. Graphs show the means ± SEM from triplicate wells. Results are representative of two independent experiments. (C) C57BL/6, _Trif_−/−, _Myd88_−/−, and _Myd88_−/− _Trif_−/− BMDMs were infected with WT C. burnetii NMII at an MOI of 100. At days 1 and 7 postinfection, bacterial uptake and replication were measured as genomic equivalents (GEs) by qPCR. Graphs show the fold change in GEs relative to the GEs measured on day 1 ± SEM from triplicate wells. (D) Seven days postinfection, BMDMs of the indicated genotypes infected with mCherry-expressing WT C. burnetii NMII at an MOI of 100 were fixed, stained with DAPI, and examined by fluorescence microscopy. The number of large NMII-containing vacuoles was determined and calculated as a percentage of the total cell number on day 7 postinfection. Graphs show the mean percentage of cells containing C. burnetii vacuoles ± SEM from triplicate coverslips. At least 300 cells were counted per coverslip. Results are representative of two independent experiments. (E) Representative fluorescence micrographs of BMDMs of the indicated genotypes infected with mCherry-expressing C. burnetii (Cb) NMII at an MOI of 100 and fixed and stained with DAPI on day 7 postinfection. Images were taken at ×40 magnification. Scale bars represent 25 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significance.

FIG 3

C. burnetii Nine Mile II T4SS activity does not enhance cytokine production or early p38 MAPK phosphorylation in C57BL/6 macrophages. (A and B) C57BL/6 BMDMs were infected with the L. pneumophila (Lp) Δ_flaA_ or Δ_dotA_ mutant at an MOI of 5 or with WT C. burnetii (Cb) or the icmL::Tn mutant NMII at an MOI of 5 or 50 for 24 h. Levels of TNF (A) and IL-6 (B) in the supernatants were measured by ELISA. (C and D) C57BL/6 and TLR2−/− BMDMs were infected with WT C. burnetii (Cb) or the icmL::Tn NMII mutant at an MOI of 50 for 24 h. Levels of TNF (C) and IL-6 (D) in the supernatants were measured by ELISA. Graphs show the means ± SEM from triplicate wells. Results are representative of three (A and B) or two (C and D) independent experiments. (E) Immunoblot analysis of phosphorylated and total p38 MAPK in cell lysates from C57BL/6 and _Myd88_−/− BMDMs that were mock infected or infected with WT C. burnetii NMII at an MOI of 50 for 0, 15, 30, or 120 min. Results are representative of two independent experiments.

FIG 4

Coxiella burnetii Nine Mile II infection does not induce inflammasome activation in C57BL/6 macrophages. (A and B) Unprimed C57BL/6 BMDMs were mock infected, infected with the L. pneumophila ΔflaA mutant at an MOI of 5, or infected with WT C. burnetii NMII at an MOI of 20, 50, or 100 for 24 h. Levels of IL-1α (A) and IL-1β (B) were measured in the supernatants by ELISA. Graphs show the means ± SEM from triplicate wells. Results are representative of three independent experiments. (C) C57BL/6 BMDMs were treated with 0.5 μg/ml LPS for 4 h followed by 2.5 mM ATP for 1 h or infected with WT C. burnetii NMII at an MOI of 50 for 24 h. Percent cytotoxicity was measured via LDH release. The graph shows the means ± SEM from triplicate wells. (D) Immunoblot analysis of pro-IL-1β and actin in cell lysates from C57BL/6 BMDMs that were mock infected or infected with the L. pneumophila Δ_flaA_ or Δ_dotA_ mutant at an MOI of 5 or WT C. burnetii NMII at an MOI of 20, 50, or 100 for 24 h. Results are representative of two independent experiments. (E, F, and G) C57BL/6 BMDMs first were primed with 0.5 μg/ml LPS for 4 h and then either mock infected, infected with the L. pneumophila Δ_flaA_ mutant at an MOI of 5, or infected with WT C. burnetii at MOIs of 20, 50, and 100 for 24 h. Levels of IL-1α (E) and IL-1β (F) were measured in the supernatants by ELISA. Graphs show the means ± SEM from triplicate wells. Results are representative of two independent experiments. (G) Immunoblot analysis of processed caspase-1 (casp-1 p10) in the supernatant and pro-IL-1β and actin in the cell lysate. Results are representative of two independent experiments. (H) C57BL/6, _Tlr2_−/−, and _Casp1_−/− _Casp11_−/− BMDMs were infected with WT NMII C. burnetii at an MOI of 100. At days 1 (black bars) and 7 (white bars) postinfection, bacterial uptake and replication were measured as genomic equivalents (GEs) by qPCR. Graphs show the fold change in GEs on day 7 relative to GEs on day 1 ± SEM from triplicate wells.

FIG 5

Type I interferons are not robustly induced and do not restrict Coxiella burnetii Nine Mile II replication. (A and B) C57BL/6 BMDMs were mock infected, infected with the L. pneumophila Δ_flaA_ mutant at an MOI of 10, or infected with WT C. burnetii NMII at an MOI of 10, 50, or 100 for 16 h. Fold induction of Ifna4 and Ifnb mRNAs was measured via qRT-PCR. Graphs show the means ± SEM from triplicate wells. Results are representative of two independent experiments. (C) Supernatants from C57BL/6 BMDMs that were mock infected, infected with the L. pneumophila Δ_flaA_ mutant at an MOI of 10, or infected with WT C. burnetii NMII at an MOI of 50 for 16 h were incubated with L2 mouse fibroblasts for 24 h. L2 cells then were infected with NDV-GFP at an MOI of 1. As a positive control, L2 cells were treated with supernatants from C57BL/6 BMDMs stimulated with poly(I·C) for 16 h. At 24 h postinfection, cells were fixed, stained with DAPI, and examined for levels of NDV-GFP replication by fluorescence microscopy. Shown are representative fluorescence micrographs. (D) Graphs of mean GFP fluorescence from each sample quantified with ImageJ software. Results are representative of two independent experiments. (E) C57BL/6, _Ifnar_−/−, or _Myd88_−/− _Trif_−/− BMDMs were infected with WT C. burnetii NMII at an MOI of 100. C. burnetii GEs were measured by qPCR on days 1 and 7 postinfection. Graphs show the fold change in GEs relative to those at day 1 ± SEM from triplicate wells. Results are representative of two independent experiments. *, P < 0.05; ns, no significance.

FIG 6

Endogenously produced TNF restricts Coxiella burnetii Nine Mile II replication in C57BL/6 macrophages. (A) C57BL/6 and _Tnf_−/− BMDMs were infected with WT C. burnetii NMII expressing mCherry at an MOI of 100. C. burnetii GEs were determined by qPCR on days 1, 3, 5, and 7 postinfection. Graphs show the fold change in GEs relative to those at day 1 ± SEM from triplicate wells. Results are representative of three independent experiments. (B) Ten ng/ml rTNF or supernatants from C57BL/6 BMDMs infected with WT C. burnetii NMII for 24 h were added to WT C. burnetii-infected _Tnf_−/− BMDMs on days 1 and 5 postinfection. C. burnetii GEs were measured by qPCR, and the fold increase in GEs on day 7 postinfection was determined relative to the GEs on day 1. Graphs show the fold change in GEs relative to those on day 1 ± SEM from triplicate wells. Results are representative of two independent experiments. (C) B6 or _Tnf_−/− BMDMs were infected with mCherry-expressing WT C. burnetii or 10 ng/ml rTNF was added to mCherry-expressing WT C. burnetii-infected _Tnf_−/− BMDMs on days 1 and 5 postinfection. On day 7 postinfection, cells were fixed, stained with DAPI, and imaged by fluorescence microscopy. The number of mCherry-expressing C. burnetii-containing vacuoles was determined and calculated as a percentage of the total cell number. Graphs show mean percentages of cells containing C. burnetii vacuoles ± SEM from triplicate coverslips. At least 300 cells were counted per coverslip. (D) Representative images (40×) of mCherry-expressing C. burnetii-containing vacuoles in infected BMDMs treated as described for panel C. Scale bars represent 25 μM. Results are representative of two independent experiments. (E) Ten ng/ml rTNF or supernatants from B6 BMDMs infected with WT C. burnetii NMII for 24 h was added to WT C. burnetii-infected _Tlr2_−/− BMDMs on days 1 and 5 postinfection. C. burnetii GEs were measured by qPCR, and the fold increase in GEs on day 7 postinfection was determined relative to the GEs on day 1. Graphs show the fold change in GEs relative to those on day 1 ± SEM from triplicate wells. Results are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significance.

References

1. Janeway CA Jr, Medzhitov R. 2002. Innate immune recognition. Annu Rev Immunol 20:197–216. doi:10.1146/annurev.immunol.20.083001.084359. - DOI - PubMed
1. Janeway CA., Jr 1989. Approaching the asymptote? Evolution and revolution in immunology. Cold Spring Harbor Symp Quant Biol 54(Part 1):1–13. doi:10.1101/SQB.1989.054.01.003. - DOI - PubMed
1. Medzhitov R. 2007. Recognition of microorganisms and activation of the immune response. Nature 449:819–826. doi:10.1038/nature06246. - DOI - PubMed
1. Akira S, Uematsu S, Takeuchi O. 2006. Pathogen recognition and innate immunity. Cell 124:783–801. doi:10.1016/j.cell.2006.02.015. - DOI - PubMed
1. Ting JP, Kastner DL, Hoffman HM. 2006. CATERPILLERs, pyrin and hereditary immunological disorders. Nat Rev Immunol 6:183–195. doi:10.1038/nri1788. - DOI - PubMed

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 - PubMed (original) (raw)