Caspase-8 activity mediates TNFα production and restricts Coxiella burnetii replication during murine macrophage infection

Abstract

Coxiella burnetii is an obligate intracellular bacteria that causes the global zoonotic disease Q Fever. Treatment options for chronic infection are limited, and the development of novel therapeutic strategies requires a greater understanding of how C. burnetii interacts with immune signaling. Cell death responses are known to be manipulated by C. burnetii, but the role of caspase-8, a central regulator of multiple cell death pathways, has not been investigated. In this research, we studied bacterial manipulation of caspase-8 signaling and the significance of caspase-8 to C. burnetii infection, examining bacterial replication, cell death induction, and cytokine signaling. We measured caspase, RIPK, and MLKL activation in C. burnetii-infected tumor necrosis factor alpha (TNFα)/cycloheximide-treated THP-1 macrophage-like cells and TNFα/ZVAD-treated L929 cells to assess apoptosis and necroptosis signaling. Additionally, we measured C. burnetii replication, cell death, and TNFα induction over 12 days in RIPK1-kinase-dead, RIPK3-kinase-dead, or RIPK3-kinase-dead-caspase-8^-/- bone marrow-derived macrophages (BMDMs) to understand the significance of caspase-8 and RIPK1/3 during infection. We found that caspase-8 is inhibited by C. burnetii, coinciding with inhibition of apoptosis and increased susceptibility to necroptosis. Furthermore, C. burnetii replication was increased in BMDMs lacking caspase-8, but not in those lacking RIPK1/3 kinase activity, corresponding with decreased TNFα production and reduced cell death. As TNFα is associated with the control of C. burnetii, this lack of a TNFα response may allow for the unchecked bacterial growth we saw in caspase-8^-/- BMDMs. This research identifies and explores caspase-8 as a key regulator of C. burnetii infection, opening novel therapeutic doors.

Keywords: apoptosis; caspases; intracellular bacteria; necroptosis; tumor necrosis factor.

Fig 1

C. burnetii inhibition of caspase-8 activation and extrinsic apoptosis. THP-1 cells were differentiated using 100 nM PMA and infected with an mCherry-expressing Nine Mile Phase II (NMII) strain of C. burnetii (mCherry-C. burnetii) at a MOI of 25 GE/cell. At 3 dpi, cells were pre-treated with 10 µg/mL CHX for 4 h, followed by overnight treatment with 20 ng/mL TNFα. (A) Representative images taken at 40× magnification. (B) Western blotting of samples as indicated. (C) Densitometry was completed in ImageJ and significance was determined by one-way ANOVA with Šίdák’s multiple comparisons test. Data are representative of three biological replicates from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 2

Necroptosis exacerbation by C. burnetii infection. L929 cells were infected with mCherry-C. burnetii at a MOI of 600 GE/cell. At 3 and 6 dpi, cells were pre-treated with 50 µM Z-VAD-FMK for 30 min, followed by 3 h incubation with 20 ng/mL TNFα. (A and D) Representative 3 and 6 dpi images taken at 40× magnification. (B and E) Western blotting of samples as indicated. (C and F) Densitometry was completed in ImageJ and significance was determined by paired t test. Data are representative of four biological replicates from four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3

Caspase-8 restricts C. burnetii replication and spread. Primary murine bone marrow-derived macrophages (BMDMs) (C57B6J WT, Ripk1^K45A/K45A, Ripk3^K51A/K51A, and Casp8^−/− Ripk3^K51A/K51A) were infected with mCherry-C. burnetii at a MOI of 100 or 300 GE/cell depending on bacterial stock infectivity to achieve approximately 10⁶ GE/µg DNA in WT BMDMs at 2 dpi. At 3, 6, 9, and 12 dpi, cells were imaged and lysed using a MP Biosciences FastPrep-24 machine and 0.1 mm zirconia beads. (A) Representative 12 dpi images taken at 40× magnification. (B) Quantification of C. burnetii genome equivalents (GE) as determined by dotA qPCR performed on whole-cell lysates. Significance was determined by mixed effects analysis with Tukey’s multiple comparisons test. Data are representative of two to three biological replicates from three independent experiments. Error bars in (B) represent SEM instead of SD. (C) Percent infection was measured at 6 and 12 dpi using a Molecular Devices ImageXpress Micro Confocal. Data represent five to six biological replicates, and 6 and 12 dpi data sets are from two independent experiments. Significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 4

BMDMs lacking caspase-8 activity have decreased cytotoxicity in response to C. burnetii infection. BMDMs were infected with mCherry-C. burnetii as in Fig. 3. At 6 dpi (A) and at 12 dpi (B), cytotoxicity was measured by SYTOX staining using a Molecular Devices ImageXpress Micro Confocal. (C) Percent SYTOX positive was normalized to the average cytotoxicity of genotype-matched mock samples. (D) Percent SYTOX positivity within the mCherry-positive population of cells. Data are representative of five to six biological replicates, and 6 and 12 dpi data sets are from two independent experiments. Significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 5

TNFα production is reduced in caspase-8^−/− BMDMs during C. burnetii infection. BMDMs were infected with mCherry-C. burnetii as in Fig. 3. (A) Relative expression of tnfa at 12 dpi was determined by qRT-PCR. Ct values were normalized first to gapdh expression, then to genotype-matched mock-infected samples. Data are representative of three biological replicates from three independent experiments, and significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. (B) Quantification of TNFα at 12 dpi in cell-free supernatant. TNFα pg/mL concentrations were normalized to the average concentrations of genotype-matched mock samples. Data are representative of five biological replicates. Significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 6

C. burnetii replication in BMDMs is increased by TNFα neutralization. BMDMs were infected with mCherry-C. burnetii as in Fig. 3 and were maintained in media containing 10 µg/mL TNFα-neutralizing antibody. (A and B) At 6 and 12 dpi, cells were lysed using a MP Biosciences FastPrep-24 machine and 0.1 mm zirconia beads, and C. burnetii GE as determined by dotA qPCR was performed on whole-cell lysates. Significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. Data represent two to three biological replicates, and 6 and 12 dpi data sets are from two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.