- Title
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15-keto-prostaglandin E2 activates host peroxisome proliferator-activated receptor gamma (PPAR-γ) to promote Cryptococcus neoformans growth during infection
- Authors
- Evans, R.J., Pline, K., Loynes, C.A., Needs, S., Aldrovandi, M., Tiefenbach, J., Bielska, E., Rubino, R.E., Nicol, C.J., May, R.C., Krause, H.M., O'Donnell, V.B., Renshaw, S.A., Johnston, S.A.
- Source
- Full text @ PLoS Pathog.
The prostaglandin E2 dependent growth defect of Δplb1 is also present in vivo. A i H99-GFP infected larvae imaged at 0, 1, 2 and 3 dpi. At least 50 larvae measured per time point across 3 biological repeats. Box and whiskers show median, 5th percentile and 95th percentile. Unpaired Mann-Whitney U tests used to compare the burden between each strain for every time point, for p values see (S1Bii Fig). B i– H99-GFP Infected larvae treated with 10 μM prostaglandin E2 or equivalent solvent (DMSO) control. At least 60 larvae measured per treatment group from 3 biological repeats. Box and whiskers show median, 5th percentile and 95th percentile. Unpaired Mann-Whitney U tests used to compare between treatments DMSO vs. 10 μM PGE2 * p = 0.0137 (threshold for significance 0.017, corrected for multiple comparisons). C i, H99-GFP Infected larvae treated with 10 μM prostaglandin D2 or equivalent solvent (DMSO) control. At least 60 larvae measured per treatment group from 4 biological repeats. Box and whiskers show median, 5th percentile and 95th percentile. Unpaired Mann-Whitney U tests used to compare between treatments DMSO vs. 10 μM PGD2., ns p = 0.8 D iΔplb1-GFP infected larvae (500 cell inoculum injected at 2 dpf) imaged at 0, 1, 2 and 3 dpi. N = 3. Box and whiskers show median, 5th percentile and 95th percentile. At least 87 larvae measured for each time point from 3 biological repeats. Unpaired Mann-Whitney U tests used to compare the burden between each strain for every time point, for p values see (S1Ai and S1Aii Fig). E i Δplb1-GFP Infected larvae treated with 10 μM prostaglandin E2 or equivalent solvent (DMSO) control. At least 35 larvae measured per treatment group from 2 biological repeats. Box and whiskers show median, 5th percentile and 95th percentile. Unpaired Mann-Whitney U tests used to compare between treatments Δplb1-GFP DMSO vs 10 μM PGE2 *** p = 0.0001 (threshold for significance 0.017, corrected for multiple comparisons). F i Δplb1-GFP Infected larvae treated with 10 μM prostaglandin D2 or equivalent solvent (DMSO) control. At least 45 larvae measured per treatment group from 3 biological repeats. Box and whiskers show median, 5th percentile and 95th percentile. Unpaired Mann-Whitney U tests used to compare between treatments DMSO vs. 10 μM PGD2. Ns p = 0.1 A ii Representative GFP images (representative = median value) H99-GFP infected larvae, untreated at 0,1,2,3 dpi. B ii, C ii Representative GFP images (representative = median value) H99-GFP infected larvae, at 2 dpi treated with 10 μM PGE2 (B ii) or PGD2 (C ii). D iiRepresentative GFP images (representative = median value) Δplb1-GFP infected larvae, untreated at 0,1,2,3 dpi. E ii, F ii Representative GFP images (representative = median value) Δplb1-GFP infected larvae, at 2 dpi treated with 10 μM PGE2 (E ii) or PGD2 (F ii).
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The observed activity of PGE2 is due to its dehydrogenated derivative 15-keto-PGE2: Fungal burden measured at 2 days post infection (2 dpi) by counting GFP positive pixels in each larvae. A i H99-GFP Infected larvae treated with 10 μM 16,16-dimethyl-prostaglandin E2 or equivalent solvent (DMSO) control. At least 75 larvae measured per treatment group from 4 biological repeats. Box and whiskers show median, 5th percentile and 95thpercentile. Unpaired Mann-Whitney U test used to compare between treatments, DMSO vs. 10 μM 16, 16-dm PGE2 ns p = 0.9954. B i H99-GFP Infected larvae treated with 10 μM 15-keto-prostaglandin E2 or equivalent solvent (DMSO) control. At least 55 larvae measured per treatment group from 3 biological repeats. Unpaired Mann-Whitney U test used to compare between treatments DMSO vs. 10 μM 15-keto = PGE2 ** p = 0.0048 (threshold for significance 0.017, corrected for multiple comparisons). C i Δplb1-GFP Infected larvae treated with 10 μM 16, 16-dimethyl prostaglandin E2 or equivalent solvent (DMSO) control. At least 45 larvae per treatment group from 3 biological repeats. Unpaired Mann-Whitney U test used to compare between treatments Δplb1-GFP DMSO vs 10 μM 16, 16-dm PGE2 ns p = 0.98. D i Δplb1-GFP Infected larvae treated with 10 μM 15-keto-prostaglandin E2 or equivalent solvent (DMSO) control. At least 35 larvae measured per treatment group from 2 biological repeats. Unpaired Mann-Whitney U test used to compare between treatments DMSO vs 10 μM 15-keto-PGE2 * p = 0.0119 (threshold for significance 0.017, corrected for multiple comparisons). A ii, B iiRepresentative GFP images (representative = median value) H99-GFP infected larvae, at 2dpi treated with 10 μM 16,16-dm-PGE2 (A ii) or 15-keto-PGE2 (B ii). C ii, D iiRepresentative GFP images (representative = median value) Δplb1-GFP infected larvae, at 2dpi treated with 10 μM 16,16-dm-PGE2 (C ii) or 15-keto-PGE2 (D ii). E H99-GFP infected larvae treated with a combination of 3 μM AH6809 and 3 μM GW627368X or equivalent solvent (DMSO) control. Box and whiskers show median, 5th percentile and 95th percentile. At least 64 larvae measured per treatment group from 4 biological repeats. Mann-Whitney U test used to compare between treatments, no significance found. |
15-keto-PGE2 promotes fungal burden by activating host PPAR- γ. A i J774 macrophages treated with Troglitazone (TLT—0.25 μM), an equivalent DMSO control or infected with H99 or Δplb1 fixed and stained at 18 hpi with Hoechst and antibody against PPAR-γ. Nuclear localization of PPAR-γ quantified by measuring nuclear grey value of at least 30 cells per condition. A single experiment is shown that is representative of n = 2. One way ANOVA with Tukey post-test used to compare all conditions. DMSO vs. TLT ** p = 0.0049. DMSO vs H99 ** p = 0.0040. TLT vs Δplb1 * p = 0.0212. H99 vs. Δplb1 * p = 0.0187. A ii Transgenic zebrafish larvae with a GFP PPAR- γ reporter treated with DMSO, 250 nM Troglitazone or 10 μM 15-keto-PGE2 + 250 nM Troglitazone overnight and imaged. Lateral views of 2 dpf embryos, anterior to the left, are shown. B J774 murine macrophages infected with Δplb1 or the parental strain H99. Infected cells treated with 25 μM GW9662 (a PPAR-γ antagonist) or equivalent solvent (DMSO) control. Mean IPR from 6 biological repeats shown with error bars representing standard deviation. An unpaired two tailed Student’s t-test was performed to compare each treatment group. H99 DMSO vs. H99 25 μM GW9662 * p = 0.026. C Δplb1-GFP infected larvae treated with 10 μM 15-keto-PGE2, 500 nM GW9662, 10 μM 15-keto-PGE2 + 500 nM GW9662 or an equivalent solvent (DMSO) control. Box and whiskers show median, 5th percentile and 95th percentile. At least 35 larvae measured per treatment group from 2 biological repeats. Mann-Whitney U test used to compare between treatments. DMSO vs. 15-keto-PGE2 **** p <0.0001 (threshold for significance 0.025, corrected for multiple comparisons), 15-keto-PGE2 vs. 15-keto-PGE2 + 500 nm GW9662 ** p = 0.005 (threshold for significance 0.025, corrected for multiple comparisons) D-F 2 day old (2 dpf) Nacre zebrafish larvae injected with 500 cell inoculum. Fungal burden measured at 2 days post infection (2 dpi) by counting GFP positive pixels within each larvae. D i H99-GFP Infected larvae treated with 0.55 μM Troglitazone (TLT) equivalent solvent (DMSO) control. Box and whiskers show median, 5th percentile and 95th percentile. At least 55 larvae measured per treatment group over 3 biological repeats. Mann-Whitney U test used to compare between treatments, DMSO vs. 0.55 μM Troglitazone ** p = 0.0044 (threshold for significance 0.025, corrected for multiple comparisons). E i Δplb1-GFP infected larvae treated with 0.55 μM Troglitazone (TLT) equivalent solvent (DMSO) control. Box and whiskers show median, 5th percentile and 95th percentile. At least 35 larvae measured per treatment group from 2 biological repeats. Mann-Whitney U test used to compare between treatments, DMSO vs. 0.55 μM Troglitazone ** p = 0.0089 (threshold for significance 0.025, corrected for multiple comparisons). F i Δlac1-GFP infected larvae treated with 0.55 μM Troglitazone (TLT) equivalent solvent (DMSO) control. At least 60 larvae measured per treatment group from 3 biological repeats. Box and whiskers show median, 5th percentile and 95th percentile, DMSO vs. 0.55 μM Troglitazone * p = 0.01 (threshold for significance 0.025, corrected for multiple comparisons). D ii, E ii and F ii,Representative GFP images (representative = median value) C. neoformans infected larvae, at 2 dpi treated with 0.55 μM Troglitazone (TLT) D ii - H99-GFP, E ii - Δplb1-GFP and F ii Δlac1-GFP. |
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A J774 cells co-infected with a 50:50 mix of Δlac1-GFP and H99. Quantification of IPR for Δlac1-GFP cells within Δlac1-GFP:H99 2:1 or 1:2 co-infected macrophages. At least 20 co-infected macrophages were analysed for each condition over 4 experimental repeats. Student’s T test performed to compare ratios– 2:1 vs 1:2 * p = 0.012. B i Δlac1-GFP infected larvae imaged at 0, 1, 2 and 3 dpi. Fungal burden measured by counting GFP positive pixels in each larvae. At least 78 larvae measured per time point across 3 biological repeats. Box and whiskers show median, 5th percentile and 95th percentile. Unpaired Mann-Whitney U tests used to compare the burden between each strain for every time point, for p values see (S2A and S2B Fig). B ii Representative GFP images (representative = median value) of 2dpi Δlac1-GFP infected larvae, untreated at 0,1,2,3 dpi C i Δlac1-GFP Infected larvae treated with 10 μM prostaglandin E2 or equivalent solvent (DMSO) control. At least 70 larvae measured per treatment group across 4 biological repeats. Box and whiskers show median, 5th percentile and 95th percentile. Unpaired Mann-Whitney U test used to compare between treatments, DMSO vs. 10 μM PGE2 * p = 0.035. D i Δlac1-GFP Infected larvae treated with 10 μM 16,16-dimethyl prostaglandin E2or equivalent solvent (DMSO) control. At least 75 larvae measured per treatment group across 4 biological repeats. Box and whiskers show median, 5th percentile and 95thpercentile. Unpaired Mann-Whitney U test used to compare between treatments, DMSO vs. 10 μM 16,16-dimethyl prostaglandin E2 ns p = 0.062. E i Δlac1-GFP Infected larvae treated with 10 μM 15-keto-prostaglandin E2 or equivalent solvent (DMSO) control. At least 58 larvae measured per treatment group across 3 biological repeats. Unpaired Mann-Whitney U test used to compare between treatments DMSO vs. 10 μM 15-keto-prostaglandin E2 ns p = 0.50. C ii, D ii, E ii Representative GFP images (representative = median value) Δlac1-GFP infected larvae, at 2 dpi treated with 10 μM PGE2 (C ii), 16,16-dm-PGE2 (D ii) or 15-keto-PGE2 (E ii). (TIF) |