Elevated prostaglandin E2 post-bone marrow transplant mediates interleukin-1β-related lung injury

Abstract

Hematopoietic stem cell transplant (HSCT) treats or cures a variety of hematological and inherited disorders. Unfortunately, patients who undergo HSCT are susceptible to infections by a wide array of opportunistic pathogens. Pseudomonas aeruginosa bacteria can have life-threatening effects in HSCT patients by causing lung pathology that has been linked to high levels of the potent pro-inflammatory cytokine, interleukin-1β (IL-1β). Using a murine bone marrow transplant (BMT) model, we show that overexpression of prostaglandin E₂ (PGE₂) post-BMT signals via EP2 or EP4 to induce cyclic adenosine monophosphate (cAMP), which activates protein kinase A or the exchange protein activated by cAMP (Epac) to induce cAMP response element binding-dependent transcription of IL-1β leading to exacerbated lung injury in BMT mice. Induction of IL-1β by PGE₂ is time and dose dependent. Interestingly, IL-1β processing post-P. aeruginosa infection occurs via the enzymatic activity of either caspase-1 or caspase-8. Furthermore, PGE₂ can limit autophagy-mediated killing of P. aeruginosa in alveolar macrophages, yet autophagy does not have a role in PGE₂-mediated upregulation of IL-1β. Reducing PGE₂ levels with indomethacin improved bacterial clearance and reduced IL-1β-mediated acute lung injury in P. aeruginosa-infected BMT mice.

Fig 1. BMT Mice are Deficient in Clearing Pseudomonas aeruginosa Infection and Experience Exacerbated Lung Tissue Injury

(a) Colony Forming Units (CFUs) were counted 24 hours after infecting non-transplanted control and BMT mice (C57BL/6J→C57BL/6J) with 5×10⁵ CFUs of Pseudomonas aeruginosa (PA01). (n=5 control; n=5 BMT). (b) Albumin measurements from bronchoalveolar lavage fluid (BALF) from PA01 infected control and BMT mice. (n=7 control; n=6 BMT) (c) Hematoxylin and Eosin stain (H&E) of lungs from saline or PA01 infected control and BMT mice; images taken at 40× magnification (representative of n=3 control; n=3 BMT). Statistics are student T test between comparative groups. *P<0.05. Data is representative of at least two independent experiments.

Fig 2. Pseudomonas aeruginosa Infection Induces Higher Levels of IL-1β and PGE₂ post-BMT Correlating with Increased Lung Injury

(a) IL-1β and (b) Prostaglandin E₂ (PGE₂) measurements from the BALF of control and BMT mice 24 hours after infection with PA01; measurements done by ELISA. (n=7 control; n=6 BMT); (c) Correlation between albumin and IL-1β in BALF 24h post-PAO1; (d) Correlation between albumin and PGE₂ in BALF 24h post-PAO1; (e) Correlation between PGE₂ and IL-1β in BALF 24h post-PAO1 ; (f) Correlation between PGE₂ and TNF-α in BALF 24h post-PAO1. In all correlations, closed symbols represent control mice whereas open circles are BMT mice. R= Pearson correlation coefficient; *P<0.05.

Fig 3. Alveolar Macrophages in BMT Mice Account for Higher IL-1β Release post-P.aeruginosa in Response to PGE₂

(a) RTqPCR measurement of relative gene expression of Cox-1, Cox-2, and IL-1β from AMs and interstitial lung from uninfected control and BMT mice normalized to GAPDH (n=3 control; n=3 BMT/group). (b) PGE₂ measurements by ELISA from overnight culture of untreated AMs from control and BMT mice (n=4 Control; n= 4 BMT). (c) IL-1β measurements by ELISA from AMs infected or not in vitro with PA01 (MOI:10), treated or not with 100nM of PGE₂ (n=3). (d) IL-1β, (e) TNF-α and (f) IL-6 measurements by ELISA from BALF of LPS (50ug)-treated control and BMT mice (n=10 control, n=10 BMT). Statistics are student T test between comparative groups. *P<0.05 ,**P<0.01, ***P<0.001, ****P<0.0001. Data is representative of at least two independent experiments.

Fig 4. PGE₂ Increases IL-1β in Bone Marrow Derived Macrophages (BMDMs) Upon Pathogenic Stimulation

(a-b) IL-1β and IL-10 measurements from supernatants of PA01 infected (MOI:10) BMDMs treated or not with 100nM PGE₂. (c) IL-1β measurements from supernatant of BMDMs treated with lipopolysaccharide (LPS) derived from P. aeruginosa (500ng/ml), heat killed PA01 (MOI:10), and (c) ATP (1mM) with LPS with or without 100nM of PGE₂. Supernatant taken 2 hours post-stimulation. One-way ANOVA with Bonferroni's post-test. *P<0.05 ,**P<0.01, ***P<0.001, ****P<0.0001. Data is representative of three independent experiments.

Fig 5. PGE₂ Mediated Increase in IL-1β is Dependent on Activation of Transcription Factor CREB by Increasing Levels of cAMP Dependent on EP2 and EP4 Signaling

IL-1β measurements from supernatant of BMDMs treated or not with PAO1 (MOI:10) with or without (a) EP2 agonist (1μM, Butaprost), EP4 agonist (500nM, ONO-AE1-329) (b) forskolin (25μM), PGE₂ (100nM), (c) PKA agonist (50μM, 6-BNZ-cAMP), Epac agonist (50μM, 8-pcpt-2′-OM-cAMP). (d) IL-1β protein measurement from supernatants of BMDMs treated or not with 100nM of PGE₂, CREB inhibitor (100μM, Naphthol AS-E phosphate) and infected or not with PA01 (MOI:10). In all cases, supernatant taken 2 hours after infection. One-way ANOVA with Bonferroni's post-test. *P<0.05 ,**P<0.01, ***P<0.001, ****P<0.0001. Data is representative of two independent experiments.

Fig 6. PAO1 can use cannonical or non-cannonical inflammasomes to make IL-1β

IL-1β measurements from supernatant of BMDMs treated or not with (a) Caspase 8 inhibitor (10nM) or Caspase 1 inhibitor (10nM) with or without PGE₂ (100nM). Supernatant taken 2 hours after infection. One-way ANOVA with Bonferroni's post-test. *P<0.05 ,**P<0.01, ***P<0.001, ****P<0.0001. Data is representative of two independent experiments.

Fig 7. Inhibition of Autophagy Impacts Bacterial Killing but Not IL-1β Release

(a) AMs were cultured in complete media or were nutrient starved in Earle's Balanced Salt Solution (EBSS) (#1) or Hanks' Balanced Salt solution (HBSS) (#2) in the presence or absence of PGE₂ (100nM) for 2 hours. (b) AMs were collected from control mice and stimulated for 1 hour in the presence of serum-free media alone with or without a dose response of PGE₂ added (50-500 nM) for 1h. Cell lysates were then analyzed for levels of LC3 and p62 by Western blot compared to GAPDH. Blot shown is representative of 3 experiments. (c) AMs were subjected to culture in complete media or were serum-starved for 1 h in the presence or absence of 100nM PGE₂ before RNA was prepared and analyzed for expression of the autophagy gene ATG5 relative to β-actin by qRT-PCR (n=4); (bottom) cell lysates were also taken from samples assessed for levels of ATG5 and β-actin by Western blot.(d) BMDMs from autophagy-deficient LC3-/- mice were stimulated with 100nM PGE₂, PAO1 (MOI:10) or the combination for 2 hours before supernatants were collected and measured for IL-1β by ELISA (n=4); Data are representative of two independent experiments and statistics were measured by one-way ANOVA with Bonferroni post-hoc test. *P<0.05 ,**P<0.01, ***P<0.001, ****P<0.0001.

Fig 8. PGE2 Elevates the Levels of IL-1β Transcripts by EP2 and EP4 Stimulation

(a) BMDMs were treated or not with 100nM PGE₂ for 1, 2, 4, and 8 hours before RNA was prepared and analyzed for expression of the IL-1β gene. (b) BMDMs were treated or not with 10nM, 100nM, and 500nM of PGE₂ during 4 hours before RNA was prepared and analyzed for expression of IL-1β. (c) BMDMs were treated with EP2 agonist (1μM, Butaprost), EP3 agonist (10nM, Sulprostone), EP4 agonist (500nM, ONO-AE1-329), and forskolin (25μM) before RNA was prepared and analyzed. All RNA data was normalized to expression levels of GAPDH. Data are representative of two independent experiments and statistics were measured by one-way ANOVA with Bonferroni post-hoc test. *P<0.05 ,**P<0.01, ***P<0.001, ****P<0.0001.

Fig 9. Human AMs and Peripheral Monocytes Upregulate IL-1β under PGE₂ stimulation

IL-1β relative expression from 100nM PGE₂ stimulated (a) Human peripheral monocytes and (b) human AMs normalized to GAPDH. (c) IL-1β protein measurements from supernatant of PGE₂-treated or not peripheral macrophages during or not PA01 infection. Statistics are student T test between comparative groups. *P<0.05 ,**P<0.01, ***P<0.001, ****P<0.0001.

Fig 10. Decreasing Levels of PGE₂ by Indomethacin Treatment Leads to Decreased IL-1β in the Lung after P. aeruginosa Infection in BMT mice

(a) IL-1β and (b) albumin measurements from the BALF of control, and BMT mice treated or not with Indomethacin (1.2mg/kg) for 24 hours after PA01 infection; measurements done by ELISA. (n=14 control; n=14 BMT; n=14 BMT treated with Indomethacin); (C) PA01 CFU measurement in BAL from infected mice, 24 hours. (n=10 control; n=10 BMT; n=10 BMT treated with Indomethacin). Data is representative of at least two independent experiments. Statistics were measured by one-way ANOVA with Bonferroni post-hoc test. *P<0.05 ,**P<0.01, ***P<0.001, ****P<0.0001; non-significant (ns).