P. aeruginosa augments irradiation injury via 15-lipoxygenase-catalyzed generation of 15-HpETE-PE and induction of theft-ferroptosis

Abstract

Total body irradiation (TBI) targets sensitive bone marrow hematopoietic cells and gut epithelial cells, causing their death and inducing a state of immunodeficiency combined with intestinal dysbiosis and nonproductive immune responses. We found enhanced Pseudomonas aeruginosa (PAO1) colonization of the gut leading to host cell death and strikingly decreased survival of irradiated mice. The PAO1-driven pathogenic mechanism includes theft-ferroptosis realized via (a) curbing of the host antiferroptotic system, GSH/GPx4, and (b) employing bacterial 15-lipoxygenase to generate proferroptotic signal - 15-hydroperoxy-arachidonoyl-PE (15-HpETE-PE) - in the intestines of irradiated and PAO1-infected mice. Global redox phospholipidomics of the ileum revealed that lysophospholipids and oxidized phospholipids, particularly oxidized phosphatidylethanolamine (PEox), represented the major factors that contributed to the pathogenic changes induced by total body irradiation and infection by PAO1. A lipoxygenase inhibitor, baicalein, significantly attenuated animal lethality, PAO1 colonization, intestinal epithelial cell death, and generation of ferroptotic PEox signals. Opportunistic PAO1 mechanisms included stimulation of the antiinflammatory lipoxin A4, production and suppression of the proinflammatory hepoxilin A3, and leukotriene B4. Unearthing complex PAO1 pathogenic/virulence mechanisms, including effects on the host anti/proinflammatory responses, lipid metabolism, and ferroptotic cell death, points toward potentially new therapeutic and radiomitigative targets.

Keywords: Bacterial infections; Chemokines; Infectious disease; Inflammation; Radiation therapy.

Figure 1. PAO1 alters the survival of irradiated mice.

(A) C57BL/6J mice after TBI (9.25 Gy) were infected with PAO1 in presence or absence of lipoxygenase inhibitor (baicalein) and monitored for more than 15 days. Unirradiated (C) and unirradiated PAO1-infected (PAO1) mice served as controls. Data are pooled from 2 independent experiments. (B) PAO1 burden in colon. Fecal samples were collected, resuspended, and serially plated on cetrimide agar and incubated at 37°C for 20 hours. PAO1 colonies were counted and represented as CFU/g. Data represent mean ± SD, n = 5 mice/group. **P < 0.01; *P < 0.05, 1-way ANOVA, Tukey’s multiple comparison test. (C) Baicalein treatment mitigated epithelial barrier disruption induced by TBI plus PAO1. Damage was assessed as discontinuity of the actin layer (green), and cell death (pink nuclei) that was particularly evident at the apex of the crypt. Dead cell (red), F-actin (green), and Hoechst (blue). Scale: 50 μm. (D) Baicalein treatment mitigated cell death induced by TBI plus PAO1. TBI, total body irradiation; PAO1, P. aeruginosa; B, baicalein. Data represent mean ± SD, n = 5 mice/group for control, TBI/PAO1/B; n = 3, for TBI, PAO1, and TBI/PAO1. ***P < 0.001, 1-way ANOVA, Tukey’s multiple comparison test.

Figure 2. PAO1-induced changes in ileal lipidome of mice exposed to TBI.

(A) Numbers of lipid species identified in mouse ileum. LPL, lysophospholipids; mCL, monolysocardiolipin; LPC, lysophospholipids; LPE, lysophosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; CL, cardiolipin; FA-OX, oxidized fatty acids; PS-OX, oxidized phosphatidylserine; PI-OX, oxidized phosphatidylinositol; PE-OX, oxidized phosphatidylethanolamine; PC-OX, oxidized phosphatidylcholine; CL-OX, oxidized cardiolipin. (B) OPLS-DA score plots showing the lipidomic differences between various groups of mice. (C) Dot plot showing variable of importance (VIP) scores of lipids that substantially contributed to the segregation between different groups in OPLS-DA. TBI, total body irradiation; PAO1, P. aeruginosa; B, baicalein.

Figure 3. Administration of PAO1 modulates immune response of irradiated mice.

(A and B) Quantitative assessment of proinflammatory lipid mediators HxA₃ (A) and LTB₄ (B) by LC-MS. (Ileal tissue homogenates from days 2 and 4 were processed and used for phospholipidomics analysis. Data represent mean ± SD, n = 5 mice/group. (C) Ileal tissue homogenates from day 2 were analyzed for cytokine and chemokine levels by Luminex assay; n = 5 mice/group. (D and E) LC-MS/MS-based quantification of proresolving lipid mediators LXA₄ (D) and resolvins RvD₁ and RvD₂ (E). Data represent mean ± SD; n = 5 mice/group, d2 and d4 represent day 2 and day 4 after TBI. C, control; TBI, total body irradiation; PAO1, P. aeruginosa; B, baicalein; HxA₃, hepoxilin A₃; LTB₄, leukotriene B₄; LXA₄, lipoxin A₄; RvD₁ and RvD₂, resolvin D₁ and D₂, respectively. ***P < 0.001, **P < 0.01, *P < 0.05, 1-way ANOVA, Tukey’s multiple comparison test.

Figure 4. PAO1 enhances radiation-induced ferroptosis.

(A) Levels of proferroptotic signal 1-stearoyl-2-15(S)-HpETE-sn-glycero-3-phosphoethanolamine (1-SA-2-15-HpETE-PE) were higher in irradiated mice after PAO1 infection. 24 hours after TBI, mice were left alone (TBI) or infected with PAO1 in the absence (TBI/PAO1) or presence of baicalein (TBI/PAO1/B). Ileum samples were collected on days 2 and 4 after TBI and processed for redox-lipidomics. Data represent mean ± SD, n = 5 mice/group; ***P < 0.0001, 1-way ANOVA, Tukey’s multiple comparison test. (B) Presence of 15-LOX, pLoxA, and GPx4 in ileum. Ileum homogenate from day 4 after radiation was tested for presence of 15-LOX, pLoxA, and GPx4 using antibody against 15-LOX and pLoxA. (C) Densitometry-based quantitative assessment of 15-LOX and pLoxA protein. Mean relative intensity of pLoxA (left) or 15-LOX (right) was normalized to actin. Data are mean ± SD, n = 5 mice/group; ***P < 0.0001; **P < 0.001; *P < 0.01, 1-way ANOVA, Tukey’s multiple comparison test. (D) Assessment of GPx4 protein and activity from day 4. Densitometry-based quantitative assessment of GPx4 (left). Mean relative intensity of GPx4 was normalized to actin. Data are mean ± SD, n = 5 mice/group; **P < 0.001; *P < 0.01, 1-way ANOVA, Tukey’s multiple comparison test. GPx4 activity measurements (middle). Ileal homogenates (15 μg of protein) after TBI were incubated in buffer containing 0.1 M Tris (pH = 8.0), 0.5 mM EDTA, 1.25% Triton X-100 with 1SA-2-15-HpETE-PE (30 μM), glutathione (150 μM), glutathione reductase (1 U/mL), and NADPH (35 μM). Activity was monitored by the disappearance of NADPH fluorescence (excitation wavelength: 340 nm; emission wavelength: 460 nm). Data are mean ± SD, n = 5 mice/group; *P < 0.05, 1-way ANOVA, Tukey’s multiple comparison test. Low-MW thiols and GSH content in gut homogenates on day 4. For calculation of GSH, fluorescence of Thiol probe IV disappearing after treatment with GPX were used. Data are mean ± SD, n = 5 mice/group; *P < 0.01 versus control, 1-way ANOVA, Tukey’s multiple comparison test.

Figure 5. PAO1 boosts the injury of intestine epithelium caused by TBI.

Left: TBI induces inflammatory response associated with accumulation of oxidized phospholipids and myeloperoxidase-specific lipid metabolites. Middle: PAO1 enhances TBI-induced recruitment and activation of innate immune cells (neutrophils and macrophages), accompanied by production and release of proinflammatory cytokines, chemokines, proinflammatory arachidonic acid–derived lipid mediators. Right: PAO1 augments generation of ferroptotic cell death signal, 15-HpETE-PE, utilizing pLoxA and suppressing GPx4 expression and enzymatic activity, hence activating ferroptotic cell death and markedly accelerating mortality of irradiated mice. PE, phosphatidylethanolamine; PC, phosphatidylcholine; PEox, oxidized phosphatidylethanolamine; PCox, oxidized phosphatidylcholine; PUFA-PLp, polyunsaturated acid containing plasmalogens; PUFA-LPL, lysophospholipids containing polyunsaturated fatty acids; 15-HpETE-PE, 15-hydroperoxyarachidonoyl-PE; 15-HETE-PE, 15-hydroxyarachidonoyl-PE; AA, arachidonic acid; LXA₄- Lipoxin A4; LTB₄, leukotriene B₄; HXA₃, hepoxilin A₃; DHA, docosahexaenoic acid; RvD₅, resolvin D₅; MPO, myeloperoxidase; 15-LOX, host 15-lipoxygenase; pLoxA, a bacterial 15-lipoxygenase; GPX₄, glutathione peroxidase 4; GSH, reduced glutathione; GSSG, oxidized glutathione. TBI, total body irradiation; PAO1, P. aeruginosa. Created with BioRender.com.