15-epi-lipoxin A5 promotes neutrophil exit from exudates for clearance by splenic macrophages

Abstract

Specialized proresolving mediators (SPMs) promote local macrophage efferocytosis but excess leukocytes early in inflammation require additional leukocyte clearance mechanism for resolution. Here, neutrophil clearance mechanisms from localized acute inflammation were investigated in mouse dorsal air pouches. 15-HEPE (15-hydroxy-5Z,8Z,11Z,13E,17Z-eicosapentaenoic acid) levels were increased in the exudates. Activated human neutrophils converted 15-HEPE to lipoxin A₅ (5S,6R,15S-trihydroxy-7E,9E,11Z,13E,17Z-eicosapentaenoic acid), 15-epi-lipoxin A₅ (5S,6R,15R-trihydroxy-7E,9E,11Z,13E,17Z-eicosapentaenoic acid), and resolvin E4 (RvE4; 5S,15S-dihydroxy-6E,8Z,11Z,13E,17Z-eicosapentaenoic acid). Exogenous 15-epi-lipoxin A₅, 15-epi-lipoxin A₄ and a structural lipoxin mimetic significantly decreased exudate neutrophils and increased local tissue macrophage efferocytosis, with comparison to naproxen. 15-epi-lipoxin A₅ also cleared exudate neutrophils faster than the apparent local capacity for stimulated macrophage efferocytosis, so the fate of exudate neutrophils was tracked with CD45.1 variant neutrophils. 15-epi-lipoxin A₅ augmented the exit of adoptively transferred neutrophils from the pouch exudate to the spleen, and significantly increased splenic SIRPa⁺ and MARCO⁺ macrophage efferocytosis. Together, these findings demonstrate new systemic resolution mechanisms for 15-epi-lipoxin A₅ and RvE4 in localized tissue inflammation, which distally engage the spleen to activate macrophage efferocytosis for the clearance of tissue exudate neutrophils.

Keywords: 15‐HEPE; air pouch; efferocytosis; resolution of inflammation; specialized proresolving mediators.

Fig. 1.. Characterization of exudates from murine air pouch model – 15-HEPE levels increase in dorsal air pouch exudates.

(A) Mouse dorsal air pouch protocol. (B) Total inflammatory cell counts in air pouch exudates up to 72 hours post-TNFα challenge; n= 4–6. (C) Macrophage and neutrophil cell count in air pouch exudates up to 72 hours post-TNFα challenge; n= 4–6. (D) Levels of IL-1β and IL-6 cytokines, and CXCL1 chemokine in air pouch exudates at 24 hours post-TNFα challenge; n = 4. (E) Levels of IL-10 cytokine in air pouch exudates at 24 hours post-TNFα challenge; n= 4. (F) Targeted lipidomics for levels of LTB₄ and PGs in air pouch exudates, using the SCIEX 5500 LC-MS/MS; n = pooled from 5 mice. (G) Levels of 15-HEPE in air pouch exudates; n = pooled from 5 mice, using LC-MS/MS. (H) Left, Identification of 15-HEPE (m/z 317>219) by LC-MS/MS multiple reaction monitoring (MRM) in negative ionization mode in exudates at 2 hours post-TNF-α with a T_R = 16.21 min and S/N = 1844.4. Right, MS/MS spectrum with a molecular ion at m/z 317 = M-H and fragment ions at m/z 299 = M-H-H₂O, m/z 273 = M-H-CO₂, m/z 255 = M-H-H₂O-CO₂, m/z 247, m/z 219, m/z 203 = 247-CO₂, and m/z 175 = 219-CO₂, with unbiased library fit score of 94.1%. Screen captures were taken in SCIEX OS software version 1.7.0.36606. Note that the accuracy for data acquisition for the QTRAP 5500 instrument is ±0.1 atomic mass units (a.m.u.). The additional digits in spectral data are due to default manufacturer software settings. Data are expressed as mean ± SEM. Similar results were obtained in 2–3 independent experiments. #, p<0.05 compared to saline control, by two-tailed unpaired Student’s t test.

Fig. 2.. 15-HEPE is converted to 15-epi-lipoxin A₅, lipoxin A₅, and resolvin E4 by human neutrophils.

(A) Human blood neutrophils were incubated with ethanol (vehicle) or 15(R/S)-HEPE (25 ng) for 30 seconds, before zymosan A (0.1 mg) was introduced for 60 mins at 37°C. Freshly isolated human neutrophils convert 15-HEPE to Lipoxin A₅, 15-epi-Lipoxin A₅, and Resolvin E4 (RvE4); n=3 [with a conversion rate of 23.9% per hour]. (B) Identification of 15-epi-LXA₅ (m/z 349>115) by LC-MS/MS in negative polarity. Left, MRM chromatogram with a T_R = 10.73 min and S/N = 256.5. Right, MS/MS spectrum with a molecular ion at m/z 349 = M-H and fragment ions at m/z 331 = M-H-H₂O, m/z 313 = M-H-2H₂O, m/z 287 = M-H-H₂O-CO₂, m/z 269 = M-H-2H₂O-CO₂, m/z 233, m/z 215 = 233-H₂O, m/z 189 = 251-H₂O-CO₂, and m/z = 115, with unbiased library fit score of 99.2%. (C) Identification of LXA₅ (m/z 349>115) by LC-MS/MS in negative polarity. Left, MRM chromatogram with a T_R = 10.51 min and S/N = 140.6. Right, MS/MS spectrum with a molecular ion at m/z 349 = M-H and fragment ions at m/z 331 = M-H-H₂O, m/z 305 = M-H-CO₂, m/z 287 = M-H-H₂O-CO₂, m/z 233, m/z 215 = 233-H₂O, m/z 189 = 251-H₂O-CO₂, and m/z = 115, with unbiased library fit score of 98.2%. (D) Identification of RvE4 (m/z 333>115) by LC-MS/MS in negative polarity. Left, MRM chromatogram with a T_R = 13.43 min and S/N = 3712.2. Right, MS/MS spectrum with a molecular ion at m/z 333 = M-H and fragment ions at m/z 315 = M-H-H₂O, m/z 297 = M-H-2H₂O, m/z 271 = M-H-H₂O-CO₂, m/z = 253 = M-H-2H₂O-CO₂, m/z 235, m/z = 217, m/z 201 = 263-H₂O-CO₂, m/z 199 = 217-H₂O, m/z 173 = 235-H₂O-CO₂, and m/z = 115, with unbiased library fit score of 98.9%. Screen captures were taken in SCIEX OS software version 1.7.0.36606. Data are expressed as mean ± SEM. Similar results were obtained in 3 independent experiments.

Fig. 3.. Lipoxin analogs promote resolution of TNF-α-induced inflammation and neutrophil clearance via apoptosis and macrophage efferocytosis.

(A) Chemical structures of 15-epi-Lipoxin A₄, O-9,12-Benzo-15-epi-Lipoxin A₄-ME (CI), and 15-epi-Lipoxin A₅-ME (CII). (B) Mouse dorsal air pouch protocol with intravenous lipoxin analogs and naproxen. (C-E) Count of total inflammatory cells (C), neutrophils (D), and macrophages (E) in air pouch exudates at 24 and 72 hours; n= 5–14. Neutrophil counts in exudates demarcated with 7AAD and AnnexinV staining. (F) 7AAD+, AnnexinV+ necroptotic/late-stage apoptotic neutrophils in air pouch exudates at 24 hours post-TNF-α challenge; n=3. (G) 7AAD-, AnnexinV+ apoptotic neutrophils in air pouch exudates at 24 hours post-TNF-α challenge; n = 3. (H) Percentages of total apoptotic neutrophils in air pouch exudates at 24 hours post-TNF-α challenge that were in a late-stage apoptosis/necroptosis state. (I) Staining protocol of macrophages performing efferocytosis of apoptotic neutrophil. (J) Percentage of macrophages performing efferocytosis of apoptotic neutrophils in exudates; n= 5–7. (K) Cell counts of neutrophils and exudate macrophages performing efferocytosis of neutrophils, n= 5–6. Data are expressed as mean ± SEM. Similar results were obtained in 3 independent experiments. #, p<0.05 compared to saline control; *, p<0.05 compared to vehicle control; and †, p<0.05 compared to naproxen, by one-way or two-way ANOVA.

Fig. 4.. Direct administration of 15-epi-Lipoxin A₅-ME (CII) into the air pouch pre- or post-TNF-α challenge is equipotent to intravenous injection

(A) Mouse dorsal air pouch protocol with intrapouch delivery of CII. (B) Levels of IL-1β and IL-6 cytokines, and CXCL1 chemokine in air pouch exudates from mice treated with intrapouch vehicle or CII 10 minutes pre-TNF-α or 2 hours post-TNF-α at 24 hours post challenge; n= 4–7. (C) Levels of IL-10 cytokine in air pouch exudates at 24 hours with the same treatment groups in (B); n= 4–7. (D) Total inflammatory cell counts in air pouch exudates at 24 hours with the same treatment groups in (B); n= 5–6. (E) Macrophage and neutrophil counts in air pouch exudates at 24 hours with the same treatment groups in (B); n= 5–6. (F) In separate animals, counts of neutrophils and exudate macrophages performing efferocytosis of neutrophils with the same treatment groups in (B); n= 5–6. Data are expressed as mean ± SEM. Similar results were obtained in 3 independent experiments. #, p<0.05 compared to saline control; and *, p<0.05 compared to vehicle control; by one-way or two-way ANOVA.

Fig. 5.. 15-epi-Lipoxin A₅-ME (CII) stimulates exit of adoptively transferred neutrophils in the air pouch exudates and clearance by splenic macrophages.

(A) Mouse dorsal air pouch protocol with administration of variant CD45.1 cells into WT CD45.2 animals, TNF-α challenge and Veh/CII treatment. (B) Total cell, macrophage, and neutrophil counts in air pouch exudates (includes both variant CD45.1 and host CD45.2 cells); n= 7. (C) FACS analysis of CD45.1 neutrophils in air pouch exudates. (D) Percentage of CD45.1 neutrophils among total inflammatory cells in air pouch exudates; n= 7. (E) FACS analysis of host macrophage performing efferocytosis on CD45.1 neutrophils in air pouch exudates. (F) Percentage of host CD45.2 macrophages performing efferocytosis on CD45.1 neutrophils in exudates; n= 7. (G-J) Number of CD45.1 neutrophils in air pouch exudates, blood, lung, and spleen, respectively; n=7. (K-N) Number of host CD45.2 macrophages performing efferocytosis on CD45.1 neutrophils in air pouch exudates, blood, lung, and spleen, respectively; n=7. (O) The number of adoptively transferred CD45.1 neutrophils remaining in air pouch exudates, macrophages performing efferocytosis on CD45.1 neutrophils in exudates, and neutrophils that exited from the air pouch; n=7. (P) Levels of IL-1β and IL-6 cytokines, and CXCL1 chemokine in air pouch exudates at 24 hours. (Q) Levels of IL-10 cytokine in air pouch exudates at 24 hours; n=7. Data are expressed as mean ± SEM. Similar results were obtained in 3 independent experiments. *p<0.05 compared to vehicle control, by one-way ANOVA or two-tailed unpaired Student’s t test.

Fig. 6.. 15-epi-Lipoxin A₅-ME (CII) activates select splenic macrophages via ALX/FPR2 receptors to clear exudate neutrophils by efferocytosis.

(A) Number of spleen macrophages that express SIRPa, MARCO and CD169; n= 6. (B) Number of host CD45.2 spleen macrophages performing efferocytosis on foreign CD45.1 neutrophil that express SIRPa, MARCO and CD169; n= 6. (C) Number of host CD45.2 spleen macrophages performing efferocytosis on host CD45.2 neutrophil that express SIRPa, MARCO and CD169; n= 6. (D) Ex-vivo protocol of spleen macrophage efferocytosis of apoptotic neutrophils. (E) Number of ex-vivo spleen macrophages performing efferocytosis on apoptotic peritoneal neutrophils that express SIRPa, MARCO or CD169 when treated with vehicle/ CII, or in the presence of competitive ALX/FPR2 receptor antagonist, WRW4; n= 5. Data are expressed as mean ± SEM. Similar results were obtained in 2–3 independent experiments. *p<0.05 compared to vehicle control, **p<0.01 compared to vehicle control, and ^#p<0.05 when compared to CII + WRW4, by two-tailed unpaired Student’s T-test or one-way ANOVA.

Fig. 7.. Resolvin E4 shares pro-resolving actions with CII in neutrophil clearance, activating macrophage efferocytosis in the exudates and the spleen.

(A) Mouse dorsal air pouch protocol with intrapouch delivery of CII or RvE4. (B) Total cell, macrophage, and neutrophil counts in air pouch exudates; n= 3. (C) Number of neutrophils in exudates and proportion of those undergoing apoptosis; n= 3. (D) Percentage of macrophages performing efferocytosis on neutrophils in exudates; n= 3. (E) Number of neutrophils in exudates and spleen macrophages performing efferocytosis of neutrophil; n=3. (F) Number of spleen macrophages that express SIRPa, MARCO and CD169; n= 3. (G) Number of spleen macrophages performing efferocytosis on neutrophils that express SIRPa, MARCO and CD169; n= 3. (H) Levels of IL-1β and IL-6 cytokines, and CXCL1 chemokine in air pouch exudates at 24 hours; n = 3 (with duplicates). (I) Levels of IL-10 cytokine in air pouch exudates at 24 hours; n= 3 (with duplicates). Data are expressed as mean ± SEM. *p<0.05 compared to vehicle control, and **p<0.01 compared to vehicle control, by one-way ANOVA.