15-epi-Lipoxin A4, Resolvin D2, and Resolvin D3 Induce NF-κB Regulators in Bacterial Pneumonia

Abstract

Specialized proresolving mediators (SPMs) decrease NF-κB activity to prevent excessive tissue damage and promote the resolution of acute inflammation. Mechanisms for NF-κB regulation by SPMs remain to be determined. In this study, after LPS challenge, the SPMs 15-epi-lipoxin A₄ (15-epi-LXA₄), resolvin D1, resolvin D2, resolvin D3, and 17-epi-resolvin D1 were produced in vivo in murine lungs. In LPS-activated human bronchial epithelial cells, select SPMs increased expression of the NF-κB regulators A20 and single Ig IL-1R-related molecule (SIGIRR). Of interest, 15-epi-LXA₄ induced A20 and SIGIRR in an lipoxin A₄ receptor/formyl peptide receptor 2 (ALX/FPR2) receptor-dependent manner in epithelial cells and in murine pneumonia. This SPM regulated NF-κB-induced cytokines to decrease pathogen-mediated inflammation. In addition to dampening lung inflammation, surprisingly, 15-epi-LXA₄ also enhanced pathogen clearance with increased antimicrobial peptide expression. Taken together, to our knowledge these results are the first to identify endogenous agonists for A20 and SIGIRR expression to regulate NF-κB activity and to establish mechanisms for NF-κB regulation by SPMs for pneumonia resolution.

Figure 1. SPMs are endogenously produced during LPS-induced lung inflammation and decrease airway epithelial inflammatory response

(A) Representative multiple reaction-monitoring (MRM) chromatogram for 15-epi-LXA₄, RvD1, 17-epi-RvD1, RvD2 and RvD3 and (B) MS/MS spectrum obtained from infected murine lung used for the identification and quantification of SPMs. (C) Calu-3 cells were exposed (24 hrs, 37°C) to LPS or vehicle (media only) in the presence of 15-epi-LXA₄ or vehicle (media only) and levels of CXCL8 in the supernatant were measured by ELISA. NF-κB activation in Calu-3 cells was detected by NF-κB phospho-p65 at 3 hrs (D) and 24 hrs (E) after LPS with 15-epi-LXA₄ or vehicle. In all experiments, 15-epi-LXA₄ (100nM) was added 30 minutes before LPS (10 μg/mL). Values represent the mean ± SEM n = 4-8 per group. * P <0.05, ** P <0.01

Figure 2. 15-epi-LXA₄ increases epithelial expression of select negative regulators of NF-κB

(A) A20, (B) ST2 and (C, D) SIGIRR expression in Calu-3 cells exposed to LPS (10 μg/mL) for 3 hrs was measured by qPCR (A-C) and Western blot (D). The time course for LPS-induced changes in relative RNA levels of (E) A20, (F) ST2 and (G) SIGIRR was measured by qPCR in Calu-3 cells incubated (30 min, 37°C) with 15-epi-LXA₄ (100 nM) or vehicle (media) prior to the addition of LPS (10 μg/mL); * P <0.05 vs baseline, # P <0.05 vs vehicle by two-way ANOVA. In some experiments, cells were exposed to an ALX/FPR2 antagonist WRW4 (210nM) and (H) A20 and (I) SIGIRR RNA levels determined by qPCR. (J) A20 and (K) SIGIRR RNA were determined in 15-epi-LXA₄- or vehicle-exposed Calu-3 cells infected with E. coli (10⁶ CFU) for 24 hrs. Results are expressed as mean ± SEM n = 3-12 per group. * P <0.05, ** P <0.01

Figure 3. SPMs selectively regulate A20 and SIGIRR

Structure-activity relationships for A20 and SIGIRR induction in airway epithelial cells were determined for (A) 15-epi-LXA₄ and select D- series resolvins that inhibit NF-κB. (B, C) Calu-3 cells were exposed to 15-epi-LXA₄, 17-epi-RvD1, RvD2, RvD3 (100nM each) or vehicle (media) prior to LPS (10 μg/mL, 3hrs). RNA levels of (B) A20 and (C) SIGIRR were measured by qPCR. (D) Calu-3 cells were treated with 15-epi-LXA₄ (10nM), a combination of 15-epi-LXA₄, 17-epi-RvD1 and RvD3 (10nM each), or vehicle (PBS) followed by exposure to IL-1β (1ng/mL, 3 hrs, 37°C), protein isolation and Western blot for phosphorylated NF-κB p65. Results are expressed as mean ± SEM, n = 4-8 per group. * P <0.05, ** P <0.01

Figure 4. 15-epi-LXA₄ enhances E. coli clearance and decreases lung inflammation

The murine left lung was inoculated with E. coli (10⁶ CFU); select animals were exposed to 15-epi-LXA₄ (100ng, IV), ciprofloxacin (0.2mg/kg, approximately IC₅₀, IP) or vehicle (0.9% saline, IV) 1 hr after E. coli infection. (A) CFU were determined in lung homogenates 6 hrs after E.coli infection. (B) Phosphorylation of NF-κB p65 was assessed in lung homogenates 6 hrs after E. coli infection. (C) A20 and (D) SIGIRR RNA in lung were determined 24 hrs after E. coli infection. (E) In BAL fluid obtained 24 hrs after infection, neutrophils were identified by flow cytometry (CD45⁺ CD11b⁺ CD11c⁻ Ly6G⁺). (F) In BAL fluid obtained 24 hrs after infection, levels of CXCL1 were determined by ELISA. Results are expressed as mean ± SEM, n = 3-12 per group. * P <0.05

Figure 5. 15-epi-LXA₄ restrains bacterial growth by increasing airway epithelial expression of antimicrobial peptides

Calu-3 cells were exposed to E. coli (MOI 10:1) for 3 hrs following 15-epi-LXA₄ (100nM) or vehicle and (A) the bacterial growth index was determined. Calu-3 cell expression of the anti-microbial peptides (B) bacterial permeability inducing protein (BPI) and (C) LL-37 was measured by qPCR. (D) Calu-3 cell expression of LL-37 protein in the setting of 15-epi-LXA₄ (100nM) or vehicle was assessed by Western blot 3 hrs after LPS stimulation. The impact of 15-epi-LXA₄ (100 ng, IV) or vehicle (0.9% saline, IV) given 1 hr after E. coli on expression of (E) mBPI and (F) mCRAMP was measured in mouse lung RNA obtained 24 hrs post-infection. (G) Mouse left lungs were inoculated with E. coli (10⁷ CFU) and CFU were determined 24 hrs after inoculation of wild type (WT) or Cnlp^−/− mice given 15-epi-LXA₄ (100 ng, IV) or vehicle (0.9% saline, IV) 1 hr after E. coli. Results are expressed as mean ± SEM, n = 4-8 per group. * P <0.05

Figure 6. ALX/FPR2^−/− mice have delayed resolution of pathogen-induced lung inflammation

(A) WT and ALX/FPR2^−/− mice were infected with E.coli (10⁶ CFU) and the time course for lung neutrophil trafficking was enumerated by flow cytometry at baseline, 6 hrs, 24 hrs, 48 hrs and 72 hrs post infection; *P <0.05 by two-way ANOVA. (B) BALF neutrophils (CD45⁺CD11B⁺CD11c⁻Ly6G⁺) from WT and ALX/FPR2^−/− mice given 15-epi-LXA₄ (100ng, IV) or vehicle (0.9% saline, IV) 1 hr after infection. (C) BALF levels of CXCL1 from WT and ALX/FPR2^−/− mice 24 hrs post-infection were measured by ELISA. (D) mCRAMP and (E) mBPI expression were measured in lung tissues by qPCR 24 hrs after infection. (F) Phosphorylation of NF-κB p65 in infected lung tissue was monitored by Western blot. Results are expressed as mean ± SEM, n = 3-12 per group. * P <0.05