Elucidation of novel 13-series resolvins that increase with atorvastatin and clear infections

Abstract

Endogenous mechanisms leading to host protection and resolution of infections without immunosuppression are of wide interest. Here we elucidate the structures of four new host-protective molecules produced in neutrophil-endothelial cocultures and present in human and mouse tissues after sterile inflammation or infection. The bioactive molecules contain conjugated triene and diene double bonds, carry an alcohol at C13 and are derived from n-3 docosapentaenoic acid (DPA, C22:5). These compounds, termed 13-series resolvins (RvTs), demonstrated potent protective actions increasing mice survival during Escherichia coli infections. RvTs also regulated human and mouse phagocyte responses stimulating bacterial phagocytosis and regulating inflammasome components. Their biosynthesis during neutrophil-endothelial cell interactions was initiated by endothelial cyclooxygenase-2 (COX-2), increased by atorvastatin via S-nitrosylation of COX-2 and reduced by COX-2 inhibitors. The actions of atorvastatin and RvTs were additive in E. coli infections in mice, where they accelerated resolution of inflammation and increased survival >60%. Taken together, these results document host-protective molecules in bacterial infections, namely RvTs, derived from n-3 DPA via transcellular biosynthesis and increased by atorvastatin. These molecules regulate key innate protective responses in the resolution of infectious inflammation.

Figure 1. Novel 13-series resolvins (RvT) from neutrophil-endothelial cell interactions are elevated in self-resolving inflammation

(a) Fractions were extracted from human neutrophil (PMN)-endothelial cell (EC) co-incubations (see methods), administered to mice via i.v. injection 5 min prior to E. coli (2.5×10⁷ CFU/mouse) inoculation and survival assessed. n=10 mice/group from three independent experiments. *p<0.05 vs. E. coli group. (b) Proposed structures from the novel 13-series resolvins (RvT) identified in human PMN-EC co-incubations. (c) Blood was collected from healthy volunteers pre- and post-exercise (see methods) and amounts of RvT assessed using LC-MS-MS. n=4 healthy volunteers from four independent experiments. *p<0.05 vs. pre-exercise values. (d) Plasma was obtained from the NIST repository (SRM 1950, d=3), collected from healthy volunteers (n=4) or patients diagnosed with sepsis (n=9), and RvT assessed using LC-MS-MS. Results are expressed as mean±s.e.m. from two independent experiments. *p<0.05, **p<0.01 vs. SRM 1950, # p<0.05, ## p<0.01 vs. healthy volunteers. (e) Mice were inoculated with E. coli (1×10⁵ CFU/mouse), blood was collected at the indicated intervals and RvT amounts assessed using LC-MS-MS. (f) Mice were inoculated with 1×10⁵ CFU/mouse E. coli (self-resolving) or 1×10⁷ CFU/mouse E. coli (delayed-resolving), blood was collected after 4h and RvT assessed using LC-MS-MS. Results for e, f are expressed as mean± s.e.m. n=4 mice per group from two independent experiments. *p<0.05 vs 0h blood levels (e) or vs. mice receiving a self-resolving inoculum (f).

Figure 2. Atorvastatin increases protective actions of neutrophil-endothelia cell fractions during mouse infections and promotes RvT formation via S-nitrosylated COX-2

(a) RvT in human neutrophil-endothelial cells co-incubations with or without atorvastatin. Mean±s.e.m.; n=4 independent cell preparations. Four independent experiments. *p<0.05 vs. EC group. (b) Fractions extracted using C18 SPE (see methods) were administered to mice (i.v.) 5 min prior to E. coli (2.5×10⁷ CFU/mouse) inoculation and survival assessed. n= 14–17 mice/group. Three independent experiments. *p<0.05 vs. E. coli, #p<0.05 vs. E. coli plus hPMN-EC, §p<0.05 vs. E. coli plus hPMN plus atorv or E. coli plus hEC plus atorv. (c, d) Endothelial cells were incubated with IL-1β and TNF-α then vehicle, celecoxib and/or atorvastatin followed by n-3 DPA and hPMN. (c–d) Fractions were extracted (c) profiled using LM metabololipidomics. Mean±s.e.m. n=4 independent cell preparations/group. Three independent experiments. *p<0.05, **p<0.01 vs. incubations with IL-1β plus TNF-α alone; #p<0.05 vs incubations with IL-1β plus TNF-α atrovastatin. (d) administered prior to E. coli (2.5×10⁷ CFU/mouse) and survival assessed. n=10 mice/group. Two independent experiments. *p<0.05 vs. E. coli; #p<0.05 vs. E. coli plus hPMN-EC plus atorv. (e–f) Mice were inoculated with E. coli (1×10⁵ CFU/mouse), 1h later L-NAME (24mg/kg) or vehicle (saline) was administered followed by atorvastatin (5μg/mouse, i.v., 5h) or vehicle (saline plus 0.01% EtOH). (e) Plasma RvT were quantified using LM metabololipidomics, (f) exudate cell numbers were enumerated. Mean±s.e.m. n=4 mice/group. Two independent experiments. *p<0.05 vs. E. coli. #p< 0.05 vs. E. coli plus atorvastatin. (g) Conversion of n-3 DPA by human recombinant (hr) COX-2 and S-nitrosylated (SNO) hr-COX2. Mean±s.e.m. n=3 incubations. Three independent experiments.

Figure 3. RvT regulate leukocyte responses and promote survival in infections

(a) Macrophages (5×10⁴ cells/well) were incubated with the indicated concentrations of RvT1, RvT2 plus RvT3 (1:1 ratio), RvT4 or vehicle (PBS containing 0.01% EtOH; 15min, 37°C, pH 7.45) then fluorescently labeled E. coli (2.5×10⁶ CFU/well). (b) Macrophages (5×10⁴ cells/well) were incubated with H₂DCFDA (5μM, 30min, 37°C, pH 7.45); cells were washed, incubated with the indicated concentrations of RvT1, RvT2 plus RvT3 (1:1 ratio), RvT4 or vehicle (15 min, 37°C, pH 7.45), E. coli (2.5×10⁶ CFU/well) were added and intracellular ROS levels determined. (c) Macrophages (5×10⁴ cells/well) were incubated as in (a) with RvT, fluorescently-labeled apoptotic PMN (2.5×10⁵ cells/well) were added and uptake assessed (see methods). Mean±s.e.m. n=4 donors. Three independent experiments. *p<0.05, **p<0.01 vs. macrophages plus vehicle. (d–g) Mice were given vehicle (saline containing 0.1% EtOH) or combination of RvT1, RvT2, RvT3 and RvT4 (ratio 2:1:1:8), each isolated and quantified by RP-UV-HPLC (see methods), via i.p. injection ~5min prior to E. coli (1×10⁷ CFU/mouse) inoculation. 12h later (d) body temperatures, (e) peritoneal exudate neutrophil counts, (f) bacterial phagocytosis by peritoneal leukocytes (%E. coli⁺ of total CD11b⁺ population), (g) Macrophage efferocytosis in peritoneal exudates were assessed. Mean± s.e.m. n=5 mice/group. Two independent experiments. *p<0.05, **p<0.01 vs. E. coli mice. #p<0.05 vs. naive mice. (h) Mice were inoculated with E. coli (2.5×10⁷ CFU/mouse); 2h later vehicle (saline+0.1% EtOH) or RvT (as in d–g; 50ng/mouse or 500ng/mouse) were administered via i.p. injection and survival assessed. n=10 mice per group. Two independent experiments *p<0.05 vs. E. coli mice.

Figure 4. Atorvastatin and RvT accelerate resolution of infections and promote survival in bacterial infections in mice

(a) Mice were inoculated with E. coli (1×10⁵ CFU/mouse) plus RvT (combination of RvT1, RvT2, RvT3 and RvT4, ratio of 1:1:1:1, isolated and quantified by RP-UV-HPLC, total 50ng/mouse). Atorvastatin (0.5μg/mouse; i.p.) or vehicle (saline containing 0.1% EtOH) was administered and exudate neutrophil counts (CD11b⁺Ly6G⁺) assessed at the indicated time points using light microscopy and flow cytometry. Results are mean±s.e.m. n=4 mice per group from two independent experiments. **p<0.01 vs. 24h vehicle group. (b) Mice were inoculated with E. coli (2.5×10⁷ CFU/mouse); after 3h administered vehicle (Saline+0.1% EtOH), 0.5μg/mouse (atorv low dose), 5μg/mouse (atorv high dose) atorvastatin, 0.5μg/mouse atorvastatin plus 50ng/mouse RvT (atorv low dose plus RvT low dose) or 5μg/mouse atorvastatin plus 500ng/mouse RvT (atorv high dose plus RvT high dose); each of the RvT was isolated as in panel (a) and survival assessed. n=10 mice per group from three independent experiments. *p<0.05, **p<0.01 vs. E. coli mice. #p<0.05 vs. E. coli plus atorv low dose mice.