Infection regulates pro-resolving mediators that lower antibiotic requirements

Abstract

Underlying mechanisms for how bacterial infections contribute to active resolution of acute inflammation are unknown. Here, we performed exudate leukocyte trafficking and mediator-metabololipidomics of murine peritoneal Escherichia coli infections with temporal identification of pro-inflammatory (prostaglandins and leukotrienes) and specialized pro-resolving mediators (SPMs). In self-resolving E. coli exudates (10(5) colony forming units, c.f.u.), the dominant SPMs identified were resolvin (Rv) D5 and protectin D1 (PD1), which at 12 h were at significantly greater levels than in exudates from higher titre E. coli (10(7) c.f.u.)-challenged mice. Germ-free mice had endogenous RvD1 and PD1 levels higher than in conventional mice. RvD1 and RvD5 (nanograms per mouse) each reduced bacterial titres in blood and exudates, E. coli-induced hypothermia and increased survival, demonstrating the first actions of RvD5. With human polymorphonuclear neutrophils and macrophages, RvD1, RvD5 and PD1 each directly enhanced phagocytosis of E. coli, and RvD5 counter-regulated a panel of pro-inflammatory genes, including NF-κB and TNF-α. RvD5 activated the RvD1 receptor, GPR32, to enhance phagocytosis. With self-limited E. coli infections, RvD1 and the antibiotic ciprofloxacin accelerated resolution, each shortening resolution intervals (R(i)). Host-directed RvD1 actions enhanced ciprofloxacin's therapeutic actions. In 10(7) c.f.u. E. coli infections, SPMs (RvD1, RvD5, PD1) together with ciprofloxacin also heightened host antimicrobial responses. In skin infections, SPMs enhanced vancomycin clearance of Staphylococcus aureus. These results demonstrate that specific SPMs are temporally and differentially regulated during infections and that they are anti-phlogistic, enhance containment and lower antibiotic requirements for bacterial clearance.

Figure 1. Profiling of SPM in E. coli infections

Mice were inoculated with E. coli at (a) 10⁵ or (b) 10⁷ CFU by intraperitoneal injection, and peritoneal leukocytes enumerated. Results are expressed as mean±s.e.m., n=4-6. See Methods for calculation of resolution indices. *p<0.05, **p<0.01, vs. 24h; ^#p<0.05, ^##p<0.01, ^###p<0.001^, vs. 48h. (c) Time course of SPM and related pathway markers. Results are expressed as mean±s.e.m. of n=5 separate time courses. *p<0.05, **p<0.01, self-resolving (10⁵ CFU) vs. higher titer (10⁷ CFU) E. coli exudates. (d) Representative MRM chromatograph of eicosanoid, resolvin and protectin pathway products from naive germ-free mice. Each LM was identified based on published LC-MS-MS (see Table S1). (e) SPM and pathway markers in colons of germ-free and conventional mice; representative of 3 mice. (f) Representative MS-MS of RvD1 (from germ-free mice) and RvD5 (from E. coli-infected mice); a=[M-H] (parent ion).

Figure 2. RvD1 and RvD5 protect mice during infection: enhancing bacterial killing and preventing hypothermia

Mice were inoculated with E. coli (10⁷ CFU) together with RvD1 or RvD5 methyl ester (100 ng), and peritoneal exudates collected 24h later. (a) Intracellular E. coli levels. MFI denotes mean fluorescence intensity. Results are expressed as mean of n=3-5. (bottom panel) Representative histograms. (b) Bacterial titers. Results are expressed as mean of n=4-5. (c) Changes in body temperatures expressed as mean of Δ°C (24h-0h) n=4-10. *p<0.05, **p<0.01, vs. E. coli alone; ##p<0.01, vs. naïve mice. (d) % survival of E. coli inoculated mice (2.5×10⁷ CFU) alone or with RvD1 (100 ng/mouse). *p<0.05 log-rank (Mantel-Cox) test, n=12 each group.

Figure 3. SPM enhance human macrophage phagocytosis of E. coli

(a-c) MΦ phagocytosis of fluorescent E. coli in the presence of PD1, RvD1 or RvD5. Results are percent increase above vehicle and expressed as mean±s.e.m., d=3-4, n=3 (PD1) or 4 (RvD1 and RvD5). *p<0.05, **p<0.01, E. coli plus SPM vs. E coli alone. (b) Venn diagrams of genes regulated by resolvins in human MΦ incubated with E. coli. CD40 (TNFRSF5, TNF receptor superfamily member 5), ITGAM (CD11b), PDE (phosphodiesterase), PLA2 (phospholipase A2), PTGS2 (cyclooxygenase-2), PTGER2 (prostaglandin E₂ receptor, EP2).

Figure 4. SPM and antibiotics accelerate resolution and enhance bacterial killing

(a-c) Mice were inoculated with E. coli (10⁵ CFU), RvD1 methyl ester (50 ng), and ciprofloxacin (Cipro, 50 ng). (a) Exudate PMN numbers and resolution indices. (b) Bacterial titers. (c) 14-HDHA and PD1 levels determined using LC-MS-MS-based LM-lipidomics. Results are expressed as mean±s.e.m. *p<0.05, **p<0.01, vs. E. coli alone; ^#p<0.05, ^##p<0.01, vs. E. coli+RvD1; ^§p<0.05, vs. E. coli+Cipro. Gray, E. coli alone; blue, E.coli+RvD1; green, E.coli+Cipro; dark blue, E.coli+RvD1+Cipro. (d) Mice were inoculated with E. coli (10⁷ CFU), SPM panel (RvD1, RvD5 and PD1; 50 ng each) and Cipro (25 μg). Body temperatures and bacterial titers were determined at 24h. Results are expressed as mean±s.e.m. *p<0.05, **p<0.01, ***p<0.001 vs. E. coli alone; ^#p<0.05, ^##p<0.01 vs. E. coli+SPM; ^§p<0.05, vs. E. coli+Cipro. (e,f) Murine dorsal pouches were given live S. aureus (10⁵ CFU) alone, plus SPM (RvD1, RvD5, PD1 at 100 ng each per mouse), vancomycin (Vanco, 2.5 μg), or both SPM and vancomycin by intra-pouch injection, pouch exudates were collected at 24h. (e) PMN and bacterial counts (CFU). Results are expressed as mean±s.e.m. *p<0.05, **p<0.01 vs. S. aureus alone; ^#p<0.05, vs. S. aureus+SPM; ^§§p<0.01, vs. S. aureus+Vanco. (f) Skin-pouch biopsies (Magnifications 40X). Arrows denote PMN infiltration into pouch linings. PC, pouch cavity. L, linings. Animal numbers are denoted within brackets (a) or bars (b-e).