Aging delays resolution of acute inflammation in mice: reprogramming the host response with novel nano-proresolving medicines

Abstract

Aging is associated with an overt inflammatory phenotype and physiological decline. Specialized proresolving lipid mediators (SPMs) are endogenous autacoids that actively promote resolution of inflammation. In this study, we investigated resolution of acute inflammation in aging and the roles of SPMs. Using a self-resolving peritonitis and resolution indices coupled with lipid mediator metabololipidomics, we found that aged mice had both delayed resolution and reduced SPMs. The SPM precursor docosahexaenoic acid accelerated resolution via increased SPMs and promoted human monocyte reprogramming. In aged mice, novel nano-proresolving medicines carrying aspirin-triggered resolvins D1 and D3 reduced inflammation by promoting efferocytosis. These findings provide evidence for age-dependent resolution pathways in acute inflammation and novel means to activate resolution.

Figure 1. Resolution of inflammation is delayed in aged mice

(A) Exudate PMN numbers in young (gray square) and aged (black square) mice subjected to zymosan challenge (0.1 mg/mouse). (B) Resolution indices were determined: Ψ_max (maximal PMN counts), T_max (the time interval when PMN reach maximum), T₅₀ (the time interval corresponding to 50% PMN reduction, or Ψ₅₀) and R_i (resolution interval, the interval between T_max and T₅₀). (C) Cytokines IL-6 and IL-12p40 in cell free supernatants and (D) monocyte/macrophage numbers (identified as CD11b⁺F4/80⁺ events) in young (gray bar) and aged (black bar) mice. * P < .05, *** P < .001 vs. young mice at indicated time interval; ^# P < .05, ^## P < .01, ^#### P < .0001 vs. 0 h within age group. (E) Uptake of fluorescently labeled apoptotic PMN (efferocytosis; 1.5×10⁵ cells) by BMDM (5×10⁴ cells, 1 h, 37°C) from young (gray square) and aged (black square) mice expressed as relative fluorescence units (RFU). * P < .05 vs. young mice. Results are mean ± s.e.m, from (A–D) 3 mice per age group at 0 h and 4 mice per age group at 4, 12 and 24 h or from (E) 4 mice per age group. Results are (A–D) combined from 3 independent experiments or (E) combined from one experiment.

Figure 2. Aged mice have reduced SPM and increased proinflammatory LM levels

(A) Representative MRM chromatograms of DHA and AA bioactive metabolomes and EPA-derived pathway markers in aged mouse peritoneal lavage before challenge (0 h). (B) Accompanying MS-MS spectra employed for identification of, e.g. RvD5, MaR1 and LXB₄ (inset, diagnostic ions; M = molecular mass). Cumulative levels of (C) D-series resolvins, protectins and maresins (left), AA-derived lipoxins (middle) and AA-derived prostaglandins and thromboxane (right) in peritoneal lavages (pg/mL) from young (gray bar) and aged (black bar) mice before challenge. *P < .05 vs. young mice. Results are (A–B) representative of 6 mice or (C) mean ± s.e.m from 3 mice per age group combined from 3 separate experiments.

Figure 3. Temporal LM levels are dysregulated in aged mice: principal component analysis

LM obtained from (A) young and (B) aged mouse peritoneal exudates at indicated time intervals following zymosan administration (0.1 mg/mouse) were identified by LM metabololipidomics (see “Methods” for details). (A–B) Score plots (left) and loading plots (right) for young and aged mice with 0h (baseline) in green, 4 h (peak of inflammation) in blue, 12 h (resolution phase) in red and 24 h (resolution phase) in aqua. Results are from 3 mice per age group at given time interval.

Figure 4. SPM precursor elevates local proresolving LM levels and shortens resolution

(A) Exudate PMN numbers and (B) resolution indices from mice administered DHA (10 μg, i.v., gray open circles) or vehicle (black squares) 1 min prior to (T₀) zymosan challenge (1 mg, i.p.). Inset, representative flow cytometry zebra plot; PMN identified as CD11b⁺Ly6G⁺ events (n=3). Resolution indices were calculated as in Figure 1. * P < .05 vs. zymosan plus vehicle for indicated time interval. (C) Shortened resolution expressed as percent change in R_i from peritonitis plus vehicle mice. (D) Increase in RvD1 and RvD3 in 12 h exudates following administration of DHA (1, 10 or 100 μg, i.v., T₀), expressed as percent increase from peritonitis plus vehicle mice. * P < .05 vs. peritonitis plus vehicle mice. Results are (A–B, D) mean ± s. e. m. or (C) mean from 3 mice in each group at each time interval.

Figure 5. Reprogrammed monocytes activate resolution

Monocytes (1×10⁶ cells) were incubated with vehicle (R0) or different ratios of DHA and EPA (R1 = 10:90, R2 = 50:50, R3 = 90:10; 10μM, 24 h, 37°C, pH 7.45). (A) R3 incubated monocytes and supernatants were collected and injected to mice (~1.5×10⁵ cells in 400μl, i.p.) 12 h after initiation of zymosan peritonitis. Peritonitis mice were injected with PBS alone. Exudate PMN were enumerated at indicated time intervals, and the resolution interval (R_i) calculated as in Figure 1. (B) Shortening of R_i following administration of indicated monocyte preparations 12 h after zymosan challenge. Results are expressed as percent change from peritonitis. Inset, correlation between increased ratio of DHA in monocyte incubations and shortening of R_i in vivo. Results are (A) mean ± s.e.m or (B) mean from 4 mice per treatment at each time interval combined from 3 independent experiments (see “Methods”). ** P < .01 vs. vehicle treated mice. (C) Cumulative DHA-derived SPM and pathway markers in indicated reprogramming monocyte incubations. Inset, correlation between increased ratio of DHA and cumulative DHA-derived SPM and pathway markers in reprogrammed monocyte supernatants. * P < .05 vs. monocytes incubated with R0. (D) Correlation between increased availability of DHA in monocyte reprogramming incubations and the levels of RvD1 (gray line) and RvD3 (black line) in these incubations. Results are (C) mean ± s.e.m or (C inset and D) mean from 5 donors combined from three independent experiments.

Figure 6. RvD3 shortens resolution of acute inflammation

(A) Exudate PMN numbers in mice administered vehicle or RvD3 (50 ng/mouse, i.p.) prior to (T₀) zymosan challenge (1 mg/mouse). Inset, representative flow cytometry zebra plot (n=4); PMN identified as CD11b⁺Ly6G⁺ events. (B) Resolution indices determined as in Figure 1. *** P < .001 vs. vehicle treated mice. Results are mean ± s.e.m from 4 mice per treatment at each time interval. (C) Enhanced uptake of fluorescently labeled apoptotic PMN (1.5×10⁵ cells) by RvD3 treated (1 nM, 15 min, 37°C) BMDM (5×10⁴ cells, 60 min, 37°C) from aged mice. **P < .01 vs. vehicle treated mice. Results are mean ± s.e.m. from 4 mice per treatment.

Figure 7. Construction of novel Resolvin-NPRM

Human monocyte-derived NPRM were obtained by energy-induced conversion of microparticles from reprogrammed monocytes that were enriched with AT-RvD1 and AT-RvD3. (A) Flow cytometry dot blot showing human monocyte derived nanoparticles. Incorporation of AT-RvD1 and AT-RvD3 into nanoparticles was determined using LC-MS-MS based LM metabololipidomics; (B) MRM chromatograms of selected ion pairs for AT-RvD1 and AT-RvD3 and (C) representative MS-MS spectra employed in the identification of AT-RvD1 and AT-RvD3 in NPRM.

Figure 8. In aged mice, monocyte-derived Resolvin-NPRM reduce inflammation and stimulate resolution

Resolvin-NPRM were prepared from human monocytes and enriched in AT-RvD1 and AT-RvD3; see “Methods”. (A) Aged mice were administered Resolvin-NPRM either prior to (group T₀; upper line) or 2 h after (group T₂; lower line) zymosan challenge (0.1 mg/mouse) and peritoneal exudates collected at 4 h. (B) Increase in macrophages containing ingested PMN (efferocytosis) in peritoneal exudates from aged mice administered Resolvin-NPRM (2×10⁵/mouse, i.p.) prior to (T₀; gray bars) or 2 h (T₂; black bars) after zymosan challenge; identified as F4/80⁺Ly6G⁺ events in peritoneal exudates using flow cytometry. (C) Reduction in exudate PMN numbers. Results are expressed as percent change from peritonitis mice. (D) Exudate LTB₄ levels in aged mice administered saline (peritonitis; open bar) or Resolvin-NPRM either prior to (T₀; gray bar) or 2 h after (T₂; black bar) zymosan challenge assessed using LM metabololipidomics following solid phase extraction (see “Methods”). Results are mean ± s.e.m from 4 mice for treatment with saline (peritonitis) and Resolvin-NPRM at 0 h and 3 mice per treatment with Resolvin-NPRM at 2 h. * P < .05 and ** P < .01 vs. peritonitis mice.