Novel Resolvin D2 Receptor Axis in Infectious Inflammation

Abstract

Resolution of acute inflammation is an active process governed by specialized proresolving mediators, including resolvin (Rv)D2, that activates a cell surface G protein-coupled receptor, GPR18/DRV2. In this study, we investigated RvD2-DRV2-dependent resolution mechanisms using DRV2-deficient mice (DRV2-knockout [KO]). In polymicrobial sepsis initiated by cecal ligation and puncture, RvD2 (∼2.7 nmol/mouse) significantly increased survival (>50%) of wild-type mice and reduced hypothermia and bacterial titers compared with vehicle-treated cecal ligation and puncture mice that succumbed at 48 h. Protection by RvD2 was abolished in DRV2-KO mice. Mass spectrometry-based lipid mediator metabololipidomics demonstrated that DRV2-KO infectious exudates gave higher proinflammatory leukotriene B₄ and procoagulating thromboxane B_2, as well as lower specialized proresolving mediators, including RvD1 and RvD3, compared with wild-type. RvD2-DRV2-initiated intracellular signals were investigated using mass cytometry (cytometry by time-of-flight), which demonstrated that RvD2 enhanced phosphorylation of CREB, ERK1/2, and STAT3 in WT but not DRV2-KO macrophages. Monitored by real-time imaging, RvD2-DRV2 interaction significantly enhanced phagocytosis of live Escherichia coli, an action dependent on protein kinase A and STAT3 in macrophages. Taken together, we identified an RvD2/DRV2 axis that activates intracellular signaling pathways that increase phagocytosis-mediated bacterial clearance, survival, and organ protection. Moreover, these results provide evidence for RvD2-DRV2 and their downstream pathways in pathophysiology of infectious inflammation.

Figure 1. RvD2 protects polymicrobial sepsis in a DRV2-dependent manner

Mice were administered with RvD2 (1μg) or vehicle i.p. following CLP. (A) % survival of (left) wild type or (right) DRV2-KO mice. **p<0.01 log-rank (Mantel-Cox) test. NS: Non significant. Results are from 10 mice in each group. (B) Changes in body temperatures 24h after CLP. Results are expressed as mean±SEM from 5-8 mice/group. *p<0.05, CLP + veh. vs. CLP + RvD2 in WT group ^#p<0.05, vs. naive mice using unpaired Student’s t test. (C) Twelve hours after CLP, exudates were collected and microbial counts were determined. Results are expressed as mean±SEM from 4-6 mice/group. *p<0.05, CLP + veh. vs. CLP + RvD2 in WT group using unpaired Student’s t test.

Figure 2. Lipid mediator metabololipidomics: DRV2-KO versus WT mice

(A) MRM chromatographs (B) Representative MS-MS of RvD1. (C) principal component analysis of exudates lipid mediator 3D Loading Plot. (D) Exudate levels of prostanoids (PGs, TxB₂), LTB₄ plus 5-HETE, and D-series resolvins (RvDs) (pg/ml) in WT and DRV2-KO mice 12h after CLP. Results are expressed as mean±SEM from 4-5 mice/group *p<0.05 WT vs. DRV2-KO using unpaired Student’s t test.

Figure 3. RvD2 regulates select proteins in DRV2-dependent manner

Proteome Profiler cytokine array was carried out using 12h CLP infectious exudates. (A) Results are expressed as relative abundance to reference spots in a heat map with 111 proteins. (B) Levels of MMP2, MMP3 and MPO; mean±SEM from 3-6 mice/group. *p<0.05, CLP vs. CLP plus RvD2 in WT group; ^##p<0.01 CLP plus RvD2 in WT vs. KO groups using unpaired Student’s t test. NS: Non significant. (Insets) Representative dot plot images.

Figure 4. RvD2-DRV2-dependent macrophage intracellular signaling

(A) Heat maps of phosphorylated signaling molecules at 0, 1, 5, 15 and 30 minutes after exposure of RvD2 (10nM) in WT and DRV2-KO mice were obtained using CyTOF (see Methods). Phosphorylation levels were calculated as the difference between the inverse hyperbolic sine (arcsinh) of the median signal intensity in RvD2-treated peritoneal resident macrophages (at 0, 1, 5, 15, 30 min) and the arcsinh of the median signal intensity in vehicle-treated macrophages at 0 min. (bottom) gating strategy for macrophages (CD11b+F4/80+) and representative histograms of pCREB. (B) Flow cytometry for pCREB: gating strategy for macrophages (CD11b+F4/80+) and representative histograms. (C) Relative intensity of pERK1/2, pSTAT3, and pCREB. Results are mean±SEM from 3 independent experiments and samples were pooled from 5 mice in each group (WT vs. DRV2-KO) for each experiment. *p<0.05, **p<0.01 WT plus RvD2 vs DRV2-KO plus RvD2 using 2-tailed Student t-test.

Figure 5. PKA and STAT3 pathways mediates RvD2-stimulated phagocytosis

(A) Differentiated mouse bone marrow macrophages collected from WT and DRV2-KO were plated onto 8-wells chamber slides (0.5 × 10⁵ cells/well) and incubated with vehicle or RvD2 (10 nM) for 15 min at 37°C, followed by addition of BacLight Green-labeled E. coli to initiate phagocytosis. Fluorescent images were then recorded every 10 min. Three separate experiments were performed. In each experiment, three fields (20x) per condition (per well) were recorded. Results are mean fluorescent intensity (MFI); mean from 3 independent experiments with 3 mice/group in triplicates (3 fields/well). *p<0.05 using unpaired Student’s t test. NS: non-significant. (Insets) Representative fluorescent images at 100 min; scale bars, 50μm. (B) Mouse resident naïve macrophages were collected from WT and DRV2-KO, and incubated with 1-100 nM of RvD2 for 15 min and cAMP levels determined. Results are expressed as % increase of cAMP. cAMP levels in the presence of forskolin (10 μM) was taken as 100%; mean±SEM from n=3. *p<0.05 WT vs DRV2-KO. (C) Mouse resident naïve macrophages collected from WT were plated onto 8-wells chamber slides (1 × 10⁵ cells/well) and incubated with vehicle or RvD2 (10 nM) in the presence or absence of a STAT3 inhibitor (NSC 74859; 100μM), PKA inhibitor (H89; 3μM) or ERK inhibitor (FR 180204; 10μM for 15 min at 37°C, followed by addition of BacLight Green-labeled E. coli to initiate phagocytosis. Fluorescent images were then recorded as in panel (A). Results are (left) mean fluorescent intensity (MFI) from a representative of n=3-4, (right) fold changes vs E. coli alone, mean±SEM from 3-4 independent experiments in triplicates or quadruplicates (3-4 fields/well). (D) Mouse resident naïve macrophages collected from WT were plated onto 96-well plates (0.5 × 10⁵ cells/well) and incubated with vehicle or RvD2 (10 nM) in the presence or absence of a STAT3 inhibitor (NSC 74859; 100μM), PKA inhibitor (H89; 3μM) or ERK inhibitor (FR 180204; 10μM for 15 min at 37°C, followed by addition of FITC-labeled serum-treated zymosan (STZ) to initiate phagocytosis. Fluorescent images were then monitored using a plate reader. Results are mean±SEM from 3-4 independent experiments in triplicates. (C,D) *p<0.05, **p<0.01, ***p<0.001 using one-way ANOVA with post hoc multiple-comparison test (Newman-Keuls).

Figure 6. Schematic representation of RvD2-DRV2-dependent signaling pathways involved in macrophage phagocytosis

RvD2-DRV2 interactions initiate (i) Gαs protein coupling, leading to activation of cAMP-PKA signaling pathway and (ii) phosphorylation of STAT3, that contributed to macrophage phagocytosis.