Cysteinyl-specialized proresolving mediators link resolution of infectious inflammation and tissue regeneration via TRAF3 activation

Abstract

The recently elucidated proresolving conjugates in tissue regeneration (CTR) maresin-CTR (MCTR), protectin-CTR (PCTR), and resolvin-CTR (RCTR), termed cysteinyl-specialized proresolving mediators (cys-SPMs) each promotes regeneration, controls infection, and accelerates resolution of inflammation. Here, we sought evidence for cys-SPM activation of primordial pathways in planaria (Dugesia japonica) regeneration that might link resolution of inflammation and regeneration. On surgical resection, planaria regeneration was enhanced with MCTR3, PCTR3, or RCTR3 (10 nM), each used for RNA sequencing. The three cys-SPMs shared up-regulation of 175 known transcripts with fold-change > 1.25 and combined false discovery rate (FDR) < 0.002, and 199 canonical pathways (FDR < 0.25), including NF-κB pathways and an ortholog of human TRAF3 (TNFR-associated factor 3). Three separate pathway analyses converged on TRAF3 up-regulation by cys-SPMs. With human macrophages, three cys-SPMs each dose-dependently increased TRAF3 expression in a cAMP-PKA-dependent manner. TRAF3 overexpression in macrophages enhanced Interleukin-10 (IL-10) and phagocytosis of Escherichia coli IL-10 also increased phagocytosis in a dose-dependent manner. Silencing of mouse TRAF3 in vivo significantly reduced IL-10 and macrophage phagocytosis. TRAF3 silencing in vivo also relieved cys-SPMs' actions in limiting polymorphonuclear neutrophil in E. coli exudates. These results identify cys-SPM-regulated pathways in planaria regeneration, uncovering a role for TRAF3/IL-10 in regulating mammalian phagocyte functions in resolution. Cys-SPM activation of TRAF3 signaling is a molecular component of both regeneration and resolution of infectious inflammation.

Keywords: chemical mediators; leukocytes; planaria; resolvins; signaling.

Fig. 1.

Cys-SPMs regulate planaria transcripts during regeneration: RNA-seq analysis. (A) Following head resection, planaria (D. japonica; 10 planarians) were incubated with MCTR3, PCTR3, RCTR3 separately (10 nM each) or vehicle (0.1% ethanol [vol/vol]) in water. Planaria were collected at 48 h and mRNA isolated for RNA-seq. (B) The total of 33,511 transcripts were obtained, of which 5,563 were up-regulated (P < 0.05) by MCTR3, PCTR3, and/or RCTR3. Among these, 542 were up-regulated by the 3 cys-SPMs; 175 are known transcripts with predicted functions with FC > 1.25 by at least 1 of the 3 cys-SPMs, and 30 of them are involved in NF-κB and/or epigenetic regulation (SI Appendix, Table S1A). (C) Venn diagram illustrates the numbers of transcripts up-regulated (P < 0.05, FC > 1.25) by MCTR3, PCTR3, and/or PCTR3. Combined FDR < 0.002 for each of the 175 transcripts. (D, Upper) Venn diagram of cys-SPM up-regulated transcripts that have predicted functions in NF-κB/TLR and/or epigenetic-related pathways. Combined FDR < 0.0005 for each of the 30 transcripts. (Lower) The heatmap depicts FCs of these transcripts. (E) Predicted protein–protein interaction network with TRAF3 using Cytoscape. Within this network, TRAF3 was predicted to interact with proteins of other cys-SPM up-regulated transcripts, including TRAF5, ABCC1, BIRC2, and USO1 (blue), and down-regulated transcripts, including USP7 and OTUD5 (red). See Insets for FC of these transcripts, and the combined FDR values are shown above each graph. Additional proteins in the BioGrid database that are predicted to interact with TRAF3 are depicted in yellow.

Fig. 2.

Cys-SPMs regulate planaria canonical pathways during regeneration. (A) Pathway analysis was carried out using CP, including Reactome, KEGG, Biocarta, and PID databases. The total of 1,245 CP were identified during regeneration, of which 502 were significantly (FDR < 0.25) regulated by MCTR3, PCTR3, and/or RCTR3. Among them, 199 CP were significantly regulated by three cys-SPMs, and within these CP, 39 are related to NF-κB and/or epigenetic pathways. (B) Venn diagram illustrates the CP that were significantly regulated (FDR < 0.25) by MCTR3, PCTR3, and/or RCTR3 during planaria regeneration. Among these, numbers of CP related to NF-κB, epigenetic modification and Wnt signaling are indicated for each database: Reactome, KEGG, Biocarta, PID, and additional databases. (C) Biocarta pathways that were significantly regulated by MCTR3, PCTR3, and RCTR3. Numbers of transcripts are expressed in blue (up-regulated) or red (down-regulated) in stacked bar graphs. (D and E) Heatmaps of significantly regulated CP by the three cys-SPMs in (D) Biocarta and (E) KEGG pathway database. For heatmaps of two pathway analysis using Reactome and PID databases, see SI Appendix, Fig. S4.

Fig. 3.

Cys-SPMs increase TRAF3 and phagocytosis with macrophages (A) Schematic representation of cys-SPMs and TRAF3 regulation of downstream effectors and phagocytosis. (B) Transcript expression. Human MΦ were incubated with STZ with each cys-SPM (10 nM) or vehicle for 6 h. Transcript levels were determined by qPCR and expressed as FCs of STZ alone; mean ± SEM, n = 5 (traf3 and il-10), 3 or 4 (traf6 and ifnγ); *P < 0.05, **P < 0.01 vs. STZ alone; two-tailed Student’s t test. (C and D) Dose–responses. Human MΦ were incubated with STZ with each cys-SPM (0.1 to 100 nM), DHA, 17S-HDHA (1 to 100 nM), or vehicle for 24 h. (C) Cells were lysed for TRAF3 ELISA and (D) supernatants collected for IL-10 ELISA. Results are percent increase compared with STZ; mean± SEM, n = 3 to 5; *P < 0.05, **P < 0.01, vs. STZ alone; one-way ANOVA; ^†P < 0.05, ^†††P < 0.001 vs. DHA; ^‡P < 0.05, ^‡‡P < 0.01 vs. 17S-HDHA at the same concentrations; two-tailed Student’s t test. (C and D, Insets) Dose–response curves of MCTR3 with DHA and/or 17S-HDHA. EC₅₀s were estimated using nonlinear regression with log (agonist) vs. response (three parameters). (E–G and I) Phagocytosis. Human MΦ were plated onto slide chambers (1 × 10⁵ cells per well) and incubated with each cys-SPM (10 nM) or vehicle control for 24 h, followed by addition of BacLight green-labeled E. coli (5 × 10⁶ CFU) to initiate phagocytosis. Fluorescent images were then recorded every 10 min for 120 min. In each experiment, four fields (20×) per condition (per well) were recorded. (E, Left) mean fluorescence intensity (MFI)/cell, average of 124 cells per field and 4 fields per condition from one representative experiment. (Right) Representative images. (Scale bar, 50 μm.) (F) Percent increase of phagocytosis compared with E. coli alone; mean± SEM, n = 5 to 7; *P < 0.05, **P < 0.01. (G and I) Cholera toxin (CTX; 1 μg/mL), PKA inhibitor (H89, 10 μM), or vehicle were incubated with MΦ together with cys-SPMs. Results are percent increase of phagocytosis compared with E. coli alone at 60 min; mean ± SEM, n = 3 or 4; *P < 0.05, **P < 0.01; (F) one-way ANOVA; (G and I) two-tailed Student’s t test. (H) cAMP. Human MΦ were incubated with each cys-SPM (0.1 to 100 nM) for 15 min. Cells were lysed and cAMP levels determined. Results are percent increase above vehicle; mean ± SEM, n = 5; *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle; one-way ANOVA. (J) TRAF3 expression was determined as in C in the absence or presence of a PKA inhibitor. Results are percent increase of STZ alone; mean ± SEM, n = 3 or 4; *P < 0.05, **P < 0.01; two-tailed Student’s t test.

Fig. 4.

Overexpression of TRAF3 enhances IL-10 and phagocytosis with human macrophages. Human MΦ were transfected with Mock or TRAF3 plasmids. (A) TRAF3 protein expression was determined using flow cytometry with a specific anti-TRAF3 Ab. (Upper) Representative flow cytometry histograms; (Lower) mean ± SEM, n = 4; *P < 0.05. (B) Cytokine levels. MΦ were incubated with STZ with each cys-SPM (10 nM) or vehicle control for 24 h. Results are picograms per milliliter or percent increase of STZ alone; mean ± SEM, n = 3; *P < 0.05. (C and D) Phagocytosis was carried out as in Fig. 3E in the presence of a cys-SPM panel (MCTR3, PCTR3, and RCTR3 1 nM each) or vehicle control. (Left) MFI per cell from one representative experiment; (C, Inset) Kinetics 30 to 60 min (MFI/min). (C, Right) Percent increase of phagocytosis; MFI (TRAF3-OE)/MFI (mock); *P < 0.05, **P < 0.01 vs. time 0. (D, Right) Percent increase of phagocytosis by the cys-SPM panel; mean ± SEM, n = 3; **P < 0.01. For kinetics from three separate donors, see SI Appendix, Fig. S7B. (E and F) Phagocytosis was carried out as in Fig. 3E in the presence of (E) IL-10 (1 pg/ml to 1,000 ng/ml), (F) IL-10 (10 ng/ml) and/or a STAT3 inhibitor (NSC, 100 μM) for 24 h. (E and F, Left) MFI per cell from one representative experiment. (E, Right) Dose–response curve; mean ± SEM, n = 4; P < 0.05. EC₅₀ was estimated using nonlinear regression with log (agonist) vs. response (three parameters). (F, Right) Percent increase of phagocytosis vs. E. coli alone; mean ± SEM, n = 3 or 4; *P < 0.05. Statistical analyses were carried out using (A, B, F) two-tailed Student’s t test, (C and E) one-way ANOVA, or (D) two-way ANOVA. (G) Proposed cys-SPMs/TRAF3/IL-10 axis.

Fig. 5.

Impact of TRAF3 on resolution of infection: In vivo silencing. (A) Timeline. Mice were injected with nontarget- or TRAF3-siRNA (10 μg per mouse, intraperitoneally). Peritoneal cells were collected at 72 h (blue bar). In separate experiments, mice were inoculated with E. coli (1 × 10⁵ CFU per mouse, intraperitoneally) with a cys-SPM panel (MCTR3, PCTR3, and RCTR3, 50 ng each) or vehicle control (0.1% ethanol [vol/vol]) in sterile saline. Exudates were collected 4 h or 24 h later (yellow bar). (B) TRAF3 protein expression at Time 0 and 24 h using flow cytometry with a specific anti-TRAF3 Ab. (Left) Representative histograms. (Right) TRAF3 expression (MFI); mean ± SEM, n = 3 (0 h) or 7 (24 h); *P < 0.05, **P < 0.01. (C) Peritoneal MΦ phagocytosis of E. coli was carried out as in Fig. 3E. (Left) Fluorescence intensities (MFI per cell); mean ± SEM, n = 5 mice; average of 195 cells per field and 4 fields per mouse; *P < 0.05, **P < 0.01, nontarget vs. TRAF3 siRNA. (Inset) Representative fluorescent images. (Scale bar, 50 μm.) See additional images in SI Appendix, Fig. S8. (Center) MFI at 2 h; mean ± SEM, n = 5; *P < 0.05. (Right) Kinetics from 0 to 60 min; mean of 5 mice per group. (D) Peritoneal MΦ were plated onto 24-well plates and incubated with STZ (100 ng/mL) for 24 h. Supernatants were collected and IL-10 levels determined; mean ± SEM, n = 3; **P < 0.01. (E and F) Infectious exudates from 24 h E. coli infection were collected; (E) PMN numbers; mean ± SEM, n = 5 to 8. (F) Exudate TNF-α levels; mean ± SEM, n = 5 or 6; *P < 0.05. (B–F) Two-tailed unpaired Student’s t test.