A neuropeptide, Substance-P, directly induces tissue-repairing M2 like macrophages by activating the PI3K/Akt/mTOR pathway even in the presence of IFNγ

Macrophage polarization plays an important role in tissue damage and repair. In this study, we show that Substance-P (SP) can directly induce M2 polarization of inflammatory macrophages. SP induced the differentiation of GM-CSF-differentiated pro-inflammatory macrophages into alternatively activated phagocytic M2 like macrophages (M2SP) through direct activation of the PI3K/Akt/mTOR/S6kinase pathway and induction of Arginase-1, CD163, and CD206, all of which were nullified by pretreatment with the neurokinin-1 receptor (NK-1R) antagonist RP67580 and specific signaling pathway inhibitors. M2SP were distinct from IL-4/IL-13-induced M2a and IL-10-induced M2c subtypes; they did not show STAT activation and exhibited high phagocytic and endothelial adhesive activity. Furthermore, SP had a dominant effect on M2 polarization over Interferon gamma (IFNγ), a potent M1-skewing cytokine, and effectively induced the M2 phenotype in monocytes and the human THP-1 cell line. Finally, adoptively transferred M2SP migrated to a spinal cord injury (SCI) lesion site and improved functional recovery. Collectively, our findings show that SP, a neuropeptide, plays a role as a novel cytokine by inducing tissue-repairing M2SP macrophages and thus may be developed for pharmacological intervention in diseases involving chronic inflammation and acute injury.


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(a-b) The phagocytic activities toward dead blood and brain cells was assessed after 24 h.

Cell immunofluorescent staining
Bone marrow-derived mononuclear cells obtained from rats were prepared for characterization of Mo BM and MΦ GM-CSF by immunofluorescent staining. Cells that were prepared following the procedures described above were immediately fixed in 3.7% formaldehyde and blocked with 5% skim milk in PBS containing 0.2% Triton X-100 (Sigma-Aldrich). They were exposed to Finally, the cell nuclei were stained with methyl green for 10 min. Each experimental condition was performed independently in duplicate.

Phagocytosis of dead cells
To assess the ability to remove dead cells and cell debris, cells activated with various cytokines and SP were incubated with PKH-labeled cells isolated from PBMCs and brain tissue. A total of 4 × 10 5 cells from PBMCs and brain were labeled with PKH67 Green (Sigma, MINI67) and PKH26 Red (Sigma, MINI26) according to the manufacturers' instructions. Dead cells were prepared as previously reported 49 . Incubation with 100 M and 500 M H2O2 for 4 h was performed to induce apoptosis and necrosis, respectively. Combined apoptotic and necrotic dead cells were used for the phagocytosis assay. MΦ GM-CSF activated with SP and various cytokines were incubated with labeled blood and brain cells for 24 h, followed by fixation in 3.7% formaldehyde. Each experimental condition was performed independently in duplicate.

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The number of positive cells was counted (5 random fields/coverslip) at 100 × magnification (0.85 mm 2 ) using the Adobe Photoshop CS6 program.

Proliferation ability
Cell proliferation was assessed by 5-bromodeoxyuridine (BrdU) incorporation into Mo BM and MΦ GM-CSF cultures. The numbers of DAPI-positive cells were counted (5 random fields/coverslip) at 100 × magnification (0.85 mm 2 ) using the Adobe Photoshop CS6 program.
Each experimental condition was performed independently in triplicate.

RT-PCR was performed after isolation of RNA from cells using TRIzol reagent (Invitrogen).
The cDNA was generated using a Random Primer DNA Labeling kit ( for 30 sec, and 72°C for 1 min. Each experimental condition was performed independently in duplicate.

THP-1 cell line culture
To examine the effect of SP on M2 polarization in human monocytes and macrophages, the RPMI1640 and 10% heat-inactivated FBS. The differentiation of THP-1 monocytes into macrophages (THP-1 MΦ) was conducted using a phorbol 12-myristate 13-acetate (PMA) concentration of 10 ng ml −1 for 24 h, followed by three washes with PBS and culturing in completed medium for another 48 h for deactivation. The cells were then treated with SP and RP67580. Each experimental condition was performed independently in duplicate.

Tissue immunofluorescent staining
The animals were anesthetized with a mixture of ketamine and Rompun, and perfused with PBS and 3.7% paraformaldehyde for blood removal and fixation. The spinal cord was collected and placed in 3.7% formaldehyde for 5 h. Then, the cord was transferred to 30% sucrose in PBS and stored for 2 d. A 15-mm section of the spinal cord, centered at the injury site, was embedded in OCT compound (Sakura), and longitudinal sections were cut at 10 or 20 µm.
Tissue was blocked with 5% skim milk in PBS containing 0.2% Triton X-100 (Sigma-Aldrich). They were exposed to the following primary antibodies at 4 °C overnight: MAP2 (Millipore,