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Cited by 142Immune cells in cancerWound Healing and TreatmentsNeuropeptides and Animal Physiology

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

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Lim, Ji Eun, Chung, Eunkyung, Son, Youngsook·2017·Scientific Reports
DOI 10.1038/s41598-017-09639-7PMC PMC5573373
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Abstract

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 (M2 SP) 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. M2 SP 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 M2 SP 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 M2 SP macrophages and thus may be developed for pharmacological intervention in diseases involving chronic inflammation and acute injury.

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Figures

Figure 1 SP induces M2 polarization through early induction of Arginase-1. (a) MoBM were collected after 1-h of attachment and differentiated into MΦGM-CSF by GM-CSF treatment for 5 d. MΦGM-CSF (CD14−CD11b+CD68+CCR7+CD206+) were defined as M1-like precursors that were differentiated from MoBM (CD14+CD11b+CD68−CCR7−CD206+). For the immunofluorescence staining results (Supplementary Fig. 1a), the number of positive-cells was counted (5 random fields/coverslip) at 100 × magnification (0.85 mm2) (n = 3). (b) CD14+ MACS-isolated monocytes were differentiated with GM-CSF treatment for 5 d, and cells were further characterized as CD11b+CD68+ macrophages. (Green = CD11b, Red = CD68, Blue = DAPI) (n = 3). (c) Proposed model for MΦGM-CSF from MoBM with 80% CD14+ and 20% CD14− phenotypes. (d) Increased Arginase-1 activity was detected at the indicated time point of SP treatment using the Arginase-1 activity assay, and the results were compared with typical M1/M2-inducing cytokines (n = 3). (e) The increased expression of Arginase-1 was detected after 6 h of SP treatment. The western blot samples derived from the same experiment and were quantified with ImageJ. Full-length blots are presented in Supplementary Figure 16a (n = 4). (f–g) Dose-dependent increased activity and expression of Arginase-1 determined after 6 h of SP treatment. This was completely blocked by pretreatment with the NK-1R antagonist RP67580 (1 μM). (f) Arginase-1 activity assay (n = 3). (g) The western blot samples derived from the same experiment and were quantified with ImageJ. Full-length blots are presented in Supplementary Figure 16b (n = 4). (h) Increased NO was detected in culture with IFNγ, GM-CSF, or LPS but not with SP, IL-4/13, or IL-10. Quantitative analysis of NO production was performed in conditioned medium from MΦGM-CSF that had been treated with SP or cytokines for 1 d. LPS treatment served as a positive control (n = 6). Data represent the mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test). Scale bar = 100 μm (b) and 10 μm (high-magnification image of inset in b).
Figure 1 SP induces M2 polarization through early induction of Arginase-1. (a) MoBM were collected after 1-h of attachment and differentiated into MΦGM-CSF by GM-CSF treatment for 5 d. MΦGM-CSF (CD14−CD11b+CD68+CCR7+CD206+) were defined as M1-like precursors that were differentiated from MoBM (CD14+CD11b+CD68−CCR7−CD206+). For the immunofluorescence staining results (Supplementary Fig. 1a), the number of positive-cells was counted (5 random fields/coverslip) at 100 × magnification (0.85 mm2) (n = 3). (b) CD14+ MACS-isolated monocytes were differentiated with GM-CSF treatment for 5 d, and cells were further characterized as CD11b+CD68+ macrophages. (Green = CD11b, Red = CD68, Blue = DAPI) (n = 3). (c) Proposed model for MΦGM-CSF from MoBM with 80% CD14+ and 20% CD14− phenotypes. (d) Increased Arginase-1 activity was detected at the indicated time point of SP treatment using the Arginase-1 activity assay, and the results were compared with typical M1/M2-inducing cytokines (n = 3). (e) The increased expression of Arginase-1 was detected after 6 h of SP treatment. The western blot samples derived from the same experiment and were quantified with ImageJ. Full-length blots are presented in Supplementary Figure 16a (n = 4). (f–g) Dose-dependent increased activity and expression of Arginase-1 determined after 6 h of SP treatment. This was completely blocked by pretreatment with the NK-1R antagonist RP67580 (1 μM). (f) Arginase-1 activity assay (n = 3). (g) The western blot samples derived from the same experiment and were quantified with ImageJ. Full-length blots are presented in Supplementary Figure 16b (n = 4). (h) Increased NO was detected in culture with IFNγ, GM-CSF, or LPS but not with SP, IL-4/13, or IL-10. Quantitative analysis of NO production was performed in conditioned medium from MΦGM-CSF that had been treated with SP or cytokines for 1 d. LPS treatment served as a positive control (n = 6). Data represent the mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test). Scale bar = 100 μm (b) and 10 μm (high-magnification image of inset in b).CC BY · PubMed Central
Figure 2 Identification of SP-induced macrophages as a distinct M2 subtype compared with IL-4/13- and IL-10-induced macrophages. (a,b) After 3 d of culture with SP or cytokines, MΦ were immunostained with specific M1/M2 markers. The induction of CD163 expression and reduction of CCR7 expression were induced to a similar extent in SP, IL-4/13 and IL-10 but not in IFNγ or GM-CSF treated with MΦGM-CSF at 3 d (Green = CCR7, Red = CD163, Blue = DAPI). The number of CCR7loCD163+ M2 cells was counted (5 random fields/coverslip) at 100 × magnification (0.85 mm2) (n = 3). (c) Decreased expression of CD68 and increased expression of CD206 were detected in response to SP treatment, which was similar to IL-4/13 treatment (Green = CD68, Red = CD206, Blue = DAPI) (n = 3). (d) MHCII was up-regulated by SP treatment, but the increased expression was much higher in response to IL-4/13 treatment (Green = MHCII, Blue = DAPI) (n = 3). (e–f) After 1 d of treatment, CD68, CD206, and MHCII were examined by western blot analysis and quantified using ImageJ. The samples derived from the same experiment and full-length blots are presented in Supplementary Figure 17 (n = 3). (g) Neither STAT3 nor STAT6 was activated by SP treatment at 6 h, which are known to involved in the IL-10 and IL-4/13-induced signaling pathway, respectively. The samples derived from the same experiment and full-length blots are presented in Supplementary Figure 18 (n = 2). Data represent the mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test). Scale bar = 100 μm (a,c,d) and 10 μm (high-magnification image inset in a,c,d).
Figure 2 Identification of SP-induced macrophages as a distinct M2 subtype compared with IL-4/13- and IL-10-induced macrophages. (a,b) After 3 d of culture with SP or cytokines, MΦ were immunostained with specific M1/M2 markers. The induction of CD163 expression and reduction of CCR7 expression were induced to a similar extent in SP, IL-4/13 and IL-10 but not in IFNγ or GM-CSF treated with MΦGM-CSF at 3 d (Green = CCR7, Red = CD163, Blue = DAPI). The number of CCR7loCD163+ M2 cells was counted (5 random fields/coverslip) at 100 × magnification (0.85 mm2) (n = 3). (c) Decreased expression of CD68 and increased expression of CD206 were detected in response to SP treatment, which was similar to IL-4/13 treatment (Green = CD68, Red = CD206, Blue = DAPI) (n = 3). (d) MHCII was up-regulated by SP treatment, but the increased expression was much higher in response to IL-4/13 treatment (Green = MHCII, Blue = DAPI) (n = 3). (e–f) After 1 d of treatment, CD68, CD206, and MHCII were examined by western blot analysis and quantified using ImageJ. The samples derived from the same experiment and full-length blots are presented in Supplementary Figure 17 (n = 3). (g) Neither STAT3 nor STAT6 was activated by SP treatment at 6 h, which are known to involved in the IL-10 and IL-4/13-induced signaling pathway, respectively. The samples derived from the same experiment and full-length blots are presented in Supplementary Figure 18 (n = 2). Data represent the mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test). Scale bar = 100 μm (a,c,d) and 10 μm (high-magnification image inset in a,c,d).CC BY · PubMed Central
Figure 3 SP activates the NK-1R/PI3K/Akt/mTOR/S6K signaling pathway, leading to the induction of Arginase-1 and CD163 expression. (a) Western blot analysis showed that SP-activated PI3K, Akt, and mTOR were detected at 5 min and 20 min. The blockade of NK-1R by pretreatment with RP67580 led to inhibition of PI3K, Akt, and mTOR. The samples derived from the same experiment and full-length blots are presented in Supplementary Figures 19, 20 (n = 3). (b–d) Relative expression of p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR was quantified with ImageJ (n = 3). (e) All inhibitors, such as the NK-1R antagonist RP67580, PI3K inhibitor LY294002, mTOR inhibitor rapamycin, and S6K inhibitor PF4708671, completely blocked the elevated SP-induced Arginase-1 activity at 6 h (n = 6). (f) All inhibitors blocked SP-induced Arginase-1 expression. The western blot results were quantified with ImageJ. The samples derived from the same experiment and full-length blots are presented in Supplementary Figure 21 (n = 3). (g) Pretreatment with RP67580 completely blocked CD163 induction in SP-induced M2 at 3 d. Inhibitors of PI3K (LY294002) and S6K (PF4708671), but not MEK/ERK (U0126), prevented the induction of CD163 expression following the administration of SP (Green = CCR7, Red = CD163, Blue = DAPI) (n = 3). (h) Rat PBMCs also showed increased expression of Arginase-1 and CD163 at 6 h and 1 d after SP treatment, respectively. Pretreatment with RP67580 blocked the increased SP-induced expression of Arginase-1 and CD163, as demonstrated by western blot analysis. The samples derived from the same experiment and full-length blots are presented in Supplementary Figure 22 (n = 2). (i) Treatment of functional blocking antibodies to IL-10 (aIL-10) and IL-6 (aIL-6) failed to block SP-induced CD163 expression at 3 d (Green = CCR7, Red = CD163, Blue = DAPI) (n = 3). (j) Schematic representation of Arginase-1+CD163+ M2 polarization in response to SP via NK-1R/PI3K/Akt/mTOR/S6K activation. Data represent the mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test). Scale bar = 100 μm (g,i) and 10 μm (high-magnification image inset in g,i).
Figure 3 SP activates the NK-1R/PI3K/Akt/mTOR/S6K signaling pathway, leading to the induction of Arginase-1 and CD163 expression. (a) Western blot analysis showed that SP-activated PI3K, Akt, and mTOR were detected at 5 min and 20 min. The blockade of NK-1R by pretreatment with RP67580 led to inhibition of PI3K, Akt, and mTOR. The samples derived from the same experiment and full-length blots are presented in Supplementary Figures 19, 20 (n = 3). (b–d) Relative expression of p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR was quantified with ImageJ (n = 3). (e) All inhibitors, such as the NK-1R antagonist RP67580, PI3K inhibitor LY294002, mTOR inhibitor rapamycin, and S6K inhibitor PF4708671, completely blocked the elevated SP-induced Arginase-1 activity at 6 h (n = 6). (f) All inhibitors blocked SP-induced Arginase-1 expression. The western blot results were quantified with ImageJ. The samples derived from the same experiment and full-length blots are presented in Supplementary Figure 21 (n = 3). (g) Pretreatment with RP67580 completely blocked CD163 induction in SP-induced M2 at 3 d. Inhibitors of PI3K (LY294002) and S6K (PF4708671), but not MEK/ERK (U0126), prevented the induction of CD163 expression following the administration of SP (Green = CCR7, Red = CD163, Blue = DAPI) (n = 3). (h) Rat PBMCs also showed increased expression of Arginase-1 and CD163 at 6 h and 1 d after SP treatment, respectively. Pretreatment with RP67580 blocked the increased SP-induced expression of Arginase-1 and CD163, as demonstrated by western blot analysis. The samples derived from the same experiment and full-length blots are presented in Supplementary Figure 22 (n = 2). (i) Treatment of functional blocking antibodies to IL-10 (aIL-10) and IL-6 (aIL-6) failed to block SP-induced CD163 expression at 3 d (Green = CCR7, Red = CD163, Blue = DAPI) (n = 3). (j) Schematic representation of Arginase-1+CD163+ M2 polarization in response to SP via NK-1R/PI3K/Akt/mTOR/S6K activation. Data represent the mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test). Scale bar = 100 μm (g,i) and 10 μm (high-magnification image inset in g,i).CC BY · PubMed Central
Figure 4 SP is a mitogen for macrophages and affects M2 polarization in a dominant manner even in the presence of the Th1 cytokine INFγ. (a) BrdU incorporation analysis revealed that SP was less potent at inducing proliferation than GM-CSF and that SP and GM-CSF had a synergistic effect on proliferation on all types of macrophages at 3 d. Fold change of BrdU incorporation in MΦGM-CSF was shown in Supplementary Figure 2a. The number of BrdU+ cells was counted (5 random fields/coverslip) at 100 × magnification (0.85 mm2) (n = 3). (b–c) The percentage of CCR7loCD163+ M2 macrophages was maintained in M2aIL-4/13 and M2cIL-10 subtypes in the presence of SP but completely diminished after 3 d of GM-CSF treatment. SP-induced M2 macrophages in M1INFγ showed similar proportions of the M2aIL-4/13 and M2cIL-10 subtypes (Green = CCR7, Red = CD163, Blue = DAPI). The number of CCR7loCD163+ M2 cells was counted (5 random fields/coverslip) at 100 × magnification (0.85 mm2) (n = 3). (d) IL-4/13-induced MGCs were still present in response to SP co-treatment at 3 d (Green = CCR7, Red = CD163, Blue = DAPI) (n = 6). (e) The activity of Arginase-1 was increased at 6 h in response to IL-4/13, IL-10 or IFNγ co-treatment with SP, as determined by Arginase-1 activity assay. SP-induced M2 polarization is dominantly working over Th1 cytokines, IFNγ (n = 6). (f) The iNOS activity assay revealed a decrease in IFNγ activity after SP co-treatment compared with that after IFNγ treatment at 1 d (n = 3). (g) SP did not activate STAT1, STAT3, STAT5, or STAT6, and SP co-treatment did not affect specific cytokine-mediated STAT expression such as STAT6 by IL-4/13, STAT3 by IL-10, STAT1 by IFNγ, and STAT5 by GM-CSF at 6 h. The western blot samples derived from the same experiment and full-length blots are presented in Supplementary Figure 23. (n = 2). Data represent the mean ± SEM (*p < 0.05, ***p < 0.001, unpaired t test). Scale bar = 500 μm (d), 100 μm (c, high-magnification image inset in d) and 10 μm (high-magnification image inset in c).
Figure 4 SP is a mitogen for macrophages and affects M2 polarization in a dominant manner even in the presence of the Th1 cytokine INFγ. (a) BrdU incorporation analysis revealed that SP was less potent at inducing proliferation than GM-CSF and that SP and GM-CSF had a synergistic effect on proliferation on all types of macrophages at 3 d. Fold change of BrdU incorporation in MΦGM-CSF was shown in Supplementary Figure 2a. The number of BrdU+ cells was counted (5 random fields/coverslip) at 100 × magnification (0.85 mm2) (n = 3). (b–c) The percentage of CCR7loCD163+ M2 macrophages was maintained in M2aIL-4/13 and M2cIL-10 subtypes in the presence of SP but completely diminished after 3 d of GM-CSF treatment. SP-induced M2 macrophages in M1INFγ showed similar proportions of the M2aIL-4/13 and M2cIL-10 subtypes (Green = CCR7, Red = CD163, Blue = DAPI). The number of CCR7loCD163+ M2 cells was counted (5 random fields/coverslip) at 100 × magnification (0.85 mm2) (n = 3). (d) IL-4/13-induced MGCs were still present in response to SP co-treatment at 3 d (Green = CCR7, Red = CD163, Blue = DAPI) (n = 6). (e) The activity of Arginase-1 was increased at 6 h in response to IL-4/13, IL-10 or IFNγ co-treatment with SP, as determined by Arginase-1 activity assay. SP-induced M2 polarization is dominantly working over Th1 cytokines, IFNγ (n = 6). (f) The iNOS activity assay revealed a decrease in IFNγ activity after SP co-treatment compared with that after IFNγ treatment at 1 d (n = 3). (g) SP did not activate STAT1, STAT3, STAT5, or STAT6, and SP co-treatment did not affect specific cytokine-mediated STAT expression such as STAT6 by IL-4/13, STAT3 by IL-10, STAT1 by IFNγ, and STAT5 by GM-CSF at 6 h. The western blot samples derived from the same experiment and full-length blots are presented in Supplementary Figure 23. (n = 2). Data represent the mean ± SEM (*p < 0.05, ***p < 0.001, unpaired t test). Scale bar = 500 μm (d), 100 μm (c, high-magnification image inset in d) and 10 μm (high-magnification image inset in c).CC BY · PubMed Central
Figure 5 M2SP reveal highly phagocytic and endothelial adhesive activity and a distinct cytokine profile. (a,b) Anti-microbial activity was determined based on phagocytic activity toward FITC-labeled E. coli particles for 30 min. Analysis of E. coli-engulfed cells demonstrated strong anti-microbial activity of all types of macrophages, M2SP, M2cIL-10, and M1INFγ-like MΦGM-CSF, except M2aIL-4/13 (Green = FITC-E.coli particles, Red = Actin, Blue = DAPI). The number of E. coli-engulfed cells was counted (10 random fields/coverslip) at 200 × magnification (2.74 mm2) (n = 3). (c) IL-4/13-induced MGCs engulfed the large microspheres (Green = 25 μm Fluoresbrite microspheres, Red = Actin, Blue = DAPI). Enlarged images were shown in Supplementary Figure 6a and engulfed microspheres were confirmed inside of the MGCs in Supplementary Figure 6b (n = 3). (d,e) The adhesive activity of M2SP to HUVECs was determined. M2SP demonstrated a high adhesion ability like M2cIL-10, which was not detected in M2aIL-4/13 (Green = VE-cadherin, Red = PKH26-MΦ, Blue = DAPI). “Bottom” image confirms the VE-cadherin boundary of HUVECs and “Top” image confirms the attachment of macrophages. “3 D integrated image” was acquired from Z stack microscopic images and was presented as cartoons (Gray = HUVECs, Black = MΦ). The number of MΦ and HUVECs was counted (5 random fields/coverslip) at 100 × magnification (0.85 mm2) (n = 2). (f–o) M2SP released IL-6, MMP-8, MCP-1, IL-10, VEGF, and Prolactin R. M2SP, M2aIL-4/13, and M2cIL-10 commonly secreted MCP-1, IL-10, VEGF and Prolactin R after 1 d of treatment. (f–n) Quantification of cytokine array density was performed with ImageJ (n = 1). (o) ELISA (n = 3). Data represent the mean ± SEM (**p < 0.01, ***p < 0.001, unpaired t test). Scale bar = 100 μm (a,c,e) and 10 μm (high-magnification image inset in a).
Figure 5 M2SP reveal highly phagocytic and endothelial adhesive activity and a distinct cytokine profile. (a,b) Anti-microbial activity was determined based on phagocytic activity toward FITC-labeled E. coli particles for 30 min. Analysis of E. coli-engulfed cells demonstrated strong anti-microbial activity of all types of macrophages, M2SP, M2cIL-10, and M1INFγ-like MΦGM-CSF, except M2aIL-4/13 (Green = FITC-E.coli particles, Red = Actin, Blue = DAPI). The number of E. coli-engulfed cells was counted (10 random fields/coverslip) at 200 × magnification (2.74 mm2) (n = 3). (c) IL-4/13-induced MGCs engulfed the large microspheres (Green = 25 μm Fluoresbrite microspheres, Red = Actin, Blue = DAPI). Enlarged images were shown in Supplementary Figure 6a and engulfed microspheres were confirmed inside of the MGCs in Supplementary Figure 6b (n = 3). (d,e) The adhesive activity of M2SP to HUVECs was determined. M2SP demonstrated a high adhesion ability like M2cIL-10, which was not detected in M2aIL-4/13 (Green = VE-cadherin, Red = PKH26-MΦ, Blue = DAPI). “Bottom” image confirms the VE-cadherin boundary of HUVECs and “Top” image confirms the attachment of macrophages. “3 D integrated image” was acquired from Z stack microscopic images and was presented as cartoons (Gray = HUVECs, Black = MΦ). The number of MΦ and HUVECs was counted (5 random fields/coverslip) at 100 × magnification (0.85 mm2) (n = 2). (f–o) M2SP released IL-6, MMP-8, MCP-1, IL-10, VEGF, and Prolactin R. M2SP, M2aIL-4/13, and M2cIL-10 commonly secreted MCP-1, IL-10, VEGF and Prolactin R after 1 d of treatment. (f–n) Quantification of cytokine array density was performed with ImageJ (n = 1). (o) ELISA (n = 3). Data represent the mean ± SEM (**p < 0.01, ***p < 0.001, unpaired t test). Scale bar = 100 μm (a,c,e) and 10 μm (high-magnification image inset in a).CC BY · PubMed Central
Figure 6 SP can activate bone marrow-derived monocytes to express an M2 phenotype. (a) Western blot analysis showed that SP increased the expression of Arginase-1 and IL-10 but not the expression of iNOS or TNF-α in MoBM after 6 h of treatment. The samples derived from the same experiment and full-length blots are presented in Supplementary Figure 24. (n = 2). (b) SP and IL-4/13 increased the expression of CD206 and CD163 but not the expression of CD68 or CCR7 after 1 d of treatment. Only IL-4/13 Strongly increased the expression of MHCII. The samples derived from the same experiment and full-length blots are presented in Supplementary Figure 25. (n = 2). (c) Immunofluorescent staining showed that MoBM differentiated into CD68+CD206+ macrophages following SP and IL-4/13 treatment at 3 d (Green = CD68, Red = CD206, Blue = DAPI). SP-primed monocytes had an elongated morphology based on confocal microscope Z stack section analysis. (d) IL-10 blocked differentiation into CD68+-activated macrophages, but SP and IL-4/13 induced CD68+CD206+CD163+ M2-phenotype macrophages at 3 d. For the immunofluorescence staining results (Fig. 6c and Supplementary Fig. 8), the number of positive-cells was counted (5 random fields/coverslip) at 100 × magnification (0.85 mm2) (n = 3). (e) Immunofluorescent staining showed that MoBM were maintained as CD14+ monocytes only by IL-10 treatment at 3 d (Green = CD14, Red = CD206, Blue = DAPI) (n = 3). (f) BrdU incorporation assays revealed a similarly potent proliferating ability of SP to that of cytokines, but a less potent proliferating ability in comparison to GM-CSF. The number of BrdU+ cells was counted (5 random fields/coverslip) at 100 × magnification (0.85 mm2) (n = 3). (g) MoSP demonstrated high adhesion ability to HUVECs, and eventually were highly migratory like MoIL-10. The numbers of MΦ and HUVECs were counted (5 random fields/coverslip) at 100 × magnification (0.85 mm2) (n = 2). Data represent the mean ± SEM (**p < 0.01, ***p < 0.001, unpaired t test). Scale bar = 20 μm (c) and 100 μm (e).
Figure 6 SP can activate bone marrow-derived monocytes to express an M2 phenotype. (a) Western blot analysis showed that SP increased the expression of Arginase-1 and IL-10 but not the expression of iNOS or TNF-α in MoBM after 6 h of treatment. The samples derived from the same experiment and full-length blots are presented in Supplementary Figure 24. (n = 2). (b) SP and IL-4/13 increased the expression of CD206 and CD163 but not the expression of CD68 or CCR7 after 1 d of treatment. Only IL-4/13 Strongly increased the expression of MHCII. The samples derived from the same experiment and full-length blots are presented in Supplementary Figure 25. (n = 2). (c) Immunofluorescent staining showed that MoBM differentiated into CD68+CD206+ macrophages following SP and IL-4/13 treatment at 3 d (Green = CD68, Red = CD206, Blue = DAPI). SP-primed monocytes had an elongated morphology based on confocal microscope Z stack section analysis. (d) IL-10 blocked differentiation into CD68+-activated macrophages, but SP and IL-4/13 induced CD68+CD206+CD163+ M2-phenotype macrophages at 3 d. For the immunofluorescence staining results (Fig. 6c and Supplementary Fig. 8), the number of positive-cells was counted (5 random fields/coverslip) at 100 × magnification (0.85 mm2) (n = 3). (e) Immunofluorescent staining showed that MoBM were maintained as CD14+ monocytes only by IL-10 treatment at 3 d (Green = CD14, Red = CD206, Blue = DAPI) (n = 3). (f) BrdU incorporation assays revealed a similarly potent proliferating ability of SP to that of cytokines, but a less potent proliferating ability in comparison to GM-CSF. The number of BrdU+ cells was counted (5 random fields/coverslip) at 100 × magnification (0.85 mm2) (n = 3). (g) MoSP demonstrated high adhesion ability to HUVECs, and eventually were highly migratory like MoIL-10. The numbers of MΦ and HUVECs were counted (5 random fields/coverslip) at 100 × magnification (0.85 mm2) (n = 2). Data represent the mean ± SEM (**p < 0.01, ***p < 0.001, unpaired t test). Scale bar = 20 μm (c) and 100 μm (e).CC BY · PubMed Central
Figure 7 SP stimulates peripheral mobilization of CD11b+CD206+ cells from bone marrow and M2SP adoptive transfer improves functional recovery from SCI. (a) At 4.5 h after SP-injection, the number of 1-h adherent cells from the blood had increased. RP67580 blocked the increased number of 1-h adherent cells in PBMCs (n = 3). (b) At 4.5 h after SP-injection, MACS analysis showed that CD11b+ cells had increased in the blood (n = 3). (c) At 4.5 h after SP-injection, MACS analysis showed that the number of CD14+ cells did not differ in the blood (n = 3). (d) At 1 dpi, CD11b+CD206+ cells among the SP-injected PBMCs increased by 1.7-fold compared with that after PBS-injection. At 5 d, CD11b+CD206+ cells in response to SP-treatment was similar to that after PBS injection (n = 3). (e) At 1 dpi, SP treatment increased CD11b+CD206+ cells in injured spinal cord by 1.7-fold compared with that after PBS treatment. At 5 dpi, the number of CD11b+CD206+ macrophages increased by 2.5-fold in response to SP compared with that after PBS injection (n = 3). (f) A schematic of the procedure used for the adoptive transfer of M2SP in a rat SCI. PKH67-labeled MΦ and M2SP were injected at 1 dpi. (g) At 2 dpi, PKH67-labeled cells were observed at the injury core-lesion site. Longitudinal tissue sections were chosen at a distance of ~ 700 μm from the dorsal surface of the injury site. White arrows represent the PKH67+CD68+ adoptive transferred cells and yellow arrows represent PKH67−CD68+ resident microglia (Green = PKH67, Red = CD68, Blue = DAPI) (n = 3). (h) At 2 dpi, PKH67-labeled cells co-localized with CD206 (Green = PKH67, Red = CD206, Blue = DAPI) (n = 3). (i) At 14 dpi, CD163+CD206+ cells were observed in the MΦ, M2SP and SP groups but not in the PBS group (Green = CD163, Red = CD206, Blue = DAPI) (n = 3). (j,k) Histology and quantitative analysis of the lesion cavity delineated by H&E staining at 14 dpi. Every 2 stained tissue samples per rat were chosen at a distance of 0.7 mm and 1.0 mm from the dorsal section and lesion cavity was analyzed using ImageJ (PBS, n = 8; MΦ, n = 8; M2SP, n = 8; SP, n = 3). (l) The BBB score was evaluated for 6 weeks (1 d ~ 2 w- PBS, n = 14; MΦ, n = 14; M2SP, n = 15; SP, n = 5/3 w ~ 6 w- PBS group, n = 6; MΦ, n = 6; M2SP, n = 6; SP, n = 5). Data represent the mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001). (a-e,k) Unpaired t test. (l) one-way ANOVA. Scale bar = 100 μm (g–i) and 1 mm (j).
Figure 7 SP stimulates peripheral mobilization of CD11b+CD206+ cells from bone marrow and M2SP adoptive transfer improves functional recovery from SCI. (a) At 4.5 h after SP-injection, the number of 1-h adherent cells from the blood had increased. RP67580 blocked the increased number of 1-h adherent cells in PBMCs (n = 3). (b) At 4.5 h after SP-injection, MACS analysis showed that CD11b+ cells had increased in the blood (n = 3). (c) At 4.5 h after SP-injection, MACS analysis showed that the number of CD14+ cells did not differ in the blood (n = 3). (d) At 1 dpi, CD11b+CD206+ cells among the SP-injected PBMCs increased by 1.7-fold compared with that after PBS-injection. At 5 d, CD11b+CD206+ cells in response to SP-treatment was similar to that after PBS injection (n = 3). (e) At 1 dpi, SP treatment increased CD11b+CD206+ cells in injured spinal cord by 1.7-fold compared with that after PBS treatment. At 5 dpi, the number of CD11b+CD206+ macrophages increased by 2.5-fold in response to SP compared with that after PBS injection (n = 3). (f) A schematic of the procedure used for the adoptive transfer of M2SP in a rat SCI. PKH67-labeled MΦ and M2SP were injected at 1 dpi. (g) At 2 dpi, PKH67-labeled cells were observed at the injury core-lesion site. Longitudinal tissue sections were chosen at a distance of ~ 700 μm from the dorsal surface of the injury site. White arrows represent the PKH67+CD68+ adoptive transferred cells and yellow arrows represent PKH67−CD68+ resident microglia (Green = PKH67, Red = CD68, Blue = DAPI) (n = 3). (h) At 2 dpi, PKH67-labeled cells co-localized with CD206 (Green = PKH67, Red = CD206, Blue = DAPI) (n = 3). (i) At 14 dpi, CD163+CD206+ cells were observed in the MΦ, M2SP and SP groups but not in the PBS group (Green = CD163, Red = CD206, Blue = DAPI) (n = 3). (j,k) Histology and quantitative analysis of the lesion cavity delineated by H&E staining at 14 dpi. Every 2 stained tissue samples per rat were chosen at a distance of 0.7 mm and 1.0 mm from the dorsal section and lesion cavity was analyzed using ImageJ (PBS, n = 8; MΦ, n = 8; M2SP, n = 8; SP, n = 3). (l) The BBB score was evaluated for 6 weeks (1 d ~ 2 w- PBS, n = 14; MΦ, n = 14; M2SP, n = 15; SP, n = 5/3 w ~ 6 w- PBS group, n = 6; MΦ, n = 6; M2SP, n = 6; SP, n = 5). Data represent the mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001). (a-e,k) Unpaired t test. (l) one-way ANOVA. Scale bar = 100 μm (g–i) and 1 mm (j).CC BY · PubMed Central
Figure 8 Summary of the characteristics of M2SP compared with other M2 and M1 subtypes.
Figure 8 Summary of the characteristics of M2SP compared with other M2 and M1 subtypes.CC BY · PubMed Central

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Metadata

Authors
Lim, J. E., Chung, E., & Son, Y.
Affiliations
Kyung Hee University (KR)
Corresponding author
  • Lim, Ji Eun
  • Chung, Eunkyung
  • Son, Youngsook
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2017-08-25
Volume
7
Issue
1
Pages
9417-9417
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-017-09639-7
PMC
PMC5573373
OpenAlex
W2750028600
Cited by
136
References
59
Citations by year
2026: 14 · 2025: 15 · 2024: 15 · 2023: 16 · 2022: 18 · 2021: 10 · 2020: 16 · 2019: 19 · 2018: 12 · 2017: 1
FWCI
5.94
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
Ministry of Science, ICT and Future Planning (HI13C1479)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Immune cells in cancer
  • Health Sciences › Medicine › Surgery › Wound Healing and Treatments
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuropeptides and Animal Physiology
Keywords
substance P, neuropeptides, M2 macrophage polarization, M2 macrophages, interferon-gamma, neurokinin-1 receptor, arginase-1, CD163, CD206, spinal cord injury, tissue repair, macrophage migration, chronic inflammation
MeSH terms
THP-1 Cells, Animals, Cell Differentiation, Humans, Interferon-gamma, Macrophage Activation, Macrophages, Male, Substance P, Signal Transduction, Cytokines, Rats, Sprague-Dawley, Neurotransmitter Agents, Phosphatidylinositol 3-Kinases, Proto-Oncogene Proteins c-akt, TOR Serine-Threonine Kinases

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