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p38/MKP-1-regulated AKT coordinates macrophage transitions and resolution of inflammation during tissue repair.

Perdiguero E, Sousa-Victor P, Ruiz-Bonilla V, Jardí M, Caelles C, Serrano AL, Muñoz-Cánoves P - J. Cell Biol. (2011)

Bottom Line: Repair of damaged tissue requires the coordinated action of inflammatory and tissue-specific cells to restore homeostasis, but the underlying regulatory mechanisms are poorly understood.Conversely, miR-21-AKT interference altered homeostasis during tissue repair.This novel regulatory mechanism involving the appropriate balance of p38, MKP-1, miR-21, and AKT activities may have implications in chronic inflammatory degenerative diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cell Biology Group, Department of Experimental and Health Sciences, Pompeu Fabra University, 08003 Barcelona, Spain.

ABSTRACT
Repair of damaged tissue requires the coordinated action of inflammatory and tissue-specific cells to restore homeostasis, but the underlying regulatory mechanisms are poorly understood. In this paper, we report new roles for MKP-1 (mitogen-activated protein kinase [MAPK] phosphatase-1) in controlling macrophage phenotypic transitions necessary for appropriate muscle stem cell-dependent tissue repair. By restricting p38 MAPK activation, MKP-1 allows the early pro- to antiinflammatory macrophage transition and the later progression into a macrophage exhaustion-like state characterized by cytokine silencing, thereby permitting resolution of inflammation as tissue fully recovers. p38 hyperactivation in macrophages lacking MKP-1 induced the expression of microRNA-21 (miR-21), which in turn reduced PTEN (phosphatase and tensin homologue) levels, thereby extending AKT activation. In the absence of MKP-1, p38-induced AKT activity anticipated the acquisition of the antiinflammatory gene program and final cytokine silencing in macrophages, resulting in impaired tissue healing. Such defects were reversed by temporally controlled p38 inhibition. Conversely, miR-21-AKT interference altered homeostasis during tissue repair. This novel regulatory mechanism involving the appropriate balance of p38, MKP-1, miR-21, and AKT activities may have implications in chronic inflammatory degenerative diseases.

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Transient PI3K–AKT inhibition in WT macrophages maintains cytokine expression and prevents cytokine silencing at late stages of the tissue repair process. (A) Gene analysis by RT-PCR in FACS-isolated macrophage populations at 8 and 10 d after injury from mice treated with wortmannin (Wort) or vehicle for the preceding 2 d. (B) Anti–IL-10 neutralizing antibody was injected twice during the later stages of muscle regeneration after CTX injury (Inj.), and muscles were isolated at day 8 and 10 postinjury (P.I.) and processed as in Fig. 4 A. IgG was used as a control. An outline of the experiment is depicted. (C, top) Apoptotic F4/80-expressing macrophages were assessed by TUNEL staining. (bottom) CTX-injured gastrocnemius muscle from WT and MKP-1−/−: F480 (red), TUNEL (green), and DAPI (blue). Regenerating fibers are indicated by dotted lines. Apoptotic nuclei are indicated by arrows. (D) Cell proliferation was assessed by Ki-67 staining as in C: Ki-67 (red) and DAPI (blue). (E) Expression of p21, p27, and β-actin (as a loading control) was analyzed by RT-PCR in FACS-isolated macrophage populations at 6 d after injury as in Fig. 3 A. Means ± SEM of at least three experiments. **, P < 0.01. Bars, 50 µm.
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fig7: Transient PI3K–AKT inhibition in WT macrophages maintains cytokine expression and prevents cytokine silencing at late stages of the tissue repair process. (A) Gene analysis by RT-PCR in FACS-isolated macrophage populations at 8 and 10 d after injury from mice treated with wortmannin (Wort) or vehicle for the preceding 2 d. (B) Anti–IL-10 neutralizing antibody was injected twice during the later stages of muscle regeneration after CTX injury (Inj.), and muscles were isolated at day 8 and 10 postinjury (P.I.) and processed as in Fig. 4 A. IgG was used as a control. An outline of the experiment is depicted. (C, top) Apoptotic F4/80-expressing macrophages were assessed by TUNEL staining. (bottom) CTX-injured gastrocnemius muscle from WT and MKP-1−/−: F480 (red), TUNEL (green), and DAPI (blue). Regenerating fibers are indicated by dotted lines. Apoptotic nuclei are indicated by arrows. (D) Cell proliferation was assessed by Ki-67 staining as in C: Ki-67 (red) and DAPI (blue). (E) Expression of p21, p27, and β-actin (as a loading control) was analyzed by RT-PCR in FACS-isolated macrophage populations at 6 d after injury as in Fig. 3 A. Means ± SEM of at least three experiments. **, P < 0.01. Bars, 50 µm.

Mentions: As shown in Fig. 1, p38 remained active in macrophages at late stages after injury when tissue is almost fully recovered (10 d), coinciding with a decline in MKP-1 expression, sustained activity of AKT, and cessation of the expression of both pro- and antiinflammatory cytokine expression. Furthermore, compared with WT mice, AKT activity was also higher in MKP1−/− macrophages at 6 d (and also at 10 d after injury; Fig. 6 A and Fig. S4 D), correlating with its unscheduled cytokine silencing (Fig. 1 C), thus suggesting that AKT, in addition to regulating the earlier pro- to antiinflammatory cytokine switching, might also be required for the final cytokine silencing in WT macrophages at advanced stages of tissue repair. To test this possibility, WT mice were transiently treated with wortmannin at 6 or 8 d after injury, and the macrophage number and activation status was analyzed 2 d later. Although macrophages in vehicle-treated WT muscles presented a silenced cytokine profile (i.e., no expression of pro- or antiinflammatory cytokines), the transient wortmannin treatment prevented full cytokine silencing, as indicated by residual expression of both pro- and antiinflammatory cytokines (Fig. 7 A). Because transient SB2013580 treatment prevented premature cytokine silencing of MKP-1−/− macrophages at 6 d after injury (Fig. 3 A), these results taken together strongly support a role for MKP-1 (through p38 neutralization and subsequent AKT down-regulation) in controlling sequential cytokine activation-silencing transitions in macrophages during tissue repair by restraining AKT activation.


p38/MKP-1-regulated AKT coordinates macrophage transitions and resolution of inflammation during tissue repair.

Perdiguero E, Sousa-Victor P, Ruiz-Bonilla V, Jardí M, Caelles C, Serrano AL, Muñoz-Cánoves P - J. Cell Biol. (2011)

Transient PI3K–AKT inhibition in WT macrophages maintains cytokine expression and prevents cytokine silencing at late stages of the tissue repair process. (A) Gene analysis by RT-PCR in FACS-isolated macrophage populations at 8 and 10 d after injury from mice treated with wortmannin (Wort) or vehicle for the preceding 2 d. (B) Anti–IL-10 neutralizing antibody was injected twice during the later stages of muscle regeneration after CTX injury (Inj.), and muscles were isolated at day 8 and 10 postinjury (P.I.) and processed as in Fig. 4 A. IgG was used as a control. An outline of the experiment is depicted. (C, top) Apoptotic F4/80-expressing macrophages were assessed by TUNEL staining. (bottom) CTX-injured gastrocnemius muscle from WT and MKP-1−/−: F480 (red), TUNEL (green), and DAPI (blue). Regenerating fibers are indicated by dotted lines. Apoptotic nuclei are indicated by arrows. (D) Cell proliferation was assessed by Ki-67 staining as in C: Ki-67 (red) and DAPI (blue). (E) Expression of p21, p27, and β-actin (as a loading control) was analyzed by RT-PCR in FACS-isolated macrophage populations at 6 d after injury as in Fig. 3 A. Means ± SEM of at least three experiments. **, P < 0.01. Bars, 50 µm.
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Related In: Results  -  Collection

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Show All Figures
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fig7: Transient PI3K–AKT inhibition in WT macrophages maintains cytokine expression and prevents cytokine silencing at late stages of the tissue repair process. (A) Gene analysis by RT-PCR in FACS-isolated macrophage populations at 8 and 10 d after injury from mice treated with wortmannin (Wort) or vehicle for the preceding 2 d. (B) Anti–IL-10 neutralizing antibody was injected twice during the later stages of muscle regeneration after CTX injury (Inj.), and muscles were isolated at day 8 and 10 postinjury (P.I.) and processed as in Fig. 4 A. IgG was used as a control. An outline of the experiment is depicted. (C, top) Apoptotic F4/80-expressing macrophages were assessed by TUNEL staining. (bottom) CTX-injured gastrocnemius muscle from WT and MKP-1−/−: F480 (red), TUNEL (green), and DAPI (blue). Regenerating fibers are indicated by dotted lines. Apoptotic nuclei are indicated by arrows. (D) Cell proliferation was assessed by Ki-67 staining as in C: Ki-67 (red) and DAPI (blue). (E) Expression of p21, p27, and β-actin (as a loading control) was analyzed by RT-PCR in FACS-isolated macrophage populations at 6 d after injury as in Fig. 3 A. Means ± SEM of at least three experiments. **, P < 0.01. Bars, 50 µm.
Mentions: As shown in Fig. 1, p38 remained active in macrophages at late stages after injury when tissue is almost fully recovered (10 d), coinciding with a decline in MKP-1 expression, sustained activity of AKT, and cessation of the expression of both pro- and antiinflammatory cytokine expression. Furthermore, compared with WT mice, AKT activity was also higher in MKP1−/− macrophages at 6 d (and also at 10 d after injury; Fig. 6 A and Fig. S4 D), correlating with its unscheduled cytokine silencing (Fig. 1 C), thus suggesting that AKT, in addition to regulating the earlier pro- to antiinflammatory cytokine switching, might also be required for the final cytokine silencing in WT macrophages at advanced stages of tissue repair. To test this possibility, WT mice were transiently treated with wortmannin at 6 or 8 d after injury, and the macrophage number and activation status was analyzed 2 d later. Although macrophages in vehicle-treated WT muscles presented a silenced cytokine profile (i.e., no expression of pro- or antiinflammatory cytokines), the transient wortmannin treatment prevented full cytokine silencing, as indicated by residual expression of both pro- and antiinflammatory cytokines (Fig. 7 A). Because transient SB2013580 treatment prevented premature cytokine silencing of MKP-1−/− macrophages at 6 d after injury (Fig. 3 A), these results taken together strongly support a role for MKP-1 (through p38 neutralization and subsequent AKT down-regulation) in controlling sequential cytokine activation-silencing transitions in macrophages during tissue repair by restraining AKT activation.

Bottom Line: Repair of damaged tissue requires the coordinated action of inflammatory and tissue-specific cells to restore homeostasis, but the underlying regulatory mechanisms are poorly understood.Conversely, miR-21-AKT interference altered homeostasis during tissue repair.This novel regulatory mechanism involving the appropriate balance of p38, MKP-1, miR-21, and AKT activities may have implications in chronic inflammatory degenerative diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cell Biology Group, Department of Experimental and Health Sciences, Pompeu Fabra University, 08003 Barcelona, Spain.

ABSTRACT
Repair of damaged tissue requires the coordinated action of inflammatory and tissue-specific cells to restore homeostasis, but the underlying regulatory mechanisms are poorly understood. In this paper, we report new roles for MKP-1 (mitogen-activated protein kinase [MAPK] phosphatase-1) in controlling macrophage phenotypic transitions necessary for appropriate muscle stem cell-dependent tissue repair. By restricting p38 MAPK activation, MKP-1 allows the early pro- to antiinflammatory macrophage transition and the later progression into a macrophage exhaustion-like state characterized by cytokine silencing, thereby permitting resolution of inflammation as tissue fully recovers. p38 hyperactivation in macrophages lacking MKP-1 induced the expression of microRNA-21 (miR-21), which in turn reduced PTEN (phosphatase and tensin homologue) levels, thereby extending AKT activation. In the absence of MKP-1, p38-induced AKT activity anticipated the acquisition of the antiinflammatory gene program and final cytokine silencing in macrophages, resulting in impaired tissue healing. Such defects were reversed by temporally controlled p38 inhibition. Conversely, miR-21-AKT interference altered homeostasis during tissue repair. This novel regulatory mechanism involving the appropriate balance of p38, MKP-1, miR-21, and AKT activities may have implications in chronic inflammatory degenerative diseases.

Show MeSH
Related in: MedlinePlus