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Kruppel-like factor KLF4 facilitates cutaneous wound healing by promoting fibrocyte generation from myeloid-derived suppressor cells.

Ou L, Shi Y, Dong W, Liu C, Schmidt TJ, Nagarkatti P, Nagarkatti M, Fan D, Ai W - J. Invest. Dermatol. (2015)

Bottom Line: Myeloid-derived suppressor cells (MDSCs) accumulate as a result of inflammation and promote cutaneous wound healing by mechanisms that are not fully understood.Recently, MDSCs have been shown to differentiate into fibrocytes, which serve as emerging effector cells that enhance cell proliferation in wound healing.In the current study, by using full-thickness and PU mouse models, we found that Kruppel-like factor 4 (KLF4) deficiency resulted in decreased accumulation of MDSCs and fibrocytes, and wound healing was significantly delayed.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, South Carolina, USA [2] Department of Biopharmaceuticals, School of Biotechnology, Southern Medical University, Guangzhou, People's Republic of China.

ABSTRACT
Pressure ulcers (PUs) are serious skin injuries whereby the wound healing process is frequently stalled in the inflammatory phase. Myeloid-derived suppressor cells (MDSCs) accumulate as a result of inflammation and promote cutaneous wound healing by mechanisms that are not fully understood. Recently, MDSCs have been shown to differentiate into fibrocytes, which serve as emerging effector cells that enhance cell proliferation in wound healing. We postulate that in wound healing MDSCs not only execute their immunosuppressive function to regulate inflammation but also stimulate cell proliferation once they differentiate into fibrocytes. In the current study, by using full-thickness and PU mouse models, we found that Kruppel-like factor 4 (KLF4) deficiency resulted in decreased accumulation of MDSCs and fibrocytes, and wound healing was significantly delayed. Conversely, KLF4 activation by the plant-derived product Mexicanin I increased the number of MDSCs and fibrocytes and accelerated the wound healing. Collectively, our study revealed a previously unreported function of MDSCs in cutaneous wound healing and identified Mexicanin I as a potential agent to accelerate PU wound healing.

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Compromised wound healing of PU in FSP-1-Cre/KLF4(flox) mice associated with decreased CCR2+MDSCs and fibrocytes(a). Similar to Figure 1c, except the WT and KLF4−/−(FSP-1) mice were used in the PU model (n=10). (b). Left: Representative images of HE staining of skin wounds in PU model. Right: Quantification of the epithelial thickness and the numbers of infiltrated lymphocytes in arbitrary red squares with the same sizes as the left (n=5). (c). Flow cytometry analysis to examine MDSCs and fibrocytes in mouse blood and skin wounds. (d). Similar to c, CD11b and Ly6C antibodies were used to examine inflammatory monocytes. (e). qRT=PCR to analyze expression of KLF4, FSP-1, CCL2, and CCR2 in the skin wounds. Scale bars: 100 μm, *p<0.05, **p<0.01.
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Figure 5: Compromised wound healing of PU in FSP-1-Cre/KLF4(flox) mice associated with decreased CCR2+MDSCs and fibrocytes(a). Similar to Figure 1c, except the WT and KLF4−/−(FSP-1) mice were used in the PU model (n=10). (b). Left: Representative images of HE staining of skin wounds in PU model. Right: Quantification of the epithelial thickness and the numbers of infiltrated lymphocytes in arbitrary red squares with the same sizes as the left (n=5). (c). Flow cytometry analysis to examine MDSCs and fibrocytes in mouse blood and skin wounds. (d). Similar to c, CD11b and Ly6C antibodies were used to examine inflammatory monocytes. (e). qRT=PCR to analyze expression of KLF4, FSP-1, CCL2, and CCR2 in the skin wounds. Scale bars: 100 μm, *p<0.05, **p<0.01.

Mentions: We used a recently established mouse PU model (Stadler et al., 2004) to further examine the role of KLF4 in wound healing with our FSP-1-Cre/KLF4(flox) mice. In this model, ulcers were typically formed at the end of the third ischemia/reperfusion (I/R) cycle and were accompanied by full-thickness loss of skin. The detached ulcerated skin was removed right after the third I/R circle. As shown in Figure 5a, one day after the ulcerated skin was removed, the open areas were increased in both WT and KLF4−/−(FSP-1) mice; in all likelihood this was related to an acute response. From Day 2 to Day 10, the wounds were gradually healed in WT mice, but the healing was significantly delayed in KLF4−/−(FSP-1) mice. HE staining showed increases in suprabasal layer of the skin and decreases in hair follicle density. In addition, the infiltrated lymphocytes in the granule tissue of the skin in KLF4−/−(FSP-1) mice were almost doubled that in WT mice, suggesting an increased inflammatory status in KLF4−/−(FSP-1) mice (Figure 5b). Consistent with decreased populations of CCR2+ MDSCs and COL1A1+CD45+CD11b+ fibrocytes in the bone marrow KLF4 knockout mice with a full-thickness wound, the numbers of these cells were also reduced in FSP-1-Cre/KLF4(flox) mice in the PU model (Figure 5c). In parallel with the increased inflammation in KLF4−/−(FSP-1) mice, the populations of CD11b+Ly6C++ cells, which may represent inflammatory monocytes (Robbins and Swirski, 2010), in blood and the skin wound were increased compared to those in WT mice (Figure 5d). In addition, the fact that FSP-1 expression was significantly reduced in the wound in KLF4−/−(FSP-1) mice (Figure 5e) is consistent with KLF4 role in regulating FSP-1 (Shi et al., 2014).


Kruppel-like factor KLF4 facilitates cutaneous wound healing by promoting fibrocyte generation from myeloid-derived suppressor cells.

Ou L, Shi Y, Dong W, Liu C, Schmidt TJ, Nagarkatti P, Nagarkatti M, Fan D, Ai W - J. Invest. Dermatol. (2015)

Compromised wound healing of PU in FSP-1-Cre/KLF4(flox) mice associated with decreased CCR2+MDSCs and fibrocytes(a). Similar to Figure 1c, except the WT and KLF4−/−(FSP-1) mice were used in the PU model (n=10). (b). Left: Representative images of HE staining of skin wounds in PU model. Right: Quantification of the epithelial thickness and the numbers of infiltrated lymphocytes in arbitrary red squares with the same sizes as the left (n=5). (c). Flow cytometry analysis to examine MDSCs and fibrocytes in mouse blood and skin wounds. (d). Similar to c, CD11b and Ly6C antibodies were used to examine inflammatory monocytes. (e). qRT=PCR to analyze expression of KLF4, FSP-1, CCL2, and CCR2 in the skin wounds. Scale bars: 100 μm, *p<0.05, **p<0.01.
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Figure 5: Compromised wound healing of PU in FSP-1-Cre/KLF4(flox) mice associated with decreased CCR2+MDSCs and fibrocytes(a). Similar to Figure 1c, except the WT and KLF4−/−(FSP-1) mice were used in the PU model (n=10). (b). Left: Representative images of HE staining of skin wounds in PU model. Right: Quantification of the epithelial thickness and the numbers of infiltrated lymphocytes in arbitrary red squares with the same sizes as the left (n=5). (c). Flow cytometry analysis to examine MDSCs and fibrocytes in mouse blood and skin wounds. (d). Similar to c, CD11b and Ly6C antibodies were used to examine inflammatory monocytes. (e). qRT=PCR to analyze expression of KLF4, FSP-1, CCL2, and CCR2 in the skin wounds. Scale bars: 100 μm, *p<0.05, **p<0.01.
Mentions: We used a recently established mouse PU model (Stadler et al., 2004) to further examine the role of KLF4 in wound healing with our FSP-1-Cre/KLF4(flox) mice. In this model, ulcers were typically formed at the end of the third ischemia/reperfusion (I/R) cycle and were accompanied by full-thickness loss of skin. The detached ulcerated skin was removed right after the third I/R circle. As shown in Figure 5a, one day after the ulcerated skin was removed, the open areas were increased in both WT and KLF4−/−(FSP-1) mice; in all likelihood this was related to an acute response. From Day 2 to Day 10, the wounds were gradually healed in WT mice, but the healing was significantly delayed in KLF4−/−(FSP-1) mice. HE staining showed increases in suprabasal layer of the skin and decreases in hair follicle density. In addition, the infiltrated lymphocytes in the granule tissue of the skin in KLF4−/−(FSP-1) mice were almost doubled that in WT mice, suggesting an increased inflammatory status in KLF4−/−(FSP-1) mice (Figure 5b). Consistent with decreased populations of CCR2+ MDSCs and COL1A1+CD45+CD11b+ fibrocytes in the bone marrow KLF4 knockout mice with a full-thickness wound, the numbers of these cells were also reduced in FSP-1-Cre/KLF4(flox) mice in the PU model (Figure 5c). In parallel with the increased inflammation in KLF4−/−(FSP-1) mice, the populations of CD11b+Ly6C++ cells, which may represent inflammatory monocytes (Robbins and Swirski, 2010), in blood and the skin wound were increased compared to those in WT mice (Figure 5d). In addition, the fact that FSP-1 expression was significantly reduced in the wound in KLF4−/−(FSP-1) mice (Figure 5e) is consistent with KLF4 role in regulating FSP-1 (Shi et al., 2014).

Bottom Line: Myeloid-derived suppressor cells (MDSCs) accumulate as a result of inflammation and promote cutaneous wound healing by mechanisms that are not fully understood.Recently, MDSCs have been shown to differentiate into fibrocytes, which serve as emerging effector cells that enhance cell proliferation in wound healing.In the current study, by using full-thickness and PU mouse models, we found that Kruppel-like factor 4 (KLF4) deficiency resulted in decreased accumulation of MDSCs and fibrocytes, and wound healing was significantly delayed.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, South Carolina, USA [2] Department of Biopharmaceuticals, School of Biotechnology, Southern Medical University, Guangzhou, People's Republic of China.

ABSTRACT
Pressure ulcers (PUs) are serious skin injuries whereby the wound healing process is frequently stalled in the inflammatory phase. Myeloid-derived suppressor cells (MDSCs) accumulate as a result of inflammation and promote cutaneous wound healing by mechanisms that are not fully understood. Recently, MDSCs have been shown to differentiate into fibrocytes, which serve as emerging effector cells that enhance cell proliferation in wound healing. We postulate that in wound healing MDSCs not only execute their immunosuppressive function to regulate inflammation but also stimulate cell proliferation once they differentiate into fibrocytes. In the current study, by using full-thickness and PU mouse models, we found that Kruppel-like factor 4 (KLF4) deficiency resulted in decreased accumulation of MDSCs and fibrocytes, and wound healing was significantly delayed. Conversely, KLF4 activation by the plant-derived product Mexicanin I increased the number of MDSCs and fibrocytes and accelerated the wound healing. Collectively, our study revealed a previously unreported function of MDSCs in cutaneous wound healing and identified Mexicanin I as a potential agent to accelerate PU wound healing.

Show MeSH
Related in: MedlinePlus