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Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, South Carolina, USADepartment of Biopharmaceuticals, School of Biotechnology, Southern Medical University, Guangzhou, People’s Republic of ChinaThese authors contributed equally to this work.
Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, South Carolina, USADepartment of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of ChinaThese authors contributed equally to this work.
Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, 6439 Garners Ferry Road, Building 2 Room B21, Columbia, South Carolina 29208, USA.
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.
Abbreviations
BM
bone marrow
EGFP
enhanced green fluorescent protein
HE stain
hematoxylin and eosin stain
IHC
immunohistochemistry
KLF4
Kruppel-like factor 4.
MDSC
myeloid-derived suppressor cell
PU
pressure ulcer
WT
wild type
INTRODUCTION
A pressure ulcer (PU) is defined as an injury caused by unrelieved pressure resulting in damage to the skin and underlying tissue (
), resulting in chronic wounds. Therefore, a better understanding of how to control inflammation in wound healing will lead to the therapeutic strategies that will potentially improve the quality of life of PU patients.
Myeloid-derived suppressor cells (MDSCs) are a heterogenous population of bone marrow (BM)-derived cells that bear monocyte markers and possess phenotypic plasticity (
). They are broadly defined as Ly6G+CD11b+ cells in mice, with a wider range of markers in humans. MDSCs are characterized by their potent ability to suppress immune responses, especially T-cell proliferation and cytokine production (
). Therefore, we postulate that in wound healing the recruited MDSCs not only execute their immunosuppressive function to keep inflammation in check but also stimulate cell proliferation once they differentiate into fibrocytes, as suggested by the presence of fibrocytes during the latter phase of the inflammatory process (
Human EZF, a Kruppel-like zinc finger protein, is expressed in vascular endothelial cells and contains transcriptional activation and repression domains.
Deficiency of Kruppel-like factor KLF4 in mammary tumor cells inhibits tumor growth and pulmonary metastasis and is accompanied by compromised recruitment of myeloid-derived suppressor cells.
Deficiency of Kruppel-like factor KLF4 in myeloid-derived suppressor cells inhibits tumor pulmonary metastasis in mice accompanied by decreased fibrocytes.
). However, whether KLF4 regulates cutaneous wound healing in an MDSC- and fibrocyte-dependent manner is not clear. In this study, using full-thickness and PU mouse models, we show that KLF4 promotes wound healing by regulating the recruitment of MDSCs and their subsequent differentiation into fibrocytes.
RESULTS
KLF4 ablation resulted in delayed wound healing in a full-thickness mouse wound healing model
To confirm our observation that KLF4 ablation-delayed cutaneous wound healing in tamoxifen-inducible KLF4-CreER/KLF4(flox) mice (
), we used RosaCre26ER/KLF4(flox) double transgenic mice, in which KLF4 was knocked out following tamoxifen induction in all cells. As shown in Figures 1a and b, successful KLF4 knockout after tamoxifen induction was confirmed by immunohistochemical staining of KLF4 in the skin and by quantitative real-time reverse-transcriptase–PCR analysis of the expression of KLF4 and its downstream target CCR2 (
) in the BM and spleen. Consistent with a growth inhibitory role of KLF4, the superbasal layer of the skin in KLF4 knockout mice (KLF4−/− mice) was significantly thicker than that in the uninduced mice (KLF4+/+ mice). We then performed the wound healing experiment in RosaCre26ER/KLF4 (flox) mice by placing full-thickness wounds (8 mm in diameter) with a puncher, as previously described (
). Wound closure in the KLF4−/− group was significantly delayed from day 3 to day 9 when compared with that in the KLF4+/+ group (Figure 1c). We also used immunohistochemistry (IHC) to examine the expression of α-SMA (a myofibroblast marker) and COL1A1 (a fibroblast marker) in granulation tissues on day 3. As expected, their immunoreactivities were both significantly reduced in the KLF4−/− group compared with the KLF4+/+ group (Figure 1d).
Figure 1Kruppel-like factor 4 (KLF4) ablation-delayed cutaneous wound healing accompanied by decreased accumulation of myeloid-derived suppressor cell (MDSCs). (a) KLF4 IHC staining in skin of RosaCreER/KLF4(flox) mice without (KLF4+/+) and with tamoxifen induction (KLF4−/−). The areas between two dotted red lines delineate skin suprabasal layers. (b) Quantitative real-time reverse-transcriptase–PCR to analyze the expression of KLF4 and CCR2. (c) Wound healing phenotypes (left, n=10) and the wound size quantification (right). (d) IHC staining of α-SMA and COL1A1 in KLF4+/+ and KLF4−/− mice at day 3 after wound induction. (e) Flow cytometry using anti-Ly6G and anti-CD11b antibodies with spleen cells and single skin cells. Representative images from one of five mice in each group are shown (a, c, and d). Scale bars: 50 μm. Mean±SEM. *P< 0.05, **P< 0.01, and ***P< 0.001. IHC, Immunohistochemistry; KLF4, Kruppel-like factor 4; and MDSCs, myeloid-derived suppressor cell.
To examine whether KLF4 deficiency reduces the recruitment of MDSCs in wound healing at day 3, we performed flow cytometric analysis by using cells purified from BM, spleen, and the wound site (specific tissue from its edge and subsurface granulation tissue). Although the percentage of MDSCs in BM showed no significant difference between the KLF4+/+ and KLF4−/−groups (data not shown), it was markedly less in the spleen and the skin wound site in the KLF4 knockout mice (KLF4−/− 8.39±0.82% vs. KLF4+/+ 14.10±0.92% in spleen, P<0.01; KLF4−/− 11.90±0.80% vs. KLF4+/+ 15.77±0.52% in skin, P<0.05) (Figure 1e).
KLF4 ablation in bone marrow-delayed cutaneous wound healing accompanied by decreased accumulation of CCR2+ MDSCs and fibrocytes in the wound site
To test the possibility that the KLF4 deficiency-induced delay of cutaneous wound healing is attributable to BM cells, we performed full-thickness wound healing experiments by using chimeric mice. These mice were generated by transplanting BM cells from RosaCre26ER/KLF4(flox)/β-actin-enhanced green fluorescent protein (EGFP) triple transgenic mice into wild-type (WT) C57BL/6 mice. The percentage of EGFP positivity in circulating leukocytes of the chimeric mice was >90%, as assessed by flow cytometry in tamoxifen-induced mice (data not shown), suggesting a successful BM reconstitution. The wound-closure kinetics showed that wound healing was significantly delayed in the BM KLF4−/− group (Figure 2a). In addition, while the total population of MDSCs in BM, spleen, and the skin wound site was not significantly different before and after KLF4 BM knockout by tamoxifen induction (data not shown), CCR2+ MDSCs in the skin wound site decreased from 3.29±0.45% in the BM KLF4+/+ group to 1.61±0.20% in the BM KLF4−/− group (P<0.05, Figure 2b).
Figure 2Delayed wound healing in bone marrow Kruppel-like factor 4 (KLF4) knockout mice and a compromised accumulation of CCR2+myeloid-derived suppressor cell (MDSCs) and fibrocytes. (a) Chimeric mice receiving bone marrow cells from Rosa26CreER/KLF4(flox)/β-actin-EGFP donor mice were used. Quantification of the wound size in each group of mice is shown (n=10). (b) Single cells from the skin wound were gated by c. They were examined by CD11b and Ly6G antibodies, followed by further analysis using a CCR2 antibody. Representative contour plots in each group are shown. (c) Similar to (b), except that COL1A1, CD45, and CD11b antibodies were used to analyze the fibrocytes. (d) Representative immunofluorescent staining of the wounds with α-SMA and COL1A1 antibodies. Yellow arrows indicate EGFP/α-SMA or EGFP/COL1A1 coexpressing cells (scale bars: 50 μm). KLF4, Kruppel-like factor 4; MDSCs, myeloid-derived suppressor cell.
The generation of fibrocytes from MDSCs shown in recent studies prompted us to examine the fibrocyte population in our wound healing model. The results from flow cytometry revealed that the percentage of BM-derived fibrocytes, characterized as EGFP+COL1A1+CD45+CD11b+, decreased from 10.62±0.94% in the KLF4+/+ group to 3.96±0.20% in the KLF4−/− group (P<0.01, Figure 2c). This finding was further confirmed by immunofluorescence staining of skin cryosections from the wound site; i.e., the numbers of COL1A1/EGFP and α-SMA/EGFP coexpressing cells in the KLF4−/− group were significantly reduced compared with those in the control group (Figure 2d).
KLF4-expressing bone marrow cells integrated into the wound healing tissue and adapted fibroblast morphology in a mouse wound healing model
To further examine whether KLF4-expressing BM cells are involved in cutaneous wound healing, we transplanted BM cells from our KLF4/EGFP transgenic mice, in which KLF4-expressing cells are labeled with EGFP (
), to WT mice and performed full-thickness wound healing experiments. In control mice, BM cells from the WT C57BL/6 mice were transplanted. As shown in Figure 3, EGFP+ cells were observed in the healing tissue of chimeric mice receiving KLF4/EGFP BM cells, but not in the control mice. Interestingly, KLF4/EGFP cells adapted an elongated morphology and expressed α-SMA, a marker of myofibroblasts which has a critical role in wound healing (
Figure 3Kruppel-like factor 4 (KLF4)-expressing bone marrow cells are integrated into the healing tissue and colocalized with α-SMA-expressing cells. Chimeric mice were generated by bone marrow transplantation using bone marrow cells from C57BL/6 mice (control) or KLF4/EGFP mice into C57BL/6 recipient mice. An 8-mm-diameter full-thickness wound was placed and the wound beds were collected 4 days later, followed by immunofluorescence staining with an anti-α-SMA antibody. Representative colocalization of EGFP cells with red α-SMA-expressing cells in the healing tissue (left) is indicated by white arrowheads (n=5). Scale bars: 25 μm. KLF4, Kruppel-like factor 4.
KLF4 deficiency in CCR2+ MDSCs decreased fibrocyte generation in FSP-1-Cre/KLF4(flox) mice
As KLF4 deficiency in BM resulted in delayed wound healing accompanied by decreased CCR2+MDSCs (Figure 2b), we postulated that KLF4 in CCR2+ MDSCs directly regulates cutaneous wound healing. To test this possibility, we generated a mouse model in which KLF4 was specifically ablated in CCR2+ MDSCs by crossing KLF4(flox) mice with FSP-1-Cre mice, because FSP-1 is highly expressed in BM CCR2+ MDSCs, as shown in our recent studies (
Deficiency of Kruppel-like factor KLF4 in myeloid-derived suppressor cells inhibits tumor pulmonary metastasis in mice accompanied by decreased fibrocytes.
FSP-1-Cre/KLF4(flox) mice showed no obvious abnormalities from birth to 8 weeks of age. After 8 weeks of age, significant hair and weight loss was observed in these KLF4-deficient mice when compared with WT mice (Figure 4a and b). Successful KLF4 knockout in monocytes in KLF4-deficient mice (designated as KLF4−/−(FSP-1)) was confirmed by quantitative real-time reverse-transcriptase–PCR (Supplementary Figure 1 Online). IHC staining showed that the number of KLF4-positive cells was decreased by 84% in KLF4−/−(FSP-1) mice (Figure 4c). Hematoxylin and eosin stain staining showed that the hair follicle density decreased by ∼50%, and the suprabasal layer was thicker in KLF4−/−(FSP-1) mice (Figure 4d). To examine the potential immunological defects in FSP-1-Cre/KLF4 (flox) mice, we measured the cytokine/chemokine levels in mouse sera. Expression levels of IL4 and IL5 were significantly increased, but the level of IL-12 (P40), a shared subunit of IL-12 and IL-23 (
), was decreased in KLF4−/−(FSP-1) mice (Figure 4e). Interestingly, expression levels of granulocyte colony-stimulating factor and macrophage inflammatory protein 1 alpha were also significantly increased in KLF4−/− (FSP-1) mice (Figure 4e). Consistent with the importance of CCR2+ MDSCs in fibrocyte generation (
Deficiency of Kruppel-like factor KLF4 in myeloid-derived suppressor cells inhibits tumor pulmonary metastasis in mice accompanied by decreased fibrocytes.
), the number of fibrocytes generated from the spleen cells decreased from 112±8 per 105 cells in the WT mice to 46±4 per 105 cells in FSP-1-Cre/KLF4 (flox) mice (Figure 4f).
Figure 4Hair loss and decreased fibrocyte generation in FSP-1-Cre/KLF4(flox) mice. (a) Representatives of the WT and FSP-1-Cre/KLF4(flox) (KLF4−/−(FSP-1)) mice. Black squares indicate an area in which severe hair loss was seen in KLF4−/−(FSP-1) mice. (b) Body weights of male and female WT and KLF4−/−(FSP-1) mice. (c) Representative images of KLF4 staining of skin (left) and measurement of KLF4-positive cells (right). (d) (left) Representative images of HE staining of skin. The areas between two dotted red lines represent skin suprabasal layers and the red arrow heads are pointing to hair follicles. (right) Measurement of epithelial thickness and hair follicles. (e) Measurement of serum cytokines by ELISA (n=3). (f) Quantification of fibrocyte generation (n=5). Scale bars: 100 μm. *P<0.05 and **P<0.01. HE staining, hematoxylin and eosin staining; KLF4, Kruppel-like factor 4; and WT, wild type.
Compromised wound healing in FSP-1-Cre/KLF4(flox) mice was associated with decreased accumulation of CCR2+ MDSCs and fibrocytes in a mouse pressure ulcer model
) 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 cycle and were accompanied by full-thickness loss of skin. The detached ulcerated skin was removed right after the third ischemia/reperfusion circle. As shown in Figure 5a, 1 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 healing was significantly delayed in KLF4−/−(FSP-1) mice. HE staining showed increases in the 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 compared with those 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 BM 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 the inflammatory monocytes (
), in blood and the skin wound were increased compared with 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 (
Deficiency of Kruppel-like factor KLF4 in myeloid-derived suppressor cells inhibits tumor pulmonary metastasis in mice accompanied by decreased fibrocytes.
Figure 5Compromised wound healing of pressure ulcer (PU) in FSP-1-Cre/KLF4(flox) mice associated with decreased CCR2+myeloid-derived suppressor cell (MDSCs) and fibrocytes. (a) Similar to Figure 1c, except that 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 the 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, except that CD11b and Ly6C antibodies were used to examine inflammatory monocytes. (e) Quantitative real-time reverse-transcriptase–PCR to analyze the expression of KLF4, FSP-1, CCL2, and CCR2 in the skin wounds. Scale bars: 100 μm, *P<0.05, **P<0.01. KLF4, Kruppel-like factor 4; MDSCs, myeloid-derived suppressor cell; and PU, pressure ulcer.
Activation of KLF4 by Mexicanin I increased CCR2+ MDSCs and fibrocytes in the mouse PU model accompanied by elevated gene expression of CCR2 and FSP-1
To further confirm the role of KLF4 in the PU healing process, we tested the plant-derived sesquiterpene lactone, Mexicanin I, which is known to be a c-Myb inhibitor (
) that may inhibit the tumor suppressor KLF4. As shown in Figure 6a, Mexicanin I, at a 50 nM concentration, promoted fibrocyte generation from mouse spleen cells, which was accompanied by the upregulation of KLF4. In the mouse PU model, we found that Mexicanin I significantly accelerated wound healing (Figure 6b). In addition, although the total populations of MDSCs or CCR2+ MDSCs in blood with and without Mexicanin I treatment in control mice did not show any significant differences (Figure 6c and data not shown), COL1A1+CD45+CD11b+ fibrocytes and CD11b+Ly6C++ inflammatory monocytes were increased following a PU induction in naïve and Mexicanin I-treated mice. Importantly, in the skin wounds, CCR2+ MDSCs and fibrocytes were increased in the Mexicanin I-treated mice from 1.36 to 2.55% for CCR2+ MDSCs and from 1.70 to 3.31% for fibrocytes. In line with the accelerated wound healing, the CD11b+Ly6C++ monocytes in the skin were decreased from 5.11% in the control mice to 2.75% in the Mexicanin I-treated mice (Figure 6c). Consistently, there was minimal change of infiltrated lymphocytes in Mexicanin I-treated mice following PU induction, but a significant increase in untreated control mice (Figure 6d). In parallel with the changes of CCR2+ MDSCs and fibrocytes, expression levels of KLF4, FSP-1, CCL2, and CCR2 were all significantly increased in the wounds after Mexicanin I treatment (Supplementary Figure 2 online). Most importantly, there were no significant differences in the wound healing kinetics, numbers of CCR2+ MDSCs, fibrocytes, and CD11b+Ly6C++ monocytes between Mexicanin I-treated mice and untreated mice in KLF4−/−(FSP-1) mice following PU induction (Figure 6e).
Figure 6Kruppel-like factor 4 (KLF4) activation by Mexicanin I accelerated pressure ulcer (PU) wound healing accompanied by increased CCR2+myeloid-derived suppressor cell (MDSCs) and fibrocytes. (a) Fibrocyte generation in the presence of 50 nM Mexicanin I (left), KLF4 expression detected by western blotting (middle) and quantitative real-time reverse-transcriptase–PCR (right). CXCR4 and β-actin were used as controls. (b) Measurement of wound healing kinetics after Mexicanin I treatment (n=10). (c) Flow cytometry analysis to examine different cell types. (d) Representatives of HE staining of the skin wounds (left) and numbers of infiltrated lymphocytes in the arbitrary squares with the same sizes from the left (right). (e) (left) Measurement of wound healing kinetics in KLF4−/−(FSP-1) mice (n=10), and (right) flow cytometry analysis. (f) Proposed function of KLF4-mediated fibrocyte generation in wound healing. Scale bar: 100 μm, *P<0.05. HE staining, hematoxylin and eosin staining; KLF4, Kruppel-like factor 4; MDSCs, myeloid-derived suppressor cell; and PU, pressure ulcer.
In this study, by using two different mouse models of wound healing, we demonstrated that KLF4 promotes healing by regulating the recruitment of monocytic MDSCs and their subsequent differentiation into fibrocytes. This concept is consistent with the current wound healing model in which the inflammatory response wanes before the proliferation of fibroblasts and keratinocytes. Thus we have shown that MDSCs have a dual role by regulating the inflammation as an immunosuppressive and by promoting cell proliferation after they differentiate into fibrocytes (Figure 6f).
Initially, the decreased recruitment of CCR2+ MDSCs into the wound of KLF4 knockout mice (Figure 2b) raised a concern that the decreased number of fibrocytes may not be because of the subsequent transdifferentiation of these MDSCs; i.e., the relative importance of transdifferentiation was questionable. However, our data suggest that KLF4 controls both the recruitment of MDSCs and their subsequent transdifferentiation. First, KLF4 deficiency in BM decreased the expression of CCR2 ((
), and it is confirmed by elevated CCL2 expression following PU induction in our study (Figure 5e). Second, we recently reported that KLF4 deficiency in CCR2+ MDSCs attenuated fibrocyte generation and resulted in compromised tumor metastasis (
Deficiency of Kruppel-like factor KLF4 in myeloid-derived suppressor cells inhibits tumor pulmonary metastasis in mice accompanied by decreased fibrocytes.
). The role of KLF4 in fibrocyte generation was also supported by Mexicainin I-mediated KLF4 activation and fibrocyte generation (Figure 6a). Third, and most importantly, in FSP-1-Cre/KLF4(flox) mice, the basal level of CCR2+ MDSCs in the blood was increased (Figure 5c). However, this population was significantly decreased in the wound. The increased basal level of CCR2+ MDSCs most likely reflected the developmental regulation of these cells by KLF4. It is also supported by our ELISA data (Figure 4e). Levels of colony-stimulating factor, macrophage inflammatory protein 1 alpha, IL4, and IL5 were increased, but IP-12(P40) level was decreased, in KLF4 knockout mice. High levels of colony-stimulating factor are correlated with the conversion of BM-derived MDSCs from CCR2+ MDSCs to neutrophils (
IL-1 activation of endothelium supports VLA-4 (CD49d/CD29)-mediated monocyte transendothelial migration to C5a, MIP-1 alpha, RANTES, and PAF but inhibits migration to MCP-1: a regulatory role for endothelium-derived MCP-1.
). Increased colony-stimulating factor and macrophage inflammatory protein 1 alpha suggests that KLF4 regulates the development of CCR2+MDSCs. In addition, given the role of IL4 and IL5 in the polarization of Th2 cells (
), alterations in the levels of these cytokines suggest that KLF4 ablation promotes Th2 cell polarization.
The critical role of KLF4 in monocytic MDSCs in cutaneous wound healing is not only supported by using the mouse model with KLF4 deficiency in the BM (Figure 2) but also by using the FSP-1-Cre/KLF4(flox) mice with KLF4 deficiency in CCR2+ MDSCs (Figure 5). However, there is one limitation in our FSP-1-Cre/KLF4(flox) mouse model. FSP-1 is expressed in multiple cell types including monocytes (
), and therefore we cannot exclude the contribution of KLF4-regulated fibroblasts in wound healing. Future experiments will be performed to specifically test the effect of BM CCR2+ MDSCs on wound healing. Similarly, we plan to test the fibroblast-specific effect of KLF4 on wound healing. In addition, the molecular mechanisms by which KLF4 regulates FSP-1 expression will be further determined.
Our data showed that Mexicanin I activated KLF4 and accelerated PU wound healing (Figure 6). Although we favored a KLF4- and fibrocyte-mediated mechanism, KLF4-independent effects of Mexicanin I should also be considered. Furthermore, pharmacological kinetics of Mexicanin I will be determined in wound healing.
Collectively, this study not only confirms a critical role of KLF4 in wound healing but also warrants the future development of KLF4-based therapeutic strategies, such as activation of KLF4 by Mexicanin I in the treatment of patients with PUs.
MATERIALS AND METHODS
Generation of KLF4-deficient mice
Rosa26CreER/KLF4(flox) mice and Fsp-1-Cre/KLF4 (flox) mice were generated by crossing Rosa26CreER (NCI, Frederick, MD, Stock#: 01XAB) and Fsp-1-Cre (Jackson, Bar Harbor, ME, Stock#: 012641) with KLF4(flox) mice, respectively. Rosa26CreER/KLF4(flox)/β-actin-EGFP mice were generated by crossing Rosa26CreER/KLF4(flox) mice with β-actin-EGFP mice (Jackson, Stock#: 006567). KLF4 knockout in RosaCre26ER/KLF4(flox) mice was induced by daily intraperitoneal injection of tamoxifen (TAM, Sigma-Aldrich, St. Louis, MO, 50 mg/kg) for 5 consecutive days. Sunflower seed oil was used as a control. Mice were bred and used in specific pathogen-free facilities according to the Animal Care and Use Committee Guidelines of the University of South Carolina. In the wound healing model, the control and mutant mice were littermates with mixed sex at a similar 6- to 8-week age range.
Bone marrow transplantation
Six-week-old C57BL/6 recipient mice were lethally irradiated (900 rad). The donor cells were harvested from the long bones of RosaCre26ER/KLF4 (flox)/β-actin-EGFP or KLF4/EGFP mice, and 3 × 106 nucleated cells were injected retro-orbitally into each recipient mouse. After repopulation success was established, KLF4 knockout was induced as described above.
Wound healing mouse models and Mexicanin I treatment
The full-thickness wound healing model was created as previously described (
). Before the placement of the wound, the mice were administered anesthesia by IP injection of ketamine (100 mg/kg) and xylazine (12.5 mg/kg). The time when tissue was removed was considered day 0 and the healing wounds were photographed on days 1, 3, 5, 7, and 9. The edges of the wound were circled and quantified by Image J and expressed as a fraction of initial area. Ten mice each in KLF4+/+ and KLF4−/− groups were used to measure the relative wound areas at different time points.
). The injection was started one day before the first ischemia/reperfusion cycle until the end of third ischemia/reperfusion when the mice were killed.
Fibrocyte generation
The splenocytes of Babl/c WT mice were purified and subjected to fibrocyte generation by using a developed approach with the application of IL-13 (50 ng/ml) and M-CSF (25 ng/ml) (
). Three days later, the cells were stained with a Hema 3 staining kit (Fisher Scientific, Waltham, MA) and the numbers of fibrocytes were counted. A concentration of 50 nM Mexicanin I was used in the experimental group and DMSO (Sigma-Aldrich) was used in the control group.
Immunohistochemistry (IHC) and immunofluorescence (IF) staining
IHC and IF were performed by using standard protocols with sections from the skin wounds. The following antibodies were used: rabbit anti-mouse KLF4 antibody (1:100), rabbit anti-mouse FSP-1 antibody (1:100) and rabbit anti-mouse α-SMA antibody (1:100, both from Abcam), and rabbit anti-mouse Collagen Type I alpha 1 antibody (COL1A1, 1:200, Rockland, Gilbertsville, PA).
Western blotting
Protein from skin tissues and cultured cells was extracted for western blotting, as described (
). Primary antibodies were rabbit anti-mouse KLF4 (1:1000, GenSpin, Milano, Italy), rabbit anti-mouse CXCR4 (1:1000, eBioscience, San Diego, CA), and rat anti-mouse β-actin (1:1000, Sigma-Aldrich).
RNA extraction and real-time PCR analysis
Total RNA was prepared using Trizol Reagent (Invitrogen, Grand Island, NY) according to the manufacturer’s instructions. First-strand cDNA synthesis and real-time PCR were carried out as described (
Deficiency of Kruppel-like factor KLF4 in mammary tumor cells inhibits tumor growth and pulmonary metastasis and is accompanied by compromised recruitment of myeloid-derived suppressor cells.
). The peripheral blood monocytes were isolated using Histoplaque (Sigma-Aldrich). Wound sites were minced and digested with 1.0 mg/ml collagenase (Sigma-Aldrich) to generate a single-cell suspension and purified by using a Percoll gradient (Sigma-Aldrich). These dissociated single cells were stained with fluorochrome-conjugated antibodies specific for mouse CD11b, Ly6G, CD45 (eBioscience), and CCR2 (R&D). For COL1A1 intracellular staining, cells were harvested, stained with surface markers, fixed with a fixation solution (eBioscience) for 15 min, permeabilized with ice-cold pure methanol for 30 min, and incubated with biotin-conjugated mouse COL1A1 antibody (Rockland) and fluorochrome-conjugated streptavidin (eBioscience). Flow cytometry was performed using FACS Aria II (BD bioscience, Franklin Lakes, NJ), and data were analyzed with Flowjo (Ashland, OR).
Quantification and statistical analysis
KLF4-positive brown cells in suprabasal layers of each arbitrarily defined high magnified field were counted by Image J. Three different high magnified fields were counted for each animal. Each group consisted of five mice. Epidermal thickness, numbers of hair follicles, and infiltrated lymphocytes were similarly quantified. Data were represented as mean±SEM. Data were analyzed using the following tests: t-test (two-group comparison) and one-way analysis of variance (multigroup comparison). A P-value <0.05 was considered to indicate statistical significance.
ACKNOWLEDGMENTS
This work was supported by NIH grant (R03AR060987) and ASPIRE-I from University of South Carolina to WA. We thank Dr Udai P. Singh and Mr Kyle McCloskey for technical assistance and Dr Joseph S. Janicki for his editing of the manuscript.
IL-1 activation of endothelium supports VLA-4 (CD49d/CD29)-mediated monocyte transendothelial migration to C5a, MIP-1 alpha, RANTES, and PAF but inhibits migration to MCP-1: a regulatory role for endothelium-derived MCP-1.
Deficiency of Kruppel-like factor KLF4 in myeloid-derived suppressor cells inhibits tumor pulmonary metastasis in mice accompanied by decreased fibrocytes.
Human EZF, a Kruppel-like zinc finger protein, is expressed in vascular endothelial cells and contains transcriptional activation and repression domains.
Deficiency of Kruppel-like factor KLF4 in mammary tumor cells inhibits tumor growth and pulmonary metastasis and is accompanied by compromised recruitment of myeloid-derived suppressor cells.
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