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. 2017 Jun 1;8:635.
doi: 10.3389/fimmu.2017.00635. eCollection 2017.

Notch Regulates Macrophage-Mediated Inflammation in Diabetic Wound Healing

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Free PMC article

Notch Regulates Macrophage-Mediated Inflammation in Diabetic Wound Healing

Andrew S Kimball et al. Front Immunol. .
Free PMC article

Abstract

Macrophages are essential immune cells necessary for regulated inflammation during wound healing. Recent studies have identified that Notch plays a role in macrophage-mediated inflammation. Thus, we investigated the role of Notch signaling on wound macrophage phenotype and function during normal and diabetic wound healing. We found that Notch receptor and ligand expression are dynamic in wound macrophages during normal healing. Mice with a myeloid-specific Notch signaling defect (DNMAMLfloxedLyz2Cre+ ) demonstrated delayed early healing (days 1-3) and wound macrophages had decreased inflammatory gene expression. In our physiologic murine model of type 2 diabetes (T2D), Notch receptor expression was significantly increased in wound macrophages on day 6, following the initial inflammatory phase of wound healing, corresponding to increased inflammatory cytokine expression. This increase in Notch1 and Notch2 was also observed in human monocytes from patients with T2D. Further, in prediabetic mice with a genetic Notch signaling defect (DNMAMLfloxedLyz2Cre+ on a high-fat diet), improved wound healing was seen at late time points (days 6-7). These findings suggest that Notch is critical for the early inflammatory phase of wound healing and directs production of macrophage-dependent inflammatory mediators. These results identify that canonical Notch signaling is important in directing macrophage function in wound repair and define a translational target for the treatment of non-healing diabetic wounds.

Keywords: Notch; diabetes; inflammation; macrophages; wound healing.

Figures

Figure 1
Figure 1
Notch signaling is dynamic in macrophages during early wound healing. C57Bl/6 mice were wounded on the back using a 4 mm punch biopsy. Wounds were collected on days 1 and 3 post-wounding. (A) Wound macrophages (CD3/CD19/Ly6G/CD11b+) were isolated using magnetic-activated cell (MAC) sorting on days 1 and 3 post-wounding. Notch 1 receptor, Notch 2 receptor, and DLL4 ligand expression quantified by RT-PCR on days 1 and 3 (n = 10 wounds in 5 mice/group; replicated 3×) (*P < 0.05; **P < 0.01). (B) Flow cytometry quantification of Notch receptors/ligands in wounds on days 1 and 3. Notch 2 and DLL4 positive cells on days 1 and 3 expressed as a percent of live, lin,Ly6G,CD11b+ cells (n = 12 wounds in 6 mice/group; replicated 2×) (*P < 0.05; **P < 0.01). (C) Hes1 gene expression was quantified by RT-PCR on days 1 and 3 (n = 10 wounds in 5 mice/group; replicated 3×) (*P < 0.05). Statistical analysis was performed using two-tailed Student’s t-test. All data are expressed as mean ± SEM.
Figure 2
Figure 2
Generation of a macrophage-specific, Notch signaling-deficient murine model (DNMAMLfloxedLyz2Cre+) with myeloid specificity in tissues. (A) Gene construct representing DNMAMLfloxed mice on a C57Bl/6 background, where crossing mice with C57Bl/6-Lyz2Cre+ will result in excision of a stop codon and transcription of DNMAML-GFP from the Rosa26 Locus in macrophages. (B) Schematic demonstrating blockade of downstream Notch signaling following receptor/ligand interaction in DNMAMLfloxedLyz2Cre+ mice. (C) Representative example of flow cytometry for green fluorescent protein (GFP) in CD11b+ Ly6Chi cells isolated from peripheral blood in the DNMAMLfloxedLyz2Cre+ mice. (D–G) Percentage of GFP+ cells in myeloid cell populations examined by flow cytometry from DNMAMLfloxedLyz2Cre+ mice and littermate controls in (D) peripheral blood, (E) spleen, (F) bone marrow, and (G) peripheral wounds (n = 5 mice/group, replicated 2×) (*P < 0.05;**P < 0.01). (H) Percentage of GFP+ cells in lymphocyte populations from spleen examined by flow cytometry from DNMAMLfloxedLyz2Cre+ mice and littermate controls (n = 5 mice/group, replicated 2×). All data are expressed as mean ± SEM.
Figure 3
Figure 3
Genetic blockade of canonical Notch signaling in macrophages results in decreased inflammatory wound macrophages and delayed early wound healing. Wounds were created using 4 mm punch biopsies on the backs of DNMAMLfloxedLyz2Cre+and littermate control mice. (A) Change in wound area was recorded daily using ImageJ Software (NIH) until complete healing was observed. Data are pooled from two experiments (n = 20 wounds in 10 mice/group, repeated 3×) (*P < 0.05; ****P < 0.0001). Histopathology (H&E) and collagen deposition of wounds of DNMAMLfloxedLyz2Cre+and littermate controls on post-injury day 4. DNMAMLfloxedLyz2Cre+wounds showed less reepithelialization, granulation formation, and collagen deposition. Data are pooled from two experiments (n = 20 wounds in 10 mice/group, repeated 2×). (B) Analytical flow cytometry of Lin/Ly6G/CD11b+/Ly6Chi wound cells in from DNMAMLfloxedLyz2Cre+ mice and littermate controls at day 3 post-injury (n = 10 wounds in 5 mice/group, repeated 2×) (*P < 0.05). Statistical analysis was performed using two-tailed Student’s t-test. All data are expressed as mean ± SEM.
Figure 4
Figure 4
Genetic and pharmacologic blockade of Notch signaling in macrophages results in decreased inflammatory cytokine production in vitro and in vivo. (A) Bone marrow-derived macrophages (BMDMs) were isolated from C57Bl/6 mice and stimulated with lipopolyssacharide (LPS) (100 ng/mL) for 12 h in the presence or absence of a gamma-secretase inhibitor (N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) (DAPT) (1 nM) or DMSO control. IL1β and TNFα gene expression was quantified by RT-PCR (n = 3 mice, repeated in triplicate) (**P < 0.01; ****P < 0.0001). (B) BMDMs were isolated from DNMAMLfloxedLyz2Cre+ and littermate control mice and stimulated with LPS (100 ng/mL) for 12 h. Surface and intracellular stains were performed and cells were then analyzed by flow cytometry for IL1β and TNFα. Results are expressed as mean fluorescence intensity (MFI) (n = 3 mice, repeated in triplicate) (**P < 0.01; ***P < 0.001). (C) Wounds were created using 4 mm punch biopsies on the backs of DNMAMLfloxedLyz2Cre+and littermate control mice. Wound macrophages (CD3/CD19/Ly6G/CD11b+) were isolated using magnetic-activated cell (MAC) sorting on day 3 post-wounding. IL1β, TNFα, and NOS2 expression in the wound macrophages was quantified by RT-PCR (n = 10 wounds in 5 mice/group, repeated 2×) (*P < 0.05). Statistical analysis was performed using analysis of variance and two-tailed Student’s t-test. All data are expressed as mean ± SEM.
Figure 5
Figure 5
Notch signaling is increased in monocytes from type 2 diabetic (T2D) patients and wound macrophages from a murine model of obesity/type 2 diabetes (T2D). Diet-inducted obesity, a physiologic model of “prediabetes,” was used to examine the effect of obesity and insulin resistance on Notch signaling in wound macrophages. C57Bl/6 mice were fed either normal diet (ND) (12% saturated fat) or a high-fat diet (HFD) (60% saturated fat) for 12–14 weeks. Mice on a HFD (diet-induced obese) were wounded on the back using a 4 mm punch biopsy. Similar to T2D patients, HFD mice have delayed wound healing and thus, wounds were collected on day 6 post-wounding. (A) Wound macrophages (CD3/CD19/Ly6G/CD11b+) were isolated using magnetic-activated cell (MAC) sorting on day 6 post-wounding. Notch 1 receptor and Notch 2 receptor expression was quantified by RT-PCR on day 6 (n = 10 wounds in 5 mice/group; replicated 2X) (**P < 0.01). (B) Notch target genes, Hes1 and Hey1 expression levels was quantified by RT-PCR in wound macrophages (CD3/CD19/Ly6G/CD11b+) on day 6 post-injury (n = 10 wounds in 5 mice/group, replicated 2×) (*P < 0.05; ***P < 0.001). (C) Peripheral blood from patients with T2D and non-diabetic controls was obtained from clinic. Age, gender and comorbid conditions were evenly distributed among the two groups. Following RBC lysis and Ficoll separation, CD14+ monocytes were isolated from the buffy coat. Notch 1 and Notch 2 receptor expression was quantified by RT-PCR (n = 12 patients, replicated 2×) (*P < 0.05). Statistical analysis was performed using two-tailed Student’s t-test. All data are expressed as mean ± SEM.
Figure 6
Figure 6
Notch signaling-deficient mice on a high-fat diet (HFD) (HFD DNMAMLfloxedLyz2Cre+) develop obesity and insulin resistance and in vitro and in vivo macrophages demonstrate decreased inflammation. DNMAMLfloxedLyz2Cre+and littermate control mice were fed either normal diet (ND) (12% saturated fat) or a HFD (60% saturated fat) for 12–14 weeks. (A) Weight (grams) of DNMAMLfloxedLyz2Cre+and littermate control mice were obtained following 14 weeks of HFD (n = 5 mice/group, repeated 2×) (****P < 0.0001). (B) Oral glucose tolerance test (OGTT) was performed in all groups. Blood glucose (milligrams/deciliter) was obtained every 15–30 min for 2 h (n = 5 mice/group) (*P < 0.05 for littermate ND versus HFD mice; ###P < 0.001, ##P < 0.01 for DNMAMLfloxedLyz2Cre+ND versus HFD mice). (C) Bone marrow-derived macrophages (BMDMs) were isolated from C57Bl/6 mice on HFD and stimulated with lipopolyssacharide (LPS) (100 ng/mL) for 12 h in the presence or absence of a gamma-secretase inhibitor (N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) (DAPT) (1 nM) or DMSO control. IL1β and TNFα gene expression was quantified by RT-PCR (n = 3 mice, repeated in triplicate) (*P < 0.05). (D) BMDMs were isolated from from DNMAMLfloxedLyz2Cre+and littermate control mice and stimulated with LPS (100 ng/mL) for 12 h and IL1β and TNFα gene expression was quantified by RT-PCR (n = 3 mice, repeated in triplicate) (*P < 0.05). (E) Wounds were created using 4 mm punch biopsies on the backs of HFD DNMAMLfloxedLyz2Cre+and littermate HFD mice. Wound macrophages (CD3/CD19/Ly6G/CD11b+) were isolated using magnetic-activated cell (MAC) sorting on day 6 post-wounding. IL1β, TNFα and NOS2 expression in the wound macrophages was quantified by RT-PCR (n = 10 wounds in 5 mice/group, repeated 2×) (*P < 0.05). Statistical analysis was performed using analysis of variance or two-tailed Student’s t-test. All data are expressed as mean ± SEM.
Figure 7
Figure 7
High-fat diet (HFD) mice deficient in Notch signaling (HFD DNMAMLfloxedLyz2Cre+) demonstrate improved late healing compared to HFD controls. DNMAMLfloxedLyz2Cre+ and littermate control mice were fed a HFD (60% fat) for 12–14 weeks. Two wounds per mouse were created using 4 mm punch biopsies on the backs of HFD DNMAMLfloxedLyz2Cre+ and HFD littermate control mice. Change in wound area was recorded daily using ImageJ Software (NIH) until complete healing was observed. Representative images of wounds from days 0 and 6 are shown (n = 20 wounds in 10 mice/group) (*P < 0.05; **P < 0.01). Statistical analysis was performed using two-tailed Student’s t-test. All data are expressed as mean ± SEM.

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