Fig 2: 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.

Fig 3: Db: VP2121-130 epitope dominance among CNS-infiltrating CD8 T cells and neutrophil infiltration during acute TMEV infection. Central nervous system (CNS)-infiltrating lymphocytes were isolated 7 days post-Theiler's murine encephalomyelitis virus (TMEV) infection. Shown is fluorescence-activated cell-sorting analysis staining for CD8 and Db: VP2121-130 peptide tetramer among CNS-infiltrating lymphocytes isolated from a representative (A) C57BL/6 mouse or (B) 129 SvIm mouse. (C) CNS-infiltrating C57BL/6 lymphocytes stained for CD8 and negative control Db: E7 peptide tetramer. (D) C57BL/6 and 129 Svlm mice display comparable levels of brain-infiltrating CD45+ cells that are CD8+ and specific for the Db: VP2121-130 epitope. (E) Equivalent capacity of CNS-infiltrating cytotoxic T lymphocytes isolated from TMEV-infected C57BL/6 and 129 SvIm mice to utilize perforin to kill VP2-transfected C57SV target cells in a chromium release assay. Untransfected C57SV cells served as a negative control. CD45hi population was gated and analyzed for the percentage of Ly6G+ cells in both (F) C57BL/6 and (G) 129 Svlm mice (n = 5 per group). (H) Bar chart showing that C57BL/6 and 129 Svlm mice display comparable levels of Ly6G+ cells, which are indicative of being a neutrophil subset. In 1 million isolated total cells recorded by flow cytometry, there were no statistically significant differences in CD45+, Ly6G+, or CD8+ Db: VP2121-130 tetramer-positive cells collected (data not shown).

Fig 4: Administration of IFN-κ improves diabetic wound healing.(A) The 4 mm punch biopsy wounds were created on DIO, and wounds were injected daily starting on day 0 for 3 days after injury with IFN-κ (0.5 μg/100 μL) or PBS control (2 wounds per mouse, n = 5 mice per group, repeated once). The arrow indicates when injections started. The change in wound area was recorded daily and analyzed with ImageJ software. Representative photographs of the wounds were taken on days 0 and 3. (B) Max wound diameter at day 3 for IFN-κ and PBS control; n = 5 per group. Trichrome staining representative picture. (C) Trichrome staining was calculated using ImageJ software (n = 5 mice/group). (D) The 4 mm punch biopsy wounds were created and splinted on DIO mice, and wounds were injected starting on day 0 for 3 days after injury with IFN-κ (0.5 μg/100 μL) or PBS control (1 wound per mouse, n = 7–8 mice per group). The arrow indicates when injections started. The change in wound area was recorded daily and analyzed with ImageJ software. Representative photographs of the wounds were taken on days 0, 3, and 5. (E) Flow cytometry quantification of Ly6Chi cells in wounds (n = 7–8 per group). (F) Wound monocyte/macrophages (MΦ) (CD3–CD19–NK1.1–Ly6G–CD11b+) were isolated from IFN-κ or PBS control mice on day 3; n = 3 per group, repeated in triplicate. Gene expression of inflammatory cytokines Tnf, Il1b, and Il12 was measured via qPCR. Data were analyzed for variances, and 2-tailed Student’s t test or 1-way ANOVA was performed. *P < 0.05, **P < 0.01, and **** P < 0.0001. Data are presented as mean ± SEM.

Fig 5: IFN-κ regulates macrophage inflammatory profile during normal tissue repair.(A) IFN-κ–KO and control wound cell isolates were processed for flow cytometry using the following gating strategy selecting for single cells, live (viability dye–), CD45+, Ly6G−, CD11b+, Ly6Chi, or Ly6Clo. (B and C) Flow cytometry quantification of Ly6Chi and Ly6Clo cells in wounds (n = 4–6 per group). (D) Wound monocyte/macrophages (MΦ) (CD3–CD19–Ly6G–CD11b+) were isolated from WT and IFN-κ–KO mice on day 3; n = 3 per group, repeated in triplicate. Gene expression of inflammatory cytokines Tnf, Il1b, and Il12 was measured via qPCR. Data were analyzed for variances, and 2-tailed Student’s t test or 1-way ANOVA was performed. *P < 0.05, **P < 0.01, and ***P < 0.001. Data are presented as mean ± SEM.