Fig 2: 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.

Fig 3: BQ123-derived PMN-MDSCs control acute inflammation in T-cell-dependent manner. Rag2 KO mice were divided in four groups, one group received neutrophils from PBS-treated mice (PBS-Neu), second group received BQ123-induced PMN-MDSCs (BQ123-MDSC), and third group is a combination of CD3+ T and PBS-Neu cells (T+PBS-Neu), fourth group are a combination of CD3+ T and BQ123-MDSC cells (T+ BQ123-MDSC). The three mouse models, as shown in Figures 3–5, respectively, were performed. About 2×106 BQ123-PMN-MDSCs or PBS-Neu were intravenously injected into recipient mice on different days among four groups, for third and fourth group, about 4 × 106 CD3+T cells from donor mice was also injected into Rag2 KO recipient mice in the same cycle. (A–F) DSS-induced acute colitis. (A) Experimental design, (B) Representative H&E staining, and CMDI score, (C) clinical symptom score (DAI) and Body weight, (D) Gross pathological appearance and Colon Length, (E) bacterial loading, and (F) The mRNA expression levels of inflammatory factors are shown, β- actin was used for normalization, and the lowest expression level sample in BQ123-MDSC group was artificially set to 1(n=4). (PBS-Neu group and BQ123-MDSCs group, n=6, T+PBS-Neu group, T+ BQ123-MDSC group, n=4). (G-M) Papain-induced acute pneumonia. (G) Experimental design, (H) Representative H&E staining, (I) Absolute number of BALF (n=6), (J) A typical example of flow cytometry and statistical results from both population and the absolute number of EOS in the BALF (All group n=6), (K) IL-5 and (L) IL-13 levels in BALF (n=6), and (M) The mRNA expression levels of inflammatory factors are presented, β- actin was used for normalization, and the lowest expression level sample in BQ123-MDSC group was artificially set to 1(n=5). (N–Q) ConA-induced acute hepatitis. (N) Experimental design, (O) Levels of AST and ALT in the serum (n=6), (P) Representative H&E staining and gross pathological appearance, and (Q) The mRNA expression levels of inflammatory factors are shown, β- actin was used for normalization, and the lowest expression level sample in BQ123-MDSC group was artificially set to 1 (n=4). Data represent the mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0005, and ns, not significant, based on the Mann-Whitney test (E, F, K) or two-tailed unpaired Student’s t test (B–D, I, J, L, M, O, Q).

Fig 4: CD28-Y170F mutants are less affected by changes in CD80 dimensions. WT CD4 T cells (A) or CD4 T cells harboring the CD28 Y170F mutant (B) were stimulated using plate-bound anti-CD3 Abs and incubated with CHO cells expressing no CD80 (control), WT CD80, CD80-CD2, or CD80-CD4. Data are a representative result from at least three experiments. Error bars represent the SEM of three replicates. (C) The integrated IL-2 response was calculated by measuring the area under the curves, such as those depicted in (A) and (B). The integrated IL-2 response for elongated CD80 molecules was expressed as a ratio of the response to corresponding normal-length (WT) CD80 molecules within the same experiment. Error bars represent the SEM from five repeat experiments. **p < 0.01, paired t test.

Fig 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.