Figure 6.

α-TREM-1 facilitates the cooperation of CD177⁺ neutrophils with intestinal epithelial cells and macrophages. (A) Representative images of CD177 immunostaining in colon tissues. Scale bar, 20 μm. (B) Cd177 expression in mouse bone marrow-derived neutrophils (BMDNs) from wild-type and Tlr4-KO mice. BMDNs and HL-60 cells pre-treated with α-TREM-1 (0.8 μg/mL) were treated with lipopolysaccharide (LPS, 200 ng/mL) for 4 h for qRT-PCR analysis. Each data represents the mean of duplicate real-time RT-PCR (n = 3). (C,D) HL-60 cells transfected with scrambled (SCR) or TREM1-specific (siT) siRNA were pre-treated with α-TREM-1 and stimulated with LPS for 1 h. (C) CD177 expression in human neutrophils treated with α-TREM-1. Each data represents the mean of duplicate real-time RT-PCR (n = 3). (D) Representative images from the neutrophil extracellular trap assay. Data are from one experiment representative of three independent experiments. Scale bar, 40 μm. (E) Modulation of CD177⁺IL-22⁺ neutrophils in the blood of inflammatory bowel disease (IBD) patients by α-TREM-1. Flow cytometric analysis was performed to evaluate the CD177⁺IL-22⁺ cells in neutrophil populations extracted from ulcerative colitis (UC) and Crohn's disease (CD) patients and healthy subjects (n = 5). Numbers indicate CD177⁺IL-22⁺ cell frequencies among neutrophils. (F) Reactive oxygen species (ROS) production in neutrophils of IBD patients. (G,H) Wound healing assay. HT-29 cells were cultured in conditioned media from HL-60 cells (G) or blood neutrophils of UC patients (H) after α-TREM-1 treatment and wound healing assays were performed. Data represent the average of four independent experiments. Statistical significance was assessed using one-way ANOVA followed by Tukey post-test. * and ^#P < 0.05, **P < 0.01, *** and ^###P < 0.005 vs. vehicle (Veh). Data are expressed as means ± S.E.M. (n = 4–5). α-T, treated with α-TREM-, treated with control antibody; Water, supplied with normal drinking water.