Figure 7. TREM-1 mediates TNF secretion and resistance to apoptosis in SCF-^condHoxb8 progenitor-derived neutrophils.

SCF-^condHoxb8 Trem1^+/+ and Trem1^−/− progenitor lines were generated by lentiviral transduction of Hoxb8 into BM-derived hematopoietic cells obtained from the respective mice and cultured in the presence of SCF and 4-hydroxytamoxifen (4-OHT). For in vitro neutrophil differentiation, SCF-^cond Hoxb8 cells were cultured for additional 5–6 days in the absence of 4-OHT. (A) H&E stained cytospins of Trem1^+/+ SCF-^condHoxb8 progenitor cells (left) and differentiated neutrophils (right). (B) FACS-based characterization of Trem1^+/+ (top panels) and Trem1^−/− (bottom panels) SCF-^condHoxb8 progenitor cells (left) and differentiated neutrophils (right). (C) TNF and iNOS mRNA expression by Trem1^+/+ and Trem1^−/− SCF-^condHoxb8 differentiated neutrophils following 2 h stimulation with plate-bound agonistic anti-TREM-1 mAb or an isotype control antibody was determined by qRT-PCR. (D) TNF secretion by Trem1^+/+ and Trem1^−/− SCF-^condHoxb8 differentiated neutrophils in response to stimulation with an agonistic anti-TREM-1 mAb or an irrelevant isotype control mAb in the presence or absence of LPS (100 ng/ml) was assessed by ELISA. (E) Spontaneous apoptosis of Trem1^+/+ and Trem1^−/− SCF-^condHoxb8 neutrophils in vitro was analysed at 5 days post differentiation with SCF (0 h) and the indicated time-points beyond by FACS-based determination of AnnexinV and DAPI double-positive cells. Bars show mean values ± SEM for n = 3 in vitro replicates from one representative experiment out of three independent experiments. (F) Caspase 3/7 activity was assessed upon 24 h stimulation of differentiated Trem1^+/+ and Trem1^−/− SCF-^condHoxb8 neutrophils with plate-bound agonistic anti-TREM-1 mAb, an isotype control antibody, or LPS (100 ng/ml) as a positive survival control. Bars show mean values ± SEM for n = 3 in vitro replicates from one representative experiment out of three independent experiments. n.d., not detected. (G) Expression of Mcl-1 mRNA was assessed by qRT-PCR 2 h post stimulation with plate-bound anti-TREM-1 or an isotype control mAb in the presence or absence of LPS. Bars show mean values ± SEM of three independent experiments for anti-TREM-1 vs. isotype control mAb stimulated cells. n.d., not detected. ****, p<0.01; **, p<0.01; *, p<0.05.