FIGURE 3.

LPS‐induced netrin‐1 expression in myeloid cells is regulated by HIF‐1α. A, Schematic illustrating the location of a hypoxia response element (HRE) 303 base pairs upstream of the transcriptional start site (TSS) of NTN1. B, Human monocyte‐derived MΦs were treated with LPS (1 μg/mL) for 8 hours and then, fixed to induce DNA‐protein cross‐linking. Chromatin Immunoprecipitation (ChIP)‐PCR targeting a sequence proximal to the HRE was performed to demonstrate fold enrichment for HIF‐1α association with the promoter of NTN1 compared with IgG isotype control. Data are normalized relative to PBS‐treated MΦs (n = 3, unpaired t test). C, Relative expression of Ntn1 in BAL cells collected from mice with conditional genetic deletion of Hif1α in myeloid cells (Hif1α ^loxp/loxp LysM Cre) 3 days after onset of LPS‐induced lung injury compared with LysM Cre mice for control (n = 4‐6 mice per group, Mann‐Whitney test). D, Representative immunohistochemistry staining for netrin‐1 protein in BAL cells isolated from Hif1α ^loxp/loxp LysM Cre mice 3 days after onset of LPS‐induced lung injury compared with LysM Cre mice (n = 3). E and F, Densitometry and western blot image and quantification for netrin‐1 protein expression in lung tissue of LysM Cre and Hif1α ^loxp/loxp LysM Cre 3 days after the onset of LPS‐induced lung injury (n = 3 mice per group, two‐way ANOVA with Bonferroni post hoc tests). All data are represented as mean ± SD; *P‐value < .05