C/EBPε Plays a Role in Immunity By Regulating Trem-1 Expression in Granulocytes

C/EBPepsilon (C/EBPε) knock-out (KO) mice have defects in granulocytic development and increased susceptibility to infection. We also observed a unique phenotype of C/EBPε KO mice showing prolonged lower grade, mild skin problems such as hair loss that progressed to severe wound problems; abdominal wall perforation to the peritoneum as well as mandibular skin perforation to the oral cavity, without significant clinical symptoms. The observation suggests inappropriate response to inflammation by loss of C/EBPε. To understand the functional defects of these granulocytes, we performed a systematic approach with ChIP-seq and microarray of bone marrow cells from C/EBPε KO mice.

Duplicate ChIP-seq libraries were prepared from bone marrow cells of wild type and C/EBPε KO mice using two different C/EBPε specific antibodies. Analysis of pooled data identified 14,253 C/EBPε binding sites, and 12,865 of these binding sites overlapped with the two antibodies. Sixteen percent of C/EBPε binding sites were in the promoter regions within 1 kb of the transcriptional start sites. Direct canonical C/EBPε target genes were defined by 1.5 log2 fold or greater increase in expression in the Gr-1+/Mac-1+ cells of wild type bone marrow cells compared to those of the KO mice. 2,224 genes met this criteria including previously identified target genes, such as ltf, camp, ngp, of which 988 genes were bound by C/EBPε. C/EBPε bound to the promoter sites of 150 genes and the enhancer region of 838 genes. The other 1,236 up-regulated genes did not have binding of C/EBPε, suggesting their up-regulation was a secondary event during terminal granulocytic differentiation. Electromobility shift assay followed by antibody supershift confirmed that C/EBPε was able to bind to upstream regions of target genes such as lft, ngp, il-1b.

Trem-1 is an amplifier of inflammatory response. Increased expression/activation of Trem-1 on neutrophil membranes takes part in cytokine production, phagocytosis, apoptosis, oxidative burst by cooperating with TLR4 in response to environmental conditions. Soluble TREM1 levels increase even in neutopenic patients, suggesting it is associated with emergent granulopoiesis in response to infection or inflammation. The expression of Trem-1 is regulated by NF-kB and PU.1. Trem-1 KO mice have reduced inflammation without defects in clearance of pathogens in mice. Considering previous reports demonstrating TLR4 increases transcription of C/EBP and potentiates transcriptional activity of C/EBP via NF-kB, regulatory loops between TLR4/NF-kB/C/EBP/Trem-1 may exist and regulate inflammatory response.

ChIP-seq illustrated a C/EBPε binding site located 92 bp upstream from transcription start site of Trem-1 (GTTGTGAAAC). Microarray comparison of Trem-1 expression in Gr-1+/Mac-1+ bone marrow cells showed 5.3 log2 fold decreased gene expression in C/EBPε KO versus wild type mice. To confirm microarray data, quantitative PCR were performed with sorted Gr-1+ bone marrow cells from wild type and KO mice. The qPCR demonstrated that trem-1 expression of C/EBPε KO cells was 60% of wild type (p=0.001). A pGL3 basic luciferase vector with the first 291bp upstream of trem-1containing a putative C/EBPε binding site was cloned and co-transfected with 2 ug of C/EBPε expressing vector into 293T cells. The dual luciferase assay, normalized by renilla luciferase, showed 8 fold increase by C/EBPε (p<0.0001).

In conclusion, we identified trem-1 as a downstream target of C/EBPε, which completes a positive regulatory loop of TLR4-NF-kB-C/EBPε-Trem1-TLR4 in response to inflammatory signal. Therefore, C/EBPε KO mice have an inappropriate inflammatory response by loss of the positive amplificatory loop, in addition to previously known defective clearance of pathogen by their inability to produce secondary granules.