In Vivo Deficiency of Both Transcription Factors, C/EBPbeta and C/EBPepsilon, Results in Lack of Cytokine Response and a High Susceptibility for Overwhelming Infections.

Abstract 3641

Poster Board III-577

Transcription factors including the CCAAT/enhancer binding protein (C/EBP) family are essential for hematopoietic cell development including myelopoiesis. In response to cytokines and/or infection, C/EBPbeta is important for emergent neutrophil production, whereas C/EBPepsilon induces terminal differentiation of granulocytes. We recently generated C/EBPbeta and C/EBPepsilon double-knockout (KO) mice and demonstrated the dependence of the hematopoietic system on both transcription factors. In order to study inflammatory response of this transgenic in vivo model, bone marrow-derived macrophages from wild-type (WT), single- as well as double-KO mice were cultured either with or without 100 ng/mL LPS and 100 ng/mL mIFNgamma as activating stimuli. After 24 hours of exposure, RNA was extracted, and gene expression difference was interrogated by whole genome microarray (Affymetrix^®, mouse genome 430 2.0). The probes were normalized with the MAS 5.0 software for processing Affymetrix oligonucleotide array data, and genes were considered to be differentially expressed with a fold change level of at least 2. Remarkably, we found more than 75 genes which were up to 20-fold downregulated in the stimulated macrophages of the double-KO mice compared to either single KO or WT mice. A pathway-based analysis indicated that these deregulated genes formed networks associated with development and function of the hematopoietic system, inflammatory response and cell-cell signaling. We confirmed the expression for some of the most highly decreased genes by RT-PCR; for example, interleukin-(IL)1beta, IL-12beta, CD38, macrophage mannose receptor (Mrc-1), toll-like receptor-2 (TLR-2), C-type lectin receptor Mincle, and insulin-like growth factor (IGF-1) were almost not detectable in the activated macrophages of the double-KO mice compared to either single-deficient or WT mice. Lacking these and other important regulators of appropriate immune responses and hematopoiesis, the double KO died between 2 - 3 months of age due to systemic infections including formation of hepatic, peritoneal and lung abscesses with Streptococcus viridians, Enterococcus species and Escherichia coli. In conclusion, phentotype as well as expression pattern of mediators of inflammatory response and hematopoiesis of the double-KO mice was distinct from either the C/EBPbeta- or C/EBPepsilon-KO mice, demonstrating that each transcription factor is important in cytokine expression and host defense, but can in part compensate for each other in the single knockout genotype.