Identification of Novel Genes That Mediate Innate Immunity Using Inbred Mice

Abstract

Innate immunity is the first line of defense against microbial infections. Although polymorphisms in toll-like receptors (TLRs) and downstream signaling molecules (CD14, TLR2, TLR4, TLR5, and IRAK4) affect the innate immune response, these variants account for only a portion of the ability of the host to respond to bacteria, fungi, and viruses. To identify other genes involved in the innate immune response, we challenged 16 inbred murine strains with lipopolysaccharide (LPS) systemically and measured serum concentrations of pro-inflammatory cytokines IL-1b, IL-6, and TNFa, and the chemokine KC 6 hr posttreatment. Loci that segregate with strain phenotypes were identified by whole genome association (WGA) mapping of cytokine concentrations. Published gene expression profiles and quantitative trait loci (QTL) were then utilized to prioritize loci and genes that potentially regulate the host response to LPS. Sixteen loci were selected for further investigation by combining WGA analysis with previously published QTL for murine response to LPS or gram negative bacteria. Thirty-eight genes within these loci were then selected for further investigation on the basis of the significance of the identified locus, transcriptional response to LPS, and biological plausibility. RNA interference-mediated inhibition of 4 of 38 candidate genes was shown to block the production of IL-6 in J774A.1 macrophages. In summary, our analysis identified 4 genes that have not previously been implicated in innate immunity, namely, 1110058L19Rik, 4933415F23Rik, Fbxo9, and Ipo7. These genes could represent potential sepsis biomarkers or therapeutic targets that should be further investigated in human populations.

Novel Innate Immune Genes Regulating the Macrophage Response to Gram Positive Bacteria

“…We used the EMMA algorithm developed by Kang et al (2008) because it implements a genetic similarity matrix and a phylogenetic control method to account for population structure among inbred strains of mice, thereby limiting false positive associations (Kang et al 2008), and we have successfully used it in our previous studies of innate immunity (Yang et al 2009). The reduction in the false positive associations achieved by implementing the EMMA algorithm to perform WGA mapping of the IL-12 cytokine response data are depicted in Figure 2, A and B, demonstrating the appropriateness of this mapping approach in our study.…”

“…We also performed permutation testing with shuffled data sets to empirically determine that the false positives rate was low (Abiola et al 2003). Pathway and network analysis was performed using Ingenuity pathway analysis (IPA) (Kramer et al 2014) RNA interference in RAW264.7 cells RNA interference (RNAi) assays were carried out as previously described (Alper et al 2008;Yang et al 2009;De Arras et al 2014a). Briefly, small interfering RNAs (siRNAs) (Dharmacon, Lafayette, CO; pools of four siRNA duplexes/gene) were transfected into the mouse macrophage cell line RAW264.7 using the Amaxa Nucleofector Shuttle according to the manufacturer's instructions.…”

“…Novel genes involved in the innate immune response to a variety of PAMPs and pathogens have been identified by our group using comparative genomics approaches (Alper et al 2008), transcriptional profiling of stimulated macrophages (Yang et al 2011b) and tissues from animal models (Yang et al 2011a), and mapping studies in inbred mice (Zaas et al 2008;Yang et al 2009). A prior study from our laboratory investigated murine antibacterial defense to streptococcal lung infection in eight strains of mice and found that TLR2-deficient mice had increased bacterial load (Hollingsworth et al 2007), and a similar study in nine inbred mouse strains identified resistant and susceptible strains of mice (Gingles et al 2001).…”

An update: Epstein-Barr virus and immune evasion via microRNA regulation

“…IL-6, a pro-inflammatory factor secreted by immune cells, is able to promote the secretion of miR-21 and miR-29b by tumor cells, and the miRNAs in turn induce the expression of IL-6 (Patel and Gooderham, 2015). Downregulation of IPO7 by miR-BART3 in macrophages has been shown to reduce IL-6 production upon lipopolysaccharide challenge (Yang et al, 2009). In the pathogenesis of inflammatory diseases (including tumors and infections), miR-21 can act as an immune response circuit switch in order to control the balance between the initial pro-inflammatory response and the anti-inflammatory response (Sheedy, 2015).…”

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

“…To overcome these technical challenges, we have optimized transfection and knockdown techniques [12][13][14][15][16] in two mouse macrophage cell lines, RAW264.7 17 and J774A.1 18 . This approach uses the Amaxa Nucleofector 96-well Shuttle System for transfection; this system uses a combination of specialized reagents and electroporation to transfect cells in 96-well format 1.…”