title: Novel Innate Immune Genes Regulating the Macrophage Response to Gram Positive Bacteria.
abstract: Host variation in Toll-like receptors and other innate immune signaling molecules alters infection susceptibility. However, only a portion of the variability observed in the innate immune response is accounted for by known genes in these pathways. Thus, the identification of additional genes that regulate the response to Gram positive bacteria is warranted. Bone marrow-derived macrophages (BMMs) from 43 inbred mouse strains were stimulated with lipotechoic acid (LTA), a major component of the Gram positive bacterial cell wall. Concentrations of the proinflammatory cytokines IL-6, IL-12, and TNF-α were measured. In silico whole genome association (WGA) mapping was performed using cytokine responses followed by network analysis to prioritize candidate genes. To determine which candidate genes could be responsible for regulating the LTA response, candidate genes were inhibited using RNA interference (RNAi) and were overexpressed in RAW264.7 macrophages. BMMs from Bdkrb1-deficient mice were used to assess the effect of Bdkrb1 gene deletion on the response to LTA, heat-killed Streptococcus pneumoniae, and heat-killed Staphylococcus aureus WGA mapping identified 117 loci: IL-6 analysis yielded 20 loci (average locus size = 0.133 Mb; 18 genes), IL-12 analysis produced 5 loci (0.201 Mb average; 7 genes), and TNF-α analysis yielded 92 loci (0.464 Mb average; 186 genes of which 46 were prioritized by network analysis). The follow-up small interfering RNA screen of 71 target genes identified four genes (Bdkrb1, Blnk, Fbxo17, and Nkx6-1) whose inhibition resulted in significantly reduced cytokine production following LTA stimulation. Overexpression of these four genes resulted in significantly increased cytokine production in response to LTA. Bdkrb1-deficient macrophages were less responsive to LTA and heat-killed S. aureus, validating the genetic and RNAi approach to identify novel regulators of the response to LTA. We have identified four innate immune response genes that may contribute to Gram positive bacterial susceptibility.
authors.affiliation: Department of Biomedical Research, National Jewish Health, Denver, Colorado 80206 Center for Genes, Environment, and Health, National Jewish Health, Denver, Colorado 80206 Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045.
authors.forename: Scott
authors.initials: S
authors.lastname: Alper
authors.affiliation: Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045.
authors.forename: Laura A
authors.initials: LA
authors.lastname: Warg
authors.affiliation: Center for Genes, Environment, and Health, National Jewish Health, Denver, Colorado 80206 Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045.
authors.forename: Lesly
authors.initials: L
authors.lastname: De Arras
authors.affiliation: Department of Biomedical Research, National Jewish Health, Denver, Colorado 80206 Center for Genes, Environment, and Health, National Jewish Health, Denver, Colorado 80206.
authors.forename: Brenna R
authors.initials: BR
authors.lastname: Flatley
authors.affiliation: Center for Genes, Environment, and Health, National Jewish Health, Denver, Colorado 80206 Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045.
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Davidson
authors.affiliation: Center for Genes, Environment, and Health, National Jewish Health, Denver, Colorado 80206.
authors.forename: Jenni
authors.initials: J
authors.lastname: Adams
authors.affiliation: Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045.
authors.forename: Keith
authors.initials: K
authors.lastname: Smith
authors.affiliation: Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242.
authors.forename: Christine L
authors.initials: CL
authors.lastname: Wohlford-Lenane
authors.affiliation: Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242.
authors.forename: Paul B
authors.initials: PB
authors.lastname: McCray
authors.affiliation: Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045.
authors.forename: Brent S
authors.initials: BS
authors.lastname: Pedersen
authors.affiliation: Center for Genes, Environment, and Health, National Jewish Health, Denver, Colorado 80206 Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045 Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045.
authors.forename: David A
authors.initials: DA
authors.lastname: Schwartz
authors.affiliation: Center for Genes, Environment, and Health, National Jewish Health, Denver, Colorado 80206 Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045 Department of Epidemiology, Colorado School of Public Health, University of Colorado Denver, Aurora, Colorado 80045 ivana.yang@ucdenver.edu.
authors.forename: Ivana V
authors.initials: IV
authors.lastname: Yang
mesh_terms: D000818:Animals; D016207:Cytokines / Q000276:immunology*; D055106:Genome-Wide Association Study; D006094:Gram-Positive Bacteria / Q000276:immunology*; D007113:Immunity, Innate; D008264:Macrophages / Q000276:immunology* / Q000382:microbiology; D051379:Mice; D008815:Mice, Inbred Strains; D034622:RNA Interference; D015398:Signal Transduction; D013203:Staphylococcal Infections / Q000276:immunology / Q000382:microbiology; D013211:Staphylococcus aureus / Q000276:immunology
chemical_list: D016207:Cytokines
keywords: Gram positive bacteria; RNA interference; inbred strains of mice; innate immunity; lipotechoic acid; whole genome association mapping