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.

Keywords: Gram positive bacteria; RNA interference; inbred strains of mice; innate immunity; lipotechoic acid; whole genome association mapping.

Figure 1

Post-LTA stimulation concentrations of IL-6 (top, red bars), IL-12 (middle, green bars), and TNF-α (bottom, blue bars) secreted by BMMs from 43 inbred mouse strains of mice. BMMs were stimulated with 0.5 µg/ml LTA for 5 hr and cytokine concentrations in the supernatant were measured by ELISA. In all three panels, means of three biological replicates with error bars representing SEMs are plotted.

Figure 2

WGA mapping analysis of IL-12 produced by BMMs from 43 strains of mice in response to LTA stimulation. Top plot shows unadjusted P-values while bottom plot shows P-values after adjustment for relatedness among inbred strains of mice. Green plus symbols designate SNPs significantly associated with LTA-induced IL-12 production.

Figure 3

The effect of RNAi-mediated inhibition of 71 genes from WGA mapping in RAW264.7 macrophages on LTA-induced IL-6 production. Pools of four siRNA duplexes per gene were transfected into RAW264.7 cells, LTA was added, and cytokine production was monitored. Shown are the results for negative control siRNA (blue; nontargeting siRNA from Dharmacon), four positive control siRNAs (red; Traf6, Tlr6, Il-6, and Map3k7), and 71 siRNAs targeting genes identified by our eQTL mapping analysis (black). All data are normalized to the NT#1 nontargeting negative control siRNA. Means of four independent measurements with error bars representing SEMs are plotted; green line indicates threshold for twofold reduction in IL-6 response as a result of gene inhibition. *P < 0.0001, ^#P < 0.001, and ^P < 0.01 by one-group two-tailed t-test testing for deviation from 1.

Figure 4

The effect of individual siRNA duplexes for nine candidate genes from the siRNA screen in RAW264.7 macrophages on LTA-induced IL-6 production. Four siRNA duplexes were transfected individually for each of the eight genes whose inhibition resulted in decreased IL-6 production and one gene whose inhibition resulted in higher IL-6 production in the screen presented in Figure 3. Means of four independent measurements with error bars representing SEMs are plotted; green lines indicate threshold for twofold reduction and red twofold increases in IL-6 response as a result of gene inhibition. ^#P < 0.001 and ^P < 0.01 by one-group two-tailed t-test testing for deviation from 1.

Figure 5

Effect of overexpression of four target genes in RAW264.7 cells on NF-κB activation. Plasmids containing full-length cDNAs for the indicated genes (or CAT negative control) cloned downstream of the CMV promoter were cotransfected with NF-kB-AP1-luc and SV40-rluc plasmids. Following transfection, cells were stimulated with 2.5 µg/ml LTA for 5 hr, and innate immune responsiveness was monitored by measuring luciferase production. Means of three independent measurements with error bars representing SEMs are plotted, and red line indicates threshold for twofold increase in IL-6 response as a result of gene overexpression. Compared to CAT (set to 1), ^P < 0.01 by unpaired two-group two-tailed t-test.

Figure 6

Effect of Bdkrb1 gene deletion on the macrophage response to Gram positive bacterial stimulation. Depicted are IL-6 (A and C) and TNF-α (B and D) production by Bdkrb1-deficient or wild-type macrophages following stimulation with LTA (A and B), HSKP (C and D), or HKSA (C and D). In all panels, means of three to eight biological replicates with error bars representing SEMs are plotted. *P < 0.0001, ^#P < 0.001, and ^P < 0.01 by two-group unpaired two-tailed t-test between Bdkrb1-deficient and wild-type macrophages.