Conservation and divergence in Toll-like receptor 4-regulated gene expression in primary human versus mouse macrophages

Abstract

Evolutionary change in gene expression is generally considered to be a major driver of phenotypic differences between species. We investigated innate immune diversification by analyzing interspecies differences in the transcriptional responses of primary human and mouse macrophages to the Toll-like receptor (TLR)-4 agonist lipopolysaccharide (LPS). By using a custom platform permitting cross-species interrogation coupled with deep sequencing of mRNA 5' ends, we identified extensive divergence in LPS-regulated orthologous gene expression between humans and mice (24% of orthologues were identified as "divergently regulated"). We further demonstrate concordant regulation of human-specific LPS target genes in primary pig macrophages. Divergently regulated orthologues were enriched for genes encoding cellular "inputs" such as cell surface receptors (e.g., TLR6, IL-7Rα) and functional "outputs" such as inflammatory cytokines/chemokines (e.g., CCL20, CXCL13). Conversely, intracellular signaling components linking inputs to outputs were typically concordantly regulated. Functional consequences of divergent gene regulation were confirmed by showing LPS pretreatment boosts subsequent TLR6 responses in mouse but not human macrophages, in keeping with mouse-specific TLR6 induction. Divergently regulated genes were associated with a large dynamic range of gene expression, and specific promoter architectural features (TATA box enrichment, CpG island depletion). Surprisingly, regulatory divergence was also associated with enhanced interspecies promoter conservation. Thus, the genes controlled by complex, highly conserved promoters that facilitate dynamic regulation are also the most susceptible to evolutionary change.

Fig. 1.

Global overview of LPS regulation between human and mouse macrophage populations. Clustered representative profiles for (A) all one-to-one orthologues significantly regulated by LPS in any macrophage population, (B) all profile-divergent orthologues, and (C) all time point-divergent orthologues. Known examples of expression divergence between the species were confirmed, including (D) INOS (P = 1.41 × 10⁻⁷), (E) IDO (P = 1.46 × 10⁻²⁴), and (F) CYP27B1 (P = 2.31 × 10⁻⁷; microarray data average fold induction ± SEM, n = 3–4, significance assessed by ANOVA). (G) Hierarchical clustering of profile-divergent orthologues involved in immune defense or antimicrobial pathways. Genes encoding cytokine, chemokine, and growth factor ligands and receptors, as well as matrix metalloproteinases, were subtracted from this list as they are considered separately in Fig. 3A. Gene fold induction is indicated by the color-code key.

Fig. 2.

Validation of human-specific TLR4 target genes and confirmed coregulation in porcine macrophages. LPS-regulated mRNA expression of selected human-specific TLR4 target genes was quantified by quantitative PCR in cells from human (HMDMs, primary CD14⁺ monocytes, PMA-differentiated THP-1 macrophage-like cells) or mouse (BMMs, TEPMs, BMMs derived from BALB/c mice, C57BL/6 BMMs primed overnight with IFN-γ, C57BL/6 splenocytes, macrophage-like cell line RAW264.7). Human-specific LPS-regulated gene expression is displayed for CCL20 (A), CXCL13 (B), IL-7R (C), P2RX7 (D), and STAT4 (E). Quantitative PCR data represent average fold induction ± range (BALB/c BMMs, n = 2) or ± SEM (n ≥ 3, all other profiles) of measurements with independent samples. Microarray data are shown only for HMDMs, BMMs, and TEPMs (dotted line, average fold induction ± SEM, n = 3–4). Expression of CCL20 (F), CXCL13 (G), and IL-7R (H) were further investigated by profiling LPS-dependent gene expression in MDMs and BMMs derived from pigs; data represent average fold induction ± SEM of measurements with three or four independently prepared samples.

Fig. 3.

Functional consequences of regulatory divergence in macrophage inputs and outputs. (A) Hierarchical clustering of DR orthologues that encode matrix metalloproteinases or cytokines, chemokines, and growth factors and their receptors. Gene fold induction is indicated by the color-code key. Human-specific production of the DR secreted proteins, CCL20 (B and D) and CXCL13 (C and E) in response to 24 h LPS treatment (B and C) or 24 h S. Typhimurium infection (D and E) was validated by ELISA. Data represent mean ± SD of technical replicates (n = 4, LPS-stimulated HMDMs or BMMs) or mean ± range of technical replicates (n = 2, LPS-stimulated TEPMs and Salmonella-infected HMDMs and BMMs). Profiles are representative of two to four experiments with independent cellular sources. (F) Cell-surface IL-7Rα/CD127 expression on HMDMs and BMMs over a time course of LPS stimulation was measured by flow cytometry [average mean fluorescence intensity (MFI) ± SD of n = 4 technical replicates, prepared, stimulated, and harvested in parallel]. Data are representative of two to five experiments with independent cellular sources. (G) The reference Kyoto Encyclopedia of Genes and Genomes pathway for TLR signaling was color-coded according to regulatory conservation (yellow) or divergence (profile or time point divergence; red) between species. LPS-regulated orthologues displaying neither striking conservation nor divergence are shaded in gray, and genes that were not significantly LPS-regulated or not represented on the focused microarray are white. This striking separation of DR genes according to pathway input/output vs. pathway core is highly unlikely to occur by chance (P = 4 × 10⁻⁷). (H) Macrophages were primed with LPS or medium for 11 h, washed, and restimulated with the TLR2/6 ligand FSL-1 for 8 h. IL-6 production was measured by ELISA. Data are average ± SD of three technical replicates, prepared, stimulated, and harvested in parallel, and are representative of three or four independent experiments.

Fig. 4.

The promoters of DR genes exhibit a higher fraction of aligning sequence and lower substitution rates than non-DR promoters. (A and B) Spatial profile of human/mouse aligning sequence. The fraction of human TSS region nucleotides aligned with mouse (A) and mouse TSS aligned with human (B) for profile-divergent genes (red) vs. non–profile-divergent subset (black), for which an orthologous macrophage TSS was defined in the other species (mouse or human) by CAGE. (C) Distributions of core promoter corrected substitution rate ratios [log10(ω_core)] for primary TSSs that are orthologous between human and mouse (n = 1,190). log10(ω_core) < 0 indicates predominantly purifying selection; log10(ω_core) > 0 indicates predominantly diversifying selection. Also shown is the distribution median (vertical black/red bar), the fraction of all TSSs represented by the distribution (red to black fraction of vertical bar), and the median of all TSSs (pale red). Distributions are shown for specific promoter types (TATA, CpG island, neither TATA nor CpG island).

Fig. 5.

DR genes are enriched for TATA box promoters, are depleted of CpG islands, and tend to exhibit a large dynamic range of expression in response to LPS. (A and B) Orthologous genes were ranked based on variation between species (SDiff or P value for significance of profile divergence; high rank is high significance and low P value) or dynamic range, such that genes with a high variation within or between species achieve a high rank, and analyzed according to percentage of genes with (A) CpG island-type orthologous gene promoters or (B) TATA box-containing orthologous gene promoters. (C and D) analyses were performed as in A and B, but considering only orthologues without a TATA box in the promoter (C) or only those that had CpG island-containing promoters (D). (E and F) Genes were ranked based on dynamic range, maximum fold suppression, or maximum fold induction and analyzed for percentage of DR genes according to (E) profile divergence or (F) time-point divergence. For A–F, 100 randomly permuted rankings of the data are shown (gray curves; nonrandom rankings protruding from the gray curves represent P < 0.01). Also indicated are confidence intervals (95%, dashed gray lines; 99%, dotted gray lines) and average for the whole dataset (solid gray lines). Sliding 300-gene windows are shown.

Fig. P1.

Typical gene, profile, and promoter characteristics associated with interspecies mRNA regulatory divergence in innate immunity. Interspecies divergence in mRNA regulation following immune stimulation was analyzed by using a custom-designed platform. Deep sequencing of mRNA 5′ ends was performed in parallel to experimentally define promoter regions, which are responsible for activating gene expression. mRNA regulation of orthologous transcripts was classified as DR or non-DR. DR orthologues were associated with specific transcript, gene, and functional properties: (i) highly regulated genes were enriched for regulatory divergence; (ii) DR gene promoters featured specific regulatory sequences, including enrichment of TATA boxes and depletion of CpG islands; (iii) DR gene promoter sequences were more highly conserved than those of non-DR genes; and (iv) DR genes were enriched for specific functions.