Gene expression during the generation and activation of mouse neutrophils: implication of novel functional and regulatory pathways

Abstract

As part of the Immunological Genome Project (ImmGen), gene expression was determined in unstimulated (circulating) mouse neutrophils and three populations of neutrophils activated in vivo, with comparison among these populations and to other leukocytes. Activation conditions included serum-transfer arthritis (mediated by immune complexes), thioglycollate-induced peritonitis, and uric acid-induced peritonitis. Neutrophils expressed fewer genes than any other leukocyte population studied in ImmGen, and down-regulation of genes related to translation was particularly striking. However, genes with expression relatively specific to neutrophils were also identified, particularly three genes of unknown function: Stfa2l1, Mrgpr2a and Mrgpr2b. Comparison of genes up-regulated in activated neutrophils led to several novel findings: increased expression of genes related to synthesis and use of glutathione and of genes related to uptake and metabolism of modified lipoproteins, particularly in neutrophils elicited by thioglycollate; increased expression of genes for transcription factors in the Nr4a family, only in neutrophils elicited by serum-transfer arthritis; and increased expression of genes important in synthesis of prostaglandins and response to leukotrienes, particularly in neutrophils elicited by uric acid. Up-regulation of genes related to apoptosis, response to microbial products, NFkB family members and their regulators, and MHC class II expression was also seen, in agreement with previous studies. A regulatory model developed from the ImmGen data was used to infer regulatory genes involved in the changes in gene expression during neutrophil activation. Among 64, mostly novel, regulatory genes predicted to influence these changes in gene expression, Irf5 was shown to be important for optimal secretion of IL-10, IP-10, MIP-1α, MIP-1β, and TNF-α by mouse neutrophils in vitro after stimulation through TLR9. This data-set and its analysis using the ImmGen regulatory model provide a basis for additional hypothesis-based research on the importance of changes in gene expression in neutrophils in different conditions.

Figure 1. Isolation of neutrophils and characterization of gene expression patterns.

A. Neutrophils were isolated from bone marrow (BM) and blood (BL) of untreated mice, from the peritoneal cavity of mice administered thioglycollate (TG) or uric acid (UA) intraperitoneally, and from the synovial fluid (SF) of mice with autoantibody-induced arthritis, on the basis of scatter patterns (which differed among conditions, left panels) and staining for CD11b and Ly6G (right panels). The population in the upper left corner of the TG plot did not express CD11b or Ly6G. B. Comparison of global gene expression patterns in neutrophils (labeled) to all of the other populations in ImmGen, using axes determined by principal components analysis (PCA). Populations in red on the right side of the diagram represent stromal cell populations; other colors represent various lymphoid and myeloid populations. To convert ImmGen nomenclature to the abbreviations used in this paper: Thio.PC = TG; UrAc.PC = UA; Arth.SynF = SF; GN.Bl = BL; GN.BM = BM = bone-marrow neutrophils from normal mice; Arth.BM = bone-marrow neutrophils from arthritic mice, note similarity to GN.BM. C. Expression of genes for components of neutrophil primary granules (top), secondary granules (middle), and 15 genes showing greater expression in neutrophils than non-neutrophils in ImmGen [mean expression among 5 neutrophil populations (BM, BL, SF, UA, and TG) being greater than 4 times the maximum expression among 198 non-neutrophil populations](bottom), during neutrophil development and activation. CMP = common myeloid precursor; GMP = granulocyte/monocyte precursor. Note that expression patterns in the “neutrophil-specific” genes as identified in this study resembled those of secondary but not primary granule components. D. Expression of groups of genes related to translation (per Gene Ontology = GO) in neutrophils (blue) and other leukocytes (red). Each bar represents mean expression among 5 neutrophil or 198 non-neutrophil populations, and error bars show standard errors.

Figure 2. Biological processes showing up-regulation or down-regulation of genes in activated neutrophils.

(A–H). Heat maps show mean expression in neutrophils from blood (BL), synovial fluid (SF), or peritonitis induced by uric acid (UA) or thioglycollate (TG). Mean expression across all four conditions was placed at the center of the gradient (white) for each gene. Red indicates increased expression, and blue indicates decreased expression. The full color gradient for each gene represents an 8-fold difference in expression. Lists of genes of interest were compiled using the KEGG and Ingenuity databases as well as literature reviews; only genes showing at least 2-fold differences in expression comparing conditions and with Q<0.05 by ANOVA are shown. In the pathway diagrams, up-regulated genes are shown in red, and down-regulated genes are shown in green. A. Uptake and metabolism of lipoproteins. B. Nr4a-family transcription factors. C. Glutathione metabolism. D. Synthesis of and response to leukotrienes and prostaglandins. E. Antigen processing and presentation via MHC class II. F. Genes related to apoptosis. G. NFκB subunits and proximal regulators of NFκB. H. Genes related to signaling by innate immune receptors for microbial products. I. Expression of H3 histone genes (Hist1h3a, b, c, d, e, g, h, I, and Hist2h3b and 3c1) in neutrophil populations. Mean ± SD of these 10 genes (black) declined after release from bone marrow (BM) to blood (BL) and further after activation (SF, UA, TG). Mean ± SD among 198 non-neutrophil populations is shown for comparison. Although it is not apparent from this plot, the lowest expression among non-neutrophils exceeded the highest expression in UA or TG neutrophils. Expression of genes for the “replacement” H3 histones, shown in red and blue, was maintained after neutrophil maturation and activation, at levels similar to non-neutrophils.

Figure 3. Regulatory genes implicated in neutrophil activation, with further focus on IRF family members.

A. Genes were placed into 25 clusters (1–128 genes each, shown as the column headings; A and B are used to identify clusters that have the same numbers of genes) based on patterns of expression in individual samples of neutrophils from blood, SF, UA, and TG, as shown in the heatmap at the top. Clusters that clearly represented up-regulated (U) or down-regulated (D) genes (relative to blood) were pooled and were used to generate a list of predicted regulatory genes (rows) showing enrichment based on the ImmGen regulatory model. Association of each of the 64 regulators with each of the 25 gene clusters was then quantified (P-value of chi-square test), and this matrix of P-values was subjected to hierarchical clustering in order to identify related regulators (rows) and related gene clusters (columns). The lower heatmap indicates these P-values (darker = lower), and the dendrogram and colored bars on the right show groups of regulators with similar patterns of association with various gene clusters. The presence of patterns in the top heatmap (e.g., clustering of clusters characterized by up-regulation in TG, SF, or UA, or by down-regulation in SF), which shows normalized average expression in the 4 neutrophil populations in each cluster, validates this method. The group of regulators shown in light blue was associated with gene clusters indicated in bold; inspection of genes in these clusters led to implication of the type 1 interferon pathway and Irf9. B. Up-regulation of genes induced by type 1 interferons via Irf9, in TG and/or UA but not SF neutrophils. The heatmap shows mean expression in blood (BL), SF, UA, and TG neutrophils. Mean expression across all four conditions was placed at the center of the gradient (white) for each gene. The full color gradient for each gene represents an 8-fold difference in expression. The list of genes of interest and the pathway diagram were generated using the KEGG and Ingenuity databases. Only genes showing at least 1.5-fold differences in expression comparing conditions are shown in the heatmap. In the pathway diagram, genes showing statistically significant (Q<0.05 by ANOVA) differences that varied 2-fold in at least one pairwise comparison of conditions are shown in red, and genes showing fold differences of 1.5–2 and/or not meeting statistical significance are shown in pink. C. Irf5 is required for production of several cytokines and chemokines by mouse neutrophils stimulated in vitro with the TLR9 ligand CpG-B, but not for production induced by the TLR2 ligand Pam3Cys nor the TLR4 ligand LPS. The panels show the mean ± SEM of 3 independent experiments using FACS-sorted Gr1^hiCD11b⁺F4/80⁻ neutrophils. Since secretion varied between experiments but reliably did so in parallel for the different analytes, data were analyzed by determining the fold difference between Irf5−/− and WT in each experiment and applying one-sample T-tests to the fold-differences for the 3 experiments. P values for cells treated with CpG were 0.014 for TNF and <0.01 for the other proteins, and 0.13–0.97 for other TLR ligands.