The early phase transcriptome of bovine monocyte-derived macrophages infected with Staphylococcus aureus in vitro

Abstract

Background: In the mammary gland, local recruitment and action of macrophages is a key immunological defence mechanism against infection. Macrophages are members of the innate immune system, serve as the first line of the defence against invading pathogens and are critical effectors and regulators of inflammation. We have examined the early phase response of bovine macrophages to infection with live Staphylococcus aureus. Genome-wide transcript profiling of blood monocyte-derived macrophages from six Norwegian Red heifers infected with live S. aureus for 2 and 6 hours in vitro was performed.

Results: About 420 of the 17 000 genes on the ARK-Genomics bovine cDNA array were differentially regulated at 6 hours post infection. Approximately 70% of the responding genes had a known identity (Entrez Gene ID) and were used in the identification of overrepresented pathways and biological functions in the dataset.Analysis of a subset of differentially regulated genes (List eQG) obtained by comparison with data from genome-wide association mapping in Norwegian Red cattle identified anti-inflammatory cytokines interleukin 4 and interleukin 13 as putative expression quantitative trait loci, suggesting that S. aureus infection triggers alternative activation of macrophages. Moreover, several classical activation pathways were found, mainly cellular immune response and cytokine signaling pathways, i.e. triggering receptor expressed on myeloid cells 1 (TREM1) and nucleotide-binding and oligomerization domain-like receptor (NLR) pathways. Tumor necrosis factor receptor superfamily member 5 (CD40 ligand) was identified as an upstream regulator which points toward CD40 likely acting as a co-stimulatory receptor during Toll-like receptor 2(TLR2)-mediated inflammatory response of bovine macrophages to S. aureus infection. Furthermore, peptidoglycan was identified as an upstream regulator in the List eQG, which indicates that this bacterial cell-wall component might be pivotal in macrophage intracellular bacterial recognition during early inflammation.

Conclusions: Here we have shown that in vitro infection of bovine macrophages with live S. aureus induced both alternative and classical activation pathways. Alternative activation of macrophages may be a mechanism contributing to intracellular persistence of S. aureus in the course of inflammation such as during mastitis in dairy cattle.

Figure 1

Hierarchical clustering of 418 genes differentially expressed in macrophages in response to Staphylococcus aureus 6 hours post infection. Letters (A-F) denote the six Norwegian Red heifers tested in the study. Uninfected control samples of each individual were used as references, i.e. control and Staphylococcus aureus infected samples from one individual were hybridized to one array. Two dye-swaps (arrays) were used per individual except for individual C, where only one array was produced.

Figure 2

Functional network over-represented in a list of differentially expressed genes on the microarray involving nuclear factor kappa B (NFKB) complex and associated molecules with a network score of 31, as identified by Ingenuity Pathway Analysis (IPA). Red denotes molecules that were up-regulated and green denotes molecules down-regulated in response to 6 hours infection with live Staphylococcus aureus in bovine macrophages.

Figure 3

Functional network over-represented in the subset of the differentially regulated genes (List eQG) involving interleukin 4 (IL-4), IL-13 and suppressor of cytokine signalling 3 (SOCS3) and associated molecules with a network score of 20, as identified by Ingenuity Pathway Analysis (IPA). List eQG consist of genes that resulted from combining of the differentially regulated genes on the microarray (n = 418) with marker positions from a study of quantitative trait loci (QTL) affecting susceptibility to mastitis in Norwegian Red cattle [26]. Red denotes molecules that were up-regulated in response to 6 hours infection with live Staphylococcus aureus in bovine macrophages.

Figure 4

Upstream regulators and their target molecules in datasets as identified by Ingenuity Pathway Analysis (IPA). A – The list of differentially regulated on microarray (n = 418) and Ensembl reannotated genes was used as an input for analysis; B – Subset of these genes (n = 28; List eQG from [18]) was used as an input. Molecules in red denote up-regulation, molecules in green denote down-regulation and molecules in orange denote predicted activation in response to 6 hours infection with live Staphylococcus aureus in bovine macrophages. Lines in orange denote predicted activation; lines in blue - predicted inhibition; lines in yellow - findings inconsistent with state of downstream molecule; and lines in grey - effect not predicted.

Figure 5

Comparison of mRNA gene expression between the microarray and reverse transcription-quantitative PCR (RT-qPCR). Genes that have shown a significant difference in expression between Staphylococcus aureus infected cells and uninfected control cells in RT-qPCR analysis are denoted with p ≤ 0.05 and non-significant genes in RT-qPCR are denoted with p > 0.05. Genes that have shown a divergent expression pattern in the RT-qPCR analysis compared to the microarray experiment are denoted with ≠. Bcl2-antagonist of cell death (BAD), caspase 1 (CASP1), chemokine c-c motif ligand 5 (CCL5), chemokine c-c motif receptor 5 (CCR5), v-fos fbj murine osteosarcoma viral oncogene homolog (FOS), intercellular adhesion molecule-1 (ICAM1), interferon beta (IFNb), interferon regulatory factor 1 (IRF1), mitogen-activated protein kinase 14, p38 (MAPK14), p21/cdc42/rac1-activated kinase 1 (PAK1), remodeling and spacing factor 1 (RSF1), Toll-like receptor 8 (TLR8) and tumor necrosis factor alpha (TNFa). Peptidylprolyl isomerase A (PPIA) was used as a reference gene.

Figure 6

Hypothetical mechanism of alternative activation of macrophages in response to Staphylococcus aureus infection. Left panel – Staphylococcus aureus enters the macrophage through the Toll-like receptor 2 (TLR2)-dependent pathway that initiates nuclear factor kappa B (NFKB)-mediated temporal inflammatory response. Triggering receptor expressed on myeloid cells 1 (TREM1) synergizes with TLR2 that stimulates intracellular signals resulting in phagocytosis and production of proinflammatory cytokines. NFKB induces expression of co-stimulatory receptor tumor necrosis factor superfamily member 5 (CD40). After phagosomal escape into the cytosol Staphylococcus aureus peptidoglycan induces nucleotide-binding and oligomerization domain 2 (NOD2) expression that in turn triggers inflammation. The macrophage is induced to produce interleukin 4 (IL-4) and IL-13 as confirmed by our study. Right panel – hypothetical alternative activation pathway triggered by IL-4 and IL-13, likely a mechanism by which Staphylococcus aureus evades the host immune response. Alternatively activated macrophage produces anti-inflammatory IL-10, which inhibits classical macrophage activation. Caspase 1(CASP1); interleukin 4 receptor (IL-4R); interleukin 13 receptor (IL-13R); Janus kinase (JAK) and signal transducer and activator of transcription (STAT).