Transcription Profiling of Monocyte-Derived Macrophages Infected In Vitro With Two Strains of Streptococcus agalactiae Reveals Candidate Pathways Affecting Subclinical Mastitis in Cattle.

Macrophages are key cells of innate immune response and serve as the first line of defense against bacteria. Transcription profiling of bacteria-infected macrophages could provide important insights on the pathogenicity and host defense mechanisms during infection. We have examined transcription profiles of bovine monocyte-derived macrophages (bMDMs) isolated from the blood of 12 animals and infected in vitro with two strains of Streptococcus agalactiae. Illumina sequencing of RNA from 36 bMDMs cultures exposed in vitro to either one of two sequence types of S. agalactiae (ST103 or ST12) for 6 h and unchallenged controls was performed. Analyses of over 1,656 million high-quality paired-end sequence reads revealed 5,936 and 6,443 differentially expressed genes (p < 0.05) in bMDMs infected with ST103 and ST12, respectively, versus unchallenged controls. Moreover, 588 genes differentially expressed between bMDMs infected with ST103 versus ST12 were identified. Ingenuity pathway analysis of the differentially up-regulated genes in the bMDMs infected with ST103 revealed significant enrichment for granulocyte adhesion and diapedesis, while significant enrichment for the phagosome formation pathway was found among down-regulated genes. Moreover, Ingenuity pathway analysis of the differentially up-regulated genes in the bMDMs infected with ST12 showed significant enrichment for type 1/type 2 T helper cell activation, while the complement activation pathway was overrepresented in the down-regulated genes. Our study identified pathogen-induced regulation of key genes and pathways involved in the immune response of macrophages against infection but also likely involved in bacterial evasion of the host immune system. These results may contribute to better understanding of the mechanisms underlying subclinical infection such as bovine streptococcal mastitis.

MicroRNA expression profiles of bovine monocyte-derived macrophages infected in vitro with two strains of Streptococcus agalactiae.

BACKGROUND: MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression at the post-transcriptional level and play a key role in the control of innate and adaptive immune responses. For a subclinical infection such as bovine streptococcal mastitis, early detection is a great challenge, and miRNA profiling could potentially assist in the diagnosis and contribute to the understanding of the pathogenicity and defense mechanisms. We have examined the miRNA repertoire and the transcript level of six key immune genes [tumor necrosis factor alpha (TNFα), interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) and transforming growth factor beta 1 (TGFß1)] during the early phase response of bovine immature macrophages to in vitro infection with live Streptococcus agalactiae. Next generation sequencing of small RNA libraries from 20 cultures of blood monocyte-derived macrophages exposed to either one of two sequence types of S. agalactiae (ST103 or ST12) for 6 h in vitro and unchallenged controls was performed. RESULTS: Analyzes of over 356 million high quality sequence reads, revealed differential expression of 17 and 44 miRNAs (P < 0.05) in macrophages infected with ST103 and ST12, respectively, versus unchallenged control cultures. We also identified the expression of 31 potentially novel bovine miRNAs. Pathway analysis of the differentially regulated miRNAs and their predicted target genes in the macrophages infected with ST12 revealed significant enrichment for inflammatory response and apoptosis, while significant enrichment for integrin and GABA signaling were found in ST103 infected macrophages. Furthermore, both bacterial strains regulated miRNAs involved in the alternative activation of macrophages. The transcript levels of TNF-α, IL-1ß, IL-6, IL-8 and IL-10 were significantly up-regulated by both bacterial strains, however the expression of TGFß1 was significantly down-regulated only by ST12. CONCLUSIONS: Our study identified pathogen-induced differential regulation of miRNAs controlling inflammation and polarization in bovine macrophages. This implies that miRNAs have potential to serve as biomarkers for early detection of bacterial infection.

Transient Migration of Large Numbers of CD14(++) CD16(+) Monocytes to the Draining Lymph Node after Onset of Inflammation.

The dynamics of skin-draining cells following infection or vaccination provide important insight into the initiation of immune responses. In this study, the local recruitment and activation of immune cells in draining lymph nodes (LNs) was studied in calves in an adjuvant-induced inflammation. A transient but remarkably strong recruitment of monocytes was demonstrated after onset of inflammation, constituting up to 41% of live cells in the draining LNs after 24 h. Numerous CD14(+) cells were visualized in subcutaneous tissues and draining LNs, and the majority of these cells did not express dendritic cell-associated markers CD205 and CD11c. In the LNs, recruited cells were predominately of a CD14(++) and CD16(+) phenotype, consistent with an intermediate monocyte subset characterized to possess a high inflammatory potential. Moreover, monocytes from the draining LN showed a high expression of genes coding for pro-inflammatory cytokines, including IL-1ß, IL-6, TNFa, and TGFß. Shortly after their appearance in the LN cortical areas, the monocytes had moved into the medulla followed by an increase in peripheral blood. In conclusion, this study provides novel information on in vivo monocyte recruitment and migration after onset of inflammation.

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

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.

Genome wide transcriptional profiling of acclimation to photoperiod in high-latitude accessions of Arabidopsis thaliana.

Three Arabidopsis thaliana accessions originating from the northernmost boundary of the species distribution in Norway (59-68°N) were used to study global wide transcriptional responses to 16 and 24 h photoperiods during flower initiation. Significant analysis of microarrays (SAM), analyses of statistically overrepresented gene ontologies (GOstat) and gene set enrichment analyses (GSEA) were used to identify candidate genes and genetic pathways underlying phenotypic adaptations of accessions to different photoperiods. Statistical analyses identified 732 and 258 differentially expressed genes between accessions in 16 and 24 h photoperiod, respectively. Among significantly expressed genes, ethylene mediated signaling pathway was significantly overrepresented in 16 h photoperiod, while genes involved in response to auxin stimulus were found to be significantly overrepresented in 24 h photoperiod. Several gene sets were found to be differentially expressed among accessions, e.g. cold acclimation, dehydration response, phytochrome signaling, vernalization response and circadian clock regulated flowering time genes. These results revealed several candidate genes and pathways likely involved in transcriptional control of photoperiodic response. In particular, ethylene and auxin signaling pathway may represent candidate genes contributing to local adaptation of high-latitude accessions of A. thaliana.