Inflammatory Pathways Affected

Several genes involved with inflammatory pathways were differential expressed. Although coherent pathways could not be identified the modulation of transcription of several key players in the immune response increases the likelihood that these DEG are relevant to the interaction of S. uberis with host cells, but further work is required to confidently define their roles.

4.6.2.1 Pro-inflammatory cytokine expression is up-regulated

Increases in cytokine expression are associated with mastitis infections, with some pathogen- specific patterns (reviewed (Bannerman, 2009)). In the present study expression of the

cytokines CXCL8, IL-12 , CCL20 CXCL5 and CCL3 were all up-regulated in ST PMNs. Inflammatory cytokines are produced by leukocytes, including neutrophils; a principal role of some cytokines is as chemokines. They attract other leukocytes to the site of inflammation and their induced expression directs the resulting immune response.

CXCL-8 (IL-8) acts as an attractant for neutrophils and monocytes and is also able to activate neutrophils; it is produced by many cells, including neutrophils, and is a key pro-inflammatory cytokine. It has previously been shown to be up-regulated in experimental infections with S. uberis 0140J (Bannerman et al., 2004a) and it was the top up-regulated DEG in mammary tissue following infection with S. uberis (Moyes et al., 2009).

IL-1β is the p40 subunit that heterodimerises with the p38 subunit to form the active dimer IL-12 and is also part of the IL-23 heterodimer. Cells of the monocyte lineage are the main producers of IL-12 , however PMNs have been shown to produce IL-12 (reviewed by (Scapini et al., 2000)). The overwhelming presence of PMNs during mastitis suggests they could significant contribute to the ongoing immune response. IL-1β triggers an IFN response in immune cells and is important in the defence against intracellular pathogens, mediating a Th1 response (Scapini et al., 2000). Bannerman and colleagues found that experimental infection with S. uberis 0140J increases levels of IL-12 which correlates with IFN production (Bannerman et al., 2004a).

IL-23, initially shown to be produced by activated dendritic cells (Oppmann et al., 2000), activates a neutrophil-mediated response via IL-17 leading to IL-1, IL-6 and TNF release (Langrish et al., 2004). Whether the IL-1β transcription detected by microarray is directed via IL-12 or IL-23 cannot be determined, in fact it is likely that both are relevant as in any inflammatory process the resulting immune response is due to the balance between many pro and anti-inflammatory molecules. In experimental challenge, IL-1β levels increases in response to S. uberis strain 0140J, but not in response to strain EF20 (Coffey et al personal communication). However, the increase in response to 0140J is much less than with the attenuated mutant strains lacking SrtA and Vru (Coffey et al personal communication). Even during infection in vivo, where numerous cytokines were measured, it is hard to definitively link the levels of IL-1β seen with biological function. As a general trend, increases in IL-1β production correlate with TNF , IL-8 and initially bacterial numbers in the udder, but IL-1β negatively correlates with IL-6, perhaps indicating an overall anti-inflammatory bias in its effect. Though some parallels may be drawn between the data generated from in vivo and in vitro studies, with regards to Il-1β production, the exact contribution of IL-12 is clouded by the fact that the IL-1β subunit is common to both IL-12 and IL-23. The contribution of IL-23 to the immune response to infection must therefore be investigated to assess the role of IL-23

CCL20, also termed macrophage inflammatory protein γ (MIP-γ ), is a chemoattractant for neutrophils and lymphocytes but not monocytes. CCL20 mRNA and protein has been detected in human neutrophils as have transcripts for CCR6, the CCL20 receptor (reviewed by (Schutyser et al., 2003)). Human CCL20 has been shown to have a direct antimicrobial action against several pathogens including streptococci (Yang et al., 2003), although the activity of bovine CCL20 against S. uberis is unknown, it is an interesting finding to explore further.

4.6.2.2 Increased Expression of Toll-Like Receptor 2 (TLR2)

TLR2 gene expression was up-regulated. Bovine TLR2 is a PRR important in binding the peptidoglycan of Gram-positive bacteria (Werling et al., 2006). Following mastitis infection due to various pathogens, TLR2 transcription has been shown to be up-regulated in infected quarters (Goldammer et al., 2004); in NEB (compared to PEB) cows TLR2 transcription also increases in circulating PMNs following intramammary infection with S. uberis (Moyes et al., 2010a). Although TLR2 activation can promote transcription of the cytokines that were also up-regulated (described previously, Section 4.6.2.1), key intermediates (i.e. MyD88) were not. It is therefore likely that increased transcription of TLR2 was in response to the stimulation of the PMNs. However due to the early time point investigated this did not yet equate to increased expression of the receptor, and thus the TLR2 pathway had not yet been activated. This illustrates the caution required when trying to relate transcriptional expression with the resulting pathway activation and protein and functional changes.

4.6.2.3 Altered Transcription of MHC in ST PMNs

Transcription of the Major Histocompatibility Group II (MHCII) transcription was increased whilst MHCI transcription decreased. MHCII is responsible for antigen presentation in professional APCs. Some recent work suggests that PMNs may be able to take up MHCII from apoptotic and necrotic cells though no evidence was found to support constitutive MHCII expression by PMNs (Whale et al., 2006a) which has been suggested by some work with human PMNs (Gosselin et al., 1993).

4.6.3 Bivalent Cations may be Important in Interactions Between S. uberis and the