Anna Düvel: Investigations on the PGE2-mediated differentiation of bovine monocytes into macrophages
Since mastitis is the consequence of a galactogen infection it was asked whether the teat as the distal compartment of the udder is mainly involved in the course of the infection, and whether this region is a prominent checkpoint for adaptive and innate immune mechanisms affecting the inflammatory process in the udder parenchyma.
Since these mechanisms are initiated and regulated by immune cells and since a deeper analysis of teat- resident leukocytic cells is lacking, the presence and distribution of cells in the teat was histologically analysed.
A particular point of interest was the demonstration of macrophages, which control a wide variety of immune mechanisms being sensor and effector cells.
Mast cells, neutrophilic granulocytes, lymphocytes, plasma cells and macrophages could be demonstrated in different relative frequencies in subepithelial and perivascular tissue of the Fürstenberg’s rosette and the teat cistern in teats of healthy heifers and lactating cows.
Regarding all leukocyte subpopulations, heifers showed the highest relative frequencies.
Compared to healthy lactating cows, experimentally E. coli infected animals (72 h or 240 h after intra-cisternal LPS application) did not show a different distribution or a different pattern of teat resident immune cell populations except for cows infected with E. coli 240 hours after LPS application where more neutrophils showed up in the tissue.
In summary, it seems that the onset of lactation rather than the more frequent contact with pathogens is the reason for a changed, in general reduced occurrence of immune cells in the teat tissue.
In all analyzed animals macrophages could be detected in the Fürstenberg’s Rosette as well as in the teat cistern. Mainly in adult lactating animals, they were present in equal frequency in the Fürstenberg’s rosette and the teat cistern.
In order to find evidence for functionally different macrophage types in healthy lactating cows, immunohistology was performed with antibodies specific for S100A8/A9 (MAC387,
indicating classical macrophages) and specific for CD163 (mAb AM-3K, indicator for alternative macrophages).
Staining of sequential sections allowed for the detection of MAC387+/AM3-K-, MAC387 -/AM-3K+ as well as MAC387+/AM-3K+ macrophages. In subepithelial tissue (Fürstenberg’s Rosette), more MAC387+/AM3-K- macrophages were detectable, while MAC387-/AM-3K+ were predominant in perivascular tissue. In the teat cistern, both populations showed an equal perivascular and subepithelial distribution.
The phenotypic macrophage heterogeneity suggested that host factors may be significantly involved in the differentiation of immigrating monocytes to macrophages.
A transcriptome analysis of teat tissue, 4 h after experimental inoculation of the udder with E.
coli, the cyclooxygenase-2 (COX2) revealed as the strongest regulated enzyme of the arachidonic acid metabolism pathway.
Therefore, prostaglandin E2 was chosen as a host factor in order to analyze its role in the in vitro differentiation of macrophages.
Bovine blood monocytes were separated by MACS and differentiated in the presence of PGE2 (1x10-12 - 1x10-6 mol/l) (PGE2-MdM) or absence of PGE2 (MdM) for seven days to macrophages. In unstimulated and LPS-stimulated cells the mRNA-expression of chemokines (CXCL8, CCL20, CCL5), Cytokines (IL-1β, TNF-α, IL-10, IL-12), the transcription factor PPARγ and the long pentraxin 3 (PTX3) was quantified by real time PCR.
MdM showed a base line expression of IL-10, CCL5, CXCL8 and PPARγ. PGE2-MdM (1x10-6 mol/l) showed a significantly lower expression of IL-10, and PPARγ. The LPS stimulation resulted in a significant upregulation of all tested genes, except for IL-12. PGE2 -MdM showed a significantly reduced augmentation of the mRNA expression of IL-10, IL-1ß, TNF-α, CCL5, CCL20 and PTX3. Interestingly, the expression of CXCL8 was not influenced in PGE2-MdM.
These results show that the endogenous host factor PGE2 affects the expression of pro- and anti-inflammatory molecules of macrophages and that in vitro PGE2 can contribute to the functional polarization of macrophages. PGE2-MdM are similar to so-called inhibited macrophages.
Since the expression of PPARγ and its dependent regulated gene PTX3 was down regulated also, it is not likely that PGE2 MdM are mainly regulatory, anti-inflammatory macrophages.
To test whether PGE2 supports the differentiation of a special macrophage phenotype, the expression of S100A8/A9 and CD163 was analyzed by immunofluorescence where 80% of the macrophages expressed S100A8/A9 (MAC387+) and 40% expressed CD163 (AM-3K+).
High and low stained cells could be clearly discriminated. Thus, the in vitro generated macrophages turned out to be phenotypically heterogeneous. In PGE2-MdM, only 23%
MAC387+ and 16% AM-3K+ cells were detectable. The relation between MAC387High and AM-3KHigh MdM was 1.9. PGE2-MdM showed a relation of 3.7.
This showed that PGE2, if present during the differentiation of macrophages, not only has inhibitory but also selective effects on gene and product expression in macrophages. It could be demonstrated that this mediator, which is produced by different cells after contact with pathogens, is able to control the differentiation of monocytes to different macrophage phenotypes.
Whether the macrophage types found in the teat have different functional capacities has to proven in future studies.
The possibility to control the differentiation of macrophages with selected factors in a well-directed way is supposed to be the basis for a prophylactic immune modulation regime in cows. The aim is to prepare the teat in a way that functionally adequate macrophages are present when bacteria are invading the udder.