Transcriptional control of macrophage function in the pig and its relationship to infectious disease susceptibility

The biology of cells of the mononuclear phagocyte system has been studied extensively in the mouse. Studies of the pig as an experimental model have commonly been consigned to specialist animal science journals. This thesis considered some of the many ways that pigs may address the shortcomings of mice as models for the study of macrophage differentiation and activation in vitro, and the biology of sepsis and other pathologies in the living animal. Flow cytometry was used initially to phenotype cells from the porcine lung, peritoneal cavity, blood and bone marrow using the LPS receptor CD14 and the FC receptor CD16, markers frequently employed to differentiate human monocytes into subsets. The expression of SIRP-alpha (SWC3a, CD172a), which is present on all cells of myeloid origin, and the haemoglobin scavenger receptor, CD163 which has previously been used to study monocyte differentiation in the pig was also studied. The findings validated previous work where blood monocytes were divided into subsets on the expression of CD14 and CD163. Furthermore, like human and mouse, pig monocytes also exhibited variation in CD16 expression, having a subset which was CD14hiCD16lo and another which was CD14loCD16hi. A whole genome approach was then used to study the differences between the monocyte subsets in the pig, using monocytes sorted into two populations based on the expression of CD14 and CD163. The gene expression profiles obtained were then compared to publically available data from monocyte subsets in human and mouse. This thesis also investigated the expression of genes that are known to be differentially expressed between human and mouse. To do this gene expression in porcine bone marrow derived macrophages was analyzed across an LPS time course. Like human macrophages, pig macrophages did not induce nitric oxide nor any arginine metabolizing genes in response to LPS. Instead they responded with robust induction of indoleamine 2,3-dioxygenase (IDO) and other enzymes of the tryptophan metabolism pathway such as kynurenine hydroxylase, kynureninase and tryptophan-tRNA synthetase. The tryptophan metabolism pathway has been implicated in sepsis in man and the absence of this pathway in the mouse may be one of the reasons why an adequate rodent model of sepsis has not been developed. The IDO inhibitor 1-methyl-tryptophan (1-MT) has been used to treat mouse macrophages where it had a protective effect after LPS administration. Similar experiments on pig macrophages did not show the same protective effect and induction of key immune genes was increased after treatment with 1-MT suggesting IDO is involved in feedback control of the immune system. With the completion of the genome sequence and the characterisation of many key regulators and markers, the pig has emerged as a tractable model of human innate immunity and disease that should address the limited predictive value of rodents in preclinical studies. This project aimed to address the gap in our knowledge of the control of innate immunity in the pig and provided further evidence that the pig can function as an ideal model to study innate immunity