Use of knockout mice to investigate immunological roles of GM-CSF and GPR18

Abstract

The use of genetically modified mice has long been used as a method for understanding the function of a gene of interest. In this thesis, we aimed to examine the immunological roles of two genes in particular, GM-CSF (Section A) and GPR18 (Section B) through the use of in vivo models and genetically modified mice. GM-CSF is a cytokine with the capacity to promote inflammation in allergic airway inflammation, which is driven by Th2 cells. Because of the importance of Th2 cells in parasite infections, the role of GM-CSF in mice infected with the nematode Nippostrongylus brasiliensis was examined using mice lacking functional GM-CSF (ΔGM-CSF mice), and mice lacking the cytokine receptor common β chain (Δβc mice), the latter being unable to signal in response to GM-CSF and IL-5. ΔGM-CSF mice showed no significant defect in parasite immunity in either primary or secondary infection. By contrast, Δβc mice showed increased parasite burden, with higher numbers of lung larvae after secondary infection and higher numbers of intestinal worms and faecal eggs after both primary and secondary infection. There were increased numbers of circulating eosinophils in the ΔGM-CSF mice, associated with significantly reduced larval numbers in the lungs. These results indicate that GM-CSF is redundant in protection against N. brasiliensis infection, and that the increased susceptibility of Δβc mice to infection is likely to be attributed to the lack of IL-5 signalling in these mice. The results suggest that clinical use of agents that neutralise GM-CSF may not be associated with increased risk of parasite infection. GPR18 is a member of G-protein coupled receptors, a family of seven-transmembrane receptors shown to demonstrate a variety of biological functions. The ligand and physiological functions of this orphan receptor remain unknown. Previous studies have shown that GPR18 is highly expressed in lymphoid tissues. Mice lacking GPR18 in all cells (ΔGPR18 mice), in T cells (GPR18ΔT mice) and B cells (GPR18ΔB mice) were generated. vi Results from this thesis did not detect a role for GPR18 in the normal functioning of B and T cells as shown by phenotyping studies in naïve and immunised GPR18ΔB and GPR18ΔT mice. On the other hand, data from the autoimmune model of experimental autoimmune encephalomyelitis (EAE) model and Klebsiella pneumoniae infection model have shown that GPR18 has a role in the regulation of these immune responses. ΔGPR18 mice were more likely to display symptoms of atypical EAE compared to WT mice, and this was associated with increased IL-17 production. During the K.pneumoniae infection, ΔGPR18 mice had decreased bacterial burden compared to control mice, which again was associated with increased IL-17 production. These experiments indicate that GPR18 has a role in downregulating IL-17 in immune responses.