SAMPLE SIMILARITY

Differential gene expression data produced by edgeR was transformed for the generation of correlation heatmaps and PCA plots.

Figure 34 shows a sample correlation matrix produced from mouse transcriptome data. Both the wild type infected and the wild type uninfected (control) samples appear highly similar (dark red/dark orange) to other samples from the same background and infection status. WT infected samples were between 97.8% and 99.0% similar, with an average of 98.5%; WT uninfected samples were all found to be 99.0% similar to each other. RAG sample transcriptomes appear less closely related (pale orange/yellow) than the wild type, with infected RAG samples ranging from 96.6% to 98.2% in similarity, and uninfected RAG samples ranging from 96.1% to 97.2% . The most dissimilar transcriptomes are displayed in yellow, showing that the wild type infected

107 However, the scale of the difference between transcriptomes is fairly minor, as the least similar transcriptomes still show 0.942 similarity out of a possible 1.000.

Figure 34. Mouse transcriptome Euclidean distance heatmap. Samples that are more similar are indicated with dark red; less similar samples are inicated with dark blue.

Two PCA plots were also generated from the mouse transcriptome data, comparing the variance of the samples the 1st_{, 2}nd _{and 3}rd _{components. The 1}st _{and 2}nd _{components, as seen in figure 35,}

do not appear to differentiate the sample clusters much, indicating that overall gene expression in the samples is similar. However, figure 36 shows that the 3rd _{component accounts for a large}

amount of variance between samples, clearly distinguishing the WT infected and uninfected samples from each other and the RAG samples. RAG infected and uninfected groups are not so easily differentiated, but this is somewhat expected given that, relative to the WT, few genes were found to be differentially expressed between the groups.

108 Figure 35. 1st _{& 2}nd _{PCA components for mouse transcriptome data. WT uninfected samples are}

represented in cyan, WT infected samples are represented in blue, RAG uninfected samples are represented in orange and RAG infected samples are in red.

109 Figure 36. 2nd _{& 3}rd _{PCA components for mouse transcriptome data. WT uninfected samples are}

represented in cyan, WT infected samples are represented in blue, RAG uninfected samples are represented in orange and RAG infected samples are in red.

3.14.1.2 GENE PANELS

Heatmaps displaying fold-changes in expression of key immunology-related genes between mouse samples were generated from edgeR data; all genes listed were significantly differentially expressed between samples (p ≤ 0.05). Panels of genes were produced for CD (cluster of

differentiation) markers genes, cytokines, and chemokines. logFC values are transformed into Z- score by the heatmap.2 function, which smooths and scales the logFC values so that they can more easily be compared between rows.

CD marker genes are used to indicate cell lineage and cell type, for example, distinguishing different types of T-cells or B-cells. Figure 37 shows the panel produced for CD marker genes. Genes related to CD 209 appear significantly more upregulated in all samples when compared with WT infected samples. Colmenares et al. 2002, found that CD 209 acts as a receptor for

Leishmania amastigotes in human dendritic cells. CD5, CD6, CD79 and CD3-related genes all

110 uninfected mice. Depletion of CD5+ B-cells was found to have no effect on the outcome of L.

major infections in different mice strains (Babai et al. 1999). CD79+ B-lymphocytes were found to

be differentially trafficked to the brain in dogs infected with Leishmania chagasi when compared with uninfected dogs (Melo et al. 2009).

Figure 37. A gene panel displaying the fold changes in key CD marker genes between samples. Genes highly upregulated in the first listed sample (L-R: WT infected, WT infected, WT uninfected) are represented in red, while those highly upregulated in the second listed sample (L-R: WT uninfected, RAG infected, RAG uninfected) are represented in blue. Comparisons for which the genes are not differentially expressed are represented with dark grey.

111 Differences in chemokine expression are displayed below in figure 38. Both RAG infected and uninfected samples appear to highly express C-C receptor 3 when compared with WT infected mice. C-X-C receptor 5 is more highly expressed in WT infected samples than in either RAG sample. A number of C-C and C-X-C receptors, such as C-X-C receptors 5 and 2, and C-C receptors 9, 7, and 5, show differing expression between WT infected and uninfected samples, with no overall pattern to expression. Sato et al. 1999 found that mice deficient in C-C R 2 had a reduced interferon gamma response when infected with L. donovani. Additional work on relative

Trypanosoma cruzi found that C-C R 5 is essential for the control of parasite replication and tissue

inflammation (Hardison et al. 2006). C-C R 7 was found to be upregulated in dendritic cells in upon interaction with Leishmania major (Steigerwald and Moll 2005).

Figure 38. A gene panel displaying the fold changes in key chemokine genes between samples. Genes highly upregulated in the first listed sample (L-R: WT infected, WT infected, WT uninfected) are represented in red, while those highly upregulated in the second listed sample (L-R: WT uninfected, RAG infected, RAG uninfected) are represented in blue. Comparisons for which the genes are not differentially expressed are represented with dark grey.

112 The relative differences in expression of cytokines are shown in figure 39 (below). Receptors of the TNF-alpha superfamily are highly expressed in WT samples when compared with RAG, and in WT infected samples when compared with WT uninfected samples. In L. donovani infections, TNF- alpha is known to be critically important in parasite control, due to its involvement in leukocyte recruitment to the liver (Engwerda et al. 2004). Interferon gamma is more highly expressed in WT infected samples than in RAG infected samples, and in WT infected samples than WT uninfected samples. The role of interferon gamma in leishmaniasis is complex; early formative studies in mice, using L. major, found that parasite control was correlated with the presence of interferon gamma, and that the absence was associated with disease progression (Kima and Soong 2013).

Figure 39. A gene panel displaying the fold changes in key cytokine genes between samples. Genes highly upregulated in the first listed sample (L-R: WT infected, WT infected, WT uninfected) are represented in red, while those highly upregulated in the second listed sample (L-R: WT uninfected, RAG infected, RAG uninfected) are represented in blue. Comparisons for which the genes are not differentially expressed are represented with dark grey.

113

3.14.2 LEISHMANIA TRANSCRIPTOME DATA