15 kDa Granulysin versus GM-CSF for monocytes differentiation: analogies and differences at the transcriptome level

Abstract

Background: Granulysin is an antimicrobial and proinflammatory protein with several isoforms. While the 9 kDa isoform is a well described cytolytic molecule with pro-inflammatory activity, the functions of the 15 kDa isoform is less well understood. Recently it was shown that 15 kDa Granulysin can act as an alarmin that is able to activate monocytes and immature dendritic cells. Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) is a growth factor widely used in immunotherapy both for in vivo and ex vivo applications, especially for its proliferative effects.

Methods: We analyzed gene expression profiles of monocytes cultured with 15 kDa Granulysin or GM-CSF for 4, 12, 24 and 48 hours to unravel both similarities and differences between the effects of these stimulators.

Results: The analysis revealed a common signature induced by both factors at each time point, but over time, a more specific signature for each factor became evident. At all time points, 15 kDa Granulysin induced immune response, chemotaxis and cell adhesion genes. In addition, only 15 kDa Granulsyin induced the activation of pathways related to fundamental dendritic cell functions, such as co-stimulation of T-cell activation and Th1 development. GM-CSF specifically down-regulated genes related to cell cycle arrest and the immune response. More specifically, cytokine production, lymphocyte mediated immunity and humoral immune response were down-regulated at late time points.

Conclusion: This study provides important insights on the effects of a novel agent, 15 kDa granulysin, that holds promise for therapeutic applications aimed at the activation of the immune response.

Figure 1

Gene expression analysis of monocytes cultured with GM-CSF or 15 kDa Granulysin. a) Principal component analysis of all samples based on the entire dataset (33757 genes); b) Dendrogram of the unsupervised cluster of 9951 genes that were present in at least 22 samples and whose expression differed in at least 5 samples by more than 1.75-fold from the median

Figure 2

Monocyte genes induced by both GM-CSF and 15 kDa granulysin. Hierarchical clustering of the 3191, 2416, 1534 and 1738 genes induced by both factors at 4 (a), 12 (b), 24 (c) and 48 (d) hours respectively (p-value < 0.001, FDR < 0.1) and the related GO analysis. The hierarchical clustering was T0 corrected; the black bar indicates T0 monocytes, the fuchsia bar GM-CSF-treated monocytes, and the light blue bar Granulysin-treated monocytes. GO analyses were made with DAVID. The bars indicate -Log10 of the p-value of the overrepresentation of genes induced in each GO family. Green bars indicate down-regulated genes, while red bars indicate up-regulated genes. The orange line indicates the threshold of statistical significance (p-value = 0.05)

Figure 3

Monocyte genes specifically induced by GM-CSF. Hierarchical clustering of the 98, 768, 756 and 467 genes induced by GM-CSF at 4 (a), 12 (b), 24 (c) and 48 (d) hours respectively (p-value < 0.001, FDR < 0.1) and the related GO analysis. The expression of each of these genes differed in GM-CSF treated monocytes compared to both time 0 monocytes and cells treated with Granulysin at the same time points. The hierarchical clustering was T0 corrected; the black bar indicates T0 monocytes, the fuchsia bar GM-CSF-treated monocytes, and the light blue bar Granulysin-treated monocytes. GO analyses were made with DAVID. The bars indicate -Log10 of the p-value of the overrepresentation of induced genes in each GO family. The green bars indicate down-regulated genes, while the red bars indicate up-regulated genes. The orange line indicates the threshold of statistical significance (p-value = 0.05)

Figure 4

Monocyte genes specifically induced by 15 kDa Granulysin. Hierarchical clustering of the 152, 498, 429 and 598 genes induced by 15 kDa Granulysin at 4 (a), 12 (b), 24 (c) and 48 (d) hours respectively (p-value < 0.001, FDR < 0.1) and the relative GO analysis. The expression of each of these genes differed in Granulysin treated monocytes compared to both time 0 monocytes and cells treated with GM-CSF at the same time points. The hierarchical cluster analysis were T0 corrected, the black bar indicates T0 monocytes, the fuchsia bar GM-CSF-treated monocytes, and the light blue bar Granulysin-treated monocytes. GO analyses were made with DAVID. The bars in the GO analysis indicate -Log10 of the p-value of the overrepresentation of the induced genes for each GO family. The green bars indicate down-regulated genes, while red ones indicate up-regulated genes. The orange line indicates the threshold of statistical significance (p-value = 0.05)

Figure 5

DC related Biocarta pathway level analysis. The percentages of genes statistically induced by each treatment and at each time point compared to time 0 monocytes are displayed in a grid (p-value < 0.05, FDR < 0.15). The position of each time-treatment in the grid is described in the bottom right corner, whereas the bottom left indicates the scale of intensity of the colors in the grid.