Lysozyme-Induced Transcriptional Regulation of TNF-α Pathway Genes in Cells of the Monocyte Lineage

Abstract

Lysozyme is one of the most important anti-bacterial effectors in the innate immune system of animals. Besides its direct antibacterial enzymatic activity, lysozyme displays other biological properties, pointing toward a significant anti-inflammatory effect, many aspects of which are still elusive. Here we investigate the perturbation of gene expression profiles induced by lysozyme in a monocyte cell line in vitro considering a perspective as broad as the whole transcriptome profiling. The results of the RNA-seq experiment show that lysozyme induces transcriptional modulation of the TNF-α/IL-1β pathway genes in U937 monocytes. The analysis of transcriptomic profiles with IPA^® identified a simple but robust molecular network of genes, in which the regulation trends are fully consistent with the anti-inflammatory activity of lysozyme. This study provides the first evidence in support of the anti-inflammatory action of lysozyme on the basis of transcriptomic regulation data resulting from the broad perspective of a whole-transcriptome profiling. Such important effects can be achieved with the supplementation of relatively low concentrations of lysozyme, for a short time of exposure. These new insights allow the potential of lysozyme in pharmacological applications to be better exploited.

Keywords: IL-1β; RNA-sequencing; TNF-α; inflammation; lysozyme; proteome profiling; whole transcriptome profiling.

Figure 1

Differentially expressed genes in U937 cells treated with lysozyme 15 μg/mL for 1 h and analysed immediately at the end of the treatment (1 h, a) or 2 h after the end of the treatment (1 h + 2, b), or treated for 24 h and analysed immediately at the end of the treatment (24 h, c). Fold change values in respect of parallel control samples are shown in ascending order. Complete gene expression data is available in Supplementary Table S1.

Figure 2

Time-trends of the three genes differentially expressed recurring at any time of analysis in U937 cells.

Figure 3

Top ten canonical pathways (a) and top ten up-stream regulators (b) detected as altered by IPA^® analysis. In (a) the ‘ratio’ value is the ratio between altered genes and total genes in the pathway, and in (b) bars represent the pathway z-score, the red line indicates the p-value of the prediction.

Figure 4

IPA^® analysis of signalling pathways of the genes differentially expressed upon lysozyme treatment. U937 cells were treated with lysozyme 15 μg/mL for 1 h and analysed immediately at the end of the treatment (1 h). Down-regulated genes by lysozyme treatment are marked in green, up-regulated genes are marked in red, orange colour indicates the prediction of an activation, blue colour the prediction of an inhibition; continuous line indicates direct relationships, dashed line indicates an indirect relationship (one or more intermediates), yellow colour indicates relationships non-consistent with the state of the down-stream molecule, grey colour indicates an unpredicted effect.

Figure 5

LC-MS/MS (liquid chromatography-tandem mass spectrometry) analysis, volcano plots show the differences of protein abundance comparing the control cells at 24 h + 24 vs. 1 h + 24 (a), and lysozyme-treated cells at 24 h + 24 vs. 1 h + 24 (b). Dashed lines mark the threshold of statistical significance with FDR < 0.05.

Figure 6

Correlation between the differential expression of proteins and of mRNAs in the control 24 h + 24 versus control 1 h + 24 (a) and in LYS 24 h + 24 versus LYS 1 h + 24 (b) in U937 cells.