## 3 Results
### Monocytes from Both Infection Models Show Significant Differences in Gene Expression  
To gain a deeper understanding of the phenotype of the Ly6 Chi monocyte subset during hepatic amebiasis and listeriosis, 3 and 5 days p.i., CD11b+Ly6C-Ly6 Chi as well as Ly6 Clo monocytes were sorted from infected livers by flow cytometry. The RNA from both populations was extracted and subjected to RNA sequencing (Figure 2A). Figure 2: Transcriptome analysis of monocytes from the livers of mice infected with E. histolytica or L. monocytogenes. **(A)** Gating and mRNA purification strategy for liver-specific Ly6 Chi and Ly6 Clo monocytes from _E. histolytica-_ and _L. monocytogenes_-infected mice. (**B**) VENN diagram of transcriptome analysis of Ly6 Chi and Ly6 Clo monocytes shows significantly regulated genes of Ly6 Chi and Ly6 Clo monocytes from both infection models. PANTHER GO-slim analysis was performed to identify biological pathways involving significantly regulated genes in both monocyte populations. **(C)** Principal component analysis of transcriptomic data from Ly6 Chi and Ly6 Clo monocytes from naïve and _E. histolytica-_ and _L. monocytogenes_-infected mice. Genes showing a significant difference in expression (adjusted \(p\) value \(<\) 0.05) in monocytes obtained from the two infection models at d3 p.i. were included in the analysis. 5486 genes were differentially expressed in Ly6 Chi monocytes from _E. histolytica_- and _L. monocytogenes_-infected mice, 54 genes were differentially expressed in Ly6 Clo cells from both models, and 194 genes were differentially expressed in both Ly6 Chi and Ly6 Clo monocytes in both infection models (Figure 2B). PANTHER GO analysis of genes differentially expressed in Ly6 Chi monocytes revealed that a small percentage of genes was included in the GO terms \"immune systems process\" (GO:0002376) and \"response to stimulus\" (GO:0050896) (Figure 2B). By contrast, we also observed the differential expression of genes associated with GO terms related to immune responses in anti-inflammatory Ly6 Clo monocytes (see Figure S2A-D for a detailed list of highly regulated genes and GO terms). Principal component analysis of Ly6 Chi and Ly6 Clo monocytes revealed clustering into different groups (Figure 2C). The day post infection did not affect the grouping, however there was a clear difference between Ly6 Chi monocytes from _L. monocytogenes_-infected mice (cluster A) and Ly6 Chi monocytes from _E. histolytica_-infected mice (cluster B): the latter clustered together with Ly6 Chi monocytes from naive mice. Additional differences between Ly6 Chi and Ly6 Clo from the _E. histolytica_ and the listeria model are also depicted by a volcano-plot and a heat map (see Figure S2E,F). For example, differences include genes involved in proinflammatory IFN-\(\gamma\) related signaling (i.e., _Iigp1_, _Gbp2_, and _Gbp8_) in Ly6 Chi monocytes from _L. monocytogenes_-infected mice, but the upregulation of genes involved in anti-inflammatory, phagocytic, or metabolic processes (_Cx3 cr1_, _Mfge8_, _Hpgd_) in Ly6 Chi monocytes from _E. histolytica_-infected mice. Overall, proinflammatory Ly6 Chi monocyte in both infection models differed significantly at the transcriptional level. Ly6 Chi monocytes from _L. monocytogenes_ infected mice presented an upregulated expression of a large number of genes, including genes with a potential function in their antibacterial response. In contrast, changes in the gene expression of Ly6 Chi monocytes from _E. histolytica_ infected mice were less pronounced and a large part of their expression profile was shared with Ly6 Chi monocytes from naive mice. Ly6 Chi Monocytes from L. monocytogenes-Infected Mice Have an Activated Phenotype and Lower M2 Polarization at the Transcriptional Level Than Ly6 Chi Monocytes from _E. histolytica_-Infected Mice  
By focusing on transcription factors with putative relevance to the polarization of monocytes towards classically activated M1 or alternatively activated M2 macrophages, we found that during infection with _L.