## 3 Results
### Monocytes from Both Infection Models Show Significant Differences in Gene Expression
monocytogenes_, the activation and development of proinflammatory monocytes is characterized by factors, such as _Irf1_, _Irf2_, _Irf204_, _Batt2_, _Mndal_, and _Irf7_[24; 25; 26; 27; 28] (Figure 3A). By contrast, during _E. histolytica_ infection, Ly6 Chi monocytes are characterized by the upregulation of transcription factors _Mafb_, _Hes1_, _Fos_, and _Tsc22d3_ (Figure 3A), which contribute to an anti-inflammatory and regenerative phenotype [29; 30; 31; 32; 33]. Time-course analysis of the expression data shows that, in addition to other genes, a selection of the above factors exhibits significantly different expression patterns between the two infection models, starting as early as d3 p.i. and remaining different until d5 p.i. (Figure 3B). Fate mapping and transfer approaches have revealed the marked plasticity of proinflammatory monocytes and identified distinct routes of monocyte polarization. Such studies suggest the existence of novel monocyte subsets, such as \"Ly6 Chi to Ly6 Clo\"-converting monocytes, nMO, dCMO, and _Cxcl10_+ and _Saa3_+ monocytes [12; 13; 14]. According to this classification, the Ly6 Chi monocytes triggered by _E. histolytica_ infection would appear to belong to the Ly6 Chi to Ly6 Clo-converting monocyte subset. By contrast, with the exception of _Csf1_ and _Ly6c2_ (_Ly6C_), the respective genes were downregulated in monocytes from the _L. monocytogenes_ infection model (Figure 3C) [35]. Relevant genes related to nMO development were upregulated in monocytes from _L. monocytogenes_-infected mice (Figure 3C). When we considered the genes that define dCMO, we found an intermediate picture. MHC-II related genes were upregulated in monocytes from _L. monocytogenes_-infected mice. However, some other hallmark genes of dcMO (i.e., _Fl13_, _Pid1_, and _Hpgd_) were strongly downregulated. Moreover, with the exception of _illb_, signature genes of _Cx110\({}^{*}\)_ and _Saa3\({}^{+}\)_ monocytes were also upregulated in Ly6 Ch\({}^{\text{th}}\) monocytes from _L. monocytogenes_-infected mice, indicating a broad repertoire of putative new Ly6 Ch\({}^{\text{th}}\) monocyte subsets during this type of infection (Figure 3C). However, several other relevant genes involved in proinflammatory or antiinflammatory immune processes are additionally upregulated in monocytes following _E. histolytica_ infection. Among these are _Cd14_, _Trem2_, a negative immune regulator and marker for M2 polarization [36], as well as _Arg1_ and _Arg2_, further indicating the transition from pro-to anti-inflammatory monocytes (Figure 3D). In summary, we were able to assign monocytes from both infection models to recently suggested subgroups with a more proinflammatory and activated phenotype in the  
Figure 3: Early M2 polarization and a less activated mRNA expression profile in Ly6 Ch\({}^{\text{th}}\) monocytes from _E. histolytica_-infected compared to _L. monocytogenes_-infected mice. (**A**) Heat map showing selected regulated genes (p\({}_{\text{ndj}}\) < 0.05; foldchange > 2) involved in monocyte/macrophage polarization and activation of Ly6 Ch\({}^{\text{th}}\) monocytes derived from the livers of _E. histolytica_ (_E. his_)- and _L. monocytogenes_ (L. m)-infected. (**B**) Time-course analysis of mRNA encoding M2 transcription factors and interferon-regulated/activated factors. (**C**) Heatmap showing classification of Ly6 Ch\({}^{\text{th}}\) monocytes derived from the livers of both infection models according to expression of genes involved in conversion from Ly6 Ch\({}^{\text{th}}\) to Ly6 Ch\({}^{\text{th}}\), neutrophil-like, dendritic cell-like, or _Cx10\({}^{+}\)_ and _Saa3\({}^{+}\)_-like monocytes. (**D**) Heat map of selected genes upregulated during ALA. All heatmaps were designed using the online tool “heatmapper” [34]. _L. monocytogenes_ model (nMO; dcMO; _Cxcl10\({}^{+}\)_ and _Saa3\({}^{+}\)_ monocytes) and a scarcely activated phenotype in the parasite model characterizing Ly6C\({}^{\mathrm{hi}}\) to Ly6C\({}^{\mathrm{lo}}\) converting monocytes.