## 3 Results
### Surface Marker Expression Implies Pathogen-Dependent Subsets of Proinflammatory Monocytes  
Next, we analyzed the differential expression of genes encoding surface markers that may be useful for further subdivision of Ly6C\({}^{\mathrm{hi}}\) monocytes. We found significant upregulation of genes encoding _Ly6c2_, _Cd38_, and _Cd74_ in Ly6C\({}^{\mathrm{hi}}\) monocytes from _L. monocytogenes_-infected mice, whereas higher expression of _Cd14_ was characteristic for Ly6C\({}^{\mathrm{hi}}\) monocytes from _E. histolytica_-infected mice (Figure 4A,B). The expression of these genes at the protein level on Ly6C\({}^{\mathrm{hi}}\) monocytes from both infection models was validated by flow cytometry. Although not significantly regulated at the transcriptional level, we included the analysis of the co-stimulatory receptor CD86 in the panel to further describe M1 polarization of inflammatory monocyte [37]. As in monocytes from naive mice and in agreement with the transcriptomic data, we found that the percentage of CD14-expressing Ly6C\({}^{\mathrm{hi}}\) monocytes was higher and remained higher from d1 p.i. on following _E. histolytica_ infection than in Ly6C\({}^{\mathrm{hi}}\) monocytes from _L. monocytogenes_-infected mice, the latter initially decreased but increased from d5 of infection (Figure 4C). This picture was mirrored by lower MFIs for CD14 on Ly6C\({}^{\mathrm{hi}}\) monocytes derived from the _L. monocytogenes_ model than on monocytes from the _E. histolytica_ model (Figure 4D). When compared with that in uninfected animals, expression of CD38 increased significantly in both models shortly after infection. However, expression was significantly stronger on monocytes from the _L. monocytogenes_ infection model (Figure 4D). Initially, the percentage of CD74-expressing monocytes remained the same as that in naive mice following _E. histolytica_ infection, but decreased on d5 p.i. (Figure 4C). During _L. monocytogenes_ infection, expression and MFI of CD74 increased on d1 p.i., but then decreased to the level observed in uninfected animals as infection progressed (Figure 4C,D). The expression and MFI level of CD86 also decreased significantly over time during infection with _L. monocytogenes_, and to a lesser extent this was also true for monocytes in the _E. histolytica_ model (Figure 4C) (expression on monocytes from spleen, blood, bone marrow see Figure S3A-C). Overall, the results of the transcriptome analysis of surface marker expression, with the exception of CD74, are reflected at the protein level in vivo. Furthermore, they suggest that CD14 in combination with CD38 may be additional putative markers for a Ly6C\({}^{\mathrm{hi}}\) monocyte subset that is very different from the conventional proinflammatory Ly6C\({}^{\mathrm{hi}}\) monocyte subset. To further differentiate Ly6C\({}^{\mathrm{hi}}\) monocytes, we selected molecules that were shown by transcriptome analysis to be highly expressed by Ly6C\({}^{\mathrm{hi}}\) monocytes after _E. histolytica_ infection (_Mafb_) or _L. monocytogenes_ infection (_Irf2_) (Figure 3B). However, MAFB1 protein was excluded from further analysis since less than 1% of Ly6C\({}^{\mathrm{hi}}\) monocytes expressed the protein (data not shown). As seen for mRNA, protein expression of IRF2 was significantly stronger in Ly6C\({}^{\mathrm{hi}}\) monocytes after _L. monocytogenes_ infection than in monocytes from naive or _E. histolytica_-infected mice (Figure 5A). On day 3 after infection, when the most severe symptoms in both models appear, the combination of antibodies against IRF2 and CD14 resulted in the detection of a significantly increased monocyte population after _E. histolytica_ infection. When IRF2 detection was combined with the detection of CD38 and CD86, Ly6C\({}^{\mathrm{hi}}\) monocytes after infection with _L. monocytogenes_ were significantly different from those of naive or _E.