## 3 Results
### Surface Marker Expression Implies Pathogen-Dependent Subsets of Proinflammatory Monocytes
histolytica_-infected animals, whereas the combination of IRF2 detection with CD74 did not reveal significantly different monocyte subpopulations between the two infection models (Figure 5B). Figure 4: Marked differences in expression of surface markers by Ly6 Chi monocytes after infection with _E. histolytica_ or _L. monocytogenes_ (**A**) Heatmap depicting differential expression of mRNA encoding selected markers on the surface of liver Ly6 Chi monocytes after infection with _E. histolytica_ (_E. his_) or _L. monocytogenes_ (_L. m_). (**B**) Normalized mRNA counts of selected surface marker genes (from transcriptome analysis). (**C**) Percentage of CD14\({}^{+}\), CD38\({}^{+}\), CD74\({}^{+}\), and CD86\({}^{+}\) Ly6 Chi monocytes during the course of infection at the indicated time points post-infection (measured by flow cytometry). (**D**) Histogram and MFI of CD14\({}^{+}\), CD38\({}^{+}\), CD74\({}^{+}\), and CD86\({}^{+}\) Ly6 Chi monocytes during the course of infection. Data in C were pooled from three independent experiments. Data in D are representative of one of these three experiments and all data are presented as the mean \(\pm\) SEM (* \(p\) \(<\) 0.05; ** \(p\) \(<\) 0.01; *** \(p\) \(<\) 0.001; _*** \(p\) \(<\) 0.0001; Mann-Whitney U test). Figure 5: New Ly6 Ch\({}^{\rm{hi}}\) monocyte subsets identified according to expression of selected surface markers and IRF2. (**A**) Gating strategy, histogram, and percentage of IRF2\({}^{+}\) Ly6 Ch\({}^{\rm{hi}}\) monocytes from livers of naïve mice and from _E. histolytica-_ and _L. monocytogenes_-infected mice on day 3 post infecion. (**B**) Gating strategy (exemplary for CD14\({}^{+}\) gated cells) based on the FMO control to determine CD14\({}^{+}\), CD38\({}^{+}\), CD74\({}^{+}\), and CD86\({}^{+}\) monocytes within the IRF2\({}^{+}\) Ly6 Ch\({}^{\rm{hi}}\) monocyte population from naïve mice and from both infection models on day 3 post infection. (**C**) UMAP plots of Ly6 Ch\({}^{\rm{hi}}\) monocytes (including CD14\({}^{+}\), CD86\({}^{+}\), CD74\({}^{+}\), CD38\({}^{+}\)) from naïve mice and from _E. histolytica-_ and _L. monocytogenes_-infected mice (down sampled to 90,000 cells per sample). Six samples per source material were included in the analysis. (**D**) Cluster heatmap table showing surface marker and IRF2 expression by \(\rm{J6 Ch}^{\rm{hi}}\) monocytes, cluster events, and integration of cluster events in the UMAP plot (* \(p<0.05\); ** \(p<0.01\); *** \(p<0.001\); *** \(p<0.0001\); Mann-Whitney U test). After analysis of the distribution of Ly6 Chi monocyte subpopulations by UMAP, initially considering only surface markers, clear demarcation of populations by _L. monocytogenes_ and _E. histolytica_ infection and by naive animals was seen, with overlapping populations of the latter (Figure 5C). When Irf2 was included, 6 distinct clusters were identified (Figure 5D). A distinct IRF2-positive monocyte population and a greater heterogeneity in the area of monocytes from the listeria model and a clear delineation of monocyte populations from naive and _E. histolytica_-infected animals could be visualized in the UMAP analysis after clustering with FlowSOM and Cluster Explorer analysis (Figure 5D). In summary, we found that the combination of antibodies against IRF2 with antibodies against CD14, CD38, or CD86 helped to distinguish proinflammatory Ly6 Chi monocytes from both infection models, supporting the results of the transcriptome study (Figure 3C) in that these monocytes are already in a transitional stage to anti-inflammatory monocytes. CD38+Ly6 Chi Monocytes Produce ROS and Contribute to Monocyte-Dependent Immunopathology during Hepatic Amebiasis  
In addition to the production of proinflammatory cytokines and chemical mediators, activated Ly6 Chi monocytes also express ROS [38]. Based on the higher mRNA expression of genes involved in ROS production and NADPH oxidase (i.e., _Sod1_, _Sod2_, _Ncf1_, _Ncf4_ as well as _Nox2 and Navxed 1_) by Ly6 Chi monocytes from _L. monocytogenes_-infected compared with _E.