## 4 Discussion
Altered genes include _Nr4a1_, a major transcription factor responsible for transition of Ly6C\({}^{\rm hi}\) to Ly6C\({}^{\rm lo}\) and survival of Ly6C\({}^{\rm lo}\) cells [30; 46], while _Csf1_ and _Ly6c2_, promoting survival and activation of Ly6C\({}^{\rm hi}\) monocytes, were downregulated [35; 47]. Their further polarization towards anti-inflammatory M2 macrophages [9] is supported by the upregulation of _MafB_, _Nr4a1_, or _Fos [29; 30; 33]. As already evident from the cluster analysis, Ly6C\({}^{\rm hi}\) monocytes from the parasite model were overall quite similar to those from naive animals. However, some genes were differentially regulated, e.g., _Arg1/Arg 2_, further suggesting an ongoing polarization into an anti-inflammatory M2 phenotype. In contrast, monocytes from the _L. monocytogenes_ infection model were characterized by a classical proinflammatory, interferon-driven transcription factor-like profile (i.e., _Mandal_, _Ifi204_ and _Inf2_) [25; 28], pointing towards an M1 phenotype [9]. A suitable antibody panel to distinguish bona fide inflammatory Ly6C\({}^{\rm hi}\) monocytes from non-inflammatory Ly6C\({}^{\rm hi}\) monocytes and to study their dynamics of Ly6C\({}^{\rm hi}\) monocytes in both infection models, was developed by selecting several surface markers that had emerged from transcriptome analysis. These included activation markers such as CD38 [39; 40; 41], CD74, a receptor for proinflammatory macrophage migration inhibitory factor involved in cell proliferation and antigen presentation [48; 49] as well as CD14, co-receptor for several Toll-like receptors involved in proinflammatory processes [50]. While CD38 and CD74 were more highly expressed in Ly6C\({}^{\rm hi}\) monocytes from the _L. monocytogenes_ infection model, CD14 was the only surface marker with higher expression in Ly6C\({}^{\rm hi}\) monocytes from the parasite model. Although not differentially expressed on the mRNA level, we included CD86 as an additional proinflammatory M1 marker [41]. The expression of CD14 and CD74 on Ly6C\({}^{\rm hi}\) monocytes from the parasite model was very similar to those from naive mice, whereas CD14 in the _L. monocytogenes_ model initially decreased and only increased toward the end of the disease course. CD38 was more highly expressed on monocytes from the parasitic model during the early phase of infection, thus describing inflammatory Ly6C\({}^{\rm hi}\) before transitioning to Ly6C\({}^{\rm lo}\) cells. As expected, CD38, CD74, and to a lesser extent CD86 remain highly expressed in Ly6C\({}^{\rm hi}\) monocytes from the _L. monocytogenes_ model over time, suggesting that these markers are useful for further distinguishing proinflammatory Ly6C\({}^{\rm hi}\) monocytes. Next, we included the transcription factor IRF2 within the panel. IRF2 in combination with CD14 best distinguishes the Ly6C\({}^{\text{hi}}\) population in the ameba and listeria model from the population in naive mice at least on day 3 post infection, the peak of liver pathology in these models (Figure 5B). Subsequent UMAP analysis based on the designated surface markers confirmed clear delineation of Ly6C\({}^{\text{hi}}\) monocyte populations between naive, _E. histolytica_- and _L. monocytogenes_-infected mice, as well as the highest diversity of proinflammatory monocyte subpopulations from the _L. monocytogenes_ model. Finally, we used ROS production as a hallmark of proinflammatory monocyte activation [38]. In contrast to the _E. histolytica_ model, where it remained stable, we observed a continuous increase in ROS-producing Ly6C\({}^{\text{hi}}\) monocytes expressing CD38 in the listeria model. Up-regulation of CD38 on monocytes during infection with listeria has been described previously [39]. Interestingly, genetic deletion of CD38 resulted in increased accumulation of inflammatory monocytes in the liver but not in the spleen and was associated with higher susceptibility to listeria infection, as observed during genetic deletion of _Ccr2_[23; 39].