## 3 Results  
Different Recruitment and Localization of Ly6C\({}^{\mathrm{hi}}\)CD11b\({}^{+}\) Monocytes in the Liver following Infection with E. histolytica or L. monocytogenes  
Proinflammatory Ly6C\({}^{\mathrm{hi}}\) monocytes exhibit opposite functions in murine models for _E. histolytica_ and _L. monocytogenes_ liver infection. In the former, their absence resulted in the amelioration of parasite-dependent liver damage, whereas in the listeria model it exacerbated liver pathology [16; 23]. To better understand the dynamics of monocyte recruitment, we examined hepatic protein concentrations of chemokines involved in these processes such as CCL2 and CCL3. We found a significant increase in CCL2 levels at d3, and an increase in CCL3 levels at d3 and d5 following parasitic infection (Figure 1A). During _L. monocytogenes_ infection, CCL2 levels were higher and increased already at d1 post infection (p.i.), while CCL3 levels were lower than during parasitic infection or in naive mice (Figure 1A). Additional cytokine analysis revealed significantly elevated expression of IL-1B, IL-10, and IL-13 during _E. histolytica_ infection, as well as increased expression of TNF-\(\alpha\) and IFN-\(\gamma\) during _L. monocytogenes_ infection (Figure S1A).  
To determine monocyte recruitment, we isolated leukocytesproes from infected livers and measured the percentage of CD11b+Ly6 Chi and CD11b+Ly6 Clo monocytes by flow cytometry. At d3 p.i., the CD11b+Ly6 Chi monocyte populations increased in both infection models, whereas the CD11b+Ly6 Clo monocyte population increased only following _E. histolytica_ infection (Figure 1B; absolute numbers see Figure S1B,C). Staining of paraffin-embedded liver sections from both models (d3p.i.) with hematoxylin and eosin (H&E), anti-CD11b, and anti-Ly6C revealed accumulation of CD11b+ and Ly6C+ cells in a dense margin around the central amebic abscess (Figure 1C) while CD11b+ and Ly6C+ cells accumulated in the center of typical _L. monocytogenes_-induced granulomas (Figure 1C; controls see Figure S1D).  
Figure 1: Recruitment of CD11b+Ly6 Chi and Ly6 Clo monocytes to the liver during infection with _E. histolytica_ and _L. monocytogenes_, and protective properties of Ly6 Chi monocytes against _E. histolytica_ infection. (**A**) Expression of CCL2 and CCL3 in liver lysates after intrahepatic _E. histolytica_ (2 \(\times\) 10\({}^{5}\)) and systemic _L. monocytogenes_ (2 \(\times\) 10\({}^{4}\)) infection, measured in a multiplex cytokine assay. (**B**) Gating scheme and percentages of CD11b+, CD11b+ Ly6 Chi, and Ly6 Clo monocytes in the liver following _E. histolytica_ or _L. monocytogenes_ infection. (**C**) Liver sections from _E. histolytica_- and _L. monocytogenes_-infected mice were stained with H&E, anti-CD11b (EPRI344), and anti-Ly6C (ER-MP20) on day 3 p.i. (**A–C**) One representative experiment of three is shown. (* \(p\) < 0.05; **p_ < 0.01; Mann-Whitney U test).  
In both infection models, the increase in CCL2 expression led to an increase in the proportion of Ly6 Chi monocytes in the liver, but with different localization in the affected tissue. The protective effect of monocytes from the listeria model already suggests heterogeneity of Ly6 Chi monocytes between the two infections.