## 3 Results and Discussion  
### Cytokine Response of Normal Urothelial Cells  
To characterize the effect of different _E. coli_ strains on the NPU cells of the in vitro model upon infection, we analyzed NPU cell cytokine production using cytokine multiplex immunoassay. For the infection of the in vitro model, we used a diverse set of _E. coli_ strains, which consisted of 24 _E. coli_ strains (10 human UPEC strains designated as DL, isolated from urine and blood of patients with UTI; 10 human commensal strains designated as BJ, isolated from feces of healthy volunteers), as well as 4 well-described human _E. coli_ strains (2 UPEC strains J96 and 536, commensal strain SE15 and laboratory strain MG1655). The results of the multiplex immunoassay for assessing the release of multiple cytokines into the cell culture supernatant after infection of the NPU cells and the obtained fold changes in the amounts of different cytokines relative to the negative control (NC) (in vitro model without bacteria) are presented in Figure 1. In Figure 2, the obtained average cytokine concentrations for each strain are plotted and Table 2 shows the calculated mean values \(\pm\) SD for the cytokine concentrations in relation to the type of _E. coli_ strain used (UPEC vs. commensal). The cytokine concentrations determined with the MagPix instrument and the calculated fold-changes can be found in the Supplementary Table S2A,B, respectively. As shown in Table 2, the NPU cells produced higher levels of cytokines in response to infection with UPEC strains, with the exception of IL-8, than in response to infection with the commensal strains, albeit the difference was not statistically significant. This is actually not surprising, since in our previous study [6], in which we used this model to assess the pathogenicity of strains, some DL strains, although isolated from the urine of UTI patients, had lower pathogenic potential, while some BJ strains, although isolated from the feces of healthy individuals, had higher pathogenic potential than would have been expected based on their isolation source. It is known that strains with a high virulence potential can be found in the intestinal microbiota of healthy individuals [20], while under certain circumstances (e.g., in immunocompromised patients), commensal _E. coli_ lacking virulence-associated genes can cause disease [2]. Figure 1: Fold change in total cytokine production by NPU cells in response to infection with different _E. coli_ strains relative to the value of cytokine production in the negative control (NC), (NC—negative control, not infected NPU cells). The experiment was performed in duplicates, fold change for each cytokine was calculated by dividing the average value of the concentration of each analyte measured by the instrument from both duplicates in the _E. coli_ infection assay with the average value of both duplicates in the negative control. Figure 2: Average value of the cytokine concentrations produced by NPU cells in the _E. coli_ infection assay. The experiment was performed in duplicates and the average value was calculated. Data for IFN-\(\alpha\) and IFN-\(\gamma\) are not shown, as cytokine production of these two analytes was below the detection limit in all strains. In the case of both uropathogenic _E. coli_ strains (J96 and 536) (Figure 1), higher levels of cytokine production were observed, compared to lower levels of cytokine production following infection with the non-pathogenic commensal strain (SE15). Compared to the control without bacteria (NC), strains J96 and 536 caused increased production of IL-10, IL-8, TNF-\(\alpha\) and IL-12p40. In addition, the strain J96 caused also an increase in the IL-1\(\beta\) production. Infection of the model with two non-pathogenic strains caused elevated production of TNF-\(\alpha\), as well as IL-12p40 (SE15), IL-10 and IL-12p40 (MG1655).