## 3 Results and Discussion
### Cytokine Response of Normal Urothelial Cells
The obvious difference between the pathogenic and non-pathogenic strains was observed in the IL-1\(\beta\) release (a high increase in IL-1\(\beta\) in the case of J96) and the higher overall number of significantly increased levels of different cytokines. The highest increase in the amount of any cytokine relative to NC was in the case of TNF-\(\alpha\), after infection of NPU cells with strains BJ30 and DL18. Considerable high fold-change in the amounts of cytokines IL-10 and IL-4 was also observed. Expression of IL-10 is highly activated during urinary infections due to its involvement in multiple biological pathways and importance in the early control of bacteria that cause urinary infections. Measurements of the concentration of IL-10 produced in individuals with cystitis showed an increase in IL-10 concentration compared to the IL-10 levels in individuals without infection, both in urine (over 40 pg/mL) and in plasma [21]. In our study, strain DL102 triggered IL-10 production even above 60 pg/mL. Pro-inflammatory cytokine IL-1\(\beta\) production is also known to be induced in epithelial cells of the urinary tract by UPEC [22]. In our model, strains DL18 and DL102 induced the greatest production of IL-1\(\beta\), reaching concentrations above 6 pg/mL (Figure 2). An IL-1\(\beta\) production of above 6 pg/mL was also detected after infection with the strain BJ30, which also exhibited a similar level of pathogenicity as DL18 and DL102 in our previous study [6]. Elevated production of IL-4 by NPU cells was induced predominantly by UPEC strains. IL-4 is involved in modulating the inflammatory responses to infection, for example, it is involved in the pathogenesis of asthma [23], allergic reactions [24] and other type 2 immune responses [25]. The urothel response to UPEC infection in vivo is also secretion of cytokine IL-6 [26]. It is known that some bacterial structures induce production of IL-6, for example P-fimbirae [27] or bacterial LPS [4], and UPEC can evade the host's immune response by suppressing the secretion of IL-6, which presents a survival advantage of UPEC [28]. In concordance, almost all UPEC strains in our model (except from DL18) caused either no change or a slight change in the secretion of IL-6. IL-8 has a pivotal role in chemotactically attracting neutrophils to the site of infection and the initiation of inflammation [29]. The most prominent increase in production of IL-8, in our model, was caused by the J96 strain, which is a potent uropathogenic _E. coli_ strain. The host's response to infection by UPEC relies on innate immune processes, for example, the activation of pathogen associated molecular pattern receptors (PAMP receptors) [30], which induces an inflammatory response, as well as the secretion of cytokines, among others TNF-\(\alpha\)[31]. Compared to the reported values of 800 pg/mL in urine of \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Cytokine** & **Average Concentration (pg/mL)** & **ANOVA** \\ \hline  & **UPEC a ** & **Commensal _E. coli b ** & _p-Value_ \\ \hline IL-10 & 32.97 \(\pm\) 15.16 & 23.14 \(\pm\) 15.12 & 0.14 \\ IL-1\(\beta\) & 3.09 \(\pm\) 1.93 & 2.50 \(\pm\) 1.41 & 0.46 \\ IL-4 & 4.02 \(\pm\) 2.49 & 2.75 \(\pm\) 2.47 & 0.21 \\ IL-6 & 4.25 \(\pm\) 0.85 & 4.17 \(\pm\) 0.57 & 0.79 \\ IL-8 & 696.88 \(\pm\) 350.29 & 736.12 \(\pm\) 228.77 & 0.29 \\ TNF-\(\alpha\) & 46.21 \(\pm\) 24.27 & 37.84 \(\pm\) 22.26 & 0.36 \\ IL-12p40 & 65.25 \(\pm\) 34.21 & 44.90 \(\pm\) 25.53 & 0.12 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Mean \(\pm\) SD concentrations of cytokines produced by NPU cells, depending on the _E. coli_ type (UPEC vs. commensal). some cytistis patients [32], in our model, the highest production of TNF-\(\alpha\) was rather low (the highest production of this cytokine was induced by the DL18 strain (87.6 pg/mL). Interestingly, both potent UPEC strains J96 and 536 caused only a slight change in the secretion of this cytokine; the concentration was even lower than that detected with the MG1655.