## 3 Results and Discussion
### Cytokine Response of Normal Urothelial Cells
Recently, we showed that TNF-\(\alpha\)-stimulated NPU cells exhibited marked inflammatory phenotypes with upregulated mRNA expression of the acute phase protein SAA3, complement component C3, chemokine CXCL10, several proinflammatory cytokines (IL1a, IL1b, IL8), and the cytokine receptor IFNGR1. In addition, protein levels of IL8 and IFNGR1 were also significantly higher in TNF-\(\alpha\)-treated NPU cells compared with the controls, as determined by ELISA and Western blot, respectively [33]. Together with these results, we can further confirm the usefulness of our urothelial model, since it does not only represent normal bladder urothel, but it can significantly respond to specific environmental conditions, as is the case in interstitial cyystitis/bladder pain syndrome and also in infectious cyystitis. Strain DL102 induced the greatest production of IL-12p40, which, among others, drives the differentiation of the immune response towards different effector pathways, as for example, the promotion of INF-\(\gamma\) secretion by natural killer cells [34]. In our study, the production of cytokines INF-\(\alpha\) and INF-\(\gamma\) was not detected, which was to be expected, as these two cytokines have not been previously associated with _E. coli_ infections. They are involved in antiviral immune responses [35] and, in addition, INF-\(\gamma\) is also involved in immunological processes in infections caused by some bacteria (_Mycobacterium tuberculosis, Salmonella, Listeria_) and some parasites (_Leishmana_, _Toxoplasma, Chlamydia_) [36]. To sum up, each _E. coli_ strain caused a distinct pattern of cytokine production by NPU cells. The majority of highly pathogenic DL and BJ strains provoked between a 3 and 4 fold change in total cytokine production (BJ30, DL18, DL102); strain DL53 provoked a 2-3 fold change. Interestingly, the two uropathogenic strains, J96 and 536, as well as the strain BJ23, which all possess many virulence-associated genes, induced much lower fold changes in total cytokine production, as would be expected. The increase in concentrations of the individual cytokines secreted by the cells of our model, in response to infection with UPEC strains, is in concordance with the findings of other researchers. Thus, [PERSON] et al. in 2016 [32] reported elevated concentrations of multiple cytokines, in comparison to the negative control, in urine samples of patients with cyystitis caused by UPEC, among others (IL-1\(\beta\), IL-4, IL-10 and TNF\(\alpha\)), as was also observed upon infection in our model with the high fold change group of _E. coli_ strains. Another study that used urine samples from patients with recurring UTs (rUTI) for the detection of multiple cytokines showed, among others, elevated concentrations of IL-1\(\beta\) and IL-10 [37], which is also in accordance with the response of our in vitro model to infection. Statistical Analysis of Correlation between Strain's Cytokine Production Group and Virulence-Associated Genes  
The 24 tested _E. coli strains_ were assigned to the following 3 different groups, according to the fold change in total cytokine production by NPU cells in response to infection with each strain: (i) the low fold change group I, containing strains BJ65, DL80, BJ51, SE15, BJ95, BJ69, 536, BJ45, J96, MG1655, BJ23 and DL95, which induced the low overall cytokine production of NPU cells in response to infection (from 1 to 1,99 fold change), (ii) moderate fold change group II, containing strains BJ97, DL75, DL1, BJ16, HS16, DL87 and DL53, which induced the moderate total cytokine production of NPU cells (from 2 to 2,99 fold change), and finally (iii) high fold change group III, containing the _E. coli_ strains DL31, BJ50, BJ30, DL18 and DL102, which induced a high total cytokine production of NPU cells in response to infection with these strains (above 3 fold change). The Fisher's exact test and Bonferroni correction were performed to reveal any statistical correlation between the assigned cytokine production groups and the presence of virulence-associated genes.