415 ± 0.071), whereas il-8 mRNA levels were not modified significantly (0.535 ± 0.211) and tnf-α mRNA remained undetectable (Fig. 6A, C, E). EPEC infection did not significantly
alter il-1β mRNA levels (E2348/69: 0.545 ± 0.069 and E22: 0.545 ± 0.115) (Fig. 6A). In the case of il-8, mRNA levels were not altered by E22 infection (0.782 ± 0.098), but E2348/69 infection resulted in decreased il-8 mRNA expression (0.396 ± 0.070) (Fig. 6C). Interestingly, in selleck chemicals cells infected with EPEC strains, tnf-α mRNA was abundantly amplified (0.751 ± 0.001 for E2348/69 infection and 0.612 ± 0.216 for E22), in contrast to undetectable levels in mock cells and cells treated with HB101 (Fig. 6E). These results Tipifarnib price state that IL-1β and IL-8 are constitutively expressed in HT-29 cells, but the synthesis of TNF-α is a consequence of EPEC infection. To analyse the impact of EPEC virulence factors in cytokine expression, we performed RT-PCR assays using RNA extracted from cells infected with EPEC E22 Δeae, ΔescN, ΔespA, or ΔfliC isogenic mutants. Infection with E22 mutants of intimin or EspA genes increased significantly il-1β
mRNA levels (E22Δeae: 0.865 ± 0.093 and E22ΔespA: 0.989 ± 0.074) compared to E22 WT (0.545 ± 0.115). In contrast, il-1β mRNA levels in cells infected with E22ΔescN or E22ΔfliC were not statistically different (0.850 ± 0.185 and 0.626 ± 0.067, respectively) from levels during E22 WT infection (Fig. 6B). Thus, E22 intimin and EspA are factors that maintain the expression of il-1β mRNA at a basal level during EPEC infection. On the other hand, il-8 mRNA expression was not altered in cells infected with any of the mutants (E22Δeae: 0.677 ± 0.211, E22ΔescN: 0.633 ± 0.002, E22ΔespA: 0.727 ± 0.206 or E22ΔfliC: 0.589 ± 0.064) (Fig. 6D) compared to E22 WT infection (0.782 ± 0.098). Interestingly, E22ΔespA infection doubled tnf-α mRNA levels (1.312 ± 0.120) in comparison with E22 WT infection (0.612 ± 0.216). The other E22 mutants activated the production of tnf-α mRNA in infected cells (E22Δeae: 0.595 ± 0.252; E22ΔescN: 0.749 ± 0.276;
Parvulin E22ΔfliC: 0.577 ± 0.179), at similar levels to those produced by cells infected with E22 WT (Fig. 6F). These results showed the effect of EPEC EspA as a negative modulator of tnf-α expression in infected cells. To quantify the secretion of proinflammatory cytokines, we established ELISA standard curves using pure IL-1β, IL-8 and TNF-α recombinant proteins to calculate the concentration of these molecules in supernatants from cells treated with HB101 or infected with EPEC E2348/69, E22 WT, E22Δeae, E22ΔescN, E22ΔespA or E22ΔfliC for 2 and 4 h (Fig. 7). Supernatants from mock-infected cells did not contain IL-1β (Fig. 7A), and this cytokine is not secreted by non-stimulated cells. In contrast to IL1β mRNA expression (Fig 6), interaction with HB101 did not activate IL-1β secretion.