Figure 4 In vitro initiation of inflammatory responses via Toll‐like receptors (TLRs) by E coli isolates from the inflamed mouse ileum. (A) Mouse commensal E coli increase nuclear factor κB (NFκB) activity via TLR4. Human embryonic kidney cells (HEK293) transiently transfected with human TLR4 were incubated with purified liposaccharide (LPS), with heat‐inactivated E coli M (EcM, isolated from inflamed mouse ileum) or the E coli reference strain ATCC29522 (Ec29522). Each bar represents mean values of two individual measurements. SD and significance levels (as compared with control cells without stimulation, w/o) determined by Student's t test are indicated (**p<0.01). Similar results were obtained for repeated analyses of four additional E coli strains isolated from the mouse ileum (not shown). (B) E coli‐mediated NFκB activation via TLR2. HEK293 cells transiently transfected with human TLR2 were incubated with the purified TLR2 ligand Pam₂Cys (P2C) or with bacterial cell lysates from EcM (isolated from inflamed mouse ileum) or from the E coli reference strain ATCC29522 (Ec29522). Each bar represents mean values of two separate measurements. SDs and significance levels (as compared with control cells without bacterial lysates) determined by Student's t test are indicated (*p<0.05; **p<0.01). Similar results were obtained for repeated analyses of four additional E coli strains isolated from the mouse ileum (not shown). (C) TLR‐dependent activation of mouse PMs by commensal bacteria. PMs isolated from wild‐type mice (WT) and from mice lacking TLR2 (TLR2^−/−), TLR4 (TLR4^−/−) or both (TLR2/4^−/−) (n = 3 each group) were incubated with purified LPS or heat‐inactivated bacteria as indicated (EcM isolated from inflamed mouse ileum; E coli reference strain ATCC29522, Ec29522; L johnsonii, Lj; L acidophilus, La). Cells without LPS treatment or bacterial stimulation served as controls (w/o). Each bar represents mean values of duplicate measurements. SDs and significance levels (if compared to WT) determined by Student's t test are indicated (*p<0.05; **p<0.01; ***p<0.001). Results are representative for two independent experiments. (D) Nitric oxide (NO) release in cultivated RAW264.7 mouse macrophages. Cells were incubated with P2C, LPS or lysates from E coli (Ec), Lj or both (EcM + Lj). Each bar represents mean values of duplicate measurements. SDs and significance levels (if compared with the non‐stimulated control (w/o)) determined by Student's t test are indicated (**p<0.01).