5 Results
5.3 Results with bacteria
5.3.3 Assessment of IL-6 and IL-8 levels
75
Figure 28. Effect of B. subtilis (Bs) on the paracellular permeability of IPEC-J2 cells treated with E. coli.
B. subtilis was added 1 h before (pre-treatment), at the same time as (co-treatment), and 1 h after (post-treatment) the addition of E. coli. Detection of the FD4 dye was performed 24 h after the treatment of E. coli.
Control: plain cell culture medium treatment; Ec: E. coli 106 CFU/ml; Bs: treatment with B. subtilis 108 CFU/ml;
Bs PRE: pre-treatment with B. subtilis 108 CFU/ml + E. coli CFU/ml; Bs CO: co-treatment with B. subtilis 108 CFU/ml + E. coli 106 CFU/ml; Bs POST: post-treatment with B. subtilis 108 CFU/ml + E. coli 106 CFU/ml.
Data are shown as means with standard deviations and expressed as relative fluorescence, considering the mean value of control as 100%. n = 6/group. Significant difference: * p < 0.05 in grey: compared with the untreated control. *** p < 0.001, in green: compared with treatment with E. coli.
76
faecium 107 CFU/ml failed to significantly decrease IL-6 secretion compared to the IL-6 production induced by S. Typhimurium (Figure 29).
Figure 29. IL-6 levels of IPEC-J2 cells after treatment with S. Typhimurium (St) and E. faecium (Ef). E.
faecium was added 1 h before (pre-treatment) or at the same time (co-treatment) of the addition of S.
Typhimurium. E. faecium was added in 108 CFU/ml or in 107 CFU/ml concentration. Control: plain cell culture medium treatment; St: S. Typhimurium 106 CFU/ml; Ef 10^8: E. faecium 108 CFU/ml; Ef 10^7: E. faecium 107 CFU/ml; Ef 10^8 PRE: pre-treatment with E. faecium 108 CFU/ml + S. Typhimurium 106 CFU/ml; Ef 10^7 PRE:
pre-treatment with E. faecium 107 CFU/ml + S. Typhimurium 106 CFU/ml; Ef 10^8 CO: co-treatment with E.
faecium 108 CFU/ml + S. Typhimurium 106 CFU/ml; Ef 10^7 CO: co-treatment with E. faecium 107 CFU/ml + S.
Typhimurium 106 CFU/ml. Data are shown as means with standard deviations and expressed as relative IL-6 concentration, considering the mean value of control as 100%. n = 6/group. Significant difference: * p < 0.05 in light grey: compared with the untreated control, * p < 0.05, in dark grey: compared with treatment with S.
Typhimurium.
Infection of IPEC-J2 cells with S. Typhimurium also increased the secretion of IL-8 (p<0.001). Treatment with the probiotic strain itself did not result in a significant change in IL-8 secretion, regardless of the applied concentration. Pre-treatment and co-treatment with
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Rela tiv e IL -6 concent ration (%)
*
*
77
E. faecium, applied at a concentration of 108 CFU/ml, significantly reduced the secretion of IL-8 compared to the amount of IL-8 secretion when IPEC-J2 cells were challenged by S.
Typhimurium (p<0.001). Pre-treatment and co-treatment with E. faecium, applied at a concentration of 107 CFU/ml, failed to decrease the IL-8 secretion in comparison to the secretion observed when cells were treated with S. Typhimurium itself (Figure 30).
Figure 30. IL-8 levels of IPEC-J2 cells after treatment with S. Typhimurium (St) and E. faecium (Ef). E.
faecium was added 1 h before (pre-treatment) or at the same time (co-treatment) of the addition of S.
Typhimurium. E. faecium was added in 108 CFU/ml or in 10^7 CFU/ml concentration. Control: plain cell culture medium treatment; St: S. Typhimurium 106 CFU/ml; Ef 10^8: E. faecium 108 CFU/ml; Ef 10^7: E. faecium 107 CFU/ml; Ef 10^8 PRE: pre-treatment with E. faecium 108 CFU/ml + S. Typhimurium 106 CFU/ml; Ef 10^7 PRE:
pre-treatment with E. faecium 107 CFU/ml + S. Typhimurium 106 CFU/ml; Ef 10^8 CO: co-treatment with E.
faecium 108 CFU/ml + S. Typhimurium 106 CFU/ml; Ef 10^7 CO: co-treatment with E. faecium 107 CFU/ml + S.
Typhimurium 106 CFU/ml. Data are shown as means with standard deviations and expressed as relative IL-8 concentration, considering the mean value of control as 100%. n = 6/group. Significant difference: *** p < 0.001 in light grey: compared with the untreated control, *** p < 0.001, in dark grey: compared with treatment with S.
Typhimurium.
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Relativ e IL -8 concent ration (%)
***
***
***
78
IL-6 secretion was induced significantly by E. coli in comparison to the control cells (p<0.05).
Neither pre-treatment nor co-treatment with E. faecium could compensate for the IL-6 elevation induced by E. coli (Figure 31).
Figure 31. IL-6 levels of IPEC-J2 cells after treatment with E. coli (Ec) and E. faecium (Ef). E. faecium was added 1 h before (pre-treatment) or at the same time (co-treatment) of the addition of E. coli. E. faecium was added in 108 CFU/ml or in 107 CFU/ml concentration. Control: plain cell culture medium treatment; Ec: E. coli 106 CFU/ml; Ef 10^8 PRE: pre-treatment with E. faecium 108 CFU/ml + E. coli 106 CFU/ml; Ef 10^7 PRE: pre-treatment with E. faecium 107 CFU/ml + E. coli 106 CFU/ml; Ef 10^8 CO: co-treatment with E. faecium 108 CFU/ml + E. coli 106 CFU/ml; Ef 10^7 CO: co-treatment with E. faecium 107 CFU/ml + E. coli 106 CFU/ml. Data are shown as means with standard deviations and expressed as relative IL-6 concentration, considering the mean value of control as 100%. n = 6/group. Significant difference: * p < 0.05 in light grey: compared with the untreated control.
Also IL-8 secretion was induced significantly by E. coli compared to the control cells (p<0.05). Pre-treatment and co-treatment with E. faecium, applied at a concentration of 108 CFU/ml further increased the secretion of IL-8 (p<0.05 for Ef 108 PRE and p<0.01 for Ef
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Relativ e IL -6 concent ration (%)
*
79
108 CO). The pre-treatment and co-treatment with E. faecium, applied at a concentration of 107 CFU/ml, failed to cause any significant effect on IL-8 secretion (Figure 32).
Figure 32. IL-8 levels of IPEC-J2 cells after treatment with E. coli (Ec) and E. faecium (Ef). E. faecium was added 1 h before (pre-treatment) or at the same time (co-treatment) of the addition of E. coli. E. faecium was added in 108 CFU/ml or in 107 CFU/ml concentration. Control: plain cell culture medium treatment; Ec: E. coli 106 CFU/ml; Ef 10^8 PRE: pre-treatment with E. faecium 108 CFU/ml + E. coli 106 CFU/ml; Ef 10^7 PRE: pre-treatment with E. faecium 107 CFU/ml + E. coli 106 CFU/ml; Ef 10^8 CO: co-treatment with E. faecium 108 CFU/ml + E. coli 106 CFU/ml; Ef 10^7 CO: co-treatment with E. faecium 107 CFU/ml + E. coli 106 CFU/ml. Data are shown as means with standard deviations and expressed as relative IL-8 concentration, considering the mean value of control as 100%. n = 6/group. Significant difference: * p < 0.05 in light grey: compared with the untreated control, * p < 0.05; ** p < 0.01, in pink: compared with treatment with E. coli.
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Relativ e IL -8 concent ration (%)
* *
**
80 5.3.3.2 Results with Lactobacillus rhamnosus
Treatment with L. rhamnosus alone did not alter the secretion of IL-6 in IPEC-J2 cells compared to the control. The pre-treatment and the post-treatment with L. rhamnosus 108 CFU/ml caused a significant decrease (p<0.05 for Lrh PRE and p<0.01 for Lrh POST) in IL-6 production as compared with the IL-6 secretion induced by S. Typhimurium, however the co-treatment with L. rhamnosus failed to decrease the IL-6 production compared with the IL-6 secretion induced by S. Typhimurium (Figure 33).
Figure 33. IL-6 levels of IPEC-J2 cells after treatment with S. Typhimurium (St) and L. rhamnosus (Lrh).
L. rhamnosus was added 1 h before (pre-treatment), at the same time as (co-treatment), or 1 h after (post-treatment) the addition of S. Typhimurium. L. rhamnosus was added in 108 CFU/ml and S. Typhimurium was added in 106 CFU/ml concentration. Control: plain cell culture medium treatment; St: S. Typhimurium 106 CFU/ml; Lrh: L. rhamnosus 108 CFU/ml; Lrh PRE: pre-treatment with L. rhamnosus 108 CFU/ml + S.
Typhimurium 106 CFU/ml; Lrh CO: co-treatment with L. rhamnosus 108 CFU/ml + S. Typhimurium 106 CFU/ml;
Lrh POST: post-treatment with L. rhamnosus 108 CFU/ml + S. Typhimurium 106 CFU/ml. Data are shown as means with standard deviations and expressed as relative IL-6 concentration, considering the mean value of control as 100%. n = 6/group. Significant difference: ** p < 0.01, in grey: compared with the untreated control. * p ≤<0.05; ** p < 0.01, in blue: compared with treatment with S. Typhimurium.
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Relativ e IL -6 concent ration (%)
** *
**
81
Treatment with L. rhamnosus alone did not result in a significant change in IL-8 secretion as compared with the control. All three treatment combinations (pre-, co-, and post-treatment) could signidicantly decrease the IL-8 secretion of IPEC-J2 cells compared with the IL-8 secretion induced by S. Typhimurium (p<0.001) (Figure 34).
Figure 34. IL-8 levels of IPEC-J2 cells after treatment with S. Typhimurium (St) and L. rhamnosus (Lrh).
L. rhamnosus was added 1 h before (pre-treatment), at the same time as (co-treatment), or 1 h after (post-treatment) the addition of S. Typhimurium. L. rhamnosus was added in 108 CFU/ml and S. Typhimurium was added in 106 CFU/ml concentration. Control: plain cell culture medium treatment; St: S. Typhimurium 106 CFU/ml; Lrh: L. rhamnosus 108 CFU/ml; Lrh PRE: pre-treatment with L. rhamnosus 108 CFU/ml + S.
Typhimurium 106 CFU/ml; Lrh CO: co-treatment with L. rhamnosus 108 CFU/ml + S. Typhimurium 106 CFU/ml;
Lrh POST: post-treatment with L. rhamnosus 108 CFU/ml + S. Typhimurium 106 CFU/ml. Data are shown as means with standard deviations and expressed as relative IL-8 concentration, considering the mean value of control as 100%. n = 6/group. Significant difference: *** p < 0.001, in grey: compared with the untreated control.
*** p < 0.001, in blue: compared with treatment with S. Typhimurium.
All three treatment combination (pre-, co-, and post-treatment with L. rhamnosus) failed to significantly alter the IL-6 secretion induced by E. coli (Figure 35).
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Relativ e IL -8 concent ration (%) ***
***
***
***
82
Figure 35. IL-6 levels of IPEC-J2 cells after treatment with E. coli (Ec) and L. rhamnosus (Lrh).
L. rhamnosus was added 1 h before (pre-treatment), at the same time as (co-treatment), or 1 h after (post-treatment) the addition of E. coli. L. rhamnosus was added in 108 CFU/ml and E. coli was added in 106 CFU/ml concentration. Control: plain cell culture medium treatment; Ec: E. coli 106 CFU/ml; Lrh PRE: pre-treatment with L. rhamnosus 108 CFU/ml + E. coli 106 CFU/ml; Lrh CO: co-treatment with L. rhamnosus 108 CFU/ml + E. coli 106 CFU/ml; Lrh POST: post-treatment with L. rhamnosus 108 CFU/ml + E. coli 106 CFU/ml; Data are shown as means with standard deviations and expressed as relative IL-6 concentration, considering the mean value of control as 100%. n = 6/group. Significant difference: ** p < 0.01, in grey: compared with the untreated control.
5.3.3.3 Results with Bacillus licheniformis and Bacillus subtilis
Infection of intestinal epithelial cells with S. Typhimurium significantly induced the secretion of IL-6 compared with control (p<0.001). In addition the treatment with B. subtilis alone also resulted in significant IL-6 secretion compared with the control (p<0.001). In comparison, treatment with only B. licheniformis did not result in a significant change in IL-6 secretion compared with the control. The pre-treatment with both B. subtilis 108 CFU/ml and B. licheniformis 108 CFU/ml caused a significant decrease in IL-6 production as compared with the IL-6 secretion induced by S. Typhimurium (p<0.001). The co- and post-treatments with B. licheniformis 108 CFU/ml also reduced the IL-6 secretion (p<0.001 for Bl CO and p<0.05 for Bl post); however, the co- and post-treatments with B. subtilis 108 CFU/ml failed
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Relativ e IL -6 concent ration (%)
**
83
to significantly decrease IL-6 secretion compared with the IL-6 production induced by S. Typhimurium (Figure 36).
Infection of IPEC-J2 cells with S. Typhimurium also triggered the secretion of IL-8 (p<0.01).
Treatment with B. licheniformis alone also resulted in a significant rise in IL-8 secretion compared with the control (p<0.001). However, the treatment with B. subtilis alone did not result in a significant change in IL-8 secretion compared with the control. With the exception of post-treatment with B. licheniformis, all other treatment combinations did not alter the IL- 8 secretion induced by S. Typhimurium. Post-treatment with B. licheniformis further increased the IL-8 secretions compared with the amount of IL-8 secretion when IPEC-J2 cells were challenged by S. Typhimurium (p<0.001) (Figure 37).
None of the pre-, co-, and post-treatments with B. licheniformis and B. subtilis had any significant effect on the IL-6 elevation induced by E. coli (Figure 38). IL-8 secretion was induced significantly by E. coli compared with control cells (p<0.05) and pre-treatment with B. licheniformis 108 CFU/ml further increased the secretion of IL-8 (p<0.001). Pre-treatment with B. subtilis and co- and post-treatments with both probiotic bacteria failed to cause any significant effect on IL-8 secretion (Figure 39).
84
Figure 36. IL-6 levels of IPEC-J2 cells after treatment with S. Typhimurium (St), B. licheniformis (Bl), and B. subtilis (Bs). B. licheniformis and B. subtilis were added 1 h before (pre-treatment), at the same time as (co-treatment), or 1 h after (post-treatment) the addition of S. Typhimurium. B. licheniformis and B. subtilis were added in 108 CFU/ml and S. Typhimurium was added in 106 CFU/ml concentration. Control: plain cell culture medium treatment; St: S. Typhimurium 106 CFU/ml; Bs: B. subtilis 108 CFU/ml; Bl: B. licheniformis 108 CFU/ml;
Bs PRE: pre-treatment with B. subtilis 108 CFU/ml + S. Typhimurium 106 CFU/ml; Bl PRE: pre-treatment with B.
licheniformis 108 CFU/ml + S. Typhimurium 106 CFU/ml; Bs CO: co-treatment with B. subtilis 108 CFU/ml + S. Typhimurium 106 CFU/ml; Bl CO: co-treatment with B. licheniformis 108 CFU/ml + S. Typhimurium 106 CFU/ml; Bs POST: post-treatment with B. subtilis 108 CFU/ml + S. Typhimurium 106 CFU/ml; Bl POST: post-treatment with B. licheniformis 108 CFU/ml + S. Typhimurium 106 CFU/ml. Data are shown as means with standard deviations and expressed as relative IL-6 concentration, considering the mean value of control as 100%. n = 6/group. Significant difference: *** p < 0.001, in grey: compared with the untreated control. * p < 0.05;
*** p < 0.001, in green: compared with treatment with S. Typhimurium.
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Relativ e IL -6 concent ration (%)
***
***
***
*
***
***
85
Figure 37. IL-8 levels of IPEC-J2 cells after treatment with S. Typhimurium (St), B. licheniformis (Bl), and B. subtilis (Bs). B. licheniformis and B. subtilis were added 1 h before (pre-treatment), at the same time as (co-treatment), or 1 h after (post-treatment) the addition of S. Typhimurium. B. licheniformis and B. subtilis were added in 108 CFU/ml and S. Typhimurium was added in 106 CFU/ml concentration. Control: plain cell culture medium treatment; St: S. Typhimurium 106 CFU/ml; Bs: B. subtilis 108 CFU/ml; Bl: B. licheniformis 108 CFU/ml;
Bs PRE: pre-treatment with B. subtilis 108 CFU/ml + S. Typhimurium 106 CFU/ml; Bl PRE: pre-treatment with B. licheniformis 108 CFU/ml + S. Typhimurium 106 CFU/ml; Bs CO: co-treatment with B. subtilis 108 CFU/ml + S. Typhimurium 106 CFU/ml; Bl CO: co-treatment with B. licheniformis 108 CFU/ml + S. Typhimurium 106 CFU/ml; Bs POST: post-treatment with B. subtilis 108 CFU/ml + S. Typhimurium 106 CFU/ml; Bl POST: post-treatment with B. licheniformis 108 CFU/ml + S. Typhimurium 106 CFU/ml. Data are shown as means with standard deviations and expressed as relative IL-8 concentration, considering the mean value of control as 100%. n = 6/group. Significant difference: ** p < 0.01, *** p < 0.001, in grey: compared with the untreated control
*** p < 0.01, in green: compared with treatment with S. Typhimurium.
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Relativ e IL -8 concent ration (%)
**
***
***
86
Figure 38. IL-6 levels of IPEC-J2 cells after treatment with E. coli, (Ec) B. licheniformis (Bl), and B. subtilis (Bs). B. licheniformis and B. subtilis were added 1 h before (pre-treatment), at the same time as (co-treatment), or 1 h after (post-treatment) the addition of E. coli. B. licheniformis and B. subtilis were added in 108 CFU/ml and E. coli was added in 106 CFU/ml concentration. Control: plain cell culture medium treatment; Ec: E. coli 106 CFU/ml; Bs PRE: pre-treatment with B. subtilis 108 CFU/ml + E. coli 106 CFU/ml; Bl PRE: pre-treatment with B. licheniformis 108 CFU/ml + E. coli 106 CFU/ml; Bs CO: co-treatment with B. subtilis 108 CFU/ml + E. coli 106 CFU/ml; Bl CO: co-treatment with B. licheniformis 108 CFU/ml + E. coli 106 CFU/ml; Bs POST: post-treatment with B. subtilis 108 CFU/ml + E. coli 106 CFU/ml; Bl POST: post-treatment with B. licheniformis 108 CFU/ml + E. coli 106 CFU/ml. Data are shown as means with standard deviations and expressed as relative IL-6 concentration, considering the mean value of control as 100%. n = 6/group. Significant difference: *** p < 0.001 in grey: compared with the untreated control.
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Relativ e IL -6 concent ration (%)
***
87
Figure 39. IL-8 levels of IPEC-J2 cells after treatment with E. coli (Ec), B. licheniformis (Bl), and B. subtilis (Bs). B. licheniformis and B. subtilis were added 1 h before (pre-treatment), at the same time as (co-treatment), or 1 h after (post-treatment) the addition of E. coli. B. licheniformis and B. subtilis were added in 108 CFU/ml and E. coli was added in 106 CFU/ml concentration. Control: plain cell culture medium treatment; Ec: E. coli 106 CFU/ml; Bs PRE: pre-treatment with B. subtilis 108 CFU/ml + E. coli 106 CFU/ml; Bl PRE: pre-treatment with B. licheniformis 108 CFU/ml + E. coli 106 CFU/ml; Bs CO: co-treatment with B. subtilis 108 CFU/ml + E. coli 106 CFU/ml; Bl CO: co-treatment with B. licheniformis 108 CFU/ml + E. coli 106 CFU/ml; Bs POST: post-treatment with B. subtilis 108 CFU/ml + E. coli 106 CFU/ml; Bl POST: post-treatment with B. licheniformis 108 CFU/ml + E. coli 106 CFU/ml. Data are shown as means with standard deviations and expressed as relative IL-8 concentration, considering the mean value of control as 100%. n = 6/group. Significant difference: * p < 0.05, in grey: compared with the untreated control. *** p < 0.001, in purple: compared with treatment with E. coli.
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Relativ e IL -8 concent ration (%)
*
***
88