Table 1.
TLR2 and TLR4 expression in the colonia mucosa of control patients according to the gender and age.
Figure 1.
Expression levels of TLR2 and TLR4 in the colonic mucosa of controls and IBS patients.
(A) Correlation between TLR2 and TLR4 mRNA expression in the whole group of IBS patients (rs = 0.78, p<0.0001). (B) Changes in mRNA expression levels of TLR2 and (C) TLR4 in controls and in patients with predominant diarrhea (IBS-D), or constipation (IBS-C) or mixed (IBS-M) assessed by real-time PCR. Values are expressed as mean ± SEM. * Represents p<0.05 using Mann-Whitney test.
Figure 2.
Correlation analysis of TLR2 and TLR4 mRNA levels and symptoms.
Correlation between TLR2 (A) and TLR4 (B) expressions in the colonic mucosa and the duration of symptoms of IBS patients (n = 46, rs = 0.34, p = 0.02 for TLR2 and rs = 0.37, p = 0.01 for TLR4). Data were correlated by using non-parametric Spearman test.
Table 2.
Correlation analysis of TLR2 and TLR4 and duration of symptoms according to the subtype of IBS.
Figure 3.
Immunofluorescence microscopy analysis of TLR2 and TLR4 in the colonic mucosa of IBS subgroups.
Representative photomicrographs show the distribution of TLR2 and TLR4 proteins in the colonic mucosa of controls and IBS patients according to the disease subtype (IBS-Constipated, IBS-Diarrhea or IBS-Mixed alternating constipation with diarrhea) Red, TLR staining; blue, DAPI nuclear staining. Original magnification, ×20.
Figure 4.
TLR2, TLR4 and CD14 expression in IECs (Epcam+ cells).
Epithelials cells obtained from the colonic mucosa were assessed by flow cytometry as described in Patients and Methods. Mucosal colonic cells were double-stained with isotype IgG control or mAbs to TLR2, TLR4 or CD14 together with APC-conjugated anti Epcam mAbs. (A) Representative histogram showing the fluorescence intensity of surface TLR4 expression in gated Epcam+ cells from all IBS subtypes and controls patients. Changes in TLR4 surface expression (B), intracellular TLR4 expression (C), CD14 expression (D) and TLR2 surface expression (E) in IECs in control and IBS subtypes. Values are expressed as mean intensities fluorescence ± SEM. (*) Represents p<0.05 and (**) represents p<0.005 using Mann-Whitney test. Kruskal-Wallis p values are *p = 0.01 (B); p = 0.5 (C); p = 0.95 (D) and *p = 0.03 (E) respectively.
Figure 5.
Relative PPARγ protein expression in colonic mucosa.
(A) Representative western blot for PPARγ expression in colonic tissue from IBS subgroups and control. (B) Western blot analysis was performed for PPARγ expression on colonic biopsy lysates of control (n = 25) and IBS-C (n = 13), or IBS-D (n = 12), or IBS-M (n = 14). Data are expressed as means ± SEM; *p = 0.0033 vs. Controls, using Mann Whitney test.
Figure 6.
Levels of pro-inflammatory cytokines and chemokines in colonic mucosa.
Cytokines were measured by multiplex cytokine bead immunoassays as described previously in Patients and Methods. Protein extracts of colonic mucosa were evaluated in duplicate for the expression of IL-8 (A), IL1β (B), TNF-α (C), IL-6 (D) and IFN γ (E) in IBS patients and controls. Results were expressed as pg/mg protein. Differences in protein secretion were determined to be statistically significant by Mann-Whitney test with a value of p<0.05 (*), p<0.005 (**) and p<0.0005 (***).