, the main cause of diarrhea in hospitalized patients, produces toxins A (TcdA) and B (TcdB), which affect intestinal epithelial cell survival, proliferation, and migration and induce an intense inflammatory response. Transforming growth factor β (TGF-β) is a pleiotropic cytokine affecting enterocyte and immune/inflammatory responses. However, it has been shown that exposure of intestinal epithelium to a low concentration of TcdA induces the release of TGF-β1, which has a protective effect on epithelial resistance and a TcdA/TGF-β signaling pathway interaction. The activation of this pathway has not been elucidated. The aim of this study was to investigate the role of the TGF-β1 pathway in TcdA-induced damage in a rat intestinal epithelial cell line (IEC-6) and in a mouse model of an ileal loop. TcdA increased the expression of TGF-β1 and its receptor, TβRII, and TcdA induced nuclear translocation of the transcription factors SMAD2/3, a hallmark of TGF-β1 pathway activation, both in IEC cells and in mouse ileal tissue. The addition of recombinant TGF-β1 (rTGF-β) prevented TcdA-induced apoptosis/necrosis and restored proliferation and repair activity in IEC-6 cells in the presence of TcdA. Together, these data show that TcdA induces TGF-β1 signaling pathway activation and suggest that this pathway might play a protective role against the effect of-toxin.

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Most contacts with food protein and microbiota antigens occur at the level of the gut mucosa. In animal models where this natural stimulation is absent, such as germ-free and antigen-free mice, the gutassociated lymphoid tissue (GALT) and systemic immunological activities are underdeveloped. We have shown that food proteins play a critical role in the full development of the immune system. C57BL/ 6 mice weaned to a diet in which intact proteins are replaced by equivalent amounts of amino acids (Aa diet) have a poorly developed GALT as well as low levels of serum immunoglobulins (total Ig, IgG, and IgA, but not IgM). In the present study, we evaluated whether the introduction of a protein-containing diet in 10 adult Aa-fed C57BL/6 mice could restore their immunoglobulin levels and whether this recovery was dependent on the amount of dietary protein. After the introduction of a casein-containing diet, Aa-fed mice presented a fast recovery (after 7 days) of secretory IgA (from 0.33 to 0.75 mg/mL, while in casein-fed mice this value was 0.81 mg/mL) and serum immunoglobulin levels (from 5.39 to 10.25 mg/mL of total Ig). Five percent dietary casein was enough to promote the restoration of secretory IgA and serum immunoglobulin levels to a normal range after 30 days feeding casein diet (as in casein-fed mice -15% by weight of diet). These data suggest that the defect detected in the immunoglobulin levels was a reversible result of the absence of food proteins as an antigenic stimulus. They also indicate that the deleterious consequences of malnutrition at an early age for some immune functions may be restored by therapeutic intervention later in life.

Clostridium difficile causes intestinal inflammation, which increases adenosine. We compared the expression of adenosine receptors (AR) subtypes A 1 , A 2A , A 2B , and A 3 in HCT-8, IEC-6 cells, and isolated intestinal epithelial cells, challenged or not with Clostridium difficile toxin A and B (TcdA and TcdB) or infection (CDI). In HCT-8, TcdB induced an early A 2B R expression at 6 h and a late A 2A R expression at 6 and 24 h. In addition, both TcdA and TcdB increased IL-6 expression at all time-points (peak at 6 h) and PSB603, an A 2B R antagonist, decreased IL-6 expression and production. In isolated cecum epithelial cells, TcdA induced an early expression of A 2B R at 2 and 6 h, followed by a late expression of A 2A R at 6 and 24 h and of A 1 R at 24 h. In CDI, A 2A R and A 2B R expressions were increased at day 3, but not at day 7. ARs play a role in regulating inflammation during CDI by inducing an early pro-inflammatory and a late anti-inflammatory response. The timing of interventions with AR antagonist or agonists may be of relevance in treatment of CDI.

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