Probiotics are considered essential tools in modulating inflammatory and infectious intestinal diseases, as well as a promising therapeutic alternative to alleviate symptoms of 5-FU induced mucositis [24]. Nonetheless, questions have been raised related to the safety of administering live bacteria in immunocompromised individuals and there is a constant search for solutions to this issue. The number of studies regarding the use of postbiotics, inactivated microorganisms and/or their metabolites, as an alternative to probiotics in these cases have risen with promising results [24, 41–43].
In this study, we have evaluated the postbiotic activity of formulations, heat-inactivated bacteria, and cell-free supernatant, derived from the E. coli strains Nissle 1917 and CEC15, in a murine model of 5-FU-induced intestinal mucositis. Beneficial activity of the two probiotic strains has been already established in vivo in various mouse models of inflammation [11, 14, 19, 44–46]. In particular, we previously compared the beneficial activity of both strains on 5-FU-induced intestinal mucositis in mice and showed the better protective effect of the CEC15 strain [20]. Here we confirmed these results, notably in showing the specific effect of CEC15, compared to EcN, in the prevention of animal weight loss, villous height reduction, goblet cell depletion, neutrophil infiltrate, intestinal permeability increases, and inflammatory related genetic markers.
Mucositis is a global inflammation in the GIT, giving rise to various symptoms such as bleeding, diarrhea, abdominal pain, fatigue, malnutrition, electrolyte imbalance, and infections. These symptoms can cause life-threatening complications of cancer chemotherapy [47–49]. The cytotoxic effects of 5-FU on epithelial cells of the GIT represent a major challenge for cancer treatment, as they impair the patient's ability to tolerate therapy, consequently reducing the quality of life and influencing treatment success [50]. The inflammation caused by 5-FU, which extends throughout the whole GIT, is highly associated with weight loss and reduced fluid and food intake. This weight loss can occur either by reduced consumption or by decreased ability to absorb nutrients [48]. As expected, the induction of intestinal mucositis with 5-FU reduced significantly the ingesta of food and liquid and, therefore, induced a considerable weight loss on mice. Here, none of the treatments was able to maintain food and liquid intake in face of 5-FU administration. This inability to attenuate the decrease of food and liquid intake has been reported for other probiotic species in this animal model, like Lactobacillus delbrueckii [51, 52], Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) [21], and Lacticaseibacillus rhamnosus (formerly Lactobacillus rhamnosus) [42]. Among the four postbiotic formulations tested, only two, heat-inactivated CEC15 (CECi) and EcN cell-free supernatant (EcNs), were able to attenuate weight loss. Similar results were found after administration of heat-inactivated L. delbrueckii CIDCA133 were the weight loss reduced from 10–6.5% from the initial weight [41], and with administration of heat-inactivated L. rhamnosus CGMCC1.3724, reducing weight loss from 2.4 g to 1.7 g [42].
Looking further on the effects of 5-FU administration, changes in the architecture of the intestinal epithelium are one of the commonly seen in the inflammatory process promoted in the intestinal mucositis [48]. We observed that all postbiotic treatments could reduce the damage associated with the intestinal mucosa by maintaining the integrity of the brush border of enterocytes and reducing the reactivity of the submucosa. The structural damage caused by the administration of 5-FU led to changes in the height of intestinal villi, the deepening of the crypts, and the reduction of goblet cells, whose main function is the production of mucin, a key component of the protective intestinal mucus layer. These are parameters for assessing the severity of the damage associated with mucositis and are also associated with the signs mentioned above [31, 53]. As expected, we could see a reduction on villus height and crypts depth, and a depletion on the number of goblet cells after 5-FU administration. All postbiotic formulations derived from both strains were able to mitigate the reduction of villus height and depletion of goblet cells. As reported before, the effectiveness of live CEC15 was confirmed as well as the absence of protection from live EcN facing 5-FU administration, the results here presented consolidate the CEC15 effect, as well as the effect of its postbiotic preparations in the protection of intestinal epithelium structure, in special the inactivated CEC15. As for EcN preparations, cell-free supernatant presented promising results, as it has already been demonstrated before [53] with the heat-inactivated form showing some level of protection in a lower extent.
The damage caused to the intestinal mucosa by 5-FU is associated with increased intestinal permeability. This last is associated with an increased risk of infection and transit of complex molecules, including toxins, through the epithelium and promotes intestinal bleeding and fluid and nutrient loss. All treatments were found to prevent increased intestinal permeability. Nevertheless, the permeability result is partially in accordance with the results on gene expression modulation of barrier related genes The groups with the lowest protection against increased permeability (EcN and EcNs, also EcNi and CECs to a lesser extent) are also the ones with reduced expression of Tjp1 and Ocln genes, highly associated with the integrity of intestinal barrier. CEC15 groups, in special the CEC and CECi ones, present the best results in both permeability and gene expression of barrier related genes with an over expression of Muc2 and Tjp1 on the CEC group and of Cldn1 on the CECi group. These results express the important role of CEC15, live or inactivated, in the maintenance of intestinal barrier integrity, by elevating the expression of tight junction proteins and the production of mucus, maintaining barrier integrity, and reducing the transit of toxic compounds and microorganisms. Previous research has highlighted the enhancement of mucins and tight junction proteins following treatment with heat-inactivated probiotics and/or their respective supernatants. An illustrative example is the supernatant of mulberry leaf extract fermented by L. acidophilus, whose consumption induced upregulation of Muc2 and Muc5ac gene expression in 5-FU-inflamed mice [54]. Additionally, the EcN-derived supernatant was found to mitigate the epithelial barrier disruption induced by enteropathogenic E. coli, in Caco-2 cells, by elevating the gene expression of tight junction proteins such as Tjp1, Cldn14, and Cldn2 [55].
All preparations of both strains promoted a protective effect on the intestinal mucosa, reducing the damage associated with mucositis. The infiltration of neutrophils in tissue is associated with inflammation as these cells are recruited in response to tissue damage [56]. The enzyme myeloperoxidase is produced exclusively by neutrophils and serves as an indirect measure of the number of cells present in the tissue [52]. MPO enzyme activity in injured tissue was attenuated by all treatments with the CEC15-derived preparations, in contrast to what is observed with those derived from the EcN strain. Similar results were seen after administration of postbiotics preparations of L. delbrueckii CIDCA133 [41]. The lower levels of MPO indicates that the CEC15 preparations are more efficient than the EcN in limiting neutrophil recruitment into the intestinal epithelium.
Mucositis is associated with altered expression of pro- and anti-inflammatory cytokine genes. These changes generate greater activation of inflammatory pathways such as NFkB and TNF, loosening of the intestinal barrier, and weakening the protection of the epithelium through the modulation of genes such as Il1b, Il17a, Nfkb1, among others [24]. Here we have seen an elevation of the expression of these three genes after administration of 5-FU, we have also observed a protection against Il1b by treatment with CECi and EcNi, and of Nfkb after treatment with EcNs in addition to live CEC15 and EcN. Expression of Il17a was only reduced by administration of live CEC15. Other groups, like EcNi and EcNs, have shown a tendency for protection (no difference to the Ct- group), however they did not present statistical difference from the MUC group. The increase on Il1b expression has been correlated to increase on permeability by degradation of the occludin mRNA in vitro and in a murine model [57] and it seems to be the case on treatments with EcN and its derivates. The Il17a gene is known to be overexpressed in intestinal inflamed tissue [58], its overexpression, however, it is not the sole responsible for the inflammatory process and there have been studies showing Il17 as an anti-inflammatory cytokine, depending on the co-expression of other cytokines such as Il10 and Inf-γ [59–62].
It's important to emphasize that not all probiotics demonstrated anti-inflammatory effects in the context of 5-FU-induced epithelial damage [53, 63]. Despite sharing similar probiotic characteristics [64], the mechanisms involved in mitigating 5-FU-induced inflammation may vary among strains or species. This variability could be attributed to factors such as the probiotic dosage, the type of antineoplastic agents, the specific experimental protocols used to induce mucositis, and the form in which beneficial microorganisms are administered, extending to their postbiotic forms (i.e., live, inactivated, or their secreted products) as we have demonstrated in this study [24, 65–67].