Intestinal mucositis is a frequently encountered adverse effects in cancer patients undergoing chemotherapy and currently there are no effective preventive and control measures. [1, 4, 5] 5-FU treatment was reported to affect the abundance of gut microbiota. In recent years, probiotics had been demonstrated therapeutic effects in chemotherapy-induced mucositis. However, the results are inconsistent. [13, 14] We previously demonstrated various Lactobacillus strains had shown beneficial effects on the mucosal barrier of intestines and could enhance tight junction integrity. [9] In this study, we hypothesize that the probiotics could preserve gut ecology, ameliorate inflammation and protect epithelium by maintaining the tight junction integrity via immune modulations of enterocytes and intestinal stem cells. Our aim is to characterize these changes and to investigate the immune modulation effects and safety of probiotic via a 5-FU-induced intestinal mucositis mouse model.
Weight loss and diarrhea score
In our mouse model study, body weight in the 5-FU group decreased considerably by day 3 after 5-FU administration. The weight of the 5-FU + Lcr35 decreased with less intensity in relation to that of the 5-FU group. On the contrary, we found that in those mice in the probiotic group, their degree in body weight loss was significantly lesser than those in the 5-FU and saline groups. Our results were similar to the findings of other studies in the literatures. [1, 15] In our experiment, no diarrhea was noted in the saline and Lcr35 groups. However, marked diarrhea developed in the two 5-FU groups 24 hours later. We demonstrated diarrhea scores improved significantly after oral Lcr35 administrations. Previous studies reported that more than one third of the oncology patients undergoing chemotherapy experienced severe intestinal mucositis. [16] Benson et al reviewed that chemotherapeutic protocol containing 5-FU has been demonstrated with a higher risk for chemotherapy-induced diarrhea. [17]
Cytokines analyses
In our study, we showed those mice in 5-FU+saline groups had significantly higher levels of pro-inflammatory cytokines. This suggested a severe pattern of intestinal mucositis in mice. However, the levels of these cytokines were significantly reversed after administration of probiotic in the 5FU+Lcr35 group. We demonstrated that the protective effects of Lcr35 on 5-FU-induced mucositis was probably by triggering Th1 immune response via down-regulations of the cytokines IFN-γ and TNF-α. In an earlier study, Justino et al reported that Saccharomyces boulardii lowered pro-inflammatory cytokine levels (TNF-α, IL-1β, and CXCL-1) in the rat jejunum and ileum induced by 5-FU. [18] The mechanism of Saccharomyces boulardii’s protective effect might be similar to the mechanism of Lcr35’s action in our study. Up to date the exact mechanism of chemotherapy-induced intestinal mucositis remains unclear. Previous studies had suggested that it involved a five-stage process. [19-21] Soares et al suggested possible pathophysiology of mucositis development including the generation of reactive oxygen species and the up-regulation of pro-inflammatory cytokines causing further mucosal injury eliciting further tissue damage. [22] Few studies have assessed the effects of Lactobacillus acidophilus on inflammation. One of these studies found lower levels of leukocyte migration in animals treated with Lactobacillus acidophilus in a model of intestinal mucositis induced by irinotecan. [23] Several studies have reported reduced inflammatory effects using other probiotic species. [18, 24]
Flow cytometry
We found there was a tremendous rise of CD3+/CD8+ lymphocyte count in the 5FU group when compared to the saline groups. However, it was reversed after probiotic administration. The CD8 T lymphocytes of 5-FU+Lcr35 group was significantly lower than 5-FU group. Besides, there was a significant increase of CD3+/CD4+ lymphocyte count in the 5FU group when compared to the saline groups. We suggested the protective effect of Lcr35 on 5-FU-induced mucositis was by down-regulations of the lymphocytes CD3+/CD8+ and CD8+/ IFN-γ cells in 5-FU+Lcr35 group. The Lcr35 could also activate the T helper cells by stimulating the CD4+/IL4+ cell maturation.
Similarly, there was a tremendous rise of CD4+/IL17A lymphocyte count in the 5FU group when compared to the saline group. Interestingly, the level of CD4+ T lymphocytes further increased after probiotic administration. The amount of CD4+/IL17A lymphocyte count in the 5-FU+Lcr35 group was significantly higher than 5-FU group. Th17 immune response was demonstrated in CD4+/IL-17A+ lymphocytes activation in 5-FU+Lcr35 group. Roles of CD4+/IL17A lymphocytes on intestinal immunity and the pathophysiology of chemotherapy-induced mucositis have been investigated recently. [25] Edelblum et al recently found that CD4+ T cells, and in particular Th17 cells, were necessary to limit acute Salmonella typhimurium invasion in CA-MLCK mice. Studies in germ free CA-MLCK mice showed that commensal bacteria are required for both CD4+ T-cell expansion and early protection against bacterial invasion. [26]
Intestinal stem cells and crypt proliferation
For further exploring the mechanism of probiotics, we also looked at the intestinal stem cells and crypt proliferation in this study. Intestinal stem cells represented by CD44 markers and crypt proliferation with Ki67 expression were shown by IHC methods. Marked CD44 expression of intestinal stem cells and Ki67 proliferation were found in immunolabelled jejunal specimens from mice after 5-FU challenge and with Lcr35 administration. In our study, 5-FU significantly stimulated the expression of CD44 and was restored by administration of Lcr35, though not to the S+S or S+Lcr35 levels. 5-FU could increase the numbers of Ki67 positive cells, but there were no significant differences between 5-FU+S and S+S groups and 5-FU+S and 5-FU+Lcr35 groups, respectively. The actual role of probiotic on stem cells proliferation remains unclear and requires further investigation.
Athiyyah et al investigated the probiotic effect of Lactobacillus plantarum IS-10506 in activating and regenerating leucine-rich repeat-containing G-protein-coupled receptor (Lgr) 5- and B lymphoma Moloney murine leukaemia virus insertion region (Bmi)1-expressing intestinal stem cells in rodents following Escherichia coli serotype O55:B5 lipopolysaccharide exposure. [27] Their results demonstrated that the probiotic Lactobacillus plantarum IS-10506 activated intestinal stem cells to counter inflammation and might be useful for maintaining intestinal health, especially when used as a prophylactic agent.
Histological analysis on villus height, crypt depth and goblet cells
In our mice model, the 5-FU + Lcr35 group experienced a significant improvement of histopathological changes, as shown by photomicrographs. Previous studies on the effects of chemotherapy-induced mucositis on villus height and crept depths were not consistent. [28, 29] This inconsistency might be due to differences in the choices of probiotic strains or regimens. Stringer et al demonstrated 5-FU could influence the mucin dynamics and might interrupt intestinal barrier function. [30] They showed a marked decrease in goblet cell number following 5-FU administration. In this study, we also demonstrated a marked decrease in goblet cell number in mice with 5-FU-induced mucositis and Lcr35 administration with or without 5-FU injection could both increase goblet cell numbers.
Safety and translocation
Probiotics are defined as living bacteria that can confer health benefits to the host. However, potential side-effects including sepsis development, presence of virulence factors and translocation of live bacteria into local tissues are possible. [31, 32] In the present study, we did identify 2 bacterial strains (E coli str. K-12; E coli O157:H7 str. Sakai; E coli UMN026) in mesentery lymph node in the saline group. Two bacterial strains (Enterococcus dispar ATCC 51266 genomic scaffold; Enterococcus faecalis; Enterococcus casseliflavus EC20) were identified in the 5-FU group. However, no bacterial translocation was found in the samples of blood, liver and spleen tissues (Suppl Table 1). Risk of systemic infection with Lcr35 administration in this mice model was not likely.
Pathophysiology of chemotherapy-induced mucositis and roles of probiotics
The pathophysiology of chemotherapy-induced mucositis is complex and most likely involves multiple different processes. [33, 34] In 2004, Sonis published the famous five-phase model theory to explain the pathophysiology of mucositis. [19] Over the past decade, this model has been built upon, with advances in our understanding in regard to cell kinetics, epithelial junctions, inflammation, the microbiome and the innate immune system. [35]
Studies have shown that chemotherapy increase intestinal permeability, induce the generation of reactive oxygen species and pro-inflammatory cytokines, and modulate gut microbiota. [19, 34] Our study showed Lcr35 could reduce levels of proinflammatory cytokines in the intestine in 5-FU–treated mice. Proinflammatory cytokines such as TNF-α and IL-6 contributed to the severity and maintenance of injury in intestinal mucositis [36] and IL-4 was found to participate as a proinflammatory cytokine in a model of 5-FU–induced intestinal damage. [37] Thus, the reduction of these cytokines suggested that the probiotic had strong anti-inflammatory activity.
We previously demonstrated Lactobacillus were associated with the maintenance of the tight junction integrity. [9] However, beneficial effects of probiotics on chemotherapy-induced mucositis were not consistent in the literature. [38, 39] In the current study, we determined the effect of probiotic treatment on the expressions of pro-inflammatory cytokines. We further explored the effects of probiotic on stem cells, T cells and cell proliferation. Our results showed convincing protective effect and safety of probiotics on the chemotherapy induced mucositis. Recently we successfully demonstrated that probiotic did modulate the abundance and diversity of gut microbiota of mice undergoing chemotherapy [12] Previous studies in the literature seldom determined the effect of probiotics treatment on the expressions of pro-inflammatory cytokines. Furthermore, the safety of probiotics administrations was rarely investigated.
Limitations
There are several limitations in this study. One limitation is that the small sample size of mice models used in this experiment. Besides, the mice used in this study were indeed normal mice without malignancy, we confessed the model could not mimic or represent the actual situation happened in the clinical patients receiving chemotherapy. The duration of the experiment should be extended in future studies to evaluate the long-term influence of probiotics on microbiota modifications, rather than only the acute changes. Nevertheless, the greatest challenge for animal model is the difficulty in translating results obtained from current model to the wide range of human patient groups, with varying ages, cancer diagnoses, and to treatments covering a wide range of drugs and doses of chemotherapy.