Mechanisms For The Protective Effect Of Probiotics On Rotavirus Infection And Diarrhea

Specific beneficial effects of probioics have been documented in a large number of clinical trials for rotavirus infection and diarrhea. However, mechanisms behind such effects are not well understood. In this study, we first evaluated a non-transformed porcine jejunum epithelial cell line (IPEC-J2) as an in-vitro model of rotavirus infection and probiotic treatment. We then studied the impact of LGG colonization on intestinal epithelial barrier function and cytokine responses in the gnotobiotic (Gn) pig model of rotavirus gastroenteritis.In the study of in-vitro IPEC-J2 cell model, porcine rotavirus (pRV) OSU strain or human rotavirus (hRV) Wa strain, along with Lactobacillus acidophilus (LA) or Lactobacillus rhamnosus GG (LGG) were used to inoculate IPEC-J2 cells. LA or LGG treatment was applied pre- or post-rotavirus infection. We demonstrated that IPEC-J2 cells were productively infected by pRV. LA or LGG treatment of the cells did not reduce virus replication. MUC3 mucin secretion was increased after pRV infection on the cells. LGG treatment post-rotavirus infection reduced the mucin secretion response induced by pRV; LGG alone increased the production of membrane-associated MUC3 mucin. LA treatment prior to rotavirus infection significantly increased pRV replication and the IL-6 response to pRV infection, which is consistent with the adjuvant effect of LA. LGG treatment after rotavirus infection down-regulated the IL-6 response, which confirmed the anti-in?ammatory effect of LGG. IPEC-J2 cells expressed TLR2, TLR3, and TLR9 constitutively. TLR2 expression was upregulated by LGG and peptidoglycan, corresponding to the decreased IL-6 response, indicating that the protective effect of LGG is associated with upregulation of TLR2 expression on intestinal epithelial cells. Trans-epithelial electrical resistance (TEER) decreased significantly at post-rotavirus inoculation 6~18 h; however, the TEER recovered to the same level as the mock-infected cells at post-rotavirus inoculation 24 h, which is consistent with the course of rotavirus infection and diarrhea in-vivo. LA, but not LGG, significantly inhibited the TEER reduction caused by pRV infection. LA or LGG treatment and/or pRV infection did not change the permeability of IPEC-J2 monolayer and the distribution of tight junction (TJ) proteins (occludin, ZO-1, claudin-1, claudin-3 and claudin-4) and aherents junction (AJ) proteins (α-catenin andβ-catenin) on the cells at post-rotavirus inoculation 24 h. However, during LA or LGG treatment and/or RV infection, whether cell monolayer permeability and TJ or AJ proteins change over the time course (post-rotavirus inoculation 6~18 h) or not need to be studied. Nevertheless, LGG significantly increased the TEER and the production ofα-catenin andβ-catenin in mock cells, indicating the protective effect of LGG on intestinal epithelia barrier. The IPEC-J2 cell model of pRV infection is a completely homologous system. It is a valuable model for studying the interactions among rotavirus-host-probiotics, and the mechanisms behind the immunomodulating effect of probiotic bacteria on innate immune responses and intestinal epithelial barrier function.In the study of in-vivo Gn pig model, we used 4 Gn pigs in each group as follows: (1) LGG colonization plus hRV inoculation (LGG+hRV+), (2) hRV only (LGG-hRV+), (3) LGG only (LGG+hRV-) or (4) mock control (LGG-hRV-). Before hRV inoculation, pigs were fed with LGG with a daily dose increase of 10-fold (103~1012 ffu [colony forming units]/mL). The intestinal LGG counts remained in the rage of 107~108 cfu/ml and did not increase with the increasing intake amount of probiotics. hRV infection significantly increased the LGG counts in intestine (108~109 cfu/ml).The mean duration of rotavirus diarrhea, fecal virus shedding and viremia was shorter, mean cumulative score of diarrhea and peak virus titer were lower, and the percentage of pigs with diarrhea was less in the LGG+hRV+ group than in the LGG-hRV+ group. Furthermore, the pigs in the LGG+hRV+ group had a delayed days-to-onset of fecal virus shedding or diarrhea compared to the LGG-hRV+ group. These results demonstrated a clear and moderate protective effect of LGG on hRV-induced diarrhea and virus replication in Gn pigs. There was a trend that pigs with diarrhea had tighter junction and more TJ and AJ proteins in intestinal epithelium than the mock control pigs at post-inoculation 3~6 d. This difference may be the compensatory effect observed after rotavirus infection. A time course study of the intestinal TJ and AJ protein expression and distribution during the first 72 h after rotavirus inoculation is needed to address the question. LGG-feeding significantly decreased the production of TJ proteins (occludin, claudin-1 and claudin-3) and AJ proteinβ-catenin in ileum from LGG+hRV+ pigs compared to LGG-hRV+ pigs, indirectly indicating that LGG colonization reduced the lesion and proliferation of intestinal epithelial cells cuased by hRV infection. Additionally, The protective effect of LGG on hRV-induced diarrhea and virus replication may be attributed to the increased MUC3 mucin production, significantly enhanced anti-inflammatory TGF-βcytokine responses to hRV infection and maintained pro-inflammatory cytokine TNF-α, IFN-γand IL-8 response. The neonatal Gn pig model of human rotavirus infection and disease provided an excellent model system for these studies because this model closely mimics the human intestinal epithelial structure, physiology and immune responses.