Characteristics of an In Vitro Mesenteric Lymph Node Cell Suspension Model and Its Possible Association with In Vivo Functional Evaluation

Abstract

In a previous study, we uncovered three immune-responsive patterns of gut microbes using an in vitro mesenteric lymph node cell suspension model, abbreviated as the MLN model hereafter. We used Akkermansia muciniphila and Clostridium butyricum as the first group directly inducing an immune response, Bifidobacterium sp. and Bacteroides sp. as the second group evoking an immune response with the help of stimuli (anti-CD3 and anti-CD28 antibodies), and Lactobacillus sp. as the third group blunting the immune response with or without stimuli. Our group previously clarified the immune-activation characteristics of A. muciniphila and linked its in vivo immune induction effect in GF and SPF mice under homeostasis. In the present study, we supplemented the characteristics of C. butyricum and B. bifidum in the in vitro MLN model and addressed the specific elements of the model. Finally, we used an in vivo TNBS-challenge model to show the functional differences between these species with different response patterns in vitro. The results showed that C. butyricum and B. bifidum evoked an immune response in vitro in a dose-dependent and strain-unique manner. Although TLR2, rather than TLR4, is indispensable for immune activation in the present in vitro model, it may not involve interaction between TLR2 and bacterial ligands. Like the PBMC model, the present in vitro MLN model is highly dependent on cell resources and should be given more attention when used to conduct a quantitative comparison. Finally, a mixture of two strong immunogenic strains, A. muciniphila and C. butyricum, significantly increased the mortality of TNBS-challenged (2,4,6-trinitrobenzene sulfonic acid, TNBS) mice, indicating a possible link between the in vitro MLN model and in vivo functional evaluation. However, more evidence is needed to clarify the associations and underlying mechanisms.

Bifidobacterium bifidum FJSWX19M5 alleviated 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced chronic colitis by mitigating gut barrier injury and increasing regulatory T cells

“…19,20 Our previous study demonstrated that treatment with B. bifidum strains reversed inflammatory responses and reduced colitis symptoms in acute 2,4,6trinitrobenzene sulfonic acid (TNBS)-treated mice. 21 We also observed that this protective effect of B. bifidum against acute colitis was strain-specific and correlated well with the immunomodulatory properties of the strains in the mesenteric lymph node (MLN) co-culture model. Based on this information, combined with our data from the in vitro discovery platform, we selected three B. bifidum strains (FJSWX19M5, FXJWS17M4 and FHENJZ3M6) and further compared the differences between those B. bifidum strains in alleviating TNBS-induced chronic colitis in mice.…”

“…Briefly, the MLN was freshly removed from male TNBS-treated BALB/c mice, mechanically disrupted, and cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 1 mM anti-CD3, and anti-CD28 (eBioscience). The cell suspensions (1 × 10 6 ) were co-cultured with viable or unviable bacterial suspension (optical density 600, 0.5) in a 96-well plate at a volume of 10 : 1 for 72 h. 21 IL-10, IL-17, and IL-22 were quantified using the corresponding mouse ELISA kits (R&D Systems) and replicated three times.…”

“…Food & Function of 10 : 1 for 72 h. 21 IL-10, IL-17, and IL-22 were quantified using the corresponding mouse ELISA kits (R&D Systems) and replicated three times.…”

Mechanistic insights into the role of probiotics in modulating immune cells in ulcerative colitis

“…69 The bioactive compounds originating from symbiotic microbiota are crucial in forming the intestinal immune system and controlling IL-10 release. 124 It is possible to stimulate the production of IL-10 in Foxp3+ cells. Treg cells are paramount in establishing immune tolerance towards antigens on mucosal surfaces, effectively avoiding inflammation in the gastrointestinal tract.…”

“…The intervention provided relief from body weight loss, mitigated colonic shortening and damage, reduced gut inflammation indicators, and restored colonic metabolism equilibrium in mice afflicted with TNBS‐induced chronic colitis 69 . The bioactive compounds originating from symbiotic microbiota are crucial in forming the intestinal immune system and controlling IL‐10 release 124 …”