Comparison of the effects of probiotic strains (Lactobacillus gasseri, Lactiplantibacillus plantarum, Lactobacillus acidophilus, and Limosilactobacillus fermentum) isolated from human and food products on the immune response of CT26 tumor-bearing mice

Abstract

This study aimed to compare the effects of the probiotic bacteria, L. gasseri (52b), L. plantarum (M11), L. acidophilus (AC2), and L. fermentum (19SH), isolated from human source and traditional food products on the modulation of the immune system and inflammatory response on BALB/c mouse model bearing CT26 tumor. Five groups of female inbred BALB/c mice were orally administered with the probiotics and their mixes (MIX, at a 1:1 ratio) at varying dosages (1.5 × 10⁸ cfu/ml and 1.2 × 10⁹ cfu/ml) before and after the injection of a subcutaneous CT26 tumor over the course of 38 days via gavage. Finally, their effects on the tumor apoptosis and the cytokine levels in spleen cell cultures were analyzed and compared. M11, MIX, and 52b groups had the greatest levels of interleukin-12 (IL-12) and interferon gamma (IFN-γ) production. The highest production level of granzyme B (GrB) was related to the MIX and 52b groups. Moreover, these groups showed the lowest production level of (IL-4) and transforming growth factor beta (TGF-β). Furthermore, the groups of MIX and 52b demonstrated the greatest amount of lymphocyte proliferation of spleen cells in response to the tumor antigen. The delayed-type hypersensitivity (DTH) response significantly increased in the groups of MIX and 52b compared with the control (p < 0.05). The findings demonstrated that the oral treatment of the human strain (52b) and the combination of these bacteria generated strong T helper type 1 (Th1) immune responses in the tumor tissue of the tumor-bearing mice, which led to the suppression of the tumor development.

Keywords: Adherence; Colon cancer; Immune system modulation; Interleukin-12; Tumor apoptosis.

Fig. 1

Model of oral administration of tested bacteria to female inbred BALB/c mouse model control group received PBS. On day 14, 2 × 10⁶ CT26 cells were injected subcutaneously. Growing tumors could be observed 3 days after inoculation of CT26 cells. The feeding process was carried out up to 24 days after the injection of the tumor

Fig. 2

A Heat-killed L. plantarum and L. gasseri from human samples and traditional foods IL-12 (p70) in spleen cell culture (in vitro). Data are presented as mean ± standard deviation, which were analyzed by one-way ANOVA and the Tukey test using Prism 8 software. A p-value of < 0.05 was considered statistically significant (****p-value < 0.0001 and *p-value < 0.0480). B IL-12 (p70) production by heat-killed L. acidophilus and L. fermentum from traditional foods in spleen cells culture (in vitro). 19 SH (**p < 0.0092) and AC2 (*p < 0.0268) compared with negative control, ns: not significant

Fig. 3

A Tumor volume. The analysis was performed using the Kruskal–Wallis test to compare the control group (PBS) with 52b (****p = 0.006) and MIX (****p = 0.002) at p < 0.05. B Tumor weight. M11 (**p = 0.0443), MIX (****p < 0.001), 52b (****p < 0.001), AC2 (**p = 0.023), and 19SH (**p = 0.042) compared with the PBS group. C Survival curve of CT26 tumor–bearing mice fed with probiotic strains. The survival curves of CT26 tumor–bearing mice fed with probiotic strains were compared using the log-rank statistical test, and statistical significance was set at p < 0.05. D Tumors were removed from the mice on the 24th day after CT26 cell line inoculation. The quality of the images was improved with CamScanner software (CamSoft, 6.23.0.2208100000)

Fig. 4

A Effects of fed probiotic strains on the production of IL-12 by tumor antigen-stimulated spleen cells. The analysis was performed using the Kruskal–Wallis test to compare the control group (PBS) with the other groups as well as pairwise differences between the fed groups at p < 0.05. M11 (****p < 0.001), 52b (**p = 0.024), and MIX (***p = 0.003) compared with the PBS group. B Effects of fed probiotic strains on IFN-γ production in spleen cells stimulated with tumor antigen. Groups M11 (****p < 0.001), 52b (**p = 0.047), MIX (****p = 0.004) compared with the PBS group, M11 compared to AC2 (**p = 0.048) and 19SH (**p = 0.011). C Effects of fed probiotic strains on TGF-β production in spleen cells stimulated with tumor antigen. 52b (****p = 0.005) and MIX (****p = 0.001) compared with the PBS group. D Effects of fed probiotic strains on GrB production in spleen cells stimulated with tumor antigen. Groups 52b (****p = 0.008), MIX (****p = 0.006), and 19SH (**p = 0.034) compared with the PBS group. E Effects of fed probiotic strains on IL-4 production in spleen cells stimulated with tumor antigen. Groups 52b (****p = 0.007) and MIX (****p = 0.001) compared with the PBS group, ns: not significant

Fig. 4

A Effects of fed probiotic strains on the production of IL-12 by tumor antigen-stimulated spleen cells. The analysis was performed using the Kruskal–Wallis test to compare the control group (PBS) with the other groups as well as pairwise differences between the fed groups at p < 0.05. M11 (****p < 0.001), 52b (**p = 0.024), and MIX (***p = 0.003) compared with the PBS group. B Effects of fed probiotic strains on IFN-γ production in spleen cells stimulated with tumor antigen. Groups M11 (****p < 0.001), 52b (**p = 0.047), MIX (****p = 0.004) compared with the PBS group, M11 compared to AC2 (**p = 0.048) and 19SH (**p = 0.011). C Effects of fed probiotic strains on TGF-β production in spleen cells stimulated with tumor antigen. 52b (****p = 0.005) and MIX (****p = 0.001) compared with the PBS group. D Effects of fed probiotic strains on GrB production in spleen cells stimulated with tumor antigen. Groups 52b (****p = 0.008), MIX (****p = 0.006), and 19SH (**p = 0.034) compared with the PBS group. E Effects of fed probiotic strains on IL-4 production in spleen cells stimulated with tumor antigen. Groups 52b (****p = 0.007) and MIX (****p = 0.001) compared with the PBS group, ns: not significant

Fig. 5

A Comparison of the treated and control groups in terms of lymphocyte proliferation of spleen cells. The analysis was performed using the Kruskal–Wallis test to compare the control group (PBS) with the other groups as well as pairwise differences between the fed groups at p < 0.05. 52b (**p = 0.031) and MIX (****p = 0.007) compared with the PBS group. B IFN-γ/IL-4. 52b (*p = 0.010), MIX (***p < 0.001), and M11 (**p = 0.008) compared with the PBS group. C The evaluation of DTH response (****p-value < 0.001 and *p-value < 0.05), ns: not significant

Fig. 6

A IL-12 (p70) production by CW components stimulation derived from the mice fed with probiotic strains. Data are presented as mean ± standard deviation, which were analyzed by one-way ANOVA and Tukey’s test using Prism 8 software. A p-value of < 0.05 was considered statistically significant (****p-value < 0.0001, ***p-value < 0.0008, **p-value < 0.004, and *p-value < 0.02). B IFN-γ production by stimulation of CW components derived from the mice fed with probiotic strains. CW M11 compared to PGN AC2 (*p = 0.0350) and PGN 19SH (*p = 0.0314), CW: cell wall, PGN: peptidoglycan, ns: not significant