Strain-Specific Identification and In Vivo Immunomodulatory Activity of Heat-Killed Latilactobacillus sakei K040706

Abstract

We previously reported that the immunostimulatory activity of heat-killed Latilactobacillus sakei K040706 in macrophages and cyclophosphamide (CTX)-treated mice. However, identification of heat-killed L. sakei K040706 (heat-killed LS06) using a validated method is not yet reported. Further, the underlying molecular mechanisms for its immunostimulatory effects in CTX-induced immunosuppressed mice remain unknown. In this study, we developed strain-specific genetic markers to detect heat-killed L. sakei LS06. The lower detection limit of the validated primer set was 2.1 × 10⁵ colony forming units (CFU)/mL for the heat-killed LS06 assay. Moreover, oral administration of heat-killed LS06 (10⁸ or 10⁹ CFU/day, p.o.) effectively improved the body loss, thymus index, natural killer cell activity, granzyme B production, and T and B cell proliferation in CTX-treated mice. In addition, heat-killed LS06 enhanced CTX-reduced immune-related cytokine (interferon-γ, interleukin (IL)-2, and IL-12) production and mRNA expression. Heat-killed LS06 also recovered CTX-altered microbiota composition, including the phylum levels of Bacteroidetes, Firmicutes, and Proteobacteria and the family levels of Muribaculaceae, Prevotellaceae, Tannerellaceae, Christensenellaceae, Gracilibacteraceae, and Hungateiclostridiaceae. In conclusion, since heat-killed L. sakei K040706 ameliorated CTX-induced immunosuppression and modulated gut microbiota composition, they have the potential to be used in functional foods for immune regulation.

Keywords: cyclophosphamide; heat-killed Latilactobacillus sakei; immune improvement; microbiota; splenocyte.

Figure 1

Animal study design. Establishment of cyclophosphamide (CTX)-induced immunosuppressed model and oral administration of heat-killed L. sakei K040706 (heat-killed LS06) 10⁸ or 10⁹ colony forming units (CFU)/day) for 20 days.

Figure 2

(A) Phylogenetic tree showing the relatedness of L. sakei strains based on ANI analysis. (B) Core and pan-genome curves show the trend of the core gene and pan-gene families with an increase in the number of genomes by Heap’s law model. (C) Pan-genome Venn diagram of L. sakei strains, including K040706, DSM 20017, DSM 15831, DS4, and DSM20019, showing the distribution of shared and unique pangenome orthologous groups (POGs) among the five strains.

Figure 2

(A) Phylogenetic tree showing the relatedness of L. sakei strains based on ANI analysis. (B) Core and pan-genome curves show the trend of the core gene and pan-gene families with an increase in the number of genomes by Heap’s law model. (C) Pan-genome Venn diagram of L. sakei strains, including K040706, DSM 20017, DSM 15831, DS4, and DSM20019, showing the distribution of shared and unique pangenome orthologous groups (POGs) among the five strains.

Figure 3

Detection of PCR product by strain-specific primers. Numbers at the top indicate the primer pairs. L, 100 bp DNA ladder; Lane 1, live LS06; Lane 2, heat-killed L. sakei LS06; Lane 3, KACC17865; Lane 4, KACC17868; Lane 5, KACC17871; Lane 6, KACC18352; Lane 7, KACC17864; Lane 8, KACC16119.

Figure 4

Real-time PCR analysis of genomic DNA, live, and heat-killed LS06 for a range of concentrations. (A) Standard curve of live L. sakei. (B) Standard curve of heat-killed LS06. (C) Melt curve generated by strain-specific primers in real-time PCR.

Figure 5

Comparison of (A) body weight and (B) thymus index between CTX and heat-killed LS06 treatment in mice. Thymus index was calculated as thymus weight (g)/body weight (kg). Data are presented as the mean ± SEM (n = 10). # p < 0.05 vs. Con group; * p < 0.05, *** p < 0.001 vs. CTX group.

Figure 6

Effects of heat-killed LS06 on the regulation of immune cells in CTX-treated mice. Heat-killed LS06 (10⁸ or 10⁹ CFU/day, p.o.) were provided for 20 days and the splenocytes were prepared from isolated spleen of sacrificed mice. (A) NK cell cytotoxic activity and (B) granzyme B production (C,D) T and B cell proliferation. Data are presented as the mean ± SEM (n = 10). # p < 0.05 vs. Con group; * p < 0.05, ** p < 0.01 vs. CTX group.

Figure 7

Effects of heat-killed LS06 on the expression level of Th1-related cytokines production and mRNA in splenocytes isolated from CTX-treated mice. (A–C) The production and (D–F) mRNA expression levels of the Th1-related cytokines. Data are presented as mean ± SEM (n = 10). # p < 0.05 vs. Con group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. CTX group.

Figure 8

Effects of heat-killed LS06 treatment on the composition of microbiota in CTX-treated mice. Genomic DNA was analyzed for bacterial composition using 16S rRNA gene sequencing. Data are presented as mean ± SEM (n = 10). # p < 0.05 vs. Con group; * p < 0.05 and *** p < 0.001 vs. CTX group.

Figure 9

Effects of heat-killed LS06 treatment on family’s composition in CTX-treated mice. Relative abundance ratio of families of (A) Bacteroidetes and (B) Firmicutes. Data are presented as mean ± SEM (n = 10). # p < 0.05 vs. Con group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. CTX group.