Page 17 - Read Online

Ding et al. Microbiome Res Rep 2024;3:16 https://dx.doi.org/10.20517/mrr.2023.64 Page 11 of 16

Bifidobacterium in female mice; (K and L) Chao 1 and Shannon index of total bacteria in male mice; (M) β-diversity of bifidobacterial
community in male mice, respectively; (N) heatmap of bifidobacterial composition in male mice. The relative abundance of
Bifidobacterium in each group was row normalization. The blue circle means lower relative abundance and the red circle means higher
*
**
***
relative abundance; (O-T) the significant difference of Bifidobacterium in male mice. P < 0.05, P < 0.01, P < 0.001, compared to
corresponding to the control group. The mean ± SEM (n = 8 per group) was used to represent the data. SEM: Error of the mean.
Similar to the effects of B. longum subsp. infantis on the bifidobacterial community in female mice, the
Chao 1 index for male mice was significantly higher in I4MI, I5TI, and I8TI groups, while the Shannon
index was significantly lower in the I4MI, I5TI, I8TI, and B6MNI groups (P < 0.05, Figure 5K and L). The
bifidobacterial profile was significantly different among the five groups (P = 0.001, Figure 5M). B. longum
subsp. infantis was the predominant Bifidobacterium species in the control (83.37%), I4MI (98.45%), I5TI
(99.14%), I8TI (99.60%), and B6MNI groups (98.21%, Figure 5N). The relative abundance of B. longum
subsp. infantis was significantly higher, but the relative abundances of B. animalis subsp. lactis, B. breve,
B. pseudocatenalatum, and B. pseudolongum were significantly lower in I4MI, I5TI, I8TI, and B6MNI
groups compared to that in the control group (P < 0.05, Figure 5O-T). Moreover, I5TI and I8TI treatments
showed a lower relative abundance of B. longum subsp. longum (P < 0.01, Figure 5R).

Effects of B. longum subsp. infantis on cytokines and immunoglobulins in serum
Th1/Th2-related cytokines in the serum were further determined to assess the regulation of strains on the
balance of Th1/Th2 in peripheral blood of the organism. Only B6MNI increased the level of IFN-γ (P < 0.05,
Figure 6A) in female mice. Four strains had no significant effects on the level of IgG2a, IL4, and IgE
[Figure 6B-D]. B6MNI increased the ratio of IgG2a/IgE (P < 0.05, Figure 6E) in female mice. In the males,
B6MNI increased the level of IFN-γ (P < 0.05, Figure 6F) and IgG2a (P < 0.05, Figure 6G). Four strains
had no significant effects on the level of IL4 and IgE [Figure 6H and I]. B6MNI increased the IgG2a/IgE
ratio (P < 0.05, Figure 6J).

DISCUSSION
In this study, we evaluated the inflammatory cytokines, genes, and proteins related to the JAK/STAT
signaling pathway that regulate the Th1/Th2 immune balance and gut microbiota in mice from 1 week-old
to 3-week-old that had been gavaged with eight different Bifidobacterium longum subsp. infantis strains
(I2MI, I4MI, I4MNI, I5TI, I6TI, I8TI, I10TI, and B6MNI). Our findings indicated that eight strains
B. longum subsp. infantis may adjust the Th1/Th2 balance in the colon and serum in female and male mice
but with strain specificity, among which B. longum subsp. infantis B6MNI was more pronounced compared
to the other seven strains.

The Th1/Th2 balance is related to the occurrence and development of allergy, asthma, and eczema .
[29]
B. longum subsp. infantis, an important microorganism in infant gut, may adjust the Th1/Th2 immune
balance in both male and female mice in this study. IFN-γ and IL-4 are the cytokines released by Th1 and
Th2 cells, respectively. Specifically, B. longum subsp. infantis B6MNI was shown to increase the level of
IFN-γ significantly in female mice but failed to increase this in male mice. Additionally, B. longum subsp.
infantis B6MNI also stimulated B cells to release Th1 type immunoglobulin IgG2a and increased the ratio of
IgG2a/IgE, which refers to the Th2 immune type transferring to the Th1 immune type. However, in males,
B. longum subsp. infantis B6MNI failed to increase the cytokines and immunoglobulin related to the Th1
immune type but decreased the IgE related to the Th2 immune type. Hence, we proposed that the
regulatory effect of B. longum subsp. infantis B6MNI on female mice was mainly based on promoting the
secretion of Th1 cytokines, while its effect on male mice was mainly based on inhibiting the secretion of Th2
cytokines, which may be influenced by gender . Currently, it has been shown that Bifidobacterium could
[30]
enhance the intestinal immune barrier, but their regulation of serum indicators is limited . The
[31]