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Page 8 of 16 Ding et al. Microbiome Res Rep 2024;3:16 https://dx.doi.org/10.20517/mrr.2023.64

B6MNI decreased the STAT6 mRNA relative expression (P < 0.05, Figure 3J) and increased the T-bet
mRNA relative expression significantly (P < 0.05, Figure 3K). No significant differences were found in the
relative expression of GATA3 in the colon of male mice [Figure 3L].

Effects of B. longum subsp. infantis on the JAK/STAT pathway in the colon of mice
The translational level of corresponding genes was verified through western blot analysis [Figure 4A and B].
The relative expression of T-bet was significantly higher only in the female mice treated with B6MNI
compared with the control group (P < 0.05, Figure 4C). No significant differences were found in the relative
expression of pSTAT6 for female mice [Figure 4D]. The relative expression of pSTAT1 was significantly
higher in the female mice fed with I10TI and B6MNI compared with the control group (P < 0.05 for all,
Figure 4E). No significant differences were found in the relative expression of GATA3 for female mice
[Figure 4F]. Additionally, B. longum subsp. infantis B6MNI and I8TI increased the relative expression of T-
bet (P < 0.05, Figure 4G). No significant differences were found in the relative expression of GATA3 in

colon tissues from either female or male mice [Figure 4H]. B. longum subsp. infantis B6MNI increased the
relative expression of pSTAT1 (P < 0.05, Figure 4I), while it decreased the relative expression of pSTAT6
(P < 0.05, Figure 4J) in male mice compared with the control group. B. longum subsp. infantis I8TI
decreased the level of pSTAT6 (P < 0.01, Figure 4J) in male mice compared with the control group.

Effects of B. longum subsp. infantis on the gut microbiota in mice
Total gut microbiota was analyzed by 16S rRNA gene sequencing. The mice of different genders showed
different results. For female mice, Chao 1 and Shannon indexes showed no significant differences among
groups for total bacteria [Supplementary Figure 6A and B]. For male mice, the Chao 1 index for total
bacteria was significantly lower in I4MI (P < 0.05), I5TI (P < 0.01), and I8TI (P < 0.01) groups compared
with that in the control [Supplementary Figure 6C]. No significant was found of Shannon index in male
mice [Supplementary Figure 6D]. PCoA revealed significantly different bacterial compositions for total
bacteria among the five groups (P = 0.001, Supplementary Figure 6E and F). The top 30 total bacteria were
presented in Supplementary Figure 6G. For total bacteria, the dominant member in the control group was
Lactobacillus (25.23%), whereas unclassified Muribaculaceae (19.07%), Bacteroides (14.39%), Enterococcus
(25.18%), and Alistipes (15.69%) became dominant in I4MI, I5TI, I10TI, and B6MNI treated groups,
respectively [Supplementary Figure 6G]. Lactobacillus and unclassified Muribaculaceae were dominant in
the control (15.98%, 15.44%) and I8TI groups (17.71%, 18.19%), Bacteroides (18.61%) and Lactobacillus
(17.34%) were predominant in the I4MI group, the I5TI group was dominated by Bacteroides (26.51%),
while the B6MNI group was dominated by Enterococcus (20.47%, Supplementary Figure 6H). The relative
abundance of Bifidobacterium in I4MI, I5TI, and B6MNI groups was significantly higher than that in the
control (P < 0.05, Supplementary Figure 6I), and the relative abundance of Enterococcus was significantly
higher in I10TI (P < 0.001) and B6MNI (P < 0.001, Supplementary Figure 6J) treated mice. Similar to that in
female mice, B. longum subsp. infantis I4MI, I5TI, I8TI, and B6MNI treatments increased the relative
abundance of total Bifidobacterium (P < 0.05, Supplementary Figure 6K), whereas the relative abundance of
Enterococcus was lower in the I8TI-treated mice but higher in the B6MNI group compared with that in the
control group (P < 0.05, Supplementary Figure 6L).

The bifidobacterial community in each group was analyzed by bifidobacterial groEL gene sequencing. For
female mice, I4MI, I5TI, I8TI, and B6MNI increased the Chao 1 index (P < 0.05), but I4MI, I5TI, and
B6MNI decreased the Shannon index (P < 0.01, Figure 5A and B). PCoA results revealed that the
Bifidobacterium composition in the five groups was significantly different (P = 0.001, Figure 5C). B. longum
subsp. infantis was dominant in the control (82.84%), I4MI (97.66%), I5TI (99.40%), I8TI (85.40%), and
B6MNI groups (96.70%, Figure 5D). Furthermore, the relative abundance of B. longum subsp. infantis was
significantly higher in I4MI, I5TI, and B6MNI groups than that in the control group (P < 0.05, Figure 5E).