a b Supplemental Figure 1 Niimi, et al IgA B220 -1 1 2 (weaning) 6 Peyer’s patches -1 1 2 (weaning) 6 weeks after parturition nullipara 102 103 104 105 106 5.5 8.9 7.8 5.1 7.7 6.7 2.9 50.9 61.1 61.5 64.5 58.1 62.0 2 (normal) 2.5 1.6 3.3 1.3 1.4 0.9 5.8 b * P<0.05 P<0.01 ** 2 weaning normal Plasma cells (B220-IgA+) 4 Number (x106) Plasmablasts (B220+IgA+) 20 10 30 Mature B cells (B220+IgA-) Total cells 60 40 Supplemental Figure 1 B cell differentiation in Peyer’s patches throughout the reproductive cycle. To isolate mononuclear cells, Peyer’s patches were individually harvested at 1 week before parturition (n = 5), just after parturition (n = 7), and 1 week (n = 7), 2 weeks (n = 7), and 6 weeks (n = 7) after parturition. Tissues were also collected two weeks after parturition from mice (n = 7) whose pups were force-weaned one week earlier. Non-pregnant nulliparous mice were used as a control group (n = 7). (a) Because Peyer’s patches, which are organized lymphoid follicles containing a large number of IgA−B220+ mature B cells, play an important role in initiating IgA production in the gut, IgA+B220+ plasmablasts that are activated by luminal antigens are noticeably found throughout the reproductive cycle. (b) Absolute numbers of IgA−B220+ mature B cells, IgA+B220+ plasmablasts and IgA+B220− plasma cells were calculated based on the number of total cells isolated and the frequency of each cell population in a flow cytometry profile. In Peyer’s patches, mature B cells had the largest population and plasmablasts were the next common subset. In contrast, the number of plasma cells were extremely low. *P < 0.05; **P < 0.01.

a b Control Infection Supplemental Figure 2 Niimi, et al HE IHC S. aureus / nucleus b Mammary gland Inguinal lymph node Left 89 % 92 % 95 % 79 % 88 % Right 96 % 94 % ND NT No.1 No.2 No.3 No.4 No.5 No.6 Animal Side S. aureus Others 93 % 0 % 87 % Supplemental Figure 2 S. aureus exposed into the mammary gland via nipples is detected in the inguinal lymph node. Mice were infected with 1 x 106 CFU/30 µL of S. aureus (n = 6) or 30 µL of just PBS (n = 6) to address whether the bacteria also colonized in the inguinal lymph node. (a) Initial pathological analysis showed that a large number of cells infiltrating not only into the stroma but also into the lumen of mammary alveoli were found in the mammary gland of infected (not control) mice. Immunohistochemical analysis with anti-S. aureus showed that the most exposed bacteria were present in the lumen of mammary alveoli; however, some were also found in the stroma. (b) 16S rRNA analysis using DNA extracted from the mammary gland and the inguinal lymph node of infected mice showed that a high frequency of S. aureus 16S rRNA was detected in both tissues. Percentage on each pie chart means how much frequency of S. aureus 16S rRNA gene was detected in each DNA sample collected from left or right side of the mammary gland or the inguinal lymph node of mice infected with S. aureus. DNA extracted from the mammary gland of one of six control mice was used as a negative control to demonstrate the absence of S. aureus 16 rRNA gene. Scale bar = 100 μm. ND; not detected, NT; not tested.

+ _ Supplemental Figure 3 Niimi, et al ccl28 / β-actin csn1 / β-actin Antibiotics NS NS (P=0.073) 6 2 4 2.0 1.0 1.5 0.5 3 1 ccl28 / β-actin pigr / β-actin iga / β-actin 6000 4000 2000 csn1 / β-actin 30000 20000 10000 csn2 / β-actin 0.15 0.05 0.10 csn3 / β-actin Supplemental Figure 3 Dispensable role of intestinal microorganisms in inducing the expression of CCL28 and pIgR in the mammary gland. Mice were freely administered antibiotics-containing water (n = 7) or just distilled water (n = 7) from one week before mating to two weeks after parturition to address the involvement of intestinal microorganisms in developing the mammary gland immune system. The expression levels of CCL28 and pIgR in addition to IgA, α-casein, β-casein and κ-casein in the mammary gland of mice administered antibiotics were identical to those of control mice.