Figure 1.
Pregnant mice were fed daily with L. paracasei in drinking water during the last week of gestation and lactation. Control mothers received drinking water without L. paracasei. Offspring mice were immunized subcutaneously (s.c.) with Bet v 1 on days 21, 35, and 49, then aerosol challenged with birch pollen (BP) on two consecutive days 1 week after the last s.c. sensitization (days 56, 57), and sacrificed on day 60.
Figure 2.
Maternal L. paracasei supplementation protects offspring from development of allergic responses.
(A) Differential leukocyte numbers in BAL fluid from offspring of L. paracasei-treated mothers or controls. Levels of IL-5 in BAL fluid (B) and production of IL-5 in Bet v 1-re-stimulated lung (C) and mediastinal lymph node (D) cell cultures were assessed by ELISA. Data is represented as mean ± SEM (n ≥8; results represent pooled data from two independently performed experiments). *p<0.05; n.d. not detected. Mac = macrophages, Lym = lymphocytes, Neu = neutrophils, Eos = eosinophils.
Figure 3.
Maternal L. paracasei supplementation reduces airway inflammation in offspring.
Representative lung tissue sections from sensitized and challenged offspring of L. paracasei-treated (B, D) or sham-treated (A, C) mothers. Samples were stained with hematoxylin and eosin (A, B) to assess inflammation/cellular infiltration or with periodic acid-Schiff stain (PAS) (C, D) to enumerate mucus-producing goblet cells. Magnification 100 x.
Figure 4.
Maternal L. paracasei supplementation results in up-regulation of regulatory markers.
(A) Expression of Foxp3 mRNA in lung tissue from offspring of L. paracasei-treated or sham-treated mothers. Foxp3 mRNA data is expressed relative to the housekeeping gene ALAS1. (B) Serum levels of TGF-β were determined by ELISA. Data is expressed as mean ± SEM (n ≥8; results represent pooled data from two independently performed experiments). **p<0.001; ***p<0.0001.
Figure 5.
L. paracasei inhibits allergen-specific recall responses in offspring both in vivo and in vitro.
(A-D) Splenocytes from Bet v 1 sensitized/challenged offspring of L. paracasei-treated or sham-treated mothers were stimulated with Bet v 1 (A-D). Splenocytes from sensitized/challenged offspring of sham-treated mothers were stimulated with Bet v 1 (Bet), L. paracasei (L.para) or Bet v 1/L. paracasei (Bet/L.para) (E, F). Cytokine production was determined by ELISA. Data is expressed as mean ± SEM (n ≥8; results represent pooled data from two independently performed experiments). *p<0.05; **p<0.01; ***p<0.001; n.d. not detected. For in vitro stimulation, L. paracasei NCC 2461 was treated with formaldehyde.
Figure 6.
Maternal L. paracasei supplementation reduces mitogen-induced immune responses.
(A-D) Splenocytes from offspring of L. paracasei-treated or sham-treated mothers were stimulated with ConA and cytokine production assessed by ELISA. Proliferation of splenocytes (E) and MLN cells (F) following stimulation with Bet v 1 or ConA was measured by 3[H]-thymidine incorporation. Data is expressed as mean ± SEM. Spleen: n≥8 per group; MLN: n ≥3 per group; (results represent pooled data from two independently performed experiments). *p<0.05; **p<0.001; ***p<0.0001.
Figure 7.
L. paracasei mediates the production of regulatory cytokines through TLRs pathways.
(A, B) Splenocytes from wild-type (WT), TLR2−/−, or TLR4−/− mice were stimulated with L. paracasei, LPS or Pam3CSK4 (Pam). Untreated cells (Med) served as a negative control. (C, D) BM-DC were stimulated with L. paracasei or LPS. Cytokine production was assessed by ELISA. Data is expressed as mean ± SEM; (results represent pooled data from two independently performed experiments). For in vitro stimulation, L. paracasei was treated with formaldehyde.