Ro stimulation of PBMCs with bacterial supernatantsPBMCs from healthy adult blood donors were thawed and washed prior to being cultured at a concentration of 106 cells/ml with 2,5 of bacterial supernatants from either S. aureus 161.2 and/or L rhamnosus GG, or left in culture medium for 6 h before addition of protein-transport inhibitor monensin (GolgiStop, BD Biosciences) over night for intracellular detection of cytokines.Early Gut Bacteria and Cytokine Responses at TwoTable 1. Percentages of infants colonized with respective bacteria.Week 1.Week 2. 9/27 10/27 7/27 9/27 19/27 33,3 37,0 25,9 33,3 70,Month 1. 8/24 9/24 8/24 8/24 18/24 33,3 37,5 33,3 33,3Month 2. 9/26 14/26 8/26 13/26 19/26 34,6 53,8 30,7 50 73,B. adolescentis B. bifidum B. breveLactobacilli11/28 8/28 8/28 6/28 15/39,3 28,6 28,6 21,4 53,S. aureusdoi:10.1371/journal.pone.0049315.tSupernatants from the same cultures were collected and stored at 285uC until cytokine analyses.FACS analysis of cytokine-producing T helper cellsA panel of FITC-, PE-, PerCP- and APC-conjugated mAbs for staining of CD4, IL-4, and IFN-c were used, all from BD Biosciences. Cells were harvested and stained according to standard procedures for surface antigens. For intracellular cytokine detection, cells were fixed and permeabilized prior tostaining. Gating was performed on the basis of forward and side scatter properties for live cells followed by specific gating of CD4+cell cytokine production within the lymphocyte gate. Data 25837696 were analyzed with FlowJo where frequencies of cytokine-producing T helper cells were determined after reduction of background percentages in cultures in medium alone. Within the live gate, cell viability was evaluated by 7AAD-binding (BD Via-ProbeTM) and did not differ significantly among donors and between stimulations.Figure 1. Lactobacilli colonization at 2 weeks of age in relation to cytokine secreting cells, after in vitro PHA stimulation at age two. Infants with (n = 9) or without (n = 18) lactobacilli at 2 weeks of age in relation to (A) IL-42, (B) IL-102 and (C) IFN-c producing cells after PHA stimulation. Boxes cover 25th to 75th percentile and the central square being the median value. Whiskers extend to non-outlier maximum and PHCCC minimum and squares represents outliers. doi:10.1371/journal.pone.0049315.gEarly Gut Bacteria and Cytokine Responses at TwoTable 2. NHS-Biotin web Relative amounts of infant gut bacterial species in relation to cytokine secreting cells at two years of age.LactobacilliSpearman R Week 1 
IL-4 IL-10 IFN-c Week 2 IL-4 IL-10 IFN-c Month 1 IL-4 IL-10 IFN-c Month 2 IL-4 IL-10 IFN-c 20.390 20.305 0.153 0.049 0.130 0.455 20.423 20.393 20.151 0.040 0.057 0.482 20.394 20.375 20.331 0.042 0.054 0.091 20.126 0.025 20.186 0.522 0.897 0.B. bifidum P-valueSpearman R P-valueB. breveSpearman R P -valueB. adolescentisSpearman RS. aureus P -valueSpearman R P -value20.334 20.238 20.0.083 0.223 0.20.202 20.325 20.0.302 0.091 0.20.001 20.099 20.0.995 0.617 0.0.064 0.257 0.0.746 0.186 0.20.353 20.299 20.0.071 0.130 0.20.269 20.364 0.0.175 0.062 0.20.016 0.014 0.0.935 0.944 0.0.332 0.462 0.0.091 0.015 0.20.310 20.249 20.0.141 0.241 0.20.226 20.325 0.0.288 0.122 0.0.059 0.149 0.0.784 0.488 0.0.181 0.323 0.0.396 0.124 0.20.243 20.357 0.0.232 0.074 0.20.301 20.351 0.0.136 0.079 0.20.066 20.072 0.0.750 0.722 0.0.334 0.381 0.0.096 0.055 0.Week 1 n = 28, Week 2 n = 27, Month 1 n = 24, Month 2 n = 26. Significant P-values are shown in bold text. doi:10.1371/journal.pone.0049315.tIL-4.Ro stimulation of PBMCs with bacterial supernatantsPBMCs from healthy adult blood donors were thawed and washed prior to being cultured at a concentration of 106 cells/ml with 2,5 of bacterial supernatants from either S. aureus 161.2 and/or L rhamnosus GG, or left in culture medium for 6 h before addition of protein-transport inhibitor monensin (GolgiStop, BD Biosciences) over night for 
intracellular detection of cytokines.Early Gut Bacteria and Cytokine Responses at TwoTable 1. Percentages of infants colonized with respective bacteria.Week 1.Week 2. 9/27 10/27 7/27 9/27 19/27 33,3 37,0 25,9 33,3 70,Month 1. 8/24 9/24 8/24 8/24 18/24 33,3 37,5 33,3 33,3Month 2. 9/26 14/26 8/26 13/26 19/26 34,6 53,8 30,7 50 73,B. adolescentis B. bifidum B. breveLactobacilli11/28 8/28 8/28 6/28 15/39,3 28,6 28,6 21,4 53,S. aureusdoi:10.1371/journal.pone.0049315.tSupernatants from the same cultures were collected and stored at 285uC until cytokine analyses.FACS analysis of cytokine-producing T helper cellsA panel of FITC-, PE-, PerCP- and APC-conjugated mAbs for staining of CD4, IL-4, and IFN-c were used, all from BD Biosciences. Cells were harvested and stained according to standard procedures for surface antigens. For intracellular cytokine detection, cells were fixed and permeabilized prior tostaining. Gating was performed on the basis of forward and side scatter properties for live cells followed by specific gating of CD4+cell cytokine production within the lymphocyte gate. Data 25837696 were analyzed with FlowJo where frequencies of cytokine-producing T helper cells were determined after reduction of background percentages in cultures in medium alone. Within the live gate, cell viability was evaluated by 7AAD-binding (BD Via-ProbeTM) and did not differ significantly among donors and between stimulations.Figure 1. Lactobacilli colonization at 2 weeks of age in relation to cytokine secreting cells, after in vitro PHA stimulation at age two. Infants with (n = 9) or without (n = 18) lactobacilli at 2 weeks of age in relation to (A) IL-42, (B) IL-102 and (C) IFN-c producing cells after PHA stimulation. Boxes cover 25th to 75th percentile and the central square being the median value. Whiskers extend to non-outlier maximum and minimum and squares represents outliers. doi:10.1371/journal.pone.0049315.gEarly Gut Bacteria and Cytokine Responses at TwoTable 2. Relative amounts of infant gut bacterial species in relation to cytokine secreting cells at two years of age.LactobacilliSpearman R Week 1 IL-4 IL-10 IFN-c Week 2 IL-4 IL-10 IFN-c Month 1 IL-4 IL-10 IFN-c Month 2 IL-4 IL-10 IFN-c 20.390 20.305 0.153 0.049 0.130 0.455 20.423 20.393 20.151 0.040 0.057 0.482 20.394 20.375 20.331 0.042 0.054 0.091 20.126 0.025 20.186 0.522 0.897 0.B. bifidum P-valueSpearman R P-valueB. breveSpearman R P -valueB. adolescentisSpearman RS. aureus P -valueSpearman R P -value20.334 20.238 20.0.083 0.223 0.20.202 20.325 20.0.302 0.091 0.20.001 20.099 20.0.995 0.617 0.0.064 0.257 0.0.746 0.186 0.20.353 20.299 20.0.071 0.130 0.20.269 20.364 0.0.175 0.062 0.20.016 0.014 0.0.935 0.944 0.0.332 0.462 0.0.091 0.015 0.20.310 20.249 20.0.141 0.241 0.20.226 20.325 0.0.288 0.122 0.0.059 0.149 0.0.784 0.488 0.0.181 0.323 0.0.396 0.124 0.20.243 20.357 0.0.232 0.074 0.20.301 20.351 0.0.136 0.079 0.20.066 20.072 0.0.750 0.722 0.0.334 0.381 0.0.096 0.055 0.Week 1 n = 28, Week 2 n = 27, Month 1 n = 24, Month 2 n = 26. Significant P-values are shown in bold text. doi:10.1371/journal.pone.0049315.tIL-4.
