Table 1.
List of antibodies used.
Fig 1.
S. aureus was identified only in AD children and correlated with high IgE levels.
(A) Ratios of average counts for bacterial species and their respective phyla found in the inflamed zones and control-matched zones of atopic dermatitis (AD) patients and their non-AD counterparts, respectively. (B) Colony-forming units (CFU/ml) of S. aureus from sampled zones in AD and non-AD subjects. Mann-Whitney, mean values with SEM are shown, p**<0.01. The black square denotes a patient with undetermined IgE values. (C) AD subjects were sorted in relation to the amounts of IgE antibodies (kU/l) measured in their blood. (B, C) Total IgE over 1000kU/l is shown in red, between 100 and 1000 kU/l in blue and below 100 kU/l in green. (D) Enterotoxins were identified in S. aureus clones from AD subjects (P02-P39) by DNA array.
Table 2.
Clinical characteristics of atopic patients (AD) and control subjects (non AD).
Fig 2.
IL-4-producing peripheral T CD4+ cells against Der p allergens are increased in AD children compared to IFN-γ- producing T CD4+ cells.
(A) IFN-γ and IL-4 ELISPot assays (spot forming units/106 T cells) were performed on peripheral blood from non-AD (N = 14) and AD (N = 15) children in response to crude extracts of Der p. (B) Ratio of IL-4 vs IFN-γ CD4+ T cell spots relative to titers of total or Der p1 specific IgE antibodies (kU/ml) in AD patients. Mann-Whitney, mean values with SEM are shown, p*<0.05, p**<0.01.
Fig 3.
Factors secreted by cutaneous S. aureus and S. epidermidis exert opposite effects on moDC.
(A) Representative activation phenotype (CD86, CD83 and HLA-DR levels) of monocyte-derived dendritic cells (moDC) exposed for 24 hours to the S. aureus (S.a, red) or S. epidermidis (S.e, blue) secretomes obtained from subject P04, or medium (NT, black) or LPS (green). (B) Secreted IFN-γ and IL-10 (pg/ml) levels in moDC as described in (A). Wilcoxon signed-rank test, mean values with SEM are shown, p*** <0.001, N = 14 independent experiments. (C) (S.e) and (S.a) were added to either moDC [moDC(S.e) and moDC(Sa)] or monocytes on day 0 of differentiation (mo(S.e)DC and mo(S.a)DC). (D) Cells were co-cultured with CFSE-labeled allogeneic naïve CD4+ T cells for 5 days at a ratio of 1:10 stimulator to T cells. The percentage of proliferating cells was then determined by flow cytometry. Mean values with +/- SEM are shown, N = 4, one-way ANOVA test p = 0.0009.
Fig 4.
S. epidermidis stimulated moDC release IL-10 that impairs their maturation and reduces S. aureus effect.
(A) Activation profile (CD86, CD83 and HLA-DR levels) of moDC exposed to either medium alone (NT), or the secretomes of S.aureus (S.a) and S.epidermidis (S.e) supplemented with anti-IL-10 or isotype-matched control (iso) antibodies. (B) MoDC were incubated with (S.a) alone or supplemented with conditioned medium (CM) from allogeneic moDC pulsed with (S.e), with the addition of anti-IL-10 or isotype-matched control antibodies. (C) Levels of secreted IFN-γ and IL-10 (ng/ml) by the same moDC presented in (A) and (B). Similar results were obtained in 3 independent experiments. (D) MoDC were exposed to a mixture of (S.a) at m.o.i of 10 (S.a 10) and increasing amounts of (S.e) co-cultured with CFSE-labeled allogeneic CD4+ T cells. The proliferation of T cells was quantified by flow cytometry (%). Paired t-test, p***<0.001 N = 4.
Fig 5.
The S. aureus and S. epidermidis secretomes exert opposite effects on the Treg suppressive function.
CFSE-labeled conventional CD4+ T cells (Tconv) were cultured in the presence of beads pre-loaded with anti-CD2, -CD3 and -CD28 antibodies and CD4+CD25+CD127dim/- T cells (Treg cells expressing Foxp3) pretreated for 24 hours with medium only (NT), or the secretomes of S. aureus (S.a) or S. epidermidis (S.e) at Tconv to Treg ratios of 1:1 (A) and 2:1 (B). Proliferation was assessed by flow cytometry after 5 days and the percentage of suppression was calculated as (1—proliferation of Tconv with Treg / proliferation of Tconv without Treg) X100. Paired t-test, p*<0.05, p**<0.01, N = 5.