Effects of the Staphylococcus aureus and Staphylococcus epidermidis Secretomes Isolated from the Skin Microbiota of Atopic Children on CD4+ T Cell Activation.

Interactions between the immune system and skin bacteria are of major importance in the pathophysiology of atopic dermatitis (AD), yet our understanding of them is limited. From a cohort of very young AD children (1 to 3 years old), sensitized to Dermatophagoides pteronyssinus allergens (Der p), we conducted culturomic analysis of skin microbiota, cutaneous transcript profiling and quantification of anti-Der p CD4+ T cells. This showed that the presence of S. aureus in inflamed skin of AD patients was associated with a high IgE response, increased expression of inflammatory and Th2/Th22 transcripts and the prevalence of a peripheral Th2 anti-Der p response. Monocyte-derived dendritic cells (moDC) exposed to the S. aureus and S. epidermidis secretomes were found to release pro-inflammatory IFN-γ and anti-inflammatory IL-10, respectively. Allogeneic moDC exposed to the S. aureus secretome also induced the proliferation of CD4+ T cells and this effect was counteracted by concurrent exposure to the S. epidermidis secretome. In addition, whereas the S. epidermidis secretome promoted the activity of regulatory T cells (Treg) in suppressing the proliferation of conventional CD4+ T cells, the Treg lost this ability in the presence of the S. aureus secretome. We therefore conclude that S. aureus may cause and promote inflammation in the skin of AD children through concomitant Th2 activation and the silencing of resident Treg cells. Commensals such as S. epidermidis may counteract these effects by inducing the release of IL-10 by skin dendritic cells

Correction: Effects of the Staphylococcus aureus and Staphylococcus epidermidis Secretomes Isolated from the Skin Microbiota of Atopic Children on CD4+ T Cell Activation.

<p>Correction: Effects of the <i>Staphylococcus aureus</i> and <i>Staphylococcus epidermidis</i> Secretomes Isolated from the Skin Microbiota of Atopic Children on CD4⁺ T Cell Activation</p

Transformation of acinetobacter baumannii: Electroporation

Although the pan and the core genome of Acinetobacter baumannii and its essential genes are relatively well characterized, functional characterization of these genes has not paralleled the genome-level studies. However, recently developed genetic tools and optimized protocols are poised to accelerate genetic manipulation of A. baumannii. Transferring exogenous DNA into the cytosol of bacteria cells is a critical step in genetic characterizations. Conjugation is restricted to the transfer of DNA from one bacterial cell to another, and only a portion of A. baumannii clinical isolates are naturally competent. Electroporation, which is thought to transiently create aqueous pores in the membrane, is a preferred method in transferring exogenous DNA as it does not have such limitations. Several factors contribute to efficiency of electroporation and often need to be empirically optimized to maximize efficiency of this procedure. Here we provide an optimized electroporation protocol and guidance for electroporation of clinical MDR isolates of A. baumannii

IL-4-producing peripheral T CD4⁺ cells against Der p allergens are increased in AD children compared to IFN-γ- producing T CD4⁺ cells.

<p>(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 <i>vs</i> 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.</p

Clinical characteristics of atopic patients (AD) and control subjects (non AD).

<p>Clinical characteristics of atopic patients (AD) and control subjects (non AD).</p

List of antibodies used.

<p>List of antibodies used.</p

The <i>S</i>. <i>aureus</i> and <i>S</i>. <i>epidermidis</i> secretomes exert opposite effects on the Treg suppressive function.

<p>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⁺CD127^dim/- T cells (Treg cells expressing Foxp3) pretreated for 24 hours with medium only (NT), or the secretomes of <i>S</i>. <i>aureus</i> (S.a) or <i>S</i>. <i>epidermidis</i> (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.</p

<i>S</i>. <i>epidermidis</i> stimulated moDC release IL-10 that impairs their maturation and reduces <i>S</i>. <i>aureus effect</i>.

<p>(A) Activation profile (CD86, CD83 and HLA-DR levels) of moDC exposed to either medium alone (NT), or the secretomes of <i>S</i>.<i>aureus</i> (S.a) and <i>S</i>.<i>epidermidis</i> (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.</p

<i>S</i>. <i>aureus</i> was identified only in AD children and correlated with high IgE levels.

<p>(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 <i>S</i>. <i>aureus</i> 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 <i>S</i>. <i>aureus</i> clones from AD subjects (P02-P39) by DNA array.</p