The extracellular serine protease from Staphylococcus epidermidis elicits a type 2-biased immune response in atopic dermatitis patients

Abstract

Background: Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disease with skin barrier defects and a misdirected type 2 immune response against harmless antigens. The skin microbiome in AD is characterized by a reduction in microbial diversity with a dominance of staphylococci, including Staphylococcus epidermidis (S. epidermidis).

Objective: To assess whether S. epidermidis antigens play a role in AD, we screened for candidate allergens and studied the T cell and humoral immune response against the extracellular serine protease (Esp).

Methods: To identify candidate allergens, we analyzed the binding of human serum IgG4, as a surrogate of IgE, to S. epidermidis extracellular proteins using 2-dimensional immunoblotting and mass spectrometry. We then measured serum IgE and IgG1 binding to recombinant Esp by ELISA in healthy and AD individuals. We also stimulated T cells from AD patients and control subjects with Esp and measured the secreted cytokines. Finally, we analyzed the proteolytic activity of Esp against IL-33 and determined the cleavage sites by mass spectrometry.

Results: We identified Esp as the dominant candidate allergen of S. epidermidis. Esp-specific IgE was present in human serum; AD patients had higher concentrations than controls. T cells reacting to Esp were detectable in both AD patients and healthy controls. The T cell response in healthy adults was characterized by IL-17, IL-22, IFN-γ, and IL-10, whereas the AD patients' T cells lacked IL-17 production and released only low amounts of IL-22, IFN-γ, and IL-10. In contrast, Th2 cytokine release was higher in T cells from AD patients than from healthy controls. Mature Esp cleaved and activated the alarmin IL-33.

Conclusion: The extracellular serine protease Esp of S. epidermidis can activate IL-33. As an antigen, Esp elicits a type 2-biased antibody and T cell response in AD patients. This suggests that S. epidermidis can aggravate AD through the allergenic properties of Esp.

Keywords: Esp; IgE; Staphylococcus epidermidis; Th2 cells; allergy; atopic dermatitis; protease.

Figure 1

Identification of Esp as an IgG4-binding protein of S. epidermidis. (A) IgG1 and IgG4 binding to ECPs of S. epidermidis. Specific IgG1 and IgG4 antibodies were measured in the sera of 50 AD patients (red) and 30 healthy individuals (black). (B) 1D-immunoblot. S. epidermidis ECPs were separated by size using a capillary-based automated immune blotting system. Signals from IgG4 binding to the ECPs were obtained. The blue arrow shows a strong signal from IgG4 binding to a protein with a molecular mass of around 35 kDa. (C) S. epidermidis ECPs were separated by 2D-gel electrophoresis and blotted onto a membrane. The blot was incubated with pooled sera from AD patients and decorated with anti-human IgG4 HRP-conjugated antibodies. The IgG4-binding spots were cut out, and the proteins were identified by LC-MS/MS. Circled spots represent Esp. AD, atopic dermatitis; ECPs, extracellular proteins of S. epidermidis; MW, molecular weight; **P≤ 0.01, ***P≤ 0.001, Mann-Whitney U-test.

Figure 2

Specific antibody measurement. Specific IgG1 and IgE antibodies against Esp and GehD were measured in the sera of 50 AD patients (red) and 30 healthy individuals (black). (A) IgG1 binding to Esp and GehD of S. epidermidis. (B) Serum IgE specific for Esp and GehD. (C) The IgE/IgG1 was calculated using the data presented in (A, B). Medians are shown. AD, atopic dermatitis; Esp, extracellular serine protease; GehD, triacylglycerol lipase; OD, optical density; ns: non-significant, P> 0.05, *P≤ 0.05, ****P≤ 0.0001. Mann-Whitney U-test.

Figure 3

Cytokine secretion by Esp-stimulated T cells. (A) T cells and monocytes were isolated from peripheral blood of AD patients (n=8) and healthy individuals (n=16) and stimulated with recombinant Esp. Culture supernatants were harvested on day 9, and cytokine concentrations were measured by cytometric bead array. The T cell response in healthy individuals was dominated by IL-17 and IL-22. T cells from AD patients responded to Esp with a Th2-biased cytokine profile: lack of IL-17 cytokines and reduced IFN-γ, IL-22 as well as IL-10 (B) Ratios were obtained by dividing values of Th2 cytokines by those of IFN-γ. Medians are shown. IL, interleukin; AD, atopic dermatitis; Esp, extracellular serine protease; Ctr., unstimulated T cells (medium control). ns: non-significant, P> 0.05, *P≤ 0.05, **P≤ 0.01, ***P≤ 0.001, ****P≤ 0.0001. Mann-Whitney U-test.

Figure 4

Proteolytic activity of Esp. (A) Mature Esp cleaves casein. Casein was incubated for 30 minutes with or without Esp. The reaction products were separated by 12% SDS-PAGE and visualised by chemiluminescence using a protein dye. (B) Proteolytic cleavage of IL-33 by Esp. Recombinant full-length IL-33 was digested with Esp for 30 minutes at 37°C. The cleavage pattern was visualised by immunoblotting, using a mouse anti-human IL-33 monoclonal antibody. (C) Mass spectrometric identification of the cleavage sites. The red arrows show the location of the Esp cleavage motifs in the IL-33 protein sequence. IL, interleukin; Esp, extracellular serine protease; MW, molecular weight.

Figure 5

IL-6 response of monocytes and monocyte-derived dendritic cells to Esp-digested IL-33. (A) Monocytes were isolated from PBMCs of healthy individuals (n=3) and stimulated with full-length human IL-33, Esp-digested IL-33, Esp alone or medium only (Ctr) for 24 hours (100 ng/mL of each stimulant was used). Secreted IL-6 was measured in the culture supernatant by ELISA. (B) Dendritic cells were generated from monocytes (moDCs) from healthy individuals (n=3) and stimulated as described in (A). Both monocytes and moDCs secreted more IL-6 when stimulated with Esp-cleaved IL-33 than in response to the full-length IL-33. moDCs, monocyte-derived dendritic cell; IL-33, interleukin 33; Esp, extracellular serine protease; Ctr, unstimulated medium control.