Elsevier

Journal of Autoimmunity

Volume 68, April 2016, Pages 14-22
Journal of Autoimmunity

Skin microbiota-associated inflammation precedes autoantibody induced tissue damage in experimental epidermolysis bullosa acquisita

https://doi.org/10.1016/j.jaut.2015.08.007Get rights and content

Highlights

  • CD11c, KC and TNFα predict outcome of experimental epidermolysis bullosa acquisita.

  • Higher richness of skin microbiota is associated with development of EBA symptoms.

  • T helper cell influence does not differ between healthy and EBA diseased mice.

Abstract

Epidermolysis bullosa acquisita (EBA) is a chronic autoimmune blistering skin disease characterized by autoantibodies against type VII collagen (COL7). Immunization of SJL/J mice with recombinant murine COL7 results in break of tolerance and skin blisters. Strikingly, despite circulating autoantibodies, the same genetic background and identical environmental conditions, 20% of mice remain healthy. To elucidate the regulation of the transition from the presence of autoantibodies to overt autoimmune disease, we characterized the innate and adaptive immune response of mice that remain healthy after immunization and compared it to mice that developed skin disease.

Both clinically healthy and diseased SJL/J mice showed circulating autoantibodies and deposition of complement-fixing IgG2c autoantibodies and C3 at the dermal–epidermal junction. However, only in diseased animals significant neutrophil infiltration and increase in FcgRIV expression were observed in the skin. In contrast, the expression of T cell signature cytokines in the T cell zone of the draining lymph node was comparable between clinically healthy and diseased animals after immunization. Surprisingly, health was associated with a decreased expression of CD11c, TNFA and KC (CXCL1) in the skin prior to immunization and could be predicted with a negative predictive value of >80%. Furthermore, mice that did not develop clinical disease showed a significantly higher richness and distinctly clustered diversity of their skin microbiota before immunization.

Our data indicate that the decision whether blisters develop in the presence of autoantibodies is governed in the skin rather than in the lymph node, and that a greater richness of cutaneous bacterial species appears to be protective.

Introduction

Epidermolysis bullosa acquisita (EBA) is a chronic, prototypic organ-specific autoimmune bullous dermatosis (AIBD) characterized by antibodies targeting type VII collagen (COL7), a major component of the dermal–epidermal junction (DEJ) [1], [2], [3], [4]. Evidence from in vitro models, animal experiments and patient material identified COL7 as the target antigen in EBA and the pathogenic relevance of anti-COL7 autoantibodies has been established both in vitro and in vivo [5], [6], [7], [8], [9]. Both innate and adaptive immune responses are involved in disease development. For example, while binding of the Fab fragment alone to its target structure cannot induce a clinically overt disease in experimental models of the disease [8], [9], several studies have shown that Fc-mediated activation of the complement system and Fc-gamma-receptor (FcgR)-dependent degranulation of neutrophils are required to produce disease symptoms [6], [10]. In addition, the adaptive immune system plays an essential role. Immunization with a recombinant fragment of murine COL7 results in autoantibody production in almost all tested strains [11]. Clinical disease with subepidermal blister formation only occurs when associated with a Th1-like immune response and is linked to the H2s haplotype [12], [13].

However, although inbred SJL/J mice carry the H2s haplotype and 100% of them produce autoantibodies after immunization with COL7, only 80% develop skin blisters. Twenty percent remain healthy despite the same genetic background and identical environmental conditions [11], [12]. The lack of disease development in a considerable proportion of immunized animals cannot be explained by the current understanding of experimental EBA pathogenesis. This suggests the presence of a so far unknown factor which may modulate innate and/or adaptive immune responses in such a way that 20% of the animals are protected from development of overt clinical disease. In this context extrinsic triggers that locally influence the development of an immune response might play a role. Indeed, it has been shown that skin microbiota are able to regulate the production of cytokines such as IL-1 by cells derived from healthy skin [14] and to modulate the clinical course of skin disorders such as psoriasis [15] and atopic dermatitis [16].

Based on these observations, we analyzed the development of clinical symptoms in EBA-susceptible SJL/J mice, studied the contribution of the innate and adaptive immune response, and investigated the role of the skin microbiota. We found that in genetically identical mice, housed under highly standardized conditions, skin blistering depends on the influence of the local skin milieu on the innate immune system. An increased richness and diversity of the skin microbiota before immunization appear to prevent blister formation. This observation underlines the importance of host–environment interaction in regulating the transition from autoimmunity to autoimmune disease.

Section snippets

Despite presence of autoantibodies, 20% of SJL/J mice do not develop skin blisters

After immunization with COL7, 100% of SJL/J mice showed circulating autoantibodies in the blood against COL7. The majority of these mice developed overt skin blistering disease. However, 20% of immunized mice remained clinically healthy lacking cutaneous inflammation (Fig. 1A). The frequency of diseased and healthy mice was randomly distributed across different cages and experiments and mice were held under identical environmental conditions. Both healthy and mice with skin blisters showed

Discussion

Despite autoantibody production in all immunized SJL/J mice, clinically overt skin blistering is observed in only 80% of mice. Interestingly, the remaining 20% of the mice do not develop skin blisters. Here, we provide evidence that blister formation in genetically identical mice housed under highly standardized conditions is controlled by the local milieu of the skin and its effects on the innate immune response.

Analysis of the skin of immunized mice revealed that both healthy mice and mice

Mice

Female SJL/J mice were obtained from Charles River Laboratories (Sulzfeld, Germany). At the beginning of the experiments mice were 8–10 weeks old. Housing facilities were according to FELASA recommendations with a dark:light cycle of 12:12 h, an ambient temperature of 23 ± 1 °C and a humidity of 65 ± 5%. Housing of the mice was performed under conventional husbandry practices in open-box cages on metal racks. SPF-conditions were ensured throughout the experiments without any detection of before

Acknowledgments

This work was supported by grants from the Excellence Cluster “Inflammation at Interfaces” (DFG, EXC306/1 and 2), Research Training Group (RTG) “Modulation of Autoimmunity” (DFG, GRK1727), and the University of Lübeck Focus Program “Autoimmunity”. We thank Lidija Gutjahr, Marie-Luise Leppin, and Karola von Lingelsheim for excellent technical assistance.

References (47)

  • D.T. Woodley

    Identification of the skin basement-membrane autoantigen in epidermolysis bullosa acquisita

    N. Engl. J. Med.

    (1984)
  • D.T. Woodley et al.

    Epidermolysis bullosa acquisita antigen is the globular carboxyl terminus of type VII procollagen

    J. Clin. Investig.

    (1988)
  • T. Hashimoto et al.

    Pathogenesis of epidermolysis bullosa acquisita, an autoimmune subepidermal bullous disease

    J. Pathol.

    (2012)
  • C. Sitaru

    Induction of dermal-epidermal separation in mice by passive transfer of antibodies specific to type VII collagen

    J. Clin. Investig.

    (2005)
  • M. Kasperkiewicz

    Genetic identification and functional validation of FcγRIV as key molecule in autoantibody-induced tissue injury

    J. Pathol.

    (2012)
  • C. Sitaru

    Induction of complement-fixing autoantibodies against type VII collagen results in subepidermal blistering in mice

    J. Immunol.

    (2006)
  • C.M. Hammers

    Complement-fixing anti-type VII collagen antibodies are induced in Th1-polarized lymph nodes of epidermolysis bullosa acquisita-susceptible mice

    J. Immunol.

    (2011)
  • S. Naik

    Compartmentalized control of skin immunity by resident commensals

    Science

    (2012)
  • A. Fahlén et al.

    Comparison of bacterial microbiota in skin biopsies from normal and psoriatic skin

    Arch. Dermatol. Res.

    (2012)
  • S. Conlan et al.

    Species-level analysis of DNA sequence data from the NIH human microbiome project

    PloS One

    (2012)
  • Ning Li et al.

    Distinct binding affinities of Mac-1 and LFA-1 in neutrophil activation

    J. Immunol.

    (2013)
  • A.S. MacLeod et al.

    Functions of skin-resident γδ T cells

    Cell. Mol. Life Sci.

    (2012)
  • J. Banchereau et al.

    Dendritic cells and the control of immunity

    Nature

    (1998)
  • Cited by (28)

    • Autoimmune pre-disease

      2023, Autoimmunity Reviews
      Citation Excerpt :

      In the skin of mice that remained healthy, decreased expression of TNF, CXCL-1 and CD11c was noted. Furthermore, mice that did not develop clinical disease showed a significantly higher richness and distinctly clustered diversity of their skin microbiota before immunization [230]. In the context of the development of autoimmune disease, a diversity-poor skin microbiome may promote clinical disease manifestation.

    • Epidermolysis bullosa acquisita

      2022, Anais Brasileiros de Dermatologia
      Citation Excerpt :

      More recently, evidence of the involvement of genes that do not belong to the major histocompatibility complex (MHC) has been described in experimental models of EBA.7 Additional studies have demonstrated the protective role of the skin microbiota diversity in the clinical manifestations of EBA.17,18 In animal-induced inflammatory EBA, T-cell-deficient mice do not develop specific autoantibodies against COLVII, demonstrating the participation of T-lymphocytes in the disease pathogenesis.

    • Bullous Diseases of the Skin and Mucous Membranes

      2022, Clinical Immunology: Principles and Practice, Sixth Edition
    • The possible role of oral microbiome in autoimmunity

      2020, International Journal of Women's Dermatology
      Citation Excerpt :

      Furthermore, the results of this research raise the possibility that the cutaneous microbiome may contribute to the pathogenesis of the disease. Ellebrecht et al. (2016) observed that 20% of immunized mice with circulating autoantibodies of epidermolysis bullosa acquisita remained healthy. The immunization was conducted under identical genetic and environmental conditions.

    • Evaluation of cutaneous, oral and intestinal microbiota in patients affected by pemphigus and bullous pemphigoid: A pilot study

      2020, Experimental and Molecular Pathology
      Citation Excerpt :

      In addition to the common skin barrier disfunctions and to the well-known immunologic disturbances, strong evidence supports the alteration of microbial composition as a common cause of skin disorders development and worsening. The healthy CM consists of both commensal and pathogenic bacteria, it affects the skin barrier, and it could be involved in antigen-driven disorders characterized by damage of the skin like psoriasis, eczema and, as recently demonstrated, in autoimmune blistering disorders (Ellebrecht et al., 2016; Miodovnik et al., 2017; De Benedetto et al., 2012). Beforehand, this research field was affected by the intrinsic limitations of conventional bacteria culture-dependent methods.

    View all citing articles on Scopus
    View full text