GM-CSF modulates autoantibody production and skin blistering in experimental epidermolysis bullosa acquisita.

GM-CSF activates hematopoietic cells and recruits neutrophils and macrophages to sites of inflammation. Inhibition of GM-CSF attenuates disease activity in models of chronic inflammatory disease. Effects of GM-CSF blockade were linked to modulation of the effector phase, whereas effects on early pathogenic events, for example, Ab production, have not been identified. To evaluate yet uncharacterized effects of GM-CSF on early pathogenic events in chronic inflammation, we employed immunization-induced epidermolysis bullosa acquisita (EBA), an autoimmune bullous disease caused by autoantibodies to type VII collagen. Compared to wild-type mice, upon immunization, GM-CSF(-/-) mice produced lower serum autoantibody titers, which were associated with reduced neutrophil numbers in draining lymph nodes. The same effect was observed in neutrophil-depleted wild-type mice. Neutrophil depletion in GM-CSF(-/-) mice led to a stronger inhibition, indicating that GM-CSF and neutrophils have additive functions. To characterize the contribution of GM-CSF specifically in the effector phase of EBA, disease was induced by transfer of anti-type VII collagen IgG into mice. We observed an increased GM-CSF expression, and GM-CSF blockade reduced skin blistering. Additionally, GM-CSF enhanced reactive oxygen species release and neutrophil migration in vitro. In immunization-induced murine EBA, treatment with anti-GM-CSF had a beneficial effect on established disease. We demonstrate that GM-CSF modulates both autoantibody production and skin blistering in a prototypical organ-specific autoimmune disease.

Fcγ receptors III and IV mediate tissue destruction in a novel adult mouse model of bullous pemphigoid.

Bullous pemphigoid (BP) and epidermolysis bullosa acquisita are subepidermal autoimmune blistering diseases mediated by autoantibodies against type XVII collagen (Col17) and Col7, respectively. For blister formation, Fc-mediated events, such as infiltration of inflammatory cells in the skin, complement activation, and release of proteases at the dermal-epidermal junction, are essential. Although in the neonatal passive transfer mouse model of BP, tissue destruction is mediated by Fcγ receptors (FcγRs) I and III, the passive transfer model of epidermolysis bullosa acquisita completely depends on FcγRIV. To clarify this discrepancy, we developed a novel experimental model for BP using adult mice. Lesion formation was Fc mediated because γ-chain-deficient mice and mice treated with anti-Col17 IgG, depleted from its sugar moiety at the Fc portion, were resistant to disease induction. By the use of various FcγR-deficient mouse strains, tissue destruction was shown to be mediated by FcγRIV, FcγRIII, and FcγRIIB, whereas FcγRI was not essential. Furthermore, anti-inflammatory mediators in already clinically diseased mice can be explored in the novel BP model, because the pharmacological inhibition of FcγRIV and depletion of granulocytes abolished skin blisters. Herein, we extended our knowledge about the importance of FcγRs in experimental BP and established a novel BP mouse model suitable to study disease development over a longer time period and explore novel treatment strategies in a quasi-therapeutic setting.

Tissue Destruction in Bullous Pemphigoid Can Be Complement Independent and May Be Mitigated by C5aR2.

Bullous pemphigoid (BP), the most frequent autoimmune bullous disorder, is a paradigmatic autoantibody-mediated disease associated with autoantibodies against BP180 (type XVII collagen, Col17). Several animal models have been developed that reflect important clinical and immunological features of human BP. Complement activation has been described as a prerequisite for blister formation, however, the recent finding that skin lesions can be induced by anti-Col17 F(ab')2 fragments indicates complement-independent mechanisms to contribute to blister formation in BP. Here, C5-/- mice injected with anti-Col17 IgG showed a reduction of skin lesions by about 50% associated with significantly less skin-infiltrating neutrophils compared to wild-type mice. Reduction of skin lesions and neutrophil infiltration was seen independently of the employed anti-Col17 IgG dose. Further, C5ar1-/- mice were protected from disease development, whereas the extent of skin lesions was increased in C5ar2-/- animals. Pharmacological inhibition of C5a receptor 1 (C5aR1) by PMX53 led to reduced disease activity when applied in a prophylactic setting. In contrast, PMX-53 treatment had no effect when first skin lesions had already developed. While C5aR1 was critically involved in neutrophil migration in vitro, its role for Col17-anti-Col17 IgG immune complex-mediated release of reactive oxygen species from neutrophils was less pronounced. Our data demonstrate that complement-dependent and -independent mechanisms coexist in anti-Col17-autoantibody-mediated tissue destruction. C5aR1 and C5aR2 seem to play opposing roles in this process with C5aR1 exerting its primary effect in recruiting inflammatory cells to the skin during the early phase of the disease. Further studies are required to fully understand the role of C5aR2 in autoantibody-mediated skin inflammation.

Whole-Genome Expression Profiling in Skin Reveals SYK As a Key Regulator of Inflammation in Experimental Epidermolysis Bullosa Acquisita.

Because of the morbidity and limited therapeutic options of autoimmune diseases, there is a high, and thus far, unmet medical need for development of novel treatments. Pemphigoid diseases, such as epidermolysis bullosa acquisita (EBA), are prototypical autoimmune diseases that are caused by autoantibodies targeting structural proteins of the skin, leading to inflammation, mediated by myeloid cells. To identify novel treatment targets, we performed cutaneous genome-wide mRNA expression profiling in 190 outbred mice after EBA induction. Comparison of genome-wide mRNA expression profiles in diseased and healthy mice, and construction of a co-expression network identified Sykb (spleen tyrosine kinase, SYK) as a major hub gene. Aligned, pharmacological SYK inhibition protected mice from experimental EBA. Using lineage-specific SYK-deficient mice, we identified SYK expression on myeloid cells to be required to induce EBA. Within the predicted co-expression network, interactions of Sykb with several partners (e.g., Tlr13, Jdp2, and Nfkbid) were validated by curated databases. Additionally, novel gene interaction partners of SYK were experimentally validated. Collectively, our results identify SYK expression in myeloid cells as a requirement to promote inflammation in autoantibody-driven pathologies. This should encourage exploitation of SYK and SYK-regulated genes as potential therapeutic targets for EBA and potentially other autoantibody-mediated diseases.

Experimental Laminin 332 Mucous Membrane Pemphigoid Critically Involves C5aR1 and Reflects Clinical and Immunopathological Characteristics of the Human Disease.

Mucous membrane pemphigoid is an autoantibody-mediated disease predominantly affecting the oral cavity, pharynx, and conjunctiva. Conjunctival lesions may lead to impaired vision and, finally, blindness. About 25% of mucous membrane pemphigoid patients generate autoantibodies against the α3 chain of laminin 332 (LAMα3), a structural protein of epidermal/epithelial basement membranes. Here, we established a mouse model by the passive transfer of rabbit IgG against the murine homologs of two immunodominant fragments in adult C57BL/6 mice (mLAMα3). After repeated subcutaneous injections of anti-mLAMα3 IgG erosions and crusts occurred predominantly around the snout, eyes, and on ears. Conjunctival and oral/pharyngeal lesions with subepithelial splitting were found in 80% and 100% of mice, respectively. In contrast, disease development was abrogated in FcRγ chain-deficient mice and markedly reduced in C5aR1-deficient mice. Furthermore, wild-type mice injected with anti-mLAMα3 F(ab')2 were completely protected. Our findings suggest a crucial codominant role of FcRγ and complement activation of the anti-mLAMα3 IgG-induced mouse model of mucous membrane pemphigoid. This model will help further discover the pathomechanisms of this devastating disease. Furthermore, it may be of use to explore the effect of urgently needed more specific anti-inflammatory mediators on mucosal and skin lesions in autoantibody-mediated diseases.

Regulatory T Cells Suppress Inflammation and Blistering in Pemphigoid Diseases.

Regulatory T cells (Tregs) are well known for their modulatory functions in adaptive immunity. Through regulation of T cell functions, Tregs have also been demonstrated to indirectly curb myeloid cell-driven inflammation. However, direct effects of Tregs on myeloid cell functions are insufficiently characterized, especially in the context of myeloid cell-mediated diseases, such as pemphigoid diseases (PDs). PDs are caused by autoantibodies targeting structural proteins of the skin. Autoantibody binding triggers myeloid cell activation through specific activation of Fc gamma receptors, leading to skin inflammation and subepidermal blistering. Here, we used mouse models to address the potential contribution of Tregs to PD pathogenesis in vivo. Depletion of Tregs induced excessive inflammation and blistering both clinically and histologically in two different PD mouse models. Of note, in the skin of Treg-depleted mice with PD, we detected increased expression of different cytokines, including Th2-specific IL-4, IL-10, and IL-13 as well as pro-inflammatory Th1 cytokine IFN-γ and the T cell chemoattractant CXCL-9. We next aimed to determine whether Tregs alter the migratory behavior of myeloid cells, dampen immune complex (IC)-induced myeloid cell activation, or both. In vitro experiments demonstrated that co-incubation of IC-activated myeloid cells with Tregs had no impact on the release of reactive oxygen species (ROS) but downregulated ß2 integrin expression. Hence, Tregs mitigate PD by altering the migratory capabilities of myeloid cells rather than their release of ROS. Modulating cytokine expression by administering an excess of IL-10 or blocking IFN-γ may be used in clinical translation of these findings.

The Leukotriene B4 and its Receptor BLT1 Act as Critical Drivers of Neutrophil Recruitment in Murine Bullous Pemphigoid-Like Epidermolysis Bullosa Acquisita.

Recruitment of neutrophils and eosinophils into the skin is a hallmark of pemphigoid diseases. The molecular cues regulating granulocyte recruitment into the skin and the individual contributions of neutrophils and eosinophils to pemphigoid diseases are, however, poorly understood. The lipid mediator leukotriene B4 (LTB4) is a potent granulocyte chemoattractant and is abundant in the skin blister fluid of bullous pemphigoid (BP) patients, but its pathogenic significance is unknown. Using mouse models of BP-like epidermolysis bullosa acquisita and of BP, we show that LTB4 and its receptor BLT1 act as critical drivers of neutrophil entry into the skin upon antibody deposition at the dermal-epidermal junction. Mice deficient in 5-lipoxygenase, a key enzyme in LTB4 biosynthesis, or in BLT1 exhibited dramatic resistance to neutrophil recruitment and, consequently, skin inflammation. Accordingly, liquid chromatography-mass spectrometry, used to comprehensively profile lipid mediator generation in the first 48 hours after antibody deposition, showed a pronounced parallel increase in LTB4 and in neutrophils in the skin. Subsequent mechanistic studies in BP-like epidermolysis bullosa acquisita uncovered that neutrophils are necessary for skin inflammation, whereas eosinophils are dispensable, thus identifying neutrophils as major culprits of blister formation. Our results highlight LTB4/BLT1 as absolutely critical drivers of murine pemphigoid disease-like skin inflammation.

IgE-mediated mechanisms in bullous pemphigoid and other autoimmune bullous diseases.

Autoimmune bullous diseases (AIBDs) are characterized by autoantibodies against structural proteins of the dermal-epidermal junction (in pemphigoid diseases) and the epidermal/ epithelial desmosomes (in pemphigus diseases). By far, the most common AIBD is bullous pemphigoid, which is immunopathologically characterized by autoantibodies against BP180 (type XVII collagen) and BP230. IgG and, to a lesser extent, IgA autoantibodies are the major autoantibody isotypes in these disorders. IgE autoantibodies are increasingly reported in particular in bullous pemphigoid. The development of specific and sensitive anti-BP180 IgE ELISA systems, the report of two experimental murine models employing IgE autoantibodies against BP180, and the successful treatment of bullous pemphigoid with the anti-IgE antibody omalizumab have raised interest in the role of IgE autoantibodies and the modulation of their production in AIBDs. Here, the relevance of IgE autoantibodies in the diagnosis, pathophysiology, and treatment decisions of AIBDs, with a focus on bullous pemphigoid, is reviewed.