CD4(+) T (helper) cells migrate in huge numbers through lymphoid organs. However, little is known about traffic routes and kinetics of CD4(+) T-cell subsets within different organ compartments. Such information is important because there are indications that CD4(+) T cells may influence the function of microenvironments depending on their developmental stage. Therefore, we investigated the migration of resting (naïve), activated, and recently activated (memory) CD4(+) T cells through the different compartments of the spleen. Resting and recently activated CD4(+) T cells were separated from thoracic duct lymph and activated CD4(+) T cells were generated in vitro by cross-linking the T-cell receptor and CD28. The present study shows that all three CD4(+) T-cell subsets selectively accumulate in the T-cell zone of the spleen. However, only activated T cells induce the formation of germinal centers (GCs) and autoantibodies in rats and mice. Our results suggest that in a two-step process they first activate B cells independent of the T-cell receptor repertoire and CD40 ligand (CD154) expression. The activated B cells then form GCs whereby CD154-dependent T-cell help is needed. Thus, activated T cells may contribute to the development of autoimmune diseases by activating autoreactive B cells in an Ag-independent manner.

T cells are key players in autoimmune diseases by supporting the production of autoantibodies. However, their contribution to the effector phase of antibody-mediated autoimmune dermatoses, i.e., tissue injury and inflammation of the skin, has not been investigated. In this paper, we demonstrate that T cells amplify the development of autoantibody-induced tissue injury in a prototypical, organ-specific autoimmune disease, namely epidermolysis bullosa acquisita (EBA) - characterized and caused by autoantibodies targeting type VII collagen. Specifically, we show that immune complex (IC)-induced inflammation depends on the presence of T cells - a process facilitated by T cell receptor (TCR)γδ and NKT cells. Because tissue damage in IC-induced inflammation is neutrophil-dependent, we further analyze the interplay between T cells and neutrophils in an experimental model of EBA. We demonstrate that T cells not only enhance neutrophil recruitment into the site of inflammation but also interact with neutrophils in lymphatic organs. Collectively, this study shows that T cells amplify the effector phase of antibody-induced tissue inflammation.

The cell stress chaperone heat shock protein 90 (Hsp90) has been implicated in inflammatory responses and its inhibition has proven successful in different mouse models of autoimmune diseases, including epidermolysis bullosa acquisita. Here, we investigated expression levels and secretory responses of Hsp90 in patients with bullous pemphigoid (BP), the most common subepidermal autoimmune blistering skin disease. In comparison to healthy controls, the following observations were made: (i) Hsp90 was highly expressed in the skin of BP patients, whereas its serum levels were decreased and inversely associated with IgG autoantibody levels against the NC16A immunodominant region of the BP180 autoantigen, (ii) in contrast, neither aberrant levels of circulating Hsp90 nor any correlation of this protein with serum autoantibodies was found in a control cohort of autoimmune bullous disease patients with pemphigus vulgaris, (iii) Hsp90 was highly expressed in and restrictedly released from peripheral blood mononuclear cells of BP patients, and (iv) Hsp90 was potently induced in and restrictedly secreted from human keratinocyte (HaCaT) cells by BP serum and isolated anti-BP180 NC16A IgG autoantibodies, respectively. Our results reveal an upregulated Hsp90 expression at the site of inflammation and an autoantibody-mediated dysregulation of the intracellular and extracellular distribution of this chaperone in BP patients. These findings suggest that Hsp90 may play a pathophysiological role and represent a novel potential treatment target in BP.

IL-17A has been identified as key regulatory molecule in several autoimmune and chronic inflammatory diseases followed by the successful use of anti-IL-17 therapy, e.g. in ankylosing spondylitis and psoriasis. Bullous pemphigoid (BP) is the most frequent autoimmune blistering disease with a high need for more specific, effective and safe treatment options. The aim of this study was to clarify the pathophysiological importance of IL-17A in BP. We found elevated numbers of IL-17A+ CD4+ lymphocytes in the peripheral blood of BP patients and identified CD3+ cells as major source of IL-17A in early BP skin lesions. IL17A and related genes were upregulated in BP skin and exome sequencing of 51 BP patients revealed mutations in twelve IL-17-related genes in 18 patients. We have subsequently found several lines of evidence suggesting a significant role of IL-17A in the BP pathogenesis: (i) IL-17A activated human neutrophils in vitro, (ii) inhibition of dermal-epidermal separation in cryosections of human skin incubated with anti-BP180 IgG and subsequently with anti-IL-17A IgG-treated leukocytes, (iii) close correlation of serum IL-17A levels and diseases activity in a mouse model of BP, (iv) IL17A-deficient mice were protected against autoantibody-induced BP, and (v) pharmacological inhibition of lL-17A reduced the induction of BP in mice. Our data give evidence for a pivotal role of IL-17A in the pathophysiology of BP and advocate IL-17A inhibition as potential novel treatment for this disease.

Systemic sclerosis (SSc) is a severe chronic autoimmune disease with high morbidity and mortality. Sera of patients with SSc contain a large variety of autoantibody (aab) reactivities. Among these are functionally active aab that bind to G protein-coupled receptors (GPCR) such as C-X-C motif chemokine receptor 3 (CXCR3) and 4 (CXCR4). Aab binding to the N-terminal portion of these two GPCRs have been shown to be associated with slower disease progression in SSc, especially deterioration of lung function. Aabs binding to GPCRs exhibit functional activities by stimulating or inhibiting GPCR signaling. The specific functional activity of aabs crucially depends on the epitopes they bind to. To identify the location of important epitopes on CXCR3 recognized by aabs from SSc patients, we applied an array of 36 overlapping 18-20mer peptides covering the entire CXCR3 sequence, comparing epitope specificity of SSc patient sera (N = 32, with positive reactivity with CXCR3) to healthy controls (N = 30). Binding of SSc patient and control sera to these peptides was determined by ELISA. Using a Bayesian model approach, we found increased binding of SSc patient sera to peptides corresponding to intracellular epitopes within CXCR3, while the binding signal to extracellular portions of CXCR3 was found to be reduced. Experimentally determined epitopes showed a good correspondence to those predicted by the ABCpred tool. To verify these results and to translate them into a novel diagnostic ELISA, we combined the peptides that represent SSc-associated epitopes into a single ELISA and evaluated its potential to discriminate SSc patients (N = 31) from normal healthy controls (N = 47). This ELISA had a sensitivity of 0.61 and a specificity of 0.85. Our data reveals that SSc sera preferentially bind intracellular epitopes of CXCR3, while an extracellular epitope in the N-terminal domain that appears to be target of aabs in healthy individuals is not bound by SSc sera. Based upon our results, we could devise a novel ELISA concept that may be helpful for monitoring of SSc patients.

Pemphigoid diseases refer to a group of severe autoimmune skin blistering diseases characterized by subepidermal blistering and loss of dermal-epidermal adhesion induced by autoantibody and immune cell infiltrate at the dermal-epidermal junction and upper dermis. Here, we explore the role of the immune cell-secreted serine protease, granzyme B, in pemphigoid disease pathogenesis using three independent murine models. In all models, granzyme B knockout or topical pharmacological inhibition significantly reduces total blistering area compared to controls. In vivo and in vitro studies show that granzyme B contributes to blistering by degrading key anchoring proteins in the dermal-epidermal junction that are necessary for dermal-epidermal adhesion. Further, granzyme B mediates IL-8/macrophage inflammatory protein-2 secretion, lesional neutrophil infiltration, and lesional neutrophil elastase activity. Clinically, granzyme B is elevated and abundant in human pemphigoid disease blister fluids and lesional skin. Collectively, granzyme B is a potential therapeutic target in pemphigoid diseases.

Heat shock proteins (Hsp) are constitutive and stress-induced molecules which have been reported to impact innate and adaptive immune responses. Here, we evaluated the role of Hsp70 as a treatment target in the imiquimod-induced, psoriasis-like skin inflammation mouse model and related in vitro assays. We found that immunization of mice with Hsp70 resulted in decreased clinical and histological disease severity associated with expansion of T cells in favor of regulatory subtypes (CD4+FoxP3+/CD4+CD25+ cells). Similarly, anti-Hsp70 antibody treatment led to lowered disease activity associated with down-regulation of pro-inflammatory Th17 cells. A direct stimulating action of Hsp70 on regulatory T cells and its anti-proliferative effects on keratinocytes were confirmed in cell culture experiments. Our observations suggest that Hsp70 may be a promising therapeutic target in psoriasis and potentially other autoimmune dermatoses.

Monocyte chemoattractant protein-1 (MCP-1) is highly expressed by lymphocytes at skin sites affected by alopecia areata (AA). Variations in MCP-1 as well as in methylene-tetrahydrofolate reductase (MTHFR), a key enzyme related to many inflammatory pathologies, have been associated with several autoimmune disorders. This study was designed to test a possible association between MCP-1 and MTHFR variations and altered expression of their genes and the risk of AA.

Epidermolysis bullosa acquisita (EBA) is an autoimmune blistering disorder characterized and caused by autoantibodies against type VII collagen (COL7). Although it has been noticed that EBA in both patients and mice is associated with an increased scratching, it is not clear whether and how the scratching contributes to disease manifestation. Hence, we here aimed to validate this clinical observation and also to investigate the potential contribution of increased scratching in EBA pathogenesis in mice. Longitudinal assessment of scratching behavior revealed an increased frequency of scratching as early as 12 hours after injection of anti-COL7 IgG into the skin of mice. Subsequently, scratching events became even more frequent in mice. In contrast, mice injected with a control antibody showed an unaltered scratching behavior throughout the observation period. Based on these observations, we hypothesized that mechanical irritation may promote the induction of inflammation in experimental EBA. To challenge this assumption, the local anesthetic dyclonine hydrochloride was topically applied before injection of anti-COL7 IgG. Dyclonine hydrochloride reduced the scratching events and impaired clinical disease manifestation. In therapeutic experimental settings, i.e. administration of the local anesthetic 24 hours after injection of anti-COL7 IgG, dyclonine hydrochloride only inhibited the scratching behavior, but had no significant effect on clinical disease development. In addition, eosinophils were detected in the skin before the injection of anti-COL7 IgG and significantly increased 48 hours after the antibody injection. Collectively, our results suggest that scratching behavior contributes to the initiation phase of disease manifestation in experimental EBA.

Onychomycosis (OM) is a common fungal nail infection. Based on the rich mycobial diversity in healthy toenails, we speculated that this is lost in OM due to the predominance of a single pathogen. We used next generation sequencing to obtain insights into the biodiversity of fungal communities in both healthy individuals and OM patients. By sequencing, a total of 338 operational-taxonomic units were found in OM patients and healthy controls. Interestingly, a classifier distinguished three distinct subsets: healthy controls and two groups within OM patients with either a low or high abundance of Trichophyton. Diversity per sample was decreased in controls compared to cases with low Trichophyton abundance (LTA), while cases with a high Trichophyton abundance (HTA) showed a lower diversity. Variation of mycobial communities between the samples showed shifts in the community structure between cases and controls-mainly driven by HTA cases. Indeed, LTA cases had a fungal β-diversity undistinguishable from that of healthy controls. Collectively, our data provides an in-depth characterization of fungal diversity in health and OM. Our findings also suggest that onychomycosis develops either through pathogen-driven mechanisms, i.e., in HTA cases, or through host and/or environmental factors, i.e., in cases with a low Trichophyton abundance.

Chronic blistering at the skin and/or mucous membranes, accompanied by a varying degree of inflammation, is the clinical hallmark of pemphigoid diseases that impose a major medical burden. Pemphigoid diseases are caused by autoantibodies targeting structural proteins of the epithelial basement membrane. One major pathogenic pathway of skin blistering and inflammation is activation of myeloid cells following Fc gamma receptor-dependent binding to the skin-bound immune complexes. This process requires activation of specific kinases, such as PI3Kδ, which have emerged as potential targets for the treatment of pemphigoid diseases. Yet, it is unknown if global cutaneous kinase activity present in lesional pemphigoid disease correlates with therapeutic effects following treatment with a given target-selective kinase inhibitor. To address this, we here first determined the kinase activity in three different mouse models of pemphigoid diseases: Antibody transfer-induced mucous membrane pemphigoid (MMP), antibody transfer-induced epidermolysis bullosa acquisita (EBA) and immunization-induced EBA. Interestingly, the kinome signatures were different among the three models. More specifically, PI3Kδ was within the kinome activation network of antibody transfer-induced MMP and immunization-induced EBA, but not in antibody transfer-induced EBA. Next, the therapeutic impact of the PI3Kδ-selective inhibitor parsaclisib was evaluated in the three model systems. In line with the kinome signatures, parsaclisib had therapeutic effects in antibody transfer-induced MMP and immunization-induced EBA, but not in autoantibody-induced EBA. In conclusion, kinase activation signatures of inflamed skin, herein exemplified by pemphigoid diseases, correlate with the therapeutic outcomes following kinase inhibition, demonstrated here by the PI3Kδ inhibitor parsaclisib.

Recently, the C-terminus of laminin γ1 has been identified as target antigen in anti-p200 pemphigoid and the disease was renamed as anti-laminin γ1 pemphigoid. However, the pathogenic relevance of these autoantibodies has not yet been demonstrated. Therefore, we employed an ex vivo model of autoantibody-mediated leukocyte-dependent neutrophil activation and dermal-epidermal separation (DES) using cryosections of human skin. We showed that anti-p200 pemphigoid sera (n = 7) induced DES in a time-dependent manner, in contrast to sera from healthy controls. Furthermore, laminin γ1-specific IgG and serum depleted from anti-laminin γ1 reactivity were generated using the recombinant C-terminus of laminin γ1 (LAMC1-term; amino acids 1364 to 1609). Interestingly, both fractions labeled the dermal-epidermal-junction (DEJ) by indirect immunofluorescence microscopy on human foreskin and recognized a 200 kDa protein by immunoblotting with dermal extract. Human and rabbit IgG against LAMC1-cterm failed to attract neutrophils at the DEJ and to induce DES. In contrast, patient serum depleted from LAMC1-cterm reactivity led to the same extent of DES as non-depleted IgG. Repeated injection of rabbit anti-murine LAMC1-cterm IgG into both neonatal and adult C57BL/6mice as well as repetitive immunization of various mouse strains with murine LAMC1-cterm failed to induce macro- and microscopic lesions. In all mice, circulating anti-LAMC1-cterm antibodies were present, but only in some mice, IgG deposits were seen at the DEJ. We conclude that autoantibodies in anti-p200 pemphigoid sera are pathogenic while pathogenicity is not mediated by autoantibodies against laminin γ1. Further studies are needed to identify the pathogenically relevant autoantigen in anti-p200 pemphigoid.

Autoimmune bullous dermatoses (AIBDs) are a group of rare chronic inflammatory skin diseases, which clinically manifest as blisters and erosions of the skin and/or mucosa. Immunologically, AIBDs are characterized and caused by autoantibodies targeting adhesion molecules in the skin and mucosa. According to the histological location of the blistering, AIBDs are classified into the following two main subtypes: pemphigus (intraepidermal blistering) and pemphigoid (subepidermal blistering). Most AIBDs were potentially life-threatening diseases before the advent of immunosuppressive drugs, especially systemic steroid therapies, which suppress pathogenic immunological activity. Although there have been recent advancements in the understanding of the pathogenesis of AIBDs, glucocorticosteroids and/or adjuvant immunosuppressive drugs are still needed to control disease activity. However, the long-term use of systemic immunosuppression is associated with major adverse events, including death. Based on the growing understanding of AIBD pathogenesis, novel treatment targets have emerged, some of which are currently being evaluated in clinical trials. Within this article, we review the current clinical trials involving pemphigus and pemphigoid and discuss the rationale that lead to these trials. Overall, we aim to foster insights into translational research in AIBDs to improve patient care.

Pemphigus is caused by autoantibodies against desmoglein (Dsg) 1, Dsg3, and/or non-Dsg antigens. Pemphigus vulgaris (PV) is the most common manifestation of pemphigus, with painful erosions on mucous membranes. In most cases, blistering also occurs on the skin, leading to areas of extensive denudation. Despite improvements in pemphigus treatment, time to achieve remission is long, severe adverse events are frequent and 20% of patients do not respond adequately. Current clinical developments focus exclusively on modulating B cell function or autoantibody half-life. However, topical modulation of PV autoantibody-induced blistering is an attractive target because it could promptly relieve symptoms.

In several autoimmune diseases, e.g., pemphigoid disease (PD), autoantibodies are the direct cause of pathology. Albeit key requirements for antibody-mediated diseases were identified, their interactions and exact temporal and spatial interactions remained elusive. The skin is easily accessible for imaging. Thus, we selected epidermolysis bullosa acquisita (EBA), a PD with autoantibodies to type VII collagen (COL7), to visualize interactions of autoantibodies, target tissue and effector cells (neutrophils). Following injection into mice, anti-COL7 IgG bound to the dermal-epidermal junction (DEJ) within minutes. We unexpectedly observed an inhomogeneous distribution of autoantibodies along the DEJ. Thus, we hypothesized that specific external triggers may affect autoantibody distribution. Indeed, mechanical irritation led to an increased autoantibody binding along the DEJ. Subsequently, anti-COL7 IgG was injected into mice expressing green fluorescent protein under the LysM promoter (LysM-eGFP) mice. This allows to visualize myeloid cells in vivo in these animals. Using multiphoton imaging, we observed a limited extravasation of LysM-eGFP+ cells into skin was observed within 24 hours. Intriguingly, LysM-eGFP+ cells did not immediately co-localize with autoantibodies, which was only noted at later time points. Of note, interactions of LysM-eGFP+ with the autoantibodies at the DEJ were short-lived. Collectively, our results define the following checkpoints for autoantibody-induced tissue injury: (i) autoantibody egress to target tissue influenced by mechanical trigger factors, (ii) neutrophil recruitment into the vicinity of autoantibody deposits and (iii) short-term neutrophil localization to these deposits, as well as (iv) delayed recruitment of neutrophils with subsequent autoantibody-induced inflammation.

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.

Pemphigus and pemphigoid diseases are organ-specific autoimmune diseases of the skin and/or mucous membranes, which are caused by autoantibodies targeting structural proteins of the skin. In other autoimmune diseases, a high prevalence of primary antibody deficiencies was noted. Conversely, a high prevalence of autoimmune diseases is reported in patients with primary antibody deficiencies. With the exception of one study, pointing toward a decrease of IgG in pemphigus patients, with a relative enrichment of IgG4, serum immunoglobulin (Ig) concentrations had not been studied in pemphigus and pemphigoid. Hence, we here aimed to investigate serum concentrations of IgM, IgA, IgG, and IgG1-4 in pemphigus and pemphigoid patients, as well as in healthy controls. Serum Ig concentrations were determined by ELISA in 105 healthy controls, 100 pemphigus vulgaris (PV), 100 pemphigus foliaceus, 99 bullous pemphigoid (BP), and 55 linear IgA bullous dermatosis (LAD) patients. In healthy controls, age had a significant impact on Ig serum concentrations: In controls at ages of 69 years or older, IgM and IgG were decreased, while all other Ig, except IgA and IgG4, were increased. When stratified by sex, lower IgM concentrations were observed in males. When corrected for age and/or sex, and compared to controls, an increase in serum IgA was noted in LAD. In almost all patient cohorts, an increase in IgG1 and IgG4 was observed, while a decrease in IgG2 or IgG3 was seen in BP or PV patients. This points toward a possible association of BP with IgG2 deficiency and warrants evaluation of IgG2 in BP patients prior to immunosuppressive therapy.

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 β2 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.

Stress-induced heat shock protein 70 (Hsp70) is a key intra- and extracellular molecular chaperone implicated in autoimmune processes. Highly immunogenic extracellular Hsp70 can activate innate and acquired (adaptive) immune responses driving the generation of anti-Hsp70 autoantibodies that are frequently observed in inflammatory/autoimmune disorders. We recently described the direct pathological role of extracellular Hsp70 in epidermolysis bullosa acquisita (EBA), an anti-type VII collagen autoantibody-mediated autoimmune blistering skin disease. Here, we determined the role of anti-Hsp70 autoantibodies in EBA. We observed that circulating anti-Hsp70 IgG autoantibodies were significantly elevated in EBA patients compared to healthy individuals and positively correlated with serum levels of pro-inflammatory interferon gamma (IFN-γ). The pathophysiological relevance of anti-Hsp70 IgG autoantibodies was demonstrated in an antibody transfer-induced EBA mouse model in which elevated serum levels of anti-Hsp70 IgG were found. In addition, anti-Hsp70 IgG-treated animals had a more intense clinical and histological disease activity, as well as upregulated nuclear factor kappa B (NF-κB) activation in skin biopsies compared to isotype-treated animals. Our results suggest that autoantibodies to Hsp70 may contribute to EBA development via enhanced neutrophil infiltration to the skin and activation of the NF-κB signaling pathway in an IFN-γ-associated manner.

Bullous pemphigoid (BP), the by far most frequent autoimmune subepidermal blistering disorder (AIBD), is characterized by the deposition of autoantibodies against BP180 (type XVII collagen; Col17) and BP230 as well as complement components at the dermal-epidermal junction (DEJ). The mechanisms of complement activation in BP patients, including the generation of C5a and regulation of its two cognate C5aRs, i.e., C5aR1 and C5aR2, are incompletely understood. In this study, transcriptome analysis of perilesional and non-lesional skin biopsies of BP patients compared to site-, age-, and sex-matched controls showed an upregulated expression of C5AR1, C5AR2, CR1, and C3AR1 and other complement-associated genes in perilesional BP skin. Of note, increased expressions of C5AR2 and C3AR1 were also observed in non-lesional BP skin. Subsequently, double immunofluorescence (IF) staining revealed T cells and macrophages as the dominant cellular sources of C5aR1 in early lesions of BP patients, while C5aR2 mainly expressed on mast cells and eosinophils. In addition, systemic levels of various complement factors and associated molecules were measured in BP patients and controls. Significantly higher plasma levels of C3a, CD55, and mannose-binding lectin-pathway activity were found in BP patients compared to controls. Finally, the functional relevance of C5aR1 and C5aR2 in BP was explored by two in vitro assays. Specific inhibition of C5aR1, resulted in significantly reduced migration of human neutrophils toward the chemoattractant C5a, whereas stimulation of C5aR2 showed no effect. In contrast, the selective targeting of C5aR1 and/or C5aR2 had no effect on the release of reactive oxygen species (ROS) from Col17-anti-Col17 IgG immune complex-stimulated human leukocytes. Collectively, this study delineates a complex landscape of activated complement receptors, complement factors, and related molecules in early BP skin lesions. Our results corroborate findings in mouse models of pemphigoid diseases that the C5a/C5aR1 axis is pivotal for attracting inflammatory cells to the skin and substantiate our understanding of the C5a/C5aR1 axis in human BP. The broad expression of C5aRs on multiple cell types critical for BP pathogenesis call for clinical studies targeting this axis in BP and other complement-mediated AIBDs.