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Inflammation is largely implicated in bullous pemphigoid (BP), the most frequent skin auto-immune blistering disease. IL-17, essentially IL-17A/F, has been involved in blister formation through regulation of protease production, and its specific serum profile within BP was related to disease outcome. However, relationships between IL-17 family ligands and receptors are quite complex with six different IL-17 isoforms, and five different receptors. We here aimed at clarifying the contribution of the IL-17 axis in BP by characterizing not only the expression of IL-17 receptor (IL-17R) members within immune cells isolated from BP patients (PMNs, n = 9; T-lymphocytes, n = 10; and monocytes, n = 10) but also the expression of IL-17 isoforms in sera ( n = 83), and blister fluid ( n = 31) of BP patients. We showed that at diagnosis, IL-17RA and IL-17RC expression were significantly increased in monocytes isolated from BP patients as compared to those from control subjects ( p = 0.006 and p = 0.016, respectively). Notably, both IL-17RA and IL-17RC mRNA expression remained elevated in BP monocytes at time of relapse. We further demonstrated a significant increase of all IL-17 isoforms tested in BP blister fluid compared with BP serum (IL-17A, p < 0.0001; IL-17A/F, p < 0.0001; IL-17B, p = 0.0023; IL-17C, p = 0.0022; IL-17E, p < 0.0001). Among all, IL-17B was the only cytokine for which a significant decreased concentration within blister fluid was observed in BP patients with severe disease compared to patients with moderate disease ( p = 0.012). We further evidenced a significant negative correlation between IL-17B levels and blister/erosion BPDAI subscore ( r = -0.52, p = 0.003). We finally identified mast cells as a potential target of IL-17B in lesional skin of BP patients. In conclusion, we showed here that IL-17RA and IL-17RC expression in monocyte was associated with disease activity and evidenced in situ a negative correlation between BP disease activity and IL-17B, whose effects could be mediated by IL-17RB expressed by mast cell in BP lesional skin., (Copyright © 2019 Nesmond, Muller, Le Naour, Viguier, Bernard, Antonicelli and Le Jan.)

Bullous Pemphigoid (BP) is a skin autoimmune blistering disease characterized by immune-mediated degradation of the dermo-epidermal junction and release of a large number of inflammatory cytokines. Interleukin-1β (IL-1β) is a pleiotropic pro-inflammatory cytokine associated with inflammasome activation and known to be pivotal in several auto-immune and auto-inflammatory diseases. We sought to clarify the presence of inflammasome-dependent IL-1β and to investigate its role in BP. Skin biopsy specimens ( n = 13), serum ( n = 60), blister fluid ( n = 26), and primary inflammatory cells from patients with BP were used to investigate inflammasome activation and function. We here highlighted a differential occurrence of a functional in situ inflammasome in patients with BP, biologically distinguished by IL-1β and NLRP3 expression. Clinically, elevated IL-1β levels were associated with the presence of erythema and urticarial plaques reflecting the inflammatory phase preceding blister formation. We further identified IL-17 and IL-23 as important molecules favoring IL-1β expression in monocyte-derived macrophages from BP patients. Finally, we demonstrated the ability of IL-1β to stimulate the release of the matrix metalloproteinase-9 in those macrophages, reinforcing the role of IL-1β in the auto-amplification loop of the inflammatory response associated to BP. However, whether this inflammasome is an epiphenomenon associated with BP disease or constitutes an amplification inflammatory step in certain patients still need to be determined. In the context of a precision medicine approach, our findings allowed us to delineate a subgroup of patients with BP that showed similarities with auto-inflammatory diseases. Subsequently, this opens up alternative therapeutic strategies targeting IL-1β pathway in the aim to control the early, pre-blistering inflammatory phase. Ultimately, this could also help in reducing the detrimental effects associated with high doses of corticosteroids treatment.

Bullous pemphigoid (BP) is a cutaneous autoimmune disease, characterized by an inflammatory cascade leading to blister formation. Although macrophages were shown to participate in BP pathophysiology, their role in the blister formation process still needs to be investigated. We here addressed the influence of serum and blister fluid (BF) from patients with BP on the polarization status of macrophages with regards to the metalloproteinase-9 (MMP-9) expression. We demonstrated that several markers related to the alternatively activated macrophage phenotype (M2) including IL-10, TARC, arginase, TNFα, and IL-1RA were meaningfully increased in BF of patients with BP. We further showed that BF, but not serum from patients with BP, significantly induced the expression of CD163, CD206, and IL-10 in BP monocyte-derived macrophages (MDMs). Notably IL-10 was the only cytokine to be correlated to the reference clinical score, BP disease activity index (BPDAI), especially to the inflammatory BPDAI subscore evaluating urticarial and erythematous skin lesions ( r = 0.57, p = 0.0004). We also found elevated levels of MMP-9 to M2-type macrophages ex vivo and highlighted the presence of CD163 + MMP-9 + macrophages histologically, at skin lesional site. Finally, we showed that methylprednisolone reduced MMP-9 levels in MDMs without modifying the other M2 markers. All together these results strongly support the presence of M2-phenotype macrophages with pro-inflammatory properties susceptible to favor blister formation in BP.

Background: DNA extracellular traps (ETs), released by neutrophils (NETs), or eosinophils (EETs), play a pathogenic role in several autoimmune disorders. However, to date, NETs have never been investigated in bullous pemphigoid (BP) with respect to clinical and immunological activities, both at baseline and at time of relapse which have been characterized with specific IL-17 and IL-23 patterns. Objective: We sought to assess whether ETs were associated with BP as well as the relative contribution of IL-17 axis cytokines to NET induction. Methods: Skin biopsy specimens were obtained from 11 patients with BP. Immuno-detection of neutrophils and eosinophils combined to DNA staining allowed us to investigate the in-situ presence of NETs and EETs using confocal scanning microscopy. NETs release was evaluated ex vivo by stimulating polymorphonuclear cells from BP patients with BP biological fluids in presence of IL-17A and IL-23 or of glucocorticoids. Results: At baseline, ETs were observed in BP lesions at the site of dermal-epidermal cleavage. Despite an important infiltrate of eosinophils, ETs were essentially associated with neutrophils in situ and were not related to BP clinical activity at diagnosis. In situ observation of NETs was associated in 6 among 8 patients with serum capacity of NET induction. Notably both blister fluid and sera from BP patients at diagnosis and at time of relapse could induce NET formation ex vivo . In contrast, a longitudinal investigation showed a decrease of NET formation with time of treatment in patients undergoing remission. Mimicking relapse, complementation of sera from BP patients with ongoing remission with either IL-17A or IL-23 increased NET formation. Conversely, IL-17A inhibited NET formation induced by serum from BP patients with relapse supplemented or not with IL-23. Finally, glucocorticoids also inhibited NET formation ex vivo in BP. Conclusion: NET formation is an associated phenomenon with BP. Furthermore, we showed that IL-23 favored NET formation, whereas the effects of IL-17A are environment dependent. Indeed, IL-17A displayed a protective effect on NET formation when associated with IL-23, showing for the first-time differential effects of these two cytokines in BP.

Bullous pemphigoid (BP) is an autoimmune bullous skin disease characterized by anti-BP180 and anti-BP230 autoantibodies (AAbs). Mucous membrane involvement is an uncommon clinical feature of BP which may evoke epidermolysis bullosa acquisita, another skin autoimmune disease characterized by anti-type VII collagen AAbs. We therefore evaluated the presence of anti-type VII collagen AAbs in the serum of BP patients with and without mucosal lesions at time of diagnosis and under therapy. Anti-BP180, anti-BP230, and anti-type VII collagen AAbs were measured by ELISA in the serum of unselected patients fulfilling clinical and histo/immunopathological BP criteria at baseline ( n  = 71) and at time of relapse ( n  = 24). At baseline, anti-type VII collagen AAbs were detected in 2 out of 24 patients with BP presenting with mucosal involvement, but not in patients without mucosal lesions ( n  = 47). At the time of relapse, 10 out of 24 BP patients either displayed a significant induction or increase of concentrations of anti-type VII collagen AAbs ( P  < 0.01), independently of mucosal involvement. Those 10 relapsing BP patients were also characterized by a sustained high concentration of anti-BP180 AAb, whereas the serum anti-BP230 AAb concentrations did not vary in BP patients with relapse according to the presence of anti-type VII collagen AAbs. Thus, our study showed that anti-type VII collagen along with anti-BP180 AAbs detection stratified BP patients at time of relapse, illustrating a still dysregulated immune response that could reflect a potential epitope spreading mechanism in those BP patients.

Bullous pemphigoid (BP) is the most common autoimmune bullous disease and typically affects the elderly. Binding of specific autoantibodies to BP180/230 hemidesmosomal components induces an inflammatory response leading to skin blister formation. Unusual manifestations of BP include additional mucous membrane involvement, without pathophysiological knowledge associated to the formation of these lesions. We here performed a prospective study on series of consecutive BP patients with ( n  = 77) and without ( n  = 18) mucosal involvements at baseline to further investigate why some BP patients display mucosal lesion and other not. Analysis of disease activity showed that BP patients with mucosal involvement displayed a higher total BP Disease Area Index (BPDAI) score ( P  = 0.008), but also higher skin and blister/erosion BPDAI scores ( P  = 0.02 and P  = 0.001, respectively). By contrast, the erythema/urticaria BPDAI score was identical between the two groups of patients. The erythema/urticaria BPDAI score, but not the blister/erosion BPDAI score, was correlated with the serum concentration of anti-BP180 NC16A autoantibodies in patients with mucosal involvement. In multivariate analysis, the absence of anti-BP230 autoantibody was the only factor independently associated with mucosal involvement (OR 7.8; 95% CI, 3.1-19.6) ( P  < 0.0001). Analysis of the distribution of BP patients according to BPDAI scores revealed a shift toward higher blister/erosion BPDAI scores for BP patients with mucosal involvement. This study indicates that mucosal lesions are clinically mainly related to disease severity and immunologically to the absence of anti-BP230 antibodies.

Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering disease in Western countries. Although topical and/or systemic glucocorticoids treatment efficacy is widely recognized, up to 30% of patients with BP may undergo a relapse during the first year of treatment. We investigated the protein expression of the total glucocorticoid receptor and GRβ isoform in the skin biopsy specimens from patients with BP and wondered whether such investigation at baseline provided a tool to predict disease outcome. Total GR and GRβ protein expressions were detected by immunohistochemistry at baseline on 12 patients who later relapse and 11 patients who remained on remission in comparison with 14 control patients. The expression of GRβ in the epidermis of patients with BP who later relapse was significantly higher than that in the epidermis of patients with BP controlled upon corticosteroid treatment, which was also higher than control patients. Thus, our results suggest that increased protein expression of GRβ in skin epithelial cells is predictive of reduced steroid treatment efficacy, and therefore of increased risk of disease relapse in patients with BP., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering disease of the skin. Investigation of the BP-associated pathophysiological processes during the last decades showed that the generation of autoantibodies directed against the hemidesmosome proteins BP180 and BP230, a hallmark of the BP-associated autoimmune response, leads to the recruitment of inflammatory immune cells at the dermal-epidermal junction, and subsequently to the release of a large amount of inflammatory molecules involved in blister formation. Analysis in transversal and longitudinal studies of autoantibodies and inflammatory molecules production both at the time of diagnosis and under treatment was mainly performed within the serum but also in the blister fluid. Some autoimmune or inflammatory molecules expression was related to the presence of clinical signs, while others were mere bystanders. In this review, we focused on the autoimmune and inflammatory molecules that have been identified as potential biomarkers of BP development and outcome., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Bullous Pemphigoid (BP) is an inflammatory rare autoimmune bullous dermatosis, which outcome cannot be predicted through clinical investigations. Eosinophils are the main immune infiltrated cells in BP. However, the release of Major Basic Protein (MBP), Eosinophil Derived Neurotoxin (EDN), and Eosinophil Cationic Protein (ECP) upon eosinophil activation has still not been evaluated with respect to BP development. MBP, EDN and ECP were measured by ELISA in serum (n = 61) and blister fluid (n = 20) of patients with BP at baseline, and in serum after 2 months of treatment (n = 41). Eosinophil activation in BP patients was illustrated at baseline by significantly higher MBP, EDN and ECP serum concentrations as compared with control subjects (n = 20), but without distinction according to disease severity or outcome. EDN and ECP values were even higher in the blister fluids (P < 0.01 and P < 0.05, respectively), whereas MBP values were lower (P < 0.001). ECP serum concentration decreased after 60 days of treatment in BP patients with ongoing remission but not in patients who later relapsed (P < 0.05). A reduction of at least 12.8 ng/mL in ECP concentrations provided a positive predictive value for remission of 81%, showing that ECP serum variation could be a useful biomarker stratifying BP patients at risk of relapse.

Background: The outcome of bullous pemphigoid (BP), the most frequent autoimmune skin-blistering disease, involves matrix metalloproteinase 9 (MMP-9), IL-17, and IL-23 release from infiltrated inflammatory cells. The chemokine CXCL10 has been associated with several autoimmune diseases, but its participation in BP pathophysiology still needs to be clarified., Objective: We sought to assess whether BP outcome was associated with different CXCL10 levels and to evaluate the contribution of CXCL10 to the described cytokine/protease inflammatory loop associated with disease outcome., Methods: Skin biopsy specimens (n = 16), serum (n = 114), blister fluid (n = 23), and primary inflammatory cells from patients with BP were used to investigate CXCL10 expression and function., Results: At baseline, both resident cells, such as keratinocytes and fibroblasts, and infiltrating immune cells expressed CXCL10 at lesional sites in skin of patients with BP. CXCL10 levels were higher in blister fluid (P < .0001) and serum (P < .005) from patients with BP than in serum from age- and sex-matched control subjects (n = 34). Furthermore, CXCL10 serum levels increased at day 60 only in patients who relapsed within the first year of treatment (n = 33, P < .005). Interestingly, CXCL10 expression could be upregulated by itself and IL-17 in inflammatory cells. Notably, neutrophils and monocytes from patients with BP, but not lymphocytes, responded to CXCL10 by increasing MMP-9 secretion through the activation of extracellular signal-regulated kinase 1/2, p38, phosphoinositide-3 kinase signaling pathways. Finally, CXCL10-increased MMP-9 secretion was inhibited by methylprednisolone and also by compound A, a novel nonsteroidal glucocorticoid receptor ligand., Conclusion: We showed that increased levels of inflammatory biomarkers in patients with BP, such as CXCL10, favor neutrophil- and monocyte-associated MMP-9 release and disease relapse and opened new therapeutic horizons in patients with this autoimmune disease., (Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Bullous pemphigoid (BP) is an inflammatory autoimmune bullous disease involving cytokines and proteases in the process of blister formation. Recently, IL-17 and IL-23 were evidenced in lesional skin and serum of BP patients at time of diagnosis, but their involvement in disease outcome has still not been investigated yet. We then analysed IL-17 and IL-23 serum levels during the first months of follow-up upon treatment. Compared with age- and sex- matched controls, high levels of IL-23 were observed at baseline in BP patients serum (P < 0.01), while IL-17 levels was not. However, some BP patients expressed high IL-17 serum level, independently of disease severity. In these patients, those with ongoing remission reduced IL-17 concentration upon treatment (P < 0.001), whereas IL-17 level remained elevated in patients who relapsed. Meanwhile, IL-23 serum levels increased during the first month of treatment in BP patients who later relapsed (P < 0.01) and MMP-9 serum level was not controlled. Accordingly, we found that both IL-17 and IL-23 increased MMP-9 secretion from leukocytes in-vitro. Then, we showed that elevated IL-17/IL-23 serum concentrations helped to discriminate BP patients who later relapsed. Such uncontrolled inflammatory response raises the question whether these molecules could become biological target for BP patients resistant to steroid treatment.

Bullous pemphigoid (BP) is an autoimmune skin disease characterized by the binding of autoantibodies to components of the hemidesmosome structure, resulting in an inflammatory response and subepidermal blister formation. To investigate the role of immune orientation in the inflammatory processes associated with disease progression, blister fluid, serum, and biopsy specimens were collected from 31 consecutive BP patients. Blister fluids displayed high levels of IL-6, IL-17, IL-22, and IL-23, whereas transforming growth factor-β was increased in BP sera. However, neither immunocytochemistry on a trans-differentiation model of IL-17-producing peripheral blood mononuclear cells nor immunohistochemistry on BP biopsy specimens could demonstrate the presence of T helper type 17 lymphocytes. Instead, innate immune cells, especially neutrophils, produced IL-17 at the skin lesional site. Of note, superpotent topical corticosteroid application quickly and markedly reduced both IL-17 expression and clinical signs of BP. Consistently, IL-17 upregulated matrix-metalloprotease-9 and neutrophil elastase expression, two proteases involved in blister formation, thereof further demonstrating its role in the progress of BP. Finally, IL-17-induced matrix degradation, originated from neutrophil activation, initiated the formation of an amplification loop of the inflammatory response that could represent the underlying phenomenon leading to the maintenance and even disease extent. Thus, our results could open new therapeutic strategies for BP patients.