Jieyu Zhang

Increased expression of NLRP3 inflammasome components and interleukin-18 in patients with bullous pemphigoid

BACKGROUND Bullous pemphigoid (BP) is the most common blistering autoimmune disease severely affecting older people. The complex interplay between innate and adaptive immunity is critical in the development of BP. Inflammasomes, which lead to cleavage-induced maturation of pro-inflammatory cytokines, have been shown to participate in a variety of autoimmune diseases. However, the role of inflammasomes in BP has not been delineated. OBJECTIVE The present work aimed to investigate whether inflammasomes are involved in the pathogenesis and progression of BP. METHODS Expressions of inflammasome components at the messenger RNA (mRNA) and protein levels in peripheral blood mononuclear cells (PBMCs) from patients with BP and from healthy controls were investigated by quantitative real-time PCR (qRT-PCR) and western blot, respectively. ELISA was employed to evaluate interleukin (IL)-1β, IL-18, lactate dehydrogenase (LDH), and high-mobility group-1 (HMGB1) levels in the serum and blister fluid of patients with BP. Immunohistochemical staining was used to detect IL-18 expression in the skin lesion of patients with BP. RESULTS The mRNA levels of NLRP3 inflammasome components (NLRP3, pro-caspase-1, and pro-IL-18) in PBMCs were significantly up-regulated in BP patients compared with those in healthy controls, and were positively correlated with the autoantibody titers for BP180-NC16A. Western Blot analysis showed that the baseline expressions of NLRP3 inflammasome components were increased in PBMCs of BP patients compared with healthy controls, and we failed to observe the mature IL-1β and IL-18. Further analysis showed that IL-18 was elevated in serum, blister fluid and lesional skin from patients with BP, and the serum IL-18 level was positively correlated with the titers of anti-BP180-NC16A autoantibody in BP patient. Most importantly, we found that mRNA expressions of the NLRP3-caspase-1-IL-18 axis components and the serum IL-18 level in BP patients decreased dramatically after effective treatment, which indicated that the up-regulation of NLRP3 inflammasome was responsible, at least to a great degree, for the enhanced IL-18 level in BP patients. CONCLUSIONS This study demonstrates that the expression of the NLRP3-caspase-1-IL-18 axis is highly expressed in the PBMCs of patients with BP, and correlated with disease activity, suggesting its involvement in the pathogenesis and progression of BP.

Proinflammatory role of blister fluid-derived exosomes in bullous pemphigoid: Blister fluid exosomes in bullous pemphigoid

Bullous pemphigoid is an autoimmune inflammatory disorder characterized by the presence of autoantibodies against bullous pemphigoid autoantigens, leading to dermal–epidermal separation with consequent blister formation. However, whether and how the components of blister fluid exacerbate the progression of bullous pemphigoid is unclear. Exosomes are nanometre‐sized vesicles released from cells into the body fluid, where they can transmit signals throughout the body. In the present study, we isolated and characterized exosomes from blister fluids of patients with bullous pemphigoid, evaluated their proinflammatory role, and identified the underlying molecular mechanisms. We found that exosomes isolated from blister fluids of patients with bullous pemphigoid showed the expected size and expressed the marker proteins CD63, CD81, and CD9. Additionally, blister fluid‐derived exosomes were internalized by human primary keratinocytes, inducing the production of critical inflammatory cytokines and chemokines. Western blotting analysis showed robust and rapid activation of the extracellular signal‐regulated kinase 1/2 and signal transducer and activator of transcription 3 signalling pathways in human primary keratinocytes after stimulation with blister fluid‐derived exosomes. We also found that blister fluid‐derived exosomes indirectly induced neutrophil trafficking by upregulating C‐X‐C motif chemokine ligand 8 in vitro. Furthermore, CD63 was localized mostly to keratinocytes and infiltrative granulocytes in skin lesions, suggesting that these cells were the possible sources of exosomes in blister fluid. Using mass spectrometry, we analysed the proteomes of blister fluid‐derived exosomes and identified a variety of proteins implicated in inflammatory and immune responses. Together, our findings provide strong evidence that blister fluid‐derived exosomes are involved in the local autoinflammatory responses of the skin associated with bullous pemphigoid. Copyright

Dysregulation of mCD46 and sCD46 contribute to the pathogenesis of bullous pemphigoid

Bullous pemphigoid (BP) is an autoimmune bullous disease caused by autoantibodies against BP180 in the epidermal basement membrane. Autoantibody-mediated complement activation is an important process in BP pathogenesis. CD46, a crucial complement regulatory protein in the complement activation, has been reported to be involved in several autoimmune diseases. In the present study, we investigated whether CD46 plays a role in BP development. We found that sCD46 expression was significantly increased in the serum and blister fluids of BP patients and correlated with the levels of anti-BP180 NC16A antibody and C3a. Otherwise, the level of mCD46 was decreased in lesions of BP patients, whereas the complement activation was enhanced. We also found that CD46 knockdown in HaCaT human keratinocytes enhanced autoantibody-mediated complement activation. Importantly, exogenous CD46 blocked complement activation in both healthy skin sections and keratinocytes induced by exposure to pathogenic antibodies from BP patients. These data suggest that CD46 deficiency is an important factor in BP pathogenesis and that increasing CD46 levels might be an effective treatment for BP.

Vascular endothelial growth factor driving aberrant keratin expression pattern contributes to the pathogenesis of psoriasis

Abstract Psoriasis is a chronic inflammatory skin disease severely affecting patients’ physical and psychological well‐being. Aberrant keratin expression in psoriasis plays a crucial role in keratinocyte dysfunction and disease development. Little is yet known about the mechanism of this keratin dysregulation. VEGF (Vascular endothelial growth factor) is significantly elevated in psoriatic patients and VEGFRs are detected on keratinocytes, leading to our hypothesis that this keratin dysregulation may be regulated by VEGF. In this study, we showed that VEGFR2 was overexpressed in psoriatic epidermis and was correlated with K (keratin) 6, K16&K17 upregulation and K1&K10 downregulation. VEGF increased both mRNA and protein levels of K6, K16&K17 and decreased those of K1&K10 in NHEKs (normal human epidermal keratinocytes). Further we identified activation of STAT3, ERK1/2, and p38 pathways in VEGF‐treated NHEKs. Using specific pathway antagonists and siRNAs we found that VEGF induced K6, K16&K17 via these three pathways and reduced K1&K10 via ERK1/2. Finally, VEGF‐induced aberrant keratin expression pattern and epidermal thickening were confirmed in a VEGF‐local‐injection mouse model. Collectively, we demonstrated that VEGF was associated with aberrant keratin expression pattern in psoriasis and provided a new insight into the role of VEGF in psoriasis pathogenesis, indicating VEGF as a potential therapeutic target. HighlightsOverexpressed VEGFR2 in psoriatic epidermis was associated with aberrant keratin expression.VEGF directly regulate keratin expression in NHEKs both in vitro and in vivo.VEGF increased K6, K16&K17 expression via STAT3, ERK1/2, and p38 pathways.VEGF decreased K1&K10 expression via ERK1/2 pathway.We first clarify a direct link between aberrant keratin expression and elevated VEGF in psoriasis.

Dysfunction of CD19+CD24hiCD27+ B regulatory cells in patients with bullous pemphigoid

Bullous pemphigoid (BP) is an autoimmune blistering skin disease characterized by the production of autoantibodies against the hemidesmosomal protein BP180. B regulatory cells (Bregs) are crucial in maintaining self-tolerance and suppressing autoantibody production. However, it is still unclear whether the dysfunctions of Bregs contributes to the autoantibody production in BP patients. In this study, we found that CD19+CD24hiCD27+ Bregs and IL-10+CD19+ Bregs were significantly increased in the peripheral blood samples of BP patients compared with that in healthy controls. Moreover, compared to Bregs from healthy individuals, we found that Bregs from BP patients fails to suppress the production of specific anti-BP180 autoantibody when co-cultured with patient-derived PBMCs. Additionally, Bregs from BP patients were defective in suppressing the CD4+ T cell proliferation and the cytokines expression (including IFN-γ, TNF-α and IL-4). Notably, we found that patient-derived Bregs produced high level of TNF-α and the TNF inhibitor etanercept could inhibit the autoantibody production in the culture system in vitro. Our results indicate that Bregs from BP patient appear phenotypically pro-inflammatory by their cytokine profile and are defective in immunosuppressive function, which suggest that Bregs play a pro-inflammatory role rather than a regulatory role in the pathogenesis of BP.

Identification of Immunodominant Th2-Cell Epitopes in Chinese Patients with Bullous Pemphigoid

Bullous pemphigoid (BP) is a subepidermal autoimmune blistering disease caused by autoantibodies targeting the juxtamembranous extracellular noncollagenous 16A (NC16A) domain of human collagen XVII (also known as BP180). Because T-helper (Th) cells are essential for antibody responses to antigens, we adopted an assay to map the immunodominant Th2-cell epitopes in NC16A. We synthesized 22 overlapping peptides spanning the entire sequence of BP180-NC16A and investigated the reactivity of Th2 cells from patients with BP to these peptides using the Enzyme-Linked ImmunoSpot (ELISPOT) assay. We screened two epitope peptides, P2 (492-506 aa: VRKLKARVDELERIR) and P5 (501-515 aa: ELERIRRSILPYGDS), and confirmed that these epitopes play a dominant role in stimulating CD4+ T-cell proliferation and Th2 IL-4 cytokine production, and activating B cells to secrete autoantibodies. These immunodominant epitopes are HLA-DR-restricted and were observed in subjects with different HLA alleles. This work contributes to elucidation of the epitope-mediated immunologic pathogenesis of BP, and the identified Th2-cell epitopes are candidates for epitope-specific therapeutic strategy.

Decreased expression levels of complement regulator CD55 contribute to the development of bullous pemphigoid

Bullous pemphigoid is a common autoimmune blistering disease of the elderly associated with autoantibody-mediated complement activation, and complement dysregulation is critical for its pathogenesis. As a crucial regulator of the complement system, CD55 has been widely studied in autoimmune diseases. Here, we investigated the involvement of CD55 in bullous pemphigoid, as little is known regarding its role in this disease. We found that CD55 levels were significantly lower in the lesions of patients with bullous pemphigoid (n = 8) compared to those in skin samples from healthy controls (n = 6). Interestingly, CD55 depletion in HaCaT human keratinocytes enhanced autoantibody-mediated complement activation. Moreover, complement activation was blocked by exogenous recombinant CD55 protein in both skin sections and keratinocytes exposed to pathogenic antibodies from patients with bullous pemphigoid. Notably, a significant increase in the expression of TNF-α and IFN-γ, administration of which downregulated CD55 levels in HaCaT cells, was observed in the sera of patients with bullous pemphigoid (n = 38) compared to that in healthy controls (n = 19). We found that ERK1/2 is involved in both TNF-α- and IFN-γ-induced CD55 downregulation. Thus, CD55 deficiency is a crucial factor in bullous pemphigoid pathogenesis, suggesting that increasing CD55 levels may exert a therapeutic effect.

Effect of Calcipotriol on IFN-γ-Induced Keratin 17 Expression in Immortalized Human Epidermal Keratinocyte Cells

Background Calcipotriol ointment has been demonstrated to be a very safe and effective topical drug for psoriasis. This study aims to investigate the effect of calcipotriol on IFN-γ-induced keratin 17 (K17) expression in a human keratinocyte cell line (HaCaT), which is a widely accepted as a mimic in vitro model for psoriasis. Material/Methods We used Western blot, immunofluorescence staining, and luciferase reporter system assays to evaluate the expression of K17 and the possible underlying mechanisms. Results Administration of IFN-γ (125–1000 U) increased K17 expression in a dose-dependent manner, and 250 U/ml IFN-γ significantly elevated K17 expression. The experimental results showed that calcipotriol at concentrations of 10−7 M and 10−5 M suppressed the IFN-γ-induced K17 expression by 58.10% and 70.68%, respectively. Through immunofluorescence staining and luciferase reporter assay, we found that Vitamin D Response Element (VDRE) affected IFN-activated site (Gamma-activated sequence, GAS) function at the transcriptional level and was involved in the inhibition of K17 expression. Conclusions Our data suggest that calcipotriol downregulates IFN-γ-mediated K17 expression in keratinocytes in a dose-dependent manner via VDRE effect GAS function. The inhibitory effect of calcipotriol on K17 expression may be a potential mechanism and function in the treatment psoriasis.

Semaphorin 4D from CD15+ Granulocytes via ADAM10-Induced Cleavage Contributes to Antibody Production in Bullous Pemphigoid

Autoreactive B-cell activation and antibody production are critical events for the development of bullous pemphigoid (BP). However, the mechanism that is involved in the modulation of B-cell activation and autoantibody generation has not been fully understood. Semaphorin 4D (Sema4D, or CD100) plays important roles in immune regulation related to B cells, but its implications in BP remain obscure. The aim of our study was to characterize Sema4D and the underlying mechanism contributing to the autoimmune features of BP. We found that soluble Sema4D (sSema4D) levels were elevated and correlated with disease severity and activity in serum and blister fluids from patients with BP. Additionally, Sema4D-expressing cells accumulated in subepidermal blisters of BP lesions. In patient-derived peripheral blood mononuclear cells, by promoting the differentiation of B cells into plasmablasts, sSema4D boosted anti-BP180/anti-BP230 antibody production in a time- and dose-dependent manner, which may be attributed to CD72-mediated activation of Akt/NF-κB phosphorylated (p-)65/ERK cascades in B cells. We determined that a disintegrin and metalloproteinase 10 is a proteolytic enzyme for the cleavage of sSema4D from CD15+ granulocytes instead of T cells, which is probably responsible for the high concentration of sSema4D in BP blister fluid and serum. These findings suggest that Sema4D is a crucial participant in BP pathogenesis.

Association of HLA class I and class II alleles with bullous pemphigoid in Chinese Hans

BACKGROUND Bullous pemphigoid (BP) is one of the most common autoimmune skin diseases. Associations of genes, especially human leukocyte antigen (HLA)-DQ alleles, with BP indicate that genetic predisposition contributes to the disease. OBJECTIVES To evaluate the association of HLA class I and HLA class II alleles with susceptibility to BP in the northern Chinese Han population. METHODS We performed genotype for HLA-A, -B, -C, -G, -DPA1, -DPB1, -DQA1, -DQB1 and -DRB1 loci in 105 patients with BP by Sanger sequence-based typing (SBT) method. These data were compared with a local control cohort of 420 age- and sex-matched cases. RESULTS Among the HLA alleles described herein, the susceptibility alleles associated with a high prevalence of BP were A*11:01 (OR = 1.9 Pc = 0.017); B*37:01 (OR = 8, Pc = 1.811 × 10-6); G*01:01 (OR = 3.61, Pc = 2.839 × 10-15) and G*01:06 (OR = 2.22, Pc = 0.025); DQA1*01:05 (OR = 4.87, Pc = 5.822 × 10-5), DQA1*05:05 (OR = 2.64, Pc = 9.114 × 10-4), and DQA1*05:08 (OR = 10.2, Pc = 0.016); DQB1*03:01 (OR = 1.69, Pc = 0.048) and DQB1*05:01 (OR = 3.42, Pc = 7.28 × 10-6); and DRB1*10:01 (OR = 6.85, Pc = 2.63 × 10-6). To the contrary, HLA-DQA1*01:02 (OR = 0.46, Pc = 8.603 × 10-4) and DQA1*01:03 (OR = 0.38, Pc = 0.048); DQB1*02:02 (OR = 0.28, Pc = 0.016); and DRB1*07:01 (OR = 0.26, Pc = 0.004) had significant associations with protection against BP. In addition, the frequency of haplotype HLA-DRB1*13-DQA1*05-DQB1*03 (OR = 12.32, Pc = 0.026) in BP patients was significantly higher than those in controls. CONCLUSION Our data demonstrated that the alleles and haplotypes found in this study may be important differential genetic markers for susceptibility to or protection against BP in individuals of northern Chinese Han population.