Erle Dang

IL-17A Upregulates Keratin 17 Expression in Keratinocytes through STAT1- and STAT3-Dependent Mechanisms

Psoriasis, an immunological skin disease, is characterized by epidermal hyperproliferation, chronic inflammation, and an accumulation of infiltrating T cells. IL-17A is a key cytokine that has a critical role in the pathogenesis of psoriasis. Keratin 17 (K17) is strongly expressed in psoriatic lesions but not in normal skin. Thus, K17 expression is regarded as a hallmark of psoriasis. We previously reported that the K17/T cells/cytokine autoimmune loop was involved in psoriasis. However, the relationship between IL-17A and K17 has yet to be determined. In the present study, IL-17A-induced K17 expression was confirmed in cultured keratinocytes in both mRNA and protein levels. In addition, increased K17 expression was found in the epidermis of IL-17A-injected mouse skin. The regulatory mechanism of K17 expression was further investigated. We found that both the signal transducer and activator of transcription (STAT) 1 and STAT3 pathways were involved in the upregulation of K17 expression induced by IL-17A, and that such regulation could be partially suppressed by STAT1 or STAT3 small interfering RNA and inhibitor. Our data suggest that IL-17A can upregulate K17 expression in keratinocytes in a dose-dependent manner through STAT1- and STAT3-dependent mechanisms. The results indicate that IL-17A might be an important cytokine in the K17/T cells/cytokine autoimmune loop associated with psoriasis.

The pro-inflammatory cytokine IL-22 up-regulates keratin 17 expression in keratinocytes via STAT3 and ERK1/2.

Background To investigate the regulation of K17 expression by the pro-inflammatory cytokine IL-22 in keratinocytes and its important role in our previously hypothesized “K17/T cell/cytokine autoimmune loop” in psoriasis. Materials and Methods K17 expression was examined in the IL-22-treated keratinocytes by real-time quantitative PCR, ELISA, Western blot and Immunofluorescence. In addition, the signaling pathways involved in K17 regulation were investigated with related inhibitors and siRNAs. In addition, K17 expression was examined in the epidermis of IL-22-injected mouse skin. Results IL-22-induced K17 expression was confirmed in keratinocytes and the epidermis of IL-22-injected mouse skin at both mRNA and protein levels, which is an important complement to the autoimmune loop. We further investigated the regulatory mechanisms and found that both STAT3 and ERK1/2 were involved in the up-regulation of K17 expression induced by IL-22. Conclusion IL-22 up-regulates K17 expression in keratinocytes in a dose-dependent manner through STAT3- and ERK1/2-dependent mechanisms. These findings indicated that IL-22 was also involved in the K17/T cell/cytokine autoimmune loop and may play an important role in the progression of psoriasis.

Follicular Helper T Cells (Tfh) and IL-21 Involvement in the Pathogenesis of Bullous Pemphigoid

The pathogenesis of bullous pemphigoid (BP) is characterized by the T cell-dependent production of autoantibodies. Recent studies have indicated that follicular T helper cells (Tfh), the key modulator of B cell activation and autoantibody production, are critical in the development of several autoimmune diseases. Tfh cells perform their functions via IL-21, their hallmark cytokine. In the present study, the frequencies of Tfh cells were investigated in the peripheral blood samples of BP patients to evaluate whether Tfh cells involve in this clinical entity. Significantly higher Tfh cell counts were observed in the peripheral blood of BP patients than those in healthy controls (median: 11.25% vs. 4.95%, respectively; P<0.001). Additionally, the serum IL-21 levels in BP patients were higher than those of the healthy controls (median: 103.98 pg/mL vs 46.77 pg/mL, respectively; P<0.001). The frequencies of Tfh cells and IL-21 levels were both positively correlated with anti-BP180-NC16A autoantibody titers (R = 0.712, P<0.01 and R = 0.578, P = 0.030, respectively). After effective therapy, the frequencies of Tfh cells as well as the serum IL-21 levels in BP patients decreased along with clinical improvement. Most importantly, Tfh depleted CD4+ T cells and anti-IL-21 neutralization antibody could inhibit the T cell-induced B cell activation and secretion of BP autoantibody in vitro. Those results suggest that Tfh cells play an important role in autoantibody production and are involved in the pathogenesis of BP.

Oxidative stress-induced calreticulin expression and translocation: new insights into the destruction of melanocytes.

Increased reactive oxygen species (ROS) contribute to melanocyte apoptosis and the development of cutaneous diseases or disorders via autoimmunity. However, the mechanisms and interrelationships between ROS and autoimmunity are unknown. This study aimed to investigate the role of calreticulin (CRT) in hydrogen peroxide (H2O2)-induced apoptosis in melanocytes. Total CRT levels increased in a time-dependent manner in human immortalized normal and vitiligo melanocytes exposed to H2O2-induced oxidative stress, and surface levels of CRT were increased. Moreover, CRT overexpression increased H2O2-induced apoptosis, whereas knockdown showed the opposite results. Furthermore, CRT-treated peripheral blood mononuclear cells (PBMCs) or stressed melanocytes expressed higher levels of IL-6 and tumor necrosis factor-α (TNF-α) than untreated cells (P<0.05); this effect was inhibited with CRT knockdown. In an in vivo model, CRT levels were positively correlated with lesion area (R=0.7582, P<0.0001) and duration of vitiligo in patients (P<0.001). ELISA analyses revealed that CRT expression was higher in vitiligo patients as compared with healthy subjects (P<0.05). These data demonstrate that CRT exposure via H2O2-induced oxidative stress plays a significant role in melanocyte apoptosis and suggest a relationship between apoptosis and immune reactions during melanocyte destruction.

Elevated serum levels of interleukin 21 are associated with disease severity in patients with psoriasis

Background Psoriasis is an immune disorder involving numerous cytokines. Recent studies have shown that interleukin (IL)‐21 plays an important role in a variety of inflammatory and autoimmune diseases. It is highly expressed in psoriatic plaques and promotes the proliferation of epidermis in mice. It seems that IL‐21 plays an important role in the pathogenesis of psoriasis. However, whether or not it is elevated in the peripheral blood of patients with psoriasis and is associated with disease severity is unclear. Therefore, our study focuses on serum IL‐21 levels and their correlation with disease severity.

Impaired function of regulatory T cells in patients with psoriasis is mediated by phosphorylation of STAT3

BACKGROUND Psoriasis is a T cell-mediated chronic inflammatory skin disease. Regulatory T cells (Tregs) are crucial in suppressing immune response to maintain the immune balance. Wheras Tregs from psoriatic patients showed poorly activity in suppressing activation of responder T cells (Tresp), the mechanisms involved in this process are still unknown. OBJECTIVES In this study, we investigated the possible role of STAT3 pathway in the pathogenesis of dysfunctional Tregs in psoriasis. METHODS The suppressive function and the proliferative activity of Tregs were detected from psoriatic patients and normal healthy controls. Expression of phospho-STAT3 in psoriatic Tregs was evaluated by flow cytometry and immunofluorescence. Furthermore, Tregs were treated with Stattic V (STAT3 inhibitor) in order to investigate the role of STAT3 pathway in the function of Tregs. In addition, IL-6, IL-21 and IL-23 treatments were performed to identify the upstream molecules of STAT3 pathway in Tregs. RESULTS Tregs from peripheral blood of psoriatic patients showed decreased suppressive function, together with phosphorylation of STAT3. In addition, Tregs isolated from psoriatic patients could produce IFN-γ, TNF-α and IL-17. In the co-culture system of Tregs and Tresp isolated from psoriatic patients, addition of STAT3 inhibitor partially restored the suppressive function of Tregs and restrained the expressions of IFN-γ, TNF-α and IL-17 in psoriatic patients. Moreover, we found that IL-6, IL-21 and IL-23 induced the phosphorylation of STAT3 in Tregs. CONCLUSIONS Our findings suggest that psoriatic Tregs experience a predominant STAT3 phosphorylation by exposure to pro-inflammatory cytokines, leading to their impaired functions in suppressing Tresp activation.

Nrf2 Promotes Keratinocyte Proliferation in Psoriasis through Up-Regulation of Keratin 6, Keratin 16, and Keratin 17

Psoriasis is a chronic inflammatory skin disease characterized by keratinocyte hyperproliferation of epidermis. Although hyperproliferation-associated keratins K6, K16, and K17 are considered to be the hallmarks of psoriasis, the molecular basis underlying the overexpression of these keratins remains unclear. Nrf2 regulates cell proliferation. Therefore, we investigated whether Nrf2 regulates keratinocyte proliferation via promoting expression of K6, K16, and K17 in psoriasis. We initially found that psoriatic epidermis exhibited elevated expression of Nrf2. Furthermore, Nrf2 promoted expression of K6, K16, and K17 in both HaCaT cells and primary human keratinocytes by binding to the ARE domains located in the promoter of these genes. Additionally, upon stimulation with IL-17 or IL-22, Nrf2 translocated to the nucleus and initiated expression of targeted keratins. In mice of imiquimod-induced psoriasis-like dermatitis, topical application of Nrf2 small interfering RNA alleviated the epidermal hyperplasia with reduced expression of these keratins. More importantly, Nrf2 promoted the proliferation of human keratinocytes through up-regulation of K6, K16, or K17. These data suggested that inflammatory cytokines promoted Nrf2 nuclear translocation in psoriatic epidermis, which led to elevated expression of K6, K16, and K17, thus promoting keratinocyte proliferation and contributing to the pathogenesis of psoriasis.

TGFβ/SMAD/microRNA-486-3p Signaling Axis Mediates Keratin 17 Expression and Keratinocyte Hyperproliferation in Psoriasis

Keratin 17 (K17) is strongly expressed in psoriatic lesions but not healthy skin, and plays a crucial role in disease pathogenesis. The mechanism of aberrant K17 expression in psoriasis has not been fully elucidated. MicroRNAs are short, single-stranded, noncoding RNAs that play important roles in regulating gene expression. Psoriasis exhibits a specific microRNA expression profile distinct from that of healthy skin. In this study, we showed that miR-486-3p was markedly reduced in psoriatic epidermis and negatively correlated with the psoriasis area and severity index score. Its expression repressed K17 protein expression and decreased proliferation in a keratinocyte cell line overexpressing K17 (LV K17) compared with controls. Our data indicated that miR-486-3p was regulated by a transforming growth factor-β (TGFβ)/SMAD pathway and possibly mediated the downregulation of K17 protein in TGFβ-treated keratinocytes. Finally, the decreased expression of TGFβ receptor I in psoriatic epidermis inactivated the TGFβ/SMAD pathway, leading to K17 overexpression and cell proliferation. Collectively, our findings demonstrated that a TGFβ/SMAD/miR-486-3p signaling axis in keratinocytes regulated K17 expression and cell proliferation. We conclude that the loss of miR-486-3p in psoriatic epidermis leads to K17 protein overexpression and contributes to the pathogenesis of psoriasis. Overexpression of miR-486-3p may therefore be a therapeutic option for psoriasis.

CD100–Plexin-B2 Promotes the Inflammation in Psoriasis by Activating NF-κB and the Inflammasome in Keratinocytes

PlxnB2 and its ligand, CD100, were originally identified as axon-guidance molecules that function during neuronal development; however, studies also showed that CD100-plexins participate in various immune responses. In this study, we found that the expression of PlxnB2 on keratinocytes was specifically increased in lesional skin of psoriasis patients but not atopic dermatitis. Levels of soluble CD100 and membrane-bound CD100 were elevated in sera of psoriasis patients and on keratinocytes of psoriatic skin, respectively. By binding to PlxnB2, soluble CD100 promoted the production of CXCL-1, CCL-20, IL-1β, and IL-18 by keratinocytes and activated the NLRP3 inflammasome. Moreover, CD100-PlxnB2 stimulated the NF-κB signaling pathway in keratinocytes through activation of small GTPase RhoA and Rac1. Our data showed that cooperation of CD100 and PlxnB2 promoted the inflammatory responses in keratinocytes by activating NF-κB and the NLRP3 inflammasome and participated in the pathogenesis of psoriasis. CD100/PlxnB2 might be a potential therapeutic target for psoriasis.

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.