Serum interleukin-34 levels in dermatomyositis: a potential biomarker for anti-MDA5-antibody-associated interstitial lung disease.

OBJECTIVES: Interleukin (IL)-34 is a hematopoietic cytokine that promotes macrophage activation. Macrophage activation in interstitial lung disease (ILD) in patients with dermatomyositis (DM), especially in those with anti-melanoma differentiation-associated gene 5 (MDA5) antibody suggests the involvement of IL-34 in the disease. However, the association between IL-34 and DM is unknown. In this study, we aimed to determine serum IL-34 levels in DM patients and evaluate their association with DM-ILD. METHODS: We measured serum IL-34 levels in 56 DM patients and 14 age- and sex- matched healthy controls by enzyme-linked immunosorbent assay, and examined their correlation with clinical parameters. In addition, pre- and post-treatment serum IL-34 levels were examined using serum samples from 7 anti-MDA5 antibody-positive DM patients. RESULTS: Serum IL-34 levels were significantly elevated in DM patients, especially in those with anti-MDA5 antibody, compared with healthy controls. In anti-MDA5-antibody-positive DM patients, serum IL-34 levels positively correlated with serum levels of ferritin and anti-MDA5 antibody, which are known biomarkers for rapidly progressive (RP)-ILD. Following combined immunosuppressive therapy, serum IL-34 levels decreased along with ferritin and anti-MDA5 antibody. CONCLUSION: These data suggest that IL-34 may be involved in the development of RP-ILD in anti-MDA5 antibody-positive DM. Serum IL-34 levels can serve as a potential biomarker for RP-ILD in this clinical entity.

Autoantibodies to nuclear valosin-containing protein-like protein: systemic sclerosis-specific antibodies revealed by in vitro human proteome.

OBJECTIVES: To identify and characterize undescribed systemic sclerosis (SSc)-specific autoantibodies targeting nucleolar antigens and to assess their clinical significance. METHODS: We conducted proteome-wide autoantibody screening (PWAS) against serum samples from SSc patients with nucleolar patterned anti-nuclear antibodies (NUC-ANAs) of specific antibodies (Abs) unknown, utilizing wet protein arrays fabricated from in vitro human proteome. Controls included SSc patients with already-known SSc-specific autoantibodies, patients with other connective tissue diseases, and healthy subjects. The selection of nucleolar antigens was performed by database search in the Human Protein Atlas. The Presence of autoantibodies was certified by immunoblots and immunoprecipitations. Indirect immunofluorescence assays on HEp-2 cells were also conducted. Clinical assessment was conducted by retrospective review of electric medical records. RESULTS: PWAS identified three candidate autoantibodies, including anti-nuclear valosin-containing protein-like (NVL) Ab. Additional measurements in disease controls revealed that only anti-NVL Abs are exclusively detected in SSc. Detection of anti-NVL Abs was reproduced by conventional assays such as immunoblotting and immunoprecipitation. Indirect immunofluorescence assays demonstrated homogeneous nucleolar patterns. Anti-NVL Ab-positive cases were characterized by significantly low prevalence of diffuse skin sclerosis and interstitial lung disease, compared with SSc cases with NUC-ANAs other than anti-NVL Abs, such as anti-U3-RNP and anti-Th/To Abs. CONCLUSION: Anti-NVL Ab is an SSc-specific autoantibody associated with a unique combination of clinical features, including limited skin sclerosis and lack of lung involvement.

Dual blockade of interleukin-17A and interleukin-17F as a therapeutic strategy for liver fibrosis: Investigating the potential effect and mechanism of brodalumab.

Liver fibrosis is a terminal manifestation of various chronic liver diseases. There are no drugs that can reverse the condition. Recently, the importance of interleukin-17 (IL17) in the pathophysiology has been revealed and has attracted attention as a therapeutic target. We aimed to reveal the roles of IL17A and IL17F in liver fibrosis, and to validate the potential of their dual blockade as therapeutic strategy. First, we retrospectively reviewed the longitudinal change of FIB-4 index, a clinical indicator of liver fibrosis, among psoriasis patients treated by brodalumab, which blocks IL17 receptor A (IL17RA). Next, we examined anti-fibrotic efficacy of anti-IL17RA antibody (Ab) in two murine liver fibrosis models by histopathological investigation and real-time reverse transcription polymerase chain reaction (RT-PCR). Finally, we analyzed the effect of IL17A and IL17F upon human hepatic stellate cells with RNA sequencing, real-time RT-PCR, western blotting, chromatin immunoprecipitation, and flow cytometry. Clinical data showed that FIB-4 index significantly decreased among psoriasis patients treated by brodalumab. In vivo studies additionally demonstrated that anti-IL17RA Ab ameliorates liver fibrosis induced by tetrachloride and methionine-choline deficient diet. Furthermore, in vitro experiments revealed that both IL17A and IL17F enhance cell-surface expression of transforming growth factor-ß receptor II and promote pro-fibrotic gene expression via the JUN pathway in human hepatic stellate cells. Our insights suggest that IL17A and IL17F share their pro-fibrotic function in the context of liver fibrosis, and moreover, dual blockade of IL17A and IL17F by anti-IL17RA Ab would be a promising strategy for the management of liver fibrosis.

Cannabinoids for the treatment of autoimmune and inflammatory skin diseases: A systematic review.

In recent years, the medical use of cannabinoids has attracted growing attention worldwide. In particular, anti-inflammatory properties of cannabinoids led to their emergence as potential therapeutic options for autoimmune and inflammatory disorders. Recent studies have also shown that cannabinoid receptors are widely expressed and have endogenous ligands in the skin, suggesting that the skin has its own endocannabinoid system. The aim of this review is to discuss the potential therapeutic effects of cannabinoids in autoimmune and inflammatory skin diseases. Following an overview of cannabinoids and the endocannabinoid system, we describe the cellular and molecular mechanisms of cannabinoids in skin health and disease. We then review the clinical studies of cannabinoids in autoimmune and inflammatory skin diseases including systemic sclerosis (SSc), dermatomyositis (DM), psoriasis (Pso) and atopic dermatitis (AD). A primary literature search was conducted in July 2023, using PubMed and Web of Science. A total of 15 articles were included after excluding reviews, non-human studies and in vitro studies from 389 non-duplicated articles. Available evidence suggests that cannabinoids may be beneficial for SSc, DM, Pso and AD. However, further studies, ideally randomized controlled trials, are needed to further evaluate the use of cannabinoids in autoimmune and inflammatory skin diseases.

Deregulated JNK signaling enhances apoptosis during hyperthermia.

PURPOSE: c-Jun N-terminal kinases (JNKs) comprise a subfamily of mitogen-activated protein kinases (MAPKs). The JNK group is known to be activated by a variety of stimuli. However, the molecular mechanism underlying heat-induced JNK activation is largely unknown. The aim of this study was to clarify how JNK activity is stimulated by heat. METHODS AND MATERIALS: The expression levels of various MAPK members in HeLa cells, with or without hyperthermia treatment, were evaluated via western blotting. The kinase activity of MAPK members was assessed through in vitro kinase assays. Cell death was assessed in the absence or presence of siRNAs targeting MAPK-related members. RESULTS: Hyperthermia decreased the levels of MAP3Ks, such as ASK1 and MLK3 which are JNK kinase kinase members, but not those of the downstream MAP2K/SEK1 and MAPK/JNK. Despite the reduced or transient phosphorylation of ASK1, MLK3, or SEK1, downstream JNK was phosphorylated in a temperature-dependent manner. In vitro kinase assays demonstrated that heat did not directly stimulate SEK1 or JNK. However, the expression levels of DUSP16, a JNK phosphatase, were decreased upon hyperthermia treatment. DUSP16 knockdown enhanced the heat-induced activation of ASK1-SEK1-JNK pathway and apoptosis. CONCLUSION: JNK was activated in a temperature-dependent manner despite reduced or transient phosphorylation of the upstream MAP3K and MAP2K. Hyperthermia-induced degradation of DUSP16 may induce activation of the ASK1-SEK1-JNK pathway and subsequent apoptosis.

Optimal treatments and outcome measures of palmoplantar pustulosis: A systematic review and network meta-analysis-based comparison of treatment efficacy.

Few studies have made direct comparisons between treatments for palmoplantar pustulosis (PPP); therefore, it is difficult to select the best treatment for each patient. To determine the best therapy and to compare reported measures of efficacy in clinical trials of systemic treatments for PPP in this systematic review and network meta-analysis. Six databases were used to perform database search on 10 July 2022. Randomized controlled trials (RCTs) were identified through a systematic literature search. The titles and abstracts of articles were initially screened for inclusion by two authors independently using our predetermined criteria. The full texts of selected articles were then independently assessed for inclusion in a blinded fashion. Disagreement between the authors was resolved by consensus. Data were abstracted in duplicate. Random-effects model was accepted to perform network meta-analysis. Assessed Grading of Recommendations Assessment, Development and Evaluation certainty of evidence were performed according to the PRISMA guidelines. The analysis was completed in July 2022. The primary outcome was the change of PPP Area and Severity Index (PPPASI) from baseline and the secondary outcome was the achievement of PPPASI-50 response. Seven RCTs with 567 patients were included. Guselkumab 100 mg was the one with the highest probability of reaching the proposed outcomes (mean difference [MD], -8.00; 95% confidence interval [CI], 4.88-11.11), while the achievement of PPPASI-50 response did not show a significant difference (odds ratio [OR], 3.79; 95% CI, 0.51-28.37). Guselkumab 200 mg was next to 100 mg of reaching the proposed outcomes (MD, -4.71; 95% CI, 2.12-7.30), while the achievement of PPPASI-50 response did not show a significant difference (OR, 2.34; 95% CI, 0.48-11.43). Network meta-analysis showed guselkumab 100 mg was the treatment with the highest probability of reaching both PPPASI and PPPASI-50 outcomes. Absolute PPPASI may be more appropriate as an outcome than PPPASI-50.

Significance of anti-transcobalamin receptor antibodies in cutaneous arteritis revealed by proteome-wide autoantibody screening.

Cutaneous arteritis (CA) is a single-organ vasculitis that exclusively affects the small to medium-sized arteries of the skin. Diagnosis depends on a histological investigation with skin biopsy, which could be burdensome for both patients and clinicians. Moreover, the pathogenesis of CA remains unstudied, and treatment has not yet been established. Herein, we applied our proteome-wide autoantibody screening method to explore autoantibodies in the serum of CA patients. As a result, anti-transcobalamin receptor (TCblR) antibodies (Abs) were specifically detected in 24% of CA patients. Patients with positive anti-TCblR Abs were spared from peripheral neuropathy compared to those with negative anti-TCblR Abs, showing characteristics as CA confined to the skin. In addition, we revealed that anti-TCblR Abs trigger the autocrine loop of interleukin-6 mediated by tripartite motif-containing protein 21 in human endothelial cells and induce periarterial inflammation in murine skin. Furthermore, we demonstrated that methylcobalamin, a ligand of TCblR, ameliorates inflammation caused by anti-TCblR Abs both in vitro and in vivo. Collectively, our investigation unveils the pathologic significance of anti-TCblR Abs in CA and their potential as a diagnostic marker and a pathophysiology-oriented therapeutic target.

Utility of nailfold capillary assessment for predicting psoriatic arthritis based on a prospective observational cohort study.

OBJECTIVES: PsA is one of the most serious comorbidities associated with psoriasis. While the early intervention in PsA is demanded, risk factors of PsA development are not well-known. This is the first prospective study to evaluate the clinical significance of nailfold capillary (NFC) changes in patients with psoriasis. METHODS: We conducted a prospective cohort study in a population of 449 psoriasis patients who had not been treated with systemic therapy or topical finger therapy. NFCs were observed by dermoscopy and capillaroscopy, and the correlation of NFC abnormalities, including nailfold bleeding (NFB) and enlarged capillaries, with the prevalence of PsA, incidence of new PsA, and serum levels of TNF-a, IL-17A and IL-23 were analysed. RESULTS: Detailed examination at the time of inclusion revealed that of 449 patients, 236 had Psoriasis vulgaris (PsV) and 213 had PsA. Both NFB and enlarged capillaries were significantly more frequent in patients with PsA (34.7% vs 84.5%, P < 0.0001; 25.4% vs 100%, P < 0.0001). In addition, PsV patients were prospectively observed before they developed PsA (mean 21 months, 95% CI 2, 77 months). Multivariate analysis suggested that the appearance of NFB and enlarged capillaries was a predictor of PsA development (HR 2.75, 95% CI 1.38, 5.47 and HR 4.49, 95% CI 2.25, 8.96, respectively). The degree of NFC abnormalities also correlated with the severity of PsA and serum cytokine levels. CONCLUSIONS: NFC abnormalities were suggested to be a predictor of PsA in psoriasis patients, and at the same time, its degree could be an indicator of disease severity.

Utility of nailfold capillary assessment for predicting pustulotic arthro-osteitis in palmoplantar pustulosis based on a prospective cohort study.

BACKGROUND: Pustulotic arthro-osteitis (PAO) is 1 of the most serious comorbidities associated with palmoplantar pustulosis (PPP). Risk factors of PAO development are not well-known. OBJECTIVE: To evaluate the clinical significance of nailfold capillary (NFC) changes in patients with PPP. METHODS: We conducted a prospective cohort study in a population of 102 PPP patients. Correlations of NFC abnormalities, including nailfold bleeding and enlarged capillaries, with the prevalence of PAO, the incidence of new PAO, and serum levels of cytokines were analyzed. RESULTS: Detailed examination revealed that of 102 PPP patients, 52 without PAO and 50 with PAO. Both nailfold bleeding and enlarged capillaries were significantly more frequent in patients with PAO (50.0% vs 92.0%, P < .0001; 50.0% vs 94.0%, P < .0001). In addition, PPP patients without PAO were prospectively observed before they developed PAO (mean 28 months [1-52 months]). Multivariate analysis suggested that these NFC abnormalities were predictors of PAO development (hazard ratio 3.37, 95% confidence interval 1.13-10.07; 3.37, 1.13-10.07) and guselkumab prevent PAO development (0.093, 0.012-0.76). The degree of NFC abnormalities correlated with the severity of PAO and serum cytokine levels. LIMITATIONS: All participants were Japanese. CONCLUSION: NFC abnormalities could be predictors of PAO in PPP patients, and their degree indicators of disease severity.

Predictors of rituximab effect on modified Rodnan skin score in systemic sclerosis: a machine-learning analysis of the DesiReS trial.

OBJECTIVES: The double-blind, parallel-group comparison, investigators initiated phase II clinical trial of IDEC-C2B8 (Rituximab) in patients with Systemic sclerosis (DesiReS) trial showed that rituximab is effective in treating skin sclerosis in SSc. However, which patient groups are likely to benefit from rituximab is unknown. METHODS: We performed post-hoc analysis of prospective data from 54 patients who received rituximab or placebo in the DesiReS trial. Twenty-seven baseline factors were used to investigate subpopulations with different magnitudes of rituximab effect on modified Rodnan skin score (mRSS) change at 24 weeks. Based on a machine-learning algorithm called the causal tree, we explored the combination of predictors needed to identify subpopulations that would respond to rituximab and have good treatment outcomes. RESULTS: Three factors were identified as branches of the decision tree: peripheral blood CD19-positive cell counts', 'mRSS', and 'serum surfactant protein D (SP-D) levels'. It was only in the subpopulation of patients with CD19-positive cell counts of <57/µl that rituximab did not show a significant improvement in mRSS vs placebo. In the subpopulation of patients with CD19-positive cell counts of ≥57/µl and mRSS ≥ 17, mRSS was most improved with rituximab [difference -17.06 (95% CI: -24.22, -9.89)]. The second greatest improvement in mRSS with rituximab was in the subpopulation with CD19-positive cell counts of ≥57/µl, mRSS < 17, and serum SP-D levels of ≥151 ng/ml [difference -10.35 (95% CI: -14.77, -5.93)]. CONCLUSION: SSc patients who have high CD19-positive cell counts and high mRSS are expected to have greater improvement in mRSS with rituximab. When the patients with high CD19-positive cell counts show low mRSS, serum SP-D levels may modify the treatment effect. TRIAL REGISTRATION: ClinicalTrials.gov, https://clinicaltrials.gov, NCT04274257 and UMIN-CTR; https://center6.umin.ac.jp, UMIN000030139.

Decrease in MAP3Ks expression enhances the cell death caused by hyperthermia.

PURPOSE: Hyperthermia is a promising anticancer treatment modality. However, the molecular mechanism underlying the thermal sensitivity of tumor cells is largely unknown. The aim of this study was to clarify how biochemical changes triggered by heat stimulate antitumor activity. METHODS AND MATERIALS: The expression levels of various MAPK members in HeLa cells with or without hyperthermia were evaluated by western blotting and RT-PCR. The intracellular Ca2+ concentration [Ca2+]i was monitored by digital imaging using CaTM-2 AM. An in vitro cleavage assay was used to determine whether calcium-dependent protease calpain cleaves MAPK components. Cell proliferation and clonogenicity were assessed in the absence or presence of siRNAs targeting MAPK members. RESULTS: Hyperthermia decreased the levels of MAP3K TAK1, RAF1 and MEKK2 but not of the downstream MAP2K and MAPK members. The hyperthermia-induced degradation of TAK1 and MEKK2 was rescued by either the proteasome inhibitor MG132 or the calpain inhibitor ALLN; however, RAF1 was not affected by the inhibitors. Heat induced down regulation of RAF1. Hyperthermia increased [Ca2+]i and calpain I expression. The calcium ionophore A23187 decreased TAK1 and MEKK2 levels. An in vitro cleavage assay demonstrated that TAK1 and MEKK2 are calpain I substrates. Knockdown of TAK1, RAF1 and MEKK2 suppressed cell proliferation and clonogenicity. CONCLUSIONS: Hyperthermia decreased the levels of MAP3K TAK1, RAF1 and MEKK2, without reduction of the downstream components in the MAP3K-MAP2K-MAPK cascade, by a calpain-dependent degradation pathway or transcriptional regulation. TAK1, RAF1 and/or MEKK2 play crucial roles in cell proliferation and clonogenicity and are potential molecular targets for hyperthermia.

Involvement of Molecular Mechanisms between T/B Cells and IL-23: From Palmoplantar Pustulosis to Autoimmune Diseases.

Palmoplantar pustulosis (PPP) is a disease that causes recurrent blisters and aseptic pustules on the palms and soles. It has been suggested that both innate and acquired immunity are involved. In particular, based on the tonsils and basic experiments, it has been assumed that T and B cells are involved in its pathogenesis. In addition, the results of clinical trials have suggested that IL-23 is closely related to the pathogenesis. This review describes PPP and the genetic background, the factors involved in the onset and exacerbation of disease and its relation to the molecular mechanism. In addition, we describe the usefulness of biological therapy and its implications in relation to the importance in pathology, the pathogenesis of PPP, the importance of the role of the IL-23-Th17 axis and IL-36 in PPP. Furthermore, we describe an animal experimental model of PPP, the efficacy and mechanism of action of guselkumab, an anti-IL-23 antibody, the latest research, and finally the possibility for it to be effective for other autoimmune diseases.

Pharmacotherapy of Itch-Antihistamines and Histamine Receptors as G Protein-Coupled Receptors.

Itching can decrease quality of life and exacerbate skin symptoms due to scratching. Itching not only contributes to disease progression but also triggers complications such as skin infections and eye symptoms. Therefore, controlling itching is very important in therapeutic management. In addition to the well-known histamine, IL-31, IL-4 and IL-13 have recently been reported as factors that induce itching. Itching may also be caused by factors other than these histamines. However, we do not know the extent to which these factors are involved in each disease. In addition, the degree of involvement is likely to vary among individuals. To date, antihistamines have been widely used to treat itching and are often effective, suggesting that histamine is more or less involved in itchy diseases. This review discusses the ligand-receptor perspective and describes the dynamics of G protein-coupled receptors, their role as biased agonists, their role as inverse agonists, proactive antihistamine therapy, and drug selection with consideration of impaired performance and anti-PAF effects.

Cytokine analysis on a countable number of molecules from living single cells on nanofluidic devices.

Analysis of proteins released from living single cells is strongly required in the fields of biology and medicine to elucidate the mechanism of gene expression, cell-cell communication and cytopathology. However, as living single-cell analysis involves fL sample volumes with ultra-small amounts of analyte, comprehensive integration of entire chemical processing for single cells and proteins into spaces smaller than single cells (pL) would be indispensable to prevent dispersion-associated analyte loss. In this study, we proposed and developed a living single-cell protein analysis device based on micro/nanofluidics and demonstrated analysis of cytokines released from living single B cells by enzyme-linked immunosorbent assay. Based on our integration method and technologies including top-down nanofabrication, surface modifications and pressure-driven flow control, we designed and prepared the device where pL-microfluidic- and fL-nanofluidic channels are hierarchically allocated for cellular and molecular processing, respectively, and succeeded in micro/nanofluidic control for manipulating single cells and molecules. 13-unit operations for pL-cellular processing including single-cell trapping and stimulation and fL-molecular processing including fL-volumetry, antigen-antibody reactions and detection were entirely integrated into a microchip. The results suggest analytical performances for countable interleukin (IL)-6 molecules at the limit of detection of 5.27 molecules and that stimulated single B cells secrete 3.41 IL-6 molecules per min. The device is a novel tool for single-cell targeted proteomics, and the methodology of device integration is applicable to other single-cell analyses such as single-cell shotgun proteomics. This study thus provides a general approach and technical breakthroughs that will facilitate further advances in micro/nanofluidics, single-cell life science research, and other fields.

Nucleosome in patients with systemic sclerosis: possible association with immunological abnormalities via abnormal activation of T and B cells.

OBJECTIVE: To determine the serum levels of nucleosome in patients with systemic sclerosis (SSc) and relate the results to the clinical features of SSc. METHODS: Serum nucleosome levels in 91 patients with SSc were examined by ELISA. The expression of Toll-like receptor (TLR) 9 in T and B cells was quantified by flow cytometric intracellular protein analysis. The effects of nucleosomes on lymphocytes were also analysed. Moreover, we assessed the effects of nucleosomes on fibrosis, using wild type and CD19-deficient bleomycin-treated mice, an experimental model for human SSc. RESULTS: Serum nucleosome levels were elevated in SSc compared with healthy controls and correlated positively with the extent of skin and pulmonary fibrosis and immunological abnormalities. The retrospective longitudinal analysis showed the serum nucleosome levels to be attenuated during the follow-up period. TLR9, which can be stimulated by nucleosome expression was upregulated in the affected T and B cells of patients with SSc. Moreover, nucleosome stimulation strongly increased interleukin (IL)-4 and IL-17 expression of T cells, B-cell IgG production and proliferation of lymphocytes in SSc compared with those in healthy controls. In bleomycin-induced SSc model mice, serum nucleosome levels were elevated compared with control mice. Furthermore, nucleosomes increased IgG production and proliferation of mouse B cells. Although TLR9 expression was similar between wild type and CD19-deficient splenic B cells, CD19 deficiency reduced these nucleosome effects. CONCLUSION: These results suggest that nucleosomes and its signalling in B and T cells contribute to disease development in SSc via TLR9.