Autoimmunity against melanoma differentiation-associated gene 5 induces interstitial lung disease mimicking dermatomyositis in mice.

Anti-melanoma differentiation-associated gene 5 (MDA5) antibody-positive dermatomyositis (DM) is characterized by amyopathic DM with interstitial lung disease (ILD). Patients with anti-MDA5 antibody-associated ILD frequently develop rapidly progression and present high mortality rate in the acute phase. Here, we established a murine model of ILD mediated by autoimmunity against MDA5. Mice immunized with recombinant murine MDA5 whole protein, accompanied with complete Freund's adjuvant once a week for four times, developed MDA5-reactive T cells and anti-MDA5 antibodies. After acute lung injury induced by intranasal administration of polyinosinic-polycytidylic acid [poly (I:C)] mimicking viral infection, the MDA5-immunized mice developed fibrotic ILD representing prolonged respiratory inflammation accompanied by fibrotic changes 2 wk after poly (I:C)-administration, while the control mice had quickly and completely recovered from the respiratory inflammation. Treatment with anti-CD4 depleting antibody, but not anti-CD8 depleting antibody, suppressed the severity of MDA5-induced fibrotic ILD. Upregulation of interleukin (IL)-6 mRNA, which was temporarily observed in poly (I:C)-treated mice, was prolonged in MDA5-immunized mice. Treatment with anti-IL-6 receptor antibody ameliorated the MDA5-induced fibrotic ILD. These results suggested that autoimmunity against MDA5 exacerbates toll-like receptor 3-mediated acute lung injury, and prolongs inflammation resulting in the development of fibrotic ILD. IL-6 may play a key role initiating ILD in this model.

Murine models of idiopathic inflammatory myopathies.

Idiopathic inflammatory myopathies (IIMs) are divided into polymyositis and dermatomyositis (DM) with specific cutaneous manifestation. Several myositis-specific autoantibodies (MSAs) have been identified in IIMs and were found to be associated with distinct clinical features, including anti-synthetase syndrome (ASyS) and immune-mediated necrotizing myopathy (IMNM). Moreover, MSA-related clinical features have been identified even within DM. Although MSAs are valuable for the diagnosis of IIMs, the pathogenic roles of these antibodies remain unknown. To investigate the pathogenesis of IIMs, classical murine models of autoimmune myositis, experimental autoimmune myositis, and C protein-induced myositis have been established by immunization with muscle-specific antigens, myosin, and myosin-binding skeletal C protein, respectively. To according to MSA-related autoimmunity, a murine model of ASyS was generated by immunization with a murine recombinant histidyl-transfer RNA (tRNA) synthetase, Jo-1, in which muscle and lung inflammation are induced depending on acquired immunity. Furthermore, it was found that the transfer of human Immunoglobulin G (IgGs) from patients with IMNM, comprising anti-signal recognition particles and anti-3-hydroxy-3-methylglutaryl coenzyme A reductase antibodies, induced complement-mediated myositis in recipient mice. We found that CD8+ T cell-mediated myositis can be established depending on autoimmunity against transcriptional intermediary factor 1γ (TIF1γ), an autoantigen for MSAs induced by recombinant human TIF1γ immunization. These new murine models reflecting MSA-associated IIMs will reveal the immunological mechanisms underlying IIMs.

Possible correlation between serum interleukin-8 levels and the activity of myositis in anti-NXP2 antibody-positive dermatomyositis.

Anti-nuclear matrix protein 2 (NXP2) antibody-positive dermatomyositis (DM) is characterized by extensive and severe myositis. In this study, we evaluated which cytokines/chemokines involved with the activity of the myositis. We performed quantitative immunoassays using the MILLIPLEX® Multiplex Assays Using Luminex to evaluate serum levels of interferon-γ, interleukin (IL)-1ß, IL-6, IL-8, IL-12p40, and tumor necrosis factor-α in samples collected over time from a 9-year-old female with anti-NXP2 antibody-positive DM. In our case, the serum level of IL-8 was elevated when the myositis worsened, and decreased in accordance with the improvement of myositis, suggesting that the serum IL-8 levels were correlated with the myositis activity. Serum levels of IL-8 in samples from five patients with anti-NXP2 antibody-positive DM and five patients with anti-transcriptional intermediary factor 1γ (TIF1γ) antibody-positive DM without both interstitial lung disease (ILD) and malignancy before starting treatments, along with five healthy controls, were also evaluate by an enzyme-linked immunosorbent assay. Serum IL-8 levels were significantly elevated in anti-NXP2 or anti-TIF1γ antibody-positive DM patients with myositis but not ILD, than healthy controls. It was suggested that serum levels of IL-8 correlate with the activity of myositis in DM including anti-NXP2 antibody-positive DM.

A case of anti-SAE1/2 antibody-positive dermatomyositis with extensive panniculitis: A possible cutaneous manifestation of treatment resistance.

Dermatomyositis constitutes a heterogeneous group of autoimmune inflammatory conditions with a wide variety of clinical outcomes. The symptomatic heterogeneity carries skin, muscle, and joint manifestations; pulmonary and cardiac involvements; and concomitant malignancy. Any of these symptoms often appear at different combinations and time courses, thus posing difficulty in early diagnosis and appropriate treatment choice. Recent progress in laboratory investigations explored the identification of several myositis-specific autoantibodies (MSAs) and myositis-associated autoantibodies, allowing precise characterization for a clinical perspective of the disease. MSAs can be detectable in approximately 80% of patients with whole dermatomyositis, some of which closely reflect unique clinical features in the particular disease subset(s), including the distribution and severity of organ involvement, treatment response, and prognosis. However, only limited evidence has been available in dermatomyositis-associated panniculitis, mostly that in anti- melanoma differentiation-associated protein 5 antibody-positive disease. We present a rare case of a patients with dermatomyositis with extensive panniculitis on the trunk whose serum IgG autoantibodies reacted with both subunits of small ubiquitin-like modifier activating enzymes (SAEs), SAE1 and SAE2. The onset of panniculitis coincided with increased disease activity, including disease-related skin manifestations, fever, dysphagia, and muscle weakness in the extremities. These symptoms responded well to a high dose of systemic steroid, but even upon receiving a high-dose intravenous immunoglobulin, the panniculitic lesions and pruritic erythema flared with tapering of steroid dose, further requiring tacrolimus and mycophenolate mofetil to achieve disease remission. To our knowledge, this is the third reported case of anti-SAE autoantibody-positive dermatomyositis with panniculitis. We aim to extend the understanding of the current limitation and further perspective in the clinical management of the extremely rare skin manifestation associated with dermatomyositis.

Blockade of CD122 on memory T cells in the skin suppresses sclerodermatous graft-versus-host disease.

BACKGROUND: Antigen-stimulated naïve T cells differentiate into effector and memory T cells, of which resident memory T (TRM) cells reside permanently in organ tissues. Involvement of TRM cells has been indicated in pathological conditions of various skin diseases. CD122, which is the ß chain subunit of interleukin (IL)- 2 and IL-15 receptors, is expressed on immune cells including TRM cells. OBJECTIVE: To investigate whether CD122 signaling in skin CD8+ TRM cells mediates the development of mucocutaneous graft-versus-host disease (GVHD). METHODS: We used a genetically modified mouse expressing a membrane-bound form of chicken ovalbumin (OVA) under the control of the keratin 14 promoter, which develops GVHD-like erosive mucocutaneous disease resulting in sclerodermatous disease after transfer of OVA-specific T cell-receptor-transgenic CD8+ OT-I cells. Mice with mucocutaneous GVHD were treated with an anti-CD122 blocking antibody. RESULTS: Administration of an anti-CD122 blocking antibody suppresses the development of acute/chronic GVHD-like mucocutaneous disease in our murine model via the reduction of CD122-expressing memory CD8+ T cells, including skin, memory autoaggressive CD8+ T cells. Moreover, blockade of CD122, even after the establishment of acute GVHD, inhibited the development of chronic GVHD-like sclerodermatous disease via the reduction of epidermal and dermal TRM autoaggressive CD8+ T cells. CONCLUSION: Skin memory CD8+ T cells in particular mediate the development of mucocutaneous GVHD, and blockade of CD122 may be an effective treatment strategy, especially for sclerodermatous GVHD.

Murine models of idiopathic inflammatory myopathy.

Idiopathic inflammatory myopathies (IIMs) are characterized by inflammation of muscles and other organs. Several myositis-specific autoantibodies (MSAs) have been identified in IIMs and were found to be associated with distinct clinical features. Although MSAs are valuable for the diagnosis of IIMs, the pathogenic roles of these antibodies remain unknown. To investigate the pathogenesis of IIMs, several animal models of experimental myositis have been established. Classical murine models of autoimmune myositis, experimental autoimmune myositis, and C protein-induced myositis are established by immunization with muscle-specific antigens, myosin, and skeletal C protein, respectively. Furthermore, a murine model of experimental myositis was generated by immunization with a murine recombinant histidyl-tRNA synthetase, Jo-1, in which muscle and lung inflammation reflecting anti-synthetase syndrome are induced depending on acquired immunity. Recently, the transfer of human IgGs from patients with immune-mediated necrotizing myopathy, comprising anti-signal recognition particles and anti-3-hydroxy-3-methylglutaryl coenzyme A reductase antibodies, was found to induce complement-mediated myositis in recipient mice. CD8+ T cell-mediated myositis can be established depending on autoimmunity against transcriptional intermediary factor 1γ (TIF1γ), an autoantigen for MSAs induced by recombinant human TIF1γ immunization. These new murine models reflecting MSA-related IIMs are useful tools for accurately understanding the pathological mechanisms underlying IIMs.

Relevance of leukaemia inhibitory factor to anti-melanoma differentiation-associated gene 5 antibody-positive interstitial lung disease.

OBJECTIVES: Anti-melanoma differentiation-associated gene 5 (MDA5) antibody-positive rapidly progressive interstitial lung disease (RP-ILD) is a life-threatening disease, the aetiology of which remains unclear. To detect potential diagnostic markers, a transcriptome analysis of the lung sample from a patient with anti-MDA5 antibody-positive RP-ILD was performed. METHODS: RNA sequencing analyses of an autopsy lung sample from a 74-year-old woman with anti-MDA5 antibody-positive RP-ILD was performed and compared with an age- and sex-matched normal lung sample. Genes with changes of gene expression ≥5-fold were considered differentially expressed genes and analysed by Metascape. The levels of leukaemia inhibitory factor (LIF) were measured in the serum samples from 12 cases of anti-MDA5 antibody-positive ILD, 12 cases of anti-aminoacyl tRNA synthetase (ARS) antibody-positive ILD, 10 cases of anti-transcription intermediary factor 1γ/anti-Mi-2 antibody DM and 12 healthy volunteers. RESULTS: Gene ontology enrichment analysis on the RNA sequencing data showed a strong association with antigen binding. Upregulated expressions of IL-1ß, IL-6 and LIF were also detected. Serum LIF levels were significantly elevated in anti-MDA5 antibody-positive ILD patients {median 32.4 pg/ml [interquartile range (IQR) 13.2-125.7]} when compared with anti-ARS antibody-positive ILD patients [4.9 pg/ml (IQR 3.1-19.7), P < 0.05] and DM patients [5.3 pg/ml (IQR 3.9-9.7), P < 0.05]. CONCLUSION: Our present study suggested that upregulation of LIF might be a new potential disease marker specific for anti-MDA5 antibody-positive ILD.

The Role of PD-L1 on Langerhans Cells in the Regulation of Psoriasis.

Langerhans cells (LCs) are skin-resident cells with potent antigen-presenting cell capabilities, which reportedly play some roles in the development of psoriasis, an inflammatory skin disease mediated by IL-17A‒producing cells, T helper 17 cells, and TCR-γδlow T cells. LCs in psoriatic skin lesions but not in normal skin express PD-L1, which binds to PD-1, an immune checkpoint molecule, to negatively regulate immune reactions. The aim of this study is to elucidate the regulatory role of LCs through the PD-1/PD-L1 axis in a murine model of imiquimod-induced psoriasis-like dermatitis. Imiquimod application on wild-type C57BL/6J mice induced PD-L1 expression on LCs both in the ear skin and skin-draining lymph nodes. To further identify the functional role of PD-L1 expressed on LCs, we generated conditional knockout mice lacking PD-L1 expression on LCs (Pd-l1-cKO mice). Pd-l1-cKO mice presented significantly more severe imiquimod-induced psoriasis-like dermatitis than their control littermates. Flow cytometric analysis showed that the frequency of activated IL-17A‒producing γδlow T cells was increased in the ear skin samples, and IL-17A production by CCR6+ migrating γδlow T cells increased in the skin-draining lymph nodes in imiquimod-applied Pd-l1-cKO mice than in control littermates. Collectively, LCs disrupt the exacerbation of psoriasis through PD-L1.

Reliability of antinuclear matrix protein 2 antibody assays in idiopathic inflammatory myopathies is dependent on target protein properties.

A line blotting assay (LB) is currently used to detect myositis-specific autoantibodies (MSAs) in patients with idiopathic inflammatory myopathies (IIMs), because of its simplicity; however, the sensitivity and specificity of this assay is low. The aim of this study is to evaluate the accuracy of the commercial LB in detection of antinuclear matrix protein 2 (NXP2) antibody. Seventy-seven serum samples from patients with IIMs, in which anti-NXP2 antibodies were detected through immunoprecipitation and western blotting (IP-WB) using K562 cell lysate, were enrolled. All samples were assessed by LB and IP-WB using recombinant human NXP2 whole protein (rNXP2) produced by insect cells, and the positive rates of each assay were compared. Thirty-two samples (41.6%) showed false-negativity by LB, which includes 11 samples with negative results by IP-WB using rNXP2. Relative intensities of IP-WB using cell lysate were significantly higher in the samples with positive results by both LB and IP-WB using rNXP2, compared to samples with positive by IP-WB using rNXP2 but negative by LB. Three of 11 samples with negative results by both LB and IP-WB using rNXP2 revealed high antibody titers. Further, differences in post-transcriptional SUMOylation were observed between recombinant and natural NXP2 proteins. In conclusion, the LB showed low sensitivity for detection of anti-NXP2 antibody, an effect exacerbated at low titers of anti-NXP2 antibodies. Moreover, there appears to be differences in the reactivities of antibodies to recombinant and natural NXP2 proteins with different post-transcriptional modifications.

Anti-nuclear matrix protein 2 antibody-positive inflammatory myopathies represent extensive myositis without dermatomyositis-specific rash.

OBJECTIVES: Myositis-specific autoantibodies (MSAs) define distinct clinical subsets of idiopathic inflammatory myopathies (IIMs). The anti-nuclear matrix protein 2 (NXP2) antibody, a MSA detected in juvenile/adult IIMs, has been reported to be associated with a high risk of subcutaneous calcinosis, subcutaneous oedema and internal malignancies. The study aimed to clarify the clinical features of anti-NXP2 antibody-positive IIMs in detail. METHODS: This was a multicentre retrospective observational study on 76 anti-NXP2 antibody-positive patients. The antibody was detected via a serological assay using immunoprecipitation and western blotting. The patients were selected from 162 consecutive Japanese patients with IIMs. RESULTS: The cohort of anti-NXP2 antibody-positive IIMs included 29 juvenile patients and 47 adult patients. Twenty-seven (35.5%) patients presented with polymyositis phenotype without dermatomyositis-specific skin manifestations (heliotrope rash or Gottron sign/papules); this was more common in the adults than children (48.9% vs 15.8%, P < 0.01). Nine (11.8%) patients had subcutaneous calcinosis, and 20 (26.3%) patients had subcutaneous oedema. In addition, the proportion of patients with muscle weakness extending to the distal limbs was high (36 patients [47.4%]) in this cohort. Adult patients had a higher prevalence of malignancy than the general population (age-standardized incidence ratio of malignancies: 22.4). CONCLUSION: Anti-NXP2 antibody-positive IIMs, which include dermatomyositis sine dermatitis, are characterized by atypical skin manifestations and extensive muscular involvement.

Evaluation of apremilast, an oral phosphodiesterase 4 inhibitor, for refractory cutaneous dermatomyositis: A phase 1b clinical trial.

Dermatomyositis, an idiopathic inflammatory myopathy, is characterized by cutaneous itchy manifestations, which are frequently refractory and recurrent even after intensive immunosuppressive treatments. To evaluate the effectiveness and safety of apremilast, an oral phosphodiesterase 4 inhibitor, in treating skin-dominant dermatomyositis in which myositis and interstitial lung disease are absent or in remission, we performed this prospective, single-arm, interventional study. A total of five Japanese patients (one male and four females, median [range] age, 64 [37-71] years) with refractory dermatomyositis-associated cutaneous manifestations were recruited and treated with a 12-week course of oral apremilast. Among five enrolled patients, three experienced diarrhea with full-dose apremilast (30 mg twice daily), two of whom withdrew from the study and recovered quickly afterwards. A total of three evaluable female patients (median [range] age, 65 [64-71] years) received apremilast treatment for 12 weeks. A 39.4% reduction from baseline Cutaneous Dermatomyositis Disease Area and Severity Index total activity score, but not the damage score, at week 12 was observed in all three patients. Visual analog scale of itching, and quality of life by Dermatology Life Quality Index were slightly improved in one and two apremilast-treated patients, respectively. As apremilast was effective, with expected and recoverable digestive adverse events (diarrhea), in patients with refractory and recurrent dermatomyositis-associated cutaneous manifestations in this first phase Ib study, it can be suggested as a possible treatment when aggressive immunosuppressive therapies with high-dose systemic corticosteroid and/or immunosuppressive agents for other manifestations, myositis, and interstitial lung disease, are not required.