Role of inter-alpha-trypsin inhibitor heavy chain 4 and its citrullinated form in experimental arthritis murine models.

Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) is a major protein in serum and reported to be upregulated at the onset of rheumatoid arthritis (RA). Its citrullinated form, cit-ITIH4, is specifically found in the serum and synovial fluid of patients with RA. However, the detailed function of ITIH4 in arthritis remains unknown. The aim of this study was to clarify the role of ITIH4 and cit-ITIH4 using experimental arthritis models. ITIH4 and cit-ITIH4 expression was examined in steady-state mice and two different arthritis models, and their pathological effects were examined in Itih4-deficient mice. In naïve C57BL/6 (WT) mice, ITIH4 was expressed as mRNA in the liver and the lung and was expressed as protein in serum and hepatocytes. In K/BxN serum transferred arthritis (K/BxN-STA) and collagen-induced arthritis (CIA), ITIH4 and cit-ITIH4 in sera were increased before the onset of arthritis, and cit-ITIH4 was further increased at the peak of arthritis. In Itih4-deficient mice, citrullinated proteins in serum and joints, especially 120 kDa protein, were clearly diminished; however, there was no significant difference in arthritis severity between WT and itih-/- mice either in the K/BxN-STA or CIA model. CIA mice also exhibited pulmonary lesions and itih4-/- mice tended to show enhanced inflammatory cell aggregation compared to WT mice. Neutrophils in the lungs of itih4-/- mice were significantly increased compared to WT mice. In summary, ITIH4 itself did not alter the severity of arthritis but may inhibit autoimmune inflammation via suppression of neutrophil recruitment.

New-onset Polymyalgia Rheumatica Following the Administration of the Pfizer-BioNTech COVID-19 Vaccine.

We herein report the case of an 80-year-old Japanese woman who presented to our hospital with bilateral pain in the shoulders and hips lasting for a month since 2 days after the second dose of the BNT162b2 COVID-19 vaccine. Her physical findings, laboratory data, and ultrasonographic findings of bilateral biceps tenosynovitis and lateral subacromial bursitis were consistent with a diagnosis of polymyalgia rheumatica (PMR). She was successfully treated with oral prednisolone 15 mg/day. Although a causal relationship could not be definitively confirmed, PMR should be considered as a differential diagnosis in cases of persistent myalgia after administration of the BNT162b2 vaccine.

Predictive Factors for Pneumomediastinum During Management of Connective Tissue Disease-related Interstitial Lung Disease: A Retrospective Study.

Objective To identify factors associated with pneumomediastinum during management of connective tissue disease (CTD)-related interstitial lung disease (ILD). Methods Patients diagnosed with pneumomediastinum after the initiation of corticosteroid therapy for their CTD-ILD were enrolled. The baseline characteristics of patients who developed pneumomediastinum after the initiation of corticosteroid therapy (n=13, all occurring within 120 days) were compared to those of patients who did not develop pneumomediastinum (n=49). A multivariate logistic regression analysis was performed to identify factors associated with pneumomediastinum. A receiver operating characteristic (ROC) curve analysis was also performed to assess the predictive performance. Results The body mass index (BMI) [odds ratio (OR) (95% confidence interval (CI)) 0.482 (0.272-0.853)] and serum lactate dehydrogenase (LDH) [OR (95% CI) 1.013 (1-1.025)] levels at baseline were identified as independent factors associated with pneumomediastinum after corticosteroid initiation. The optimal cut-off points of the BMI and LDH levels for predicting pneumomediastinum development, as estimated by the Youden index, were 20.2 kg/m2 and 378 U/L, respectively. LDH showed a sensitivity of 61.5% and the highest specificity of 87.8%. Importantly, combining these markers resulted in the highest sensitivity of 100% and a specificity of 71.4%. Conclusion A low BMI and high serum LDH levels at baseline are useful predictive factors for pneumomediastinum development in CTD-ILD patients.

Specific Increase in Joint Neutrophil Extracellular Traps and Its Relation to Interleukin 6 in Autoimmune Arthritis.

Neutrophils and their extracellular traps have been shown to play an important role in the pathogenesis of rheumatoid arthritis (RA), but the detailed mechanisms in joints are still unclear, and their regulation remains to be solved. Here, we explored neutrophil extracellular trap (NET)osis in experimental models of arthritis and further investigated the effects of interleukin-6 (IL-6) inhibition in neutrophils and NETosis. In skins of peptide GPI-induced arthritis (pGIA), citrullinated protein was detected as well as citrullinated histone expression in immunized skin but this was not specific to pGIA. Citrullinated histone expression in pGIA joints was specific to pGIA and was merged with neutrophil elastase, suggesting NETosis. Neutrophils in joints tend to upregulate IL-6 receptors when compared with bone marrow neutrophils. Administration of mouse anti-IL-6 receptor antibodies in pGIA suppressed arthritis in association with a decrease in neutrophil infiltration and NETosis in joints. In the plasma of RA patients, citrullinated protein was significantly reduced after tocilizumab treatment. Our results suggest that IL-6 enhances neutrophil chemotaxis and NETosis in inflammatory joints and could be the source of citrullinated proteins.

Impaired function of PD-1+ follicular regulatory T cells in systemic lupus erythematosus.

Aberrant autoantibody production is characteristic of systemic lupus erythematosus (SLE), but follicular regulatory T (TFR) cells can potentially suppress this abnormality. We investigate functional changes in TFR cells from SLE patients. Circulating TFR cells were collected from 19 SLE patients and 14 healthy controls (HC) to compare molecular expression and in-vitro suppressive capacity of follicular helper T (TFH) cell proliferation. To reveal the stability of forkhead box protein 3 (FoxP3) in TFR, pyrosequencing of conserved non-coding sequence (CNS) 2 at the FoxP3 gene locus was performed. We then tested interleukin (IL)-2 in SLE-TFR cells to check restoration of suppressor function. Programmed cell death 1 (PD-1) expression in SLE-TFR cells was positively correlated with anti-DNA antibody levels and disease activity. These cells had impaired suppressive function for TFH cells with decreased expression of suppression mediators FoxP3, cytotoxic T lymphocyte antigen 4 (CTLA-4) and IL-2 receptor alpha (IL-2Rα). Pyrosequencing identified hyper-methylation in CNS2 region of SLE-TFR cells comparing to HC. With in-vitro IL-2 stimulation, PD-1 expression of TFR cells significantly decreased, together with increased expression of FoxP3 and CTLA-4, especially at a low dose. Thus, SLE-TFR cells have functionally defective to TFH suppression, but low-dose IL-2 therapy might be useful to restore this ability.

QTc interval prolongation in patients with systemic lupus erythematosus treated with hydroxychloroquine.

OBJECTIVES: The primary objective is to reveal the effect of hydroxychloroquine (HCQ) treatment on corrected QT (QTc) interval in patients with systemic lupus erythematosus (SLE). The secondary objective is to investigate factors that affect QTc prolongation. METHODS: SLE patients who had electrocardiograms between 2015 and 2020 were recruited and assigned to two groups based on whether they were treated with HCQ (HCQ group) or not (control group). Change of QTc before and after HCQ administration in the HCQ group was measured and compared with the control group. Patients treated with HCQ were further divided into two groups based on presence or absence of QTc prolongation and the characteristics were compared. RESULTS: In total, 126 patients were recruited, of whom 42 were treated with HCQ. In the HCQ group, the mean QTc significantly increased (p < .001), while there was no significant difference of mean QTc in the control group. Moreover, those in the HCQ group with QTc prolongation showed a significantly higher proportion of hypertension and longer SLE duration compared to those without QTc prolongation. However, the multiple logistic regression analysis showed that there were no significant differences among them. CONCLUSION: HCQ could induce QTc prolongation in SLE patients. It might be better that the possibility of QTc prolongation was taken into consideration when HCQ was administered in the patients with longer disease duration of SLE and coincidence of hypertension.

Clinical features and prognosis of nocardiosis in patients with connective tissue diseases.

OBJECTIVES: To investigate the clinical features and prognosis of nocardiosis complicated by connective tissue diseases (CTDs). METHODS: We examined patients with CTDs who were diagnosed with nocardiosis from October 2004 to 2019. We retrospectively investigated patient characteristics and therapeutic outcomes. We then performed a comparison between survivors and non-survivors. RESULTS: Fourteen patients were examined. Underlying CTDs were systemic lupus erythematosus (28.6%), vasculitis syndrome (28.6%), rheumatoid arthritis (21.4%), adult Still disease (14.3%) and dermatomyositis (7.1%). Infected organs were lung (85.7%), brain (42.9%), skin/cutaneous lesions (28.6%) and muscle (7.1%). Disseminated infections were seen in nine patients (64.3%). At the onset of nocardiosis, all patients were given prednisolone (23.2 ± 11.9 mg/day). Only two patients (14.3%) were given TMP-SMX for prophylaxis of pneumocystis pneumonia. Relapse occurred in one patient (7.1%) and four patients (28.6%) died from nocardiosis for a cumulative survival rate at 52 weeks of 76.9%. In a comparison of survivors (71.4%) and non-survivors (28.6%), cutaneous lesions were significantly more frequent in the latter (10 vs 75%, p = .04) with an odds ratio of 27.0 (95% CI: 1.7-453.4). CONCLUSION: Cutaneous lesions as a result of dissemination might be a risk factor for nocardiosis mortality in patients with CTDs.

IgG4-related pleural disease with aortitis and submandibular glands involvement successfully treated with corticosteroid: case-based review.

IgG4-related disease (IgG4-RD) is a fibro-inflammatory condition characterized by increased serum IgG4 level, infiltration of lymphocytes and IgG4-positive (IgG4+) plasma cells and fibrosis. It can occur in almost all organs, commonly affecting the pancreas, biliary tract, salivary and lacrimal glands and kidneys. However, reports of IgG4-RD accompanied by pathologically confirmed, IgG4-related pleural disease are scarce. Here, we present a case of a 64-year-old man with suspected malignant pleural mesothelioma based on imaging findings but finally diagnosed with IgG4-RD (including pleuritis, periaortitis and bilateral submandibular gland enlargement) based on a high serum IgG4 level and pleural histopathological findings such as lymphoplasmacytic infiltration including IgG4+ plasma cells and fibrosis. Systemic corticosteroid therapy was effective at reducing serum IgG4, improving bilateral submandibular gland enlargement, and regressing pleural thickening and periaortic soft tissue. We also discuss clinical characteristics and pleural pathological features of previously reported cases with IgG4-related pleural disease based on a comprehensive literature review. Our case of IgG4-RD with pleura, aorta and submandibular gland involvement, pathologically confirmed by pleural specimen might be unique and very rare.

Human leukocyte antigen in Japanese patients with idiopathic inflammatory myopathy.

Objective: The human leukocyte antigen (HLA) is the strongest genetic risk factor for idiopathic inflammatory myopathy (IIM), and different HLA alleles have been reported to be associated with IIM susceptibility among different ethnic groups. In this study, we have investigated HLA alleles associated with IIM in Japanese patients.Methods: Genotyping of HLA-DRB1 and DPB1 were performed in 252 Japanese IIM patients (166 dermatomyositis [DM] and 86 polymyositis [PM] patients) and the association was analyzed with comparison to controls (n = 1026 for DRB1 and n = 413 for DPB1).Results: DRB1*08:03 was associated with IIM (p = 1.60 × 10-5, pc = .0005, odds ratio [OR] 2.11, 95% confidence interval [CI] 1.52-2.92) and DM (p = .0004, pc = .0128, OR 2.06, 95%CI 1.40-3.02). DPB1*05:01 was also associated with IIM (p = .0001, pc = .0021, OR 1.96, 95%CI 1.38-2.77) and DM (p = .0005, pc = .0075, OR 2.05, 95%CI 1.37-3.08). DRB1*09:01 (p = .0012, pc = .0368, OR 0.35, 95% CI 0.18-0.69) and DPB1*04:01(p = .0004, pc = .0057, OR 0.05, 95% CI 0.00-0.85) were protectively associated with PM. Two locus analyses suggested that DRB1*09:01 and DPB1*04:01 were independently associated with PM.Conclusion: Protective associations of HLA were detected in Japanese PM patients.

Revisit of autoimmunity to glucose-6-phosphate isomerase in experimental and rheumatoid arthritis.

Rheumatoid arthritis (RA) is an inflammatory disorder characterized by synovial inflammation in multiple joints. Autoantibodies (Abs) are the hallmark of RA, and as disease-specific and diagnostic markers, rheumatoid factor and anti-citrullinated protein antibody (ACPA) are produced pre-clinically, but their pathogenic roles in RA remain elusive. In this review, we focus on one of the candidate autoantigens in RA; glucose-6-phosphate isomerase (GPI). The arthritogenic role of GPI has been confirmed in two different mouse models: the K/BxN- and GPI-induced arthritis models. Both anti-GPI Abs and citrullinated-GPI peptide Abs have been detected in human RA. Studies conducted in these rodent models have confirmed that the pathogenesis of arthritis involves the localization of autoantigens not only in the joints but also in the circulation. In this review, we revisit and summarize the arthritogenic relevance of GPI in animal RA models and in human RA, and extend the discussion to joint-specific inflammation.

Potential involvement of OX40 in the regulation of autoantibody sialylation in arthritis.

OBJECTIVE: An increased proportion of circulating follicular helper T (Tfh) cells was reported in rheumatoid arthritis (RA), but it remains uncertain how Tfh cells affect antibody hyposialylation. We investigated the regulation of autoantibody hyposialylation by Tfh cells in RA using murine model. METHODS: Behaviours of Tfh cells and their function on B cell promotion were analysed. Change of arthritogenicity and sialylation of autoantibodies during the course of arthritis was examined by mass spectrometry. Tfh-mediated regulation of hyposialylation was investigated, and the responsible cell surface molecule was specified both in vitro and in vivo. The relation between circulating Tfh cells and hyposialylation was analysed in patients with RA. RESULTS: An increase in Tfh, particularly interleukin-17 producing Tfh (Tfh17) cells, at the onset of arthritis and their enhancement of autoantibody production were found. Autoantibodies at the onset phase demonstrated stronger inflammatory properties than those at the resolution phase, and mass spectrometric analysis revealed their difference in sialylation. In vitro coculture showed enhanced hyposialylation by the Tfh cells via OX40, which was highly expressed in the Tfh and Tfh17 cells. Blockade of OX40 prevented the development of arthritis with reduction in Tfh17 cells and recovery of autoantibody sialylation. Analysis of patients with RA showed abundance of OX40-overexpressing Tfh17 cells, and their proportion correlated negatively with the expression of α2,6-sialyltransferase 1, an enzyme responsible for sialylation. CONCLUSIONS: OX40 expressed on Tfh cells can regulate autoantibody sialylation and play a crucial role in the development of autoimmune arthritis.

Effect of Biological Disease-modifying Anti-rheumatic Drugs on Airway and Interstitial Lung Disease in Patients with Rheumatoid Arthritis.

Objective Biological disease-modifying anti-rheumatic drugs (bDMARDs) represent an important advance in alleviating rheumatoid arthritis (RA), but their effect on rheumatic airway disease (AD) and interstitial lung disease (ILD) is still unclear. This study was performed to evaluate the association of the use of different bDMARDs with new-onset or worsening of RA-AD/ILD. Methods We performed a retrospective cohort study of RA patients who received bDMARDs and assessed their AD/ILD before and after drug initiation in our hospital over the past 10 years. We evaluated the serial changes in computed tomography (CT), classified patients according to AD/ILD progression, and analyzed associations between clinical characteristics and outcomes. Results We enrolled 49 patients. Thirty patients received tumor necrosis factor inhibitors (TNFis), 12 received abatacept (ABT), and the remaining 7 received tocilizumab (TCZ). Seventeen patients had ILD, 10 had AD, and 6 had both AD and ILD before the initiation of bDMARDs. New emergence or exacerbation of AD/ILD was observed in 18 patients after drug initiation, while the remaining 31 remained stable or improved. Multiple logistic regression analyses revealed that pre-existing AD was an independent risk factor against the emergence or exacerbation of RA-AD/ILD, and ABT use was a protective factor against it. Conclusion Our study showed that pre-existing RA-AD is associated with future worsening of RA-AD/ILD, and ABT over other bDMARDs was associated with a better prognosis. Future studies to confirm our results are needed.

Peptidyl arginine deiminase inhibition suppresses arthritis via decreased protein citrullination in joints and serum with the downregulation of interleukin-6.

Objective: To explore the relevance of citrullinated proteins and anti-citrullinated protein antibodies (ACPA) via protein arginine deiminase (PAD) inhibition in peptide glucose-6-phosphate isomerase-induced arthritis (pGIA).Methods: Cl-amidine, a PAD inhibitor, was injected into pGIA. Clinical scores and histopathological findings of ankle joints were assessed. Serum ACPA titers were analyzed using ELISA. Citrullinated protein expression in joints and sera were examined with immunohistochemistry and Western blot analysis, respectively. Serum levels of IL-6, TNFα, and IL-1ß were measured with cytometric bead array (CBA). Gene expression levels of IL-6 and TNFα in joints, lymph nodes, and spleens were analyzed with quantitative PCR. GPI-specific productions of IFNγ and IL-17 from T cells in lymph nodes were evaluated.Results: Cl-amidine treatment significantly reduced arthritis severity while ACPA titers tended to be lower, but not significantly different compared to the control. Citrullinated proteins in joints and sera from treated mice were clearly decreased. With Cl-amidine treatment, serum IL-6 levels were significantly decreased, and IL-6 and TNFα gene expression were significantly reduced in joints. IL-17 production from GPI-specific T cells tended to be lower in Cl-amidine-treated mice, but not significantly different.Conclusion: Our results suggested that PAD-mediated citrullinated protein was involved in the pathogenesis of arthritis via IL-6.

Identification of novel biomarker as citrullinated inter-alpha-trypsin inhibitor heavy chain 4, specifically increased in sera with experimental and rheumatoid arthritis.

BACKGROUND: Anticitrullinated protein antibodies (ACPA) and citrullinated proteins play key roles in the pathogenesis of rheumatoid arthritis (RA). Many candidate citrullinated antigens have been identified in joints, but citrullinated proteins in sera are mostly uncertain in patients with RA. We explored the expression of citrullinated proteins in joints and sera of experimental arthritis, and we further investigated their specific expression correlated with the disease activity in patients with RA. METHODS: Citrullinated protein expression in tissues was examined by IHC in peptide glucose-6-phosphate isomerase-induced arthritis (pGIA). Serum citrullinated proteins from pGIA were examined by Western blotting, and the sequence was identified by MS. With the same methods, serum citrullinated proteins were analyzed in patients with RA, primary Sjögren's syndrome, systemic lupus erythematosus, and osteoarthritis as well as in healthy subjects, by Western blotting and MS. In patients with RA, the relationship between the expression of the identified protein (inter-alpha-trypsin inhibitor heavy chain 4 [ITIH4]) and clinical features was evaluated, and the levels of citrullinated ITIH4 were compared before and after biological treatment. The antibody response against citrullinated ITIH4 peptide was measured by enzyme-linked immunosorbent assay. RESULTS: Citrullinated proteins were detected specifically in arthritic joints and sera from pGIA relative to controls. In sera, a common band of citrullinated protein at 120 kDa was revealed, and it fluctuated in parallel with arthritis score of pGIA by Western blotting. Interestingly, in 82% of RA patient sera, similar bands of citrullinated protein were specifically detected. These proteins were identified as citrullinated ITIH4, and especially the R438 site was commonly citrullinated between mice and humans. Citrullinated ITIH4 levels were associated with clinical parameters such as C-reactive protein (CRP), rheumatoid factor, and Disease Activity Score in 28 joints as measured by CRP in patients with RA. Its levels were decreased in correlation with the reduction of disease activity score after effective treatment in patients with RA. Moreover, antibody response to citrullinated epitope in ITIH4 was specifically observed in patients with RA. CONCLUSIONS: Our results suggest that serum citrullinated ITIH4 was specifically increased in patients with RA and could be a novel biomarker for assessing disease activity in patients with RA.

Gastrocnemius Myalgia as a Rare Initial Manifestation of Crohn's Disease.

The initial symptoms of Crohn's disease (CD) sometimes present as extraintestinal lesions, which can be a diagnostic challenge for physicians. Painful legs, known as "gastrocnemius myalgia syndrome", are rare complications that often precede abdominal manifestations. We herein report the case of a 38-year-old man who presented with bilateral leg myalgia lasting for 4 months. Magnetic resonance imaging showed abnormal intensity, and a muscle biopsy revealed inflammatory cell infiltration. Abdominal symptoms appeared three months after the myalgia onset, and the diagnosis of CD was confirmed later by endoscopic and radiological findings. To our knowledge, this is the first description of gastrocnemius myalgia syndrome in Japan.

Human Leukocyte Antigen and Systemic Sclerosis in Japanese: The Sign of the Four Independent Protective Alleles, DRB1*13:02, DRB1*14:06, DQB1*03:01, and DPB1*02:01.

OBJECTIVE: Several studies on associations between human leukocyte antigen (HLA) allele frequencies and susceptibility to systemic sclerosis (SSc) have been reported. Anti-centromere antibodies (ACA) and anti-topoisomerase I antibodies (ATA) are found in SSc patients. Here, we sought to identify HLA alleles associated with SSc in Japanese, and explored their associations with SSc phenotypes including the presence of autoantibodies. METHODS: Associations of HLA-DRB1, DQB1, and DPB1 were analyzed in 463 Japanese SSc patients and 413 controls. RESULTS: We found that DRB1*13:02 (P = 0.0011, Pc = 0.0319, odds ratio [OR] 0.46, 95% confidence interval [CI] 0.29-0.73), DRB1*14:06 (P = 6.60X10-5, Pc = 0.0020, OR 0.05, 95%CI 0.01-0.41), DQB1*03:01 (P = 0.0009, Pc = 0.0150, OR 0.56, 95%CI 0.40-0.79), and DPB1*02:01 (P = 5.16X10-6, Pc = 8.77X10-5, OR 0.52, 95%CI 0.39-0.69) were protectively associated with SSc. In addition, these four alleles seemed to be independently associated with the protection against the susceptibility of SSc. On the other hand, we could not find predisposing alleles for overall SSc. With respect to SSc subsets, a tendency for these four alleles to be protectively associated was observed. However, there was a significant association between DRB1*01:01, DRB1*10:01, DQB1*05:01, and DPB1*04:02 and the susceptibility to SSc with ACA. On the other hand, the presence of DRB1*15:02, DQB1*06:01, DPB1*03:01, and DPB1*09:01 was associated with SSc with ATA. CONCLUSION: Thus, the present study has identified protective associations of the four HLA class II alleles with overall Japanese SSc and predisposing associations of HLA class II alleles with Japanese SSc subsets.