Immunomics analysis of rheumatoid arthritis identified precursor dendritic cells as a key cell subset of treatment resistance.

OBJECTIVES: Little is known about the immunology underlying variable treatment response in rheumatoid arthritis (RA). We performed large-scale transcriptome analyses of peripheral blood immune cell subsets to identify immune cells that predict treatment resistance. METHODS: We isolated 18 peripheral blood immune cell subsets of 55 patients with RA requiring addition of new treatment and 39 healthy controls, and performed RNA sequencing. Transcriptome changes in RA and treatment effects were systematically characterised. Association between immune cell gene modules and treatment resistance was evaluated. We validated predictive value of identified parameters for treatment resistance using quantitative PCR (qPCR) and mass cytometric analysis cohorts. We also characterised the identified population by synovial single cell RNA-sequencing analysis. RESULTS: Immune cells of patients with RA were characterised by enhanced interferon and IL6-JAK-STAT3 signalling that demonstrate partial normalisation after treatment. A gene expression module of plasmacytoid dendritic cells (pDC) reflecting the expansion of dendritic cell precursors (pre-DC) exhibited strongest association with treatment resistance. Type I interferon signalling was negatively correlated to pre-DC gene expression. qPCR and mass cytometric analysis in independent cohorts validated that the pre-DC associated gene expression and the proportion of pre-DC were significantly higher before treatment in treatment-resistant patients. A cluster of synovial DCs showed both features of pre-DC and pro-inflammatory conventional DC2s. CONCLUSIONS: An increase in pre-DC in peripheral blood predicted RA treatment resistance. Pre-DC could have pathophysiological relevance to RA treatment response.

A case of sepsis-induced cardiomyopathy successfully treated with venoarterial extracorporeal membrane oxygenation.

Introduction: Sepsis with concomitant acute pyelonephritis, secondary to urolithiasis, is common. We report a case of sepsis-induced cardiomyopathy with acute pyelonephritis, successfully managed with venoarterial extracorporeal membrane oxygenation. Case presentation: A 64-year-old woman presented with fever and disturbed consciousness. Abdominal computed tomography revealed right hydronephrosis with ipsilateral ureteral stone. Despite ureteral stent placement and antibiotic treatment, her hemodynamics worsened. She was diagnosed with sepsis-induced cardiomyopathy and underwent venoarterial extracorporeal membrane oxygenation. Her hemodynamics improved rapidly; venoarterial extracorporeal membrane oxygenation was withdrawn on postoperative day-3. She was discharged from our hospital after sufficient antibiotic treatment. Conclusion: Venoarterial extracorporeal membrane oxygenation may be initiated in patients with sepsis-induced cardiomyopathy. Evaluation of left ventricular ejection fraction via echocardiography is important to determine the indication for venoarterial extracorporeal membrane oxygenation.

Transcriptome Profiling of Immune Cell Types in Peripheral Blood Reveals Common and Specific Pathways Involved in the Pathogenesis of Myositis-Specific Antibody-Positive Inflammatory Myopathies.

OBJECTIVE: Idiopathic inflammatory myopathies (IIM) demonstrate characteristic clinical phenotypes depending on the myositis-specific antibody (MSAs) present. We aimed to identify common or MSA-specific immunological pathways in different immune cell types from peripheral blood by transcriptome analysis. METHODS: We recruited 33 patients with IIM who were separated into the following groups: 15 patients with active disease at onset and 18 with inactive disease under treatment. All patients were positive for MSAs: anti-melanoma differentiation-associated gene 5 (MDA5) antibody (Ab) in 10 patients, anti-Mi-2 Ab in 7, and anti-aminoacyl-transfer RNA synthetase (ARS) Ab in 16. The patients were compared with 33 healthy controls. Twenty-four immune cell types sorted from peripheral blood were analyzed by flow cytometry, RNA sequencing, and differentially expressed gene analysis combined with pathway analysis. RESULTS: The frequencies of memory B cell types were significantly decreased in active patients, and the frequency of plasmablasts was prominently increased in active patients with anti-MDA5 Ab in comparison with healthy controls. The expression of type I interferon (IFN)-stimulated genes of all immune cell types was increased in the active, but not inactive, patients. Endoplasmic reticulum stress-related genes in all IIM memory B cells and oxidative phosphorylation-related genes in inactive IIM double negative B cells were also increased, suggesting prominent B cell activation in IIM. Furthermore, active patients with anti-MDA5 Ab, anti-Mi-2 Ab, or anti-ARS Ab were distinguished by IFN-stimulated and oxidative phosphorylation-related gene expression in plasmablasts. CONCLUSION: Unique gene expression patterns in patients with IIM with different disease activity levels and MSA types suggest different pathophysiologies. Especially, B cells may contribute to common and MSA-specific immunological pathways in IIM.

Effect of TNF inhibitors with bisphosphonates vs bisphosphonates alone on bone mineral density and bone and cartilage biomarkers at 1 year in patients with rheumatoid arthritis: A prospective study.

BACKGROUND: The present study aimed prospectively to investigate the effect of a combination of tumour necrosis factor inhibitors and bisphosphonates (TNFi with BP) on bone mineral density (BMD) and bone and cartilage biomarkers compared to that of BP alone at 1 year in patients with rheumatoid arthritis (RA). METHODS: Two groups of patients with RA and osteoporosis were enrolled. One group (37 patients) had already received BP, while the other group (37 patients) had already received TNFi with BP. The serum bone resorption and formation markers, cartilage markers, BMD in the lumbar spine, femoral neck, and distal radius were prospectively investigated at the beginning of the study and at 6 and 12 months. RESULTS: The percentages of change recorded for the various assessment categories were as follows in the TNFi with BP group: (1) tartrate-resistant acid phosphatase-5b had significantly decreased and osteocalcin had increased; (2) matrix metalloproteinase-3 and cartilage oligomeric matrix protein had significantly decreased; and (3) each BMD did not differ significantly between the groups. CONCLUSION: Our data suggested that TNFi with BP therapy not only suppressed cartilage degradation and bone resorption but also increased bone formation; however, this treatment did not affect the BMD at 1 year.

An association analysis of HLA-DRB1 with systemic lupus erythematosus and rheumatoid arthritis in a Japanese population: effects of *09:01 allele on disease phenotypes.

Objective. To re-evaluate the roles of HLA-DRB1 alleles in susceptibility to SLE and RA and their effects on autoantibody status in large-scale Japanese cohorts. Methods. A total of 656 SLE, 2410 RA and 911 control subjects, who were all Japanese, were genotyped for HLA-DRB1 alleles using sequence-specific oligonucleotide probes. The association of alleles with disease susceptibility was tested by logistic regression analysis and by the relative predispositional effect method. The association with autoantibody status was examined by the standard χ(2) test. Results. HLA-DRB1*15:01, *09:01, *08:02 and *04:01 were significantly associated with SLE susceptibility, while shared epitope (SE) alleles and DRB1*09:01 were associated with RA susceptibility. The compound heterozygote of DRB1*09:01/*15:01 conferred an increased risk for SLE compared with the homozygotes for DRB1*09:01 and *15:01 and was associated with earlier onset of disease, whereas the compound effect of DRB1-SE/*09:01 was not clear in RA. DRB1*09:01 was significantly associated with the appearance of anti-Sm antibody in SLE as well as ACPA in RA, while protectively associated with anti-dsDNA antibody in SLE. No significant interaction was observed between DRB1*09:01 and smoking status for the appearance of ACPA, unlike that observed in SE alleles in RA. Conclusion. We identified HLA-DRB1 alleles associated with SLE and RA in a Japanese population and demonstrated a shared susceptibility of DRB1*09:01 between the diseases as well as its effect on autoantibody production.

ANCA-associated vasculitis with dual ANCA positivity in coexistence with mixed connective tissue disease.

We here report a rare case of dual antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) in a 38-year-old Japanese woman previously diagnosed with mixed connective tissue disease. The patient was found to be positive for myeloperoxidase- and proteinase 3-ANCA, and was diagnosed with AAV following admission to hospital with fervescence, polyarthralgia, purpura, and asymmetric numbness of the extremities. Examination of her genetic background revealed that she carried HLA-DR9, which confers risk of both diseases in Japanese populations.

Osteogenesis and osteoclast inhibition in rheumatoid arthritis patients treated with bisphosphonates alone or in combination with pitavastatin over an 18-month follow-up after more than 4 years of treatment with bisphosphonates.

INTRODUCTION: To investigate the effects of bisphosphonates (Bis) (etidronate, alendronate, and risedronate), alone and in combination with statin, on the BMD (bone mineral density) and bone metabolism of rheumatoid arthritis (RA) patients. METHODS: Seventy-seven RA patients who had been receiving prednisolone (PSL) and Bis for over 4 years were divided into two groups: Bis and Bis + statin (n = 42 and 35; average age, 66.4 and 65.3 years; average disease duration, 24.9 and 20.8 years; average PSL dose, 2.4 and 2.7 mg, respectively). Serum levels of NTX (N-terminal telopeptide of type I collagen), TRACP-5b (tartrate-resistant acid phosphate-5b), PICP (C-terminal propeptide of type I procollagen), and RANKL (receptor activator of NF-κB ligand) were measured over an 18-month period of treatment and follow-up. The BMD levels of the two groups at the radius, lumbar spine, and femoral neck were compared using DXA (dual-energy x-ray absorptiometry). RESULTS: A significant increase was only observed in the BMD of the lumbar spine at 18-months, but the BMDs of the radius and femoral neck decreased during the follow-up period in the Bis group. Meanwhile, a significant increase was observed in the BMD of the lumbar spine in the Bis + statin group during administration and the BMDs of the radius and femoral neck stayed at baseline. Among the markers of bone metabolism, serum NTX was up-regulated after 6 months in the Bis + statin group. Serum TRACP-5b was significantly increased during the follow-up period in the Bis + statin group, but only at 18 months in the Bis group. Serum PICP recovered to base line in the Bis + statin group, whereas that in the Bis group did not observably recover during the post-administration follow-up, but rather decreased. CONCLUSION: Our findings suggest that both bone resorption and bone formation were inhibited by long-term administration of Bis alone, whereas combination therapy with Bis + statin may be associated with a less marked inhibition of bone metabolism. Cardiovascular disease is highly prevalent in RA patients and some patients are prescribed statins and bisphosphonate. Bis + statin may confer more benefit to the bone metabolism of these patients compared to Bis alone.

A genome-wide association study identified AFF1 as a susceptibility locus for systemic lupus eyrthematosus in Japanese.

Systemic lupus erythematosus (SLE) is an autoimmune disease that causes multiple organ damage. Although recent genome-wide association studies (GWAS) have contributed to discovery of SLE susceptibility genes, few studies has been performed in Asian populations. Here, we report a GWAS for SLE examining 891 SLE cases and 3,384 controls and multi-stage replication studies examining 1,387 SLE cases and 28,564 controls in Japanese subjects. Considering that expression quantitative trait loci (eQTLs) have been implicated in genetic risks for autoimmune diseases, we integrated an eQTL study into the results of the GWAS. We observed enrichments of cis-eQTL positive loci among the known SLE susceptibility loci (30.8%) compared to the genome-wide SNPs (6.9%). In addition, we identified a novel association of a variant in the AF4/FMR2 family, member 1 (AFF1) gene at 4q21 with SLE susceptibility (rs340630; P = 8.3×10(-9), odds ratio = 1.21). The risk A allele of rs340630 demonstrated a cis-eQTL effect on the AFF1 transcript with enhanced expression levels (P<0.05). As AFF1 transcripts were prominently expressed in CD4(+) and CD19(+) peripheral blood lymphocytes, up-regulation of AFF1 may cause the abnormality in these lymphocytes, leading to disease onset.

Regulatory polymorphisms in EGR2 are associated with susceptibility to systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease induced by the combinations of environmental and genetic factors. Recently, mice in which the early growth response 2 (EGR2) gene, a zinc-finger transcription factor, is conditionally knocked out in CD2(+) T cells have been shown to develop a lupus-like autoimmune disease. Here, we evaluated if polymorphisms in the EGR2 gene influence SLE susceptibility in humans. We first analyzed the effect of SNPs in the EGR2 region on EGR2 expression, and a significant positive correlation with expression was identified in an SNP located at the 5' flanking region of EGR2 (rs10761670, R=0.23, P=0.00072). We then performed a case-control association study using three sets of SLE cohorts by genotyping 14 tag SNPs in the EGR2 gene region. A peak of association with SLE susceptibility was observed for rs10761670 [Pooled: OR = 1.23 (95% CI 1.10-1.37), P=0.00023). This SNP was also associated with susceptibility to rheumatoid arthritis (RA) [OR = 1.15 (95% CI 1.05-1.26), P = 0.0019), suggesting that EGR2 is a common risk factor for SLE and RA. Among the SNPs in complete linkage disequilibrium with rs10761670 (r(2) = 1.0), two SNPs (rs1412554 and rs1509957) affected the binding of transcription factors and transcriptional activity in vitro, suggesting that they may be candidates of causal regulatory variants in this region. Therefore, EGR2 is a genetic risk factor for SLE, in which increased gene expression may contribute to SLE pathogenesis.

The association of a nonsynonymous single-nucleotide polymorphism in TNFAIP3 with systemic lupus erythematosus and rheumatoid arthritis in the Japanese population.

OBJECTIVE: Genome-wide association (GWA) studies in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in Caucasian populations have independently identified risk variants in and near the tumor necrosis factor alpha (TNFalpha)-induced protein 3 gene (TNFAIP3), which is crucial for the regulation of TNF-mediated signaling and Toll-like receptor signaling. The aim of this study was to assess the role of TNFAIP3 in the development of SLE and RA in Japanese subjects. METHODS: We selected 2 single-nucleotide polymorphisms (SNPs) from previous GWA studies. Rs2230926 is a nonsynonymous SNP in TNFAIP3 and is associated with SLE, while rs10499194 is an intergenic SNP associated with RA. We then performed 2 independent sets of SLE case-control comparisons (717 patients and 1,362 control subjects) and 3 sets of RA case-control comparisons (3,446 patients and 2,344 control subjects) using Japanese subjects. We genotyped SNPs using TaqMan assays. RESULTS: We observed a significant association between rs2230926 and an increased risk of SLE and RA in the Japanese population (for SLE, odds ratio [OR] 1.92, 95% confidence interval [95% CI] 1.53-2.41, P = 1.9 x 10(-8); for RA, OR 1.35, 95% CI 1.18-1.56, P = 2.6 x 10(-5)). The intergenic SNP rs10499194 was also associated with SLE and RA, while the risk allele for RA in Caucasians was protective against the diseases in our population. CONCLUSION: We demonstrated a significant association between the nonsynonymous variant in TNFAIP3 and the risk for SLE and RA in the Japanese population. TNFAIP3, similar to STAT4 and IRF5, may be a common genetic risk factor for SLE and RA that is shared between the Caucasian and Japanese populations.

Contribution of a haplotype in the HLA region to anti-cyclic citrullinated peptide antibody positivity in rheumatoid arthritis, independently of HLA-DRB1.

OBJECTIVE: To examine the risk of anti-cyclic citrullinated peptide (anti-CCP) antibody positivity in rheumatoid arthritis (RA) patients carrying certain haplotypes in the HLA region. METHODS: A total of 1,389 Japanese patients with RA were genotyped for 30 single-nucleotide polymorphisms (SNPs) in the HLA region using commercial oligonucleotide arrays (from Perlegen or Affymetrix) as well as for HLA-DRB1 alleles using a sequence-specific polymerase chain reaction method. Stepwise logistic regression was used to select from among the 30 SNPs the ones that represented a risk of anti-CCP antibody positivity. Haplotypes of the selected SNPs were inferred using an expectation-maximization algorithm. Associations of individual SNPs were evaluated with the Cochran-Armitage test for trend. DRB1 alleles and haplotypes were evaluated with the chi-square test. Heterogeneities of risks among the shared epitope (SE) and non-SE HLA-DRB1 alleles were examined using the exact test. Haplotype associations that were independent of individual HLA-DRB1 alleles were evaluated using the likelihood ratio test. RESULTS: Significant associations were found for 9 SNPs (smallest P value being 2.4x10(-8)) and in 4 HLA-DRB1 alleles (smallest P value being 2.0x10(-10) in DRB1*0405). Stepwise logistic regression selected 4 SNPs (rs9262638, rs7775228, rs4713580, and rs9277359). Among the 16 inferred haplotypes of these 4 SNPs, 6 indicated significant associations (smallest P value being 1.9x10(-11)). Risks among SE and non-SE alleles were significantly heterogeneous (P=0.0095 and P=9.8x10(-9), respectively), indicating the importance of stratification with individual DRB1 alleles rather than SE alleles. Conditional analysis of the risk associated with individual DRB1 alleles identified a risk haplotype that was independent of DRB1 (odds ratio 2.00 [95% confidence interval 1.44-2.79], P=2.6x10(-5)). CONCLUSION: Heterogeneous risks of anti-CCP antibody positivity were confirmed among SE and non-SE alleles in our patient population. A risk haplotype in the HLA region that is independent of HLA-DRB1 was confirmed.

Functional SNPs in CD244 increase the risk of rheumatoid arthritis in a Japanese population.

Rheumatoid arthritis is a chronic autoimmune inflammatory disease with a complex genetic etiology. Members of the signaling lymphocyte activation molecule (SLAM) family carry out pivotal functions in innate immunity and in conventional lymphocytes. We identified a linkage disequilibrium block associated with rheumatoid arthritis in the chromosome 1q region containing multiple SLAM family genes. In this block, the association peaked at two functional SNPs (rs3766379 and rs6682654) in CD244 in two independent rheumatoid arthritis cohorts from Japan (P = 3.23 x 10(-8) and P = 7.45 x 10(-8)). We also identified a Japanese cohort with systemic lupus erythematosus that had a similar genotype distribution as the rheumatoid arthritis cohorts. We demonstrated that the rheumatoid arthritis-susceptible alleles of rs3766379 and rs6682654 and their haplotype increased their expression in luciferase and allele-specific transcript quantification assays. CD244 is a genetic risk factor for rheumatoid arthritis and may have a role in the autoimmune process shared by rheumatoid arthritis and systemic lupus erythematosus.

Association of STAT4 with susceptibility to rheumatoid arthritis and systemic lupus erythematosus in the Japanese population.

OBJECTIVE: STAT4 encodes a transcriptional factor that transmits signals induced by several key cytokines, and it might be a key molecule in the development of autoimmune diseases. Recently, a STAT4 haplotype was reported to be associated with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) in Caucasian populations. This was replicated in a Korean RA population. Interestingly, the degree of risk of RA susceptibility with the STAT4 haplotype was similar in the Caucasian and Korean populations. The present study was undertaken to investigate the effect of STAT4 on susceptibility to RA and SLE in the Japanese. METHODS: We performed an association study using 3 independent Japanese RA case-control populations (total 3,567 cases and 2,199 controls) and 3 independent Japanese SLE populations (total 591 cases). All samples were genotyped using the TaqMan fluorogenic 5' nuclease assay for single-nucleotide polymorphism (SNP) rs7574865, which tags the susceptibility haplotype. The association of the SNP with disease susceptibility in each case-control study was calculated using Fisher's exact test, and the results were combined, using the Mantel-Haenszel method, to obtain combined odds ratios (ORs). RESULTS: We observed a significant association of the STAT4 polymorphism with susceptibility to both RA and SLE. The combined ORs for RA and SLE, respectively, were 1.27 (P = 8.4 x 10(-9)) and 1.61 (P = 2.1 x 10(-11)) for allele frequency distribution; these ORs were quite similar to those previously observed in the Caucasian population. CONCLUSION: We conclude that STAT4 is associated with RA and SLE in the Japanese. Our results indicate that STAT4 is a common genetic risk factor for autoimmune diseases, with similar strength across major racial groups.

[Genomic approaches to bone and joint diseases. Current status of human disease genomics in rheumatoid arthritis].

Rheumatoid arthritis (RA) is an autoimmune/inflammatory disorder which causes progression of polyarticular destruction. RA is a multifactorial disorder, in which genetic and environmental factors contribute to the onset of diseases. Linkage analyses and case-control association studies in RA have identified several susceptible genes including genetic variation in human leukocyte antigen region. Here, we review major genes susceptible to RA, and discuss effects of these genes on production of anti-cyclic citrullinated peptides antibody.

A functional SNP in the NKX2.5-binding site of ITPR3 promoter is associated with susceptibility to systemic lupus erythematosus in Japanese population.

Systemic lupus erythematosus (SLE) is one of the common autoimmune diseases with complex genetic components. To identify a gene(s) susceptible to SLE, we performed a case-control association study using genome-wide gene-based single nucleotide polymorphisms (SNPs) in Japanese population. Here we report that an SNP (rs3748079) located in a promoter region of the inositol 1,4,5-triphosphate receptor type 3 (ITPR3) gene on chromosome 6p21 was significantly associated with SLE in two independent Japanese case-control samples [P=0.0000000178 with odds ratio of 1.88, 95% confidence interval (CI) of 1.51-2.35]. This particular SNP also revealed associations with rheumatoid arthritis (RA) (P=0.0084 with odds ratio of 1.23, 95% CI of 1.05-1.43) and with Graves' disease (GD) (P=0.00036 with odds ratio of 1.57, 95% CI of 1.22-2.02). We found the binding of NKX2.5 specific to the non-susceptible T allele in the region including this SNP. Furthermore, an SNP in NKX2.5 also revealed an association with SLE (P=0.0037 with odds ratio of 1.74, 95% CI of 1.19-2.55). Individuals with risk genotype of both ITPR3 and NKX2.5 loci have higher risk for SLE (odds ratio=5.77). Our data demonstrate that genetic and functional interactions of ITPR3 and NKX2.5 play a crucial role in the pathogenesis of SLE.

CD64 on neutrophils is a sensitive and specific marker for detection of infection in patients with rheumatoid arthritis.

OBJECTIVE: In inflammatory diseases, differentiation between infection and disease flares is often clinically difficult because of similar signs and symptoms, such as fever and elevation of inflammatory markers. In rheumatoid arthritis (RA), infection is not only one of the major complications but also one of the frequent causes of death. Use of biologic agents such as tumor necrosis factor-a blockers has been reported to increase the incidence of tuberculosis or opportunistic infections. We examined the utility of CD64 (FcgRI) expressed on neutrophils as a marker for detection of infection complicated with RA. METHODS: We measured the expression level of CD64 per neutrophil quantitatively by flow cytometry in 279 samples from 237 patients with RA with various levels of disease activity or types of infection, and in 52 samples from 36 controls including subjects with infection. RESULTS: CD64 expression was significantly higher among RA patients with infection (median 4156 molecules per neutrophil, interquartile range 2583-8587) than in those without infection (884, IQR 670-1262) (p < or = 0.001). The sensitivity of CD64 on neutrophils for the diagnosis of infection (using a cutoff value of 2000 molecules per cell) was 92.7% and specificity was 96.5%. CD64 expression was not affected by the disease activity of RA or the use of corticosteroids, disease modifying antirheumatic drugs, and biologic agents. CD64 was upregulated in infection by bacteria, viruses, fungi, and mycobacteria. CONCLUSION: Our results suggest that quantitative measurement of CD64 expression on neutrophils can be used as a sensitive and specific marker to detect infection complicating RA.