Similarity and difference between systemic lupus erythematosus and NZB/W F1 mice by multi-omics analysis.

OBJECTIVES: Several animal disease models have been used to understand the mechanisms of systemic lupus erythematosus (SLE); however, the translation of findings from animals to humans has not been sufficiently examined in drug development. To confirm the validity of New Zealand black x New Zealand white (NZB/W) F1 mice as an SLE model, we extensively characterized SLE patients and NZB/W F1 mice by omics analysis. METHODS: Peripheral blood from patients and mice and spleen and lymph node tissue from mice were analysed using cell subset analysis, cytokine panel assays, and transcriptome analysis. RESULTS: CD4+ effector memory T cells, plasmablasts, and plasma cells were increased in both SLE patients and NZB/W F1 mice. Levels of tumor necrosis factor-α, interferon gamma induced protein-10, and B cell activating factor in plasma were significantly higher in SLE patients and NZB/W F1 mice than in their corresponding controls. Transcriptome analysis revealed an upregulation of genes involved in the interferon signalling pathway and T-cell exhaustion signalling pathway in both SLE patients and the mouse model. In contrast, death receptor signalling genes showed changes in the opposite direction between patients and mice. CONCLUSION: NZB/W F1 mice are a generally suitable model of SLE for analysing the pathophysiology and treatment response of T/B cells and monocytes/macrophages and their secreted cytokines.

Verification of optimal conditions for the scattering correction of 123I-FP-CIT SPECT on a single-photon emission tomography system with a two-detector whole-body cadmium-zinc-telluride semiconductor detector.

To identify the optimal scattering correction for 123I-FP-CIT SPECT (DAT-SPECT) using a two-detector whole-body cadmium-zinc-telluride semiconductor detector (C-SPECT) system with a medium-energy high-resolution sensitivity (MEHRS) collimator. The C-SPECT system with the MEHRS collimator assessed image quality and quantification using a striated phantom. Different reconstruction methods and scattering correction settings were compared, including filtered back projection (FBP) and ordered subset expectation maximization (OSEM). Higher %contrast and %CV values were observed > 10% subwindow (SW) for all conditions, with no significant differences between images without scattering correction and those < 7% SW. The FBP images show a greater increase in %CV > 10% SW than the OSEM images. The specific binding ratio in the radioactivity ratio of 8:1 was higher than the true value under all conditions. The C-SPECT system with an MEHRS collimator provided accurate scattering suppression and enabled high-quality imaging for DAT-SPECT. Careful setting of the scattering correction is essential for total count accuracy.

Expression of S100A8 protein on B cells is associated with disease activity in patients with systemic lupus erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is an intractable disease characterized by autoantibody production and autoreactive B and T cell proliferation. Although several studies have revealed multiple genetic and environmental associations, the underlying mechanisms remain unknown. METHODS: We performed proteomics and transcriptomics using liquid chromatography-mass spectrometry and DNA microarray, using peripheral blood B cells from patients with SLE, and healthy controls (HC). We explored molecules associated with the pathophysiology of SLE by flow cytometry and B cell stimulation assay. RESULTS: We identified for the first time that expression of both S100A8 protein and mRNA were markedly upregulated in SLE B cells. The results obtained using flow cytometry showed that S100A8 was highly expressed on the surface of B cells of patients with active SLE (MFI; HC 102.5 ± 5.97, stable SLE 111.4 ± 12.87, active SLE 586.9 ± 142.9), and S100A8 on the cell surface was decreased after treatment (MFI; pre-treat 1094.5 ± 355.38, post-treat 492.25 ± 247.39); therefore, it is suggested that S100A8 may be a marker for disease activity. The mRNA expression of S100A8 was particularly upregulated in memory B cells of SLE (56.68 fold higher than HC), suggesting that S100A8 may be mainly secreted by memory B cells in the pathogenesis of SLE. CONCLUSIONS: Our results imply that the S100A8 proteins secreted from memory B cells may stimulate granulocytes and monocytes through pattern recognition receptors, activate the innate immune system, and are involved in the pathogenesis of SLE.

Dynamics of Type I and Type II Interferon Signature Determines Responsiveness to Anti-TNF Therapy in Rheumatoid Arthritis.

The factors influencing long-term responses to a tumor necrosis factor inhibitor (TNFi) in rheumatoid arthritis (RA) patients currently remain unknown. Therefore, we herein conducted a multi-omics analysis of TNFi responses in a Japanese RA cohort. Blood samples were collected from 27 biological disease-modifying antirheumatic drug (DMARD)-naive RA patients at the initiation of and after three months of treatment with TNFi. Treatment responses were evaluated at one year. Differences in gene expression levels in peripheral blood mononuclear cells (PBMCs), plasma protein levels, drug concentrations, and the presence/absence of anti-drug antibodies were investigated, and a cell phenotypic analysis of PBMCs was performed using flow cytometry. After one year of treatment, thirteen patients achieved clinical remission (responders), while the others did not or switched to other biologics (non-responders). Differentially expressed genes related to treatment responses were enriched for the interferon (IFN) pathway. The expression of type I IFN signaling-related genes was higher in non-responders than in responders before and after treatment (P = 0.03, 0.005, respectively). The expression of type II IFN signaling-related genes did not significantly differ before treatment; however, it increased in non-responders and decreased in responders, with a significant difference being observed after three months of treatment (P = 1.2×10-3). The total number of lymphocytes and C-X-C Motif Chemokine Ligand 10 (CXCL10) protein levels were associated with the type I IFN signature (P = 6.7×10-7, 6.4×10-3, respectively). Hepatocyte growth factor (HGF) protein levels before treatment predicted fold increases in type II IFN (P = 0.03). These IFN signature-related indices (the number of lymphocytes, CXCL10, and HGF) significantly differed between responders and non-responders (P = 0.01, 0.01, and 0.04, respectively). A single-cell analysis revealed that the type I IFN signature was more highly enriched in monocytes than in other cell types. A deconvolution analysis of bulk-RNA sequence data identified CD4+ and CD8+ T cells as the main sources of the type II IFN signature in non-responders. Collectively, the present results demonstrated that the dynamics of the type I and II IFN pathways affected long-term responses to TNFi, providing information on its biological background and potential for clinical applications.

Metabolomic approach to the exploration of biomarkers associated with disease activity in rheumatoid arthritis.

We aimed to investigate metabolites associated with the 28-joint disease activity score based on erythrocyte sedimentation rate (DAS28-ESR) in patients with rheumatoid arthritis (RA) using capillary electrophoresis quadrupole time-of-flight mass spectrometry. Plasma and urine samples were collected from 32 patients with active RA (DAS28-ESR≥3.2) and 17 with inactive RA (DAS28-ESR<3.2). We found 15 metabolites in plasma and 20 metabolites in urine which showed a significant but weak positive or negative correlation with DAS28-ESR. When metabolites between active and inactive patients were compared, 9 metabolites in plasma and 15 in urine were found to be significantly different. Consequently, we selected 11 metabolites in plasma and urine as biomarker candidates which significantly correlated positively or negatively with DAS28-ESR, and significantly differed between active and inactive patients. When a multiple logistic regression model was built to discriminate active and inactive cohorts, three variables-histidine and guanidoacetic acid from plasma and hypotaurine from urine-generated a high area under the receiver operating characteristic (ROC) curve value (AUC = 0.8934). Thus, this metabolomics approach appeared to be useful for investigating biomarkers of RA. Combination of plasma and urine analysis may lead to more precise and reliable understanding of the disease condition. We also considered the pathophysiological significance of the found biomarker candidates.

Increase of MZB1 in B cells in systemic lupus erythematosus: proteomic analysis of biopsied lymph nodes.

BACKGROUND: Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease in which dysregulation of B cells has been recognized. Here, we searched for potential biomarkers of SLE using liquid chromatography-tandem mass spectrometry (LC-MS). METHODS: Lymph nodes from SLE patients and controls were analyzed by LC-MS. To validate the identified molecules, immunoblotting and immunohistochemistry were performed and B cells from SLE patients were analyzed by quantitative RT-PCR. B-cell subsets from NZB/W F1 mice, which exhibit autoimmune disease resembling human SLE, were analyzed by flow cytometry. Endoplasmic reticulum (ER) stress was induced by tunicamycin and the serum concentration of anti-dsDNA antibodies was determined by ELISA. TUNEL methods and immunoblotting were used to assess the effect of tunicamycin. RESULTS: MZB1, which comprises part of a B-cell-specific ER chaperone complex and is a key player in antibody secretion, was one of the differentially expressed proteins identified by LC-MS and confirmed by immunoblotting. Immunohistochemically, larger numbers of MZB1+ cells were located mainly in interfollicular areas and scattered in germinal centers in specimens from SLE patients compared with those from controls. MZB1 colocalized with CD138+ plasma cells and IRTA1+ marginal zone B cells. MZB1 mRNA was increased by 2.1-fold in B cells of SLE patients with active disease (SLE Disease Activity Index 2000 ≥ 6) compared with controls. In aged NZB/W F1 mice, splenic marginal zone B cells and plasma cells showed elevated MZB1 levels. Tunicamycin induced apoptosis of MZB1+ cells in target organs, resulting in decreased serum anti-dsDNA antibody levels. Additionally, MZB1+ cells were increased in synovial tissue specimens from patients with rheumatoid arthritis. CONCLUSIONS: MZB1 may be a potential therapeutic target in excessive antibody-secreting cells in SLE.

Transgelin-2 is upregulated on activated B-cells and expressed in hyperplastic follicles in lupus erythematosus patients.

Transgelin-2 (TAGLN2) is an actin-binding protein that controls actin stability and promotes T cell activation. TAGLN2 is also expressed on B-cells but its function in B-cells is unknown. We found that TAGLN2-expressing B-cells were localized in the germinal center (GC) of secondary lymphoid tissues and TAGLN2 mRNA was significantly upregulated after IgM+IgG stimulation in primary human B-cells, suggesting that TAGLN2 was upregulated upon B-cell activation. In support of this, lymph nodes (LNs) from patients with systemic lupus erythematosus (SLE), in which the intense GC activity have been recognized, showed increased TAGLN2 expression in B-cells compared to control LNs. Moreover, TAGLN2+B-cells were distributed widely not only in the GC but also in the perifollicular areas in SLE LNs. In contrast, CD19+ B-cells and CD19+CD27+ memory-B cells in peripheral blood of SLE patients showed no increase in TAGLN2 mRNA. Two-photon excitation microscopy of Raji cells demonstrated that TAGLN2 colocalized with F-actin and moved together to the periphery upon stimulation. TAGLN2-knockdown in Raji cells resulted in impaired phosphorylation of PLCγ2 leading to inhibition of cell migration. Microarray analysis of TAGLN2-knockdown Raji cells showed decreased expression of the genes associated with immune function including CCR6 and as well as of those associated with regulation of the actin cytoskeleton including ABI2, compared to controls. These results suggest that TAGLN2 might regulate activation and migration of B-cells, in particular, the entry of activated B-cells into the follicle. We also suggest that TAGLN2 could be used as a marker for activated B-cells.

Cleaved Form of Osteopontin in Urine as a Clinical Marker of Lupus Nephritis.

We assessed the utility of two forms of osteopontin (OPN), OPN full and its cleaved form (OPN N-half), in plasma and urine as markers of disease activity in lupus nephritis (LN). Samples were collected from patients with systemic lupus erythematosus (SLE) (LN: N = 29, non-LN: N = 27), IgA nephropathy (IgAN) (N = 14), minimal change nephrotic syndrome (MCNS) (N = 5), diabetic nephropathy (DN) (N = 14) and healthy volunteers (HC) (N = 17). While there was no significant difference in urine OPN full concentration between groups, urine OPN N-half concentration was significantly higher in patients with LN than HC (p < 0.05). Moreover, urine OPN N-half was higher in LN patients with overt proteinuria (urine protein/creatinine ratio: P/C > 0.5) than LN patients with minimal proteinuria (P/C < 0.5, p < 0.0001), and also higher than in DN patients with overt proteinuria (P/C > 0.5, p < 0.01). Urine thrombin activity correlated with urine OPN N-half concentration (p < 0.0001), but not with urine OPN full concentration. These results suggest that urine OPN N-half concentration reflects renal inflammation. Thus, urine OPN N-half may be a novel disease activity marker for LN.

Human T cells expressing BEND3 on their surface represent a novel subpopulation that preferentially produces IL-6 and IL-8.

BEN domain-containing protein 3 (BEND3) has no transmembrane region, is localized in the cytoplasm, and is involved in chromatin function and transcription. We here identified a novel subpopulation of human T cells that expressed BEND3 on their cell surface (BEND3(+) T cells). BEND3(+) T cells consisted of approximately 3% of T cells in the peripheral blood, were present in both CD4(+) and CD8(+) T cells, and were also observed in cord blood. The stimulation of BEND3(+) T cells through the TCR/CD3 complex led to the production of various kinds of cytokines; however, the levels of IL-6 and IL-8 produced by BEND3(+) T cells were higher than those by BEND3(-) T cells. The proportion of BEND3(+) T cells was also increased in some patients with inflammatory diseases. Taken together, these results indicate that BEND3(+) T cells are a new subpopulation of T cells in terms of their cytokine profile. Further analyses on BEND3(+) T cells may be of importance and useful in understanding human T cell immunology.

Multiple modes of action of tacrolimus (FK506) for neuroprotective action on ischemic damage after transient focal cerebral ischemia in rats.

While the immunosuppressant tacrolimus (FK506) is known to be neuroprotective following cerebral ischemia, the mechanisms underlying its neuroprotective properties are not fully understood. To determine the mode of action by which tacrolimus ameliorates neurodegeneration after transient focal ischemia, we therefore evaluated the effect of tacrolimus on DNA damage, release of cytochrome c, activation of microglia and infiltration of neutrophils following a 60-min occlusion of the middle cerebral artery (MCA) in rats. In this model, cortical brain damage gradually expanded until 24 h after reperfusion, whereas brain damage in the caudate putamen was fully developed within 5 h. Tacrolimus (1 mg/kg) administered immediately after MCA occlusion significantly reduced ischemic damage in the cerebral cortex, but not in the caudate putamen. Tacrolimus decreased both apoptotic and necrotic cell death at 24 h and reduced the number of cytochrome c immunoreactive cells at 8 h after reperfusion in the ischemic penumbra in the cerebral cortex. In contrast, tacrolimus did not show significant neuroprotection for necrotic cell death and reduction of cytochrome c immunoreactive cells in the caudate putamen. Tacrolimus also significantly decreased microglial activation at 8 h and inflammatory markers (cytokine-induced neutrophil chemoattractant and myeloperoxidase [MPO] activity) at 24 h after reperfusion in the ischemic cortex but not in the caudate putamen. These results collectively suggest that tacrolimus ameliorates the gradually expanded brain damage by inhibiting both apoptotic and necrotic cell death, as well as suppressing inflammatory reactions.