Interleukin 4-driven reversal of self-reactive B cell anergy contributes to the pathogenesis of systemic lupus erythematosus.

OBJECTIVES: Reactivation of anergic autoreactive B cells (BND cells) is a key aetiological process in systemic lupus erythematosus (SLE), yet the underlying mechanism remains largely elusive. This study aimed to investigate how BND cells participate in the pathogenesis of SLE and the underlying mechanism. METHODS: A combination of phenotypical, large-scale transcriptome and B cell receptor (BCR) repertoire profiling were employed at molecular and single cell level on samples from healthy donors and patients with SLE. Isolated naïve B cells from human periphery blood were treated with anti-CD79b mAb in vitro to induce anergy. IgM internalisation was tracked by confocal microscopy and was qualified by flow cytometer. RESULTS: We characterised the decrease and disruption of BND cells in SLE patients and demonstrated IL-4 as an important cytokine to drive such pathological changes. We then elucidated that IL-4 reversed B cell anergy by promoting BCR recycling to the cell surface via STAT6 signalling. CONCLUSIONS: We demonstrated the significance of IL-4 in reversing B cell anergy and established the scientific rationale to treat SLE via blocking IL-4 signalling, also providing diagnostic and prognostic biomarkers to identify patients who are most likely going to benefit from such treatments.

Identification and validation of hub genes and pathways associated with mitochondrial dysfunction in hypertrophy of ligamentum flavum.

Background: Lumbar spinal stenosis which can lead to irreversible neurologic damage and functional disability, is characterized by hypertrophy of ligamentum flavum (HLF). Recent studies have indicated that mitochondrial dysfunction may contribute to the development of HLF. However, the underlying mechanism is still unclear. Methods: The dataset GSE113212 was obtained from the Gene Expression Omnibus database, and the differentially expressed genes were identified. The intersection of DEGs and mitochondrial dysfunction-related genes were identified as mitochondrial dysfunction-related DEGs. Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and Gene Set Enrichment Analysis were performed. Protein-protein interaction network was constructed, and miRNAs and transcriptional factors of the hub genes were predicted via the miRNet database. Small molecule drugs targeted to these hub genes were predicted via PubChem. Immune infiltration analysis was performed to evaluate the infiltration level of immune cells and their correlation with the hub genes. In final, we measured the mitochondrial function and oxidative stress in vitro and verified the expression of hub genes by qPCR experiments. Results: In total, 43 genes were identified as MDRDEGs. These genes were mainly involved in cellular oxidation, catabolic processes, and the integrity of mitochondrial structure and function. The top hub genes were screened, including LONP1, TK2, SCO2, DBT, TFAM, MFN2. The most significant enriched pathways include cytokine-cytokine receptor interaction, focal adhesion, etc. Besides, SP1, PPARGC1A, YY1, MYC, PPARG, and STAT1 were predicted transcriptional factors of these hub genes. Additionally, increased immune infiltration was demonstrated in HLF, with a close correlation between hub genes and immune cells found. The mitochondrial dysfunction and the expression of hub genes were validated by evaluation of mitochondrial DNA, oxidative stress markers and quantitative real-time PCR. Conclusion: This study applied the integrative bioinformatics analysis and revealed the mitochondrial dysfunction-related key genes, regulatory pathways, TFs, miRNAs, and small molecules underlying the development of HLF, which improved the understanding of molecular mechanisms and the development of novel therapeutic targets for HLF.

Causality of genetically determined metabolites and metabolic pathways on osteoarthritis: a two-sample mendelian randomization study.

BACKGROUND: Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases and is the leading cause of pain and disability in the aged population. However, the underlying biological mechanism has not been fully understood. This study aims to reveal the causal effect of circulation metabolites on OA susceptibility. METHODS: A two-sample Mendelian Randomization (MR) analysis was performed to estimate the causality of GDMs on OA. A genome-wide association study (GWAS) of 486 metabolites was used as the exposure, whereas 8 different OA phenotypes, including any-site OA (All OA), knee and/or hip OA (knee/hip OA), knee OA, hip OA, spine OA, finger and/or thumb OA (hand OA), finger OA, thumb OA, were set the outcomes. Inverse-variance weighted (IVW) was used for calculating causal estimates. Methods including weight mode, weight median, MR-egger, and MR-PRESSO were used for the sensitive analysis. Furthermore, metabolic pathway analysis was performed via the web-based Metaconflict 4.0. All statistical analyses were performed in R software. RESULTS: In this MR analysis, a total of 235 causative associations between metabolites and different OA phenotypes were observed. After false discovery rate (FDR) correction and sensitive analysis, 9 robust causative associations between 7 metabolites (e.g., arginine, kynurenine, and isovalerylcarnitine) and 5 OA phenotypes were finally identified. Additionally, eleven significant metabolic pathways in 4 OA phenotypes were identified by metabolic pathway analysis. CONCLUSION: The finding of our study suggested that identified metabolites and metabolic pathways can be considered useful circulating metabolic biomarkers for OA screening and prevention in clinical practice, and can also serve as candidate molecules for future mechanism exploration and drug target selection.

MYC promotes fibroblast osteogenesis by regulating ALP and BMP2 to participate in ectopic ossification of ankylosing spondylitis.

BACKGROUND: Ectopic ossification is an important cause of disability in patients with ankylosing spondylitis (AS). Whether fibroblasts can transdifferentiate into osteoblasts and contribute to ossification remains unknown. This study aims to investigate the role of stem cell transcription factors (POU5F1, SOX2, KLF4, MYC, etc.) of fibroblasts in ectopic ossification in patients with AS. METHODS: Primary fibroblasts were isolated from the ligaments of patients with AS or osteoarthritis (OA). In an in vitro study, primary fibroblasts were cultured in osteogenic differentiation medium (ODM) to induce ossification. The level of mineralization was assessed by mineralization assay. The mRNA and protein levels of stem cell transcription factors were measured by real-time quantitative PCR (q-PCR) and western blotting. MYC was knocked down by infecting primary fibroblasts with lentivirus. The interactions between stem cell transcription factors and osteogenic genes were analysed by chromatin immunoprecipitation (ChIP). Recombinant human cytokines were added to the osteogenic model in vitro to evaluate their role in ossification. RESULTS: We found that MYC was elevated significantly in the process of inducing primary fibroblasts to differentiate into osteoblasts. In addition, the level of MYC was remarkably higher in AS ligaments than in OA ligaments. When MYC was knocked down, the expression of the osteogenic genes alkaline phosphatase (ALP) and bone morphogenic protein 2 (BMP2) was decreased, and the level of mineralization was reduced significantly. In addition, the ALP and BMP2 were confirmed to be the direct target genes of MYC. Furthermore, interferon-γ (IFN-γ), which showed high expression in AS ligaments, was found to promote the expression of MYC in fibroblasts in the process of ossification in vitro. CONCLUSIONS: This study demonstrates the role of MYC in ectopic ossification. MYC may act as the critical bridge that links inflammation with ossification in AS, thus providing new insights into the molecular mechanisms of ectopic ossification in AS.

Clinicopathologic predictors of central lymph node metastases in clinical node-negative papillary thyroid microcarcinoma: a systematic review and meta-analysis.

BACKGROUND: The presence of central lymph node metastases (CLNM) has been suggested as a risk factor for poorer prognosis and recurrence in papillary thyroid microcarcinoma (PTMC). However, the clinicopathologic factors for CLNM in clinical node-negative (CN0) PTMC were not well defined. This study aimed to perform a systematic review and meta-analysis to investigate the significant clinicopathologic predictors of CLNM in CN0 PTMC. METHODS: A systematic literature search was performed in PubMed, Embase, Cochrane Library, and Web of Science. Case-control studies on the association of clinicopathologic risk factors with CLNM in CN0 PTMC were included. RESULTS: Thirteen eligible studies involving 6068 patients with CN0 PTMC were included. From the pooled analyses, male (odds ratio [OR]: 2.07, 95% CI: 1.49-2.87, P < 0.001), multifocality (OR: 1.88, 95% CI: 1.54-2.29, P < 0.001), tumor size > 5 mm (OR: 1.84, 95% CI: 1.55-2.18, P < 0.001), and extrathyroidal extension (OR: 1.96, 95% CI: 1.30-2.95, P = 0.001) are significantly associated with increased risk of CLNM in CN0 PTMC. A sample size with a cutoff point of 200 was identified as the source of heterogeneity for sex according to meta-regression (t = 3.18, P = 0.033). Then, the subgroup analysis of male was performed, which illustrated that male increased the risk of CLNM in the small sample group (SG) and the large sample group (LG) by 6.11-folds and 2.01-folds, respectively (SG: OR, 6.11, 95% CI, 3.16-11.81, P < 0.001; LG: OR, 2.01, 95% CI, 1.65-2.46, P < 0.001). CONCLUSIONS: Male, multifocality, tumor size > 5 mm, and extrathyroidal extension may be reliable clinical predictors of CLNM in CN0 PTMC. Moreover, prophylactic central lymph node dissection should be considered in surgical decision-making for CN0 PTMC patients, who are male, multifocal, with tumor size > 5 mm, and with extrathyroidal extension. TRIAL REGISTRATION: CRD42021242211 (PROSPERO).

Thyroid Antibody Status is Associated with Central Lymph Node Metastases in Papillary Thyroid Carcinoma Patients with Hashimoto's Thyroiditis.

OBJECTIVE: The aim of this study was to explore the impact of thyroid antibody status on central lymph node metastases (CLNM) in papillary thyroid carcinoma (PTC) patients with Hashimoto's thyroiditis (HT). METHODS: A retrospective analysis was performed on 346 PTC patients with HT who underwent thyroidectomy and ipsilateral central lymph node dissection (CLND). Histopathological characteristics of the tumor and serum levels of thyroid hormone, as well as antibodies, were collected and analyzed. RESULTS: The multivariate logistic regression analysis showed that being male [odds ratio (OR) 3.269, 95% confidence interval (CI) 1.240-8.619], tumor size > 1 cm [1 cm < diameter (D) ≤ 2 cm: OR 6.947, 95% CI 2.886-16.722; 2 cm < D: OR 5.880, 1.937-17.846], and antibody status [thyroid peroxidase antibody (TPOAb) and thyroglobulin antibody (TgAb) double negative: OR 3.791, 95% CI 1.391-10.331; TPOAb and TgAb double positive: OR 4.047, 95% CI 1.509-10.856; TgAb single positive: OR 6.024, 95% CI 2.019-17.970] were independent risk factors for CLNM. Additionally, a risk-score scale, including sex, antibody status, and tumor size, was established to predict CLNM. The sensitivity, specificity, positive predictive value, and negative predictive value were 55.7%, 84.4%, 74.4%, and 70%, respectively, when the cut-off point was chosen as 3. CONCLUSIONS: Antibody status is a critical independent risk factor for CLNM in PTC patients with HT. For the CLND strategy, a more conservative option could be considered in a low-risk cohort with the following characteristics: female sex, smaller tumor size, and TPOAb single positive.

Hyperprolactinemia is associated with a high prevalence of serum autoantibodies, high levels of inflammatory cytokines and an abnormal distribution of peripheral B-cell subsets.

PURPOSE: Hyperprolactinemia (HPRL) has been reported in many autoimmune diseases. However, the serum autoantibody profile and peripheral B-cell subset distribution in women with HPRL are largely unknown. The current study aimed to investigate the autoantibody prevalence and cytokine levels as well as to further explore the B-cell subset distribution in women with HPRL. METHODS: Sera from 202 women with HPRL and 97 healthy women were included in this study. All sera were examined for prolactin (PRL), anti-nuclear antibody (ANA), rheumatoid factor, anticardiolipin (ACL), immunoglobulin G, immunoglobulin M, complement 3, complement 4, interleukin 4 (IL-4) and interleukin 6 (IL-6). Peripheral blood was collected from 22 women with HPRL and 19 healthy women, and B-cell subsets were measured by flow cytometry. RESULTS: At least one autoantibody was found in 47 out of 202 women with HPRL compared with 9 of 97 healthy women (p < 0.001). The levels of IL-4 (p < 0.0001) and IL-6 (p < 0.0001) were significantly higher in women with HPRL than in healthy women. The percentages of naive IgD+IgM- B cells (BND cells, p < 0.0001), antibody-secreting cells (p = 0.007) and unswitched memory B cells (p = 0.004) among the total B cells from HPRL women were significantly higher than those from healthy women. CONCLUSIONS: Women with HPRL had a higher prevalence of autoantibodies, higher serum levels of IL-4 and IL-6, and more BND cells, antibody-secreting B cells and unswitched memory B cells than healthy women. These data imply that a high level of PRL is associated with autoimmune diseases.