Blockade of IL-11 Trans-Signaling or JAK2/STAT3 Signaling Ameliorates the Profibrotic Effect of IL-11.

OBJECTIVES: Systemic sclerosis (SSc) is a rare rheumatic disease characterized by vascular damage, dysregulated immune response, and fibrosis. Interleukin-11 (IL-11) is upregulated in SSc. This study aimed to investigate the pathological and therapeutic role of the IL-11 trans-signaling pathway in SSc. METHODS: Plasma IL-11 level was evaluated in 32 patients with SSc and 15 healthy controls, while the expression levels of ADAM10, ADAM17, IL-11, IL-11 Rα, or IL-11 co-stained with CD3 or CD163 in the skin of SSc patients and healthy controls were analyzed. Fibroblasts were treated with IL-11 and ionomycin to evaluate the profibrotic effect of IL-11 trans-signaling pathway. TJ301 (sgp130Fc) and WP1066 (a JAK2/STAT3 inhibitor) intervention groups were set up to investigate the antifibrotic effect of targeting IL-11. RESULTS: Levels of plasma IL-11 were extremely low in most SSc patients and healthy controls. In contrast, levels of IL-11, IL-11 Rα, and ADAM10, but not ADAM17, were significantly elevated in the skin of SSc patients. Moreover, the numbers of IL-11+ CD3+ cells and IL-11+ CD163+ cells were increased in the skin of SSc patients. Besides, IL-11 and ADAM10 were also elevated in the skin and pulmonary of bleomycin-induced SSc mouse. Fibroblasts co-stimulated with IL-11 and ionomycin showed increased expression of COL3 and phosphorylation of STAT3, which could be inhibited by TJ301 or WP1066. TJ301 also ameliorated skin and lung fibrosis in BLM-induced SSc mouse. CONCLUSIONS: IL-11 induces fibrosis in SSc by regulating the trans-signaling pathway. Blockage of sgp130Fc or inhibition of the JAK2/STAT3 pathway could ameliorate the profibrotic effect of IL-11.

Determination of local chromatin interactions using a combined CRISPR and peroxidase APEX2 system.

The architecture and function of chromatin are largely regulated by local interacting molecules, such as transcription factors and noncoding RNAs. However, our understanding of these regulatory molecules at a given locus is limited because of technical difficulties. Here, we describe the use of Clustered Regularly Interspaced Short Palindromic Repeats and an engineered ascorbate peroxidase 2 (APEX2) system to investigate local chromatin interactions (CAPLOCUS). We showed that with specific small-guide RNA targets, CAPLOCUS could efficiently identify both repetitive genomic regions and single-copy genomic locus with high resolution. Genome-wide sequencing revealed known and potential long-range chromatin interactions for a specific single-copy locus. CAPLOCUS also identified telomere-associated RNAs. CAPLOCUS, followed by mass spectrometry, identified both known and novel telomere-associated proteins in their native states. Thus, CAPLOCUS may be a useful approach for studying local interacting molecules at any given chromosomal location.

Skin telocytes versus fibroblasts: two distinct dermal cell populations.

It is already accepted that telocytes (TCs) represent a new type of interstitial cells in human dermis. In normal skin, TCs have particular spatial relations with different dermal structures such as blood vessels, hair follicles, arrector pili muscles or segments of sebaceous and/or eccrine sweat glands. The distribution and the density of TCs is affected in various skin pathological conditions. Previous studies mentioned the particular (ultra)structure of TCs and also their immunophenotype, miR imprint or proteome, genome or secretome features. As fibroblast is the most common intersitital cell (also in human dermis), a dedicated comparison between human skin TCs and fibroblasts (Fbs) was required to be performed. In this study, using different techniques, we document several points of difference between human dermis TCs and Fbs. By transmission electron microscopy (TEM) and scanning electron microscopy (SEM), we demonstrated TCs with their hallmark cellular prolongations - telopodes. Thus, we showed their ultrastructural distinctiveness from Fbs. By RayBio Human Cytokine Antibody Array V analyses performed on the supernatant from separately cultured TCs and Fbs, we detected the cytokine profile of both cell types, individually. Two of 79 detected cytokines - epithelial-derived neutrophil-activating peptide 78 and granulocyte chemotactic protein-2 - were 1.5 times higher in the supernatant of TCs (comparing with Fbs). On the other hand, 37 cytokines were at least 1.5 higher in Fbs supernatant (comparing with TCs), and among them six cytokines - interleukin 5, monocyte chemotactic protein-3 (MCP-3), MCP-4, macrophage inflammatory protein-3, angiogenin, thrombopoietin - being 9.5 times higher (results also confirmed by ELISA testing). In summary, using different techniques, we showed that human dermal TCs and Fbs are different in terms of ultrastructure and cytokine profile.

NRBP1 modulates uric acid transporter ABCG2 expression by activating the Wnt/ß-catenin pathway in HK-2 cells.

BACKGROUND: Nuclear receptor binding protein 1 (NRBP1) and ATP-binding cassette subfamily G member 2 (ABCG2) was the gout risk gene and high-capacity urate exporter respectively. However, the relationship between NRBP1 and ABCG2 and the underlying molecular mechanism contributing to these associations are unknown. METHODS: Firstly, the efficiency of the overexpression and knockdown of NRBP1 was confirmed by western blot. Next, the effect of NRBP1 overexpression and knockdown on the expression of ABCG2, organic anion transporter 1 (OAT1), glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1) was detected by qRT-PCR and western blot. At the same time, the cellular location of ABCG2 and its expression after NRBP1 overexpression and knockdown was tested by immunofluorescence (IF) staining. Then, the mechanism of NRBP1 modulates ABCG2 expression was evaluated by western blot with or without the ß-catenin inhibitor (21H7). RESULTS: The lentivirus system was used to generate stable NRBP1 overexpression, while the plasmids carrying a NRBP1 siRNA was generated to knockdown NRBP1 expression in HK-2 cells. Meanwhile, the overexpression of NRBP1 significantly decreased the mRNAs and proteins expression of GLUT9 and URAT1, while the knockdown of NRBP1 increased the mRNAs and proteins expression of ABCG2 significantly. In addition, the NRBP1 modulates the expression of ABCG2 was by ctivating the Wnt/ß-catenin pathway in HK-2 cells according to the IF and western blot results. CONCLUSION: Taken together, our study demonstrated that NRBP1 inhibition played an essential role in attenuating hyperuricemia and gout by upregulation of ABCG2 via Wnt/ß-catenin signaling pathway in HK-2 cells.

A Common Functional Variant at the Enhancer of the Rheumatoid Arthritis Risk Gene ORMDL3 Regulates its Expression Through Allele-Specific JunD Binding.

Genome-wide association studies (GWASs) have identified over 100 loci associated with rheumatoid arthritis (RA); however, the functionally affected genes and the underlying molecular mechanisms contributing to these associations are often unknown. In this study, we conducted an integrative genomic analysis incorporating multiple "omics" data and identified a functional regulatory DNA variant, rs56199421, and a plausible mechanism by which it regulates the expression of a putative RA risk gene, ORMDL Sphingolipid Biosynthesis Regulator 3 (ORMDL3). The T allele of rs56199421, located in the enhancer region of ORMDL3, exhibited stronger direct binding ability than the other C allele of rs56199421 did in vitro with the transcription factor JunD and demonstrated higher transcriptional activity. Moreover, the T allele of rs56199421 is associated with elevated RA risk, and ORMDL3 expression is increased in RA patients. Thus, these findings suggest that the T allele of rs56199421 enhances JunD transcription factor binding, increases enhancer activity, and elevates the expression of the RA risk gene ORMDL3. Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-023-00107-z.

Ultrasound assessment of skin thickness and stiffness: the correlation with histology and clinical score in systemic sclerosis.

BACKGROUND: Ultrasound is a useful tool to evaluate and quantify skin lesions. Few studies have assessed the criterion validity of skin ultrasound in systemic sclerosis (SSc). The aims of the study were to investigate skin thickness and stiffness using ultrasound and shear wave elastography (SWE) in SSc and to validate skin ultrasound measurements against histological skin thickness. METHODS: A total of 22 patients with diffuse cutaneous SSc (dcSSc), 22 with limited cutaneous SSc (lcSSc), and 22 age- and gender-matched healthy controls were enrolled. Skin thickness and stiffness were measured by B-mode ultrasound with SWE imaging on the bilateral fingers and hands. Additional ultrasound evaluation was carried out in 13 patients (9 dcSSc and 4 lcSSc) on their dorsal forearms, followed by skin biopsy conducted in the same skin areas. Correlations between ultrasound measurements and histological skin thickness and modified Rodnan skin score (mRSS) were investigated using Spearman's correlation. RESULTS: Compared with controls, ultrasound-measured skin thickness and skin stiffness were significantly higher in patients with SSc (p < 0.001) and even higher in those with dcSSc. No clear correlation could be established between ultrasound-determined skin thickness and stiffness at the same site. Ultrasound-measured skin thickness correlated well with histological skin thickness (r = 0.6926, p = 0.009). A weaker association was also observed between histological skin thickness and local mRSS (r = 0.5867, p = 0.050). CONCLUSIONS: Ultrasound is a reliable tool for quantifying skin involvement in SSc. Ultrasound-measured skin thickness showed good agreement with histological skin thickness.

Ligustrazine attenuates inflammation and oxidative stress in a rat model of arthritis via the Sirt1/NF-κB and Nrf-2/HO-1 pathways.

Inflammation responses and oxidative stress are closely involved in the pathogenesis of arthritis. Ligustrazine (Lig), a natural four methyl which is isolated from Chinese herb ligusticum chuanxiong hort, has been proved significantly anti-inflammation and anti-oxidative stress effects. The present study aimed to evaluate the effect of Lig on inflammation and oxidative stress in Freund's complete adjuvant (FCA)-induced arthritis in rats. The treatment of Lig significantly decreased the hind-paw volume change and alleviated the histopathological changes in sections of rat paws induced by arthritis. Lig also reduced the serum levels of pro-inflammatory cytokines (interleukin [IL]-6, IL-1 beta, and tumor necrosis factor-alpha), increased the activity of superoxide dismutase (SOD) and reduced the concentration of malondialdehyde (MDA). Besides that, the protein expressions of the sirtuin 1 (Sirt1)/nuclear factor kappa B (NF-κB) and nuclear factor (erythroid-derived 2)-like-2 factor (Nrf-2)/heme oxygenase (HO)-1 pathways determined by western bolt further confirmed that Lig effectively inhibited the Sirt1/NF-κB pathway and activated the Nrf-2/HO-1 pathway. Taken together, our results suggest Lig has therapeutic potential for arthritis, which might be via the regulation of Sirt1/NF-κB and Nrf-2/HO-1 pathways.

Protective Effect of Tetrandrine in a Rabbit Model of Osteoarthritis.

OBJECTIVES: This study aims to investigate the antiinflammatory effect of tetrandrine in a rabbit model of osteoarthritis. PATIENTS AND METHODS: The cruciate ligaments and medial menisci of 36 female New Zealand white rabbits (each weighing 2.0-3.0 kg) were resected. Six weeks after surgery, the animals were randomly divided into three groups (n=12 in each): tetrandrine (20 mg/kg/day), indomethacin (3 mg/kg/day) and control (saline 10 mL/day) groups. After 14 days of treatment, the results were assessed by macroscopic observation, histological evaluation, measurement of the levels of interleukin-1 beta, tumor necrosis factor-alpha and nitric oxide in the synovial fluid and messenger ribonucleic acid expression of metalloproteinase-3 in synovia. RESULTS: In the tetrandrine group, the pathological changes were more attenuated than in the control group. Levels of interleukin-1 beta, tumor necrosis factor-alpha, nitric oxide and metalloproteinase-3 in the synovial fluid or synovium were all significantly suppressed in the tetrandrine group compared with the control group. There were no significant differences between the tetrandrine and indomethacin groups. CONCLUSION: The results of this study demonstrate that tetrandrine may protect against the development of experimentally induced osteoarthritis.

Clinical characteristics of infectious ulceration over tophi in patients with gout.

Objective The aim of this study was to investigate the clinical characteristics of infectious ulceration over tophi in patients with gout. Methods Participants were recruited from the First Affiliated Hospital of Wenzhou Medical University. The clinical characteristics of the patients and wound characteristics were recorded. Results Of the 38 enrolled patients, 18 were found to have infectious ulceration over tophi. Staphylococcus aureus was the most common pathogen and was identified in nine patients. Patients with infection were significantly older (69.6 vs. 60.1 years) and had a worse quality of life than those without infection. Patients with infection also had a significantly longer ulcer duration (125.6 vs. 54.2 days), larger ulcer size (2.47 vs. 1.99 cm2), a higher rate of tissue necrosis in the ulcer bed (55.6% vs. 20.0%), a lower rate of callus at the edge (27.8% vs. 70.0%), and a higher moisture level than did patients without infection. Additionally, patients with infection had significantly delayed wound healing (35.3 vs. 20.3 days) compared with patients without infection. Conclusions Older patients with a long ulcer duration and larger ulcer size are more susceptible to infection. Infection can lower patients' quality of life and delay wound healing.

NRBP1 modulates uric acid transporter ABCG2 expression by activating the Wnt/B-catenin pathway in HK-2 cells / NRBP1 regula la expresión del transportador de ácido úrico ABCG2 en las células HK-2 mediante la activación de la vía Wnt/B-catenina

Background: Nuclear receptor binding protein 1 (NRBP1) and ATP-binding cassette subfamily G member 2 (ABCG2) was the gout risk gene and high-capacity urate exporter respectively. However, the relationship between NRBP1 and ABCG2 and the underlying molecular mechanism contributing to these associations are unknown. Methods: Firstly, the efficiency of the overexpression and knockdown of NRBP1 was confirmed by western blot. Next, the effect of NRBP1 overexpression and knockdown on the expression of ABCG2, organic anion transporter 1 (OAT1), glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1) was detected by qRT-PCR and western blot. At the same time, the cellular location of ABCG2 and its expression after NRBP1 overexpression and knockdown was tested by immunofluorescence (IF) staining. Then, the mechanism of NRBP1 modulates ABCG2 expression was evaluated by western blot with or without the β-catenin inhibitor (21H7). Results: The lentivirus system was used to generate stable NRBP1 overexpression, while the plasmids carrying a NRBP1 siRNA was generated to knockdown NRBP1 expression in HK-2 cells. Meanwhile, the overexpression of NRBP1 significantly decreased the mRNAs and proteins expression of GLUT9 and URAT1, while the knockdown of NRBP1 increased the mRNAs and proteins expression of ABCG2 significantly. In addition, the NRBP1 modulates the expression of ABCG2 was by ctivating the Wnt/β-catenin pathway in HK-2 cells according to the IF and western blot results. Conclusion: Taken together, our study demonstrated that NRBP1 inhibition played an essential role in attenuating hyperuricemia and gout by upregulation of ABCG2 via Wnt/β-catenin signaling pathway in HK-2 cells. (AU)

Antecedentes: La proteína de unión al receptor nuclear 1 (NRBP1) y el miembro G de la subclase ATP binding Box 2 (ABCG2) son los genes de riesgo de gota y los genes de salida de urato de alto rendimiento, respectivamente. Sin embargo, se desconoce la relación entre NRBP1 y ABCG2, y los posibles mecanismos moleculares que conducen a estas asociaciones. Métodos: En primer lugar, la sobreexpresión y el knockout de NRBP1 fueron confirmados por Western-blot. Los efectos de la sobreexpresión y knockout de NRBP1 en la expresión de ABCG2, transportador de aniones orgánicos 1 (OAT1), transportador de glucosa 9 (GLUT9) y transportador de ácido úrico 1 (URAT1) fueron detectados por qRT-PCR y Western-blot. Mientras tanto, la localización y expresión de ABCG2 después de la sobreexpresión y knockout de NRBP1 fueron detectadas por inmunofluorescencia (IF). Luego, el efecto regulador de NRBP1 sobre la expresión de ABCG2 fue estudiado por Western-blot y comparado con el inhibidor de la β-catenina (21H7). Resultados: El sistema lentiviral indujo una sobreexpresión estable de NRBP1, mientras que el plásmido portador de SiRNA NRBP1 inhibió la expresión de NRBP1 en las células HK-2. Mientras tanto, la sobreexpresión de NRBP1 redujo significativamente la expresión de ARNm y proteínas de GLUT9 y URAT1, mientras que el knockout de NRBP1 aumentó significativamente la expresión de ARNm y proteínas de ABCG2. Además, de acuerdo con los resultados de IF y Western-blot, NRBP1 regula la expresión de ABCG2 activando la vía Wnt/β-catenina en las células HK-2. Conclusión: La inhibición del NRBP1 aumenta la regulación de ABCG2 a través de la vía de señalización Wnt/β-catenina, que desempeña un papel importante en la reducción de la hiperuricemia y la gota. (AU)

DNA hypomethylation of a transcription factor binding site within the promoter of a gout risk gene NRBP1 upregulates its expression by inhibition of TFAP2A binding.

BACKGROUND: Genome-wide association studies (GWASs) have identified dozens of loci associated with gout, but for most cases, the risk genes and the underlying molecular mechanisms contributing to these associations are unknown. This study sought to understand the molecular mechanism of a common genetic variant, rs780093, in the development of gout, both in vitro and in vivo. RESULTS: Nuclear receptor binding protein 1 (NRBP1), as a gout risk gene, and its regulatory region, 72 bp upstream of the transcription start site, designated as B1, were identified through integrative analyses of genome-wide genotype and DNA methylation data. We observed elevated NRBP1 expression in human peripheral blood mononuclear cells (PBMCs) from gout patients. In vitro luciferase reporter and protein pulldown assay results showed that DNA methylation could increase the binding of the transcription factor TFAP2A to B1, leading to suppressed gene expression. There results were further confirmed by in vivo bisulfite pyrosequencing showing that hypomethylation on B1 is associated with increased NRBP1 expression in gout patients. CONCLUSIONS: Hypomethylation at the promoter region of NRBP1 reduces the binding of TFAP2A and thus leads to elevated NRBP1 expression, which might contribute to the development of gout.

DNA methylation mediates genotype and smoking interaction in the development of anti-citrullinated peptide antibody-positive rheumatoid arthritis.

BACKGROUND: Multiple factors, including interactions between genetic and environmental risks, are important in susceptibility to rheumatoid arthritis (RA). However, the underlying mechanism is not fully understood. This study was undertaken to evaluate whether DNA methylation can mediate the interaction between genotype and smoking in the development of anti-citrullinated peptide antibody (ACPA)-positive RA. METHODS: We investigated the gene-smoking interactions in DNA methylation using 393 individuals from the Epidemiological Investigation of Rheumatoid Arthritis (EIRA). The interaction between rs6933349 and smoking in the risk of developing ACPA-positive RA was further evaluated in a larger portion of the EIRA (1119 controls and 944 ACPA-positive patients with RA), and in the Malaysian Epidemiological Investigation of Rheumatoid Arthritis (MyEIRA) (1556 controls and 792 ACPA-positive patients with RA). Finally, mediation analysis was performed to investigate whether DNA methylation of cg21325723 mediates this gene-environment interaction on the risk of developing of ACPA-positive RA. RESULTS: We identified and replicated one significant gene-environment interaction between rs6933349 and smoking in DNA methylation of cg21325723. This gene-smoking interaction is a novel interaction in the risk of developing ACPA-positive in both Caucasian (multiplicative P value = 0.056; additive P value = 0.016) and Asian populations (multiplicative P value = 0.035; additive P value = 0.00027), and it is mediated through DNA methylation of cg21325723. CONCLUSIONS: We showed that DNA methylation of cg21325723 can mediate the gene-environment interaction between rs6933349 and smoking, impacting the risk of developing ACPA-positive RA, thus being a potential regulator that integrates both internal genetic and external environmental risk factors.

X-linked agammaglobulinemia combined with juvenile idiopathic arthritis and invasive Klebsiella pneumoniae polyarticular septic arthritis.

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disease caused by mutations in the Bruton's tyrosine kinase (BTK) gene. XLA can also present in combination with juvenile idiopathic arthritis (JIA), the major chronic rheumatologic disease in children. We report herein the first known case of a juvenile patient diagnosed with XLA combined with JIA that later developed into invasive Klebsiella pneumoniae polyarticular septic polyarthritis. An additional comprehensive review of XLA combined with JIA and invasive K. pneumoniae septic arthritis is also presented. XLA was identified by the detection of BTK mutations while the diagnosis of JIA was established by clinical and laboratory assessments. Septic arthritis caused by invasive K. pneumoniae was confirmed by culturing of the synovia and gene detection of the isolates. Invasive K. pneumoniae infections can not only result in liver abscesses but also septic arthritis, although this is rare. XLA combined with JIA may contribute to invasive K. pneumoniae infection.