Angiogenesis, integral to the pathogenesis of rheumatoid arthritis (RA), contributes to the advancement of synovial hyperplasia, pannus formation, and bone destruction. The precise effect of the Huayu Tongbi formula (HT), a proven traditional Chinese medicine for RA treatment, on angiogenesis remains uncertain. To investigate the effects of HT on RA angiogenesis and the potential underlying molecular mechanisms. The target spectrum and pharmacological effects of HT were screened using network pharmacology. In vitro, RA-FLS were stimulated with CoCl₂ to simulate hypoxic conditions. Human umbilical vein endothelial cells (HUVECs) were cultured with conditioned medium (CM) from RA-FLS supplemented with HT or the HIF1A inhibitor LW-6 to investigate the impact of synovial cell secretions on angiogenesis, encompassing proliferation, migration, adhesion, and lumen formation. Western blot was employed to assess protein levels of hypoxia-inducible factor 1 subunit alpha (HIF1A), vascular endothelial growth factor receptor 2 (VEGFR2), and phosphorylated VEGFR2 (p-VEGFR2). Enzyme-linked immunosorbent assay (ELISA) was utilized to quantify VEGFA secretion, while quantitative real-time PCR (qRT‒PCR) measured mRNA expression of VEGFA, Angiopoietin 1 (ANGPT1), and ANGPT2. In vivo, a collagen-induced arthritis (CIA) model was established. Pathological changes and vessel counts were observed via hematoxylin-eosin (HE) staining, bone structure was evaluated using X-ray, mature and immature vessels were classified via immunofluorescence, and the mechanism was further verified using immunohistochemistry and ELISA. Liquid chromatography‒mass spectrometry (LC‒MS) was employed to identify the active components of HT drug-containing serum, and the potential metabolites were identified by bioinformatics analysis. Network pharmacology analysis indicated potential targets of HT in angiogenesis, correlating with HIF1A and VEGF pathways. In vitro, HT reversed proliferation, migration, and adhesion of the cell lines and suppressed luminal formation by HUVECs. HT (10 % and 15 %) effectively reduced HIF1A and VEGFA expression, attenuated VEGFR2 levels and its phosphorylation, and downregulated VEGFA, ANGPT1, and ANGPT2 mRNA levels. In vivo, HT mitigated inflammation and bone destruction, reduced immature (CD31+/α-SMA-) and mature (CD31+/α-SMA+) vessel counts in the synovium, and inhibited the HIF1A-VEGFA-ANGPT axis. Finally, 19 active components from HT drug-containing serum were successfully identified, and we also unexpectedly discovered that HT might affect metabolic pathways such as proline and tyrosine metabolism, which have been reported to be involved in angiogenesis. HT has a promising effect on alleviating RA angiogenesis, at least in part through HIF1A-VEGFA-ANGPT axis inhibition, which reduces the cross-talk between RA-FLS and HUVECs. Our results suggest that HT is a drug candidate for the treatment of RA angiogenesis.

Gout is a chronic disease caused by monosodium urate crystal deposition. Previous studies have focused on the resident macrophage, infiltrating monocyte, and neutrophil responses to monosodium urate crystal, yet the mechanisms of the potential involvement of other immune cells remain largely unknown. In this study, we enrolled seven gout patients and five age-matched healthy individuals and applied single-cell mass cytometry to study the distribution of immune cell subsets in peripheral blood. To our knowledge, our study reveals the immune cell profiles of gout at different stages for the first time. We identified many immune cell subsets that are dysregulated in gout and promote gouty inflammation, especially those highly expressing CCR4 and OX40 (TNFR superfamily member 4), including CCR4+OX40+ monocytes, CCR4+OX40+CD56high NK cells, CCR4+OX40+CD4+ NK T cells, and CCR4+CD38+CD4+ naïve T cells. Notably, the plasma levels of CCL17 and CCL22, measured by ELISA, increased in the acute phase of gout and declined in the interval. We also found a clue that Th2-type immune responses may participate in gout pathology. Moreover, the subset of granzyme B+ (GZMB+) CD38+ NK cells is positively correlated with serum urea acid level, and another two γδT subsets, GZMB+CD161+ γδT cells and GZMB+CCR5+ γδT cells, are negatively correlated with erythrocyte sedimentation rate. In sum, gouty arthritis is not a disease simply mediated by macrophages; multiple types of immune cell may be involved in the pathogenesis of the disease. Future research needs to shift attention to other immune cell subsets, such as NK cells and T cells, which will facilitate the identification of novel therapeutic targets.

Besides genetic alterations, the cellular environment also determines disease onset and progression. When different cell types contribute to disease outcome, this imposes environmental challenges as different cell types likely differ in their extracellular dependencies. Hsa‐microRNA‐31‐5p (miR‐31) is highly expressed in keratinocytes of psoriatic skin, and we show that expression in keratinocytes is induced by limited glucose availability and enables increased survival under limiting glucose conditions by increasing glutamine metabolism. In addition, miR‐31 expression results in not only secretion of specific metabolites (aspartate and glutamate) but also secretion of immunomodulatory factors. We show that this miR‐31‐induced secretory phenotype is sufficient to induce Th17 cell differentiation, a hallmark of psoriasis. Inhibitors of miR31‐induced metabolic rewiring and metabolic crosstalk with immune cells alleviate psoriasis pathology in a mouse model of psoriasis. Together our data illustrate an emerging concept of metabolic interaction across cell compartments that characterizes disease development, which can be employed to design effective treatment options for disease, as shown here for psoriasis.

A considerable body of evidence supports a role for type-I IFN in the pathogenesis of primary SS (pSS). As plasmacytoid dendritic cells (pDCs) are a major source of type-I IFN, we investigated their molecular regulation by measuring expression of a large set of miRNAs. pDCs were isolated from peripheral blood of pSS patients (n = 30) and healthy controls (n = 16) divided into two independent cohorts (discovery and replication). Screening of 758 miRNAs was assessed by an OpenArray quantitative PCR-based technique; replication of a set of identified miRNAs was performed by custom array. Functional annotation of miRNA targets was performed using pathway enrichment. Novel targets of miR-29a and miR-29c were identified using a proteomic approach (stable isotope labelling with amino acids in cell culture). In the discovery cohort, 20 miRNAs were differentially expressed in pSS pDCs compared with healthy control pDCs. Of these, differential expression of 10 miRNAs was confirmed in the replication cohort. The dysregulated miRNAs were involved in phosphoinositide 3-kinase-Ak strain transforming and mammalian target of rapamycin signalling, as well as regulation of cell death. In addition, a set of novel protein targets of miR-29a and miR-29c were identified, including five targets that were regulated by both miRs. The dysregulated miRNome in pDCs of patients with pSS is associated with aberrant regulation of processes at the centre of pDC function, including type-I IFN production and cell death. As miR-29a and miR-29c are pro-apoptotic factors and several of the novel targets identified here are regulators of apoptosis, their downregulation in patients with pSS is associated with enhanced pDC survival.

To assess whether a Methotrexate-based therapy could achieve more clinical benefit, we arranged a Simon 2-Stage Phase 1 Trial. Single-cell RNA sequencing and lipidomic profiling were performed to reveal the potential mechanisms. Patients were enrolled in an open-label, Simon 2-stage, single-center, single-arm trial at Guangdong Provincial Hospital of Chinese Medicine. Main inclusion criteria were defined as follows: Aged 18 to 70, low to medium disease activity, fulfilled the RA classification criteria of EULAR/ACR 2010. Patients received the oral medication of MTX 10-15 mg weekly and natural product granules twice a day. Primary outcome was the American College of Rheumatology (ACR) 20% preliminary definition of improvement. Single-cell RNA sequencing(scRNA-seq) on peripheral blood mononuclear cells (PBMCs) was used to show the aberrant metabolism before and after the trial. Plasma lipidomic profiling quantified the lipid changes caused by this MTX-based therapy. Finally, post-hoc analysis on responders and non-responders were used for further analysis. Between October 2020 and June 2022, 46 patients received treatment, while 42 finished follow-ups. 27 of 46 (58.70%) patients achieved ACR20, and significant changes were observed in several secondary outcomes. Comparative scRNA-seq analysis before and after the treatment revealed that lipidomic metabolism was broadly downregulated. Plasma lipidomic profiling reveals that 40 lipids were observed significantly changed. Post-hoc analysis showed the lipid changes were separately linked to clinical parameters in responders and non-responders. The study reveals that the combination therapy of HQT+MTX is effective and has a certain correlation with lipid metabolism, but more rigorous study design is still needed to confirm this speculation.