Precision application of targeted therapies is urgently needed to improve long-term clinical outcomes for children affected by inflammatory arthritis, known as juvenile idiopathic arthritis (JIA). Progress has been hampered by our limited understanding of the cellular basis of inflammation in the target tissue of the disease, the synovial membrane. Here, we analyzed biopsies from the inflamed joints of treatment-naïve children with JIA, early in the course of their disease, using single-cell RNA sequencing, multiplexed immunofluorescence, and spatial transcriptomics to establish a cellular atlas of the JIA synovium. We identified distinct spatial tissue niches, composed of specific stromal and immune cell populations. In addition, we localized genes linked to arthritis severity and disease risk to effector cell populations, including tissue resident SPP1 ⁺ macrophages and fibrin-associated myeloid cells. Combined analyses of synovial fluid and peripheral blood from matched individuals revealed differences in cellular composition, signaling pathways, and transcriptional programs across these distinct anatomical compartments. Furthermore, our analysis revealed several pathogenic cell populations that are shared with adult-onset inflammatory arthritis, as well as age-associated differences in tissue vascularity, prominence of innate immunity, and enrichment of TGF-β–responsive stromal subsets that up-regulate expression of disease risk–associated genes. Overall, our findings demonstrate the need for age-specific analyses of synovial tissue pathology to guide targeted treatment strategies in JIA.

Digestive organ expansion factor (Def) is a nucleolar protein that plays dual functions: it serves as a component of the ribosomal small subunit processome for the biogenesis of ribosomes and also mediates p53 degradation through the cysteine proteinase calpain-3 (CAPN3). However, nothing is known about the exact relationship between Def and CAPN3 or the regulation of the Def function. In this report, we show that CAPN3 degrades p53 and its mutant proteins p53^A138V, p53^M237I, p53^R248W, and p53^R273P but not the p53^R175H mutant protein. Importantly, we show that Def directly interacts with CAPN3 in the nucleoli and determines the nucleolar localisation of CAPN3, which is a prerequisite for the degradation of p53 in the nucleolus. Furthermore, we find that Def is modified by phosphorylation at five serine residues: S50, S58, S62, S87, and S92. We further show that simultaneous phosphorylations at S87 and S92 facilitate the nucleolar localisation of Capn3 that is not only essential for the degradation of p53 but is also important for regulating cell cycle progression. Hence, we propose that the Def-CAPN3 pathway serves as a nucleolar checkpoint for cell proliferation by selective inactivation of cell cycle-related substrates during organogenesis. The nucleolus is primarily known as the subcellular organelle for the biogenesis of the ribosomal small and large subunits in eukaryotic cells. However, it is also increasingly evident that many nucleolar proteins also play essential roles in other biological processes. Here, we show that Digestive organ expansion factor (Def), a nucleolar protein, determines the nucleolar localisation of the cysteine proteinase CAPN3, and they form a complex that degrades the tumor suppressor protein p53, an essential regulator of the cell cycle, in the nucleolus. Furthermore, we show that Def is modified by phosphorylation at five serine residues and that Def phosphorylation modification facilitates the nucleolar localization of Capn3, which is also important for regulating cell cycle progression. Hence, we propose that the Def-CAPN3 pathway serves as a nucleolar checkpoint of cell proliferation by selective inactivation of cell cycle-related substrates during organogenesis, revealing a novel biological role for the nucleolus.

Spatial transcriptomics and proteomics have enabled profound insights into tissue organization, yet these technologies remain largely disparate, and emerging same-slide multiomics approaches are limited in plex, spatial resolution, signal retention, and integrative analytics. We introduce IN-situ DEtailed Phenotyping To High-resolution transcriptomics (IN-DEPTH), a streamlined, resource-efficient, commercially compatible workflow using single-cell spatial proteomics-derived imaging to guide transcriptomic capture on the same slide without RNA signal loss. To integrate modalities beyond niche-level mapping, we developed Spectral Graph Cross-Correlation (SGCC), a proteomic–transcriptomic framework resolving spatially coordinated functional state changes across interacting cell populations. Applied to diffuse large B-cell lymphoma (DLBCL), IN-DEPTH and SGCC enabled stepwise discovery from Epstein–Barr virus (EBV)–positive and EBV-negative tumor comparisons with single-cell resolution, revealing coordinated tumor–macrophage–CD4 T-cell remodeling, immunosuppressive C1Q macrophage enrichment, CD4 T-cell dysfunction, and a candidate IL27–STAT3 signaling axis. Collectively, IN-DEPTH enables scalable spatial multiomics to uncover clinically relevant microenvironmental mechanisms and toward robust spatial multimodal AI models. Significance: IN-DEPTH enables same-slide spatial multiomics across commercial platforms via a protein-first strategy preserving protein epitopes, RNA quality, and tissue integrity. Coupled with SGCC, it resolves coordinated spatial immune remodeling, revealing EBV/LMP1-driven C1Q macrophage polarization and CD4 T-cell dysfunction in DLBCL, with broad applicability to other diseases.

In chronic inflammatory diseases, maladaptive tissue remodeling is driven by a complex interplay of resident cells, immune infiltrates, and the extracellular matrix. In the autoimmune disorder rheumatoid arthritis (RA), synovial tissue undergoes massive expansion to form an invasive pannus that drives the erosion of cartilage and bone. The mechanisms mediating this aggressive growth are incompletely defined. Using spatial transcriptomic profiling of patient tissue, we detected an abundance of proliferating fibroblasts near the synovial tissue lining surface and adjacent to macrophages expressing high levels of secreted phosophoprotein-1 (encoding osteopontin; SPP1 ^hi macrophages). These synovial lining regions were also distinctly marked by deposits of the clot-forming protein fibrin. Although the SPP1 ^hi macrophages phenotypically resembled the profibrotic macrophages that drive lung and liver fibrosis, these niches were devoid of the dense highly ordered collagen that marks fibrosis. Functionally, we found that SPP1 ^hi macrophages degraded and phagocytosed fibrin matrices and promoted fibroblast proliferation. Given that fibrin provides transient matrices for de novo tissue generation in the context of wound healing, these data support a model of hyperplastic tissue outgrowth involving SPP1 ^hi macrophages, fibroblasts, and fibrin matrices adhered to the exterior synovial tissue surface. Whereas current RA therapies primarily aim to dampen proinflammatory responses, our findings provide the rationale for targeting progenerative pathways and SPP1 ^hi macrophages.

Myofibroblast differentiation, essential for driving extracellular matrix synthesis in pulmonary fibrosis, requires increased glycolysis. Although glycolytic cells must export lactate, the contributions of lactate transporters to myofibroblast differentiation are unknown. In this study, we investigated how monocarboxylate transporters (MCTs) 1 and 4, key pulmonary lactate transporters, influence myofibroblast differentiation and experimental pulmonary fibrosis. Our findings revealed that inhibiting MCT1 or MCT4 using RNA interference or small molecules reduced transforming growth factor–β1 (TGFβ)–stimulated myofibroblast differentiation in lung fibroblasts from healthy donors and patients with idiopathic pulmonary fibrosis. Small-molecule MCT inhibitors also decreased bleomycin-induced pulmonary fibrosis in C57Bl6/N mice aged 10 to 12 weeks. Through bioenergetic analyses, stable isotope tracing, metabolomics, and imaging mass spectrometry in both human cells and mice, we demonstrate that inhibiting lactate transport enhanced oxidative phosphorylation, reduced reactive oxygen species production, and diminished glucose metabolite incorporation into fibrotic lung regions. Furthermore, we introduce VB253, an MCT4 inhibitor, which ameliorates pulmonary fibrosis in both young and aged mice, with comparable efficacy to established antifibrotic therapies. These results underscore the necessity of lactate transport for myofibroblast differentiation, identify MCT1 and MCT4 as promising pharmacologic targets in pulmonary fibrosis, and support further evaluation of lactate transport inhibitors as a therapy for patients with limited treatment options.