The complement system drives local inflammatory tissue priming by metabolic reprogramming of synovial fibroblasts.

Arthritis typically involves recurrence and progressive worsening at specific predilection sites, but the checkpoints between remission and persistence remain unknown. Here, we defined the molecular and cellular mechanisms of this inflammation-mediated tissue priming. Re-exposure to inflammatory stimuli caused aggravated arthritis in rodent models. Tissue priming developed locally and independently of adaptive immunity. Repeatedly stimulated primed synovial fibroblasts (SFs) exhibited enhanced metabolic activity inducing functional changes with intensified migration, invasiveness and osteoclastogenesis. Meanwhile, human SF from patients with established arthritis displayed a similar primed phenotype. Transcriptomic and epigenomic analyses as well as genetic and pharmacological targeting demonstrated that inflammatory tissue priming relies on intracellular complement C3- and C3a receptor-activation and downstream mammalian target of rapamycin- and hypoxia-inducible factor 1α-mediated metabolic SF invigoration that prevents activation-induced senescence, enhances NLRP3 inflammasome activity, and in consequence sensitizes tissue for inflammation. Our study suggests possibilities for therapeutic intervention abrogating tissue priming without immunosuppression.

Notch signalling drives synovial fibroblast identity and arthritis pathology.

The synovium is a mesenchymal tissue composed mainly of fibroblasts, with a lining and sublining that surround the joints. In rheumatoid arthritis the synovial tissue undergoes marked hyperplasia, becomes inflamed and invasive, and destroys the joint1,2. It has recently been shown that a subset of fibroblasts in the sublining undergoes a major expansion in rheumatoid arthritis that is linked to disease activity3-5; however, the molecular mechanism by which these fibroblasts differentiate and expand is unknown. Here we identify a critical role for NOTCH3 signalling in the differentiation of perivascular and sublining fibroblasts that express CD90 (encoded by THY1). Using single-cell RNA sequencing and synovial tissue organoids, we found that NOTCH3 signalling drives both transcriptional and spatial gradients-emanating from vascular endothelial cells outwards-in fibroblasts. In active rheumatoid arthritis, NOTCH3 and Notch target genes are markedly upregulated in synovial fibroblasts. In mice, the genetic deletion of Notch3 or the blockade of NOTCH3 signalling attenuates inflammation and prevents joint damage in inflammatory arthritis. Our results indicate that synovial fibroblasts exhibit a positional identity that is regulated by endothelium-derived Notch signalling, and that this stromal crosstalk pathway underlies inflammation and pathology in inflammatory arthritis.

The effect of 1.5 T cardiac magnetic resonance on human circulating leucocytes.

Aims: Investigators have proposed that cardiovascular magnetic resonance (CMR) should have restrictions similar to those of ionizing imaging techniques. We aimed to investigate the acute effect of 1.5 T CMR on leucocyte DNA integrity, cell counts, and function in vitro, and in a large cohort of patients in vivo. Methods and results: In vitro study: peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers, and histone H2AX phosphorylation (γ-H2AX) expression, leucocyte counts, and functional parameters were quantified using flow cytometry under the following conditions: (i) immediately following PBMC isolation, (ii) after standing on the benchside as a temperature and time control, (iii) after a standard CMR scan. In vivo study: blood samples were taken from 64 consecutive consenting patients immediately before and after a standard clinical scan. Samples were analysed for γ-H2AX expression and leucocyte counts. CMR was not associated with a significant change in γ-H2AX expression in vitro or in vivo, although there were significant inter-patient variations. In vitro cell integrity and function did not change with CMR. There was a significant reduction in circulating T cells in vivo following CMR. Conclusion: 1.5 T CMR was not associated with DNA damage in vitro or in vivo. Histone H2AX phosphorylation expression varied markedly between individuals; therefore, small studies using γ-H2AX as a marker of DNA damage should be interpreted with caution. Cardiovascular magnetic resonance was not associated with loss of leucocyte viability or function in vitro. Cardiovascular magnetic resonance was associated with a statistically significant reduction in viable leucocytes in vivo.

Fibroblasts Derived From Vestibular Schwannoma Express Protumorogenic Markers.

BACKGROUND AND AIM: Vestibular schwannomas (VSs), despite being histologically benign, cause significant morbidity because of their challenging intracranial location and the propensity for growth. The role of the stroma and particularly fibroblasts, in the progression of VS, is not completely understood. This study examines the profile of fibroblasts in VS. METHODS: Seventeen patients undergoing surgical excision of VS were recruited into the study. Reverse transcription with quantitative polymerase chain reaction (RT-qPCR) was performed on VS tissue samples and fibroblast-associated molecules examined. Immunofluorescence and immunohistochemistry in VS tissue were used to study the expression of fibroblast markers CD90 and podoplanin in situ. Fibroblast cultures were established from VS, and RT-qPCR analysis was performed on a panel of fibroblast markers on VS and control tissue fibroblasts. RESULTS: Several fibroblast-associated molecules including members of galectin family and matrix metalloproteinases were found to be expressed in VS tissue on RT-qPCR analysis. In situ, expression of CD90 and podoplanin was observed in VS tissue both on immunohistochemistry and immunofluorescence. RT-qPCR analysis of fibroblasts from VS and control vestibular neuroepithelium (NE) showed a higher expression of several molecules of the galectin and matrix metalloproteinases family on VS fibroblasts compared with NE fibroblasts. CONCLUSION: This work examines fibroblasts from VS and shows qualitative differences from NE fibroblasts on RT-qPCR. Further understanding of the fibroblast function in the progression of VS will potentially unveil new targets to manage VS growth.

Tissue-resident, extravascular Ly6c- monocytes are critical for inflammation in the synovium.

Monocytes are abundant immune cells that infiltrate inflamed organs. However, the majority of monocyte studies focus on circulating cells, rather than those in tissue. Here, we identify and characterize an intravascular synovial monocyte population resembling circulating non-classical monocytes and an extravascular tissue-resident monocyte-lineage cell (TR-MC) population distinct in surface marker and transcriptional profile from circulating monocytes, dendritic cells, and tissue macrophages that are conserved in rheumatoid arthritis (RA) patients. TR-MCs are independent of NR4A1 and CCR2, long lived, and embryonically derived. TR-MCs undergo increased proliferation and reverse diapedesis dependent on LFA1 in response to arthrogenic stimuli and are required for the development of RA-like disease. Moreover, pathways that are activated in TR-MCs at the peak of arthritis overlap with those that are downregulated in LFA1-/- TR-MCs. These findings show a facet of mononuclear cell biology that could be imperative to understanding tissue-resident myeloid cell function in RA.

Cross-tissue, single-cell stromal atlas identifies shared pathological fibroblast phenotypes in four chronic inflammatory diseases.

BACKGROUND: Pro-inflammatory fibroblasts are critical for pathogenesis in rheumatoid arthritis, inflammatory bowel disease, interstitial lung disease, and Sjögren's syndrome and represent a novel therapeutic target for chronic inflammatory disease. However, the heterogeneity of fibroblast phenotypes, exacerbated by the lack of a common cross-tissue taxonomy, has limited our understanding of which pathways are shared by multiple diseases. METHODS: We profiled fibroblasts derived from inflamed and non-inflamed synovium, intestine, lungs, and salivary glands from affected individuals with single-cell RNA sequencing. We integrated all fibroblasts into a multi-tissue atlas to characterize shared and tissue-specific phenotypes. FINDINGS: Two shared clusters, CXCL10+CCL19+ immune-interacting and SPARC+COL3A1+ vascular-interacting fibroblasts, were expanded in all inflamed tissues and mapped to dermal analogs in a public atopic dermatitis atlas. We confirmed these human pro-inflammatory fibroblasts in animal models of lung, joint, and intestinal inflammation. CONCLUSIONS: This work represents a thorough investigation into fibroblasts across organ systems, individual donors, and disease states that reveals shared pathogenic activation states across four chronic inflammatory diseases. FUNDING: Grant from F. Hoffmann-La Roche (Roche) AG.

Pro-IL-1ß Is an Early Prognostic Indicator of Severe Donor Lung Injury During Ex Vivo Lung Perfusion.

BACKGROUND: Ex vivo lung perfusion (EVLP) is used to evaluate and recondition extended criteria donor lungs for transplantation. Interleukin-1ß (IL-1ß) has been identified as a prognostic indicator of nonrecovery during EVLP. This may be an effect of inflammasome activation or cellular necrosis following donation and graft preservation. Delineating the mechanism of IL-1ß release is required. METHODS: The inactive intracellular precursor molecule, pro-IL-1ß, was characterized along with the pro-IL-1ß processing enzyme, caspase-1, in the perfusate of n = 20 human lungs that had undergone EVLP (n = 10 lungs that failed to recover and were discarded versus n = 10 lungs that reconditioned and were transplanted). In an experimental porcine model, n = 8 lungs underwent EVLP and were randomized to receive either a specific NLRP3 inflammasome inhibitor or control. RESULTS: Significant increases in pro-IL-1ß and caspase-1 were observed in the perfusate from human lungs that did not recondition during EVLP compared with those that successfully reconditioned and were used for transplantation. Within the porcine EVLP, NLRP3 inflammasome inhibition reduced IL-1ß within the perfusate compared with controls, but this had no impact on lung function, hemodynamics, or inflammation. CONCLUSIONS: Our data suggest that pro-IL-1ß is passively released following cellular necrosis of the donor lung.

Inflammation causes remodeling of mitochondrial cytochrome c oxidase mediated by the bifunctional gene C15orf48.

Dysregulated mitochondrial function is a hallmark of immune-mediated inflammatory diseases. Cytochrome c oxidase (CcO), which mediates the rate-limiting step in mitochondrial respiration, is remodeled during development and in response to changes of oxygen availability, but there has been little study of CcO remodeling during inflammation. Here, we describe an elegant molecular switch mediated by the bifunctional transcript C15orf48, which orchestrates the substitution of the CcO subunit NDUFA4 by its paralog C15ORF48 in primary macrophages. Expression of C15orf48 is a conserved response to inflammatory signals and occurs in many immune-related pathologies. In rheumatoid arthritis, C15orf48 mRNA is elevated in peripheral monocytes and proinflammatory synovial tissue macrophages, and its expression positively correlates with disease severity and declines in remission. C15orf48 is also expressed by pathogenic macrophages in severe coronavirus disease 2019 (COVID-19). Study of a rare metabolic disease syndrome provides evidence that loss of the NDUFA4 subunit supports proinflammatory macrophage functions.

New Developments in Transcriptomic Analysis of Synovial Tissue.

Transcriptomic technologies are constantly changing and improving, resulting in an ever increasing understanding of gene expression in health and disease. These technologies have been used to investigate the pathological changes occurring in the joints of rheumatoid arthritis patients, leading to discoveries of disease mechanisms, and novel potential therapeutic targets. Microarrays were initially used on both whole tissue and cell subsets to investigate research questions, with bulk RNA sequencing allowing for further elaboration of these findings. A key example is the classification of pathotypes in rheumatoid arthritis using RNA sequencing that had previously been discovered using microarray and histology. Single-cell sequencing has now delivered a step change in understanding of the diversity and function of subpopulations of cells, in particular synovial fibroblasts. Future technologies, such as high resolution spatial transcriptomics, will enable step changes integrating single cell transcriptomic and geographic data to provide an integrated understanding of synovial pathology.

Ex Vivo Normothermic Perfusion Induces Donor-Derived Leukocyte Mobilization and Removal Prior to Renal Transplantation.

INTRODUCTION: Ex vivo normothermic perfusion offers an alternative method of organ preservation, allowing donor kidneys to be reanimated and evaluated prior to transplantation. Beyond preservation, it can be used to characterize the immunological contribution of the donor kidney in isolation. Furthermore, it has the potential to be used as an immunomodulatory strategy to manipulate donor kidneys prior to transplantation. METHODS: Explanted porcine kidneys underwent 6 hours of perfusion. Sequential perfusate samples were collected and leukocytes characterized via flow cytometry. An inflammatory profile was generated via cytokine quantification. Cell-free DNA was also determined as markers of cell death. RESULTS: All kidneys functioned within normal parameters and met the criteria for transplantation at the end of perfusion. Throughout perfusion there were continuous increases in pro-inflammatory cytokines, including large concentrations of interferon-γ, suggesting that perfusion drives a significant inflammatory response. Increasing concentrations in cell-free DNA were also observed, suggesting cell death. During perfusion there was a marked cellular diapedesis of T cells, B cells, natural killer (NK) cells, and monocytes from the kidney into the circuit. Minor populations of granulocytes and macrophages were also detected. DISCUSSION: We demonstrate that ex vivo normothermic perfusion initiates an inflammatory cytokine storm and release of mitochondrial and genomic DNA. This is likely to be responsible for immune cell activation and mobilization into the circuit prior to transplantation. Interestingly this did not have an impact on renal function. These data therefore suggest that normothermic perfusion can be used to immunodeplete and to saturate the pro-inflammatory capacity of donor kidneys prior to transplantation.

Human mesenchymal stem cells stimulate EaHy926 endothelial cell migration: combined proteomic and in vitro analysis of the influence of donor-donor variability.

Mesenchymal stem cells (MSCs) stimulate angiogenesis within a wound environment and this effect is mediated through paracrine interactions with the endothelial cells present. Here we report that human MSC-conditioned medium (n=3 donors) significantly increased EaHy-926 endothelial cell adhesion and cell migration, but that this stimulatory effect was markedly donor-dependent. MALDI-TOF/TOF mass spectrometry demonstrated that whilst collagen type I and fibronectin were secreted by all of the MSC cultures, the small leucine rich proteoglycan, decorin was secreted only by the MSC culture that was least effective upon EaHy-926 cells. These individual extracellular matrix components were then tested as culture substrata. EaHy-926 cell adherence was greatest on fibronectin-coated surfaces with least adherence on decorin-coated surfaces. Scratch wound assays were used to examine cell migration. EaHy-926 cell scratch wound closure was quickest on substrates of fibronectin and slowest on decorin. However, EaHy-926 cell migration was stimulated by the addition of MSC-conditioned medium irrespective of the types of culture substrates. These data suggest that whilst the MSC secretome may generally be considered angiogenic, the composition of the secretome is variable and this variation probably contributes to donor-donor differences in activity. Hence, screening and optimizing MSC secretomes will improve the clinical effectiveness of pro-angiogenic MSC-based therapies.