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Preclinical and clinical studies suggest that consumption of long chain omega-3 polyunsaturated fatty acids (PUFAs) reduces severity of chronic inflammatory and autoimmune diseases. While these ameliorative effects are conventionally associated with downregulated expression of proinflammatory cytokine and chemokine genes, our laboratory has recently identified Type 1 interferon (IFN1)-regulated gene expression to be another key target of omega-3 PUFAs. Here we used single cell RNA sequencing (scRNAseq) to gain new mechanistic perspectives on how the omega-3 PUFA docosahexaenoic acid (DHA) influences TLR4-driven proinflammatory and IFN1-regulated gene expression in a novel self-renewing murine fetal liver-derived macrophage (FLM) model. FLMs were cultured with 25 µM DHA or vehicle for 24 h, treated with modest concentration of LPS (20 ng/ml) for 1 and 4 h, and then subjected to scRNAseq using the 10X Chromium System. At 0 h (i.e., in the absence of LPS), DHA increased expression of genes associated with the NRF2 antioxidant response (e.g. Sqstm1, Hmox1, Chchd10) and metal homeostasis (e.g.Mt1, Mt2, Ftl1, Fth1), both of which are consistent with DHA-induced polarization of FLMs to a more anti-inflammatory phenotype. At 1 h post-LPS treatment, DHA inhibited LPS-induced cholesterol synthesis genes (e.g. Scd1, Scd2, Pmvk, Cyp51, Hmgcs1, and Fdps) which potentially could contribute to interference with TLR4-mediated inflammatory signaling. At 4 h post-LPS treatment, LPS-treated FLMs reflected a more robust inflammatory response including upregulation of proinflammatory cytokine (e.g. Il1a, Il1b, Tnf) and chemokine (e.g.Ccl2, Ccl3, Ccl4, Ccl7) genes as well as IFN1-regulated genes (e.g. Irf7, Mx1, Oasl1, Ifit1), many of which were suppressed by DHA. Using single-cell regulatory network inference and clustering (SCENIC) to identify gene expression networks, we found DHA modestly downregulated LPS-induced expression of NF-κB-target genes. Importantly, LPS induced a subset of FLMs simultaneously expressing NF-κB- and IRF7/STAT1/STAT2-target genes that were conspicuously absent in DHA-pretreated FLMs. Thus, DHA potently targeted both the NF-κB and the IFN1 responses. Altogether, scRNAseq generated a valuable dataset that provides new insights into multiple overlapping mechanisms by which DHA may transcriptionally or post-transcriptionally regulate LPS-induced proinflammatory and IFN1-driven responses in macrophages.

International audience; Chronic low back pain is the number one cause of years lived with disability. In about 40% of patients, chronic lower back pain is related to intervertebral disc (IVD) degeneration. The standard-of-care focuses on symptomatic relief, while surgery is the last resort. Emerging therapeutic strategies target the underlying cause of IVD degeneration and increasingly focus on the relatively overlooked notochordal cells (NCs). NCs are derived from the notochord and once the notochord regresses they remain in the core of the developing IVD, the nucleus pulposus. The large vacuolated NCs rapidly decline after birth and are replaced by the smaller nucleus pulposus cells with maturation, ageing, and degeneration. Here, we provide an update on the journey of NCs and discuss the cell markers and tools that can be used to study their fate and regenerative capacity. We review the therapeutic potential of NCs for the treatment of IVD-related lower back pain and outline important future directions in this area. Promising studies indicate that NCs and their secretome exerts regenerative effects, via increased proliferation, extracellular matrix production, and anti-inflammatory effects. Reports on NC-like cells derived from embryonic- or induced pluripotent-stem cells claim to have successfully generated NC-like cells but did not compare them with native NCs for phenotypic markers or in terms of their regenerative capacity. Altogether, this is an emerging and active field of research with exciting possibilities. NC-based studies demonstrate that cues from developmental biology can pave the path for future clinical therapies focused on regenerating the diseased IVD.

Supplemental material, sj-docx-2-car-10.1177_19476035221081469 for Folate Receptor Expression by Human Monocyte���Derived Macrophage Subtypes and Effects of Corticosteroids by Kelly Warmink, Michiel Siebelt, Philip S. Low, Frank M. Riemers, Bingbing Wang, Saskia G. M. Plomp, Marianna A. Tryfonidou, P. Ren�� van Weeren, Harrie Weinans and Nicoline M. Korthagen in CARTILAGE

Objective Folate receptor beta (FR-β) has been used as a clinical marker and target in multiple inflammatory diseases, including osteoarthritis (OA) and rheumatoid arthritis (RA). However, the conditions under which FR-β+ macrophages arise remain unclear and could be affected by corticosteroids. Therefore, we studied FR-β expression in vitro in macrophage subtypes and determined their response to triamcinolone acetonide (TA), a clinically often-used corticosteroid. Design Human monocyte–derived macrophages were differentiated to the known M0, M1, or M2 macrophage phenotypes. The phenotype and FR-β expression and plasticity of the macrophage subtypes were determined using flow cytometry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA). Results FR-β expression was low in granulocyte-macrophage colony-stimulating factor (GM-CSF)-generated (M1-like) macrophages and high in macrophage colony-stimulating factor (M-CSF)-generated (M0 and M2-like) macrophages. FR-β expression remained high once the M0 or M2 macrophages were stimulated with pro-inflammatory stimuli (interferon-γ plus lipopolysaccharide) to induce M1-like macrophages. On the contrary, anti-inflammatory TA treatment skewed GM-CSF macrophage differentiation toward an M2 and FR-β+ phenotype. Conclusions As corticosteroids skewed monocytes toward an FR-β-expressing, anti-inflammatory phenotype, even in an M1 priming GM-CSF environment, FR-β has potential as a biomarker to monitor success of treatment with corticosteroids. Without corticosteroid treatment, M-CSF alone induces high FR-β expression which remains high under pro-inflammatory conditions. This explains why pro-inflammatory FR-β+ macrophages (exposed to M-CSF) are observed in arthritis patients and correlate with disease severity.

Supplemental material, sj-docx-1-car-10.1177_19476035221081469 for Folate Receptor Expression by Human Monocyte���Derived Macrophage Subtypes and Effects of Corticosteroids by Kelly Warmink, Michiel Siebelt, Philip S. Low, Frank M. Riemers, Bingbing Wang, Saskia G. M. Plomp, Marianna A. Tryfonidou, P. Ren�� van Weeren, Harrie Weinans and Nicoline M. Korthagen in CARTILAGE

Objective Joint distraction triggers intrinsic cartilage repair in animal models of osteoarthritis (OA), corroborating observations in human OA patients treated with joint distraction. The present study explores the still largely elusive mechanism initiating this repair process. Design Unilateral OA was induced in the knee joint of 8 dogs using the groove model; the contralateral joint served as a control. After 10 weeks, 4 animals received joint distraction, the other 4 serving as OA controls. Halfway the distraction period (after 4 weeks of a standard 8-week distraction treatment), all animals were euthanized, and joint tissues were collected. A targeted quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis was performed of commonly involved processes including matrix catabolism/anabolism, inflammation, and known signaling pathways in OA. In addition, cartilage changes were determined on tissue sections using the canine OARSI (Osteoarthritis Research Society International) histopathology score and collagen type II (COL2A1) immunostaining. Results Midway distraction, the distracted OA joint showed an upregulation of proteolytic genes, for example, ADAMTS5, MMP9, MMP13, compared to OA alone and the healthy joints, which correlated with an increased OARSI score. Additionally, genes of the transforming growth factor (TGF)-β and Notch pathway, and markers associated with progenitor cells were increased. Conclusions Joint distraction initiates both catabolic and anabolic transcriptional responses. The enhanced turnover, and thereby renewal of the matrix, could be the key to the cartilage repair observed in the months after joint distraction.