Effects of Notch signaling on the lineage commitment of human peripheral blood monocyte trilineage progenitor under inflammatory conditions

Effects of Notch signaling on the lineage commitment of human peripheral blood monocyte trilineage progenitor under inflammatory conditions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Effects of Notch signaling on the lineage commitment of human peripheral blood monocyte trilineage progenitor under inflammatory conditions Danka Grcevic, Sara Aničić, Maša Filipović, Ivo Krešić, Ozana Jakšić, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6843542/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Nov, 2025 Read the published version in Cell Death Discovery → Version 1 posted 11 You are reading this latest preprint version Abstract Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease, causing significant morbidity and disability. Inflammation-induced activation of myeloid cells is involved in disease pathogenesis and contributes to joint destruction. Due to the significant plasticity of myeloid lineage, peripheral blood (PBL) monocytes have the potential to differentiate into a variety of mature cells, including macrophages, osteoclasts and dendritic cells (DCs), depending on the environmental cues and activated signaling pathways. Therefore, we aimed to determine how the Notch-pathway affects tripotent differentiation of human PBL monocyte progenitor under inflammatory conditions. After obtaining ethical approval and informed consent, we first determined the frequency of common trilineage monocyte progenitors (TMPs), expressing the phenotype CD45+CD15−CD3−CD19−CD56−CD11b+CD16−CD14+CCR2+, in the PBL of healthy controls and RA patients. To assess the effect of Notch-pathway activation on TMPs differentiation potential, we then coated culture plates with the immobilized Notch-ligands Jagged-1 (JAG1) and Delta-like ligand-1 (DLL1). Macrophages, osteoclasts and DCs were induced by the appropriate cytokines (M-CSF, M-CSF and RANKL or GM-CSF and IL-4, respectively), whereas the addition of bacterial lipopolysaccharides mimicked an inflammatory environment. We observed that TMP population is expanded in RA PBL and expresses Notch receptors, implicating its susceptibility to Notch-regulation. Our results further suggest that in the context of inflammation, Notch-signaling, especially via DLL1, polarizes TMPs toward the pro-inflammatory and antigen-presenting capacity of DCs and macrophages, in contrast to suppressing phagocytosis and matrix degradation by macrophages and osteoclasts. These Notch effects are seen as higher IL1B expression and enhanced T lymphocyte stimulation by DCs, higher HLA-DR expression and suppressed phagocytosis by macrophages as well as lower CTSK expression and suppressed TRAP activity by osteoclasts. In conclusion, we demonstrated that the Notch axis effectively regulates the behavior of mature myeloid lineages derived from common TMP. Therefore, modulation of Notch-signaling may be an important complementary approach to treating RA pathogenesis. Biological sciences/Immunology/Inflammation Health sciences/Pathogenesis/Immunopathogenesis/Innate immunity Biological sciences/Immunology/Osteoimmunology Full Text Additional Declarations Table 1 is available in the Supplementary Files section. There is no conflict of interest Supplementary Files Table1.docx Table 1 Cite Share Download PDF Status: Published Journal Publication published 10 Nov, 2025 Read the published version in Cell Death Discovery → Version 1 posted Editorial decision: revise 02 Jul, 2025 Review # 3 received at journal 30 Jun, 2025 Review # 1 received at journal 17 Jun, 2025 Review # 2 received at journal 12 Jun, 2025 Reviewer # 3 agreed at journal 11 Jun, 2025 Reviewer # 2 agreed at journal 10 Jun, 2025 Reviewer # 1 agreed at journal 10 Jun, 2025 Reviewers invited by journal 10 Jun, 2025 Submission checks completed at journal 10 Jun, 2025 First submitted to journal 07 Jun, 2025 Editor assigned by journal 07 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6843542","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":469259228,"identity":"2e540fbd-32fe-45c0-8dd7-743341898fb1","order_by":0,"name":"Danka 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Inflammation-induced activation of myeloid cells is involved in disease pathogenesis and contributes to joint destruction. Due to the significant plasticity of myeloid lineage, peripheral blood (PBL) monocytes have the potential to differentiate into a variety of mature cells, including macrophages, osteoclasts and dendritic cells (DCs), depending on the environmental cues and activated signaling pathways. Therefore, we aimed to determine how the Notch-pathway affects tripotent differentiation of human PBL monocyte progenitor under inflammatory conditions. After obtaining ethical approval and informed consent, we first determined the frequency of common trilineage monocyte progenitors (TMPs), expressing the phenotype CD45+CD15−CD3−CD19−CD56−CD11b+CD16−CD14+CCR2+, in the PBL of healthy controls and RA patients. To assess the effect of Notch-pathway activation on TMPs differentiation potential, we then coated culture plates with the immobilized Notch-ligands Jagged-1 (JAG1) and Delta-like ligand-1 (DLL1). Macrophages, osteoclasts and DCs were induced by the appropriate cytokines (M-CSF, M-CSF and RANKL or GM-CSF and IL-4, respectively), whereas the addition of bacterial lipopolysaccharides mimicked an inflammatory environment. We observed that TMP population is expanded in RA PBL and expresses Notch receptors, implicating its susceptibility to Notch-regulation. Our results further suggest that in the context of inflammation, Notch-signaling, especially via DLL1, polarizes TMPs toward the pro-inflammatory and antigen-presenting capacity of DCs and macrophages, in contrast to suppressing phagocytosis and matrix degradation by macrophages and osteoclasts. These Notch effects are seen as higher IL1B expression and enhanced T lymphocyte stimulation by DCs, higher HLA-DR expression and suppressed phagocytosis by macrophages as well as lower CTSK expression and suppressed TRAP activity by osteoclasts. In conclusion, we demonstrated that the Notch axis effectively regulates the behavior of mature myeloid lineages derived from common TMP. 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