An in vitro model for osteoarthritis using long-cultured inflammatory human macrophages repeatedly stimulated with
Aldo Ummarino1,2, Alba Pensado-López1, Roberta Migliore2
1Department of Biomedical Sciences, Humanitas University, Milan, Italy.
Abstract:
Osteoarthritis (OA) is characterized by an abundance of inflammatory M1-like macrophages damaging local tissues. The search for new potential drugs for OA suffers from the lack of appropriate methods of long-lasting inflammation. Here we developed and characterized an in vitro protocol of long-lasting culture of primary human monocyte-derived macrophages differentiated with a combination of M-CSF+GM-CSF that optimally supported long-cultured macrophages (LC-Mϕs) for up to 15 days, unlike their single use. Macrophages repeatedly stimulated for 15 days with the TLR2 ligand Pam3CSK4 (LCS-Mϕs), showed sustained levels over time of IL-6, CCL2, and CXCL8, inflammatory mediators that were also detected in the synovial fluids of OA patients. Furthermore, macrophages isolated from the synovia of two OA patients showed an expression profile of inflammation-related genes similar to that of LCS-Mϕs, validating our protocol as a model of chronically activated inflammatory macrophages. Next, to confirm that these LCS-Mϕs could be modulated by anti-inflammatory compounds, we employed dexamethasone and/or celecoxib, two drugs widely used in OA treatment, that significantly inhibited the production of inflammatory mediators. This easy-to-use in vitro protocol of long-lasting inflammation with primary human macrophages could be useful for the screening of new compounds to improve the therapy of inflammatory disorders.
Insights
Researchers developed a new in vitro method to culture inflammatory macrophages for up to 15 days, mimicking chronic inflammation seen in osteoarthritis (OA). This model aids in screening potential anti-inflammatory drugs for OA therapy.
Area of Science:
- Immunology
- Cell Biology
- Pharmacology
Background:
- Osteoarthritis (OA) is linked to M1-like macrophages causing tissue damage.
- Current drug discovery for OA is hindered by a lack of suitable long-lasting inflammation models.
Purpose of the Study:
- To develop and characterize an in vitro protocol for long-lasting culture of primary human monocyte-derived macrophages.
- To establish a model of chronically activated inflammatory macrophages for OA research.
- To validate the model's utility in screening anti-inflammatory compounds.
Main Methods:
- Primary human monocytes differentiated with M-CSF+GM-CSF into long-cultured macrophages (LC-Mϕs).
- Repeated stimulation with TLR2 ligand Pam3CSK4 for 15 days to create LCS-Mϕs.
- Analysis of inflammatory mediators (IL-6, CCL2, CXCL8) and gene expression.
- Treatment of LCS-Mϕs with dexamethasone and celecoxib.
Main Results:
- The M-CSF+GM-CSF combination supported LC-Mϕs for up to 15 days.
- LCS-Mϕs exhibited sustained production of IL-6, CCL2, and CXCL8.
- Gene expression profiles of LCS-Mϕs mirrored those from OA patient synovia.
- Dexamethasone and celecoxib significantly inhibited inflammatory mediator production in LCS-Mϕs.
Conclusions:
- A novel, easy-to-use in vitro protocol for long-lasting macrophage inflammation was established.
- This model effectively mimics chronic inflammation relevant to osteoarthritis.
- The protocol is valuable for screening novel anti-inflammatory compounds for OA and other inflammatory disorders.
