Basal and IL-1\u03b2 enhanced chondrocyte chemotactic activity on monocytes are co-dependent on both IKK\u03b1 and IKK\u03b2 NF-\u03baB activating kinases

Eleonora Olivotto,Spartaco Santi,Manuela Minguzzi,Stefania D’Adamo,Rosa Maria Borzì,Mariagrazia Uguccioni,Kenneth B. Marcu,Annalisa Astolfi

doi:10.1038/s41598-021-01063-2

Abstract

IKKα and IKKβ are essential kinases for activating NF-κB transcription factors that regulate cellular differentiation and inflammation. By virtue of their small size, chemokines support the crosstalk between cartilage and other joint compartments and contribute to immune cell chemotaxis in osteoarthritis (OA). Here we employed shRNA retroviruses to stably and efficiently ablate the expression of each IKK in primary OA chondrocytes to determine their individual contributions for monocyte chemotaxis in response to chondrocyte conditioned media. Both IKKα and IKKβ KDs blunted both the monocyte chemotactic potential and the protein levels of CCL2/MCP-1, the chemokine with the highest concentration and the strongest association with monocyte chemotaxis. These findings were mirrored by gene expression analysis indicating that the lowest levels of CCL2/MCP-1 and other monocyte-active chemokines were in IKKαKD cells under both basal and IL-1β stimulated conditions. We find that in their response to IL-1β stimulation IKKαKD primary OA chondrocytes have reduced levels of phosphorylated NFkappaB p65pSer536 and H3pSer10. Confocal microscopy analysis revealed co-localized p65 and H3pSer10 nuclear signals in agreement with our findings that IKKαKD effectively blunts their basal level and IL-1β dependent increases. Our results suggest that IKKα could be a novel OA disease target.

Highlights

IKKα and IKKβ are essential kinases for activating NF-κB transcription factors that regulate cellular differentiation and inflammation
We analysed at the same time the IL-1β dependent increased expression of 128 genes corresponding to chemokines and their receptors in one patient
We focused our attention to the four chemokines active on monocytes investigated in the Bioplex assay (evidenced by red arrows in the membranes of Fig. 1a that shows the microarray results for un-stimulated (US), left and IL-1β stimulated conditions

Summary

Introduction

IKKα and IKKβ are essential kinases for activating NF-κB transcription factors that regulate cellular differentiation and inflammation. We employed shRNA retroviruses to stably and efficiently ablate the expression of each IKK in primary OA chondrocytes to determine their individual contributions for monocyte chemotaxis in response to chondrocyte conditioned media Both IKKα and IKKβ KDs blunted both the monocyte chemotactic potential and the protein levels of CCL2/MCP-1, the chemokine with the highest concentration and the strongest association with monocyte chemotaxis. Defined as a non-inflammatory disease, OA is considered a condition involving persistent low-grade inflammation, oxidative s tress[3] and activation of innate inflammatory pathways with the recruitment of monocytes, lymphocytes, and other leukocytes in the synovial t issue[4] This condition contributes to perpetuate and enhance the pathophysiology of the disease[5]. Factor kappa-light-chain-enhancer of activated B cells (NF-κB) is reported to play a prominent role in OA8,9 both in cartilage and synovial t issue[10]

Methods

Results

Conclusion