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. 2022 Aug 29:9:904721.
doi: 10.3389/fmed.2022.904721. eCollection 2022.

Typical response of CD14++CD16- monocyte to knee synovial derived mediators as a key target to overcome the onset and progression of osteoarthritis

Nik Syazana Izyan Saffery  1 , Krishnamurithy Genasan  1   2 , Chee Ken Chan  3 , Khairul Anwar Ayob  1 , Seow Hui Teo  1 , Mohamed Zubair Mohamed Al-Fayyadh  1 , Iekhsan Othman  4 , Syafiq Asnawi Zainal Abidin  4 , Murali Malliga Raman  1 , Hanumantha Rao Balaji Raghavendran  5 , Tunku Kamarul  1   6
Affiliations

Typical response of CD14++CD16- monocyte to knee synovial derived mediators as a key target to overcome the onset and progression of osteoarthritis

Nik Syazana Izyan Saffery et al. Front Med (Lausanne). .

Abstract

Objective: Synovitis with increased infiltration of immune cells is observed in osteoarthritis (OA). Given the inflammatory condition of synovitis, we explored the protein profile of OA synovium (OAS) and its effect on circulating monocytes activation, migration, and functional commitments.

Methods: Knee-synovium was acquired from end-stage OA (N = 8) and trauma patients (Trauma baseline control: TBC; N = 8) for characterization using H&E histology, IHC (iNOS), LCMS-QTOF, and MALDI-imaging. Response of peripheral blood monocytes to OAS conditioned-media (OACM) was observed using transwell (n = 6). The migrated cells were captured in SEM, quantified using phase-contrast microphotographs, and their activation receptors (CCR2, CXCR2, CX3CR1, and CD11b), pro-inflammatory genes, and phagocytic potential were studied using flow cytometry, gene expression array/qPCR, and latex beads (LB) phagocytosis assay, respectively.

Results: The Venn diagram displayed 119 typical proteins in OAS, while 55 proteins in TBCS. The STRING protein network analysis indicated distinctive links between proteins and gene ontology (GO) and revealed proteins associated with leukocyte-mediated immunity in OAS as compared to TBC. The MALDI-imaging showed typical localized proteins at 2234.97, 2522.61, 2627.21, 3329.50, and 3539.69 m/z and IHC confirmed pro-inflammatory iNOS expression in OA synovium. CD14++CD16- classical monocytes significantly migrated in OACM and expressed CCR2, CXCR2, and CD11b receptors, TNFRSF11A, MAPK1, S100A8, HSPB1, ITGAL, NFATC1, IL13RA1, CD93, IL-1β, TNF-α, and MYD88 genes and increased LB uptake as compared to SFM.

Conclusion: Our findings suggest that the differential protein profile of OA synovium and the classical monocytes migrated, activated, and functionally committed in response to these mediators could be of therapeutic advantage.

Keywords: MALDI-imaging; inflammation; monocytes; osteoarthritis; proteomics; synovium.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
The profile of OA synovium (OAS) and trauma baseline control synovium (TBCS). (A) Synovium from the end-stage knee OA patient underwent knee arthroplasty. (B) Synovium from the sports trauma patient underwent arthroscopic surgery for anterior cruciate ligament (ACL) repair. (C,D) H&E stained microphotographs. (E,F) SDS-PAGE protein bands of pooled OAS or TBCS. (G,H) False discovery rate (FDR) curve of peptides matched with the corresponding ESI-LCMS/MS QTOF spectra of peptides. (I,J) Peptide spectrum matches (PSMs) score distribution of target (blue square—OAS or TBCS) and decoy (amber square). (K,L) LCMS distribution profile and 3D intensity peaks of peptide and de novo sequences.
FIGURE 2
FIGURE 2
The differential protein analysis in OAS and TBCS. (A) Venn diagram; (B) Heatmap of overlapped proteins expressed at different magnitudes; (C,D) STRING protein–protein interaction networks.
FIGURE 3
FIGURE 3
Treemap visualization of the enriched biological process obtained from REVIGO analysis of differential proteins. (A) OAS and (B) TBCS. The most enriched physiological processes corresponded with larger components on the map.
FIGURE 4
FIGURE 4
The localized protein profile in OAS and TBCS. (A) MALDI spectra; (B) H&E staining of histological section and its corresponding MALDI imaging; (C) Expression of the pro-inflammatory marker, iNOS in mononuclear cells.
FIGURE 5
FIGURE 5
The migration potential of monocytes in response to SFM, MCP-1, and OACM. (A) Transwell migration model; (B) SEM micrographs of cells on the 3-μm pores insert membrane at basolateral site (post-migration); (C) Phase contrast photomicrographs of the migrated cells; (D) Enumeration of migrated cells from the phase contrast photomicrographs using Image-J; (E) The gating strategy in FACs analysis using CD45, Lineage negative (Lin-), CD14, and CD16 markers; (F) Percentage count of CD14+ (monocytes) and CD14– (non-monocytes) gated migrated cell population; (G) Percentage count of migrated classical monocytes (CM: CD14++CD16–), (H) intermediate monocytes (ITM: CD14++CD16+)%; and (I) Non-classical monocytes (NCM: CD14+dimCD16++). PBMNCs were used as pre-migration baseline control when compared with migrated monocytes (not significant: ns). The assessments were reported to be statistically significant if *p < 0.05 and ***p < 0.001.
FIGURE 6
FIGURE 6
The chemotaxis and the pro-inflammatory response of monocytes to SFM, MCP-1, and OACM. (A) Transwell migration model of monocytes using CCR2 receptor binding with CCL2 in concentration gradient. (B) The flow cytometry histogram profile of CCR2, CXCR2, and CX3CR1 receptors. (C) The statistical analysis of the expression of CCR2, CXCR2, and CX3CR1. (D) Heatmap of gene expression array. (E) The fold-change in gene expression array. (F–H) Fold-change of gene expression in qPCR. The assessments were reported to be statistically significant if *p < 0.05, **p < 0.01 and ***p < 0.001.
FIGURE 7
FIGURE 7
The phagocytosis potential of migrated monocytes in response to SFM, MCP-1, and OACM. (A) Heatmap of gene expression array of NFATC1, IL13RA1, and CD93 genes. (B) The fold-change of genes against monocytes migrated in SFM. (C) FACs analysis of CD11b/ITGAM receptor. (D) Confocal photomicrographs of monocytes internalized with latex beads at 10× and 40× magnification. (E) The quantitative analysis of latex beads uptake/cell by migrated cells in response to MCP-1 and OACM against SFM. The assessments were reported to be statistically significant if *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.
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