Background/Purpose:  TNF has been shown to contribute to osteoclastogenesis independently and in conjunction with M-CSF or RANKL, two key cytokines involved in osteoclast (OC) development. We have previously demonstrated that TNF enhances the kinetics of RANKL-induced human osteoclastogenesis and that its effects are mitigated more effectively by the anti-TNF biologic adalimumab (ADA) as compared to etanercept (ETN). To determine the mechanism responsible for the difference in effectiveness between the two biologics, a label‐free quantitative proteomics study was conducted on TNF-activated human OC upon biologic treatment using an EASY Nano LC1000/ QExactive Plus LC/MS system.

Methods:  Human OC precursors (OCP) were exposed for up to 5 days to M-CSF, M-CSF+RANKL (RANKL) alone or in combination with 100 ng/mL TNF +/- 5 ug/mL ADA, ETN or human IgG1 (IGG) as a pre-formed complex. OC differentiation was confirmed by measuring tartrate-resistant acid phosphatase 5b (TRAP 5b) activity. Peptides from cell lysates were generated using modified Filter Aid Sample Preparation. Sample pooling (sham) was used to minimize false discovery by applying a cut off value (ratio >150% or <66.7%) based on reproducibility between sham samples. Data was analyzed with a 3 database search algorithm through Proteome Discoverer (PD) 2.0 and a 4th algorithm using MaxQuant. Protein IDs were aggregated in Scaffold software. Relative quantification was achieved using precursor ion area extraction function within PD. Data is expressed as percentage based on ratio of ion counts of each condition to that of RANKL with differences considered significant if >150% only for proteins whose percentage between TNF and IGG conditions are similar. Flow cytometry analysis for monocyte-related markers was performed on day 4 OCP cultures.

Results:  Ten OC-related proteins were identified (e.g. OSTF1, NFATC1, ACP5, CTSK, MMP9, TCIRG1, ATP6V0D2), 5 of which (underlined) were induced by TNF (>161%), decreased by ADA (<77%), but unaffected by ETN. Overall, TNF upregulated the levels of 110 proteins as compared to RANKL. A greater proportion of these proteins had levels reduced following ADA treatment as compared to ETN (69% and 20%, respectively) concordant with a greater reduction in OC maturation with ADA as to that of ETN (TRAP 5b levels of 11 and 40U/L, respectively). In addition to lowering TNF-induced protein levels concordant with neutralization by ADA, we observed indications for OCP development toward an alternative myeloid lineage based on its >1.5-fold induction of 25 proteins that are typically expressed in monocytic cells (e.g. PSMB9, DPP7 & IFI30) including 2 proteins reportedly shown to negatively regulate OC development (LGMN & ZFAND5). Moreover, several monocyte-related markers (CD14, CD163 & CD206) were increased by the ADA:TNF complex as compared to RANKL based on flow cytometry.

Conclusion:  Our in vitro findings demonstrate that ADA treatment, unlike exposure to ETN, dramatically reduces the TNF-induced protein levels in human OC and moreover, that the ADA:TNF complex may potentially alter their proteome to a profile resembling that of a monocytic cell through an increase in negative regulators of OC development.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/adalimumabtnf-complexes-induce-a-divergent-proteomic-profile-in-human-osteoclast-precursors-to-that-resembling-a-monocytic-cell/