Background/Purpose: Exacerbated differentiation of monocytes into osteoclasts (OC) contributes to the pathogenesis of rheumatoid arthritis (RA) resulting in severe bone erosion and functional damage. Osteoclastogenesis is initiated by M-CSF and RANKL signalization through their respective receptor CSF-1R and RANK. In vitro, it has been demonstrated that Human Cytomegalovirus (HCMV) infection inhibits macrophage differentiation through a down regulation of numerous receptor, including CSF-1R [Frascaroli et al., J. Immunol., (2009)]. As CSF-1R signaling is essential to induce RANK expression, the key receptor for osteoclast differentiation, we studied the effect of HCMV infection on osteoclastogenesis and evaluated the consequences of HCMV seropositivity on bone erosion evolution in a RA cohort.

Methods: Blood monocytes from healthy donors were purified by adherence selection and differentiated in osteoclast by recombinant M-CSF and RANKL (both at 50 ng/mL) during 12 days. After 24 hours of differentiation, pre-osteoclasts were infected with a HCMV clinical strain (VHL/E) at the m.o.i. of 3. Expression of cellular proteins CSF-1R, RANK, and “X-protein” (patent being filed) and viral protein (IE) expressions were studied by RT-qPCR, Western Blot, Flow Cytometry and fluorescence microscopy. OC differentiation was evaluated by manual counting after TRAP staining. Lentiviral transduction and Amaxa transfections were performed to over-express or inhibit the expression of “X-protein” and confirm its role.

We then analyzed patients from the French « ESPOIR » cohort, fulfilling the 2010 ACR/EULAR criteria for RA. We evaluated the correlation between HCMV serology status and structural involvement assessed by the modified Sharp score, at baseline and 1 year.

Results: We demonstrated for the first time that HCMV infection inhibits osteoclastogenesis. No osteoclast was observed in HCMV infected wells in vitro. We observed that HCMV infection inhibits CSF-1R and RANK mRNA and proteins expression in a viral replication dependent manner. We found that viral IE protein expression was followed by an increase of expression of the cellular “X-protein”. To confirm these observations, we over-expressed the “X protein” in monocytes and observed that CSF-1R, RANK and, as a result, osteoclastogenesis were completely inhibited. Knock-down experiment of “X protein” with shRNA lentiviral vectors blocked the inhibitory effect of HCMV on CSF-1R and RANK expression.

Analysis of the ESPOIR cohort, including 273 HCMV+ and 214 HCMV- RA patients, demonstrated that, although there was no difference at inclusion, HCMV seropositive patients displayed less severe bone erosion score in comparison with HCMV negative ones after one year (p=0.0151),thus providing a pathophysiological counterpart of our in vitro observations.

Conclusion: HCMV infection inhibits osteoclastogenesis through a mechanism involving “X-protein” and results in a protective effect on bone erosion during RA. Over expression of this “X-protein” inhibited OC differentiation and could be a new therapeutic target to limit bone erosion during chronic inflammatory disease such as RA and in osteoporosis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/evidence-for-inhibition-of-osteoclastogenesis-by-cytomegalovirus-infection-implication-in-ra-bone-erosion-and-identification-of-a-cellular-protein-as-a-therapeutic-target/