NF-κB/MAPK activation underlies ACVR1-mediated inflammation in human heterotopic ossification
Emilie Barruet, Blanca M. Morales, Corey J. Cain, Amy N. Ton, Kelly L. Wentworth, Tea V. Chan, Tania A. Moody, Mariëlle C. Haks, Tom H.M. Ottenhoff, Judith Hellman, Mary C. Nakamura, Edward C. Hsiao
Emilie Barruet, Blanca M. Morales, Corey J. Cain, Amy N. Ton, Kelly L. Wentworth, Tea V. Chan, Tania A. Moody, Mariëlle C. Haks, Tom H.M. Ottenhoff, Judith Hellman, Mary C. Nakamura, Edward C. Hsiao
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Clinical Research and Public Health Bone biology Inflammation

NF-κB/MAPK activation underlies ACVR1-mediated inflammation in human heterotopic ossification

Abstract

BACKGROUND. Inflammation helps regulate normal growth and tissue repair. Although bone morphogenetic proteins (BMPs) and inflammation are known contributors to abnormal bone formation, how these pathways interact in ossification remains unclear. METHODS. We examined this potential link in patients with fibrodysplasia ossificans progressiva (FOP), a genetic condition of progressive heterotopic ossification caused by activating mutations in the Activin A type I receptor (ACVR1/ALK2). FOP patients show exquisite sensitivity to trauma, suggesting that BMP pathway activation may alter immune responses. We studied primary blood, monocyte, and macrophage samples from control and FOP subjects using multiplex cytokine, gene expression, and protein analyses; examined CD14+ primary monocyte and macrophage responses to TLR ligands; and assayed BMP, TGF-β activated kinase 1 (TAK1), and NF-κB pathways. RESULTS. FOP subjects at baseline without clinically evident heterotopic ossification showed increased serum IL-3, IL-7, IL-8, and IL-10. CD14+ primary monocytes treated with the TLR4 activator LPS showed increased CCL5, CCR7, and CXCL10; abnormal cytokine/chemokine secretion; and prolonged activation of the NF-κB pathway. FOP macrophages derived from primary monocytes also showed abnormal cytokine/chemokine secretion, increased TGF-β production, and p38MAPK activation. Surprisingly, SMAD phosphorylation was not significantly changed in the FOP monocytes/macrophages. CONCLUSIONS. Abnormal ACVR1 activity causes a proinflammatory state via increased NF-κB and p38MAPK activity. Similar changes may contribute to other types of heterotopic ossification, such as in scleroderma and dermatomyositis; after trauma; or with recombinant BMP-induced bone fusion. Our findings suggest that chronic antiinflammatory treatment may be useful for heterotopic ossification.

Authors

Emilie Barruet, Blanca M. Morales, Corey J. Cain, Amy N. Ton, Kelly L. Wentworth, Tea V. Chan, Tania A. Moody, Mariëlle C. Haks, Tom H.M. Ottenhoff, Judith Hellman, Mary C. Nakamura, Edward C. Hsiao

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Figure 6

Increased NF-κB activity in FOP monocytes.

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Increased NF-κB activity in FOP monocytes. (A) Representative Western bl...
(A) Representative Western blots (repeated for at least 3 different biological samples) showing activation of TGF-β activated kinase 1 (TAK1) and NF-κBp65 phosphorylation, as well as IkBa degradation (top) in control and FOP monocytes. The SMAD1/5/9 pathway is not activated in FOP monocytes (bottom). (B) TAK1 phosphorylation is increased upon LPS stimulation (10 ng/ml) in both control (n ≥ 5) and FOP (n ≥ 4) monocytes. There were no significant differences in total lysate NF-κBp65 phosphorylation upon LPS stimulation. (C) Time course of LPS-induced NF-κBp65 nuclear translocation in cells stimulated with LPS (10 ng/ml), BMP4 (50 ng/ml), or Activin A (50 ng/ml). Immunofluorescence staining of NF-κBp65 and quantification of nuclear staining (NF-κBp65:Dapi) shows a significant increase of NF-κBp65 nuclear translocation in FOP monocytes when stimulated with LPS and Activin A for 3 hours (control, n ≥ 3; FOP, n ≥ 4). Scale bars: 50μm. *P < 0.05 and ***P < 0.005 by multiple comparison Student’s t test. Error bars represent means ± 1 SD. At least 100 nuclei were evaluated per condition. (D) mRNA levels of IRF3 were increased with Activin A stimulation in FOP monocyte (control, n ≥ 3; FOP, n ≥ 3). *P < 0.05, by multiple comparison Student’s t test. Error bars represent mean ± 1 SD. The distribution of the subjects is described in Supplemental Tables 7 and 8.