MK2 signaling is essential for Aggregatibacter actinomycetemcomitans inflammatory-induced bone loss.

Enterprise Lithuania MAP Kinase ResourceLSB2014

Bethany A Herbert,Medical University of South Carolina, USA

Bethany A Herbert, Michael S Valerio, Keith L Kirkwood

Aggregatibacter actinomycetemcomitans (A.a.) is associated with aggressive periodontal disease (PD) pathogenesis, which is characterized by inflammation coupled with alveolar bone loss. Chemokines are up regulated during aggressive PD promoting inflammatory cell recruitment, including macrophages. Interestingly, preclinical periodontal disease studies support that inhibition of MAP kinase-activated protein kinase 2 (MK2), a p38 regulated mitogen activated protein kinase, attenuates A.a.-LPS induced inflammatory cell infiltration and bone loss. We hypothesize that MK2 signaling is critical for chemokine expression during A.a. infection. To determine the role MK2 signaling in macrophages during A.a. infection, bone marrow cells were harvested from 8-12 week old mice. Magnetic bead sorting was used to isolate CD11b+ cells that were differentiated into macrophages (BMDMs) for 6 days with M-CSF. Mk2+/+ and Mk2-/- mice were treated with live A.a. at the mid-sagittal suture daily for 3-5 days. Protein and RNA were isolated from tissue at the injection site. Immunoblot analysis showed that A.a. induced phosphorylation of MK2 in BMDMs occurs within 10 minutes. RT-qPCR of Mk2-/- BMDMs treated with A.a. exhibited a reduction in gene expression of Ccl3 (P<0.001) and Ccl4 (P<0.05) in comparison to A.a. treated Mk2+/+ CD11b+ BMDMs. Immunoblot results from calvarial tissues showed an increase in MK2 (P<0.001) during A.a. challenge. Nanostring analysis of calvarial tissue RNA revealed that gene expression of Ccl3, Ccl4, Ccl12, and Cxcl1 were attenuated in Mk2-/- compared to Mk2+/+ mice during infection. Using a multiplex analysis, Mk2-/- mice that showed a reduction in CCL2 (P<0.05), CCL3 (P<0.05), and CXCL1 (P<0.001) protein in after A.a challenge. Resorption pits in mouse calvaria in vivo observed by microcomputed tomography indicated a reduced amount of calvarial resorption seen in Mk2-/- compared to Mk2+/+ mice post 5 days of A.a. infection (P<0.01). These data suggest that MK2 signaling in macrophages contributes to regulation of inflammatory chemokines during A.a. challenge.