Therapeutic Delivery of CCL2 Modulates Immune Response and Restores Host-microbe Homeostasis

The oral cavity is an easily accessible environment that allows for protective interventions aiming at modulating the immune response to control disease processes driven by a breakdown of host- microbe homeostasis. Periodontal disease (PD) is a prevalent condition in which quantitative and qualitative changes of the oral microbiota (dysbiosis) trigger non-resolving chronic inflammation, progressive bone loss and ultimately tooth loss. Here, we demonstrate the therapeutic benefit of local sustained delivery of the myeloid-recruiting chemokine CCL2 in murine ligature-induced PD using clinically relevant models as a preventive, interventional or reparative therapy. Local delivery of CCL2 into the periodontium inhibited bone loss and accelerated bone gain that could be ascribed to reduced osteoclasts numbers. CCL2 treatment upregulated M2-macrophage and downregulated pro-inflammatory and pro-osteoclastic markers. Furthermore, single cell RNA sequencing indicated that CCL2 therapy reversed disease-associated transcriptomic profiles of murine gingival macrophages via inhibiting TREM-1 signaling in classically activated macrophages and inducing PKA signaling in infiltrating macrophages. Finally, 16S rRNA sequencing showed mitigation of microbial dysbiosis in the periodontium that correlated with a reduction in microbial load in CCL2- treated mice. This study reveals a novel protective effect of CCL2 local delivery in PD as a model for chronic inflammatory diseases caused by a disturbance in host-microbe homeostasis. PD was induced for 7 days in 6-8 weeks Balb/C mice using silk ligatures around the second maxillary molar. Polymer (PLGA) microparticles (MPs) loaded with CCL2 chemokine, IL-4 (positive control) or blank MPs were locally delivered at the same time of ligature placement. Healthy mice without PD induction or mice with PD induction only (untreated) were used as controls. At the end of the disease induction period, the gingival tissues around the ligated molar in each treatment group were dissected and enzymatically digested to obtain a single cell suspension. The CD45 positive cells were isolated from the cell suspesions using column based magnetic cell isolation and magnetic CD45 microbeads. The viability of the isolated CD45+ cells was assessed using an auto cell counter before proceeding to the Sc-RNA Seq workflow.