Statins Modulate Microenvironmental Cues Driving Macrophage Polarization in Simulated Periodontal Inflammation

Abstract

Periodontal disease (PD) is a chronic inflammatory disorder characterized by the destruction of connective tissue, tooth loss, and systemic infections. Clinically, treatment of PD includes control of the etiologic factors via several modalities: initial therapy including scaling and root planing (SRP), corrective phase of surgical treatment, both with and without adjunct antimicrobial/pharmacological agents, followed by a maintenance/supportive periodontal therapy phase. Each treatment phase aims to control oral biofilm by addressing risk factors and etiology. Monotherapy of systemic antibiotics is insufficient compared to their use as an adjunct to SRP. The critical issue of systemic antimicrobial usage includes adverse patient outcomes and increased bacterial resistance. Therefore, alternative adjuncts to periodontal therapy have been sought. Statins are widely prescribed for the treatment of hypercholesterolemia and cardiovascular disease. Statins have demonstrated anti-inflammatory properties and immunomodulatory effects, and a few retrospective studies showed that statin patients exhibit fewer signs of periodontal inflammation than subjects without the medication. Despite the available clinical studies on the local administration of statins for PD, no studies have reported the macrophage polarization response. We have developed a gingival fibroblast-macrophage co-culture model to track macrophage response when exposed to a battery of microenvironmental cues mimicking macrophage polarization/depolarization observed in vivo. Using our model, we demonstrate that simvastatin suppresses macrophage inflammatory response and upregulates tissue homeostasis and M2 macrophage markers. Our findings support the usage of statins to mitigate periodontal inflammation as a valid strategy.

Keywords: adjunctive therapy; host immune response; macrophage polarization; periodontal disease; statins.

Figure 1

Validation of plasticity in THP-1-derived M1/M2 macrophages. (A) A schematic representation of treatments to differentiate THP-1 monocytes into macrophages (M0–M1–M2). (B) Gene expression principal component analysis clustering M0 (CCL2), M1 (LPS, IFNγ, TNFα), and M2 (IL4, IL10, TGFβ) macrophages. (n = 3) preparations per treatment group. (C) Heat map of averaged gene expression profiles (n = 3; data found to be significantly different were plotted as heat maps). (D) The proteome profiler on culture supernatants assessed chemokine secretion to validate RT-PCR analysis. Supernatants pooled from 3 cell preparations.

Figure 2

Simvastatin is a modulator of macrophage plasticity. (A) A schematic representation of simvastatin treatment and THP-1-macrophages (M0–M1–M2). (B) Morphological characteristics observed. (C) Unsupervised clustering and heat maps summarizing chemokine, TLR, NFkB, IFN-I signaling from M0 (CCL2), M1 (LPS, IFNγ, TNFα), and M2 (IL4, IL10, TGFβ) macrophages and were plotted as heat maps (n = 3 preparations per treatment group). See Supplementary Figure S2 for more information.

Figure 3

Simvastatin promotes M1 to M2 switch. RT-PCR validation of NanoString data summarizing chemokine, TLR, NFkB, signaling from M0 (CCL2), M1 (LPS, IFNγ, TNFα), and M2 (IL4, IL10, TGFβ) macrophages presented as heat maps. (n = 3) preparations per treatment group, and data found to be significantly different were plotted as heat maps.

Figure 4

MyD88−dependent modulation of M2 polarization by simvastatin. (A) A schematic representation of simvastatin, MyD88 inhibitor treatment, and THP-1-macrophages (M0–M1–M2). (B) Cell surface antigen analysis of M2 macrophage markers CD68, CD163, and CD206 was conducted using flow cytometry. Expression levels were normalized using IgG isotype control, and values were expressed in percentage of positive cells (mean ± SD) from three independent preparations. (C) RT-PCR analysis and heat maps summarizing TLR and NFkB signaling from M0 (CCL2), M1 (LPS, IFNγ, TNFα), and M2 (IL4, IL10, TGFβ) macrophages. (n = 3) preparations per treatment group, and data found to be significantly different were plotted as heat maps. (D) Graphical summary of statin-mediated suppression of TLR and NFkB signaling.