(M1130-09-48) CCL2 Microparticles Modulate Host Immune Response in Periodontal Disease

Monday, October 21, 2024

11:30 AM - 12:30 PM MT

Presenting Author(s)

Julie Kobyra, BS

Graduate Student
University of Pittsburgh
Pittsburgh, Pennsylvania, United States

Main Author(s)

Julie Kobyra, BS

Graduate Student
University of Pittsburgh
Pittsburgh, Pennsylvania, United States

Co-Author(s)

MS

Mostafa Shehabeldin, Ph.D.

Graduate Student Researcher
University of Pittsburgh
Pittsburgh, Pennsylvania, United States
YC

Yejin Cho, BS

Graduate Student Researcher
University of Pittsburgh
Pittsburgh, Pennsylvania, United States
NL

Nursima Lacin, BS

Graduate Student Researcher
University of Pittsburgh
Pittsburgh, Pennsylvania, United States
JW

Jiefei Wang, BS

Bioinformatics Analyst
University of Pittsburgh
Pittsburgh, Pennsylvania, United States
LL

Lu Li, Ph.D.

Post Doctoral Researcher
University of Buffalo
Buffalo, New York, United States
PD

Patricia Diaz, Ph.D.

Director UB Microbiome Center
University of Buffalo
Buffalo, New York, United States
UC

Uma Chandran, Ph.D.

Director
University of Pittsburgh
Pittsburgh, Pennsylvania, United States
CS

Charles Sfeir, Ph.D.

Associate Professor
University of Pittsburgh
Pittsburgh, Pennsylvania, United States
SL

Steven Little, Ph.D.

Distinguished Professor and Department Chair
University of Pittsburgh
Pittsburgh, Pennsylvania, United States

Purpose: Periodontal disease (PD) is extremely prevalent in the United States affecting an estimated 42 percent of American adults, with the highest incidences in older adults [1]. Characterized by destructive inflammation of the periodontium, periodontitis is thought to be initiated by a dysbiotic biofilm and perpetuated by an ineffective host immune response. Current periodontal therapy begins with physical debridement (mechanical removal) of bacteria biofilms from around the tooth, gingiva, and subgingival periodontal pocket via scaling and root planing (SRP). While SRP is efficacious for some if followed by rigorous oral hygiene regimens, it is often followed by adjunct therapies to improve clinical outcomes. Current adjunct therapies are focused on decreasing bacterial load through local, sustained release of antibiotics; however, these therapies fail to address the underlying immune imbalance. To this end, our group has focused on developing novel drug delivery strategies to restore immune homeostasis within the oral cavity. We have previously demonstrated that local, sustained delivery of CCL2, a chemokine known to recruit macrophages and polarize them towards an anti-inflammatory phenotype, promotes repair in murine periodontal disease [2,3]. Here, we further investigated the mechanisms in which CCL2 microparticles (MPs) are working to resolve inflammation.

Methods: CCL2 MPs were fabricated using a double emulsion solvent evaporation method as previously described [2]. These MPs were then utilized in a ligature induced murine model of periodontitis (LIP). Male Balb/c mice ages 6 to 8 weeks were used for induction of periodontitis. Briefly, a silk 6-0 suture was tied around the second left maxillary molar, while the right remained unligated as a control. Four days following ligature placement and subsequently the onset of inflammation, CCL2 MPs or Blank MPs were injected into the soft tissue surrounding the ligated molar. Mice were then kept alive until day 10 at which point maxillae were harvested for downstream analysis. Analysis focused on investigating the impact on bone resorption (microCT), changes in both pro- and anti-inflammatory gene expression (qRT-PCR), microbiome analysis, and single cell sequencing.

Results: Encapsulation of CCL2 within PLGA MPs resulted in spherical particles that exhibit sustained release over the course of 30 days (Figure 1). Local administration of CCL2 MPs as an interventional therapy in murine PD significantly reduced bone resorption when compared to the controls (Figure 2). Additionally, CCL2 MPs promoted the presence of anti-inflammatory macrophages, as seen by an increase in Arg1 expression, and decreased several markers associated with inflammation and bone resorption. Given this, we sought to further characterize the impact of local CCL2 delivery. Using 16S rRNA sequencing, we looked at how the microbiome changed throughout the experimental timeline. We found that there were no differences in the species present when comparing the CCL2 MP treated group to Blank MP treated and the untreated groups. Furthermore, compared to the healthy control, all mice with PD had an expansion of enterococcus faecalis bacteria. Additionally, there was no significant differences in the species diversity as depicted by comparable Chao1 and Shannon indices (Figure 3). To further probe the mechanisms behind CCL2 MPs, we utilized single cell sequencing to investigate changes in different immune cell populations. Analysis regarding the single cell data is ongoing and emphasis is being placed on investigating the changes that occur within the monocyte/macrophage cluster.

Conclusion: Local, sustained delivery of CCL2 MPs in a ligature induced murine model of periodontitis may be a viable therapeutic option. CCL2 MPs were found to significantly inhibit bone resorption and promote the presence of anti-inflammatory macrophages. Despite the fact the CCL2 MPs did not impact the oral microbiome, they still provided a therapeutic benefit. This suggests that CCL2 MPs are primarily working to promote resolution of disease through their immunomodulatory capabilities. Continued analysis of the single cell data will hopefully provide further insight into the breadth of CCL2 MPs immunomodulatory effects.

References: 1. Eke PI, Thornton-Evans GO, Wei L, Borgnakke WS, Dye BA, Genco RJ. Periodontitis in US Adults. J Am Dent Assoc. 2018;149:576-588.e6.
2. Zhuang Z, Yoshizawa-Smith S, Glowacki A, Maltos K, Pacheco C, Shehabeldin M, et al. Induction of M2 Macrophages Prevents Bone Loss in Murine Periodontitis Models. J Dent Res. 2019;98:200–8.
3. Shehabeldin M, Gao J, Ki Y, Chong R, Tabib T, Li L, Smardz M, Gaffen S, Diaz P, Lafyatis R, Little S, Sfeir S. Therapeutic Delivery of CCL2 Modulates Immune Response and Restores Host-microbe Homeostasis. Accepted to PNAS. Anticipated Publication Sept. 2024.

Figure 1: Characterization of CCL2 microparticles (MPs) (A) Scanning electron microscopy image of CCL2 MPs (B) Volume impedance measurements of CCL2 MPs via Beckman coulter counter. Average microparticle size was 21.49  10.02 m. (C) Cumulative release profile of CCL2 from MPs over the course of 30 days.

Figure 2: Sustained release of CCL2 MPs decreases bone loss in established murine periodontal disease. (A) Quantification of interdental bone loss as measured by the distance between the cementoenamel junction (CEJ) and alveolar bone crest (ABC) from microCT images. Gene expression analysis of (B) anti-inflammatory macrophage marker Arg1 (C) common pro-inflammatory cytokine IL-6, and (D) bone resorption marker TNFSF11.

Figure 3: Characterization of oral microbiome composition using 16S rRNA sequencing
(A) Identification of top bacterial species presence and their relative abundance in each experimental group. Assessment of alpha diversity of the microbiome by (B) Chao1 diversity index and (C) Shannon evenness index.