Macrophage Polarization and Alveolar Bone Healing Outcome: Despite a Significant M2 Polarizing Effect, VIP and PACAP Treatments Present a Minor Impact in Alveolar Bone Healing in Homeostatic Conditions

Abstract

Host inflammatory immune response comprises an essential element of the bone healing process, where M2 polarization allegedly contributes to a favorable healing outcome. In this context, immunoregulatory molecules that modulate host response, including macrophage polarization, are considered potential targets for improving bone healing. This study aims to evaluate the role of the immunoregulatory molecules VIP (Vasoactive intestinal peptide) and PACAP (Pituitary adenylate cyclase activating polypeptide), which was previously described to favor the development of the M2 phenotype, in the process of alveolar bone healing in C57Bl/6 (WT) mice. Experimental groups were submitted to tooth extraction and maintained under control conditions or treated with VIP or PACAP were evaluated by microtomographic (µCT), histomorphometric, immunohistochemical, and molecular analysis at 0, 3, 7, and 14 days to quantify tissue healing and host response indicators at the healing site. Gene expression analysis demonstrates the effectiveness of VIP or PACAP in modulating host response, evidenced by the early dominance of an M2-type response, which was paralleled by a significant increase in M2 (CD206+) in treated groups. However, despite the marked effect of M1/M2 balance in the healing sites, the histomorphometric analysis does not reveal an equivalent/corresponding modulation of the healing process. µCT reveals a slight increase in bone matrix volume and the trabecular thickness number in the PACAP group, while histomorphometric analyzes reveal a slight increase in the VIP group, both at a 14-d time-point; despite the increased expression of osteogenic factors, osteoblastic differentiation, activity, and maturation markers in both VIP and PACAP groups. Interestingly, a lower number of VIP and PACAP immunolabeled cells were observed in the treated groups, suggesting a reduction in endogenous production. In conclusion, while both VIP and PACAP treatments presented a significant immunomodulatory effect with potential for increased healing, no major changes were observed in bone healing outcome, suggesting that the signals required for bone healing under homeostatic conditions are already optimal, and additional signals do not improve an already optimal process. Further studies are required to elucidate the role of macrophage polarization in the bone healing process.

Microtomographic, histomorphometric, and molecular features show a normal alveolar bone healing process in iNOS-deficient mice along a compensatory upregulation of eNOS and nNOS isoforms

“…Accordingly, previous studies describe the upregulation of iNOS in similar contexts, such as in bone fracture sites 13 . When iNOS expression profile is compared with the migration pattern of M1 cells in healing sites, 1 , 4 , 16 , 17 we can observe a similar kinetics pattern between iNOS levels and CD80+ cells, reinforcing the possible iNOS/M1 interconnection along the bone healing process. Potential iNOS inducers, such as pro-inflammatory cytokines TNF, IL1B and IL6, and M1-markers, 1 , 4 , 16 - 18 were expressed in bone healing sites with profiles resembling that observed in iNOS, also strengthening the putative involvement of iNOS as part of the inflammatory process that precedes bone healing.…”

“…Notably, bone healing in both the iNOS-KO and WT strains was similar to that previously described in the WT C57Bl/6 strain. 1 , 4 , 16 , 17 Since only mineralized tissue features are subjected to the μCT analysis, we performed a histological and histomorphometric comparative analysis of healing sites. Nevertheless, similarly to the μCT analysis, the overall histological and histomorphometric features showed a similarity between bone healing features in the iNOS-KO and WT strains.…”

Vasoactive intestinal peptide blockade suppresses tumor growth by regulating macrophage polarization and function in CT26 tumor-bearing mice

“…Studies have demonstrated that VIP plays a crucial role in promoting M2 macrophage polarization in inflammatory environments induced by LPS and/or IFNγ 11,15,35,36 . Similarly, VPAC2 agonist induced M2-related gene markers but reduced M1-related gene markers in macrophages derived from human monocytes by GM-CSF (GM-MØ) stimulated with LPS.…”

The potential role of cfDNA-related innate immune responses in postoperative bone loss after alveolar bone grafting

“…Macrophage polarization could be affected by the microenvironment in the periodontal tissues, and the phenotypes of macrophages could determine the final osteogenesis of the alveolar bone ( 29 ). Thus, we carried out a preliminary experiment by observing the morphological changes of macrophages by the stimuli of preoperative and postoperative GCF following the published protocol ( 17 ).…”