Oral Microbiota and Immune System Crosstalk: A Translational Research

Abstract

Background: Oral pathogens may exert the ability to trigger differently the activation of local macrophage immune responses, for instance Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans induce predominantly pro-inflammatory (M1-like phenotypes) responses, while oral commensal microbiota primarily elicits macrophage functions consistent with the anti-inflammatory (M2-like phenotypes).

Methods: In healthy individuals vs. periodontal disease patients' blood samples, the differentiation process from monocyte to M1 and M2 was conducted using two typical growth factors, the granulocyte/macrophage colony stimulating factor (GM-CSF) and the macrophage colony stimulating factor (M-CSF).

Results: In contrast with the current literature our outcomes showed a noticeable increase of macrophage polarization from healthy individuals vs. periodontal patients. The biological and clinical significance of these data was discussed.

Conclusions: Our translational findings showed a significant variance between control versus periodontal disease groups in M1 and M2 marker expression within the second group significantly lower skews differentiation of M2-like macrophages towards an M1-like phenotype. Macrophage polarization in periodontal tissue may be responsible for the development and progression of inflammation-induced periodontal tissue damage, including alveolar bone loss, and modulating macrophage function may be a potential strategy for periodontal disease management.

Keywords: clinical biochemistry; clinical microbiology; immune system; lymphocytes; macrophage polarization; microbiota; oral pathology; oral-systemic disease; periodontal disease; translational research.

Figure 1

Oral dysbiosis and central nervous system (CNS) diseases: The described mechanisms may be involved in complex interactions among oral dysbiosis and CNS diseases. The central theme of CNS diseases pathogenesis is the presence of brain inflammation as illustrated by increases in pro-inflammatory cytokines, LPS and macrophages polarization. The periodontal-derived pro-inflammatory molecules and bacterial products may reach the brain via systemic circulation and/or neural pathways, contributing to brain inflammation and inflammatory vicious cycle.

Figure 2

Monocyte-derived macrophages presented a unique morphology dependent on performed stimulatory effect. Granulocyte/macrophage colony stimulating factor (GM-CSF) led to a majority of elongated, fibroblast- spindle like shaped cells (c), and (d) similar to macrophages existent in lung alveoli, in contrast the presence of macrophage colony-stimulating factor (M-CSF) induced a majority of round or oval macrophages (fried eggs) like peritoneal macrophages (Axio observer Z1, Zeiss-AxioCamMR5 resolution 100×) (a,b). Human monocytes were cultured in RPMI-1640 with 20% heat-inactivated fetal calf serum supplemented with 5 ng/mL GM-CSF (granulocyte-macrophage colony-stimulating factor) and 5 ng/mL M-CSF (macrophage colony- stimulating factor). Monocytes were differentiated for 10 days in the presence of M-CSF and GM-CSF cytokines. (a) At day 3, (b) at day 4 and (c,d) at day 7 and 10. After seven days monocytes differentiated into macrophages.

Figure 3

The positive expression of CD14 by immune-fluorescence analysis, which is considered to be a typical marker for the identification of macrophages. After a 7-day culture, the cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with PBS (1×) containing either 0.1% Triton X-100, labeled with anti-CD14 (green stain CD14 expression with Alexa Fluor 488) polyclonal antibody at a dilution of 1:50, followed by a goat anti rabbit Alexa Fluor^® 488 secondary antibody to visualize the membrane. Images were assessed by inverted microscopy (Axio Observer Z1, Zeiss, resolution 100×). Nuclei were stained with DAPI. As a consequence of the growth factors used, GM-CSF led to a majority of elongated, fibroblast-spindle like shaped cells, and similar to macrophages existent in lung alveoli, in contrast the presence of M-CSF induced a majority of round or oval macrophages (fried eggs) like peritoneal macrophages.

Figure 4

Gating strategy used for the analysis of M1 and M2 phenotype. For the analysis of human macrophage differentiation phenotype, cells were harvested and analyzed by flow cytometry. Morphological gating (top left) was used to exclude cell debris from analysis. The CD14+ monocyte differ from CD14-, in order to identify macrophages, CD14+ cells were identified (top middle). Next, CD14+ cells were analyzed for their expression of CD80 and CD86 (top right red and yellow), HLA-DR and CD68 (bottom right light green and orange), CD16 and HLA-DR (bottom middle blue) and CD163 and CD206 (bottom left green/light blue). Median fluorescence intensity of each marker was extracted by using the FlowLogic software, low-auto fluorescent cells that strongly express MHCII and CD14+ hi, high-auto fluorescent cells are marked as macrophages.

Figure 5

Histogram overlays obtained by using the FlowLogic software. Representative data obtained from healthy donor-derived macrophage culture (blue histograms) and patient-derived macrophage culture (red histogram). Histogram overlays were obtained by using the FlowLogic software.

Figure 6

Expression of M1 markers in healthy donors and periodontal disease (PD) patients. Representative data obtained from healthy donor-derived macrophage culture (blue histograms) and patient-derived macrophage culture (red histogram). In the healthy control subjects showed a major expression of CD68 and CD86 (p = 0.012103) as compared to the periodontal patients, similarly for the CD80 and the HLA-DR marker but in this instance, there was not a significant difference (p > 0.05).

Figure 7

Expression of M2 markers in healthy donors and PD patients. Representative data obtained from one healthy donor-derived macrophage culture (blue histograms) and one patient-derived macrophage culture (red histogram). For the M2 markers a similar trend was observed: the healthy control subjects show a major expression of CD163 (p = 0.009405). For the CD206 there was a major expression, but not statistically significant (p = 0.063356).