(E-167) Characterization of initial immune response by oral macrophages and their role in host-pathogen interactions

Introduction:: The human oral cavity polymicrobial populations and their interactions with the host help shape oral health. However, understanding the ways in which these communities cooperate and how these interactions influence host physiology remains a challenge. Current models to investigate host-pathogen interactions remain clinical and in vitro models. Clinical studies, although informative, lack the ability to study the progression from healthy to dysbiotic state due to their complexity, challenges to independently control experimental variables and identification of specific triggers. On the other hand, in-vitro strategies are limited to immortalized two-dimensional cell culture, functional for short periods of time, and lacking the complexity of the polymicrobial organization due to absence of oxygen and metabolic conditions. Particularly, the introduction of immunocompetent cells in existing models is challenging because of 1) short lifespan and lack of appropriate culture conditions for immune cells and 2) inability to recapitulate the roles of the immune system (homeostatic balance between the host and the microbial communities and contributions to the ecosystem stability in healthy condition). Thus, there is a need for in-vitro models that can support long-term investigations of host-pathogens with the incorporation of immune components to understand disease trajectory and the role that host immunity plays in these interactions. The aim of this work is to characterize the initial immune response of monocytes and oral macrophages after exposure to spent artificial saliva, collected from a previously validated long-term 3D gingival tissue model that recapitulated host-subgingival plaque microbiome interactions in-vitro.

Materials and Methods:: To understand oral macrophages and monocyte-macrophages differentiation as a result of innate immune response, THP-1-monocytes were differentiated to M0 macrophages via phorbol 12-myristate 13-acetate (PMA 185ng/ml) then exposed to spent artificial saliva, which was collected from a previously validated long term 3D gingival tissue model that recapitulated host-subgingival plaque microbiome interactions in-vitro. M0 macrophages were exposed to spent artificial saliva from pre-host microbiome inoculum (7 days in culture with no microbiome) and post-host microbiome inoculum (7 days in culture with host-subgingival plaque microbiome). Samples were then assessed through cell surface makers expression (CD68 for M0, CD80 for M1, and CD206 for M2) via flow cytometry and confocal microscopy then compared against controls (M0, M1 and M2 obtained via cytokines treatment). To confirm macrophages phenotype (M0, M1 and M2), THP-1 controls were differentiated via pro/anti-inflammatory cytokines. Pro-inflammatory response was induced through INF-y (20ng/ml) and lipopolysaccharide portion form P. Gingivilis (100 ng/ml) for 48 hours. In contrast, anti-inflammatory response was induced via IL-4 (20 ng/ml). Samples were then assessed as described above.

Results, Conclusions, and Discussions:: Our studies have shown the ability to modulate an immune response from THP1-derived macrophages after exposure to pre- and post-microbiome inoculum spent artificial saliva from 3D gingival tissue model. An initial morphological change was observed for the sample exposed spent artificial saliva (fig.1D), similar to what was observed in cytokine-induced polarization controls indicating a response to the treatment (pre- or post-microbiome inoculated spent artificial saliva). This initial change indicated sample initial polarization which corroborates what was observed on flow cytometry. However, to confirm the immune response exposed to spent artificial saliva, we first characterized the response from controls obtained via cytokine induced polarization through flow cytometry and confocal imaging. After differentiation from THP1-monocyte to M0 macrophage through exposure to PMA, samples were stained with CD68 (highly express protein in macrophages) to confirm M0 phenotype (Fig. 1A). Samples showed a shift in fluorescent signal compared to negative control confirming M0 phenotype (Fig. 1 AIV). Once we confirmed M0 phenotype, we then tested samples treated with pro- (INF-y and LPS) and anti- (IL-4) inflammatory cytokine by staining for CD80 and CD206 corresponding to M1 and M2 phenotype respectively. Fluorescent microscopy revealed the expression of CD80 and CD206 from our samples as expected (Fig. 1 A&B). However, flow cytometry analysis shows a small change in fluorescence intensity in shift in CD80 compared to not stained control (Fig. 1BIV). In contrast, the opposite scenario was observed in the case of CD206 (Fig. 1CIV). This could be attributed to a low immune response to the treatment or short exposure time. However, an initial change can be observed in pro- and anti- inflammation samples as observed from confocal imaging and morphological change observed in bright field. Further characterization of the THP-1 derived macrophage response such as cytokine profile and gene expression will be done in the future to further to establish a strong baseline to test their transition to pro- or anti- inflammation.

Acknowledgements (Optional): : RO3DE030224 and RO3RO3DE030224-S1 Research to promote Diversity in Health-Related Research.



References (Optional): :