Immunomodulatory effects of eubiotic and dysbiotic multi-species biofilms on oral keratinocytes

TL;DR

This study shows how healthy and unhealthy oral biofilms affect keratinocytes and immune cells, contributing to periodontal disease.

Contribution

The study introduces a co-culture model to investigate host responses to eubiotic and dysbiotic oral biofilms.

Findings

Dysbiotic biofilms showed higher proteolytic activity, likely due to Porphyromonas gingivalis.

Keratinocytes responded more pro-inflammatorily to dysbiotic biofilms, releasing cytokines like MIP-3a and IL-8.

Both biofilm types activated monocytes and neutrophils, with dysbiotic biofilms having a stronger effect on monocytes.

Abstract

Elucidating host‒microbe interactions is essential for understanding oral health and disease. In periodontitis, the host inflammatory response to accumulated plaque shifts eubiotic biofilm communities toward dysbiosis, with enrichment of proteolytic bacterial species. The first line of host defence in the subgingival niche involves oral keratinocytes, which communicate with immune cells in the mucosa. Host responses to individual bacterial species have been widely characterized, but in this study, we used a co-culture model to better understand how changes in the multispecies biofilm phenotype affect keratinocyte effector function as well as the effects on inflammatory cells.

Biofilms representative of eubiotic (HA) or dysbiotic (DA) bacterial communities were developed on nitro-cellulose membranes over 7 days and then co-cultured with oral keratinocytes for 6 h. Biofilm proteolytic activity was measured with a fluorescent substrate. Multiplex cytokine analysis of the co-culture medium was used to study keratinocyte responses and the activation of inflammatory cells was investigated via flow cytometry.

Proteolytic activity was greater in the DA biofilms than in the HA biofilms, most likely due to gingipains from Porphyromonas gingivalis. Keratinocytes released a range of cytokines, chemokines and growth factors, and the response to DA was more pro-inflammatory than that to HA biofilms, with relatively high levels of factors such as MIP-3a, IL-8, GM-CSF and IL-17 C. Co-culture medium from both the HA and DA biofilms elicited strong monocyte and neutrophil activation responses, although the effect of the DA biofilms was greater for monocytes than for neutrophils.

In this study, we show that keratinocytes have distinct response profiles to HA as compared to DA periodontal biofilm communities. The combination of products from the biofilm and the activated keratinocytes generated significant activation of inflammatory cells. This in vitro model thus provides insight on the complex host-microbiome interactions during development of periodontal disease.

The online version contains supplementary material available at 10.1186/s12903-025-07576-w.

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