Oral mucositis is still one of the most painful side effects of chemotherapeutic treatment and a mounting body of evidence suggests a key role for the oral microbiome in mucositis development. However, the underlying mechanisms remain elusive. In this work, we have investigated the interactions between the host, the microbiome, and chemotherapeutic treatments in more detail. The effect of 5-fluorouracil, commonly inducing mucositis, was assessed on a co-culture model that consists of an epithelial cell layer and a biofilm derived from oral microbiota from different types of samples (saliva, buccal swabs and tongue swabs) and donors (healthy individuals and patients suffering from mucositis). After 24 h co-incubation, all oral microbial samples were found to reduce wound healing capacity with 26 ± 15% as compared with untreated condition. Compared with saliva and tongue samples, buccal samples were characterized by lower bacterial cell counts and hence higher wound healing capacity. For samples from healthy individuals, an inverse correlation was observed between bacterial cell counts and wound healing capacity, whereas for patients suffering from mucositis no correlation was observed. Moreover, patient-derived samples had a less diverse microbial community and higher abundances of pathogenic genera. No major impact of 5-fluorouracil on wound healing capacity or the composition of the microbiome was seen at physiologically relevant concentrations in the mouth. In conclusion, bacterial cell count is inversely correlated with wound healing capacity, which emphasizes the importance of oral hygiene during oral wound healing in healthy individuals. However, future research on extra measures besides oral hygiene is needed to assure a good wound healing during mucositis, as for patients the bacterial composition seems also crucial. The direct effect of 5-fluorouracil on both the microbiome and wound healing is minimal, pointing to the importance of the host and its immune system in chemotherapy-induced microbial shifts. Impact statement Chemotherapy-induced oral mucositis has a major impact on the quality of life of patients. The additional costs and treatment time associated with this pathology are significant. Although the pathology of the disease is well understood, the role and importance of oral microbiota currently are less clear. In this study, we focused on the effect of oral microbiota on wound healing, the final phase of oral mucositis, during 5-FU exposure. We show that the bacterial load and composition have a major impact on the healing process in contrast to 5-FU which only marginally slows down healing. This emphasizes the importance of good oral health care during oral mucositis to minimize bacterial load around the oral lesions. However, since we show that also the composition of the oral microbiome plays a role in wound recovery, the identification of specific pathogenic species or their metabolites might be worthwhile to allow proper treatment.

The colonic epithelial barrier is vital to preserve gut and host health by maintaining the immune homeostasis between host and microbes. The mechanisms underlying beneficial or harmful host‐microbe interactions are poorly understood and impossible to study in vivo given the limited accessibility and ethical constraints. Moreover, existing in vitro models lack the required cellular complexity for the routine, yet profound, analysis of the intricate interplay between different types of host and microbial cells. We developed and characterized a broadly applicable, easy‐to‐handle in vitro triple coculture model that combines chemically‐induced macrophage‐like, goblet and epithelial cells covered by a mucus layer, which can be coincubated with complex human‐derived gut microbiota samples for 16 h. Comparison with a standard epithelial monolayer model revealed that triple cocultures produce thicker mucus layers, morphologically organize in a network and upon exposure to human‐derived gut microbiota samples, respond via pro‐inflammatory cytokine production. Both model systems, however, were not suffering from cytotoxic stress or different microbial loads, indicating that the obtained endpoints were caused by the imposed conditions. Addition of the probiotic Lactobacillus rhamnosus GG to assess its immunomodulating capacity in the triple coculture slightly suppressed pro‐inflammatory cytokine responses, based on transcriptomic microarray analyses. TNF conditioning of the models prior to microbial exposure did not cause shifts in cytokines, suggesting a strong epithelial barrier in which TNF did not reach the basolateral side. To conclude, the triple coculture model is tolerable towards manipulations and allows to address mechanistic host‐microbe research questions in a stable in vitro environment.

Spondyloarthritis is a group of chronic inflammatory diseases that primarily affects axial or peripheral joints and is frequently associated with inflammation at non-articular sites. The disease is multifactorial, involving genetics, immunity and environmental factors, including the gut microbiota. In vivo, microbiome contributions are difficult to assess due to the multifactorial disease complexity. In a proof-of-concept approach, we therefore used a triple coculture model of immune-like, goblet and epithelial cells to investigate whether we could detect a differential impact from spondyloarthritis- vs. healthy-derived gut microbiota on host cell response. Despite their phylogenetic resemblance, flow cytometry-based phenotypic clustering revealed human-derived gut microbiota from healthy origin to cluster together and apart from spondyloarthritis donors. At host level, mucus production was higher upon exposure to healthy microbiota. Pro-inflammatory cytokine responses displayed more inter-individual variability in spondyloarthritis than in healthy donors. Interestingly, the high dominance in the initial sample of one patient of Prevotella, a genus previously linked to spondyloarthritis, resulted in the most differential host response upon 16 h host-microbe coincubation. While future research should further focus on inter-individual variability by using gut microbiota from a large cohort of patients, this study underscores the importance of the gut microbiota during the SpA disease course.

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