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Abstract Background 3D cell culture models have recently garnered increasing attention for replicating organ microarchitecture and eliciting in vivo-like responses, holding significant promise across various biological disciplines. Broadly, 3D cell culture encompasses organoids as well as single- and multicellular spheroids. While the latter have found successful applications in tumor research, there is a notable scarcity of standardized intestinal models for infection biology that mimic the microarchitecture of the intestine. Hence, this study aimed to develop structured multicellular intestinal spheroids (SMIS) specifically tailored for studying molecular basis of infection by intestinal pathogens. Results We have successfully engineered human SMIS comprising four relevant cell types, featuring a fibroblast core enveloped by an outer monolayer of enterocytes and goblet cells along with monocytic cells. These SMIS effectively emulate the in vivo architecture of the intestinal mucosal surface and manifest differentiated morphological characteristics, including the presence of microvilli, within a mere two days of culture. Through analysis of various differentiation factors, we have illustrated that these spheroids attain heightened levels of differentiation compared to 2D monolayers. Moreover, SMIS serve as an optimized intestinal infection model, surpassing the capabilities of traditional 2D cultures, and exhibit a regulatory pattern of immunological markers similar to in vivo infections after Campylobacter jejuni infection. Notably, our protocol extends beyond human spheroids, demonstrating adaptability to other species such as mice and pigs. Conclusion Based on the rapid attainment of enhanced differentiation states, coupled with the emergence of functional brush border features, increased cellular complexity, and replication of the intestinal mucosal microarchitecture, which allows for exposure studies via the medium, we are confident that our innovative SMIS model surpasses conventional cell culture methods as a superior model. Moreover, it offers advantages over stem cell-derived organoids due to scalability and standardization capabilities of the protocol. By showcasing differentiated morphological attributes, our model provides an optimal platform for diverse applications. Furthermore, the investigated differences of several immunological factors compared to monotypic monolayers after Campylobacter jejuni infection underline the refinement of our spheroid model, which closely mimics important features of in vivo infections.

Abstract Background Zoonotic microorganisms are increasingly impacting human health worldwide. Due to the development of the global population, humans and animals live in shared and progressively crowded ecosystems, which enhances the risk of zoonoses. Although Campylobacter species are among the most important bacterial zoonotic agents worldwide, the molecular mechanisms of many host and pathogen factors involved in colonisation and infection are poorly understood. Campylobacter jejuni colonises the crypts of the human colon and causes acute inflammatory processes. The mucus and associated proteins play a central host-protective role in this process. The aim of this study was to explore the regulation of specific glycosyltransferase genes relevant to differential mucin-type O-glycosylation that could influence host colonisation and infection by C. jejuni. Results Since microRNAs are known to be important regulators of the mammalian host cell response to bacterial infections, we focussed on the role of miR-125a-5p in C. jejuni infection. Combining in vitro and in vivo approaches, we show that miR-125a-5p regulates the expression of the sialyltransferase ST3GAL1 in an infection-dependent manner. The protein ST3GAL1 shows markedly increased intestinal levels in infected mice, with enhanced distribution in the mucosal epithelial layer in contrast to naïve mice. Conclusion From our previous studies and the data presented here, we conclude that miR-125a-5p and the previously reported miR-615-3p are involved in regulating the glycosylation patterns of relevant host cell response proteins during C. jejuni infection. The miRNA-dependent modulation of mucin-type O-glycosylation could be part of the mucosal immune response, but also a pathogen-driven modification that allows colonisation and infection of the mammalian host.