Trusting a gut feeling: the potential of a newly refined human intestinal enteroid model to evaluate viral‐host interactions

The intestinal epithelium (IE) functions as a physical barrier between the host and the external environment. In an immunological balancing act, the IE must remain tolerant of resident microbial organisms, recognize opportunistic pathogens, and absorb essential nutrients to maintain homeostasis. The establishment of molecular mechanisms of physiological importance between the IE, the microbiome and the immune system have become an emerging area of research. Yet substantial work remains to understand the microbial-host axis, including critical work on understudied viral-host interactions. The comprehension of microbial-host tropism and intracellular signalling will provide significant insight into health and disease, which is fundamental for developing therapeutic targets to address the functional cellular outcomes that have an impact on physiological homeostasis. However, modelling the human IE can be challenging, as interactions between host and both commensal and pathogenic organisms are not always reproducible in animal systems or transformed cell lines (Saxena et al. 2015). Various tissue explants and primary cell cultures are also limited to short-term practice and require continual replenishment from primary sources. To better recapitulate intestinal structure, function and cellular diversity, human intestinal enteroid (HIE) models have been established. HIEs are constructed from intestinal epithelial crypts that are extracted from individual participants and cultured under ex vivo conditions. These crypts contain Lgr5+ stem cells that are exploited for their differentiation capacity; subsequently, the stem cells are used to generate both two-dimensional (2D) and three-dimensional (3D) cellular models that comprise functional intestinal cells (enterocytes, goblet cells, Paneth cells, tuft cells and enteroendocrine cells). However, HIEs are limited by a relative lack of cytokine production in response to microbial and host stimuli, which makes innate immune studies difficult. In a recent issue of The Journal of Physiology, Ruan et al. (2020) report technical refinements to a previously established HIE culture media. In an attempt to increase cellular response to pro-inflammatory microbial and host stimuli, antioxidants were removed from complete medium with growth factors (CMGF+). This was speculated to enhance generation of reactive oxygen species (ROS), vital regulators for pro-inflammatory cell signalling pathways. As hypothesized, the creation of a simplified cell culture medium (CMGF−) by the removal of antioxidants resulted in a more responsive HIE culture system that has the potential to more effectively mimic the human IE. The present study found that HIEs cultured in CMGF− media were significantly more responsive to IL-1α, TNF and flagellin stimuli. This was demonstrated by an increase in IL-8 production, which has a significant role in neutrophil migration. Contrary to other IE models, LPS and LTA were not potent activators of IL-8. This had functional consequences, as LPS and LTA did not promote neutrophil migration as effectively as TNF or flagellin. A robust increase in pro-inflammatory cytokine production of MCP-1 and GRO was caused by TNF, IL-1α and flagellin stimulation, whereas LPS was not a potent activator. IL-1α, TNF and flagellin were also reported as the only stimuli associated with an increase in relative ROS production, demonstrating the role of ROS in the enhancement of cellular responsiveness. The experiments from the current study revealed that the microbial stimuli LPS and LTA were responsible for a relatively limited pro-inflammatory cellular response in this HIE model; this may be indicative of the immunological tolerance that these jejunal human enteroids have towards non-specific pathogen-associated molecular patterns derived from bacteria that inhabit the gut. RNA-seq analysis of HIEs identified low levels of RNA transcripts for TLR2 or TLR4, the quintessential receptors for LTA and LPS, respectively, when compared to transcript copies for host stimuli receptors TNFRSF1A and 1B. However, LPS was a potent activator of TNF, which was shown to have significant pro-inflammatory potential in this model. HIEs stimulated with TNF and/or IL-1α were also found to mildly secrete an expansive repertoire of pro-inflammatory mediators: GM-CSF, MCP-3, EGF, MDC, IL-7 and IL-15. Additionally, both host stimuli were capable of secreting significant levels of IFN-γ, which is a key regulator of anti-viral immune coordination. The work conducted by Ruan et al. (2020) is the first to systematically characterize HIE cytokine production in response to relevant pro-inflammatory host and microbial stimuli. With regard to evaluating functional responses of the IE, the authors identified two major roadblocks that have impeded the dissection of mechanisms contributing to inflammatory responses mediated by the intestinal epithelium. The first is the types of models available. Previous work investigating crosstalk between the IE, microbiome and the immune system has been severely limited to animal models and immortal cell lines. Although these models are significant ethical checkpoints, each is associated with numerous limitations. Importantly, neither is fully representative of human physiology, ultimately limiting the ability to truly evaluate treatment effectiveness and the progression of various pathologies. Prior establishment of the HIE model was instrumental in providing a physiologically relevant model that is only surpassed by human in vivo studies. Still, the investigation of communication networks between the microbiome, immune system and the IE has been challenging due to the second drawback: poor cytokine response elicited by various pro-inflammatory stimuli. In an attempt to decode inflammatory communication networks that perpetuate various intestinal pathologies, the current study strategically manipulated the HIE model through refinements of the media composition to appropriately respond to various host- and microbial-derived stimuli, allowing for the quantification of various cytokines and chemokines. Overall, this study has provided significant insight into the functional responses of the IE following various stimuli. It will be an invaluable tool in elucidating the molecular mechanisms that are present in health and disease, in addition to establishing a model that can more effectively evaluate the functional outcomes associated with interactions between the IE, microbiome and the immune system. Using a simplified culture media, Ruan et al. (2020) state that HIEs are a biologically relevant system for examining microbial- and immune-mediated epithelial responses. To truly establish the fundamental utility of this HIE model as a system for examining these responses, we suggest future studies use a more comprehensive variety of microbial stimuli. The microbial analogues used by the authors were limited to bacterial stimuli, but substantial evidence exists to support the expansion of this model to include other microorganisms, such as viruses. The present study found that RNA-seq analysis clearly demonstrated TLR3 transcripts as the most abundant TLR, but the potential for viral-TLR3 activation was not explored in this study. In brief, TLR3 receptors positioned in the endosomal compartment sense dsRNA viruses, such as human rotavirus (HRV). HRV is a gastrointestinal virus and a major cause of diarrhoea-associated morbidity and mortality in young children (Saxena et al. 2015). Unfortunately, our understanding of HRV is limited, as it poorly replicates in classical IE cellular models. Newly established 3D spheroid HIEs are susceptible to HRV infection (Saxena et al. 2015) but have not been studied in the context of inflammatory mediator production with this CMGF− enhanced model. Research has demonstrated that TLR3-induced ROS production is required for the activation of essential transcriptional regulators NF-κB and IRF-3, which are needed for the subsequent release of an expansive pro-inflammatory mediator profile (Yang et al. 2013). Similar to the response observed following host- and microbial-derived stimulation of the HIE model, TLR3 activation has been found to potently induce the release of IL-8, TNF-α and IL-1α in various models of airway epithelium, further warranting the investigation of pro-inflammatory mediator release in HIE following relevant viral stimuli (Lever et al. 2015; Royer et al. 2017). Therefore, using HIEs that are within CMGF− media to validate the response of TLR3 to a viral synthetic analogue, such as poly(I:C) (Fig. 1), is critical for both the characterization of this model as a valuable tool for investigating disease therapeutics and for the understanding of intestinal pathophysiology pertinent to dsRNA enteric viruses. Most recently, the gastrointestinal tract was shown to be susceptible to SARS-CoV-2 (Lamers et al. 2020). Following infection, it was determined that SARS-CoV-2 increased gene expression and subsequent release of a large array of pro-inflammatory mediators from human small intestinal organoids (Lamers et al. 2020). Due to the potentially fatal inflammatory manifestations observed in severe cases of SARS-CoV-2, infecting an established IE model that fully recapitulates the structure and functionality of the intestine could significantly enhance our understanding of whether ROS-dependent cytokine production driven by intracellular TLR activation is involved in the pathogenesis of the novel SARS-CoV-2. This further validates the urgent need to establish the proposed model by Ruan et al. (2020) for viral-host interactions, as it has potential for urgent real-world application. In summary, the removal of antioxidant components from HIE culture medium to enhance ROS signalling is essential for appropriate pro-inflammatory responses following exposure to various host- and microbial-derived stimuli. This simple refinement of the HIE medium composition addressed major limitations of the previous HIE model, improving its physiological relevance for future studies investigating immunological and/or epithelial cell function in conjunction with the microbiome during physiological and pathological states. We emphasize that this model will be instrumental in the evaluation of viral-host interactions due to the vast number of viruses that constitute the microbiome, in addition to viruses that infiltrate the gastrointestinal system and cause disease. The authors declare no conflicts of interest. All authors contributed to the conception or design of the work and drafting the work or revising it critically for important intellectual content. All authors have read and approved the final version of the manuscript and have agreed to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. The authors have not received funding for this work. We would like to thank Dr Adam J. MacNeil and Grant C. Hayward for their comments and feedback on this manuscript.

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