Growing evidence supports that microorganisms and their byproducts can affect an individual’s phenotype and vice versa (1). With advances in high-throughput sequencing technology in the last decade, great efforts have been devoted to understanding host-microbiome interactions. Numerous works have demonstrated that the microbiome not only shapes the host immune system, but also correlates with tissue homeostasis and pathophysiology of diseases (2). Of note, the gut is the most heavily colonized organ. It contains over 70% of total symbionts. Significant dysbiosis has been found in gut luminal and fecal microbiota according to disease cohort studies, suggesting a causal relationship between the gut and its microbiota in host health (3-5). For a mechanistic study, a germ-free mouse model has been widely used to assess impact of the microbiome on disease progression. However, significant differences in microbial tropism, cellular composition, and microenvironment cues such as metabolic pathways between human and mouse often hinder interpretation of results (6). In this aspect, organoid technology has brought great advances in modeling of host-microbiome interaction
To investigate interactions between IOs and microbes, it is essential to mimic the naïve gut environment harboring microbes. In the gastrointestinal tract, microbes exist within the lumen and directly interact with the intestinal epithelium through the apical side. In contrast, typical IOs have basal-out structures. Therefore, co-culture methods that allow physiologically relevant interaction are required for modeling microbial infections.
Microinjection of bacteria directly into the lumen of IOs can facilitate bacterial contact with the apical side of the epithelium (11, 12). Since the closed lumen has low oxygen tension, microinjection can improve the infection efficiency of anaerobic bacteria (13). Given that manual injection is a highly labor-intensive, time-consuming procedure (14, 15), a high-throughput organoid microinjection platform has been developed (16). However, since it is not a perfect anaerobic co-culture system, maintenance of a long-term culture of IOs with anaerobic bacteria is limited.
Microbes can be simply treated to organoid growth media or embedded with ECM during IO culture. It is the most common method to study host-microbe interaction so far. Organoids can be cultured with live- and heat-killed (HK) bacteria or with conditioned media containing their byproducts, including bacterial toxins and metabolites (17-19). However, this method restricts the access of bacteria to the apical side of IOs. To overcome this limitation, IOs can be mechanically shredded to expose the luminal side and then re-seeded into ECM following co-culture with live bacteria (20, 21).
ODMs are established by seeding dissociated 3D-grown IOs on a Transwell plate to expose the apical surface upward with media (22, 23). ODMs can recapitulate cell compositions of gastrointestinal epithelium such as enterocytes, goblet cells, Paneth cells, and other cell populations (24). This monolayer culture provides practical advantages of easy microbial access to the luminal side and convenient sample collection compared to ECM-embedded classical IOs. However, 2D-grown epithelial stem cells usually undergo differentiation. They cannot be sub-cultured or maintained for a long time. Thus, many cells are needed each time. In addition, it is hard to obtain morphological information with ODM method (25). ODM culture technique can be further modified by exposing the upper side of the layer to air to generate an oxygen gradient. With the air-liquid interface (ALI) culture method, in which the basolateral side and the apical side contact with media and surrounding air, respectively, ODMs can differentiate into more mature epithelial cells such as mucus-secreting goblet cells than 3D organoids (26, 27). In addition, IHACS (intestinal hemi-anaerobic co-culture system), composed of a hypoxic apical chamber sealed with a rubber plug and a basal chamber in normal oxygen concentration can facilitate the survival of both epithelial cells and microbes (28, 29).
The apical-out IO model is an alternative to microinjection which has limitations of laborious processes and requirement of special equipment. Reversion of epithelial polarity is performed by removing ECM and maintaining suspended IOs in the low-attachment plate, where spontaneous polarity changes from basal-out to apical-out occur (30). Apical-out IOs allow direct interactions between the epithelium and microbes. Functional assays for nutrient uptake and epithelial barrier integrity can also be performed since epithelial cells within apical-out IOs can differentiate into a more mature state than conventional IOs (30).
Pathobiont has a significant impact on host health. Enteric infections by bacterial pathogens are responsible for various diarrheal diseases, particularly in developing countries. Dysbiosis in the gut microbiome can lead to several enteric and systemic disorders (31). Moreover, the rapid increase of antibiotic-resistant pathogens has become a critical threat in recent years (32). Thus, numerous efforts have been made to evaluate and understand the detrimental impact of pathobionts on the gut using IOs (Table 1).
Recent studies have shown that genotoxic colibactin-secreting
Other pathogenic bacteria: A positive correlation between the abundance of
Since the gut is part of the digestive tract, oral pathogenic bacteria can be detrimental to the intestine. For instance,
Recent evidence has shown that probiotics can provide beneficial effects on the host by improving the balance of gut microbiota composition and promoting intestinal mucosal barrier function, indicating their therapeutic potential to treat a variety of intestinal disorders as shown below (Table 2) (76, 77).
The protective effect of
Organoid technology holds great potential to overcome limitations of conventional models such as 2D cell lines and experimental animals for modeling human anatomy and physiology. However, several challenging issues have to be solved to achieve advanced modeling of host-microorganism interactions with IOs. First, the absence of other cellular components except for epithelial cells is the main limitation of present IOs. Compared to pluripotent stem cell-derived organoids, which exhibit diverse cellular complexity, most adult stem cell-derived organoids like IOs consist of restricted lineage-derived cells that impede modeling of naïve microenvironment (89). Considering that microorganisms usually elicit an immune response and a repair process, which are predominantly mediated by regional immune cells and stromal cells respectively, the addition of these cells to an IO-microbe co-culture system would be necessary to recapitulate
This study was supported by the National Research Foundation of Korea (NRF), funded by the Ministry of Science and ICT (2018-R1A5A2023879, 2019-R1A2C2085876) and the Ministry of Education (2021-R1I1A1A01055654). Korean Fund for Regenerative Medicine (KFRM) grant funded by the Ministry of Science and ICT and the Ministry of Health & Welfare (22A0205L1) also supported this project.
The authors have no conflicting interests.
Studies reporting host-pathogenic bacteria interactions using organoids
|Bacteria||Source of organoid||Culture system||Key findings||References|
|Human small intestine, colon||ODM||↑IL-8 secretion||(24)|
|Human (unspecified)||ODM||↑IL-8 secretion||(34)|
|Human small intestine||ODM||Basolateral infection,||(35)|
|Human small intestine, colon||ODM||Testing the therapeutic effect of bacteriophage||(36)|
|Human small intestine||3D-microinjection||↓Organoid growth||(38)|
|Human/mouse small intestine||3D-microinjection||Recapitulation of early infection cycle, TTSS-1 is required for colonization||(39)|
|Human iPSC||3D-microinjection||↑Proinflammatory cytokines, InvA-dependent invasion||(40)|
|Human ESC||3D-microinjection||T3SS-1-dependent invasion,||(41)|
|Human small intestine, colon||Apical-out||Cytoskeletal rearrangement||(30)|
|Human small intestine||ODM||YrbE-dependent inflammatory response||(42)|
|EHEC||Human colon||ODM||↓Colonic mucus||(45)|
|Brush border damage|
|Human colon||ODM||Change in active ion transport||(47)|
|ETEC||Human small intestine||ODM||PDE5-mediated restriction of intracellular cGMP accumulation||(50)|
|Pig small intestine||ODM||F4-mediated adhesion||(51)|
|Human colon||ODM||Long-term exposure caused mutational signature||(54)|
|Human/mouse colon||ODM/Shredded 3D||↑Proliferation Wnt-independent growth||(21)|
|Human iPSC, ESC||3D-microinjection||↓Epithelial barrier function||(11)|
|Human iPSC||3D-microinjection||↓NHE3 expression||(56)|
|Human/mouse colon||3D derived from infected mice/3D-toxin treatment||↓Adherens junction,||(57)|
|Human small intestine||ODM||Adherence mechanism in human ODM model||(58)|
|Human iPSC||3D-toxin treatment||↓Transmembrane adhesion protein||(18)|
|Human iPSC||3D-toxin treatment||Protective effect of HSA||(59)|
|Human (unspecified)||3D-toxin treatment||Protective effect of antibiotic Bacitracin||(60)|
|Human/mouse iPSC||3D-microinjection||Protective effect of Paneth cells on
|Mouse small intestine||3D-toxin treatment||↑cAMP pathway||(62)|
|Human small intestine||3D-toxin treatment||Testing CT inhibitor with swelling assay||(63)|
|Human small intestine||3D-toxin treatment||O-blood group exhibited different responses to CT||(64)|
|Mouse small intestine||Shredded 3D||↑Organoid growth||(20)|
|Mouse small intestine||Shredded 3D||↑TNFa||(66)|
|↑Paneth cell, goblet cell|
|Mouse small intestine||Shredded 3D||↑TLR 2/4 signaling||(67)|
|Human small intestine, colon||Apical-out||Binding with basolateral receptor||(30)|
|Mouse small intestine||3D-microinjection||InlA-Ecad-dependent translocation through goblet cells||(68)|
|Mouse (unspecified)||Shredded 3D||TMT-based quantitative proteomic analysis in different strains||(69)|
|Mouse small intestine||3D-bacterial lysate||DNA damage||(72)|
|Human (unspecified)||ODM-OMV treatment||↑TNF, NF-κB, MAPK signaling||(74)|
|Mouse small intestine||3D||Regulation of cell composition||(75)|
Studies reporting host-probiotic bacteria interactions using organoids
|Bacteria||Source of organoid||Culture system||Key findings||References|
|Mouse small intestine||3D||↑Intestinal epithelial regeneration||(79)|
|Mouse small intestine||3D||↑Proliferation of intestinal epithelial stem cells||(80)|
|Mouse small intestine||3D||↑Dendritic cell maturation and IL-10 production||(81)|
|Mouse small intestine||3D||↑Expression of TLR3||(82)|
|Mouse small intestine, colon||3D-microinjection||↑Epithelial barrier function||(83)|
|Mouse small intestine||3D||↑Protects the intestinal mucosa against pathogen||(84)|
|Mouse small intestine||3D -HK bacteria||↑Intestinal epithelial function and differentiation||(85)|
|Human colon||ODM||↑Differentiation of goblet cell and stem cell||(29)|
|Human small intestine||3D-microinjection||↑Epithelial barrier function||(87)|
||Human small intestine||3D-microinjection||↑Epithelial proliferation & secretion of anti-microbial peptide||(88)|