Pathogen-Epithelium Interactions and Inflammatory Responses in Salmonella Dublin Infections through Bovine Ileal Monolayer Models Derived from Adult Organoids

doi:10.21203/rs.3.rs-4132778/v1

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Pathogen-Epithelium Interactions and Inflammatory Responses in Salmonella Dublin Infections through Bovine Ileal Monolayer Models Derived from Adult Organoids

https://doi.org/10.21203/rs.3.rs-4132778/v1

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Salmonella enterica serovar Dublin (S. Dublin) is an important enteric pathogen affecting cattle and poses increasing public health risks. Understanding the pathophysiology and host-pathogen interactions of S. Dublin infection is critical for developing effective control strategies, yet studies are hindered by the lack of physiologically relevant in vitro models. This study aimed to generate a robust bovine monolayer derived from adult ileal organoids, validate its feasibility as an in vitro infection model with S. Dublin, and evaluate the epithelial response to infection. A stable, confluent monolayer with a functional epithelial barrier was established under optimized culture conditions. The model's applicability for studying S. Dublin infection was confirmed by documenting intracellular bacterial invasion and replication and the resultant impacts on epithelial integrity, showing significant disruption of the monolayer, and a specific inflammatory response, providing insights into the pathogen-epithelium interactions. The study underscores the utility of organoid-derived monolayers in advancing our understanding of enteric infections in livestock and highlights implications for therapeutic strategy development and preventive measures, with potential applications extending to both veterinary and human medicine. The established bovine ileal monolayer offers a novel and physiologically relevant in vitro platform for investigating enteric pathogen-host interactions, particularly for pathogens like S. Dublin.

Biological sciences/Microbiology/Bacteria/Bacterial host response

Biological sciences/Biological techniques/Biological models/Gastrointestinal models

Adult stem cells

bovine ileum

in vitro infection model

organoid-derived monolayer

Salmonella

S. Dublin

Salmonella enterica subspecies enterica serovar Dublin (S. Dublin) is a cattle-adapted serovar that infect not only bovine but also other animals including humans. It is a globally important enteric pathogen with increasing public health risks [1]. Outcome of S. Dublin infection in cattle range from subclinical, leading to latent carrier state, to clinical, causing systemic illness such as enteritis and sepsis [2, 3]. In humans, S. Dublin infection usually carries more devastating health impact, causing severe bloodstream infections such as septicemia, osteomyelitis, meningitis, and even death in susceptible populations [1, 4]. A recent study has documented an increasing incidence of S. Dulin infection and resulting disease clinical severity in humans as well as an emerging antimicrobial drug resistance among S. Dublin isolates [1].

Many of the human infections with S. Dublin are transmitted through direct contact with infected animals or consumption of contaminated beef and dairy products [1, 5]. It has been postulated that development of antimicrobial resistance in S. Dublin could be associated with the use of such drugs in cattle [1]. Therefore, development of effective disease control strategies in cattle, hence better understanding of the pathophysiology and host-pathogen interactions, are critical in limiting economic and public health impacts associated with S. Dublin infection. Nonetheless, such studies have been hampered by the lack of physiologically relevant in vitro infection models which allow effective evaluations of host-pathogen interactions at the intestinal luminal interface.

Historically, impacts of Salmonella infection on intestinal epithelial cells have been investigated extensively using in vivo murine and immortalized cell line-based in vitro models [6]. Focusing specifically on bovine, in vivo models have been utilized to explore host-pathogen interactions and host response to Salmonella infection [7, 8]. More recently, with the emergence of organoid technology, in vitro organoid-based infection models have been developed in bovine using various pathogens including Salmonella [9–11]. Three-dimensional (3D) organoid models are considered superior to traditional cell line-based models due to their structural, cellular and functional resemblance to in vivo tissue [10]. However, accessibility to the apical surface of the epithelial cells where host-pathogen interaction actually takes place is limited due to their luminal structure, posing a significant challenge to such studies [6, 12, 13].

To overcome this limitation, organoid-derived 2D monolayer system, and various infection models thereof, have been developed and utilized to investigate host response to and interactions with pathogens mainly in humans and murine [14–16]. While such models offer unique opportunities to better understand pathophysiology of enteric infections, studies on bovine counterparts remain limited [11, 17, 18]. The development and validation of the bovine ileal monolayer model for studying S. Dublin infection represent a significant advancement with far-reaching implications for public health. By facilitating a deeper understanding of the pathophysiology of S. Dublin infections and the host-pathogen interactions at the intestinal epithelium, this model directly contributes to the development of more effective strategies for disease control and prevention in cattle, which is crucial for mitigating the risk of zoonotic transmission to humans. Given that many human infections with S. Dublin are linked to direct contact with infected animals or consumption of contaminated animal products, improving disease management in cattle can significantly reduce the incidence of such infections in humans, thereby protecting public health.

Therefore, the present study aimed to (1) describe a robust technique to generate bovine monolayers derived from adult ileal organoids, (2) confirm feasibility of the ileal organoid-derived monolayers as an in vitro infection model with S. Dublin, and (3) evaluate the impact of S. Dublin infection on the epithelial cells and their response to S. Dublin infection. By highlighting the utility of organoid-derived monolayers in studying species specific host-microbial interactions, this research underscores the importance of adopting a One Health approach. This perspective encourages collaborative efforts across disciplines to achieve the best health outcomes for people, animals, and the environment, recognizing that the health of each is interconnected and dependent on the others. Therefore, the contributions of this study extend beyond the immediate field of veterinary science, offering valuable insights and tools that can be leveraged to protect and improve human health as part of a comprehensive public health strategy.

Generation of stable bovine ileal monolayers derived from adult organoids

A stable intestinal epithelial monolayer was generated using adult bovine ileal organoids, which were developed according to a previous report [19]. The optimum culture conditions for the ileal monolayer were determined by varying the culture medium compositions and cell seeding densities (Supplementary Fig. S1). Confluent monolayers were consistently formed within 2–3 days of culture when the cells were seeded at a density of 5x10⁵ cells/well in a 24-well cell culture insert and cultured in an optimized monolayer culture medium, which consists of the organoid culture medium supplemented with rho-associated kinase (ROCK) and TGF-β inhibitors and 20% fetal bovine serum (FBS). Coinciding with this, a stable trans-epithelial electrical resistance (TEER) was also reached and consistently maintained for up to 9 days. In contrast, the time required to form a confluent monolayer was more variable from 2–3 days to up to 6 days and a stable TEER was maintained only for 2 days when the cells were seeded at a lower density (3x10⁵ cells/well). Similarly, formation of a confluent monolayer was delayed or its stability was breached shortly when the cells were cultured in the other medium tested which included a commercially available organoid culture medium (IntestiCult) and DMEM/F12-based organoid culture medium with or without supplementations and less than 20% FBS. Therefore, bovine ileal organoid-derived monolayers were generated by seeding the cells at a density of 5x10⁵ cells/well and using the optimized monolayer culture medium which consisted of the organoid culture medium supplemented with 20% FBS along with ROCK and TGFβ inhibitors in the subsequent study.

The monolayers cultured under the optimized conditions as determined above exhibited uniform cobblestone morphology on a phase-contrast microscopy (Fig. 1a). Scanning electron microscopy (SEM) revealed densely packed microvilli which uniformly covered the entire surface of the monolayer (Fig. 1b&c). Both of these structures are characteristic of intestinal epithelial cells and in accordance with previous observations [20, 21]. Daily evaluation of transepithelial electrical resistance (TEER) across the monolayer documented significant increase relative to the previous day until Day 4 of culture (p < 0.01), reaching the value of 412.6 ± 82.7 Ω*cm² from 84.4 ± 7.9 Ω*cm² measured at Day 1. Subsequently, a plateau of approximately 400 Ω*cm² was maintained for more than 7 days (Fig. 1d). Permeability assay using 4 kDa fluorescein isothiocyanate (FITC)-dextran also documented initial significant decline in the apparent permeability (P_app) from Day 1 to 3 of culture (p < 0.001), which was maintained at Day 5. These observations indicated the establishment and maintenance of functional and stable epithelial barrier integrity throughout the culture period.

Furthermore, immunofluorescence staining of confluent monolayers against F-actin and E-cadherin demonstrated uniform expressions of these proteins at cell-to-cell junctions (Fig. 1e&f). Z-stack images of these markers revealed strong apical expression of F-actin and basolateral expression of E-cadherin together with basally located DAPI-stained nuclei. These findings are consistent with cellular structures noted in a polarized epithelium, which is characterized with F-actin-rich apical brush border, E-cadherin-containing basolateral adherens junctions and basal nuclei [17, 22]. Staining against Sambucus nigra agglutinin (SNA), a sialic acid-specific lectin which binds to mucin produced by goblet cells, confirmed presence of functioning goblet cells within the monolayers (Fig. 1g). The cells within the monolayers also exhibited expression of stem and epithelial lineage cell marker genes as confirmed by RT-qPCR analysis (Fig. 1h). Taken together, these findings support the presence of multilineage cell population within the bovine ileal organoid-derived monolayers.

Establishment of bovine ileal organoid-derived monolayers as an in vitro infection model

Once the ileal organoid-derived monolayers have grown to confluency and reached stable state, S. Dublin was inoculated to the apical compartment. Various infection doses and initial incubation time before treating with gentamicin were tested to optimize the experimental conditions (Supplementary Fig. S2). Consequently, the monolayers were cocultured with S. Dublin at an infection dose of 1x10⁶ CFU/well for 1 hour in antibiotic-free monolayer culture medium to allow bacterial adhesion and invasion to the epithelial cells. The infected monolayers were then treated with 50 µg/mL gentamicin for 1 hour to kill extracellular bacteria. Subsequently, the monolayers were incubated with 10 µg/mL gentamicin till 24 hours post infection to examine survival and replication of internalized bacteria (Fig. 2). Feasibility of adult ileal organoid-derived monolayers as an in vitro infection model was confirmed by documenting interactions between S. Dublin and the ileal epithelial cells with multi-modal analyses.

Scanning electron microscopy (SEM) imaging at 24 hours post infection revealed bacterial adhesion and microvillous effacement on the apical surface of the epithelial cells in the S. Dublin infected monolayers comparing with uniform microvilli noted in the control monolayers (Fig. 3a&b). Transmission electron microscopy (TEM) imaging revealed intracellular bacteria encapsulated within vesicles, or endosomes, in the cytoplasm of the infected monolayers. Furthermore, the interactions between the epithelial cells and S. Dublin were observed as bacteria adhering to mucus as well as the disruption of the apical membrane in association with intracellular invasion. These observations were consistent with those reported previously using traditional in vivo and in vitro models [8, 15, 23–25].

Enumeration of intracellular bacteria at 2 and 24 hours post infection revealed that efficiency of intracellular invasion during the first hour of exposure to the monolayer was 1.9 ± 0.9%, and that of the intracellular survival over the 24-hour study period was 451.8 ± 175.8% of the inoculum, respectively (Fig. 3c). The total number of bacteria within the cells and those released to the apical compartment increased by more than 900 folds over the 24-hour period relative to that detected at 2 hours post infection. Overnight cultures of the culture medium collected from the apical and basolateral compartments at 2 hours post infection grew no identifiable bacterial colonies (Supplementary Table S1). These results confirmed that S. Dublin can invade, replicate and survive in the adult bovine ileal epithelial cells, indicating that the current monolayer system recreated the environment that allows S. Dublin to establish an intracellular infection.

Impact of S. Dublin infection on epithelial barrier integrity

Phase-contrast microscopy at 24 hours post infection with S. Dublin revealed extensive epithelial damage as noted by disruption of the uniform monolayer accompanied by cellular extrusion, while the uninfected control maintained undisrupted monolayer with uniform cobblestone morphology (Fig. 4a&b). Immunofluorescence staining revealed disruption of F-actin and formation of contractile actin rings in the infected monolayers, which are similar to the previous observations using human colorectal cell lines [23, 26]. Extrusion of epithelial cells from the apical surface of the monolayers was also evident in z-stack image of the infected monolayer. Additionally, S. Dublin infection caused distinct accumulation and redistribution of adherens junction protein E-cadherin, contrasting to the uniform distribution observed in the controls as reported previously [24, 27]. Morphological distortion of epithelial cells, observed as the uneven cell shape, was also evident in the S. Dublin infected monolayers, comparing with more regular cell morphology noted in the uninfected controls. The measurement of TEER before and after S. Dublin infection over 24 hours documented significant decline in the infected monolayers comparing with the uninfected controls at 24 hours post infection (p < 0.01) (Fig. 4c).

Immune response of ileal epithelial cells to S. Dublin infection

Host response of the ileal monolayers to S. Dublin infection was evaluated using RT-qPCR and ELISA against inflammatory cytokines. RT-qPCR revealed significant upregulation of TNF-α (p < 0.01) and IL-8 (p < 0.05), but not IL-6 (p = 0.22), genes in the infected monolayers compared to the uninfected controls (Fig. 5a). Levels of TNF-α released into the culture medium fell below the assay range (< 0.123 ng/mL) in both apical and basolateral compartments using a commercial bovine TNF-α ELISA kit. Levels of IL-8 was higher in both apical and basolateral culture medium collected from the S. Dublin infected wells relative to those detected in the control wells, with significant difference being noted in the apical medium (p < 0.001) (Fig. 5b). Approximately 2/3 of the total IL-8 secreted from the cells were detected in the basolateral culture medium in both the control and infected wells, indicating preferential secretion of IL-8 toward the basolateral direction as described previously [28, 29].

The present study described the establishment of a stable adult bovine ileal organoid-derived monolayer and confirmed feasibility of the system as a valuable in vitro model to study S. Dublin infection and host response to the bacteria. Exploiting a major advantage of 3D organoids which retain cellular heterogeneity derived from intestinal stem cells similar to the native intestinal tissue, the organoid-derived monolayer system is superior to the conventional cell-based monolayer models. Comparing with 3D organoid models, the organoid-derived monolayer system is better suited for in vitro studies of host-pathogen interactions due to its 2D structure, which allows direct access to the apical surface of the epithelial cells, overcoming difficulties faced when using 3D organoids as an infection model [9, 11]. Successful use of DMEM/F12-based culture medium in both organoids and monolayer cultures improved cost-efficiency of the technique, hence accessibility to the technology, comparing with when using commercially available organoid culture medium as described previously [11]. Furthermore, the infection model described here successfully recapitulated multiple observations reported in in vivo and in vitro studies which investigated host-pathogen interactions and host response to Salmonella infection in the intestine. These observations include intracellular invasion, survival and replication of bacteria, effacement and perturbation of microvilli on the apical surface of the epithelial cells, disruption of an intact monolayer accompanied by extrusion of infected epithelial cells, rearrangement of inter-cellular tight junctions and decline in the TEER value, and increased inflammatory response [15, 23–25, 30–33]. Robustness of the technique, as confirmed by employing multimodal imaging and analytical techniques, not only offers a valuable tool for investigation of bovine intestinal epithelial cell biology but also contributes to broadening the currently limited knowledge of bovine organoids and associated technologies.

The present study successfully established and maintained a stable monolayer most consistently and for longer culture duration when the cells were seeded at a higher density (5x10⁵ cells/well). This observation was different from the previous studies which reported higher success rate with much lower seeding density (2.5x10⁴ cells/well vs 1 ~ 2.5x10⁵ cells/well) using calf and porcine ileal organoids [17, 34]. Another difference is that culture medium supplemented with 1% FBS has been reported effective with calf ileal monolayers, while 20% FBS, consistent with the present study, was used for porcine ileal monolayers. It is difficult to ascertain factors contributing to these discrepant observations since culture medium compositions were not identical among these studies. However, it is possible that age of donors at the time of tissue sampling might have influenced on cell survival and organization to form confluent monolayers. Furthermore, stability of the adult monolayers which was reached earlier and maintained longer with slightly higher TEER value in the present culture condition comparing with those reported with calves suggests that adult cells mature faster and be more stable than calf cells. These features could provide additional advantage to some studies where a wider window for subsequent experiments is ideal.

The percentage of S. Dulin that invaded epithelial cells during the first hour of infection was approximately 2% of the inoculum. This result was slightly greater than the previous observations (0.7–1.5%) using human ileal organoid-derived monolayer and cell line-based culture models infected with various strains of S. Typhimurium or S. Typhi [8, 15, 35]. Intriguingly, despite this apparent greater efficiency of S. Dublin to invade the adult bovine ileal epithelial cells, significant disruption of the epithelial barrier integrity was only detected at 24 hours post infection, which was much slower than what has been reported in human colonic cell lines [33]. Less striking and gradual impact of S. Dublin infection in the present model could be associated with potentially superior local immune response generated by the bovine ileal organoid-derived monolayers. For instance, an increased inflammatory cytokine release and mucus secretion to protect epithelial cells from bacterial invasion have been suggested by previous studies which documented lower levels of infection in multi-cellular models relative to single cell culture models [8, 35]. It has also been proposed that expression of genes or cell surface receptors which influence bacterial invasion or host immune response could have been altered in these models [8, 35]. These potential explanations are also applicable to the organoid-derived monolayer systems due to their physiological resemblance to in vivo tissue attributable to their multi-cellular nature as demonstrated by the present and several other studies [11, 17, 36].

Bacterial factors, such as host-species and segmental tropism, could also have contributed to the variations among different studies as noted in studies utilizing different segments of human- and chicken-derived intestinal organoids or various strains of Salmonella pathovars [15, 35, 37]. Although S. Dublin is a cattle-adapted serotype and could cause tissue lesions in ileum, cecum and colon of infected animals, it could also establish life-long infection in cattle leading to an asymptomatic carrier status especially in healthy mature individuals [3]. It would be interesting to compare and contrast if S. Dublin infection to calf-derived ileal monolayers would result in similar observations to the present adult-derived monolayer model. Nonetheless, greater than 900 folds increase in the number of bacteria recovered after 24 hours of incubation relative to that recovered at 2 hours post infection is far greater than the previous observation in chicken intestinal organoids [37]. This result suggests that the present ileal monolayer system recreates an in vivo environment which allows bacterial survival and efficient replication, suggesting the usefulness of the system as an effective in vitro infection model to study host-pathogen interactions and host response to S. Dublin infection.

Redistribution of adherens junction protein and rearrangement of cytoskeleton actin filament as well as a significant decline in the TEER following exposure to S. Dublin were all in line with various in vivo and in vitro studies [23, 24, 26, 27, 33, 38]. Both contraction of actin filaments and redistribution of junctional proteins have been described to occur when a dying cell is pushed out of the monolayer and the monolayer tries to maintain its integrity as a part of normal physiological turnover of intestinal epithelial cells [23]. These observations also characterize a process of Salmonella-induced barrier dysfunction, which facilitates bacterial translocation across the gut epithelium and subsequent bacterial dissemination [27]. Taken together, these results further confirm that the adult bovine ileal organoid-derived monolayer system presented here is a viable in vitro infection model which can be adopted in various studies to better understand pathophysiology and host-pathogen interactions that occurs during challenges with S. Dublin and other enteric pathogens in bovine.

S. Dublin infection of the adult bovine ileal monolayers resulted in significant upregulation of chemokine IL-8 and proinflammatory cytokines TNF-α expressions and secretion of IL-8 to both apical and basolateral directions compared with the uninfected controls. IL-8 is a major chemoattractant for neutrophils and plays an important role in the pathogenesis of Salmonella infection by inducing an early mucosal inflammatory response at the site of infection. Increased expression and secretion of IL-8 has been reported upon invasion of epithelial cells by bacteria including Salmonella using various in vivo and in vitro models such as human colonic epithelial cells and bovine ligated ileal loop models [39–41]. The response noted in the present study was in agreement with these observations and was also in line with histopathological observations of suppurative enteritis in calves spontaneously infected with S. Dublin [42]. These evidence support that the cellular response presented here are likely representing normal physiologic responses of in vivo intestine upon challenges with S. Dublin.

Contrasting to IL-8, upregulation of TNF-α expression was not paralleled by increased TNF-α secretion. This observation partially agrees with the previous studies, which did not observe significant alterations in expression and secretion of TNF-α upon infection with Salmonella [39, 40]. TNF-α is a potent proinflammatory mediator and it also plays a crucial role in host defense against Salmonella infection [41]. It is known to be expressed by some epithelial cells and upregulation of TNF-α has been reported in human intestinal epithelial cells and murine ligated ileal loop models infected with Salmonella [28, 43]. Explanations for these discrepancies between studies, even when using the models derived from the same species, require further investigation. However, these findings underscore the importance of developing in vitro models which exhibit similar response to the species of interest at cellular levels besides clinical and gross pathological similarities to better understand pathogenesis of Salmonella-induced enterocolitis.

The present study successfully established a stable, confluent monolayer derived from adult bovine ileal organoids under optimized culture conditions, introducing a novel in vitro platform for investigating intestinal epithelial dynamics and host-pathogen interactions. The high-density cell seeding and supplementation with ROCK and TGF-β inhibitors, along with 20% FBS, enabled the formation of a functional epithelial barrier, demonstrating uniform cobblestone morphology, densely packed microvilli, and robust TEER and P_app values. The model’s application to S. Dublin infection offered insights into bacterial adhesion, invasion, and intracellular replication, leading to significant epithelial disruption and a specific inflammatory response marked by TNF-α and IL-8 upregulation, thereby highlighting its physiological relevance and suitability for studying enteric pathogens. The findings reveal the potential of organoid-derived monolayers to advance our understanding of infectious diseases in livestock and suggest implications for developing therapeutic strategies and preventive measures. The model also serves as a valuable tool for evaluating drug efficacy and studying host-microbial interactions, relevant to both veterinary and human medicine. Future research should explore the model’s application to other gastrointestinal pathogens, interactions between the intestinal epithelium and the microbiota, immune responses, drug absorption, and metabolism, with comparative studies on monolayers from different intestinal sections and species, to broaden our understanding of intestinal physiology and disease.

Development and maintenance of bovine ileal organoids

Ileal tissue samples were freshly obtained from four approximately 15- to 18-month-old cattle at a local slaughterhouse. The tissue was sampled and processed for crypt isolation and organoid development following the previously described protocol [19]. Briefly, pieces of ileal tissue were sampled using biopsy forceps, rinsed with ice-cooled Dulbecco’s phosphate-buffered saline (PBS) containing 1x penicillin/streptomycin and 25 µg/mL gentamicin, minced to small fragments and incubated in 20 mM ethylenediaminetetraacetic acid (EDTA) solution at 4^oC for 15 minutes on a tube rotator to isolate ileal crypts. The crypts were collected, resuspended in Matrigel and seeded onto a 48-well plate in 30 µL per well. Subsequently, each well received 300 µL of organoid culture medium once Matrigel was polymerized at 37^oC for 10–15 minutes. The organoid culture medium was prepared according to the previous study [19] (Supplementary Table S2). The medium was changed every other day for growth and maintenance of ileal organoids. The organoids were passaged every 6 to 8 days according to the reported protocol [19]. Briefly, organoids were recovered from Matrigel using Cell Recovery Solution, dissociated with TrypLE Express, resuspended in Matrigel at an expansion ratio of approximately 1:6 and cultured as described above.

Generation of ileal organoid-derived monolayers

Ileal organoids which demonstrated stable growth and expansion following at least three passages were used to generate monolayers. Ileal organoids were harvested from Matrigel using Cell Recovery Solution and dissociated with TrypLE Express supplemented with 10 µM Y-27632 at 37^oC for 10 minutes. Dissociated organoids were filtered through a 70 µm cell strainer to obtain single cell suspension. Subsequently, the cells were resuspended in the optimized monolayer culture medium and seeded onto a 24-well cell culture insert at 5x10⁵ cells/well. The cell culture insert was precoated with 2% (v/v) Matrigel in Advanced DMEM/F12 supplemented with 2 mM GlutaMAX and 10 mM HEPES at 37^oC for 1 hour. The monolayer culture medium was prepared by adding 10 µM Y-27632, 500 nM LY2157299, and 20% (v/v) FBS to the organoid culture medium without antibiotics (Supplementary Table S2). Each well received 200 µL and 500 µL of the monolayer culture medium in apical and basolateral compartments, respectively, and the medium was changed every other day. Formation of confluent monolayers was monitored daily using phase-contrast microscopy (DMi1, Leica). Epithelial barrier integrity of the monolayers was evaluated through TEER measurement and paracellular permeability assay. Structural and cellular characteristics of the monolayers were evaluated using SEM imaging, immunofluorescence staining, and RT-qPCR analyses.

Bacterial culture and infection of monolayers

S. Dublin isolated from bovine was grown in 3 mL of Luria-Bertani (LB) broth overnight at 37^oC with shaking at 200 rpm. The freshly saturated culture was diluted 1:10 into fresh LB broth and cultured for 2 hours to late log phase. Bacteria were harvested, washed with PBS, and resuspended to a concentration of 1x10⁷ CFU/mL in the monolayer culture medium. The monolayer was infected apically by replacing 100 µL of culture medium with the same volume of bacterial suspension and incubated for 1 hour in antibiotic-free culture medium to allow bacterial invasion to the epithelial cells. Subsequently, the monolayer was incubated with the medium containing 50 µg/mL gentamicin for 1 hour to kill extracellular bacteria, and then in the medium containing 10 µg/mL gentamicin till the end of the experiment, i.e. 24 hours post infection.

Infection of the ileal epithelial cells and intracellular multiplication of S. Dubin was confirmed by enumerating intracellular bacteria following treatment with 50 µg/mL gentamicin and at 24 hours post infection and by electron microscopies at the end of experiment. Epithelial cell integrity and structure were evaluated by measuring the TEER at 0, 4, 8, 12, and 24 hours post infection and by immunofluorescence staining of structural and junctional proteins at 24 hours post infection. Response of bovine ileal epithelial cells to S. Dublin infection was evaluated by RT-qPCR and ELISA of cytokines at the end of experiment.

Bacterial enumeration

Infected monolayers were rinsed with PBS and lysed with 1% Triton X-100 at 2 and 24 hours post infection. The culture medium in the apical and basolateral compartments and the PBS used to rinse the apical compartment were also collected at each time point. Serial dilutions of the lysate, apical medium and PBS rinse, and basolateral medium were plated on LB agar in triplicates and incubated at 37^oC overnight to determine CFU per well. Percent internalization and intracellular survival of S. Dublin were calculated by normalizing the CFU/well obtained from the cell lysate at 2 and 24 hours post infection to the initial bacterial inoculum, respectively [35]. The replication rate of S. Dublin was calculated by normalizing the total CFU/well obtained from the cell lysate, apical medium and PBS rinse, and basolateral medium at 24 hours post infection to that obtained at 2 hours post infection. Enumeration was performed in three independent experiments using three biological replicates, with at least two technical replicates per experiment.

TEER measurement

The TEER of ileal organoid-derived monolayers was monitored daily and at 0 (pre-) and 4, 8, 12 and 24 hours post infection using an Epithelial Voltohmmeter (EVOM) (Millicell ERS-2, Millipore) connected with Ag/AgCl electrodes. The measurement and calculation of TEER followed previously described techniques [44]. Briefly, the resistance across the semi-permeable membrane was measured in wells of both blank (TEER_blank) and the ileal monolayers (TEER_monolayer) by placing one electrode in the apical compartment and the other in the basolateral compartment without touching the membrane. The measured TEER in units of Ω was converted to reported TEER in units of Ωཥcm² by multiplying the difference of these values (TEER_monolayer - TEER_blank) by the surface area of the cell culture insert where the cells are cultured in cm². The measurement was obtained in at least three independent experiments using three biological replicates, with at least two technical replicates per experiment to assess reproducibility in various bovine ileal organoid-derived monolayers.

Permeability assay

The epithelial barrier integrity of the monolayers was evaluated by performing permeability assay on Days 1, 3 and 5 of culture following the previously described protocol [36, 45]. Briefly, 0.5 mg/mL of 4 kDa FITC-dextran was applied to the apical compartment at time 0, and the fluorescence intensity of the culture medium in the basolateral compartment was determined every 20 minutes over 120 minutes using SpectraMax i3x microplate reader (Molecular Devices). The measurements were obtained with excitation and emission wavelengths of 495 and 535 nm, respectively. The P_app (cm/s) was calculated by dividing the amount of FITC-dextran that passed through the cell layer over a fixed time period (µg/s) by the initial FITC-dextran concentration in the apical compartment (µg/mL) and the surface area (cm²) of the cell culture insert. The assay was performed in three independent experiments using three biological replicates, with at least two technical replicates per experiment.

Immunofluorescence staining

Monolayers were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.3% Triton X-100 for 10 minutes and blocked with 2% bovine serum albumin (BSA) for 60 minutes, respectively, at room temperature. A primary antibody and fluorescence probes against E-cadherin (BD Biosciences, 1:200), SNA (Vector Laboratories, 1:100), F-actin (Phalloidin) (Invitrogen, 1:400) and nuclei (DAPI) (Thermo Scientific, 1:1000) were diluted in 2% BSA and incubated for 1 hour at room temperature in dark. Subsequently, the membrane was cut from the inserts and mounted on glass slides using ProLong Gold Antifade reagent. Fluorescence images were captured using a white-light point scanning confocal microscope (SP8-X, Leica) and images were processed using LAS X (Leica).

Electron microscopy

Monolayers were fixed and processed for scanning and transmission electron microscopy as described previously [36, 46]. Briefly, monolayers were fixed with 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer overnight at 4^oC, rinsed with 0.1 M cacodylate buffer, and post-fixed with 0.5% osmium tetroxide overnight at 4^oC. Samples were serially dehydrated in 30 to 100% ethanol and finally in hexamethyldisilazane (HMDS) for SEM or in propylene oxide for TEM. SEM samples were coated with Pt/Pd sputter coater (Cressington High Resolution Sputter Coater) and imaged using Quanta 200F SEM (FEI). TEM samples were embedded in Spurrs resin, sectioned and stained with uranyl acetate, potassium permanganate and Raynold’s lead, and imaged using Tecnai G2 20 Twin TEM (FEI).

RT-qPCR

RT-qPCR was performed as described previously [19]. Briefly, total RNA was extracted from the control and S. Dublin-infected monolayers at the end of experiment using RNeasy Plus Mini Kit (Qiagen). High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) was used to synthesize cDNA. RT-qPCR was performed using PowerUp SYBR Green Master Mix (Applied Biosystems), with the primer sequence being amplified at 60°C for 40 cycles. Expression levels of stem (LGR5) and epithelial lineage cell (CHGA, LYZC, MUC2, FABP2) marker genes were evaluated using the cDNA synthesized from the uninfected control samples for characterization of the monolayers. Gene expression levels of cytokines (TNF-α, IL-6, IL-8) were compared between the uninfected control and infected monolayers. Primers used in this study were adopted from previous studies and summarized in Supplementary Table S3 [10, 17, 47–49]. Internal controls, namely GAPDH, RPL0, and ACTB [10, 49, 50], were used to normalize relative expressions of the target genes, which were calculated by applying the standard curve method. RT-qPCR reactions were carried out in duplicate from three biological replicates with two technical replicates per experiment.

ELISA

Culture medium of apical and basolateral compartments was collected at 24 hours post infection, centrifuged and supernatant was stored at -80^oC until the analysis was carried out. Commercially available sandwich ELISA kits, namely bovine TNF-α ELISA kit (Thermo Scientific) and human IL-8 ELISA kit (R&D Systems), were used as reported previously [51, 52]. The measurement was obtained with SpectraMax i3x microplate reader (Molecular Devices) according to the manufacture’s protocol. The assays were performed in duplicate from three biological replicates with two technical replicates per experiment.

Statistical analyses

Quantitative data were analyzed using R v.3.4.1 (The R foundation). The normality of each dataset was evaluated using Shapiro-Wilk test. Subsequently, either paired t-tests or Wilcoxon’s signed rank tests were employed to compare data between the control and S. Dublin-infected monolayers. The results were presented as mean +/- standard error of the mean (s.e.m.), with p < 0.05 being considered statistically significant.

Acknowledgement

The authors would like to thank the participating slaughterhouse for supplying donor cattle. The authors also would like to thank Itsuma Nagao, Takashi Goyama, Yurika Tachibana, Gerald D. Dykstra and Nao Nagao-Akiyama for their help in tissue sampling, Lindsay Parrish for her technical help in bacterial preparations, and Dr. Valerie Lynch-Holm and Dr. Brittney Wager from Franceschi Microscopy and Imaging Center at Washington State University for their technical support in electron microscopy. This study was supported in part by the Office of the Director National Institutes of Health (K01OD030515 and R21OD031903 to Y.M.A.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author contributions

Conceptualization: MK and YMA

Data curation: MK

Formal analysis: MK and YMA

Funding acquisition: YMA

Investigation: MK

Methodology: MK and YMA

Project administration: YMA

Resources: CSM, CRB, and YMA

Software: YMA

Supervision: YMA

Validation: MK

Visualization: MK

Writing – original draft: MK

Writing – review & editing: MK and YMA

Data availability statement

All data relevant to this study are included in this published article and its Supplementary Information files.

Competing interests statement

The authors declare no competing interests.

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Pathogen-Epithelium Interactions and Inflammatory Responses in Salmonella Dublin Infections through Bovine Ileal Monolayer Models Derived from Adult Organoids

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Version 1

Abstract

Figures

Introduction

Results

Generation of stable bovine ileal monolayers derived from adult organoids

Establishment of bovine ileal organoid-derived monolayers as an in vitro infection model

Impact of S. Dublin infection on epithelial barrier integrity

Immune response of ileal epithelial cells to S. Dublin infection

Discussion

Conclusion

Materials and methods

Development and maintenance of bovine ileal organoids

Generation of ileal organoid-derived monolayers

Bacterial culture and infection of monolayers

Bacterial enumeration

TEER measurement

Permeability assay

Immunofluorescence staining

Electron microscopy

RT-qPCR

ELISA

Statistical analyses

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1