Abstract
Intestinal epithelial cells (IECs) are at the forefront of host-pathogen interactions, coordinating a cascade of immune responses to confer protection against pathogens. Here we show that IEC-intrinsic vitamin A signaling restricts pathogen invasion early in the infection and subsequently activates immune cells to promote pathogen clearance. Mice blocked for retinoic acid receptor (RAR) signaling selectively in IECs (stopΔIEC) showed significantly higher Salmonella burden in colonic tissues early in the infection associated with higher luminal and systemic loads of the pathogen at later stages. Higher susceptibility of stopΔIEC mice correlated with attenuated mucosal interferon gamma (IFNγ) production by underlying immune cells. We found that, at homeostasis, the intestinal epithelium of stopΔIEC mice produced significantly lower amounts of interleukin 18 (IL-18), a potent inducer of IFNγ. Regulation of IL-18 by vitamin A was also observed in a dietary model of vitamin A supplementation. IL-18 reconstitution in stopΔIEC mice restored resistance to Salmonella by promoting epithelial cell shedding to eliminate infected cells and limit pathogen invasion early in infection. Further, IL-18 augmented IFNγ production by underlying immune cells to restrict pathogen burden and systemic spread. Our work uncovers a critical role for vitamin A in coordinating a biphasic immune response to Salmonella infection by regulating IL-18 production by IECs.
Author Summary Epithelial cells line the intestinal lumen, forming a barrier between the body and dietary and microbial contents in the lumen. Apart from absorbing nutrients from diet, these epithelial cells help mediate a stable, symbiotic relationship between microbes in the gut and the immune cells. During infection, they help co-ordinate the immune response to counter the infection. How dietary micronutrients, such as vitamin A, inform epithelial cell function during infection is poorly understood. Using a model where epithelial cells in the gut cannot respond to vitamin A signals, we find that epithelial vitamin A signaling promotes resistance to Salmonella infection. We show that, vitamin A increases the production of a key cytokine, interleukin 18, by epithelial cells. IL-18 promotes shedding of infected epithelial cells to reduce the pathogen invasion while also inducing the production of interferon gamma by immune cells to mediate pathogen clearance. Thus, epithelial cells dynamically respond to dietary vitamin A to regulate interleukin 18 production and potentiate resistance to infection.
Introduction
Resistance to an invasive pathogen involves coordination between the early and late phase of the immune response so as to achieve pathogen clearance without excess collateral damage to the host. The intestinal epithelium is at the forefront of host-microbial interactions and is critical for orchestrating these immune responses during infection. Chemokines secreted by the epithelium are responsible for immune cell recruitment and activation [1]. T cells, NK cells as well as neutrophils are recruited to the colon during infection and secrete pro-inflammatory cytokines such as interferon gamma (IFNγ) to promote bacterial clearance and halt systemic spread of the infection [2, 3]. Moreover, the epithelium itself undergoes cell shedding in the early stages of infection as an innate defense mechanism to clear intracellular pathogens [4, 5]. Mechanisms that orchestrate such diverse functions of intestinal epithelial cells (IECs) during an infection remain poorly studied.
Vitamin A is an important dietary nutrient absorbed in the form of carotenoids and retinyl esters by intestinal epithelial cells and metabolized into its active form retinoic acid (RA). The heterodimeric nuclear receptor complex, retinoic acid receptor (RAR) and retinoid X receptor (RXR), is activated by RA binding to induce target gene expression [6]. Retinoic acid signaling is a potent regulator of gut immune make-up, affecting both the recruitment as well as activity of dendritic cells [7], T cells [8], B cells [9] and innate lymphoid cells (ILCs) [10] in the gut. The retinoic acid synthesized by epithelial cells, while available to underlying immune cells, is also capable of initiating a signaling response within IECs themselves. Retinoic acid signaling in intestinal epithelial cells (IECs) regulates epithelial lineage specification and promotes small intestinal T helper 17 (Th17) responses [11, 12]. Dietary vitamin A deficiency markedly increases susceptibility to enteric pathogens [13], however, relatively little is known about the contribution of IEC-intrinsic retinoic acid signaling in the context of infection.
In this study we use a mouse model expressing IEC-specific dominant negative retinoic acid receptor (stopΔIEC) to investigate the role of retinoic acid signaling during infection. stopΔIEC mice are more susceptible to luminal and systemic colonization by Salmonella. This is associated with abrogated shedding of infected epithelial cells as well as a blunted interferon gamma (IFNγ) response. We find that expression of interleukin-18, a known inducer of IFNγ, is dependent on retinoic acid signaling in intestinal epithelial cells. RAR signaling-dependent IL-18 promotes epithelial cell shedding as well as mucosal IFNγ production to orchestrate resistance to infection. Our results thus reveal a novel regulatory axis in the gut, wherein epithelial-intrinsic signaling in response to vitamin A, sequentially triggers IL-18 dependent mechanisms to first limit tissue invasion and then trigger an IFNγ response to promote pathogen clearance.
Results
Epithelial-intrinsic RAR signaling is protective against Salmonella colonization
Vitamin A deficiency results in increased susceptibility to infection by Salmonella and other enteric pathogens [13, 14]. Vitamin A deficiency causes immune dysregulation in the gut, including improper lymphoid recruitment, maturation and functional potential [15, 16]. These sweeping changes obscure the finer details of how vitamin A regulates infection outcome. Dietary vitamin A is sequentially absorbed, metabolized and distributed by intestinal epithelial cells [17]. Retinoic acid synthesized by intestinal epithelial cells regulates immune cell recruitment and cytokine production [18, 19]. We therefore hypothesized that vitamin A signaling in IECs regulates the mucosal immune response to mediate infection susceptibility. To address this question, we chose a mouse model wherein vitamin A signaling was specifically abrogated in intestinal epithelial cells while metabolism of vitamin A was unchanged. This was done through the Villin-Cre dependent overexpression of a dominant negative form of retinoic acid receptor alpha [20, 21], found to be the most dominant isoform expressed in intestinal epithelial cells (Fig 1A). Mice overexpressing this dominant negative RAR (stopΔIEC) were defective in the expression of the RA-responsive gene isx compared to their wild type littermates (stopflox) (Fig 1B and 1C) [22]. At homeostasis, stopΔIEC mice displayed an increase in goblet cell differentiation as well as overall thickness of the mucus barrier (S1A and S1B Fig), corroborating the results of a study using RARaΔIEC mice [11]. However, stopΔIEC mice showed no significant differences in epithelial permeability or turnover (S1C and S1D Fig).
The role of epithelial-intrinsic vitamin A signaling during infection was assessed using a gastroenteritis infection model of non-typhoidal Salmonella Typhimurium (Fig 1D) [23]. Bacterial burden in the feces, colon and mesenteric lymph nodes was determined at early (18 hours post infection; hpi) and late (72 hpi) time points to assess if vitamin A signaling modulates the kinetics of the pathogen colonization. At early time points, stopΔIEC mice and stopflox littermate controls had similar luminal burdens of the pathogen. However, stopΔIEC mice showed significantly higher loads of the bacterium within colon tissues (Fig 1E). This advantage in initial tissue invasion bolstered pathogen burdens at later time points, with higher loads found in the feces, colon as well as mesenteric lymph nodes of stopΔIEC mice (Fig 1F). These results show that loss of RAR signaling in the intestinal epithelium phenocopies dietary vitamin A deficiency in the context of enteric infection. Further, the changes in infection kinetics suggest that epithelial-intrinsic vitamin A signaling co-ordinates early and late immune mechanisms to promote resistance to infection.
Epithelial-intrinsic RAR signaling promotes mucosal IFNγ response during infection
Vitamin A metabolism and signaling in the epithelium is a key determinant of the intestinal immune make up. In the gut, retinoic acid influences the balance between Th1 and Th17 cells, both of which are important for controlling infection [8, 24, 25]. IEC-intrinsic vitamin A metabolism promotes mucosal IL-22 production, which in turn induces dysbiosis and aids pathogen colonization [18]. We therefore analyzed the colonic lamina propria populations in our mouse model to check if the increased susceptibility of stopΔIEC mice is due to a dysregulated immune response. While stopΔIEC and stopflox mice showed similar colonic immune make-up at homeostasis (S2A-S2D Fig), on day 3 of Salmonella infection, stopΔIEC mice displayed a defect in interferon gamma production (Fig 2A). CD4 and CD8 T cells were recruited to the colon to similar extents in stopΔIEC and stopflox mice, however activation of these cells to produce IFNγ was defective in stopΔIEC mice (Fig 2B-2D). No significant differences in mucosal IL-17 and IL-22 production were observed between stopΔIEC and stopflox mice during infection (S3A and S3B Fig).
Previous studies, both in vitro and in vivo, have shown that interferon gamma can be regulated by vitamin A, with retinoic acid promoting IFNγ production in intestinal T cells [26, 27]. Interferon gamma plays an important role in resistance to Salmonella infection. IFNγ knockout mice are more susceptible to Salmonella, both at intestinal and systemic level [28]. Mice with distinct microbiome compositions were found to be differentially susceptible to Salmonella; a function of the IFNγ induction by these communities [2]. Mechanistically, IFNγ promotes phagocytosis, oxidative/nitrosative response and cellular autophagy to clear the pathogen [29, 30]. To assess if the defect in IFNγ production was responsible for the increased susceptibility in stopΔIEC mice, we performed IFNγ feedback experiments (Fig 2E). Reconstitution of IFNγ in stopΔIEC mice led to a rescue of susceptibility, with bacterial burdens returning to wild type levels (Fig 2F). These results suggest, that epithelial-intrinsic RAR signaling primes mucosal IFNγ production to restrict Salmonella infection.
Intestinal epithelium-intrinsic RAR signaling regulates interleukin-18
Interleukin-18 was first discovered as an interferon gamma inducing molecule [31, 32]. Upon transcription and translation, it is present within the cell as a precursor form (pro-IL18) which is proteolytically processed by caspases into its mature, secreted form [33]. This IL-1 family cytokine, unlike its close cousin IL 1β, is constitutively expressed by a wide variety of cell types in the body. In the gut, intestinal epithelial cells form the main source of IL-18 [34, 35]. IL-18 is microbially regulated. It accumulates in the intestine after microbial colonization and is responsive to microbial metabolites such as butyrate and taurine [36, 37]. Further, IL-18 is known to be up-regulated at the early stages of Salmonella infection [3]. A study with human neuroblastoma cells identified induction of IL-18 by all trans retinoic acid in vitro [38]. Further, serum IL-18 levels have been shown to increase during vitamin A supplementation in obese mice; an effect dependent on active vitamin A metabolism [39]. However, regulation of homeostatic IL-18 levels in the gut by vitamin A has not been previously reported. We hypothesized that interleukin-18 might be the mechanistic link between IEC-intrinsic RAR signaling and mucosal IFNγ response.
We assessed the levels of IL-18 at homeostasis between stopΔIEC and stopflox mice. Colon whole tissue (Fig 3A) as well as colonocytes (Fig 3B) of stopΔIEC mice showed reduction in the protein levels of the precursor form of IL-18. These results suggest that RAR signaling in intestinal epithelial cells promotes il18 gene expression in the gut. In order to confirm that this phenomenon is not restricted to our stopΔIEC mouse model, we confirmed the same in a dietary model where wild type mice were fed a diet spiked with retinyl acetate for 3 days. Compared to mice receiving vehicle control, mice fed excess retinyl acetate had increased levels of IL-18 in colonocytes (Fig 3C). These results suggest that dietary vitamin A can dynamically modulate the levels of IL-18 in the colon.
RAR signaling-dependent IL-18 orchestrates early resistance to Salmonella invasion
Our results in stopΔIEC mice demonstrated a direct correlation between intestinal IL-18 levels and resistance to Salmonella infection. In order to establish a causal relationship between IL-18 and infection outcome, we reconstituted IL-18 levels in stopΔIEC mice and compared their susceptibility to stopflox and stopΔIEC mice at early time points (18 hpi) of Salmonella infection. We found that while IL-18 left luminal colonization unaffected, IL-18 feedback in stopΔIEC mice completely rescued tissue burdens of the pathogen to levels lower than wild type (Fig 4A-4C). This was further confirmed using confocal microscopy where staining for Salmonella revealed significantly higher levels of bacteria within stopΔIEC colonic tissues compared to stopflox and IL-18 feedback mice (Fig 4D-4F; S1-S3 Movies). Feedback with IFNγ, however, failed to rescue early tissue invasion in stopΔIEC mice (Fig 4A-4C). This suggests that epithelial RAR signaling promotes early resistance to bacterial invasion in an IL-18 dependent, but IFNγ independent, manner.
The epithelium is the first point of contact with adherent and invasive pathogens. Early in the infection, Salmonella uses epithelial invasion as a strategy to induce tissue inflammation, which endows it a selective advantage over gut commensals [40, 41]. Restricting this invasion is important to limit luminal expansion and systemic spread of the pathogen. Recently, epithelial cell death has been identified as a strategy to eliminate infected cells in the gut and limit tissue colonization. While cell shedding routinely occurs as a part of epithelial turnover, the rate of shedding increases dramatically during pathological insult to the epithelium [42, 43]. In the context of infection, inflammasome-mediated cell death pathways have been implicated in this epithelial-intrinsic response [4, 44]. The activation of these cell death pathways is associated with IL-18 cleavage and secretion [42]. We hypothesized that RAR signaling in IECs modulates cell death response during Salmonella infection via IL-18. Cell death has been reported to occur as an early response to the infection, especially in the cecum which is more permissive to bacterial invasion [4]. We, therefore, assessed the extent of cecal epithelial cell shedding in stopΔIEC mice at 18 hpi using cleaved caspase-3 staining as a marker for dying epithelial cells. We observed significantly higher numbers of dying cells in stopflox mice compared to stopΔIEC mice. This defect was rescued completely upon IL-18 reconstitution (Fig 4G-4J). This suggests that RAR signaling mediated IL-18 production promotes an epithelial cell shedding response which is associated with restricted tissue invasion by Salmonella.
Intestinal epithelium-intrinsic RAR signaling regulates pathogen colonization via interleukin-18
Our results with IFNγ feedback showed that at day 3 of infection, IFNγ is successfully able to rescue pathogen colonization in stopΔIEC mice (Fig 2F). IL-18, but not IFNγ, feedback was required to rescue early tissue invasion susceptibility in stopΔIEC mice (Fig 4C). Therefore, we hypothesized that IL-18 levels in the gut promoted early innate defenses to infection, while priming mucosal IFNγ to mediate pathogen clearance in the later stages. To evaluate this, we analyzed the effect of IL-18 reconstitution in stopΔIEC mice on day 3 of infection (Fig 5A). Feedback of IL-18 was sufficient to bring down bacterial loads in fecal, MLN as well as colon samples of stopΔIEC mice back to wild type levels (Fig 5B and 5C). Assessment of the colonic lamina propria revealed that this feedback effectively rescued IFNγ production by mucosal T cells (Fig 5D and 5E). These results suggest that epithelial RAR signaling modulates the mucosal IFNγ response via IL-18 to mediate resistance to pathogen. These results align with a study where epithelial IL-18 expression, regulated by histone deacetylase 3 activity, primes IFNγ response in intraepithelial lymphocytes to restrict colonization by Citrobacter [45]. IL-18 induction by protozoan colonization in the gut exerts a similar protective effects against Salmonella by promoting mucosal Th1 and Th17 response [46]. Our results corroborate the importance of IL-18 as a determinant of infection susceptibility and reveal a previously unappreciated pathway for IL-18 regulation via vitamin A signaling and dietary vitamin A.
Discussion
Vitamin A is a potent dietary micronutrient and vitamin A deficiency causes susceptibility to a spectrum of infectious diseases, especially in developing countries. A large body of work has contributed to our understanding of the immunomodulatory potential of vitamin A and its metabolite retinoic acid. However, pleiotropic cellular effects, complex metabolic and distribution pathways as well as source- and concentration-dependent functional effects have made dietary vitamin A models of infection difficult to interpret [47]. In this study, we employ a tissue-specific signaling abrogation model to elucidate the specific role of the vitamin A signaling pathway in the intestinal epithelium during infection. A key feature of this model is the absence of interference with the vitamin A metabolic machinery, avoiding any differences in gut immune trafficking mediated by epithelium-derived RA. Specifically, this study extends our understanding of homeostatic functions of vitamin A signaling in the intestine and reveals a previously unappreciated regulatory role for this pathway during Salmonella infection in the gut. In addition to describing the dynamic regulation of homeostatic IL-18 levels in the gut by vitamin A, we elucidate the functional role of the vitamin A-IL18 axis in restricting early tissue invasion by Salmonella and priming mucosal IFNγ production to mediate pathogen clearance (Fig 6).
Cytokines secreted by epithelial cells are important for immune co-ordination in the gut. Vitamin A is known to induce the signaling of important cytokines such as IL-22 in the gut [26]. A previous study from our group has shown that epithelial-intrinsic retinoic acid synthesis promotes IL-22 expression in underlying immune cells which in turn promotes Salmonella induced dysbiosis and gut colonization [18]. In contrast, the current study shows that epithelial-intrinsic RA signaling regulates IL-18 expression which protects against pathogen colonization and systemic spread. This highlights that retinoic acid synthesized by epithelial cells has distinct autocrine and paracrine functions, each having differential effects on infection outcome.
Interleukin-18 is set apart from other members of the IL1 family by being constitutively expressed by wide range of cell types throughout the body [48]. While both IL1β and IL-18 have pro-inflammatory functions, the classic fever response associated with IL1β is absent with IL-18. TLR ligands (LPS, poly (I:C), pam3CSK4) and type I interferons induce expression of IL-18, which is then proteolytically processed via the inflammasome pathway [49]. While induction of IL-18 during inflammatory conditions is well documented, relatively little is known about homeostatic regulators of IL-18. Studies have shown that late in gestation, inactive IL-18 starts accumulating in the intestine and active IL-18 is detectable postnatally [50]. Microbial colonization of the gut induces an upregulation in IL-18 expression [37] and the microbial metabolite butyrate is implicated in transcriptional regulation of IL-18 [36]. This study is the first to demonstrate that homeostatic IL-18 levels in the gut are regulated by IEC-intrinsic vitamin A signaling. Further, we show that vitamin A supplementation in the diet is also capable of inducing IL-18 in the gut, suggesting a dynamic regulation of epithelial IL-18 levels by dietary vitamin A.
Although our results clearly demonstrate that IL-18 levels in the gut are regulated by vitamin A, the exact mechanism of this regulation remains unresolved. Studies mining transcriptional targets of RAR signaling by in silico and ChIP-seq techniques have not identified IL-18 as a candidate, suggesting that retinoic acid receptor does not directly bind the il18 promoter to induce its expression [51, 52]. IL-18 expression is regulated by a host of transcription factors such as NF-κB, PU.1, Stat1, AP-1 and Bcl6 [49, 53, 54]. Both Stat1 and Bcl6 are reported to be regulated by retinoic acid [55–57]. Further, a post-transcriptional regulators of IL-18, Bruton’s tyrosine kinase, also has a predicted RAR binding site [58, 59]. We hypothesize that vitamin A regulation of IL-18 occurs by an indirect mechanism involving an interplay between one or more direct targets of RAR signaling. Uncovering the mechanistic link between vitamin A and IL-18 will be the focus of future investigations.
IL-18 and IFNγ are implicated in a host of gastrointestinal and systemic infections caused Toxoplasmas gondii [60], C. rodentium [45], Listeria monocytogenes [61, 62] among others. These pathogens represent a diversity of adhesion/invasion strategies as well as potential for systemic spread and toxicity. It remains to be seen how epithelial-intrinsic vitamin A signaling and IL-18 modulate infection outcome in these contexts.
Materials and Methods
Mice
All mice were bred in the SPF barrier facility at Brown University. Wild type stopflox mice were a kind gift from Dr. S. Sockanathan (Johns Hopkins University School of Medicine). Villin-Cre mice in a C56BL/6J background were purchased from Jackson Laboratories. stopflox mice were bred to Villin-Cre mice to get stopflox (Cre negative) or stopΔIEC (Cre positive) mice. All mice used in the study were 6-10 weeks old and were gender-matched across different groups. Littermates or co-housed mice were used to minimize microbiome mediated effects on the study.
Bacterial strains and maintenance
All infection experiments were carried out using Salmonella Typhimurium SL1344 GFP strain (kind gift of Dr. Vanessa Sperandio, UT Southwestern). Strain was routinely maintained on Luria Bertani agar plates containing 100 μg/ml ampicillin.
Mouse infections
For a gastroenteritis model of Salmonella infection protocol outlined by Barthel et al. was followed [23]. Briefly, mice were deprived of food and water for 4 hours and then orally gavaged with 20 mg streptomycin. After 20 hours, mice were again deprived of food and water for 4 hours. Overnight culture of Salmonella was subcultured for 4 hours, following which mice were infected with 108 bacteria by oral gavage. Mice were sacrificed 72 hours post infection to harvest organs and assess bacterial burden. For bacterial burden in distal colon, tissues were harvested, fileted and washed twice in phosphate-buffered saline (PBS). Tissues were then incubated in PBS containing 400 μg/ml gentamicin for 30 min at room temperature with shaking. Tissues were then rinsed with PBS, weighed, homogenized and plated to determine bacterial burden.
In order to assess the infection at early time points, protocol outlined by Sellin et al was followed [4]. The streptomycin treatment and Salmonella subculture was done as described above and mice were infected with 5×107 bacteria by oral gavage. Assessment of cell death as well as tissue burden were performed in the cecum and proximal colon respectively which are the primary sites infected at this early time point. For cell death assessment, cecal tissues were harvested and fixed in 4% paraformaldehyde/4% sucrose overnight. Fixed tissues were then saturated in PBS containing 20% sucrose, embedded in optimal cutting temperature (OCT) medium, frozen over dry ice and stored at −80°C. Proximal colon tissues were washed and gentamycin-treated for assessment of bacterial burden.
For reconstitution experiments, mouse recombinant IFNγ (Sino Biological; Cat. #50709-MNAH) and mouse recombinant IL-18 (Sino Biological; Cat. # 50073-MNCE) were used. Since IFNγ production defect in stopΔIEC mice was observed only after infection, reconstitution was performed 0 hr and 48hr post infection with 10 μg recombinant IFNγ intraperitoneally. Since stopΔIEC mice had homeostatic deficiency in IL-18 production, they were reconstituted with 2.5 μg recombinant IL-18 intraperitoneally 24 hours before and 24 hours after infection. For the early time point experiments, stopΔIEC mice received a single dose of 5 μg recombinant IL-18 intraperitoneally 24 hours before infection or a single dose of 15 μg recombinant IFNγ intraperitoneally 0 hours post infection.
Colonic lamina propria lymphocyte isolation and analysis
Colonic lamina propria lymphocytes were isolated as described by Kim et al [63]. Briefly, colons were flushed to remove luminal content, fileted, cut into three pieces and stored in ice-cold PBS. Tissues were washed by vigorous shaking, followed by sequential 10 min digestions at 37°C in HBSS containing 3% FCS, 1mM dithiothreitol and 30mM ethylene diamine tetraacetic acid (EDTA) and HBSS containing 3% FCS and 30mM EDTA. Tissues were vigorously shaken between treatments to dislodge epithelial cells. Lymphocytes were liberated from the lamina propria by digesting tissues in RPMI complete containing 40 μl/ml collagenase (Sigma-Aldrich, stock solution 5 mg/ml) and 5 μl/ml DNase (Sigma-Aldrich, stock solution 0.5 mg/ml) for one hour at 37°C followed by vigorous shaking. Cells were strained through a 70 μm filter and separated on a discontinuous 40%/80% Percoll (GE Healthcare) gradient. Cells at the interface were harvested and processed for flow cytometry analysis.
Flow cytometry analysis
Cells were stimulated for 4 hours at 37°C in RPMI complete containing 1X cell stimulation cocktail (eBioscience) and protein transport inhibitor cocktail (eBioscience). Stimulated cells were harvested and stained for surface markers and viability for 30 min at room temperature. Samples were stained with viability dye APCef780 (ThermoFisher), CD45 evolve655 (ThermoFisher), CD4 BV785 (BioLegend), CD3 eF450 (ThermoFisher), CD8a BV605 (BioLegend). Following overnight fixation using the Fixation/Permeabilization solution (eBioscience Foxp3 Staining Buffer Set), cells were permeabilized and stained for cytokines IL-17A AF488 (eBioscience), IL-22 PE (eBioscience) and IFNγ PE (eBioscience). Cells were analyzed using the Aria IIIu cytometer and data was analyzed using the FlowJo software.
Laser capture microdissection and quantitative real time PCR
Intestinal tissues were flushed with PBS and OCT, embedded in OCT, frozen on dry ice and stored at −80°C. Cryosections (10 μm thick) were stained with methyl green and eosin. Intestinal epithelial cells were selectively captured using the Arcturus Laser capture Microdissection system. RNA was isolated from IECs using the RNAqueous-Micro Total RNA kit (Ambion) and converted into cDNA using the iScript cDNA synthesis kit (BioRad). Whole tissue samples were homogenized, RNA was extracted using the PureLink RNA isolation kit (Life Technologies) and converted to cDNA using MMLV reverse transcriptase. Quantitative real time PCR was carried out using SYBR green master mix (Maxima). Expression was normalized using gapdh as a housekeeping gene. Details of primers are provided in Table 1.
Barrier function analysis
BrdU incorporation assay was used for assessment of barrier turnover. Mice were injected intraperitoneally with 1 mg of bromodeoxyuridine. Mice were sacrificed 2 hours and 24 hours post injection, distal colon tissues were fixed in methacarn fixative and embedded in paraffin blocks. Tissue sections (7 μm thick) were stained with anti-BrdU antibody (Novus Biologicals; #NBP2-14890) and visualized. For mucus staining, colon tissues samples were fixed in methacarn and embedded in paraffin blocks. Tissue sections (7 μm thick) were stained with alcian blue-periodic acid Schiff’s reagent to analyze mucus thickness and goblet cell numbers in the crypt.
Immunofluorescence staining and confocal microscopy
For visualization of cell shedding and intracellular bacterial loads, PFA-fixed, OCT-embedded samples of cecum and proximal colon were cryosectioned to 10 μm thickness. Sections were air dried, permeabilized with 0.5% Triton X-100 and blocked with 10% donor goat serum. Sections were stained with anti-Salmonella LPS (Difco; #DF2659-47-5)), anti-Cleaved Caspase-3 (Cell Signaling; #9661S) and anti-Epcam (BioLegend; #118201) antibody. DAPI was used to counterstain nuclei. Tissues were visualized using the Olympus FV3000 microscope.
Western Blot
For analysis of protein levels specifically in intestinal epithelial cells, colons were harvested and flushed with PBS. The colon epithelial cells were lysed in situ with tissue protein extraction reagent (TPER, Thermo Fisher) containing protease inhibitor cocktail. After 5 min incubation, lysate was recovered, centrifuged at 10,000 g for 3 min to remove debris and stored at −80°C [64]. Whole tissue lysates were obtained by homogenizing tissue samples in 500 μl of TPER containing protease inhibitors and incubating on ice for 20 min. Lysates were centrifuged as above and stored at −80°C. Protein in the samples were quantified using the DC protein assay (BioRad) and approximately 50 μg of protein was loaded for SDS-PAGE. Prestained protein ladder (BioRad) was loaded as a reference. Proteins were transferred on to PVDF membranes and blocked with 4% bovine serum albumin in TBST buffer for one hour. Blots were stained overnight at 4°C using primary antibodies against IL-18 (Abcam; #ab71495) followed by incubation with appropriate secondary HRP-conjugated antibodies. Beta-actin (Santa Cruz; #sc-47778-HRP) levels in the sample were used for normalization. Blots were analyzed using ImageJ software to calculate relative protein levels.
Dietary intervention
6-8 weeks old stopflox mice were fed regular mouse chow coated with retinyl acetate (500 IU/g) or equivalent amount of vehicle control (corn oil) for 3 days. Chow intake was monitored to ensure both groups of mice consumed similar amounts. Mice were sacrificed and colon lysates were prepared to analyze IL-18 levels using western blot.
Microscope image acquisition
Image acquisition was performed on the Olympus FV3000 confocal microscope. Images were acquired using a 60X oil immersion lens. For intracellular bacterial data, images were acquired as a Z-stack. All images processing was performed using the Fiji software with an Olympus plugin. Channel color for DAPI was changed to greyscale post-processing. Original 16-bit stacks were converted into RGB format before exporting as a video.
Statistical analysis
Data shown represent means ± SEM. Data was plotted and analyzed using GraphPad Prism software. For comparison of two groups, Student’s t test was employed. Comparison of two or more groups was performed using One-way ANOVA. Two-way ANOVA was used to compare gene expression across multiple groups.
Ethics statement
All experiments were approved by and carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee at Brown University (Protocol # 1803000345).
Acknowledgments
We thank Kevin Carlson for assistance with flow cytometry and Geoff Williams for maintaining the Leduc Bioimaging facility.