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RESEARCH ARTICLE Host-Microbe Biology crossm Intestinal Epithelial Cells and the Microbiome Undergo Swift Reprogramming at the Inception of Colonic Citrobacter rodentium Infection Eve G. D. Hopkins,a Theodoros I. Roumeliotis,b Caroline Mullineaux-Sanders,a Jyoti S. Choudhary,b Gad Frankela a Centre for Molecular Microbiology and Infection, Department of Life Sciences, Imperial College, London, United Kingdom b Functional Proteomics Group, Chester Beatty Laboratories, Institute of Cancer Research, London, United Kingdom ABSTRACT We used the mouse attaching and effacing (A/E) pathogen Citrobacter rodentium, which models the human A/E pathogens enteropathogenic Escherichia coli and enterohemorrhagic E. coli (EPEC and EHEC), to temporally resolve intestinal epithelial cell (IEC) responses and changes to the microbiome during in vivo infection. We found the host to be unresponsive during the first 3 days postinfection (DPI), when C. rodentium resides in the caecum. In contrast, at 4 DPI, the day of colonic colonization, despite only sporadic adhesion to the apex of the crypt, we observed robust upregulation of cell cycle and DNA repair processes, which were associated with expansion of the crypt Ki67-positive replicative zone, and downregulation of multiple metabolic processes (including the tricarboxylic acid [TCA] cycle and oxidative phosphorylation). Moreover, we observed dramatic depletion of goblet and deep crypt secretory cells and an atypical regulation of cholesterol homeostasis in IECs during early infection, with simultaneous upregulation of cholesterol biogenesis (e.g., 3-hydroxy-3-methylglutaryl– coenzyme A reductase [Hmgcr]), import (e.g., low-density lipoprotein receptor [Ldlr]), and efflux (e.g., AbcA1). We also detected interleukin 22 (IL-22) responses in IECs (e.g., Reg3␥) on the day of colonic colonization, which occurred concomitantly with a bloom of commensal Enterobacteriaceae on the mucosal surface. These results unravel a new paradigm in host-pathogen-microbiome interactions, showing for the first time that sensing a small number of pathogenic bacteria triggers swift intrinsic changes to the IEC composition and function, in tandem with significant changes to the mucosaassociated microbiome, which parallel innate immune responses. IMPORTANCE The mouse pathogen C. rodentium is a widely used model for colonic infection and has been a major tool in fundamental discoveries in the fields of bacterial pathogenesis and mucosal immunology. Despite extensive studies probing acute C. rodentium infection, our understanding of the early stages preceding the infection climax remains relatively undetailed. To this end, we apply a multiomics approach to resolve temporal changes to the host and microbiome during early infection. Unexpectedly, we found immediate and dramatic responses occurring on the day of colonic infection, both in the host intestinal epithelial cells and in the microbiome. Our study suggests changes in cholesterol and carbon metabolism in epithelial cells are instantly induced upon pathogen detection in the colon, corresponding with a shift to primarily facultative anaerobes constituting the microbiome. This study contributes to our knowledge of disease pathogenesis and mechanisms of barrier regulation, which is required for development of novel therapeutics targeting the intestinal epithelium. KEYWORDS cholesterol homeostasis, Citrobacter rodentium, host response to infection, intestinal epithelial cells, the microbiome March/April 2019 Volume 10 Issue 2 e00062-19 Citation Hopkins EGD, Roumeliotis TI, Mullineaux-Sanders C, Choudhary JS, Frankel G. 2019. Intestinal epithelial cells and the microbiome undergo swift reprogramming at the inception of colonic Citrobacter rodentium infection. mBio 10:e00062-19. 10.1128/mBio.00062-19. Editor Rino Rappuoli, GSK Vaccines Copyright © 2019 Hopkins et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Jyoti S. Choudhary,, or Gad Frankel, Received 14 January 2019 Accepted 21 February 2019 Published 2 April 2019 ® 1 Hopkins et al. ® T he intestinal epithelium serves a dual role as it enables nutrient absorption while simultaneously providing a barrier to commensal bacteria and pathogens (1). Constant renewal of the epithelium every 5 to 7 days is enabled by LGR5⫹ stem cells at the base of the crypts, where they lie intermingled with deep crypt secretory (DCS) cells called Reg4⫹ cells (2). Transit-amplifying (TA) cells, arising from proliferation and partial differentiation of LGR5⫹ cells, rapidly divide in the lower half of the crypt a number of times before arresting their cell cycle and differentiating into mature cell types as they migrate to the crypt’s upper surface. These differentiated cell types include absorptive enterocytes, goblet cells, enteroendocrine cells, and tuft cells. In addition to providing a barrier, intestinal epithelial cells (IECs) also have immunoregulatory properties enabling them to detect invading pathogens, e.g., they are able to express pattern recognition receptors, and subsequently influence development of the mucosal immune cell response (3). Citrobacter rodentium, an extracellular mouse pathogen, is a physiologically relevant model for the human clinical pathogens enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC), and it has been widely used to probe mucosal responses to colonic infection (4, 5). Hallmarks of C. rodentium infection include tissue regeneration via colonic crypt hyperplasia (CCH), which results from increased amplification of TA cells in conjunction with inhibition of both anoikis and cell detachment. (5). Furthermore, the host has been shown to mount a robust nutritional immune response to C. rodentium infection, manifested by secretion of lipocalin-2 (LCN-2) and calprotectin (a heterodimer of subunits S100A8 and S100A9), which sequester the trace minerals Fe (LCN-2) and Mn and Zn (calprotectin) (6). Following oral inoculation, C. rodentium initially colonizes the cecal patch, a major lymphoid structure in the cecum, where it adapts to the gut environment (7). We define these first few days as the establishment phase, during which most of the inoculum passes straight through the intestinal tract and is shed in the feces (8). C. rodentium spreads from the cecal patch to the colon at 4 days postinfection (DPI), enabling rapid bacterial proliferation as it penetrates the mucosa and intimately attaches to IECs, which we define as the expansion phase. Colonization levels plateau at 108 to 109 CFU/g of feces between 8 to 12 DPI, before bacterial shedding decreases as the infection starts to clear between 12 to 16 DPI, which we define as the steady-state and clearance phases, respectively (5, 8). Mice develop colitis during C. rodentium infection (4), and dysbiosis is induced in the large intestine, resulting in a reduction in the overall abundance and diversity of commensal bacteria (9). IECs express the receptor for interleukin 22 (IL-22), which plays an essential role during C. rodentium infection, as it fortifies the intestinal barrier and restricts the pathogen to the gut (10). IL-22 also promotes production of antimicrobial peptides (AMPs), e.g., Reg3␤ and Reg3␥, LCN-2, calprotectin, and mucins (11–14). IL-22 is produced by innate class 3 lymphoid cells (ILC3s) during the establishment and expansion phases of infection, followed by CD11b⫹ Ly6C⫹ Ly6G⫹ neutrophils (15) and Th17 and Th22 T cells (16, 17), which contribute to IL-22 production during the steady-state and clearance phases. Despite IL-22 promoting epithelial regeneration in a STAT-3-dependent manner, IL-22–/– mice exhibit increased CCH and tissue damage at peak C. rodentium infection compared to that of wild-type mice (18), suggesting that in the absence of IL-22 there is an uncoordinated damage repair response to infection. Furthermore, IL-22 has also been implicated in regulating tight junctions and the permeability of IECs, including upregulation of the paracellular water and Na⫹ channel Claudin-2 (19, 20). Recently, we reported that C. rodentium subverts IEC metabolism in order to evade innate immune responses and establish a favorable niche in the colon (6, 21). Using multiplex proteomics, we uncovered a significant downregulation of host metabolic pathways, including gluconeogenesis, lipid metabolism, the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS), with a simultaneous upregulation of cell cycle and DNA replication pathways. At this stage, the IECs seemed to rely on aerobic glycolysis, fueled by a robust upregulation of the basolateral glucose transMarch/April 2019 Volume 10 Issue 2 e00062-19 2 Swift Host and Microbiome Responses to Gut Infections ® porter Slc5A9 (6). Presumably, this metabolic reprogramming occurs to meet the increased cellular energetic demands mediating tissue repair responses to the infection. Furthermore, our proteomics data, validated with a fecal cholesterol quantification assay, highlighted upregulation of both cholesterol biogenesis and efflux/influx pathways (6), processes which are usually regulated antagonistically. These observations are consistent with cholesterol being an essential ingredient of new membranes formed in proliferating cells and having a role in innate immunity. Importantly, while studies of pathogen-host interactions are usually conducted at the acute phase of the infection, little is currently known about the temporal host responses culminating before the pathogen burden peaks. The aim of this study was to temporally resolve IEC and microbiome responses at the expansion phase of C. rodentium infection (4 and 6 DPI). This revealed a dramatic reprogramming of the crypt cellular composition, metabolism, and DNA replication and repair immediately as the pathogen starts to colonize the colon, which coincided with the expansion of mucosaassociated Enterobacteriaceae. RESULTS Sporadic mucosal association of C. rodentium at the onset of colonic colonization. We recently reported IEC responses to C. rodentium at the steady-state phase of the infection (8 DPI), including reprogramming of bioenergetics and metabolism (6). Here, we aimed to track the progression of C. rodentium infection-induced alterations to the gut microenvironment, using multiplexed quantitative proteomics, transcriptomics, enzyme-linked immunosorbent assay (ELISA), and 16S rRNA gene sequencing. We first performed temporal profiling of C. rodentium shedding and tissue association during the expansion phase of infection. This revealed that shedding reached an average of 6.5 ⫻ 107 CFU/g by 4 DPI and 6.9 ⫻ 108 CFU/g by 6 DPI (Fig. 1A). Importantly, we recorded larger standard errors of the means at 2 to 4 DPI than at 5 and 6 DPI, suggesting that while migrating from the cecum to the colon, C. rodentium is more sensitive to variable host environments in individual mice (e.g., the composition of the microbiota) but is able to adapt once initial colon colonization has occurred, thus reaching homogenous levels of colonization from 5 DPI onwards. Comparing tissue distributions of C. rodentium at 4 DPI (i.e., on the day of colonic colonization) and 6 DPI (i.e., the intermediate time between the expansion and steady-state phases) by immunohistochemistry revealed distinct differences in bacterial abundance and distribution (Fig. 1B). Notably, no C. rodentium was detected in colonic sections at 3 DPI (see Fig. S1 in the supplemental material). While binding of C. rodentium to the colonic mucosa was scarce and highly varied between different mice at 4 DPI, a uniform distribution of the pathogen along the entire colonic mucosa was seen at 6 DPI (Fig. S1). These data suggest that despite 3 ⫻ 107 CFU/g being shed in the feces at 3 DPI, C. rodentium is not yet visibly detected in the large intestine; however, on the 4th day, C. rodentium is seeded in the colon, which is followed by rapid expansion. C. rodentium triggers rapid polarization of metabolic and cell proliferation processes. In order to determine the temporal impact of C. rodentium infection on IECs during the expansion phase, we performed proteomic analysis of colonic IECs isolated from C. rodentium-infected mice at 4 and 6 DPI using mock-infected mice as controls. We included 5 mice per time point and monitored colonization levels up until and including the day of extraction; mice that did not reach the minimum number of CFU per gram of stool thresholds (1 ⫻ 107 CFU/g stool by 4 DPI or 1 ⫻ 108 CFU/g stool by 6 DPI) were excluded from further processing to reduce heterogeneity within each condition (Fig. S2). Protein extracts from IECs enriched from mice within the same condition of a single biological repeat were pooled at a 1:1 ratio and labeled with tandem mass tags (TMT) before undergoing liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis (Fig. S3A; Table S1). To generate a robust data set, three biological repeats were included within the same multiplex experiment. We quantified a total of 10,418 proteins, of which 9,127 were mapped to Mus musculus and 1,290 to C. rodentium (peptide false-discovery rate [FDR], ⬍1%). Statistical March/April 2019 Volume 10 Issue 2 e00062-19 3 Hopkins et al. ® FIG 1 Sporadic C. rodentium colonization at 4 DPI induces upregulation of the cell cycle and downregulation of metabolic processes. (A) Line graph showing average numbers of CFU/g feces with standard error of the mean bars over the time course of C. rodentium infection (n ⫽ 10). (B) Representative images of immunostaining of C. rodentium (white) and DNA (blue) on colonic sections (n ⫽ 10) from mock-infected (Mock) or infected mice at 4 DPI or 6 DPI, as indicated. (Continued on next page) March/April 2019 Volume 10 Issue 2 e00062-19 4 Swift Host and Microbiome Responses to Gut Infections ® analysis considering both the 4 and the 6 DPI data as infected samples identified 587 upregulated and 446 downregulated proteins compared to protein expression in the mock-infected samples (Fig. S3B). Further analysis of the most significantly changed proteins upon infection revealed two protein subsets: those that decrease during the course of infection, which highlighted processes, including carbohydrate and pyruvate metabolism, as downregulated, and those that increase, which showed upregulation of processes, including response to stress, DNA replication, and ribosome biogenesis (Fig. 1C). Bioinformatic analysis of the data set as a whole identified additional enriched pathways, including significant downregulation of further metabolic processes, such as the TCA cycle, OXPHOS, propanoate, pyruvate, and starch and glucose metabolism, and upregulation of cell cycle and DNA repair pathways (mismatch repair, homologous recombination, and nucleotide excision repair) (Fig. 1D). Unexpectedly, many of these processes correlate with those identified as significantly altered at the steady-state phase of infection (6), suggesting that a significant response to pathogen infection is mounted in IECs during the expansion phase. Interestingly, Forkhead box O3 (FOXO3), a transcription factor involved in modulating the metabolic state and cellular apoptosis, was predicted to be significantly inactivated during C. rodentium infection (enrichment score, ⫺0.31; Benjamini-Hochberg FDR, 8.16E– 03), correlating with a previous in vitro study that showed that C. rodentium infection led to inactivation of FOXO3 in intestinal epithelia (Fig. S4) (22). To further resolve temporal changes, one-dimensional (1D) enrichment analysis was applied to 4 and 6 DPI samples separately, revealing early onset of a number of DNA repair pathways, with base excision repair specific to 4 DPI only (Fig. 1E). Significant upregulation of a number of DNA repair pathways at 4 DPI, in addition to upregulation of cell cycle and DNA replication processes, strongly suggests that proliferation pathways are activated even when C. rodentium colonization is low, sporadic, and restricted to the upper surface of the crypt. While both expansion phase time points show downregulation of the TCA cycle, the pentose phosphate pathway, and pyruvate metabolism, only 6 DPI additionally shows upregulation of steroid hormone biosynthesis, suggesting that changes to IEC metabolism are initiated as early as the day of colonic colonization (4 DPI), but further aspects of metabolism become significantly affected as pathogen levels in the colon increase. Furthermore, downregulation of the TCA cycle and OXPHOS suggests a progressive shift of cellular bioenergetics during infection to aerobic glycolysis, which coincides with an increase in cell proliferation. On a protein-specific level, among the proteins ranked in the top 100 for most changed in abundance, we found a number of innate immunity and nutritional immunity proteins, including matrix metallopeptidase 9 (MMP9), Reg3␤, Reg3␥, inducible nitric oxide synthase (iNOS), LCN-2, DMBT1, S100A8, and S100A9 (Fig. 1F). Furthermore, the neutrophil chemoattractant CXCL5, the glucose transporter that fuels glycolysis, Slc5A9, the rate-limiting enzyme in the cholesterol biogenesis pathway, Hmgcr (3-hydroxy-3-methylglutaryl– coenzyme A reductase), and the basolateral cholesterol efflux transporter Abca1 were also found among top 100 proteins with significantly increased abundance during infection. Together, these data show that a significant change in both the metabolic and proliferative states of IECs is induced on the day of colonic colonization (4 DPI), coinciding with innate and nutritional immunity responses. FIG 1 Legend (Continued) Sections from 4 DPI were highly varied; thus, the image with the average level of C. rodentium staining was selected. Scale bar ⫽ 500 ␮m. (C) Heat map showing proteins with significantly altered abundances at 6 DPI compared to abundances in mock-infected mice and filtered for the 30% most altered proteins upon infection. Scaled abundances for 3 biological repeats of each time point are shown (R1 to R3). Profile plots show significant downregulated proteins in blue and upregulated proteins in red. The right-most plot shows relative enrichments of named KEGG and GOBP pathways. Fischer exact test FDR, ⬍0.05. Benj. Hoch., Benjamini-Hochberg. (D) KEGG pathway enrichment analysis. Proteins are ranked according to log2 values on the x axis, with increasingly negative log2FC values on the far left (blue) to increasingly positive log2FC values during infection on the far right (red). 1D annotation enrichment of KEGG pathways during infection are highlighted in the heat map on the y axis, with the most downregulated pathways at the top to the most upregulated pathways at the bottom. FDR ⬍ 0.05; t test (n ⫽ 3). (E) Venn diagram showing pathways that are upregulated (red) or downregulated (blue) at 4 DPI, 6 DPI, or both time points of infection. (F) Heat map of selected proteins from the top 100 proteins when statistically significantly altered abundances are ranked based on the highest fold change at 6 DPI compared to abundances in mock-infected controls. March/April 2019 Volume 10 Issue 2 e00062-19 5 Hopkins et al. ® FIG 2 Cholesterol homeostasis is perturbed during early infection. (A and B) Log2FCs in infected samples (4 or 6 DPI, as indicated) compared with samples from mock-infected mice of named SREBP2-induced proteins (A) and LXR-induced proteins (B). The dotted line at log2FC value 0.6 represents the implicated cutoff for an upregulated protein. (C to H) A qRT-PCR analysis of Hmgcr (C), Ldlr (D), Pcsk9 (E), Abca1 (F), Abcg5 (G), and Idol (H) revealed expression levels ...
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