Colonizing bacteria around aggregated lymphoid tissue of the rat ascending colon change diurnally and affect the host local transcriptome

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Numerous bacteria colonize between the mucosal folds of the ascending colon in rodents; however, the rhythm of bacteria colonizing the ascending colon remains to be clarified. Therefore, we first aimed to elucidate the circadian rhythms of bacteria colonizing in the rat ascending colon. The settlement levels of indigenous bacteria were significantly higher at zeitgeber time (ZT) 18 (dark phase) than at ZT6 (light phase) in the region encompassing the aggregated lymphoid tissue in the ascending colon (ALT-AC). The bacterial composition around the ALT-AC was dominated by the phylum Firmicutes and the family Lachnospiraceae , displaying notable distinctions from the compositions found in cecal contents and feces. The relative abundance of some bacterial species around the ALT-AC, such as Mucispirillum schaedleri , changed significantly between ZT6 and ZT18. Furthermore, we explored the effect of bacterial expansion on gene expression in the ALT-AC at ZT18 by administrating antibiotics for 1 day to inihibit bacterial growth. The antibiotic-treated group exhibited significant downregulation of multiple genes, including those associated with cell proliferation ( Plk3 ), differentiation into goblet cells ( Spdef , Atoh1 , Bhlha15 ), and Golgi organization ( Gorasp2 ). These results suggested that indigenous bacteria around the rat ALT-AC undergo diurnal changes in both settlement levels, peaking at the dark phase, and bacterial composition. In addition, bacterial expansion during the dark phase can induce changes in the expression of diverse genes, including genes associated with goblet cell differentiation. Ascending colon Circadian rhythm Indigenous bacteria Aggregated lymphoid tissue Histological analysis Transcriptome Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Numerous indigenous bacteria settle in the animal alimentary tract. The amounts and compositions of bacteria in the digestive tract are known to vary throughout the day according to their circadian rhythms. For example, bacterial compositions show circadian rhythms in the gastrointestinal contents of mice, such as cecal contents and feces (Heddes et al. 2022 ; Wu et al. 2018 ). On the other hand, we have documented in rats a region-specific circadian rhythm in the numbers of bacteria colonizing on the mucosal surfaces of the esophagus, the non-glandular part of the stomach, the ileal ordinary mucosa, and Peyer's patch (Sakata et al. 2022 ; Mantani et al. 2023 ). Such indigenous bacteria also have a variety of effects on the host intestine. In mice, the settlement of segmented filamentous bacteria in the small intestine induces the expression of antibacterial peptides (Reg3γ, LCN2, S100A8) (Brooks et al. 2021 ) and the differentiation of Th17 cells (Atarashi et al. 2015 ). The colonization of Clostridium spp ., one of the bacteria indigenous to the murine gastrointestinal tract, induces the differentiation of regulatory T cells in the murine colon (Atarashi et al. 2011 ). Bacterial settlement also promotes intestinal epithelial cell proliferation in the murine small intestine via the production of short-chain fatty acids (SCFA) (Park et al. 2016 ). Considering these effects of the indigenous bacteria on the homeostasis of the intestinal mucosa, it is important to clarify their circadian rhythm to understand how indigenous bacteria affect the host. However, studies of the circadian rhythms in indigenous bacteria have just begun, and some regions, including the ascending colon, remain to be explored. In general, the colon is the region where colonizing bacteria abound in many animal species. Mucosal folds develop in the ascending colon of various rodents, such as mice (Nava et al. 2011 ), rats (Mantani et al. 2013 ), cotton rats (Chuluunbaatar et al. 2020 ), African mole rats (Kotzé et al. 2009 ), guinea pigs, and chinchillas (Holtenius and Björnhag 1985 ). Large amounts of bacteria colonize the spaces between mucosal folds in the ascending colon of mice (Nava et al. 2011 ) and rats (Mantani et al. 2013 ). Our prior study has revealed circadian rhythms in the abundance of colonizing bacteria in various regions of the rat gastrointestinal tract (Sakata et al. 2022 ), however, we could not examine the circadian rhythm of bacterial colonization in the ascending colon because conventional paraffin sectioning caused cracks in the bacterial layer between the mucosal folds. Therefore, our primary objective in this study was to elucidate the pattern of diurnal changes in the amount and composition of indigenous bacteria in the ascending colon by histological analysis and 16S rRNA amplicon sequencing analyses. Furthermore, given that inhibiting diurnal bacterial growth with 1-day antibiotic treatment changed the gene expression profile in the rat ileal Peyer’s patch in our previous study (Mantani et al. 2023 ), we hypothesized that expansion of indigenous bacteria in the rat ascending colon might have an important role in regulating gene expression in the aggregated lymphoid tissues there. We therefore tested this hypothesis with transcriptome analysis as the second aim. MATERIALS AND METHODS Animals A total of 39 male Wistar rats aged 9 weeks (Japan SLC Inc., Shizuoka, Japan) were maintained under specific pathogen-free conditions in individual ventilated cages (Sealsafe Plus; Techniplast S.p.a, Buguggiate, Italy) with controlled temperature (23 ± 2°C) and humidity (50 ± 10%) on a 12/12-h light/dark cycle at the Kobe University Life-Science Laboratory. The rats had free access to water and food (Lab RA-2; Nosan, Kanagawa, Japan). This study was approved by the Institutional Animal Care and Use Committee (permission numbers: 2023-04-01, 2019-09-01). All procedures in studies involving animals were performed in accordance with the ethical standards of the institution (the Kobe University Animal Experimentation Regulations) or at the practice at which the studies were conducted. Tissue preparation for light microscopy Twenty-five rats aged 9 weeks were divided into four groups according to the sampling time: zeitgeber time (ZT) 0 (n = 6), which corresponds to the beginning of the light phase, ZT 6 (n = 7), ZT 12 (n = 6), and ZT 18 (n = 6). After euthanasia by inhalation of isoflurane (FUJIFILM Wako Pure Chemical, Osaka, Japan), the ascending colons were dissected and immediately fixed in Carnoy’s fixative for 12 h at room temperature. The regions containing the aggregated lymphoid tissue (ALT-AC) located in a specific area of the ascending colon (Crouse et al. 1989 ), as well as the region adjacent to the ALT-AC on its proximal side, were taken from each fixed ascending colon (Supplementary Fig. 1) and dehydrated. In this study, we used methacrylate resin (Technovit 8100; Kulzer, Hanau, Germany) to embed the blocks, because methacrylate resin is effective for preserving luminal contents in intestinal blocks (Hasegawa et al. 2017 ) and was expected to be effective for preserving indigenous bacteria that colonize on the mucosal epithelium as well. Then, 4-µm-thick sections were cut and placed on CREST-coated glass slides (Matsunami Glass, Osaka, Japan). These tissue sections were washed with distilled water and used for Mayer’s hematoxylin and eosin (HE) staining. Histological quantification of settlement level of indigenous bacteria and statistical analysis The area (mm 2 ) of the bacterial layer between the intestinal superficial epithelial cells and the luminal content was calculated in a single cross-section using ImageJ2 (ver. 2.3.0/1.53f), as was the length (mm) of the outermost circumference of the transverse sections of the ascending colon (Supplementary Fig. 2). The normality of distribution was first assessed by the Kolmogorov‒Smirnov test. For parametric variables in multiple comparisons, one-way ANOVA was performed, followed by the Bonferroni test for post hoc comparisons. For nonparametric variables in multiple comparisons, the Kruskal‒Wallis test was performed, followed by the Steel‒Dwass test for post hoc comparisons. P values less than 0.05 were considered statistically significant. Statistical analyses were conducted with BellCurve for Excel (ver. 4.07). Tissue preparation for 16S rRNA amplicon sequencing Eight rats aged 9 weeks were equally divided into two groups based on the sampling time: ZT6 and ZT18 (4 rats per ZT). After euthanasia by inhalation of isoflurane (FUJIFILM Wako Pure Chemical), the area containing the ALT-AC, the cecal contents, and the feces were dissected. The luminal content was then carefully removed from the ascending colon by flushing with 4.0% paraformaldehyde (PFA) fixative in 0.1 M phosphate buffer (PB; pH 7.4), then fixed with 4.0% PFA fixative in 0.1M PB for 6 h at 4℃ and snap-frozen in liquid nitrogen as described previously (Mantani et al. 2018 ). Serial 10-µm-thick sections were cut using a Cryotome Leica CM1950 (Leica Microsystems, Nussloch, Germany). The tissue sections for the 16S rRNA amplicon sequencing were placed in Eppendorf tubes, whereas the tissue sections just before and after each tissue section for the 16S rRNA amplicon sequencing were stained with HE staining (see also Supplementary Fig. 3). These sections were stored at − 30°C until use. After confirming that the luminal content had been mostly removed from each tissue block using HE-stained sections, the sections cut from the same frozen block for 16S rRNA amplicon sequencing were washed with diethylpyrocarbonate (DEPC)-treated water. Total DNA was extracted with the RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. DNA from cecal contents and feces was extracted using Nucleospin Tissue kits (Macherey‒Nagel, Düren, Germany). Libraries for sequencing were then prepared by a two-step tailed PCR method with TaKaRa Ex Taq Hot Start Version (Takara Bio, Shiga, Japan) as described previously (Sakata et al. 2022 ). Briefly, the sequences of 16S rRNA regions were first amplified using PCR with primers for the V4 region (F 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNGTGCCAGCMGCCGCGGTAA-3’ R 5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNGGACTACHVGGGTWTCTAAT-3’) and then amplified with the index primer. Each PCR product was sequenced on an Illumina Miseq system (Illumina, San Diego, CA, USA). The reads were processed with QIIME2 (Bolyen et al. 2019 ) as described previously (Sakata et al. 2022 ). The relative abundance of bacteria colonizing the ascending colon was compared with that of cecal contents and feces at taxonomic levels, from phylum to species, using LEfSe (Segata et al. 2011 ). Statistical analysis of the Chao1 index and the weighted UniFrac distances was performed using the Kruskal‒Wallis and PERMANOVA tests combined with the false discovery rate (FDR) method of Benjamini‒Hochberg, respectively. q -values less than 0.05 were considered statistically significant. One-day antibiotic treatment for transcriptome analysis To investigate how the diurnal change in indigenous bacteria affects the ALT-AC transcriptome, we impaired the diurnal growth of indigenous bacteria by administering antibiotics for 1 day as described previously (Mantani et al. 2023 ). Briefly, 6 male Wistar rats aged 9 weeks were equally divided into a control group and an antibiotic-treated (Abx) group. The drinking water was then replaced with a 1% sucrose solution for the control group and with an Abx solution (vancomycin (0.5 g/L; FUJIFILM Wako Pure Chemical), polymyxin B (0.1 g/L; FUJIFILM Wako Pure Chemical), and 1% sucrose) for the Abx group 1 day before euthanasia at ZT18 (see also Supplementary Fig. 4). Sucrose was added to the solution to prevent the rats from avoiding drinking. After euthanasia at ZT18 by inhalation of isoflurane, the ALT-ACs were dissected from 3 rats in the control group and 3 rats in the Abx group. Total RNA was extracted from each sample with NucleoSpin RNA Plus kits (Macherey‒Nagel) according to the manufacturer’s instructions and used for transcriptome analysis as follows. Transcriptome analysis We controlled the quality of RNA-seq data using fastp (version 0.23.2) (Chen et al. 2018 ) with default options to discard low-quality reads. We aligned the remaining reads to the rat reference genome (rn6) using HISAT2 (version 2.2.1) (Kim et al. 2019 ) with the “–no-mixed –dta” options. To quantify gene-level expressions, we counted reads for each transcript using featureCounts (version 2.0.6) (Liao et al. 2014 ) with the RefSeq gene annotation (rn6.ncbiRefSeq.gtf) and the “-p -B -t exon -g gene_id” options. Differential expression analysis was performed using the DESeq2 method (Love et al. 2014 ). Adjusted P values less than 0.05 and absolute fold changes greater than two were considered statistically significant. We conducted GO enrichment analysis with the identified differentially expressed genes using Metascape (Zhou et al. 2019 ). The results of a volcano plot and principal component analysis were plotted in Python (version 3.8.5). RESULTS Diurnal change in the settlement levels of indigenous bacteria in the rat ascending colon Regardless of regions in the ascending colon or ZT, a bacterial layer densely stained with hematoxylin was observed between the mucosal folds of the rat ascending colon (Fig. 1 a-h), and this bacterial layer was a continuous thin layer even at the tip of the mucosal folds. The bacterial layer was clearly distinguishable from the luminal contents because the luminal contents were less strongly stained than the bacterial layer due to the low density of bacteria. Fibers from the diet, but not the luminal content, were occasionally mixed inside this bacterial layer. Furthermore, bacilli or rod-shaped bacteria, which exhibited the same shape as bacteria identified in the space of mucosal folds under electron microscopy in a previous study (Mantani et al. 2015 ), were observed in the bacterial layer under high magnification regardless of ZT or regions in the ascending colon (Fig. 1 a’-h’). In the region containing the ALT-AC, the bacterial layer between the mucosal folds tended to be thicker at ZT18 (Fig. 1 d) than at the other ZTs (Fig. 1 a-c). Conversely, the luminal content frequently penetrated deeply into the space between mucosal folds where the bacterial layer was thinned at ZT6 (Fig. 1 b) and ZT12 (Fig. 1 c). The histological measurement revealed that the area of the bacterial layer per the length of the outermost circumference of the ascending colon at ZT18 was significantly larger than that at ZT6 in the ALT-AC-containing regions (Fig. 1 i; P < 0.05). On the other hand, no significant differences were found between any of the ZTs in the region without the ALT-AC, although three of the six samples at ZT18 showed higher values than any of the samples at ZT6 (Fig. 1 j). Diurnal changes in bacterial composition on the mucosal surface of the ascending colon including the ALT-AC Histological analysis showed that the settlement level of indigenous bacteria was high at ZT18 and low at ZT6 in the ALT-AC-containing region. Next, we performed tissue-section 16S rRNA amplicon sequencing analysis (Sakata et al. 2022 ) (Supplementary Fig. 3) at ZT6 and ZT18 to characterize the specificity in the bacterial composition of the indigenous bacteria settled around the ALT-AC and its diurnal change. First, principal coordinate analyses based on the weighted UniFrac distance revealed that the bacterial composition statistically exhibited distinct characteristics among the ascending colon, cecal contents, and feces, but not between ZTs (Fig. 2 a, b; q < 0.05). On the other hand, there was no significant difference between ZT6 and ZT18 in the weighted UniFrac distance of bacteria colonizing the ascending colon. The Chao1 index, which is used to evaluate the richness of bacterial species, showed significantly fewer bacterial species in the mucosal surface of the ascending colon compared to the cecal contents and feces at both ZTs (Fig. 2 c, d; q < 0.05). Next, the relative abundance of each bacterium at phylum level (Fig. 3 a) and family level (Fig. 3 b) in each sample was analyzed. The relative abundance of Firmicutes and Bacteroidetes was almost at the same level in the cecal content. On the other hand, their relative frequencies differed in the feces and ascending colon; Bacteroidetes dominated in feces, whereas Firmicutes accounted for more than 80% of the indigenous bacteria in the ascending colon (Fig. 3 a). At the family level, two bacterial families, Lachnospiraceae and Ruminococcaceae , predominated in the indigenous bacteria in the ascending colon, and the relative abundance of Lachnospiraceae was significantly higher in the ascending colon than in the cecal content and feces at both ZTs (Fig. 3 b; Supplementary Figs. 5, 6). The relative abundance of several bacterial species colonizing the ascending colon differed significantly between ZT6 and ZT18 (Fig. 3 c). A species belonging to the genus Anaerotruncus was significantly more abundant at ZT6 than at ZT18, whereas 5 bacterial species— Mucispirillum schaedleri , 2 Lachnospiraceae species (PAC001459_s, PAC001126_s), a Caproiciproducens species (PAC001505_s), and a Dehalobacterium species (PAC001221_s)—were more abundant at ZT18 than at ZT6 (Fig. 3 c). These findings suggest that the indigenous bacteria around the ALT-AC are unique compared to those in cecal contents or feces, and that their composition changes slightly within a day. Effect of suppressing diurnal expansion of bacterial colonies on the ALT-AC transcriptome at ZT18 Histological analysis suggested that the settlement level of indigenous bacteria, especially around the ALT-AC, changes diurnally with a peak at ZT18. Therefore, to explore what genes could be regulated by the diurnal expansion of bacterial colonies in the ALT-AC and the surrounding mucosa at ZT18, when the bacteria were most abundant, we performed 1-day Abx treatment (Mantani et al. 2023 ) (Supplemantary Fig. 4 ) and transcriptome analysis. Principal component analysis showed that plots of the control group formed small clusters, whereas those of the Abx group were dispersed, indicating distinct gene expression profiles between the control and the Abx groups (Fig. 4 a). We identified a total of 146 differentially expressed genes (DEGs) between the control and the Abx group in the ALT-AC; 107 were upregulated and 39 were downregulated (Fig. 4 b). The downregulated genes were enriched in the following cascades: epithelial cell development, Golgi organization, positive regulation of growth, monocarboxylic acid transport, positive regulation of inflammatory response, cellular response to starvation, positive regulation of apoptotic process, lipid homeostasis, protein phosphorylation, regulation of metal ion transport, and tissue homeostasis (Fig. 4 c). On the other hand, the upregulated genes were enriched in 17 cascades, such as biological oxidations, phospholipid efflux, bile acid secretion, positive regulation of interferon-beta production, xenobiotic catabolic process, response to toxic substance, organic hydroxy compound metabolic process, regulation of cellular respiration, benzene-containing compound metabolic process, and negative regulation of cell‒cell adhesion (Fig. 4 d). Downregulated genes in the Abx group included several transcription factors ( Spdef , Atoh1 , Bhlha15 ), a Golgi reassembly stacking protein ( Gorasp2 : LOC103690018 ), and a cell-cycle-related gene ( Plk3 ) (Fig. 4 b, Supplementary Table 1). Upregulated genes in the Abx group were associated with lipid metabolism, including apolipoproteins ( Apoa1 , Apoa4 , Apoc3 ) and the cytochrome P450 family (e.g., Cyp2b1 , Cyp3a62 , Cyp3a9 , Cyp8b1 ), as along with genes involved in bile acid transport, Slc51a and Slc51b (Fig. 4 b, Supplementary Table 2). Genes involved in viral RNA degradation ( Oas1a, Oas2 , Oas3 ) and Granzymes ( Gzma, Gzmf ) known to induce cell death in virus-infected cells were also upregulated in the Abx group (Fig. 4 b, Supplementary Table 2). DISCUSSION Our previous studies have demonstrated a circadian rhythm in the settlement level of indigenous bacteria across various regions of the rat alimentary tract, peaking at ZT12 in the esophagus, ZT6 in the non-glandular part of the stomach, ZT0 in the ileal villi, and from ZT18 to ZT0 in the most distal ileal Peyer’s patch (Sakata et al. 2022 ; Mantani et al. 2023 ). Focusing on the ascending colon, this study showed that the bacterial quantity between the mucosal folds was significantly higher at ZT18 than at ZT6 in the region including the ALT-AC, but not in that without ALT-AC. This finding suggests a diurnal change, with a peak at ZT18, in bacterial abundance in the rat ascending colon, especially around the ALT-AC. Tissue section 16S rRNA amplicon sequencing in this study revealed significantly less bacterial species richness in the indigenous bacteria around the ALT-AC than in cecal contents and feces. It also showed that a unique bacterial flora, characterized by Firmicutes and Lachnospiraceae and differing from that of the cecal contents and feces, colonized the mucosal surface of the rat ascending colon. This finding supports a previous study that showed similar bacteria are dominant between mucosal folds in the mouse ascending colon (Nava et al. 2011 ). Further, comparing the composition of indigenous bacteria in the ascending colon between ZT6 and ZT18 suggested that the composition of colonizing bacteria around the ALT-AC underwent diurnal changes, possibly induced by the diurnal variation in the relative abundance of Mucispirillum schaedleri and Anaerotruncus . Most of bacteria on the mucosal surface of the ascending colon at both ZT6 and ZT18 in this study were classified into Lachnospiraceae or Ruminococcaceae , which produce SCFA (e.g., acetate, butyrate, propionate) by fermentation of carbohydrates, including cellulose (Barcenilla et al. 2000 ; Biddle et al. 2013 ). In addition, the relative abundance of two Lachnospiraceae species differed significantly between ZT6 and ZT18 in the present study, suggesting the hypothesis that this diurnal change might be accompanied by a circadian rhythm in SCFA production. SCFA derived from the intestinal microflora play various physiological roles related to the intestinal immune system. For example, IgA secretion is facilitated by acetate (Takeuchi et al. 2021 ) and butyrate (Isobe et al. 2020 ) in the murine intestine. Butyrate and propionate promote the differentiation of regulatory T cells in the murine colon (Furusawa et al. 2013 ). Therefore, the hypothesis concerning the circadian rhythm in SCFA production due to diurnal changes in Lachnospiraceae species in the ascending colon is an important topic for understanding the temporal regulation of the intestinal immune system and should be tested in future studies. In the present study, we explored which genes are regulated by the expansion of indigenous bacterial colonies at ZT18 in the rat ALT-AC by administering nonabsorbable antibiotics in the drinking water for 1 day. Through transcriptome analysis, we identified 146 DEGs whose expression might be affected by colonizing bacteria in the rat ALT-AC in daily life. These DEGs contained more than twice as many upregulated genes downregulated ones. Considering that the numbers of upregulated and downregulated genes after the 1-day antibiotic treatment in the rat ileum were almost equal (Mantani et al. 2023 ), the host gene expression regulated by bacterial growth on the ALT-AC may be biased toward repression. In general, aggregated lymphoid tissues in the intestine are the specific sites for inducing immune responses against luminal antigens. In our previous study, inhibition of bacterial expansion by 1-day Abx treatment showed the downregulation of immune-associated genes, such as cytokines and chemokines, at ZT0 in the ileal Peyer’s patch (Mantani et al. 2023 ). In the present study, on the other hand, the expression of immune-related genes, at least cytokines and chemokines, did not decrease in ALT-AC, in contrast to the ileal Peyer’s patch (Mantani et al. 2023 ). Instead, genes associated with epithelial cell differentiation, monocarboxylic acid transport, and the positive regulation of inflammatory response were downregulated. In addition, genes related to various cascades, such as lipid metabolism, antiviral immune responses, and xenobiotic metabolism, which are unexpected as host responses to bacteria, were identified among the upregulated genes in the ALT-AC. These results imply that the expansion of indigenous bacteria during the dark phase is involved in regulating multifaceted functions of the ALT-AC other than the well-known antibacterial immune response. In the present study, a 1-day antibiotic treatment from ZT18 to inhibit bacterial growth reduced the expression of several transcription factors ( Spdef , Atoh1 , and Bhlha15 ) associated with epithelial cell differentiation in the ALT-AC. In mice, Atoh1 and Spdef are expressed in goblet cells and Paneth cells of the ileum and in goblet cells of the colon (Lo et al. 2017 ; Noah et al. 2010 ). Spdef promotes the differentiation of secretory progenitor cells into goblet cells in the ileum, and goblet cells are almost absent in Atoh1 -deficient mice (Lo et al. 2017 ; Noah et al. 2010 ). Bhlha15 -positive cells reside at the base of the colonic crypt in mice and differentiate into goblet or tuft cells (Hayakawa et al. 2019 ). These findings suggest that the bacterial growth on the ALT-AC during dark phase promotes epithelial stem cells to differentiate into secretory lineage, such as goblet cells. This idea is also supported by another observation: the downregulation of Gorasp2 , which is essential for Golgi cisternal stacking of the Golgi apparatus (an abundant organelle in goblet cells) (Xiang et al. 2010) within the antibiotic-treated group in this study. However, it is unclear whether indigenous bacteria directly regulate the differentiation of epithelial cells because the expression level of cell differentiation-related genes depends in part on cell proliferation activity. In the present study, the downregulated genes in the 1-day Abx group included Plk3 , which is necessary for the transition from G1 to the S phase of the cell cycle (Zimmerman et al. 2007), suggesting that bacterial depletion for 1 day might also affect cell proliferation. Such a relationship exists between cell proliferation and intestinal microflora in the colonic epithelium, as observed by comparing germ-free rats and conventional rats (Alam et al. 1994 ). Therefore, we could not exclude the possibility that the downregulation of genes involved in goblet cell differentiation in the 1-day Abx group is a consequence of the suppression of epithelial cell proliferation owing to bacterial depletion. This hypothesis needs to be tested in the future. In conclusion, this study provided findings suggesting that the settlement level of indigenous bacteria around the ALT-AC changes diurnally, peaking at the dark phase, and that this change is associated with the diurnal change in the bacterial composition at the species level, characterized by changes in the relative abundance of Mucispirillum schaedleri and Anaerotruncus . In addition, the expansion of the bacterial colony during the dark phase around the ALT-AC can contribute to regulating the expression of various genes, including those involved in goblet cell differentiation. Declarations Author contributions The authors have contributed to this study as follows: conceptualization (YM), investigation (AS, RM), formal analysis (AS, NK, YM), visualization (AS, NK, YM), funding acquisition (AS, SZ, YM), supervision (SZ, TY, NH, YM), writing original draft (AS), critically reviewing and editing manuscript (NK, RM, TY, NH, YM). All authors have read and approved the final manuscript. Research involving Animals This study was approved by the Institutional Animal Care and Use Committee (permission number 2023-04-01, 2019-09-01). All procedures in studies involving animals were performed in accordance with the ethical standards of the institution (the Kobe University Animal Experimentation Regulations) or at the practice at which the studies were conducted. Competing interests The authors declare that they have no conflict of interest associated with this manuscript. Data availability RNAseq data were deposited into the Gene Expression Omnibus database under accession number GSE284464 and are available at the following URL: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE284464 References Alam M, Midtvedtm T, Uribe A (1994) Differential Cell Kinetics in the Ileum and Colon of Germfree Rats. Scand J Gastroenterol 29:445–451 Atarashi K, Tanoue T, Ando M, Kamada N, Nagano Y, Narushima S, Suda W, Imaoka A, Setoyama H, Nagamori T, Ishikawa E, Shima T, Hara T, Kado S, Jinnohara T, Ohno H, Kondo T, Toyooka K, Watanabe E, Yokoyama S, Tokoro S, Mori H, Noguchi Y, Morita H, Ivanov I, Sugiyama T, Nuñez G, Camp J, Hattori M, Umesaki Y, Honda K (2015) Th17 cell induction by adhesion of microbes to intestinal epithelial cells. 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Supplementary Files ShimadaetalSupplymentalfigure.pdf Cite Share Download PDF Status: Published Journal Publication published 26 Aug, 2025 Read the published version in Cell and Tissue Research → Version 1 posted Editorial decision: Revision requested 24 Mar, 2025 Reviews received at journal 23 Mar, 2025 Reviews received at journal 23 Mar, 2025 Reviewers agreed at journal 10 Mar, 2025 Reviewers agreed at journal 03 Mar, 2025 Reviewers invited by journal 03 Mar, 2025 Editor assigned by journal 20 Feb, 2025 Submission checks completed at journal 20 Feb, 2025 First submitted to journal 18 Feb, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6052953","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":433297449,"identity":"dd0921bf-f0fe-4858-a8b9-882dd7317160","order_by":0,"name":"Asaka SHIMADA","email":"","orcid":"","institution":"Kobe University","correspondingAuthor":false,"prefix":"","firstName":"Asaka","middleName":"","lastName":"SHIMADA","suffix":""},{"id":433297450,"identity":"00d1fd6b-243a-474e-b15d-10db7a6e334d","order_by":1,"name":"Naoto KUBOTA","email":"","orcid":"","institution":"University of California, Riverside","correspondingAuthor":false,"prefix":"","firstName":"Naoto","middleName":"","lastName":"KUBOTA","suffix":""},{"id":433297451,"identity":"1e2849f8-063b-45ec-a49a-c9a6aface466","order_by":2,"name":"Sika Zheng","email":"","orcid":"","institution":"University of California, Riverside","correspondingAuthor":false,"prefix":"","firstName":"Sika","middleName":"","lastName":"Zheng","suffix":""},{"id":433297452,"identity":"78ad85e0-3e43-45ef-9ba0-996128eea40f","order_by":3,"name":"Rinako MORISHITA","email":"","orcid":"","institution":"Kobe University","correspondingAuthor":false,"prefix":"","firstName":"Rinako","middleName":"","lastName":"MORISHITA","suffix":""},{"id":433297453,"identity":"1d0c5d49-87d5-451a-929e-2eee4063626b","order_by":4,"name":"Toshifumi YOKOYAMA","email":"","orcid":"","institution":"Kobe University","correspondingAuthor":false,"prefix":"","firstName":"Toshifumi","middleName":"","lastName":"YOKOYAMA","suffix":""},{"id":433297455,"identity":"bc5738a7-00e2-4c52-a15a-0997d36837ad","order_by":5,"name":"Nobuhiko HOSHI","email":"","orcid":"","institution":"Kobe University","correspondingAuthor":false,"prefix":"","firstName":"Nobuhiko","middleName":"","lastName":"HOSHI","suffix":""},{"id":433297457,"identity":"bdeaa0fd-33c1-456d-9e56-ab96a0d697c5","order_by":6,"name":"Youhei MANTANI","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYPACCTkQycxgACR5GNgIKWdsAGoxJlkLQ2IDWAsDEVp0288ef/Bzh0X6/Pazxx4XFNyTY+A5fOzBBzxazM7kJTb2npHI3XAmL914hkGxMQNvW7rhDHxaDuQYNvC2AbUw5JhJ8xgkJDbw8wAZ+LScf2PY+LdNIl2+/w1YSz1Yyx98Wm7kGDYDbUlguAGxJYGBt8dMGp/3zW68MZwt2yZhuOHGu3RjoBbDNp5j6YY9eB2WY/DxbVudvHx/7rHHPH8S5Pl5ko89+IHPGgTggUQHwXjE1DIKRsEoGAWjAB0AABmqSFCxXAawAAAAAElFTkSuQmCC","orcid":"","institution":"Kobe University","correspondingAuthor":true,"prefix":"","firstName":"Youhei","middleName":"","lastName":"MANTANI","suffix":""}],"badges":[],"createdAt":"2025-02-18 06:08:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6052953/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6052953/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00441-025-04000-1","type":"published","date":"2025-08-26T15:56:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83542810,"identity":"a5413f9c-f9b3-401e-baba-63aeb8780e8d","added_by":"auto","created_at":"2025-05-28 08:33:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2622620,"visible":true,"origin":"","legend":"\u003cp\u003eDiurnal changes in the quantity of indigenous bacteria in the ascending colon.\u0026nbsp; Bacterial layers strongly stained with hematoxylin are observed between the mucosal folds at all ZTs and are clearly distinguished from the luminal contents.\u003cstrong\u003e\u0026nbsp; \u003c/strong\u003ea-d) Bacterial quantity around the aggregated lymphoid tissues in the ascending colon (ALT-AC) at ZT0 (a), ZT6 (b), ZT12 (c), and ZT18 (d).\u0026nbsp; While the bacterial layer is thinner and the luminal content\u003cdel\u003e \u003c/del\u003epenetrates deeply into the region between the mucosal folds at ZT6 (b) and ZT12 (c), there is less penetration of luminal content and a thicker bacterial layer at ZT18 (d) than in the other ZTs (a-c).\u0026nbsp; e-h) Bacterial quantity in the region without the ALT-AC at ZT0 (e), ZT6 (f), ZT12 (g), and ZT18 (h). \u0026nbsp;No remarkable difference is found in the bacterial layer among ZTs.\u0026nbsp; a-h) The dotted line indicates a boundary between the luminal contents and the bacterial layer.\u0026nbsp; MF, mucosal fold.\u0026nbsp; a’-h’ are high-magnification images of the square areas in a-h, respectively.\u0026nbsp; Rod-shaped bacteria and bacilli accumulate in the bacterial layer, which is stained strongly with hematoxylin (arrows).\u0026nbsp; i, j) Area of the bacterial layer (mm\u003csup\u003e2\u003c/sup\u003e) per circumference of the analyzed transverse section (mm) of the region with (i) or without (j) the ALT-AC at each ZT (see also Supplementary Fig. 2).\u0026nbsp; The bars at the bottom of each graph indicate light/dark phase. \u0026nbsp;Each number in parentheses indicates the number of rats.\u0026nbsp; (a-h) Bar=100 µm.\u0026nbsp; (a’-h’) Bar=10 µm.\u0026nbsp; (i) Each value represents the mean±SD.\u0026nbsp; One-way ANOVA with Bonferroni adjustment.\u0026nbsp; Asterisks, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. \u0026nbsp;(j) Horizontal bar, median.\u0026nbsp; Kruskal‒Wallis test with Steel‒Dwass test\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6052953/v1/558808eff97169414c4b9a96.jpg"},{"id":83542612,"identity":"0a86aad8-dd8b-4448-b402-571cb6594cfa","added_by":"auto","created_at":"2025-05-28 08:25:49","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":583396,"visible":true,"origin":"","legend":"\u003cp\u003e16S rRNA sequencing analysis on the bacteria colonizing around the aggregated lymphoid tissues in the ascending colon (ALT-AC), the cecal contents, and feces. \u0026nbsp;a) Principal coordinate analysis based on weighted UniFrac distance. At both ZT6 and ZT18, indigenous bacteria in the ascending colon (A. colon) and bacteria in the cecal contents (Cecum) and feces form distinct clusters. b) Comparison of the weighted UniFrac distances between the bacteria which settle in the ascending colon, bacteria in the cecal contents, and feces at each ZT. \u0026nbsp;c) Rarefaction curve of alpha diversity with the Chao1 index. d) Comparison of alpha diversity with the Chao1 index among the bacteria which settle around ALT-AC, bacteria in the cecal contents, and feces at each ZT. (b, d) There is statistical significance (\u003cem\u003eq \u003c/em\u003e\u0026lt;0.05) between values with different letters. \u0026nbsp;(b) PERMANOVA with the FDR method of Benjamini‒Hochberg. (d) Horizontal bar, median. \u0026nbsp;Kruskal‒Wallis test with the FDR method of Benjamini‒Hochberg\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6052953/v1/40a241209ce7a8d575552c82.jpg"},{"id":83542611,"identity":"ae52f28b-f481-4691-9282-b0e836e8aa24","added_by":"auto","created_at":"2025-05-28 08:25:49","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":923903,"visible":true,"origin":"","legend":"\u003cp\u003ea, b) Taxonomic composition of bacteria from the region with aggregated lymphoid tissues in the ascending colon (ALT-AC) (A. colon), cecal contents (Cecum), and feces at the phylum (a) and family (b) levels. \u0026nbsp;c) Comparison of the relative abundance of bacteria which settle around ALT-AC by LEfSe analysis. \u0026nbsp;Each bar indicates the bacterial taxa with significantly higher relative abundances at ZT6 (green) or ZT18 (red)\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6052953/v1/e50bfa73e84b9191421c3c97.jpg"},{"id":83542618,"identity":"7a673d15-72b9-43d1-a38c-b30697951007","added_by":"auto","created_at":"2025-05-28 08:25:49","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":874311,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of suppressing the expansion of bacterial colonies by a 1-day treatment with antibiotics (Abx) on the local transcriptome in the aggregated lymphoid tissues in the ascending colon (ALT-AC). a) Principal component analysis for the transcriptomes. Samples from the control group (blue) form a small cluster, while those from the Abx group (orange) are scattered. \u0026nbsp;b) Volcano plots. \u0026nbsp;Genes with significantly abundant expression in the Abx group (adjusted \u003cem\u003eP\u003c/em\u003e-value \u0026lt; 0.05 and Log2 (fold change) \u0026gt; 1) are shown as plots in red, while those in the control group (adjusted \u003cem\u003eP\u003c/em\u003e-value \u0026lt; 0.05 and Log2 (fold change) \u0026lt; -1) are shown as plots in blue. c, d) Enrichment analysis by Metascape for differentially expressed genes (DEG) downregulated (c) and upregulated (d) in the Abx group compared to the control group in the ALT-AC\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6052953/v1/f085eb81b6c5b1b243adb3a5.jpg"},{"id":90344780,"identity":"e71da5f8-2b71-4e52-a9b6-c3b7a8e0aad2","added_by":"auto","created_at":"2025-09-01 16:00:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5465144,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6052953/v1/20fb6c33-b543-4ffc-80a8-66c281650030.pdf"},{"id":83542621,"identity":"8e0eb776-9b86-4bf2-bb69-b34420394293","added_by":"auto","created_at":"2025-05-28 08:25:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":10329075,"visible":true,"origin":"","legend":"","description":"","filename":"ShimadaetalSupplymentalfigure.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6052953/v1/7925c5fbe6f42b9e3cefc7f5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Colonizing bacteria around aggregated lymphoid tissue of the rat ascending colon change diurnally and affect the host local transcriptome","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eNumerous indigenous bacteria settle in the animal alimentary tract. The amounts and compositions of bacteria in the digestive tract are known to vary throughout the day according to their circadian rhythms. For example, bacterial compositions show circadian rhythms in the gastrointestinal contents of mice, such as cecal contents and feces (Heddes et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). On the other hand, we have documented in rats a region-specific circadian rhythm in the numbers of bacteria colonizing on the mucosal surfaces of the esophagus, the non-glandular part of the stomach, the ileal ordinary mucosa, and Peyer's patch (Sakata et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mantani et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Such indigenous bacteria also have a variety of effects on the host intestine. In mice, the settlement of segmented filamentous bacteria in the small intestine induces the expression of antibacterial peptides (Reg3γ, LCN2, S100A8) (Brooks et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and the differentiation of Th17 cells (Atarashi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The colonization of \u003cem\u003eClostridium spp\u003c/em\u003e., one of the bacteria indigenous to the murine gastrointestinal tract, induces the differentiation of regulatory T cells in the murine colon (Atarashi et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Bacterial settlement also promotes intestinal epithelial cell proliferation in the murine small intestine via the production of short-chain fatty acids (SCFA) (Park et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Considering these effects of the indigenous bacteria on the homeostasis of the intestinal mucosa, it is important to clarify their circadian rhythm to understand how indigenous bacteria affect the host. However, studies of the circadian rhythms in indigenous bacteria have just begun, and some regions, including the ascending colon, remain to be explored.\u003c/p\u003e \u003cp\u003eIn general, the colon is the region where colonizing bacteria abound in many animal species. Mucosal folds develop in the ascending colon of various rodents, such as mice (Nava et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), rats (Mantani et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), cotton rats (Chuluunbaatar et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), African mole rats (Kotz\u0026eacute; et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), guinea pigs, and chinchillas (Holtenius and Bj\u0026ouml;rnhag \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). Large amounts of bacteria colonize the spaces between mucosal folds in the ascending colon of mice (Nava et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and rats (Mantani et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Our prior study has revealed circadian rhythms in the abundance of colonizing bacteria in various regions of the rat gastrointestinal tract (Sakata et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), however, we could not examine the circadian rhythm of bacterial colonization in the ascending colon because conventional paraffin sectioning caused cracks in the bacterial layer between the mucosal folds. Therefore, our primary objective in this study was to elucidate the pattern of diurnal changes in the amount and composition of indigenous bacteria in the ascending colon by histological analysis and 16S rRNA amplicon sequencing analyses. Furthermore, given that inhibiting diurnal bacterial growth with 1-day antibiotic treatment changed the gene expression profile in the rat ileal Peyer\u0026rsquo;s patch in our previous study (Mantani et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), we hypothesized that expansion of indigenous bacteria in the rat ascending colon might have an important role in regulating gene expression in the aggregated lymphoid tissues there. We therefore tested this hypothesis with transcriptome analysis as the second aim.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003eAnimals\u003c/p\u003e \u003cp\u003eA total of 39 male Wistar rats aged 9 weeks (Japan SLC Inc., Shizuoka, Japan) were maintained under specific pathogen-free conditions in individual ventilated cages (Sealsafe Plus; Techniplast S.p.a, Buguggiate, Italy) with controlled temperature (23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and humidity (50\u0026thinsp;\u0026plusmn;\u0026thinsp;10%) on a 12/12-h light/dark cycle at the Kobe University Life-Science Laboratory. The rats had free access to water and food (Lab RA-2; Nosan, Kanagawa, Japan). This study was approved by the Institutional Animal Care and Use Committee (permission numbers: 2023-04-01, 2019-09-01). All procedures in studies involving animals were performed in accordance with the ethical standards of the institution (the Kobe University Animal Experimentation Regulations) or at the practice at which the studies were conducted.\u003c/p\u003e \u003cp\u003eTissue preparation for light microscopy\u003c/p\u003e \u003cp\u003eTwenty-five rats aged 9 weeks were divided into four groups according to the sampling time: zeitgeber time (ZT) 0 (n\u0026thinsp;=\u0026thinsp;6), which corresponds to the beginning of the light phase, ZT 6 (n\u0026thinsp;=\u0026thinsp;7), ZT 12 (n\u0026thinsp;=\u0026thinsp;6), and ZT 18 (n\u0026thinsp;=\u0026thinsp;6). After euthanasia by inhalation of isoflurane (FUJIFILM Wako Pure Chemical, Osaka, Japan), the ascending colons were dissected and immediately fixed in Carnoy\u0026rsquo;s fixative for 12 h at room temperature. The regions containing the aggregated lymphoid tissue (ALT-AC) located in a specific area of the ascending colon (Crouse et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), as well as the region adjacent to the ALT-AC on its proximal side, were taken from each fixed ascending colon (Supplementary Fig.\u0026nbsp;1) and dehydrated. In this study, we used methacrylate resin (Technovit 8100; Kulzer, Hanau, Germany) to embed the blocks, because methacrylate resin is effective for preserving luminal contents in intestinal blocks (Hasegawa et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and was expected to be effective for preserving indigenous bacteria that colonize on the mucosal epithelium as well. Then, 4-\u0026micro;m-thick sections were cut and placed on CREST-coated glass slides (Matsunami Glass, Osaka, Japan). These tissue sections were washed with distilled water and used for Mayer\u0026rsquo;s hematoxylin and eosin (HE) staining.\u003c/p\u003e \u003cp\u003eHistological quantification of settlement level of indigenous bacteria and statistical analysis\u003c/p\u003e \u003cp\u003eThe area (mm\u003csup\u003e2\u003c/sup\u003e) of the bacterial layer between the intestinal superficial epithelial cells and the luminal content was calculated in a single cross-section using ImageJ2 (ver. 2.3.0/1.53f), as was the length (mm) of the outermost circumference of the transverse sections of the ascending colon (Supplementary Fig.\u0026nbsp;2). The normality of distribution was first assessed by the Kolmogorov‒Smirnov test. For parametric variables in multiple comparisons, one-way ANOVA was performed, followed by the Bonferroni test for post hoc comparisons. For nonparametric variables in multiple comparisons, the Kruskal‒Wallis test was performed, followed by the Steel‒Dwass test for post hoc comparisons. \u003cem\u003eP\u003c/em\u003e values less than 0.05 were considered statistically significant. Statistical analyses were conducted with BellCurve for Excel (ver. 4.07).\u003c/p\u003e \u003cp\u003eTissue preparation for 16S rRNA amplicon sequencing\u003c/p\u003e \u003cp\u003eEight rats aged 9 weeks were equally divided into two groups based on the sampling time: ZT6 and ZT18 (4 rats per ZT). After euthanasia by inhalation of isoflurane (FUJIFILM Wako Pure Chemical), the area containing the ALT-AC, the cecal contents, and the feces were dissected. The luminal content was then carefully removed from the ascending colon by flushing with 4.0% paraformaldehyde (PFA) fixative in 0.1 M phosphate buffer (PB; pH 7.4), then fixed with 4.0% PFA fixative in 0.1M PB for 6 h at 4℃ and snap-frozen in liquid nitrogen as described previously (Mantani et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Serial 10-\u0026micro;m-thick sections were cut using a Cryotome Leica CM1950 (Leica Microsystems, Nussloch, Germany). The tissue sections for the 16S rRNA amplicon sequencing were placed in Eppendorf tubes, whereas the tissue sections just before and after each tissue section for the 16S rRNA amplicon sequencing were stained with HE staining (see also Supplementary Fig.\u0026nbsp;3). These sections were stored at \u0026minus;\u0026thinsp;30\u0026deg;C until use.\u003c/p\u003e \u003cp\u003eAfter confirming that the luminal content had been mostly removed from each tissue block using HE-stained sections, the sections cut from the same frozen block for 16S rRNA amplicon sequencing were washed with diethylpyrocarbonate (DEPC)-treated water. Total DNA was extracted with the RecoverAll\u0026trade; Total Nucleic Acid Isolation Kit for FFPE (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer\u0026rsquo;s instructions. DNA from cecal contents and feces was extracted using Nucleospin Tissue kits (Macherey‒Nagel, D\u0026uuml;ren, Germany). Libraries for sequencing were then prepared by a two-step tailed PCR method with TaKaRa Ex Taq Hot Start Version (Takara Bio, Shiga, Japan) as described previously (Sakata et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Briefly, the sequences of 16S rRNA regions were first amplified using PCR with primers for the V4 region (F 5\u0026rsquo;-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNGTGCCAGCMGCCGCGGTAA-3\u0026rsquo; R 5\u0026rsquo;-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNGGACTACHVGGGTWTCTAAT-3\u0026rsquo;) and then amplified with the index primer. Each PCR product was sequenced on an Illumina Miseq system (Illumina, San Diego, CA, USA). The reads were processed with QIIME2 (Bolyen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) as described previously (Sakata et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The relative abundance of bacteria colonizing the ascending colon was compared with that of cecal contents and feces at taxonomic levels, from phylum to species, using LEfSe (Segata et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Statistical analysis of the Chao1 index and the weighted UniFrac distances was performed using the Kruskal‒Wallis and PERMANOVA tests combined with the false discovery rate (FDR) method of Benjamini‒Hochberg, respectively. \u003cem\u003eq\u003c/em\u003e-values less than 0.05 were considered statistically significant.\u003c/p\u003e \u003cp\u003eOne-day antibiotic treatment for transcriptome analysis\u003c/p\u003e \u003cp\u003eTo investigate how the diurnal change in indigenous bacteria affects the ALT-AC transcriptome, we impaired the diurnal growth of indigenous bacteria by administering antibiotics for 1 day as described previously (Mantani et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Briefly, 6 male Wistar rats aged 9 weeks were equally divided into a control group and an antibiotic-treated (Abx) group. The drinking water was then replaced with a 1% sucrose solution for the control group and with an Abx solution (vancomycin (0.5 g/L; FUJIFILM Wako Pure Chemical), polymyxin B (0.1 g/L; FUJIFILM Wako Pure Chemical), and 1% sucrose) for the Abx group 1 day before euthanasia at ZT18 (see also Supplementary Fig.\u0026nbsp;4). Sucrose was added to the solution to prevent the rats from avoiding drinking. After euthanasia at ZT18 by inhalation of isoflurane, the ALT-ACs were dissected from 3 rats in the control group and 3 rats in the Abx group. Total RNA was extracted from each sample with NucleoSpin RNA Plus kits (Macherey‒Nagel) according to the manufacturer\u0026rsquo;s instructions and used for transcriptome analysis as follows.\u003c/p\u003e \u003cp\u003eTranscriptome analysis\u003c/p\u003e \u003cp\u003eWe controlled the quality of RNA-seq data using fastp (version 0.23.2) (Chen et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) with default options to discard low-quality reads. We aligned the remaining reads to the rat reference genome (rn6) using HISAT2 (version 2.2.1) (Kim et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) with the \u0026ldquo;\u0026ndash;no-mixed \u0026ndash;dta\u0026rdquo; options. To quantify gene-level expressions, we counted reads for each transcript using featureCounts (version 2.0.6) (Liao et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) with the RefSeq gene annotation (rn6.ncbiRefSeq.gtf) and the \u0026ldquo;-p -B -t exon -g gene_id\u0026rdquo; options. Differential expression analysis was performed using the DESeq2 method (Love et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Adjusted \u003cem\u003eP\u003c/em\u003e values less than 0.05 and absolute fold changes greater than two were considered statistically significant. We conducted GO enrichment analysis with the identified differentially expressed genes using Metascape (Zhou et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The results of a volcano plot and principal component analysis were plotted in Python (version 3.8.5).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eDiurnal change in the settlement levels of indigenous bacteria in the rat ascending colon\u003c/p\u003e \u003cp\u003eRegardless of regions in the ascending colon or ZT, a bacterial layer densely stained with hematoxylin was observed between the mucosal folds of the rat ascending colon (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-h), and this bacterial layer was a continuous thin layer even at the tip of the mucosal folds. The bacterial layer was clearly distinguishable from the luminal contents because the luminal contents were less strongly stained than the bacterial layer due to the low density of bacteria. Fibers from the diet, but not the luminal content, were occasionally mixed inside this bacterial layer. Furthermore, bacilli or rod-shaped bacteria, which exhibited the same shape as bacteria identified in the space of mucosal folds under electron microscopy in a previous study (Mantani et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), were observed in the bacterial layer under high magnification regardless of ZT or regions in the ascending colon (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea\u0026rsquo;-h\u0026rsquo;). In the region containing the ALT-AC, the bacterial layer between the mucosal folds tended to be thicker at ZT18 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed) than at the other ZTs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-c). Conversely, the luminal content frequently penetrated deeply into the space between mucosal folds where the bacterial layer was thinned at ZT6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) and ZT12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). The histological measurement revealed that the area of the bacterial layer per the length of the outermost circumference of the ascending colon at ZT18 was significantly larger than that at ZT6 in the ALT-AC-containing regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). On the other hand, no significant differences were found between any of the ZTs in the region without the ALT-AC, although three of the six samples at ZT18 showed higher values than any of the samples at ZT6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDiurnal changes in bacterial composition on the mucosal surface of the ascending colon including the ALT-AC\u003c/p\u003e \u003cp\u003eHistological analysis showed that the settlement level of indigenous bacteria was high at ZT18 and low at ZT6 in the ALT-AC-containing region. Next, we performed tissue-section 16S rRNA amplicon sequencing analysis (Sakata et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) (Supplementary Fig.\u0026nbsp;3) at ZT6 and ZT18 to characterize the specificity in the bacterial composition of the indigenous bacteria settled around the ALT-AC and its diurnal change. First, principal coordinate analyses based on the weighted UniFrac distance revealed that the bacterial composition statistically exhibited distinct characteristics among the ascending colon, cecal contents, and feces, but not between ZTs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b; \u003cem\u003eq\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). On the other hand, there was no significant difference between ZT6 and ZT18 in the weighted UniFrac distance of bacteria colonizing the ascending colon. The Chao1 index, which is used to evaluate the richness of bacterial species, showed significantly fewer bacterial species in the mucosal surface of the ascending colon compared to the cecal contents and feces at both ZTs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d; \u003cem\u003eq\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Next, the relative abundance of each bacterium at phylum level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) and family level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) in each sample was analyzed. The relative abundance of \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eBacteroidetes\u003c/em\u003e was almost at the same level in the cecal content. On the other hand, their relative frequencies differed in the feces and ascending colon; \u003cem\u003eBacteroidetes\u003c/em\u003e dominated in feces, whereas \u003cem\u003eFirmicutes\u003c/em\u003e accounted for more than 80% of the indigenous bacteria in the ascending colon (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). At the family level, two bacterial families, \u003cem\u003eLachnospiraceae\u003c/em\u003e and \u003cem\u003eRuminococcaceae\u003c/em\u003e, predominated in the indigenous bacteria in the ascending colon, and the relative abundance of \u003cem\u003eLachnospiraceae\u003c/em\u003e was significantly higher in the ascending colon than in the cecal content and feces at both ZTs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb; Supplementary Figs.\u0026nbsp;5, 6). The relative abundance of several bacterial species colonizing the ascending colon differed significantly between ZT6 and ZT18 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). A species belonging to the genus \u003cem\u003eAnaerotruncus\u003c/em\u003e was significantly more abundant at ZT6 than at ZT18, whereas 5 bacterial species\u0026mdash;\u003cem\u003eMucispirillum schaedleri\u003c/em\u003e, 2 \u003cem\u003eLachnospiraceae\u003c/em\u003e species (PAC001459_s, PAC001126_s), a \u003cem\u003eCaproiciproducens\u003c/em\u003e species (PAC001505_s), and a \u003cem\u003eDehalobacterium\u003c/em\u003e species (PAC001221_s)\u0026mdash;were more abundant at ZT18 than at ZT6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). These findings suggest that the indigenous bacteria around the ALT-AC are unique compared to those in cecal contents or feces, and that their composition changes slightly within a day.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEffect of suppressing diurnal expansion of bacterial colonies on the ALT-AC transcriptome at ZT18\u003c/p\u003e \u003cp\u003eHistological analysis suggested that the settlement level of indigenous bacteria, especially around the ALT-AC, changes diurnally with a peak at ZT18. Therefore, to explore what genes could be regulated by the diurnal expansion of bacterial colonies in the ALT-AC and the surrounding mucosa at ZT18, when the bacteria were most abundant, we performed 1-day Abx treatment (Mantani et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) (Supplemantary Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and transcriptome analysis. Principal component analysis showed that plots of the control group formed small clusters, whereas those of the Abx group were dispersed, indicating distinct gene expression profiles between the control and the Abx groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). We identified a total of 146 differentially expressed genes (DEGs) between the control and the Abx group in the ALT-AC; 107 were upregulated and 39 were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The downregulated genes were enriched in the following cascades: epithelial cell development, Golgi organization, positive regulation of growth, monocarboxylic acid transport, positive regulation of inflammatory response, cellular response to starvation, positive regulation of apoptotic process, lipid homeostasis, protein phosphorylation, regulation of metal ion transport, and tissue homeostasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). On the other hand, the upregulated genes were enriched in 17 cascades, such as biological oxidations, phospholipid efflux, bile acid secretion, positive regulation of interferon-beta production, xenobiotic catabolic process, response to toxic substance, organic hydroxy compound metabolic process, regulation of cellular respiration, benzene-containing compound metabolic process, and negative regulation of cell‒cell adhesion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Downregulated genes in the Abx group included several transcription factors (\u003cem\u003eSpdef\u003c/em\u003e, \u003cem\u003eAtoh1\u003c/em\u003e, \u003cem\u003eBhlha15\u003c/em\u003e), a Golgi reassembly stacking protein (\u003cem\u003eGorasp2\u003c/em\u003e: \u003cem\u003eLOC103690018\u003c/em\u003e), and a cell-cycle-related gene (\u003cem\u003ePlk3\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, Supplementary Table\u0026nbsp;1). Upregulated genes in the Abx group were associated with lipid metabolism, including apolipoproteins (\u003cem\u003eApoa1\u003c/em\u003e, \u003cem\u003eApoa4\u003c/em\u003e, \u003cem\u003eApoc3\u003c/em\u003e) and the cytochrome P450 family (e.g., \u003cem\u003eCyp2b1\u003c/em\u003e, \u003cem\u003eCyp3a62\u003c/em\u003e, \u003cem\u003eCyp3a9\u003c/em\u003e, \u003cem\u003eCyp8b1\u003c/em\u003e), as along with genes involved in bile acid transport, \u003cem\u003eSlc51a\u003c/em\u003e and \u003cem\u003eSlc51b\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, Supplementary Table\u0026nbsp;2). Genes involved in viral RNA degradation (\u003cem\u003eOas1a, Oas2\u003c/em\u003e, \u003cem\u003eOas3\u003c/em\u003e) and Granzymes (\u003cem\u003eGzma, Gzmf\u003c/em\u003e) known to induce cell death in virus-infected cells were also upregulated in the Abx group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur previous studies have demonstrated a circadian rhythm in the settlement level of indigenous bacteria across various regions of the rat alimentary tract, peaking at ZT12 in the esophagus, ZT6 in the non-glandular part of the stomach, ZT0 in the ileal villi, and from ZT18 to ZT0 in the most distal ileal Peyer\u0026rsquo;s patch (Sakata et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mantani et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Focusing on the ascending colon, this study showed that the bacterial quantity between the mucosal folds was significantly higher at ZT18 than at ZT6 in the region including the ALT-AC, but not in that without ALT-AC. This finding suggests a diurnal change, with a peak at ZT18, in bacterial abundance in the rat ascending colon, especially around the ALT-AC.\u003c/p\u003e \u003cp\u003eTissue section 16S rRNA amplicon sequencing in this study revealed significantly less bacterial species richness in the indigenous bacteria around the ALT-AC than in cecal contents and feces. It also showed that a unique bacterial flora, characterized by \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eLachnospiraceae\u003c/em\u003e and differing from that of the cecal contents and feces, colonized the mucosal surface of the rat ascending colon. This finding supports a previous study that showed similar bacteria are dominant between mucosal folds in the mouse ascending colon (Nava et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Further, comparing the composition of indigenous bacteria in the ascending colon between ZT6 and ZT18 suggested that the composition of colonizing bacteria around the ALT-AC underwent diurnal changes, possibly induced by the diurnal variation in the relative abundance of \u003cem\u003eMucispirillum schaedleri\u003c/em\u003e and \u003cem\u003eAnaerotruncus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eMost of bacteria on the mucosal surface of the ascending colon at both ZT6 and ZT18 in this study were classified into \u003cem\u003eLachnospiraceae\u003c/em\u003e or \u003cem\u003eRuminococcaceae\u003c/em\u003e, which produce SCFA (e.g., acetate, butyrate, propionate) by fermentation of carbohydrates, including cellulose (Barcenilla et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Biddle et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In addition, the relative abundance of two \u003cem\u003eLachnospiraceae\u003c/em\u003e species differed significantly between ZT6 and ZT18 in the present study, suggesting the hypothesis that this diurnal change might be accompanied by a circadian rhythm in SCFA production. SCFA derived from the intestinal microflora play various physiological roles related to the intestinal immune system. For example, IgA secretion is facilitated by acetate (Takeuchi et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and butyrate (Isobe et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) in the murine intestine. Butyrate and propionate promote the differentiation of regulatory T cells in the murine colon (Furusawa et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Therefore, the hypothesis concerning the circadian rhythm in SCFA production due to diurnal changes in \u003cem\u003eLachnospiraceae\u003c/em\u003e species in the ascending colon is an important topic for understanding the temporal regulation of the intestinal immune system and should be tested in future studies.\u003c/p\u003e \u003cp\u003eIn the present study, we explored which genes are regulated by the expansion of indigenous bacterial colonies at ZT18 in the rat ALT-AC by administering nonabsorbable antibiotics in the drinking water for 1 day. Through transcriptome analysis, we identified 146 DEGs whose expression might be affected by colonizing bacteria in the rat ALT-AC in daily life. These DEGs contained more than twice as many upregulated genes downregulated ones. Considering that the numbers of upregulated and downregulated genes after the 1-day antibiotic treatment in the rat ileum were almost equal (Mantani et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), the host gene expression regulated by bacterial growth on the ALT-AC may be biased toward repression. In general, aggregated lymphoid tissues in the intestine are the specific sites for inducing immune responses against luminal antigens. In our previous study, inhibition of bacterial expansion by 1-day Abx treatment showed the downregulation of immune-associated genes, such as cytokines and chemokines, at ZT0 in the ileal Peyer\u0026rsquo;s patch (Mantani et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In the present study, on the other hand, the expression of immune-related genes, at least cytokines and chemokines, did not decrease in ALT-AC, in contrast to the ileal Peyer\u0026rsquo;s patch (Mantani et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Instead, genes associated with epithelial cell differentiation, monocarboxylic acid transport, and the positive regulation of inflammatory response were downregulated. In addition, genes related to various cascades, such as lipid metabolism, antiviral immune responses, and xenobiotic metabolism, which are unexpected as host responses to bacteria, were identified among the upregulated genes in the ALT-AC. These results imply that the expansion of indigenous bacteria during the dark phase is involved in regulating multifaceted functions of the ALT-AC other than the well-known antibacterial immune response.\u003c/p\u003e \u003cp\u003eIn the present study, a 1-day antibiotic treatment from ZT18 to inhibit bacterial growth reduced the expression of several transcription factors (\u003cem\u003eSpdef\u003c/em\u003e, \u003cem\u003eAtoh1\u003c/em\u003e, and \u003cem\u003eBhlha15\u003c/em\u003e) associated with epithelial cell differentiation in the ALT-AC. In mice, \u003cem\u003eAtoh1\u003c/em\u003e and \u003cem\u003eSpdef\u003c/em\u003e are expressed in goblet cells and Paneth cells of the ileum and in goblet cells of the colon (Lo et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Noah et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). \u003cem\u003eSpdef\u003c/em\u003e promotes the differentiation of secretory progenitor cells into goblet cells in the ileum, and goblet cells are almost absent in \u003cem\u003eAtoh1\u003c/em\u003e-deficient mice (Lo et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Noah et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). \u003cem\u003eBhlha15\u003c/em\u003e-positive cells reside at the base of the colonic crypt in mice and differentiate into goblet or tuft cells (Hayakawa et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These findings suggest that the bacterial growth on the ALT-AC during dark phase promotes epithelial stem cells to differentiate into secretory lineage, such as goblet cells. This idea is also supported by another observation: the downregulation of \u003cem\u003eGorasp2\u003c/em\u003e, which is essential for Golgi cisternal stacking of the Golgi apparatus (an abundant organelle in goblet cells) (Xiang et al. 2010) within the antibiotic-treated group in this study. However, it is unclear whether indigenous bacteria directly regulate the differentiation of epithelial cells because the expression level of cell differentiation-related genes depends in part on cell proliferation activity. In the present study, the downregulated genes in the 1-day Abx group included \u003cem\u003ePlk3\u003c/em\u003e, which is necessary for the transition from G1 to the S phase of the cell cycle (Zimmerman et al. 2007), suggesting that bacterial depletion for 1 day might also affect cell proliferation. Such a relationship exists between cell proliferation and intestinal microflora in the colonic epithelium, as observed by comparing germ-free rats and conventional rats (Alam et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Therefore, we could not exclude the possibility that the downregulation of genes involved in goblet cell differentiation in the 1-day Abx group is a consequence of the suppression of epithelial cell proliferation owing to bacterial depletion. This hypothesis needs to be tested in the future.\u003c/p\u003e \u003cp\u003eIn conclusion, this study provided findings suggesting that the settlement level of indigenous bacteria around the ALT-AC changes diurnally, peaking at the dark phase, and that this change is associated with the diurnal change in the bacterial composition at the species level, characterized by changes in the relative abundance of \u003cem\u003eMucispirillum schaedleri\u003c/em\u003e and \u003cem\u003eAnaerotruncus\u003c/em\u003e. In addition, the expansion of the bacterial colony during the dark phase around the ALT-AC can contribute to regulating the expression of various genes, including those involved in goblet cell differentiation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have contributed to this study as follows: conceptualization (YM), investigation (AS, RM), formal analysis (AS, NK, YM), visualization (AS, NK, YM), funding acquisition (AS, SZ, YM), supervision (SZ, TY, NH, YM), writing original draft (AS), critically reviewing and editing manuscript (NK, RM, TY, NH, YM).\u0026nbsp;\u0026nbsp;All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResearch involving Animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional Animal Care and Use Committee (permission number 2023-04-01, 2019-09-01). \u0026nbsp;All procedures in studies involving animals were performed in accordance with the ethical standards of the institution (the Kobe University Animal Experimentation Regulations) or at the practice at which the studies were conducted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest associated with this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNAseq data were deposited into the Gene Expression Omnibus database under accession number GSE284464 and are available at the following URL:\u0026nbsp;https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE284464\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlam M, Midtvedtm T, Uribe A (1994) Differential Cell Kinetics in the Ileum and Colon of Germfree Rats. 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Proc Natl Acad Sci USA 104:1847\u0026ndash;1852\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-and-tissue-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ctre","sideBox":"Learn more about [Cell and Tissue Research](https://link.springer.com/journal/441)","snPcode":"441","submissionUrl":"https://submission.springernature.com/new-submission/441/3","title":"Cell and Tissue Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Ascending colon, Circadian rhythm, Indigenous bacteria, Aggregated lymphoid tissue, Histological analysis, Transcriptome","lastPublishedDoi":"10.21203/rs.3.rs-6052953/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6052953/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe settlement levels of indigenous bacteria show circadian rhythms in various regions of the rat alimentary tract. Numerous bacteria colonize between the mucosal folds of the ascending colon in rodents; however, the rhythm of bacteria colonizing the ascending colon remains to be clarified. Therefore, we first aimed to elucidate the circadian rhythms of bacteria colonizing in the rat ascending colon. The settlement levels of indigenous bacteria were significantly higher at zeitgeber time (ZT) 18 (dark phase) than at ZT6 (light phase) in the region encompassing the aggregated lymphoid tissue in the ascending colon (ALT-AC). The bacterial composition around the ALT-AC was dominated by the phylum \u003cem\u003eFirmicutes\u003c/em\u003e and the family \u003cem\u003eLachnospiraceae\u003c/em\u003e, displaying notable distinctions from the compositions found in cecal contents and feces. The relative abundance of some bacterial species around the ALT-AC, such as \u003cem\u003eMucispirillum schaedleri\u003c/em\u003e, changed significantly between ZT6 and ZT18. Furthermore, we explored the effect of bacterial expansion on gene expression in the ALT-AC at ZT18 by administrating antibiotics for 1 day to inihibit bacterial growth. The antibiotic-treated group exhibited significant downregulation of multiple genes, including those associated with cell proliferation (\u003cem\u003ePlk3\u003c/em\u003e), differentiation into goblet cells (\u003cem\u003eSpdef\u003c/em\u003e, \u003cem\u003eAtoh1\u003c/em\u003e, \u003cem\u003eBhlha15\u003c/em\u003e), and Golgi organization (\u003cem\u003eGorasp2\u003c/em\u003e). These results suggested that indigenous bacteria around the rat ALT-AC undergo diurnal changes in both settlement levels, peaking at the dark phase, and bacterial composition. In addition, bacterial expansion during the dark phase can induce changes in the expression of diverse genes, including genes associated with goblet cell differentiation.\u003c/p\u003e","manuscriptTitle":"Colonizing bacteria around aggregated lymphoid tissue of the rat ascending colon change diurnally and affect the host local transcriptome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-28 08:25:43","doi":"10.21203/rs.3.rs-6052953/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-24T15:51:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-23T12:31:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-23T07:53:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"256711003225470355280220812902239933507","date":"2025-03-10T13:35:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"144239115807114172646797331534174238540","date":"2025-03-04T00:10:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-03T13:58:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-21T04:24:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-02-21T04:23:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell and Tissue Research","date":"2025-02-18T06:01:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-and-tissue-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ctre","sideBox":"Learn more about [Cell and Tissue Research](https://link.springer.com/journal/441)","snPcode":"441","submissionUrl":"https://submission.springernature.com/new-submission/441/3","title":"Cell and Tissue Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"244e21f0-27fa-4179-a838-d2c55ffcb3a1","owner":[],"postedDate":"May 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-01T15:58:38+00:00","versionOfRecord":{"articleIdentity":"rs-6052953","link":"https://doi.org/10.1007/s00441-025-04000-1","journal":{"identity":"cell-and-tissue-research","isVorOnly":false,"title":"Cell and Tissue Research"},"publishedOn":"2025-08-26 15:56:51","publishedOnDateReadable":"August 26th, 2025"},"versionCreatedAt":"2025-05-28 08:25:43","video":"","vorDoi":"10.1007/s00441-025-04000-1","vorDoiUrl":"https://doi.org/10.1007/s00441-025-04000-1","workflowStages":[]},"version":"v1","identity":"rs-6052953","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6052953","identity":"rs-6052953","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}