Gentamicin alleviates intestinal graft-versus-host disease by modulating butyrate-associated gut microbiota

Gentamicin alleviates intestinal graft-versus-host disease by modulating butyrate-associated gut microbiota | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Gentamicin alleviates intestinal graft-versus-host disease by modulating butyrate-associated gut microbiota linlin shao, Ye Zhang, Yuefen Hu, Zhengyu Dao, Hailei Zhang, Shaoqiu Leng, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7181845/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Intestinal graft-versus-host disease (GVHD) is a common complication following allogeneic hematopoietic cell transplantation (allo-HSCT) and is commonly treated with antibiotics. Additionally, certain antibiotics used for gut-decontamination prophylaxis and neutropenic fever could affect GVHD-related mortality in human patients and mice. Objectives This study aimed to investigate the role of gentamicin and butyrate in mitigating intestinal GVHD and improving the prognosis post allo-HSCT. Study Design: An allo-HSCT mouse model was prepared to assess the effects of gentamicin and sodium butyrate supplementation. 16S rRNA sequencing was performed for microbiota analysis using fecal samples from mice. The effects of sodium butyrate on cell proliferation and apoptosis were analyzed using LS174T human goblet cells. Protein extracts from LS174T cells and mouse intestinal epithelial cells (IECs) were analyzed using western blotting. Samples from the small intestine and colon were evaluated using hematoxylin & eosin (H&E) and periodic acid-Schiff (PAS) staining. Finally, intestine slices were evaluated for Lgr5 and Muc2 expression. A Wilcoxon rank-sum test was used for microbiome analysis and survival was analyzed using Kaplan–Meier curves. The survival curves were compared using a log-rank test. Statistical significance was set at P < 0.05. Results We observed that the intestinal barrier was compromised in mice with GVHD. Gentamicin treatment after allo-HSCT significantly reduced the mortality and GVHD scores in recipient mice. Additionally, 16S rRNA gene sequencing showed that gentamicin altered the gut microbiota composition and decreased Clostridium levels. However, sodium butyrate supplementation in allo-HSCT mice after treatment with gentamicin significantly increased the mortality and intestinal GVHD severity, shortened the length of the colon, decreased colonic mucus layer thickness, and aggravated epithelial barrier damage in aGVHD mice. Further investigation revealed that sodium butyrate induced the apoptosis of goblet cells and inhibited the expression of Muc2 in vivo and in vitro . In addition, sodium butyrate inhibited the proliferation of intestinal stem cells. Interestingly, concurrent supplementation of gentamicin and sodium butyrate before transplantation significantly relieved GVHD. Conclusion Our results show that gentamicin alleviates GVHD by modulating butyrate associated gut microbiotas, while sodium butyrate weakens the benefit of gentamicin on GVHD by inducing goblet cell apoptosis, reducing Muc2 expression, and inhibiting intestinal stem cell proliferation. Thus, butyrate may have a double-edged effect on GVHD based on the exposure timing. Biological sciences/Stem cells/Haematopoietic stem cells Biological sciences/Cell biology/Cell death/Apoptosis Intestinal graft-versus-host disease (GVHD) allogeneic hematopoietic cell transplantation (allo-HSCT) gentamicin (GM) sodium butyrate (SB) Mucin 2 (Muc2) intestinal stem cells (ISCs) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Hematopoietic stem cell transplantation (HSCT) is a cornerstone in the treatment of numerous hematological disorders [ 1 ] . However, disease relapse and graft-versus-host disease (GVHD) are the two primary causes of mortality following allogeneic hematopoietic cell transplantation (allo-HSCT) [ 2 ] . GVHD mainly affects the gastrointestinal system, and severe intestinal GVHD is associated with a poor prognosis in patients post-HSCT [ 3 ] . Antibiotics are crucial for managing infections in patients who undergo allo-HSCT, in addition, recent study reported that gut-decontaminating prophylactic antibiotics can affect the severity of intestinal GVHD [ 4 ] . Gentamicin, an aminoglycoside antibiotic, is commonly used to inhibit the growth of gram-negative bacteria [ 5 ] , moreover, a multicenter clinical study found that it decreased the morbidity of gastrointestinal GVHD in children [ 6 ] . However, the underlying mechanism of post-HSCT intestinal GVHD modulation by gentamicin remains unclear. Additionally, despite recent research suggesting that inhibiting Clostridium proliferation in the intestinal tract can aid in maintaining intestinal homeostasis or treat intestinal diseases [ 7 ] , the role of Clostridium in GVHD requires further investigation. Moreover, increasing evidence shows that short-chain fatty acids (SCFAs) [ 8 ] play a vital role in maintaining intestinal metabolic homeostasis [ 9 – 10 ] . Among these, butyrate is a key metabolite produced by bacterial fermentation in the intestine [ 11 ] . Clostridium is a primary producer of butyrate [ 12 ] , of which sodium butyrate is a salt form [ 13 ] . Butyrate produced in the large intestine by intestinal bacteria is essential for fueling IECs and promoting mucin production [ 14 ] . While some studies have reported protective effects of butyrate on GVHD [ 15 – 16 ] , Golob et al. showed that butyrate has a double-edged effect on GVHD based on the exposure time. They reported that butyrate inhibited intestinal stem cells from forming an intact epithelial monolayer in addition to providing a protective effect [ 17 ] . Furthermore, the colon and intestinal mucus layers are rich in antibacterial peptides and proteins that safeguard the epithelium from harmful agents, viruses, and pathogenic bacteria [ 18 – 19 ] . Additionally, goblet cells secrete Muc2 [ 20 ] , a critical secretory protein in the human intestine [ 21 – 22 ] , which is essential for protecting against bacterial infections by limiting the number of pathogens and commensal bacteria on the colonic mucosal surface [ 23 ] . This action mitigates tissue damage and prevents the translocation of bacteria across the epithelium [ 24 ] . Consequently, the role of Muc2 in intestinal mucosal barrier dysfunction has garnered increasing attention, suggesting it may be pivotal in treating such damage [ 25 ] . In this study, we investigated the effects of gentamicin and sodium butyrate supplementation in allo-HSCT mice. In addition, we managed to explore the underlying mechanism of gentamicin and sodium butyrate affecting intestinal GVHD. Methods Murine aGVHD model Specific pathogen-free (SPF) BALB/c mice (GemPharmatech Co., Ltd, China) were housed in a pathogen-free facility at the Laboratory Animal Centre of Shandong University. Female BALB/c mice (6–8 weeks old) were maintained on a 12-h dark/light cycle at an ambient temperature of 22°C ± 2°C with controlled humidity (~ 45%). Lethally irradiated (750cGy) BALB/c mice were intravenously injected with 5 × 10⁶ bone marrow (BM) cells and 5 × 10⁶ splenocytes (SP) from major histocompatibility complex (MHC)-mismatched male C57BL/6J donors on day 0. Body weights and clinical scores [ 26 ] were recorded every other day. The mice were divided into seven subgroups. The GVHD GM group represented GVHD mice treated with gentamicin from 7 days before transplantation to 21 days after transplantation. The GVHD no antibiotics group represented GVHD mice treated without gentamicin or sodium butyrate. The GVHD GM + SB group represented GVHD mice treated with gentamicin 7 days before bone marrow transplantation (BMT) to 21 days after BMT, and sodium butyrate from day 1 to day 21 after BMT. The GVHD GM + SB(-7d) group represented GVHD mice treated with gentamicin and sodium butyrate 7 days before BMT to 21 days after BMT. The GVHD SB group represented GVHD mice treated with SB from day 1 to day 21 after BMT. The non-GVHD group represented syngeneic control mice without GVHD. The non-GVHD SB group represented syngeneic control mice without GVHD treated with sodium butyrate to detect the toxicity. Sodium butyrate and gentamicin treatment Up till day 7 before the injection of BM cells and splenocytes, mice received gentamicin (0.2 µg/mL) [ 27 ] in their drinking water daily. Allogeneic mice were administered sodium butyrate 150 mM in their drinking water from day 1 to day 21 [ 28 ] , followed by autoclaved free drinking water on other days. Gentamicin (Fuan Pharmaceutical Co., LTD, China) and sodium butyrate (MP Biomedicals, USA) were dissolved in double-distilled water immediately prior to drinking. Cell culture LS174T human goblet cells (Bio-Channel, China) were cultured in RPMI 1640 culture medium (Gibco, USA) supplemented with 10% fetal bovine serum (Tocyto, USA) and 1% penicillin/streptomycin (Gibco, USA). The cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂. Sodium butyrate was dissolved and diluted in the RPMI 1640 medium immediately before use. Cell proliferation assays LS174T human goblet cells were cultured in the presence of 0, 0.1, 1, 10, and 50 mM sodium butyrate. Cells were incubated with 10 µL of CCK-8 (Beyotime, China) for 3 h, and absorbance was measured at 450 nm. Each sample was measured in triplicate. Apoptosis assays Apoptosis was assessed using an Annexin V/7-AAD apoptosis detection kit (BestBio, Shanghai, China) according to the manufacturer’s protocol. LS174T cells were harvested after different treatments, washed twice with phosphate buffer saline (PBS; Beyotime, C0221A), and resuspended in 500 µL of binding buffer. Subsequently, cells were stained with 5 µL Annexin V for 15 min followed by 5 µL 7-AAD for 15 min in the dark at 4°C. The percentage of apoptotic cells was analyzed immediately using a Galios flow cytometer (Beckman Coulter, CA, USA). RNA extraction and quantitative real-time PCR Total RNA was extracted from cells using an RNA Rapid Extraction Kit (Invitrogen, USA). RNA concentration and purity were measured using a spectrophotometer (Eppendorf, Germany). Reverse transcription was performed using the Prime Script RT reagent Perfect Real Time Kit (Vazyme, 7E711J3) at 37°C for 15 min, followed by at 85°C for 10 s. Quantitative PCR was conducted in duplicate on a LightCycler 480II real-time PCR system (Roche, Switzerland) using the SYBR Green Real-time PCR Master Mix kit (Vazyme, 7E782J3). Primer sequences used were as follows: β-actin F (5’-GAAGAGCTACGAGCTGCCTGA-3’) and R (5’-CAGACAGCACTGTGTTGGCG-3’), Muc2 F (5’-AAGTGCTCCTGTTACCACCG-3’), and R (5’-CGGCACACACATCGTTCTTC-3’). Melting curves were analyzed to determine PCR specificity. All experiments were conducted according to the manufacturer’s instructions. Relative mRNA levels were expressed relative to β-actin using the 2 −ΔCT method. Western blotting analysis Primary antibodies against Muc2 (ab272692) and antibodies against β-actin (ab227387) were purchased from Abcam (Cambridge, UK). All reagents were prepared and stored according to the manufacturer’s instructions. LS174T cells and mouse IECs were collected, washed twice with PBS, and lysed with RIPA buffer (Bestbio, BB-3101) containing a protease inhibitor on ice. Protein concentrations were measured using a bicinchoninic acid protein assay kit (Beyotime, P0009). Protein extracts (20 µg) were loaded onto 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis gels and electrotransferred onto nitrocellulose membranes (BioTrace, USA). After blocking with an efficient blocking solution for 15 min at room temperature, membranes were incubated overnight with specific primary antibodies at 4°C, followed by incubation with HRP-conjugated secondary antibodies at room temperature for 1 h. Protein bands were detected using a FluorChem E Chemiluminescent imaging system (Protein Simple, San Jose, CA, USA). Histological analysis Samples from the small intestine and colon were fixed in 10% formalin and 4% paraformaldehyde, embedded in paraffin, sectioned (3–5 µm), and stained with hematoxylin & eosin (H&E). Colonic sections were stained with the periodic acid-Schiff (PAS) staining to evaluate mucus thickness. Sections were imaged and measured using a 3DHISTECH system. Five measurements were obtained per image and averaged over the entire usable colonic surface. Pathology scores [ 29 ] were quantified by two blinded pathologists. Extraction of intestinal epithelial cells IECs were isolated as previously described [ 30 – 31 ] . Briefly, the small and large intestines were harvested individually from 8–10 mice and rinsed extensively with RPMI 1640 (Gibco, Grand Island, New York, USA) after Peyer’s patches were removed (for small intestine). The rinsed intestines were opened longitudinally and macerated; the tissue pieces were shaken gently in RPMI-1640 containing 2 mM EDTA and 10% fetal bovine serum (FBS). The tissue preparations were passed through 70-µm mesh filters, and the resulting single-cell suspensions were separated using Percoll (GE Healthcare, Little Chalfont, United Kingdom) density gradients of 25%, 40%, and 75%. After centrifugation at 2,000 × g for 20 min, the interface between the 25% and 40% layers was collected to obtain IECs. The cells were stained with antibodies against epithelial cell adhesion molecule (EpCAM; Biolegend, San Diego, California, USA), or CD45 (Biolegend) and nucleic acid dye (Via-Probe; Becton Dickinson, Franklin Lakes, New Jersey, USA). The EpCAM + cells were further confirmed to be IECs via intracellular staining with a pan-cytokeratin antibody (Abcam, Cambridge, Massachusetts, USA) and fixation and permeability reagents (Invitrogen, Carlsbad, California, USA). The IECs were sorted using FACS (Aria III, Becton Dickinson) and analyzed using the FlowJo software (Ashland, Oregon, USA). Immunofluorescence The intestine slices were fixed with 4% formaldehyde (Beyotime, China) at 37℃ for 30 min. After permeabilizing with 0.1% Triton X-100 (Solarbio, China) for 5 min, samples were blocked with goat serum (Beyotime, China) at 37℃ for 30 min. Sections were dewaxed and rehydrated, followed by antigen retrieval. Subsequently, the intestine slices were labeled with Lgr5 (Proteintech, Chicago, USA, 1:50) and Muc2 (Abcam, cat# ab254183, 1:100) antibodies at 4℃ overnight. The following day, slides were incubated with a secondary antibody (Abcam, cat# ab150077, 1:1000) at 37℃ for 1 h and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Abcam, cat# ab285390). The staining was evaluated using the Olympus SLIDEVIEW VS200. Stool sample collection and microbial diversity analysis Fecal samples from mice were collected and stored at − 80°C. Subsequently, 16S rRNA sequencing was performed for microbiota analysis. Total genomic DNA samples were extracted using the OMEGA Soil DNA Kit (M5635-02, Omega Bio-Tek, Norcross, GA, USA), according to the manufacturer’s instructions, and stored at − 20°C until further analysis. The quantity and quality of extracted DNAs were measured using a NanoDrop NC2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively. PCR amplification of the bacterial 16S rRNA genes V3–V4 region was performed using primers F (5’-ACTCCTACGGGAGGCAGCA-3’) and R (5’-GGACTACHVGGGTWTCTAAT-3’). Sample-specific 7-bp barcodes were incorporated into the primers for multiplex sequencing. The PCR components contained 5 µL of buffer (5×), 0.25 µL of Fast pfu DNA Polymerase (5 U/µL), 2 µL (2.5 mM) of dNTPs, 1 µL (10 µM) of each Forward and Reverse primer, 1 µL of DNA Template, and 14.75 µL of ddH2O. PCR was conducted as follows: 2 min of initial denaturation at 98°C, 15 s of denaturation at 98°C, 30 s of annealing at 55°C, 30 s of extension at 72°C, and a final extension at 72°C for 5 min. PCR was performed in triplicate using a 20 µL mixture containing 5 µL 5 × reaction buffer, 5 µL 5 × GC buffer, 2 µL 2.5 mM dNTPs, 8.75 µL ddH2O, 1 µL each primer (10 µM), 0.25 µL Q5 DNA Polymerase, and 2 µL template DNA. PCR amplicons were purified with Vazyme VAHTSTM DNA Clean Beads (Vazyme, Nanjing, China) and quantified using the Quant-i T PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). After the individual quantification step, amplicons were pooled in equal amounts, and pair-end 2×250 bp sequencing was performed using the Illlumina NovaSeq platform with NovaSeq 6000 SP Reagent Kit (500 cycles) by Shanghai Bioprofile Technology Company Ltd (Shanghai, China). Microbiome bioinformatics analyses were performed with QIIME2 2019.4 with slight modification according to the official tutorials ( https://docs.qiime2.org/2019.4/tutorials/ ). Briefly, raw sequence data were demultiplexed using the demux plugin following by primer cutting with the cutadapt plugin. Sequences were then quality filtered, denoised, merged, and chimera removed using the DADA2 plugin. Non-singleton amplicon sequence variants (ASVs) were aligned with mafft and used to construct a phylogeny with fasttree2. Alpha (Chao1, ACE, Shannon) and beta diversity metrics (weighted UniFrac, unweighted UniFrac and Bray – Curtis dissimilarity) were estimated using the diversity plugin, with samples rarefied to sequences per sample. Taxonomy was assigned to ASVs using the classify-sklearn naïve Bayes taxonomy classifier in the feature-classifier plugin against the Greengenes Database. Alcian blue staining Paraffin sections were dewaxed in xylene twice, 10 min each. The sections were then hydrated by treatment with an ethanol gradient ethanol: 100% ethanol → 95% → 80% → 70% → distilled water for 1–2 min per step. The tissue section was covered with a drop of Alcian blue dye (pH 2.5) and stained at room temperature for 30 min. The sections were rinsed under running water for 5 min to remove excess dye. The nucleus was stained using neutral red for 1–2 min followed by rinsing with water for 30 s or with hematoxylin for 3–5 min followed by differentiation, which returned to blue. The specimens were dehydrated and cleared by passing through an ethanol gradient of 70% → 80% → 95% → 100% ethanol for 1 min per step. Subsequently, specimen were passed through xylene twice for 5 min each. Finally neutral gum was added, the specimens were covered with cover glass and observed under the Olympus SLIDEVIEW VS200. Statistical analysis Between-group comparisons were conducted using Student’s t-test for normally distributed data. The Wilcoxon rank-sum test was used for microbiome analysis. Progression-free survival and overall survival rates are presented using Kaplan–Meier curves. The log survival curves were compared using a log-rank test. Statistical significance was set at P < 0.05. ImageJ v.1.50 was used for western blot image processing, and Kaluza 2.1.1 was used for flow cytometry analysis. Statistical analysis was conducted using GraphPad Prism 8.0.2. *P < 0.05, **P < 0.01, and ***P < 0.0001 were considered statistically significant. Results Alteration of gut microbiota diversity and composition in the gentamicin-treated GVHD mice We assessed the impact of gentamicin on aGVHD (Fig. 1 A) and found that allogeneic mice without gentamicin treatment experienced more severe GVHD compared with those treated with gentamicin (P < 0.05, Fig. 1 B). Additionally, mice in the GVHD GM group lost significantly less weight than those in the GVHD no antibiotics group from the second week after the transplantation (Fig. 1 C). Meanwhile, allogeneic mice treated with gentamicin had lower GVHD scores than mice treated without antibiotics, suggesting that aGVHD was milder in the GVHD GM group (Fig. 1 D). Therefore, we isolated total bacterial DNA from mouse fecal samples at 14 days post-transplantation and sequenced the 16S rRNA genes using Illumina to further explore this mechanism. Principal co-ordinates analysis (PCoA) revealed a significant difference in the Euclidean distance between the GVHD GM group and the GVHD no antibiotics group (confidence interval: 90%), indicating a substantial shift in the microbial composition with respect to operational taxonomic units (Fig. 1 E). Stool samples from mice in the GVHD GM group and the GVHD no antibiotics group were analyzed for Chao1 (P = 0.0198) and ACE (P = 0.0193) indices. A significant reduction was observed in intestinal microbiota diversity in the GVHD GM group compared with that in the GVHD no antibiotics group (Fig. 1 F and 1 G). At the genus level, Clostridium was significantly depleted in the GM group compared with that in the GVHD no antibiotics group (P = 0.0269, Fig. 1 H and 1 I). However, no difference was observed in intestinal microbiota diversity and composition between the GVHD GM group and the GVHD no antibiotics group at day 0 (Fig. 1 K–N), which suggests that gentamicin decreased Clostridium mainly in GVHD mice instead of normal ones. Additionally, intestinal microbiota diversity and composition at day 0 and day 14 presented no differences in the GVHD no antibiotics group (Fig. 1 P–S). Intervention of sodium butyrate after allo-HSCT increased the mortality and aggravated GVHD of mice We examined the effects of sodium butyrate in an allo-HSCT mouse model treated with gentamicin to investigate the role of Clostridium in aGVHD and determine whether Clostridium exerts its effect by producing butyrate. The workflow for assessing the pharmacodynamics of sodium butyrate is shown in Fig. 2 A. The non-GVHD SB group allowed an assessment the toxicities of sodium butyrate without induction of an allogeneic response. We found that sodium butyrate had no effect on the survival (Fig. 2 B), body weight (Fig. 2 C), or GVHD score of normal mice (Fig. 2 D). In contrast, the GVHD SB group experienced the most severe GVHD among all groups. Whereas mice in the GVHD GM + SB group exhibited more severe aGVHD than those in the GVHD GM group (Fig. 2 B and 2 C), suggesting that supplementation of sodium butyrate might invalidate the beneficial effect of gentamicin. Conversely, concurrent administration of gentamicin and sodium butyrate pre-transplantation showed a more obvious protective effect, accompanied by the least weight loss and the lowest GVHD score, which suggests that applying sodium butyrate only after transplant might not demonstrate the beneficial effects on GVHD priming. Additionally, body weight comparisons showed that allo-HSCT mice with aGVHD lost more weight compared with that of the mice in the non-GVHD group. Mice in the GVHD GM + SB group showed significantly decreased weights compared with those in the GVHD GM group (Fig. 2 C), meanwhile GVHD scores for the GVHD GM group were lower than those for the GVHD GM + SB group (Fig. 2 D). Alpha diversity of mice feces in the GVHD GM group at day 14, the GVHD GM + SB group at day 14, and the GVHD no antibiotics group at day 0 showed significant differences (Fig. 2 F and 2 G). The intestinal microbiota diversity reduced significantly in the GVHD GM + SB group at day 14 compared with that in the GVHD no antibiotics group at day 0 (Chao 1 P = 0.037, Shannon P = 0.016). Similarly, beta diversity indices in the GVHD GM group at day 14, the GVHD GM + SB group at day 14, and the GVHD no antibiotics group at day 0 showed significant differences (P = 0.002; Fig. 2 H–J). At the genus level, The abundance of Clostridium was significantly elevated in the GVHD GM + SB group (Fig. 2 L and 2 M). Sodium butyrate aggravates the thinning of the colon mucus layer and epithelial barrier damage We examined the histological effects of gentamicin and sodium butyrate on aGVHD. The length of mice colons in the GVHD GM + SB group were shorter than those in the GVHD GM group (Fig. 3 A). Furthermore, mice in the GVHD GM group had significantly longer colons than those in the GVHD no antibiotics group. In contrast, mice in the GVHD SB group experienced the most severe reduction in colon length. Whereas the length of colons in GVHD mice treated with gentamicin and sodium butyrate 7 days before transplantation were similar to those of the non-GVHD group. No statistically significant difference was observed between the GVHD GM + SB group compared with the GVHD no antibiotics group. PAS staining showed that the colonic mucus layer was thinner in GVHD GM + SB mice compared with mice in the GVHD GM group (Fig. 3 B). Additionally, mice in the GVHD SB group had the thinnest mucus layer among all groups, followed by the GVHD no antibiotics group. Meanwhile, mice in the GVHD GM + SB (-7d) group retained the thickest mucus layer. H&E staining revealed more severe epithelial damage in the colon (Fig. 3 C) and higher GVHD histological scores in the small intestines (Fig. 3 D) of mice in the GVHD GM + SB group compared with allogeneic mice in the GVHD GM group. Conversely, mice in the GVHD SB group exhibited the most severe damage in both the colon and small intestine, followed by the GVHD no antibiotics group. Whereas mice in the GVHD GM + SB (-7d) group exhibited the most intact intestinal mucosal structure in all groups. The proportion of matured goblet cells, defined as those with a diameter > 10µm [ 32 – 33 ] , was significantly reduced at 18 days after allo-HSCT. Mice in the GVHD GM + SB (-7d) group retained the most matured goblet cells, while GVHD mice treated with sodium butyrate only had minimal mature goblet cells, followed by the GVHD no antibiotics group. In contrast, the GVHD GM group had more matured goblet cells than the GVHD GM + SB group, suggesting that sodium butyrate may inhibit goblet cell maturation or promote the apoptosis of mature goblet cells. This trend was consistent in the colon (Fig. 3 E) and small intestine (Fig. 3 F). Sodium butyrate inhibits LS174T cell proliferation and increases apoptosis LS174T cells, a human goblet cell line, were used to elucidate the biological role of sodium butyrate in goblet cells in vitro . LS174T cells were intervened with varying concentrations of sodium butyrate (0, 0.1, 1, 10, and 50 mM) for 96 h, and the optical density (OD) value was measured. Sodium butyrate at 10 mM significantly inhibited the proliferation of LS174T cells compared with 0.1 and 1 mM, while proliferation at 50 mM showed no significant difference compared with that at 10 mM (Fig. 4 A). LS174T cell apoptosis was assessed by flow cytometry using Annexin V/7-AAD staining. Cells were exposed to sodium butyrate at a concentration gradient of 0, 0.1, 1, and 10 mM for 24 h and irradiated with 750cGy X-ray. The percentage of apoptotic cells was evaluated after an additional 48 h of culture. Compared with the control group, sodium butyrate (10 mM) markedly increased the percentage of apoptotic cells (Fig. 4 B and 4 C). In particular, the sodium butyrate (10 mM) intervened group had higher BAX expression, cleaved Caspase-8, cleaved Caspase-9, and had lower BCL-2 expression (Fig. 4 D) compared with those of the control group. Sodium butyrate decreases the secretion of Muc2 and inhibits the proliferation of intestinal stem cells As Muc2 is the primary product of goblet cells, we hypothesized that sodium butyrate supplementation accelerates the thinning of colonic mucus by suppressing the secretion of Muc2. Therefore, LS174T cells were intervened with 0, 1, and 10 mM sodium butyrate for 24 h, irradiated with 750cGy X-ray, and cultured for another 48 h before RNA and protein extraction to validate our hypothesis. Both the mRNA (Fig. 5 A) and protein (Fig. 5 B) expression levels of Muc2 in the sodium butyrate (10 mM)-intervened groups were significantly decreased compared with those in the control group. These results indicate that sodium butyrate effectively inhibits the expression of Muc2 in LS174T cells. Subsequently, we isolated IECs from the colon and small intestine in all groups of mice and detected the mRNA (Fig. 5 C and 5 E) and protein (Fig. 5 D and 5 F) expression levels of Muc2. We found that the GVHD GM + SB group had lower Muc2 expression compared with the GVHD GM group, both in the colon and small intestine. Particularly, the GVHD SB group had the lowest Muc2 expression among all groups, followed by the GVHD no antibiotics group. The GVHD GM + SB(-7d) group retained the most Muc2 in mice that developed GVHD. We further validated above results through intestinal immunofluorescence staining in the colon (Fig. 5 G) and small intestine (Fig. 5 J). Compared with the non-GVHD group, the number of Lgr5 + cells in the colon (Fig. 5 H) and small intestine (Fig. 5 K) of mice decreased after transplantation. The number of Lgr5 + cells in the GVHD SB group were the lowest among all groups, followed by the GVHD no antibiotics group. Conversely, the GVHD GM + SB (-7d) group retained the most Lgr5 + cells in mice that developed GVHD. The numbers of Lgr5 + cells in the GVHD GM group were significantly higher than those in the GVHD GM + SB group, which suggests that butyrate accumulation may inhibit the proliferation of intestinal stem progenitor cells. Discussion Allo-HSCT is an established treatment for various benign and malignant hematopoietic diseases. However, GVHD remains the principal hurdle in achieving favorable patient outcomes following allo-HSCT [ 34 ] . GVHD can affect nearly every tissue, including the skin and liver, with the intestines being the primary target of allogeneic donor T cells. Additionally, studies on the effects of antibiotics on GVHD have shown varying results [ 35 ] . Decontamination therapy was shown to offer a protective effect against gastrointestinal acute GVHD in children undergoing allo-HSCT [ 6 ] . In particular, gentamicin used in oral decontamination therapy contributed to a lower rate of aGVHD. This may be because of the depletion of Gram-negative bacteria and abrogation of the pro-inflammatory effect of intestinal microbiota inducing gut GVHD. Accordingly, in this study, mice treated without antibiotics experienced more severe aGVHD than did those treated with gentamicin, suggesting that post-HSCT mice could benefit from gentamicin. Although broad-spectrum antibiotics are crucial for treating infections in patients who undergo allo-HSCT [ 36 – 37 ] , some antibiotics have been linked to increased intestinal GVHD and shorter OS [ 38 – 39 ] . Therefore, we analyzed the 16S rRNA sequences of mice fecal samples to further investigate the effect of gentamicin on GVHD, which revealed a significant depletion of Clostridium in the gentamicin group; notably, butyrate is a primary metabolite of Clostridium in the intestine. SCFAs are significant microbial metabolites [ 40 – 41 ] with the primary SCFAs being acetate (C2), propionate (C3), and butyrate (C4) [ 42 ] . Butyrate serves as the main energy source for IECs and stimulates goblet cells to produce mucin [ 43 ] . Additionally, butyrate inhibits the differentiation, maturation, and function of dendritic cells and macrophages, reduces the production of IL-12 and interferon-γ (IFN-γ), blocks the NF-kappa B signaling pathway, and promotes IL-10 production [ 44 ] . Moreover, previous studies have suggested that sodium butyrate inhibits histone deacetylase (HDAC), which improves the survival of GVHD mice [ 45 – 47 ] . However, contrary to these studies, our results show that sodium butyrate decreased the survival rate of GVHD mice and aggravated gastrointestinal mucosal destruction. In the context of colitis resulting in the loss of crypt architecture, Kaiko et al. [ 48 ] found that butyrate accumulation inhibits the proliferation of intestinal stem progenitor cells and delays wound repair. Additionally, Fredricks et al. [ 17 ] reported that the loss of epithelial architecture results in the exposure of colonic stem cells to microbially produced butyrate after the onset of severe aGVHD of the gut. This exposure may impair colonic mucosal recovery from aGVHD, leading to increased risk for refractory and chronic GVHD. Lgr5+, also known as Gpr49, marks mitotically active intestinal stem cells that exhibit exquisite sensitivity to canonical Wnt modulation, and contributes to homeostatic regeneration [ 49 – 50 ] . Therefore, we assessed the effects of sodium butyrate on the proliferation and differentiation ability of intestinal crypt stem cells via immunofluorescence staining. Accordingly, we found a significant reduction in Lgr5 + intestinal stem cells in the GVHD GM + SB group compared with that in GVHD mice treated with gentamicin only. Thus, butyrate potentially exhibits a biphasic response that may be helpful in protecting against the onset of aGVHD, however, once aGVHD of the gut has occurred, it may impair recovery and lead to worsened outcomes. Although Reddy et al. [ 16 ] suggest that butyrate has direct salutary effects on IECs in the context of inflammation and GVHD, their study did not directly address the impact of butyrate on various other cells that make up gastrointestinal epithelium besides IECs. Moreover, the SCFA–GPR43–ERK–NLRP3 axis in non-hematopoietic cells, such as IECs, plays a role in mitigating the severity of target tissue damage in GVHD [ 15 ] . Additionally, the NLRP3 inflammasome has previously been demonstrated to enhance GVHD in host hematopoietic antigen-presenting cells [ 51 ] ; however, the underlying mechanism of action of butyrate on the stem cells in intestinal GVHD requires further investigation. In this regard, we found that the crypt structure was destroyed, and the mucosal injury aggravated such that the overlying colonocytes were damaged as colitis progressed. Thus, we hypothesized that the exposure of the stem cell niche to butyrate resulted in an anti-proliferative effect, thereby exacerbating the manifestation of GVHD. Subsequently, we observed increased apoptosis of goblet cells, which synthesize and secrete mucus, protecting mucosal tissue from potential pathogens, mutagens, and physical or chemical damage, thereby preventing inflammation and inhibiting the development of diseases [ 52 ] . A previous study showed that intestinal goblet cells could protect mice from GVHD following allogeneic stem cell transplantation [ 53 ] . Thus, our results suggested that sodium butyrate might aggravate GVHD by damaging goblet cells. Therefore, we investigated the mechanism through which the goblet cells affect GVHD. Mucins (MUC) are a heterogeneous family of large complex glycoproteins [ 54 ] . Muc2, the main component of the intestinal mucus layer, is found at the top of IECs [ 22 ] while goblet cells continuously secrete, store, and release Muc2 to maintain mucus layers [ 55 – 57 ] . Additionally, recent studies have indicated that Muc2 plays an important role in protecting the gut barrier, regulating microbiome homeostasis, and preventing diseases [ 58 ] . Moreover, the loss of Muc2 during GVHD results in increased epithelial injury [ 59 ] . Accordingly, using LS174T cells [ 60 – 61 ] , we found that sodium butyrate not only inhibited the proliferation of LS174T cells and promoted their apoptosis but also reduced the expression of Muc2 in these cells, which might be one of the reasons for the aggravation of GVHD by sodium butyrate. In conclusion, our findings provide new insights into the mechanisms by which antibiotics and SCFAs influence GVHD pathogenesis. Gentamicin administration before transplantation significantly prolonged the survival of GVHD mice and reduced the severity of GVHD. Fecal sequencing revealed a significant reduction in the abundance of butyric-producing bacteria in the gentamicin-treated group. In contrast GVHD mice administered sodium butyrate showed that the protective effect of gentamicin could be invalidated by sodium butyrate supplementation after transplantation. Therefore, we speculate that sodium butyrate levels may affect the progression of intestinal GVHD. The GVHD mice intervened with sodium butyrate only after transplantation showed the most severe disease in all the study groups, while the normal mice treated with sodium butyrate under the same conditions (non-GVHD SB group) showed no abnormal performance compared with that of the control mice (non-GVHD group), indicating that sodium butyrate is not toxic if GVHD does not occur. However, sodium butyrate intervention before transplantation showed that the protective effect was even more significant in GVHD mice. This suggests that the intervention time of sodium butyrate in GVHD is crucial; supplementing before the priming of GVHD can prevent and delay the occurrence and progression of GVHD, whereas supplementing after the priming of GVHD has inhibited effects on goblet cells and intestinal stem cells. Thus, our study provides novel insights into the role and mechanism of gentamicin and SCFAs in GVHD, which should be further explored in future studies. Declarations Competing Interests The authors declare no competing interests. Ethical approval This study was approved by the Medical Ethical Committee of Qilu Hospital of Shandong University (DWLL-2024-369). The study did not involve any interventional clinical trial. Funding This work was supported by grants from the National Key R&D Program of China (No. 2023YFC2507804), National Natural Science Foundation of China (No. 82170124, No. 82030005), ECCM Program of Clinical Research Centre of Shandong University (No. 2021SDUCRCB008), and Taishan Scholar Foundation of Shandong Province (No. tstp20221157). Acknowledgements We thank the Translational Medicine Core Facility of Shandong University for consultation and instrument availability that supported this work. Data Availability Statement The data analyzed in this study are provided within the paper. References Jenq RR, van den Brink MR. Allogeneic haematopoietic stem cell transplantation: individualized stem cell and immune therapy of cancer. Nat Rev Cancer 2010;10:213-21. https://doi.org/10.1038/nrc2804. Malard F, Holler E, Sandmaier BM, Huang H, Mohty M. Acute graft-versus-host disease. Nat Rev Dis Primers 2023;9:27. https://doi.org/10.1038/s41572-023-00438-1. Hill GR, Ferrara JL. 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The Milk Active Ingredient, 2'-Fucosyllactose, Inhibits Inflammation and Promotes MUC2 Secretion in LS174T Goblet Cells In Vitro. Foods 2023;12:186. https://doi.org/10.3390/foods12010186. Olivo-Martínez Y, Bosch M, Badia J, Baldomà L. Modulation of the Intestinal Barrier Integrity and Repair by Microbiota Extracellular Vesicles through the Differential Regulation of Trefoil Factor 3 in LS174T Goblet Cells. Nutrients 2023;15:2437. https://doi.org/10.3390/nu15112437. Additional Declarations The authors have declared there is NO conflict of interest to disclose. Cite Share Download PDF Status: Posted Version 1 posted 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-7181845","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":491888589,"identity":"3990bebf-bfd2-4ccf-acca-4ce91fdbb14e","order_by":0,"name":"linlin 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and composition in the gentamicin-treated GVHD mice\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic of the pharmacodynamic study of GVHD. HSCT mice were provided drinking water supplemented with gentamicin. (B) Survival curves of the non-GVHD, GVHD no antibiotics, and GVHD GM groups. Kaplan–Meier survival curves of mice (n = 5–10 per group). Experiments were performed in triplicate. (C, D) Body weights (C) and GVHD scores (D) of the non-GVHD, GVHD no antibiotics, and GVHD GM groups (n = 5–10 per group). (E) PCoA of Euclidean distance analysis. (F, G) Significant differences are observed in metagenomic functions between the GVHD GM and GVHD no antibiotics groups. (H) Abundance of \u003cem\u003eClostridium\u003c/em\u003e. (I) Wilcoxon rank-sum test bar plot at the genus level. (J) PCoA of Euclidean distance analysis. (K, L) Alpha diversity analysis. (M, N) Beta diversity analysis (p = 0.828). (O) Non-metric multidimensional scaling (NMDS) of Euclidean distance analysis. (P, Q) Alpha diversity analysis. (R, S) Beta diversity analysis (p = 0.073).\u003c/p\u003e","description":"","filename":"17.22.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7181845/v1/576373bdb95ab3b21c816104.jpg"},{"id":88037776,"identity":"11e65f35-eb14-42ba-b4a4-bc014ad4f7cc","added_by":"auto","created_at":"2025-07-31 16:33:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1436350,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIntervention of sodium butyrate after allo-HSCT increased the mortality and aggravated GVHD of mice\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic of the pharmacodynamic study of sodium butyrate. (B) Survival curves of the non-GVHD, non-GVHD SB, GVHD SB, GVHD GM, GVHD no antibiotics, GVHD GM+SB, and GVHD GM + SB (-7d) groups (n = 5–10 per group). Data presented are from three independent experiments.\u003cstrong\u003e \u003c/strong\u003e(C, D) Body weights (C) and GVHD scores (D) of the non-GVHD, non-GVHD SB, GVHD SB, GVHD GM, GVHD no antibiotics, GVHD GM+SB, and GVHD GM + SB (-7d) groups (n = 5–10 per group). (E) PCoA of Euclidean distance analysis. (F, G) Alpha diversity analysis. (H, I, J) Beta diversity analysis (p = 0.002). (K) Venn diagrams of community analysis. (L, M) \u003cem\u003eClostridium \u003c/em\u003eis more abundant in the GVHD GM+SB group.\u003c/p\u003e","description":"","filename":"27.22.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7181845/v1/84b750307869105c59ec7948.jpg"},{"id":88037027,"identity":"148a56ba-07ee-443b-923a-740bd4e3324b","added_by":"auto","created_at":"2025-07-31 16:25:09","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2354494,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSodium butyrate aggravates the thinning of the colon mucus layer and epithelial barrier damage\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Macroscopic appearance and length of the colon (n = 3 per group). (B) Colon mucosal thickness on day 18 post-transplantation (n = 5 – 10 per group, scale bars = 20 µm). (C) H\u0026amp;E staining of histological sections of the colon on day 18 (n = 5–10 per group, scale bars = 100 µm). (D) H\u0026amp;E staining of small intestinal sections on day 18 (n = 5 – 10 per group, scale bars= 100 µm). (E) Representative images of Alcian blue staining of colon. Proportion of matured goblet cells defined as those with diameter \u0026gt; 10 mm (n = 3 per group, scale bars = 100 µm). (F) Alcian blue staining and frequency of goblet cells in the small intestines (n = 3 per group, scale bars = 100 µm).\u003c/p\u003e","description":"","filename":"37.22.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7181845/v1/8f247fbe852d03659b218bf8.jpg"},{"id":88035677,"identity":"a16e716f-4664-404f-81fc-036db1cbe22b","added_by":"auto","created_at":"2025-07-31 16:17:09","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":654152,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSodium butyrate inhibits LS174T cell proliferation and increases apoptosis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) CCK-8 analysis of the proliferation of LS174T cells exposed to sodium butyrate at 0, 0.1, 1, 10, and 50 mM for 24, 48, 72, and 96 h (n = 3). (B, C) Gating strategy used to identify apoptotic LS174T cells exposed to sodium butyrate at 0, 1, and 10 mM for 24 h, which were then irradiated with 750cGy X-ray and cultured for an additional 48 h. Annexin V was used to label early apoptotic cells, meanwhile Annexin V and 7-AAD were used to label late apoptotic cells (n = 3). (D) Expression of apoptosis-related proteins (BAX, BCL-2, cleaved Caspase-8, and cleaved Caspase-9, n = 3).\u003c/p\u003e","description":"","filename":"47.22.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7181845/v1/3d60d9fceb0af3df0aa7bf67.jpg"},{"id":88035681,"identity":"dd5bb7b8-c024-4b49-81ac-0de6c72c8f18","added_by":"auto","created_at":"2025-07-31 16:17:09","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2542063,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSodium butyrate decreases the secretion of Muc2 and inhibits the proliferation of intestinal stem cells\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) mRNA expression levels of Muc2 in LS174T cells intervened with 0, 1, and 10 mM sodium butyrate (n = 3). (B) Western blot analysis of Muc2 levels in LS174T cells intervened with 0, 1, and 10 mM sodium butyrate (n = 3). (C, E) Muc2 mRNA expression levels in mice colon (C) and small intestinal (E) epithelial cells (n = 3). (D, F) Western blot analysis of Muc2 levels in mice colon (D) and small intestinal (F) epithelial cells (n = 3). (G–I) Representative immunofluorescence staining of Muc2 (red) and Lgr5 (green) in the colon sections of mice; n = 3, scale bar: 100 µm. (J–L) Representative immunofluorescence staining of Muc2 (red) and Lgr5 (green) in the small intestine sections of mice; n = 3, scale bar: 100 µm.\u003c/p\u003e","description":"","filename":"57.22.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7181845/v1/85762cc2be462ca0f677232a.jpg"},{"id":90519505,"identity":"b091fb6c-6ec8-4062-9dab-933d4744533e","added_by":"auto","created_at":"2025-09-03 15:14:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8051212,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7181845/v1/3565f721-096f-4507-9bb8-67240e3f039a.pdf"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Gentamicin alleviates intestinal graft-versus-host disease by modulating butyrate-associated gut microbiota","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHematopoietic stem cell transplantation (HSCT) is a cornerstone in the treatment of numerous hematological disorders \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. However, disease relapse and graft-versus-host disease (GVHD) are the two primary causes of mortality following allogeneic hematopoietic cell transplantation (allo-HSCT) \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. GVHD mainly affects the gastrointestinal system, and severe intestinal GVHD is associated with a poor prognosis in patients post-HSCT \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Antibiotics are crucial for managing infections in patients who undergo allo-HSCT, in addition, recent study reported that gut-decontaminating prophylactic antibiotics can affect the severity of intestinal GVHD \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Gentamicin, an aminoglycoside antibiotic, is commonly used to inhibit the growth of gram-negative bacteria \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e, moreover, a multicenter clinical study found that it decreased the morbidity of gastrointestinal GVHD in children \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. However, the underlying mechanism of post-HSCT intestinal GVHD modulation by gentamicin remains unclear.\u003c/p\u003e\u003cp\u003eAdditionally, despite recent research suggesting that inhibiting \u003cem\u003eClostridium\u003c/em\u003e proliferation in the intestinal tract can aid in maintaining intestinal homeostasis or treat intestinal diseases \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e, the role of \u003cem\u003eClostridium\u003c/em\u003e in GVHD requires further investigation. Moreover, increasing evidence shows that short-chain fatty acids (SCFAs) \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e play a vital role in maintaining intestinal metabolic homeostasis \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e–\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Among these, butyrate is a key metabolite produced by bacterial fermentation in the intestine \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eClostridium\u003c/em\u003e is a primary producer of butyrate \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e, of which sodium butyrate is a salt form \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Butyrate produced in the large intestine by intestinal bacteria is essential for fueling IECs and promoting mucin production \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. While some studies have reported protective effects of butyrate on GVHD \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e–\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, Golob et al. showed that butyrate has a double-edged effect on GVHD based on the exposure time. They reported that butyrate inhibited intestinal stem cells from forming an intact epithelial monolayer in addition to providing a protective effect \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFurthermore, the colon and intestinal mucus layers are rich in antibacterial peptides and proteins that safeguard the epithelium from harmful agents, viruses, and pathogenic bacteria \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e–\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Additionally, goblet cells secrete Muc2 \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e, a critical secretory protein in the human intestine \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e–\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e, which is essential for protecting against bacterial infections by limiting the number of pathogens and commensal bacteria on the colonic mucosal surface \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. This action mitigates tissue damage and prevents the translocation of bacteria across the epithelium \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Consequently, the role of Muc2 in intestinal mucosal barrier dysfunction has garnered increasing attention, suggesting it may be pivotal in treating such damage \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, we investigated the effects of gentamicin and sodium butyrate supplementation in allo-HSCT mice. In addition, we managed to explore the underlying mechanism of gentamicin and sodium butyrate affecting intestinal GVHD.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cem\u003eMurine aGVHD model\u003c/em\u003e\u003c/p\u003e\u003cp\u003eSpecific pathogen-free (SPF) BALB/c mice (GemPharmatech Co., Ltd, China) were housed in a pathogen-free facility at the Laboratory Animal Centre of Shandong University. Female BALB/c mice (6–8 weeks old) were maintained on a 12-h dark/light cycle at an ambient temperature of 22°C ± 2°C with controlled humidity (~ 45%). Lethally irradiated (750cGy) BALB/c mice were intravenously injected with 5 × 10⁶ bone marrow (BM) cells and 5 × 10⁶ splenocytes (SP) from major histocompatibility complex (MHC)-mismatched male C57BL/6J donors on day 0. Body weights and clinical scores \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e were recorded every other day. The mice were divided into seven subgroups. The GVHD GM group represented GVHD mice treated with gentamicin from 7 days before transplantation to 21 days after transplantation. The GVHD no antibiotics group represented GVHD mice treated without gentamicin or sodium butyrate. The GVHD GM + SB group represented GVHD mice treated with gentamicin 7 days before bone marrow transplantation (BMT) to 21 days after BMT, and sodium butyrate from day 1 to day 21 after BMT. The GVHD GM + SB(-7d) group represented GVHD mice treated with gentamicin and sodium butyrate 7 days before BMT to 21 days after BMT. The GVHD SB group represented GVHD mice treated with SB from day 1 to day 21 after BMT. The non-GVHD group represented syngeneic control mice without GVHD. The non-GVHD SB group represented syngeneic control mice without GVHD treated with sodium butyrate to detect the toxicity.\u003c/p\u003e\u003cp\u003e\u003cem\u003eSodium butyrate and gentamicin treatment\u003c/em\u003e\u003c/p\u003e\u003cp\u003eUp till day 7 before the injection of BM cells and splenocytes, mice received gentamicin (0.2 µg/mL) \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e in their drinking water daily. Allogeneic mice were administered sodium butyrate 150 mM in their drinking water from day 1 to day 21 \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e, followed by autoclaved free drinking water on other days. Gentamicin (Fuan Pharmaceutical Co., LTD, China) and sodium butyrate (MP Biomedicals, USA) were dissolved in double-distilled water immediately prior to drinking.\u003c/p\u003e\u003cp\u003e\u003cem\u003eCell culture\u003c/em\u003e\u003c/p\u003e\u003cp\u003eLS174T human goblet cells (Bio-Channel, China) were cultured in RPMI 1640 culture medium (Gibco, USA) supplemented with 10% fetal bovine serum (Tocyto, USA) and 1% penicillin/streptomycin (Gibco, USA). The cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂. Sodium butyrate was dissolved and diluted in the RPMI 1640 medium immediately before use.\u003c/p\u003e\u003cp\u003e\u003cem\u003eCell proliferation assays\u003c/em\u003e\u003c/p\u003e\u003cp\u003eLS174T human goblet cells were cultured in the presence of 0, 0.1, 1, 10, and 50 mM sodium butyrate. Cells were incubated with 10 µL of CCK-8 (Beyotime, China) for 3 h, and absorbance was measured at 450 nm. Each sample was measured in triplicate.\u003c/p\u003e\u003cp\u003e\u003cem\u003eApoptosis assays\u003c/em\u003e\u003c/p\u003e\u003cp\u003eApoptosis was assessed using an Annexin V/7-AAD apoptosis detection kit (BestBio, Shanghai, China) according to the manufacturer’s protocol. LS174T cells were harvested after different treatments, washed twice with phosphate buffer saline (PBS; Beyotime, C0221A), and resuspended in 500 µL of binding buffer. Subsequently, cells were stained with 5 µL Annexin V for 15 min followed by 5 µL 7-AAD for 15 min in the dark at 4°C. The percentage of apoptotic cells was analyzed immediately using a Galios flow cytometer (Beckman Coulter, CA, USA).\u003c/p\u003e\u003cp\u003e\u003cem\u003eRNA extraction and quantitative real-time PCR\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTotal RNA was extracted from cells using an RNA Rapid Extraction Kit (Invitrogen, USA). RNA concentration and purity were measured using a spectrophotometer (Eppendorf, Germany). Reverse transcription was performed using the Prime Script RT reagent Perfect Real Time Kit (Vazyme, 7E711J3) at 37°C for 15 min, followed by at 85°C for 10 s. Quantitative PCR was conducted in duplicate on a LightCycler 480II real-time PCR system (Roche, Switzerland) using the SYBR Green Real-time PCR Master Mix kit (Vazyme, 7E782J3). Primer sequences used were as follows: β-actin F (5’-GAAGAGCTACGAGCTGCCTGA-3’) and R (5’-CAGACAGCACTGTGTTGGCG-3’), Muc2 F (5’-AAGTGCTCCTGTTACCACCG-3’), and R (5’-CGGCACACACATCGTTCTTC-3’). Melting curves were analyzed to determine PCR specificity. All experiments were conducted according to the manufacturer’s instructions. Relative mRNA levels were expressed relative to β-actin using the 2\u003csup\u003e−ΔCT\u003c/sup\u003e method.\u003c/p\u003e\u003cp\u003e\u003cem\u003eWestern blotting analysis\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePrimary antibodies against Muc2 (ab272692) and antibodies against β-actin (ab227387) were purchased from Abcam (Cambridge, UK). All reagents were prepared and stored according to the manufacturer’s instructions. LS174T cells and mouse IECs were collected, washed twice with PBS, and lysed with RIPA buffer (Bestbio, BB-3101) containing a protease inhibitor on ice. Protein concentrations were measured using a bicinchoninic acid protein assay kit (Beyotime, P0009). Protein extracts (20 µg) were loaded onto 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis gels and electrotransferred onto nitrocellulose membranes (BioTrace, USA). After blocking with an efficient blocking solution for 15 min at room temperature, membranes were incubated overnight with specific primary antibodies at 4°C, followed by incubation with HRP-conjugated secondary antibodies at room temperature for 1 h. Protein bands were detected using a FluorChem E Chemiluminescent imaging system (Protein Simple, San Jose, CA, USA).\u003c/p\u003e\u003cp\u003e\u003cem\u003eHistological analysis\u003c/em\u003e\u003c/p\u003e\u003cp\u003eSamples from the small intestine and colon were fixed in 10% formalin and 4% paraformaldehyde, embedded in paraffin, sectioned (3–5 µm), and stained with hematoxylin \u0026amp; eosin (H\u0026amp;E). Colonic sections were stained with the periodic acid-Schiff (PAS) staining to evaluate mucus thickness. Sections were imaged and measured using a 3DHISTECH system. Five measurements were obtained per image and averaged over the entire usable colonic surface. Pathology scores \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e were quantified by two blinded pathologists.\u003c/p\u003e\u003cp\u003e\u003cem\u003eExtraction of intestinal epithelial cells\u003c/em\u003e\u003c/p\u003e\u003cp\u003eIECs were isolated as previously described \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e–\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Briefly, the small and large intestines were harvested individually from 8–10 mice and rinsed extensively with RPMI 1640 (Gibco, Grand Island, New York, USA) after Peyer’s patches were removed (for small intestine). The rinsed intestines were opened longitudinally and macerated; the tissue pieces were shaken gently in RPMI-1640 containing 2 mM EDTA and 10% fetal bovine serum (FBS). The tissue preparations were passed through 70-µm mesh filters, and the resulting single-cell suspensions were separated using Percoll (GE Healthcare, Little Chalfont, United Kingdom) density gradients of 25%, 40%, and 75%. After centrifugation at 2,000 × g for 20 min, the interface between the 25% and 40% layers was collected to obtain IECs. The cells were stained with antibodies against epithelial cell adhesion molecule (EpCAM; Biolegend, San Diego, California, USA), or CD45 (Biolegend) and nucleic acid dye (Via-Probe; Becton Dickinson, Franklin Lakes, New Jersey, USA). The EpCAM + cells were further confirmed to be IECs via intracellular staining with a pan-cytokeratin antibody (Abcam, Cambridge, Massachusetts, USA) and fixation and permeability reagents (Invitrogen, Carlsbad, California, USA). The IECs were sorted using FACS (Aria III, Becton Dickinson) and analyzed using the FlowJo software (Ashland, Oregon, USA).\u003c/p\u003e\u003cp\u003e\u003cem\u003eImmunofluorescence\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe intestine slices were fixed with 4% formaldehyde (Beyotime, China) at 37℃ for 30 min. After permeabilizing with 0.1% Triton X-100 (Solarbio, China) for 5 min, samples were blocked with goat serum (Beyotime, China) at 37℃ for 30 min. Sections were dewaxed and rehydrated, followed by antigen retrieval. Subsequently, the intestine slices were labeled with Lgr5 (Proteintech, Chicago, USA, 1:50) and Muc2 (Abcam, cat# ab254183, 1:100) antibodies at 4℃ overnight. The following day, slides were incubated with a secondary antibody (Abcam, cat# ab150077, 1:1000) at 37℃ for 1 h and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Abcam, cat# ab285390). The staining was evaluated using the Olympus SLIDEVIEW VS200.\u003c/p\u003e\u003cp\u003e\u003cem\u003eStool sample collection and microbial diversity analysis\u003c/em\u003e\u003c/p\u003e\u003cp\u003eFecal samples from mice were collected and stored at − 80°C. Subsequently, 16S rRNA sequencing was performed for microbiota analysis. Total genomic DNA samples were extracted using the OMEGA Soil DNA Kit (M5635-02, Omega Bio-Tek, Norcross, GA, USA), according to the manufacturer’s instructions, and stored at − 20°C until further analysis. The quantity and quality of extracted DNAs were measured using a NanoDrop NC2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively. PCR amplification of the bacterial 16S rRNA genes V3–V4 region was performed using primers F (5’-ACTCCTACGGGAGGCAGCA-3’) and R (5’-GGACTACHVGGGTWTCTAAT-3’). Sample-specific 7-bp barcodes were incorporated into the primers for multiplex sequencing. The PCR components contained 5 µL of buffer (5×), 0.25 µL of Fast pfu DNA Polymerase (5 U/µL), 2 µL (2.5 mM) of dNTPs, 1 µL (10 µM) of each Forward and Reverse primer, 1 µL of DNA Template, and 14.75 µL of ddH2O. PCR was conducted as follows: 2 min of initial denaturation at 98°C, 15 s of denaturation at 98°C, 30 s of annealing at 55°C, 30 s of extension at 72°C, and a final extension at 72°C for 5 min. PCR was performed in triplicate using a 20 µL mixture containing 5 µL 5 × reaction buffer, 5 µL 5 × GC buffer, 2 µL 2.5 mM dNTPs, 8.75 µL ddH2O, 1 µL each primer (10 µM), 0.25 µL Q5 DNA Polymerase, and 2 µL template DNA. PCR amplicons were purified with Vazyme VAHTSTM DNA Clean Beads (Vazyme, Nanjing, China) and quantified using the Quant-i T PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). After the individual quantification step, amplicons were pooled in equal amounts, and pair-end 2×250 bp sequencing was performed using the Illlumina NovaSeq platform with NovaSeq 6000 SP Reagent Kit (500 cycles) by Shanghai Bioprofile Technology Company Ltd (Shanghai, China). Microbiome bioinformatics analyses were performed with QIIME2 2019.4 with slight modification according to the official tutorials (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://docs.qiime2.org/2019.4/tutorials/\u003c/span\u003e\u003cspan address=\"https://docs.qiime2.org/2019.4/tutorials/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Briefly, raw sequence data were demultiplexed using the demux plugin following by primer cutting with the cutadapt plugin. Sequences were then quality filtered, denoised, merged, and chimera removed using the DADA2 plugin. Non-singleton amplicon sequence variants (ASVs) were aligned with mafft and used to construct a phylogeny with fasttree2. Alpha (Chao1, ACE, Shannon) and beta diversity metrics (weighted UniFrac, unweighted UniFrac and Bray – Curtis dissimilarity) were estimated using the diversity plugin, with samples rarefied to sequences per sample. Taxonomy was assigned to ASVs using the classify-sklearn naïve Bayes taxonomy classifier in the feature-classifier plugin against the Greengenes Database.\u003c/p\u003e\u003cp\u003e\u003cem\u003eAlcian blue staining\u003c/em\u003e\u003c/p\u003e\u003cp\u003eParaffin sections were dewaxed in xylene twice, 10 min each. The sections were then hydrated by treatment with an ethanol gradient ethanol: 100% ethanol → 95% → 80% → 70% → distilled water for 1–2 min per step. The tissue section was covered with a drop of Alcian blue dye (pH 2.5) and stained at room temperature for 30 min. The sections were rinsed under running water for 5 min to remove excess dye. The nucleus was stained using neutral red for 1–2 min followed by rinsing with water for 30 s or with hematoxylin for 3–5 min followed by differentiation, which returned to blue. The specimens were dehydrated and cleared by passing through an ethanol gradient of 70% → 80% → 95% → 100% ethanol for 1 min per step. Subsequently, specimen were passed through xylene twice for 5 min each. Finally neutral gum was added, the specimens were covered with cover glass and observed under the Olympus SLIDEVIEW VS200.\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eBetween-group comparisons were conducted using Student’s t-test for normally distributed data. The Wilcoxon rank-sum test was used for microbiome analysis. Progression-free survival and overall survival rates are presented using Kaplan–Meier curves. The log survival curves were compared using a log-rank test. Statistical significance was set at P \u0026lt; 0.05. ImageJ v.1.50 was used for western blot image processing, and Kaluza 2.1.1 was used for flow cytometry analysis. Statistical analysis was conducted using GraphPad Prism 8.0.2. *P \u0026lt; 0.05, **P \u0026lt; 0.01, and ***P \u0026lt; 0.0001 were considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eAlteration of gut microbiota diversity and composition in the gentamicin-treated GVHD mice\u003c/em\u003e\u003c/p\u003e\u003cp\u003eWe assessed the impact of gentamicin on aGVHD (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and found that allogeneic mice without gentamicin treatment experienced more severe GVHD compared with those treated with gentamicin (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Additionally, mice in the GVHD GM group lost significantly less weight than those in the GVHD no antibiotics group from the second week after the transplantation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Meanwhile, allogeneic mice treated with gentamicin had lower GVHD scores than mice treated without antibiotics, suggesting that aGVHD was milder in the GVHD GM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Therefore, we isolated total bacterial DNA from mouse fecal samples at 14 days post-transplantation and sequenced the 16S rRNA genes using Illumina to further explore this mechanism. Principal co-ordinates analysis (PCoA) revealed a significant difference in the Euclidean distance between the GVHD GM group and the GVHD no antibiotics group (confidence interval: 90%), indicating a substantial shift in the microbial composition with respect to operational taxonomic units (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Stool samples from mice in the GVHD GM group and the GVHD no antibiotics group were analyzed for Chao1 (P\u0026thinsp;=\u0026thinsp;0.0198) and ACE (P\u0026thinsp;=\u0026thinsp;0.0193) indices. A significant reduction was observed in intestinal microbiota diversity in the GVHD GM group compared with that in the GVHD no antibiotics group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). At the genus level, \u003cem\u003eClostridium\u003c/em\u003e was significantly depleted in the GM group compared with that in the GVHD no antibiotics group (P\u0026thinsp;=\u0026thinsp;0.0269, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). However, no difference was observed in intestinal microbiota diversity and composition between the GVHD GM group and the GVHD no antibiotics group at day 0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK\u0026ndash;N), which suggests that gentamicin decreased \u003cem\u003eClostridium\u003c/em\u003e mainly in GVHD mice instead of normal ones. Additionally, intestinal microbiota diversity and composition at day 0 and day 14 presented no differences in the GVHD no antibiotics group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eP\u0026ndash;S).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eIntervention of sodium butyrate after allo-HSCT increased the mortality and aggravated GVHD of mice\u003c/em\u003e\u003c/p\u003e\u003cp\u003eWe examined the effects of sodium butyrate in an allo-HSCT mouse model treated with gentamicin to investigate the role of \u003cem\u003eClostridium\u003c/em\u003e in aGVHD and determine whether \u003cem\u003eClostridium\u003c/em\u003e exerts its effect by producing butyrate. The workflow for assessing the pharmacodynamics of sodium butyrate is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. The non-GVHD SB group allowed an assessment the toxicities of sodium butyrate without induction of an allogeneic response. We found that sodium butyrate had no effect on the survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), or GVHD score of normal mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). In contrast, the GVHD SB group experienced the most severe GVHD among all groups. Whereas mice in the GVHD GM\u0026thinsp;+\u0026thinsp;SB group exhibited more severe aGVHD than those in the GVHD GM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), suggesting that supplementation of sodium butyrate might invalidate the beneficial effect of gentamicin. Conversely, concurrent administration of gentamicin and sodium butyrate pre-transplantation showed a more obvious protective effect, accompanied by the least weight loss and the lowest GVHD score, which suggests that applying sodium butyrate only after transplant might not demonstrate the beneficial effects on GVHD priming. Additionally, body weight comparisons showed that allo-HSCT mice with aGVHD lost more weight compared with that of the mice in the non-GVHD group. Mice in the GVHD GM\u0026thinsp;+\u0026thinsp;SB group showed significantly decreased weights compared with those in the GVHD GM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), meanwhile GVHD scores for the GVHD GM group were lower than those for the GVHD GM\u0026thinsp;+\u0026thinsp;SB group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Alpha diversity of mice feces in the GVHD GM group at day 14, the GVHD GM\u0026thinsp;+\u0026thinsp;SB group at day 14, and the GVHD no antibiotics group at day 0 showed significant differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). The intestinal microbiota diversity reduced significantly in the GVHD GM\u0026thinsp;+\u0026thinsp;SB group at day 14 compared with that in the GVHD no antibiotics group at day 0 (Chao 1 P\u0026thinsp;=\u0026thinsp;0.037, Shannon P\u0026thinsp;=\u0026thinsp;0.016). Similarly, beta diversity indices in the GVHD GM group at day 14, the GVHD GM\u0026thinsp;+\u0026thinsp;SB group at day 14, and the GVHD no antibiotics group at day 0 showed significant differences (P\u0026thinsp;=\u0026thinsp;0.002; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH\u0026ndash;J). At the genus level, The abundance of \u003cem\u003eClostridium\u003c/em\u003e was significantly elevated in the GVHD GM\u0026thinsp;+\u0026thinsp;SB group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eSodium butyrate aggravates the thinning of the colon mucus layer and epithelial barrier damage\u003c/em\u003e\u003c/p\u003e\u003cp\u003eWe examined the histological effects of gentamicin and sodium butyrate on aGVHD. The length of mice colons in the GVHD GM\u0026thinsp;+\u0026thinsp;SB group were shorter than those in the GVHD GM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Furthermore, mice in the GVHD GM group had significantly longer colons than those in the GVHD no antibiotics group. In contrast, mice in the GVHD SB group experienced the most severe reduction in colon length. Whereas the length of colons in GVHD mice treated with gentamicin and sodium butyrate 7 days before transplantation were similar to those of the non-GVHD group. No statistically significant difference was observed between the GVHD GM\u0026thinsp;+\u0026thinsp;SB group compared with the GVHD no antibiotics group. PAS staining showed that the colonic mucus layer was thinner in GVHD GM\u0026thinsp;+\u0026thinsp;SB mice compared with mice in the GVHD GM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Additionally, mice in the GVHD SB group had the thinnest mucus layer among all groups, followed by the GVHD no antibiotics group. Meanwhile, mice in the GVHD GM\u0026thinsp;+\u0026thinsp;SB (-7d) group retained the thickest mucus layer. H\u0026amp;E staining revealed more severe epithelial damage in the colon (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) and higher GVHD histological scores in the small intestines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) of mice in the GVHD GM\u0026thinsp;+\u0026thinsp;SB group compared with allogeneic mice in the GVHD GM group. Conversely, mice in the GVHD SB group exhibited the most severe damage in both the colon and small intestine, followed by the GVHD no antibiotics group. Whereas mice in the GVHD GM\u0026thinsp;+\u0026thinsp;SB (-7d) group exhibited the most intact intestinal mucosal structure in all groups. The proportion of matured goblet cells, defined as those with a diameter\u0026thinsp;\u0026gt;\u0026thinsp;10\u0026micro;m \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e, was significantly reduced at 18 days after allo-HSCT. Mice in the GVHD GM\u0026thinsp;+\u0026thinsp;SB (-7d) group retained the most matured goblet cells, while GVHD mice treated with sodium butyrate only had minimal mature goblet cells, followed by the GVHD no antibiotics group. In contrast, the GVHD GM group had more matured goblet cells than the GVHD GM\u0026thinsp;+\u0026thinsp;SB group, suggesting that sodium butyrate may inhibit goblet cell maturation or promote the apoptosis of mature goblet cells. This trend was consistent in the colon (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) and small intestine (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eSodium butyrate inhibits LS174T cell proliferation and increases apoptosis\u003c/em\u003e\u003c/p\u003e\u003cp\u003eLS174T cells, a human goblet cell line, were used to elucidate the biological role of sodium butyrate in goblet cells \u003cem\u003ein vitro\u003c/em\u003e. LS174T cells were intervened with varying concentrations of sodium butyrate (0, 0.1, 1, 10, and 50 mM) for 96 h, and the optical density (OD) value was measured. Sodium butyrate at 10 mM significantly inhibited the proliferation of LS174T cells compared with 0.1 and 1 mM, while proliferation at 50 mM showed no significant difference compared with that at 10 mM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). LS174T cell apoptosis was assessed by flow cytometry using Annexin V/7-AAD staining. Cells were exposed to sodium butyrate at a concentration gradient of 0, 0.1, 1, and 10 mM for 24 h and irradiated with 750cGy X-ray. The percentage of apoptotic cells was evaluated after an additional 48 h of culture. Compared with the control group, sodium butyrate (10 mM) markedly increased the percentage of apoptotic cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In particular, the sodium butyrate (10 mM) intervened group had higher BAX expression, cleaved Caspase-8, cleaved Caspase-9, and had lower BCL-2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) compared with those of the control group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eSodium butyrate decreases the secretion of Muc2 and inhibits the proliferation of intestinal stem cells\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAs Muc2 is the primary product of goblet cells, we hypothesized that sodium butyrate supplementation accelerates the thinning of colonic mucus by suppressing the secretion of Muc2. Therefore, LS174T cells were intervened with 0, 1, and 10 mM sodium butyrate for 24 h, irradiated with 750cGy X-ray, and cultured for another 48 h before RNA and protein extraction to validate our hypothesis. Both the mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) expression levels of Muc2 in the sodium butyrate (10 mM)-intervened groups were significantly decreased compared with those in the control group. These results indicate that sodium butyrate effectively inhibits the expression of Muc2 in LS174T cells. Subsequently, we isolated IECs from the colon and small intestine in all groups of mice and detected the mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE) and protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF) expression levels of Muc2. We found that the GVHD GM\u0026thinsp;+\u0026thinsp;SB group had lower Muc2 expression compared with the GVHD GM group, both in the colon and small intestine. Particularly, the GVHD SB group had the lowest Muc2 expression among all groups, followed by the GVHD no antibiotics group. The GVHD GM\u0026thinsp;+\u0026thinsp;SB(-7d) group retained the most Muc2 in mice that developed GVHD. We further validated above results through intestinal immunofluorescence staining in the colon (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG) and small intestine (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). Compared with the non-GVHD group, the number of Lgr5\u0026thinsp;+\u0026thinsp;cells in the colon (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH) and small intestine (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK) of mice decreased after transplantation. The number of Lgr5\u0026thinsp;+\u0026thinsp;cells in the GVHD SB group were the lowest among all groups, followed by the GVHD no antibiotics group. Conversely, the GVHD GM\u0026thinsp;+\u0026thinsp;SB (-7d) group retained the most Lgr5\u0026thinsp;+\u0026thinsp;cells in mice that developed GVHD. The numbers of Lgr5\u0026thinsp;+\u0026thinsp;cells in the GVHD GM group were significantly higher than those in the GVHD GM\u0026thinsp;+\u0026thinsp;SB group, which suggests that butyrate accumulation may inhibit the proliferation of intestinal stem progenitor cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAllo-HSCT is an established treatment for various benign and malignant hematopoietic diseases. However, GVHD remains the principal hurdle in achieving favorable patient outcomes following allo-HSCT \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. GVHD can affect nearly every tissue, including the skin and liver, with the intestines being the primary target of allogeneic donor T cells.\u003c/p\u003e\u003cp\u003eAdditionally, studies on the effects of antibiotics on GVHD have shown varying results \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Decontamination therapy was shown to offer a protective effect against gastrointestinal acute GVHD in children undergoing allo-HSCT \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. In particular, gentamicin used in oral decontamination therapy contributed to a lower rate of aGVHD. This may be because of the depletion of Gram-negative bacteria and abrogation of the pro-inflammatory effect of intestinal microbiota inducing gut GVHD. Accordingly, in this study, mice treated without antibiotics experienced more severe aGVHD than did those treated with gentamicin, suggesting that post-HSCT mice could benefit from gentamicin. Although broad-spectrum antibiotics are crucial for treating infections in patients who undergo allo-HSCT \u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e, some antibiotics have been linked to increased intestinal GVHD and shorter OS \u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Therefore, we analyzed the 16S rRNA sequences of mice fecal samples to further investigate the effect of gentamicin on GVHD, which revealed a significant depletion of \u003cem\u003eClostridium\u003c/em\u003e in the gentamicin group; notably, butyrate is a primary metabolite of \u003cem\u003eClostridium\u003c/em\u003e in the intestine.\u003c/p\u003e\u003cp\u003eSCFAs are significant microbial metabolites \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003ewith the primary SCFAs being acetate (C2), propionate (C3), and butyrate (C4) \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. Butyrate serves as the main energy source for IECs and stimulates goblet cells to produce mucin\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Additionally, butyrate inhibits the differentiation, maturation, and function of dendritic cells and macrophages, reduces the production of IL-12 and interferon-γ (IFN-γ), blocks the NF-kappa B signaling pathway, and promotes IL-10 production \u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Moreover, previous studies have suggested that sodium butyrate inhibits histone deacetylase (HDAC), which improves the survival of GVHD mice \u003csup\u003e[\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. However, contrary to these studies, our results show that sodium butyrate decreased the survival rate of GVHD mice and aggravated gastrointestinal mucosal destruction.\u003c/p\u003e\u003cp\u003eIn the context of colitis resulting in the loss of crypt architecture, Kaiko et al. \u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e found that butyrate accumulation inhibits the proliferation of intestinal stem progenitor cells and delays wound repair. Additionally, Fredricks et al. \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e reported that the loss of epithelial architecture results in the exposure of colonic stem cells to microbially produced butyrate after the onset of severe aGVHD of the gut. This exposure may impair colonic mucosal recovery from aGVHD, leading to increased risk for refractory and chronic GVHD. Lgr5+, also known as Gpr49, marks mitotically active intestinal stem cells that exhibit exquisite sensitivity to canonical Wnt modulation, and contributes to homeostatic regeneration \u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. Therefore, we assessed the effects of sodium butyrate on the proliferation and differentiation ability of intestinal crypt stem cells via immunofluorescence staining. Accordingly, we found a significant reduction in Lgr5\u0026thinsp;+\u0026thinsp;intestinal stem cells in the GVHD GM\u0026thinsp;+\u0026thinsp;SB group compared with that in GVHD mice treated with gentamicin only. Thus, butyrate potentially exhibits a biphasic response that may be helpful in protecting against the onset of aGVHD, however, once aGVHD of the gut has occurred, it may impair recovery and lead to worsened outcomes. Although Reddy et al. \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e suggest that butyrate has direct salutary effects on IECs in the context of inflammation and GVHD, their study did not directly address the impact of butyrate on various other cells that make up gastrointestinal epithelium besides IECs. Moreover, the SCFA\u0026ndash;GPR43\u0026ndash;ERK\u0026ndash;NLRP3 axis in non-hematopoietic cells, such as IECs, plays a role in mitigating the severity of target tissue damage in GVHD \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Additionally, the NLRP3 inflammasome has previously been demonstrated to enhance GVHD in host hematopoietic antigen-presenting cells \u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e; however, the underlying mechanism of action of butyrate on the stem cells in intestinal GVHD requires further investigation. In this regard, we found that the crypt structure was destroyed, and the mucosal injury aggravated such that the overlying colonocytes were damaged as colitis progressed. Thus, we hypothesized that the exposure of the stem cell niche to butyrate resulted in an anti-proliferative effect, thereby exacerbating the manifestation of GVHD. Subsequently, we observed increased apoptosis of goblet cells, which synthesize and secrete mucus, protecting mucosal tissue from potential pathogens, mutagens, and physical or chemical damage, thereby preventing inflammation and inhibiting the development of diseases \u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e. A previous study showed that intestinal goblet cells could protect mice from GVHD following allogeneic stem cell transplantation \u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e. Thus, our results suggested that sodium butyrate might aggravate GVHD by damaging goblet cells. Therefore, we investigated the mechanism through which the goblet cells affect GVHD.\u003c/p\u003e\u003cp\u003eMucins (MUC) are a heterogeneous family of large complex glycoproteins \u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e. Muc2, the main component of the intestinal mucus layer, is found at the top of IECs \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e while goblet cells continuously secrete, store, and release Muc2 to maintain mucus layers \u003csup\u003e[\u003cspan additionalcitationids=\"CR56\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e. Additionally, recent studies have indicated that Muc2 plays an important role in protecting the gut barrier, regulating microbiome homeostasis, and preventing diseases \u003csup\u003e[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e. Moreover, the loss of Muc2 during GVHD results in increased epithelial injury \u003csup\u003e[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]\u003c/sup\u003e. Accordingly, using LS174T cells \u003csup\u003e[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]\u003c/sup\u003e, we found that sodium butyrate not only inhibited the proliferation of LS174T cells and promoted their apoptosis but also reduced the expression of Muc2 in these cells, which might be one of the reasons for the aggravation of GVHD by sodium butyrate.\u003c/p\u003e\u003cp\u003eIn conclusion, our findings provide new insights into the mechanisms by which antibiotics and SCFAs influence GVHD pathogenesis. Gentamicin administration before transplantation significantly prolonged the survival of GVHD mice and reduced the severity of GVHD. Fecal sequencing revealed a significant reduction in the abundance of butyric-producing bacteria in the gentamicin-treated group. In contrast GVHD mice administered sodium butyrate showed that the protective effect of gentamicin could be invalidated by sodium butyrate supplementation after transplantation. Therefore, we speculate that sodium butyrate levels may affect the progression of intestinal GVHD. The GVHD mice intervened with sodium butyrate only after transplantation showed the most severe disease in all the study groups, while the normal mice treated with sodium butyrate under the same conditions (non-GVHD SB group) showed no abnormal performance compared with that of the control mice (non-GVHD group), indicating that sodium butyrate is not toxic if GVHD does not occur. However, sodium butyrate intervention before transplantation showed that the protective effect was even more significant in GVHD mice. This suggests that the intervention time of sodium butyrate in GVHD is crucial; supplementing before the priming of GVHD can prevent and delay the occurrence and progression of GVHD, whereas supplementing after the priming of GVHD has inhibited effects on goblet cells and intestinal stem cells. Thus, our study provides novel insights into the role and mechanism of gentamicin and SCFAs in GVHD, which should be further explored in future studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eEthical approval\u003c/h2\u003e\u003cp\u003e This study was approved by the Medical Ethical Committee of Qilu Hospital of Shandong University (DWLL-2024-369). The study did not involve any interventional clinical trial.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by grants from the National Key R\u0026amp;D Program of China (No. 2023YFC2507804), National Natural Science Foundation of China (No. 82170124, No. 82030005), ECCM Program of Clinical Research Centre of Shandong University (No. 2021SDUCRCB008), and Taishan Scholar Foundation of Shandong Province (No. tstp20221157).\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eWe thank the Translational Medicine Core Facility of Shandong University for consultation and instrument availability that supported this work.\u003c/p\u003e\u003ch2\u003eData Availability Statement\u003c/h2\u003e\u003cp\u003eThe data analyzed in this study are provided within the paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJenq RR, van den Brink MR. 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Proc Natl Acad Sci U S A 2014;111:2247-52. https://doi.org/10.1073/pnas.1322269111.\u003c/li\u003e\n\u003cli\u003eLohova E, Pilmane M. Expression of MUC-2, MUC-6, NAPE-PLD, IL-6 and IL-13 in Healthy and Metaplastic Bronchial Epithelium. Diseases 2022;11:5. https://doi.org/10.3390/diseases11010005.\u003c/li\u003e\n\u003cli\u003eYao Q, Li H, Gao Y, Zheng N, Delcenserie V, Wang J. The Milk Active Ingredient, 2\u0026apos;-Fucosyllactose, Inhibits Inflammation and Promotes MUC2 Secretion in LS174T Goblet Cells In Vitro. Foods 2023;12:186. https://doi.org/10.3390/foods12010186.\u003c/li\u003e\n\u003cli\u003eOlivo-Mart\u0026iacute;nez Y, Bosch M, Badia J, Baldom\u0026agrave; L. Modulation of the Intestinal Barrier Integrity and Repair by Microbiota Extracellular Vesicles through the Differential Regulation of Trefoil Factor 3 in LS174T Goblet Cells. Nutrients 2023;15:2437. https://doi.org/10.3390/nu15112437.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Intestinal graft-versus-host disease (GVHD), allogeneic hematopoietic cell transplantation (allo-HSCT), gentamicin (GM), sodium butyrate (SB), Mucin 2 (Muc2), intestinal stem cells (ISCs)","lastPublishedDoi":"10.21203/rs.3.rs-7181845/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7181845/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIntestinal graft-versus-host disease (GVHD) is a common complication following allogeneic hematopoietic cell transplantation (allo-HSCT) and is commonly treated with antibiotics. Additionally, certain antibiotics used for gut-decontamination prophylaxis and neutropenic fever could affect GVHD-related mortality in human patients and mice.\u003c/p\u003e\u003cp\u003e\u003cb\u003eObjectives\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis study aimed to investigate the role of gentamicin and butyrate in mitigating intestinal GVHD and improving the prognosis post allo-HSCT.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStudy Design:\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAn allo-HSCT mouse model was prepared to assess the effects of gentamicin and sodium butyrate supplementation. 16S rRNA sequencing was performed for microbiota analysis using fecal samples from mice. The effects of sodium butyrate on cell proliferation and apoptosis were analyzed using LS174T human goblet cells. Protein extracts from LS174T cells and mouse intestinal epithelial cells (IECs) were analyzed using western blotting. Samples from the small intestine and colon were evaluated using hematoxylin \u0026amp; eosin (H\u0026amp;E) and periodic acid-Schiff (PAS) staining. Finally, intestine slices were evaluated for Lgr5 and Muc2 expression. A Wilcoxon rank-sum test was used for microbiome analysis and survival was analyzed using Kaplan\u0026ndash;Meier curves. The survival curves were compared using a log-rank test. Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe observed that the intestinal barrier was compromised in mice with GVHD. Gentamicin treatment after allo-HSCT significantly reduced the mortality and GVHD scores in recipient mice. Additionally, 16S rRNA gene sequencing showed that gentamicin altered the gut microbiota composition and decreased \u003cem\u003eClostridium\u003c/em\u003e levels. However, sodium butyrate supplementation in allo-HSCT mice after treatment with gentamicin significantly increased the mortality and intestinal GVHD severity, shortened the length of the colon, decreased colonic mucus layer thickness, and aggravated epithelial barrier damage in aGVHD mice. Further investigation revealed that sodium butyrate induced the apoptosis of goblet cells and inhibited the expression of Muc2 \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. In addition, sodium butyrate inhibited the proliferation of intestinal stem cells. Interestingly, concurrent supplementation of gentamicin and sodium butyrate before transplantation significantly relieved GVHD.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur results show that gentamicin alleviates GVHD by modulating butyrate associated gut microbiotas, while sodium butyrate weakens the benefit of gentamicin on GVHD by inducing goblet cell apoptosis, reducing Muc2 expression, and inhibiting intestinal stem cell proliferation. Thus, butyrate may have a double-edged effect on GVHD based on the exposure timing.\u003c/p\u003e","manuscriptTitle":"Gentamicin alleviates intestinal graft-versus-host disease by modulating butyrate-associated gut microbiota","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-31 16:17:04","doi":"10.21203/rs.3.rs-7181845/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8c78bdf3-de5d-453f-8f48-abfe33c0420c","owner":[],"postedDate":"July 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":52220948,"name":"Biological sciences/Stem cells/Haematopoietic stem cells"},{"id":52220949,"name":"Biological sciences/Cell biology/Cell death/Apoptosis"}],"tags":[],"updatedAt":"2025-09-03T15:05:57+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-31 16:17:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7181845","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7181845","identity":"rs-7181845","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}