Gut microbiota regulates AML via alternation of intestinal barrier function mediated by butyrate

The gut microbiota has been linked to many cancers, yet the role of intestinal microbes 30 in acute myeloid leukemia (AML) progression remains unclear. Here, we observed a 31 significant shift in the gut microbiota in AML patients, characterized by reduced 32 Faecalibacterium abundance. According to a murine AML model, we found that 33 intestinal microbial diversity decreased as the disease progressed. On the other side, gut 34 microbiota dysbiosis induced by antibiotic treatment accelerated AML progression with 35 a higher leukemia cell burden and shorter overall survival (OS), while fecal microbiota 36 transplantation altered this process. Metabolome analyses showed that microbiota- 37 derived butyrate concentration obviously decreased in AML patient feces, and butyrate 38 gavage postponed AML progression in a mouse model. Moreover, our study revealed 39 that intestinal barrier function is decreased in AML mice which may be related to the 40 microbiota disorder caused by AML. Lower intestinal barrier function increased the 41 bacterial-associated lipopolysaccharide (LPS) concentration in the peripheral blood of 42 AML patients or mice through enhancing intestinal permeability. Butyrate repaired the 43 intestinal barrier damage and inhibited LPS absorption in AML mice. Collectively, 44 these findings demonstrate that the gut microbiota promotes AML progression in a 45 metabolite dependent manner, and targeting the gut microbiota might provide a novel therapeutic option for AML.


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Acute myeloid leukemia (AML) is a hematological malignancy characterized by 51 excessive proliferation of immature myeloid cells. Although chemotherapy has been 52 proven to be effective against this malignancy 1 , long-term survival is modest 2 . The 53 on SCFAs in AML is still lacking. 79 The existence of the gut microbiota, along with its nutritious SCFA metabolites, can   Among the 61 individuals, all were chosen for stool 16S ribosomal RNA sequencing. 119 The study was performed with the patients' written informed consent at the beginning  All mice were six to eight weeks-old and were maintained in an SPF environment.

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Animal protocols were approved by the Animal Ethics Committee of Qilu Hospital, 154 Shandong University. 156 Fresh feces were weighed and diluted with normal saline to adjust the volume of normal 157 saline so that the fecal suspension concentration was approximately 150 mg/mL. Then, 158 the fecal suspension was filtered to remove large particles, the filtrate was collected, 159 glycerol was added to a concentration 20%, and the samples were stored at -80 ℃.

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Fecal hydration liquid was prepared from human samples for rodent gavage. For fecal microbiota transplantation (FMT), mice were randomized into the following 163 groups (n = 5 per group): AML-FMT (antibiotic-treated AML mice followed by FMT 164 with fecal hydration liquid from AML patients) and Con-FMT (antibiotic-treated AML 165 mice followed by FMT with fecal hydration liquid from heathy people). An antibiotic 166 mix (ABX) was administered by oral gavage. Briefly, the antibiotic was used as follows: 167 on days 21-7 before MLL-AF9 cell injection, mice received a daily gavage of 168 metronidazole (100 mg/kg), while the antibiotic mix (ampicillin (1 g/L), vancomycin 169 (0.5 g/L) and neomicin (1 g/L)) was added to the drinking water. Then, FMT was carried 170 out via oral gavage with a fecal suspension (150 mg/mL) in a final volume of 250 uL.

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FMT was performed daily from 14 to 35 days after the start of the antibiotic regimen.      Trimmomatic and merged by FLASH with the following criteria: (i) the reads were 253 truncated at any site receiving an average quality score <20 over a 50 bp sliding window;

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(ii) primers were exactly matched, allowing 2 nucleotide mismatches, and reads 255 containing ambiguous bases were removed; And (iii) sequences whose overlap was 256 longer than 10 bp were merged according to their overlap sequence. Operational 257 taxonomic units (OTUs) were clustered with a 97% similarity cutoff using UPARSE 258 (version 7.1 http://drive5.com/uparse/), and chimeric sequences were identified and 259 removed using UCHIME. The taxonomy of each 16S rRNA gene sequence was      (Table 1). Importantly, the abundance of Faecalibacterium and 355 number of OTUs were much higher in the favorable-risk of AML patients ( Figure 1F      However, butyrate-treated AML mice showed significantly ameliorated intestinal 477 damage as compared to the control AML mice and only mildly increased FITC-dextran 478 permeability as compared to normal mice ( Figure 6A). Next, we examined the intestinal 479 cell-cell junction integrity. We utilized transmission electron microscopy (TEM) to 480 examine the ability of butyrate to preserve cellular junctions in AML mice. As we 481 expected, significantly larger gap between intestinal epithelial cells was found in AML 482 mice (Fig. 6D, middle panel). In contrast, the integrity of the intestinal epithelial cell's 483 junction was preserved at both normal ( Figure 6D, left panel) and butyrate-treated mice 484 ( Figure 6D, right panel). Moreover, we evaluated the expression of ZO-1, claudin-1 and  Intestinal barrier is the key to protect the body from the harmful effects of gut 502 microbiota, and the impairment of its function may lead to the displacement of intestinal 503 harmful substances to the circulatory system. LPS is the main harmful product of gut 504 microbiota and the intestinal barrier is the only way for LPS to enter the blood. We used 505 ELISA to detect the LPS concentration in the plasma of peripheral blood, and the results 506 showed that the LPS concentration in the plasma of the AML group was significantly 507 higher than that in the plasma of the control group in humans ( Figure 7A). Moreover, 508 we also determined the LPS concentration in the plasma of butyrate-treated AML mice 509 and FMT-treated AML mice, and the results showed that butyrate-treatment and Con-  (Supplementary figure 5f). Our results showed that the LPS-treated 528 AML mice presented with more severe splenomegaly than the control AML mice, and 529 the results showed that the percentage of GFP + leukemia cells in the peripheral blood, 530 spleen and bone marrow of LPS-treated AML mice was higher than that in the 531 peripheral blood, spleen and bone marrow of control AML mice ( Figure 7H and 7I).  which accelerates AML cell proliferation and disease progression. We believe that the 552 discovery of this mechanism may help discover new targets for AML treatment. 553 We found that there are significant alterations in gut microbiota diversity in AML 554 and that this change is related to the risk stratification of the disease. This indicated that 555 while AML causes the imbalance of gut microbiota, the disordered microbiota also 556 affects the progress of the disease. To better serve clinical treatment, it is particularly 557 important to study the mechanism of the flora in the progression of AML. Blocking the 558 positive feedback between AML and bacterial dysbiosis in mechanism will help to solve  clinical studies. Therefore, we will gradually solve these problems and conduct deep 618 insight to the relationship between AML and gut microbiota.

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In conclusion, our results suggest that the imbalance in the gut microbiota as a Some or all data, models, or code generated or used during the study are available from 635 the corresponding author by request.

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The authors declare no competing financial interests.      and healthy controls (n=17). (B) Heatmap of the relative abundances of the top 26 most abundant metabolites which signi cantly changed in AML group. The color bar indicates z score, which represents the relative abundance. Z score < 0 (>0) meant the relative abundance was lower (higher) than the mean. (C) The concentrations of propionic acid, butyric acid, and acetic acid in fecal samples of AML patients (n=22) and controls (n=22) were determined by GC-MS. (D) Heatmap of spearman correlation analysis between the gut microbiota and the metabolite. (E) The functional abundance distribution histogram of samples from AML patients and healthy controls in the COG database using PICRUSt software (the top 35 is selected by the maximum sorting method). (F) The signi cant differences in metagenomics functions in AML patients compared to that in healthy controls (corrected P < 0.05 and con dence intervals = 95%).

Figure 6
Butyrate reverses intestinal barrier damage in mice with AML(A) The concentration of FITC-dextran in the peripheral blood after FITC-dextran gavage for 6 hours. Data represent the mean SEM (n = 5). (B) The mRNA expression levels of tight junction protein components claudin-1, claudin-2 and ZO-1 in intestinal epithelial cells of AML, control and butyrate-treatment mice (n=3). (C) The protein levels of claudin-1, claudin-2 and ZO-1 in intestinal epithelial cells were determined by Western blot. GAPDH was used as the control (n=3). (D). Transmission electron microscopy (TEM) of intestines, isolated from normal, AML, and butyrate-treated AML mice for duration of experiment; arrows indicate cell-cell interface. (E) Immuno uorescence analysis of small intestine tissue from normal, AML and butyrate-treated mic. Cells were xed and stained with a rabbit polyclonal antibody. APC (red) goat anti-rabbit IgG was used as a secondary antibody. Immuno uorescence indicated the expression quantity and localization of claudin-1, claudin-2 and ZO-1.