Multi-omics Reveal the Enabling Factors of Aplastic Anaemia

Background: Aplastic anemia is a kind of anemia caused by bone marrow failure due to autoimmune abnormalities. Numerous studies have shown that autoimmunity is regulated by intestinal microora, and that most intestinal microora regulates immunity through short chain fatty acids. At the same time, almost all patients with aplastic anemia will have bone marrow adipose tissue, and the process of bone marrow adipose tissue is regulated by medium and long chain fatty acids. Our previous studies have found that the intestinal ora of aplastic anemia patients is different from that of normal people. Therefore, this study aims to conduct a comprehensive detection of the intestinal ora and fatty acid metabolism in patients with aplastic anemia and to study their correlation. Methods: Fatty acid compositions in the peripheral plasma and bone marrow supernatants of 12 newly diagnosed aplastic anaemia patients and 10 normal controls were comprehensively analysed by CG-MS. Gene sequencing of faecal samples from both groups was also performed with macrogene sequencing technology. Results: Based on the metagenomic sequencing technology, we analyzed the difference of intestinal ora between group AA and group NC. We found that the main difference between the two groups existed in the bacterial community, and the abundance of most microbial species showed a downward trend. Based on the CG-MS lipid detection technique, we found that there were differences in lipid metabolism between the AA group and the NC group, whether short chain fatty acids or medium and long chain fatty acids. 8. The most attractive ndings are interrelationships between the Citrobacter spp (cid:0) stearic acid (c18:0), isobutyric acid, and lysine degradation and betaine biosynthesis pathway and the interrelationship between catenibacterium_mitsuokai species at Catenibacterium, cis-7,10,13,16-docosatetraenoic acid (c22:4), ubiquinone and other terpenoid quinones pathway. We also found the intercorrelation between the abundance of Enhydrobacter genus and Enhydrobacter_aerosaccus species, cis-13,16-docosadienoic acid in peripheral plasma, cis-7,10,13,16-docosatetraenoic acid in bone marrow supernatant, tyrosine and tryptophan biosynthesis pathways. Conclusions: Our results show that Citrobacter infection may be a driving factor for aplastic anaemia, identifying a potential role for stearic acid in the immunopathogenesis of aplastic anaemia. Our study also demonstrates the potential role of 22-carbon long-chain polyunsaturated fatty acids, which may not only be involved in the metabolism of broblasts in bone marrow but also inuence the formation of the bone marrow microenvironment, in aplastic anaemia. the change in acid in anaemia The correlation between lipid in short-chain acid production and metabolism 7 metabolizes acid 8 . Bacteroides vulgatus Bacteroides thetaiotaomicron through the pathway. anaerobes, pathway regulation and through 10 deacetylases the G study, we found there are higher of and citroniae but lower of copri, bromii fatty acid levels in supernatants and in normal differences in the levels of microorganisms, metabolic pathways and metabolites between the two groups analysed by metagenomic sequencing. aim study to elucidate the changes in fatty acid metabolism in patients with aplastic anaemia and to determine how they might be related to gut microbes. The abundance of the uncultured_ actinobacterium_ HF0500_ 35g12 species in the bacterial kingdom was positively correlated with the valeric acid content, which is negatively correlated with the microbial abundances of the Candidatus_ Zambryskibacteria phylum, the genus Cruoricaptor, the Cruoricaptor_ ignavus species, the genus drancourtella, the Bidobacterium_ Saeculare species, the Drancourtella_ Massiliensis species, the Pseudomonas_ Syringae species, the Sulfurospirillum_ Halorespirans species and the Staphylococcus_phage_phiSA_BS2 species in the viral kingdom. There as a positive correlation between isobutyric acid and the microbial abundances of the Sphingobacteriia class, Sphingobacteriales order, Sphingobacteriaceae family, Afella , and Enterococcus_sp._4E1_DIV0656 species. The abundance of microbes in the bacterial kingdom that showed a negative correlation with the isobutyrate content included those in the Absiella genus, Absiella_dolichum species, Cruoricaptor genus, Cruoricaptor_ignavus species , Mycoavidus genus , Mycoavidus_cysteinexigens species and 8 other microbial species. The abundance of Catenibacterium genus and Catenibacterium_mitsuokai speices in the bacterial kingdom was positively correlated with the content of Docosatetraenoate, which is negatively correlated with the microbial abundance of the Enhydrobacter genus, Enhydrobacter_aerosaccus species, Smithella genus, Bidobacterium_sp._MSTE12 genus and 7 other species. The content of isovaleric acid showed a negative correlation with the abundances of There were positive correlations between the abundances of the Citrobacter_rodentium species and Dysgonomonas_sp._BGC7 species in the bacterial kingdom and the content of stearate. The abundances of the Sphingobacteriia class, Sphingobacteriaceae order, Sphingobacteriales family, and Sphingobacterium_gobiense species were proportional to the contents of 10-heptadecenoate, petroselaidate, nervonoate and trans 11-eicosenoate. The abundances of the rst three were also proportional to the contents of linoelaidate, and the latter was related to the contents of eicosenoate, rucate, brassidate and docosadienoate. There was a positive correlation between the abundances of the Catenibacterium genus, Catenibacterium_mitsuokai and Ralstonia_pickettii species and the content of docosatetraenoate. The abundance of Enterobacter_sp._50793107, Lactobacillus_equigenerosi, and Lactobacillus_pobuzihii species and the content of nervonoate showed the same correlation. The abundance of the Enterococcus_phage_EFDG1 species in the viral kingdom was also positively correlated with the contents of transvaccenate, linoelaidate, 7-transnonadecenoate and nervonoate. In the AA group(cid:0)there was a negative correlation between the abundance of microbes in the faeces and fatty acid content in the plasma, which showed a correlation between the abundances of the Desulfosarcina genus, species, Succinispira Succinispira_mobiliz species and 9 other species in the bacterial kingdom and the content of caproate. There was also a correlation between the content of docosadienoate and the abundances of the Enhydrobacter genus, Enhydrobacter_aerosaccus species, Mycoaviduss genus, Mycoavidus_cysteinexigens species and 5 other species in the bacterial kingdom. Therefore, there was a correlation between the content of isobutyric acid and the abundances of the Citrobacter genus, Citrobacter pasteurii species and Citrobacter sp. MGH106 species. The species abundances of Chloroexi_ bacterium_ GWB2_ 49_ 20, Kocuria_ kristinae, and Lactobacillus_ Equigenerosi were negatively correlated with the content of petroselaidate, the latter two with the content of transvaccenate and the latter with the content of linoelaidate. The species abundances of Chloroexi_bacterium_GWB2_49_20, Enterobacter_sp._50793107, Lactobacillus_equigenerosi, and Serratia_sp._TEL were negatively correlated with the contents of trans 11-eicosenoate, linoelaidate, brassidate, and erucate, the former 1 with the content of linoelaidate. There were also negative correlations between microbial abundance and the contents of various fatty acids, which showed a correlation between the abundance of the Lactobacillus_equigenerosi species and the contents of palmitelaidate, 10-heptadecenoate, 10-transsheptadecenoate, petroselaidate, transvaccenate, linoelaidate, 7-transnonadecenoate, eicosenoate, trans 11-eicosenoate, erucate, brassidate, docosadienoate, docosatetraenoate, nervonoate, and caproate. There was also a correlation between the abundances of the Cruoricaptor genus and Cruoricaptor_ignavus species and the contents of palmitelaidate, transsheptadecenoate, stearate, and caproate; a correlation between the abundance of the Lactobacillus_pobuzihii species the contents of 7-transnonadecenoate, nervonoate, and isobutyric acid; and a correlation between the abundance of Serratia_sp._TEL and the contents of 11-eicosenoate, trans 11-eicosenoate, erucate, brassidate, docosadienoate, and isobutyric acid. A negative correlation was also found between the species abundance of Enterobacter_sp._50793107 and the contents of trans 11-eicosenoate, erucate, brassidate, nervonoate and isobutyric acid. There was a negative correlation between the genus abundance of Lentzea and the contents of linoelaidate, 7-transnonadecenoate, and 11-eicosenoate; a negative correlation between the species abundance of Chloroexi_bacterium_GWB2_49_20C and the contents of petroselaidate, linoelaidate, and 11-eicosenoate; a negative correlation between the species abundance of Bidobacterium_saeculare and the contents of docosatetraenoate and caproate; a negative correlation between the species abundance of Gulbenkiania_indica and the contents of caproate and isobutyric acid; a negative correlation between the species abundance of Kocuria_kristinae and the contents of petroselaidate and transvaccenate; and a negative correlation between the species abundance of Xenorhabdus_innexi and the contents of docosadienoate and isobutyric acid. There was also a negative correlation between microbial abundance in the eukaryotic kingdom and fatty acid content in the plasma, which showed a negative correlation between the genus abundance of Arabidopsis and the content of 11-eicosenoate; and negative correlations between the species abundance of Candida_maltosa and the content of stearate(cid:0) the genus abundance of Cucurbita and the contents of 10-heptadecenoate, petroselaidate, transvaccenate, linoelaidate, docosatetraenoate and the species Correlations between microbial abundance alterations and KEGG signalling pathways in the AA group The abundances of the Mycoavidus genus, Mycoavidus_cysteinexigens species, Achromobacter_sp._ATCC35328 species and Xenorhabdus_innexi species in the bacterial kingdom were negatively correlated with type I diabetes mellitus and positively correlated with the abundances of the Geobacillus_thermodenitricans species and Kocuria_kristinae species in the bacterial kingdom and the abundance of the Methanobacterium_congolense species in the archaea kingdom. Ubiquinone and other terpenoid-quinone biosynthesis was positively correlated with the abundances of the Catenibacterium genus, Catenibacterium_mitsuokai and Ralstonia_pickettii species and negatively correlated with the abundances of the Desulfosarcina genus, Bidobacterium_saeculare, Desulfosarcina_cetonica and Megasphaera_massiliensis species in the bacterial kingdom. The abundance of Planctomycetes_bacterium_TMED75 in the bacterial kingdom and Sphingobacterium_gobiense species was positively correlated with N-glycan biosynthesis, which was also negatively correlated with the abundances of Enhydrobacter, the Sulfuricurvum genus, Enhydrobacter_aerosaccus and 6 other species in the bacterial kingdom and the Nematoda phylum, Enoplea class, Trichinellida order, Trichurida family, Arabidopsis, and Trichuris genus in the eukaryotic kingdom. A positive correlation was also found between the abundances of Enhydrobacter and the Sulfuricurvum genera, Enhydrobacter aerosaccus, Enterobacter sp. CC120223-11 and 5 other species in the bacterial kingdom and the phenylalanine, tyrosine and tryptophan biosynthesis promote cell metabolism enhance the memory potential of activated CD8+ T cells 33 . Patients with aplastic anaemia show downregulation of Treg cells and upregulation of Th17 cells 21,34 Previous aplastic anaemia to Citrobacter, our Citrobacter abundance was downregulated; the immune changes Citrobacter consistent, Citrobacter infection be the driving factor of aplastic anaemia. Citrobacter in in with aplastic anaemia gut microenvironment of aplastic anaemia. NOX1 intestinal epithelium Citrobacter, epithelial cells 36 . Lysine fatty acid Citrobacter, of energy lysine acetyl-coA. biosynthesis betaine an independent biosynthesis process that focuses on plants. Betaine the end product of choline oxidation in the body. Methionine in the biosynthesis of the gut microenvironment, and its role in the formation of the gut microenvironment correlated with the contents of eicosenoate, docosatetraenoate and 10-transsheptadecenoate in the plasma. In addition, the number of Mycoavidus species 37 in the nematode-related bacterial community was decreased and negatively correlated with docosadienoate and docosatetraenoate. Previous studies have shown that nematodes are primarily associated with unsaturated fatty acids, consistent with our ndings. The nematode Caenorhabditis elegans stores unsaturated fatty acids in droplets in its subcutaneous and intestinal cells 38 . Additionally, 18-carbon PUFAs affect basal innate immune function, the p38 MAP kinase pathway and the transcription of fat-3-regulated genes through the nematode Caenorhabditis elegans to modulate intestinal infection and the expression of genes involved in the stress response, thereby affecting the organism's ability to defend against bacterial infection 39 . Helminth-induced chronic infection can increase anti-inammatory cytokine secretion and suppress Treg cell activity and increase short-chain fatty acid (SCFA) production 40 . The loss of C. elegans fat-1 expression inhibits lipid droplet formation and selectively disrupts peroxisomes and apical endosomes. Lipid analysis in fat-1-decient nematodes revealed a signicant reduction in heptadecaenoic acid, while other major FAs were unaffected 41 . Caenorhabditis elegans intestinal colonization causes protein homeostasis disruption, which can be improved by butyrate 42 . Two peptides, ACAN1 and NAK1, derived from the nematode phylum, have immunomodulatory functions and can inhibit the proliferation of CD4+ T cells and the production of IL-2 and TNF 43 . Therefore, we can conclude that the nematode phylum acts as a mediator in the human body. On the one hand, the nematode phylum can directly participate in the synthesis and storage of fatty acids; on the other hand, it serves as the link between fatty acid metabolism and immune regulation. The abundance of nematode phylum microbes in the faeces of patients with aplastic anaemia was decreased, as were the levels of several fatty acids that showed bone marrow microenvironment. Therefore, the downregulation of docosatetraenoate may be involved in both immune response enhancement and the process of bone destruction and marrow steatosis in aplastic anaemia patients. Although no specic studies have been performed, we believe that unsaturated fatty acids have immense potential in the study of the pathogenesis of aplastic anaemia. The abundance of Enhydrobacter species was increased in the AA group and was positively correlated with docosadienoatein in the plasma and docosatetraenoate in bone marrow supernatants. Moreover, the abundance of Enhydrobacter aerosaccus species in the Enhydrobacter genus had a positive correlation with phenylalanine, tyrosine and tryptophan biosynthesis but a negative correlation with N-glycan biosynthesis. Previous studies have shown that the Enhydrobacter genus is also abundant in gastrointestinal metaplasia and associated with carcinoma of the head of pancreas (CHP) 59,60 . In in vitro synthesis experiments, an aminotransferase derived from the bacterium Enhydrobacter catalysed the synthesis of L-phenylalanine by using 3-GABA as an amino donor 61 , as conrmed by our results in patients with aplastic anaemia. Studies have shown that L-phenylalanine metabolism is associated with fatty acid synthesis 62 . Il-10 has been shown to directly inhibit the function of CD8+ T cells by increasing the number of n-glycan branches to decrease antigenic sensitivity 63 . Additionally, CD8+ cell function was upregulated in patients with aplastic anaemia, and our study showed that the abundance of species in the n-branching glycan biosynthesis pathway was decreased in the AA group, consistent with previous ndings. N-glycan biosynthesis is also involved in the migration of bone marrow-derived mesenchymal stem cells 64 . Downregulation of N-glycan biosynthesis may inhibit the migration of MSCs outside the bone marrow in aplastic anaemia patients, thus prompting the MSC source to differentiate into adipocytes directly inside the bone marrow and complete the marrow lipidation process. Although there is no research to prove this hypothesis, ubiquinone and other terpenoid quinones pathway suggests that Catenibacterium negatively regulate cis-7,10,13,16-docosatetraenoic acid by biosynthesis of ubiquinone and other terpenoid quinones.The intercorrelation between the abundance of Enhydrobacter genus and Enhydrobacter_aerosaccus species, cis-13,16-docosadienoic acid in peripheral plasma, cis-7,10,13,16-docosatetraenoic acid in bone marrow supernatant, tyrosine and tryptophan biosynthesis pathway suggests that the genus aquabacterium is associated with two long-chain fatty acids through tyrosine and tryptophan biosynthetic pathway modulation in the disease group.From the point of view of immunity, citreobacilli plays an important role in the pathogenesis of aplastic anemia. We speculate that it may act as a driving factor for aplastic anemia.From the perspective of lipid metabolism, docosanpoly unsaturated fatty acids are of signicant relevance to the microbiota in the patient group, and we speculate that docosanpoly unsaturated fatty acids may serve as mediators of microbiota regulated medium - and long-chain fatty acid metabolism. Overall, our study not only sheds light on the possibility that Citrobacter infection may function as an aplastic anaemia agent but also revealed the potential role of stearate in the immunopathogenesis of aplastic anaemia. In addition, our study demonstrates the potential roles of 22 unsaturated fatty acids in aplastic anaemia. Unsaturated fats are not only involved in the metabolism of broblasts in the bone marrow, thus affecting the formation of the bone marrow microenvironment and participation in the process of bone marrow steatosis, but may also be involved in the regulation of immune cells in the peripheral blood to participate in disease progression in aplastic anaemia patients. Our study provides insights into the pathogenesis of aplastic anaemia and, more importantly, sheds light on the aetiology of aplastic anaemia.

treatment. The admission criteria were as follows: patients who did not take antibiotics, probiotics or other haematologic diseases within 3 months prior to admission. Bone marrow supernatant, peripheral blood plasma and stool samples were also collected from ten healthy volunteers. This study was approved by the Ethics Committee of Tianjin Medical University General Hospital, and informed written consent was obtained from all patients or their parents according to the Declaration of Helsinki.

DNA extraction
Total DNA was extracted from the faeces with a QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany). DNA completeness and purity were assessed by running the samples on 1.2% agarose gels. The concentration of DNA was determined on a Qubit uorometer.

Metagenomic library preparation and sequencing
Extracted DNA was sheared with a Covaris M220 (Covaris, Woburn, MA, USA) programmed to generate 300-bp fragments. The sequencing libraries were constructed with a NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (NEB, USA). The products were puri ed using AgarosAgencourt AMPure XP (Beckman, USA) and quanti ed using a GenNextTM NGS Library Quanti cation Kit (Toyobo, Japan). The libraries were sequenced using Illumina NovaSeq 6000 and 150-bp paired-end technology at TinyGen Bio-Tech (Shanghai) Co., Ltd.
The medium/long chain fatty acids were synthesized as follows: 52 fatty acid methyl ester solutions were mixed with positive hexane to make 10 standard concentration gradients (1 sg/mL, 5 sg/mL, 10 sg/mL, 25 sg/mL, 50 sg/mL, 100 μg/mL, 250 μg/mL, 500 μg/mL, 1000 μg/mL, and 2000 μg/mL). The concentration is the total concentration of each component (51 standard fatty acid methyl esters). The stock solution was stored at -80 °C and is now available as a standard solution.

Sample pretreatment
The short-chain fatty acids were synthesized as follows: the sample was transferred to a 2 mL centrifuge tube; 50 μL of 15 % phosphoric acid, 10 μL of 75 μg/mL of the internal label (iso-acid) solution and 140 μL of ether were added; the mixture was subjected to vortex oscillation for 1 min and centrifugation for 10 min at 4 °C (12000 rpm) for liquidation detection.
The medium/long-chain fatty acids were synthesized as follows: the appropriate amount of sample was transferred to a 15 mL centrifuge tube; 2 mL 1% methanol sulfate solution was added and mixed for 1 min; the mixture was esteri ed for 30 min in a water bath at 80 °C; the solution was removed and cooled; 1 mL positive hexane extraction was added, the mixture was washed with 5 mL H 2 O (4 °C), centrifuged at 12000 rpm at 4 °C for 10 min, placed in an oscillating mixer for 30 s, centrifuged at 12000 rpm for 5 min, and subjected to precise absorption of 300 sL of upper liquid in a 2 mL centrifuge tube; 15 sl 500 ppm salicylic acid was added as the internal label; and the mixture was placed in an oscillating mixer for 10 sand subjected to precise absorption of 250 sL (added to the test bottle).

GC-MS detection
The chromatographic conditions were as follows: an Agilent HP-INNOWAX capillary column (30 m x 0.25 mm ID x 0.25 μm) was used; the inlet temperature was 250 °C, the ion source temperature was 230 °C, and the transmission line temperature was 250 °C for the four poles; the bar temperature was 150 °C.
The program began at a starting temperature of 90 °C, was increased to 120 °C, was increased to 150 °C at a rate of 5 °C/min, and then was increased to 250 °C at a rate of 25 °C/min. The helium carrier had a carrier ow rate of 1.0 mL/min. MS conditions were as follows: electron blast ionization (EI) source, SIM scan mode, and electron energy: 70 eV.
Data analysis: The raw fastq les were demultiplexed based on the index. The raw, paired-end reads were trimmed and quality controlled using Trimmomatic

Results
Bone marrow fatty acid metabolism in patients with aplastic anaemia: Changes in supernatant fatty acids in the bone marrow of patients with aplastic anaemia: Orthogonal least squares discriminant analysis (OPLS-DA) were used to analyse the major fatty acids in the AA and NC groups (Tab 1, Fig 1). In the bone marrow supernatant, there was a signi cant difference in the short-chain fatty acid compositions, while there were no signi cant differences in the mediumand long-chain fatty acid compositions. There were signi cant differences in 3 of the 7 short-chain fatty acids tested. Among the 52 types of medium-and long-chain fatty acids detected, only one showed a signi cant difference. The major difference between patients with aplastic anaemia and normal controls was short-chain fatty acid metabolism. OPLS-DA were used to analyse the major fatty acids in the AA and NC groups (Tab 3 and Figs 3). In the peripheral plasma, there were signi cant differences in medium-and long-chain fatty acid compositions, while there were no signi cant differences in short-chain fatty acid copositions. Among the 52 types of medium-and long-chain fatty acids detected, there were signi cant differences in the contents of 16. Only one of the seven short-chain fatty acids evaluated showed a signi cant difference. The major difference between aplastic anaemia patients and normal controls is short-chain fatty acid metabolism. Metagenomic sequencing showed that the microbial abundance in the faecal micro ora of the AA group was changed at six levels: phylum, class, order, family, genus and species.
Species abundance changes in the AA group β diversity analysis showed no signi cant difference in microbial abundance between the AA and NC groups, but the tendency of grouping is obvious. (Fig 5).
Phylum level: According to the α diversity analysis, there was no difference in coverage, sobs, or the Chao, ACE or Simpson index between the AA and NC groups, but the difference in the Shannon index, which was a parameter of colony diversity, was signi cant (p = 0.010) (Fig 6). The analysis of group differences con rmed that the overall microbial abundances of bacteria and eukaryotes at the phylum level were decreased in the AA group. The microbial abundances of Candidatus_ Zambryskibacteria, Candidatus_ Falkowbacteria, Candidatus_ Yanofskybacteria and Nematoda were increased in the AA group (Tab 5, Fig 7). Class level In the AA group, there were no signi cant differences in α diversity (Fig 8). The results showed no signi cant changes in the sequencing depth, species quantity, distribution uniformity, colony abundance, diversity, or microbial diversity in the AA group. The differential analysis showed that the abundances of Sphingobacteriia (bacteria), Oomycetes and Enoplea (eukaryotes) were decreased in the AA group (Tab 6, Fig 9). Family level: At the family level, the α diversity analysis also showed no signi cant difference between the two (Fig 12). The microbial abundances of Peronosporaceae, the Trichuridae family, alcaligenaceae in the eukaryotic kingdom, Rhodobacteraceae, Ruminococcaceae, Sphingobacteriaceae and Yersiniaceae family in the bacterial kingdom were decreased in the AA group. The microbial abundance of the Enterocytozoonidae family of the eukaryotic kingdom was elevated in the AA group (Tab 8, Fig 13). Species level: Similarly, there were no signi cant differences observed in the α diversity analysis at the species level (Fig 16). However, in the AA group, the abundances of 84 species were increased, including 2 species in the viral kingdom (Staphylococcus_phage_phiSA_BS2, Streptococcus_phage_P0092), 2 species in the eukaryotic kingdom (Candida_maltosa, Enterocytozoon_bieneusi), 2 species in the archaea kingdom (Methanobacterium_congolense, Compared with the normal control group, there were signi cant differences in the abundances of genes involved in 12 signalling pathways in the AA group. Among them, the abundances of genes involved in 2 metabolism-related pathways were upregulated (penicillin and cephalosporin biosynthesis and tyrosine and tryptophan biosynthesis). In addition, the abundances of genes involved in eight metabolism-related pathways, including nonribosomal peptides, betalain biosynthesis, and lysine degradation, showed the opposite trend. We also observed the downregulation of genes involved in pathways related to type I diabetes mellitus in humans (Fig 18, Tab 11).   There was a positive correlation between isovaleric acid, which was elevated in the bone marrow supernatant of individuals in the AA group, and the abundance of genes involved in the type I diabetes mellitus pathway. The lysine degradation pathway was positively correlated with the content of isobutyric acid, which was upregulated in both the peripheral plasma and bone marrow supernatant from the AA group but negatively correlated with stearate, which was downregulated in the peripheral plasma. The downregulated levels of docosadioate, 11-eicosenoate, stearate and docosatetraenoate in the peripheral plasma in the AA group were negatively associated with phenylalanine, tyrosine, and tryptophan biosynthesis, N-glycan biosynthesis, and betaine biosynthesis, respectively. The same correlation was also found between the content of docosadienoate and N-glycan biosynthesis (Tab 14, Fig 21). in the bacterial kingdom were negatively correlated with type I diabetes mellitus and positively correlated with the abundances of the Geobacillus_thermodenitri cans species and Kocuria_kristinae species in the bacterial kingdom and the abundance of the Methanobacterium_congolense species in the archaea kingdom. Ubiquinone and other terpenoid-quinone biosynthesis was positively correlated with the abundances of the Catenibacterium genus, Catenibacterium_mitsuokai and Ralstonia_pickettii species and negatively correlated with the abundances of the Desulfosarcina genus, Bi dobacterium_saeculare, Desulfosarcina_cetonica and Megasphaera_massiliensis species in the bacterial kingdom. The abundance of Planctomycetes_bacterium_TMED75 in the bacterial kingdom and Sphingobacterium_gobiense species was positively correlated with N-glycan biosynthesis, which was also negatively correlated with the abundances of Enhydrobacter, the Sulfuricurvum genus, Enhydrobacter_aerosaccus and 6 other species in the bacterial kingdom and the Nematoda phylum, Enoplea class, Trichinellida order, Trichurida family, Arabidopsis, and Trichuris genus in the eukaryotic kingdom. A positive correlation was also found between the abundances of Enhydrobacter and the Sulfuricurvum genera, Enhydrobacter aerosaccus, Enterobacter sp. CC120223-11 and 5 other species in the bacterial kingdom and the phenylalanine, tyrosine and tryptophan biosynthesis signalling pathways, as well as the abundances of the Citrobacter genus, Citrobacter_pasteurii, Citrobacter_rodentium, Citrobacter_sp._MGH106 and 3 other species in the bacterial kingdom and the lysine degradation signalling pathway. The correlation between the abundances of the Cucurbita genus and Cucurbita moschata species in eukaryotes and the phenylalanine, tyrosine and tryptophan biosynthesis signalling pathways was negative. A positive correlation was also found in the betalain biosynthesis signalling pathway and the abundances of the Citrobacter genus, Citrobacter_pasteurii, Citrobacter_rodentium, Citrobacter_sp._MGH106 and 4 other species (Tab 15, Fig 22). Tab 15.the correlation between the microbial abundance and the KEGG pathway abundance.

Discussion
Aplastic anaemia is de ned as pancytopenia with hypocellular bone marrow in the absence of abnormal in ltrate and no increase in reticulin. To diagnose aplastic anaemia, there must be at least two of the following criteria: (i) haemoglobin <100 g/l, (ii) platelet count <50 × 10 9 /l, and (iii) neutrophil count <1·5 × 10 9 /l 13 . Most cases of aplastic anaemia are characterized as a bone marrow failure disorder caused by immune cells attacking their own haematopoietic stem cells, and the main effector cells identi ed are CD8+ T cells (also called cytotoxic T cells, CTLs) that express interferon γ 14 . However, the mechanism of CD8+ T cell activation is not clear. In addition to cytotoxic T cells, AA patients have immune disorders caused by a large number of other immune molecules. Th1 and Th2 cells are upregulated in aplastic anaemia patients, while Tregs (regulatory T cells) are downregulated in both quantity and function, triggering an autoimmune escape mechanism that drives disease progression 15,16 . The decrease in the number of Tregs may be due to infection-  21 . In addition, the pathogenesis of aplastic anaemia has been proven to be related to the bone marrow haematopoiesis microenvironment, in which AA patients contain fewer endosteal, vascular, and perivascular cells than healthy controls 22 . Moreover, MSCs from AA patients exhibit weaker proliferative capacity and modulate the bone marrow immune microenvironment by secreting multiple cytokines 23 while also having a stronger tendency to differentiate into adipocytes 24 , which are increased in the haematopoietic microenvironment and lead to the inhibition of HSC differentiation and development 25 . In conclusion, aplastic anaemia is an immune disease with a complicated pathogenesis; however, the factors involved in the onset of aplastic anaemia are unclear. Our study revealed the role of lipid metabolism in the pathogenesis of aplastic anaemia through a large number of correlation analyses between macrogenes and lipid metabonomics and showed that Citrobacter rodentium may act as a motile factor in aplastic anaemia.
The species abundances of Citrobacter pasteurii, Citrobacter sp. MGH, and Citrobacter rodentium in the Citrobacter genus in the bacterial kingdom were downregulated in the AA group and had a positive correlation and negative correlation with the contents of stearate and isobutyric acid, respectively (Fig 23). While lysine degradation and the betalain biosynthesis pathway were positively correlated with Citrobacter species abundance, the former had a positive correlation and negative correlation with the contents of stearate and isobutyric acid in the plasma, respectively. Furthermore, the isobutyric acid content increased while the stearic acid (C18:0) content was decreased in the AA group. Citrobacter has been identi ed as a microorganism that can cause intestinal in ammation; thus, Citrobacter rodentium is inextricably linked to human immune cells, especially dendritic cells and CD4+ T cells 26 . Citrobacter koseri, a subspecies of Citrobacter, stimulates dendritic cells to induce IL-33 through massive ATP production 27 . Plasmacytoid dendritic cells (pDCs) are the main dendritic cells upregulated upon stimulation in Citrobacter rodentium infection 28 . Upregulation of the mDC/pDC ratio in aplastic anaemia patients has also been validated, whereby the dendritic cell response due to the downregulation of Citrobacter abundance and immune cell changes in the AA group in the barrier coincide 29 . The involvement of short-chain fatty acids can enhance the induction of Th1 and Th17 cells during Citrobacter rodentium infection in mice 30 . In our study, the isobutyric acid content was upregulated in the AA group and negatively correlated with Citrobacter abundance, coinciding with the upregulation of Th17 cells in AA patients. Downregulation of murine Treg cells enhances Citrobacter rodentium susceptibility, whereas infection with Citrobacter rodentium in the intestinal cells of mice lacking Treg cells elicits a strong Th17 response 31 . Moreover, the branched palmitic acid esters of hydroxy stearic acids (PAHSAs), which induce colonic T cell activation and inhibit proin ammatory cytokine and chemokine expression in mice, attenuate dendritic cell activation and subsequent T cell proliferation and Th1 polarization in vitro 32 . In our study, the stearic acid content and Citrobacter level were downregulated at the same time, and both were positively correlated, in accordance with the conclusion of the above study. Taken together, these results suggest that the unusual presentation of stearic and isobutyric acids in patients with aplastic anaemia is most likely related to its immunopathogenic mechanisms. Although little research has pointed out the correlation between isobutyric acid and human immunity, studies on short-chain fatty acids (SCFAs) derived from microbes are not rare. Studies have shown that SCFAs promote cell metabolism and enhance the memory potential of activated CD8+ T cells 33 . Patients with aplastic anaemia show downregulation of Treg cells and upregulation of Th17 cells 21,34 . Previous studies have suggested that aplastic anaemia patients are susceptible to Citrobacter, but in our study, Citrobacter abundance was downregulated; however, the immune changes induced by Citrobacter infection were consistent, which suggested that Citrobacter infection might be the driving factor of aplastic anaemia. Citrobacter infection may also be involved in energy metabolism in patients with aplastic anaemia and thus in the gut microenvironment of aplastic anaemia. Citrobacter controls the production of H2O2 by the NADPH oxidase NOX1 to provide growth conditions for other aerobic bacteria early after infection 35 . The intestinal epithelium absorption of acylcarnitine is impaired by Citrobacter, and then the aerobic metabolism of intestinal epithelial cells is affected 36 . Lysine degradation, which has the same fatty acid correlation with Citrobacter, is a process of energy metabolism that refers to the conversion of lysine to acetyl-coA. Another closely related biosynthesis pathway of betaine is an independent biosynthesis process that focuses on plants. Betaine is the end product of choline oxidation in the human body. Methionine is involved in the biosynthesis of the gut microenvironment, and its role in the formation of the gut microenvironment is unknown.
In conclusion, it is reasonable to hypothesize that Citrobacter and its related fatty acids mainly in uence the immunopathogenesis of aplastic anaemia and greatly in uence the formation of the intestinal microenvironment; therefore, Citrobacter is very likely to be a pathogenic factor of aplastic anaemia and has great potential and research value in aplastic anaemia.
We also noted a decrease in the abundance of Nematoda in eukaryotes identi ed in aplastic anaemia patients and a decrease in the abundances of Enoplea, Trichinellida, Trichuridae, Enterocytozoon and Trichuris within this phylum, which were negatively correlated with the contents of eicosenoate, docosatetraenoate and 10-transsheptadecenoate in the plasma. In addition, the number of Mycoavidus species 37  protein homeostasis disruption, which can be improved by butyrate 42 . Two peptides, ACAN1 and NAK1, derived from the nematode phylum, have immunomodulatory functions and can inhibit the proliferation of CD4+ T cells and the production of IL-2 and TNF 43 . Therefore, we can conclude that the nematode phylum acts as a mediator in the human body. On the one hand, the nematode phylum can directly participate in the synthesis and storage of fatty acids; on the other hand, it serves as the link between fatty acid metabolism and immune regulation. The abundance of nematode phylum microbes in the faeces of patients with aplastic anaemia was decreased, as were the levels of several fatty acids that showed negative correlations with it, although there was a negative correlation between the two. There should be a complex interaction mechanism among them. First, the decline in nematode microbiome levels may be due to an increased proportion of T cells in the peripheral blood of immunocompromised patients with aplastic anaemia. Inhibition of the nematode phyla decreased the colonization of long-chain fatty acids in the intestinal epithelium and increased the contents of long-chain fatty acids in the peripheral blood, such that there was a negative correlation between them. We hypothesize that the decrease in fatty acid contents in the peripheral blood may be due to other microorganisms or microbial associations. Docosadienoate was negatively correlated with N-glycan biosynthesis, and N-glycan biosynthesis was negatively correlated with nematode-related microorganisms. N-glycan biosynthesis is associated with weight loss 44 ; in other words, there may be a negative correlation with the biosynthesis of fatty acids, which can con rm the negative change in the fatty acids described above and nematode microbial abundance.
The decrease in microbial abundance of the Catenibacterium genus, including Catenibacterium mitsuokaiin species, in the bacterial kingdom was also noTab. In the AA group, there was a positive correlation between the docosatetraenoate content in both the plasma and supernatant, and the docosatetraenoate content was also positively correlated with downregulated ubiquinone and other terpenoid-quinone biosynthesis pathways. Previous studies have shown that the microbial abundance of this genus is similarly downregulated in in ammatory bowel disease patients 45 and in aplastic anaemia patients in our study, suggesting that it may have some relevance for immunity. In a study of fructose fermentation by faecal microorganisms in vitro, the abundance of Catenibacterium increased with increasing SCFA contents, especially with the increase in butyric acid 46,47 . A study of the gut ora in obese patients found an elevated microbial abundance of Catenibacterium and high plasma levels of short-chain fatty acids, corroborating the above ndings 48 . As mentioned earlier, the impact of short-chain fatty acid synthesis on the immune response is gradually being recognized. Valeric acid and butyric acid enhance the antitumour activity of cytotoxic T lymphocytes (CTLs) and chimeric antigen receptor (car) T cells through metabolic and epigenetic reprogramming 49 . SCFAs promote apoptosis by promoting aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor 1α (HIF1α) expression to upregulate IL-22 production by CD4+ T cells 50 . Microbiota-derived metabolites can modulate the suppressive function of Bregs 51 . Butyrate produced by the intestinal ora regulates antigen presentation and radiotherapy following DC-induced antitumour immune responses 52 . As previously mentioned, these immune cells have been implicated in the pathogenesis of aplastic anaemia 53 . Furthermore, docosatetraenoate is positively correlated with ubiquinone and other terpenoid quinone biosynthesis pathways, and recent studies have shown that ubiquinone and other terpenoid quinone biosynthesis pathways are often linked to the synthesis of unsaturated fatty acids 54 . We speculate that the immune abnormalities caused by the decline in the abundance of Catenibacterium mitsuokai species at the Catenibacterium genus may be involved in the pathogenesis of aplastic anaemia; furthermore, the biotransformation of docosatetraenoate is in uenced by ubiquinone and other terpenoid quinone biosynthesis pathways. Docosatetraenoate, a long-chain unsaturated fatty acid that cannot be synthesized in the body, has various functions and is involved in the regulation of in ammation and bone destruction through the cyclooxygenase and lipoxygenase pathways 55 . Long-chain unsaturated fatty acids are also involved in the regulation of bone marrow-derived macrophage activity by regulating long-chain acyl-coenzyme A synthetases (ACSLs) 56 and have a protective effect on drug-induced bone destruction 57,58 . In conclusion, unsaturated fatty acids may be involved in the regulation of the in ammatory response and may affect the bone marrow microenvironment.
Therefore, the downregulation of docosatetraenoate may be involved in both immune response enhancement and the process of bone destruction and marrow steatosis in aplastic anaemia patients. Although no speci c studies have been performed, we believe that unsaturated fatty acids have immense potential in the study of the pathogenesis of aplastic anaemia.
The abundance of Enhydrobacter species was increased in the AA group and was positively correlated with docosadienoatein in the plasma and docosatetraenoate in bone marrow supernatants. Moreover, the abundance of Enhydrobacter aerosaccus species in the Enhydrobacter genus had a positive correlation with phenylalanine, tyrosine and tryptophan biosynthesis but a negative correlation with N-glycan biosynthesis. Previous studies have shown that the Enhydrobacter genus is also abundant in gastrointestinal metaplasia and associated with carcinoma of the head of pancreas (CHP) 59,60 . In in vitro synthesis experiments, an aminotransferase derived from the bacterium Enhydrobacter catalysed the synthesis of L-phenylalanine by using 3-GABA as an amino donor 61 , as con rmed by our results in patients with aplastic anaemia. Studies have shown that L-phenylalanine metabolism is associated with fatty acid synthesis 62 . Il-10 has been shown to directly inhibit the function of CD8+ T cells by increasing the number of n-glycan branches to decrease antigenic sensitivity 63 . Additionally, CD8+ cell function was upregulated in patients with aplastic anaemia, and our study showed that the abundance of species in the n-branching glycan biosynthesis pathway was decreased in the AA group, consistent with previous ndings. N-glycan biosynthesis is also involved in the migration of bone marrow-derived mesenchymal stem cells 64 . Downregulation of N-glycan biosynthesis may inhibit the migration of MSCs outside the bone marrow in aplastic anaemia patients, thus prompting the MSC source to differentiate into adipocytes directly inside the bone marrow and complete the marrow lipidation process. Although there is no research to prove this hypothesis, we still believe there is great research potential. As previously mentioned, long-chain unsaturated fatty acids may be involved in regulating the immune response and preventing bone destruction. However, here, we believe that longchain unsaturated fatty acids primarily affect the process of steatosis in the bone marrow through their role in the bone marrow microenvironment in aplastic anaemia patients. Enhydrobacter (also associated with the presence of long-chain fatty acids) was positively and negatively associated with two biosynthetic pathways, both of which were positively associated with docosadienoate in the plasma and docosatetraenoate in the bone marrow, respectively. Therefore, it is reasonable to hypothesize that this microbe is more likely to affect the aplastic anaemia process than the immune response.

Conclusion
Our study is the rst time that apply Metagenomic to the study of aplastic anemia.Based on the CG-MS lipid detection technique, we found that there were differences in lipid metabolism between the AA group and the NC group, whether short chain fatty acids or medium and long chain fatty acids;Differences in fatty acid metabolism are re ected both in bone marrow supernatants and peripheral plasma, with more signi cant differences in long-chain fatty acids in peripheral plasma than in short chain fatty acids in bone marrow supernatants;Most fatty acids, whether medium and long chain fatty acids or short chain fatty acids showed a decreasing trend in the disease group, demonstrating that the metabolism of the disease group as a whole was attenuated; Microbial metabolic alterations are associated with alterations in fatty acids, not only in short but also in long-chain fatty acids, demonstrating that microbial metabolism similarly affects long-chain fatty acid synthesis and metabolism;The interrelationship between Citrobacter spp stearic acid (c18:0), isobutyric acid, and lysine degradation and betaine biosynthesis pathway suggests that Citrobacter positively regulated stearic acid biosynthesis and precisely negatively regulated isobutyric acid production by these two pathways.The interrelationship between catenibacterium_mitsuokai species at Catenibacterium, cis-7,10,13,16-docosatetraenoic acid (c22:4), ubiquinone and other terpenoid quinones pathway suggests that Catenibacterium negatively regulate cis-7,10,13,16-docosatetraenoic acid by biosynthesis of ubiquinone and other terpenoid quinones.The intercorrelation between the abundance of Enhydrobacter genus and Enhydrobacter_aerosaccus species, cis-13,16-docosadienoic acid in peripheral plasma, cis-7,10,13,16-docosatetraenoic acid in bone marrow supernatant, tyrosine and tryptophan biosynthesis pathway suggests that the genus aquabacterium is associated with two long-chain fatty acids through tyrosine and tryptophan biosynthetic pathway modulation in the disease group.From the point of view of immunity, citreobacilli plays an important role in the pathogenesis of aplastic anemia. We speculate that it may act as a driving factor for aplastic anemia.From the perspective of lipid metabolism, docosanpoly unsaturated fatty acids are of signi cant relevance to the microbiota in the patient group, and we speculate that docosanpoly unsaturated fatty acids may serve as mediators of microbiota regulated medium -and long-chain fatty acid metabolism.
Overall, our study not only sheds light on the possibility that Citrobacter infection may function as an aplastic anaemia agent but also revealed the potential role of stearate in the immunopathogenesis of aplastic anaemia. In addition, our study demonstrates the potential roles of 22 unsaturated fatty acids in aplastic anaemia. Unsaturated fats are not only involved in the metabolism of broblasts in the bone marrow, thus affecting the formation of the bone marrow microenvironment and participation in the process of bone marrow steatosis, but may also be involved in the regulation of immune cells in the peripheral blood to participate in disease progression in aplastic anaemia patients. Our study provides insights into the pathogenesis of aplastic anaemia and, more importantly, sheds light on the aetiology of aplastic anaemia. Figure 1 A. OPLS-DA analysis of medium and long-chain fatty acids in the bone marrow;B. OPLS-DA analysis of short-chain fatty acids in the bone marrow   PCoA and NMDS analysis showed no signi cant community differences between AA and NC groups              A. the correlation between microbial abundance and long-chain fatty acids in the plasm in AA group. B. the correlation between microbial abundance and ahort-chain fatty acids in the plasm in AA group. (only the signi cant correlation showed here)

Figure 21
A.the correlation between the abundance of KEGG pathway and the long-chain fatty acids. B. the correlation between abundance of KEGG pathway and the short-chain fatty acids Figure 22 the correlation between the microbial abundance and the KEGG pathway abundance.

Figure 23
Citrobacter infection may affect the immune status of the body directly or by adjusting the content of isobutyric acid in the intestines, thereby inducing aplastic anemia.