Gut microbiota derived L-ornithine promotes resistance to obesity through metabolites mediated immunosuppressive macrophages

Gut microbiota derived L-ornithine promotes resistance to obesity through metabolites mediated immunosuppressive macrophages | 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 Research Article Gut microbiota derived L-ornithine promotes resistance to obesity through metabolites mediated immunosuppressive macrophages Yuanyuan Li, Yuqing Liu, Juanjuan Wang, YuanHuan Gao, Yuan Zhang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6652808/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Nov, 2025 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted 5 You are reading this latest preprint version Abstract Gut microbiota can affect the occurrence and development of obesity. But the exact mechanism(s) by which obesity is prevented is still not fully understood. In this study, we found that L-ornithine (L-orn) from the gut microbiota lactobacillus helps mice to resist to high-fat diet (HFD) mediated obesity through its metabolite spermine (SPM) and spermindine (SPD) in the macrophages. SPM reduced inflammatory cytokines in the macrophages by inhibiting NF-κB and AKT (protein kinase B) signal pathways, while SPD activated Src and induced indoleamine 2, 3-dioxygenase 1 (IDO-1) to promote immunosuppressive IDO-1 macrophages. Notably, L-orn was inversely associated with body mass index (BMI) in obese patients. Sc-RNA sequencing data also showed that the NF-κB and AKT pathways were significantly up-regulated and the Src signaling pathway was significantly down-regulated in the inflammatory macrophages of adipose tissues. Thus, our results suggest that gut microbiota derived L-orn can control the occurrence and development of obesity through metabolites mediated anti-inflammatory macrophages. L-ornithine obesity spermine spermindine Indoleamine 2 3-dioxygenase 1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Obesity is a global epidemic and a major risk factor for cardiovascular disease, stroke and all-cause mortality [ 1 , 2 ]. So far, the method of prevention and treatment of obesity is unsatisfactory. The prevalence of obesity is still increasing in many populations around the world. It is urgent to look for an effective method to prevent and therapy against obesity. Studies have shown that gut microbiota can effectively affect the occurrence and development of obesity [ 3 – 5 ]. But the exact mechanism(s) by which can prevent obesity still is incompletely clear. Chronic low-grade inflammation is characteristic of obese tissue [ 6 , 7 ]. Adipose tissue macrophages (ATMs) play an important role in chronic adipose tissue inflammation [ 8 ]. Single-cell RNA sequences (sc-RNA Seq) divide macrophages in adipose tissue into multiple subpopulations [ 9 – 12 ], suggesting that macrophage subpopulations constitute a continuous cellular state, some of which are more pro-inflammatory while others are more metabolically active. However, inflammatory macrophage (IM) subpopulations and inflammatory gene programs has been shown to be positively correlated with obesity [ 9 , 12 ]. IMs are involved in complex paracrine and autocrine signaling to maintain a pro-inflammatory microenvironment [ 4 , 6 ]. Key signaling pathways in IMs include the NF-κB and AKT signaling pathways, which play a key role in regulating macrophage function in adipose tissue, especially affecting inflammation [ 13 , 14 ]. In addition, lipopolysaccharide (LPS) from the gut microbiota can stimulate macrophages to produce pro-inflammatory mediators such as TNF-α, IL-6, IL-1β, and nitric oxide (NO) via TLR4, as well as M1 macrophage-associated genes (TNFα, IL-6, IL-1β, iNOS, and MCP1), and also amplify inflammation via NF-κB [ 15 ]. The gut microbiota can resist inflammation in individuals through its metabolites [ 16 ] [ 17 ]. It regulates immunity by releasing ligands and metabolites that are transferred from the gut to local and systemic circulation [ 18 ]. Disturbances of gut microbiota are considered to be characteristic of a variety of metabolic diseases that can affect host metabolic and immune function [ 19 ]. They can increase gram-negative bacteria and raises LPS levels. LPS binds TLR4 on macrophages and triggers M1 polarization and systemic inflammation, leading to insulin resistance. Regardless, microbial metabolites (such as short-chain fatty acids (SCFA)) promote anti-inflammatory M2 macrophages, enhance insulin sensitivity, and reduce inflammation [ 20 ]. Gut bacteria can metabolize primary bile acids into secondary forms that activate receptors on macrophages (FXR, TGR5) and inhibit pro-inflammatory M1 polarization [ 21 ]. Here we demonstrate that gut microbiota lactobacillus derived L-orn, which is associated with enteric human homologous protein REG3α, protects against obesity through L-orn metabolite mediated anti-inflammatory macrophages. Materials and methods Reagents and oligoes used in this study were listed in supplementary Table S1 . Mice, huREG3α transgenic mice Four-to six-week-old male or female C57BL/6 mice were obtained from Nanjing Animal Center. Human REG3α transgenic mice ( huREG3αtg) mice were prepared by Nanjing Animal Center [ 22 ]. All experimental mice were bred and maintained under specific pathogen-free conditions in the Animal Center of Nankai University. Experiments were carried out using age- and gender- matched mice. All procedures were conducted according to the Institutional Animal Care and Use Committee of the Model Animal Research Center. Animal experiments were approved by the Institute’s Animal Ethics Committee of Nankai University. All experimental variables such as husbandry, parental genotypes and environmental influences were carefully controlled. Mouse models For high-fat diet model, 6- to 8-week-old male mice and their control littermates were fed using high-fat diet (D12492, protein 26.2%, carbohydrate 26.3%, and fat 34.9%) and control diets (D12450B, 60% of calories may be derived from fat), which was from Research Diets, Inc. (NJ, USA). For lactobacillus transplantation, mice were first treated with ampicillin (A, 1 g/L, Sigma), vancomycine (V, 0.5g/L), neomycin sulfate (N, 1 g/L), and metronidazole (M, 1g/L) via the drinking water for two weeks. To confirm the elimination of bacteria, stools were collected from antibiotic-treated and untreated mice and cultured in anaerobic and aerobic condition. Mice were orally administered 200 µl of 1×10 9 lactobacillus (once/week). For administering L-orn, eflornithine (DFMO), SPM and SPD infusion, mice were randomly assigned to two different treatment groups (n = 6/group), and then mice were administered in drinking distilled H 2 O. The mean L-Orn, SPM and SPD consumption of mice was ∼3.3 g/kg/d [ 23 ]. The mean DFMO consumption of mice was ∼1.5 g/kg/d. Mice fed with H 2 O without L-orn, SPM, SPD and DFMO were used as control. Preparation of chimeric mice. Recipient mice were irradiated with a single dose of 8 Gy of irradiation using a Shepherd Mark I caesium irradiator (J.L. Shepherd and Associates). Then, bone marrow cells (BMCs) collected from wild-type (WT) or IDO-1 KO mice were subsequently injected into the irradiated WT or IDO-1 KO recipient mice (2 × 10 6 cells per mouse) via the tail vein. Human samples For human serum collection, 208 adult participants, among which 42 with BMI < 18.5 kg/cm2, 41 with BMI 18.5–24 kg/cm2, 43 with BMI 24–27 kg/cm2, 42 with BMI 27–30 kg/cm2 and 40 with BMI ≥ 30 kg/cm2 were selectively recruited. More than half (54.3%) were males and the mean age was 42 years (SD = 13 years). The mean BMI was 25.05 ± 5.07 kg/cm2. All participants were free of acute stress conditions such as fever and diarrhea. Height and weight were measured to the nearest 0.1cm and 0.1 kg without shoes or heavy clothing using a calibrated stadiometer (GL-310, Seoul, Korea). Participants were instructed to fast for ≥ 12 hours before blood sampling in the next morning. This study was conducted with approval from the Institutional Review Board at Nankai University, Tianjin Union Medical Center and Tianjin First Central Hospital. Participants were recruited from the health screening centers of Tianjin Union Medical Center and Tianjin First Central Hospital. All participants provided written informed consent. Gut microbiota analyses Gut microbiota was analyzed by Majorbio Biotechnology Company (Shanghai, China) using primers that target to the V3-V4 regions of 16S rRNA according to previous reported method [ 22 ]. The samples were clustered at genus and OTU levels using the sample-genus and sample-OTU count matrices respectively. For each clustering, Morisita-Horn dissimilarity was used to compute a sample distance matrix from the initial count matrix, and the distance matrix was subsequently used to generate a hierarchical clustering using Ward’s minimum variance method. The Wilcoxon Rank Sum test was used to identify OTUs that had differential abundance in the different sample groups. For the absolute numbers of gut bacteria, 16s rRNAs were extracted, and then amplified using genus or strain specific primers. The concentration of each product was detected and then exchanged into copy numbers using a standard curve. Lactobacillus isolation and culture For lactobacillus isolation, 100 mg fresh faecal samples were collected and diluted in 2 ml BPS solution, and cultured on Rogosa SL selective medium (Sigma-Aldrich) for lactobacillus enumeration, and then colonies were identified and purified using 16s ribosomal RNA sequence analyses. Lactobacilli were cultured in deMan, Rogosa, Sharpe (MRS; 3 M Health Care, St. Paul, MN) media and also grown on MRS agar containing 10% sucrose. Anaerobic conditions were generated with the sachets of AnaeroPack-Anaero (Mitsubishi Gas Chemical, Japan) in an air-tight jar. After 24 h of cultivation in liquid media, lactobacilli could reach 1 × 10 9 CFU/ml. Ex vivo macrophages stimulation Peritoneal macrophages were collected on day 3 after intraperitoneal injection of 2 mL/mouse 4% w/v thioglycolate. Peritoneal macrophages isolated from C57BL/6 mice were cultured overnight in 6-well microplates with 1640 containing 10% FBS, at 37°C in a humidified 5% CO 2 atmosphere, and then L-orn, SPM or SPD was added into culture at the indicated concentration and time, and expression of cytokine was analyzed using qRT-PCR, RNA-seq or ELISA. RNA-seq analysis RNA-seq analyses were done according to reported method [ 24 ]. Based on the manual of TRIzol®reagent (Invitrogen, Shanghai, China), total RNAs from cells were extracted. Library was prepared and transcriptome was sequenced on an MGISEQ-2000 platform to generate 100-bp paired-end reads. Data were analyzed on http://biosys.bgi.com . Flow cytometry analyses For adipose tissue (AT) digestion, transfer AT to 50 ml conical tubes by pouring the homogenate and rinsing the weigh boat with 1 ml DPBS (0.5% BSA) and 3 ml collagenase II digest solution. Incubate AT homogenate in a rotational shaker (200 rpm) at 37°C for 20 min. Add 10 ml DPBS (0.5% BSA) to conical tubes and place on ice. Triturate homogenate numerous times using a 10 ml serological pipette, and pass cell suspensions through 100 µm filter in to a new 50 ml conical tube. Centrifuge cell suspension at 500 × g for 10 min at 4°C. Decant supernatant and resuspend SVF cell pellet in 3 ml ACK buffer to lyse contaminating erythrocytes. Add 12 ml FACS buffer and centrifuge cell suspension at 500 × g for 10 min at 4°C. Decant supernatant and resuspend SVF cell pellet in FACS buffer. The cells were collected at the interphase of the Percoll gradient, washed and resuspended in medium, and then stained and analyzed by flow cytometry. Dead cells were eliminated through 7-AAD staining. L-ornithine analyses L-orn concentration were detected using the Ion Chromatography system. Serum samples were extracted in 5% trichloroacetic acid (TCA, typically by mixing with equal volume of 10% TCA) by vigorous vortexing. Serum extracts were centrifuged at 13 000 rpm in table top micro centrifuge. 100 mg adipose tissues were thawed and minced in 5% TCA. The mixture was homogenized by sonication (Fisher Sonic Dismembrator Model 300). Tissue homogenates were centrifuged in a micro-centrifuge at 10 000 × g for 10 min at 4°C. 100 mg fresh faecal samples were collected and extracted in 5% TCA (typically by mixing with equal volume of 10% TCA) by vigorous vortexing. The supernatants were extracted twice with diethyl ether, after which the extracts were diluted in water (1:9) before being applied to the column, and eluted by increasing concentration of methanesulfonic acid (3–45 mM). L-orn were detected with an on-line ion conductivity detector coupled to an SRS 300 suppressor for cations (which eliminates all anions including the mobile phase ion, methanesulfonic acid, allowing only cations to be detected). Quantification of L-orn was done by measuring the (conductivity in µSiemens) peak areas of individual L-orn based on the standard curve derived from analyzing a dilution series of L-orn. Others ELISA, Western blotting, RT-PCR and qRT-PCR were performed according to our previous methods[ 25 , 26 ]. Statistical analyses Unpaired Student’s t test, two –ways ANOVA and one-way ANOVA followed by Tukey post hoc test were used to determine significance. A 95% confidence interval was considered significant and was defined as p < 0.05. * indicates p < 0.05, ** p < 0.01, *** p < 0.001. Results HuREG3α tgIEC mice are resistant to high-fat diet mediated obesity Our previous studies showed that mice with intestinal epithelial cells expressing human REG3α (huREG3α tgIEC mice) were resistant to DSS mediated colitis by altering gut microbiota [ 22 ]. Since the occurrence and development of obesity is also associated with alterations of the gut microbiota [ 3 – 5 ], we adopted a high-fat diet (HFD) mediated obesity model to investigate the effect of human REG3α expressed in mouse intestinal epithelial cells on obesity. Compared with control wild-type (WT) mice, male and female huREG3α tgIEC mice were observed to had significantly lower body weight and fat pad weight (Fig. 1 A, C), suggesting that huREG3α tgIEC mice are resistance to HFD-mediated obesity. Insulin sensitive and glucose tolerance tests also showed that huREG3α tgIEC mice were less sensitive to insulin and less tolerant to glucose (Fig. 1 B). Inflammatory macrophages (F4/80 + , CD11C + , INOS + ) and immunosuppressive macrophages (F4/80 + , CD206 + , and arginine (Arg)-1 + ) are associated with the onset and progression of obesity [ 4 , 6 ]. Data showed a significant decrease in inflammatory macrophages and an increase in anti-inflammatory macrophages in the adipose tissues of huREG3α tgIEC mice (Fig. 1 D, supplementary Figure S1 ). Inflammatory cytokines also markedly decreased in the adipose tissue of huREG3α tgIEC mice (Fig. 1 E). Notably, there showed an increased IDO-1 + macrophages in huREG3α tgIEC mice as compared to control WT mice (Fig. 1 F), imply that this macrophage subpopulation might also play a role in resistance to HFD-mediated obesity in huREG3α tgIEC mice. In addition, when fed normally, the mice showed no differences in weight, insulin sensitivity and glucose tolerance (supplementary Figure S2). Taken together, huREG3α tgIEC mice are resistant to HFD mediated obesity. There were high levels of L-ornithine in adipose tissues of huREG3α tgIEC mice HuREG3α tgIEC -associated gut microbiota plays a role in resistance to DSS- mediated colitis in huREG3α tgIEC mice [ 22 ]. REG3, an antimicrobial peptide secreted by intestinal epithelial cells, can directly kill bacterium [ 27 ], which potentially induce dysregulation of gut microbiota [ 27 ]. Here we again analyzed the composition of gut microbiota and further confirmed the previous results (Fig. 2 A, supplementary Figure S3). Interestingly, lactobacillus numbers were significantly increased not only in huREG3α tgIEC mice, but also in HFD fed huREG3α tgIEC mice (Fig. 2 A, supplementary Figure S3), implying that increased lactobacillus may play a role in the resistance of huREG3α tgIEC mice to HFD-mediated obesity. Thus, we further analyzed the composition of lactobacillus in HFD fed HuREG3α tgIEC mice. The increased lactobacillus in human HuREG3α tgIEC was named Lactobacillus NK2 (L. NK2) [ 22 ]. Interestingly, L. NK2 also significantly increased in HFD fed HuREG3α tgIEC mice (Fig. 2 B, C). It is worth noting that REG3 may kill some gram-positive bacteria. But, gram-positive lactobacillus are not sensitive to REG3 [ 28 , 29 ]. We previously found that L. NK2 could produce large amounts of L-orn [ 22 ]. Data here showed that HFD fed huREG3α tgIEC mice not only had increased L-orn in stool and serum, but also in adipose tissue (Fig. 2 D). Thus, L-orn is significantly elevated in the adipose tissue, peripheral blood and stool of HFD-fed huREG3α tgIEC mice. Gut microbiota derived L-ornithine is involved in resistant to HFD mediated obesity Chronic inflammation is characteristic of obese tissue [ 6 , 7 ]. Since L-orn has anti-inflammatory effects [ 16 , 17 ], this suggests that lactobacillus-derived L-orn may be involved in resistance to HFD-mediated obesity. Next, we investigated the effects of L-orn on obesity. The data showed that L-orn-fed mice also showed significant resistance to HFD-mediated obesity and reduced sensitivity to insulin and tolerant to glucose compared to control mice (supplementary Figure S4A, C). L-orn-fed mice also had a lighter fat pat weights (supplementary Figure S4B). Anti-inflammatory F4/80 + CD206 + and F4/80 + IL-10 + macrophages significantly increased in adipose tissue of L-orn-fed mice (supplementary Figure. S4D). Thus, these data suggest that L-orn can protect against HFD mediated obesity. To further determine the effect(s) of gut microbiota derived L-orn on obesity, we also used Lactobacillus reuteriΔOTC (MutLac) which does not produce L-orn (22) to further investigate the role of L-orn in obesity. Arginine can be metabolized into L-orn in the gut microbiota via catabolic pathways such as arginine deiminase pathway (ADI) [ 30 , 31 ]. Mice colonized with L. reuteri which could produce L-orn (22), showed significant resistance to HFD mediated obesity and reduced sensitivity to insulin and tolerant to glucose compared to mice colonized with MutLac (Fig. 3 A, B). MutLac colonized mice were heavier in body weight as compared with control mice colonized L. reuteri after feeding HFD (Figs. 3 A). The data also showed a significant decrease in the number of anti-inflammatory F4/80 + CD206 + , F4/80 + IL-10 + , and F4/80 + Arg-1 + macrophages in adipose tissue of the mice colonized with MutLac while a significant increase could be detected in inflammatory macrophages (F4/80 + CD11C + and F4/80 + TNFa + ) (Figs. 3 C, D). Analysis of inflammatory cytokines in adipose tissue also showed that MutLac colonized mice had significantly higher levels of inflammatory cytokines in the adipose tissues than those in L. reuteri colonized mice (Fig. 3 E). Again, there had more IDO-1 + macrophages in the adipose tissues of mice colonized L. reuteri than MutLac (Fig. 3 F). L-orn concentration in the adipose tissues, peripheral sera and feces of MutLac colonized mice was lower than in L. reuteri colonized mice or WT mice (Fig. 3 J). These data suggest that MutLac can reduce the ability of lactobacillus to resist HFD mediated obesity. Taken together, lactobacillus derived L-orn plays a key role in the resistance to HFD mediated obesity. Gut microbiota L-ornithine reduces macrophage inflammation Anti-inflammatory macrophages increased significantly in L-orn fed mice, suggesting that the effect of L-orn on obesity may be through reducing macrophage inflammation. Since obesity is associated with LPS-mediated inflammation [ 15 ], we next looked at the role of L-orn in LPS-mediated inflammatory cytokines. The data showed that inflammatory cytokines were significantly reduced after exposure to L-orn (Fig. 4 A). RNA-seq analysis showed that macrophage inflammation genes treated with L-orn were significantly reduced upon exposure to LPS (supplementary Figure S5). Several downregulated inflammatory signaling pathways in L-orn treated macrophages were also observed, including TLR, NOD-like receptor signaling, cytokine-cytokine receptor, and IL-17 signaling (Fig. 4 B), which are related to NF-κB and AKT [ 13 , 14 ]. Indeed, L-orn not only failed to activate NF-κB and AKT (Fig. 4 C), but also effectively inhibited AKT activation (Fig. 4 C) and LPS-mediated NF-κB activation (Fig. 4 D). L-orn was able to further inhibit the production of inflammatory cytokines, which could be saved by the L-orn inhibitor difluoromethylornithine (DFMO) (Fig. 4 E). In addition, L-orn significantly also promoted the expression of CD206, which are expressed by immunosuppressive macrophages, suggesting that L-orn can also promote the differentiation of immunosuppressive macrophages (Fig. 4 F). Taken together, L-orn can significantly inhibit intracellular AKT activity and LPS-mediated NF-κB activity to reduce inflammatory cytokines in macrophages. Decreased inflammation is derived from L-ornithine metabolite SPM in macrophages Spermine is a polyamine synthesized from ornithine via the polyamine pathway (ornithine→humutine→spermidine→spermine) that plays a key role in adipose tissue biology (Fig. 5 A) [ 32 ]. Therefore, we next investigated the effects of L-orn metabolite SPM on macrophage-mediated inflammation. When macrophages were exposed to SPM, SPM alone did not affect phosphorylation of AKT and NF-κB (Fig. 5 B), while LPS did (Fig. 5 C). Notably, SPM could inhibit LPS-mediated phosphorylation of NF-κBp65 and AKT, and reduce inflammatory cytokines; whereas SPD, another intracellular metabolite of L-orn, did not do so (Figs. 5 C, D). We also further analyzed whether SPM could also inhibit HFD mediated obesity. After feeding SPM, SPM not only inhibited the development of obesity, including reduced body weight and fat pads, reduced sensitivity to insulin and tolerant to glucose (Fig. 5 E-G), but also reduced inflammatory cytokines in obese tissue (Fig. 5 H). Thus, L-orn metabolite SPM can inhibit inflammatory macrophages to resist to obesity. L-ornithine metabolite SPD mediated IDO-1 macrophages are involved in resistance to obesity Previous studies have shown that L-orn metabolite SPD is able to reprogram mouse conventional dendritic cells to an immunomodulatory phenotype through Src kinase-dependent phosphorylation of IDO-1 [ 33 ]. The data show that SPD, but not SPM, could cause Src phosphorylation (Fig. 6 A, B). As controls, L-orn also promoted the phosphorylation of Src (Fig. 6 A, B). Importantly, SPD could also promote the phosphorylation of IDO-1(Fig. 6 C) and the expression of IDO-1 (Fig. 6 D-E). Furthermore, SPD mediated IDO-1 was a time- and dose- dependent (Fig. 6 D-E). IDO-1 is a key regulator of immune homeostasis, balance tolerance, and inflammation. Src may regulate IDO-1 through transcriptional control, such as Src-dependent signaling pathways that enhance IDO-1 expression and post-translational modification, and Src-mediated phosphorylation that also regulates IDO-1 enzyme activity [ 34 ]. Next, we investigated the effect of IDO-1 + macrophages on obesity by establishing a macrophage transplantation model. Data showed that mice transplanted with IDO-1 knockout (KO) macrophages were more sensitive to HFD mediated obesity. These transplanted mice not only had increased body and Fat pad weights, but also had reduced glucose tolerance and insulin sensitivity (Fig. 6 F-H). Thus, IDO-1 + macrophages are required for L-orn mediated resistance to HFD mediated obesity. Gut microbiota L-ornithine is related to obesity in humans Finally, we investigated the potential effects of gut microbiota derived L-orn on human obesity. We first studies the relevance of gut microbiota–derived L-orn to the body weights of 208 individuals with different body mass indices (BMIs), which were used previously [ 35 ]. Lower levels of L-orn were observed in overweight and obese individuals (Fig. 7 A). Furthermore, there existed a negative relationship between the concentrations of the microbiota metabolite L-orn and BMIs of individuals (Fig. 7 B), implying that L-orn potentially was related to the resistant role in the occurrence and development of obesity in human. We next also used published sc-RNA seq data [ 9 ] to analyze the signal pathway in the IM of adipose tissues in patients with obesity. There indeed had increased NF-κB and AKT signal pathways, and decreased Src pathways in the IM of adipose tissues of patients with obesity (Fig. 7 C, D). Notably, there also had upregulated inflammatory signal pathways in IM of adipose tissues of patients with obesity as compared to the IM in lean individuals (Fig. 7 E), suggesting the increased inflammatory responses in the IM of adipose tissues of patients with obesity. We finally investigated the effects of the SPM on the NF-κB and inflammatory cytokines, and roles of the SPD in the IDO expression and Src phosphorylation in human macrophages. Data exhibited that SPM but not SPD could affect the activation of NF-κB and reduce the expression of inflammatory cytokines, and SPD but not SPM could induce the phosphorylation of Src (Fig. 7 F, G). SPD also promoted the differentiation of IDO + macrophages (Fig. 7 H). Taken together, similar to the effects of L-orn on the macrophages in mice, obesity also is related to gut microbiota derived L-orn in humans. Discussion We demonstrate here that gut microbiota derived L-orn, which is associated with human REG3α, can protect against HFD mediated obesity through SPM mediated anti-inflammatory and SPD-induced IDO-1 macrophages. SPM can inhibit NF-κB and AKT to reduce inflammatory cytokines in the macrophages, while SPD can promote the expression and phosphorylation of IDO-1 to cause IDO-1 immunosuppressive macrophages, thereby inhibiting HFD mediated obesity. Importantly, we also found that BMI in obese patients was inversely associated with serum L-orn. Inflammatory macrophages from human obese tissue also exhibit enhanced NF-κB and AKT and reduced Src signaling pathways These results provide a strong foundation for L-orn as a tool for preventing and treating obesity. Gut microbiota derived L-orn can protect against HFD mediated obesity through metabolites mediated immunosuppressive macrophages. Previous studies also found that L-orn was related to obesity [ 36 , 37 ]. Our data showed negative relationship between L-orn and obesity. L-arginine, which can be metabolized into L-orn in gut microbiota contributes significantly to reducing inflammation and infection complications [ 30 , 31 ]. In preclinical models, dietary supplementation with L-arginine improves and faster resolution of DSS-induced colitis [ 38 ]. However, it is not fully understood how L-orn reduces inflammation. Here we demonstrate that gut microbiota L-orn metabolite SPM can reduce the expression of inflammatory cytokines via inhibiting NF-κB and AKT. Other studies also found that SPM can suppress the immune response of activated macrophages by inhibiting the expression of NOS2 [ 39 ]. In a mouse model of acute liver injury, SPM also induced M2 polarization in tumor-associated macrophages [ 40 , 41 ]. Increased SPM also inhibited NLRP3 inflammasome assembly and subsequent pyroptosis by inhibiting K + efflux. In addition, SPM also significantly reduced p-JAK1, p-tyrosine kinase 2 (TYK2), p-STAT1, and p-STAT2 after IFN-β stimulation [ 42 ]. In diet-induced obesity mouse models, large daily doses of SPM are an effective strategy for weight loss and improved glucose status [ 43 , 44 – 45 ]. Spermidine is a central polyamine synthesized from putrescine that plays a key role in adipose tissue homeostasis, affecting metabolism, inflammation and cellular resilience. Our data show that SPD plays a role in resistance of mice to HFD mediated obesity via Src/IDO-1 mediated IDO-1immunosuppressive macrophages. Indeed, IDO-1 −/− mice fed HFD gained less weight, had lower fat mass, and had better glucose and insulin resistance as compared to WT mice [ 46 ]. Loss or inhibition of IDO-1 improved insulin sensitivity, protected the intestinal mucosal barrier, reduced endotoxemia and chronic inflammation, and regulated lipid metabolism in liver and adipose tissue [ 46 ]. SPD could also confer a tolerance phenotype on conventional dendritic cells, which depends on the expression of IDO-1 and the activity of Src kinase. Interestingly, activation of Src and IDO-1 was also detected in SPD-treated macrophages after exposure to L-orn [ 47 ]. Inhibitors of Src (e.g., dasatinib) or IDO-1 (e.g., epacadostat) also showed potential in preclinical models to reduce adipose inflammation and improve insulin sensitivity [ 48 ]. Numerous studies have endeavored to identify the microbiota signatures associated with obesity [ 49 ], including decreased diversity, altered levels of specific bacterial taxa (e.g., Akkermansia spp., Christensenella spp., Bacteroides spp., Prevotella spp. Blautia spp., etc.) and also changes in metabolic pathways or products. Some gut bacteria such as Lactobacillus and Enterococcus , which utilize the ADI to convert dietary or host-derived arginine into L-orn, have shown strong potential in fighting obesity-related inflammation and metabolic issues [ 50 ]. Studies in animals also demonstrate that lactobacillus can reduce body weight, fat, and inflammation [ 50 , 51 ]. These results suggest that lactobacillus could be an effective way to manage obesity and related health problems [ 50 ]. Declarations Supplementary information The online version contains supplementary material available at: Conflict of interests The authors have no relevant financial or nonfinancial Ethics approval The animal study protocol was approved by the Laboratory AnimalWelfare and Animal Experiment Ethics Review Committee of Nankai University (Approval Number: NK-20190912). Consent for publication. All authors agree to the publication of this study. Funding This research was supported by NSFC grants 91842302, 82271779, 81901677, 31470876, 91629102, ISF-NSFC program 31461143010; Tianjin science and technology commission (18JCZDJC35300); CAMS Innovation Fund for Medical Science (CIFMS2017-12M-2-005); a Ministry of Science and Technology grant (2016YFC1303604); the State Key Laboratory of Medicinal Chemical Biology; The Fundamental Research Funds for the Central University, Nankai university(Grant number 63191724). Data Availability Raw 16S rRNA gene sequence data for the feces microbiota were deposited in the NCBI Short Read Archive under BioProject Accession Number PRJNA326574. Author Contributions R. Y. designed the research and wrote the paper; Y. L, J. W., Y. G., conducted in vivo and in vitro experiments; Y. Z offered an assistance to the experiments.All authors read and approved the final manuscript. Acknowledgment Not applicable. 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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-6652808","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":459222859,"identity":"11fb9160-ee7d-49b7-9f27-9dcd5a4bac28","order_by":0,"name":"Yuanyuan Li","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"Yuanyuan","middleName":"","lastName":"Li","suffix":""},{"id":459222860,"identity":"47824307-bd8d-4404-b188-eafbb572e218","order_by":1,"name":"Yuqing Liu","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"Yuqing","middleName":"","lastName":"Liu","suffix":""},{"id":459222861,"identity":"fc292503-3077-4497-bb78-4ecf9c711b2c","order_by":2,"name":"Juanjuan Wang","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"Juanjuan","middleName":"","lastName":"Wang","suffix":""},{"id":459222862,"identity":"59e090ed-c69d-4c9f-8bc0-b7c19baa0261","order_by":3,"name":"YuanHuan Gao","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"YuanHuan","middleName":"","lastName":"Gao","suffix":""},{"id":459222863,"identity":"af2ebfd5-d519-4862-833a-3d62a3bacb00","order_by":4,"name":"Yuan Zhang","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Zhang","suffix":""},{"id":459222864,"identity":"41b51c6b-4bde-45f9-ac58-acd62b355bbe","order_by":5,"name":"Rongcun Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYNCCCiDmYWA4wMDATKyWMyRrYWyDaGEgSov5jORnD7/OuyNvznP44QGGCuvEBvazB/BqkbmRZm4su+2Z4c7eNoMDDGfSExt48hLwapGQSDCTltx2mHHDeQaDA4xthxMbJHgMCGhJ/yYtOeew/Ybz7B8OMP4jSkuOmeTHhsOJG872AG1pIEYLz5syaYZjh5M3nDlTcCDhWLpxG08OAS3s6dskf9Qctt1wJn3zhw811rL97Gfwa2EQSGBg5oFxEoCYDb96IOA/wMD4g6CqUTAKRsEoGNEAAEOrSUwPz5e7AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-5826-4493","institution":"Nankai University","correspondingAuthor":true,"prefix":"","firstName":"Rongcun","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-05-13 07:52:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6652808/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6652808/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00018-025-05882-8","type":"published","date":"2025-11-26T15:57:42+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83437424,"identity":"37fa1655-c2e4-40d1-ac8d-99da08607d92","added_by":"auto","created_at":"2025-05-26 08:47:04","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":319396,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHuman Reg3a promotes resistance of mice to high-fat diet induced obesity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Body weight changes in male (right) or female (left) wild-type (WT) and HuREG3α\u003csup\u003etgIEC \u003c/sup\u003e(REG3a) mice fed high-fat diet (HFD) (n=15). These mice are no differences at baseline before feeding HFD.\u003c/p\u003e\n\u003cp\u003e(B) Glucose tolerance and insulin sensitivity of WT\u003cem\u003e \u003c/em\u003eand HuREG3α\u003csup\u003etgIEC \u003c/sup\u003e(REG3a) mice fed HFD for three months (n=15).\u003c/p\u003e\n\u003cp\u003e(C) Typical phenotype of WT and HuREG3α\u003csup\u003etgIEC \u003c/sup\u003e(REG3a) mice fed HFD for three months (n=15). One representative of 15 mice, and analyses of fat-pad weights.\u003c/p\u003e\n\u003cp\u003e(D) Flow cytometry of F4/80\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e, F4/80\u003csup\u003e+\u003c/sup\u003eArg-1\u003csup\u003e+\u003c/sup\u003e, F4/80+CD11C\u003csup\u003e +\u003c/sup\u003e and F4/80\u003csup\u003e+\u003c/sup\u003e iNOS cells in the fat tissues of HuREG3α\u003csup\u003etgIEC\u003c/sup\u003e (REG3a+HFD) and their control WT (WT+HFD) mice fed HFD for three months.\u003c/p\u003e\n\u003cp\u003e(E) ELISA of IL-β, TNFa\u0026nbsp; IL-6 in the mixed adipose tissues of WT\u003cem\u003e \u003c/em\u003eand HuREG3α\u003csup\u003etgIEC \u003c/sup\u003e(REG3a) mice fed HFD (n=15).\u003c/p\u003e\n\u003cp\u003e(F) Flow cytometry of F4/80\u003csup\u003e+\u003c/sup\u003eIDO-1\u003csup\u003e+\u003c/sup\u003ecells in the fat tissues of huREG3\u003csup\u003eIECtg\u003c/sup\u003e (REG3a+HFD) and their control (WT+HFD) mice fed HFD for three months.\u003c/p\u003e\n\u003cp\u003eOne sample (D-F) from mixed five mice.\u003c/p\u003e\n\u003cp\u003eError bars indicate mean ± SD;\u003c/p\u003e\n\u003cp\u003eStatistic test: Two-way ANOVA test (A and B); Unpaired Student’s t test (C-G); *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eData are a representative of three independent experiments.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6652808/v1/6877273a4570d8c73f28aef7.jpg"},{"id":83436961,"identity":"c371b644-662a-4df6-ba39-6732ea191ae0","added_by":"auto","created_at":"2025-05-26 08:39:03","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":465399,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHuman Reg3a affects composition of gut microbiota.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Comparison of gut bacteria after 16S rRNA-seq of gut microbiota from pooled ileum content (S.I) and colon\u0026nbsp;content (Co) samples of WT and\u0026nbsp;huREG3\u003csup\u003eIECtg\u003c/sup\u003e (HuREGIIIa) mice before (6 mice (male)/group) and after (15 mice (male)/group) giving HFD for three months.\u003c/p\u003e\n\u003cp\u003e(B) Proportion of different lactobacillus after sequencing analyses from pooled ileum content (SI) and colon content (colon) samples of WT (Wt/SI/HFD, Wt/Colon/HFD) and huREG3\u003csup\u003eIECtg\u003c/sup\u003e (Reg3a/SI/HFD, Reg3a/Colon/HFD) mice (15 mice/group) fed HFD for three months.\u003c/p\u003e\n\u003cp\u003e(C) Numbers of Lactobacillus genus and Lactobacillus NK2 in colon content (Co) samples of WT and huREG3\u003csup\u003eIECtg\u003c/sup\u003e (REG3a) mice fed HFD for three months.\u003c/p\u003e\n\u003cp\u003e(D) L-ornithine concentrations of intestinal contents (SI), colon contents, sera and adipose tissues in \u003cem\u003ehuREG4\u003c/em\u003e\u003csup\u003e\u003cem\u003eIECtg\u003c/em\u003e\u003c/sup\u003e (Reg3a/HFD) and their control WT (WT/HFD) mice fed HFD for three months (n=15).\u003c/p\u003e\n\u003cp\u003eError bars indicate mean ± SD;\u003c/p\u003e\n\u003cp\u003eStatistic test: Unpaired Student’s t test (C and E); *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6652808/v1/a79e60df846b8b01d0848c85.jpg"},{"id":83436053,"identity":"7bd450eb-6628-4770-8adc-0b9e4c412489","added_by":"auto","created_at":"2025-05-26 08:31:03","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":456418,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eL-ornithine from human Reg3a associated gut microbiota promotes resistance of mice to HFD-mediated obesity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Body weight changes of mice fed HFD (n=15).\u003c/p\u003e\n\u003cp\u003e(B)\u0026nbsp;Glucose tolerance and insulin sensitivity of mice fed HFD (n=15). Mice were treated via L. reuteri (WT+Lac) or Mutant L. reuteri (WT+MutLac) gavage.\u003c/p\u003e\n\u003cp\u003e(C) Flow cytometry of F4/80\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e, F4/80\u003csup\u003e+\u003c/sup\u003eIL-10\u003csup\u003e+\u003c/sup\u003e, F4/80\u003csup\u003e+\u003c/sup\u003eArg-1\u003csup\u003e+\u003c/sup\u003e, F4/80\u003csup\u003e+\u003c/sup\u003eCD11C\u003csup\u003e +\u003c/sup\u003e and F4/80\u003csup\u003e+\u003c/sup\u003e TNFa cells in the adipose tissues of mice fed HFD.\u003c/p\u003e\n\u003cp\u003e(D) Immunostaining of F4/80CD206 in the adipose tissues of mice fed HFD (n=15). One representative of 15 mice.\u003c/p\u003e\n\u003cp\u003e(E)\u0026nbsp;ELISA of IL-6, TNFa and IL-1β in in the adipose tissues of mice fed HFD.\u003c/p\u003e\n\u003cp\u003e(F) Flow cytometry of F4/80\u003csup\u003e+\u003c/sup\u003eIDO-1\u003csup\u003e+\u003c/sup\u003e cells in the adipose tissues of mice fed HFD. \u0026nbsp;(J) L-orn concentration in colon contents, blood and adipose tissues of mice fed HFD.\u003c/p\u003e\n\u003cp\u003eMice were treated via L. reuteri (Lac) or Mutant L. reuteri (MutLac) gavage.\u003c/p\u003e\n\u003cp\u003eWT, untreated control; One sample (C, E and F) from 5 mice.\u003c/p\u003e\n\u003cp\u003eError bars indicate mean ± SD;\u003c/p\u003e\n\u003cp\u003eStatistic test: Two-way ANOVA test (A and B); One-way ANOVA followed by Tukey post hoc test (C, D, E and F). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eData are a representative of three independent experiments.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6652808/v1/56fbcff6f035741fd144666b.jpg"},{"id":83436965,"identity":"720f9ec6-4376-407f-a367-a5ca793646de","added_by":"auto","created_at":"2025-05-26 08:39:04","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":511538,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eL-ornithine reduces inflammatory cytokines in macrophages.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) ELISA of IL-6, TNFaand IL-1β in the supernatants of macrophages after exposure to LPS, L-ornithine (L-orn) or LPS + L-orn;\u003c/p\u003e\n\u003cp\u003e(B) Downregulated gene signaling pathways in macrophages after exposed to L-ornithine. RNA-sequencing of the macrophages was done after exposure to L-ornithine (L-orn);\u003c/p\u003e\n\u003cp\u003e(C) Immunoblotting of P-AKT, AKT, p-p65 and p65 after exposure to LPS (upper) or L-ornithine (L-orn, lower) at the indicated time.\u003c/p\u003e\n\u003cp\u003e(D) Immunoblotting of p-p65 and p65 after exposure to LPS, LPS + L-ornithine (L-orn) or LPS+L-orn+DFMO；\u003c/p\u003e\n\u003cp\u003e(E) ELISA of TNFa and IL-6 in the supernatants of macrophages after exposure to LPS, L-ornithine (L-orn), DFMO, LPS + L-orn, LPS + DFMO or LPS + L-orn + DFMO;\u003c/p\u003e\n\u003cp\u003e(F) Flow cytometry of CD206 of the macrophages after exposure to L-ornithine (L-orn) for 24 hs.\u003c/p\u003e\n\u003cp\u003eError bars indicate mean ± SD;\u003c/p\u003e\n\u003cp\u003eStatistic test: One-way ANOVA followed by Tukey post hoc test (A and E);\u003c/p\u003e\n\u003cp\u003e*\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003eData are a representative of three independent experiments.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6652808/v1/327e8df417b1a6ff9679cf89.jpg"},{"id":83436055,"identity":"15332625-5be5-4060-848b-9d496aa958ba","added_by":"auto","created_at":"2025-05-26 08:31:04","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":540390,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eL-ornithine metabolite SPM reduces inflammatory cytokines through inhibiting NF-kB and AKT signal pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic illustration showing the metabolism pathway of L-ornithine (-Lorn) in the macrophages(Mac); ODC, Ornithine decarboxylase; OAT, ornithine aminotransferase; SRM, spermidine synthase; SMS, spermine synthase.\u003c/p\u003e\n\u003cp\u003e(B) \u0026nbsp;Immunoblotting of p-AKT, AKT, p-P65 and P65 after exposure to SPD (right) or SPM (left) at the indicated time;\u003c/p\u003e\n\u003cp\u003e(C) Immunoblotting of p-AKT, AKT, p-P65 and P65 after exposure to LPS, SPD and LPS+SPD (right) or LPS, SPM and LPS+SPM (left);\u003c/p\u003e\n\u003cp\u003e(D) QRT-PCR of IL-6, TNF-a and IL-1β after exposure to LPS+SPM at the indicated time；R. E., relative expression.\u003c/p\u003e\n\u003cp\u003e(E) Body weight changes of mice fed HFD (n=15) after exposure to SPM or SPM inhibitor (SPMinh). These mice are no differences at baseline before feeding HFD;\u003c/p\u003e\n\u003cp\u003e(F) Glucose tolerance and insulin sensitivity of mice fed HFD after exposure to SPM or SPM inhibitor (SPMinh, n=15);\u003c/p\u003e\n\u003cp\u003e(G) Fat pad weights of mice fed HFD (n=15) upon exposure to SPM or SPM inhibitor (SPMinh, n=15);\u003c/p\u003e\n\u003cp\u003e(H) ELISA of IL-6, TNFa and IL-1β in the adipose tissues of mice fed HFD (n=15) upon exposure to SPM or SPM inhibitor (SPMinh);\u003c/p\u003e\n\u003cp\u003eError bars indicate mean ± SD;\u003c/p\u003e\n\u003cp\u003eStatistic test: Two-way ANOVA test (E and F); One-way ANOVA followed by Tukey post hoc test (D, G and H);\u003c/p\u003e\n\u003cp\u003e*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eData are a representative of three independent experiments.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6652808/v1/6752ce37d5ace7325eacfeeb.jpg"},{"id":83436962,"identity":"40ceb839-dc92-4fc2-bf4c-455b2d91fd15","added_by":"auto","created_at":"2025-05-26 08:39:04","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":338479,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eL-ornithine metabolite SPD promotes differentiation of IDO-1 macrophages.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic illustration showing the metabolism pathway of L-ornithine (L-orn) in the macrophages (Mac); ODC, Ornithine decarboxylase; OAT, ornithine aminotransferase; SRM, spermidine synthase; SMS, spermine synthase.\u003c/p\u003e\n\u003cp\u003e(B) Immunoblotting of p-Src or Src after exposure to L-orn, SPD or SPM at the indicated time;\u003c/p\u003e\n\u003cp\u003e(C) Immunoblotting of p-IDO-1 and IDO-1 upon exposure to SPD;\u003c/p\u003e\n\u003cp\u003e(D) QRT-PCR (upper) and immunoblotting (lower) of IDO-1 after exposure to SPD in the indicated concentrations;\u003c/p\u003e\n\u003cp\u003e(E) QRT-PCR (upper) and immunoblotting (lower) of IDO-1 after exposure to SPD in the indicated times;\u003c/p\u003e\n\u003cp\u003e(F) Body weight changes in IDO-1 KO (IDO1KO mac) or WT macrophage (WT mac) transplantation mice fed HFD (n=15) upon exposure to SPD. These mice are no differences at baseline before feeding HFD.\u003c/p\u003e\n\u003cp\u003e(G) Glucose tolerance and insulin sensitivity in IDO-1 KO (IDO1KO mac) or WT macrophage (WT mac) transplantation mice fed HFD (n=15) upon exposure to SPD (n=15).\u003c/p\u003e\n\u003cp\u003e(H) Fat pad weights in IDO-1 KO (IDO1KO mac) or WT macrophage (WT mac) transplantation mice fed HFD (n=15) upon exposure to SPD.\u003c/p\u003e\n\u003cp\u003eWT in (F-H), un-transplant mice.\u003c/p\u003e\n\u003cp\u003eError bars indicate mean ± SD;\u003c/p\u003e\n\u003cp\u003eStatistic test: Two-way ANOVA test (F and G); One-way ANOVA followed by Tukey post hoc test (D, E and H);\u003c/p\u003e\n\u003cp\u003e*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eData are a representative of three independent experiments.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6652808/v1/5d0fa81a0933e1233be61268.jpg"},{"id":83436060,"identity":"243ed5b9-3f4a-4564-a3cf-f2d9833dbbb6","added_by":"auto","created_at":"2025-05-26 08:31:04","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":378842,"visible":true,"origin":"","legend":"\u003cp\u003ePublished sc-RNA seq data [9] in C, D and E were analyzed in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGut microbiota L-ornithine is related to obesity in humans.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) L-ornithine (L-orn) levels in the sera of different groups in the patients with obesity, BMI\u0026lt; or = 18.5 (n=42), 18.5-23.9 (n=41), 24-26.9 (n=43), 27-29.9 (n=42), or \u0026gt; or = 30 (n=40). ‘\u003c/p\u003e\n\u003cp\u003e(B) Negative relationship between BMI (body mass indices) and L-orn in the sera of patients with obesity according to analyses of linear regression. R=0.777;\u003c/p\u003e\n\u003cp\u003e(C) GSEA data showing enrichment of NF-kB and AKT signal pathway sets in inflammatory macrophages VS anti-inflammatory macrophages in the adipose tissues of patients with obesity.\u003c/p\u003e\n\u003cp\u003e(D) GSEA data showing enrichment of SRC signal pathway sets in inflammatory macrophages VS anti-inflammatory macrophages in the adipose tissues of patients with obesity.\u003c/p\u003e\n\u003cp\u003e(E) Upregulated signal pathways in inflammatory macrophages of the adipose tissues of patients with obesity VS inflammatory macrophages in the adipose tissues of lean individuals.\u003c/p\u003e\n\u003cp\u003e(F) Immunoblotting of p-p-65, p65, p-Src and Src in human monocytes derived macrophages after exposure to SPM or SPD.\u003c/p\u003e\n\u003cp\u003e(G) QRT-PCR of IL-6, TNFa and IL-1β in the supernatants of human monocytes derived macrophages after exposure to SPM or SPD.\u003c/p\u003e\n\u003cp\u003e(H) Flow cytometry of F4/80\u003csup\u003e+\u003c/sup\u003eIDO-1\u003csup\u003e+\u003c/sup\u003e macrophages after exposure to SPD.\u003c/p\u003e\n\u003cp\u003eError bars indicate mean ± SD (B and F);\u003c/p\u003e\n\u003cp\u003eStatistic test: One-way ANOVA followed by Tukey post hoc test (G); Unpaired Student’s t test (B, G, and H); *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6652808/v1/c4d915dcbd1219901cc8352c.jpg"},{"id":97179294,"identity":"ba81af3e-1ff0-425c-bc80-4ef8b403a408","added_by":"auto","created_at":"2025-12-01 16:14:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4174775,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6652808/v1/a665d12f-9142-4029-9036-0342fbdf1f7c.pdf"},{"id":83437423,"identity":"fb379485-02e9-46b4-95a7-0908f0d1aa13","added_by":"auto","created_at":"2025-05-26 08:47:04","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1214050,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"Graph.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6652808/v1/130026e5c11ec72ed64e519c.jpeg"},{"id":83436062,"identity":"c45ded10-4b57-4962-a727-88a1f2d0d492","added_by":"auto","created_at":"2025-05-26 08:31:04","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":7435714,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-6652808/v1/94e5d2a64bd2cd27c53c6f56.docx"}],"financialInterests":"","formattedTitle":"Gut microbiota derived L-ornithine promotes resistance to obesity through metabolites mediated immunosuppressive macrophages","fulltext":[{"header":"Introduction","content":"\u003cp\u003eObesity is a global epidemic and a major risk factor for cardiovascular disease, stroke and all-cause mortality [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. So far, the method of prevention and treatment of obesity is unsatisfactory. The prevalence of obesity is still increasing in many populations around the world. It is urgent to look for an effective method to prevent and therapy against obesity. Studies have shown that gut microbiota can effectively affect the occurrence and development of obesity [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. But the exact mechanism(s) by which can prevent obesity still is incompletely clear.\u003c/p\u003e \u003cp\u003eChronic low-grade inflammation is characteristic of obese tissue [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdipose tissue macrophages (ATMs) play an important role in chronic adipose tissue inflammation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Single-cell RNA sequences (sc-RNA Seq) divide macrophages in adipose tissue into multiple subpopulations [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], suggesting that macrophage subpopulations constitute a continuous cellular state, some of which are more pro-inflammatory while others are more metabolically active. However, inflammatory macrophage (IM) subpopulations and inflammatory gene programs has been shown to be positively correlated with obesity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. IMs are involved in complex paracrine and autocrine signaling to maintain a pro-inflammatory microenvironment [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Key signaling pathways in IMs include the NF-κB and AKT signaling pathways, which play a key role in regulating macrophage function in adipose tissue, especially affecting inflammation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In addition, lipopolysaccharide (LPS) from the gut microbiota can stimulate macrophages to produce pro-inflammatory mediators such as TNF-α, IL-6, IL-1β, and nitric oxide (NO) via TLR4, as well as M1 macrophage-associated genes (TNFα, IL-6, IL-1β, iNOS, and MCP1), and also amplify inflammation via NF-κB [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe gut microbiota can resist inflammation in individuals through its metabolites [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It regulates immunity by releasing ligands and metabolites that are transferred from the gut to local and systemic circulation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Disturbances of gut microbiota are considered to be characteristic of a variety of metabolic diseases that can affect host metabolic and immune function [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. They can increase gram-negative bacteria and raises LPS levels. LPS binds TLR4 on macrophages and triggers M1 polarization and systemic inflammation, leading to insulin resistance. Regardless, microbial metabolites (such as short-chain fatty acids (SCFA)) promote anti-inflammatory M2 macrophages, enhance insulin sensitivity, and reduce inflammation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Gut bacteria can metabolize primary bile acids into secondary forms that activate receptors on macrophages (FXR, TGR5) and inhibit pro-inflammatory M1 polarization [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Here we demonstrate that gut microbiota lactobacillus derived L-orn, which is associated with enteric human homologous protein REG3α, protects against obesity through L-orn metabolite mediated anti-inflammatory macrophages.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eReagents and oligoes used in this study were listed in supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMice, huREG3α transgenic mice\u003c/h2\u003e \u003cp\u003eFour-to six-week-old male or female C57BL/6 mice were obtained from Nanjing Animal Center. Human REG3α transgenic mice ( huREG3αtg) mice were prepared by Nanjing Animal Center [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. All experimental mice were bred and maintained under specific pathogen-free conditions in the Animal Center of Nankai University. Experiments were carried out using age- and gender- matched mice. All procedures were conducted according to the Institutional Animal Care and Use Committee of the Model Animal Research Center. Animal experiments were approved by the Institute\u0026rsquo;s Animal Ethics Committee of Nankai University. All experimental variables such as husbandry, parental genotypes and environmental influences were carefully controlled.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMouse models\u003c/h3\u003e\n\u003cp\u003eFor high-fat diet model, 6- to 8-week-old male mice and their control littermates were fed using high-fat diet (D12492, protein 26.2%, carbohydrate 26.3%, and fat 34.9%) and control diets (D12450B, 60% of calories may be derived from fat), which was from Research Diets, Inc. (NJ, USA).\u003c/p\u003e \u003cp\u003eFor lactobacillus transplantation, mice were first treated with ampicillin (A, 1 g/L, Sigma), vancomycine (V, 0.5g/L), neomycin sulfate (N, 1 g/L), and metronidazole (M, 1g/L) via the drinking water for two weeks. To confirm the elimination of bacteria, stools were collected from antibiotic-treated and untreated mice and cultured in anaerobic and aerobic condition. Mice were orally administered 200 \u0026micro;l of 1\u0026times;10\u003csup\u003e9\u003c/sup\u003e lactobacillus (once/week).\u003c/p\u003e \u003cp\u003eFor administering L-orn, eflornithine (DFMO), SPM and SPD infusion, mice were randomly assigned to two different treatment groups (n\u0026thinsp;=\u0026thinsp;6/group), and then mice were administered in drinking distilled H\u003csub\u003e2\u003c/sub\u003eO. The mean L-Orn, SPM and SPD consumption of mice was \u0026sim;3.3 g/kg/d [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The mean DFMO consumption of mice was \u0026sim;1.5 g/kg/d. Mice fed with H\u003csub\u003e2\u003c/sub\u003eO without L-orn, SPM, SPD and DFMO were used as control.\u003c/p\u003e \u003cp\u003ePreparation of chimeric mice. Recipient mice were irradiated with a single dose of 8 Gy of irradiation using a Shepherd Mark I caesium irradiator (J.L. Shepherd and Associates). Then, bone marrow cells (BMCs) collected from wild-type (WT) or \u003cem\u003eIDO-1 KO\u003c/em\u003e mice were subsequently injected into the irradiated WT \u003cem\u003eor IDO-1 KO\u003c/em\u003e recipient mice (2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per mouse) via the tail vein.\u003c/p\u003e\n\u003ch3\u003eHuman samples\u003c/h3\u003e\n\u003cp\u003eFor human serum collection, 208 adult participants, among which 42 with BMI\u0026thinsp;\u0026lt;\u0026thinsp;18.5 kg/cm2, 41 with BMI 18.5\u0026ndash;24 kg/cm2, 43 with BMI 24\u0026ndash;27 kg/cm2, 42 with BMI 27\u0026ndash;30 kg/cm2 and 40 with BMI\u0026thinsp;\u0026ge;\u0026thinsp;30 kg/cm2 were selectively recruited. More than half (54.3%) were males and the mean age was 42 years (SD\u0026thinsp;=\u0026thinsp;13 years). The mean BMI was 25.05\u0026thinsp;\u0026plusmn;\u0026thinsp;5.07 kg/cm2. All participants were free of acute stress conditions such as fever and diarrhea. Height and weight were measured to the nearest 0.1cm and 0.1 kg without shoes or heavy clothing using a calibrated stadiometer (GL-310, Seoul, Korea). Participants were instructed to fast for \u0026ge;\u0026thinsp;12 hours before blood sampling in the next morning. This study was conducted with approval from the Institutional Review Board at Nankai University, Tianjin Union Medical Center and Tianjin First Central Hospital. Participants were recruited from the health screening centers of Tianjin Union Medical Center and Tianjin First Central Hospital. All participants provided written informed consent.\u003c/p\u003e\n\u003ch3\u003eGut microbiota analyses\u003c/h3\u003e\n\u003cp\u003eGut microbiota was analyzed by Majorbio Biotechnology Company (Shanghai, China) using primers that target to the V3-V4 regions of 16S rRNA according to previous reported method [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The samples were clustered at genus and OTU levels using the sample-genus and sample-OTU count matrices respectively. For each clustering, Morisita-Horn dissimilarity was used to compute a sample distance matrix from the initial count matrix, and the distance matrix was subsequently used to generate a hierarchical clustering using Ward\u0026rsquo;s minimum variance method. The Wilcoxon Rank Sum test was used to identify OTUs that had differential abundance in the different sample groups.\u003c/p\u003e \u003cp\u003eFor the absolute numbers of gut bacteria, 16s rRNAs were extracted, and then amplified using genus or strain specific primers. The concentration of each product was detected and then exchanged into copy numbers using a standard curve.\u003c/p\u003e\n\u003ch3\u003eLactobacillus isolation and culture\u003c/h3\u003e\n\u003cp\u003eFor lactobacillus isolation, 100 mg fresh faecal samples were collected and diluted in 2 ml BPS solution, and cultured on Rogosa SL selective medium (Sigma-Aldrich) for lactobacillus enumeration, and then colonies were identified and purified using 16s ribosomal RNA sequence analyses. Lactobacilli were cultured in deMan, Rogosa, Sharpe (MRS; 3 M Health Care, St. Paul, MN) media and also grown on MRS agar containing 10% sucrose. Anaerobic conditions were generated with the sachets of AnaeroPack-Anaero (Mitsubishi Gas Chemical, Japan) in an air-tight jar. After 24 h of cultivation in liquid media, lactobacilli could reach 1 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e CFU/ml.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEx vivo macrophages stimulation\u003c/h2\u003e \u003cp\u003ePeritoneal macrophages were collected on day 3 after intraperitoneal injection of 2\u003c/p\u003e \u003cp\u003emL/mouse 4% w/v thioglycolate. Peritoneal macrophages isolated from C57BL/6\u003c/p\u003e \u003cp\u003emice were cultured overnight in 6-well microplates with 1640 containing 10% FBS,\u003c/p\u003e \u003cp\u003eat 37\u0026deg;C in a humidified 5% CO 2 atmosphere, and then L-orn, SPM or SPD was added into culture at the indicated concentration and time, and expression of cytokine was analyzed using qRT-PCR, RNA-seq or ELISA.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRNA-seq analysis\u003c/h3\u003e\n\u003cp\u003eRNA-seq analyses were done according to reported method [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Based on the manual of TRIzol\u0026reg;reagent (Invitrogen, Shanghai, China), total RNAs from cells were extracted. Library was prepared and transcriptome was sequenced on an MGISEQ-2000 platform to generate 100-bp paired-end reads. Data were analyzed on \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://biosys.bgi.com\u003c/span\u003e\u003cspan address=\"http://biosys.bgi.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eFlow cytometry analyses\u003c/h3\u003e\n\u003cp\u003eFor adipose tissue (AT) digestion, transfer AT to 50 ml conical tubes by pouring the homogenate and rinsing the weigh boat with 1 ml DPBS (0.5% BSA) and 3 ml collagenase II digest solution. Incubate AT homogenate in a rotational shaker (200 rpm) at 37\u0026deg;C for 20 min. Add 10 ml DPBS (0.5% BSA) to conical tubes and place on ice. Triturate homogenate numerous times using a 10 ml serological pipette, and pass cell suspensions through 100 \u0026micro;m filter in to a new 50 ml conical tube. Centrifuge cell suspension at 500 \u0026times; g for 10 min at 4\u0026deg;C. Decant supernatant and resuspend SVF cell pellet in 3 ml ACK buffer to lyse contaminating erythrocytes. Add 12 ml FACS buffer and centrifuge cell suspension at 500 \u0026times; g for 10 min at 4\u0026deg;C. Decant supernatant and resuspend SVF cell pellet in FACS buffer. The cells were collected at the interphase of the Percoll gradient, washed and resuspended in medium, and then stained and analyzed by flow cytometry. Dead cells were eliminated through 7-AAD staining.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eL-ornithine analyses\u003c/h2\u003e \u003cp\u003eL-orn concentration were detected using the Ion Chromatography system. Serum samples were extracted in 5% trichloroacetic acid (TCA, typically by mixing with equal volume of 10% TCA) by vigorous vortexing. Serum extracts were centrifuged at 13 000 rpm in table top micro centrifuge. 100 mg adipose tissues were thawed and minced in 5% TCA. The mixture was homogenized by sonication (Fisher Sonic Dismembrator Model 300). Tissue homogenates were centrifuged in a micro-centrifuge at 10 000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min at 4\u0026deg;C. 100 mg fresh faecal samples were collected and extracted in 5% TCA (typically by mixing with equal volume of 10% TCA) by vigorous vortexing. The supernatants were extracted twice with diethyl ether, after which the extracts were diluted in water (1:9) before being applied to the column, and eluted by increasing concentration of methanesulfonic acid (3\u0026ndash;45 mM). L-orn were detected with an on-line ion conductivity detector coupled to an SRS 300 suppressor for cations (which eliminates all anions including the mobile phase ion, methanesulfonic acid, allowing only cations to be detected). Quantification of L-orn was done by measuring the (conductivity in \u0026micro;Siemens) peak areas of individual L-orn based on the standard curve derived from analyzing a dilution series of L-orn.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eOthers\u003c/h2\u003e \u003cp\u003eELISA, Western blotting, RT-PCR and qRT-PCR were performed according to our previous methods[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eUnpaired Student\u0026rsquo;s t test, two \u0026ndash;ways ANOVA and one-way ANOVA followed by Tukey post hoc test were used to determine significance. A 95% confidence interval was considered significant and was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. * indicates p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHuREG3α\u003csup\u003etgIEC\u003c/sup\u003e mice are resistant to high-fat diet mediated obesity\u003c/h2\u003e \u003cp\u003eOur previous studies showed that mice with intestinal epithelial cells expressing human REG3α (huREG3α\u003csup\u003etgIEC\u003c/sup\u003e mice) were resistant to DSS mediated colitis by altering gut microbiota [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Since the occurrence and development of obesity is also associated with alterations of the gut microbiota [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], we adopted a high-fat diet (HFD) mediated obesity model to investigate the effect of human REG3α expressed in mouse intestinal epithelial cells on obesity. Compared with control wild-type (WT) mice, male and female huREG3α\u003csup\u003etgIEC\u003c/sup\u003e mice were observed to had significantly lower body weight and fat pad weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, C), suggesting that huREG3α\u003csup\u003etgIEC\u003c/sup\u003e mice are resistance to HFD-mediated obesity. Insulin sensitive and glucose tolerance tests also showed that huREG3α\u003csup\u003etgIEC\u003c/sup\u003e mice were less sensitive to insulin and less tolerant to glucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Inflammatory macrophages (F4/80\u003csup\u003e+\u003c/sup\u003e, CD11C\u003csup\u003e+\u003c/sup\u003e, INOS\u003csup\u003e+\u003c/sup\u003e) and immunosuppressive macrophages (F4/80\u003csup\u003e+\u003c/sup\u003e, CD206\u003csup\u003e+\u003c/sup\u003e, and arginine (Arg)-1\u003csup\u003e+\u003c/sup\u003e) are associated with the onset and progression of obesity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Data showed a significant decrease in inflammatory macrophages and an increase in anti-inflammatory macrophages in the adipose tissues of huREG3α\u003csup\u003etgIEC\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Inflammatory cytokines also markedly decreased in the adipose tissue of huREG3α\u003csup\u003etgIEC\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Notably, there showed an increased IDO-1\u003csup\u003e+\u003c/sup\u003e macrophages in huREG3α\u003csup\u003etgIEC\u003c/sup\u003e mice as compared to control WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), imply that this macrophage subpopulation might also play a role in resistance to HFD-mediated obesity in huREG3α\u003csup\u003etgIEC\u003c/sup\u003e mice. In addition, when fed normally, the mice showed no differences in weight, insulin sensitivity and glucose tolerance (supplementary Figure S2). Taken together, huREG3α\u003csup\u003etgIEC\u003c/sup\u003e mice are resistant to HFD mediated obesity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eThere were high levels of L-ornithine in adipose tissues of huREG3α\u003csup\u003etgIEC\u003c/sup\u003e mice\u003c/h2\u003e \u003cp\u003eHuREG3α\u003csup\u003etgIEC\u003c/sup\u003e-associated gut microbiota plays a role in resistance to DSS- mediated colitis in huREG3α\u003csup\u003etgIEC\u003c/sup\u003e mice [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. REG3, an antimicrobial peptide secreted by intestinal epithelial cells, can directly kill bacterium [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], which potentially induce dysregulation of gut microbiota [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Here we again analyzed the composition of gut microbiota and further confirmed the previous results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, supplementary Figure S3). Interestingly, lactobacillus numbers were significantly increased not only in huREG3α\u003csup\u003etgIEC\u003c/sup\u003e mice, but also in HFD fed huREG3α\u003csup\u003etgIEC\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, supplementary Figure S3), implying that increased lactobacillus may play a role in the resistance of huREG3α\u003csup\u003etgIEC\u003c/sup\u003e mice to HFD-mediated obesity. Thus, we further analyzed the composition of lactobacillus in HFD fed HuREG3α\u003csup\u003etgIEC\u003c/sup\u003e mice. The increased lactobacillus in human HuREG3α\u003csup\u003etgIEC\u003c/sup\u003e was named \u003cem\u003eLactobacillus NK2\u003c/em\u003e (L. NK2) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Interestingly, L. NK2 also significantly increased in HFD fed HuREG3α\u003csup\u003etgIEC\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). It is worth noting that REG3 may kill some gram-positive bacteria. But, gram-positive lactobacillus are not sensitive to REG3 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. We previously found that L. NK2 could produce large amounts of L-orn [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Data here showed that HFD fed huREG3α\u003csup\u003etgIEC\u003c/sup\u003e mice not only had increased L-orn in stool and serum, but also in adipose tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Thus, L-orn is significantly elevated in the adipose tissue, peripheral blood and stool of HFD-fed huREG3α\u003csup\u003etgIEC\u003c/sup\u003e mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eGut microbiota derived L-ornithine is involved in resistant to HFD mediated obesity\u003c/h2\u003e \u003cp\u003eChronic inflammation is characteristic of obese tissue [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Since L-orn has anti-inflammatory effects [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], this suggests that lactobacillus-derived L-orn may be involved in resistance to HFD-mediated obesity. Next, we investigated the effects of L-orn on obesity. The data showed that L-orn-fed mice also showed significant resistance to HFD-mediated obesity and reduced sensitivity to insulin and tolerant to glucose compared to control mice (supplementary Figure S4A, C). L-orn-fed mice also had a lighter fat pat weights (supplementary Figure S4B). Anti-inflammatory F4/80\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e and F4/80\u003csup\u003e+\u003c/sup\u003eIL-10\u003csup\u003e+\u003c/sup\u003e macrophages significantly increased in adipose tissue of L-orn-fed mice (supplementary Figure. S4D). Thus, these data suggest that L-orn can protect against HFD mediated obesity.\u003c/p\u003e \u003cp\u003eTo further determine the effect(s) of gut microbiota derived L-orn on obesity, we also used \u003cem\u003eLactobacillus reuteriΔOTC\u003c/em\u003e (MutLac) which does not produce L-orn (22) to further investigate the role of L-orn in obesity. Arginine can be metabolized into L-orn in the gut microbiota via catabolic pathways such as arginine deiminase pathway (ADI) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Mice colonized with \u003cem\u003eL. reuteri\u003c/em\u003e which could produce L-orn (22), showed significant resistance to HFD mediated obesity and reduced sensitivity to insulin and tolerant to glucose compared to mice colonized with MutLac (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). MutLac colonized mice were heavier in body weight as compared with control mice colonized \u003cem\u003eL. reuteri\u003c/em\u003e after feeding HFD (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The data also showed a significant decrease in the number of anti-inflammatory F4/80\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e, F4/80\u003csup\u003e+\u003c/sup\u003eIL-10\u003csup\u003e+\u003c/sup\u003e, and F4/80\u003csup\u003e+\u003c/sup\u003eArg-1\u003csup\u003e+\u003c/sup\u003e macrophages in adipose tissue of the mice colonized with MutLac while a significant increase could be detected in inflammatory macrophages (F4/80\u003csup\u003e+\u003c/sup\u003eCD11C\u003csup\u003e+\u003c/sup\u003e and F4/80\u003csup\u003e+\u003c/sup\u003eTNFa\u003csup\u003e+\u003c/sup\u003e) (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). Analysis of inflammatory cytokines in adipose tissue also showed that MutLac colonized mice had significantly higher levels of inflammatory cytokines in the adipose tissues than those in \u003cem\u003eL. reuteri\u003c/em\u003e colonized mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Again, there had more IDO-1\u0026thinsp;+\u0026thinsp;macrophages in the adipose tissues of mice colonized \u003cem\u003eL. reuteri\u003c/em\u003e than MutLac (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). L-orn concentration in the adipose tissues, peripheral sera and feces of MutLac colonized mice was lower than in \u003cem\u003eL. reuteri\u003c/em\u003e colonized mice or WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). These data suggest that MutLac can reduce the ability of lactobacillus to resist HFD mediated obesity. Taken together, lactobacillus derived L-orn plays a key role in the resistance to HFD mediated obesity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eGut microbiota L-ornithine reduces macrophage inflammation\u003c/h2\u003e \u003cp\u003eAnti-inflammatory macrophages increased significantly in L-orn fed mice, suggesting that the effect of L-orn on obesity may be through reducing macrophage inflammation. Since obesity is associated with LPS-mediated inflammation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], we next looked at the role of L-orn in LPS-mediated inflammatory cytokines. The data showed that inflammatory cytokines were significantly reduced after exposure to L-orn (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). RNA-seq analysis showed that macrophage inflammation genes treated with L-orn were significantly reduced upon exposure to LPS (supplementary Figure S5). Several downregulated inflammatory signaling pathways in L-orn treated macrophages were also observed, including TLR, NOD-like receptor signaling, cytokine-cytokine receptor, and IL-17 signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), which are related to NF-κB and AKT [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Indeed, L-orn not only failed to activate NF-κB and AKT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), but also effectively inhibited AKT activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) and LPS-mediated NF-κB activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). L-orn was able to further inhibit the production of inflammatory cytokines, which could be saved by the L-orn inhibitor difluoromethylornithine (DFMO) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). In addition, L-orn significantly also promoted the expression of CD206, which are expressed by immunosuppressive macrophages, suggesting that L-orn can also promote the differentiation of immunosuppressive macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Taken together, L-orn can significantly inhibit intracellular AKT activity and LPS-mediated NF-κB activity to reduce inflammatory cytokines in macrophages.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eDecreased inflammation is derived from L-ornithine metabolite SPM in macrophages\u003c/h2\u003e \u003cp\u003eSpermine is a polyamine synthesized from ornithine via the polyamine pathway (ornithine\u0026rarr;humutine\u0026rarr;spermidine\u0026rarr;spermine) that plays a key role in adipose tissue biology (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Therefore, we next investigated the effects of L-orn metabolite SPM on macrophage-mediated inflammation. When macrophages were exposed to SPM, SPM alone did not affect phosphorylation of AKT and NF-κB (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), while LPS did (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Notably, SPM could inhibit LPS-mediated phosphorylation of NF-κBp65 and AKT, and reduce inflammatory cytokines; whereas SPD, another intracellular metabolite of L-orn, did not do so (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). We also further analyzed whether SPM could also inhibit HFD mediated obesity. After feeding SPM, SPM not only inhibited the development of obesity, including reduced body weight and fat pads, reduced sensitivity to insulin and tolerant to glucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-G), but also reduced inflammatory cytokines in obese tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Thus, L-orn metabolite SPM can inhibit inflammatory macrophages to resist to obesity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eL-ornithine metabolite SPD mediated IDO-1 macrophages are involved in resistance to obesity\u003c/h2\u003e \u003cp\u003ePrevious studies have shown that L-orn metabolite SPD is able to reprogram mouse conventional dendritic cells to an immunomodulatory phenotype through Src kinase-dependent phosphorylation of IDO-1 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The data show that SPD, but not SPM, could cause Src phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). As controls, L-orn also promoted the phosphorylation of Src (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). Importantly, SPD could also promote the phosphorylation of IDO-1(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) and the expression of IDO-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-E). Furthermore, SPD mediated IDO-1 was a time- and dose- dependent (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-E). IDO-1 is a key regulator of immune homeostasis, balance tolerance, and inflammation. Src may regulate IDO-1 through transcriptional control, such as Src-dependent signaling pathways that enhance IDO-1 expression and post-translational modification, and Src-mediated phosphorylation that also regulates IDO-1 enzyme activity [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Next, we investigated the effect of IDO-1\u003csup\u003e+\u003c/sup\u003e macrophages on obesity by establishing a macrophage transplantation model. Data showed that mice transplanted with IDO-1 knockout (KO) macrophages were more sensitive to HFD mediated obesity. These transplanted mice not only had increased body and Fat pad weights, but also had reduced glucose tolerance and insulin sensitivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-H). Thus, IDO-1\u003csup\u003e+\u003c/sup\u003e macrophages are required for L-orn mediated resistance to HFD mediated obesity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eGut microbiota L-ornithine is related to obesity in humans\u003c/h2\u003e \u003cp\u003eFinally, we investigated the potential effects of gut microbiota derived L-orn on human obesity. We first studies the relevance of gut microbiota\u0026ndash;derived L-orn to the body weights of 208 individuals with different body mass indices (BMIs), which were used previously [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Lower levels of L-orn were observed in overweight and obese individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Furthermore, there existed a negative relationship between the concentrations of the microbiota metabolite L-orn and BMIs of individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), implying that L-orn potentially was related to the resistant role in the occurrence and development of obesity in human.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next also used published sc-RNA seq data [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] to analyze the signal pathway in the IM of adipose tissues in patients with obesity. There indeed had increased NF-κB and AKT signal pathways, and decreased Src pathways in the IM of adipose tissues of patients with obesity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D). Notably, there also had upregulated inflammatory signal pathways in IM of adipose tissues of patients with obesity as compared to the IM in lean individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE), suggesting the increased inflammatory responses in the IM of adipose tissues of patients with obesity.\u003c/p\u003e \u003cp\u003eWe finally investigated the effects of the SPM on the NF-κB and inflammatory cytokines, and roles of the SPD in the IDO expression and Src phosphorylation in human macrophages. Data exhibited that SPM but not SPD could affect the activation of NF-κB and reduce the expression of inflammatory cytokines, and SPD but not SPM could induce the phosphorylation of Src (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF, G). SPD also promoted the differentiation of IDO\u003csup\u003e+\u003c/sup\u003e macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). Taken together, similar to the effects of L-orn on the macrophages in mice, obesity also is related to gut microbiota derived L-orn in humans.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe demonstrate here that gut microbiota derived L-orn, which is associated with human REG3α, can protect against HFD mediated obesity through SPM mediated anti-inflammatory and SPD-induced IDO-1 macrophages. SPM can inhibit NF-κB and AKT to reduce inflammatory cytokines in the macrophages, while SPD can promote the expression and phosphorylation of IDO-1 to cause IDO-1 immunosuppressive macrophages, thereby inhibiting HFD mediated obesity. Importantly, we also found that BMI in obese patients was inversely associated with serum L-orn. Inflammatory macrophages from human obese tissue also exhibit enhanced NF-κB and AKT and reduced Src signaling pathways These results provide a strong foundation for L-orn as a tool for preventing and treating obesity.\u003c/p\u003e \u003cp\u003eGut microbiota derived L-orn can protect against HFD mediated obesity through metabolites mediated immunosuppressive macrophages. Previous studies also found that L-orn was related to obesity [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Our data showed negative relationship between L-orn and obesity. L-arginine, which can be metabolized into L-orn in gut microbiota contributes significantly to reducing inflammation and infection complications [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In preclinical models, dietary supplementation with L-arginine improves and faster resolution of DSS-induced colitis [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. However, it is not fully understood how L-orn reduces inflammation. Here we demonstrate that gut microbiota L-orn metabolite SPM can reduce the expression of inflammatory cytokines via inhibiting NF-κB and AKT. Other studies also found that SPM can suppress the immune response of activated macrophages by inhibiting the expression of NOS2 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In a mouse model of acute liver injury, SPM also induced M2 polarization in tumor-associated macrophages [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Increased SPM also inhibited NLRP3 inflammasome assembly and subsequent pyroptosis by inhibiting K\u003csup\u003e+\u003c/sup\u003e efflux. In addition, SPM also significantly reduced p-JAK1, p-tyrosine kinase 2 (TYK2), p-STAT1, and p-STAT2 after IFN-β stimulation [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In diet-induced obesity mouse models, large daily doses of SPM are an effective strategy for weight loss and improved glucose status [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSpermidine is a central polyamine synthesized from putrescine that plays a key role in adipose tissue homeostasis, affecting metabolism, inflammation and cellular resilience. Our data show that SPD plays a role in resistance of mice to HFD mediated obesity via Src/IDO-1 mediated IDO-1immunosuppressive macrophages. Indeed, IDO-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice fed HFD gained less weight, had lower fat mass, and had better glucose and insulin resistance as compared to WT mice [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Loss or inhibition of IDO-1 improved insulin sensitivity, protected the intestinal mucosal barrier, reduced endotoxemia and chronic inflammation, and regulated lipid metabolism in liver and adipose tissue [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. SPD could also confer a tolerance phenotype on conventional dendritic cells, which depends on the expression of IDO-1 and the activity of Src kinase. Interestingly, activation of Src and IDO-1 was also detected in SPD-treated macrophages after exposure to L-orn [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Inhibitors of Src (e.g., dasatinib) or IDO-1 (e.g., epacadostat) also showed potential in preclinical models to reduce adipose inflammation and improve insulin sensitivity [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNumerous studies have endeavored to identify the microbiota signatures associated with obesity [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], including decreased diversity, altered levels of specific bacterial taxa (e.g., \u003cem\u003eAkkermansia\u003c/em\u003e spp., \u003cem\u003eChristensenella\u003c/em\u003e spp., \u003cem\u003eBacteroides\u003c/em\u003e spp., \u003cem\u003ePrevotella\u003c/em\u003e spp. \u003cem\u003eBlautia\u003c/em\u003e spp., etc.) and also changes in metabolic pathways or products. Some gut bacteria such as \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eEnterococcus\u003c/em\u003e, which utilize the ADI to convert dietary or host-derived arginine into L-orn, have shown strong potential in fighting obesity-related inflammation and metabolic issues [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Studies in animals also demonstrate that lactobacillus can reduce body weight, fat, and inflammation [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. These results suggest that lactobacillus could be an effective way to manage obesity and related health problems [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eSupplementary information\u003c/h2\u003e \u003cp\u003eThe online version contains supplementary material available at:\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConflict of interests\u003c/strong\u003e \u003cp\u003eThe authors have no relevant financial or nonfinancial\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthics approval\u003c/h2\u003e \u003cp\u003e The animal study protocol was approved by the Laboratory AnimalWelfare and Animal Experiment Ethics Review Committee of Nankai University (Approval Number: NK-20190912).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication.\u003c/strong\u003e \u003cp\u003eAll authors agree to the publication of this study.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported by NSFC grants 91842302, 82271779, 81901677, 31470876, 91629102, ISF-NSFC program 31461143010; Tianjin science and technology commission (18JCZDJC35300); CAMS Innovation Fund for Medical Science (CIFMS2017-12M-2-005); a Ministry of Science and Technology grant (2016YFC1303604); the State Key Laboratory of Medicinal Chemical Biology; The Fundamental Research Funds for the Central University, Nankai university(Grant number 63191724).\u003c/p\u003e \u003cp\u003e \u003cb\u003eData Availability\u003c/b\u003e Raw 16S rRNA gene sequence data for the feces microbiota were deposited in the NCBI Short Read Archive under BioProject Accession Number PRJNA326574.\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eR. Y. designed the research and wrote the paper; Y. L, J. W., Y. G., conducted \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments; Y. Z offered an assistance to the experiments.All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgment\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFont-Burgada J, Sun B, Karin M (2016) Obesity and Cancer: The Oil that Feeds the Flame. Cell Metabolism 23(1): 48-62.\u003c/li\u003e\n\u003cli\u003eGrover, Steven A, Kaouache, Mohammed, Rempel, Philip, Joseph, Lawrence, Dawes, Martin (2015) Years of life lost and healthy life-years lost from diabetes and cardiovascular disease in overweight and obese people: a modelling study. Lancet Diabetes Endocrinol 3(2): 114-122.\u003c/li\u003e\n\u003cli\u003eDeehan EC, Mocanu V, Madsen KL (2024) Effects of dietary fibre on metabolic health and obesity. 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Adv Nutr 16(2): 100362. https://doi.org/10.1016/j.advnut.2024.100362\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"L-ornithine, obesity, spermine, spermindine, Indoleamine 2, 3-dioxygenase 1","lastPublishedDoi":"10.21203/rs.3.rs-6652808/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6652808/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGut microbiota can affect the occurrence and development of obesity. But the exact mechanism(s) by which obesity is prevented is still not fully understood. In this study, we found that L-ornithine (L-orn) from the gut microbiota lactobacillus helps mice to resist to high-fat diet (HFD) mediated obesity through its metabolite spermine (SPM) and spermindine (SPD) in the macrophages. SPM reduced inflammatory cytokines in the macrophages by inhibiting NF-κB and AKT (protein kinase B) signal pathways, while SPD activated Src and induced indoleamine 2, 3-dioxygenase 1 (IDO-1) to promote immunosuppressive IDO-1 macrophages. Notably, L-orn was inversely associated with body mass index (BMI) in obese patients. Sc-RNA sequencing data also showed that the NF-κB and AKT pathways were significantly up-regulated and the Src signaling pathway was significantly down-regulated in the inflammatory macrophages of adipose tissues. Thus, our results suggest that gut microbiota derived L-orn can control the occurrence and development of obesity through metabolites mediated anti-inflammatory macrophages.\u003c/p\u003e","manuscriptTitle":"Gut microbiota derived L-ornithine promotes resistance to obesity through metabolites mediated immunosuppressive macrophages","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-26 08:30:59","doi":"10.21203/rs.3.rs-6652808/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2025-06-14T03:07:08+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-05-20T09:41:31+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-20T09:36:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-14T13:26:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2025-05-13T18:59:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"68a896db-c402-4acb-a4c6-1174c84cff97","owner":[],"postedDate":"May 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:09:26+00:00","versionOfRecord":{"articleIdentity":"rs-6652808","link":"https://doi.org/10.1007/s00018-025-05882-8","journal":{"identity":"cellular-and-molecular-life-sciences","isVorOnly":false,"title":"Cellular and Molecular Life Sciences"},"publishedOn":"2025-11-26 15:57:42","publishedOnDateReadable":"November 26th, 2025"},"versionCreatedAt":"2025-05-26 08:30:59","video":"","vorDoi":"10.1007/s00018-025-05882-8","vorDoiUrl":"https://doi.org/10.1007/s00018-025-05882-8","workflowStages":[]},"version":"v1","identity":"rs-6652808","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6652808","identity":"rs-6652808","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}