IL4I1-catalyzed tryptophan metabolites mediate the anti-inflammatory function of cytokine-primed human muscle stem cells

IL4I1-catalyzed tryptophan metabolites mediate the anti-inflammatory function of cytokine-primed human muscle stem cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article IL4I1-catalyzed tryptophan metabolites mediate the anti-inflammatory function of cytokine-primed human muscle stem cells Changshun Shao, Muqiu Zuo, Jiankai Fang, Peiqing Huang, Shisong Liu, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2618290/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Jul, 2023 Read the published version in Cell Death Discovery → Version 1 posted 9 You are reading this latest preprint version Abstract Muscle stem cells (MuSCs) have been demonstrated to exert impressive therapeutic efficacy in disease settings through orchestrating inflammatory microenvironments. Nevertheless, the mechanisms underlying the immunoregulatory property of MuSCs remain largely uncharacterized. Here, we showed that interleukin-4-induced-1 (IL4I1), an essential enzyme that catalyzes indole metabolism in humans, was highly expressed in human MuSCs exposed to IFN-γ and TNF-α. Functionally, the MuSCs were found to inhibit the infiltration of neutrophils into sites of inflammation in a IL4I1-dependent manner and thus ameliorate acute lung injury in mice. Mechanistically, the indole metabolites, including indole-3-pyruvic acid (I3P) and indole-3-aldehyde (I3A), produced by IL4I1, acted as ligands to activate aryl hydrocarbon receptor (AHR), leading to augmented expression of TNF-stimulated gene 6 (TSG-6) in inflammatory cytokine-primed MuSCs. Furthermore, I3P administration alone suppressed neutrophil infiltration in damaged lungs. I3P could also reduce the level of reactive oxygen species in neutrophils. Therefore, our study has uncovered a novel mechanism by which MuSCs acquire their immunoregulatory property and may help to develop or optimize MuSC-based therapies for inflammatory diseases. Biological sciences/Immunology/Immunotherapy/Immunosuppression Biological sciences/Stem cells/Muscle stem cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Muscle stem cells (MuSCs), also known as satellite cells, reside in an unique niche between the muscle sarcolemma and the basal lamina of individual myofibres, and contribute to the maintenance and repair of tissue architectural structures through both self-renewal and myogenic differentiation [ 1 , 2 ]. While MuSCs are essential for muscle tissue regeneration, recent evidence has shown that MuSCs also act as critical regulators in the resolution of inflammation through releasing various paracrine factors [ 3 ]. This paracrine immunomodulatory property of MuSCs is akin to what has been well established in mesenchymal stem cells (MSCs) [ 4 , 5 ]. As an example, MuSCs can act on maturing macrophages and confer them with oxidative phosphorylation-dependent anti-inflammatory properties via insulin-like growth factor-2 (IGF-2), thus ameliorating dextran sulfate sodium (DSS)-induced colitis [ 6 ]. In addition, MuSCs primed with inflammatory cytokines also reduce inflammation and promote tissue repair by producing TNF-stimulated gene 6 (TSG-6) [ 7 ]. The induction of TSG-6 by inflammatory cytokines is mediated by tryptophan metabolites, including kynurenine (KYN) and kynurenic acid (KYNA), that are catalyzed by indoleamine 2,3-dioxygenase (IDO). IGF-2 and TSG-6 function as effectors of immunoregulation for both MuSCs and MSCs. Whether or not MuSCs can acquire their immunomodulatory property via pathways distinct from those in MSCs has not been explored. Interleukin-4-induced-1 (IL4I1), a L-amino acid oxidase, was very recently identified as a novel potential therapeutic target of cancer treatment [ 8 ]. IL4I1 promoted aryl hydrocarbon receptor (AHR)-driven malignant properties and suppressed anti-tumor immunity, even in the presence of immune checkpoint blockade (ICB) and IDO1 inhibition [ 9 ]. Specifically, indole-3-pyruvic acid (I3P) and indole-3-aldehyde (I3A) catalyzed by IL4I1 in tumor activated AHR signaling through receptor-ligand binding, leading to increased motility of cancer cells and diminished proliferation of CD8 + T cells [ 9 ]. Unlike arginase and IDO that function intracellularly, IL4I1, as a secretory enzyme, can modulate the functions of neighboring immune cells in the inflamed tissue microenvironment [ 10 ]. On the one hand, IL4I1 can directly reduce the stability of immune synapses between T cell and dendritic cells (DCs), thereby elevating the threshold of T cell activation and thus dampening their activation [ 11 ]. IL4I1 also regulates the polarization of macrophages, as reflected by the progressive infiltration of anti-inflammatory macrophages and the displacement of proinflammatory macrophages when IL4I1 was overexpressed [ 12 ]. In adaptive immunity, IL4I1 can suppress the proliferation of TH17 cells and promote the differentiation and activity of CD4 + CD25 + FOXP3 + regulatory T cells [ 13 , 14 ], thus improving the neurological severity score and alleviating experimental autoimmune encephalomyelitis in vivo [ 15 ]. Thus, there is growing evidence that IL4I1 is a vital metabolic enzyme mediating the immunosuppressive effects in disease settings. TSG-6, a 30-kDa secreted glycoprotein, influences intercellular 3D structures and remodels the extracellular matrix (ECM) through binding with hyaluronic acid, chondroitin sulfate and proteoglycan [ 16 ]. More importantly, it possesses the prominent anti-inflammatory capacity and instructs the immunoregulatory properties of MSCs in an array of diseases, including myocardial infarction, acute lung injury (ALI) and psoriasis [ 17 – 19 ]. In one instance, TSG-6 competitively conjugates the cell-surface glycosaminoglycan (GAG) binding site of CXCL8 against heparin, thereby inhibiting the infiltration of neutrophils into sites of inflammation [ 20 ]. The extravasation of leukocytes (mainly neutrophils and macrophages) is also restrained by TSG-6 released by MSCs through binding to CD44 [ 21 ]. Furthermore, TSG-6 administration promoted the phenotypic polarization of macrophages towards an anti-inflammatory state, thereby reducing inflammation in a lipopolysaccharide (LPS)-induced ALI model [ 22 ]. Although TSG-6 can potently suppress aberrant immune responses in the inflamed tissue microenvironment, how TSG-6 expression is regulated remains to be fully characterized. In this study, we found that MuSCs primed with IFN-γ and TNF-α exerted impressive therapeutic efficacy for LPS-induced ALI through IL4I1-mediated immunometabolism. Mechanistically, I3P and I3A produced by IL4I1 not only inhibited the infiltration of neutrophils into sites of inflammation through AHR-driven TSG-6 expression but also weakened the pathogenic phenotypes of neutrophils in damaged lungs. Our study reveals a novel indole metabolism-dependent immunosuppressive function of MuSCs and may aid the development of MuSC-based cell therapies for inflammatory diseases. Results IL4I1 is upregulated in MuSCs primed with IFN-γ and TNF-α through NF-κB and STAT6 pathways Inflammatory cytokines have been demonstrated to drastically augment the expression of immunomodulatory and regenerative factors and thus boost the therapeutic function of stem cells in disease settings [ 3 , 4 ]. To interrogate the changes in transcriptional landscape induced by inflammatory cytokines, we performed RNA sequencing (RNA-seq) analysis of MuSCs and MSCs stimulated by IFN-γ and TNF-α. We noted that the expression of IL4I1, which catalyzes IDO1-independent tryptophan metabolism and possesses strong immunoregulatory functions, was significantly increased in both IFN-γ/TNF-α-primed MuSCs and MSCs (Fig. 1 A, B). Interestingly, the induction of IL4I1 appeared to be more pronounced in MuSCs than in MSCs. This was confirmed using qRT-PCR (Fig. 1 C). These results suggested that different kinds of tissue stem cells may respond to inflammatory cytokines via common mechanisms. The enhanced production of IL4I1 at protein levels in inflammatory cytokine-stimulated MuSCs was further confirmed using ELISA and Western blot analysis (Fig. 1 D, E). IL4I1 expression is classically induced by IL-4 in B cells through activating STAT6 pathway [ 23 ]. Given that IFN-γ and TNF-α treatment can activate NF-κB and STAT6 pathways and drive the expression of various immunomodulatory molecules, we employed their specific inhibitors to explore the potential regulation mechanism of IL4I1 expression. Consistent with this idea, the expression of IL4I1 was dramatically decreased in IFN-γ/TNF-α-primed MuSCs after AS1517499 (an inhibitor of STAT6) and BAY117082 (an inhibitor of NF-κB) treatment, respectively (Fig. 1 F-H). These results were consistent with the previous study showing that IL4I1 expression is increased in response to NF-κB activation and the stimulation of IL-4/STAT6 axis [ 24 ]. Taken together, these findings indicated that IFN-γ and TNF-α can stimulate IL4I1 expression in MuSCs through activating NF-κB and STAT6 pathways. IL4I1 is vital for the therapeutic effects of MuSCs on LPS-induced ALI To further investigate the immunoregulatory function of IL4I1 in IFN-γ/TNF-α-primed MuSCs, we established stable IL4I1 knockdown cell line (IL4I1-KD-hMuSCs) using lentivirus transfection and injected intravenously IL4I1-KD-hMuSCs into mice suffering from LPS-induced ALI (Fig. 2 A, B). Hematoxylin and eosin (H&E) staining of lung tissue sections revealed widespread septal thickening, significantly increased air-space cellularity and exudation, and enhanced interstitial immune cell infiltration in mice treated with LPS. Importantly, administration of Ctrl-hMuSCs resulted in significant amelioration. In contrast, the therapeutic effect of IL4I1-KD-hMuSCs was noticeably compromised (Fig. 2 C). Furthermore, the inhibitory effect on IL-6 level, an important indicator for the progression of ALI [ 25 ], was impaired with the IL4I1-KD-hMuSC administration (Fig. 2 D, E). Consistently, the expression of chemokines, including Cxcl1 , Ccl5 , and Mcp1 , that are responsible for the recruitment of inflammatory cells, was markedly reduced in lung tissues after Ctrl-hMuSC infusion. IL4I1 knockdown abolished the suppressive effects of MuSCs on the expression of these chemokines (Fig. 2 F-H). We next performed immunohistochemical staining for CXCL1 that mediates the infiltration of neutrophils. As expected, IL4I1-KD-hMuSC administration failed to decrease CXCL1 expression in damaged lungs of mice suffering ALI (Fig. 2 I). Taken together, these data demonstrated that the beneficial effects of MuSCs in ALI mice depend on IL4I1. IL4I1 decreases the infiltration of neutrophils in lung tissues Increased infiltration of neutrophils characterizes the development and progression of ALI and excessive and prolonged activation of neutrophils can lead to the destruction of basement membranes, thereby increasing permeability of the alveolar capillary barrier in lungs [ 26 ]. To further determine the role of IL4I1 in regulating the dynamics of inflammatory cells in ALI mice, we performed flow cytometry analysis of immune cell populations in lungs, blood and bronchoalveolar lavage (BAL) fluid. As expected, Ctrl-hMuSC administration significantly diminished the proportion and number of infiltrated neutrophils in lung tissues (Fig. 3 A, B). Furthermore, the proportion of circulating neutrophils in total immune cell populations was obviously decreased in the blood of ALI mice (Fig. 3 C). In addition, infusion of Ctrl-hMuSCs reduced the number of immune cells, particularly neutrophils, in BAL fluid of ALI mice (Fig. 3 D, E). Consistent with these results, immunofluorescence staining of Ly6G also showed decreased neutrophil infiltration in lung tissues of these mice (Fig. 3 F). However, IL4I1 knockdown rendered MuSCs less effective in inhibiting the infiltration of neutrophils (Fig. 3 A-F). Collectively, these results indicated that IL4I1 in MuSCs decreases inflammatory response in ALI partially through inhibiting the recruitment of neutrophils into damaged lung tissues. IL4I1 metabolites I3P and I3A promote the production of TSG-6 in MuSCs TSG-6 possesses strong tissue-protective and anti-inflammatory properties in ALI through various mechanisms of action [ 27 – 30 ]. Our previous studies also found that inflammatory cytokine-primed MSCs and MuSCs can respectively alleviate LPS-induced ALI and DSS-induced colitis through TSG-6 production mediated by IDO-driven tryptophan metabolism [ 7 , 18 ]. Paralleling to IDO, IL4I1-catalyzed indole metabolism is another important approach of tryptophan catabolism in mice and humans. Thus, we further determined whether IL4I1 could regulate the expression of TSG-6 in MuSCs through this modality of action. IL4I1 knockdown was found to dramatically reduce TSG-6 mRNA and protein levels in IFN-γ/TNF-α-primed MuSCs (Fig. 4 A, B). As a L-amino acid oxidase independent of KYN pathway initiated by IDO1/tryptophan-2,3-dioxgenase 2 (TDO2), IL4I1 converts tryptophan into indole metabolites I3P and I3A (Fig. 4 C), which exhibit powerful anti-inflammatory functions during the development and progression of various tumors [ 9 ]. To explore whether IL4I1 could promote the expression of TSG-6 through its downstream metabolites I3P and I3A, we conducted a rescue experiment in IL4I1-depleted MuSCs with indole metabolites. Supplementation with I3P or I3A indeed restored TSG-6 expression in IFN-γ/TNF-α-primed MuSCs with IL4I1 knockdown (Fig. 4 D-F). While TSG-6 expression was greatly induced in MuSCs stimulated with inflammatory cytokines, exogenous I3P and I3A could further increase TSG-6 production in these MuSCs (Fig. 4 G-J). Together, these data suggested that IL4I1 upregulates TSG-6 expression through indole metabolites I3P and I3A in inflammatory cytokine-primed MuSCs. I3P and I3A promote TSG-6 production in MuSCs via AHR signaling We previously found that AHR is highly expressed in inflammatory cytokine-primed MuSCs [ 7 ]. It is one of the most recognized members of the basic helix-loop-helix-per-arnt-Sim (bHLH-PAS) transcription factor superfamily. When translocated to the nucleus after binding to ligands, AHR will form heterodimers with the AHR nuclear translocation (ARNT), which can initiate the transcription machinery of multiple genes involved in various cellular processes through binding to distinct biological response element (XRE) sequences [ 31 ]. TSG-6, in particular, is a target gene of AHR [ 18 ]. Thus, we next attempted to determine whether I3P and I3A could promote TSG-6 expression in MuSCs through AHR signaling. When CH-223191 was added to antagonize the nuclear translocation of AHR, the induction of CYP1B1 , a classical target gene of AHR signaling, was indeed blocked in IFN-γ/TNF-α-primed MuSCs (Fig. 5 A). Consistent with this, AHR blockade led to diminished TSG-6 expression in MuSCs (Fig. 5 B, C). Furthermore, I3P and I3A significantly increased the accumulation of AHR in the nucleus, indicating that I3P and I3A could activate AHR signaling in activated MuSCs (Fig. 5 D, E). The enhanced AHR activity was further supported by the increased expression of CYP1B1 by I3P or I3A administration (Fig. 5 F). However, AHR blocking through CH-223191 abolished the enhancement of TSG-6 expression caused by I3P and I3A in IFN-γ/TNF-α-primed MuSCs (Fig. 5 G-J). Collectively, these results demonstrated that I3P and I3A promote the expression of TSG-6 in MuSCs by activating AHR. I3P administration alleviates ALI To further investigated the therapeutic potential of I3P in disease settings, we intraperitoneally injected I3P into mice suffering from LPS-induced ALI (Fig. 6 A). Consistent with MuSC therapy, I3P administration resulted in significant amelioration, as reflected by reduced septal thickening, significant decreases in air-space cellularity and exudation, and diminished interstitial immune cell infiltration in ALI mice (Fig. 6 B). Flow cytometry analysis also showed that the number of infiltrating neutrophils in damaged lungs was obviously reduced in ALI mice treated with I3P (Fig. 6 C). In addition, there was decreased expression of Cxcl1 in mice treated with I3P (Fig. 6 D). While reactive oxygen species (ROS) production is essential for neutrophils to combat bacteria and other invading pathogens, excessive ROS can induce tissue damage and exacerbate lung inflammation [ 32 – 34 ]. Given the recent report that I3P can activate an anti-oxidative gene expression program [ 35 ], we speculated that I3P might also directly act on neutrophils and reduce their tissue damaging activity mediated by ROS. Indeed, I3P administration induced the expression of anti-oxidative genes nuclear factor erythroid 2-related factor 2 ( Nfe2l2 , also known as Nrf2 ) and heme oxygenase 1 ( Ho-1 ) in infiltrating neutrophils in lungs (Fig. 6 E). In neutrophils treated with PMA, ROS level was reduced by concomitant I3P addition (Fig. 6 F). Collectively, these data suggested that I3P administration can inhibit the infiltration of neutrophils and protect the lungs against ROS-mediated oxidative damage from neutrophils, thereby exerting the therapeutic efficacy for ALI. Discussion Tissue stem cells are generally known for their ability to differentiate into various types of daughter cells that participate in organogenesis, maintain tissue integrity or carry out other functions. The premise of stem cell-based therapy is largely based on the assumption that tissue stem cells may replace the missing or damaged functional cells in the targeted organ. However, only few transplanted stem cells are capable of differentiating into the desired cells to complete local physical reconstitution through their ‘stemness’ referred to the ability of lineage commitments [ 36 ]. Emerging evidence suggests that the therapeutic effects of stem cell transplantation may rely on their ability to enable damaged or dysfunctional tissues to form a balanced inflammatory and regenerative microenvironment, a paradigm also known as ‘empowerment’ [ 4 , 37 , 38 ]. A recent study found that the beneficial therapeutic effect of cardiac stem cell (CSC) therapy was attributed to CSC-mediated immunomodulation for CCR2 + and CX3CR1 + macrophages rather the production of new cardiomyocytes through lineage differentiation. These macrophages helped to decrease cardiac fibroblast activities and promote remodeling of ECM, thereby halting progressive heart fibrosis [ 39 ]. We recently showed that MuSCs harbor potent immunoregulatory properties in disease settings [ 6 ]. Importantly, these abilities can be further strengthened through preconditioning with inflammatory cytokines. Mechanistically, priming with IFN-γ and TNF-α enhanced TSG-6 production through IDO-mediated tryptophan catabolism along the KYN pathway, thereby endowing MuSCs more powerful therapeutic potency in DSS-induced colitis mice [ 7 ]. These results demonstrated that the immunosuppressive properties of MuSCs are governed by tryptophan metabolism. KYN pathway initiated by IDO1/2 or TDO2 utilizes tryptophan to produce KYNA and other bioactive molecules that function as endogenous AHR ligands [ 40 , 41 ]. High expression of IDO in inflammatory cytokine-primed stem cells is essential for their therapeutic effects in disease settings [ 3 , 4 ]. Therein, KYNA activates AHR signaling, and thus upregulates the transcriptional expression of TSG-6 in IFN-γ/TNF-α-activated human MSCs and MuSCs [ 7 , 18 ]. Interestingly, IL4I1-catalyzed indole generation is recently emerging as an alternate route of tryptophan catabolism in humans. Indole metabolites, including I3P and I3A, also function as potent AHR agonists. This route enables cancer cells to circumvent the anti-tumor immunity unleased by IDO1 inhibitors in cancer therapy [ 9 ]. It also suggests that indole metabolites and their effects on AHR activity are not solely attributed to microbial metabolism. We found that IL4I1 is upregulated to a greater extent than IDO1 in inflammatory cytokine-primed human MuSCs, suggesting that whereas there are two routes along which tryptophan is metabolized to generate bioactive small molecules that modulate immune functions, the preferred route may vary depending on cell types. In addition to activating AHR to promote the expression of TSG-6 in MuSCs, the extracellular I3P catalyzed by IL4I1 can also influence the functions of neighboring immune cells. Actually, I3P administration could directly curb disease symptoms in ALI mice through distinct mechanisms of action. Specifically, there was decreased infiltration of neutrophils into damaged lung tissues of diseased mice after I3P treatment, in which I3P inhibited CXCL1-mediated chemotaxis by neutrophils towards lungs. Moreover, ROS-mediated pathogenic effects of neutrophils were significantly restrained through increased expression of anti-oxidative genes when I3P administration. Collectively, these results suggested that I3P therapy may be explored for the treatment of inflammatory diseases. In summary, we have discovered a novel mechanism by which MuSCs function as immunoregulators. When stimulated by inflammatory cytokines, MuSCs yield I3P and I3A via IL4I1-catalyzed indole metabolism. As AHR ligands, they activate AHR, thereby upregulating TSG-6 expression. The indole metabolites can also directly act on immune cells to exert their anti-inflammatory effects (Fig. 7 ). These results not only highlight the importance of IL4I1 and its downstream metabolites in shaping the immunoregulatory function of MuSCs, but also provide a basis for developing indole metabolism-based new strategies for the treatment of inflammatory diseases. Materials And Methods MuSC culture MuSCs were as previously described [ 7 ]. MuSCs were cultured in myogenic growth medium containing 1:1 mixture of DMEM Low medium and MCDB 131 medium, 20% fetal bovine serum (FBS), 1% penicillin-streptomycin (all from Gibco, MA, USA), 1% insulin-transferrin-selenium (Invitrogen, Carlsbad, CA, USA), and 10 µM p38 MAPK inhibitor (SB203580, Selleck, Houston, TX, USA), which functions to block the differentiation of MuSCs and thus maintain their self-renewal properties [ 42 ]. Cell plates and slides were pre-coated with ECM (Sigma-Aldrich, St Louis, MO, USA). All details regarding the characterization of cultured MuSCs were shown in Figure S1 . ALI model Male C57BL/6 mice (6–8 weeks old) were purchased from Charles River Experimental Animal Technology Co. Ltd. (Beijing, China) and maintained under specific pathogen-free conditions of the Laboratory Animal Center of Soochow University. The experiments were approved by the Institutional Animal Care and Use Committee of Soochow University (SUDA20210916A07). The mice were housed in a 12 h light-dark cycle, and fed with irradiated food and sterile water ad libitum . All mice were acclimatized to the housing for 2 weeks before starting the experimental procedures. In this study, these mice were randomly divided into control, LPS, LPS + NC-hMuSCs and LPS + IL4I1-shRNA-hMuSCs groups. The LPS-induced ALI model was established by an airway administration of LPS (2 mg/kg, per mouse) into mice. 1 h later, hMuSCs (5 × 10 5 cells) and I3P (20 mg/kg) were intravenously injected to mice with ALI. After 24 h LPS challenge, mice were sacrificed and BAL fluid was collected through the tracheal cannula. Blood and lung tissue samples were collected for subsequent analyses. Western blotting analysis Western blotting was performed to measure the protein level of IL4I1 in hMuSCs. Total proteins were extracted using RIPA lysis buffer (Beyotime, Shanghai, China). Protein concentration was determined using a BCA protein assay kit (Bio-Rad, Hercules, CA, USA). 40 µg of Protein was separated by a 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride membrane (Millipore, Temecula, CA, USA). After blocking with 5% BSA for 2 h, the membrane was incubated with primary antibodies overnight at 4°C. The primary antibodies included anti-human IL4I1 (Abcam, MA, USA) and anti-human β-ACTIN (Abcam). The membrane was then washed three times with TBST and respectively incubated with horseradish peroxidase-conjugated anti-rabbit and anti-rat antibodies (Abcam) at room temperature for 1 h. Finally, the protein levels were detected by the chemiluminescence reagent. qRT-PCR Total RNA was extracted using RNAprep Pure Cell Kit (Feijie, Shanghai, China), and reverse-transcribed into cDNA with PrimeScript™ RT Master Mix (TaKaRa, Dalian, China). The levels of mRNA expression were analyzed by QuantStudio™ 6 Flex System according to the manufacturer’s instructions. The total reaction volume of 10 µl was comprised of 1 ng cDNA, 3 µl DNAase/RNAse-free water (TaKaRa), 1 µl special primers (GENEWIZ, Suzhou, Jiangsu, China), and 5 µl SYBR qPCR SuperMixplus with ROX (Novoprotein, Shanghai, China). The real-time PCR program was as follows: pre-denaturation at 95°C for 30 s; 40 cycles of denaturation at 95°C for 5 s; annealing and extension at 60°C for 30 s. The total amount of mRNA was compared with endogenous β-ACTIN mRNA. Finally, the relative expression of mRNA was calculated using 2 –ΔΔCt method. Primer sequences were shown in Supplementary Table S1 . Elisa The supernatants of MuSCs were collected 24 h after distinct treatments to detect the concentration of TSG-6 through ELISA as previously described [ 7 ]. Briefly, a 96-well plate was pre-coated with 50 µl of 10 µg/ml TSG-6 antibody (Santa Cruz, Dallas, Texas, USA) overnight, washed with PBS and blocked with the buffer containing 0.25% BSA and 0.05% Tween 20. After 1 hour, the excess blocking buffer was washed away. Biotinylated anti-human TSG-6 antibody (R&D Systems, Minneapolis, MN, USA) was added and incubated for 2 h at room temperature. After washing with PBS, streptavidin-horseradish peroxidase (R&D Systems) was added for 30 min at room temperature, and developed using substrate solutions (R&D Systems). Immunofluorescence assay For immunofluorescence staining of lung sections, the hydrated slides were subjected to antigen retrieval in Tris-EDTA, pH 9.0 (Yuanye, Shanghai, China), at 96°C for 30 min. Then, lung sections were incubated with 3% bovine serum albumin (Amresco, OH, USA) for 1 h to prevent non-specific staining before incubating with anti-mouse Ly6G (Abcam) overnight at 4°C. Finally, lung sections were incubated with anti-rat IgG (H + L) (Alexa Fluo 594, Abcam) for 1 h and Hoechst 33324 (Beyotime) for 10 min at room temperature. MuSCs were seeded on eight-chamber slide (Thermo Fisher) at the density of 1 × 10 4 cells per chamber. After indicated treatment, cells were fixed with paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 for 10 min, and blocked with 3% BSA in PBS for 1 h. The cells were then incubated with 10 µg/ml anti-human AHR (Abcam) overnight at 4°C. Then, cells were washed with PBS and incubated with anti-Mouse IgG (H + L) (Alexa Fluor 647, Thermo Fisher) for 1 h at room temperature. After PBS washing, cell nucleus was stained with 1 µg/ml Hoechst 33324 (Beyotime) for 8 min at room temperature. Finally, images were taken with the Nikon microscope. Flow cytometry Mice were sacrificed 24 h after LPS exposure. Lung tissues and peripheral blood mononuclear cells were collected. To obtain BAL fluid, lungs were washed three times with 1 ml PBS. Cells for flow cytometric analysis were pre-incubated with anti-CD16/CD32 (BioLegend, San Diego, CA, USA) to block Fc receptors, and then stained with anti-mouse CD45 (BioLegend), anti-mouse CD11b (BioLegend), anti-mouse Ly6C (BioLegend) and anti-mouse Ly6G (BioLegend) at 4°C for 20 min. Then, the total cell number in BAL fluid was counted and neutrophil numbers were determined on a Cytoflex Flow Cytometer (Beckman Coulter, CA, USA). Histological analysis Isolated lung tissues were fixed in 4% paraformaldehyde, dehydrated using graded ethanol, embedded in paraffin, cut into 5-µm-thick sections, and then stained with hematoxylin and eosin (H&E). For the immunohistochemistry of CXCL1, the lung samples were stained with anti-mouse CXCL1 antibody (Abcam), followed by HRP-linked anti-rat secondary antibody (Maixin, Fuzhou, Fujian, China) and diaminobenzidine (Maixin). Images were taken with the Nikon microscope. More than three fields from each mouse were selected and the images are representative for each experiment repeats. Statistical analysis Statistical analyses were performed using GraphPad Prism 9 (GraphPad Prism Software Inc., CA, USA). All data in this article are presented as the mean ± SEM. For two-group comparison, two-tailed unpaired t test was performed. For multiple group comparison, one-way analysis of variance test was performed. Notably, P values less than 0.05 were statistically considered significant. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Declarations DATA AVAILABILITY The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request AUTHOR CONTRIBUTIONS M.Z. and J.F. designed the project, performed the experiments, analyzed the data, and prepared the manuscript. P.H., S.L., P.H., S.W., Z.L., C.F., L.C. and P.L. performed the experiments and provided advice for data analysis. Y.S. and C.S. conceived and supervised the project and revised the manuscript. FUNDING This study was supported by grants from the National Key R&D Program of China (2021YFA1100600 and 2022YFA0807300), the National Natural Science Foundation of China (32150710523 and 82202032), Jiangsu Province International Joint Laboratory for Regenerative Medicine Fund, Suzhou Foreign Academician Workstation Fund (SWY202202) and Suzhou Science and Technology Initiative Fund (SYS2020087). COMPETING INTERESTS The authors declare no competing interests. References Sousa-Victor P, Garcia-Prat L, Munoz-Canoves P. Control of satellite cell function in muscle regeneration and its disruption in ageing. Nat Rev Mol Cell Biol. 2022;23:204–226. Brunet A, Goodell MA, Rando TA. Ageing and rejuvenation of tissue stem cells and their niches. Nat Rev Mol Cell Biol. 2023;24:45–62. Fang J, Feng C, Chen W, Hou P, Liu Z, Zuo M, et al. Redressing the interactions between stem cells and immune system in tissue regeneration. Biol Direct. 2021;16:18. Wang Y, Fang J, Liu B, Shao C, Shi Y. 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Additional Declarations (Not answered) Supplementary Files Fulllengthwesternblots.docx Supplementaryinformation.docx Cite Share Download PDF Status: Published Journal Publication published 28 Jul, 2023 Read the published version in Cell Death Discovery → Version 1 posted Editorial decision: revise 27 Mar, 2023 Review # 2 received at journal 22 Mar, 2023 Reviewer # 2 agreed at journal 08 Mar, 2023 Review # 1 received at journal 07 Mar, 2023 Reviewer # 1 agreed at journal 07 Mar, 2023 Reviewers invited by journal 05 Mar, 2023 Submission checks completed at journal 23 Feb, 2023 Editor assigned by journal 22 Feb, 2023 First submitted to journal 22 Feb, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2618290","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":180882498,"identity":"b11afa7c-a834-449a-b67c-f91bab21b319","order_by":0,"name":"Changshun 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University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Peishan","middleName":"","lastName":"Li","suffix":""},{"id":180882509,"identity":"49ed4d1c-7e11-4685-abfa-d9042f8799c6","order_by":11,"name":"Y Shi","email":"","orcid":"","institution":"University of Chinese Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Y","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2023-02-23 01:06:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2618290/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2618290/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41420-023-01568-x","type":"published","date":"2023-07-28T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":34042734,"identity":"2d8c1162-651c-40c5-ab9a-de115d62c725","added_by":"auto","created_at":"2023-03-10 00:11:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":176913,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIFN-γ and TNF-α increase the expression of IL4I1 in hMuSCs through NF-kB and STAT6 pathway.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003eVolcano plot of differentially expressed genes in MuSCs after the stimulation of IFN-γ and TNF-α (10 ng/ml each) for 24 husing RNA-seq. \u003cstrong\u003eB\u003c/strong\u003eVolcano plot of differentially expressed genes in MSCs after the stimulation of IFN-γ and TNF-α (10 ng/ml each) for 24 h using RNA-seq. \u003cstrong\u003eC\u003c/strong\u003e The mRNA expression of IL4I1 in MSCs and MuSCs after the stimulation of IFN-γ and TNF-α (10 ng/ml each) for 24 h was assayed by qRT-PCR. \u003cstrong\u003eD \u003c/strong\u003eThe concentration of IL4I1 in the supernatant of MuSCs after the stimulation of IFN-γ and TNF-α (10 ng/ml each) for 24 h was measured by ELISA. \u003cstrong\u003eE\u003c/strong\u003e The protein expression of IL4I1 and β-ACTIN (loading control) in MuSCs after the stimulation of IFN-γ and TNF-α (10 ng/ml each) for 24 h were determined by western blotting. \u003cstrong\u003eF, G\u003c/strong\u003e The mRNA and protein expression levels of IL4I1 in MuSCs stimulated with IFN-γ and TNF-α (10 ng/ml each) for 24 h in the presence or absence of AS1517499 were respectively assayed by qRT-PCR and western blotting. \u003cstrong\u003eH \u003c/strong\u003eThe protein expression levels of IL4I1 in MuSCs stimulated with IFN-γ and TNF-α (10 ng/ml each) for 24 h in the presence or absence of BAY117082 were assayed by western blotting. Data were shown as means ± SEM. Data are representative of three experiments with similar results. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2618290/v1/20b7aa9f5c6ab5e0cc6285b5.png"},{"id":34042735,"identity":"85dc8212-06b5-4c0e-aa30-8d65b716cff9","added_by":"auto","created_at":"2023-03-10 00:11:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1225241,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL4I1 mediates the therapeutic effect of MuSCs on ALI.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e The therapeutic strategy of MuSCs in the LPS-induced ALI model. Mice were treated with 2 mg/kg LPS through endotracheal infusion. 1 hour later, ctrl-hMuSCs or IL4I1-KD-hMuSCs (5 × 10\u003csup\u003e5\u003c/sup\u003e cells) pretreated with IFN-γ and TNF-α (10 ng/ml each) for 24 h were intravenously injected into mice. Then, all experimental mice were euthanized after 23 h, and the lung samples were collected for further processing. \u003cstrong\u003eB\u003c/strong\u003e The efficiency of IL4I1 knockdown measured by western blotting analysis. \u003cstrong\u003eC\u003c/strong\u003e Lung tissues after the various treatments were fixed for H\u0026amp;E staining. Scale bars, 249 μm. \u003cstrong\u003eD, E\u003c/strong\u003e The expression levels of IL-6 mRNA (left) and protein (right) in the lung tissue homogenates of ALI mice were determined by qRT-PCR and ELISA. \u003cstrong\u003eF-H\u003c/strong\u003e The expression levels of chemokines in the lung tissue homogenates of ALI mice were determined by qRT-PCR.\u003cstrong\u003e I\u003c/strong\u003e Neutrophil infiltration in lung tissues was evaluated by immunofluorescence staining of Ly6G (red). Scale bars, 115.9 μm. Data were shown as means ± SEM. Data are representative of three experiments with similar results. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2618290/v1/6227a3b285de4d0a144b77cb.png"},{"id":34041967,"identity":"7c7dcdc8-17d8-439a-95fe-cc7cc1b6f01a","added_by":"auto","created_at":"2023-03-10 00:03:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":585985,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL4I1 from MuSCs suppresses neutrophil infiltration into lung tissues in ALI mice. A, B\u003c/strong\u003e Mice were treated with 2 mg/kg LPS through endotracheal infusion. 1 hour later, ctrl-hMuSCs or IL4I1-KD-hMuSCs (5 × 10\u003csup\u003e5\u003c/sup\u003e cells) pretreated with IFN-γ and TNF-α (10 ng/ml each) for 24 h were intravenously injected into mice. The proportion and absolute number of neutrophils in the left lung tissues of ALI mice were determined by flow cytometry analysis. \u003cstrong\u003eC\u003c/strong\u003e The proportion of neutrophils in the blood of ALI mice was measured by flow cytometry analysis. \u003cstrong\u003eD\u003c/strong\u003e The proportion of neutrophils in the BAL fluid of ALI mice was measured by flow cytometry analysis. \u003cstrong\u003eE\u003c/strong\u003e The absolute numbers of total immune cells and neutrophils in the BAL fluid of ALI mice were determined by flow cytometry analysis. \u003cstrong\u003eF\u003c/strong\u003e Lung tissues in mice with the various treatments were stained with CXCL1 antibody. Scale bars, 249 μm. Data were shown as means ± SEM. Data are representative of three experiments with similar results. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2618290/v1/0f82133c8dc4573a8c52059b.png"},{"id":34041966,"identity":"1b0cdd21-7c5c-41cd-ab09-be6fd96fb51a","added_by":"auto","created_at":"2023-03-10 00:03:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":175727,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eI3P and I3A promote TSG-6 production in MuSCs. A \u003c/strong\u003e\u003cem\u003eTSG-6\u003c/em\u003e expression in ctrl-hMuSCs and IL4I1-KD-hMuSCs stimulated with IFN-γ and TNF-α (10 ng/ml each) for 24 h was measured by qRT-PCR. \u003cstrong\u003eB\u003c/strong\u003eThe concentration of TSG-6 in the supernatants of ctrl-hMuSCs and IL4I1-KD-hMuSCs stimulated with IFN-γ and TNF-α (10 ng/ml each) for 24 h was determined by ELISA.\u003cstrong\u003e C\u003c/strong\u003e Schematic diagram illustrating tryptophan metabolism in humans via different catabolic routes. \u003cstrong\u003eD \u003c/strong\u003e\u003cem\u003eTSG-6\u003c/em\u003e expression in ctrl-hMuSCs and IL4I1-KD-hMuSCs stimulated with IFN-γ and TNF-α (10 ng/ml each) for 24 h in the presence or absence of I3P (50 μM) was measured by qRT-PCR. \u003cstrong\u003eE\u003c/strong\u003e The concentration of TSG-6 in the supernatants of ctrl-hMuSCs and IL4I1-KD-hMuSCs stimulated with IFN-γ and TNF-α (10 ng/ml each) for 24 h in the presence or absence of I3P (50 μM) was determined by ELISA. \u003cstrong\u003eF\u003c/strong\u003e The concentration of TSG-6 in the supernatants of ctrl-hMuSCs and IL4I1-KD-hMuSCs stimulated with IFN-γ and TNF-α (10 ng/ml each) for 24 h in the presence or absence of I3A (100 μM)was determined by ELISA. \u003cstrong\u003eG, I\u003c/strong\u003e \u003cem\u003eTSG-6\u003c/em\u003eexpression in hMuSCs stimulated with IFN-γ and TNF-α (10 ng/ml each) for 24 h in the presence or absence of I3P (50 μM) and I3A (100 μM) was measured by qRT-PCR. \u003cstrong\u003eH, J \u003c/strong\u003eThe concentration of TSG-6 in the supernatants of hMuSCs stimulated with IFN-γ and TNF-α (10 ng/ml each) for 24 h in the presence or absence of I3P (50 μM) and I3A (100 μM) was determined by ELISA. Data were shown as means ± SEM. Data are representative of three experiments with similar results. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2618290/v1/9220cd64988b7f5540f5a2f4.png"},{"id":34041972,"identity":"0ffac534-14cc-4d85-8f78-6f83891609b2","added_by":"auto","created_at":"2023-03-10 00:03:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":573103,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eI3P and I3A activates AHR signaling pathway to enhance TSG-6 production in MuSCs.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e \u003cem\u003eCYP1B1\u003c/em\u003e expression in MuSCs stimulated with IFN-γ and TNF-α (10 ng/ml each) in the presence or absence of CH-223191(CH, 100 μM) for 24 h was measured by qRT-PCR. \u003cstrong\u003eB\u003c/strong\u003e \u003cem\u003eTSG-6\u003c/em\u003e expression in MuSCs stimulated with IFN-γ and TNF-α (10 ng/ml each) in the presence or absence of CH-223191 (CH, 100 μM) for 24 h was measured by qRT-PCR. \u003cstrong\u003eC\u003c/strong\u003e The concentration of TSG-6 in the supernatants of MuSCs stimulated with IFN-γ and TNF-α (10 ng/ml each) in the presence or absence of CH-223191 (CH, 100 μM) for 24 h was determined by ELISA. \u003cstrong\u003eD\u003c/strong\u003e MuSCs treated with IFN-γ and TNF-α (10 ng/ml each) in the presence or absence of I3P (50 μM) and I3A (100 μM) were subjected to immunofluorescence. Scale bars, 36.8 μm. \u003cstrong\u003eE\u003c/strong\u003e MuSCs treated with IFN-γ and TNF-α (10 ng/ml each) in the presence or absence of I3P (50 μM) or I3A (100 μM) as well as CH-223191 (CH, 100 μM) were subjected to cytoplasmic and nuclear extraction, and the distribution of AHR was analyzed by western blot analysis. β-actin and laminB1 were served as loading controls for cytoplasmic and nuclear proteins, respectively. \u003cstrong\u003eF\u003c/strong\u003e \u003cem\u003eCYP1B1\u003c/em\u003eexpression in MuSCs stimulated with IFN-γ and TNF-α (10 ng/ml each) in the presence or absence of I3P (50 μM) and I3A (100 μM) for 24 h was measured by qRT-PCR. \u003cstrong\u003eG, I\u003c/strong\u003e \u003cem\u003eTSG-6\u003c/em\u003e expression in MuSCs stimulated with IFN-γ and TNF-α (10 ng/ml each) in the presence or absence of I3P (50 μM) or I3A (100 μM) and CH-223191 (CH, 100 μM) for 24 h was measured by qRT-PCR. \u003cstrong\u003eH, J\u003c/strong\u003e The concentration of TSG-6 in the supernatants of MuSCs stimulated with IFN-γ and TNF-α (10 ng/ml each) in the presence or absence of I3P (50 μM) or I3A (100 μM) and CH-223191 (CH, 100 μM) for 24 h was determined by ELISA. Data were shown as means ± SEM. Data are representative of three experiments with similar results. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2618290/v1/38400a4d85e3501e412a4280.png"},{"id":34041964,"identity":"0b743bb1-fad1-4e90-865d-8a5d7db7359a","added_by":"auto","created_at":"2023-03-10 00:03:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":437617,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eI3P administration suppresses neutrophil infiltration and enhances their oxidation resistance in damaged lungs. A \u003c/strong\u003eThe therapeutic strategy of I3P in the LPS-induced ALI model. Mice were treated with 2 mg/kg LPS through endotracheal infusion. 1 hour later, I3P (20 mg/kg) were intraperitoneally injected into mice. Then, all experimental mice were euthanized after 23 h, and the lung samples were collected for further processing. \u003cstrong\u003eB\u003c/strong\u003e Lung tissues after the various treatments were fixed for H\u0026amp;E staining. Scale bars, 249 μm. \u003cstrong\u003eC\u003c/strong\u003e The number of neutrophils in the lungs of ALI mice was measured by flow cytometry analysis. \u003cstrong\u003eD\u003c/strong\u003e The expression levels of \u003cem\u003eCxcl1\u003c/em\u003e in the lung tissue homogenates of ALI mice were determined by qRT-PCR. \u003cstrong\u003eE\u003c/strong\u003e The expression levels of \u003cem\u003eNrf2 \u003c/em\u003eand\u003cem\u003e Ho-1\u003c/em\u003e in neutrophils in the bone marrow and lungs of ALI mice were determined by qRT-PCR. \u003cstrong\u003eF\u003c/strong\u003e ROS levels in PMA-activated neutrophils treated with I3P (200 μM) were measured by flow cytometry analysis. Data were shown as means ± SEM. Data are representative of three experiments with similar results. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2618290/v1/e963802052dfaed5d5f6ada1.png"},{"id":34041968,"identity":"e840c1e9-bb6a-4034-8abf-c68550d07b89","added_by":"auto","created_at":"2023-03-10 00:03:47","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":399853,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA schematic model of the anti-inflammatory effects of IL4I1 on LPS induced-ALI. \u003c/strong\u003eIL4I1 induced by IFN-γ and TNF-α catalyzes tryptophan metabolism to produce indole metabolites I3P and I3A in MuSCs, which promote TSG-6 production through activating AHR signaling pathway, and consequently alleviate ALI. Additionally, extracellular I3P can suppress the infiltration of neutrophils into damaged lungs and reduce ROS-mediated oxidative damage from neutrophils.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-2618290/v1/67cc929830408be0eaf3289a.png"},{"id":40752406,"identity":"fe33a9d9-8bda-463c-8702-111a199b2d55","added_by":"auto","created_at":"2023-07-29 07:07:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4265547,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2618290/v1/c1cdf845-ad75-451b-a972-484198eb6a26.pdf"},{"id":34041971,"identity":"bc46c943-50b4-45be-b8e3-7135700dc235","added_by":"auto","created_at":"2023-03-10 00:03:47","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":446680,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fulllengthwesternblots.docx","url":"https://assets-eu.researchsquare.com/files/rs-2618290/v1/91831a9352f55aff1bcd0227.docx"},{"id":34042736,"identity":"bc5f9d7b-9e80-4809-8f2d-ef2908ccfba5","added_by":"auto","created_at":"2023-03-10 00:11:47","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":209300,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-2618290/v1/6f47b5950e01615956d90782.docx"}],"financialInterests":"(Not answered)","formattedTitle":"IL4I1-catalyzed tryptophan metabolites mediate the anti-inflammatory function of cytokine-primed human muscle stem cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMuscle stem cells (MuSCs), also known as satellite cells, reside in an unique niche between the muscle sarcolemma and the basal lamina of individual myofibres, and contribute to the maintenance and repair of tissue architectural structures through both self-renewal and myogenic differentiation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. While MuSCs are essential for muscle tissue regeneration, recent evidence has shown that MuSCs also act as critical regulators in the resolution of inflammation through releasing various paracrine factors [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This paracrine immunomodulatory property of MuSCs is akin to what has been well established in mesenchymal stem cells (MSCs) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. As an example, MuSCs can act on maturing macrophages and confer them with oxidative phosphorylation-dependent anti-inflammatory properties via insulin-like growth factor-2 (IGF-2), thus ameliorating dextran sulfate sodium (DSS)-induced colitis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In addition, MuSCs primed with inflammatory cytokines also reduce inflammation and promote tissue repair by producing TNF-stimulated gene 6 (TSG-6) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The induction of TSG-6 by inflammatory cytokines is mediated by tryptophan metabolites, including kynurenine (KYN) and kynurenic acid (KYNA), that are catalyzed by indoleamine 2,3-dioxygenase (IDO). IGF-2 and TSG-6 function as effectors of immunoregulation for both MuSCs and MSCs. Whether or not MuSCs can acquire their immunomodulatory property via pathways distinct from those in MSCs has not been explored.\u003c/p\u003e \u003cp\u003eInterleukin-4-induced-1 (IL4I1), a L-amino acid oxidase, was very recently identified as a novel potential therapeutic target of cancer treatment [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. IL4I1 promoted aryl hydrocarbon receptor (AHR)-driven malignant properties and suppressed anti-tumor immunity, even in the presence of immune checkpoint blockade (ICB) and IDO1 inhibition [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Specifically, indole-3-pyruvic acid (I3P) and indole-3-aldehyde (I3A) catalyzed by IL4I1 in tumor activated AHR signaling through receptor-ligand binding, leading to increased motility of cancer cells and diminished proliferation of CD8\u003csup\u003e+\u003c/sup\u003e T cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Unlike arginase and IDO that function intracellularly, IL4I1, as a secretory enzyme, can modulate the functions of neighboring immune cells in the inflamed tissue microenvironment [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. On the one hand, IL4I1 can directly reduce the stability of immune synapses between T cell and dendritic cells (DCs), thereby elevating the threshold of T cell activation and thus dampening their activation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. IL4I1 also regulates the polarization of macrophages, as reflected by the progressive infiltration of anti-inflammatory macrophages and the displacement of proinflammatory macrophages when IL4I1 was overexpressed [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In adaptive immunity, IL4I1 can suppress the proliferation of TH17 cells and promote the differentiation and activity of CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003eFOXP3\u003csup\u003e+\u003c/sup\u003e regulatory T cells [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], thus improving the neurological severity score and alleviating experimental autoimmune encephalomyelitis \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Thus, there is growing evidence that IL4I1 is a vital metabolic enzyme mediating the immunosuppressive effects in disease settings.\u003c/p\u003e \u003cp\u003eTSG-6, a 30-kDa secreted glycoprotein, influences intercellular 3D structures and remodels the extracellular matrix (ECM) through binding with hyaluronic acid, chondroitin sulfate and proteoglycan [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. More importantly, it possesses the prominent anti-inflammatory capacity and instructs the immunoregulatory properties of MSCs in an array of diseases, including myocardial infarction, acute lung injury (ALI) and psoriasis [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In one instance, TSG-6 competitively conjugates the cell-surface glycosaminoglycan (GAG) binding site of CXCL8 against heparin, thereby inhibiting the infiltration of neutrophils into sites of inflammation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The extravasation of leukocytes (mainly neutrophils and macrophages) is also restrained by TSG-6 released by MSCs through binding to CD44 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Furthermore, TSG-6 administration promoted the phenotypic polarization of macrophages towards an anti-inflammatory state, thereby reducing inflammation in a lipopolysaccharide (LPS)-induced ALI model [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Although TSG-6 can potently suppress aberrant immune responses in the inflamed tissue microenvironment, how TSG-6 expression is regulated remains to be fully characterized.\u003c/p\u003e \u003cp\u003eIn this study, we found that MuSCs primed with IFN-γ and TNF-α exerted impressive therapeutic efficacy for LPS-induced ALI through IL4I1-mediated immunometabolism. Mechanistically, I3P and I3A produced by IL4I1 not only inhibited the infiltration of neutrophils into sites of inflammation through AHR-driven TSG-6 expression but also weakened the pathogenic phenotypes of neutrophils in damaged lungs. Our study reveals a novel indole metabolism-dependent immunosuppressive function of MuSCs and may aid the development of MuSC-based cell therapies for inflammatory diseases.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIL4I1 is upregulated in MuSCs primed with IFN-γ and TNF-α through NF-κB and STAT6 pathways\u003c/h2\u003e \u003cp\u003eInflammatory cytokines have been demonstrated to drastically augment the expression of immunomodulatory and regenerative factors and thus boost the therapeutic function of stem cells in disease settings [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. To interrogate the changes in transcriptional landscape induced by inflammatory cytokines, we performed RNA sequencing (RNA-seq) analysis of MuSCs and MSCs stimulated by IFN-γ and TNF-α. We noted that the expression of IL4I1, which catalyzes IDO1-independent tryptophan metabolism and possesses strong immunoregulatory functions, was significantly increased in both IFN-γ/TNF-α-primed MuSCs and MSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Interestingly, the induction of IL4I1 appeared to be more pronounced in MuSCs than in MSCs. This was confirmed using qRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These results suggested that different kinds of tissue stem cells may respond to inflammatory cytokines via common mechanisms. The enhanced production of IL4I1 at protein levels in inflammatory cytokine-stimulated MuSCs was further confirmed using ELISA and Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E). IL4I1 expression is classically induced by IL-4 in B cells through activating STAT6 pathway [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Given that IFN-γ and TNF-α treatment can activate NF-κB and STAT6 pathways and drive the expression of various immunomodulatory molecules, we employed their specific inhibitors to explore the potential regulation mechanism of IL4I1 expression. Consistent with this idea, the expression of IL4I1 was dramatically decreased in IFN-γ/TNF-α-primed MuSCs after AS1517499 (an inhibitor of STAT6) and BAY117082 (an inhibitor of NF-κB) treatment, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-H). These results were consistent with the previous study showing that IL4I1 expression is increased in response to NF-κB activation and the stimulation of IL-4/STAT6 axis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Taken together, these findings indicated that IFN-γ and TNF-α can stimulate IL4I1 expression in MuSCs through activating NF-κB and STAT6 pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIL4I1 is vital for the therapeutic effects of MuSCs on LPS-induced ALI\u003c/h2\u003e \u003cp\u003eTo further investigate the immunoregulatory function of IL4I1 in IFN-γ/TNF-α-primed MuSCs, we established stable IL4I1 knockdown cell line (IL4I1-KD-hMuSCs) using lentivirus transfection and injected intravenously IL4I1-KD-hMuSCs into mice suffering from LPS-induced ALI (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). Hematoxylin and eosin (H\u0026amp;E) staining of lung tissue sections revealed widespread septal thickening, significantly increased air-space cellularity and exudation, and enhanced interstitial immune cell infiltration in mice treated with LPS. Importantly, administration of Ctrl-hMuSCs resulted in significant amelioration. In contrast, the therapeutic effect of IL4I1-KD-hMuSCs was noticeably compromised (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Furthermore, the inhibitory effect on IL-6 level, an important indicator for the progression of ALI [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], was impaired with the IL4I1-KD-hMuSC administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E). Consistently, the expression of chemokines, including \u003cem\u003eCxcl1\u003c/em\u003e, \u003cem\u003eCcl5\u003c/em\u003e, and \u003cem\u003eMcp1\u003c/em\u003e, that are responsible for the recruitment of inflammatory cells, was markedly reduced in lung tissues after Ctrl-hMuSC infusion. IL4I1 knockdown abolished the suppressive effects of MuSCs on the expression of these chemokines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-H). We next performed immunohistochemical staining for CXCL1 that mediates the infiltration of neutrophils. As expected, IL4I1-KD-hMuSC administration failed to decrease CXCL1 expression in damaged lungs of mice suffering ALI (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). Taken together, these data demonstrated that the beneficial effects of MuSCs in ALI mice depend on IL4I1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eIL4I1 decreases the infiltration of neutrophils in lung tissues\u003c/h2\u003e \u003cp\u003eIncreased infiltration of neutrophils characterizes the development and progression of ALI and excessive and prolonged activation of neutrophils can lead to the destruction of basement membranes, thereby increasing permeability of the alveolar capillary barrier in lungs [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. To further determine the role of IL4I1 in regulating the dynamics of inflammatory cells in ALI mice, we performed flow cytometry analysis of immune cell populations in lungs, blood and bronchoalveolar lavage (BAL) fluid. As expected, Ctrl-hMuSC administration significantly diminished the proportion and number of infiltrated neutrophils in lung tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). Furthermore, the proportion of circulating neutrophils in total immune cell populations was obviously decreased in the blood of ALI mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In addition, infusion of Ctrl-hMuSCs reduced the number of immune cells, particularly neutrophils, in BAL fluid of ALI mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E). Consistent with these results, immunofluorescence staining of Ly6G also showed decreased neutrophil infiltration in lung tissues of these mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). However, IL4I1 knockdown rendered MuSCs less effective in inhibiting the infiltration of neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-F). Collectively, these results indicated that IL4I1 in MuSCs decreases inflammatory response in ALI partially through inhibiting the recruitment of neutrophils into damaged lung tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eIL4I1 metabolites I3P and I3A promote the production of TSG-6 in MuSCs\u003c/h2\u003e \u003cp\u003eTSG-6 possesses strong tissue-protective and anti-inflammatory properties in ALI through various mechanisms of action [\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Our previous studies also found that inflammatory cytokine-primed MSCs and MuSCs can respectively alleviate LPS-induced ALI and DSS-induced colitis through TSG-6 production mediated by IDO-driven tryptophan metabolism [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Paralleling to IDO, IL4I1-catalyzed indole metabolism is another important approach of tryptophan catabolism in mice and humans. Thus, we further determined whether IL4I1 could regulate the expression of TSG-6 in MuSCs through this modality of action. IL4I1 knockdown was found to dramatically reduce TSG-6 mRNA and protein levels in IFN-γ/TNF-α-primed MuSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). As a L-amino acid oxidase independent of KYN pathway initiated by IDO1/tryptophan-2,3-dioxgenase 2 (TDO2), IL4I1 converts tryptophan into indole metabolites I3P and I3A (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), which exhibit powerful anti-inflammatory functions during the development and progression of various tumors [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. To explore whether IL4I1 could promote the expression of TSG-6 through its downstream metabolites I3P and I3A, we conducted a rescue experiment in IL4I1-depleted MuSCs with indole metabolites. Supplementation with I3P or I3A indeed restored TSG-6 expression in IFN-γ/TNF-α-primed MuSCs with IL4I1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F). While TSG-6 expression was greatly induced in MuSCs stimulated with inflammatory cytokines, exogenous I3P and I3A could further increase TSG-6 production in these MuSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-J). Together, these data suggested that IL4I1 upregulates TSG-6 expression through indole metabolites I3P and I3A in inflammatory cytokine-primed MuSCs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eI3P and I3A promote TSG-6 production in MuSCs via AHR signaling\u003c/h2\u003e \u003cp\u003eWe previously found that AHR is highly expressed in inflammatory cytokine-primed MuSCs [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. It is one of the most recognized members of the basic helix-loop-helix-per-arnt-Sim (bHLH-PAS) transcription factor superfamily. When translocated to the nucleus after binding to ligands, AHR will form heterodimers with the AHR nuclear translocation (ARNT), which can initiate the transcription machinery of multiple genes involved in various cellular processes through binding to distinct biological response element (XRE) sequences [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. TSG-6, in particular, is a target gene of AHR [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Thus, we next attempted to determine whether I3P and I3A could promote TSG-6 expression in MuSCs through AHR signaling. When CH-223191 was added to antagonize the nuclear translocation of AHR, the induction of \u003cem\u003eCYP1B1\u003c/em\u003e, a classical target gene of AHR signaling, was indeed blocked in IFN-γ/TNF-α-primed MuSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Consistent with this, AHR blockade led to diminished TSG-6 expression in MuSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). Furthermore, I3P and I3A significantly increased the accumulation of AHR in the nucleus, indicating that I3P and I3A could activate AHR signaling in activated MuSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, E). The enhanced AHR activity was further supported by the increased expression of \u003cem\u003eCYP1B1\u003c/em\u003e by I3P or I3A administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). However, AHR blocking through CH-223191 abolished the enhancement of TSG-6 expression caused by I3P and I3A in IFN-γ/TNF-α-primed MuSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-J). Collectively, these results demonstrated that I3P and I3A promote the expression of TSG-6 in MuSCs by activating AHR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eI3P administration alleviates ALI\u003c/h2\u003e \u003cp\u003eTo further investigated the therapeutic potential of I3P in disease settings, we intraperitoneally injected I3P into mice suffering from LPS-induced ALI (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Consistent with MuSC therapy, I3P administration resulted in significant amelioration, as reflected by reduced septal thickening, significant decreases in air-space cellularity and exudation, and diminished interstitial immune cell infiltration in ALI mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Flow cytometry analysis also showed that the number of infiltrating neutrophils in damaged lungs was obviously reduced in ALI mice treated with I3P (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). In addition, there was decreased expression of \u003cem\u003eCxcl1\u003c/em\u003e in mice treated with I3P (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). While reactive oxygen species (ROS) production is essential for neutrophils to combat bacteria and other invading pathogens, excessive ROS can induce tissue damage and exacerbate lung inflammation [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Given the recent report that I3P can activate an anti-oxidative gene expression program [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], we speculated that I3P might also directly act on neutrophils and reduce their tissue damaging activity mediated by ROS. Indeed, I3P administration induced the expression of anti-oxidative genes nuclear factor erythroid 2-related factor 2 (\u003cem\u003eNfe2l2\u003c/em\u003e, also known as \u003cem\u003eNrf2\u003c/em\u003e) and heme oxygenase 1 (\u003cem\u003eHo-1\u003c/em\u003e) in infiltrating neutrophils in lungs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). In neutrophils treated with PMA, ROS level was reduced by concomitant I3P addition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Collectively, these data suggested that I3P administration can inhibit the infiltration of neutrophils and protect the lungs against ROS-mediated oxidative damage from neutrophils, thereby exerting the therapeutic efficacy for ALI.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTissue stem cells are generally known for their ability to differentiate into various types of daughter cells that participate in organogenesis, maintain tissue integrity or carry out other functions. The premise of stem cell-based therapy is largely based on the assumption that tissue stem cells may replace the missing or damaged functional cells in the targeted organ. However, only few transplanted stem cells are capable of differentiating into the desired cells to complete local physical reconstitution through their \u0026lsquo;stemness\u0026rsquo; referred to the ability of lineage commitments [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Emerging evidence suggests that the therapeutic effects of stem cell transplantation may rely on their ability to enable damaged or dysfunctional tissues to form a balanced inflammatory and regenerative microenvironment, a paradigm also known as \u0026lsquo;empowerment\u0026rsquo; [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. A recent study found that the beneficial therapeutic effect of cardiac stem cell (CSC) therapy was attributed to CSC-mediated immunomodulation for CCR2\u003csup\u003e+\u003c/sup\u003e and CX3CR1\u003csup\u003e+\u003c/sup\u003e macrophages rather the production of new cardiomyocytes through lineage differentiation. These macrophages helped to decrease cardiac fibroblast activities and promote remodeling of ECM, thereby halting progressive heart fibrosis [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. We recently showed that MuSCs harbor potent immunoregulatory properties in disease settings [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Importantly, these abilities can be further strengthened through preconditioning with inflammatory cytokines. Mechanistically, priming with IFN-γ and TNF-α enhanced TSG-6 production through IDO-mediated tryptophan catabolism along the KYN pathway, thereby endowing MuSCs more powerful therapeutic potency in DSS-induced colitis mice [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These results demonstrated that the immunosuppressive properties of MuSCs are governed by tryptophan metabolism.\u003c/p\u003e \u003cp\u003eKYN pathway initiated by IDO1/2 or TDO2 utilizes tryptophan to produce KYNA and other bioactive molecules that function as endogenous AHR ligands [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. High expression of IDO in inflammatory cytokine-primed stem cells is essential for their therapeutic effects in disease settings [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therein, KYNA activates AHR signaling, and thus upregulates the transcriptional expression of TSG-6 in IFN-γ/TNF-α-activated human MSCs and MuSCs [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Interestingly, IL4I1-catalyzed indole generation is recently emerging as an alternate route of tryptophan catabolism in humans. Indole metabolites, including I3P and I3A, also function as potent AHR agonists. This route enables cancer cells to circumvent the anti-tumor immunity unleased by IDO1 inhibitors in cancer therapy [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It also suggests that indole metabolites and their effects on AHR activity are not solely attributed to microbial metabolism. We found that IL4I1 is upregulated to a greater extent than IDO1 in inflammatory cytokine-primed human MuSCs, suggesting that whereas there are two routes along which tryptophan is metabolized to generate bioactive small molecules that modulate immune functions, the preferred route may vary depending on cell types.\u003c/p\u003e \u003cp\u003eIn addition to activating AHR to promote the expression of TSG-6 in MuSCs, the extracellular I3P catalyzed by IL4I1 can also influence the functions of neighboring immune cells. Actually, I3P administration could directly curb disease symptoms in ALI mice through distinct mechanisms of action. Specifically, there was decreased infiltration of neutrophils into damaged lung tissues of diseased mice after I3P treatment, in which I3P inhibited CXCL1-mediated chemotaxis by neutrophils towards lungs. Moreover, ROS-mediated pathogenic effects of neutrophils were significantly restrained through increased expression of anti-oxidative genes when I3P administration. Collectively, these results suggested that I3P therapy may be explored for the treatment of inflammatory diseases.\u003c/p\u003e \u003cp\u003eIn summary, we have discovered a novel mechanism by which MuSCs function as immunoregulators. When stimulated by inflammatory cytokines, MuSCs yield I3P and I3A via IL4I1-catalyzed indole metabolism. As AHR ligands, they activate AHR, thereby upregulating TSG-6 expression. The indole metabolites can also directly act on immune cells to exert their anti-inflammatory effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These results not only highlight the importance of IL4I1 and its downstream metabolites in shaping the immunoregulatory function of MuSCs, but also provide a basis for developing indole metabolism-based new strategies for the treatment of inflammatory diseases.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMuSC culture\u003c/h2\u003e \u003cp\u003eMuSCs were as previously described [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. MuSCs were cultured in myogenic growth medium containing 1:1 mixture of DMEM Low medium and MCDB 131 medium, 20% fetal bovine serum (FBS), 1% penicillin-streptomycin (all from Gibco, MA, USA), 1% insulin-transferrin-selenium (Invitrogen, Carlsbad, CA, USA), and 10 \u0026micro;M p38 MAPK inhibitor (SB203580, Selleck, Houston, TX, USA), which functions to block the differentiation of MuSCs and thus maintain their self-renewal properties [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Cell plates and slides were pre-coated with ECM (Sigma-Aldrich, St Louis, MO, USA). All details regarding the characterization of cultured MuSCs were shown in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eALI model\u003c/h2\u003e \u003cp\u003eMale C57BL/6 mice (6\u0026ndash;8 weeks old) were purchased from Charles River Experimental Animal Technology Co. Ltd. (Beijing, China) and maintained under specific pathogen-free conditions of the Laboratory Animal Center of Soochow University. The experiments were approved by the Institutional Animal Care and Use Committee of Soochow University (SUDA20210916A07). The mice were housed in a 12 h light-dark cycle, and fed with irradiated food and sterile water \u003cem\u003ead libitum\u003c/em\u003e. All mice were acclimatized to the housing for 2 weeks before starting the experimental procedures. In this study, these mice were randomly divided into control, LPS, LPS\u0026thinsp;+\u0026thinsp;NC-hMuSCs and LPS\u0026thinsp;+\u0026thinsp;IL4I1-shRNA-hMuSCs groups. The LPS-induced ALI model was established by an airway administration of LPS (2 mg/kg, per mouse) into mice. 1 h later, hMuSCs (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells) and I3P (20 mg/kg) were intravenously injected to mice with ALI. After 24 h LPS challenge, mice were sacrificed and BAL fluid was collected through the tracheal cannula. Blood and lung tissue samples were collected for subsequent analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting analysis\u003c/h2\u003e \u003cp\u003eWestern blotting was performed to measure the protein level of IL4I1 in hMuSCs. Total proteins were extracted using RIPA lysis buffer (Beyotime, Shanghai, China). Protein concentration was determined using a BCA protein assay kit (Bio-Rad, Hercules, CA, USA). 40 \u0026micro;g of Protein was separated by a 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride membrane (Millipore, Temecula, CA, USA). After blocking with 5% BSA for 2 h, the membrane was incubated with primary antibodies overnight at 4\u0026deg;C. The primary antibodies included anti-human IL4I1 (Abcam, MA, USA) and anti-human β-ACTIN (Abcam). The membrane was then washed three times with TBST and respectively incubated with horseradish peroxidase-conjugated anti-rabbit and anti-rat antibodies (Abcam) at room temperature for 1 h. Finally, the protein levels were detected by the chemiluminescence reagent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eqRT-PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using RNAprep Pure Cell Kit (Feijie, Shanghai, China), and reverse-transcribed into cDNA with PrimeScript\u0026trade; RT Master Mix (TaKaRa, Dalian, China). The levels of mRNA expression were analyzed by QuantStudio\u0026trade; 6 Flex System according to the manufacturer\u0026rsquo;s instructions. The total reaction volume of 10 \u0026micro;l was comprised of 1 ng cDNA, 3 \u0026micro;l DNAase/RNAse-free water (TaKaRa), 1 \u0026micro;l special primers (GENEWIZ, Suzhou, Jiangsu, China), and 5 \u0026micro;l SYBR qPCR SuperMixplus with ROX (Novoprotein, Shanghai, China). The real-time PCR program was as follows: pre-denaturation at 95\u0026deg;C for 30 s; 40 cycles of denaturation at 95\u0026deg;C for 5 s; annealing and extension at 60\u0026deg;C for 30 s. The total amount of mRNA was compared with endogenous \u003cem\u003eβ-ACTIN\u003c/em\u003e mRNA. Finally, the relative expression of mRNA was calculated using 2\u003csup\u003e\u0026ndash;ΔΔCt\u003c/sup\u003e method. Primer sequences were shown in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eElisa\u003c/h3\u003e\n\u003cp\u003eThe supernatants of MuSCs were collected 24 h after distinct treatments to detect the concentration of TSG-6 through ELISA as previously described [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Briefly, a 96-well plate was pre-coated with 50 \u0026micro;l of 10 \u0026micro;g/ml TSG-6 antibody (Santa Cruz, Dallas, Texas, USA) overnight, washed with PBS and blocked with the buffer containing 0.25% BSA and 0.05% Tween 20. After 1 hour, the excess blocking buffer was washed away. Biotinylated anti-human TSG-6 antibody (R\u0026amp;D Systems, Minneapolis, MN, USA) was added and incubated for 2 h at room temperature. After washing with PBS, streptavidin-horseradish peroxidase (R\u0026amp;D Systems) was added for 30 min at room temperature, and developed using substrate solutions (R\u0026amp;D Systems).\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence assay\u003c/h2\u003e \u003cp\u003eFor immunofluorescence staining of lung sections, the hydrated slides were subjected to antigen retrieval in Tris-EDTA, pH 9.0 (Yuanye, Shanghai, China), at 96\u0026deg;C for 30 min. Then, lung sections were incubated with 3% bovine serum albumin (Amresco, OH, USA) for 1 h to prevent non-specific staining before incubating with anti-mouse Ly6G (Abcam) overnight at 4\u0026deg;C. Finally, lung sections were incubated with anti-rat IgG (H\u0026thinsp;+\u0026thinsp;L) (Alexa Fluo 594, Abcam) for 1 h and Hoechst 33324 (Beyotime) for 10 min at room temperature.\u003c/p\u003e \u003cp\u003eMuSCs were seeded on eight-chamber slide (Thermo Fisher) at the density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per chamber. After indicated treatment, cells were fixed with paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 for 10 min, and blocked with 3% BSA in PBS for 1 h. The cells were then incubated with 10 \u0026micro;g/ml anti-human AHR (Abcam) overnight at 4\u0026deg;C. Then, cells were washed with PBS and incubated with anti-Mouse IgG (H\u0026thinsp;+\u0026thinsp;L) (Alexa Fluor 647, Thermo Fisher) for 1 h at room temperature. After PBS washing, cell nucleus was stained with 1 \u0026micro;g/ml Hoechst 33324 (Beyotime) for 8 min at room temperature. Finally, images were taken with the Nikon microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eMice were sacrificed 24 h after LPS exposure. Lung tissues and peripheral blood mononuclear cells were collected. To obtain BAL fluid, lungs were washed three times with 1 ml PBS. Cells for flow cytometric analysis were pre-incubated with anti-CD16/CD32 (BioLegend, San Diego, CA, USA) to block Fc receptors, and then stained with anti-mouse CD45 (BioLegend), anti-mouse CD11b (BioLegend), anti-mouse Ly6C (BioLegend) and anti-mouse Ly6G (BioLegend) at 4\u0026deg;C for 20 min. Then, the total cell number in BAL fluid was counted and neutrophil numbers were determined on a Cytoflex Flow Cytometer (Beckman Coulter, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eHistological analysis\u003c/h2\u003e \u003cp\u003eIsolated lung tissues were fixed in 4% paraformaldehyde, dehydrated using graded ethanol, embedded in paraffin, cut into 5-\u0026micro;m-thick sections, and then stained with hematoxylin and eosin (H\u0026amp;E). For the immunohistochemistry of CXCL1, the lung samples were stained with anti-mouse CXCL1 antibody (Abcam), followed by HRP-linked anti-rat secondary antibody (Maixin, Fuzhou, Fujian, China) and diaminobenzidine (Maixin). Images were taken with the Nikon microscope. More than three fields from each mouse were selected and the images are representative for each experiment repeats.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism 9 (GraphPad Prism Software Inc., CA, USA). All data in this article are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. For two-group comparison, two-tailed unpaired \u003cem\u003et\u003c/em\u003e test was performed. For multiple group comparison, one-way analysis of variance test was performed. Notably, \u003cem\u003eP\u003c/em\u003e values less than 0.05 were statistically considered significant. *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.Z. and J.F. designed the project, performed the experiments, analyzed the data, and prepared the manuscript. P.H., S.L., P.H., S.W., Z.L., C.F., L.C. and P.L. performed the experiments and provided advice for data analysis. Y.S. and C.S. conceived and supervised the project and revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the National Key R\u0026amp;D Program of China (2021YFA1100600 and 2022YFA0807300), the National Natural Science Foundation of China (32150710523 and 82202032), Jiangsu Province International Joint Laboratory for Regenerative Medicine Fund, Suzhou Foreign Academician Workstation Fund (SWY202202) and Suzhou Science and Technology Initiative Fund (SYS2020087).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSousa-Victor P, Garcia-Prat L, Munoz-Canoves P. Control of satellite cell function in muscle regeneration and its disruption in ageing. Nat Rev Mol Cell Biol. 2022;23:204\u0026ndash;226.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrunet A, Goodell MA, Rando TA. Ageing and rejuvenation of tissue stem cells and their niches. Nat Rev Mol Cell Biol. 2023;24:45\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFang J, Feng C, Chen W, Hou P, Liu Z, Zuo M, et al. Redressing the interactions between stem cells and immune system in tissue regeneration. Biol Direct. 2021;16:18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Fang J, Liu B, Shao C, Shi Y. Reciprocal regulation of mesenchymal stem cells and immune responses. Cell Stem Cell. 2022;29:1515\u0026ndash;1530.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan Y, Yang J, Fang J, Zhou Y, Candi E, Wang J, et al. The secretion profile of mesenchymal stem cells and potential applications in treating human diseases. Signal Transduct Target Ther. 2022;7:92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFang J, Zhang S, Liu Z, Pan Y, Cao L, Hou P, et al. Skeletal muscle stem cells confer maturing macrophages anti-inflammatory properties through insulin-like growth factor-2. Stem Cells Transl Med. 2020;9:773\u0026ndash;785.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang S, Fang J, Liu Z, Hou P, Cao L, Zhang Y, et al. Inflammatory cytokines-stimulated human muscle stem cells ameliorate ulcerative colitis via the IDO-TSG6 axis. Stem Cell Res Ther. 2021;12:50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Z, Li T, Mao C, Liu W, Tao Y. IL4I1-driven AHR signature: a new avenue for cancer therapy. 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Stem Cell Reports. 2015;5:621\u0026ndash;632.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2618290/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2618290/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMuscle stem cells (MuSCs) have been demonstrated to exert impressive therapeutic efficacy in disease settings through orchestrating inflammatory microenvironments. Nevertheless, the mechanisms underlying the immunoregulatory property of MuSCs remain largely uncharacterized. Here, we showed that interleukin-4-induced-1 (IL4I1), an essential enzyme that catalyzes indole metabolism in humans, was highly expressed in human MuSCs exposed to IFN-γ and TNF-α. Functionally, the MuSCs were found to inhibit the infiltration of neutrophils into sites of inflammation in a IL4I1-dependent manner and thus ameliorate acute lung injury in mice. Mechanistically, the indole metabolites, including indole-3-pyruvic acid (I3P) and indole-3-aldehyde (I3A), produced by IL4I1, acted as ligands to activate aryl hydrocarbon receptor (AHR), leading to augmented expression of TNF-stimulated gene 6 (TSG-6) in inflammatory cytokine-primed MuSCs. Furthermore, I3P administration alone suppressed neutrophil infiltration in damaged lungs. I3P could also reduce the level of reactive oxygen species in neutrophils. Therefore, our study has uncovered a novel mechanism by which MuSCs acquire their immunoregulatory property and may help to develop or optimize MuSC-based therapies for inflammatory diseases.\u003c/p\u003e","manuscriptTitle":"IL4I1-catalyzed tryptophan metabolites mediate the anti-inflammatory function of cytokine-primed human muscle stem cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-10 00:03:42","doi":"10.21203/rs.3.rs-2618290/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2023-03-27T14:43:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2023-03-22T16:43:47+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2023-03-08T22:08:02+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2023-03-07T20:30:54+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2023-03-07T20:26:09+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2023-03-05T09:51:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-02-23T10:30:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-02-23T01:03:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death Discovery","date":"2023-02-23T01:03:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"db2a8521-ad23-4f37-8f39-a6d9d4eb33c4","owner":[],"postedDate":"March 10th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":19674350,"name":"Biological sciences/Immunology/Immunotherapy/Immunosuppression"},{"id":19674351,"name":"Biological sciences/Stem cells/Muscle stem cells"}],"tags":[],"updatedAt":"2023-07-29T07:06:59+00:00","versionOfRecord":{"articleIdentity":"rs-2618290","link":"https://doi.org/10.1038/s41420-023-01568-x","journal":{"identity":"cell-death-discovery","isVorOnly":false,"title":"Cell Death Discovery"},"publishedOn":"2023-07-28 04:00:00","publishedOnDateReadable":"July 28th, 2023"},"versionCreatedAt":"2023-03-10 00:03:42","video":"","vorDoi":"10.1038/s41420-023-01568-x","vorDoiUrl":"https://doi.org/10.1038/s41420-023-01568-x","workflowStages":[]},"version":"v1","identity":"rs-2618290","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2618290","identity":"rs-2618290","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}