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

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 into 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.

Fig. 1. IFN-γ and TNF-α increase the expression of IL4I1 in MuSCs through NF-κB and STAT6 pathway.

A Volcano plot of differentially expressed genes in MuSCs after the stimulation of IFN-γ and TNF-α (10 ng/ml each) for 24 h using RNA-seq. B Volcano 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. C 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. D The concentration of IL4I1 in the supernatants of MuSCs after the stimulation of IFN-γ and TNF-α (10 ng/ml each) for 24 h was measured by ELISA. E 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 was determined by western blotting. F 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. G The 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 were representative of three experiments with similar results. For two-group comparison, statistical analysis was performed by Student’s t test. ***P < 0.001; ****P < 0.0001.

Fig. 2. IL4I1 mediates the therapeutic effect of MuSCs on ALI.

A The therapeutic strategy of MuSCs in the LPS-induced ALI model. Mice were treated with 2 mg/kg LPS through endotracheal infusion. 1 h later, Ctrl-shRNA MuSCs or IL4I1-shRNA MuSCs (5 × 10⁵) 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. B The efficiency of IL4I1 knockdown measured by western blotting analysis. C The total amount of IL4I1 protein in the lung tissue homogenates of ALI mice was determined by ELISA (PBS: n = 3, LPS: n = 3, LPS+Ctrl-shRNA MuSCs: n = 3, LPS + IL4I1-shRNA MuSCs: n = 3). D The expression levels of IL-6 mRNA (left) and protein (right) in the lung tissue homogenates of ALI mice were respectively determined by qRT-PCR and ELISA. E Lung tissues of mice with various treatments were fixed for H&E staining. Yellow arrowhead, area of widespread septal thickening with increased air-space cellularity and exudation and enhanced interstitial immune cell infiltration in the damaged lungs of ALI mice. Scale bars, 250 μm. F The expression levels of chemokines in the lung tissue homogenates of ALI mice were determined by qRT-PCR. G Lung tissues of mice with various treatments were stained with CXCL1 antibody. Scale bars, 250 μm (PBS: n = 3, LPS: n = 4, LPS+Ctrl-shRNA MuSCs: n = 5, LPS + IL4I1-shRNA MuSCs: n = 5). Data were shown as means ± SEM. Data were representative of three experiments with similar results. For multiple group comparison, statistical analysis was performed by one-way ANOVA test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3. Secreted IL4I1 from MuSCs suppresses neutrophil infiltration into lung tissues in ALI mice.

A, B Mice were treated with 2 mg/kg LPS through endotracheal infusion. 1 h later, Ctrl-shRNA MuSCs or IL4I1-shRNA MuSCs (5 × 10⁵) pretreated with IFN-γ and TNF-α (10 ng/ml each) for 24 h were intravenously injected into mice. The proportion and absolute numbers of neutrophils in the left lung tissues of ALI mice were determined by flow cytometry analysis. C The proportion of neutrophils in the blood of ALI mice was measured by flow cytometry analysis (PBS: n = 3, LPS: n = 5, LPS+Ctrl-shRNA MuSCs: n = 5, LPS + IL4I1-shRNA MuSCs: n = 5). D The proportion of neutrophils in the BAL fluid of ALI mice was measured by flow cytometry analysis. E The absolute numbers of total immune cells including neutrophils in the BAL fluid of ALI mice were determined by flow cytometry analysis (PBS: n = 2, LPS: n = 5, LPS+Ctrl-shRNA MuSCs: n = 5, LPS + IL4I1-shRNA MuSCs: n = 4). F The neutrophil infiltration into lung tissues was evaluated by immunofluorescence staining of Ly6G (red) and Hoechst (blue). Scale bars, 120 μm. Data were shown as means ± SEM. Data were representative of three experiments with similar results. For multiple group comparison, statistical analysis was performed by one-way ANOVA test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4. I3P and I3A promote TSG-6 production in MuSCs.

A The mRNA and protein levels of TSG-6 in Ctrl-shRNA MuSCs and IL4I1-shRNA MuSCs stimulated with IFN-γ and TNF-α (10 ng/ml each) for 24 h were respectively measured by qRT-PCR and ELISA. B A schematic diagram illustrating tryptophan metabolism in humans via different catabolic routes. C The mRNA and protein levels of TSG-6 in Ctrl-shRNA MuSCs and IL4I1-shRNA MuSCs stimulated with IFN-γ and TNF-α (10 ng/ml each) for 24 h in the presence or absence of I3P (50 μM) were respectively measured by qRT-PCR and ELISA. D The concentration of TSG-6 in the supernatants of Ctrl-shRNA MuSCs and IL4I1-shRNA MuSCs 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. E The mRNA and protein levels of TSG-6 in MuSCs stimulated with IFN-γ and TNF-α (10 ng/ml each) for 24 h in the presence or absence of I3P (50 μM) were respectively measured by qRT-PCR and ELISA. F The mRNA and protein levels of TSG-6 in MuSCs stimulated with IFN-γ and TNF-α (10 ng/ml each) for 24 h in the presence or absence of I3A (100 μM) were respectively measured by qRT-PCR and ELISA. Data were shown as means ± SEM. Data were representative of three experiments with similar results. For two-group comparison, statistical analysis was performed by Student’s t test. For multiple group comparison, statistical analysis was performed by one-way ANOVA test. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 5. I3P and I3A activate AHR signaling pathway to enhance TSG-6 production in MuSCs.

A The CYP1B1 expression in MuSCs stimulated with IFN-γ and TNF-α (10 ng/ml each) in the presence or absence of CH223191 (100 μM) for 24 h was measured by qRT-PCR. B The mRNA and protein levels of TSG-6 in MuSCs stimulated with IFN-γ and TNF-α (10 ng/ml each) in the presence or absence of CH223191 (100 μM) for 24 h were respectively measured by qRT-PCR and ELISA. C Immunofluorescence staining of AHR in MuSCs treated with IFN-γ (I, 10 ng/ml) and TNF-α (T, 10 ng/ml) in the presence or absence of I3P (50 μM) and I3A (100 μM) for 24 h. Scale bars, 40 μm. D MuSCs treated with IFN-γ (I, 10 ng/ml) and TNF-α (T, 10 ng/ml) in the presence or absence of I3P (50 μM), I3A (100 μM) and CH223191 (CH, 100 μM) were subjected to the cytoplasmic and nuclear extraction, and the distribution of AHR in cytoplasm and nucleus was analyzed by western blotting analysis. β-ACTIN and LAMINIB1 were served as loading controls for cytoplasmic and nuclear proteins, respectively. E The CYP1B1 expression 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. F The mRNA and protein levels of TSG-6 in MuSCs stimulated with IFN-γ and TNF-α (10 ng/ml each) in the presence or absence of I3P (50 μM) and CH223191 (CH, 100 μM) for 24 h were respectively measured by qRT-PCR and ELISA. G The mRNA and protein levels of TSG-6 in MuSCs stimulated with IFN-γ and TNF-α (10 ng/ml each) in the presence or absence of I3A (100 μM) and CH223191 (CH, 100 μM) for 24 h were respectively measured by qRT-PCR and ELISA. Data were shown as means ± SEM. Data were representative of three experiments with similar results. For two-group comparison, statistical analysis was performed by Student’s t test. For multiple group comparison, statistical analysis was performed by one-way ANOVA test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 6. I3P administration suppresses neutrophil infiltration and enhances oxidation resistance in damaged lungs.

A The therapeutic strategy of I3P in the LPS-induced ALI model. Mice were treated with 2 mg/kg LPS through endotracheal infusion. 1 h 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. B Lung tissues of mice with various treatments were fixed for H&E staining. Yellow arrowhead, area of widespread septal thickening with increased air-space cellularity and exudation and enhanced interstitial immune cell infiltration in the damaged lungs of ALI mice. Scale bars, 250 μm. C The number of neutrophils in the lungs of ALI mice was measured by flow cytometry analysis (PBS: n = 5, LPS: n = 8, LPS + I3P: n = 10). D The expression levels of Cxcl1 in the lung tissue homogenates of ALI mice were determined by qRT-PCR (PBS: n = 2, LPS: n = 8, LPS + I3P: n = 7). E The expression levels of Ho1 and Nrf2 in neutrophils infiltrating into the lungs were determined by qRT-PCR (PBS: n = 2, LPS: n = 4, LPS + I3P: n = 4). F ROS levels in PMA-activated neutrophils treated with I3P (200 μM) were measured by flow cytometry analysis. Data were shown as means ± SEM. Data were representative of three experiments with similar results. For multiple group comparison, statistical analysis was performed by one-way ANOVA test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 7. A schematic model of the anti-inflammatory effects of IL4I1 on LPS induced-ALI.

IL4I1 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.