The mammalian immune system uses various pattern recognition receptors to recognize invaders and host damage and transmits this information to downstream immunometabolic signalling outcomes. Laccase domain-containing 1 (LACC1) protein is an enzyme highly expressed in inflammatory macrophages and serves a central regulatory role in multiple inflammatory diseases such as inflammatory bowel diseases, arthritis and clearance of microbial infection1,2,3,4. However, the biochemical roles required for LACC1 functions remain largely undefined. Here we elucidated a shared biochemical function of LACC1 in mice and humans, converting l-citrulline to l-ornithine (l-Orn) and isocyanic acid and serving as a bridge between proinflammatory nitric oxide synthase (NOS2) and polyamine immunometabolism. We validated the genetic and mechanistic connections among NOS2, LACC1 and ornithine decarboxylase 1 (ODC1) in mouse models and bone marrow-derived macrophages infected by Salmonella enterica Typhimurium. Strikingly, LACC1 phenotypes required upstream NOS2 and downstream ODC1, and Lacc1–/– chemical complementation with its product l-Orn significantly restored wild-type activities. Our findings illuminate a previously unidentified pathway in inflammatory macrophages, explain why its deficiency may contribute to human inflammatory diseases and suggest that l-Orn could serve as a nutraceutical to ameliorate LACC1-associated immunological dysfunctions such as arthritis or inflammatory bowel disease.
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Supplementary information and source data are provided with this paper. Additional data that support the findings of this study are available from the corresponding author on reasonable request.
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We thank J. Alderman, B. Cadugan, C. Lieber, C. Hughes, L. Evangelisti, E. Hughes-Picard, F. Zhang, P. Ranney, W. Philbrick and P. Maher-Rivera for help with mouse gene-editing projects and in overall administration, as well as J. Galan for the gift of S. Typhimurium strain SL1344. We thank T. Wu at Yale West Campus Analytical Core for assistance with MS analysis and J. Karosas at the Yale Analytical and Stable Isotope Center for assistance with ICP–MS analysis. This work was supported by the Howard Hughes Medical Institute (to R.A.F.), the Burroughs Wellcome Fund (no. 1016720 to J.M.C.) and Yale University. Z.W. was supported by the China Scholarship Council.
R.A.F. is a recipient of a grant from AbbVie, Inc., is a founder of Rheos Biomedicines and is a consultant to GSK and Zai laboratories. The remaining authors declare no competing interests.
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Extended data figures and tables
a, ICP-MS assays showed the enrichment of Zn64 and Zn66 isotopes in recombinant mLACC1 relative to vector control. b, A quantitative ICP-MS assay for Zn showed the average Zn:mLACC1 ratio as 0.94, suggesting a 1:1 ratio in the isolated enzyme. c-d, Volcano plots from UPLC-QTOF-MS experiments showing the fold change (FC) (substrate with mLACC1 versus substrate with heat inactivated mLACC1) and false discovery rate (FDR) values collected in positive ion mode (c) and negative ion mode for the specific detection of ribose-1-phosphate (d). e, Rates of L-Orn production in enzymatic assays at different pH conditions. f, Rates of L-Orn production in enzymatic assays with the supplementation of EDTA or TPEN in the reaction buffer. g, The standard curve used for quantifying L-Orn in enzymatic assays. h, The standard curve used for quantifying 2-aminobenzoate-HNCO carbamoylation products in enzymatic assays. i, The LC-MS trace of 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA, Marfey’s reagent)-coupled L-Orn in enzymatic assays, supporting the stereoconfiguration. j, Expanded view 13C-NMR spectroscopic data of LACC1 reaction using 6-13C1-L-Cit as substrate, showing L-Orn and (OCN)– as major products. The mean and SEM (error bars) are derived from three biological replicates (n = 3). Statistical significance (two-tailed t-test) compared to control (Ctrl): *P < 0.05; **P < 0.01; ***P < 0.001; nd, not detectable.
Velocity plots of isolated enzyme reactions at different concentrations of L-Cit (5 μM, 10 μM, 25 μM, 50 μM, 100 μM, 200 μM and 500 μM) in enzyme kinetic analysis of WT mLACC1 (a) and hLACC1 (b) and the polymorphic human enzyme variants: K38E (c), I254V (d) and C284R (e). f, Michaelis-Menten curves of mLACC1, hLACC1, hLACC1 K38E, hLACC1 I254V, and hLACC1 C284R. The mean and SEM (error bars) are derived from three biological replicates (n = 3). Statistical significance (two-tailed t-test) compared to control (Ctrl): *P < 0.05; **P < 0.01; ns, not significant.
Extended Data Fig. 3 Lacc1C284R/C284R shows an intermediate defect in antibacterial protection against S. Typhimurium infection.
a, Body weight of each mouse was monitored daily after S. Typhimurium infection (n = 10). b, CFUs in fecal pellets were measured at day 4 after infection. c, d, e, CFUs in caecum, spleen and liver, respectively, were measured at day 6 after infection. f–h, ELISA measurements of TNFα, IL6 and IL12b secreted by BMDMs in the presence or absence (NT, non-treatment) of immunostimulant (LPS + IFNγ). i–k, ELISA measurements of TNFα, IL6 and IL12b secreted by BMDMs in the presence or absence (NT) of immunostimulant (LPS + IFNγ), exogenously supplied LACC1 product L-Orn (100 μM, 1 mM, 5 mM, 10 mM), and/or the conjugate base of LACC1 product HNCO, NaOCN (10 μM, 100 μM, 500 μM, 1 mM). The mean and SEM (error bars) are derived from three (f–k) or ten (a–e) biological replicates (n = 3 or n = 10). Statistical significance (two-tailed t-test) compared to control (Ctrl): *P < 0.05; **P < 0.01; ns, not significant.
Body weight of each mouse in the absence of S. Typhimurium infection was monitored daily with or without 1% L-Orn administration in drinking water, showing no significant change in body weight. The mean and SEM (error bars) are derived from ten biological replicates (n = 10). Statistical significance (two-tailed t-test) compared to control (Ctrl): ns, not significant.
Extended Data Fig. 5 LACC1 bridges L-Arg metabolism with polyamine synthesis in inflammatory macrophages.
a, b, MS intensity of SAM and dcSAM, respectively, in inflammatory BMDMs from WT and Lacc1−/− mice. Lacc1–/– BMDMs show an elevated but non statistically significant level of dcSAM. c–e, UPLC-QTOF-MS intensity of 13C-labeled metabolites in L-Arg metabolism and polyamine synthesis with 13C-Arg feeding (c), 13C-Cit feeding (d) and 13C-ArgSuc feeding (e), respectively. These data complement LC-QQQ-MS experimental measurements shown in Fig. 3a. The mean and SEM (error bars) are derived from three biological replicates (n = 3). Statistical significance (two-tailed t-test) compared to control (Ctrl): *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
Extended Data Fig. 6 Irreversible inhibition of ODC1 phenocopied exacerbated S. Typhimurium induced cell death from Lacc1−/− inflammatory BMDMs, but exogenous putrescine could not complement activity.
a, b, c, ELISA measurements of TNFα, IL6, and IL12b, respectively, secreted by BMDMs stimulated by LPS and IFNγ with or without 1 mM DFMO and/or 500 μM putrescine (Put) treatment. d, CFUs of intracellular S. Typhimurium in inflammatory BMDMs. e, LDH assay in S. Typhimurium infected inflammatory BMDMs. The mean and SEM (error bars) are derived from three biological replicates (n = 3). Statistical significance (two-tailed t-test) compared to control (Ctrl): *P < 0.05; **P < 0.01; ns, not significant.
a, LACC1’s newly described role in bridging L-Arg and polyamine metabolism in inflammatory macrophages. The carbons are highlighted with consistency to our 1,2,3,4,5-13C5-L-Cit (red) and 6-13C1-L-Cit (blue) feeding studies in inflammatory BMDMs. Carbons in spermidine and spermine highlighted in green derive from dcSAM. b, The involvement of LACC1 (a.k.a., FAMIN) in the purine nucleotide cycle as a purine nucleoside enzyme18. ADSL, adenylosuccinate lyase; ADSS, adenylosuccinate synthase; AMP, adenosine monophosphate; AMPD, AMP deaminase; GDP, guanosine diphosphate; GMP, guanosine monophosphate; GTP, guanosine triphosphate; IMP, inosine monophosphate; XMP, xanthosine monophosphate.
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Wei, Z., Oh, J., Flavell, R.A. et al. LACC1 bridges NOS2 and polyamine metabolism in inflammatory macrophages. Nature 609, 348–353 (2022). https://doi.org/10.1038/s41586-022-05111-3