Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

LACC1 bridges NOS2 and polyamine metabolism in inflammatory macrophages

This article has been updated

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: LACC1 is an endogenous isocyanic acid synthase, converting l-Cit to l-Orn and HNCO.
Fig. 2: LACC1 inhibits proinflammatory cytokine signalling and protects from S. Typhimurium infection.
Fig. 3: LACC1 bridges arginine metabolism with polyamine synthesis in inflammatory macrophages.
Fig. 4: NOS2 is indispensable for the antibacterial function of LACC1.

Data availability

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. 

Change history

  • 19 August 2022

    In the version of this article initially published, the surname of Peter Murray in the Peer review information was misspelt and has now been amended.

References

  1. Heng, T. S. & Painter, M. W., Consortium, I. G. P. The Immunological Genome Project: networks of gene expression in immune cells. Nat. Immunol. 9, 1091–1094 (2008).

    Article  CAS  PubMed  Google Scholar 

  2. Skon-Hegg, C. et al. LACC1 regulates TNF and IL-17 in mouse models of arthritis and inflammation. J Immunol. 202, 183–193 (2019).

    Article  CAS  PubMed  Google Scholar 

  3. Kang, J. W. et al. Myeloid cell expression of LACC1 is required for bacterial clearance and control of intestinal inflammation. Gastroenterology 159, 1051–1067 (2020).

    Article  CAS  PubMed  Google Scholar 

  4. Mogensen, T. H. Pathogen recognition and inflammatory signaling in innate immune defenses. Clin. Microbiol. Rev. 22, 240–273 (2009).

    Article  CAS  PubMed Central  Google Scholar 

  5. Schieber, M. & Chandel, N. S. ROS function in redox signaling and oxidative stress. Curr. Biol. 24, R453–R462 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Mittler, R. ROS are good. Trends Plant Sci. 22, 11–19 (2017).

    Article  CAS  PubMed  Google Scholar 

  7. Martínez, M. C. & Andriantsitohaina, R. Reactive nitrogen species: molecular mechanisms and potential significance in health and disease. Antioxid. Redox Signal. 11, 669–702 (2009).

    Article  PubMed  CAS  Google Scholar 

  8. Marletta, M. A., Yoon, P. S., Iyengar, R., Leaf, C. D. & Wishnok, J. S. Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate. Biochemistry 27, 8706–8711 (1988).

    Article  CAS  PubMed  Google Scholar 

  9. Nathan, C. & Shiloh, M. U. Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc. Natl Acad. Sci. USA 97, 8841–8848 (2000).

    Article  PubMed Central  Google Scholar 

  10. Qualls, J. E. et al. Sustained generation of nitric oxide and control of mycobacterial infection requires argininosuccinate synthase 1. Cell Host Microbe 12, 313–323 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Omarjee, O. et al. LACC1 deficiency links juvenile arthritis with autophagy and metabolism in macrophages. J. Exp. Med. 218, e20201006 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Huang, C. et al. Genetic risk for inflammatory bowel disease is a determinant of Crohn’s disease development in chronic granulomatous disease. Inflamm. Bowel Dis. 22, 2794–2801 (2016).

    Article  PubMed  Google Scholar 

  13. Assadi, G. et al. LACC1 polymorphisms in inflammatory bowel disease and juvenile idiopathic arthritis. Genes Immun. 17, 261–264 (2016).

    Article  CAS  PubMed  Google Scholar 

  14. Assadi, G. et al. Functional analyses of the Crohn’s disease risk gene LACC1. PLoS ONE 11, e0168276 (2016).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  15. Kallinich, T. et al. Juvenile arthritis caused by a novel FAMIN (LACC1) mutation in two children with systemic and extended oligoarticular course. Pediatr. Rheumatol. Online J. 14, 63 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Wakil, S. M. et al. Association of a mutation in LACC1 with a monogenic form of systemic juvenile idiopathic arthritis. Arthritis Rheumatol. 67, 288–295 (2015).

    Article  CAS  PubMed  Google Scholar 

  17. Grant, A. V. et al. Crohn’s disease susceptibility genes are associated with leprosy in the Vietnamese population. J. Infect. Dis. 206, 1763–1767 (2012).

    Article  CAS  PubMed  Google Scholar 

  18. Cader, M. Z. et al. FAMIN is a multifunctional purine enzyme enabling the purine nucleotide cycle. Cell 180, 278–295 (2020).

    Article  CAS  Google Scholar 

  19. Szymanski, A. M. & Ombrello, M. J. Using genes to triangulate the pathophysiology of granulomatous autoinflammatory disease: NOD2, PLCG2 and LACC1. Int. Immunol. 30, 205–213 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Murray, P. J. et al. Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity 41, 14–20 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Pearce, E. L. & Pearce, E. J. Metabolic pathways in immune cell activation and quiescence. Immunity 38, 633–643 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Delanghe, S., Delanghe, J. R., Speeckaert, R., Van Biesen, W. & Speeckaert, M. M. Mechanisms and consequences of carbamoylation. Nat. Rev. Nephrol. 13, 580–593 (2017).

    Article  CAS  PubMed  Google Scholar 

  23. Lundquist, P., Backman-Gullers, B., Kågedal, B., Nilsson, L. & Rosling, H. Fluorometric determination of cyanate in plasma by conversion to 2,4(1H,3H)-quinazolinedione and separation by high-performance liquid chromatography. Anal. Biochem. 211, 23–27 (1993).

    Article  CAS  PubMed  Google Scholar 

  24. Lahiri, A., Hedl, M., Yan, J. & Abraham, C. Human LACC1 increases innate receptor-induced responses and a LACC1 disease-risk variant modulates these outcomes. Nat. Commun. 8, 15614 (2017).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  25. Minois, N., Carmona-Gutierrez, D. & Madeo, F. Polyamines in aging and disease. Aging (Albany NY) 3, 716–732 (2011).

    Article  Google Scholar 

  26. Hardbower, D. M. et al. Arginase 2 deletion leads to enhanced M1 macrophage activation and upregulated polyamine metabolism in response to Helicobacter pylori infection. Amino Acids 48, 2375–2388 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Cheng, Y. et al. Helicobacter pylori-induced macrophage apoptosis requires activation of ornithine decarboxylase by c-Myc. J. Biol. Chem. 280, 22492–22496 (2005).

    Article  CAS  PubMed  Google Scholar 

  28. Shiloh, M. U. et al. Phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase. Immunity 10, 29–38 (1999).

    Article  CAS  PubMed  Google Scholar 

  29. Roberts, J. M. et al. Isocyanic acid in the atmosphere and its possible link to smoke-related health effects. Proc. Natl Acad. Sci. USA 108, 8966–8971 (2011).

    PubMed Central  Google Scholar 

  30. Cordes, T. & Metallo, C. M. M. Itaconate alters succinate and coenzyme A metabolism via inhibition of mitochondrial complex II and methylmalonyl-CoA mutase. Metabolites 11, 117 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Murphy, M. P. & O’Neill, L. A. J. Krebs cycle reimagined: the emerging roles of succinate and itaconate as signal transducers. Cell 174, 780–784 (2018).

    Article  CAS  PubMed  Google Scholar 

  32. Viola, A., Munari, F., Sánchez-Rodríguez, R., Scolaro, T. & Castegna, A. The metabolic signature of macrophage responses. Front. Immunol. 10, 1462 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Chen, M. et al. Itaconate is an effector of a Rab GTPase cell-autonomous host defense pathway against Salmonella. Science 369, 450–455 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  34. Saveljeva, S. et al. A purine metabolic checkpoint that prevents autoimmunity and autoinflammation. Cell Metab. 34, 106–124 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Hardbower, D. M. et al. Ornithine decarboxylase regulates M1 macrophage activation and mucosal inflammation via histone modifications. Proc. Natl Acad. Sci. USA 114, E751–E760 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Nakamura, A. et al. Symbiotic polyamine metabolism regulates epithelial proliferation and macrophage differentiation in the colon. Nat. Commun. 12, 2105 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  37. Gaboriau, F., Vaultier, M., Moulinoux, J.-P. & Delcros, J.-G. Antioxidative properties of natural polyamines and dimethylsilane analogues. Redox Rep. 10, 9–18 (2005).

    Article  CAS  PubMed  Google Scholar 

  38. Ghosh, I., Sankhe, R., Mudgal, J., Arora, D. & Nampoothiri, M. Spermidine, an autophagy inducer, as a therapeutic strategy in neurological disorders. Neuropeptides 83, 102083 (2020).

    Article  CAS  PubMed  Google Scholar 

  39. Jeong, J.-W. et al. Spermidine protects against oxidative stress in inflammation models using macrophages and zebrafish. Biomol. Ther. (Seoul) 26, 146–156 (2018).

    Article  CAS  Google Scholar 

  40. Eisenberg, T. et al. Induction of autophagy by spermidine promotes longevity. Nat. Cell Biol. 11, 1305–1314 (2009).

    Article  CAS  PubMed  Google Scholar 

  41. Yang, Q. et al. Spermidine alleviates experimental autoimmune encephalomyelitis through inducing inhibitory macrophages. Cell Death Differ. 23, 1850–1861 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Puleston, D. J. et al. Polyamines and eIF5A hypusination modulate mitochondrial respiration and macrophage activation. Cell Metab. 30, 352–363 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Delporte, C. et al. Myeloperoxidase-catalyzed oxidation of cyanide to cyanate: a potential carbamylation route involved in the formation of atherosclerotic plaques? J. Biol. Chem. 293, 6374–6386 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Laubach, V. E., Shesely, E. G., Smithies, O. & Sherman, P. A. Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death. Proc. Natl Acad. Sci. USA 92, 10688–10692 (1995).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  45. Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Nowarski, R. et al. Epithelial IL-18 equilibrium controls barrier function in colitis. Cell 163, 1444–1456 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Jackson, R. et al. The translation of non-canonical open reading frames controls mucosal immunity. Nature 564, 434–438 (2018).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  48. Chen, L. M., Kaniga, K. & Galán, J. E. Salmonella spp. are cytotoxic for cultured macrophages. Mol. Microbiol. 21, 1101–1115 (1996).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

R.A.F. and J.M.C. conceived the project. Z.W. designed and performed all experiments and analysed data. J.O. contributed to NMR experiments and MS analysis. Z.W., R.A.F. and J.M.C. wrote the manuscript with input from all authors. R.A.F. supervised the mouse model and cytokine studies. J.M.C. supervised the biochemistry and metabolism studies.

Corresponding authors

Correspondence to Richard A. Flavell or Jason M. Crawford.

Ethics declarations

Competing interests

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.

Peer review

Peer review information

Nature thanks Alexandre Belot, Kivanc Birsoy, Michael Murphy and Peter Murray for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 In vitro biochemical analysis of isolated LACC1 variants.

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.

Source data

Extended Data Fig. 2 Kinetic analysis of isolated enzymes.

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.

Source data

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.

Source data

Extended Data Fig. 4 Supplementation of L-Orn control under homeostatic conditions.

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.

Source data

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.

Source data

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.

Source data

Extended Data Fig. 7 Summary of the enzymatic functions of LACC1 in inflammatory macrophages.

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.

Supplementary information

Source data

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-022-05111-3

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research