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C13orf31 (FAMIN) is a central regulator of immunometabolic function

Nature Immunology volume 17, pages 1046–1056 (2016)Cite this article

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Abstract

Single-nucleotide variations in C13orf31 (LACC1) that encode p.C284R and p.I254V in a protein of unknown function (called 'FAMIN' here) are associated with increased risk for systemic juvenile idiopathic arthritis, leprosy and Crohn's disease. Here we set out to identify the biological mechanism affected by these coding variations. FAMIN formed a complex with fatty acid synthase (FASN) on peroxisomes and promoted flux through de novo lipogenesis to concomitantly drive high levels of fatty-acid oxidation (FAO) and glycolysis and, consequently, ATP regeneration. FAMIN-dependent FAO controlled inflammasome activation, mitochondrial and NADPH-oxidase-dependent production of reactive oxygen species (ROS), and the bactericidal activity of macrophages. As p.I254V and p.C284R resulted in diminished function and loss of function, respectively, FAMIN determined resilience to endotoxin shock. Thus, we have identified a central regulator of the metabolic function and bioenergetic state of macrophages that is under evolutionary selection and determines the risk of inflammatory and infectious disease.

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Figure 1: FAMIN interacts with fatty acid synthase and localizes to peroxisomes.
Figure 2: FAMIN promotes glycolysis and flux of glucose into de novo lipogenesis.
Figure 3: FAMIN augments FAO.
Figure 4: FAMIN controls FAO of endogenously synthesized lipids.
Figure 5: FAMIN-deficient macrophages have impaired mROS production and exhibit features of mitochondrial injury and remodeling.
Figure 6: FAMIN(p.I254V) is hypomorphic and C284R) leads to complete loss of function.
Figure 7: FAMIN deficiency causes defective bacterial clearance and inflammasome activation in vitro.
Figure 8: FAMIN deficiency causes dysregulated response to endotoxin in vivo.

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Acknowledgements

We thank G. Brown for help with laccase assays; J. Murkin and M. Deery for proteomics; L. Porter for metabolic-flux assays; J. Skepper for electron microscopy; I. Purvis for help with in vivo procedures; R. Rodrigues, M. Md-Ibrahim and J. Jones for cellular assays; S. Dhillon for the generation of constructs; T. Lawley, M. Pardo, J. Choudhary, K. Smith, J. Lee, D. Thomas, G. Schneditz, L. Haag, M. Parkes and R. Blumberg for discussions; all National Institute for Health Research Cambridge BioResource volunteers for the participation; the Cambridge BioResource staff for help with volunteer recruitment; members of the Cambridge BioResource SAB and Management Committee for support of this study; and the National Institute for Health Research Cambridge BRC Cell Phenotyping Hub for expertise and help. Access to Cambridge BioResource volunteers and their data and samples is governed by the Cambridge BioResource SAB (documents on access arrangements and contact details, http://www.cambridgebioresource.org.uk/). Supported by the European Research Council under the European Community's Seventh Framework Programme (FP7/2007-2013)/ERC Grant agreement 260961 (A.K.), the Wellcome Trust (investigator award 106260/Z/14/Z to A.K.; a PhD fellowship for clinicians to M.Z.C.; and a Career Re-Entry Fellowship to N.C.K.), the Wellcome Trust Sanger Institute (G.D., A.B., S.M., S.C. and K.B.), the US National Institutes of Health (5U420D011174 and 5U54HG006348 to A.B. and K.B.), the Biotechnology and Biological Sciences Research Council (M.J.O.W.), the National Institute for Health Research Cambridge Biomedical Research Centre, the European Crohn's and Colitis Organisation (M.T.) and the Swedish Medical Research Council and the Olle Engkvist foundation (M.D'A.).

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Authors and Affiliations

  1. Division of Gastroenterology and Hepatology, Department of Medicine, Addenbrooke's Hospital, University of Cambridge, Cambridge, UK

    M Zaeem Cader, Sarah L Kempster, Gavin W Sewell, Svetlana Saveljeva, Jonathan W Ashcroft, Markus Tschurtschenthaler, Tim Raine, Nicole C Kaneider & Arthur Kaser

  2. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, UK

    Katharina Boroviak, Simon Clare, Subhankar Mukhopadhyay, Brendan Doe, Allan Bradley & Gordon Dougan

  3. Signalling Programme, Babraham Institute, Babraham Research Campus, Cambridge, UK

    Qifeng Zhang & Michael J O Wakelam

  4. Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden

    Ghazaleh Assadi & Mauro D'Amato

  5. Cambridge Institute for Medical Research, University of Cambridge, Cambridge Centre for Lung Infection, Cambridge, UK

    Karen P Brown & R Andres Floto

  6. Division of Respiratory Medicine, Department of Medicine, Addenbrooke's and Papworth Hospitals, University of Cambridge, Cambridge, UK

    Edwin R Chilvers & R Andres Floto

  7. Department of Biochemistry, University of Cambridge, Cambridge, UK

    Jules L Griffin

  8. BioDonostia Health Research Institute San Sebastian and Ikerbasque, Basque Foundation for Science, Bilbao, Spain

    Mauro D'Amato

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  1. M Zaeem Cader
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Contributions

M.Z.C., together with S.L.K., G.W.S., S.S., J.W.A., M.T., T.R. and N.C.K., designed and performed most of the experiments; K.B., B.D. and A.B. designed, generated and confirmed the genotype of CRISPR-Cas9–generated mouse lines; Q.Z. and M.J.O.W. provided lipidomics experiments and analysis; G.A. and M.D'A. identified the cellular localization of FAMIN; S.C., S.M. and G.D. contributed Salmonella and part of the in vivo experimentation; K.P.B. and R.A.F. contributed to mycobacterial experiments; E.R.C. helped with metabolic-flux assays and ROS experimentation; J.L.G. contributed metabolomics experimentation and analysis; and A.K. devised and coordinated the project and, together with M.Z.C. and G.D., and with contributions from all authors, designed experiments, interpreted data and wrote the manuscript.

Corresponding author

Correspondence to Arthur Kaser.

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Integrated supplementary information

Supplementary Figure 1 Expression of Strep-tagged FAMIN in HEK293T cells.

Immunoblots (IB) of HEK293T lysates expressing N- and C-terminally Strep-tagged FAMIN(p.254I) and FAMIN(p.254V) variants for FAMIN and Strep-tag; β-actin loading control. Data are representative of three independent experiments.

Supplementary Figure 2 FAMIN localizes to peroxisomes.

(a) Co-localization by immunofluorescence (IF) of FAMIN (red) with PMP70 (green) in primary human macrophages (MΦ). DAPI nuclear staining, blue; scale bar = 10 μm. White box corresponds to enlarged images shown in Fig. 1d. (b–e) Co-localization by IF of FAMIN (red) with PMP70 (green), catalase (green), cytochrome oxidase IV (green) and calreticulin (green) in THP-1 macrophages. DAPI, blue; scale bar = 10 μm for original images and 5 μm for enlarged images. Upper panels: original image, middle panels: enlargement of the area shown in the white box, lower panels: further enlargement as indicated. No significant co-localization of FAMIN was detected with CENP-A, centromere; caveolin-2, cholesterol/sphingolipid enriched plasma membrane; EEA1 and RAB5, early endosome; LAMP1, lysosomes; NUP98, nuclear envelope; fibrillin, nucleolus and syntaxin 6, trans-Golgi network (data not shown). (f) Proximity-ligation assay (PLA) of FAMIN in combination with PMP70, Catalase (red) or centromere protein A (red) as negative control in THP-1 macrophages. DAPI, blue; scale bar = 10 μm. White box corresponds to enlarged images shown in Fig. 1e. Data are representative of three independent experiments.

Supplementary Figure 3 Generation of mFamin–/–, mFaminp254I and mFaminp284R mice.

Schematic representation of the targeting strategy for creating a mFamin–/– allele in murine ES cells. The targeting vector, L1L2_Bact_P (International Mouse Knockout Consortium), contains a reading frame-independent LacZ gene trap cassette consisting of an En2 splice acceptor (SA), an internal ribosomal entry site (IRES), LacZ gene and a polyadenylation site (PA). The vector also contains a selectable marker consisting of a human β-actin promoter (hBactP), neomycin resistance gene (neo) and a PA site. The vector is flanked by flippase recognition target (FRT) sites to allow removal of the targeting cassette and conditional mFamin deletion upon Cre-mediated recombination of the loxP sites. mFaminp254I and mFaminp284R mice were generated by CRISPR/Cas9 genome editing to introduce nucleotide changes to encode p.254I or p.284R amino acids, respectively, at indicated positions. Nucleotides in mFamin exon 5 were targeted using guide RNAs, ‘line’ 7 and ‘line’ 9, respectively as outlined in the methods. Two different targeting oligodeoxynucleotides were used for each: one containing only the nucleotide changes leading to the amino acid substitutions, the other containing 2 additional synonymous nucleotide changes (‘wobble’) in the underlined region.

Supplementary Figure 4 Famin expression is highest in M1 macrophages, and FAMIN deficiency does not affect FASN expression.

(a) mRNA expression of Famin in M0, M1 and M2 macrophages (MΦ). (b) mRNA expression of Fasn in M0, M1 and M2 macrophages. (c) Immunoblots (IB) of FASN in M1 and M2 macrophage cell lysates; β-actin loading control. Data are from one experiment with three mice representative of two (b,c; mean ± S.E.M.) or three independent experiments (c).

Supplementary Figure 5 FAMIN does not directly affect the Krebs cycle.

(a) Schematic representation of 1,2-13C-glucose incorporation, via citrate, into fatty acyl species. (b) Basal extracellular acidification rate (ECAR) of mFamin–/– and mFamin+/+ M1 and M2 macrophages (MΦ) in the presence of exogenous pyruvate (n = 5/14). (c) Relative levels of malate, fumarate, succinate and α-ketoglutarate in M1 and M2 macrophages. (d) Oxygen consumption rate (OCR) of mFamin–/– and mFamin+/+ M2 macrophages treated as indicated with 40 μM etomoxir (ETO) 1 h prior to OCR measurement and followed by sequential treatment (dotted vertical lines) with oligomycin (Oligo), FCCP, and rotenone
plus antimycin (Rot + ant). *P < 0.05, **P < 0.01 (Unpaired, two-tailed Student’s t-test). Data are pooled from three independent experiments (b; mean ± S.E.M.), from one experiment with seven mice per group (c; mean ± S.E.M.) or from one experiment with three mice representative of two independent experiments (d; mean ± S.E.M.).

Supplementary Figure 6 FAMIN-deficient M2 macrophages exhibit impaired mitochondrial ROS- and FAO-dependent production of extracellular ROS.

(a,b) Intracellular ROS measurement in unstimulated M1 and M2 mFamin–/– and mFamin+/+ macrophages (MΦ) stained with the cytosolic ROS indicator, 5-(and-6)-chloromethyl-2-7-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and measured in relative fluorescence units, RFU. (c–i) Zymosan induced eROS production in M1 and M2 macrophages treated as indicated for 16 h with 20 μM C75; or 1 h 40 μM etomoxir (ETO) or 500 μM mitoTEMPO prior to stimulation; or silenced for Fasn (Fasn siRNA), Cpt1a (Cpt siRNA) or Cybb (Cybb siRNA) or scrambled siRNA (Ctrl siRNA); Left, eROS kinetic plots measured in relative light units, RLU and right, area under curve, AUC. (j) NADPH quantification in M1 and M2 macrophages cell lysates. (k) PMA-induced eROS production in mFamin–/– and mFamin+/+ neutrophils. *P < 0.05, **P < 0.01 (Unpaired, two-tailed Student’s t-test). Data are from one experiment with three mice representative of two independent experiments (a–k; mean ± S.E.M.).

Supplementary Figure 7 FAMIN p.I254V and p.C284R M2 macrophage have impaired eROS production.

(a) Zymosan-stimulated eROS production in mFamin–/–, mFaminp254V, mFaminp254I and mFaminp284R murine M2 macrophages (MΦ). (b) Zymosan induced eROS production in mFaminp254I and mFaminp254V M2 macrophages treated as indicated for 16 h with 20 μM C75. (c) FAMIN mRNA expression in M2 macrophages and neutrophils from healthy donors homozygous for the Crohn’s disease and leprosy risk (‘rs3764147G/G’) and non-risk (‘rs3764147A/A’) haplotypes. Data are from one experiment with three mice representative of three independent experiments (a,b; mean ± S.E.M.) or pooled from ten independent experiments (c; mean ± S.E.M.).

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Cader, M., Boroviak, K., Zhang, Q. et al. C13orf31 (FAMIN) is a central regulator of immunometabolic function. Nat Immunol 17, 1046–1056 (2016). https://doi.org/10.1038/ni.3532

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