IRF5 promotes inflammatory macrophage polarization and TH1-TH17 responses

Journal name:
Nature Immunology
Volume:
12,
Pages:
231–238
Year published:
DOI:
doi:10.1038/ni.1990
Received
Accepted
Published online

Abstract

Polymorphisms in the gene encoding the transcription factor IRF5 that lead to higher mRNA expression are associated with many autoimmune diseases. Here we show that IRF5 expression in macrophages was reversibly induced by inflammatory stimuli and contributed to the plasticity of macrophage polarization. High expression of IRF5 was characteristic of M1 macrophages, in which it directly activated transcription of the genes encoding interleukin 12 subunit p40 (IL-12p40), IL-12p35 and IL-23p19 and repressed the gene encoding IL-10. Consequently, those macrophages set up the environment for a potent T helper type 1 (TH1)-TH17 response. Global gene expression analysis demonstrated that exogenous IRF5 upregulated or downregulated expression of established phenotypic markers of M1 or M2 macrophages, respectively. Our data suggest a critical role for IRF5 in M1 macrophage polarization and define a previously unknown function for IRF5 as a transcriptional repressor.

At a glance

Figures

  1. High expression of IRF5 in M1 macrophages and upregulation by GM-CSF.
    Figure 1: High expression of IRF5 in M1 macrophages and upregulation by GM-CSF.

    (a) Immunoblot analysis of total protein extracts from monocytes collected at day 0 (Mono) or differentiated for 5 d into M1 macrophages with GM-CSF (50 ng/ml) or into M2 macrophages with M-CSF (100 ng/ml), then left untreated (−) or simulated for 24 h with LPS (+). Actin serves as a loading control throughout. (b) RT-PCR analysis of IRF5 mRNA in monocytes left untreated (0) or stimulated for 2, 4, 8, 24 or 48 h with GM-CSF (50 ng/ml) or M-CSF (100 ng/ml); results are presented relative to those of untreated monocytes, set as 1. *P < 0.001 (two-way analysis of variance (ANOVA)). (c) Immunoblot analysis of total protein extracts from M2 macrophages left untreated (−) or treated (+) for 24 h with GM-CSF (50 ng/ml) for M2-to-M1 polarization. (d) Immunoblot analysis of total protein extracts from M1 macrophages left untreated or treated for 24 h with M-CSF (100 ng/ml) for M1-to-M2 polarization. Results are from at least four independent experiments, each with cells derived from a different donor (a,c,d), or are from five independent experiments, each with cells derived from a different donor (b; mean and s.e.m.).

  2. IRF5 influences the production of macrophage lineage-specific cytokines.
    Figure 2: IRF5 influences the production of macrophage lineage–specific cytokines.

    (a) Enzyme-linked immunosorbent assay (ELISA) of the secretion of IL-12p70, IL-23, IL-12p40 and IL-10 by M2 macrophages infected with adenoviral vector encoding IRF5 or IRF3 or empty vector (pENTR) and stimulated for 24 h with LPS. *P < 0.01 and **P < 0.001 (one-way ANOVA with Dunnett's multiple-comparison post-test). (b) ELISA of the secretion of IL-12p70, IL-23, IL-12p40 and IL-10 by M1 macrophages transfected with siRNA targeting IRF5 (siIRF5) or nontargeting (control) siRNA (siC) and stimulated for 24 h with LPS (10 ng/ml) plus IFN-γ (50 ng/ml). *P < 0.01 and **P < 0.001 (Student's t-test). Data are representative of seven to nine (a) or six to eight (b) independent experiments, each with cells derived from a different donor (mean and s.e.m.).

  3. IRF5 promotes lymphocyte proliferation and TH1-TH17 responses.
    Figure 3: IRF5 promotes lymphocyte proliferation and TH1-TH17 responses.

    (a) Immunocytochemical staining of IFN-γ in T lymphocytes cultured for 4 d together with M2 macrophages obtained from mismatched donors and infected with adenoviral vector encoding IRF5 or IRF3 or empty vector, then stimulated for 3 h with PMA, ionomycin and brefeldin A; results are presented as mean fluorescence intensity (MFI). (b) IFN-γ in supernatants of the cells in a after 4 d of coculture. (c) Expression of T-bet mRNA in T lymphocytes cultured for 2 d together with M2 macrophages obtained from mismatched donors and infected with adenoviral vectors as in a; results are presented in arbitrary units (AU) relative to those obtained with empty vector control. (d) Immunocytochemical staining of IL-17A in the T lymphocytes in a. (e) IL-17A in supernatants of the cells in a after 4 d of coculture. (f) Expression of RORγt mRNA, assessed and presented as in c. *P < 0.05, **P < 0.01 and ***P < 0.001 (one-way ANOVA with Dunnett's multiple-comparison post-test). Data are representative of seven (a,d), six (b,c,f) or four (e) independent experiments, each with cells derived from a different donor (mean and s.e.m.).

  4. IRF5 regulates the expression of mRNA for macrophage lineage-specific cytokines.
    Figure 4: IRF5 regulates the expression of mRNA for macrophage lineage–specific cytokines.

    (a) Quantitative PCR analysis of mRNA for IL-12p40, IL-12p35, IL-23p19 and IL-10 in M2 macrophages infected with adenoviral vector encoding IRF5 or IRF3; basal expression is presented relative to that of control cells infected with empty vector. *P < 0.05, **P < 0.01 and ***P < 0.001 (one-way ANOVA with Dunnett's multiple-comparison post-test). (b) Expression of mRNA for IL-12p40, IL-12p35, IL-23p19 and IL-10 in M1 macrophages transfected with siRNA targeting IRF5 and left untreated or stimulated for 8 h with with LPS (10 ng/ml); results are presented (as percent inhibition) relative to those of control cells transfected with nontargeting control siRNA. *P < 0.01 and **P < 0.001 (Student's t-test). (c) Global mRNA expression (for sets of M1- and M2-specific genes21, 27; above plot) by unstimulated M2 macrophages infected with adenoviral vector encoding IRF5, presented relative to that of unstimulated M2 macrophages infected with empty vector. Results are presented as higher (red) or lower (green) expression after infection with vector encoding IRF5 (key (below), log2 fold change). Data are from three to six independent experiments, each with cells derived from a different donor (a; mean and s.e.m.), are from five to six independent experiments (b; mean and s.e.m.) or are representative of one experiment with four different donors (c).

  5. IRF5 is directly involved in the transcriptional regulation of lineage-specific cytokines.
    Figure 5: IRF5 is directly involved in the transcriptional regulation of lineage-specific cytokines.

    Recruitment of proteins to the promoters of genes encoding IL-12p40 (IL12B; a), IL-12p35 (IL12A; b), IL-23p19 (IL23A; c) or IL-10 (d) in M1 macrophages left unstimulated (0) or stimulated for 1, 2, 4, 8 or 24 h with LPS (10 ng/ml), assessed by chromatin immunoprecipitation with antibody to IRF5, antibody to RNA polymerase II (PolII) or immunoglobulin G (IgG; control); results are presented relative to those obtained with genomic DNA (input). Data are from one experiment representative of three (error bars, s.d.).

  6. IRF5 inhibits the transcriptional activation of human IL10.
    Figure 6: IRF5 inhibits the transcriptional activation of human IL10.

    (a) Luciferase activity of M2 macrophages infected for 24 h with a wild-type IL10 luciferase reporter plasmid (WT), plus a construct encoding IRF5 or an IRF5 mutant lacking the DNA-binding domain (IRF5ΔDBD) or empty vector, then left unstimulated (US) or stimulated for 4 h with LPS (10 ng/ml). (b) Luciferase activity of M2 macrophages infected for 24 h with the wild-type reporter in a or an IL10 luciferase reporter plasmid with site-specific mutations in the ISRE site at positions −180 to −173 (ISREmut), plus the IRF5 constructs in a or empty vector, then stimulated with LPS as in a. *P < 0.01 (one-way ANOVA with Dunnett's multiple-comparison post-test). Data are from three independent experiments, each with cells derived from a different donor (mean and s.e.m.).

  7. Impaired production of M1 and TH1-TH17 cytokines in Irf5-/- mice.
    Figure 7: Impaired production of M1 and TH1-TH17 cytokines in Irf5−/− mice.

    (a) Immunoblot analysis of total protein extracts from wild-type C57BL/6 bone marrow cells differentiated for 8 d into M1 macrophages with GM-CSF (20 ng/ml) or into M2 macrophages with M-CSF (100 ng/ml); extracts of adherent cells were probed with antibody to IRF5. Data are representative of three experiments. (b) ELISA of IL-12p70, IL-23 and IL-10 secreted by M1 macrophages obtained from C57BL/6 mice (n = 8) and stimulated for 24 h with LPS (100 ng/ml). *P < 0.05 and **P < 0.01 (Student's t-test). Data are representative of three experiments (mean and s.e.m. of one sample per mouse analyzed in triplicate). (c) ELISA (IL-12p40 and IL-23) or cytrometric bead assay (IL-10) of the serum concentrations of cytokines in Irf5−/− mice (n = 10) and their wild-type littermates (n = 10) injected intraperitoneally with LPS (20 μg), assessed 3 h later. *P < 0.05 and **P < 0.01 (Student's t-test). Data are from three independent experiments (mean and s.e.m. of eight to ten serum samples). (d) Expression of mRNA for M1 and M2 markers (horizontal axis) in peritoneal cells from the LPS-injected mice in c. *P < 0.05, **P < 0.01 and ***P < 0.001 (Student's t-test). Data are from three independent experiments (mean and s.e.m. of ten samples). (e) ELISA of IFN-γ and IL-17A in spleen cells obtained from the LPS-injected mice in c and cultured for 48 h in the presence of antibody to CD3. *P < 0.05 and **P < 0.01 (Student's t-test). Data are from two independent experiments (mean and s.e.m. of four to five spleen cultures).

References

  1. Gordon, S. & Taylor, P.R. Monocyte and macrophage heterogeneity. Nat. Rev. Immunol. 5, 953964 (2005).
  2. Mosser, D.M. & Edwards, J.P. Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 8, 958969 (2008).
  3. Gordon, S. Alternative activation of macrophages. Nat. Rev. Immunol. 3, 2335 (2003).
  4. Romagnani, P., Annunziato, F., Piccinni, M.P., Maggi, E. & Romagnani, S. TH1/TH2 cells, their associated molecules and role in pathophysiology. Eur. Cytokine Netw. 11, 510511 (2000).
  5. Martinez, F.O., Sica, A., Mantovani, A. & Locati, M. Macrophage activation and polarization. Front. Biosci. 13, 453461 (2008).
  6. Korn, T., Bettelli, E., Oukka, M. & Kuchroo, V.K. IL-17 and TH17 Cells. Annu. Rev. Immunol. 27, 485517 (2009).
  7. Tamura, T. et al. IFN regulatory factor-4 and -8 govern dendritic cell subset development and their functional diversity. J. Immunol. 174, 25732581 (2005).
  8. Porta, C. et al. Tolerance and M2 (alternative) macrophage polarization are related processes orchestrated by p50 nuclear factor κB. Proc. Natl. Acad. Sci. USA 106, 1497814983 (2009).
  9. Ruffell, D. et al. A CREB-C/EBPβ cascade induces M2 macrophage-specific gene expression and promotes muscle injury repair. Proc. Natl. Acad. Sci. USA 106, 1747517480 (2009).
  10. Satoh, T. et al. The Jmjd3-Irf4 axis regulates M2 macrophage polarization and host responses against helminth infection. Nat. Immunol. 11, 936944 (2010).
  11. Ouyang, X. et al. Cooperation between MyD88 and TRIF pathways in TLR synergy via IRF5 activation. Biochem. Biophys. Res. Commun. 354, 10451051 (2007).
  12. Takaoka, A. et al. Integral role of IRF-5 in the gene induction programme activated by Toll-like receptors. Nature 434, 243249 (2005).
  13. Mancl, M.E. et al. Two discrete promoters regulate the alternatively spliced human interferon regulatory factor-5 isoforms. Multiple isoforms with distinct cell type-specific expression, localization, regulation, and function. J. Biol. Chem. 280, 2107821090 (2005).
  14. Dideberg, V. et al. An insertion-deletion polymorphism in the interferon regulatory factor 5 (IRF5) gene confers risk of inflammatory bowel diseases. Hum. Mol. Genet. 16, 30083016 (2007).
  15. Dieguez-Gonzalez, R. et al. Association of interferon regulatory factor 5 haplotypes, similar to that found in systemic lupus erythematosus, in a large subgroup of patients with rheumatoid arthritis. Arthritis Rheum. 58, 12641274 (2008).
  16. Graham, R.R. et al. A common haplotype of interferon regulatory factor 5 (IRF5) regulates splicing and expression and is associated with increased risk of systemic lupus erythematosus. Nat. Genet. 38, 550555 (2006).
  17. Kristjansdottir, G. et al. Interferon regulatory factor 5 (IRF5) gene variants are associated with multiple sclerosis in three distinct populations. J. Med. Genet. 45, 362369 (2008).
  18. Miceli-Richard, C. et al. Association of an IRF5 gene functional polymorphism with Sjogren's syndrome. Arthritis Rheum. 56, 39893994 (2007).
  19. Fleetwood, A.J., Lawrence, T., Hamilton, J.A. & Cook, A.D. Granulocyte-macrophage colony-stimulating factor (CSF) and macrophage CSF-dependent macrophage phenotypes display differences in cytokine profiles and transcription factor activities: implications for CSF blockade in inflammation. J. Immunol. 178, 52455252 (2007).
  20. Hoeve, M.A. et al. Divergent effects of IL-12 and IL-23 on the production of IL-17 by human T cells. Eur. J. Immunol. 36, 661670 (2006).
  21. Verreck, F.A., de Boer, T., Langenberg, D.M., van der Zanden, L. & Ottenhoff, T.H. Phenotypic and functional profiling of human proinflammatory type-1 and anti-inflammatory type-2 macrophages in response to microbial antigens and IFN-γ- and CD40L-mediated costimulation. J. Leukoc. Biol. 79, 285293 (2006).
  22. Krausgruber, T. et al. IRF5 is required for late-phase TNF secretion by human dendritic cells. Blood 115, 44214430 (2010).
  23. Hammer, M. et al. Dual specificity phosphatase 1 (DUSP1) regulates a subset of LPS-induced genes and protects mice from lethal endotoxin shock. J. Exp. Med. 203, 1520 (2006).
  24. Fleetwood, A.J., Dinh, H., Cook, A.D., Hertzog, P.J. & Hamilton, J.A. GM-CSF- and M-CSF-dependent macrophage phenotypes display differential dependence on type I interferon signaling. J. Leukoc. Biol. 86, 411421 (2009).
  25. Ahern, P.P. et al. Interleukin-23 drives intestinal inflammation through direct activity on T cells. Immunity 33, 279288 (2010).
  26. Nistala, K. et al. TH17 plasticity in human autoimmune arthritis is driven by the inflammatory environment. Proc. Natl. Acad. Sci. USA 107, 1475114756 (2010).
  27. Martinez, F.O., Gordon, S., Locati, M. & Mantovani, A. Transcriptional profiling of the human monocyte-to-macrophage differentiation and polarization: new molecules and patterns of gene expression. J. Immunol. 177, 73037311 (2006).
  28. Ziegler-Heitbrock, L. et al. IFN-α induces the human IL-10 gene by recruiting both IFN regulatory factor 1 and Stat3. J. Immunol. 171, 285290 (2003).
  29. Hamilton, J.A. Colony-stimulating factors in inflammation and autoimmunity. Nat. Rev. Immunol. 8, 533544 (2008).
  30. Medzhitov, R. & Horng, T. Transcriptional control of the inflammatory response. Nat. Rev. Immunol. 9, 692703 (2009).
  31. Ghisletti, S. et al. Identification and characterization of enhancers controlling the inflammatory gene expression program in macrophages. Immunity 32, 317328 (2010).
  32. Negishi, H. et al. Negative regulation of Toll-like-receptor signaling by IRF-4. Proc. Natl. Acad. Sci. USA 102, 1598915994 (2005).
  33. El Chartouni, C., Schwarzfischer, L. & Rehli, M. Interleukin-4 induced interferon regulatory factor (Irf) 4 participates in the regulation of alternative macrophage priming. Immunobiology 215, 821825 (2010).
  34. Sanjabi, S., Hoffmann, A., Liou, H.C., Baltimore, D. & Smale, S.T. Selective requirement for c-Rel during IL-12 P40 gene induction in macrophages. Proc. Natl. Acad. Sci. USA 97, 1270512710 (2000).
  35. Mise-Omata, S. et al. A proximal κB site in the IL-23 p19 promoter is responsible for RelA- and c-Rel-dependent transcription. J. Immunol. 179, 65966603 (2007).
  36. Saraiva, M. & O'Garra, A. The regulation of IL-10 production by immune cells. Nat. Rev. Immunol. 10, 170181 (2010).
  37. Fiorentino, D.F., Zlotnik, A., Mosmann, T.R., Howard, M. & O'Garra, A. IL-10 inhibits cytokine production by activated macrophages. J. Immunol. 147, 38153822 (1991).
  38. Wing, K. & Sakaguchi, S. Regulatory T cells exert checks and balances on self tolerance and autoimmunity. Nat. Immunol. 11, 713 (2010).
  39. Mosser, D.M. & Zhang, X. Interleukin-10: new perspectives on an old cytokine. Immunol. Rev. 226, 205218 (2008).
  40. Schneemann, M. & Schoeden, G. Macrophage biology and immunology: man is not a mouse. J. Leukoc. Biol. 81, 579 (2007).
  41. Ponting, C.P. The functional repertoires of metazoan genomes. Nat. Rev. Genet. 9, 689698 (2008).
  42. Oppmann, B. et al. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 13, 715725 (2000).
  43. Romagnani, S., Maggi, E., Liotta, F., Cosmi, L. & Annunziato, F. Properties and origin of human TH17 cells. Mol. Immunol. 47, 37 (2009).
  44. Murphy, C.A. et al. Divergent pro- and antiinflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation. J. Exp. Med. 198, 19511957 (2003).
  45. Yen, D. et al. IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6. J. Clin. Invest. 116, 13101316 (2006).
  46. Shen, H. et al. Gender-dependent expression of murine Irf5 gene: implications for sex bias in autoimmunity. J Mol Cell Biol 2, 284290 (2010).
  47. Campbell, I.K. et al. Protection from collagen-induced arthritis in granulocyte-macrophage colony-stimulating factor-deficient mice. J. Immunol. 161, 36393644 (1998).
  48. Cook, A.D., Braine, E.L., Campbell, I.K., Rich, M.J. & Hamilton, J.A. Blockade of collagen-induced arthritis post-onset by antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF): requirement for GM-CSF in the effector phase of disease. Arthritis Res. 3, 293298 (2001).
  49. Lacaze, P. et al. Combined genome-wide expression profiling and targeted RNA interference in primary mouse macrophages reveals perturbation of transcriptional networks associated with interferon signalling. BMC Genomics 10, 372 (2009).
  50. Liu, J., Cao, S., Herman, L.M. & Ma, X. Differential regulation of interleukin (IL)-12 p35 and p40 gene expression and interferon (IFN)-γ-primed IL-12 production by IFN regulatory factor 1. J. Exp. Med. 198, 12651276 (2003).

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Affiliations

  1. Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College of Science, Technology and Medicine, London, UK.

    • Thomas Krausgruber,
    • Katrina Blazek,
    • Tim Smallie,
    • Saba Alzabin,
    • Marc Feldmann &
    • Irina A Udalova
  2. Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.

    • Helen Lockstone &
    • Natasha Sahgal
  3. National Heart and Lung Institute, Faculty of Medicine, Imperial College of Science, Technology and Medicine, South Kensington Campus, London, UK.

    • Tracy Hussell

Contributions

T.K., T.S., K.B. and S.A. did research; T.K., H.L., N.S. and I.A.U. designed research and analyzed data; and T.K., M.F., T.H. and I.A.U. wrote the paper.

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The authors declare no competing financial interests.

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