Article | Published:

Infection drives meningeal engraftment by inflammatory monocytes that impairs CNS immunity

Nature Immunologyvolume 20pages407419 (2019) | Download Citation

Abstract

Tissue macrophages have an embryonic origin and can be replenished in some tissues under steady-state conditions by blood monocytes. However, little is known about the residency and properties of infiltrating monocytes after an inflammatory challenge. The meninges of the central nervous system (CNS) are populated by a dense network of macrophages that act as resident immune sentinels. Here we show that, following lymphocytic choriomeningitis virus infection, resident meningeal macrophages (MMs) acquired viral antigen and interacted directly with infiltrating cytotoxic T lymphocytes, which led to macrophage depletion. Concurrently, the meninges were infiltrated by inflammatory monocytes that engrafted the meningeal niche and remained in situ for months after viral clearance. This engraftment led to interferon-γ-dependent functional changes in the pool of MMs, including loss of bacterial and immunoregulatory sensors. Collectively, these data indicate that peripheral monocytes can engraft the meninges after an inflammatory challenge, imprinting the compartment with long-term defects in immune function.

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Data availability

The microarray datasets generated during the current study are available in the NCBI GEO database (accession no. GSE108648). In addition, all primary data that support the findings of this study are available from the corresponding author upon request.

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References

  1. 1.

    Mowat, A. M., Scott, C. L. & Bain, C. C. Barrier-tissue macrophages: functional adaptation to environmental challenges. Nat. Med. 23, 1258–1270 (2017).

  2. 2.

    Okabe, Y. & Medzhitov, R. Tissue biology perspective on macrophages. Nat. Immunol. 17, 9–17 (2016).

  3. 3.

    Perdiguero, E. G. & Geissmann, F. The development and maintenance of resident macrophages. Nat. Immunol. 17, 2–8 (2016).

  4. 4.

    Prinz, M., Erny, D. & Hagemeyer, N. Ontogeny and homeostasis of CNS myeloid cells. Nat. Immunol. 18, 385–392 (2017).

  5. 5.

    Laurent, P. et al. Immune-mediated repair: a matter of plasticity. Front. Immunol. 8, 454 (2017).

  6. 6.

    Butler, S. J. & Bronner, M. E. From classical to current: analyzing peripheral nervous system and spinal cord lineage and fate. Dev. Biol. 398, 135–146 (2015).

  7. 7.

    Song, I. & Dityatev, A. Crosstalk between glia, extracellular matrix and neurons. Brain Res. Bull. 136, 101–108 (2018).

  8. 8.

    Haddad-Tovolli, R., Dragano, N. R. V., Ramalho, A. F. S. & Velloso, L. A. Development and function of the blood-brain barrier in the context of metabolic control. Front. Neurosci. 11, 224 (2017).

  9. 9.

    Coles, J. A., Myburgh, E., Brewer, J. M. & McMenamin, P. G. Where are we? The anatomy of the murine cortical meninges revisited for intravital imaging, immunology, and clearance of waste from the brain. Prog. Neurobiol. 156, 107–148 (2017).

  10. 10.

    Rua, R. & McGavern, D. B. Advances in meningeal immunity. Trends Mol. Med. 24, 542–559 (2018).

  11. 11.

    Galea, I., Bechmann, I. & Perry, V. H. What is immune privilege (not)? Trends Immunol. 28, 12–18 (2007).

  12. 12.

    Kipnis, J. Multifaceted interactions between adaptive immunity and the central nervous system. Science 353, 766–771 (2016).

  13. 13.

    Klein, R. S., Garber, C. & Howard, N. Infectious immunity in the central nervous system and brain function. Nat. Immunol. 18, 132–141 (2017).

  14. 14.

    Ransohoff, R. M. Animal models of multiple sclerosis: the good, the bad and the bottom line. Nat. Neurosci. 15, 1074–1077 (2012).

  15. 15.

    Herz, J., Filiano, A. J., Smith, A., Yogev, N. & Kipnis, J. Myeloid cells in the central nervous system. Immunity 46, 943–956 (2017).

  16. 16.

    Goldmann, T. et al. Origin, fate and dynamics of macrophages at central nervous system interfaces. Nat. Immunol. 17, 797–805 (2016).

  17. 17.

    Nayak, D., Roth, T. L. & McGavern, D. B. Microglia development and function. Annu. Rev. Immunol. 32, 367–402 (2014).

  18. 18.

    Roth, T. L. et al. Transcranial amelioration of inflammation and cell death after brain injury. Nature 505, 223–228 (2014).

  19. 19.

    Mrdjen, D. et al. High-dimensional single-cell mapping of central nervous system immune cells reveals distinct myeloid subsets in health, aging, and disease. Immunity 48, 380–395 (2018).

  20. 20.

    Ginhoux, F., Schultze, J. L., Murray, P. J., Ochando, J. & Biswas, S. K. New insights into the multidimensional concept of macrophage ontogeny, activation and function. Nat. Immunol. 17, 34–40 (2016).

  21. 21.

    Louveau, A. et al. Structural and functional features of central nervous system lymphatic vessels. Nature 523, 337–341 (2015).

  22. 22.

    Galea, I. et al. Mannose receptor expression specifically reveals perivascular macrophages in normal, injured, and diseased mouse brain. Glia 49, 375–384 (2005).

  23. 23.

    McGavern, D. B. & Kang, S. S. Illuminating viral infections in the nervous system. Nat. Rev. Immunol. 11, 318–329 (2011).

  24. 24.

    Kang, S. S. et al. Migration of cytotoxic lymphocytes in cell cycle permits local MHC I-dependent control of division at sites of viral infection. J. Exp. Med. 208, 747–759 (2011).

  25. 25.

    Kim, J. V., Kang, S. S., Dustin, M. L. & McGavern, D. B. Myelomonocytic cell recruitment causes fatal CNS vascular injury during acute viral meningitis. Nature 457, 191–195 (2009).

  26. 26.

    Kang, S. S. & McGavern, D. B. Lymphocytic choriomeningitis infection of the central nervous system. Front. Biosci. 13, 4529–4543 (2008).

  27. 27.

    Emonet, S. F., Garidou, L., McGavern, D. B. & de la Torre, J. C. Generation of recombinant lymphocytic choriomeningitis viruses with trisegmented genomes stably expressing two additional genes of interest. Proc. Natl Acad. Sci. USA 106, 3473–3478 (2009).

  28. 28.

    Moseman, E. A., Wu, T., de la Torre, J. C., Schwartzberg, P. L. & McGavern, D. B. Type I interferon suppresses virus-specific B cell responses by modulating CD8(+) T cell differentiation. Sci. Immunol. 1, eaah3565 (2016).

  29. 29.

    Gossa, S., Nayak, D., Zinselmeyer, B. H. & McGavern, D. B. Development of an immunologically tolerated combination of fluorescent proteins for in vivo two-photon imaging. Sci. Rep. 4, 6664 (2014).

  30. 30.

    Perdiguero, E. G. et al. The origin of tissue-resident macrophages: when an erythro-myeloid progenitor is an erythro-myeloid progenitor. Immunity 43, 1023–1024 (2015).

  31. 31.

    Park, C. G. et al. Five mouse homologues of the human dendritic cell C-type lectin, DC-SIGN. Int. Immunol. 13, 1283–1290 (2001).

  32. 32.

    Takahara, K. et al. Difference in fine specificity to polysaccharides of Candida albicans mannoprotein between mouse SIGNR1 and human DC-SIGN. Infect. Immun. 80, 1699–1706 (2012).

  33. 33.

    Takahara, K., Tokieda, S., Nagaoka, K. & Inaba, K. Efficient capture of Candida albicans and zymosan by SIGNR1 augments TLR2-dependent TNF-alpha production. Int. Immunol. 24, 89–96 (2012).

  34. 34.

    Takahara, K. et al. C-type lectin SIGNR1 enhances cellular oxidative burst response against C. albicans in cooperation with Dectin-1. Eur. J. Immunol. 41, 1435–1444 (2011).

  35. 35.

    Kato, C. & Kojima, N. SIGNR1 ligation on murine peritoneal macrophages induces IL-12 production through NFkappaB activation. Glycoconj. J. 27, 525–531 (2010).

  36. 36.

    Koppel, E. A. et al. Specific ICAM-3 grabbing nonintegrin-related 1 (SIGNR1) expressed by marginal zone macrophages is essential for defense against pulmonary Streptococcus pneumoniae infection. Eur. J. Immunol. 35, 2962–2969 (2005).

  37. 37.

    Nagaoka, K. et al. Association of SIGNR1 with TLR4-MD-2 enhances signal transduction by recognition of LPS in Gram-negative bacteria. Int. Immunol. 17, 827–836 (2005).

  38. 38.

    Pavlov, V. A. & Tracey, K. J. The vagus nerve and the inflammatory reflex–linking immunity and metabolism. Nat. Rev. Endocrinol. 8, 743–754 (2012).

  39. 39.

    Stein, M., Keshav, S., Harris, N. & Gordon, S. Interleukin 4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunologic macrophage activation. J. Exp. Med. 176, 287–292 (1992).

  40. 40.

    Mosser, D. M. & Edwards, J. P. Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 8, 958–969 (2008).

  41. 41.

    Linehan, S. A., Martinez-Pomares, L., Stahl, P. D. & Gordon, S. Mannose receptor and its putative ligands in normal murine lymphoid and nonlymphoid organs: in situ expression of mannose receptor by selected macrophages, endothelial cells, perivascular microglia, and mesangial cells, but not dendritic cells. J. Exp. Med. 189, 1961–1972 (1999).

  42. 42.

    Faraco, G., Park, L., Anrather, J. & Iadecola, C. Brain perivascular macrophages: characterization and functional roles in health and disease. J. Mol. Med. 95, 1143–1152 (2017).

  43. 43.

    Derecki, N. C. et al. Regulation of learning and memory by meningeal immunity: a key role for IL-4. J. Exp. Med. 207, 1067–1080 (2010).

  44. 44.

    Derecki, N. C., Quinnies, K. M. & Kipnis, J. Alternatively activated myeloid (M2) cells enhance cognitive function in immune compromised mice. Brain Behav. Immun. 25, 379–385 (2011).

  45. 45.

    Italiani, P. & Boraschi, D. Development and functional differentiation of tissue-resident versus monocyte-derived macrophages in inflammatory reactions. Results Probl. Cell. Differ. 62, 23–43 (2017).

  46. 46.

    Scott, C. L. et al. Bone marrow-derived monocytes give rise to self-renewing and fully differentiated Kupffer cells. Nat. Commun. 7, 10321 (2016).

  47. 47.

    van de Laar, L. et al. Yolk sac macrophages, fetal liver, and adult monocytes can colonize an empty niche and develop into functional tissue-resident macrophages. Immunity 44, 755–768 (2016).

  48. 48.

    Gentek, R., Molawi, K. & Sieweke, M. H. Tissue macrophage identity and self-renewal. Immunol. Rev. 262, 56–73 (2014).

  49. 49.

    Misharin, A. V. et al. Monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span. J. Exp. Med. 214, 2387–2404 (2017).

  50. 50.

    Gundra, U. M. et al. Vitamin A mediates conversion of monocyte-derived macrophages into tissue-resident macrophages during alternative activation. Nat. Immunol. 18, 642–653 (2017).

  51. 51.

    Cronk, J. C. et al. Peripherally derived macrophages can engraft the brain independent of irradiation and maintain an identity distinct from microglia. J. Exp. Med. 215, 1627–1647 (2018).

  52. 52.

    Tebruegge, M. & Curtis, N. Epidemiology, etiology, pathogenesis, and diagnosis of recurrent bacterial meningitis. Clin. Microbiol. Rev. 21, 519–537 (2008).

  53. 53.

    Netea, M. G. et al. Trained immunity: a program of innate immune memory in health and disease. Science 352, aaf1098 (2016).

  54. 54.

    Rosas-Ballina, M. et al. Acetylcholine-synthesizing T cells relay neural signals in a vagus nerve circuit. Science 334, 98–101 (2011).

  55. 55.

    Herz, J., Johnson, K. R. & McGavern, D. B. Therapeutic antiviral T cells noncytopathically clear persistently infected microglia after conversion into antigen-presenting cells. J. Exp. Med. 212, 1153–1169 (2015).

  56. 56.

    Manglani, M. & McGavern, D. B. Intravital imaging of neuroimmune interactions through a thinned skull. Curr. Protoc. Immunol. 120, 24.2.1–24.2.12 (2018).

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Acknowledgements

The study was supported by the National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH).

Author information

Affiliations

  1. Viral Immunology and Intravital Imaging Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA

    • Rejane Rua
    • , Jane Y. Lee
    • , Alexander B. Silva
    • , Isabella S. Swafford
    • , Dragan Maric
    • , Kory R. Johnson
    •  & Dorian B. McGavern

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Contributions

R.R. conceived the project, designed, executed and interpreted the animal studies, analyzed data and wrote the manuscript. J.Y.L., A.B.S. and I.S.S. executed animal studies and analyzed the data. D.M. executed animal studies. K.R.J. analyzed the data. D.B.M. conceived the project, designed and interpreted the animal studies and wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Dorian B. McGavern.

Integrated supplementary information

  1. Supplementary Figure 1 Gating strategy for meningeal macrophages (MMs).

    Myeloid and dendritic cell populations were analyzed by flow cytometry 4 days after PBS, r3LCMV or WT LCMV infection. (a) Gating strategy for MMs (gated on LiveDead CD45.2+ CD11b+ Thy1.2 Ly6C Ly6G F4/80+ CD206+), Ly6c+ monocytes (gated on LiveDead CD45.2+ CD11b+ Thy1.2 Ly6C+ Ly6G), cDC (gated on LiveDead CD45.2+ MHC-II+ Thy1.2 CD11c+ CD11b-), moDC (gated on LiveDead CD45.2+ MHC-II+ Thy1.2 CD11c+ CD11b+). Data are representative of two independent experiments with six (PBS) and ten (LCMV) mice per group. (b) MMs, Ly6C+ monocytes, cDC and moDC were analyzed for the expression of CD206, CD11c, MerTK and CD64. CD206+ MMs express the macrophage markers CD64 and MerTK. Data are representative of two independent experiments with six (PBS) and ten (LCMV) mice per group. (c) MMs and Ly6C+ monocytes were analyzed for the expression of Ly6C, TimD4 and Lyve-1. Most MMs express the resident macrophages markers TimD4 and Lyve-1. Data are representative of two independent experiments with six (PBS) and ten (LCMV) mice per group.

  2. Supplementary Figure 2 MMs up-regulate activation markers after infection.

    (a) Microarray analysis was performed on sorted MMs RNA extracted from mice injected with r3LCMV-TFP four days earlier and compared to PBS-injected controls (four independent experiments with ninety mice per group). MMs were gated on LiveDead Thy1.2 CD45.2+ CD11b+ Gr1 F4/80+ CD206+. Heat maps show expression of differentially regulated genes that were statistically different (p < 0.05, Welch-modified t-test under Benjamini-Hochberg (BH) false discovery rate (FDR) multiple comparison correction conditions) after infection and exceeded a threshold of 1.5-fold. Each column represents the relative mean expression levels of the denoted genes in the PBS vs. LCMV groups. Data are representative of four independent experiments with ninety mice per group. Genes in the heat maps were selected based on their transmembrane localization. See corresponding Supplementary Table 3 for full list. (b) MMs (gated on LiveDead CD45.2+ CD11b+ Thy1.2 Ly6C Ly6G F4/80+ CD206+) were analyzed by flow cytometry 4 days after PBS or WT LCMV infection for surface expression of activation markers (red markers indicate ones detected by microarray). Histograms are representative of three independent experiments with five mice per group.

  3. Supplementary Figure 3 MMs disappear following wild type LCMV infection.

    Localization of the different areas (frontal, parietal, occipital, sutures) in a meningeal whole mount stain of CD206+ cells of PBS (a) or LCMV-infected (b) mice 6 days post-infection and quantification of cell density (c). Cell density was calculated by histocytometry based on four regions (frontal, parietal, occipital, sutures). Bar graphs show the mean ± SEM for the indicated groups, and asterisks denote statistical significance (****p < 0.0001, unpaired two-tailed Student’s t test). Data are representative of two independent experiments with six (lobes) and nine (suture) regions per group. (d,e) Disappearance of MMs was confirmed by an additional gating strategy. MMs (based on CD206+ or Lyve-1+TimD4+ staining) were analyzed by flow cytometry 6 days after WT LCMV infection and compared to controls. (d) Gating strategy and (e) counts of MMs identified either by CD206+ staining (gated on LiveDead CD45.2+ CD11b+ Thy1.2 Ly6C+ Ly6G F4/80+ CD206+) or Lyve-1+TimD4+ staining (gated on LiveDead CD45.2+ CD11b+ Thy1.2 Ly6C+ Ly6G F4/80+ Lyve-1+ TimD4+) are shown. Bar graphs show the mean ± SEM for the indicated groups, and asterisks denote statistical significance (***p < 0.001, unpaired two-tailed Student’s t test). Data are representative of two independent experiments with five (PBS) and eight (r3LCMV) mice per group.

  4. Supplementary Figure 4 MMs disappear while myelomonocytic cells are recruited after r3LCMV infection.

    (a) MMs (gated on LiveDead CD45.2+ CD11b+ Thy1.2 Ly6C+ Ly6G F4/80+ CD206+) were enumerated by flow cytometry 4, 6, and 8 days after r3LCMV infection and compared with controls. Bar graphs show the mean ± SEM for the indicated groups, and asterisks denote statistical significance (NS: not significant; **p < 0.01, unpaired two-tailed Student’s t test). Data are representative of three independent experiments with six (D4, D8), seven (PBS D6) and ten (r3LCMV D6) mice per group. (b,c) Additional gating strategy validates the loss of MMs 8 days post-infection. (b) Gating strategy and (c) counts of MMs identified either by CD206+ staining (gated on LiveDead CD45.2+ CD11b+ Thy1.2 Ly6C+ Ly6G F4/80+ CD206+) or Lyve-1+TimD4+ staining (gated on LiveDead CD45.2+ CD11b+ Thy1.2 Ly6C+ Ly6G F4/80+ Lyve-1+ TimD4+) are shown. Bar graphs depict the mean ± SEM for the indicated groups, and asterisks denote statistical significance (**p < 0.01, unpaired two-tailed Student’s t test). Data are representative of two independent experiments with six mice per group. (d,e) Myeloid cell populations were analyzed by flow cytometry at 4 and 6 days after PBS, r3LCMV or WT LCMV infection. (d) Gating strategy is shown for Ly6C+ monocytes (gated on LiveDead CD45.2+ CD11b+ Thy1.2 Ly6C+ Ly6G) that were differentiated from neutrophils (gated on LiveDead CD45.2+ CD11b+ Thy1.2 Ly6C- Ly6G+) and MMs (gated on LiveDead CD45.2+ CD11b+ Thy1.2 Ly6C Ly6G F4/80+ CD206+). (e) Meningeal monocyte cell counts are provided. Bar graphs show the mean ± SEM for the indicated groups, and asterisks denote statistical significance (**p < 0.01, ****p < 0.0001, unpaired two-tailed Student’s t test). Data are representative of three independent experiments with thirteen (PBS), six (r3LCMV) and twelve (WT LCMV) mice per group or two independent experiments with fifteen (PBS), eleven (r3LCMV) and four (WT LCMV) mice per group.

  5. Supplementary Figure 5 Engrafted monocytes remain in the meninges for several weeks.

    (a-c) Head-shielded C57BL/6 recipients were irradiated, reconstituted with actin-TFP+ bone-marrow, and infected at 6 weeks with r3LCMV. Thirty days post-infection, MMs (gated on LiveDead- CD45+ Thy1.2- CD11b+ Ly6C- Ly6G- F4/80+ CD206+ cells) were analyzed flow cytometrically for TFP expression. (a) Representative gating strategy, (b) proportion of TFP+ donor-derived cells and (c) number of MMs are provided. Bar graphs show the mean ± SEM for the indicated groups, and asterisks denote statistical significance (***p < 0.001; unpaired two-tailed Student’s t test). Data are representative of two independent experiments with five mice per group. (d,e) Cx3cr1CreER/+ x Stopfl/fl TdTomato mice were pulsed with tamoxifen for a week. Three, 40, and 70 days after treatment, the percentage of TdTomato+ MMs (gated on LiveDead- CD45+ Thy1.2- CD11b+ Ly6C- Ly6G- F4/80+ CD206+ cells) was analyzed flow cytometrically. (d) Representative gating strategy and (e) proportion of TdTomato+ cells indicate near complete initial labeling of MMs followed by progressive turnover. Bar graphs show the mean ± SEM for the indicated groups, and asterisks denote statistical significance (**p < 0.01; unpaired two-tailed Student’s t test). Data are representative of two independent experiments with nine (D3), six (D40) and 4 (D70) mice per group. (f,g) Cx3cr1CreER/+ x Stopfl/fl TdTomato mice were pulsed with tamoxifen 40 days prior to infection. At 2 (f) and 6 (g) months after r3LCMV infection, the percentage of TdTomato+ MMs (gated on LiveDead- CD45+ Thy1.2- CD11b+ Ly6C- Ly6G- F4/80+ CD206+ cells) was analyzed flow cytometrically and compared to PBS-infected controls. The proportion of TdTomato- MMs remained significantly elevated r3LCMV-infected mice relative to controls at both time points analyzed. Bar graphs show the mean ± SEM for the indicated groups, and asterisks denote statistical significance (**p < 0.01; ***p < 0.001; unpaired two-tailed Student’s t test). Data are representative of two independent experiments with four (PBS 2 months) and six (LCMV 2 months, and 6 months) mice per group.

  6. Supplementary Figure 6 Gating strategy for sorting MHC II+ and MHC II- MMs, and lack of lysozyme M-GFP expression by microglia.

    (a) Representative FACS plots show the gating strategy used to sort MHC II+ and MHC II- MMs (LiveDead CD45.2+ CD11b+ Thy1.2 Gr1 F4/80+ CD206+). Data are representative of two independent experiments with four mice per group. (b-e) Myeloid cell populations were analyzed by flow cytometry in LysM-GFP mice infected with r3LCMV or PBS, or in C57BL/6 (B6) mice as a control. (b) Gating strategy for brain leukocytes. (c) Expression of LysM-GFP in brain monocytes (gated on LiveDead CD45.2hi CD11b+ Thy1.2 Ly6C+ Ly6G) and microglia (gated on LiveDead CD45.2low CD11b+ Thy1.2 Ly6C Ly6G). Data are representative of two independent experiments with two mice per group. (d) Gating strategy for meningeal cells. (e) Expression of LysM-GFP in meningeal monocytes (gated on LiveDead CD45.2hi CD11b+ Thy1.2 Ly6C+ Ly6G) and MMs (gated on LiveDead CD45.2+ CD11b+ Thy1.2 Ly6C Ly6G F4/80+ CD206+) Data are representative of two independent experiments with four mice per group.

  7. Supplementary Figure 7 Recombination efficiency in LysMCre/+ Ifngr1fl/fl mice.

    Myeloid cell populations were analyzed in LysMCre/+ Ifngr1fl/fl and control mice. MMs (gated on LiveDead CD45.2+ CD11b+ Thy1.2 Ly6C Ly6G F4/80+ CD206+), blood Ly6C+ monocytes (gated on LiveDead CD45.2+ CD11b+ Thy1.2 CD115+ Ly6C+ Ly6G) and blood Ly6C- monocytes (gated on LiveDead CD45.2+ CD11b+ Thy1.2 CD115+ Ly6C- Ly6G) were analyzed by flow cytometry for expression of IFNγR. (a) Representative histogram showing IFNγR expression on MM. (b) IFNγR expression was reduced by > 50% on MMs in LysMCre/+ Ifngr1fl/fl mice relative to controls as indicated by the proportion of IFNγR+ cells as well as IFNγR gMFI. Bar graphs show the mean ± SEM for the indicated groups, and asterisks denote statistical significance (***p < 0.001, ****p < 0.0001, unpaired two-tailed Student’s t test). Data are representative of two independent experiments with six mice per group. (c) Representative histogram of IFNγR staining on CD115+ Ly6C+ monocytes. (d) IFNγR expression was reduced by > 50% on blood monocytes in LysMCre/+ Ifngr1fl/fl mice as indicated by the proportion of IFNγR+ cells as well as IFNγR gMFI. Bar graphs show the mean ± SEM for the indicated groups, and asterisks denote statistical significance (****p < 0.0001, unpaired two-tailed Student’s t test). Data are representative of two independent experiments with four mice per group.

  8. Supplementary Figure 8 Profile and number of MMs in LysMCre/+ Ifngr1fl/fl mice.

    (a-c) MMs were analyzed in LysMCre/+ Ifngr1fl/fl mice vs. controls during steady state and 30 days following PBS injection. MMs (gated on LiveDead CD45.2+ CD11b+ Thy1.2 Ly6C Ly6G F4/80+ CD206+) were enumerated (a) and analyzed by flow cytometry for the expression of MHC II (b) and MHC I (c). Bar graphs show the mean ± SEM for the indicated groups, and asterisks denote statistical significance (NS: not significant, unpaired two-tailed Student’s t test). Data are representative of three independent experiments with seven, four, seventeen and five mice per group (a), seven, four, seven and five mice per group (b), six, four, six and four mice per group (c), respectively, for the following four groups: naïve controls and LysMCre/+ Ifngr1fl/fl mice, PBS-injected controls and LysMCre/+ Ifngr1fl/fl mice. (d) MMs were analyzed in LysMCre/+ Ifngr1fl/fl mice 30 days after PBS or r3LCMV injection. MMs (gated on LiveDead CD45.2+ CD11b+ Thy1.2 Ly6C Ly6G F4/80+ CD206+) were enumerated. Bar graphs show the mean ± SEM for the indicated groups, and asterisks denote statistical significance (NS: not significant, unpaired two-tailed Student’s t test). Data are representative of three independent experiments with six (PBS) and seven (r3LCMV) mice per group.

  9. Supplementary Figure 9 MMs have altered gene expression after LCMV infection.

    Microarray analysis was performed on RNA extracted from sorted MHC II+ (a) vs. MHC II- (b) MMs from mice injected with r3LCMV-TFP 30 days prior and compared to PBS-injected mice. Data are representative of four independent experiments, with ninety mice per group. MMs were gated on LiveDead Thy1.2 CD45.2+ CD11b+ Gr1 F4/80+ CD206+ and I-Ab/I-Eb positive (MHC II+ MM) or I-Ab/I-Eb negative (MHC II- MM). Heat maps show expression of differentially regulated genes that were statistically different (p < 0.05, Welch-modified t-test under BH FDR multiple comparison correction conditions) after infection and exceeded a threshold of 1.5-fold. Each column represents the mean relative expression levels of the denoted genes in the PBS vs. LCMV groups. Data are representative of four independent experiments with ninety mice per group. Genes in the heat maps were clustered together based on immune sensing (sensome) and other inflammatory processes (inflammatory molecules). See corresponding Supplementary Table 3 for full list.

  10. Supplementary Figure 10 Analysis of SIGN-R1 expression on engrafted MMs and its contribution to neutrophil recruitment after intracranial LPS challenge.

    (a,b) Cx3cr1CreER/+ x Stopfl/fl TdTomato mice were pulsed with tamoxifen 40 days prior to infection. (a,b) Thirty days after infection, SIGN-R1 expression was quantified flow cytometrically on Tdtomato- vs. TdTomato- MMs (gated on LiveDead- CD45+ Thy1.2- CD11b+ Ly6C- Ly6G- F4/80+ CD206+ cells) and compared to PBS-injected controls. Representative FACS plots showing MMs SIGN-R1 expression are provided in (a). Bar graphs in (b) depict the mean ± SEM for the indicated groups, and asterisks denote statistical significance (NS: not significant; ***p < 0.001; unpaired two-tailed Student’s t test). Data are representative of two independent experiments with four (PBS) and five (r3LCMV) mice per group. (c,d) 100 µg of SIGN-R1 blocking antibody or isotype control antibody was injected twice i.v. (16 h and 1 h) before i.c. LPS challenge. (c) 6 h post-LPS challenge, MMs (gated on LiveDead Thy1.2 CD45.2+ CD11b+ Ly6C Ly6G F4/80+ CD206+) were analyzed by flow cytometry for the presence of SIGN-R1. Bar graphs in depict the mean ± SEM for the indicated groups, and asterisks denote statistical significance (****p < 0.0001, unpaired two-tailed Student’s t test). Data are representative of three independent experiments with eight mice per group. (d) Infiltrating neutrophils (gated on LiveDead- CD45+ Thy1.2- CD11b+ Ly6C- Ly6G+ cells) from SIGN-R1-blocked mice and controls were enumerated by flow cytometry 6 h post-LPS challenge. Data are represented as a percentage of control mice. SIGN-R1-blocked mice show a significant decrease in meningeal neutrophil infiltration. Bar graphs in depict the mean ± SEM for the indicated groups, and asterisks denote statistical significance (*p < 0.05; unpaired two-tailed Student’s t test). Data are representative of three independent experiments with eight mice per group.

Supplementary information

  1. Supplementary Figures 1–10 and Supplementary Table 4

  2. Reporting Summary

  3. Supplementary Table 1: Differentially regulated genes in MMs 4 d after LCMV infection

    Microarray analysis was performed on RNA extracted from the MM of LCMV-TFP-infected mice at 4 d post-infection and compared to mock-infected mice. Data are representative of four independent experiments with 90 mice per group. Supplementary Table 1 (‘Gene_Expression’ tab) provides a list of the 215 genes that were differentially regulated in LCMV-infected and mock-infected mice (P < 0.05, Welch-modified t-test under BH FDR multiple comparison correction conditions). The annotated data (‘Enriched_Pathways’ and ‘Upstream_Regulator_Analysis’ tabs) were used to generate the illustrations shown in Figure 2d,e. Fold changes relative to mock-infected controls are shown (‘Gene_Expression’ tab). Positive numbers indicate an increase in expression, whereas negative numbers represent decreased expression (relative to mock). On the basis of differentially expressed genes, IPA software was used to generate the ‘Enriched_Pathways’ and ‘Upstream_Regulator_Analysis’ tabs, which indicate the pathways up-regulated upon LCMV infection and the upstream regulators of MM transcriptome after LCMV infection.

  4. Supplementary Table 2: Differentially regulated genes in MHC II+ MMs vs. MHC II– MMs

    Microarray analysis was performed on RNA extracted from the MHC II+ and MHC II MM of LCMV-TFP-infected mice at 30 d post-infection and mock-infected mice. Data are representative of four independent experiments with 90 mice per group. Supplementary Table 2 (‘Gene_Expression_PBS’ tab) provides a list of the 233 genes that were differentially regulated in MHC II+ and MHC II MM in mock-infected mice, 30 d after injection (P < 0.05, Welch-modified t-test under BH FDR multiple comparison correction conditions). Fold changes of MHC II+ MM relative to MHC II MM, both collected 30 d after PBS injection, are shown. Positive numbers indicate an increase in expression, whereas negative numbers represent decreased expression (relative to MHC II MM). On the basis of differentially expressed genes, IPA software was used to generate the ‘MHC II+_upstream_regulatorsPBS’ tab, which indicate the molecules putatively involved in changes in the MHC II+ transcriptome compared to the MHC II transcriptome, in the context of a mock-infection. The annotated data (‘MHC II+_upstream_regulatorsPBS’ tab) were used to generate the illustration shown in Fig. 6f. Similarly, Supplementary Table 2 (‘Gene_Expression_LCMV’ tab) provides a list of the 277 genes that were differentially regulated in MHC II+ and MHC II MM in LCMV-infected mice at 30 d post-infection (P < 0.05, Welch-modified t-test under BH FDR multiple comparison correction conditions). Fold changes of MHC II+ MM relative to MHC II MM, both collected 30 d after LCMV injection, are shown (‘Gene_Expression_LCMV’ tab). Positive numbers indicate an increase in expression, whereas negative numbers represent decreased expression (relative to MHC II MM). Based on differentially expressed genes, IPA software was used to generate the ‘MHC II+_upstream_regulatorsLCMV’ tab, which indicate the molecules putatively involved in changes in the MHC II+ transcriptome compared to the MHC II transcriptome in the context of LCMV infection. The annotated data (‘MHC II+_upstream_regulatorsLCMV’ tab) was used to generate the illustration shown in Fig. 6g.

  5. Supplementary Table 3: Differentially regulated genes in MM 30 d after LCMV infection

    Microarray analysis was performed on RNA extracted from the MHC II+ and MHC II MM of LCMV-TFP-infected mice at 30 d post-infection and mock-infected mice. Data are representative of four independent experiments with 90 mice per group. Comparison was performed for each macrophage population after mock versus LCMV infection. Supplementary Table 3 (‘Gene_Expression_ MHC II+’ tab) provides a list of the 74 genes that were differentially regulated in MHC II+ MM from LCMV-infected mice, compared to mock-infected mice, as well as fold changes (P < 0.05, Welch modified t-test under BH FDR multiple-comparison correction conditions). Positive numbers indicate genes upregulated in LCMV infected-mice. Supplementary Table 3 (‘Gene_Expression_ MHC II’ tab) provides a list of the 39 genes that were differentially regulated in MHC II MM from LCMV-infected mice compared to mock-infected mice, as well as fold changes. Positive numbers indicate genes upregulated in LCMV infected-mice.

  6. Supplementary Video 1: Behavior of MMs

    Representative time lapses of 3D reconstructions show the dynamics of Cx3cr1gfp/+ labelled myeloid cells (green) captured by TPLSM through a thinned skull of naive Cx3cr1-GFP+/− mice. The maximal projections represent z-stacks (60 µm in depth) imaged over a 12-min time period. The blood vessels are labelled with Evans blue (red). Representative of 12 movies from six independent experiments.

  7. Supplementary Video 2: Positioning of MMs

    Representative time lapses of 3D reconstructions show the dynamics of Cx3cr1gfp/+ parenchymal microglia and superficial meningeal macrophages (green) captured by TPLSM through a thinned skull of naive Cx3cr1gfp/+ mice. The maximal projections represent z-stacks (60 µm in depth) imaged over a 26-min time period. The skull is seen with second harmonic signal (blue). Representative of 12 movies from six independent experiments.

  8. Supplementary Video 3: Interactions between MMs and CTL at the peak of the disease

    Representative time lapses of 3D reconstructions show the dynamics of Cx3cr1gfp/+ labelled myeloid cells (green) captured by TPLSM through a thinned skull of LCMV-infected Cx3cr1gfp/+ mice at the peak of the disease (6 d post-infection). LCMV-specific CTL express a fluorescent protein (pink). The maximal projections represent z-stacks (60 µm in depth) imaged over a 40-min time period. The blood vessels are labelled with Evans blue (red). Note the short- and long-term interactions between MM and CTL. Representative of 16 movies from eight independent experiments.

  9. Supplementary Video 4: Behavior of MMs 30 d after LCMV or mock infection

    Representative time lapses of 3D reconstructions show the dynamics of Cx3cr1gfp/+ labelled myeloid cells (green) captured by TPLSM through a thinned skull of LCMV-infected Cx3cr1gfp/+ mice at the recovery phase of the disease (30 d post-infection of LCMV, or PBS-injected control). The maximal projections represent z-stacks (60 µm in depth) imaged over a 38-min time period. Note the similar density and scanning behavior of MM after both PBS (mock) or LCMV-TFP infection. Representative of six movies from three independent experiments (PBS) or fourteen movies from seven independent experiments (LCMV).

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https://doi.org/10.1038/s41590-019-0344-y