Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative disorders that overlap in their clinical presentation, pathology and genetic origin. Autoimmune disorders are also overrepresented in both ALS and FTD, but this remains an unexplained epidemiologic observation1,2,3. Expansions of a hexanucleotide repeat (GGGGCC) in the C9orf72 gene are the most common cause of familial ALS and FTD (C9-ALS/FTD), and lead to both repeat-containing RNA and dipeptide accumulation, coupled with decreased C9orf72 protein expression in brain and peripheral blood cells4,5,6. Here we show in mice that loss of C9orf72 from myeloid cells alone is sufficient to recapitulate the age-dependent lymphoid hypertrophy and autoinflammation seen in animals with a complete knockout of C9orf72. Dendritic cells isolated from C9orf72−/− mice show marked early activation of the type I interferon response, and C9orf72−/− myeloid cells are selectively hyperresponsive to activators of the stimulator of interferon genes (STING) protein—a key regulator of the innate immune response to cytosolic DNA. Degradation of STING through the autolysosomal pathway is diminished in C9orf72−/− myeloid cells, and blocking STING suppresses hyperactive type I interferon responses in C9orf72−/− immune cells as well as splenomegaly and inflammation in C9orf72−/− mice. Moreover, mice lacking one or both copies of C9orf72 are more susceptible to experimental autoimmune encephalitis, mirroring the susceptibility to autoimmune diseases seen in people with C9-ALS/FTD. Finally, blood-derived macrophages, whole blood and brain tissue from patients with C9-ALS/FTD all show an elevated type I interferon signature compared with samples from people with sporadic ALS/FTD; this increased interferon response can be suppressed with a STING inhibitor. Collectively, our results suggest that patients with C9-ALS/FTD have an altered immunophenotype because their reduced levels of C9orf72 cannot suppress the inflammation mediated by the induction of type I interferons by STING.
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The RNA sequencing data reported here have been deposited in Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov) under accession number GSE151936. Data from GSE151936 were used for Figs. 1h, i, 2k, 4a, b, i and Extended Data Figs. 4a, b, 5a, b, 6a–g, 7c and 8a–f. Human whole-blood RNA sequencing data reported here can be accessed from the database of Genotypes and Phenotypes (dbGaP, https://www.ncbi.nlm.nih.gov/gap) using accession number phs002055.v1.p1. Data from dbGaP with accession number phs002055.v1.p1 were used for Fig. 4d, e. Human cerebellum RNA-seq data can be found in ref. 30. Source data are provided with this paper.
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We thank R. Jeroen Pasterkamp for providing C9orf72fl/fl mice; M. Vasquez for maintaining the animal colony; H. Maghzi for assisting with EAE experiments; G. Martins for running the flow cytometry core; and and M. Maniex for assistance with tissue collection. A.Y. is the recipient of a Ramón y Cajal fellowship from the Ministerio de Ciencia, Innovación y Universidades, Spain. This work was supported by NIH grant NS069669 (to R.H.B), the Robert and Louise Schwab family, the Cedars-Sinai ALS Research Fund (to R.H.B.), Arthritis Foundation grant AF2017-433570 (to C.J), and NIH grant 1R21AI126368-01 (to M.A.).
The authors declare no competing interests.
Peer review information Nature thanks Tony Wyss-Coray, Aaron D. Gitler and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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 DCs and T cells develop normally in C9orf72−/− mice, but markers of immune activation and inflammatory cytokine production are seen from a young age.
a, Percentage of DC populations in total splenocytes of 8-week-old mice (n = 5). b, MFI measured via flow cytometry for MHC class II and costimulatory molecules (CD80, CD86 and CD40) of three splenic DC populations (CD11b, CD8a and pDC) from 8-week-old mice (n = 5). c, Percentage of DC populations from total splenocytes of 8-month-old mice (n = 6). d, MFI measured via flow cytometry for MHC II and costimulatory molecules of splenic DC populations from 8-month-old mice (n = 6). e, Intracellular TNF staining of splenic DCs from 10-week-old mice after stimulation with LPS (100 ng ml−1) (n = 3). f, Quantification of double-negative (DN), double-positive (DP) and single-positive CD4 and CD8 T-cell populations in the thymus of 8-week-old mice (n = 3). g, Percentage of Foxp3+ CD4 T cells in the spleen of 8-week-old mice (n = 3), showing no change in T-regulatory cell populations. h, ELISA of supernatants collected from isolated CD4 T cells from the spleen of 6-month-old mice after anti-CD3/anti-CD28 treatment for 72 h. Graphs show a representative experiment with biological replicates, which was repeated twice. a–h, One-way ANOVA. e, i, Data shown as means ± s.e.m. h, Data shown as means ± s.d. Source data
Extended Data Fig. 2 Profiling of splenocytes from myeloid-specific deletion of C9orf72 via Cx3cr1Cre.
a, Proportions of immune-cell populations in 12-month-old C9orf72fl/fl:Cx3cr1Cre mice. iMono, inflammatory monocytes; Macs, macrophages; Neut., neutrophils; pMono, patrolling monocytes. b, Proportions of splenic DC populations in 5-month-old C9orf72fl/fl:Cx3cr1Cre mice (n = 7). c, C9orf72fl/fl;Cx3cr1Cre CD11b splenic DCs from 5-month-old mice have increased MFI for CD86 compared with C9orf72fl/fl mice (n = 4). d, MFI for splenic DC MHC II and costimulatory molecules in 5-month-old C9orf72fl/fl:Cx3cr1Cre mice (n = 7). Unpaired two-tailed Student’s t-test. b, c, Data shown as means ± s.e.m. Source data
a, Left, gross images of splenomegaly in C9orf72fl/fl:LysMCre mice. Right, spleen weights from C9orf72fl/fl and C9orf72fl/fl;LysMCre mice (in milligrams) normalized to body weight (in grams) at 5 months (n = 4). b, Proportions of splenic DC populations in 5-month-old C9orf72fl/fl;LysMCre mice (n = 4). c, Proportions of splenic DC populations in 12-month-old C9orf72fl/fl;LysMCre mice (n = 4). d, MFI of splenic DC MHC II and costimulatory molecules in 5-month-old C9orf72fl/fl;LysMCre mice (n = 4). e, MFI measured via flow cytometry for MHC II and costimulatory molecules of splenic DC populations from 12-month-old C9orf72fl/fl;LysMCre mice (n = 4). f, Percentages of CD4 and CD8 T cells in splenocytes. g, h, CD4 T cell (g) and CD8 T cell (h) activation states in 5-month-old C9orf72fl/fl;LysMCre mice. i, Percentages of CD4 and CD8 T cells amongst splenocytes in 12-month-old mice. j, k, CD4 T cell (j) and CD8 T cell (k) activation states in 12-month-old C9orf72fl/fl;LysMCre mice (n = 4). Unpaired two-tailed Student’s t-test. a–c, Data shown as means ± s.e.m. Source data
Extended Data Fig. 4 RNA-seq values for genes stimulated by type I interferon in different immune populations following total-body versus myeloid-cell-specific deletion of C9orf72.
a, b, RNA-seq of CD11b cells (a) and B cells (b) from wild-type (WT; n = 4), total knockout (C9(−/−); n = 3) and C9orf72fl/fl;Cx3cr1Cre (n = 4) mice. The results are similar to those observed in DCs of total-body nulls (Fig. 2), and show activation of ISGs. Results from T cells of the same dataset are shown in Fig. 1. Two-way ANOVA; data shown as means ± s.e.m.
Extended Data Fig. 5 RNA-seq of C9orf72-deficient DCs, responses of ISGs in BMDMs to TLR agonists, and amelioration of type I interferon responses by deletion of STING.
a, PCA plot from RNA-seq of freshly isolated splenic classical DCs from 10-week-old mice (n = 4). b, TPM values for the indicated cytokines and inflammatory genes (WT, n = 4; C9(−/−), n = 4 biologically independent samples). c–e, qRT–PCR analysis of Ifnb1, Mx1 and Cxcl10 in WT and C9(−/−) BMDMs after stimulation with CpG (c), poly I:C (d) and LPS (e) (representative of three experiments). f, The STING antagonist H151 blocks the hyperactive ISG response to stimulation with cGAMP (representative of three experiments). g, qRT–PCR analysis of IFNβ in total BMDMs of WT, Gt(−/−), C9(−/−) and C9(−/−):Gt(−/−) mice after stimulation with cGAMP. Representative of three independent experiments. b–e, Unpaired two-tailed t-test. f, g, One-way ANOVA. b, Data shown as means ± s.e.m. c–g, Data shown as means ± s.d. Source data
Extended Data Fig. 6 Deletion of STING mitigates the increased gene-expression response to type I interferon in C9orf72−/− immune cells.
a–d, Heat maps showing ISGs from RNA-seq of isolated splenocyte populations from 3-month-old mice. Cell types are indicated and include: a, CD11b myeloid cells; b, CD4 T cells; c, CD8 T cells; d, B cells, from WT, C9(−/−) and C9orf72;STING double knockout (C9(−/−)/Gt(−/−)) mice. e–g, TPM values of indicated ISGs in: e, CD4 T cells (WT, n = 4; C9(−/−), n = 3; C9(−/−)/Gt(−/−), n = 4); f, CD8 T cells (WT, n = 4; C9(−/−), n = 3; C9(−/−)/Gt(−/−), n = 4); and g, B cells (WT, n = 4; C9(−/−), n = 3; C9(−/−)/Gt(−/−), n = 4). Deletion of STING leads to a marked rescue of ISG expression across all cell types. e–g, Two-way ANOVA; data shown as means ± s.e.m.
a, b, Results from flow cytometry analysis of splenic CD11c costimulatory markers in 3-month-old WT (n = 4), C9(−/−) (n = 5) and C9(−/−)/Gt(−/−) (n = 4) mice. c, RNA-seq results for splenic CD11b cells from 3-month-old WT (n = 4), C9(−/−) (n = 3) and C9(−/−)/Gt(−/−) (n = 4) mice. d, e, Results from flow cytometry analysis of splenic CD4 T cells (d) and CD8 T cells (e) from 3-month-old mice. One-way ANOVA. a, b, d, e, Data shown as means ± s.e.m. Source data
a–c, Heat map from RNA-seq data for isolated CD4 T cells (a), CD8 T cells (b) and B cells (c) from spleens of 3-month-old WT (n = 4) and C9(−/−) (n = 3) mice. d–f, Markers of TH1 (d), TH2 (e) and TH17 (f) polarization genes from CD4 T cells. Two-way ANOVA. d–f, Data shown as means ± s.e.m.
Extended Data Fig. 9 C9orf72−/− mice are more susceptible than wild-type mice to EAE and show increased infiltration of TH1-polarized T cells into nervous tissue.
a, Representative LFB staining of spinal cord from WT (n = 3), C9(+/−)(n = 3) and C9(−/−) (n = 3) mice. b, Representative H&E staining of spinal cords from WT (n = 3), C9(+/−) (n = 3) and C9(−/−) (n = 3) mice at end stage. c, IBA1 staining of spinal cord from the indicated genotypes (n = 3). d, e, Splenic C9(−/−) CD4 (d) and CD8 (e) T cells produce increased levels of IFNγ during pre-onset (day 9) (WT, n = 4; C9(+/−), n = 4; C9(−/−), n = 3 biologically independent samples) and peak (day 15) (WT, n = 3; C9(+/−), n = 3; C9(−/−), n = 3 biologically independent samples) of EAE. f–i, Total number of CD3+ T cells (f), IFNγ+ CD4 T cells (g), IL-17+ CD4 T cells (h) and IFNγ+ IL-17+ CD4 T cells (i) in whole brain of WT (n = 2), C9(+/−) (n = 3) and C9(−/−) (n = 3) mice during the peak (day 15) of disease. f–i, One-way ANOVA; data shown as means ± s.e.m. Source data
Extended Data Fig. 10 C9orf72−/− mice that are resistant to B16 melanoma tumours show an enhanced cytotoxic T cell response.
a, Percentages of CD44+ CD4 T cells (left) and CD8 T cells (right) in the lungs of WT (n = 2), C9(+/−) (n = 3) and C9(−/−) (n = 3) mice on day 14 of B16 melanoma challenge. b, c, Percentages of naive CD4 T cells (b) and memory CD4 T cells (c) in the spleens of WT, C9(+/−) and C9(−/−) mice with and without inoculation with B16 cells (n = 5). d–f, Percentages of naive CD8 T cells (d), memory CD8 T cells (e) and effector memory CD8 T cells (f) in the spleens of WT, C9(+/−) and C9(−/−) mice with and without inoculation with B16 cells (n = 5). One-way ANOVA; data shown as means ± s.e.m. Source data
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McCauley, M.E., O’Rourke, J.G., Yáñez, A. et al. C9orf72 in myeloid cells suppresses STING-induced inflammation. Nature 585, 96–101 (2020). https://doi.org/10.1038/s41586-020-2625-x
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