Modifying macrophages at the periphery has the capacity to change microglial reactivity and to extend ALS survival

Abstract

Microglia and peripheral macrophages have both been implicated in amyotrophic lateral sclerosis (ALS), although their respective roles have yet to be determined. We now show that macrophages along peripheral motor neuron axons in mouse models and patients with ALS react to neurodegeneration. In ALS mice, peripheral myeloid cell infiltration into the spinal cord was limited and depended on disease duration. Targeted gene modulation of the reactive oxygen species pathway in peripheral myeloid cells of ALS mice, using cell replacement, reduced both peripheral macrophage and microglial activation, delayed symptoms and increased survival. Transcriptomics revealed that sciatic nerve macrophages and microglia reacted differently to neurodegeneration, with abrupt temporal changes in macrophages and progressive, unidirectional activation in microglia. Modifying peripheral macrophages suppressed proinflammatory microglial responses, with a shift toward neuronal support. Thus, modifying macrophages at the periphery has the capacity to influence disease progression and may be of therapeutic value for ALS.

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Fig. 1: Peripheral nerve macrophages are activated over the course of disease in fast-progression SOD1G93A and slow-progression SOD1G37R ALS mice and in patients with ALS.
Fig. 2: Infiltration of peripheral myeloid cells into the spinal cord of mutant SOD1-expressing ALS mice is weak and depends on the rate of disease progression.
Fig. 3: Replacement of peripheral macrophages with SOD1WT/GFP or Nox2 KO BM transplantation decreases peripheral nerve macrophage and CNS microglial activation in ALS mice.
Fig. 4: BM transplantation with SOD1WT/GFP cells to replace peripheral nerve macrophages at a presymptomatic stage delays the symptomatic disease phase of mutant SOD1-expressing ALS mice.
Fig. 5: SOD1WT/GFP BM transplantation at disease onset to replace peripheral nerve macrophages increases survival of mutant SOD1-expressing ALS mice.
Fig. 6: RNA-seq analysis reveals profound differences between reaction profiles of sciatic nerve peripheral macrophages and spinal cord microglia during disease in SOD1G93A ALS mice.
Fig. 7: RNA-seq analysis of sciatic nerve peripheral macrophages and microglia isolated from SOD1WT/GFP-grafted ALS mice with increased survival reveals a switch in macrophage-activation profiles and modifications of microglial reactivity toward neuronal support.

Data availability

The raw RNA-seq data were uploaded to the NCBI GEO under the accession code GSE156202.

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Acknowledgements

We acknowledge the patients and their families and the Brainbank NeuroCEB Neuropathology Network. We thank the following ICM core facilities (Paris, France): iGenSeq, Histomics, Celis, iCONICS and ICM-Quant (which received funding from the program “Investissements d’avenir” ANR-10-IAIHU-06); the Pitié-Salpêtrière Post-genomic P3S core facility; and the technical staff from our animal housing facility, UMS28 CEF (Centre d’expérimentation fonctionnelle). We thank N. Robil from GenoSplice, D. Bouteiller, E. Mundwiller and Y. Marie from iGenSeq, and J. Guegan from ICONICS for RNA-seq sample preparations and advice, and J. Dumont and A. Millecamps from ICM-Quant for assistance with image analyses. We thank L. Bernard for ALS mouse muscle analysis. We thank E. Pamer (University of Chicago, USA) for providing the Ccr2–GFP mice. This work was supported by the Thierry Latran foundation (Microglia in ALS to S.B., PeriMAC to C.S.L. and S.B.), NRJ-Institut de France (to S.B.), European FP7 International Reintegration Marie Curie Grant (IRG230978 to S.B.), ERANET-NEURON (TracInflam, ANR-14-NEUR-0003-02 to S.B.), the ALS association (ALSA, 16-IIP-258 and 20-IIA-530 to S.B.), the associations Aide à la Recherche des Maladies du Cerveau (ARMC), La longue route des malades de la SLA, the SLAFR, un pied devant l'autre, and private donors to the ALS at the ICM. A.C. and F.B. were supported by fellowships from the French ministry of research and A.C. from the Association pour la Recherche sur la Sclérose Latérale Amyotrophique et autres Maladies du Motoneurone (ARSLA). We would like to thank A. Gervais, C. Colin, J. Lameth, P. Mesci, M. Baratin, C. Delarasse and V. Zujovic for help, advice and tools.

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Authors

Contributions

A.C., S.Z., C.I., M.R., F.B. and L.S. performed experiments. A.J., P.d.l.G., F.B., A.C., C.S.L. and S.B. performed RNA-seq analyses. M.M. and C.S.L. provided mouse lines. A.C., S.Z. and C.I. carried out analyses. M.R. provided important advice on cytometry experiment design, data analysis and interpretation. A.C. and S.B. wrote the manuscript. D.S. provided neuropathological expertise and samples from patients with ALS. M.M., D.B., S.M., D.S. and C.S.L. brought key advice and revised the manuscript. S.B. directed the study.

Corresponding author

Correspondence to Séverine Boillée.

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The authors declare no competing interests.

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Peer review information Nature Neuroscience thanks Hande Ozdinler 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

Extended Data Fig. 1 Staining of macrophages in ALS mouse sciatic nerve and human control spinal cord, root and peripheral nerve tissues.

(Additional data to Fig. 1). a-e, Immunostaining of sciatic nerves for the macrophage markers CD11b, CD68, F4/80 in 100 day-old control C57Bl6 (Bl6, n = 3 mice) (a) and SOD1G93A (b-e) mice at different disease stages (n = 4 mice at each stage). Scale bar: 100μm (a-e). f-j, Immunostaining against the microglia/macrophage marker CD68 (in brown) of human control spinal cord ventral horn (f), dorsal root (containing sensory neuron axons) (g), ventral root (containing motor neuron axons) (h) and control peripheral nerves (i,j) Scale bars: 40μm (f-j). Data associated to the n = 11 ALS patients and n = 4 controls in Supplementary Table 1.

Extended Data Fig. 2 Scheme of the different bone-marrow (BM) transplantation protocols in control and mutant SOD1 ALS mice.

a, Protocols used in control C57Bl6 mice showing the ages at treatments and follow-ups. b, Protocol used for busulfan myeloablation and BM transplantation in the slow progressing SOD1G37R mice, indicating the time points when blood GFP+ cells were monitored, and the disease stages. c-c’, Protocols used for busulfan myeloablation and BM transplantation in the fast progressing SOD1G93A mice, executed at two different stages, pre-symptomatic (c), or at disease onset (c’), displaying the time points when blood GFP+ cells were monitored, and the disease stages.

Extended Data Fig. 3 Analysis of cell replacement in myeloablated and bone-marrow (BM) grafted mice.

(Additional data to Fig. 2). a, Number of white blood cells /ml of blood in non-treated, busulfan-treated or irradiated C57Bl6 (Bl6) mice; Bars: means±SEM for n = 8–12 mice. ****: p < 0.0001, one-way ANOVA followed by a Dunnett’s post-hoc analysis. b, Proportion of GFP+ cells in the blood of actin-GFP or Bl6 treated mice from D12 to D370 after BM graft. Bars: means±SEM for n = 3-4 mice. c-d, Proportion of GFP+ cells in the different blood cell types (CD3+, CD19+,GR1+, CD11b+ and F4/80+ cells) in actin-GFP BM grafted busulfan-treated (n = 4) (c) or irradiated (n = 7) (d) Bl6 mice. Bars: means±SEM. e, Number of white blood cells / ml of blood after busulfan treatment and before BM graft; bars: means±SEM for n = 7 SOD1G37R mice and n = 15 Bl6 or SOD1G93A mice. ****: p < 0.0001, one-way ANOVA followed by a Dunnett’s post-hoc analysis. f-g, Proportion of GFP+ cells in blood CD11b+ cells of GFP->SOD1G93A mice, 12 and 60 days (corresponding to disease onset) after BM graft (f) and of GFP->SOD1G37R mice at several time points after BM graft (g); bars: means±SEM for n = 21 (f) and n = 8 (g) mice. h, Proportion of GFP+ macrophages in GFP->SOD1G93A gastrocnemius (Gastroc.) muscles. Bars: means±SEM for n = 4 (onset) or n = 4 (end-stage) mice. Detailed n and statistics in Supplementary Table 7.

Extended Data Fig. 4 Additional analysis for peripheral cell infiltration in the spinal cord of SOD1G93A mice using the Siglec1/CD169 marker previously proposed as peripheral monocyte/macrophage specific.

a-f SOD1G93A mouse spinal cord sections immunostained against Iba1 (green; a-c) and CD169 (red; d-f) at disease onset (n = 4 mice) (a,d), early symptomatic stage (n = 4 mice) (b,e) and end-stage (n = 4 mice) (c,f). Note that CD169 immunostaining was absent at disease onset, as expected, but that CD169 staining increased in Iba1+ cells over the disease course. Scale bar: 50μm (a-f). g-k, GFP->SOD1G93A mouse spinal cord section at the late symptomatic stage (displaying a higher rate of infiltrated GFP+ cells, n = 3 mice) stained against CD169 (red, g) and Iba1 (blue, h) and showing GFP+ cells originating from the periphery (green, i). j, overlay of (g-i), white arrows show GFP+/CD169+/Iba1+ cells (originating from the periphery), and white arrowheads show non-GFP endogenous microglial cells positive for Iba1 and CD169. k, lower magnification of (j), white rectangle shows (j). Note that CD169 was also expressed by activated microglial cells and that it could therefore not be used to differentiate endogenous microglia from infiltrated peripheral cells. Scale bar: 50μm (for g-j), and 100μm (k).

Extended Data Fig. 5 Additional analysis for peripheral cell infiltration in the spinal cord of ALS mice using Ccr2-GFP and Ccr2-RFP reporter mice.

a–f, Characterization of Ccr2+ cells after sciatic nerve crush-induced inflammation. (a-c) Ccr2-GFP mouse sciatic nerves (n = 3 mice). Blue circles show GFP+ (Ccr2+, green) and (CD11b-CD68-F4/80)+ (red) monocytes and red circles show GFP+/(CD11b, CD68, F4/80)+ differentiated macrophages (identified by their morphology). (d-f) Ccr2-RFP mouse sciatic nerves (n = 4 mice). (d) Blue circles show (CD11b-CD68-F4/80)+ (green)/RFP (Ccr2)+ (red) monocytes, yellow circles show RFP (Ccr2)+ cells but not expressing the macrophage markers. (e-f) To identify these cells, Ccr2-RFP sciatic nerves were stained against the pan T cell marker CD3 (e) and against RFP (f) showing that most of the RFP+ (Ccr2+) cells in Ccr2-RFP mouse sciatic nerves, after a lesion, are T lymphocytes (yellow circle). Scale bars: 100 μm (a,d) and (b-c, e-f). g-h, Characterization of Ccr2+ cells in spinal cords of SOD1G93A mice crossed with Ccr2-GFP mice (n = 3) (g) or Ccr2-RFP mice (n = 4) (h) at disease end-stage. g, white arrow points to an Iba1+ (red) Ccr2+ (anti-GFP - green) cell. h, White arrowheads: CD3+/RFP+ cells (lymphocytes expressing Ccr2), white arrows: CD3-/ RFP+ monocytes showing that most of the RFP+ (Ccr2 positive) cells in Ccr2-RFPxSOD1 mice were T lymphocytes. Scale bar: 50μm (g,h). i-j, Quantifications of CCR2+Iba+ and CD3- monocytes/ macrophages or CD3+ lymphocytes originating from the periphery in Ccr2-GFPxSOD1G93A (i, n = 3-4 mice/time point) and Ccr2-GFPxSOD1G37R (j, n = 4 mice/ time point) mice. k, Proportion of Iba1+ myeloid cells expressing Ccr2 (GFP+) in Ccr2-GFPxSOD1G93A (n = 3 mice/time point) and Ccr2-GFPxSOD1G37R (n = 4 mice/time point) mice. Bars: means± SEM. Detailed n and statistics in Supplementary Table 7.

Extended Data Fig. 6 Further characterization of the bone-marrow (BM) transplanted ALS mice and the pathology in their tissues.

(Additional data to Figs. 3 and 4). a, Number of white blood cells per ml of blood in non-treated C57Bl6 (Bl6) mice and busulfan-treated mice before BM transplantation. Bars represent means±SEM for n = 15 mice per group. ****: p < 0.0001, one-way ANOVA followed by a Dunnett’s post-hoc analysis. b, As Nox2 KO cells do not express human SOD1, cells originating from the Nox2 KO BM were indirectly identified by measuring, in the blood cells of Nox2 KO->SOD1G93A mice, the levels of human SOD1 transgenes by a quantitative PCR, 12 days and 60 days after BM transplantation. Bars: means±SEM for n = 12 mice/time point. Lower levels of human SOD1 in the blood revealed higher levels of Nox2 KO cells. c, Proportion of GFP+ cells in CD11b+ myeloid cells in the blood of SOD1WT/GFP->SOD1G93A mice 12 days and 60 days after BM transplantation. Bars: means±SEM for n = 14 mice at each time point. d, Proportion of GFP+ macrophages among all (CD11b-CD68-F4/80) + macrophages in the sciatic nerves of SOD1WT/GFP->SOD1G93A mice. Means±SEM for n = 4 (onset) and n = 8 (end-stage) mice. e, f, Quantification of GFP+cells originating from the periphery in the spinal cord of SOD1WT/GFP->SOD1G93A mice at the different disease stages. e, Area occupied by GFP fluorescence and f, proportion of Iba+ microglial cells expressing GFP. Bars represent means±SEM for n = 3–8 mice per time point and genotype. g, Macrophage activation measured by CD11b-CD68-F4/80 immunoreactive area in sciatic nerves of non-grafted SOD1G93A (grey, same data as Fig. 1g) and SOD1G93A->SOD1G93A (red, same data as Fig. 3a) mice. Bars represent means±SEM for n = 4-5 mice per genotype and time point. Detailed n and statistics in Supplementary Table 7.

Extended Data Fig. 7 Additional analysis of pre-symptomatic grafted SOD1G93A mice.

(Additional data to Figs. 3 and 4). a, Density of macrophages (in cells/mm2) in sciatic nerves of non-grafted SOD1G93A and pre-symptomatic grafted SOD1G93A mice, at the different disease stages. b, motor neuron numbers per spinal cord section in C57Bl6 (Bl6), SOD1WT, non-grafted SOD1G93A and pre-symptomatic grafted SOD1G93A mice. c, Number of T lymphocytes (CD3+) per lumbar spinal cord section of non-grafted and pre-symptomatic grafted SOD1G93A mice. Bars: means±SEM; n = 3–8 mice per genotype and time-point (a-c). d, Ratio of CD4+ and CD8+T cells in the spinal cord of non-grafted and pre-symptomatic grafted SOD1G93A mice at end-stage. Note that Nox2 KO->SOD1G93A mice were the only ones showing increased number of lymphocytes and that CD4+ cells were predominant. Bars: means±SEM for n = 4–8 mice/group. e, Proportion of T lymphocytes (CD3+) in the blood of control Bl6 and Nox2 KO mice. Bars: means±SEM for n = 4 mice/ group. f, Cybb (encoding for Nox2) mRNA levels in blood cells of Nox2 KO, Bl6 and symptomatic SOD1G93A mice. Bars: means±SEM for n = 4 mice/group. Note that T lymphocytes barely expressed Nox2 in control and symptomatic SOD1G93A mice - deletion of Nox2 in T lymphocytes was unlikely to influence their infiltration, which was rather linked to a non-cell autonomous effect. g, Proportion of regulatory T lymphocytes (FoxP3+ and CD3+) among T lymphocytes (CD3+) in the spinal cord of non-grafted SOD1G93A mice at onset and end-stage. Bars: means±SEM for n = 4 mice/group. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001, one-way ANOVA followed by a Tukey’s post-hoc analysis (b, c, f), and two tailed student t-test (g). Detailed n and statistics in Supplementary Table 7.

Extended Data Fig. 8 SOD1G93A bone-marrow transplantation at the pre-symptomatic stage or at onset does not influence disease progression of SOD1G93A ALS mice, showing no influence of the transplantation protocol, per se.

(Additional data to Figs. 3, 4 and 5). a-c, Kaplan-Meier plots of ages reached at onset [at weight peak (a) or at grip strength peak (a’)], early disease [at 10% of weight loss (b) or at 35% of grip strength loss (b’)] and end-stage [at complete hind limb paralysis (c)], in non-grafted SOD1G93A mice (grey), SOD1G93A bone-marrow grafted SOD1G93A (SOD1G93A->SOD1G93A) mice, grafted at the pre-symptomatic stage (red) or at disease onset (pink). Mean ages±SEM are indicated with animal numbers in brackets. No statistical significant differences were found between the groups at any time-point, according to log-rank test.

Extended Data Fig. 9 Additional analysis of RNAseq data obtained from sciatic nerve macrophages and spinal cord microglia.

(Additional data to Figs. 6 and 7). a, Scheme depicting the different groups and numbers of samples analyzed by RNA sequencing (RNAseq). Microglial cells (MG) were isolated from SOD1G93A, SOD1WT, C57Bl6 (Bl6) and onset grafted SOD1WT->SOD1G93A and SOD1G93A->SOD1G93A mice. Sciatic nerve peripheral macrophages (MP) were isolated from SOD1G93A mice at the 4 stages, onset grafted SOD1WT->SOD1G93A and SOD1G93A->SOD1G93A at 2 stages and from SOD1WT mice at the age corresponding to disease end-stage in SOD1G93A mice, but could not be recovered from Bl6 mice probably due to their limited numbers. b, Percentage of sequences specific to microglia (blue) or sciatic nerve peripheral macrophages (red) or common to both (purple), at the different disease stages. Only genes expressed in at least two samples per group were considered. c (right panel), Table summarizing the number of regulated genes (up-regulated, yellow; down-regulated, purple) between the different disease stages in SOD1G93A microglial cells (MG) or sciatic nerve peripheral macrophages (MP), and at equivalent stages between SOD1G93A microglial cells and sciatic nerve peripheral macrophages (grey boxes). c (left panel), Table summarizing the number of regulated genes (up-regulated, yellow; down-regulated, purple) in microglial cells (MG) or sciatic nerve peripheral macrophages (MP), between onset grafted SOD1WT->SOD1G93A and SOD1G93A->SOD1G93A mice at early symptomatic stage or disease end-stage. ES: end-stage.

Extended Data Fig. 10 Analysis of microglia and macrophage specific genes in RNAseq data obtained from spinal cord microglia and sciatic nerve macrophages.

(Additional data to Figs. 6 and 7). Violin plots of all values at all the control/disease stages for nerve macrophages (red) or microglia (blue) of 15 microglia and 5 macrophage signature genes. Signature gene references: Chiu, I. M. et al. A neurodegeneration-specific gene-expression signature of acutely isolated microglia from an amyotrophic lateral sclerosis mouse model. Cell Rep. 4, 385–401 (2013)22; Bennett, F. C. et al. A Combination of Ontogeny and CNS Environment Establishes Microglial Identity. Neuron 98, 1170–1183 (2018)23; Bennett, M. L. et al. New tools for studying microglia in the mouse and human CNS. Proc. Natl. Acad. Sci. 12, E1738–E1746 (2016)24; Zondler, L. et al. Peripheral monocytes are functionally altered and invade the CNS in ALS patients. Acta Neuropathol. 132, 391–411 (2016)25; Van Hove, H. et al. A single-cell atlas of mouse brain macrophages reveals unique transcriptional identities shaped by ontogeny and tissue environment. Nat. Neurosci. 22, (2019)62; Saederup, N. et al. Selective chemokine receptor usage by central nervous system myeloid cells in CCR2-red fluorescent protein knock-in mice. PLoS One 5, (2010)54, Lund, H. et al. Competitive repopulation of an empty microglial niche yields functionally distinct subsets of microglia-like cells. Nat. Commun. 9, 4845 (2018)63; Gosselin, D. et al. An environment-dependent transcriptional network specifies human microglia identity. Science. 3222, 33–35 (2017)64.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, legends for Supplementary Tables 1–7, and Supplementary Figs. 1 and 2.

Reporting Summary

Supplementary Table 3

Lists of sciatic nerve peripheral macrophage regulated genes over the disease course in SOD1G93A mice.

Supplementary Table 4

Lists of spinal cord microglia regulated genes over the disease course in SOD1G93A mice.

Supplementary Table 5

Lists of sciatic nerve peripheral macrophage compared to spinal cord microglia regulated genes.

Supplementary Table 6

Lists of sciatic nerve peripheral macrophage or microglial cell regulated genes in SOD1WT→SOD1G93A versus SOD1G93A→SOD1G93A onset-grafted mice at the early-symptomatic stage or disease end-stage.

Supplementary Table 7

Information on sample sizes and statistics for all figures.

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Chiot, A., Zaïdi, S., Iltis, C. et al. Modifying macrophages at the periphery has the capacity to change microglial reactivity and to extend ALS survival. Nat Neurosci 23, 1339–1351 (2020). https://doi.org/10.1038/s41593-020-00718-z

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