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Cited by 170Amyotrophic Lateral Sclerosis ResearchNeuroinflammation and Neurodegeneration MechanismsImmune Cell Function and Interaction

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Natural killer cells modulate motor neuron-immune cell cross talk in models of Amyotrophic Lateral Sclerosis

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Garofalo, Stefano, Cocozza, Germana, Porzia, Alessandra, Inghilleri, Maurizio, Raspa, Marcello, Scavizzi, Ferdinando, Aronica, Eleonora, Bernardini, Giovanni, Peng, Ling, Ransohoff, Richard M., Santoni, Angela, Limatola, Cristina·2020·Nature Communications
DOI 10.1038/s41467-020-15644-8PMC PMC7156729
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Abstract

In amyotrophic lateral sclerosis (ALS), immune cells and glia contribute to motor neuron (MN) degeneration. We report the presence of NK cells in post-mortem ALS motor cortex and spinal cord tissues, and the expression of NKG2D ligands on MNs. Using a mouse model of familial-ALS, hSOD1 G93A , we demonstrate NK cell accumulation in the motor cortex and spinal cord, with an early CCL2-dependent peak. NK cell depletion reduces the pace of MN degeneration, delays motor impairment and increases survival. This is confirmed in another ALS mouse model, TDP43 A315T . NK cells are neurotoxic to hSOD1 G93A MNs which express NKG2D ligands, while IFNγ produced by NK cells instructs microglia toward an inflammatory phenotype, and impairs FOXP3 + /Treg cell infiltration in the spinal cord of hSOD1 G93A mice. Together, these data suggest a role of NK cells in determining the onset and progression of MN degeneration in ALS, and in modulating Treg recruitment and microglia phenotype.

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Figures

Fig. 1 NK cells infiltrate the CNS affected regions in ALS patients. a NKp46+ cells (in red) in slices of the spinal cord and motor cortex obtained from sALS patients, not present in control patients. 3d reconstruction of one representative NK cell on the right. Scale bar: 20 μm. (n = 8 controls, 12 sALS patients **P < 0.01 power 0.999 two-tailed Student’s t-test). Each dot represents the number of NKp46+ cells per patient. Only 3 representative black squares are reported in the graph for Controls, in which were no detected NK cells. b Percentage of CD56+/high/low/CD3− cells obtained from peripheral blood of control or sALS patients (n = 14 controls, 15 sALS patients. **P < 0.01 power 0.860 two-tailed Student’s t-test). Each dot represents the % of NK cells per patient. Error bars show mean ± SEM. Source data are provided as a Source Data file.
Fig. 1 NK cells infiltrate the CNS affected regions in ALS patients. a NKp46+ cells (in red) in slices of the spinal cord and motor cortex obtained from sALS patients, not present in control patients. 3d reconstruction of one representative NK cell on the right. Scale bar: 20 μm. (n = 8 controls, 12 sALS patients **P < 0.01 power 0.999 two-tailed Student’s t-test). Each dot represents the number of NKp46+ cells per patient. Only 3 representative black squares are reported in the graph for Controls, in which were no detected NK cells. b Percentage of CD56+/high/low/CD3− cells obtained from peripheral blood of control or sALS patients (n = 14 controls, 15 sALS patients. **P < 0.01 power 0.860 two-tailed Student’s t-test). Each dot represents the % of NK cells per patient. Error bars show mean ± SEM. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 2 NK cells infiltrate the CNS and affect survival time in ALS models. a Left: NKp46+ cells (in red) in cerebral slices of the lumbar spinal cord and motor cortex of hSOD1G93A mice (13 weeks), not present in wt mice. Scale bar: 10 μm. 3d reconstruction of one representative NK cell on the right. Right: time course analysis of NK1.1+/CD3− cells in the lumbar spinal cord and the cerebral motor cortex of hSOD1G93A mice (mice at 8 and 19 weeks, n = 4; mice at 10, 13 and 16 weeks, n = 6. **P < 0.01, power 0.999, one-way ANOVA vs 8 weeks). b Frequency of NK1.1+/CD3− cells in the spleen of wt and hSOD1G93A mice (wt, n = 5; hSOD1G93A n = 8). c Expression of GZMb, PRF and CD69 in the spleen and spinal cord of wt and hSOD1G93A mice (wt, n = 3; hSOD1G93A, n = 5, **P < 0.01, power 0.841, one-way ANOVA). d Distances of NKp46+ cells from vessel endothelial cells (CD31+ cells) in the spinal cord of hSOD1G93A (13 weeks) mice (n = 4 mice/21 cells). Scale bar: 20 μm. Representative immunofluorescence is shown. e NK1.1+/CD3− cells frequency in the motor cortex (top) and in the spinal cord (bottom) of hSOD1G93A mice treated with Ab-CCL2 (n = 6, *P < 0.05 power 0.916 two-tailed Student’s t-test). Representative density plots are shown on the right. f Scheme of Ab-NK1.1 administration. g Kaplan–Meier curve of hSOD1G93A and TDP43A315T mice treated with vehicle or Ab-NK1.1 (hSOD1G93A, vehicle: 136.8 ± 1.7 days; Ab-NK1.1: 147.2 ± 2.4 days; n = 5. TDP43A315T, vehicle: 78.5 ± 1.5 days; Ab-NK1.1: 88.7 ± 1.9 days, n = 4. Data are the mean ± s.e.m. **P < 0.01, two-sided log-rank test). h Age of paralysis, calculated as the absence of hindlimb movement, upon Ab-NK1.1 treatment in hSOD1G93A mice; vehicle: 18.3 ± 0.2 weeks; Ab-NK1.1: 19.5 ± 0.2 weeks (n = 7, data are the mean ± s.e.m. **P < 0.01 power 0.921 two-tailed Student’s t-test). For boxplots (a, c, e), the center line, boxes and whiskers represent the median, inner quartiles, and rest of the data distribution, respectively.
Fig. 2 NK cells infiltrate the CNS and affect survival time in ALS models. a Left: NKp46+ cells (in red) in cerebral slices of the lumbar spinal cord and motor cortex of hSOD1G93A mice (13 weeks), not present in wt mice. Scale bar: 10 μm. 3d reconstruction of one representative NK cell on the right. Right: time course analysis of NK1.1+/CD3− cells in the lumbar spinal cord and the cerebral motor cortex of hSOD1G93A mice (mice at 8 and 19 weeks, n = 4; mice at 10, 13 and 16 weeks, n = 6. **P < 0.01, power 0.999, one-way ANOVA vs 8 weeks). b Frequency of NK1.1+/CD3− cells in the spleen of wt and hSOD1G93A mice (wt, n = 5; hSOD1G93A n = 8). c Expression of GZMb, PRF and CD69 in the spleen and spinal cord of wt and hSOD1G93A mice (wt, n = 3; hSOD1G93A, n = 5, **P < 0.01, power 0.841, one-way ANOVA). d Distances of NKp46+ cells from vessel endothelial cells (CD31+ cells) in the spinal cord of hSOD1G93A (13 weeks) mice (n = 4 mice/21 cells). Scale bar: 20 μm. Representative immunofluorescence is shown. e NK1.1+/CD3− cells frequency in the motor cortex (top) and in the spinal cord (bottom) of hSOD1G93A mice treated with Ab-CCL2 (n = 6, *P < 0.05 power 0.916 two-tailed Student’s t-test). Representative density plots are shown on the right. f Scheme of Ab-NK1.1 administration. g Kaplan–Meier curve of hSOD1G93A and TDP43A315T mice treated with vehicle or Ab-NK1.1 (hSOD1G93A, vehicle: 136.8 ± 1.7 days; Ab-NK1.1: 147.2 ± 2.4 days; n = 5. TDP43A315T, vehicle: 78.5 ± 1.5 days; Ab-NK1.1: 88.7 ± 1.9 days, n = 4. Data are the mean ± s.e.m. **P < 0.01, two-sided log-rank test). h Age of paralysis, calculated as the absence of hindlimb movement, upon Ab-NK1.1 treatment in hSOD1G93A mice; vehicle: 18.3 ± 0.2 weeks; Ab-NK1.1: 19.5 ± 0.2 weeks (n = 7, data are the mean ± s.e.m. **P < 0.01 power 0.921 two-tailed Student’s t-test). For boxplots (a, c, e), the center line, boxes and whiskers represent the median, inner quartiles, and rest of the data distribution, respectively.CC BY · PubMed Central
Fig. 3 NK cell–MN communication in ALS. a Expression of Mult-1 in the spinal cord of wt and hSOD1G93A mice (13 weeks) (n = 5). Scale bar: 20 μm. b Colocalization of Smi32 and mult-1 covered area in spinal cord of wt and hSOD1G93A mice (n = 5 data are the mean ± s.e.m. **P < 0.01 power 1 two-tailed Student’s t-test). For boxplots, the center line, boxes and whiskers represent the median, inner quartiles, and rest of the data distribution, respectively. c RT-PCR of mult-1, rae-1, nectin-2 and pvr genes in NeuN+ cells sorted from lumbar spinal cord of wt and hSOD1G93A mice (13 weeks; n = 5, *P < 0.05, **P < 0.01 power >0.85 vs wt mice, one-way ANOVA). d NK cells, isolated from the spleen of wt mice, were incubated with primary cultures of neurons obtained from the spinal cord of wt or hSOD1G93A (13 weeks) mice (left), in the presence of a specific Ab against NKG2D, of dynasore, wiskostatin or vehicle; degranulation of NK cells (having subtracted basal degranulation) in a 2.5:1 ratio with neurons was assessed by FACS analysis of CD107a+ cells (middle panel); cell viability in neuron cultures is shown in the right panel (wt, n = 7. hSOD1G93A, vehicle n = 6, treated with AbNKG2D, dynasore and wiskostatin n = 4, PRF−/− n = 4, **P < 0.01 power 0.999 one-way ANOVA). Error bars show mean ± SEM. e Representative image of NK cell—MN contacts in the spinal cord of hSOD1G93A mice (original magnification, ×600). Scale bar: 20 μm. (hSOD1G93A mice, n = 6) f Quantification of MNs (counted as Smi32+ cells in the ventral horns of the spinal cord) in wt (n = 5) and hSOD1G93A mice treated with vehicle (n = 6) or Ab-NK1.1 (n = 5), and dendrimer or dendrimer-loaded with scrambled siRNA or siRNA-NKG2D41 (n = 4). Data are shown as mean ± s.e.m. **P < 0.01 power >0.9 one-way ANOVA). Representative immunofluorescence images are shown on the right. Scale bar: 20 μm. Source data are provided as a Source Data file.
Fig. 3 NK cell–MN communication in ALS. a Expression of Mult-1 in the spinal cord of wt and hSOD1G93A mice (13 weeks) (n = 5). Scale bar: 20 μm. b Colocalization of Smi32 and mult-1 covered area in spinal cord of wt and hSOD1G93A mice (n = 5 data are the mean ± s.e.m. **P < 0.01 power 1 two-tailed Student’s t-test). For boxplots, the center line, boxes and whiskers represent the median, inner quartiles, and rest of the data distribution, respectively. c RT-PCR of mult-1, rae-1, nectin-2 and pvr genes in NeuN+ cells sorted from lumbar spinal cord of wt and hSOD1G93A mice (13 weeks; n = 5, *P < 0.05, **P < 0.01 power >0.85 vs wt mice, one-way ANOVA). d NK cells, isolated from the spleen of wt mice, were incubated with primary cultures of neurons obtained from the spinal cord of wt or hSOD1G93A (13 weeks) mice (left), in the presence of a specific Ab against NKG2D, of dynasore, wiskostatin or vehicle; degranulation of NK cells (having subtracted basal degranulation) in a 2.5:1 ratio with neurons was assessed by FACS analysis of CD107a+ cells (middle panel); cell viability in neuron cultures is shown in the right panel (wt, n = 7. hSOD1G93A, vehicle n = 6, treated with AbNKG2D, dynasore and wiskostatin n = 4, PRF−/− n = 4, **P < 0.01 power 0.999 one-way ANOVA). Error bars show mean ± SEM. e Representative image of NK cell—MN contacts in the spinal cord of hSOD1G93A mice (original magnification, ×600). Scale bar: 20 μm. (hSOD1G93A mice, n = 6) f Quantification of MNs (counted as Smi32+ cells in the ventral horns of the spinal cord) in wt (n = 5) and hSOD1G93A mice treated with vehicle (n = 6) or Ab-NK1.1 (n = 5), and dendrimer or dendrimer-loaded with scrambled siRNA or siRNA-NKG2D41 (n = 4). Data are shown as mean ± s.e.m. **P < 0.01 power >0.9 one-way ANOVA). Representative immunofluorescence images are shown on the right. Scale bar: 20 μm. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 4 NK cells modulate microglia phenotype in hSOD1G93A mice. a Quantification of soma and scanning domain area, branch numbers and length of Iba1+ cells in slices obtained from the spinal cord of wt and hSOD1G93A mice (13 weeks) treated with vehicle or Ab-NK1.1. Right: representative images (original magnification, ×600; Scale bar: 10 μm) of the maximal intensity projections of confocal z-stack images of Iba1+ cells in the different conditions, converted to binary images and then skeletonized. (n = 3 mice per treatment, 30 cells per mice in at least six slices. **P < 0.01 power 0.921 one-way ANOVA). Error bars show mean ± SEM. b Mean (±s.e.m.) area of Iba1+ cells (expressed as % of spinal cord area) in wt or hSOD1G93A mice (13 weeks) treated with vehicle or Ab-NK1.1 (n = 4, **P < 0.01 power 1 one-way ANOVA). Right: representative immunofluorescence image of Iba1+ cells in spinal cord sections. Scale bar: 50 μm. c–g RT-PCR of il-6, il-1β, tnfα, nos2, nox2, p47phox, chil3, socs3, arg-1, tgfb, msod1, p2yr12, trem2, kcnn4, bdnf, il-15 gene expression in microglial cells isolated from the lumbar spinal cord of wt or hSOD1G93A mice (13 weeks) treated with vehicle or Ab-NK1.1 (n = 5 mice. Data are the mean ± s.e.m. *P < 0.05 **P < 0.01 power >0.85 one-way ANOVA). Source data are provided as a Source Data file.
Fig. 4 NK cells modulate microglia phenotype in hSOD1G93A mice. a Quantification of soma and scanning domain area, branch numbers and length of Iba1+ cells in slices obtained from the spinal cord of wt and hSOD1G93A mice (13 weeks) treated with vehicle or Ab-NK1.1. Right: representative images (original magnification, ×600; Scale bar: 10 μm) of the maximal intensity projections of confocal z-stack images of Iba1+ cells in the different conditions, converted to binary images and then skeletonized. (n = 3 mice per treatment, 30 cells per mice in at least six slices. **P < 0.01 power 0.921 one-way ANOVA). Error bars show mean ± SEM. b Mean (±s.e.m.) area of Iba1+ cells (expressed as % of spinal cord area) in wt or hSOD1G93A mice (13 weeks) treated with vehicle or Ab-NK1.1 (n = 4, **P < 0.01 power 1 one-way ANOVA). Right: representative immunofluorescence image of Iba1+ cells in spinal cord sections. Scale bar: 50 μm. c–g RT-PCR of il-6, il-1β, tnfα, nos2, nox2, p47phox, chil3, socs3, arg-1, tgfb, msod1, p2yr12, trem2, kcnn4, bdnf, il-15 gene expression in microglial cells isolated from the lumbar spinal cord of wt or hSOD1G93A mice (13 weeks) treated with vehicle or Ab-NK1.1 (n = 5 mice. Data are the mean ± s.e.m. *P < 0.05 **P < 0.01 power >0.85 one-way ANOVA). Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 5 IFNγ mediates the effects of NK cells in ALS. a Expression of IFNγ in the spinal cord of wt or hSOD1G93A mice (13 weeks) treated with control IgG or Ab-NK1.1 (n = 5, **P < 0.01 power 1 one-way ANOVA). Error bars show mean ± SEM. b Frequency of IFNγ+/NK1.1+/CD3− cell population isolated from the spinal cord of hSOD1G93A mice (n = 5). Representative FACS analysis is shown on the right. c Scheme of mice treatment with XMG1.2. d Kaplan–Meier curve of hSOD1G93A mice treated with control IgG or XMG1.2 (hSOD1G93A n = 7 vehicle: 138.7 ± 2.5 days; n = 6 XMG1.2: 149.7 ± 2.3 days, data are the mean ± s.e.m. **P < 0.01 two-sided log-rank test). e Age of paralysis, considered as the absence of hindlimb movement, upon XMG1.2 treatment, in hSOD1G93A mice (n = 7 vehicle: 18.6 ± 0.3 weeks; n = 6 XMG1.2: 20.0 ± 0.3 weeks. Data are the mean ± s.e.m. **P < 0.01 power 0.80 one-way ANOVA). RT-PCR analysis of il-6, il-1β, tnfα, nos2 (f); and bdnf, chil3, socs3, arg-1 (g) gene expression in microglial cells isolated from the lumbar spinal cord of wt or hSOD1G93A mice (13 weeks) treated with control IgG or XMG1.2 (n = 5. Data are the mean ± s.e.m. wt vs hSOD1G93A and hSOD1G93A vs hSOD1G93A – XMG1.2. *P < 0.05 **P < 0.01 power >0.9 one-way ANOVA). h RT-PCR of ccl2 expression in NeuN+ cells isolated from the lumbar spinal cord of wt and hSOD1G93A mice (13 weeks) treated with control IgG, Ab-NK1.1 or XMG1.2 (n = 5, **P < 0.01 power 0.859 vs wt mice, one-way ANOVA). i RT-PCR of ccl2 expression in microglia isolated from the lumbar spinal cord of wt and hSOD1G93A mice (13 weeks) treated with control IgG or Ab-NK1.1 (n = 5, **P < 0.01 power 0.859 vs wt mice, one-way ANOVA). Each dot (h, i) represents the qPCR analysis of ccl2 per mice. Source data are provided as a Source Data file.
Fig. 5 IFNγ mediates the effects of NK cells in ALS. a Expression of IFNγ in the spinal cord of wt or hSOD1G93A mice (13 weeks) treated with control IgG or Ab-NK1.1 (n = 5, **P < 0.01 power 1 one-way ANOVA). Error bars show mean ± SEM. b Frequency of IFNγ+/NK1.1+/CD3− cell population isolated from the spinal cord of hSOD1G93A mice (n = 5). Representative FACS analysis is shown on the right. c Scheme of mice treatment with XMG1.2. d Kaplan–Meier curve of hSOD1G93A mice treated with control IgG or XMG1.2 (hSOD1G93A n = 7 vehicle: 138.7 ± 2.5 days; n = 6 XMG1.2: 149.7 ± 2.3 days, data are the mean ± s.e.m. **P < 0.01 two-sided log-rank test). e Age of paralysis, considered as the absence of hindlimb movement, upon XMG1.2 treatment, in hSOD1G93A mice (n = 7 vehicle: 18.6 ± 0.3 weeks; n = 6 XMG1.2: 20.0 ± 0.3 weeks. Data are the mean ± s.e.m. **P < 0.01 power 0.80 one-way ANOVA). RT-PCR analysis of il-6, il-1β, tnfα, nos2 (f); and bdnf, chil3, socs3, arg-1 (g) gene expression in microglial cells isolated from the lumbar spinal cord of wt or hSOD1G93A mice (13 weeks) treated with control IgG or XMG1.2 (n = 5. Data are the mean ± s.e.m. wt vs hSOD1G93A and hSOD1G93A vs hSOD1G93A – XMG1.2. *P < 0.05 **P < 0.01 power >0.9 one-way ANOVA). h RT-PCR of ccl2 expression in NeuN+ cells isolated from the lumbar spinal cord of wt and hSOD1G93A mice (13 weeks) treated with control IgG, Ab-NK1.1 or XMG1.2 (n = 5, **P < 0.01 power 0.859 vs wt mice, one-way ANOVA). i RT-PCR of ccl2 expression in microglia isolated from the lumbar spinal cord of wt and hSOD1G93A mice (13 weeks) treated with control IgG or Ab-NK1.1 (n = 5, **P < 0.01 power 0.859 vs wt mice, one-way ANOVA). Each dot (h, i) represents the qPCR analysis of ccl2 per mice. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 6 NK cells mediate Treg recruitment in the spinal cord of hSOD1G93A mice. a Analysis of Foxp3+ cells in the spinal cord of hSOD1G93A mice (13 weeks) treated with vehicle or Ab-NK1.1, expressed as % of spinal cord area ± s.e.m. (n = 5, **P < 0.01 power 1 two-tailed Student’s t-test). Representative immunofluorescence on the right. Error bars show mean ± SEM. Scale bar: 20 μm. b RT-PCR analysis of foxp3 expression in the lumbar spinal cord of hSOD1G93A mice (13 weeks) treated with control IgG (n = 5), Ab-NK1.1 (n = 6) or XMG1.2 (n = 5), (**P < 0.01 power 921 vs control, one-way ANOVA). Source data are provided as a Source Data file.
Fig. 6 NK cells mediate Treg recruitment in the spinal cord of hSOD1G93A mice. a Analysis of Foxp3+ cells in the spinal cord of hSOD1G93A mice (13 weeks) treated with vehicle or Ab-NK1.1, expressed as % of spinal cord area ± s.e.m. (n = 5, **P < 0.01 power 1 two-tailed Student’s t-test). Representative immunofluorescence on the right. Error bars show mean ± SEM. Scale bar: 20 μm. b RT-PCR analysis of foxp3 expression in the lumbar spinal cord of hSOD1G93A mice (13 weeks) treated with control IgG (n = 5), Ab-NK1.1 (n = 6) or XMG1.2 (n = 5), (**P < 0.01 power 921 vs control, one-way ANOVA). Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 7 NK cell analyses in sALS patients. a Expression of ULBP-3 in the spinal cord and cerebral motor cortex of control or sALS patients, showing co-staining with Smi32+ cells (n = 8 controls, 12 sALS patients). Scale bar: 20 μm. b Colocalization of Smi32 and ULBP-3 covered area in the spinal cord and cerebral motor cortex of control or sALS patients (n = 8 controls, 12 sALS patients **P < 0.01 power 1 two-tailed Student’s t-test). c Representative image of NK cell–MN contacts in the spinal cord of ALS patient (original magnification, x600). Scale bar: 10 μm. (n = 8 ALS patients). For boxplots, the center line, boxes and whiskers represent the median, inner quartiles, and rest of the data distribution, respectively. d Distances of NKp46+ cells from vessel endothelial cells (CD31+ cells) in the spinal cord of ALS patient (n = 12 sALS patients). Scale bar: 20 μm. Representative immunofluorescence is shown. e Expression of IFNγ in the spinal cord of control and sALS patients (n = 3, **P < 0.01 power 0.871 two-tailed Student’s t-test). Error bars show mean ± SEM. f Frequency of IFNγ+ cells in the CD56+/CD3− cell populations (NK cells) isolated from the peripheral blood of control or sALS patients (n = 6 controls, 9 sALS patients. *P < 0.05 two-tailed Student’s t-test). Note that when the highest value among sALS patients was excluded, the difference among the two groups remains significant (p < 0.034 power 0.716). g NK cells, isolated from the peripheral blood of control or sALS patients, were incubated with human K562 cells. Degranulation was assessed by FACS analysis of CD107+ cells (%, minus degranulation in the absence of targets) (n = 14 controls, 11 sALS patients). h, i Frequency of GZMb+ (h) and PRF+ (i) cells in the CD56+/CD3- cell populations (NK cells) isolated from the peripheral blood of control or sALS patients (n = 8 controls, 15 sALS patients). Source data are provided as a Source Data file.
Fig. 7 NK cell analyses in sALS patients. a Expression of ULBP-3 in the spinal cord and cerebral motor cortex of control or sALS patients, showing co-staining with Smi32+ cells (n = 8 controls, 12 sALS patients). Scale bar: 20 μm. b Colocalization of Smi32 and ULBP-3 covered area in the spinal cord and cerebral motor cortex of control or sALS patients (n = 8 controls, 12 sALS patients **P < 0.01 power 1 two-tailed Student’s t-test). c Representative image of NK cell–MN contacts in the spinal cord of ALS patient (original magnification, x600). Scale bar: 10 μm. (n = 8 ALS patients). For boxplots, the center line, boxes and whiskers represent the median, inner quartiles, and rest of the data distribution, respectively. d Distances of NKp46+ cells from vessel endothelial cells (CD31+ cells) in the spinal cord of ALS patient (n = 12 sALS patients). Scale bar: 20 μm. Representative immunofluorescence is shown. e Expression of IFNγ in the spinal cord of control and sALS patients (n = 3, **P < 0.01 power 0.871 two-tailed Student’s t-test). Error bars show mean ± SEM. f Frequency of IFNγ+ cells in the CD56+/CD3− cell populations (NK cells) isolated from the peripheral blood of control or sALS patients (n = 6 controls, 9 sALS patients. *P < 0.05 two-tailed Student’s t-test). Note that when the highest value among sALS patients was excluded, the difference among the two groups remains significant (p < 0.034 power 0.716). g NK cells, isolated from the peripheral blood of control or sALS patients, were incubated with human K562 cells. Degranulation was assessed by FACS analysis of CD107+ cells (%, minus degranulation in the absence of targets) (n = 14 controls, 11 sALS patients). h, i Frequency of GZMb+ (h) and PRF+ (i) cells in the CD56+/CD3- cell populations (NK cells) isolated from the peripheral blood of control or sALS patients (n = 8 controls, 15 sALS patients). Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 8 Graphic summary. (Left) NK cells’ effects in the spinal cord of hSOD1G93A mice at the early symptomatic stage. NK cells directly exert cytotoxic activity against MNs through the NKG2D-Mult-1 axis. Furthermore, IFNγ released by NK cells: (i) increases the release of CCL2 by damaged neurons; (ii) reduces the number of Treg Foxp3+ cells; (iii) shapes microglia toward a pro-inflammatory phenotype. (Right) NK cell depletion, in hSOD1G93A mice, induces neuroprotective microglia phenotype, increases the number of Treg Foxp3+ cells, protecting the MNs.
Fig. 8 Graphic summary. (Left) NK cells’ effects in the spinal cord of hSOD1G93A mice at the early symptomatic stage. NK cells directly exert cytotoxic activity against MNs through the NKG2D-Mult-1 axis. Furthermore, IFNγ released by NK cells: (i) increases the release of CCL2 by damaged neurons; (ii) reduces the number of Treg Foxp3+ cells; (iii) shapes microglia toward a pro-inflammatory phenotype. (Right) NK cell depletion, in hSOD1G93A mice, induces neuroprotective microglia phenotype, increases the number of Treg Foxp3+ cells, protecting the MNs.CC BY · PubMed Central

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  1. Ludolph, A. Amyotrophic lateral sclerosis & frontotemporal degeneration. A Revis. El Escorial criteria 16, 291–292 (2015).

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Authors
Garofalo, S., Cocozza, G., Porzia, A., Inghilleri, M., Raspa, M., Scavizzi, F., Aronica, E., Bernardini, G., Peng, L., Ransohoff, R. M., Santoni, A., & Limatola, C.
Affiliations
  • Sapienza University of Rome (IT)
  • Istituto Neurologico Mediterraneo (IT)
  • Institute of Cell Biology and Neurobiology (IT)
  • Netherlands Institute for Neuroscience (NL)
  • Istituto Pasteur (IT)
  • Centre National de la Recherche Scientifique (FR)
Corresponding author
  • Garofalo, Stefano
  • Limatola, Cristina
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2020-04-14
Volume
11
Issue
1
Pages
1773-1773
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-020-15644-8
PMC
PMC7156729
OpenAlex
W3015391013
Cited by
170
References
50
Citations by year
2026: 24 · 2025: 25 · 2024: 30 · 2023: 36 · 2022: 25 · 2021: 19 · 2020: 11
FWCI
9.85
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journal article
Language
en
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  • Pure Amsterdam UMC (publishedVersion, cc-by)
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  • Data Archiving and Networked Services (DANS) (submittedVersion, other-oa)
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  • HAL (submittedVersion, other-oa)
  • RePEc: Research Papers in Economics (submittedVersion)
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Funders
  • Agence Nationale de la Recherche (ANR-16-ENM2-0004)
  • Ministero dell’Istruzione, dell’Università e della Ricerca (PRIN 2015, H2020)
  • Associazione Italiana per la Ricerca sul Cancro (PRIN 2015)
  • Fondazione Italiana per la Ricerca sul Cancro (22329)
  • Horizon 2020 Framework Programme
Topics
  • Health Sciences › Medicine › Clinical Neurology › Amyotrophic Lateral Sclerosis Research
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Cell Function and Interaction
Keywords
amyotrophic lateral sclerosis, natural killer cells, motor neuron degeneration, NKG2D ligands, MCP-1, microglial activation, interferon-gamma, regulatory T cells, neuroinflammation, motor cortex, spinal cord, neurotoxicity
MeSH terms
Adult, Aged, Amyotrophic Lateral Sclerosis, Animals, Disease Models, Animal, Female, Humans, Killer Cells, Natural, Male, Middle Aged, Motor Cortex, Motor Neurons, Spinal Cord, Disease Progression, Mice, Natural Cytotoxicity Triggering Receptor 1

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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