We need both, do you have ANY CONFIDENCE AT ALL that your stroke medical 'professionals' will get human testing going?
Do you prefer your doctor, hospital and board of director's incompetence NOT KNOWING? OR NOT DOING? Your choice; let them be incompetent or demand action!
Targeting immune cells in the aged brain reveals that engineered cytokine IL-10 enhances neurogenesis and improves cognition
Under a Creative Commons license
Open access
Introduction
Brain function deteriorates with age. Additionally, age-related neurodegenerative diseases (e.g., Alzheimer’s disease and Parkinson’s disease) surge in the elderly population. While many pathways are remodeled in the brain during aging,1,2,3,4 a key feature of old brains is immune cell infiltration and inflammation. Aging leads to a pronounced increase in the number of T cells across several brain regions in mice and humans,5,6,7 including neural stem cell (NSC) niches8,9,10,11 and white matter tracts.12,13,14,15,16 T cells from old brains are clonally expanded,8,17 suggesting they have encountered antigens. T cell infiltration and clonal expansion are further accentuated in age-related neurodegenerative diseases.18,19,20,21,22,23,24,25,26 In parallel, pro-inflammatory pathways are strongly upregulated during aging in many cell types, and this is exacerbated in Alzheimer’s disease.8,13,27,28,29,30,31,32,33,34,35,36 A systematic understanding of age-related changes in brain immune cells may inform strategies to mitigate brain aging.
Could immune-based interventions be designed to counter brain aging? Modulating immune cells and inflammatory pathways can affect the old brain.12,13,28,30,37,38,39 But previous studies have relied on broad interventions, often performed in the periphery13 and in young or disease model mice.18,24,40,41,42,43,44,45 We lack strategies to target specific immune cell subsets and pathways within the old brain to determine their mode of action. Moreover, identifying such brain immune interventions could help develop “aging immunotherapies” and would be critical to counter aspects of brain aging and age-related brain diseases.
Here, we developed a platform that leverages engineered proteins and direct brain delivery to test immune cell-specific interventions in old mice. We identified T cells with an exhaustion signature and targeted them with a potent engineered checkpoint inhibitor, leading to T cell expansion and strong pro-inflammatory responses in many cell types, including microglia. To rescue age-related inflammatory balance in microglia, we used an engineered interleukin (IL)-10 variant that uncouples pro- and anti-inflammatory responses and found that it had beneficial effects on multiple cell types and cognition in aged mice. Our findings pave the way for novel immunotherapies for the aged brain.
Results
Characterization of immune cells in the aged brain
We systematically characterized immune cells in the aging brain, focusing on the subventricular zone (SVZ) neurogenic niche—a region that contains adult NSCs and declines during aging.46,47,48,49 Leveraging previously published single-cell RNA-sequencing (scRNA-seq) datasets,8,50 we found that SVZ neurogenic niches contained microglia (the brain-resident myeloid population), macrophages, CD8+ T cells, CD4+ T cells, and low numbers of other immune cells (Figures 1A and S1A). CD8+ T cells were not very numerous but significantly increased in number with age (Figure 1A, upper and middle panel). Microglia were the most abundant immune cell type and showed the largest age-related changes in gene expression (Figure 1A, lower panel). We thus focused on CD8+ T cells and microglia for further characterization and targeting in the old brain.
Figure 1. Characterization of immune cells in the old brain
(A) Analysis of immune cell populations in the SVZ neurogenic niche of young (3–4 months, n = 7) and old (22–29 months, n = 6) male mice in two merged scRNA-seq published datasets.8,50 Top panel, quantification of the proportion of different immune cell types relative to the total number of cells. Data are mean ± SEM; each dot represents one mouse. Middle panel, age-dependent changes (log2[old/young]) in the abundance of each cell type. Bottom panel, age-dependent changes in gene expression per cell type. Dots represent differential expression MAST Z score for each gene. Genes significantly changed with age (Bonferroni-corrected p < 0.05) are in color.
(B) scRNA-seq analysis of CD8+ T cells freshly isolated from the brain and lung of old male mice (22–25 months, n = 11 mice, pooled). Uniform manifold approximation and projection (UMAP) clustering of all T cells colored by organ of origin, downsampled to 3,724 cells per organ. One independent experiment.
(C) UMAP as in (B) colored by cell type.
(D) Changes in the percentage of T cell subtypes in old brains vs. old lungs.
(E) Heatmap of top six marker genes for each T cell subtype in the old brain.
(F) FACS quantification of percent PD1+ and CD44+CD69+ of CD8+ T cells freshly isolated from the brains and lungs of old male mice (24 months, n = 6). One independent experiment.
(G) Heatmap of T cell marker genes in T cells from same dataset as (A).
(H) Left, representative images showing exhaustion scores for T cells in coronal brain sections of young (6 months) and old (28 months) male mice in a spatial transcriptomics (MERFISH) dataset.7 Right, quantification of exhaustion scores in T cells from young (3–6 months, n = 5) and old (26–34 months, n = 6) male mice across four brain regions, cortex (CTX), striatum and adjacent regions (STR), white matter tracts of the corpus callosum and anterior commissure (CC/ACO), and the ventricle (VEN). One coronal section per mouse. Boxplots of median and lower and upper quartile values.
(I) Left, representative immunofluorescence images of SVZ from young (4 months) and old (28 months) male mice. Red, CD8+ (T cells); green, PD1+ (checkpoint protein); blue, DAPI (nuclei). Scale bar, 10 μm. Right, number of CD8+ and CD8+ PD1+ T cells in the SVZ of young (3–4 months, n = 7) and old (28–32 months, n = 7) male mice. Each dot represents one mouse (average of two sections per mouse). One independent experiment.
(J) FACS quantification of the percentage of CD8+PD1+ and CD8+CD44+CD69+ T cells of live/CD45+ cells freshly isolated from the brain of young (6 months, n = 6) and old (24 months, n = 6) male mice. One independent experiment.
(K) Average changes in gene expression in microglia of the SVZ neurogenic niche of young and old male mice from three published scRNA-seq datasets.8,50,51 Red, upregulated; blue, downregulated genes.
(L) Heatmap of log-normalized counts for pro- and anti-inflammatory genes in microglia from the SVZ in a published dataset.8
(M) Log-normalized expression values of Socs3 (left) and combined genes in the IL-10 pathway in microglia from the SVZ in a published dataset.8 Horizontal lines, median.
(N) IL-10 signaling scores for microglia in spatial transcriptomics dataset.7
(O and P) FACS histograms and quantification of inflammation proteins in microglia (CD45+CD11b+) freshly isolated from the SVZ of young (3 months) and old (24 months) male mice (n = 6). Each dot represents one mouse (mean fluorescence intensity [MFI] values from ∼500 microglia per mouse). One independent experiment.
Data are mean ± SEM. p values, two-sided Wilcoxon rank-sum test. NS, not significant.
See also Figure S1.
More at link.
No comments:
Post a Comment