Fig 1: Elevated CCL11 levels associated with cognitive impairment induce hippocampal dysregulation(A) Plasma levels of CCL11 in people experiencing long COVID with (n = 48 human subjects, brain fog [+]) and without (n = 15 human subjects, brain fog [−]) cognitive symptoms.(B) History of autoimmune disease in patients reporting “brain fog” (n = 37 human subjects without history of autoimmune disease, n = 11 human subjects with history of autoimmune disease).(C) Timeline of CCL11 challenge in CD1 strain mice. Brains were collected 24 h after last injection (day 11 post-injection, 11DPI). Created with biorender.com.(D and E) Reactive microglia (IBA1+ CD68+) quantification 11 days post-CCL11 injection in the cortical gray matter (D) and cingulum of the corpus callosum (E) of mice (n = 9 control, n = 10 CCL11-treated mice).(F) Reactive microglia (IBA1+ CD68+) quantification 11 days post-CCL11 injection in the dentate gyrus of mice (n = 9 control, n = 10 CCL11-treated mice).(G) Representative confocal micrographs of activated microglia (IBA1, white; CD68, magenta) in the dentate gyrus of mice 11 days post-CCL11 injection.(H) Neuroblast (DCX+) quantification 11 days post-injection in the dentate gyrus of mice (n = 9 mice/group).(I) Representative confocal micrographs of neuroblasts (DCX, magenta; DAPI, cyan) in the dentate gyrus of mice 11 days post-CCL11 injection.Data shown as mean ± SEM. (A, B, D–F, and H) unpaired two-tailed t test. ns, p > 0.05. p values shown in figure panels. Each dot represents one mouse or human subject. Scale bars, 50 μm. DG, dentate gyrus.See also Figures S2 and S6 and Data S1.
Fig 2: Mild respiratory COVID mouse model exhibits CSF cytokine/chemokine elevations(A) Schematic of experimental paradigm for respiratory system-restricted SARS-CoV-2 infection in mice and experimental workflow. Created with biorender.com.(B) Body weight (% of day 0 weight) of control and mild COVID mice. Data shown as mean ± SEM; n = 24 mice per group; ns, p > 0.05 by two-way ANOVA with multiple comparisons.(C) Confocal micrograph of SARS-CoV-2 nucleocapsid protein (SARS-CoV-2-N) 7 days post-infection (7DPI). SARS-CoV-2-N, magenta; DAPI, cyan. Scale bars, 1 mm.(D and E) Cytokine analyses of serum in control and mild COVID mice 7 days post-infection (D) and 7 weeks post-infection (7WPI) (E). Data shown as fold change (FC) median fluorescence intensity compared with control group; n = 5–7 (CD1 strain) mice per group.See Table S1 for individual statistics.(F and G) Cytokine analyses of CSF in control and mild COVID mice 7 days post-infection (F) and 7 weeks post-infection (G). Data shown as fold change (FC) median fluorescence intensity compared with control group; n = 6–7 mice (CD1 strain) per group.See Table S1 for individual statistics.(H and I) CSF levels of CCL7 from mice 7 days post-infection (H) and 7 weeks post-infection (I). n = 7 mice per group.(J and K) CCL11 levels in CSF of mice 7 days post-infection (J) and 7 weeks post-infection (K). n = 6 mice per control group and n = 7 mice per mild COVID group in (J). n = 7 mice per group in (K).Data shown as mean ± SEM; each dot represents an individual mouse; p values shown in figure panels; ns, p > 0.05; two-tailed unpaired t test.See also Figure S1, Table S1, and Data S1.
Fig 3: Microglial reactivity, CCL11 levels, and microglial CCL11 receptor expression, related to Figure 5(A) Correlation between neuroblasts (DCX+) and activated microglia (IBA1+ CD68+) in the dentate gyrus of CD1 strain mice 7 days post-infection (7DPI). Line fitted with simple linear regression (n = 5 mice per control group; n = 4 mice per mild COVID group), related to Figure 4.(B) Plasma levels of people experiencing long COVID with “brain fog” broken down by sex (n = 16 subjects per male group; n = 32 subjects per female group), related to Figure 5.(C and D) Serum levels of CCL11 from CD1 strain mice 7 days post-infection and 7 weeks post-infection (7WPI) (D) n = 7 mice per group, related to Figure 1.(E–G) CCR2, CCR3, and CCR5 transcriptional expression in mouse neural cell types (E), human neural cells (F), and mouse reactive microglia and astrocytes after lipopolysaccharide (LPS) stimulation (G). (E–G) Data are expressed as row-scaled FPKM. Rows are centered; unit variance scaling is applied to rows. Columns are clustered using correlation distance and average linkage.Data in (B–D) shown as mean ± SEM; each dot represents an individual mouse or human subject; p values shown in figure panels; ns p > 0.05; two-tailed unpaired t test.
Fig 4: Total microglia counts, related to Figures 2, 4, 5, and 7(A–S) Total microglial counts (IBA1+ cells), assessed at 7-days or 7-weeks following mild respiratory COVID in cortex or subcortical white matter of BALB/c (A–D) and CD1 (E–H) mouse strains and in hippocampal white matter of the dentate gyrus (DG) of CD1 mice (I and J), or assessed in cortex, subcortical white matter and hippocampal white matter at the end of a CCL11 systemic administration paradigm (11 days post-injection [DPI], K–M), or assessed in cortex, subcortical white matter and hippocampal white matter following (N–S).Data shown as mean ± SEM; each dot represents an individual mouse; p values shown in figure panels; ns p > 0.05; two-tailed unpaired t test.
Fig 5: CCL11 is a SASP factor driving remyelination impairment.a Immunofluorescent staining of p16tdt+ cells (RFP) and CCL11 in 12 months old demyelinated lesions. Arrows indicate p16tdt + CCL11+ colocalization (n = 4 mice). b Immunofluorescent staining of microglia (IBA1) and CCL11 in young (3 months) and aged (18–22 months) demyelinated lesions. c Quantification of CCL11 in b (n = 2 per group). d Protein levels of CCL11 in young, aged vehicle treated, and aged AP treated lesions at 20 dpl (n = 3 per group; each n denotes biologically independent replicate). e CCL11 plasma concentrations in healthy control, Relapse Remitting MS, and Primary Progressive MS samples (n = 15 C, 19 RRMS, 8 PPMS, uncorrected for age; each n denotes biologically independent replicate). f Experimental schematic of IgG vehicle (50 µg/kg), CCL11 neutralizing antibody (CCL11na) (50 µg/kg), or recombinant CCL11 (rCCL11) (10 µg/kg) treatment of young lesions. g, h Immunofluorescent staining of mature oligodendrocytes with CC1 and OLIG2 and myelin with MBP in vehicle, CCL11na, and rCCL11 treated lesions at 20 dpl. i, j Quantification of MBP+ area and CC1 + OLIG2+ cells in (g, h) (n = 5 V, 4 CCL11na, 3 rCCL11; each n denotes biologically independent replicate). k Schematic of in vitro experiment timeline. l Immunofluorescent staining of primary rat oligodendrocytes treated with vehicle or recombinant CCL11 (100 ng/µL dosed at DIV2, DIV4) for MBP and alpha/beta tubulin. Red arrows denote low MBP cells. m Quantification of DIV5 oligodendrocytes demonstrate higher percentage of CCL11-treated cells have low MBP expression (20.7 ± 2.2%) compared to control cells (10.5 ± 2.5%) n, Quantification of DIV5 oligodendrocytes demonstrate lower percentage of CCL11-treated cells have high MBP expression (79.3 ± 2.2%) compared to control cells (89.5 ± 2.5%) (for m, n: n = 30 FOVs for control group and n = 63 FOVs for rCCL11-treated group across cultures from 2 biological replicates, 3 experiments, 1–2 coverslips per condition per experiment. Data points refer to fields of view). Images represent half field of view. Data are mean ± SEM (c–e, i, j, m, n). Scale bar, 50 µm (a, b, g, h), 20 µm (i), 10 µm (a, b insets). P-values derived from two-sided Kolmogorov-Smirnov t-test (m, n), one-way ANOVA with Tukey corrected multiple comparisons (d, i, j), or one-way ANOVA with FDR corrected multiple comparisons (e). Select figures created in BioRender (2025) (f) https://BioRender.com/x25z952. Source data are provided as a Source Data file.
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