Journal: Cell

Article Title: Mild respiratory COVID can cause multi-lineage neural cell and myelin dysregulation

doi: 10.1016/j.cell.2022.06.008

Figure Lengend Snippet: 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 .

Article Snippet: CD1 mice were weighed and injected intraperitoneally with recombinant mouse CCL11 (10μg/kg in PBS, R&D systems 420-ME) or PBS vehicle control four times over a ten-day period at D1, D4, D7, and D10, and then perfused 24 hours after the final injection on D11, as described in .

Techniques: Infection, Control, Fluorescence, Two Tailed Test

Journal: Cell

Article Title: Mild respiratory COVID can cause multi-lineage neural cell and myelin dysregulation

doi: 10.1016/j.cell.2022.06.008

Figure Lengend Snippet: 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.

Article Snippet: CD1 mice were weighed and injected intraperitoneally with recombinant mouse CCL11 (10μg/kg in PBS, R&D systems 420-ME) or PBS vehicle control four times over a ten-day period at D1, D4, D7, and D10, and then perfused 24 hours after the final injection on D11, as described in .

Techniques: Expressing, Infection, Control, Clinical Proteomics, Two Tailed Test

Journal: Cell

Article Title: Mild respiratory COVID can cause multi-lineage neural cell and myelin dysregulation

doi: 10.1016/j.cell.2022.06.008

Figure Lengend Snippet: Total microglia counts, related to Figures 2 , , , and (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.

Article Snippet: CD1 mice were weighed and injected intraperitoneally with recombinant mouse CCL11 (10μg/kg in PBS, R&D systems 420-ME) or PBS vehicle control four times over a ten-day period at D1, D4, D7, and D10, and then perfused 24 hours after the final injection on D11, as described in .

Techniques: Injection, Two Tailed Test

Journal: Cell

Article Title: Mild respiratory COVID can cause multi-lineage neural cell and myelin dysregulation

doi: 10.1016/j.cell.2022.06.008

Figure Lengend Snippet: 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 and .

Article Snippet: CD1 mice were weighed and injected intraperitoneally with recombinant mouse CCL11 (10μg/kg in PBS, R&D systems 420-ME) or PBS vehicle control four times over a ten-day period at D1, D4, D7, and D10, and then perfused 24 hours after the final injection on D11, as described in .

Techniques: Clinical Proteomics, Injection, Control, Two Tailed Test

Journal: Cell

Article Title: Mild respiratory COVID can cause multi-lineage neural cell and myelin dysregulation

doi: 10.1016/j.cell.2022.06.008

Figure Lengend Snippet:

Article Snippet: CD1 mice were weighed and injected intraperitoneally with recombinant mouse CCL11 (10μg/kg in PBS, R&D systems 420-ME) or PBS vehicle control four times over a ten-day period at D1, D4, D7, and D10, and then perfused 24 hours after the final injection on D11, as described in .

Techniques: Virus, Recombinant, Electron Microscopy, Plasmid Preparation, Sequencing, Software

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: Characterization of CAFs and NFs obtained from clinical surgical tissues from patients with head and neck cancer (HNC) ( a ) Morphological comparisons between CAFs and NFs from a representative HNC case showed that CAFs (right panel) consisted of more cytoplasmic protrusions than NFs (left panel). Photographs were captured at 40× magnification. ( b ) Quantitative PCR (left panel) of the culture medium showed a significantly higher expression of vimentin and α-SMA in CAFs than in NFs. Western blot analysis (right panel) also demonstrated that levels of vimentin and α-SMA were significantly higher in CAFs than in NFs. ( c ) Flow cytometric analysis of the cell surface markers, CD10 and GPR77, showed a marked increase in their expression in CAFs compared to NFs. ( d ) A heat map of the gene microarray of NFs and CAFs showed that there were several differences in the expression profile of the secreted genes. The arrow indicates a marked discrepancy of the relative mRNA levels of CCL11 in CAF compared with NF. ( e ) The RT-PCR (left panel) and ELISA (right panel) analysis showed an increased expression of CCL11 in CAFs compared with that in NFs. ( f ) Western blot analysis showed that the protein level of CCL11 was higher in CAFs than in NFs in cell lysates. ( g ) Western blot analysis showed a higher CCL11 expression in CAF-CM compared to NF-CM. The asterisk indicated a significant difference (*: p < 0.05; **: p < 0.01) between experimental and control groups.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Microarray, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Control

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: CCL11 produced by CAFs causes increased migration and invasion, and the EMT of HNC cells. ( a ) Comparative analysis of the migration and invasion of HNC cells associated with CCL11. Four test groups were classified for comparative analysis of migration and invasive abilities. FaDu and NPC204 cells cultured with medium containing CAF-induced CCL11 presented greater abilities of migration and invasion, with a statistically significant difference, than three other groups: NF, NF with CCL11, and CAFs treated with CCL11 antibody. The asterisk indicates a significant difference (*: p < 0.05; **: p < 0.01) ( b ) Comparative photographs of the infiltrating behavior of FaDu and NPC204 cells in an organotypic culture in four groups seeded onto a mixture layer containing NFs or CAFs with CCL11 or CCL11 antibody. The arrow(s) indicate infiltration buds from the HNC cells seeded above. ( c ) Representative blots of the EMT-associated markers in FaDu and NPC204 cells, as observed upon Western blotting analysis in five groups, showed that treatment with CAF-conditioned medium or the application of rCCL11 decreased the expression of epithelial-type markers (E-cadherin), and increased the expression of mesenchymal-type markers (fibronectin) and EMT regulators (Snail and Twist). In addition, increased expression of invasion-related MMP2 and MMP9 was also seen in those two groups, compared with other groups. The asterisk indicates a significant difference ( p < 0.05) between experimental and control groups. Results are expressed as mean ± SD.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Produced, Migration, Cell Culture, Western Blot, Expressing, Control

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: Comparative analysis of induction of CSC properties and drug resistance in HNC cells associated with CCL11. ( a ) Four groups were classified for comparative analysis of the ability of sphere formation. Increased ability of sphere formation in two test groups of HNC cells exposed to a CAF medium and the group with treatment of rCCL11 was noted. ( b ) Flow cytometric analysis showed a significant increase in CD44 and CD44/CD24, as well as in CD133 in HNC cells exposed to rCCL11, compared to control HNC cells ( p < 0.05). ( c ) Flow cytometric analysis showed a marked increase in ALDH-1 activity in HNC cells exposed to rCCL11 compared to control HNC cells. ( d ) Western blot analysis showed that CSC-representative markers, Oct-4, Nanog, and Sox-2, were also overexpressed in addition to the increased expression of two important drug resistance genes, ABCG-2 and MDR-1 , in HNC cells exposed to rCCL11. ( e ) Treatment with Cisplatin at 24 h showed a significant increase in chemoresistance in both FaDu and NPC204 cells exposed to rCCL11 compared with control HNC cells. The asterisk indicates a significant difference (*: p < 0.05; **: p < 0.01) between experimental and control groups.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Control, Activity Assay, Western Blot, Expressing

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: CCL11 and CCR3 expression with associated signal pathway in HNC cell lines and their correlation to clinical outcomes in 104 HNC patients. ( a ) Confocal microscopic images showed CCL11 (green) localized to both cell and nuclear membranes, while CCR3 (red) localized only to the cell membrane in FaDu cells; CCL11 and CCR3 co-localized at the cell membrane (yellow). In NPC204 cells, CCL11 (green) and CCR3 (red) were found to co-localize at protrusions polarized to the cells (yellow). ( b ) Using the crisp technique, higher expression of CCR3, MMP2, and MMP3 was found in over-expressed CCL11 cloned-FaDu and NPC204 cells. Cloned CCL11-overexpressed cells were abolished by adding eotaxin siRNA or CCR3 antibody, which reversed the expression of CCR3 and invasion-related MMP2 and MMP9. ( c ) Higher phosphorylation levels of p38 MAPK and ERK were found in cloned CCL11-overexpressed FaDu and NPC204 cells and were reversed by treatment of the p38 MAPK inhibitor (SB203580) and ERK inhibitor (FR180204), respectively. The phosphorylation level of JNK was kept in low condition before and after treatment of the JNK inhibitor (SP600125). ( d ) Photomicrographs of immunohistochemical staining from tissue microarray showing CCL11 and CCR3 expression in three different representative groups of HNC patients (magnification, ×200). ( e ) Kaplan–Meier survival analysis of patients showed that overexpression of CCL11 and CCR3 were statistically associated with poor overall survival).

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Expressing, Membrane, Clone Assay, Phospho-proteomics, Immunohistochemical staining, Staining, Microarray, Over Expression

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: The diagrammatic illustration demonstrates the major mechanism that CAFs secreting CCL11 promotes HNC cell migration and invasion and induces properties of drug resistance and stemness, shown as follows. CAFs in TME secret CCL11 binding to the CCR3 receptors on HNC cells via the paracrine effect. The signal induces overexpression of transcriptional factors, such as Snail and Twist, which regulate EMT and are also responsible for self-induction of CCL11 in an autocrine fashion. As a result, CCL11, via paracrine or autocrine signaling when targeting CCR3 receptors, play a functional role in the induction of EMT and CSC properties, for further tumor progression.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Migration, Binding Assay, Over Expression, Functional Assay

Journal: The Journal of Experimental Medicine

Article Title: ICOS signaling limits regulatory T cell accumulation and function in visceral adipose tissue

doi: 10.1084/jem.20201142

Figure Lengend Snippet: Increased VAT-T R accumulation in the absence of ICOS signaling is associated with elevated CCR3 expression. (A) ST2 expression in splenic T R s ( n = 3–5 per group from eight independent experiments). (B) ST2 expression in CD45.1 + and CD45.2 + donor splenic T R s in WT:YF and WT:KO chimeric mice. Lines connect CD45.1 + and CD45.2 + donor splenic T R s within the same chimera ( n = 3–5 chimeric mice per group from three independent experiments). (C) Expression of CCR3 in VAT-T R s in mice ≤8 wk and >8 wk of age ( n = 3–5 per group from seven independent experiments). (D) Expression of CCR2 and CCR3 by VAT-T R s ( n = 3–5 per group from two independent experiments). DP, CCR2 + CCR3 + ; DN, CCR2 − CCR3 − . (E) Expression of indicated CCR3 ligands in total VAT normalized to Tbp as measured by qPCR (n.d. indicates not detected; n = 5 per group). (F) Schematic of in vitro culture experiments examining the impact of ICOS signaling on CCR3 expression (left). Graphs indicating fold change in T R frequency of CD4 + cells and %CCR3 + of T R s between individual culture samples stimulated (stim) with or without αICOS for 2 d (middle). Representative flow cytometry plots with frequency of CCR3 + T R s after 2 d in specified culture conditions (right; n = 1–3 per group from three independent experiments). (G) Left: Representative flow cytometry plots indicating VAT-T R frequency with or without CCL11/24 blockade. Graphs summarize T R frequencies in indicated tissues after 2 wk ( n = 3–4 per group). Mice were age matched within independent experiments and collectively; pooled data are from experiments using male mice aged 8–16 wk unless otherwise indicated. Statistical significance was determined using one-way ANOVA with Tukey’s post-test (A and C); two-tailed, paired Student’s t test for expression in donor cells within the same chimeric mouse (B); and two-tailed Student’s t test (F and G). All data are presented as mean values ± SD.

Article Snippet: Mice aged 8–10 wk were given 0.75 µg/g body weight αCCL11 and αCCL24 (MAB420 and MAB528; R&D Systems) or an equivalent amount of rat IgG (Sigma) diluted in PBS by i.p. injection on days 0, 5, and 10 and sacrificed for analysis on day 13.

Techniques: Expressing, In Vitro, Flow Cytometry, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: ICOS signaling limits regulatory T cell accumulation and function in visceral adipose tissue

doi: 10.1084/jem.20201142

Figure Lengend Snippet: Increased accumulation of CCR3 + T R s in the absence of ICOS signaling is specific to VAT (goes with ). (A) CCR2 expression by splenic (top) and VAT-T R s (bottom) as measured by flow cytometry ( n = 3–5 per group from two independent experiments). (B) Expression of CCR3 in splenic T R s in mice ≤8 wk and >8 wk of age ( n = 3–5 per group from seven independent experiments). (C) Expression of CCR3 by gated CD45.1 + and CD45.2 + donor VAT-T R s in chimeric mice. Graphs summarize CCR3 expression by donor T R s in VAT and spleen. Line connects point representing CD45.1 + and CD45.2 + cells within the same chimeric mouse ( n = 2–4 per group from two independent experiments). (D) CCR3 expression by tissue-localized skin T R s as measured by flow cytometry ( n = 2–4 per group from two independent experiments). (E) CCR3 expression by CD45.1 + and CD45.2 + donor T R s within the same chimeric mouse in indicated tissues. Line connects CD45.1 + and CD45.2 + cells within the same chimeric mouse ( n = 2–4 per group from two independent experiments). (F) Frequency of tissue-restricted VAT eosinophils with and without in vivo CCL11/24 blockade as measured by flow cytometry ( n = 3 or 4 mice per group). Mice were age matched within individual experiments, and pooled data are from experiments using 8–16-wk-old male mice. Statistical significance was determined using one-way ANOVA with Tukey’s post-test (A, B, and D); two-tailed, paired Student’s t test for expression in donor cells within the same chimeric mouse (C and E); and two-tailed Student’s t test (F). All data are presented as mean values ± SD. LPL, lamina propria lymphocyte.

Article Snippet: Mice aged 8–10 wk were given 0.75 µg/g body weight αCCL11 and αCCL24 (MAB420 and MAB528; R&D Systems) or an equivalent amount of rat IgG (Sigma) diluted in PBS by i.p. injection on days 0, 5, and 10 and sacrificed for analysis on day 13.

Techniques: Expressing, Flow Cytometry, In Vivo, Two Tailed Test