Journal: The European Respiratory Journal

Article Title: GPR183 antagonism reduces macrophage infiltration in influenza and SARS-CoV-2 infection

doi: 10.1183/13993003.01306-2022

Figure Lengend Snippet: IAV infection leads to upregulation of CH25H and CYP7B1 expression in the lung and production of the oxysterols 25-OHC and 7α,25-OHC. a) The biosynthetic pathway of 25-OHC and 7α,25-OHC. b) Experimental design; C57BL/6J mice were infected intranasally with 5500 PFU of IAV and mRNA expression of (c) Ch25h, Cyp7b1 and Hsd3b7 were measured by qRT-PCR at 3 dpi and 7 dpi normalised to Hprt (upper panel). Quantitative analysis of CH25H, CYP7B1 and HSD3B7 protein labelling by IHC (lower panel). Representative IHC images of CH25H, CYP7B1 and HSD3B7 in lung sections of uninfected or IAV-infected mice. d) Concentrations of 25-OHC and 7α,25-OHC in the lungs at 3 dpi and 7 dpi expressed in pmol per gram lung tissue (left panel) or in pmol per mL BALF (right). Data are presented as mean } sd of n=4 uninfected and n=6–10 infected mice per timepoint.

Article Snippet: Immunohistochemistry (IHC) was performed on deparaffinised/rehydrated lung sections by immunolabelling with antibodies against SARS-CoV-2 nucleocapsid protein (40143-R040 Sino Biological), IBA1 (019-19741; NovaChem), CH25H (BS-6480R, Bioss Antibodies), CYP7B1 (BS-5052R, Bioss Antibodies) and isotype control (rabbit IgG 31235, Thermo Fisher Scientific) diluted in Da Vinci Green Diluent (PD900, Biocare Medical) followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit Ig antibody (1:200) (ab6721, Abcam).

Techniques: Infection, Expressing, Quantitative RT-PCR

Journal: The European Respiratory Journal

Article Title: GPR183 antagonism reduces macrophage infiltration in influenza and SARS-CoV-2 infection

doi: 10.1183/13993003.01306-2022

Figure Lengend Snippet: SARS-CoV-2 infection leads to upregulation of CH25H and CYP7B1 expression in the lung and production of the oxysterols 25-OHC and 7α,25-OHC. C57BL/6J mice were infected intranasally with approximately 8×104 PFU of mouse49 adapted SARS-CoV-2. mRNA expression of (a) Ch25h , Cyp7b1 and Hsd3b7 was measured by qRT-PCR at 2 dpi and 5 dpi normalised to Hprt . b) Quantitative analysis of CH25H, CYP7B1 and HSD3B7 protein by IHC labelling and (c) representative IHC images of CH25H, CYP7B1 and HSD3B7 in lung sections in uninfected mice and 2 and 5 dpi. d) Concentrations of 25-OHC and 7a,25-OHC in the lungs at 2 dpi and 5 dpi expressed in pmol per gram lung tissue (top panel) or in pmol per mL BALF (bottom panel). Data are presented as mean } sd of n=3 uninfected mice and n=9–10 infected mice per timepoint. Scale Bar=50 μm; U/I=mock infected; dpi=days post-infection; ns=not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 indicate significant differences.

Article Snippet: Immunohistochemistry (IHC) was performed on deparaffinised/rehydrated lung sections by immunolabelling with antibodies against SARS-CoV-2 nucleocapsid protein (40143-R040 Sino Biological), IBA1 (019-19741; NovaChem), CH25H (BS-6480R, Bioss Antibodies), CYP7B1 (BS-5052R, Bioss Antibodies) and isotype control (rabbit IgG 31235, Thermo Fisher Scientific) diluted in Da Vinci Green Diluent (PD900, Biocare Medical) followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit Ig antibody (1:200) (ab6721, Abcam).

Techniques: Infection, Expressing, Quantitative RT-PCR

Journal: The European Respiratory Journal

Article Title: GPR183 antagonism reduces macrophage infiltration in influenza and SARS-CoV-2 infection

doi: 10.1183/13993003.01306-2022

Figure Lengend Snippet: Single cell RNASeq expression analysis of cells collected by bronchoalveolar lavage from healthy controls and COVID19 patients. Summary heatmap (left panels) showing average normalised expression level of gene (a) Ch25h (b) Cyp7b1 , (c) Hsd3b7 and (d) Gpr183 per individual per cell type cluster. Summary heatmap (right panels) show the average log fold change (logFC) in expression of each gene in 24 cell type clusters between: (1) moderate COVID-19 cases (“M”) and healthy controls (“H”); (2) severe COVID-19 cases (“S”) and healthy controls; (3) COVID-19 cases (“M&S””) and healthy controls; and (4) severe and moderate COVID-19 cases, respectively. The logFC values were estimated using (1) negative binomial generalised linear models applied to raw UMI counts, adjusting for total UMI counts per cell, number of genes detected per cell and percent mitochondrial counts per cell (“NB regression”); or (2) non-parametric Wilcoxon rank sum test applied to normalised counts. Significant associations are highlighted with a single asterisk if they surpass Bonferroni significance (p<1.30×10 −4 ) or a double asterisk if they were further expressed in at least 5% of cells in both groups with an absolute value of logFC greater than 0.25. A logFC greater than zero suggests the expression level of the gene is higher among the focal group ( e.g. , moderate COVID-19 cases) compared to the other group ( e.g. , healthy controls), or vice versa.

Article Snippet: Immunohistochemistry (IHC) was performed on deparaffinised/rehydrated lung sections by immunolabelling with antibodies against SARS-CoV-2 nucleocapsid protein (40143-R040 Sino Biological), IBA1 (019-19741; NovaChem), CH25H (BS-6480R, Bioss Antibodies), CYP7B1 (BS-5052R, Bioss Antibodies) and isotype control (rabbit IgG 31235, Thermo Fisher Scientific) diluted in Da Vinci Green Diluent (PD900, Biocare Medical) followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit Ig antibody (1:200) (ab6721, Abcam).

Techniques: Expressing

Journal: bioRxiv

Article Title: 25-hydroxycholesterol dysregulates brain endothelial cell function and exacerbates cerebral haemorrhage

doi: 10.1101/2024.05.10.590792

Figure Lengend Snippet: A Representative images of control (ICH-) and haemorrhagic (ICH+) Tg(fli1:EGFP)/Tg(gata1a:DsRed) 3 days post-fertilization (dpf) larvae, 24 hours after SARS-CoV-2 spike protein (spike) injection into the hindbrain (0.25 mg/ml, 2 nl). Red indicates erythrocytes (gata1a positive) and cyan endothelial cells (fli1+ positive). Dotted lines indicate brain area. Scale bars 250 µm. B Time course of ICH+ frequency in Tg(fli:EGFP)/Tg(gata1a:DsRed) larvae in uninjected, injected with BSA control, injected with pre-heated spike at 80 °C for 30 min (spike-80) or injected with spike conditions (n=4, 11-14 embryos per experiment). C Haematoma size (gata positive area in brain region) in larvae 24 h post-injection of BSA or spike (n=146-147 embryos, 3 independent experiments). D-E Gene expression was analysed in larval heads 8 and 14 hours after BSA or spike injections, for ch25h ( D ), ch25hl1.1 , ch25hl2 and ch25hl3 ( E ). Data are mean ± S.E.M ( B , C , E ) or median ± quartiles ( C ). *, p < 0.05; **, p < 0.01; ns, non-significant, determined by repeated measures ANOVA with Dunnet’s post-hoc test compared to uninjected ( B ), Mann-Whitney test ( C ), or randomized block two-way ANOVA with Sidak’s post hoc analysis compared to BSA ( D, E ).

Article Snippet: For CH25H immunohistochemistry, antigen retrieval was performed by placing slides in 97.5 °C water bath in Tris-EDTA (pH 9.0) for 20 min. Non-specific binding was blocked with PBS with 5% goat serum, 0.3% Triton X-100 and 0.1% Tween for 1 h. CH25H antibody (Aviva system biology, OABF01697) was diluted at 1:2000 blocking solution and incubated on slide overnight at 4 °C.

Techniques: Control, Injection, Gene Expression, MANN-WHITNEY, Blocking Assay

Journal: bioRxiv

Article Title: 25-hydroxycholesterol dysregulates brain endothelial cell function and exacerbates cerebral haemorrhage

doi: 10.1101/2024.05.10.590792

Figure Lengend Snippet: A Representative images of microbleeds found in human foetal cortex by H&E staining. Bleeds were classified into small, medium and large sizes according to area. Scale bar size is shown in each image. B Samples were categorised according to a bleeding score, based on bleed density. Control samples were classified in score 0, and haemorrhagic samples were classified in scores 1 and 2. C Density of small, medium, and large bleeds in samples with bleeding scores 0 to 2. D Representative images for CH25H staining (pink) and bleeds (yellow) from non-haemorrhagic or haemorrhagic samples, proximal and distal to bleeds. Scale bars 30 µm. E CH25H+ cell density was quantified in samples with bleeding scores 0 to 2. F Distance of CH25H+ cells to the nearest bleed was quantified in samples with bleeding scores 1 and 2 (n=626-702 cells). G Ratio of immunofluorescence integrated density of the SARS-CoV-2 spike protein relative to its isotype control, data obtained from , in samples with bleeding scores 0 to 2. Data are mean ± S.E.M ( B , C , E , F ) or median ± quartiles ( G ). **, p < 0.01; ***, p < 0.001; ***, p < 0.0001; ns, nonsignificant, determined by one-way ANOVA with Kruskal-Wallis post hoc test compared to score 0 ( B , E, G ), or two-way ANOVA with Tukey’s post hoc analysis multiple comparisons between scores ( C ), or Mann-Whitney test ( F ).

Article Snippet: For CH25H immunohistochemistry, antigen retrieval was performed by placing slides in 97.5 °C water bath in Tris-EDTA (pH 9.0) for 20 min. Non-specific binding was blocked with PBS with 5% goat serum, 0.3% Triton X-100 and 0.1% Tween for 1 h. CH25H antibody (Aviva system biology, OABF01697) was diluted at 1:2000 blocking solution and incubated on slide overnight at 4 °C.

Techniques: Staining, Control, Immunofluorescence, MANN-WHITNEY

Journal: Cell Cycle

Article Title: Exosomes-carried microRNA-26b-5p regulates microglia M1 polarization after cerebral ischemia/reperfusion

doi: 10.1080/15384101.2020.1743912

Figure Lengend Snippet: Antibodies used for western blot analysis.

Article Snippet: Antibody Item number Provider Dilution ratio β-actin ab179467 Abcam 1: 5000 CH25H ab191406 Abcam 1: 1000 TLR-2 ab35962 Abcam 1: 500 TLR-4 ab185145 Abcam 1: 1000 TLR-6 ab59479 Abcam 1: 1000 CD63 ab53277 Abcam 1: 1000 CD9 ab74032 Abcam 1: 1000 CD81 ab9643 Abcam 1: 50 Alix ab32503 Abcam 1: 5000 IgG ab205718 Abcam 1:2000 Open in a separate window CH25H, Cholesterol 25-hydroxylase; TLR, Toll-like receptor.

Techniques: Western Blot

Journal: The European Respiratory Journal

Article Title: GPR183 antagonism reduces macrophage infiltration in influenza and SARS-CoV-2 infection

doi: 10.1183/13993003.01306-2022

Figure Lengend Snippet: IAV infection leads to upregulation of CH25H and CYP7B1 expression in the lung and production of the oxysterols 25-OHC and 7α,25-OHC. a) The biosynthetic pathway of 25-OHC and 7α,25-OHC. b) Experimental design; C57BL/6J mice were infected intranasally with 5500 PFU of IAV and mRNA expression of (c) Ch25h, Cyp7b1 and Hsd3b7 were measured by qRT-PCR at 3 dpi and 7 dpi normalised to Hprt (upper panel). Quantitative analysis of CH25H, CYP7B1 and HSD3B7 protein labelling by IHC (lower panel). Representative IHC images of CH25H, CYP7B1 and HSD3B7 in lung sections of uninfected or IAV-infected mice. d) Concentrations of 25-OHC and 7α,25-OHC in the lungs at 3 dpi and 7 dpi expressed in pmol per gram lung tissue (left panel) or in pmol per mL BALF (right). Data are presented as mean } sd of n=4 uninfected and n=6–10 infected mice per timepoint.

Article Snippet: Immunohistochemistry (IHC) was performed on deparaffinised/rehydrated lung sections by immunolabelling with antibodies against SARS-CoV-2 nucleocapsid protein (40143-R040 Sino Biological), IBA1 (019-19741; NovaChem), CH25H (BS-6480R, Bioss Antibodies), CYP7B1 (BS-5052R, Bioss Antibodies) and isotype control (rabbit IgG 31235, Thermo Fisher Scientific) diluted in Da Vinci Green Diluent (PD900, Biocare Medical) followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit Ig antibody (1:200) (ab6721, Abcam).

Techniques: Infection, Expressing, Quantitative RT-PCR

Journal: The European Respiratory Journal

Article Title: GPR183 antagonism reduces macrophage infiltration in influenza and SARS-CoV-2 infection

doi: 10.1183/13993003.01306-2022

Figure Lengend Snippet: SARS-CoV-2 infection leads to upregulation of CH25H and CYP7B1 expression in the lung and production of the oxysterols 25-OHC and 7α,25-OHC. C57BL/6J mice were infected intranasally with approximately 8×104 PFU of mouse49 adapted SARS-CoV-2. mRNA expression of (a) Ch25h , Cyp7b1 and Hsd3b7 was measured by qRT-PCR at 2 dpi and 5 dpi normalised to Hprt . b) Quantitative analysis of CH25H, CYP7B1 and HSD3B7 protein by IHC labelling and (c) representative IHC images of CH25H, CYP7B1 and HSD3B7 in lung sections in uninfected mice and 2 and 5 dpi. d) Concentrations of 25-OHC and 7a,25-OHC in the lungs at 2 dpi and 5 dpi expressed in pmol per gram lung tissue (top panel) or in pmol per mL BALF (bottom panel). Data are presented as mean } sd of n=3 uninfected mice and n=9–10 infected mice per timepoint. Scale Bar=50 μm; U/I=mock infected; dpi=days post-infection; ns=not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 indicate significant differences.

Article Snippet: Immunohistochemistry (IHC) was performed on deparaffinised/rehydrated lung sections by immunolabelling with antibodies against SARS-CoV-2 nucleocapsid protein (40143-R040 Sino Biological), IBA1 (019-19741; NovaChem), CH25H (BS-6480R, Bioss Antibodies), CYP7B1 (BS-5052R, Bioss Antibodies) and isotype control (rabbit IgG 31235, Thermo Fisher Scientific) diluted in Da Vinci Green Diluent (PD900, Biocare Medical) followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit Ig antibody (1:200) (ab6721, Abcam).

Techniques: Infection, Expressing, Quantitative RT-PCR

Journal: The European Respiratory Journal

Article Title: GPR183 antagonism reduces macrophage infiltration in influenza and SARS-CoV-2 infection

doi: 10.1183/13993003.01306-2022

Figure Lengend Snippet: Single cell RNASeq expression analysis of cells collected by bronchoalveolar lavage from healthy controls and COVID19 patients. Summary heatmap (left panels) showing average normalised expression level of gene (a) Ch25h (b) Cyp7b1 , (c) Hsd3b7 and (d) Gpr183 per individual per cell type cluster. Summary heatmap (right panels) show the average log fold change (logFC) in expression of each gene in 24 cell type clusters between: (1) moderate COVID-19 cases (“M”) and healthy controls (“H”); (2) severe COVID-19 cases (“S”) and healthy controls; (3) COVID-19 cases (“M&S””) and healthy controls; and (4) severe and moderate COVID-19 cases, respectively. The logFC values were estimated using (1) negative binomial generalised linear models applied to raw UMI counts, adjusting for total UMI counts per cell, number of genes detected per cell and percent mitochondrial counts per cell (“NB regression”); or (2) non-parametric Wilcoxon rank sum test applied to normalised counts. Significant associations are highlighted with a single asterisk if they surpass Bonferroni significance (p<1.30×10 −4 ) or a double asterisk if they were further expressed in at least 5% of cells in both groups with an absolute value of logFC greater than 0.25. A logFC greater than zero suggests the expression level of the gene is higher among the focal group ( e.g. , moderate COVID-19 cases) compared to the other group ( e.g. , healthy controls), or vice versa.

Article Snippet: Immunohistochemistry (IHC) was performed on deparaffinised/rehydrated lung sections by immunolabelling with antibodies against SARS-CoV-2 nucleocapsid protein (40143-R040 Sino Biological), IBA1 (019-19741; NovaChem), CH25H (BS-6480R, Bioss Antibodies), CYP7B1 (BS-5052R, Bioss Antibodies) and isotype control (rabbit IgG 31235, Thermo Fisher Scientific) diluted in Da Vinci Green Diluent (PD900, Biocare Medical) followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit Ig antibody (1:200) (ab6721, Abcam).

Techniques: Expressing