Journal: Stress Biology

Article Title: An asymptomatic geminivirus activates autophagy and enhances plant defenses against diverse pathogens

doi: 10.1007/s44154-024-00176-8

Figure Lengend Snippet: Establishment of an AGV inoculation method through Agrobacterium -mediated inoculation on root plants. A . Illustration describing the step-by-step procedure for inoculating AGV on the roots of various solanaceous plants using Agrobacterium cultures carrying an infectious cDNA clone of AGV. B . Efficiency of AGV infection through the inoculation method described in A. AGV DNA was detected in upper systemic leaves by PCR. C and E . Autophagic activity in leaf ( C ) and root ( E ) tissues of AGV-infected tomato revealed by visualizing autophagy vesicles through fluorescent dye monodansylcadaverine (MDC) staining and confocal laser scanning microscopy. Scale bars, 20 μm. D and F . Quantification of autophagic activity based on the numbers of MDC-stained autophagic structures in the cells of the leaf ( D ) and root ( F ) tissue described in C and E . The mock-inoculated sample was set to a value of 1.0. Vertical lines on the bars represent the SD. “**” and “***” indicates a significant difference at P < 0.01 and 0.001, respectively (Student’s t- test)

Article Snippet: The fluorescence signals of GFP-ATG8f and MDC staining were observed using a confocal microscope (FC3000, Olympus) as previously described (Zhao et al. ).

Techniques: Infection, Activity Assay, Staining, Confocal Laser Scanning Microscopy

Journal: Journal of Translational Medicine

Article Title: METTL3-mediated m6A modification of SIRT1 mRNA affects the progression of diabetic cataracts through cellular autophagy and senescence

doi: 10.1186/s12967-024-05691-w

Figure Lengend Snippet: HG-induced impaired autophagic flux and accelerated senescence in HLEB3 cells, and knockdown of METTL3 restored autophagic flux and delayed senescence. ( A - C ) Assessment of METTL3 silencing effectiveness in siMETTL3-transfected HLEB3 cells at mRNA level using RT-qPCR and protein level using western blot analysis ( n = 3). ( D ) m6A level in siMETTL3-transfected HLEB3 cells with an anti-m6A antibody. ( E - F ) HLEB3 cells were cultured in HG for 72 h post-transfection. Autophagy expression following various treatments using western blot ( n = 3). ( G-H ) Ad-mCherry-GFP-LC3B transfection into HLEB3 cells post-various treatments. Representative confocal microscopy images and quantification of fluorescent puncta per cell are shown. Data were quantitatively analyzed using randomized fields of view from 3 independent experiments. Scale bar = 10 μm. ( I ) Representative images of autophagosomes visualized via the MDC method; Scale bar = 50 μm. ( J-K ) Senescence biomarkers levels in HLEB3 cells subjected to different treatments using western blot ( n = 3). ( L-M ) SA-β-Gal staining was used to detect the senescence levels in differently treated HLEB3 cells. Data were quantitatively analyzed using randomized fields of view from 3 independent experiments. Scale bar = 100 μm. Statistical significance was determined by two-sided Student’s t-test or one-way ANOVA with Bonferroni’s multiple comparisons, as appropriate

Article Snippet: After treating the cells in groups, MDC staining solution (C3018, Beyotime, China) was added to each group.

Techniques: Knockdown, Transfection, Quantitative RT-PCR, Western Blot, Cell Culture, Expressing, Confocal Microscopy, Staining

Journal: Journal of Translational Medicine

Article Title: METTL3-mediated m6A modification of SIRT1 mRNA affects the progression of diabetic cataracts through cellular autophagy and senescence

doi: 10.1186/s12967-024-05691-w

Figure Lengend Snippet: Overexpression of METTL3 further impairs HG-induced autophagic flux in HLEB3 cells and accelerates senescence. ( A - C ) Assessment of METTL3 overexpressing effectiveness in OE-METTL3-transfected HLEB3 cells at mRNA level using RT-qPCR and protein level using western blot analysis ( n = 3). ( D ) m6A level in OE-METTL3-transfected HLEB3 cells with an anti-m6A antibody. (E - F ) Autophagy-related biomarker expression in HLEB3 cells under different treatments using western blot ( n = 3). ( G - H ) Ad-mCherry-GFP-LC3B was transfected into HLEB3 cells, followed by various treatments. Representative confocal microscopy images and quantification of fluorescent puncta per cell are shown. Data were quantitatively analyzed using randomized fields of view from 3 independent experiments ( n = 3). Scale bar = 10 μm. ( I ) Representative images of autophagosomes visualized via the MDC method. Scale bar = 50 μm. ( J - K ) Senescence biomarkers levels in HLEB3 cells subjected to different treatments using western blot ( n = 3). ( L - M ) SA-β-Gal staining was used to detect the senescence levels in differently treated HLEB3 cells. Data were quantitatively analyzed using randomized fields of view from 3 independent experiments. Scale bar = 100 μm. Statistical significance was determined by two-sided Student’s t-test or one-way ANOVA with Bonferroni’s multiple comparisons, as appropriate

Article Snippet: After treating the cells in groups, MDC staining solution (C3018, Beyotime, China) was added to each group.

Techniques: Over Expression, Transfection, Quantitative RT-PCR, Western Blot, Biomarker Assay, Expressing, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: Monocyte-derived cells but not Microglia cause Oxidative Tissue Damage in Neuroinflammation

doi: 10.1101/2024.09.18.612891

Figure Lengend Snippet: a, Schematic illustration of cluster selection and data integration of multiple human snRNA-seq datasets (author and date depicted) b, Dot plot illustrating “ROS” module score in mononuclear phagocytes (MPs) from control and MS patient samples. c, Dot plot of the “ROS” module score values in MPs across different tissues from control and MS patients. Control tissues: GM (Gray Matter), WM (White Matter); MS tissues: NAGM (Normal Appearing GM), NAWM (Normal Appearing WM); R (Remyelination), LE (Lesion Edge), CILE (Chronic Inactive LE), Lesion GM, Chronic Lesion, Active Lesion. d, UMAP of the two clusters of MPs: damage-associated MPs (DA-MPs) and homeostatic-associated MPs. e, Bar graph of the percentage of the MP clusters in control and MS samples. f, Dot plot illustrating “ROS” module score values in the two MPs clusters. g, UMAP of immune clusters found in Mendiola et al . h, Bar graph of the percentage of the immune clusters found in control and EAE CNS. i, Dot plot illustrating “ROS” module score (mouse GO term: 0072593) in homeostatic microglia (hMicro), damage-associated microglia (DAMs), and monocyte-derived cell clusters (MdCs). k, UMAP of the CNS immune cell populations found during peak EAE k, Lollipop plot of DCFDA expression across different immune clusters in the inflamed CNS (peak EAE CNS day 15 post-immunization). l, Histogram showing DCFDA expression in MdCs and microglia. m, Violin plot of DCFDA expression in MdCs and microglia (control n=6, EAE n=6). (unpaired two-tailed t-test; **P = 0.0071). Pooled data from three experiment. For b,c,f,i, Circle size is depicting % of normalized expression detected per cluster and color is depicting expression level as Average Module Score or Average Expression.

Article Snippet: Total RNA was isolated from sorted microglia and MdCs using the QuickRNA Microprep Kit (R1051, Zymo Research) according to the manufacturer’s instructions.

Techniques: Selection, Control, Derivative Assay, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Monocyte-derived cells but not Microglia cause Oxidative Tissue Damage in Neuroinflammation

doi: 10.1101/2024.09.18.612891

Figure Lengend Snippet: a Heatmap of markers expressed by the different clusters and their annotations. b, Harmony-integrated Louvain clusters identified in the dataset. c, UMAP of the annotated immune clusters. d, UMAPs separated by control and MS with overlay of the “ROS” module score. e, UMAP overlay of the genes used to annotate Homeostatic Mononuclear Phagocytes (MPs), Damage-associated (DA) MPs, and Monocyte-derived MPs. f, UMAP and g, Heatmap from the Mendiola et al. 3 dataset. h , UMAP and i, Heatmap from the Jordao et al dataset. j , UMAP and k, Heatmap of cluster expression for the annotated clusters in the Peruzzotti-Jametti et al 6 dataset. l, Dot plot of the “ROS” module score at different stages of EAE from the Jordao et al dataset. m, Dot plot of the “ROS” module score during control, peak, and chronic EAE stages in the Peruzzotti-Jametti et al 6 dataset. n , Clustered heatmap of the 20 most expressed ROS-associated genes in MPs found in the Peruzzotti-Jametti et al 6 dataset. o , Heatmap of marker expression from annotated immune clusters during peak EAE. p, Overlayed ROS (DCFDA) expression. q, Dot plots depict the gating strategy used to calculate the infiltrated immune cell counts, Live CD45+CD44+, DCFDA and Mitosox expression in Microglia (Microglia+BAMs) and Monocyte derived cells (MdCs). r, Biaxal contour plot of CD44 (y-axis) and DCFDA (x-axis) expression in MdCs and microglia. For panels l and m , circle size depicts the percentage of expression detected per cluster, and color indicates the expression level as the average module score or average expression.

Article Snippet: Total RNA was isolated from sorted microglia and MdCs using the QuickRNA Microprep Kit (R1051, Zymo Research) according to the manufacturer’s instructions.

Techniques: Control, Derivative Assay, Expressing, Marker

Journal: bioRxiv

Article Title: Monocyte-derived cells but not Microglia cause Oxidative Tissue Damage in Neuroinflammation

doi: 10.1101/2024.09.18.612891

Figure Lengend Snippet: a, Dot plot illustrating Cybb expression in homeostatic microglia (hMicro), damage-associated microglia (DAMs), and monocyte-derived cell clusters (MdCs) from Peruzzotti-Jametti et al during control (non-immunized), peak and chronic phases of EAE. Circle size is depicting % expression detected per cluster and color is depicting expression level as Average Module Score or Average Expression. b, Relative gene expression Cybb measured by qPCR of FC-sorted Microglia (n=6) and MdCs (n=5) during peak EAE. (Mann–Whitney test; **P = 0.0022). c,d, Passive EAE was induced by adoptive transfer of primed lymphocytes from actively immunized donor mice. Plots depict mean clinical score ( c ) overtime (two-way ANOVA, and Sidak’s post hoc test) and ( d ) cumulative clinical score Ccr2 +/+ Cybb fl /fl (n=5, f) and Ccr2 CreERT2/+ Cybb fl /fl (n=6, f) mice (unpaired two-tailed t-test). e, ROS production measured by DCFDA median expression in manually gated (FlowJo) microglia and MdC from Ccr2 +/+ Cybb fl /fl (n=7, m/f) and Ccr2 CreERT2/+ Cybb fl /fl (n=8, m/f) mice (unpaired two-tailed t-test). f, ROS production measured by DCFDA median expression in manually gated (FlowJo) microglia and MdC from Cx3cr1 +/+ Cybb fl /fl (n=4, m/f) and Cx3cr1 CreERT2/+ Cybb fl /fl (n=5, m/f) mice. Tamoxifen regimen corresponds to early tamoxifen treatment (unpaired two-tailed t-test). g, ROS production measured by DCFDA median expression in microglia and MdC clusters (generated in R) in Cx3cr1 +/+ Cybb fl /fl (n=6, m/f) and Cx3cr1 CreERT2/+ Cybb fl /fl (n=6, m/f) mice (unpaired two-tailed t-test).

Article Snippet: Total RNA was isolated from sorted microglia and MdCs using the QuickRNA Microprep Kit (R1051, Zymo Research) according to the manufacturer’s instructions.

Techniques: Expressing, Derivative Assay, Control, MANN-WHITNEY, Adoptive Transfer Assay, Two Tailed Test, Generated

Journal: bioRxiv

Article Title: Monocyte-derived cells but not Microglia cause Oxidative Tissue Damage in Neuroinflammation

doi: 10.1101/2024.09.18.612891

Figure Lengend Snippet: a, Schematic illustration of the tamoxifen treatment strategy to target Cybb in MdCs during neuroinflammation. b , Relative gene expression of Cybb / Pol2 in CNS microglia and MdCs in Ccr2 +/+ Cybb fl /fl (n=3, f) and Ccr2 CreERT2/+ Cybb fl /fl (n=4, f) mice (unpaired two-tailed t-test, **p=0.0043 in MdCs). c-d , Active EAE was induced by MOG 35-55 /CFA/PT immunization. Plots depict mean clinical score ( c ) over time (two-way ANOVA, and Sidak’s post hoc test) and ( d ) cumulative clinical score Ccr2 +/+ Cybb fl /fl (n=11, m/f) and Ccr2 CreERT2/+ Cybb fl /fl (n=10, m/f) mice (unpaired two-tailed t-test). e, Absolute counts of CNS infiltrating immune cells (gated in FlowJo CD45+CD44+, gating strategy ) at 19 d.p.i. in Cx3cr1 +/+ Cybb fl /fl (n=5, f) and Cx3cr1 CreERT2/+ Cybb fl /fl (n=4,f) mice. Unpaired two-tailed t-test. f, Schematic representation of tamoxifen treatment to target embryonic hematopoiesis-derived macrophages, but not bone marrow derived MdCs. g, Relative gene expression of Cybb / Pol2 in CNS microglia and MdCs in Cx3cr1 +/+ Cybb fl /fl (Microglia n=6, MdCs=2 f) and Cx3cr1 CreERT2/+ Cybb fl /fl (Microglia n=5, MdCs n=2, f) mice (unpaired two-tailed t-test, **p=0.0043 in Microglia). h-i, Active EAE was induced by MOG 35-55 /CFA/PT immunization. Plots show mean clinical score over time (Two-way ANOVA, and Sidak’s post hoc test) ( h ) and cumulative clinical score ( i ) in Cx3cr1 +/+ mCAT fl/wt (n=15, m/f) and Cx3cr1 CreERT2/+ mCAT fl/wt (n=15, m/f) mice (unpaired two-tailed t-test). j, Absolute counts of CNS infiltrating immune cells (gated in flowjo CD45+CD44+, gating strategy ) at 19 d.p.i. in Cx3cr1 +/+ Cybb fl /fl (n=6, f) and Cx3cr1 CreERT2/+ Cybb fl /fl (n=10, f) mice. Unpaired two-tailed t-test. k , Schematic illustration of the tamoxifen treatment strategy to target Cybb in microglia and MdCs during neuroinflammation. l , Relative gene expression of Cybb / Pol2 in CNS microglia and MdCs in Cx3cr1 +/+ Cybb fl /fl (n=4, f) and Cx3cr1 CreERT2/+ Cybb fl /fl (n=4, f) mice (unpaired two-tailed t-test, **p=0.0036 in Microglia and **p=0.0063 in MdCs). m-n , Active EAE was induced by MOG 35-55 /CFA/PT immunization. Plots depict mean clinical score ( m ) over time (two-way ANOVA, and Sidak’s post hoc test) and ( n) cumulative clinical score Cx3cr1 +/+ Cybb fl /fl (n=11, m/f) and Cx3cr1 CreERT2/+ Cybb fl /fl (n=8, m/f) mice (unpaired two-tailed t-test). o, Absolute counts of CNS infiltrating immune cells (gated in FlowJo CD45+CD44+, gating strategy ) at 19 d.p.i. in Cx3cr1 +/+ Cybb fl /fl (n=6, f) and Cx3cr1 CreERT2/+ Cybb fl /fl (n=6, f) mice.

Article Snippet: Total RNA was isolated from sorted microglia and MdCs using the QuickRNA Microprep Kit (R1051, Zymo Research) according to the manufacturer’s instructions.

Techniques: Expressing, Two Tailed Test, Derivative Assay

Journal: bioRxiv

Article Title: Monocyte-derived cells but not Microglia cause Oxidative Tissue Damage in Neuroinflammation

doi: 10.1101/2024.09.18.612891

Figure Lengend Snippet: a , DotPlots illustrating mitochondria complex I module score in CNS microglia and MdCs in the Peruzzotti-Jametti et al . dataset in peak and chronic EAE compared to control CNS. Circle size is depicting % expression detected per cluster and color is depicting expression level as Average Module Score. b , Clustered heatmap of the expression of the mitochondria complex I genes in MPs found in the Peruzzotti-Jametti et al . dataset. c , Immunoblot showing HA-tag and actin bands, in FACs sorted microglia and MdCs from Cx3cr1 +/+ mCAT fl/wt and Cx3cr1 CreERT2/+ mCAT fl/wt mice (pooled cells from n=3 mice, m/f). d , Immunoblot showing HA-tag and actin bands, in FACs sorted microglia and MdCs early treatment tamoxifen regimen Cx3cr1 CreERT2/+ mCAT fl/wt mice (pooled cells from n=2 mice, m/f). e-f , Representative MBP staining (left) and the percentage of myelin loss quantified in lumbar spinal cord sections in early treatment tamoxifen regimen Cx3cr1 +/+ mCAT fl/wt (n=5, f) and Cx3cr1 CreERT2/+ mCAT fl/wt (n=4, f) mice (right). Unpaired two-tailed t-test.

Article Snippet: Total RNA was isolated from sorted microglia and MdCs using the QuickRNA Microprep Kit (R1051, Zymo Research) according to the manufacturer’s instructions.

Techniques: Control, Expressing, Western Blot, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Monocyte-derived cells but not Microglia cause Oxidative Tissue Damage in Neuroinflammation

doi: 10.1101/2024.09.18.612891

Figure Lengend Snippet: a, Schematic illustration of the tamoxifen treatment strategy employed to reduce mtROS in CX3CR1 expressing MPs. b , ROS production measured by normalized DCFDA expression in microglia and MdC clusters (generated in R) of Cx3cr1 +/+ mCAT fl/wt (n=6, f) and Cx3cr1 CreERT2/+ mCAT fl/wt (n=4, f). Pooled data from two experiments (unpaired two-tailed t-test, *P=0.04 in Microglia and **P=0.0071 in MdCs). c , Mitochondrial ROS (mtROS) production measured by mitosox expression in manually gated (FlowJo) microglia and MdC of Cx3cr1 +/+ mCAT fl/wt (n=4, f) and Cx3cr1 CreERT2/+ mCAT fl/wt (n=3 f). Unpaired two-tailed t-test (*P=0.0237 in Microglia and *P=0.0363 in MdCs). d - e , Active EAE was induced by MOG 35-55 /CFA/PT immunization. ( d ) Plots show mean clinical score over time (two-way ANOVA, and Sidak’s post hoc test, day 13 **P=0.0022, day 14 **P=0.0023, day 15 **P=0.0031, day 16 **P=0.0059, day 18 *P=0.0453, day 19 *P=0.0109) and ( e ) cumulative clinical score in Cx3cr1 +/+ mCAT fl/wt (n=8, f) and Cx3cr1 CreERT2/+ mCAT fl/wt (n=8, f) mice. Unpaired two-tailed t-test (*P=0.0405). f, Absolute counts of CNS infiltrating immune cells (gated in FlowJo CD45+CD44+, gating strategy ) at 19 d.p.i. in Cx3cr1 +/+ mCAT fl/wt (n=5, f) and Cx3cr1 CreERT2/+ mCAT fl/wt (n=4, f) mice. Unpaired two-tailed t-test (*P=0.0091). g, Schematic representation of tamoxifen treatment to target embryonic hematopoiesis-derived macrophages, but not bone marrow derived MdCs . h , ROS production measured by DCFDA median expression in manually gated (FlowJo) microglia from Cx3cr1 +/+ mCAT fl/wt (n=4, f) and Cx3cr1 CreERT2/+ mCAT fl/wt (n=4, f)) in steady state CNS (unpaired two-tailed t-test; *P=0.0135). i , mtROS production measured as mitosox median expression in manually gated (FlowJo) microglia from Cx3cr1 +/+ mCAT fl/wt (n=4, f) and Cx3cr1 CreERT2/+ mCAT fl/wt (n=4, f) in steady state CNS (unpaired two-tailed t-test; *P=0.0423). j-k, Active EAE was induced by MOG 35-55 /CFA/PT immunization. Plots show mean clinical score over time (Two-way ANOVA, and Sidak’s post hoc test) ( j ) and cumulative clinical score ( k ) in Cx3cr1 +/+ mCAT fl/wt (n=10, m/f) and Cx3cr1 CreERT2/+ mCAT fl/wt (n=7, m/f) mice. Unpaired two-tailed t-test. Pooled data from two experiments. l , Absolute counts of CNS infiltrating immune cells (gated in FlowJo CD45+CD44+, gating strategy ) at 19 d.p.i. in Cx3cr1 +/+ mCAT fl/wt (n=5, f) and Cx3cr1 CreERT2/+ mCAT fl/wt (n=4, f) mice. Unpaired two-tailed t-test.

Article Snippet: Total RNA was isolated from sorted microglia and MdCs using the QuickRNA Microprep Kit (R1051, Zymo Research) according to the manufacturer’s instructions.

Techniques: Expressing, Generated, Two Tailed Test, Derivative Assay

Journal: bioRxiv

Article Title: Monocyte-derived cells but not Microglia cause Oxidative Tissue Damage in Neuroinflammation

doi: 10.1101/2024.09.18.612891

Figure Lengend Snippet: a , Schematic representation of tamoxifen treatment to target bone marrow derived MdCs during EAE disease onset. b , ROS production measured by normalized DCFDA median expression in microglia and MdC clusters (generated in R) Ccr2 +/+ mCAT fl/ wt (n=5, m/f) and Ccr2 CreERT2/+ mCAT fl/ wt (n=5, m/f) mice. Pooled data from two experiments (unpaired two-tailed t-test; **P=0.0054 in MdCs). c , mtROS production measured as mitosox median expression in manually gated (FlowJo) microglia and MdCs from Ccr2 +/+ mCAT fl/wt (n=6, m/f) and Ccr2 CreERT2/+ mCAT fl/ wt (n=5, m/f) mice. Pooled data from two experiments (unpaired two-tailed t-test, **P=0.003 in MdCs). d - e , Active EAE was induced by MOG 35-55 /CFA/PT immunization. Plots show mean clinical score over time (Two-way ANOVA, and Sidak’s post hoc test, day 19 **P=0.0078) ( d ) and cumulative clinical score ( e ) in Ccr2 +/+ mCAT fl/wt (n=14, m/f) and Ccr2 CreERT2/+ mCAT fl/wt (n=14, m/f) mice (unpaired two-tailed t-test; *P = 0.0339). Pooled data from two experiments. f, Representative MBP staining (left) and g , the percentage of myelin loss quantified in lumbar spinal cord sections in Ccr2 +/+ mCAT fl/wt (n=9, m/f) and Ccr2 CreERT2/+ mCAT fl/wt (n=8, m/f) mice (unpaired two-tailed t-test; **P = 0.0163). Data pooled from two independent experiments. h, Absolute counts of CD45+CD44+ CNS infiltrating immune cells (gated in FlowJo) at day 17 d.p.i. (n=5 per group, f). Unpaired two-tailed t-test (*P = 0.0164). i, UMAP from CNS leukocytes clustered by cell type and labelled based on surface marker expression. j, Bar graph of the percentage of the immune clusters found in in Ccr2 +/+ mCAT fl/wt and Ccr2 CreERT2/+ mCAT fl/wt (n=5 per group, f). k , Absolute counts of CNS infiltrating immune CD4 T cells (left) and Ly6Chi monocytes (right) (analysed in R) at day 17 d.p.i. (n=5 per group, f), (unpaired two-tailed t-test, *P=0.0386 in CD4 and *P=0.0403 in Ly6cHi). l, Radar plot showing changes in the marker expression of CNS immune cell clusters in Ccr2 CreERT2/+ mCAT fl/wt relative to Ccr2 +/+ mCAT fl/wt and (n=5 per group). Values correspond to −log10(p value) and were adjusted with the Benjamini-Hochberg correction. Bar color denotes the upregulation or downregulation of the respective marker. Marker order is consistent across subsets, left to right: CD38, MHCII, CD44, CX3CR1, F480, CD11C, and MerTK. Dashed line denotes −log10(0.05) cutoff for p value. m-n Passive EAE was induced by adoptive transfer of primed lymphocytes from actively immunized donor mice. Plots show ( m ) mean clinical score over time (two way ANOVA, and Sidak’s post hoc test, day 10 *P=0.0254, day 11 **P=0.0020, day 12 **P=0.0030, day 13 **P=0.0025, day 14 ***P=0.0006, day 15 **P=0.0076, day 16 **P=0.0019, day 17 **P=0.0020, day 18 **P=0.0013, day 19 **P=0.0039, day 20 ***P=0.0007) and ( n ) cumulative clinical score Ccr2 +/+ mCAT fl/wt (n=8, m/f) and Ccr2 CreERT2/+ mCAT fl/wt (n=9, m/f) mice (pooled data from two experiments, unpaired two-tailed t-test; ***P = 0.0009).

Article Snippet: Total RNA was isolated from sorted microglia and MdCs using the QuickRNA Microprep Kit (R1051, Zymo Research) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Expressing, Generated, Two Tailed Test, Staining, Marker, Adoptive Transfer Assay

Journal: bioRxiv

Article Title: Monocyte-derived cells but not Microglia cause Oxidative Tissue Damage in Neuroinflammation

doi: 10.1101/2024.09.18.612891

Figure Lengend Snippet: a , Immunoblot showing HA-tag and Actin bands, in FACS sorted microglia and MdCs from Ccr2 CreERT2/+ mCAT fl/ wt (pooled cells from n=3 mice). b , Heatmap showing the median marker expression across the identified main clusters, UMAP is shown in . c, Absolute counts of CNS infiltrating Neutrophils, CD8 T cells, B cells, Monocyte derived cells (MdCs), Dendritic cells (DCs) and CNS resident immune Microglia (R generated). d-k Median fluorescent intensity (MFI) of activation markers (R generated) in ( d ) MdCs, ( e ) Neutrophils, ( f ) Ly6cHi Monocytes, ( g ) Microglia, ( h ) Dendritic cells, ( i ) B cells, ( j ) CD4 T cells and ( k ) CD8 T cells. (For MFI of CD11C in Ly6cHi Monocytes, unpaired two-tailed t-test. **P=0.0066). Mice analyzed at 17 d.p.i (n=5 per group, f), (unpaired two-tailed t-test).

Article Snippet: Total RNA was isolated from sorted microglia and MdCs using the QuickRNA Microprep Kit (R1051, Zymo Research) according to the manufacturer’s instructions.

Techniques: Western Blot, Marker, Expressing, Derivative Assay, Generated, Activation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Monocyte-derived cells but not Microglia cause Oxidative Tissue Damage in Neuroinflammation

doi: 10.1101/2024.09.18.612891

Figure Lengend Snippet: a, Schematic illustration of cluster selection and data integration of multiple human snRNA-seq datasets (author and date depicted) b, Dot plot illustrating “ROS” module score in mononuclear phagocytes (MPs) from control and MS patient samples. c, Dot plot of the “ROS” module score values in MPs across different tissues from control and MS patients. Control tissues: GM (Gray Matter), WM (White Matter); MS tissues: NAGM (Normal Appearing GM), NAWM (Normal Appearing WM); R (Remyelination), LE (Lesion Edge), CILE (Chronic Inactive LE), Lesion GM, Chronic Lesion, Active Lesion. d, UMAP of the two clusters of MPs: damage-associated MPs (DA-MPs) and homeostatic-associated MPs. e, Bar graph of the percentage of the MP clusters in control and MS samples. f, Dot plot illustrating “ROS” module score values in the two MPs clusters. g, UMAP of immune clusters found in Mendiola et al . h, Bar graph of the percentage of the immune clusters found in control and EAE CNS. i, Dot plot illustrating “ROS” module score (mouse GO term: 0072593) in homeostatic microglia (hMicro), damage-associated microglia (DAMs), and monocyte-derived cell clusters (MdCs). k, UMAP of the CNS immune cell populations found during peak EAE k, Lollipop plot of DCFDA expression across different immune clusters in the inflamed CNS (peak EAE CNS day 15 post-immunization). l, Histogram showing DCFDA expression in MdCs and microglia. m, Violin plot of DCFDA expression in MdCs and microglia (control n=6, EAE n=6). (unpaired two-tailed t-test; **P = 0.0071). Pooled data from three experiment. For b,c,f,i, Circle size is depicting % of normalized expression detected per cluster and color is depicting expression level as Average Module Score or Average Expression.

Article Snippet: Cell sorting of live CD11b + CD45 + CX3CR1 + CD44 - microglia and CD11b + CD45 + CX3CR1 + CD44 + Ly6C + Ly6G - MdCs was performed using a BD FACS Aria III (FACS DIVA Software v9).

Techniques: Selection, Control, Derivative Assay, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Monocyte-derived cells but not Microglia cause Oxidative Tissue Damage in Neuroinflammation

doi: 10.1101/2024.09.18.612891

Figure Lengend Snippet: a Heatmap of markers expressed by the different clusters and their annotations. b, Harmony-integrated Louvain clusters identified in the dataset. c, UMAP of the annotated immune clusters. d, UMAPs separated by control and MS with overlay of the “ROS” module score. e, UMAP overlay of the genes used to annotate Homeostatic Mononuclear Phagocytes (MPs), Damage-associated (DA) MPs, and Monocyte-derived MPs. f, UMAP and g, Heatmap from the Mendiola et al. 3 dataset. h , UMAP and i, Heatmap from the Jordao et al dataset. j , UMAP and k, Heatmap of cluster expression for the annotated clusters in the Peruzzotti-Jametti et al 6 dataset. l, Dot plot of the “ROS” module score at different stages of EAE from the Jordao et al dataset. m, Dot plot of the “ROS” module score during control, peak, and chronic EAE stages in the Peruzzotti-Jametti et al 6 dataset. n , Clustered heatmap of the 20 most expressed ROS-associated genes in MPs found in the Peruzzotti-Jametti et al 6 dataset. o , Heatmap of marker expression from annotated immune clusters during peak EAE. p, Overlayed ROS (DCFDA) expression. q, Dot plots depict the gating strategy used to calculate the infiltrated immune cell counts, Live CD45+CD44+, DCFDA and Mitosox expression in Microglia (Microglia+BAMs) and Monocyte derived cells (MdCs). r, Biaxal contour plot of CD44 (y-axis) and DCFDA (x-axis) expression in MdCs and microglia. For panels l and m , circle size depicts the percentage of expression detected per cluster, and color indicates the expression level as the average module score or average expression.

Article Snippet: Cell sorting of live CD11b + CD45 + CX3CR1 + CD44 - microglia and CD11b + CD45 + CX3CR1 + CD44 + Ly6C + Ly6G - MdCs was performed using a BD FACS Aria III (FACS DIVA Software v9).

Techniques: Control, Derivative Assay, Expressing, Marker

Journal: bioRxiv

Article Title: Monocyte-derived cells but not Microglia cause Oxidative Tissue Damage in Neuroinflammation

doi: 10.1101/2024.09.18.612891

Figure Lengend Snippet: a, Dot plot illustrating Cybb expression in homeostatic microglia (hMicro), damage-associated microglia (DAMs), and monocyte-derived cell clusters (MdCs) from Peruzzotti-Jametti et al during control (non-immunized), peak and chronic phases of EAE. Circle size is depicting % expression detected per cluster and color is depicting expression level as Average Module Score or Average Expression. b, Relative gene expression Cybb measured by qPCR of FC-sorted Microglia (n=6) and MdCs (n=5) during peak EAE. (Mann–Whitney test; **P = 0.0022). c,d, Passive EAE was induced by adoptive transfer of primed lymphocytes from actively immunized donor mice. Plots depict mean clinical score ( c ) overtime (two-way ANOVA, and Sidak’s post hoc test) and ( d ) cumulative clinical score Ccr2 +/+ Cybb fl /fl (n=5, f) and Ccr2 CreERT2/+ Cybb fl /fl (n=6, f) mice (unpaired two-tailed t-test). e, ROS production measured by DCFDA median expression in manually gated (FlowJo) microglia and MdC from Ccr2 +/+ Cybb fl /fl (n=7, m/f) and Ccr2 CreERT2/+ Cybb fl /fl (n=8, m/f) mice (unpaired two-tailed t-test). f, ROS production measured by DCFDA median expression in manually gated (FlowJo) microglia and MdC from Cx3cr1 +/+ Cybb fl /fl (n=4, m/f) and Cx3cr1 CreERT2/+ Cybb fl /fl (n=5, m/f) mice. Tamoxifen regimen corresponds to early tamoxifen treatment (unpaired two-tailed t-test). g, ROS production measured by DCFDA median expression in microglia and MdC clusters (generated in R) in Cx3cr1 +/+ Cybb fl /fl (n=6, m/f) and Cx3cr1 CreERT2/+ Cybb fl /fl (n=6, m/f) mice (unpaired two-tailed t-test).

Article Snippet: Cell sorting of live CD11b + CD45 + CX3CR1 + CD44 - microglia and CD11b + CD45 + CX3CR1 + CD44 + Ly6C + Ly6G - MdCs was performed using a BD FACS Aria III (FACS DIVA Software v9).

Techniques: Expressing, Derivative Assay, Control, MANN-WHITNEY, Adoptive Transfer Assay, Two Tailed Test, Generated

Journal: bioRxiv

Article Title: Monocyte-derived cells but not Microglia cause Oxidative Tissue Damage in Neuroinflammation

doi: 10.1101/2024.09.18.612891

Figure Lengend Snippet: a, Schematic illustration of the tamoxifen treatment strategy to target Cybb in MdCs during neuroinflammation. b , Relative gene expression of Cybb / Pol2 in CNS microglia and MdCs in Ccr2 +/+ Cybb fl /fl (n=3, f) and Ccr2 CreERT2/+ Cybb fl /fl (n=4, f) mice (unpaired two-tailed t-test, **p=0.0043 in MdCs). c-d , Active EAE was induced by MOG 35-55 /CFA/PT immunization. Plots depict mean clinical score ( c ) over time (two-way ANOVA, and Sidak’s post hoc test) and ( d ) cumulative clinical score Ccr2 +/+ Cybb fl /fl (n=11, m/f) and Ccr2 CreERT2/+ Cybb fl /fl (n=10, m/f) mice (unpaired two-tailed t-test). e, Absolute counts of CNS infiltrating immune cells (gated in FlowJo CD45+CD44+, gating strategy ) at 19 d.p.i. in Cx3cr1 +/+ Cybb fl /fl (n=5, f) and Cx3cr1 CreERT2/+ Cybb fl /fl (n=4,f) mice. Unpaired two-tailed t-test. f, Schematic representation of tamoxifen treatment to target embryonic hematopoiesis-derived macrophages, but not bone marrow derived MdCs. g, Relative gene expression of Cybb / Pol2 in CNS microglia and MdCs in Cx3cr1 +/+ Cybb fl /fl (Microglia n=6, MdCs=2 f) and Cx3cr1 CreERT2/+ Cybb fl /fl (Microglia n=5, MdCs n=2, f) mice (unpaired two-tailed t-test, **p=0.0043 in Microglia). h-i, Active EAE was induced by MOG 35-55 /CFA/PT immunization. Plots show mean clinical score over time (Two-way ANOVA, and Sidak’s post hoc test) ( h ) and cumulative clinical score ( i ) in Cx3cr1 +/+ mCAT fl/wt (n=15, m/f) and Cx3cr1 CreERT2/+ mCAT fl/wt (n=15, m/f) mice (unpaired two-tailed t-test). j, Absolute counts of CNS infiltrating immune cells (gated in flowjo CD45+CD44+, gating strategy ) at 19 d.p.i. in Cx3cr1 +/+ Cybb fl /fl (n=6, f) and Cx3cr1 CreERT2/+ Cybb fl /fl (n=10, f) mice. Unpaired two-tailed t-test. k , Schematic illustration of the tamoxifen treatment strategy to target Cybb in microglia and MdCs during neuroinflammation. l , Relative gene expression of Cybb / Pol2 in CNS microglia and MdCs in Cx3cr1 +/+ Cybb fl /fl (n=4, f) and Cx3cr1 CreERT2/+ Cybb fl /fl (n=4, f) mice (unpaired two-tailed t-test, **p=0.0036 in Microglia and **p=0.0063 in MdCs). m-n , Active EAE was induced by MOG 35-55 /CFA/PT immunization. Plots depict mean clinical score ( m ) over time (two-way ANOVA, and Sidak’s post hoc test) and ( n) cumulative clinical score Cx3cr1 +/+ Cybb fl /fl (n=11, m/f) and Cx3cr1 CreERT2/+ Cybb fl /fl (n=8, m/f) mice (unpaired two-tailed t-test). o, Absolute counts of CNS infiltrating immune cells (gated in FlowJo CD45+CD44+, gating strategy ) at 19 d.p.i. in Cx3cr1 +/+ Cybb fl /fl (n=6, f) and Cx3cr1 CreERT2/+ Cybb fl /fl (n=6, f) mice.

Article Snippet: Cell sorting of live CD11b + CD45 + CX3CR1 + CD44 - microglia and CD11b + CD45 + CX3CR1 + CD44 + Ly6C + Ly6G - MdCs was performed using a BD FACS Aria III (FACS DIVA Software v9).

Techniques: Expressing, Two Tailed Test, Derivative Assay

Journal: bioRxiv

Article Title: Monocyte-derived cells but not Microglia cause Oxidative Tissue Damage in Neuroinflammation

doi: 10.1101/2024.09.18.612891

Figure Lengend Snippet: a , DotPlots illustrating mitochondria complex I module score in CNS microglia and MdCs in the Peruzzotti-Jametti et al . dataset in peak and chronic EAE compared to control CNS. Circle size is depicting % expression detected per cluster and color is depicting expression level as Average Module Score. b , Clustered heatmap of the expression of the mitochondria complex I genes in MPs found in the Peruzzotti-Jametti et al . dataset. c , Immunoblot showing HA-tag and actin bands, in FACs sorted microglia and MdCs from Cx3cr1 +/+ mCAT fl/wt and Cx3cr1 CreERT2/+ mCAT fl/wt mice (pooled cells from n=3 mice, m/f). d , Immunoblot showing HA-tag and actin bands, in FACs sorted microglia and MdCs early treatment tamoxifen regimen Cx3cr1 CreERT2/+ mCAT fl/wt mice (pooled cells from n=2 mice, m/f). e-f , Representative MBP staining (left) and the percentage of myelin loss quantified in lumbar spinal cord sections in early treatment tamoxifen regimen Cx3cr1 +/+ mCAT fl/wt (n=5, f) and Cx3cr1 CreERT2/+ mCAT fl/wt (n=4, f) mice (right). Unpaired two-tailed t-test.

Article Snippet: Cell sorting of live CD11b + CD45 + CX3CR1 + CD44 - microglia and CD11b + CD45 + CX3CR1 + CD44 + Ly6C + Ly6G - MdCs was performed using a BD FACS Aria III (FACS DIVA Software v9).

Techniques: Control, Expressing, Western Blot, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Monocyte-derived cells but not Microglia cause Oxidative Tissue Damage in Neuroinflammation

doi: 10.1101/2024.09.18.612891

Figure Lengend Snippet: a, Schematic illustration of the tamoxifen treatment strategy employed to reduce mtROS in CX3CR1 expressing MPs. b , ROS production measured by normalized DCFDA expression in microglia and MdC clusters (generated in R) of Cx3cr1 +/+ mCAT fl/wt (n=6, f) and Cx3cr1 CreERT2/+ mCAT fl/wt (n=4, f). Pooled data from two experiments (unpaired two-tailed t-test, *P=0.04 in Microglia and **P=0.0071 in MdCs). c , Mitochondrial ROS (mtROS) production measured by mitosox expression in manually gated (FlowJo) microglia and MdC of Cx3cr1 +/+ mCAT fl/wt (n=4, f) and Cx3cr1 CreERT2/+ mCAT fl/wt (n=3 f). Unpaired two-tailed t-test (*P=0.0237 in Microglia and *P=0.0363 in MdCs). d - e , Active EAE was induced by MOG 35-55 /CFA/PT immunization. ( d ) Plots show mean clinical score over time (two-way ANOVA, and Sidak’s post hoc test, day 13 **P=0.0022, day 14 **P=0.0023, day 15 **P=0.0031, day 16 **P=0.0059, day 18 *P=0.0453, day 19 *P=0.0109) and ( e ) cumulative clinical score in Cx3cr1 +/+ mCAT fl/wt (n=8, f) and Cx3cr1 CreERT2/+ mCAT fl/wt (n=8, f) mice. Unpaired two-tailed t-test (*P=0.0405). f, Absolute counts of CNS infiltrating immune cells (gated in FlowJo CD45+CD44+, gating strategy ) at 19 d.p.i. in Cx3cr1 +/+ mCAT fl/wt (n=5, f) and Cx3cr1 CreERT2/+ mCAT fl/wt (n=4, f) mice. Unpaired two-tailed t-test (*P=0.0091). g, Schematic representation of tamoxifen treatment to target embryonic hematopoiesis-derived macrophages, but not bone marrow derived MdCs . h , ROS production measured by DCFDA median expression in manually gated (FlowJo) microglia from Cx3cr1 +/+ mCAT fl/wt (n=4, f) and Cx3cr1 CreERT2/+ mCAT fl/wt (n=4, f)) in steady state CNS (unpaired two-tailed t-test; *P=0.0135). i , mtROS production measured as mitosox median expression in manually gated (FlowJo) microglia from Cx3cr1 +/+ mCAT fl/wt (n=4, f) and Cx3cr1 CreERT2/+ mCAT fl/wt (n=4, f) in steady state CNS (unpaired two-tailed t-test; *P=0.0423). j-k, Active EAE was induced by MOG 35-55 /CFA/PT immunization. Plots show mean clinical score over time (Two-way ANOVA, and Sidak’s post hoc test) ( j ) and cumulative clinical score ( k ) in Cx3cr1 +/+ mCAT fl/wt (n=10, m/f) and Cx3cr1 CreERT2/+ mCAT fl/wt (n=7, m/f) mice. Unpaired two-tailed t-test. Pooled data from two experiments. l , Absolute counts of CNS infiltrating immune cells (gated in FlowJo CD45+CD44+, gating strategy ) at 19 d.p.i. in Cx3cr1 +/+ mCAT fl/wt (n=5, f) and Cx3cr1 CreERT2/+ mCAT fl/wt (n=4, f) mice. Unpaired two-tailed t-test.

Article Snippet: Cell sorting of live CD11b + CD45 + CX3CR1 + CD44 - microglia and CD11b + CD45 + CX3CR1 + CD44 + Ly6C + Ly6G - MdCs was performed using a BD FACS Aria III (FACS DIVA Software v9).

Techniques: Expressing, Generated, Two Tailed Test, Derivative Assay

Journal: bioRxiv

Article Title: Monocyte-derived cells but not Microglia cause Oxidative Tissue Damage in Neuroinflammation

doi: 10.1101/2024.09.18.612891

Figure Lengend Snippet: a , Schematic representation of tamoxifen treatment to target bone marrow derived MdCs during EAE disease onset. b , ROS production measured by normalized DCFDA median expression in microglia and MdC clusters (generated in R) Ccr2 +/+ mCAT fl/ wt (n=5, m/f) and Ccr2 CreERT2/+ mCAT fl/ wt (n=5, m/f) mice. Pooled data from two experiments (unpaired two-tailed t-test; **P=0.0054 in MdCs). c , mtROS production measured as mitosox median expression in manually gated (FlowJo) microglia and MdCs from Ccr2 +/+ mCAT fl/wt (n=6, m/f) and Ccr2 CreERT2/+ mCAT fl/ wt (n=5, m/f) mice. Pooled data from two experiments (unpaired two-tailed t-test, **P=0.003 in MdCs). d - e , Active EAE was induced by MOG 35-55 /CFA/PT immunization. Plots show mean clinical score over time (Two-way ANOVA, and Sidak’s post hoc test, day 19 **P=0.0078) ( d ) and cumulative clinical score ( e ) in Ccr2 +/+ mCAT fl/wt (n=14, m/f) and Ccr2 CreERT2/+ mCAT fl/wt (n=14, m/f) mice (unpaired two-tailed t-test; *P = 0.0339). Pooled data from two experiments. f, Representative MBP staining (left) and g , the percentage of myelin loss quantified in lumbar spinal cord sections in Ccr2 +/+ mCAT fl/wt (n=9, m/f) and Ccr2 CreERT2/+ mCAT fl/wt (n=8, m/f) mice (unpaired two-tailed t-test; **P = 0.0163). Data pooled from two independent experiments. h, Absolute counts of CD45+CD44+ CNS infiltrating immune cells (gated in FlowJo) at day 17 d.p.i. (n=5 per group, f). Unpaired two-tailed t-test (*P = 0.0164). i, UMAP from CNS leukocytes clustered by cell type and labelled based on surface marker expression. j, Bar graph of the percentage of the immune clusters found in in Ccr2 +/+ mCAT fl/wt and Ccr2 CreERT2/+ mCAT fl/wt (n=5 per group, f). k , Absolute counts of CNS infiltrating immune CD4 T cells (left) and Ly6Chi monocytes (right) (analysed in R) at day 17 d.p.i. (n=5 per group, f), (unpaired two-tailed t-test, *P=0.0386 in CD4 and *P=0.0403 in Ly6cHi). l, Radar plot showing changes in the marker expression of CNS immune cell clusters in Ccr2 CreERT2/+ mCAT fl/wt relative to Ccr2 +/+ mCAT fl/wt and (n=5 per group). Values correspond to −log10(p value) and were adjusted with the Benjamini-Hochberg correction. Bar color denotes the upregulation or downregulation of the respective marker. Marker order is consistent across subsets, left to right: CD38, MHCII, CD44, CX3CR1, F480, CD11C, and MerTK. Dashed line denotes −log10(0.05) cutoff for p value. m-n Passive EAE was induced by adoptive transfer of primed lymphocytes from actively immunized donor mice. Plots show ( m ) mean clinical score over time (two way ANOVA, and Sidak’s post hoc test, day 10 *P=0.0254, day 11 **P=0.0020, day 12 **P=0.0030, day 13 **P=0.0025, day 14 ***P=0.0006, day 15 **P=0.0076, day 16 **P=0.0019, day 17 **P=0.0020, day 18 **P=0.0013, day 19 **P=0.0039, day 20 ***P=0.0007) and ( n ) cumulative clinical score Ccr2 +/+ mCAT fl/wt (n=8, m/f) and Ccr2 CreERT2/+ mCAT fl/wt (n=9, m/f) mice (pooled data from two experiments, unpaired two-tailed t-test; ***P = 0.0009).

Article Snippet: Cell sorting of live CD11b + CD45 + CX3CR1 + CD44 - microglia and CD11b + CD45 + CX3CR1 + CD44 + Ly6C + Ly6G - MdCs was performed using a BD FACS Aria III (FACS DIVA Software v9).

Techniques: Derivative Assay, Expressing, Generated, Two Tailed Test, Staining, Marker, Adoptive Transfer Assay

Journal: bioRxiv

Article Title: Monocyte-derived cells but not Microglia cause Oxidative Tissue Damage in Neuroinflammation

doi: 10.1101/2024.09.18.612891

Figure Lengend Snippet: a , Immunoblot showing HA-tag and Actin bands, in FACS sorted microglia and MdCs from Ccr2 CreERT2/+ mCAT fl/ wt (pooled cells from n=3 mice). b , Heatmap showing the median marker expression across the identified main clusters, UMAP is shown in . c, Absolute counts of CNS infiltrating Neutrophils, CD8 T cells, B cells, Monocyte derived cells (MdCs), Dendritic cells (DCs) and CNS resident immune Microglia (R generated). d-k Median fluorescent intensity (MFI) of activation markers (R generated) in ( d ) MdCs, ( e ) Neutrophils, ( f ) Ly6cHi Monocytes, ( g ) Microglia, ( h ) Dendritic cells, ( i ) B cells, ( j ) CD4 T cells and ( k ) CD8 T cells. (For MFI of CD11C in Ly6cHi Monocytes, unpaired two-tailed t-test. **P=0.0066). Mice analyzed at 17 d.p.i (n=5 per group, f), (unpaired two-tailed t-test).

Article Snippet: Cell sorting of live CD11b + CD45 + CX3CR1 + CD44 - microglia and CD11b + CD45 + CX3CR1 + CD44 + Ly6C + Ly6G - MdCs was performed using a BD FACS Aria III (FACS DIVA Software v9).

Techniques: Western Blot, Marker, Expressing, Derivative Assay, Generated, Activation Assay, Two Tailed Test