Journal: JCI Insight

Article Title: Blocking IL-6 signaling prevents astrocyte-induced neurodegeneration in an iPSC-based model of Parkinson’s disease

doi: 10.1172/jci.insight.163359

Figure Lengend Snippet: ( A ) Representative immunocytochemistry (ICC) images of CTL (SP09) and L2-PD astrocytes (SP13) expressing DAPI (blue) and GFAP (green) after 14 days in culture. Scale bar: 100 μm. CTL astrocytes (SP09) treated for 48 hours with C1q, TNF-α, and IL-1α were used as positive control (CTL+CS). Images on the right show a magnification of the area boxed in the left images. Scale bar: 10 μm. Yellow arrowheads indicate high GFAP staining in hypertrophic astrocytes. ( B ) Mean intensity of GFAP staining. ( C ) Form factor of GFAP + cells calculated as: FF = 4 Pi number (π) (area/perimeter 2 ). ( D ) Representative ICC images of CTL, L2-PD, and activated CTL astrocytes expressing Vimentin (CTL: SP09; L2-PD: SP13), AQP4 (CTL: SP17; L2-PD: SP06), and C3 (CTL: SP09; L2-PD: SP13). Scale bar: 100 μm. Images on the right show a magnification of the area boxed in the left images. Scale bar: 10 μm. Yellow arrowheads indicate high expression of the specific marker shown in the images. ( E ) Mean intensity of C3 staining with respect to CTL. ( F ) Relative mRNA expression of panreactive, A1-specific and A2-specific transcripts in L2-PD astrocytes with respect to CTL. Box-and-whisker plots show median, 25th and 75th percentiles, minimum, and maximum values ( n = 3 experiments; form factor, mean GFAP, and C3 intensity were performed from 30 astrocytes per experiment per condition). One-way ANOVA was used, with Bonferroni as post hoc. * P < 0.05; ** P < 0.01; *** P < 0.001. ( G ) Heatmap of differentially expressed genes (DEG) of CTL and L2-PD astrocytes. ( H ) Enrichment plot showing the enrichment score for the selected GO BP gene sets (e.g., antigen processing and presentation of exogenous peptide antigen via MHC class II, B cell mediated immunity, complement activation, humoral immune response mediated by circulating immunoglobulin, immunoglobulin production, negative regulation of neuron differentiation, peptide antigen assembly with MHC protein complex, response to IL-6).

Article Snippet: Antibodies used were: rabbit polyclonal antibodies against IL-6 (Abcam, ab6672) diluted 1:100; rabbit polyclonal anti–IL-6R (R&D Systems, MAB2271) diluted at 1:50; anti-GFAP antibody (Diagnostic BioSystems, Mob064) diluted at 1:1,000, and rabbit anti-TH (MilliporeSigma, T8700) diluted 1:100.

Techniques: Immunocytochemistry, Expressing, Positive Control, Staining, Marker, Whisker Assay, Activation Assay

Journal: JCI Insight

Article Title: Blocking IL-6 signaling prevents astrocyte-induced neurodegeneration in an iPSC-based model of Parkinson’s disease

doi: 10.1172/jci.insight.163359

Figure Lengend Snippet: ( A ) Schematic experimental set up to perform a cytokine array from astrocyte conditioned medium (ACM). ( B ) Cytokine IL-6 levels released by CTL, L2-PD, and isogenic (L2-PD corr ) astrocytes after 2 weeks in culture. CTL astrocytes were treated for 48 hours with C1q, TNF-α, and IL-1α as positive control. ( C ) Representative ICC images of L2-PD (SP13) and L2-PD corr (SP13wt/wt) astrocytes staining positive for: top panels, DAPI (blue), GFAP (white), and AQP4 (red); bottom panels, DAPI (blue), GFAP (white), and C3 (red). Scale bar: 100 μm. Images on the right show a magnification of the area boxed in the left images. Scale bar: 10 μm. ( D ) Mean intensity of GFAP staining. ( E ) Form factor of GFAP + cells calculated as: FF = 4 pi (area/perimeter 2 ). ( F ) Mean intensity of C3 staining with respect to L2-PD astrocytes. ( G ) Representative ICC images of L2-PD (SP13) treated with either DMSO or LRRK2-kinase inhibitor (1 μM) astrocytes staining positive for: top panels, DAPI (blue), GFAP (white), and AQP4 (red); bottom panels, DAPI (blue), GFAP (white), and C3 (red). Scale bar: 100 μm. Images on the right show a magnification of the area boxed in the left images. Scale bar: 10 μm. ( H ) Mean intensity of GFAP staining. ( I ) Form factor of GFAP + cells calculated as: FF = 4 pi (area/perimeter 2 ). ( J ) Mean intensity of C3 staining with respect to L2-PD astrocytes treated with DMSO. Box-and-whisker plots show median, 25th and 75th percentiles, minimum, and maximum values ( n = 3 experiments; form factor, mean GFAP, and C3 intensity were performed from 30 astrocytes per experiment per condition). One-way ANOVA was used with Bonferroni as post hoc. Student t test or Mann-Whitney U test for nonparametric conditions were used when only 2 groups were compared. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Antibodies used were: rabbit polyclonal antibodies against IL-6 (Abcam, ab6672) diluted 1:100; rabbit polyclonal anti–IL-6R (R&D Systems, MAB2271) diluted at 1:50; anti-GFAP antibody (Diagnostic BioSystems, Mob064) diluted at 1:1,000, and rabbit anti-TH (MilliporeSigma, T8700) diluted 1:100.

Techniques: Positive Control, Staining, Whisker Assay, MANN-WHITNEY

Journal: JCI Insight

Article Title: Blocking IL-6 signaling prevents astrocyte-induced neurodegeneration in an iPSC-based model of Parkinson’s disease

doi: 10.1172/jci.insight.163359

Figure Lengend Snippet: ( A ) Schematic representation of the experimental procedure to analyze the effect of astrocyte conditioned medium (ACM) and IL-6 involvement on neuronal survival and degeneration. ACM was collected after 14 days in culture and added to iPSC-derived DAn from both CTL and patients with L2-PD for 1 week. Anti–IL-6R antibody Tocilizumab (10 μg/mL) was added to ACM. ( B ) Representative ICC images of iPSC-derived CTL (SP11) and L2-PD neurons (SP12) expressing tyrosine hydroxylase (TH, black) treated for 1 week with CTL-ACM, L2-PD ACM and L2-PD ACM + anti–IL-6R antibody. Scale bar: 100 μm. CTL neurons are derived from SP11 iPSC line. L2-PD neurons are derived from SP12, SP06, and SP13 iPSC lines. ( C ) Percentage of TH + cells respect to DAPI. ( D ) Representative ICC images of iPSC-derived CTL and L2-PD neurons staining for TH (black). Scale bar: 20 μm. ( E and F ) Number of branches and neurite length of CTL and L2-PD TH + neurons treated for 1 week with ACM. ( G ) Representative ICC images of DAn (TH, red) expressing IL-6R (green) from CTL (SP11) and L2-PD (SP13) iPSC-derived DAn after 35 days of differentiation. Scale bar: 20 μm. ( H ) Orthogonal views show colocalization between IL-6R and TH in L2-PD (SP13) DAn. ( I ) Representative Western blot for IL-6R of iPSC-derived CTL neurons (SP11) and L2-PD neurons (SP12). ( J ) Quantification of protein IL-6R respect to β-actin. ( K ) Relative IL-6R mRNA expression from iPSC-derived CTL neurons (SP11 and SP17) and L2-PD neurons (SP12 and SP13). Box-and-whisker plots show median, 25th and 75th percentiles, minimum, and maximum values ( n = 3 experiments; 30 neurons per experiment per condition for each line). One-way ANOVA was used with Bonferroni as post hoc ( C , E , F ). Student t test or Mann-Whitney U test ( J and K ) for nonparametric conditions were used when only 2 groups were compared. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Antibodies used were: rabbit polyclonal antibodies against IL-6 (Abcam, ab6672) diluted 1:100; rabbit polyclonal anti–IL-6R (R&D Systems, MAB2271) diluted at 1:50; anti-GFAP antibody (Diagnostic BioSystems, Mob064) diluted at 1:1,000, and rabbit anti-TH (MilliporeSigma, T8700) diluted 1:100.

Techniques: Derivative Assay, Expressing, Staining, Western Blot, Whisker Assay, MANN-WHITNEY

Journal: JCI Insight

Article Title: Blocking IL-6 signaling prevents astrocyte-induced neurodegeneration in an iPSC-based model of Parkinson’s disease

doi: 10.1172/jci.insight.163359

Figure Lengend Snippet: ( A ) Representative ICC images of CTL (SP09) or ID-PD (ID-PD1: SP04; ID-PD2: SP08; ID-PD3: SP16) astrocytes staining positive for: top panels, DAPI (blue) and GFAP (green); bottom panels, DAPI (blue) and C3 (red). CTL astrocytes treated for 48 hours with cytokines were used as positive control. Scale bar: 100 μm. ( B ) Form factor of GFAP + cells calculated as: FF = 4 pi (area/perimeter 2 ). ( C ) Mean intensity of GFAP staining. ( D ) Mean intensity of C3 staining with respect to CTL. ( E ) IL-6 protein levels released by CTL and ID-PD ACM after 2 weeks in culture. Box-and-whisker plots show median, 25th and 75th percentiles, minimum, and maximum values ( n = 3 experiments; form factor, mean GFAP, and C3 intensity were performed from 30 astrocytes per experiment per condition). ( F ) Representative ICC images of iPSC-derived CTL (SP11) and L2-PD (SP12) neurons expressing tyrosine hydroxylase (TH, black) treated for 1 week with CTL astrocyte conditioned medium (ACM), idiopathic (ID-PD) ACM, and ID-PD ACM + anti–IL-6R Tocilizumab (10 μg/mL). Scale bar: 100 μm. ( G ) Percentage of TH + cells respect to DAPI. ( H and I ) Number of branches and neurite length of TH + neurons cultured with ACM for 1 week. Box-and-whisker plots show median, 25th and 75th percentiles, minimum, and maximum values ( n = 3 experiments; 30 neurons per experiment per condition for each line). One-way ANOVA was used with Bonferroni as post hoc. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Antibodies used were: rabbit polyclonal antibodies against IL-6 (Abcam, ab6672) diluted 1:100; rabbit polyclonal anti–IL-6R (R&D Systems, MAB2271) diluted at 1:50; anti-GFAP antibody (Diagnostic BioSystems, Mob064) diluted at 1:1,000, and rabbit anti-TH (MilliporeSigma, T8700) diluted 1:100.

Techniques: Staining, Positive Control, Whisker Assay, Derivative Assay, Expressing, Cell Culture

Journal: JCI Insight

Article Title: Blocking IL-6 signaling prevents astrocyte-induced neurodegeneration in an iPSC-based model of Parkinson’s disease

doi: 10.1172/jci.insight.163359

Figure Lengend Snippet: ( A ) Representative immunohistochemical sections of the mesencephalon including the substantia nigra pars compacta from control and PD cases at early (Braak stages 1–3) and late stages (Braak stages 4 and 5) of the disease. IL-6 (visualized with DAB) is present in astrocytes (GFAP + cells; nickel). Scale bar: 25 μm. Images on the upper right show a magnification of the area boxed in the main images. Scale bar: 10 μm. ( B ) Percentage of GFAP + cells expressing IL-6 over total GFAP + cells. ( C ) IL-6R (green) is localized in DAn in PD cases (Braak stage 2) as visualized by TH immunofluorescence (red); autofluorescence is blocked by preincubating the sections with Sudan black. Scale bar: 100 μm. Asterisks show colocalization of TH + cells expressing IL-6R. ( D ) Representative immunofluorescence images showing increased IL-6R immunoreactivity in PD substantia nigra pars compacta (Braak stage 2) as compared with controls. Scale bar: 25 μm. Asterisks show neurons with IL-6R expression. ( E ) Percentage of IL-6R + staining area per image/total image area. Postmortem brain samples include: 3 healthy donors, 1 PD (Braak stage 1), 2 PD (Braak stage 2), 2 PD (Braak stage 3), 1 PD (Braak stage 4), and 2 PD (Braak stage 5). We counted 50 astrocytes per individual (GFAP + ) from 6–8 images each. One-way ANOVA was used with Bonferroni as post hoc. Student t test was used when only 2 groups were compared. ** P < 0.01; *** P < 0.001.

Article Snippet: Antibodies used were: rabbit polyclonal antibodies against IL-6 (Abcam, ab6672) diluted 1:100; rabbit polyclonal anti–IL-6R (R&D Systems, MAB2271) diluted at 1:50; anti-GFAP antibody (Diagnostic BioSystems, Mob064) diluted at 1:1,000, and rabbit anti-TH (MilliporeSigma, T8700) diluted 1:100.

Techniques: Immunohistochemical staining, Expressing, Immunofluorescence, Staining

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Panax notoginseng Alleviates Sepsis-Induced Acute Kidney Injury by Reducing Inflammation in Rats

doi: 10.1155/2022/9742169

Figure Lengend Snippet: PNP markedly reduced the secretion of peripheral blood cytokines induced by CLP in vivo . IL-18, IL-1 β , TNF- α , and IL-6 levels in kidney tissues were determined by ELISA. The results shown are representative of at least three independent experiments. Each value represents the mean ± SD.

Article Snippet: After that, TMA sections were incubated with rabbit antihuman primary polyclonal antibodies against IL-1 β , TNF- α , and IL-6 (Cell Signaling Technology, CST) overnight at 4°C and then incubated with a biotin-labeled secondary antibody (Invitrogen, Carlsbad, CA) at room temperature for 15 min, followed by incubation with HRP-conjugated streptavidin (Invitrogen, Carlsbad, CA) at room temperature for 15 min. Then, the color development was performed with a DAB Substrate Kit (Dako, Glostrup, Denmark).

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Panax notoginseng Alleviates Sepsis-Induced Acute Kidney Injury by Reducing Inflammation in Rats

doi: 10.1155/2022/9742169

Figure Lengend Snippet: PNP attenuated the expression of cytokines in the kidney tissues induced by CLP in vivo . The expression of IL-1 β , TNF- α , and IL-6 levels in kidney tissues was determined by immunohistochemical staining. The results shown are representative of at least three independent experiments. Scale bar: 10 μ m.

Article Snippet: After that, TMA sections were incubated with rabbit antihuman primary polyclonal antibodies against IL-1 β , TNF- α , and IL-6 (Cell Signaling Technology, CST) overnight at 4°C and then incubated with a biotin-labeled secondary antibody (Invitrogen, Carlsbad, CA) at room temperature for 15 min, followed by incubation with HRP-conjugated streptavidin (Invitrogen, Carlsbad, CA) at room temperature for 15 min. Then, the color development was performed with a DAB Substrate Kit (Dako, Glostrup, Denmark).

Techniques: Expressing, In Vivo, Immunohistochemical staining, Staining