Journal: PLoS Pathogens

Article Title: Synergistic Induction of Interferon α through TLR-3 and TLR-9 Agonists Identifies CD21 as Interferon α Receptor for the B Cell Response

doi: 10.1371/journal.ppat.1003233

Figure Lengend Snippet: (A) Spleen cells from cotton rats were stimulated with Concanavalin A for 72 hours and lymphoblasts purified by centrifugation on a ficoll-gradient. Subsequently, cells were stained with antisera specific for the human interferon receptor to test for crossreactive binding to cotton rat cells and analyzed by flow cytometry. Antibody specific for human interferon alpha receptor-1 (IFNAR-1) did not react with cotton rat cells (left) whereas antibody specific for human interferon alpha receptor-2 (IFNAR-2) did (right). IFNR specific antibody is shown as black line, controls as gray areas. B. IFNAR-2 antibody was added to cotton rat spleen cells. One hour later different concentrations of a combination of ODN2216 and poly I:C was added. 24 hours later supernatant was harvested and tested for the presence of type I interferon. The data is combined from two independent experiments. Significant differences were seen between cells treated with the combination of ODN2216 and poly I:C alone, or the combination of ODN2216 and poly I:C with antibody. IFNAR-1 antibody (not reactive with cotton rat cells) had no effect. C. Cotton rat spleen cells were stimulated with ODN 2216 or poly I:C, with or without the addition of recombinant cotton rat interferon alpha. (One unit equals one picogram of type I interferon.) 24 hours later supernatant was harvested and tested for the presence of type I interferon. The data is combined from two independent experiments, and significance was compared to cells without type I interferon treatment. Statistical analysis was done by ANOVA (* p<0.05, ** p<0.01, *** p<0.001).

Article Snippet: Neutralizing goat antibodies against cotton rat interferon alpha and IL-6 were purchased from R&D systems.

Techniques: Purification, Centrifugation, Staining, Binding Assay, Flow Cytometry, Recombinant

Journal: PLoS Pathogens

Article Title: Synergistic Induction of Interferon α through TLR-3 and TLR-9 Agonists Identifies CD21 as Interferon α Receptor for the B Cell Response

doi: 10.1371/journal.ppat.1003233

Figure Lengend Snippet: A) The number of MeV-specific B cells was measured from bone marrow cells of MeV-immune cotton rats. The addition of ODN 2216 and poly I:C individually and in combination increased B cell numbers whereas the addition of sera neutralizing cotton rat interferon alpha and IL-6 reduced this stimulation. Each bar graph represents the mean ± SD of triplicate wells. B) Cotton rats were immunized intranasally (upper panel) or subcutaneously (lower panel) with MeV, or MeV with ODN 2216 and/or pI:C in the presence or absence of human MeV-specific IgG (neutralization titer of 100) which had been injected intraperitoneally one day before immunization. Sera were collected at seven weeks post vaccination and the titer of neutralizing antibody was determined by neutralization assay. Each bar graph represents the average titer of four animals ± SD. The experiment is representative of three experiments. Statistical analysis was done by ANOVA (* p<0.05, ** p<0.01, *** p<0.001).

Article Snippet: Neutralizing goat antibodies against cotton rat interferon alpha and IL-6 were purchased from R&D systems.

Techniques: Neutralization, Injection

Journal: Nature medicine

Article Title: cGAS drives non-canonical inflammasome activation in age-related macular degeneration

doi: 10.1038/nm.4450

Figure Lengend Snippet: ( a ) Top, fundus photographs of eyes of WT (n = 7 eyes) and Ifnar −/− (n = 14 eyes) mice subretinally injected with Alu expression plasmid (pAlu) or empty vector (pNull). Bottom, immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of the above eyes showing RPE cell boundaries. ( b ) Immunoblot of pro-caspase-11 (pro-Casp1) and the p30 cleavage product of caspase-11 (Casp11 p30) in WT and Ifnar −/− mouse RPE cells mock transfected or transfected with Alu RNA. ( c ) Immunoblot of pro-caspase-4 in IFN-β-treated human RPE cells. ( d ) IFN-β secretion by human RPE cells transfected with Alu expression plasmid (pAlu) or empty vector (pNull). Data presented are mean ± SD; n = 3 independent experiments; * P = 0.0012, two-tailed t test. ( e ) Immunoblot of phosphorylated STAT2 (pSTAT2) and total STAT2 in human RPE cells transfected with Alu expression plasmid (pAlu) or empty vector (pNull) or DICER1 or control (Ctr) anti-sense oligonucleotides (AS). ( f ) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of WT mice subject to Alu RNA co-administration with IFN-β neutralizing antibody (n = 6 eyes) or Isotype IgG (n = 4 eyes). ( g ) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of WT (n = 6 eyes) or Irf3 −/− (n = 6 eyes) or Stat2 −/− (n = 7 eyes) mice subretinally injected with Alu expression plasmid (pAlu) or empty vector (pNull). ( h ) Immunolocalization of IFN-β in the RPE of human geographic atrophy eyes and age-matched unaffected controls. Representative image from control and Dry AMD eyes are presented, n = 4 eyes. ( i ) Abundance of IFN-β mRNA in the RPE of the human geographic atrophy eyes compared to age-matched healthy controls, (Data presented are mean ± SEM; n = 4 eyes; * P = 0.018, two-tailed t test). For all immunoblots, cropped gel image of bands of interest of representative immunoblots of three independent experiments and densitometric analysis (mean (SEM)) are shown. In a , f and g , binary (Healthy %) and morphometric (PM, polymegethism (mean (SEM)) quantification of RPE degeneration are shown (Fisher’s exact test for binary; two-tailed t test for morphometry; * P < 0.05; * *P < 0.01; ** *P < 0.001). Loss of regular hexagonal cellular boundaries in ZO-1 stained flat mounts is indicative of degenerated RPE. The degenerated retinal area is outlined by blue arrowheads in the fundus images.

Article Snippet: Briefly, immunohistochemical staining of fixed human eyes was performed with rabbit antibody against cGAS (0.1 μg/ml, Sigma-Aldrich, Cat# HPA031700) or interferon β (0.2 μg/ml, Santa Cruz Biotechnology, Cat# sc-20107) and mouse antibody against gasdermin D (1.5 μg/ml, Abcam, Cat# ab57785).

Techniques: Injection, Expressing, Plasmid Preparation, Immunofluorescence, Staining, Western Blot, Transfection, Two Tailed Test, Control

Journal: Nature medicine

Article Title: cGAS drives non-canonical inflammasome activation in age-related macular degeneration

doi: 10.1038/nm.4450

Figure Lengend Snippet: ( a ) Relative abundance of Ifnb mRNA in WT and Mb21d1 −/− mouse RPE cells mock-transfected or transfected with Alu RNA. Data presented are mean ± SEM ; n = 4 cell culture replicates; * P = 0.0001, two-tailed t test. ( b ) Immunoblots of pro-caspase-1 (pro-Casp1) and the p20 cleavage product of caspase-1 (Casp1 p20) in WT and Mb21d1 −/− mouse RPE cells transfected with Alu expression plasmid (pAlu) or empty vector control (pNull). ( c ) Immunoblots of pro-caspase-11 (pro-Casp1) and the p30 cleavage product of caspase-1 (Casp11 p30) in WT and Mb21d1 −/− mouse RPE cells transfected with Alu expression plasmid (pAlu) or empty vector control (pNull). ( d ) IL-18 secretion by WT and Mb21d1 −/− mouse RPE cells mock transfected or transfected with Alu RNA. Data presented are mean ± SD ; n = 3 independent experiments; * P = 0.032, two-tailed t test. ( e ) Relative abundance of IFNB mRNA in control (sh Scramble) or cGAS shRNA knockdown human RPE cells transfected with or DICER1 or control (Ctr) anti-sense oligonucleotides (AS). Data presented are mean ± SEM ; n = 3 cell culture replicates; * P = 0.0002, two-tailed t test. ( f ) Immunoblot of phosphorylated STAT2 (pSTAT2); pro-caspase-4 and casp4 p30; pro-caspase-1 and p20 cleavage casp1 p20 in control (sh Scramble) or cGAS shRNA knockdown human RPE cells mock-transfected or transfected with Alu RNA. Knockdown efficiency of cGAS is shown by cGAS immunoblot and tubulin was used as a loading control. ( g ) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of WT (n = 6 eyes) and Mb21d1 −/− (n = 8 eyes) mice subretinally injected with vehicle or Alu RNA. ( h ) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of Mb21d1 −/− (n = 7 eyes) mice reconstituted by in vivo transfection of cGAS expression plasmid (pFlag-cGAS) or control GFP expression plasmid (pFlag-GFP), subretinally injected with Alu RNA. ( i ) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of Mb21d1 −/− mice subretinally co-administered Alu RNA with recombinant IFN-β (n = 6 eyes), vehicle control (n = 6 eyes), IFN-β expression plasmid (pIFNB; n = 5 eyes) or empty vector control (pNull; n = 5 eyes). For all immunoblots, cropped gel image of bands of interest of representative immunoblots of three independent experiments and densitometric analysis (mean (SEM)) are shown. In g , h and i , binary (Healthy %) and morphometric (PM, polymegethism (mean (SEM)) quantification of RPE degeneration are shown (Fisher’s exact test for binary; two-tailed t test for morphometry; * P < 0.05; * *P < 0.01; ** *P < 0.001). Loss of regular hexagonal cellular boundaries in ZO-1 stained flat mounts is indicative of degenerated RPE. The degenerated retinal area is outlined by blue arrowheads in the fundus images.

Article Snippet: Briefly, immunohistochemical staining of fixed human eyes was performed with rabbit antibody against cGAS (0.1 μg/ml, Sigma-Aldrich, Cat# HPA031700) or interferon β (0.2 μg/ml, Santa Cruz Biotechnology, Cat# sc-20107) and mouse antibody against gasdermin D (1.5 μg/ml, Abcam, Cat# ab57785).

Techniques: Transfection, Cell Culture, Two Tailed Test, Western Blot, Expressing, Plasmid Preparation, Control, shRNA, Knockdown, Immunofluorescence, Staining, Injection, In Vivo, Recombinant

Journal: Nature medicine

Article Title: cGAS drives non-canonical inflammasome activation in age-related macular degeneration

doi: 10.1038/nm.4450

Figure Lengend Snippet: ( a ) Immunolocalization of cGAS in the RPE of human geographic atrophy eyes and age-matched unaffected controls. Representative image from control and Dry AMD eyes are presented, n = 4 eyes. ( b ) Immunoblots of pro-caspase-1 and p20 cleavage product of caspase-1 (Casp1 p20) in WT and Tmem173 −/− mouse RPE cells transfected with Alu expression plasmid (pAlu) or empty vector control plasmid (pNull). ( c ) Immunoblots of pro-caspase-11 and p30 cleavage product of caspase-11 (Casp11 p30) in WT and Tmem173 −/− mouse RPE cells mock transfected or transfected with Alu RNA. ( d ) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of WT (n = 6 eyes) and Tmem173 −/− (n = 10 eyes) mice subretinally injected with vehicle or Alu RNA. ( e ) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of Tmem173 −/− mice subretinally co-administered Alu RNA with recombinant IFN-β (n = 4 eyes) or vehicle control (n = 4 eyes); or IFN-β expression plasmid (pIFNB; n = 4 eyes) or empty vector control (pNull; n = 4 eyes). For all immunoblots, cropped gel image of bands of interest of representative immunoblots of three independent experiments and densitometric analysis (mean (SEM)) are shown. In d and e , binary (Healthy %) and morphometric (PM, polymegethism (mean (SEM)) quantification of RPE degeneration are shown (Fisher’s exact test for binary; two-tailed t test for morphometry; * P < 0.05; * *P < 0.01; ** *P < 0.001). Loss of regular hexagonal cellular boundaries in ZO-1 stained flat mounts is indicative of degenerated RPE. The degenerated retinal area is outlined by blue arrowheads in the fundus images.

Article Snippet: Briefly, immunohistochemical staining of fixed human eyes was performed with rabbit antibody against cGAS (0.1 μg/ml, Sigma-Aldrich, Cat# HPA031700) or interferon β (0.2 μg/ml, Santa Cruz Biotechnology, Cat# sc-20107) and mouse antibody against gasdermin D (1.5 μg/ml, Abcam, Cat# ab57785).

Techniques: Control, Western Blot, Transfection, Expressing, Plasmid Preparation, Immunofluorescence, Staining, Injection, Recombinant, Two Tailed Test

Journal: Nature medicine

Article Title: cGAS drives non-canonical inflammasome activation in age-related macular degeneration

doi: 10.1038/nm.4450

Figure Lengend Snippet: ( a ) Relative abundance of cytosolic mitochondrial DNA (mtDNA) in human RPE cells mock-transfected or transfected with Alu RNA (Data presented are mean ± SEM; n = 3 independent experiments; * P = 0.0018, two-tailed t test). ( b ) Relative enrichment of mtDNA in cGAS immunoprecipitate in ChIP like pull-down assay. Mock or Alu RNA transfected, indicated mouse embryonic fibroblast (MEF) were analyzed upon HA-cGAS immunoprecipitation with anti-HA antibody or isotype conrol. Data presented are mean ± SEM; n = 3; * P = 0.008, two-tailed t test. ( c ) Relative abundance of cytosolic mitochondrial DNA (mtDNA) in WT and Ppif −/− mouse RPE cells mock transfected (n = 4 cell culture replicates) or transfected with Alu RNA (Data presented are mean ± SEM; n = 6 cell culture replicates; * P = 0.004, two-tailed t test). ( d ) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of WT (n = 6 eyes) and Ppif −/− (n = 12 eyes) mice subretinally injected with vehicle or Alu RNA. ( e ) Immunoblot for procaspase-1 (procasp-1) and p20 cleavage product of caspase-1 in WT and Ppif −/− mouse RPE cells mock transfected or transfected with Alu RNA. ( f ) Immunoblot for procaspase-11 (procasp-11) and p30 cleavage product of caspase-11 (Casp11 p30) in WT and Ppif −/− mouse RPE cells mock transfected or transfected with Alu RNA. ( g ) Immunoblot for procaspase-4 (procasp-4) and p30 cleavage product of caspase-4 (Casp4 p30) in WT and mitochondrial DNA deficient Rho 0 ARPE19 human RPE cells mock-transfected or transfected with Alu RNA. ( h , i ) WT and mitochondrial DNA deficient Rho 0 ARPE19 human RPE cells mock-transfected or transfected with Alu RNA. (h) IL-18 secretion; data presented are mean ± SD; n = 4 independent experiments; * P = 0.0001, two-tailed t test. ( i ) IFN- β secretion; data presented are mean ± SD; n = 4 independent experiments; * P = 0.004, two-tailed t test. ( j ) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of the Ppif −/− mice subretinally co-administered Alu RNA with recombinant IFN-β (n = 5 eyes) or vehicle control (n = 5 eyes); or IFN-β expression plasmid (pIFNB; n = 6 eyes) or empty vector control (pNull; n = 5 eyes). For all immunoblots, cropped gel image of bands of interest of representative immunoblots of three independent experiments and densitometric analysis (mean (SEM)) are shown. In d and j , binary (Healthy %) and morphometric (PM, polymegethism (mean (SEM)) quantification of RPE degeneration are shown (Fisher’s exact test for binary; two-tailed t test for morphometry; * P < 0.05; * *P < 0.01; ** *P < 0.001). Loss of regular hexagonal cellular boundaries in ZO-1 stained flat mounts is indicative of degenerated RPE. The degenerated retinal area is outlined by blue arrowheads in the fundus images.

Article Snippet: Briefly, immunohistochemical staining of fixed human eyes was performed with rabbit antibody against cGAS (0.1 μg/ml, Sigma-Aldrich, Cat# HPA031700) or interferon β (0.2 μg/ml, Santa Cruz Biotechnology, Cat# sc-20107) and mouse antibody against gasdermin D (1.5 μg/ml, Abcam, Cat# ab57785).

Techniques: Transfection, Two Tailed Test, Pull Down Assay, Immunoprecipitation, Cell Culture, Immunofluorescence, Staining, Injection, Western Blot, Recombinant, Control, Expressing, Plasmid Preparation

Journal: Arthritis Research & Therapy

Article Title: Circ-CAMTA1 regulated by Ca 2+ influx inhibited pyruvate carboxylase activity and modulate T cell function in patients with systemic lupus erythematosus

doi: 10.1186/s13075-024-03422-6

Figure Lengend Snippet: The expression levels of circ-CAMTA1, circ-ASH1L, and circ-ASAP1 in Jurkat cells after stimulation. Jurkat cells were stimulated with PMA 20 ng/mL + Ionomycin (Iono) 500 ng/mL for 24 h. ( A ) The expression levels of circ-CAMTA. ( B ) The expression levels of circ-ASH1L. ( C ) The expression levels of circ-ASAP1. Jurkat cells were stimulated with 1000 IU/mL recombinant human interferon-alpha (IFN-α) for 72 h. ( D ) The expression levels of circ-CAMTA1. ( E ) The expression levels of circ-ASH1L. ( F ) The expression levels of circ-ASAP1. Data was presented as mean ± SD. (ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001)

Article Snippet: To analyzed circRNA expression levels in Jurkat cells after stimulation, cells were treated with phorbol 12-myristate 13-acetate (PMA; 20 ng/mL; Sigma-Aldrich) and ionomycin (500 ng/mL; Sigma-Aldrich, ) for 24 h. For analysis of circRNA expression in response to interferon-alpha (IFN-α), Jurkat cells were treated with 1000 IU/mL recombinant human IFN-α for 72 h. The IFN-alpha we used was purchased from Proteintech (Rosemont, IL, USA) and was produced in HEK293.

Techniques: Expressing, Recombinant