Journal: Frontiers in Immunology

Article Title: Impaired B Cell Apoptosis Results in Autoimmunity That Is Alleviated by Ablation of Btk

doi: 10.3389/fimmu.2021.705307

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: IFNα , FITC , RMMA-1 , PBL assay science , 22100-3 , 100x.

Techniques: Immunohistochemistry

Journal: Cellular & molecular immunology

Article Title: Plasmacytoid dendritic cells promote acute kidney injury by producing interferon-α.

doi: 10.1038/s41423-019-0343-9

Figure Lengend Snippet: Fig. 2 Adoptive transfer of pDCs aggravates AKI. pDCs were isolated from spleens and transferred via adoptive i.v. injection into WT and DTRBDCA2 mice (2 × 106 cells/mouse) on day 2 after cisplatin injection. a The representative dot plots for labeled cells in the kidneys 20 h after transfer are shown in the left panel. Labeled cells in the kidneys were counted (right panel). b Blood urea nitrogen (BUN) and serum creatinine (SCr) levels were measured on day 3. c Kidney KIM-1 expression was measured on day 3. d Representative photomicrographs of kidney sections stained with periodic acid-Schiff were examined at a magnification of ×400. Scale bar: black 100 μm. The kidney pathology was evaluated and is shown in the right panel. e The concentration of IFN-α, TNF-α and IL-1β in recipient mice after adoptive transfer of pDCs was assessed by ELISA. The kidney expression of Irf7, Mx1, and Isg15 was assessed by RT-PCR. The fold induction was determined relative to the vehicle control. f The percentages of neutrophils and mononuclear phagocytes in recipient kidneys are shown (upper panel). The representative dot plots are shown in the lower panel. The data are expressed as the mean ± s.e.m. The results shown are one representative experiment of at least two independent experiments with 4–6 mice per group. *P < 0.05, and **P < 0.01. Statistical comparisons among groups were calculated by ANOVA followed by Tukey’s test.

Article Snippet: The supernatants of stimulated pDCs were then applied to TECs cultured with or without 2 μg/ml anti-IFN-α mAb (#22100–1; PBL Interferon Source). rmIFN-α (2000 U/well) was used as a positive control.

Techniques: Adoptive Transfer Assay, Isolation, Injection, Labeling, Expressing, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Cellular & molecular immunology

Article Title: Plasmacytoid dendritic cells promote acute kidney injury by producing interferon-α.

doi: 10.1038/s41423-019-0343-9

Figure Lengend Snippet: Fig. 3 pDCs aggravate AKI through IFN-α. a The kidney mRNA expression levels of IFN-α and IRF-7 at various times were examined by RT- PCR. b Renal CD45+PDCA-1+ cells, CD31+ cells and EpCAM+ cells were sorted from mouse kidneys using a fluorescence-activated cell sorter, and IFN-α expression was examined. c Cisplatin-injured WT mice received an i.v. injection of anti-IFN-α beginning the day of cisplatin treatment, with an additional dose 2 days later. Blood urea nitrogen (BUN) and serum creatinine (SCr) levels and kidney KIM-1 expression were measured on day 3. d The kidney pathology was evaluated and is shown at a magnification of ×400. The histopathological score of the kidneys was evaluated and is shown in the right panel. e Flow cytometric analysis of kidney immune cells after IFN-α neutralization is shown. f The kidney expression of TNF-α and IL-1β was examined. g AKI was induced in DTRBDCA2 and Ctrl mice. pDCs were depleted with DT. Mice then received the indicated doses of IFN-α on days 0–2. h BUN and serum creatinine (SCr) were examined on day 3. i Representative photomicrographs of kidney sections were examined at a magnification of ×400. The histopathological score of the kidneys was evaluated and is shown on the right. j Flow cytometric analysis of kidney immune cells after IFN-α administration. k GEO database expression data for IFN-α in control subjects (n = 11) and in subjects with acute injury (n = 27) are shown in the left panel. Linear regression analysis of the IFN-α expression value with TNF-α values in the AKI group is shown in the right panel. Scale bar: black 100 μm in d and i. The data are expressed as the mean ± s.e.m. The results shown are one representative experiment of at least two independent experiments with 4–6 mice per group. *P < 0.05, **P < 0.01, and ***P < 0.001. Statistical comparisons were calculated by a 2-tailed Student’s t-test in k and by ANOVA followed by Tukey’s test in other panels.

Article Snippet: The supernatants of stimulated pDCs were then applied to TECs cultured with or without 2 μg/ml anti-IFN-α mAb (#22100–1; PBL Interferon Source). rmIFN-α (2000 U/well) was used as a positive control.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Injection, Neutralization, Control

Journal: Cellular & molecular immunology

Article Title: Plasmacytoid dendritic cells promote acute kidney injury by producing interferon-α.

doi: 10.1038/s41423-019-0343-9

Figure Lengend Snippet: Fig. 4 pDCs induce apoptosis of TECs through IFN-α. Cleaved caspase 3 expression in mouse kidneys after adoptive transfer (a), pDC depletion (b), IFN-α administration (c), and IFN-α neutralization (d) was examined. Densitometric analysis of western blots of cleaved caspase 3 was performed by ratioing the observed value to that of the average control mouse titer (arbitrary units [a.u.]), and the results are shown in the lower panel. ** P < 0.01 vs. WT Cis, DTRBDCA2 Veh, and Veh. ***P < 0.001 vs. control. e Purified TECs were cultured with the supernatant from pDC cultures in the presence or absence of an anti-IFN-α antibody for 24 h, followed by evaluation of the caspase 3 activity level. *** P < 0.001 vs. control. The data are expressed as the mean ± s.e.m. The pictures shown are one representative experiment of at least two independent experiments. The densitometric analysis results shown are representative of the analysis of two western blots. Statistical comparisons among groups were calculated by ANOVA followed by Tukey’s test.

Article Snippet: The supernatants of stimulated pDCs were then applied to TECs cultured with or without 2 μg/ml anti-IFN-α mAb (#22100–1; PBL Interferon Source). rmIFN-α (2000 U/well) was used as a positive control.

Techniques: Expressing, Adoptive Transfer Assay, Neutralization, Western Blot, Control, Cell Culture, Activity Assay

Journal: Cellular & molecular immunology

Article Title: Plasmacytoid dendritic cells promote acute kidney injury by producing interferon-α.

doi: 10.1038/s41423-019-0343-9

Figure Lengend Snippet: Fig. 5 pDCs are activated by factors released by apoptotic TECs. a TECs were stimulated with 100 μm cisplatin with or without a 50 μm concentration of the caspase 3 inhibitor Z-DEVD-FMK. The supernatants containing cisplatin and inhibitor were replaced and then collected after another 24 h of culture. pDCs were isolated and incubated with TEC supernatant for 24 h. The representative histograms of the flow cytometric analysis of CD80, CD86, CD40, and MHC class II expression on ex vivo-stimulated pDCs are shown in the left panel. The mean fluorescence intensity (MFI) values are given in the right panel. **P < 0.01 and ***P < 0.001 vs. Veh. b The IFN-α concentration in the supernatant of ex vivo-stimulated pDCs was analyzed with ELISA. ***P < 0.001 vs. Veh. The results shown are from one representative experiment of at least two independent experiments in triplicate cultures with 4–5 samples per group. The data are expressed as the mean ± s.e.m. Statistical comparisons among groups were calculated by ANOVA followed by Tukey’s test.

Article Snippet: The supernatants of stimulated pDCs were then applied to TECs cultured with or without 2 μg/ml anti-IFN-α mAb (#22100–1; PBL Interferon Source). rmIFN-α (2000 U/well) was used as a positive control.

Techniques: Concentration Assay, Isolation, Incubation, Expressing, Ex Vivo, Enzyme-linked Immunosorbent Assay

Journal: Cellular & molecular immunology

Article Title: Plasmacytoid dendritic cells promote acute kidney injury by producing interferon-α.

doi: 10.1038/s41423-019-0343-9

Figure Lengend Snippet: Fig. 6 Depletion of pDCs prevents IR-induced AKI. a The percentage of pDCs in kidney CD45+ cells 24 h after IR was measured by flow cytometry (left panel). A representative flow cytometry plot for pDCs in injured kidneys is shown in the right panel. b The MFI of cell surface phenotype markers on intrarenal pDCs after IR is shown. c DTRBDCA2 mice and control mice were treated with DT 24 h before IR. Blood urea nitrogen (BUN) and serum creatinine (SCr) levels and kidney expression of KIM-1 were measured 24 h after IR. d The kidney pathology was evaluated and is shown at a magnification of ×400. Scale bar: black 100 μm. The histopathological score of the kidneys was evaluated and is shown in the right panel. e Flow cytometric analysis of kidney immune cells after IR-induced AKI was performed, and the results are shown. f The kidney expression of TNF-α and IL-1β after IR-induced AKI was analyzed and is shown. g The kidney IFN-α concentration was measured by ELISA. The results are expressed in pg/mg total protein. h Cleaved caspase 3 expression in mouse kidneys was examined and is shown in the upper panel. Densitometric analysis of western blots is shown in the lower panel. i Schematic representation showing that pDCs exacerbate AKI via IFN-α. In this study, TECs were shown to contribute to pDC recruitment and activation after AKI. Then, kidney-infiltrating pDCs lead to the release of IFN-α, which exacerbates interstitial inflammation and tubular epithelial cell damage. The data are expressed as the mean ± s.e.m. The results shown are from one representative experiment of at least two independent experiments with 4–6 mice per group or triplicate cultures with 4–5 samples per group. *P < 0.05, **P < 0.01, and ***P < 0.001. Statistical comparisons among groups were calculated by ANOVA followed by Tukey’s test.

Article Snippet: The supernatants of stimulated pDCs were then applied to TECs cultured with or without 2 μg/ml anti-IFN-α mAb (#22100–1; PBL Interferon Source). rmIFN-α (2000 U/well) was used as a positive control.

Techniques: Cytometry, Control, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay