Journal: bioRxiv

Article Title: Single-cell sequencing reveals clonally expanded plasma cells during chronic viral infection produce virus-specific and cross-reactive antibodies

doi: 10.1101/2021.01.29.428852

Figure Lengend Snippet: Experimental overview of immunization schemes and antibody repertoire statistics. A. Serial immunization scheme with ovalbumin (OVA) and subsequent bone marrow plasma cell (BM PC) isolation using CD138+ magnetic beads. Subsequent antibody repertoire libraries were prepared using the 5’ single-cell immune profiling kit from 10X Genomics. B. Serial immunization scheme with the extracellular region of human tumor necrosis factor receptor 2 (TNFR2) and subsequent bone marrow plasma cell (BM PC) isolation using flow cytometry. BM PCs were sorted based on CD138 hi , TACI hi , B220 lo , CD19 lo . Subsequent antibody repertoire and transcriptome libraries were prepared using the 5’ single-cell immune profiling kit from 10X Genomics. C. The total number of cells recovered in each infected or immunized mouse. Only cells containing exactly one variable heavy (V H ) and variable light (V L ) chain were considered. D. The total number of clones recovered in each infected or immunized mouse. Clone was determined by grouping those B cells containing identical CDRH3+CDRL3 amino acid sequences. Only cells containing exactly one variable heavy (V H ) and variable light (V L ) chain were considered. E-F. Fraction of clones (E) and cells (F) corresponding to a given isotype in each infected or immunized mouse. Clone was determined by grouping those B cells containing identical CDRH3+CDRL3 amino acid sequences. Only cells containing exactly one variable heavy (V H ) and variable light (V L ) chain were considered.

Article Snippet: For the OVA immunized mouse, BM PCs were isolated using a CD138+ plasma cell isolation kit (Miltenyi, 130-092-530) following the instructions of the manufacturer.

Techniques: Clinical Proteomics, Isolation, Magnetic Beads, Flow Cytometry, Infection, Clone Assay

Journal: bioRxiv

Article Title: Single-cell sequencing reveals clonally expanded plasma cells during chronic viral infection produce virus-specific and cross-reactive antibodies

doi: 10.1101/2021.01.29.428852

Figure Lengend Snippet: Differentially expressed genes between the bone marrow plasma cells from mice either chronically infected with LCMV or immunized with human TNFR2. (A) Normalized expression of plasma cell genes Sdc1 ( CD138 ), Tnfrsf13b ( TACI ), Slamf7 , and Ptprc ( B220 ) for either mice infected with LCMV or immunized with human TNFR2. (B) Uniform manifold approximation project (UMAP) plots showing normalized gene expression for selected genes.

Article Snippet: For the OVA immunized mouse, BM PCs were isolated using a CD138+ plasma cell isolation kit (Miltenyi, 130-092-530) following the instructions of the manufacturer.

Techniques: Clinical Proteomics, Infection, Expressing, Gene Expression

Journal: International Journal of Biological Sciences

Article Title: Tubule-mitophagic secretion of SerpinG1 reprograms macrophages to instruct anti-septic acute kidney injury efficacy of high-dose ascorbate mediated by NRF2 transactivation

doi: 10.7150/ijbs.74430

Figure Lengend Snippet: High-dose ascorbate favors tubular secretion of SerpinG1 in a mitophagy-dependent fashion under inflammatory stress. (A) Heatmap showing levels of downregulated genes encoding secreted proteins in SVCT-1 and -2 knockout (KO) RTECs with LPS stimuli ( n = 5 per group). L, LPS. (B) Western-blotting analyses comparing abundance of DAB2, IGF1, SerpinG1, SerpinB2, TNC and VEGFC protein in cell lysates or conditioned medium (CM) from SVCT-1 and/or -2 knockout (KO) RTECs with LPS stimuli. CB: coomassie blue. (C) ELISA testing secretion of SerpinG1 in SVCT-1 and/or -2 knockout (KO) RTECs with LPS and/or high-dose ascorbate costimuli ( n = 4 per group). Data are expressed as mean ± s.d. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (D) Western-blotting analyses and ELISA determining abundance and secretion of SerpinG1 in cell lysates or conditioned medium (CM) from SVCT-1 and -2 shRNA-transfected HK-2 cells with LPS and/or high-dose ascorbate costimuli in the presence or absence of reconstituted SVCT-1 plus -2 expression ( n = 5 per group). Data are expressed as mean ± s.d. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. OE: overexpression. (E and F) Representative images and quantification of immunohistochemistry (IHC) staining for SerpinG1 and p-ULK1_Ser555 in renal sections from LIE mice receiving PBS (P) or high-dose ascorbate (A2) therapy at the indicated times ( n = 3 per group). Data are expressed as mean ± s.d. Scale bar: 100 μm. (G) ELISA evaluating secretion of SerpinG1 in the LPS and/or high-dose ascorbate-costimulated RTECs with scrambled shRNA (Scr) or Atg7 shRNA (sh. Atg7 ) transfection ( n = 4 per group). Data are expressed as mean ± s.d. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (H) ELISA assessing secretion of SerpinG1 in the LPS and high-dose ascorbate-costimulated RTECs in the presence or absence of SVCT-1 plus -2 KO and/or Atg7 shRNA (sh. Atg7 ) transfection ( n = 3 per group). Data are expressed as mean ± s.d. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value.

Article Snippet: The high-dose ascorbate (b.i.d) was then given to mice for 72 h. To evaluate the therapeutic value of SerpinG1, after 4 h of i.p. injection of LPS, mice were randomized to repetitive administration (q.d) of vehicle mixture (10 mg/mL glycine, 2.9 mg/mL sodium citrate and 8.5 mg/mL sodium chloride, pH 7.0) or 800 μg recombinant SerpinG1 (OPCA00220, Aviva Systems Biology) by tail-vein for 72 h. To explore the role of macrophages in anti-septic AKI efficacy of rSerpinG1, the clodronate liposomes-pretreated mice were subjected to LIE challenge for 4 h and then received 800 μg rSerpinG1 treatment.

Techniques: Knock-Out, Western Blot, Enzyme-linked Immunosorbent Assay, shRNA, Transfection, Expressing, Over Expression, Immunohistochemistry

Journal: International Journal of Biological Sciences

Article Title: Tubule-mitophagic secretion of SerpinG1 reprograms macrophages to instruct anti-septic acute kidney injury efficacy of high-dose ascorbate mediated by NRF2 transactivation

doi: 10.7150/ijbs.74430

Figure Lengend Snippet: NRF2 transactivation contributes to the high-ascorbate-inducible SerpinG1 secretion mediated by tubular mitophagy. (A) Top panel: GSEA comparing correlation between renal transcriptomics of renal tissues from the LIE-challenged Atg7 flox/flox or Atg7 ΔTE mice receiving high-dose ascorbate therapy and NRF2 gene signature. Bottom panel: western-blotting analyses assessing abundance of SerpinG1 in renal tissues from Atg7 flox/flox and Atg7 ΔTE mice ( n = 3 per group). (B and C) Representative images and quantification of SOD2 + and NRF2 + staining in renal sections from LIE mice with PBS or high-dose ascorbate therapy ( n = 7 per group). Data are expressed as mean ± s.d. Two-sided Student's t test was used to calculate the P value. Scale bar: 50 μm. (D) RT-qPCR analysis assessing mRNA expression of SerpinG1 in renal tissues from LIE mice with PBS or high-dose ascorbate therapy ( n = 5 per group). Data are expressed as mean ± s.d. Experiments were performed five times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Two-sided Student's t test was used to calculate the P value. (E) Left panel: ELISA measuring secretion of SerpinG1 in the LPS-stimulated or LPS plus high-dose ascorbate-costimulated RTECs with NRF2.shRNA (sh.NRF2) transfection ( n = 3 per group). Data are expressed as mean ± s.d. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. Right panel: western-blotting analyses detecting levels of SerpinG1 in conditioned medium (CM) from LPS plus high-dose ascorbate-costimulated RTECs with NRF2.shRNA (sh.NRF2) transfection. (F) Left panel: RT-qPCR analysis evaluating mRNA expression of SerpinG1 in the LPS-stimulated RTECs in the presence of HA-tagged NRF2 expression ( n = 5 per group). Data are expressed as mean ± s.d. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. Un: untransfected; EV: empty vector. Right panel: western-blotting analyses examining levels of SerpinG1 in conditioned medium (CM) from the LPS-stimulated RTECs in the presence of HA-tagged NRF2 expression. (G) ChIP analyses for NRF2 binding at SerpinG1 promoter in the LPS-stimulated or LPS plus high-dose ascorbate-costimulated RTECs with HA-tagged NRF2 expression ( n = 3 per group). Data are expressed as mean ± s.d. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (H) Luciferase assays of pGL3-ARE-luc activity in the LPS-stimulated or LPS plus high-dose ascorbate-costimulated HK-2 cells with or without liensinine (50 µmol/L) treatment ( n = 4 per group). Data are expressed as mean ± s.d. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (I) RT-qPCR analysis evaluating mRNA expression of SerpinG1 in the LPS plus high-dose ascorbate-costimulated RTECs transfected with ATG7 shRNA (sh. Atg7 ) in the presence or absence of NRF2 depletion ( n = 5 per group). Data are expressed as mean ± s.d. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value.

Article Snippet: The high-dose ascorbate (b.i.d) was then given to mice for 72 h. To evaluate the therapeutic value of SerpinG1, after 4 h of i.p. injection of LPS, mice were randomized to repetitive administration (q.d) of vehicle mixture (10 mg/mL glycine, 2.9 mg/mL sodium citrate and 8.5 mg/mL sodium chloride, pH 7.0) or 800 μg recombinant SerpinG1 (OPCA00220, Aviva Systems Biology) by tail-vein for 72 h. To explore the role of macrophages in anti-septic AKI efficacy of rSerpinG1, the clodronate liposomes-pretreated mice were subjected to LIE challenge for 4 h and then received 800 μg rSerpinG1 treatment.

Techniques: Western Blot, Staining, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, shRNA, Transfection, Plasmid Preparation, Binding Assay, Luciferase, Activity Assay

Journal: International Journal of Biological Sciences

Article Title: Tubule-mitophagic secretion of SerpinG1 reprograms macrophages to instruct anti-septic acute kidney injury efficacy of high-dose ascorbate mediated by NRF2 transactivation

doi: 10.7150/ijbs.74430

Figure Lengend Snippet: SerpinG1 perpetuates anti-inflammatory macrophages and prevents septic AKI. (A) Representative contour plots (left panel) and quantification (right panel) of FACS assessing CD206 + and CD206 + /CD86 - populations in the LPS-stimulated or LPS plus rSerpinG1-costimulated BMDMs ( n = 3 per group). Data are expressed as mean ± s.d. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. D, DMSO. (B) Western-blotting analyses (top panel) and quantification (bottom panel) measuring abundance of iNOS and ARG1 protein in LPS-stimulated or LPS plus rSerpinG1-costimulated BMDMs ( n = 3 per group). (C) ELISA assessing secretion of interleukin-1β (IL-1β), interleukin-18 (IL-18), C-C motif chemokine ligand 3 (CCL3) and interferon-γ (IFN-γ) in the LPS-stimulated or LPS plus rSerpinG1-costimulated BMDMs ( n = 4 per group). Data are expressed as mean ± s.d. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (D) Representative images (top panel) and quantification (bottom panel) of CD206 + /F4/80 + , p-ULK1_Ser555 and H&E staining in renal sections from LPS-induced endotoxemia (LIE) mice receiving rSerpinG1 (800 μg) administration ( n = 8 per group). Data are expressed as mean ± s.d. Two-sided Student's t test was used to calculate the P value. Scale bar: 50 μm and 100 μm. (E) ELISA comparing interleukin-1β (IL-1β) and interleukin-18 (IL-18) production in kidney homogenate of LPS-induced endotoxemia (LIE) mice receiving rSerpinG1 administration ( n = 8 per group). Two-sided Student's t test was used to calculate the P value. (F) Kaplan-Meier curves determining survivals of LPS-induced endotoxemia (LIE) mice receiving rSerpinG1 administration ( n ≥ 12 mice per group). Log-rank t test was used to calculate the P value.

Article Snippet: The high-dose ascorbate (b.i.d) was then given to mice for 72 h. To evaluate the therapeutic value of SerpinG1, after 4 h of i.p. injection of LPS, mice were randomized to repetitive administration (q.d) of vehicle mixture (10 mg/mL glycine, 2.9 mg/mL sodium citrate and 8.5 mg/mL sodium chloride, pH 7.0) or 800 μg recombinant SerpinG1 (OPCA00220, Aviva Systems Biology) by tail-vein for 72 h. To explore the role of macrophages in anti-septic AKI efficacy of rSerpinG1, the clodronate liposomes-pretreated mice were subjected to LIE challenge for 4 h and then received 800 μg rSerpinG1 treatment.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Staining

Journal: International Journal of Biological Sciences

Article Title: Tubule-mitophagic secretion of SerpinG1 reprograms macrophages to instruct anti-septic acute kidney injury efficacy of high-dose ascorbate mediated by NRF2 transactivation

doi: 10.7150/ijbs.74430

Figure Lengend Snippet: SerpinG1 is required for the anti-septic AKI efficacy of high-dose ascorbate. (A) Experimental scheme illustrating therapeutic regimen of high-dose ascorbate for LIE mice that were infected with AAV9-sh.SerpinG1 or the control vector AAV9-Scr at the indicated time points. (B) Representative images of SerpinG1 and GFP staining in renal sections from mice with AAV9-sh.SerpinG1 or AAV9-Scr infection. Scale bar: 100 μm. (C) RT-qPCR analysis comparing mRNA expression of SerpinG1 in renal tissues from mice with AAV9-sh.SerpinG1 or AAV9-Scr infection ( n = 5 per group). Data are expressed as mean ± s.d. Experiments were performed five times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (D) Representative images (top panel) and quantification (bottom panel) of CD206 + /F4/80 + , p-ULK1_Ser555 and H&E staining in renal sections from LIE mice with AAV9-sh.SerpinG1 or AAV9-Scr infection in the presence of PBS (P) or high-dose ascorbate (A2) therapy ( n = 4 per group). Data are expressed as mean ± s.d. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (E) ELISA measuring secretion of interleukin-1β (IL-1β) and interleukin-18 (IL-18) in kidney homogenate of LIE mice with AAV9-sh.SerpinG1 or AAV9-Scr infection in the presence of PBS (P) or high-dose ascorbate (A2) therapy ( n ≥ 5 per group). Data are expressed as mean ± s.d. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (F) Serum creatinine (Scr) and blood urea nitrogen (BUN) levels in LIE mice with AAV9-sh.SerpinG1 or AAV9-Scr infection in the presence of PBS (P) or high-dose ascorbate (A2) therapy ( n = 6 per group). Data are expressed as mean ± s.d. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (G) Schematic models of the role of tubular mitophagy-dependent SerpinG1 in the high-dose ascorbate-inducible M2 macrophages polarization mediated by NRF2 transactivation.

Article Snippet: The high-dose ascorbate (b.i.d) was then given to mice for 72 h. To evaluate the therapeutic value of SerpinG1, after 4 h of i.p. injection of LPS, mice were randomized to repetitive administration (q.d) of vehicle mixture (10 mg/mL glycine, 2.9 mg/mL sodium citrate and 8.5 mg/mL sodium chloride, pH 7.0) or 800 μg recombinant SerpinG1 (OPCA00220, Aviva Systems Biology) by tail-vein for 72 h. To explore the role of macrophages in anti-septic AKI efficacy of rSerpinG1, the clodronate liposomes-pretreated mice were subjected to LIE challenge for 4 h and then received 800 μg rSerpinG1 treatment.

Techniques: Infection, Control, Plasmid Preparation, Staining, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay