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. 2021 Sep:105:103152.
doi: 10.1016/j.dnarep.2021.103152. Epub 2021 Jun 24.

DNA glycosylase deficiency leads to decreased severity of lupus in the Polb-Y265C mouse model

Sesha L Paluri  1 , Matthew Burak  2 , Alireza G Senejani  3 , Madison Levinson  4 , Tania Rahim  5 , Kaylyn Clairmont  5 , Michael Kashgarian  6 , Isabel Alvarado-Cruz  4 , Rithy Meas  4 , Marina Cardó-Vila  4 , Caroline Zeiss  7 , Stephen Maher  8 , Alfred L M Bothwell  8 , Erdem Coskun  9 , Melis Kant  10 , Pawel Jaruga  10 , Miral Dizdaroglu  10 , R Stephen Lloyd  11 , Joann B Sweasy  12
Affiliations

DNA glycosylase deficiency leads to decreased severity of lupus in the Polb-Y265C mouse model

Sesha L Paluri et al. DNA Repair (Amst). 2021 Sep.

Abstract

The Polb gene encodes DNA polymerase beta (Pol β), a DNA polymerase that functions in base excision repair (BER) and microhomology-mediated end-joining. The Pol β-Y265C protein exhibits low catalytic activity and fidelity, and is also deficient in microhomology-mediated end-joining. We have previously shown that the PolbY265C/+ and PolbY265C/C mice develop lupus. These mice exhibit high levels of antinuclear antibodies and severe glomerulonephritis. We also demonstrated that the low catalytic activity of the Pol β-Y265C protein resulted in accumulation of BER intermediates that lead to cell death. Debris released from dying cells in our mice could drive development of lupus. We hypothesized that deletion of the Neil1 and Ogg1 DNA glycosylases that act upstream of Pol β during BER would result in accumulation of fewer BER intermediates, resulting in less severe lupus. We found that high levels of antinuclear antibodies are present in the sera of PolbY265C/+ mice deleted of Ogg1 and Neil1 DNA glycosylases. However, these mice develop significantly less severe renal disease, most likely due to high levels of IgM in their sera.

Keywords: Base excision repair; DNA glycosylase; DNA polymerase beta; Oxidative DNA damage; Systemic lupus erythematosus.

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Conflict of interest statement

Declaration of Competing Interest

There are no competing interests.

Figures

Figure 1.
Figure 1.. MEFs isolated from the PolbY265C/+ Ogg1−/− Neil1−/− (C/+ ΔON) mice are sensitive to menadione.
The cell survival assay was conducted as described in Methods.
Figure 2.
Figure 2.. Alkaline DNA Olive moments for Polb+/+ Ogg1+/+ Neil1+/+ (+/+), Polb+/+ Ogg1−/− Neil1−/− (+/+ ΔON), PolbY265C/+ Ogg1+/+ Neil1+/+ (C/+), and Polb+/+ Ogg1−/− Neil1−/− (C/+ ΔON) MEFs treated with menadione.
Single-strand breaks resulting from menadione treatment from were visualized with PicoGreen and quantified using CometScore (TriTeck) software. +/+ and C/+ MEFs accumulate significantly more single-strand breaks with compared to +/+ ΔON and C/+ ΔON. T-tests were used to determine if the means of each of the two groups were significantly different from each other.
Figure 3.
Figure 3.. MEFs isolated from the PolbY265C/+ Ogg1+/+ Neil1+/+ (C/+) mice have high levels of double-strand breaks after three hours of recovery from menadione treatment.
Cells were treated or not with 30 μM menadione for 1 h, followed by recovery in fresh media for 3 hours. Western blot analysis was then performed as described in Methods. Statistical analysis was performed in Graph-Pad Prism using 1-way ANOVA. A. Quantification of levels of γH2AX from 3 experiments. The fold-change is relative to untreated control. B. A representative western blot is shown.
Figure 4.
Figure 4.. Anti-nuclear antibodies present in Polb+/+ Ogg1−/− Neil1−/− (+/+ ΔON) and PolbY265C/+ Ogg1−/− Neil1−/− (C/+ ΔON) mouse serum.
A. ANA was visualized with fluorescein (FITC)–conjugated goat anti–mouse IgG and scored from 1 (low) to 4 (high). C/+ ΔON serum contained higher levels of ANA with respect to +/+ ΔON. A t-test was used to determine if the means of the two groups were significantly different from each other. B. Quantification of germinal centers per field as described in methods. At least 50 fields were imaged. C. An example of the germinal center from the spleens of PolbY265C/+ Ogg1−/− Neil1−/− mice. PNA stains activated B cells, IgD labels immature B cells, and CD4 labels T cells.
Figure 5.
Figure 5.. Serum from PolbY265C/+ Ogg1−/− Neil1−/− (C/+ ΔON) mice has high levels of IgM.
Elisa assays were used to quantify the levels of IgM and IgG in the sera. A. IgM. B. IgG. Serum from an MRL/lpr mouse is used as a control. A t-test was used to determine if the means of the two groups were significantly different from each other.
Figure 6.
Figure 6.. Renal disease severity is lower in the PolbY265C/+ Ogg1−/− Neil1−/− (C/+ ΔON) mice.
Kidney tissue from mice was fixed in histological formalin solution fixative, embedded in paraffin and sectioned. H&E staining was subsequently performed with the tissue sections. The severity of kidney lesions was scored on a scale from 1 (least severe) to 20 (most severe) as described in methods. Scores of kidneys from Polb+/+ Ogg1+/+ Neil1+/+ (+/+), Polb+/+ Ogg1−/− Neil1−/− (+/+ ΔON), PolbY265C/+ Ogg1+/+ Neil1+/+ (C/+), and PolbY265C/+ Ogg1−/− Neil1−/− (C/+ ΔON) are shown. Significant renal disease, as determined by a t-test, was observed with sections from C/+ mice, whereas kidney scores from +/+, +/+ ΔON, and C/+ ΔON mice were comparable.
Figure 7.
Figure 7.. The PolbY265C/+ Ogg1−/− Neil1−/− (C/+ ΔON) have less immune complex deposition.
Formalin-fixed paraffin embedded kidney sections from Polb+/+ Ogg1+/+ Neil1+/+ (+/+), PolbY265C/+ Ogg1+/+ Neil1+/+ (C/+), Polb+/+ Ogg1−/− Neil1−/− (+/+ ΔON), PolbY265C/+ Ogg1−/− Neil1−/− (C/+ ΔON) were stained with FITC–conjugated goat anti–mouse IgG. The number of IgG-positive glomeruli was then normalized to the total number of glomeruli present in the tissue section. Significantly higher levels of IgG-positive cells were present in C/+ kidney sections.
Figure 8.
Figure 8.. The PolbY265C/+ Ogg1−/− Neil1−/− (C/+ ΔON) have fewer TUNEL positive cells.
Paraffin embedded kidney sections from Polb+/+ Ogg1+/+ Neil1+/+ (+/+), PolbY265C/+ Ogg1+/+ Neil1+/+ (C/+), Polb+/+ Ogg1−/− Neil1−/− (+/+ ΔON), PolbY265C/+ Ogg1−/− Neil1−/− (C/+ ΔON) were stained with TUNEL. The total number of TUNEL-positive cells was then normalized to the total cells in each field. Significantly higher levels of TUNEL positive cells were present in C/+ kidney sections.
Figure 9.
Figure 9.. Class switch recombination is not altered in the PolbY265C/+ Ogg1−/− Neil1−/− mice.
Representative flow cytometric analysis of LPS/IL-4-stimulated mouse splenic B cells surface stained with anti-CD45R/B220-PB, anti-IgG1-PE and live/dead fixability dye from: (A) Polb+/+ Ogg1+/+ Neil1+/+ (+/+), (B) PolbY265C/+ Ogg1+/+ Neil1+/+ (C/+), (C) Polb+/+ Ogg1−/− Neil1−/− (+/+ ΔON), (D) PolbY265C/+ Ogg1−/− Neil1−/− (C/+ ΔON). The percentage of IgG1+B220+ cells represent CSR to IgG1 at 64 hr. (E) Statistical analyses of three independent flow cytometric analysis experiments of LPS/IL-4-stimulated splenic B cells switching to IgG1 at 64 hr from +/+, C/+, +/+ ΔON and C/+ ΔON mice. A Student’s t test was used to calculate statistical significance. (F) Representative cell proliferation curves of LPS/IL-4-stimulated splenic B cells from +/+, C/+, +/+ ΔON and C/+ ΔON mice. B cells were stained with CFSE, harvested at 64 h and analyzed by flow cytometry.
Figure 10:
Figure 10:. The PolbY265C/+ Ogg1−/− Neil1−/− (C/+ ΔON) mice have increased percentage of 7-AAD+ IgG1+ cells.
Representative flow cytometric analysis of LPS/IL-4-stimulated mouse splenic B cells stained with anti-CD45R/B220-PB, anti-IgG1-PE, 7-AAD and live/dead fixability dye from: (A) Polb+/+ Ogg1+/+ Neil1+/+ (+/+), (B) PolbY265C/+ Ogg1+/+ Neil1+/+ (C/+), (C) Polb+/+ Ogg1−/− Neil1−/− (+/+ ΔON), (D) PolbY265C/+ Ogg1−/− Neil1−/− (C/+ ΔON). The percentage of IgG1+7-AAD+ cells represent switched IgG1 B cells that were dying at 64 hr. (E) Statistical analysis of three independent flow cytometric analysis experiments of dying IgG1 cells from +/+, C/+, +/+ ΔON and C/+ ΔON mice at 64 hr. A Student’s t test was used to calculate statistical significance. Significantly higher levels of 7-AAD+ IgG1+ cells were observed in C/+ ΔON mice. (F) TUNEL-positive cells in spleens of the mouse genotypes shown. TUNEL staining in five sections of the spleens of each of the mice were quantified. Each mark represents the average number of TUNEL stained cells per 0.09 mm2 from one mouse.
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