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ATR-mediated DNA damage response underlies abnormal B cell activity in systemic lupus erythematosus

 Last Update: 2022-11-04
 Source: Internet
 Author: User

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Summary

In patients with systemic lupus erythematosus (SLE), B cells are involved in the autoimmune response, but broad-based B-cell-directed therapies have shown only modest efficacy
while attenuating the humoral immune response to the vaccine and inducing immunosuppression.
The development of more effective treatments for pathogenic cloning was an unmet need
.
Here, we demonstrate enhanced activation
of the ATR/Chk1 pathway in the B-cell DNA damage response (DDR) in patients with active SLE.
B cells are treated with type I IFN (a key driver of SLE immunity) to induce ATR expression
by binding of interferon regulator 1 to its gene promoter.
Pharmacological targeting of ATR in B cells by a specific inhibitor (VE-822) attenuates its immunogenicity, including pro-inflammatory cytokine secretion, plasma albumin formation, and antibody production
.
Taken together, these findings identify the ATR-mediated DDR axis as a coordinator of type I IFN-mediated B cell responses in SLE and a potential new therapeutic target
.

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Brief introduction

The DNA damage response (DDR) mechanism is a dynamic feature of
immune system development and function.
The key role of DDR includes monitoring the genomic rearrangement required by lymphocytes to form antigen receptors, produce antibodies, mature, and rapidly divide in response to infection (1,2).
Persistent DDR due to genetic or epigenetic factors is a driving feature
of malignant disease and developmental syndromes.
Notably, abnormal DDR has recently been reported in a range of autoimmune diseases, including systemic lupus erythematosus (SLE), multiple sclerosis (MS), rheumatoid arthritis (RA), and Ecardy-Gutier syndrome (1, tri–6).
However, the molecular complexity of the DDR pathway, as well as the lack of understanding of environmental and cell-dependent DDR involvement, hinders the therapeutic targeting of
DDR in these diseases.

The main pathways of DDR signaling are the three protein kinases ATM (ataxia telangiectasia mutation), ATR (ATR and Rad3 related), and DNA-PKcs (DNA-dependent protein kinase catalytic sub)) (7).
ATM and DNA-PKcs are considered to be the pinnacle of the double-stranded DNA break signal cascade, while ATR is primarily activated during single-stranded DNA breakage and responds to factors that cause DNA replication stress, such as ultraviolet light (8,9).
Once activated, these DDR components can be phosphorylated alone or synergistically including checkpoint kinase 2 (Chk2; Thr), including many proteins ⁶⁸, via ATM), Chk1 (service ³⁴⁵, via ATR), H2AX (serial number ¹³⁹ γΗ2ΑΧ) and p53 (serial ^{number 15}), which determine cellular response and fate (8).
Notably, p95/NBS1 (also known as nibrin) is typically colocalized with γΗ2αχ at damaged genomic loci and can propagate ATR and ATM-dependent signals (10, 11).
Although ATM, ATR, and DNA-PKcs share some common functions, their roles and phenotypes associated with their perturbations differ (7,9).
Unlike ATM and DNA-PKcs, ATR is essential in proliferating cells; Thus, ATR knockout can lead to cell proliferation failure and early embryonic lethality (12,13).

SLE is a heterogeneous and potentially serious autoimmune disease where the interaction between environmental and genetic factors leads to complex biological network disturbances that ultimately lead to immune dysregulation (14).
B-cell abnormalities are closely related to the pathophysiology of SLE and contribute to the deregulation of immune regulation (15–18).
More specifically, the main pathogenic features of B cells of SLE include: (i) hyperactivity with increased antigen presentation capacity; (ii) increased number of cytoplasmic layer, memory, double negative memory and transitional cells; (iii) abnormal secretion of pro-inflammatory and anti-inflammatory cytokines, highlighted by increased interleukin-10 (IL-10), IL-6, IL-4 and tumor necrosis factor-β (TNF-β) and decreased IL-2 production; and (iv) increasing the secretion of pathogenic autoantibodies (15, 18, 19).
This abnormal B cell function in SLE patients is affected by type I interferon (IFN) associated with the molecular signature of IFN, especially in patients with moderate to severe lupus (20, 21).
Although B cells play an important role in the pathogenesis of SLE, their regulatory mechanism is only partially understood; Current treatments targeting B cells [such as rituximab and belimumab (22)] eliminate the entire B cell population, leading to immunosuppression and weakening the humoral immune response to the vaccine (23).

On the basis of the above, the currently unmet need is to understand the molecular events in pathogenic B cell clones, which will allow them to be selectively targeted so that healthy B cell sequences are not affected, thereby mediating immune protection
.
Although B cells are prone to DDR errors because DDR is selected in antibody diversification (8) there is growing evidence that leukocyte DDR is inefficient in SLE patients, and the molecular link between B cell DDR and the pathophysiology of autoimmune diseases is unclear (III–5, 24–26).
B-cell ATM down-regulation in patients with rheumatoid arthritis mediates pro-stomatomy B-cell phenotype leading to erosive disease (6).
To this end, elucidating the molecular pathways of pathogenic B cell DDR may contribute to the design
of rational treatment options for SLE patients.

Here, we demonstrated dominant DDR in SLE B cells
using proteomics and transcriptomic analyses from SLE patients and healthy human B cells.
Experimental screening of major DDR pathways revealed that the ATR/Chk1 axis was deeply activated
in SLE B cells compared to healthy B cells.
IFN-α exposure of healthy B cells (SLE-like environment) mimics the DDR signature of SLE B cells, while in vitro inhibition of ATR activity reverses the pathogenic phenotype
of the cells.
We also provide evidence that IFN regulator 1 (IRF1) is an important contributor to SLEI.
type IFN signaling and is part of the DDR mechanism that mediates activation of
the ATR pathway in B cells that mediate IFN-α processing.
Our data suggest that IFN-α-mediated, IRF1-driven ATR mechanisms are involved in the pathogenesis of SLE by promoting B cell overactivity, and demonstrate a previously unrecognized pathway
in which type I IFN promotes humoral immune responses.

Results The B cells of SLE patients showed abundant DDR

To determine the unknown mechanism of functional importance associated with the pathogenic role of B cells in SLE, we first evaluated the proteomic profile of total B cells isolated from the peripheral blood of patients with active SLE (SLEDAI) ≥8) age/sex matched healthy controls (HCs) (Figure 1A along with the demographics of Table S1).

The resulting dataset contains 6449 master proteomes that are further filtered based on the presence of at least two peptides, resulting in a protein list
of 4995 proteins.
Principal component analysis of total protein showed strong separation between the two groups (Figure 1B).
Ingenuity enrichment analysis (IPA) and collusion pathway analysis reveal key known features related to the pathogenesis of SLE, such as complement activation, circulating antibody regulation, cytokines (IL-1, IL-4, IL-6, and IL-10), antigen presentation, inflammatory response, etc.
, nuclear factor κB (NF-κΒ) activation and mammalian targets transducted by rapamycin (mTOR) signaling (Figure 1C, Figure S1 and Table S2)
。 Notably, B cells from SLE patients showed an abundant response
to DNA damage stimuli.
In particular, DNA repair, p53 signal transduction, G1--proteomic features of SLE B cells are disproportionately proportioned in S-checkpoint regulation and other DDR-related pathways compared to HC (Figure 1C as well as Figure _S1).

To verify enriched DDR, we performed flow cytometry assays on γH2AX, a classic indicator of DDR activation (27).
We detected higher levels of γH2AX in human and mouse (NZB/W-F1) sle peripheral blood B cells than HC, further confirming the aggravation of DDR in SLE B cells (Figure 1D as well as Figure S2).

Next, we asked whether DDR enrichment in SLE is a specific feature of B cells or a general feature
of immune cell populations.
Thus, the expression level of γH2AX was also evaluated in peripheral helper T cells (CD4) level ⁺), T cytotoxicity (CD8+) and T modulatory (CD4⁺CD25⁺Foxp3⁺), classical monocytes (HLA-DR⁺CD14 ⁺CD16^? ), intermediate monocytes (HLA-DR⁺CD14 ^{type +} CD16⁺), non-classical monocytes (HLA-DR ⁺CD14^? CD16⁺) and neutrophil (CD66b⁺) SLE and HC patients
.
Detected by flow cytometry and confocal microscopy, B cells exhibited the most significant increase in γΗ2ΑΧ expression compared to HC among a variety of SLE-derived immune cells detected (Figures S3 and S4).

Figure 1 Proteomics and transcriptomics analysis determined that DDR is a feature of
SLE B cells.

(A) Isolation of peripheral blood B cells (n=11 individuals/group) from SLE and HC patients by magnetic bead method for proteomic analysis
.
(B) Principal component analysis (PCA) uses expression techniques
for all proteins in SLE and HC patients.
(C) IPA
using 1094 differentially expressed proteins of SLE and HC [false discovery rate (FDR) <0.
05].
A select typical path (FDR<0.
2)
is shown.
DDR correlation paths are shown
in bold.
(D) Flow cytometric analysis of CD19-gated peripheral blood mononuclear cells ⁺ detection of B cell DDR activity in SLE and HC patients (n=7 individuals/group).

Representative frequency plots and histograms show overlap
of unstained cells (gray), stained SLE (red), and HC (blue) cells.
Unpaired students' t-test*P<0.
05(E) flow cytometry analysis of γΗ2ΑΧ expression across cell cycles in bead-isolated B cells from SLE and HC patients (a study ( n = 6 people per group).

Cell cycle analysis uses Ki67 and 7-AAD
.
The same stage
of the two groups (SLE and HC) was compared.
There was no significant significance at any cell stage between the two groups [bidirectional analysis of variance (ANOVA)].

(F) Gene ensemble enrichment analysis (GSEA) to analyze the changes in DDR (n=9) in B cell subsets (resting phase, transitional phase 3, isotype conversion memory, double negative type 2 and antibody secretion type) in SLE patients compared with HC (n =12) Using the published RNA sequencing (RNA seq) dataset (15).
liquid chromatography; tandem mass spectrometry; ES, enrichment scores; normalized enrichment score; FDR < 0.
25 cutoff
.
MFI, average fluorescence intensity
.
Results are expressed
as averages ± SEMs.

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Since DDR includes cell cycle checkpoint control, we sought to investigate whether elevated expression of γΗ2ΑΧ in SLE B cells is related
to a specific stage of the cell cycle.
Therefore, we performed cell cycle analysis
by flow cytometry on Ki67 proliferation-specific markers, 7-AAD cell DNA content markers, and γΗ2ΑΧ in freshly isolated B cells from SLE and HC patients.
Although γΗ2ΑΧ is significantly overexpressed in SLE, no statistically significant differences were found at any cell stage, suggesting that the upregulation of γΗ2ΑΧ associated with SLE may be independent of the cell cycle (Figure 1E as well as Figures S5, A and B).

Finally, to investigate whether proteomic data for fully deregulated DDR can be extrapolated to the transcriptome, we performed gene collection enrichment analysis (GSEA) for B cell subsets of SLE and HC patients if specific B cell subsets are present in DDR signatures, utilizing a publicly available RNA sequencing (RNA-seq) dataset (15).
The results showed that almost all major SLE B cell subsets were involved in alterations in DDR, including resting phase, transition phase3, homotype-switched memory, double negative 2, and antibody-secretory B cells (Figure 1F).
Together, these data suggest that B cells exhibit a profound DDR
in SLE patients.

Activates the ATR pathway to drive SLE B cell DDR

Next, we sought to identify the specific molecular mechanisms of DDR by detecting the main kinase proteins and components of DDR, namely ATR, ATM, DNA-PKcs, Chk1, Chk2, p53, and p95/NBS1 (7), in SLE and hcb cells (Figure 2A).
Since protein phosphorylation is often required for activation and conduction of DDR signaling, most targets are studied in phosphorylated form (28).
We found elevated levels of pATR (Thr) ¹⁹⁸⁹), pChk1 (serial number ³⁴⁵), p-p53 (serial ^{number 15}) compared to HC-B cells, SLE p95/NBS1 is no different from pATM (Ser¹⁹⁸¹), pChk2 (thrust ⁶⁸), and pDNA PKcs (serial ^{number 2056} Assay by immunofluorescence microscopy or flow cytometry (Figure 2B).
Activation of the ATR pathway in SLE B cells was also confirmed by Western blot analysis of individual individuals (Figure S6).

These data suggest that the ATR/Chk1 pathway is mainly activated in SLE B cells, while ATM/Chk2 and DNA-PKcs do not play a key role
.
Furthermore, to further demonstrate that the ATR-mediated DDR pathway is specific for SLE and not secondary to the inflammatory environment of any autoimmune disease, we measured pATR protein levels in B cells isolated around patients with ankylosing spondylitis (AS) (Demographic Table S1).

AS is an autoinflammatory disease that, unlike SLE, B cells play a major role in pathogenesis and lack type I IFN signaling (29).
There was no significant change in pATR protein levels in AS-derived B cells compared to HC (Figure S7).

Figure 2.
The ATR/Chk1-DDR pathway is activated
in SLE B cells.

(A) The schematic diagrams of the main DDR pathways are ATR/Chk1, ATM/Chk2 and DNA-PKC
.
(B) Study of DDR contributors and immunofluorescence confocal microscopy images or representative histograms staining SLE and HC B cells [CD19⁺, cell sorting by fluorescence activation (FACS) or magnetic bead separation].

Representative confocal image of pATR (Thr) ¹⁹⁸⁹; Green; n = 5 people per group), pChk1 (Ser³⁴⁵; Green; n = 5 people per group), p95/NBS1 (red; n = 5 people per group), pATM (Ser¹⁹⁸¹; n=7 people/group), pDNA-PKcs (Ser²⁰⁵⁶; Green; n=5 people/group), pChk2 (Thr⁶⁸; Green; n = 6 people/group), and p-p53 (Ser¹⁵; Green; n = 5 people per group).

Analysis results for pATR, pChk1, pDNA-PKcs, pChk2, and p-p53 are expressed as mean spots/cells per individual, pATM represents MFI for each individual, and p95/NBS1 represents the average intensity/cell
for each individual.
For pATM, a representative histogram shows overlap
of unstained cells (gray), stained SLE (red), and stained HC cells (blue).
Scale bar, 2 μm
.
(C) Diffusion assessment (Ki67) and (D) Flow cytometry detection of B cell apoptosis (PARP1 and caspase-3 breaks) isolated by SLE and HC magnetic beads (n = 7 individuals per group) and/ or confocal microscopy
.
Representative histograms show overlap of unstained cells (gray), stained SLE cells (red), and stained HC cells (blue).

Lysis of caspase-3 staining, positive signals indicated in green, cells stained with 4′,6-diamino-2-phenylindole (DAPI) nuclei (blue; n = 3 people per group).

Scale bar, 2 μm
.
Results for all cases were expressed as mean ± scanning electron microscopy, statistically significant t-test for unpaired students, P≥0.
05 [not significant (ns)]*P< 0.
05, and **P<0.
01
.

DDR/，ATR/Chk1(30,31).
，HC，SLE B（Ki67）[（ADP）1（PARP1）caspase-3]，ATR/Chk1SLE，(2，CD).
SLEhcb(2C)G₁B（S5B），G₁-SG₂-M(1CS1）（S）（G₂-M）B
。，pATRS（EdU）⁺)G₂-（pH3⁺)，（IL-21/CpGb/sCD40L）(32–38)SLEHCBEdU（S8A）
。，SLEHC，EdUATR⁺EdU^?B，pH3⁺pH3，pATR^?
。，pATREdU⁺SLE BEdU⁺HC-B，pH3⁺SLE BpH3^?SLE B
。，，ATRSLEBHC（S8、BC）
。

ATRIFN-αB

ATRDDRSLEB，B，IIFN-α(3A).
ATR mRNA、pATRpChk1IFN-α，ATM mRNA、pATM、pChk2pDNA-PKcs(3，BD).
，IFN-αBSGATR₂-MSLE B（S8，AC）
。，BIFN-αSLEATRDDR，，SLE
。

3-αB
。

B，IL-21/CpGb/sCD40L（）IFN-α（850U/ml），，ATRi（5μΜ）（DMSO），
。(A)-αB
。(B)（RT-PCR）IFN-αATRATM mRNA；n=3；t）
。(C)-αB（/）pATR（Thr¹⁹⁸⁹;），pChk2（⁶⁸;），pDNA PKcs（Ser²⁰⁵⁶;）DAPI（；n=3；t）
。，2μm
。(D)pChk1（Ser）³⁴⁵)pATM（Ser¹⁹⁸¹)-αB，，MFIMFI（t）
。CD19⁺.
(E)ATRi（berzosertib）IFN-αB
。2
。(F)D2（IL-10、IL-6、IL-4、TNF-βIL-2）(n=10；t）
。±SEM
。P≥0.
05（）*P<0.
05**P<0.
01，****P<0.
0001
。

ATRSLE，IFN-αBATR，B，SLE
。，ATRDDR，
。，berzosertib（ATRi），Chk1ATR(39)，IFN-αB(3ES9）
。，IFN-αB[TNF-α，IL-13，IL-4，IL-10，IL-6，IL-2，TNF-β，IFN-γ，IL-17A，IL-12p70，（APRIL）、B（BAFF）CD40（CD40L）]B
。IL-10、IL-6、IL-4TNF-β(3F)IL-2、IL-12p70、CD40L、BAFF、APRIL、IFN-γ(3F，S10）
。，ATRiSLEB
。

ATRIFN-αB

，ATRDDRSLEB，SLE
。，，SLE B，(15,18,40–42).
，IFN-αBATR（CD40、CD80BAFF）[-DR（HLA-DR）](4，AC，S11、AB）
。ATR，IFN-αBHLA-DR，ATR（S12）
。，ATRChk1，ATR-Chk1CHR-124（Chk1Chk1i）（S12CS13）
。，ATRIFN-αB，ATRB
。

4ATRiIFN-αB
。

B cells are isolated from the peripheral blood of healthy people by magnetic bead method and cultured for 2 (D2), 3 (D3), and 7 (D7) days with IL-21/CpGb/sCD40L survival and mild proliferation mixed culture medium in the presence or absence of IFN-α (850 U/ml) and ATRi or DMSO (control), depending on the experiment
.
(A) IFN-α-treated B cells in vitro ATRi assay for assessing cell activation status (ATRi, 5 μΜ).

Flow cytometry quantitatively detects CD19 gated data ⁺ (B)CD40 (n=5 individuals per condition), CD80 (n= 5 people per situation), BAFF (n = 5 people per situation), and (C) HLA-DR (n = 4 people per situation).

The trial
was used in all cases of paired students.
(D) Schematic of the ATRi experiment (ATRi, 2 μμ) to evaluate surface and released immunoglobulins
in vitro with IFN-α treated B cells.
(E) Representative flow cytometry gated surface IgM, IgD, and IgG quantified at D7 and D3 and D7 (n = 5 to 7 individuals per condition; Bidirectional ANOVA).

(F) Detection of immunoglobulins (IgM, IgD, IgA, IgE, IgG1, IgG2, IgG3, and IgG4) released from cell culture supernatants by flow cytometry using LEGENDplex technology (n = 8 people per condition; Paired students' t-test).

Results are expressed
as mean ± SEM.
P≥0.
05 (nanoseconds)*P<0.
05***P<0.
001, and ****P< 0.
0001
。

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Next, we investigate whether the production of antibodies (i.
e.
, immunoglobulins) is affected, as their production may be affected with the release of cytokines, such as CD40L, BAFF, and APRIL, but on the other hand, may also be weakened
by a decrease in pro-inflammatory cytokines such as IL-6 and TNF-β.
The results showed that on the third day of culture, surface immunoglobulin M (sIgM) was significantly reduced.
On day 7, sIgD decreases while IFN-α-treated B cells exposed to ATRi do not have any significant differences compared to the unexposed group (Figure 4, D and E).
In addition, IFN-α treated atrial myocytes released significantly reduced IgM, IgA, IgE, IgG1, IgG2, IgG3, and IgG4 (Figures 4, D, and F), suggesting that the ATR-DDR pathway plays an important role
in antibody formation.
Finally, we expect a decrease
in isotypic switching and antibody secretion of B cells due to reduced surface IgM and IgD levels of IFN-α treated B cells and release of immunoglobulins.
Therefore, we tried to study the formation of B cell subsets (Figure 5A).
On day 7 of culture, IFN-α-treated ATRi-treated ATRi-treated ATRi B cells had significantly reduced levels of isotype conversion memory (SWME) cells (CD19) ⁺ immunoglobulin^? Chuandi 27+), total memory unit (CD19 ⁺ Chuandi 27⁺), transitional cells (CD19⁺ immunoglobulins ^{dim CD38}+), and plasma layers (CD19⁺ immunoglobulins^? Chuandi 27^+CD38+), while homotype switchless memory (UNSWME) (CD19⁺ immunoglobulin ⁺Kawachi 27⁺), naïve (CD19 ⁺ immunoglobulin⁺ Kawachi 27^? Double-negative cells (CD19 ⁺ immunoglobulin^? Kawachi 27^? There was little to no impact compared to patients who did not receive treatment for atrial fibrillation (Figures 5, A and B).

5IFN-αB
。

B(n=5～7），，IL-21/CpGb/sCD40L，IFN-α（850 U/ml）ATRiDMSO（）3（D3）7（D7）
。(A)IFN-αBATRi（ATRi，2μΜ），B
。(B)D7D3D7（D3，n=5；7，n=7）
。B：（UNSWME）CD19⁺⁺27⁺，CD19⁺⁺27^?，（SWME）CD19⁺^?27⁺，（DN）CD19⁺^?27^?，CD19⁺27⁺CD19⁺CD38⁺，CD19⁺^?27⁺CD38⁺
。±SEM
。P≥0.
05（）**P<0.
01，***P<0.
001（）
。

，ATRiSLE B
。IFN-αB，ATRi，G₀-（DMSO）（），B（S14）
。，SLEB（G₀)，
。

，，ATRIFN-αB：（i）IL-10、IL-6、IL-4TNF-β，IL-12p70IL-2；（ii）；（iii）；（iv）；（）
。

IRF1IFN-αBATR

IFN-αSLEBATR，IRF1（ChIP），【（EPD）】(6A).
SLEBATRIRF1 mRNA(6B)BIFN-α(3B6).
IFN-αBIRF1IgG，IRF1（
。，1)IRF1(6D).
IRF1
。

6.
IRF1IFN-αBATR
。

(A) Schematic of the automatic barcode reader transcription start site around the gene locus (TSS, arrow; Coding area, black box).

The numbers above the schematic represent the distance
to the bound site predicted by TSS and IRF1 (EPD database).
(B) B cell IRF1 and ATR mRNA levels (n=6/group) isolated by magnetic beads from SLE and HC patients, by quantitative real-time RT-PCR (t-test of unpaired students).

(C) Real-time RT-PCR detection of IRF1 mRNA levels (n = 4 individuals per case) in interferon-α treated B cells (paired student's t-test).

Cells were cultured with IL-21/CpGb/sCD40L Survival and Light Proliferation Mix and IFN-α (850 u/ml) for 2 days
.
(D) Chip analysis of IRF1-to binding sites for automatic barcode reader gene locus
.
Array-on-chip experiments were performed using anti-IRF1 antibody (a-IRF1) or control antibody (IgG) in chromatin isolated from interferon-α treated B cells (n=4 healthy individuals; t-test of unpaired students).

(E) Schematic of inhibition of
IRF1 expression in IFN-α treated B cells with siIRF1 or siNeg (control).
(F) Real-time quantitative RT-PCR to detect the relative expression levels of IRF1, ATR and ATM mRNA under siNeg and siIRF1 conditions (n = 3 people per case).

(G) pATR (Thr) staining and quantification (puncta/cell) of SiNeg and siIRF1 B cells ¹⁹⁸⁹; green) or pChk1 (Ser³⁴⁵; red) and marked with DAPI (blue; n = 3 people per situation; t-test of unpaired students).

Scale bar, 2 μm
.
(H) Flow cytometry analysis of transition period (CD19+ immunoglobulin ^dim CD38⁺and plasmablast (CD19⁺ immunoglobulin^?).
B cells on Sichuan 27 + CD38 ⁺) siNeg and siIRF1 (n = 3 people per condition; Paired students' t-test).

For all cases, P≥0.
05 (nanoseconds)*P<0.
05**P<0.
01, and ****P <0.
0001
.
For (B) to (D) and (F), the results are expressed
as averages ± SEMs.

IRF1，IRF1 RNA（siIRF1）（siNeg）IFN-αBIRF1，ATR mRNA、ATR、pATR（Thr）¹⁹⁸⁹)pChk1（Ser³⁴⁵)(6，EG，S15）
。ATMmRNA，IRF1ATR(6F).
，IRF1ATR mRNA、ATR、pATRpChk1，ATM mRNA(6，FG，S15）
。IRF1B，ATR(6HS16）
。，IRF1ATR，IFN-αSLE BDDR
。

Although DDR is associated with various aspects of inflammatory states, little is known about how its relaxation regulation makes specific immune cell populations pathogenic
.
The molecular mechanisms of DDR-driven in autoimmune diseases are unclear
.
B cells play a key role in the pathogenesis of systemic lupus erythematosus (SLE), but the drivers of their pathogenicity are unknown
.
In this study, through proteomic and transcriptomic analysis of B cells from SLE patients, we revealed a deregulated DDR
.
More specifically, we describe a previously unrecognized mechanism associated with B cell dysfunction, meaning that ATR-mediated pathways are involved in the specific enrichment
of SLE B cells.
Our in vitro data show that the ATR/Chk1 pathway is triggered during the type I interferon-driven autoimmune response, and targeted drug inhibition of ATR activity inhibits key features of SLE pathophysiology, including B cell activation, plasma albumin formation, antibody production, and pro-inflammatory cytokine release
.
Finally, we provide evidence that this ATR overactivation is mediated by direct molecular interactions with IRF1, which link the type I IFN signature signal of SLE to upregulation of the ATR/Chk1 pathway and IRF1-mediated induction of pathogenic B cells
.
The pharmacological targeting of ATR mitigates the pathogenic cell characteristics
of the ATR pathway as a potential therapeutic target for SLE.

，SLE-TDDR(43,44)BDDR
。，SLE
。DDRSLE，V（D）J、(,5,26)，B
。，ATRDDRB，，
。Taher，SLE BATR/Chk1，ATM/Chk2DNA-PKcs
。(45)SLEBATRATM
。，，DDR
。，eb（EBV），BATR/Chk1B(46–50).
，EBVBDDR，ATR/Chk1，ATM/Chk2，ATRB(50).
SLEB，EBV，(51).
，ATR/Chk1B
。，ATM/Chk2B，，RA，B(6).
DDR，ATMIFN(52,53).
，DDR
。ATR，SLEB，
。

BBSLE(54).
SLEBAFFIIFN（IFN-αIFN-β）IIFN(55).
SLE（CD20）B，B，2019(56).
SLE、，
。，BDDR，
。SLE BATRDDRB，
。

Our data suggest that in IFN-α treated B cells, after ATR activity is blocked, B cell characteristics do not easily lead to SLE, such as decreased antibody production; plasma layer; Soluble IL-10, IL-6, IL-4, TNF-β, and soluble IL-2 levels increased, but BAFF release increased and membrane-bound BAFF decreased
.
Current anti-BAFF treatments for systemic lupus erythematosus (SLE) target both soluble BAFF and membrane-bound BAFF, with higher potency for soluble BAFF (57, 58).
While both forms of BAFF are biologically active, it has not been determined
whether membrane-bound and soluble BAFF's specific targeting will produce different clinical outcomes.
These findings highlight ATR as a potential therapeutic target for SLE, revealing the important role
of the ATR-DDR pathway in the production of cytokines by B cells.
It is unclear whether this is a direct effect between ATR activity and cytokine production signaling, or due to altered
B cell differentiation status.
Rodia and so on
.
(59) demonstrated that ATM activity is essential for fibroblasts to produce IL-6; However, when ATR is inhibited, IL-6 production is reduced
.
In addition, in a recent study (6) increased secretion of interleukin 6 was associated
with a defect in B cell activation of ATM in patients with rheumatoid arthritis.
Therefore, we conclude that in SLE B cells, cytokine release may be affected by abnormal ATR-mediated DDR, and that the specific DDR signals that regulate cytokine secretion vary
depending on the cell type and immune microenvironment.

Although our results suggest that both ATR and Chk1 are hyperphosphorylated in SLE B cells compared to HC, only when ATR is inhibited does it affect the activation and antigen presentation status of SLE-like B cells, while Chk1 is not inhibited
.
Downregulation of ATR or Chk1 is expected to cause similar changes, since activation of Chk1 requires phosphorylation
of ATR.
However, it is possible that these two components do not always function as a linear pathway, as supported by several studies, suggesting differences in the effects of atrial fibrillation and Chk1i (60–63).
In this direction, there is evidence that ATR-driven switch recombination, and Chk1 is more involved in the growth of normal B cells (64–66), indicating that they have a significant contribution
to B cell physiology.
Speroni and so on
.
(67) The results showed that Chk1, rather than ATR, drives the replication process
of U2OS cells after ultraviolet irradiation.
The independent role of ATR mediated by Chk1 through its specific interaction with proliferating nuclear antigens has also been described earlier (68, 69), and Luciani et al
.
(62) An ATR-dependent, Chk1-independent, S-period checkpoint was reported, which suppressed the initiation
of replication.
In addition, in a groundbreaking study (63) the authors revealed the role of ATR in interacting with replication protein A (RPA), a key sensor for inducing DDR after exposure of cells to genotoxic stress (70), independent
of Chk1-mediated replication stress responses.
Finally, the possibility of different time point-dependent activation between ATR and Chk1 was studied.
However, this has not been confirmed, suggesting that the two components have different roles (60).
Our results reveal a novel mechanism
by which type I IFN is involved in the pathogenesis of SLE by inducing ATR-mediated DDR in B cells by IRF1.
To support this, the findings (i) showed normal ATR activation levels in B cells from patients with AS disease without IFN markers, and (ii) increased ATR activation levels in B cells treated with IFN-α, suggesting that type I IFN is necessary
to enhance ATR activity.
IRF1 is considered one of the potential drivers of common B-cell lymphomas and interacts with multiple myeloma oncogene 1 protein (MUM1), which plays a role in the progression of B-cell lymphoma (71–74).
According to the EPD database, molecules other than IRF1 may also interact directly with ATR regulatory regions, suggesting that further studies may reveal other mechanisms
that drive the ATR pathway of autoreactive B cells in B-cell-mediated diseases.

In our study, IFN-α-treated B cells did not adequately differentiate into plasma proteins when ATR or IRF1 were inhibited, suggesting that the IRF1-ATR axis is critical
for plasma albumin formation.
In systemic lupus erythematosus, the amount of plasma albumin is usually significantly increased and is associated with disease activity and flares (18).
Plasma albumin/plasma cell depletion is currently used in therapeutic interventions for systemic lupus erythematosus (54).
However, it has recently been reported that IRF1 is involved in B cell differentiation to plasma cells after lipopolysaccharide stimulation (75), and the special role of ATR in B cell lines has not been described
.
Taken together, these findings suggest that the IRF1-ATR axis is important for B cell differentiation and plays a role
in SLE abnormal plasma protein formation.

In summary, we reported overactivation of a specific DDR pathway in B cells of SLE patients, one of
the most overreactive cell populations in SLE.
We found that ATR-mediated DDR signaling is abnormally regulated in B cells via IRF1, driving the response
of pathogenic cells.
While this is relevant to understanding the pathogenesis of SLE and B-cell-mediated autoimmune diseases, it may also provide new insights
into the specific regulatory role of ATR signaling in autoimmunity and its coordination with IFN signaling.
Overall, our findings suggest that targeting the IRF1-ATR axis that manipulates SLE-like B cells may have therapeutic effects on
SLE.
For this purpose, McNally and so on
.
(1) In human autoimmune diseases of hemophagocytic lymphohistiocytosis and multiple sclerosis, the use of chemical DDR inhibitors to suppress the immune response
of antigen-activated T cells is proposed.
In this direction, various selective ATR inhibitors have been tested in phase 1/2 clinical trials for the treatment of solid tumors (39, 76).
VE-822, marketed as berzosertib, has demonstrated acceptable safety and early efficacy and is currently being evaluated in 18 ongoing clinical trials (e.
g.
, NCT04266912, NCT03641313, and NCT04052555).

Limitations of the study

Targeting ATR-mediated DDR pathways to pathogenic B cells may improve the autoimmune response, but it also impairs the immune response to pathogens, although this can be overcome
, at least in part, through the use of approved vaccinations.
In addition, the treatment implementation we found may be hampered
by the lack of a protocol for ATR pathways that target autoreactive B cells rather than broad B cell directed inhibition.
In addition, establishing a suitable in vitro experimental pipeline with the patient's own generated B cells that preserves their disease characteristics so that the atrial effect can be studied is an important question for
future research.

Materials and methods for human subjects

Peripheral blood was collected from SLE patients [n=51, according to the 1997 American College of Rheumatology Standard Classification (77)] and healthy people (n=103).

At the time of sampling, all patients had moderate to high disease activity (SLEDAI≥ and the vast majority had not received cytotoxic drugs
in the 12 months prior to donation.
All patients and healthy people were recruited through the Department of Rheumatology and Clinical Immunology of the Fourth Department of Internal Medicine, the University Hospital "Atikon" and the General Hospital "Aslipoeen" (all in Athens, Greece
).
Obtain the informed consent of all individuals prior to sample collection (Athens, Greece, Option 10/22-6-2017).

All patients did not take any therapeutic dose
for at least 24 hours before blood draw.
To rule out nonspecific effects of the inflammatory environment, we used peripheral blood samples (n=4) from AS patients as an additional control as they showed a broad inflammatory response but no pathogenic B-cell response and autoantibody production
.
Table S1 summarizes the clinical and demographic characteristics
.

Animal studies

All procedures for mice comply with institutional guidelines and are reviewed and approved
by the Hellenic Federal Veterinary Service (1044/1319; Athens, Greece).
New Zealand black ♀× New Zealand white wine ♂F1 mice (i.
e.
, NZB/W-F1) spontaneously developed an autoimmune syndrome similar to human SLE (78).
NZB (NZB/OlaHsd) and NZW (NZW/OlaHsd) mice are purchased
from Envigo.
NZB/W-F1 presents with a disease of ≥ 100 ng/dL proteinuria (after 6 months of age) that pre-adapts
at 10 weeks of age.
Repeat each experiment three times
.
All animals are kept at the Institute of Biomedical Research Athens Zoological Institute
.
The NZB/W-F1 mice used in the experiment were all female
.
Each cage of 6 mice is kept in a temperature (21° to 23°C) and humidity controlled colony chamber, maintained with a 12 h light/12 h dark cycle (07:00 to 19:00 light), standard food (4RF21, Musedola Srl, Italy) and water provided ad libitum
.
According to the European Federation of Scientific Associations for Laboratory Animals, all rats in animal facilities are regularly inspected by a health monitoring program and are free of pathogens
.

Proteomics sample preparation

Simultaneous isolation of B cells from freezing [stored in 90% fetal bovine serum (FBS)/10% dimethyl sulfoxide] (to avoid bulk effects).
Peripheral blood mononuclear cells (PBMCs) from SLE and HC patients (n=11/condition) were treated
with PBMCs using the EasySep Human B Cell Isolation Kit (catalog number 17954, STEMCELL Technologies) for deoxyribonuclease.
Complete cell lysis of the purified cell population using a buffer consisting of 4% sodium lauryl sulfate, 100 mM tris/HCl, and 100 mM dithiothreitol (pH 7.
6) and incubate at 95 °C for 5 min
.
Further sonicate the lysed samples in a water bath for 30 min
.
Centrifuge at 17,000 for 20 min, purify the protein extract from the debris.
Transfer the supernatant to a clean tube and process according to Hughes' single-tank solid-phase enhanced sample preparation (SP3) method, etc
.
(79), non-acidification, including protein alkylation step
in 100 mM iodoacetamide.
Digestion using 0.
25-μg of trypsin/endoprotease Lys-C (from Lysobacter enzymogenes (LysC) mixture) in 25 mM ammonium bicarbonate buffer for continuous shaking
at 1400 rpm.
The next day, remove the beads, further purify the peptide sample by SP3 peptide purification, and evaporate in
a vacuum centrifuge.
The dried sample is dissolved in buffer A, sonicated for 5 min, absorbance at 280 nm is measured with nanodrop technique, and the peptide concentration
is determined.

Ultra-high pressure nano-liquid crystal

Each sample is analyzed three times (technical replicates).

Use C18 capture columns (aclaim PepMap100; 100μm×2 cm； Thermo Fisher Scientific) pre-concentrates approximately 0.
5 μg of peptide at a flow rate of 10 μl/min and loads it onto a 50 cm long C18 column (inner diameter 75 μm; Particle size 2 μm; 100 feet; Acclaim PepMap100 RSLC，Thermo Fisher Scientific）
。 The binary pump of high performance liquid chromatography (RSLCnano, Thermo Fisher Scientific) consists of solution A [2% (v/v) acetonitrile (ACN) dissolved in 0.
1% (v/v) formic acid] and solution B [80% (v/v) ACN in 0.
1% (v/v) formic acid
].
Separate the peptides with a linear gradient, progressing from 5% B to 27.
5% B in 58 min to 40% B in 2 min to reach 99% B in 2 min and stay there for 5 min, then allow equilibration for 20 min with a flow rate of 300 ml/min
.
Place the column in
an oven at 50 °C.

Tandem mass spectrometry

The eluted peptides are ionized by a nanospray source and detected
by a Q-Exactive HF-X mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) in a data-dependent mode.
The mass/charge ratio of the peptide is 350~1500 (m/z) The resolution of MS1 is 120000, the automatic gain control (AGC) is 3×10⁶, and the maximum injection time is 100ms, This is followed by the 12 tandem mass spectrometry with the most abundant 2+-tetra⁺ charged ions with a resolution of 15000, AGC of 1×10⁵, and a maximum injection time of 22ms, The isolation window is 1.
2 m/z under 28 standardized collision energy (NCE) and 30 s dynamic exclusion conditions, using the software Xcalibur (Thermo Fisher Scientific) control system and acquiring the original file, and using meters /z445.
12003.

data analysis

Searching raw files using Proteomic Finder 2.
4 The reference proteome FASTA database (containing 9595959 protein sequences) downloaded from UniProt on September 19, 2019 and the protein metering tool _HCD28_PD spectral library using the peptide search option activate and continuously use mspeSearch and SequestHT nodes
。 Dynamic modification of proteins to oxidation, +15.
995da(M); Deamidation, +0.
984da(N,Q); N-terminal acetylated modification, +42.
011da; Suffer losses, ? 131.
040 dam（M）； and Met loss+acetyl? 89.
030 Dami
.
Carbamimethyl/+57.
021da(C) is a static modifier
.
Filter the results for high-confidence peptides and add enhanced peptides and protein annotations
.
Only the master protein is evaluated
.
Quantitative abundance is based on intensity values and normalized to total peptide amounts
.
Statistical evaluation of protein normalized abundance in healthy individuals and SLE patients using proteomic discovery software (background-based pairing).

The minimum percentage for replication features is set to 60%.

A total of 4995 proteins were identified containing at least two peptides
.
Mass spectrometry proteomics data have been deposited via PRIDE into the Protein Exchange Society (80) dataset identifier PXD031389
.

Enrichment analysis of proteomic data

1094 differentially expressed proteins and at least two expressed peptides based on FDR (false discovery rate) < 0.
05 were used as inputs for STRING enrichment analysis (81) and QIAGEN IPA (Chagen Company
.
; https://digitalinsights.
qiagen.
com/IPA) (82) Tools
.
FDR<0.
05 was used to determine the significance of rich gene ontobiological processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) terms in string analysis, and FDR<0.
2 was used to determine the significance of enrichment typical pathways in non-directed IPA analysis
.
A complete list of differentially expressed proteins and enrichment terms is provided in Table S2
.

RNA sequence data

Data published in FastQ format (15) is downloaded and processed as described
below.
Evaluation of sequencing quality with FastQC software (83).
The raw reads in fastq format are cropped to adapter content and low-quality bases (Q<30), using Cutapt (84) on the 3' end and consistent with the human genome (hg38 version) using the star2.
6 algorithm (85).
Gene quantification with HTSeq (86).
GENCODE COMMENT FILE VERSION 29 IS USED FOR ANNOTATIONS
.
In R(87).

Enrichment analysis of RNA sequence data

GSEA (88) has enriched our gene set based on the molecular signature database v.
7.
0 Log ₁₀ converts the P-value multiplied by the fold change
.
Significantly upregulated genes ranked first, while significantly downregulated genes ranked last
.
The GSEA pre-ranked analysis
is then performed using the default settings.
When FDR, enrichment is considered significant (question value) < 25%.

Isolation of human cells from peripheral blood

Heparinized blood (10 ml) was collected from healthy people and SLE or AS patients and PBMCs
were isolated using Histopaque-1077 (Sigma-Aldrich) density gradient.
Briefly, the blood is diluted 1:1 with phosphate-buffered saline (PBS) and layered
on Histopaque-1077 solution (Sigma-Aldrich).
Centrifuge the tubes at 500 °C for 30 min
at room temperature.
Collect the PBMC layer and rinse the cells
with PBS.
If neutrophils are required in addition to PBMCs, cell isolation is performed using the Histopaque-1077/Histopaque-1119 (Sigma-Aldrich) double gradient method
.
Briefly, blood is diluted 1:2 with PBS and layered in Histopaque-1077/Histopaque-1119 (1:1).

Centrifuge the tubes at 1200 g for 30 min
at room temperature.
Neutrophils are collected at the interface of the Histopaque-1119 and Histopaque-1077 layers and rinsed
with PBS.
For the isolation of total B cells, the EasySep Human B Cell Isolation Kit (catalog number 17954, STEMCELL Technologies) is also used
in most experiments following the Histopaque-1077 protocol.

Flow cytometry and cell sorting

To analyze and isolate immune cells, single-cell suspensions isolated from fresh human PBMCs or neutrophils are stained
with anti-CD19, CD4, CD8, CD25, HLA-DR, CD14, CD16, and CD66b.
All classified cells were 7-AAD negative (420404, BioLegend).

Intracellular staining includes Foxp3, γΗ2ΑΧ (Ser¹³⁹), Κi67, pATM (serial ^{number 1981}), and cPARP1 (Asp²¹⁴).
Fix and stain
cells using the Foxp3 staining device (catalog number: 00-5523-00, eBioscience) according to the manufacturer's instructions.
Cells are classified
on FACSAria III (BD Biosciences) using BD FACSDiva v8.
0.
1 software (BD Biosciences).
To analyze cells cultured after isolation of EasySep human B cells, cells were stained
with binding antibodies against CD19, CD40, CD80, BAFF, HLA-DR, IGD, CD27, CD38, IgM, and IgG.
Apoptosis testing is performed after administration of DDR drug inhibitors, and cultured B cells are further stained
with anti-CD19, annexin V (catalog number 640908, BioLegend), and 7-AAD as recommended by the manufacturer.
To analyze mouse peripheral blood B cells, PBMCs
were isolated using Histopaque-1077 density gradient.
PBMCs are then stained with binding antibodies against B220/CD45R and γΗ2ΑΧ(Ser)¹³⁹).
Data acquisition was performed
on FACSAria III (BD Biosciences), BD Facselesta, and BD FACSDiva v8.
0.
1 software (BD Biosciences).
Analysis
with FlowJo software.
Table S3 includes details of the antibody (fluorescent pigment, clone, vendor, catalog number, and concentration).

Cell cycle assessment

For cell cycle analysis of flow cytometry, first stain 200,000-500,000 B cells per sample with anti-CD19 antibodies at room temperature in 200 μl of 5% FBS/PBS buffer to ensure B cell purity, then wash with PBS, fix the cells using a Foxp3 staining device and stain
them for Ki67 and γΗ2ΑΧ according to the manufacturer's instructions.
Finally, stain with 7-AAD cell DNA content markers (5 μl per sample) in 200 μl of 5% FBS/PBS buffer for 15 min at room temperature, wash with PBS, and resuspend
in 300 μl of 5% FBS/PBS.
Cell analysis uses BD-facselesta and BD-FACSDiva v8.
0.
1 software
.
7-AAD uses a linear scale
.
This method can assess the expression of γΗ2ΑΧ during apoptosis in the ₀, gram ₁, S, and_G2-M stages
.
To analyze B cells on ATRi, B cells were cultured in P96 circular wells (200,000 cells per well) for 3 days under IFN-α (850 U/ml), IL-21 (50 ng/ml)/CpGb (2.
5 μg/ml)/sCD40L (1 μg/ml) survival and mild proliferation stimulation, followed by staining and analysis
.

For cell cycle analysis by confocal microscopy, culture B cells for 45 h under the following conditions: (i) SLE B cells of the IL-21/CpGb/sCD40L cocktail for 45 h as described above for survival and mild proliferation induction, and (ii) HC B cells treated with IFN-α and IL-21/CpGb/sCD40L cocktail (SLE-like B cells).
and (iii) hcb cells with IL-21/CpGb/sCD40L cocktail (control conditions).

After 45 h of culture, cells are exposed to an additional 3 h culture containing EdU (5 μμ) to capture cells in the S phase of the cell cycle, then, after EdU rinsing, we continue to use the Click-iT-EdU cell proliferation protocol for imaging (catalog number:
.
C10337, Thermo Fisher Scientific), simultaneous pH3 staining (1:100; for the detection of_G2-mitotic cells), pATR (Thr¹⁹⁸⁹; 1: 100), as well as Hoechst 33342 (nuclear anti-tank).

At least 80 cells
were analyzed per subject.
The secondary antibody for pH3 was Alexa Fluor 555 against murine IgG (1/500) and the pATR was Alexa Fluor 647 against rabbit IgG (1/200) (Table S3).

Immunofluorescence confocal microscopy

In the cellular immunofluorescence experiment, cells (separated by cell sorting or EasySep Human B Cell Isolation Kit) are implanted in poly pretreated coverslips-I-lysine, fixed with 4% paraformaldehyde for 15 min at room temperature, washed twice
with PBS.
Cells are blocked and penetrated
with 1% bovine serum albumin in PBS (blocking buffer) containing 0.
1% tritonx-100 for 30 min at room temperature.
Then, incubate with primary antibody and 4′,6-diamino-2-phenylindole (DAPI) in blocking buffer for 1 h at room temperature, then wash three times with PBS containing 0.
1% Triton X-100, and then incubate with secondary antibody for 45 min
at room temperature in the dark.
Finally, cells are mounted using an extended diamond corrosion protection patch (catalog number: P36961, Thermo Fisher Scientific Corporation) and visualized using
an inverted or vertical confocal imaging system, Leica SP5.
Calculate spots/cells or intensity/cells using macros developed in Fiji (89).
The main antibody in immunofluorescence is anti-γΗ2ΑΧ (Ser¹³⁹; 1/200), pATR (thrust ¹⁹⁸⁹; 1/100), pChk1 (serial ^{number 345}; 1/50), p-p53 (serial ^{number 15}; 1/100), pChk2 (thrust ⁶⁸; 1.
2/100), p95/NBS1 (1/100), pyrolysis caspase-3 (Asp¹⁷⁵; 1/800) and pDNA PKcs (serial ^{number 2056}; 1/150), secondary antibodies are Alexa-Fluor 555 against mouse IgG (1/500), Alexa-Fluor 488 against rabbit IgG (1/500), and CF 555 against rabbit IgG (1/1000; Table S3).

For analysis, 60 to 100 cells (corresponding to at least four different regions of the coverslip)
were observed per human subject under a confocal microscope.

Cell culture and chemical inhibition

After isolating healthy human B cells from fresh PBMCs (EasySep Human B Cell Isolation Kit), culture 2 in a humidified incubator at 37 °C and plate 5 cell numbers per well in a 96-well round bottom plate (Sarstedt) at a concentration of 1.
5×10
。 Cells were cultured in RPMI 1640 with glutamine (catalog number 61870036, Gibco) with 10% (v/v) heat-inactivated FBS (catalog number 10270106, Gibco), penicillin streptomycin (100 U/ml and 100 μg/ml, respectively; catalog number 15140m, Gibco), sodium pyruvate (1 mm; Catalog number 11360070, Gibco), Hepes (10 mM; catalog number 15630106, Gibco), and 2-mercaptoethanol (0.
05 mM; Catalog number 31350010, Gibco).

In addition, all cultured B cells, regardless of the experiment, were supplemented with IL-21 (50 ng/ml; catalog number 200-21, PeproTech), CpG-B (2.
5 μg/ml; catalog numbers HC4039, Hycult Biotech) and sCD40L/CD154 (1 μg/ml; Catalog number 11343345, Immunization tools) of the "Survival/Light Proliferation" mixture
.
To mimic the DDR of SLE B cells, IFN-α (catalog number 11200-1, PBL Analytical Sciences) was added to the
cells at 850 u/ml.
For chemical ATR inhibition, berzosertib (i.
e.
ATRi; catalog number S7102, Selleckchem) added to cells at 2 or 5 μμ; For Chk1 inhibition, CHR-124 (i.
e.
, Chk1i; catalog number HY-13263, Selleckchem) was added to the cells at 50 nM (details on the legend).

Determination of immunoglobulins and cytokines

After collecting the supernatant from cultured B cells treated with IFN-α (850 U/ml) on day 7, measure the released immunoglobulin with or without atria (2 μμ)
using the LEGENDplex human immunoglobulin isotyping panel (8-plex) according to the manufacturer's instructions.
After collecting the supernatant (5 μμ) of cultured B cells treated with IFN-α (850 u/ml) on day 2, the cytokines
released were determined with the LEGENDplex human B cell plate (13plex).
Data acquisition is done
on FACSAria III (BD Biosciences) and BD FACSDiva v8.
0.
1 software (BD Biosciences).
The analysis was carried out
using LEGENDplex data analysis software.

IRF1 knockout test

For IRF1 knockout, siRNA (i.
e.
, siIRF1) is selected using a silencer (100 nM; catalog number 4392420, Ambion), while the silencer selects a negative control siRNA (i.
e.
, siNeg) as a control (100 nM; Catalog number 4390843, Ambion).

Transfect cells
with small interfering RNAs (siRNAs) using liposome 2000 (catalog number 11668019, Invitrogen) according to the manufacturer's protocol.
Briefly, 3×105 wells per well in a 96-well flat-bottomed plate in Opti-MEM I reduced serum medium (100 μl per well; catalog number 31985070, Gibco) plated cells per well and added appropriate siRNA and transfection reagent for 5 h
.
After 5 h, discard the supernatant and add the above supplemental medium and viability/light proliferation cocktail (five _final = 200 μλ per well).

After 42 h of initial plating, IFN-α (850 u/ml)
is added to all wells.
Gene expression analysis: RNA was isolated after 2 days of initial plating; For protein expression analysis, cells are collected 4 days after primary plating and 5 days after primary plating for cell subset and proliferation
assays.

Chip experiments

To analyze the interaction between IFN-α stimulated B cell ATR molecules, microarray experiments
were performed.
Briefly, 4×105 cells are plated in a 96-well circular bottom plate in medium containing a viability/light proliferation cocktail and
IFN-α.
Cells are collected after 6 h of culture with the above reagents and tested on a chip using the Magna ChIP A/G chromatin immunoprecipitation kit (catalog number 17-10085, Merck, Inc
.
) according to the manufacturer's protocol.
The antibodies used are against IRF1 (catalog number 8478, cell signaling) and control IgG (catalog number 3900, cell signaling techniques; Table S3).

Chip pellet detection and analysis uses the promoter region of real-time quantitative polymerase chain reaction (qPCR) as a primer automatic barcode reader
.
In all cases, the data (value derived from the input sample) were normalized by the "input percentage (%IP)" method and expressed
as multiples of change relative to the control anti-IgG IPs.
The sequence of IRF1 core consistent response elements in an automatic barcode reader utilizes the gene transcription regulatory database of promoter sequences (90) and EPD(91).
The primer sequences are as follows: automatic barcode reader loc1 forward, 5'-tgctgtaatcttgtgaggtagagaca-3'; Automatic barcode reader loc1 reverse, 5'-GGGATTGGTTACAGGCC-3'; ATR loc2 forward, 5′-TCTTGCTTCTCTGTGCCTCC-3′; Automatic barcode reader loc2 reverse, 5'-GGCTTCTTTCGCACCGA-3'; Automatic barcode reader loc3 forward, 5′-cactagtcacacgccaac-3′; and automatic barcode reader loc3 reverse, 5'-CCCGGGTCTATGCAGAAA-3'
.

Quantitative PCR analysis (real-time RT-qPCR)

Lyse the cells in RA1 buffer (Macherey-Nagel) and extract the RNA using the NuclearSpin RNAxS isolation kit according to the
manufacturer's instructions.
The first complementary DNA synthesis was performed
using the PrimeScript RT-PCR kit (catalog number RR037A, Takara).
qPCR was performed
using the KAPA-SYBR Rapid Universal Kit (catalog number KK4602, KAPA Biosystems).
The relative expression of the target gene is calculated by comparing the expression of the target gene with the housekeeping gene (GAPDH).

The primer sequences for real-time reverse transcription qPCR (RT-qPCR) are as follows: automatic barcode reader forward, 5′-ggagatttcctgagcatgtcgg-3′; Automatic barcode reader reverse, 5′-ggcttttactccagaaccatc-3′; ATM forward, 5′-TGTCCAGGACACGAAGGAGA-3′; ATM reverse, 5′-caggttctctcagcataggga-3′; IRR 1 forward, 5′-ccaaggaagtcatgtg-3′; IRR 1 reverse, 5′-TAGCCTGGAACTGTGTAG-3′; Before actin, 5′-ctcttccagctctcttcct-3′; actin reverse, 5′-agactgttggcgtacag-3′; GAPDH forward, 5′-GCACCACCAACTGCTTAG-3′; and the opposite of GAPDH, 5′-GCCCACACAGTCTTCTG-3′
.

Western blotting

After the addition of proteases (1×; catalog number 1183617001, Roche) and a radioimmunoprecipitation test mixture of phosphatases [150 mM sodium chloride, 50 mM tris-HCl (pH 8.
0), 1% Nonidet P-40, 0.
5% sodium deoxycholate, and 0.
1% sodium lauryl sulfate], lysis was performed with vortexing (20 min every 5 min with 1 min interval in between, in ice) (1:100; catalog number P5726, Sigma-Aldrich) inhibitors; Then, centrifuge at full speed for 10 min at 4 °C and collect the supernatant (protein lysate).

Determination of protein content
using detergent compatibility (DC) protein analysis method (catalog number 5000112, Bio-Rad) according to the manufacturer's instructions.
Add β-mercaptoethanol (6×) to the sample and heat at 95 °C for 5 min
.
Break down the cell extracts using a 4% to 20% precast polyacrylamide gel (catalog number: 4561094, Bio-Rad) and transfer the cell extracts to a nitrocellulose membrane (Bio-Rad)
using a transfer device according to the manufacturer's instructions.
The protein load per well is determined to be 20 μg
.
Saturation the membrane with 5% skim milk diluted with TRIS buffered saline (0.
1% Tween 20) for 1 h and incubate for 1 h
at 4 °C with primary antibodies and anti-mouse horseradish peroxidase (HRP) or anti-rabbit HRP secondary antibodies.
According to the manufacturer's instructions, blots
were prepared by enhanced chemiluminescence (catalog number 34580, Thermo Fisher Scientific Corporation).
For analysis, phosphoprotein expression is quantified by normalizing the corresponding total protein: phosphorylation and total protein are first normalized
with housekeeping protein expression.
Antibodies are shown
in Table S3.

Statistical analysis

Statistical analysis using experimental setups with paired or unpaired students is tested and one-way or two-way ANOVA (ANOVA) is performed in the GraphPad Prism v8.
0.
1 software, as shown
in the figure.
Data are expressed
as mean ± SEM.
P<0.
05 was considered statistically significant
.
All P values and n are reported
in the graphical legend.
Investigators did not turn a blind eye to the identity of the samples
.
Compare samples
were collected and analyzed under the same conditions.
Each experiment is repeated at least three times
.
For the LEGENDplex experiment, each test consists of three independent experiments where samples
are collected.

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