AIM2 deficiency in B cells ameliorates systemic lupus erythematosus by regulating Blimp-1–Bcl-6 axis-mediated B-cell differentiation

Absent in melanoma 2 (AIM2) has been reported to be a component of inflammasomes in innate immune cells. Surprisingly, AIM2 is expressed by B cells, and higher AIM2 expression is observed in the B cells from lupus patients. To date, the inflammasome-independent function of AIM2 in B cells remains unclear. Here, we report increased expression of AIM2 in human tonsil memory and germinal center (GC) B cells and in memory B cells and plasma cells from the circulation and skin lesions of lupus patients. Conditional knockout of AIM2 in B cells reduces the CD19+ B-cell frequency in lymph nodes and spleens, and dampens KLH-induced IgG1-antibody production. In a pristane-induced mouse model of lupus, AIM2 deficiency in B cells attenuates lupus symptoms and reduces the frequency of GC B cells, T follicular helper (Tfh) cells, plasmablast cells, and plasma cells. Furthermore, the loss of AIM2 in human B cells leads to the increased expression of Blimp-1 and reduces the expression of Bcl-6. However, the silencing of Blimp-1 and Bcl-6 has no significant effect on AIM2 expression, indicating that AIM2 might be the upstream regulator for Blimp-1 and Bcl-6. In addition, IL-10 is found to upregulate AIM2 expression via DNA demethylation. Together, our findings reveal that AIM2 is highly expressed in the B cells of lupus patients and promotes B-cell differentiation by modulating the Bcl-6–Blimp-1 axis, providing a novel target for SLE treatment.


Pristane-induced lupus-like mouse model:
Female CD19 cre AIM2 f/f and AIM2 f/f mice aged 8-10 weeks were intraperitoneally injected with 500μl pristane (Sigma) per mouse. Urine samples were collected from the pristane-treated mice, and proteinuria was assessed by a colorimetric assay strip (URIT). The serum samples of pristane-treated mice were collected at the beginning and the end of the observation period. The serum anti-dsDNA IgG and ANA IgG levels were detected by ELISA. Draining lymph nodes (dLNs) and spleens from two models were used to execute immune cell analysis by flow cytometry. Using multi-IHC staining technique to analyze the C3 and IgG deposition in kidneys of pristane model.

Co-IP and western blotting
Nuclear and cytosolic proteins were extracted in CD19 + B cells from human tonsils and spleens of animal model by using a kit (Invent SC-003), and used to analyze the location of AIM2 by western blotting. Additionally, extracting the total proteins of CD19 + B cells from human tonsils to explore the interaction of AIM2 with Blimp-1, Bcl-6 and ASC by Co-IP.

Silencing AIM2 or Blimp-1 in vitro
First, we sorted CD19 + B cells from healthy subjects with magnetic beads (Miltenyi Biotec,. Second, 2x10 6 cells were resuspended in 100 µL electroporation liquid (Lonza, V4XP-3024) and then mixed with 2.5 µL AIM2 ASO (Ruibo, 20 µM) or 2.5 µL Prdm1 siRNA (Ruibo, 20 µM). All of the cells were transferred into electroporation cups to be transfected following the human B cell protocol in an electrical transfection instrument (Lonza, 4D-Nuclefector TM ). Finally, all the transfected cells were transferred into 1 mL of 1640 complete culture medium (90% 1640 with 10% FBS) containing IL-10 as a stimulator (the final concentration was 20 ng/ml) and cultured in an incubator (37°C, 5% CO2). After 48 hours, total RNA from all the samples was collected by TRIzol for subsequent qRT-PCR analysis.
All methods are described in detail in the supplementary material.

Statistical analysis
We used SPSS 18.0 to perform the statistical analyses. All the data were presented as the mean ± SEM and were assessed for normal distribution and similar variance between groups. Statistical significance of groups was assessed using two-tailed unpaired Student's t-tests for comparisons between two groups and one-way analysis of variance (ANOVA) with relevant post-hoc tests for multiple comparisons. We used the 2-tailed Mann-Whitney U test for statistical analyses when the data were not normally distributed or displayed unequal variances between two groups. The correlation analysis of two indexes was performed using Pearson's r test or Spearman's r test (for abnormally distributed data). No statistical method was used to predetermine the sample size. All the animals were randomly divided into the treatment groups.

Study approval
All human sample studies were approved by the ethics committee of the Second Xiangya Hospital of Central South University. We obtained written informed consent from all the subjects. All the animal care protocols and experiments were reviewed and approved by the Animal Care and Use Committee of the Laboratory Animal Research Center at the Second Xiangya Hospital of Central South University.  Table 3.

Supplementary Methods Subjects
Lupus skin lesion samples: 3 SLE and 3 DLE patients' skin lesion tissues were collected from the department of dermatopathology of the Second Xiangya Hospital of Central South University. The detailed information of SLE patients and HCs is listed in Supplemental Table 4.

Mice
AIM2 f/f mice and AIM2 -/mice were purchased from the Shanghai Model Organisms Company. C57BL/6J mice were purchased from the Slack Company. CD19 cre mice were donated by Professor Wang Honglin of Shanghai Jiaotong University. AIM2 f/f mice were bred with CD19 cre mice to generate control AIM2 f/f and CD19 cre AIM2 f/f (CKO) mice.

Human serum IL-10 ELISA analysis
Collecting the serum samples of 56 SLE patients who were recruited from outpatient clinics of the Second Xiangya Hospital of Central South University and 24 sex-and age-matched healthy controls (HC) who were recruited from the Physical Examination Center at the Second Xiangya Hospital of Central South University. Serum IL-10 from SLE patients and HCs were analyzed by ELISA according to the protocol (4Abio, cat. CHE0013-096). The detailed information of SLE patients and HCs is listed in Supplemental Table5.

Human naï ve B cells were stimulated with IL-10 in vitro
Naï ve B cells from peripheral blood of healthy people were isolated by human naï ve B cell magnetic beads (Miltenyi Biotec,. 2x10 6 cells per well of 24-well plate were stimulated with IL-10 (Sino Biological, cat. 10947-HNAE, 20ng/ml) respectively. After 48 hours of stimulation, samples were collected to analyze the expression levels of AIM2 in each subgroup of B cells (including naï ve B cells, memory B cells and plasma cells) by flow cytometry. The detailed information of healthy donors is listed in Supplemental Table6.

RNA isolation and qRT-PCR
Total RNA was extracted using TRIzol reagent (Invitrogen). RNA quality control and concentration were detected by NanoDrop spectrophotometer (ND-2000, Thermo). mRNA was reverse-transcribed by the PrimeScript RT reagent Kit with a gDNA Eraser (TaKaRa, cat. RR047A) according to the manufacturer's protocols. qRT-PCR was carried out with TB Green Premix Ex Taq TM (Tli RNaseH Plus) (TaKaRa, cat. RR420A) by using thermocycler (Roche, LightCycler 96). The fold change of target gene expression was calculated as 2 -ΔΔCt (ΔΔCt=ΔCt of experimental group -ΔCt of control group), which was normalized to the control group. mRNA primers for AIM2, Blimp-1, XBP1, Bcl-6, IRF4, MTA3, CD27, CD38 and GAPDH were purchased from Tsingke Biotechnology Co.. All gene primers are shown in Supplemental Table 8.

Pyrosequencing
Genome DNA of SLE patient CD19 + B cells, healthy human CD19 + B cells and healthy human CD19 + B cells stimulated with IL-10 were isolated using TIANamp Genomic DNA kit (TIANGEN, Cat. DP304-02). Approximately 20ng genome DNA was conversed by sulfite according to the manufacturer's protocols (Zymo research, EZ DNA Methylation TM kit, cat. D5002). Conversed genome DNA was used as amplification template. The targeted fragment from promoter of AIM2 was amplified using special primers and then was sequenced by Pyrophosphate sequencer (QIAGEN, PyroMark Q24). Primers for AIM2 were purchased from Tsingke Biotechnology Co.. Primer sequences are shown in Supplemental Table 9.

Histology
Mouse kidneys from pristane models were fixed by paraformaldehyde of which the concentration was 4% and then embedded in paraffin. Sliced sections of 5 μm thickness were affixed into HistoBond® adhesive microscopic slides. These slides were stained with H&E (hematoxylin and eosin) and then stored at room temperature. For the immunofluorescence (IF) assay, slides were incubated at 70°C for one hour to dissolve the wax. The slides were further dewaxed by followed sequential incubation: xylene for 10 minutes, 95% ethanol for 15 minutes, and 70% ethanol for 15 minutes. And then, the slides were incubated in blocking buffer (5% FBS in PBST) for 30 minutes at room temperature. Sections were incubated with primary and secondary antibodies at 4°C overnight and 30 minutes, respectively. The following antibodies were used: anti-C3 (Abcam, cat. ab11862), HRP-conjugated Affinipure Goat anti-rabbit IgG (H+L) (Proteintech, cat. SA00001-2) and Alexa Fluor® 488 anti-mouse IgG antibody (Abcam, ab150117). Immune-complex (IC) depositions in the mouse kidneys were analyzed and evaluated using a digital fluorescence microscope (Leica). We used NLS Elements Basic Research Imaging Software (Leica) to record and analyze fluorescent staining for presentation.
Multi-IHC of skin tissue was optimized by performing a duplex experiment (CD19, Opal 570 and Aim2, Opal 650). All TSA staining of tissues were finished with the fluorophores DAPI counterstain.

IHC Tissue imaging and analysis
We used the PerkinElmer Vectra multispectral imaging system (PerkinElmer, Vectra 3.0.3) to scan all slides. All multispectral images were deconvoluted using spectral libraries built from images of single stained tissues for each reagent by inForm Advanced Image Analysis software (PerkinElmer, inForm 2.3.1).

qRT-PCR for CD19 + B cell from from healthy human and SLE patients
We sorted CD19 + B cells from healthy human and SLE patients by magnetic beads (Miltenyi Biotec, 130-050-301). Total RNA were extracted by TRIzol reagent (Invitrogen), and then we got cDNA by RT(TaKaRa, cat. RR047A). qPCR was carried out with TB Green Premix Ex Taq TM (Tli RNaseH Plus) (TaKaRa, cat. RR420A) by using thermocycler (Bio-Rad CFX). The fold change of target gene expression was calculated as 2 -ΔΔCt (ΔΔCt=ΔCt of experimental group -ΔCt of control group), which was normalized to the control group. mRNA primers for ASC, NLRP1, NLRP3, NLRP6, NLRP12, IFI16, NLRC4 and GAPDH were purchased from Tsingke Biotechnology Co.. Clinical sample information were listed in supplemental Table10. All gene primers are shown in supplemental Table11.

CD19 + B cell were stimulated by steroids Dexamethason(DEX) and qRT-PCR
We sorted CD19 + B cells from healthy human and SLE patients by magnetic beads (Miltenyi Biotec, 130-050-301). 2x10 6 CD19 + B cells from healthy human were cultured by complete medium (90%1640 with 10%FBS) in 24 pore plate, and stimulated by Dexamethason with different concentration(Final concentration: 0μg/ml, 5μg/ml,10μg/ml). All sample were collected after 48h by stimulated by DEX. Total RNA were extracted by TRIzol reagent (Invitrogen), and then we got cDNA by RT(TaKaRa, cat. RR047A). qPCR was carried out with TB Green Premix Ex Taq TM (Tli RNaseH Plus) (TaKaRa, cat. RR420A) by using thermocycler (Bio-Rad CFX). The fold change of target gene expression was calculated as 2 -ΔΔCt (ΔΔCt=ΔCt of experimental group -ΔCt of control group), which was normalized to the control group. mRNA primers for AIM2, ASC, NLRP1, NLRP3, NLRP6, NLRP12, IFI16, NLRC4 and GAPDH were purchased from Tsingke Biotechnology Co.. All gene primers are shown in supplemental Table11. Sample information from healthy donor was listed in supplemental Table12.

CHIP-qRT-PCR
PBMCs of healthy volunteers and SLE patients were sorted by Ficoll-Paque Plus (GE healthcare, cat. 17-1440-03) , and CD19 + B cells from PBMCs were sorted with magnetic beads (Miltenyi Biotec,. 1.5×10 6 CD19 + B cells stimulated with IL-10 (20ng/ml) were collected after 72h. After cross-linking with 1% formaldehyde, stopping the cross-linking with glycine, and performing cell lysis to obtain nuclear lysate according to the kit instructions (Abcam, ab500). 1.5×10 6 cells/CHIP was incubated with antibodies at 4°C overnight, and the amount of H3AC (Active Motif, 61637), H3K4me3 (Active Motif, 61379), H3K9me3 (Active Motif, 61013) and H3K27me (Active Motif, 61017) antibody is 5μg/CHIP equally. The next day, after adding 40μl protein A agarose beads, rotating at 4°C for 1h. The magnetic bead-antibody-antigen complex was washed for 4 times using the 1×CHIP buffer in the kit to remove unbound proteins, and then de-crosslinked and digested with proteinase K. The magnetic bead-antibody-antigen was separated by heating at 98°C for 10 minutes, and centrifuged to obtain the DNA supernatant, and finally the DNA concentration was determined. The primers for different positions in the AIM2 promoter region were designed and specific identification was performed, and then the reaction was performed according to the qRT-PCR system and the relative quantitative value was calculated. Sample information from healthy donor and SLE patients were listed in supplemental Table13. Primers for AIM2 promoter CHIP-qRT-PCR were listed in supplemental Table14.

Statistical analysis
We used the software GraphPad Prism 8.0. to perform statistical analysis. All data was presented as the mean ± SEM and assessed for normal distribution and similar variance between groups. Statistical significance of groups was assessed using a two-tailed unpaired Student's t-tests for comparisons between two groups and one-way analysis of variance (ANOVA) with relevant post hoc tests for multiple comparisons. We used the 2-tailed Mann-Whitney U test for statistical analysis, when the data were not normally distributed or displayed unequal variances between two groups. The correlation analysis of two indexes was performed using Pearson's r test or Spearman's r test (for abnormally distributed data). No statistical method was used to predetermine the sample size. All animals were allocated randomly to treatment groups.

Study approval
All samples studies from human were approved by the ethics committee of the Second Xiangya Hospital of Central South University. We obtained the written informed consent from all subjects.     Table S7. Sample information of CD19 + B cells of peripheral blood mononuclear cells (PBMCs) from healthy donors which were stimulated with IL-10.    Table S10. qRT-PCR sample information of CD19 + B cells of PBMCs from healthy donors and SLE patients.  Reverse: GGCAATGTGGTTCACGTACT Table S14. Primers for AIM2 promoter Chip-qRT-PCR