NLRP12 is an innate immune checkpoint for repressing IFN signatures and attenuating lupus nephritis progression

Signaling driven by nucleic acid sensors participates in interferonopathy-mediated autoimmune diseases. NLRP12, a pyrin-containing NLR protein, is a negative regulator of innate immune activation and type I interferon (IFN-I) production. Peripheral blood mononuclear cells (PBMCs) derived from systemic lupus erythematosus (SLE) patients expressed lower levels of NLRP12, with an inverse correlation with IFNA expression and high disease activity. NLRP12 expression was transcriptionally suppressed by runt-related transcription factor 1–dependent (RUNX1-dependent) epigenetic regulation under IFN-I treatment, which enhanced a negative feedback loop between low NLRP12 expression and IFN-I production. Reduced NLRP12 protein levels in SLE monocytes was linked to spontaneous activation of innate immune signaling and hyperresponsiveness to nucleic acid stimulations. Pristane-treated Nlrp12–/– mice exhibited augmented inflammation and immune responses; and substantial lymphoid hypertrophy was characterized in NLRP12-deficient lupus-prone mice. NLRP12 deficiency mediated the increase of autoantibody production, intensive glomerular IgG deposition, monocyte recruitment, and the deterioration of kidney function. These were bound in an IFN-I signature–dependent manner in the mouse models. Collectively, we reveal a remarkable link between low NLRP12 expression and lupus progression, which suggests the impact of NLRP12 on homeostasis and immune resilience.


Ligand Stimulation and Virus Infection
THP-1, human CD14 + monocytes and mouse BMDC were applied for the ligand stimulation. Poly (dA:dT), poly (dC:dG), LMW poly(I:C) were suspended in LyoVec ™ transfection reagent (InvivoGen) in a concentration of 0.01 mg/ml for 15 min and then was transfected into cells (1μg ligand for 1×10 5 cells). Human CD14 + monocytes or THP-1 were infected with Vesicular stomatitis Virus-Indiana strain (ATCC ® VR-1419™) or human herpesvirus 1 (ATCC ® VR-1493™) at the multiplicity of infection (MOI) of 0.1. For cytokine treatment, human CD14 + monocytes or THP-1 cells were treated with IFNα2 (3,000 U) for 4 or 8 hours as mention in legends. In the study of the inhibitors to the epigenetic regulation, 3 μM or10 μM those inhibitors were used to treat the cells, followed by stimulating cells with ligands.
Dual-luciferase reporter assay HEK293T (3×10 5 cells in a 24-well plates) were transfected using lipofectamine TM 2000 (Cat.11668019, Thermo Scientific, Inc.). Ten ng of pRT-TK Renilla luciferase reporter plasmid and 100 ng of firefly luciferase reporter plasmids were co-transfected with indicated plasmids. For NLRP12 promoter, 50 ng of pRT-TK Renilla luciferase reporter plasmid and 500 ng of NLRP12 promoter firefly luciferase reporter plasmids (pLG4) were co-transfected with RUNX1 plasmids. Luciferase activity was measured 24 h after transfection using the Dual-Glo Luciferase Assay System (Cat.E2920, Promega, Inc.). Renilla luciferase reporter plasmid as an internal control was transfected in all the setting of luciferase reporter assay.

EMSA
Cytoplasmic and nuclear fractions were extracted using NE-PER nuclear and cytoplasmic extraction reagents. Gel shift assay was conducted by LightShift ® Chemiluminescent EMSA kit (ThermoFisher). Eight μg of nuclear extract was incubated with serial of reaction components. Biotin-labeled DNA probe (hot probe) containing consensus RUNX1 binding sequences (500 fmol) was added to the reaction mixed followed by adding a non-labeled DNA probe (cold probe, 30 pmol) to compete the DNA (hot probe)-protein interaction. The DNA-protein complexes were resolved on a non-denaturing polyacrylamide gel prior to chemiluminescent graphic imaging. DNA probe was generated by annealing the oligonucleotides and its complementary strands.

Chromatin immunoprecipitation (ChIP)
Cells (3×10 7 ) per reaction were fixed with formaldehyde to crosslink the protein-DNA complexes, and DNA in lysed cells was sonicated to lengths of 500-1000 bp in a

Cells isolation and Flow Cytometry Analysis
In pristane-treated mice, peritoneal cells were harvested by lavage of the peritoneal cavity with 8 mL of PBS; kidney was minced and digested with the HBSS buffer containing 1 mg/ml collagenase type IV (Sigma C5138) at 37°C for 10 min as previous description. The tissue homogenates were passed through cell strainer (40 μm) and lysed with RBC lysis buffer to remove the remaining red blood cells. Cells were then re-suspended in 40% (w/v) Percoll-HBSS solution and overlaid onto 70% (w/v)

Immunofluorescence analysis
Frozen sections with 3-4 µm were fixed in acetone -20℃ for 7 minutes followed by blocking with 10% bovine serum albumin (BSA) for 1 hour in room temperature.

Immunohistochemistry (IHC) staining and histology analysis
Paraffin-embedded kidney section was used for immunohistochemistry staining.