Abstract
Rare genetic variants in toll-like receptor 7 (TLR7) are known to cause lupus in humans and mice. UNC93B1 is a transmembrane protein that regulates TLR7 localization into endosomes. In the present study, we identify two new variants in UNC93B1 (T314A, located proximally to the TLR7 transmembrane domain, and V117L) in a cohort of east Asian patients with childhood-onset systemic lupus erythematosus. The V117L variant was associated with increased expression of type I interferons and NF-κB-dependent cytokines in patient plasma and immortalized B cells. THP-1 cells expressing the variant UNC93B1 alleles exhibited exaggerated responses to stimulation of TLR7/-8, but not TLR3 or TLR9, which could be inhibited by targeting the downstream signaling molecules, IRAK1/-4. Heterozygous mice expressing the orthologous Unc93b1V117L variant developed a spontaneous lupus-like disease that was more severe in homozygotes and again hyperresponsive to TLR7 stimulation. Together, this work formally identifies genetic variants in UNC93B1 that can predispose to childhood-onset systemic lupus erythematosus.
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Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that typically develops in adults but can affect around 1 in 100,000 children1. There is a significant genetic contribution to the condition, ranging from common variants with small effects, through to fully penetrant disease-causing alleles2. One example of this is TLR7, which marks a genetic interval that is a risk factor for developing SLE3, but can also drive a monogenic form of the disease as a result of gain-of-function mutations4. TLR7 typically functions as a sensor of viral single-stranded (ss)RNA in endosomes, to which it is trafficked by the transmembrane protein UNC93B1 (ref. 5). TLR7 then signals through Myd88 to interleukin-1 receptor-associated kinase 1/-4 (IRAK1/-4), leading to nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and type I interferon (IFN) expression programs that are typically associated with SLE6, but critically are required to fight viral infection. Consequently, people with loss-of-function mutations in TLR7 or UNC93B1 are immunodeficient7,8. On the other hand, again similar to TLR7, UNC93B1 expression is increased in patients with SLE and active disease9, and mutations in murine UNC93B1 can cause a lupus-like disease10,11. There is also a sporadic lupus-like disease in dogs that is the result of a mutation in UNC93B1 (ref. 12). Despite all of these compelling data linking UNC93B1 to disease, there was still no genetic variant in the human population that formally validated its role in SLE pathogenesis.
Rare UNC93B1 variants in patients with childhood-onset SLE
Given the recent observation that rare genetic variants in TLR7 can cause childhood-onset SLE4, we searched in this patient population for variants that may influence the interaction between TLR7 and the transmembrane (TM) protein that regulates its cellular localization, UNC93B1 (ref. 13). We identified a new variant in UNC93B1 (c.A940G p.T314A) using whole-exome sequencing (WES) in patient 1 (P1), which is not present in the general population and is highly conserved (Fig. 1a). Based on a published structure of UNC93B1 in complex with TLR7 (ref. 13), the affected amino acid is at the start of helix 2 (H2) between TM helices TM6 and TM7 of UNC93B1 (Fig. 1b). H2 is positioned on top of the TM helix of TLR7 responsible for the UNC93B1–TLR7 interaction. In addition, Thr314 is in close proximity to the carboxy terminus of the protein, a disordered region that has been shown to have two phosphorylation sites (Ser547 and Ser550) that regulate TLR7 activation11. Looking more broadly in a cohort of 272 patients with lupus recruited at the Guangzhou Women’s and Children’s Medical Centre, we found that seven (P2–P8) encode UNC93B1 (c.G349T p.V117L), a highly conserved variant (Fig. 1a). Structurally, this residue lies at the interface between UNC93B1 subunits (protomers) in the published dimeric assembly (Fig. 1b). Although these protomers are close to each other, there are no noticeable interactions that would prevent dimerization of TLR7, similar to what occurs for TLR3 (ref. 13). Instead, V117L may act in a similar way to K333R, which is also located at an UNC93B1 protomer interface and results in increased TLR7 activation. However, as Lys333 is ubiquitinated, and not very close to V117L, this would represent a different mechanism of activation11. UNC93B1 V117L variant is present in the general east Asian population and is most prevalent in south coast Han individuals (Fig. 1c)14. We calculate that it confers a 17.9-fold increased risk of developing childhood-onset SLE (Supplementary Table 1). For all patients with SLE identified as carrying this allele, there were no immediate reports of affected family members. The presence of heterozygous UNC93B1 V117L was confirmed for the father of P2 and the mother of P3, both of whom are currently unaffected. The two UNC93B1 variants were nominated as potential effectors for systemic inflammation in our subjects according to the well-known biological role of UNC93B1 in autoimmunity15, predicted pathogenicity scores (Supplementary Table 2) and lack of other likely pathogenic variants found in primary immunodeficiency genes based on another early onset lupus cohort16.
a, Amino acid conservation across five species, as indicated, for residues surrounding the variants of interest in UNC93B1. b, UNC93B1 (Protein Data Bank (PDB) 7CYN) shown in light green and green (surface and cartoon representations) with Thr314 shown as blue sticks on H2. TLR7 protomers are shown in purple and magenta (surface and cartoon representations). Val117 and Leu117 are shown as red sticks between the interface of two UNC93B1 protomers. The structural analysis software used was Pymol v.2.5.8. c, UNC93B1 V117L variant local geographical distribution.
Clinical parameters for patients encoding UNC93B1 T314A (P1) and V117L (P2–P7) were available. These include elevated titers of serum autoantibodies to double-stranded DNA (anti-dsDNA) and anti-nuclear antibodies (ANAs), with increased protein in 24 h of collected urine and depleted serum complement 3/4 (Fig. 2a and Extended Data Figs. 1a–g and 2a–f). Comprehensive electron microscopy, light microscopy and immunofluorescence studies for kidney tissues of P1 are consistent with diffuse proliferative lupus nephritis with membranous lupus nephritis, IV −G (A) +V. The mesangial cells are aggregated. Electron densification was deposited in stromal hyperplasia, in the subepithelial, intrabasement, subendothelial and mesangial zones. In the tubulointerstitium, epithelial cells were cavitated with a small amount of renal tubular atrophy and inflammatory cells infiltrated into the renal interstitium. In renal interstitial vessels, there was red blood cell (RBC) aggregation in the loops of little capillaries and thickening of the arteriole wall (Fig. 2b–f). The complete blood count from noncoagulated whole blood for P1–P3 and P5–P7 was generally normal, with periods of leukocytosis (P1–P3 and P5), neutrophilia and monocytosis, corresponding to periods of lymphocytopenia (Extended Data Fig. 1a–c,e–g). The overall disease score was clinically evaluated using SLEDAI and SLEDAI-2k scoring systems, in which results ranged from 2 to 20 (Fig. 2a and Extended Data Fig. 1a–d,f). Other clinical parameters and treatments are reported in Supplementary Table 3. We isolated peripheral blood mononuclear cells (PBMCs) from patients with UNC93B1V117L, P2 and P3, using a Ficoll–Hypaque density gradient separation method and found spontaneous expression of interleukin (IL)-8, MX1 and IFN-stimulated gene 15 (ISG-15) genes in P2 (Fig. 2g and Extended Data Fig. 3a), and IL-8, MX1, IFN-induced proteins with tetratricopeptide repeat (IFIT)1, IFIT3 and ISG-15 in P3 (Fig. 2h and Extended Data Fig. 3b). Indeed, we observed induced secretion of typical TLR7-induced cytokines, IL-8, IP-10, IFNγ, IFNλ1/2/3, IL-12p70, IL-10, IFNα, IFNβ and IL-6, in P2 (Fig. 2i and Extended Data Fig. 3c,d), and IL-8, IFNα2, IFNγ, IFNλ1/2/3, IL-12p70 and IL-10 in P3 (Fig. 2j and Extended Data Fig. 3e). Furthermore, we detected elevated levels of IP-10, IL-8 and tumor necrosis factor (TNF) in the plasma of P2 and P3 (Fig. 2k) and IFNα2, IFNγ, IFNλ1/2/3, IL-6, IL-12p70, IL-1β, granulocyte–macrophage colony-stimulating factor (GM-CSF) and IL-10 in plasma of P2 (Extended Data Fig. 3f). These data identify rare UNC93B1 variants in patients with childhood-onset SLE, associated with typical clinical parameters and elevated levels of lupus-associated cytokines, similar to patients with mutations in TLR7 (ref. 4).
a, Anti-dsDNA autoantibodies (normal range (NR), <18 IU l−1), ANA autoantibodies (NR, <18 IU l−1), 24-h urinary protein (NR, 0–0.1 g per 24 h) and SLEDAI score of disease for P1 (T314A), P2, P3 and P4 (all V117L). Data are for monthly visits postdiagnosis. NR is highlighted in gray. b–f, Kidney pathology for patient with UNC93B1 T314A. b, Transmission electron microscopy showing two glomeruli, capillary endothelial cell proliferation, vacuolar degeneration, RBCs, monocytes and neutrophil aggregation in little vascular loops. The podocyte foot process has diffuse effacement (yellow arrows), with the cell swollen and cavitated. The basement membrane has diffuse thickening up to 1,300 nm (red arrows), P1. Scale bar, 4 μm. c, Periodic acid–Schiff (PAS) stain showing thickening of the basement membrane and mesangial and endothelial cell proliferation (white arrows), P1. Scale bar, 50 μm. d, PAS methenamine (PASM) stain showing thickening of the basement membrane and a small number of platinum ear-like structures (white arrows), P1. Scale bar, 50 μm. e, Immunofluorescence for collagen IV of the glomerular basement membrane showing α3+ve, α5+ve, P1. Scale bar, 20 μm. f, Immunofluorescence of kidney tissue showing complement 3 deposition, P1. Scale bar, 20 μm. g, RT–qPCR analysis of IL-8 and MX1 mRNA expression in the PBMCs of P2 compared with healthy controls. h, RT–qPCR analysis of IL-8 and MX1 mRNA expression in the PBMCs of P3 compared with healthy controls. i, Production of IL-8 and IP-10 in the supernatant of PBMCs isolated from P2 and healthy controls measured by CBA. j, Production of IL-8 and IFNα2 in the supernatant of PBMCs isolated from P3 and healthy controls measured by CBA. PBMCs in g–j were isolated from patients’ whole blood and incubated in media for 12 h before analysis without stimulation. k, Levels of IP-10, IL-8 and TNF in the plasma from P2 and P3 compared with healthy controls measured by CBA. For g–k, n = 3 biological samples; indicated P values were determined by two-way ANOVA, multiple comparisons, Padj value; data are presented as mean with s.d.
UNC93B1 variants drive inflammation via TLR7/-8 and IRAK1/-4
To study the direct effect of these rare UNC93B1 variants, we created an in vitro model with THP-1 monocytes overexpressing wild-type (WT), V117L or T314A using UNC93B1 lentivirus constructs. The patient mutations trigger spontaneous upregulation of transcripts for IFNβ and the ISGs, IFIT3, ISG-15 and ISG-20L2 (Fig. 3a and Extended Data Fig. 4a). Upregulated messenger RNA levels of inflammatory cytokines, IL-8, IL-12a and TNF, were also observed (Extended Data Fig. 4a). In addition, the mutated UNC93B1-induced phosphorylation of IFN regulatory factor (IRF)5, NF-κB and MAPK (JNK and P38) is shown by immunoblotting (Fig. 3b). Overexpression of UNC93B1 V117L and T314A resulted in excess secretion of IFNα, IFNβ and IL-6 as detected by ELISA (Fig. 3c) and IL-8 and IP-10 as detected by cytokine bead array (CBA) (Extended Data Fig. 4b). For an unbiased comparison, we performed RNA sequencing (RNA-seq) in THP-1 cells overexpressing UNC93B1 V117L or T314A. From the most significantly upregulated genes, at least six are known biomarkers for disease activity in lupus (DEFB1 (ref. 17), PRLR (ref. 18), S100A8/-9 (ref. 19), FCER2 (ref. 20) and PRG2 (ref. 21)) (Fig. 3d,e). Further gene set and pathway analysis pointed toward programs related to innate immune response, phagosome activity and antigen processing and presentation (Extended Data Figs. 4c–f and 5a,b). Owing to the core role of B cells in the pathogenesis of SLE22, we generated immortalized B cell lines from patients with UNC93B1V117L variants (P2 and P3) and healthy controls. At baseline, B cell lines from the patients revealed induced expression of IL-6, IL-8, TNF, IL-12a and ISG-20L2 genes compared with healthy controls (Fig. 3f and Extended Data Fig. 5c). Increased secretion of IL-6, IL-8, IL-12p70, IL-10, TNF and GM-CSF in the supernatant of the unstimulated B cells from patients with the UNC93B1 V117L variant was also observed (Fig. 3g and Extended Data Fig. 5d,e). Collectively, these results indicate that the UNC93B1 variants identified can intrinsically promote inflammation and immune dysfunction associated with type I IFN and NF-κB signaling pathways.
a, RT–qPCR analysis of IFNβ, IFIT3 and ISG-15 expression in the indicated THP-1 cell lines (n = 3 biological replicates). There were three independent experiments. b, Levels of phosphorylated NF-κB, JNK, p38 and IRF5, as measured by immunoblotting, in lysates of the indicated THP-1 cells. Data are representative of three independent experiments. c, Production of IL-6, IFNα and IFNβ in the indicated THP-1 cell lines (n = 3, three independent experiments measured by ELISA). The indicated P values in a and c were determined using unpaired, two-tailed Student’s t-test; the data are presented as the mean with s.d. d,e, RNA-seq was performed for UNC93B1V117L (d) and UNC93B1T314A (e) THP-1 cells compared with WT. DEGs are presented as a volcano plot and mRNA was extracted from THP-1 of UNC93B1WT, UNC93B1V117L and UNC93B1T314A (n = 3 biological replicates). f, RT–qPCR analysis of IL-6, IL-8 and TNF expression in the indicated immortalized B cell lines (baseline) (n = 3 biological replicates). g, Production of IL-6, IL-8 and IL-12p70 in the indicated immortalized B cell lines (baseline) (n = 3 biological replicates, measured by CBA). The indicated P values in f and e were determined by two-way ANOVA, multiple comparison, Padj value; data are presented as mean with s.d.
So far, the main function for UNC93B1 is to regulate trafficking and localization of nucleic acid-sensing TLRs (NAS-TLRs), TLR3, TLR7, TLR8 and TLR9 (refs. 5,23,24). Thus, we next investigated which could be involved in this phenotype by stimulating patients’ PBMCs using poly(I:C), R848 and CPG-C, synthetic ligands for TLR3, TLR7/-8 and TLR9, respectively. The results revealed that patient PBMCs stimulated by R848 showed induced expression of IFIT1 and TNF compared with healthy controls, but not when stimulated by poly(I:C) and CPG-C (Fig. 4a). In addition, secretion of TNF, IL-1β and IL-6 in the supernatant of PBMCs from P2 were increased specifically after stimulation by R848, but not poly(I:C) or CPG-C (Fig. 4b and Extended Data Fig. 6a). These results were independently confirmed using PBMCs of P3 (Fig. 4c and Extended Data Fig. 6b). As R848 is a dual TLR7 and TLR8 synthetic agonist, we next stimulated PBMCs, THP-1 and/or immortalized B cell lines with a variety of specific agonists for TLR7 (guanosine, loxoribine and/or R837), or a specific agonist of TLR8 (TL8-506). Guanosine and TL8-506-stimulated PBMCs reveal that both upregulate IL-6, IL-8, IL-1β, IL-10, IL-12p70 and TNF in patients compared with healthy controls (Fig. 4d and Extended Data Fig. 6c). Although stimulated THP-1 showed the specific upregulation of the TLR7 pathway (guanosine and R848) but not TLR3 (poly(I:C)) or TLR9 (CPG-C) pathways, stimulation by TL8-506 showed induced IL-18 production in V117L and T314A cells, and IL-1β, IL-23 and IL-10 in V117L but not T314A cells (Fig. 4e and Extended Data Fig. 6d). Stimulation of UNC93B1V117L-immortalized B cells also demonstrates upregulation of the TLR7 pathway, compared with healthy control cells, whether as a result of dual agonist, R848- or TLR7-specific agonists, guanosine, loxoribine or R837 stimulation. But no obvious stimulation was achieved by the TLR8-specific agonist, TL8-506, except a slight increase in some but not all tested cytokines, because human B cells are not as naturally predominant in expressing TLR8 as myeloid cells25,26 (Fig. 4f and Extended Data Fig. 6e). Gene variants, V117L and T314A, did not affect the phosphorylation of the UNC93B1 protein (Supplementary Fig. 1), suggesting the presence of another mechanism involved in the upregulation of TLR7/-8 signaling.
a, RT–qPCR analysis of IFIT1 and TNF mRNA expression in the PBMCs of P3 compared with healthy controls after stimulation by 10 μg ml−1 of HMW poly(I:C), 1 μg ml−1 of R848 and 4 μg ml−1 of CPG-C for 12 h (n = 3 biological samples). Unstim, Unstimulated. b, Production of TNF and IL-1β in the supernatant of PBMCs of P2 compared with healthy controls after stimulation by 10 μg ml−1 of HMW poly(I:C), 1 μg ml−1 of R848 and 4 μg ml−1 of CPG-C for 8 h (n = 3 biological samples). c, Production of TNF in the supernatant of PBMCs of P3 compared with healthy controls after stimulation by 10 μg ml−1 of HMW poly(I:C), 1 μg ml−1 of R848 and 4 μg ml−1 of CPG-C for 12 h (n = 3 biological samples). d, Production of IL-6 and IL-8 in the supernatant of PBMCs of P2 and P3 compared with healthy controls after stimulation by 1 mM guanosine and 200 ng ml−1 of TL8-506 for 24 h (n = 3 biological samples). The P value in IL-6, P3 (guanosine set), was 0.0001 compared with HC1 and <0.0001 with HC2 and HC3, and in the TL8-506 set it was 0.0034 compared with HC1 and <0.0001 with HC3. The P value in IL-8, P3 (guanosine set), was 0.0053 compared with HC1, 0.0065 with HC2 and 0.0008 with HC3 and, in the TLR8-506 set, was 0.0001 compared with HC1, 0.0003 with HC2 and <0.0001 with HC3. The indicated P values in a–d were determined by two-way ANOVA, multiple comparisons, Padj value; data are presented as the mean with s.d. e, Production of IL-18 in the supernatant of THP-1 of indicated variants after stimulation by 0.7 mM guanosine, 1 μg ml−1 of R848, 200 ng ml−1 of TL8-506, 100 μg ml−1 of HMW poly(I:C) and 5 μg ml−1 of CPG-C for 24 h (n = 3 biological replicates); the indicated P values were determined by unpaired, two-tailed Student’s t-test and data are presented as the mean with s.d. f, Production of IL-12p70, IFNα2, IL-6 and IL-8 in the supernatant of indicated immortalized B cell lines after stimulation by 0.5 mM guanosine, 0.1 mM loxoribine, 2.5 μg ml−1 of R837, 1 μg ml−1 of R848 and 200 ng ml−1 of TL8-506 for 24 h (n = 3 biological replicates). The indicated P values in f were determined by two-way ANOVA, multiple comparisons, Padj value; data are presented as the mean with s.d. Production of cytokines in the cells supernatant was measured by CBA.
Some of the most attractive therapeutic targets downstream of UNC93B1 and the NAS-TLRs are the signaling adapter kinases IRAK1 and IRAK4 (ref. 27). To start, we found that the phosphorylation of IRAK4 was increased owing to overexpression of mutated UNC93B1 (Extended Data Fig. 7a). Transcriptionally, IRAK4 inhibition using zimlovisertib (PF-06650833), a potent selective inhibitor, was efficacious, returning THP-1 R848-induced TNF, IL-6, IL-8 and the ISGs IFIT1, IFIT3, ISG-15 and ISG-54 to baseline in a dose-dependent fashion (Fig. 5a and Extended Data Fig. 7b). In agreement, the downstream cytokines produced by UNC93B1 V117L and T314A R848-stimulated Thp-1 cells (for example, IL-6, IL-8, IL-23, IL-1β and IFNα2) were significantly blunted in a dose-dependent pattern (Fig. 5b and Extended Data Fig. 7c). The pathway was confirmed by immunoblotting cell lysates, where R848-induced phosphorylation of NF-κB and MAPK (ERK and P38) was also downregulated as a result of IRAK4 inhibition dose dependently (Fig. 5c). We also tested a dual targeting IRAK1/-4 inhibitor, a new benzimidazole, which was efficacious in downregulating ISG-4, TNF, IL-6 and IL8 transcription in R848-stimulated Thp-1 cells in a dose-dependent fashion (Fig. 5d and Extended Data Fig. 7d). Consistently, production of IL-6, IL-8, IL-23 and monocyte chemoattractant protein 1 (MCP-1) was also downregulated by the IRAK1/-4 inhibitor (Fig. 5e and Extended Data Fig. 7e). We were able to confirm these results in patient cells in which we found that the IRAK4 inhibitor reduced IL-6, TNF, IL-12p70, IFNγ and IL-10 to baseline in UNC93B1 V117L loxoribine-stimulated immortalized B cells, in a dose-dependent manner (Fig. 5f and Extended Data Fig. 7f). The IRAK4 inhibitor was also efficacious in returning IL-6, IL-8 and MCP-1 in P3 R848-stimulated PBMCs to the baseline (Fig. 5g and Extended Data Fig. 7g). In addition, UNC93B1 V117L loxoribine-stimulated, immortalized B cells experienced suppressed IFNγ and IL-8 owing to the IRAK1/-4 inhibitor (Fig. 5h). Overall, these findings point toward IRAK1/-4 as the dominant signaling pathway triggered by variants in UNC93B1 found in patients with childhood-onset SLE. Taken together, genetic variation in UNC93B1 regulates inflammation in patients with childhood-onset SLE through a TLR7/-8–IRAK1/-4 pathway.
a, RT–qPCR analysis of IFIT1 and TNF expression in the indicated THP-1 cell lines, stimulated by R848 for 24 h after being incubated with IRAK4 inhibitor (inh.) for 30 min. ***P = 0.0002, ****P < 0.0001. b, Production of IL-6 and IL-8 in the supernatant of the indicated THP-1 cell lines, stimulated by R848 for 24 h after being incubated with IRAK4 inhibitor for 30 min. ****P < 0.0001, **P = 0.0021, ***P = 0.0003 (20 nM set), ***P = 0.0001 (50 nM set). c, Levels of phosphorylated NF-κB, ERK and P38, as measured by immunoblotting using lysates of the indicated THP-1 cells, stimulated by 1 μg ml−1 of R848 for 24 h after being incubated with IRAK4 inhibitor for 30 min. Data are representative of three independent experiments. d, RT–qPCR analysis of ISG54 and TNF expression in the indicated THP-1 cell lines, stimulated by R848 for 24 h after being incubated with IRAK1/-4 inhibitor for 30 min. In ISG54, **P = 0.0025, ***P = 0.0001, ****P < 0.0001, and in TNF, ***P = 0.0002, ***P = 0.0003, ****P < 0.0001, respectively. e, Production of IL-6 and IL-8 in the supernatant of the indicated THP-1 cell lines, stimulated by R848 for 24 h after being incubated with IRAK1/-4 inhibitor for 30 min. In IL-6: *P = 0.0212, ***P = 0.0002, ***P = 0.0008, ***P = 0.0001, ***P = 0.0009, ****P < 0.0001, respectively, and in IL-8, *P = 0.0334, *P = 0.0107, **P = 0.0041, ***P = 0.0005, **P = 0.0024, ***P = 0.0002, respectively. f, Production of IL-6 and TNF in the supernatant of the indicated immortalized B cell lines, stimulated by loxoribine for 24 h after being incubated with IRAK4 inhibitor for 30 min. g, Production of IL-6 and IL-8 in the supernatant of the indicated PBMCs, stimulated by R848 for 24 h after being incubated with and without IRAK4 inhibitor for 30 min. The indicated P values were determined by unpaired, two-tailed Student’s t-test (n = 3 biological samples). h, Production of IFNγ and IL-8 in the supernatant of the indicated immortalized B cell lines, stimulated by loxoribine for 24 h after being incubated with IRAK1/-4 inhibitor for 30 min. Production of cytokines in the cell supernatant was measured by CBA. For all experiments, data are presented as the mean with s.d. For a, b, d–f and h, n = 3 biological replicates; the indicated P values were determined by two-way ANOVA, multiple comparisons, Padj value.
Lupus-like disease in mice with the mutation UNC93B1V117L
Given that UNC93B1V117L is a highly significant risk factor for childhood-onset SLE, but also present in the general population, we sought to confirm pathogenicity in vivo and created mice with the orthologous mutation V117L. The mutation, UNC93B1V117L, was introduced to the germline of mice using CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 genome editing technology (Extended Data Fig. 8). UNC93B1V117L mice were born at normal Mendelian ratios and initially appear healthy; however, both heterozygous and homozygous mice lose weight over time (Fig. 6a), develop splenomegaly with increased spleen cellularity (Fig. 6b) and have a small kidney size (Fig. 6c). In addition, lupus-associated serum anti-dsDNA and anti-Smith autoantibodies were increased in knock-in mice (Fig. 6d), but no change was noted for total immunoglobulin G (IgG) (Supplementary Fig. 2). Bone marrow (BM) populations of CD45+ immune cells, macrophages and CD11b+/Gr-1+ cells were increased along with CD16+/CD14− cells, regulatory T cells and activated T cells (Fig. 6e). Functionally, the intracellular production of IFNγ in CD4+ T cells and CD3+/CD4− cells of BM was also upregulated in the mutant mice (Fig. 6f). In the spleen of UNC93B1V117L mice, there were increased B cells and a decreased CD4 T cell:B cell ratio, along with elevated regulatory T cells, activated T cells, germinal center B cells, CD45+/CD19+/CD3−/CD95+/CXCR5+ cells and CD45+/CD4+/CD44+ cells (Fig. 6g). Serum cytokine analysis demonstrated that UNC93B1V117L leads to upregulation of IL-12p70, IP-10, IL-6 and IFNγ (Fig. 7a). Histopathological studies were done for hematoxylin and eosin (H&E)-stained tissues sections from kidneys, spleens, lungs and pancreata. Histologically, there was overt pathology in the kidney (Fig. 7b), spleen (Fig. 7c), lung (Fig. 7d) and pancreas (Fig. 7e), where disease scores of mesangial expansion, extramedullary hematopoietic cell hyperplasia and inflammatory cell infiltrate, respectively, were elevated in mice with the UNC93B1V117L variant. Molecularly, increased mRNA of the ISGs, MX1, ISG-20L2, IFIT1 and IRF7 were observed in kidney tissue homogenate (Fig. 7f).
a, Appearance and weight of UNC93B1WT/WT, UNC93B1WT/V117L and UNC93B1V117L/V117L mice. b, Spleen weight (data pooled from three independent experiments) and splenocyte count of indicated mice. Mice were age matched to 8–14 months. c, Kidney weight of indicated mice. Mice were age matched to 8–14 months and the data pooled from two independent experiments. d, Serum autoantibodies to dsDNA or Smith for the indicated mice. Mice were age matched to 8–14 months and the data pooled from three independent experiments. e, Flow cytometric analysis of indicated immune cell populations in BM of UNC93B1WT/WT, UNC93B1WT/V117L and UNC93B1V117L/V117L mice. Mice were age matched to 8 months. f, Intracellular cytokine staining of IFNγ in BM CD4+ T cells and CD3+/CD4− cells of the indicated mice. MFI, mean fluorescence intensity. Mice were age matched to 8 months. g, Flow cytometric analysis of indicated immune cell populations in spleen of UNC93B1WT/WT, UNC93B1WT/V117L and UNC93B1V117L/V117L mice (n as indicated). Mice were age matched to 8 months (for B cells, CD4 T cell:B cell ratio, regulatory T cells and CD45+/CD4+/CD44+ cells) or 14 months (for activated T cells, germinal center B cells and CD45+/CD19+/CD3−/CD95+/CXCR5+ cells). n as indicated. Statistical analysis was done using unpaired, two-tailed Student’s t-tests and data are presented as the mean with s.d. The exact P values are shown. Abs, absorbance; OD, optical density.
a, Serum IL-12p70, IP-10, IL-6 and IFNγ of the indicated mice (n = 5 for IL-12p70 and IP-10; n = 4 for IL-6 and IFNγ). IP-10 mice were aged 8 months and IL-12p70, IL-6 and IFNγ mice 10 months. b, H&E staining of the kidneys from indicated mice (n as indicated). The green arrows indicated mesangial stromal hyperplasia and the red arrows endothelial and mesangial proliferation. Mice were age matched to 8–14 months and the data pooled from three independent experiments. c, H&E staining of the spleens from the indicated mice (n = 5). The green arrows indicated the extramedullary hematopoietic cells and the red arrows the inflammatory cells. Mice were age matched to 8 months. d, H&E staining of the lungs from the indicated mice (n = 3). The yellow arrows (granulocytes) and red arrow (lymphocytes) indicated inflammatory cell infiltration; mice were aged 14 months. e, H&E staining of the pancreata from the indicated mice (n = 3). The red arrows indicate inflammatory cell infiltration (granulocytes or lymphocytes) and the mice were aged 14 months. The graphs show the pathological disease scores. f, RT–qPCR analysis of Mx1, Isg20l2, Ifit1 and Irf7 mRNA in kidney tissues from the indicated mice (n = 3). Mice were age matched to 8 months. Statistical analysis was done using unpaired, two-tailed Student’s t-tests and data are presented as mean with s.d. The exact P values are shown.
BM-derived macrophages (BMDMs) isolated from UNC93B1V117L mice experienced increased mRNA of the ISGs, IRF7 and IFIT1, when compared with BMDMs of UNC93B1WT mice (Fig. 8a). Meanwhile, phosphorylation of the UNC93B1/TLR7 signaling pathway, including IRF5, IRF7, NF-κB and MAPK (JNK and P38), was also observed to be activated in UNC93B1V117L BMDMs (Fig. 8b). In addition, UNC93B1V117L BMDMs exhibit elevated intrinsic intracellular production of TNF (Extended Data Fig. 9a). Unbiased RNA-seq indicates that all of the most highly upregulated genes are known to be inducible by type I or type II IFN28 and include the central TLR signaling molecule IRF7, as well as IRF3 and TBK1 (TANK-binding kinase 1)29 (Extended Data Fig. 9b,c). Consistently, this was associated with pathway analysis implicating TLR signaling, TNF signaling, antigen processing and presentation (Extended Data Fig. 9d–f). Overall, there is a very significant association with genes listed in the Kyoto Encyclopedia of Genes and Genomes (KEGG) under the disease: Systemic lupus erythematosus (Fig. 8c). To investigate the specificity of NAS-TLR signaling in this mouse model, BMDMs were stimulated by NAS-TLR ligands and tested for inflammatory markers. As in human samples, data from these experiments showed the involvement of TLR7 in the mouse lupus-like phenotype. IFIT1, IRF7, ISG-15 and TNF genes were more highly expressed in BMDMs of mice with the UNC93B1V117L variant after stimulation with R848 but not poly(I:C) or CPG-C (Fig. 8d). Also, secretion of CXCL1, CCL5 and IP-10 in the supernatant of UNC93B1V117L BMDMs was elevated after R848 stimulation but not after stimulation by poly(I:C) or CPG-C (Fig. 8e). These findings implicate enhanced TLR7 signaling as the pathway driving inflammation and a lupus-like phenotype in mice with the equivalent gene variant to humans, contributing to childhood-onset SLE.
a, RT–qPCR analysis of Irf7 and Ifit1 mRNA in BMDMs from the indicated mice (n = 3). Mice were age matched to 8 months. Statistical analysis was done using unpaired Student’s t-tests. The exact P values are shown. b, Levels of phosphorylated NF-κB, JNK, P38, IRF5 and IRF7, as measured by immunoblotting, in lysates of BMDMs from the indicated mice (n = 3). Mice were age matched to 8 months. c, RNA-seq performed for UNC93B1V117L BMDMs compared with controls (UNC93B1WT) (n = 3 biological replicates). Mice were age matched to 8 months. Gene set enrichment analysis significantly associated with SLE. d, RT–qPCR analysis of Ifit1, Irf7, Isg-15 and Tnf mRNA expression in the BMDMs from the indicated mice (n = 3) after stimulation by 40 μg ml−1 of HMW poly(I:C), 2 μg ml−1 of R848 and 10 μg ml−1 of CPG-C for 24 h. Mice were age matched to 14 months. Statistical analysis was done using unpaired Student’s t-tests. The exact P values are shown. e, Production of CXCL1, CCL5 and IP-10 in the supernatant of mice BMDMs isolated from the indicated mice (n = 3), measured by CBA after stimulation by 40 μg ml−1 of HMW poly(I:C), 2 μg ml−1 of R848 and 10 μg ml−1 of CPG-C for 24 h. Mice were age matched to 14 months. Statistical analysis was done using unpaired, two-tailed Student’s t-tests and data are presented as the mean with s.d. The exact P values are shown.
Therefore, UNC93B1V117L drives a lupus-like disease in mice at a cellular level, with relevant end-organ damage. This is associated with activation of the TLR7 signaling pathway and inflammatory gene expression that is consistent with the clinical presentation of patients with the orthologous variant, who suffer from childhood-onset SLE.
Discussion
Our discovery of rare genetic changes in UNC93B1 that predispose to childhood-onset SLE was facilitated by a large body of work culminating in the discovery of lupus-causing variants in TLR7 (ref. 4). It is interesting that all patients with UNC93B1 variants identified in the present study were female, which could relate to a lack of TLR7 X-chromosome inactivation30; however, it could also reflect the strong gender predisposition of this disease in general. We found that the spontaneous inflammatory signaling via IRAK1/-4, due to the variants in UNC93B1 identified here, is associated with increased responses to stimulation of TLR7/-8. This seems logical based on the current literature; however, further work is required to formally delete or inhibit TLR7 and resolve pathology from the UNC93B1V117L mouse model that we generated. Theoretically, there should also be an endogenous ligand to stimulate TLR7 in this context, which could be guanosine or another nucleic acid31. Furthermore, although the structural location of UNC93B1 T314A presents a logical mechanism to impact TLR7, the molecular effect of UNC93B1 V117L is not yet clear. Given that the phosphorylation of UNC93B1 appeared unaltered, this could also suggest an area in which new findings for the gene variants could be made. Mechanistic insight into this process will be extremely useful and potentially clinically actionable in the future as TLR7 inhibitors are being developed32.
Not only are TLR7 inhibitors in clinical trials, but also IRAK1/-4 inhibitors could have therapeutic benefit for the patients identified, based on our results. So far, none of the patients characterized in the present study have gone on to receive JAK inhibitors or biological therapeutics that would also target type I IFN signaling. Those approaches should be beneficial and this could be particularly important given that UNC93B1 V117L is present in the general east Asian population, where it could be a contributor to the incidence of SLE33. Within this population there is a strong geographical bias (Fig. 1c), for which the underlying basis is unknown. Given that the family members of the affected individuals in the present study were all apparently healthy, we currently consider that UNC93B1 V117L is not a monogenic disease-causing allele with incomplete penetrance, but rather a very strong risk factor for developing childhood-onset SLE. This is consistent with other gene mutations in lupus, for example, DNase1, for which healthy carriers have been observed34.
Our mouse model represents an important confirmation of the patient findings and shows that a gene dosage effect of the gain-of-function variant is present. Although no homozygous humans have been identified so far, we can speculate that they may have more severe, or a greater likelihood to develop, disease. As UNC93B1 V117L is present in the general population as a rare but highly significant risk factor for disease, the preclinical efficacy of new therapeutic modalities can be accurately modeled for the resulting patient population using our mice avatars. The mouse model should also be useful to determine the cellular contribution of UNC93B1 to lupus-like disease, either intrinsically in B cells or with activation of innate immune signaling from antigen-presenting cells, and the distinct contribution of type I IFNs compared with other inflammatory programs. We would expect this to be similar to other gain-of-function UNC93B1 mouse models of lupus that have been published35.
Overall, these findings are not just the first patients with childhood-onset SLE caused by variants in UNC93B1, but also bridge the gap from rare monogenic diseases to show that this signaling pathway is relevant to the incidence of disease more generally. Moreover, therapeutics targeting this specific pathway are being developed and can be tested first in a mouse model for which there is a corresponding patient population.
Methods
Patients
Female patients P1–P8, aged 8, 9, 8, 10, 10, 12, 13 and 26 years, respectively, presented symptoms of systemic inflammation associated with lupus nephritis. Patients’ blood and other samples were collected at Guangzhou Women and Children’s Medical Centre, Guangzhou, China. No compensation was provided. All patients were diagnosed as patients with SLE according to the European League against Rheumatism/American College of Rheumatology (EULAR/ACR) classification criteria. Clinical data for P1–P7 are presented (Fig. 2a–f, Extended Data Figs. 1a–g and 2a–f and Supplementary Table 3). Written informed consent has been obtained from the authorized individual for all participating patients. The Guangzhou Women and Children’s Medical Centre Medical Ethics Committee approved the study procedures ((2021)073B00) consistent with the Helsinki Declaration about ethics in using human samples.
DNA sequencing
WES and Sanger sequencing were performed at Novogene. The frequency of variant V117L in the east Asian population was obtained from PGC.Han 2.0 (ref. 14).
Human PBMC isolation and plasma separation
Noncoagulated blood samples from the patients with SLE and healthy controls were diluted in an equal volume of sterile phosphate-buffered saline (PBS), and dispensed slowly and gently along the side of a 15-ml conical tube containing 2 ml of Ficoll–Hypaque density gradient. This was followed by centrifugation at 800g for 20 min, 4 Acc (acceleration), 4 Dec (deceleration) and 4 °C. The cloud-like cell layer of cells was collected from the interphase into a new tube and washed twice with sterile PBS by centrifuging at 300g for 5 min. The pellet was resuspended in complete culture medium consisting of 90% Roswell Park Memorial Institute (RPMI)-1640, 10% fetal bovine serum (FBS) and 1% (v/v) penicillin–streptomycin (Gibco) and then incubated at 37 °C, 5% CO2 and 95% humidity. The human PBMCs were used in experiments immediately after isolation or stored FBS with 10% dimethyl sulfoxide (DMSO) at −80 °C. All of the above procedures were performed in an aseptically controlled area using sterilized tools.
For plasma separation, noncoagulated blood samples from the patients with SLE and healthy controls were centrifuged at 1,500g for 10 min; then the upper yellow fluid was separated into a new tube by pipetting without disturbing the layer of buffy coat. Samples were stored at −40 °C up to the time of analysis.
Immortalized B cell line generation
Immortalized B cells were generated using noncoagulated blood samples from the patients with SLE, P2 and P3 and healthy controls through infection by Epstein–Barr virus according to a previous protocol36.
Mice
Animal studies were approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University (protocol no. GY2022-035). Mice lines (UNC93B1WT/V117L and UNC93B1V117L/V117L) were designed, developed and housed by Shanghai Model Organisms Center, Inc. under specific pathogen–free conditions with normal diet, 12-h light:12-h dark cycle, 20–26 °C and humidity of 40–70%. The point mutation mice model at exon3 (117V to L) of the UNC93B1 gene was generated via CRISPR–Cas9 technology. Briefly, Cas9 mRNA and guide RNA were produced by in vitro transcription, an oligonucleotide donor DNA was synthesized and the mixture of Cas9 mRNA, gRNA and donor DNA was microinjected into fertilized eggs (C57BL/6J). Then three positive F0 mice were identified by PCR and sequencing, F0 mice were crossed with WT C57BL/6J mice to generate F1 mice and then four positive F1 mice were identified by PCR and sequencing. The gRNA used was: GTGTAGAGCAGGGCAGCGATAGG. The recombinant strategy, including the knock-in locus and the oligonucleotide donor DNA sequence, and the site of target for UNC93B1WT/WT, UNC93B1WT/V117L and UNC93B1V117L/V117L genotypes are shown in Extended Data Fig. 8. All mice used for analysis in this project are females within the age indicated for each experiment independently in ʻResultsʼ. Within genotypes, animals were randomly allocated in all experiments. Data collection and analysis were not performed blind to the conditions of the experiments, except for histopathology. No animals or data points were excluded except if an animal was injured as a result of fighting or contaminated samples.
Mouse serum collection
Mice orbital whole-blood samples were collected into tubes without anticoagulant and incubated at 25 °C for at least 15 min to coagulate the blood. The coagulated blood samples were centrifuged at 1,500g for 10 min; then the upper yellow fluid was separated into a new tube using a pipette. Samples were stored at −40 °C up to the time of analysis.
Tissue digestion and flow cytometry
The whole spleen was minced into tiny pieces in a six-well plate on ice using the plunger end of the syringe and filtered using a 70-µm strainer with rinsing by 5 ml of PBS with 2% FBS. The collected tissue filtrates were centrifuged at 500g for 5 min at 4 °C. The cell pellet was suspended in RBC lysis buffer, incubated for 5 min at 25 °C and then mixed with a double volume of PBS with 2% FBS. The immune cells were collected in the pellet after centrifugation at 500g for 5 min at 4 °C.
BM was collected from mouse femur bones by centrifuging using a technique of two layers of tubes and incubated with RBC lysis buffer for 5 min at 25 °C, and then mixed with a double volume of PBS with 2% FBS. The immune cells were collected in the pellet after centrifugation at 500g for 5 min at 4 °C.
For flow cytometry, 1 million cells were incubated with Zombie Aqua-A (BioLegend) for 15 min in the dark, washed for staining by antibodies for cell-surface markers, incubated for 30 min at 4 °C and then washed. A fixation and permeabilization kit (Thermo Fisher Scientific, cat. no. 88-8824-00) was used for fixation and intracellular permeabilization for intracellular and nuclear markers, according to the manufacturer’s protocol. Cells were analyzed using CYTEK NL-CLC. Data were collected using CYTEK NL-CLC software and SpectroFlo and analyzed using FlowJo_v.10.8.1. A gating strategy is shown in Extended Data Fig. 10.
Isolation of mouse BMDMs
The BM of mice femur bones was collected aseptically as mentioned above. BMDMs were generated by incubating the BM immune cells in a complete medium, RPMI-1640, 10% FBS, 2 mM l-glutamine and 1% (v/v) penicillin–streptomycin, containing 50 ng ml−1 of macrophage-CSF (Peprotech) for 6–8 d at 37 °C with 5% CO2 and 100% humidity.
Intracellular staining for TNF and IFNγ
BMDMs or BM immune cells were stimulated by a 1× cocktail of phorbol 12-myristate 13-acetate, ionomycin, Brefeldin A and monensin from Invitrogen (cat. no. 00-4975-93) for 4–6 h at 37 °C with 5% CO2 and 100% humidity, and then stained using PE-Cy7 TNF and BV-421 IFNγ antibody with a fixation and permeabilization kit (Thermo Fisher Scientific, cat. no. 88-8824-00) according to the manufacturer’s protocol. Cells were analyzed using CYTEK NL-CLC. Data were collected using CYTEK NL-CLC software and SpectroFlo and analyzed using FlowJo_v.10.8.1.
Histopathology
The kidney, spleen, lung and pancreas tissues were preserved in 4% paraformaldehyde. Tissue processing and histopathology reporting were performed by Wuhan Servicebio Technology Laboratory. For disease scoring (blinded) of tissue sections stained by H&E, 0 = within the normal range, 1 = very slight (the changes that appear just exceeded the normal range), 2 = slight (lesions may be observed but not severe), 3 = medium (lesions are obvious and likely to be more severe) and 4 = severe (lesions have taken up the entire tissue and organs). For kidney pathology of mesangial stromal hyperplasia and mesangial and endothelial cell proliferation, the score is calculated for around 50 glomeruli per mouse.
Antibodies and reagents
For immunoblots, the following antibodies were used: anti-FLAG (Sigma-Aldrich, cat. no. F1804), anti-NF-κB p65 (Cell Signaling, cat. no. 8242S), anti-phospho-NF-κB p65 (Ser468) (Cell Signaling, cat. no. 3039S), anti-JNK (Abcam, cat. no. ab179461), anti-phospho-JNK (Abcam, cat. no. ab124956), anti-P38 (Cell Signaling, cat. no. 8690), anti-phospho-P38 (Cell Signaling, cat. no. 9211S), anti-IRF5 (Cell Signaling, cat. no. 20261), anti-phospho-IRF5 (Ser437) (Invitrogen, cat. no. PA5-64760), anti-IRAK4 (Cell Signaling, cat. no. 4363), anti-phospho-IRAK4 (Thr345/Ser346) (Cell Signaling, cat. no. 11927), anti-p44/42 MAPK (ERK1/2) (Cell Signaling, cat. no. 9102S), anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (Cell Signaling, cat. no. 9101S), anti-IRF7 (Cell Signaling, cat. no. 39659), anti-phospho-IRF7 (Ser437/438) (Cell Signaling, cat. no. 24129), anti-p-Ser/phosphoserine (Santa Cruz Biotechnology, cat. no. sc-81514), anti-β-actin (ABclonal, cat. no. AC026), anti-rabbit IgG horseradish peroxidase (HRP)-linked antibody (Cell Signaling, cat. no. 7074) and anti-mouse IgG HRP-linked antibody (Cell Signaling, cat. no. 7076). The dilution of anti-β-actin, anti-rabbit IgG HRP-linked antibody, anti-mouse IgG HRP-linked antibody and anti-FLAG was 1:5,000. The dilution of anti-p-Ser/phosphoserine was 1:1,000. The dilution for other primary antibodies was 1:1,500. For flow cytometry, the following antibodies and reagents were used: anti-mouse TNF (BioLegend, cat. no. 506324, 1:200), anti-mouse IFNγ (BioLegend, cat. no. 505830, 1:400), anti-mouse CD45 (BioLegend, cat. no. 103132, 1:200), Zombie Aqua-A (BioLegend, cat. no. 423101, 1:500), anti-mouse F4/80 (BioLegend, cat. no. 123137, 1:200), anti-mouse/human CD11b (BioLegend, cat. no. 101205, 1:500), anti-mouse CD14 (BioLegend, cat. no. 123335, 1:200), anti-mouse CD16 (BioLegend, cat. no. 158004, 1:100), anti-mouse CD3 (BioLegend, cat. no. 100248, 1:100), anti-mouse CD4 (BioLegend, cat. no. 100406, 1:400), anti-mouse FOXP3 (BioLegend, cat. no. 126407, 1:200), anti-mouse CD25 (BioLegend, cat. no. 102043, 1:300), anti-mouse/human CD44 (BioLegend, cat. no. 103059, 1:100), anti-mouse CD69 (BioLegend, cat. no. 104507 or 104510, 1:200), anti-mouse Gr-1 (BioLegend, cat. no. 108416, 1:200), anti-mouse CD19 (BioLegend, cat. no. 159812, 1:400), anti-mouse CD95 (BioLegend, cat. no. 152612, 1:200), anti-mouse/human Bcl-6 (BioLegend, cat. no. 358510, 1:100), anti-mouse/human CD45R/B220 (BioLegend, cat. no. 103222, 1:200), anti-mouse CXCR5 (BioLegend, cat. no. 145517, 1:100), and fixation and permeabilization kit (Thermo Fisher Scientific, cat. no. 88-8824-00). For TLR stimulation, the following ligands were used: high molecular weight (HMW) poly(I:C), a TLR3 ligand (tlrl-pic, InvivoGen), guanosine, a TLR7 ligand (Sigma-Aldrich, cat. no. G6264-1G) resuspended in DMSO (freshly prepared before each experiment), loxoribine, a guanosine analog, a TLR7 ligand (tlrl-lox, InvivoGen), imiquimod (R837), an imidazoquinoline amine analog to guanosine, a TLR7 ligand (tlrl-imqs-1, InvivoGen), TL8-506, a benzazepine compound and an analog of the TLR8 agonist VTX-2337, a TLR8 ligand (tlrl-tl8506, InvivoGen) and R848 (also known as resiquimod, an imidazoquinoline and dual TLR7 and TLR8 synthetic agonist) (tlrl-r848, InvivoGen), CPG-C, synthetic oligonucleotides that contain unmethylated CpG dinucleotides in particular sequence contexts (CpG motifs), a TLR9 ligand (tlrl-m362, InvivoGen) used in human experiments and CPG-C, ODN 2395 VacciGrade (InvivoGen, cat. no. vac-2395-1) (used in mice BMDM stimulation).
UNC93B1 lentivirus constructs and transduction
For human UNC93B1 (NM_030930) overexpression, constructs of WT or mutated, V117L or T314A UNC93B1 were inserted into Ubi-MCS-3FLAG-SV40-Cherry-IRES-puromycin. Site-directed mutagenesis technology was used to generate c.G349T:p.V117L and c.A940G:p.T314A mutation using a WT UNC93B1 construct. These plasmids, along with PSPAX2 and pMD2G, were used to produce lentiviruses in the supernatant. The design and synthesis of plasmids and lentivirus production were done at Genechem Laboratory, Shanghai, China. THP-1 cells were transduced by indicated lentiviruses to generate stable cell lines overexpressing UNC93B1 WT, V117L and T314A. Puromycin, 2 µg ml−1 (Santa Cruz Biotechnology), treatment and FACS sorting by BD FACSAria lll were performed for positive cell selection lasting 2 weeks after infection. The infection efficiency and stable overexpression were verified by immunoblotting assay for flag expression and flow cytometry for mCherry by CYTEK NL-CLC.
Cell culture
THP-1 cells were obtained from American Type Culture Collection and cultured accordingly. THP-1 and human PBMCs and immortalized B cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 2 mM l-glutamine and 1% (v/v) penicillin–streptomycin. All cells were cultured at 37 °C with 5% CO2 and 100% humidity. THP-1, immortalized B cells and/or PBMCs incubated with zimlovisertib (PF-06650833) (MedChemExpress, cat. no. HY-19836) (in the present study referred to as IRAK4 inhibitor) and/or with IRAK1/4 inhibitor I, a new benzimidazole (Sigma-Aldrich, cat. no. 15409) (in the present study referred to as IRAK1/-4 inhibitor) as indicated in ʻResultsʼ.
Messenger RNA extraction and qPCR
Extraction of mRNA from THP-1, human PBMCs, immortalized B cells, BMDMs or mice kidney tissue homogenate was performed using EZ-press RNA Purification Kit and converted to complementary DNA using the 4× Reverse Transcription Master Mix Kit (with genomic DNA Remover) from EZ Bioscience and 1,000 ng of mRNA per sample according to the manufacturer’s instructions. The quantification of RNA concentration was done using Varioskan LUX (Thermo Fisher Scientific). Quantitative real-time PCR (RT–qPCR) was conducted using the 2× Color SYBR Green qPCR Master Mix (ROX2 plus) from EZ Bioscience, cDNA, RNase-free dH2O and primers shown in Supplementary Table 4 by LightCycler 480 ll (Roche, 96 or 384), according to the manufacturer’s instructions.
Immunoprecipitation
Cell lysates were extracted using a low-salt lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl2, 10% glycerol and 1% Triton X-100) supplemented with 5 mg ml−1 of protease and a phosphatase inhibitor cocktail (Roche). Cell lysates were incubated with washed anti-Flag M2 magnetic beads (Sigma-Aldrich, cat. no. M8823) at 4 °C overnight with mixing using an end-over-end rotator. Then, the beads were washed 4× by low-salt lysis buffer. The protein eluted by resuspending washed beads with 2× sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer and boiling for 10 min.
Immunoblotting
Cell lysates were extracted using a low-salt lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100) supplemented with 5 mg ml−1 of a protease and phosphatase inhibitor cocktail (Roche). Protein quantification was performed using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, cat. no. 23227) according to the manufacturer’s instructions. Then, 10% or 12% SDS–PAGE was used for protein lysates (20 μg) with loading buffer and transferred to polyvinylidene fluoride membrane (Millipore Co.). For the phos-tag gel, 12% SDS–PAGE was prepared by adding 5 mM phosbind acrylamide (ABExBIO, cat. no. F4002) and 10 mM MnCl2 and processed according to the manufacturer’s instructions. Membranes were blocked with 5% skimmed milk in Tris-buffered saline–Tween-20 (TBST) and then incubated overnight at 4 °C with primary antibodies. After washing, the membranes were incubated for 1 h at 25 °C with secondary antibody conjugated with HRP. Finally, TBST-washed membranes were treated with enhanced chemiluminescence for detection using a Molecular Imager ChemiDoc XRS+ imaging system (BioRad). Developed membranes were imaged using the Image Lab detection system (BioRad).
ELISA and CBA
ELISA and CBA experiments were carried out using serum/plasma and/or cell supernatants according to the manufacturer’s instructions. ELISA kits for humans, IFNα (CSB-E08636h), IFNβ (CSB-E09889h) and IL-6 (CSB-E04638h) were purchased from Cusabio. ELISA kits for mice, LBIS mouse anti-dsDNA (FUJIFILM Wako Shibayagi Corp., cat. no. 637-02691), mouse anti-Sm Igs (total (A + G +M)) (Alpha Diagnostic International, cat. no. 5405) and mouse IgG (Alpha Diagnostic International, cat. no. 6320) were purchased as indicated. Absorbance were measured using Varioskan LUX (Thermo Fisher Scientific). Human (cat. no. 740390) and mouse (cat. no. 740621) LEGENDplex 13-plex CBA Anti-Virus Response Panel kits, or LEGENDplex 13-plex CBA Human Inflammation Panel 1 (cat. no. 740809) from BioLegend was used in cytokine quantification using a V-bottomed 96-plate protocol. Beads were analyzed using CYTEK NL-CLC. Data were collected using CYTEK NL-CLC software and SpectroFlo and analyzed using the LEGENDplex Data Analysis Software Suite (BioLegend).
RNA-seq and analysis
THP-1 cells or mice BMDMs were suspended in Trizol and sent to the Beijing Genomic Institute (BGI) laboratory for RNA-seq. For RNA-seq, total RNA was first extracted from human THP-1 cells or mice BMDMs and the mRNA was first enriched using poly(dT), then fragmented and reverse transcribed into cDNA and subjected to end-repair and adapter ligation processes. The resulting mRNA library was paired-end sequenced (2× 150 bp) with the sequencer DNBSEQ-T7 (BGI Genomics).
The sequencing reads for human THP-1 cells were then aligned to human reference genome GRCh38 using STAR aligner (v.2.7.9a)37 to generate the binary alignment file (.bam), which was then passed to HTSeq for gene expression quantification using the htseq-count function (v.0.11.1)38. The resulting gene expression matrix was then normalized and differentially expressed genes (DEGs) were identified using R package DESeq2 (v.1.34.0)39. Genes with adjusted P (Padj) value <0.05 and absolute fold-change (log2(space)) > 1 were considered to be DEGs. This analysis pipeline was used for the volcano plots in Fig. 3. Other analysis was performed using BGI software (https://biosys.bgi.com/#/report/login).
Statistical analysis
Statistical analysis was performed using Prism 9 software (GraphPad Software Inc.). Contingency analysis, Fisher’s exact test, Bapista–Pike test, χ2, unpaired Student’s t-test or analysis of variance (ANOVA: one-way or two-way) was used to compare differences between groups. P < 0.05 was considered significant.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
The RNA-seq raw sequencing data for the THP-1 cell line reported in the present study are deposited under supervision and control of the Genome Sequence Archive (GSA) of the BGI, Chinese Academy of Sciences (https://ngdc.cncb.ac.cn/gsa-human/s/uExs2NxY), under accession no. HRA005592. THP-1 RNA-seq data for this project could be accessed after an approval application. Please refer to the GSA (https://ngdc.cncb.ac.cn/gsa-human/) for detailed application guidance. The RNA-seq raw sequencing data for the mouse model reported in the present study are deposited under the supervision and control of the GSA of the BGI, Chinese Academy of Sciences (https://ngdc.cncb.ac.cn/gsa/s/1dDv8y6X), under accession no. CRA012690. For access to mouse RNA-seq data of this project, please contact the corresponding authors for approval. All other data are available in the main text or supplementary materials. Source data are provided with this paper.
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Acknowledgements
We are grateful to all members of the laboratories of Yuxia Zhang and S.L.M. for supplying reagents and technical advice or assistance. We also thank S. Adlat for his kind help during the drafting of this manuscript. The present study is supported by the National Natural Science Foundation of China (grant nos 81873869 to S.L.M., 32250410295 to E.I., 82125015 to Yuxia Zhang and 82101911 to Z.Z.), Guangzhou Science and Technology Key R&D Plan (grant no. 202206010002 to Yuxia Zhang), Guangzhou Science and Technology Bureau (grant no. 2024A03J0806 to P.Z.), Research Fund of Guangzhou Women and Children’s Medical Center (grant nos 3001124 to M.A. and 2180129 to S.L.M.) and Research Fund of Guangzhou Women and Children’s Medical Center (grant no. 3001032 to Yuxia Zhang).
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S.L.M. and Yuxia Zhang conceived the project. M.A. conducted all experiments with assistance from E.I. M.A. performed data analysis, drafted figures and wrote the first version of the manuscript. K.H.-S. performed structural analysis. Z.L. assisted with bioinformatics analysis. J.Z. assisted with animal experiments. M.L., H.X., M.Y, B.L., Z.Z., Yiyi Liu and Jun Cui were involved in experiments and data analysis. Jingjie Chang, X.L. and C.G. assisted with provision of human samples. C.G. assisted with immortalized B cell generation. P.Z., X.G., Yunfeng Liu, Q.W, Jiazhang Chen and C.L. were involved in clinical management. W.Z. obtained funding to support research. Yan Zhang was involved in WES analysis. S.L.M. supervised the project. M.A. wrote the manuscript with input from all the authors.
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S.L.M. is a scientific advisor for Odyssey therapeutics and NRG therapeutics. The other authors declare no competing interests.
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Nature Immunology thanks Yusuke Murakami, George Tsokos, and Ali Zarrin for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: L. A. Dempsey, in collaboration with the Nature Immunology team.
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Extended data
Extended Data Fig. 1 Clinical characteristics associated with UNC93B1 T314A and V117L variants.
(a–g) Complement 3 (NR, 0.80-1.50 g/L)/4 (NR, 0.13-0.43 g/L) levels for patients with UNC93B1 T314A, P1 and V117L, P2-P7. (a-d) SLE score (SLEDAI-2K), P1-P4. (f) SLEDAI score, P6. (a-c, e-g) Circulating white blood cells (WBC) (NR, 5-12 × 109/L), neutrophils% (NR, 40-60%), monocytes% (NR, 1-8%), lymphocytes% (NR, 31-40%), and platelets count (NR, 140-440 × 109/L) for patients with UNC93B1 T314A, P1 and V117L, (P2, P3, and P5-P7). (e-g) Anti-dsDNA autoantibodies (NR, <18IU/L) and ANA autoantibodies (NR, <18IU/L) for P5-P7 (all V117L). (e, f) 24hrs. urine protein (NR, 0-0.1 g/24 h) for P5 and P6 (all V117L). Data is for monthly visits post diagnosis. NR is highlighted in gray.
Extended Data Fig. 2 Clinical characteristics associated with UNC93B1 V117L variant.
(a–f) ANA autoantibodies by HEp-2 cells, (a) for healthy control, (b, c) for P2 (b) and P3 (c) showing mixed patterns in which the speckled pattern is predominant, (d, e) for P4 (d) and P5 (e) showing speckled pattern, and (f) for P6 showing cytoplasmic pattern. The scale bars were supplied in source data of this figure where these images were magnified from.
Extended Data Fig. 3 Patients with UNC93B1 V117L variant display elevated interferons and inflammatory cytokines.
(a) Quantitative RT-PCR analysis of ISG-15 mRNA expression in the PBMCs of P2 compared to healthy controls. p value was determined by one-way ANOVA, data are presented as mean with SD, n = 3 biological samples. (b) Quantitative RT-PCR analysis of IFIT1, IFIT3, and ISG-15 mRNA expression in the PBMCs of P3 compared to healthy controls, n = 3 biological samples. (c) Production of IFNγ, IFNλ2/3, IL-12P70, and IL-10 in the supernatant of PBMCs isolated from P2 and healthy controls measured by CBA, n = 3 biological samples. (d) Production of IFNα, IFNβ, and IL-6 in the supernatant of PBMCs isolated from P2 and healthy controls measured by ELISA, n = 3 biological samples. (e) Production of IFNγ, IFNλ2/3, IL-10, and IL-12P70 in the supernatant of PBMCs isolated from P3 and healthy controls measured by CBA, n = 3 biological samples. Indicated p values in b–e were determined by two-way ANOVA, multiple comparisons, adjusted p value, data are presented as mean with SD. PBMCs in a-e isolated from patients’ whole blood and incubated in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and 1% (v/v) Penicillin-Streptomycin for 12hrs before analysis without stimulation. (f) Levels of IFN- α2, IFNγ, IFNλ2/3, IL-6, IL-12P70, IL-1β, GM-CSF, and IL-10 in the plasma from P2 compared to healthy control measured by CBA, p values were determined by unpaired t-test, two-tailed, data are presented as mean with SD, n = 3 biological samples.
Extended Data Fig. 4 Pathway analysis of inflammation in UNC93B1V117L and UNC93B1T314A THP-1 cells.
(a) Quantitative RT-PCR analysis of ISG20L2, IL-8, IL-12a and TNF expression in the indicated THP-1 cell lines, n = 3 biological replicates. Three independent experiments. (b) Production of IP-10 and IL-8 in the indicated THP-1 cell lines measured by CBA. n = 3 biological replicates. Indicated p values in a,b were determined by unpaired t-test, two-tailed, data are presented as mean with SD. (c–f) Gene Set Enrichment Analysis (GSEA) with WT as control groups and UNC93B1V117L or UNC93B1T314A as treatment groups which shows a significant association for genes relating to (c) antigen processing and cross presentation, (d,e) cross presentation of soluble exogenous antigens and (f) ER/Phagosome function. mRNA was extracted from THP-1 of UNC93B1WT, UNC93B1V117L, and UNC93B1T314A, n = 3 biological replicates.
Extended Data Fig. 5 V117L and T314A UNC93B1 variants associated inflammation features.
(a, b) DEGs and pathway analysis of inflammation in UNC93B1V117L and UNC93B1T314A THP-1 cells. (a) RNA sequencing was performed for UNC93B1V117L THP-1 cells compared to control (UNC93B1WT), reactome Enrichment analysis of significantly upregulated genes highlights innate immune system and antigen processing/presentation. (b) RNA sequencing was performed for UNC93B1T314A THP-1 cells compared to control (UNC93B1WT), reactome Enrichment analysis of significantly upregulated genes highlights innate immune system and ER/Phagosome pathways. mRNA was extracted from THP-1 of UNC93B1WT, UNC93B1V117L, and UNC93B1T314A, n = 3 biological replicates. (c) Quantitative RT-PCR analysis of IL-12a and ISG20L2 expression in the indicated immortalized B cell lines (baseline), n = 3 biological replicates, indicated p values were determined by unpaired t-test, two-tailed, data are presented as mean with SD. (d, e) Production of IL-10 (d), TNF and GM-CSF (e) in the indicated immortalized B cell lines (baseline). n = 3 biological replicates, measured by CBA. Indicated p values were determined by two-way ANOVA, multiple comparisons, adjusted p value, data are presented as mean with SD.
Extended Data Fig. 6 UNC93B1 genetic variation drives inflammation via TLR7/8.
(a) Production of IL-6 in the supernatant of PBMCs of P2 compared to healthy controls after stimulation by 10 μg/mL HMW Poly I:C, 1 μg/mL R848, and 4 μg/mL CPG-C for 8hrs, n = 3 biological samples. (b) Production of IP-10, IL-6, and IL-1β in the supernatant of PBMCs of P3 compared to healthy controls after stimulation by 10 μg/mL HMW Poly I:C, 1 μg/mL R848, and 4 μg/mL CPG-C for 12hrs, n = 3 biological samples. Indicated p values in a, b were determined by two-way ANOVA, multiple comparisons, adjusted p value, data are presented as mean with SD. (c) Production of IL-1β, IL-10, IL12P70, and TNF in the supernatant of PBMCs of P2 compared to healthy controls after stimulation by 1 mM guanosine and 200 ng/mL TL8-506 for 24hrs, indicated p values were determined by two-way ANOVA, multiple comparisons, adjusted p value, data are presented as mean with SD, p value of TNF (guanosine set) determined by unpaired t-test, two-tailed, data are presented as mean with SD, n = 3 biological samples. (d) Production of IL-1β, IL-23, and IL-10 in the supernatant of THP-1 of indicated variants after stimulation by 0.7 mM guanosine, 1 μg/mL R848, 200 ng/mL TL8-506, 100 μg/mL HMW Poly I:C, and 5 μg/mL CPG-C for 24hrs, indicated p values were determined by unpaired t-test, two-tailed, data are presented as mean with SD, n = 3 biological replicates. (e) Production of IL-10 and IFN-γ in the supernatant of indicated immortalized B cell lines after stimulation by 0.5 mM guanosine, 0.1 mM loxoribine, 2.5 μg/mL R837, 1 μg/mL R848, and 200 ng/mLTL8-506 for 24 hrs, indicated p values were determined by two-way ANOVA, multiple comparisons, adjusted p value, data are presented as mean with SD, n = 3 biological replicates. Production of cytokines in the cells supernatant was measured by CBA.
Extended Data Fig. 7 IRAK1/4 adaptors involved in UNC93B1 genetic variation-induced inflammation.
(a) Levels of phosphorylated IRAK4 as measured by immunoblot using lysates of the indicated THP-1 cells. Data are representative of three independent experiments. (b) Quantitative RT-PCR analysis of IL-6, IL-8, IFIT3, ISG-15, and ISG-54 expression in the indicated THP-1 cell lines, stimulated by 2 μg/mL R848 for 24hrs after being incubated with 0, 1, 10, 50 nM IRAK4 inhibitor for 30 minutes, n = 3 biological replicates. ***p = 0.0003, ****p < 0.0001. (c) Production of IL-1β, IL-23 and IFN-α2 in the supernatant of the indicated THP-1 cell lines, stimulated by 1 μg/mL R848 for 24hrs after being incubated with 0, 10, 20, 50 nM IRAK4 inhibitor for 30 minutes, n = 3 biological replicates. In IL-1β, **p = 0.0012, **p = 0.0022, ***p = 0.0005, ***p = 0.0005, ***p = 0.0002 respectively. In IFN-α2, *p = 0.0380, ***p = 0.0008, *p = 0.0291, ***p = 0.0007, **p = 0.0092, ***p = 0.0003 respectively. (d) Quantitative RT-PCR analysis of IL-6 and IL-8 expression in the indicated THP-1 cell lines, stimulated by 2 μg/mL R848 for 24hrs after being incubated with 0, 0.1, 1, 5 µM IRAK1/4 inhibitor for 30 minutes, n = 3 biological replicates. In IL-6, *p = 0.0114, **p = 0.0033, **p = 0.0022, ***p = 0.0003, ****p < 0.0001 respectively. In IL-8, *p = 0.0327, ****p < 0.0001. (e) Production of IL-23 and MCP-1 in the supernatant of the indicated THP-1 cell lines, stimulated by 2 μg/mL R848 for 24hrs after being incubated with 0, 0.1, 1, 5 µM IRAK1/4 inhibitor for 30 minutes, n = 3 biological replicates. In IL-23, **p = 0.0023, ***p = 0.0001, ***p = 0.0005, ***p = 0.0002 respectively. MCP-1, **p = 0.0037, *p = 0.0131, **p = 0.0089, **p = 0.0011, *p = 0.0296 respectively. (f) Production of IL-12P70, IFN-γ and IL-10 in the supernatant of the indicated immortalized B cell lines, stimulated by 0.1 mM loxoribine for 24hrs after being incubated with 0, 10, 20, 50 nM IRAK4 inhibitor for 30 minutes, n = 3 biological replicates. Indicated p values in b-f were determined by two-way ANOVA, multiple comparisons, adjusted p value, data are presented as mean with SD. (g) Production of MCP-1 in the supernatant of the indicated PBMCs, stimulated by 2 μg/mL R848 for 24hrs after being incubated with and without 25 nM IRAK4 inhibitor for 30 minutes, indicated p values were determined by unpaired t-test, two-tailed, data are presented as mean with SD, n = 3 biological samples. Production of cytokines in the cells supernatant was measured by CBA.
Extended Data Fig. 8 Schematic for the CRISPR/Cas9 strategy to generate UNC93B1 V117L knock-in mice.
(a) The recombinant strategy, the underlined letters are the GuideRNA target site, and the blue font is the mutant site, the red bold font is the mutant base, and the green bold font is the synonymous mutation made to prevent the gRNA from being cut twice. Target sites for, UNC93B1WT/WT (b), UNC93B1WT/V117L (c), and UNC93B1V117L/V117L (d) mice genotypes. HE, heterozygous; HO, homozygotes.
Extended Data Fig. 9 DEGs and upregulated pathways of inflammation in UNC93B1V117L homozygous mice compared to wild type.
(a) Intracellular cytokine staining of TNF in bone marrow-derived macrophage (BMDM) of indicated mice (n = 3). Red histograms are unstained controls. Statistical analysis was done using unpaired t-tests, two-tailed, data are presented as mean with SD. The exact p values are shown. (b–f) RNA sequencing was performed for UNC93B1V117L BMDM compared to control, n = 3 biological replicates. (b) Differentially expressed genes presented as a volcano plot. (c) msigdb_m2_reactome enrichment analysis of significantly upregulated genes highlights innate immune system and toll-like receptor pathways. (d-f) GSEA significantly associated with TLR signaling (d), TNF signaling (e) and antigen processing/presentation (f). Mice were 8 months old. Ho, homozygotes.
Extended Data Fig. 10 Gating strategies.
Representative gating strategies for myeloid cells in bone marrow, macrophage, monocytes, inflammatory Gr-1+ cells, and IFN-γ producing CD4 T cells and CD3+/CD4− cells, lymphocytes subsets, B cells, CD4 T cells, T regulatory cells, activated T cells, or/and germinal center B cells in bone marrow or/and spleen. These strategies used for data presented in Fig. 6.
Supplementary information
Supplementary Information
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Al-Azab, M., Idiiatullina, E., Liu, Z. et al. Genetic variants in UNC93B1 predispose to childhood-onset systemic lupus erythematosus. Nat Immunol 25, 969–980 (2024). https://doi.org/10.1038/s41590-024-01846-5
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DOI: https://doi.org/10.1038/s41590-024-01846-5
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