Publications

OBJECTIVE: Podocytes are integral to the maintenance of the glomerular filtration barrier. Their injury results in proteinuria and disease progression in lupus nephritis (LN). Aberrant IgG glycosylation drives podocyte injury in LN and leads to cytoskeletal rearrangement, motility changes, and decreased nephrin production. Based on these findings, we hypothesized that IgG glycosylation patterns differentiate systemic lupus erythematosus (SLE) with and without LN and that this aberrant glycosylation reprograms podocyte metabolism.

METHODS: IgG was isolated from 40 pediatric SLE and from 7 healthy control samples. N-glycan analysis was performed using mass spectrometry. IgG deglycosylation was performed through enzymatic treatment by Peptide N-Glycosidase F for functional studies in podocytes. Untargeted metabolomics was performed in cultured podocytes after exposure to healthy IgG, LN-derived IgG, or deglycosylated LN-IgG and analyzed by metabolite set enrichment analysis. Digital droplet polymerase chain reaction was used to evaluate urine cells and podocytes in culture for pyruvate kinase expression.

RESULTS: The glycosylation pattern of IgG from children with LN was different from that in children with SLE without kidney involvement. Successful treatment led to normalization of IgG glycosylation. Cultured podocytes treated with LN-derived IgG had a lower rate of glycolysis compared to podocytes incubated with deglycosylated LN-IgG or IgG from healthy volunteers. Untargeted metabolomics of podocytes revealed glycolysis as the most enriched pathway in LN and identified five key metabolites (pyruvic acid, phosphoenolpyruvic acid, 2-phosoglycerate, 3 phosphoglycerate, and fructose 1,6 bisphosphate) in which their levels significantly differed among podocytes exposed to LN-derived IgG (LN-IgG) compared to healthy IgG and deglycosylated LN-IgG. This analysis also revealed clustering around a rate limiting step of glycolysis catalyzed by PKM (Pyruvate Kinase M). Urine analyses revealed elevated pyruvic acid and greater expression of pyruvate kinase in podocytes shed in urine in patients with LN compared to levels in patients with SLE without kidney involvement. Podocytes in culture had elevated PKM levels when exposed to LN-IgG compared to IgG from patients with nonrenal SLE and LN in remission.

CONCLUSION: Aberrant IgG glycosylation develops in children with LN and adversely alters podocyte metabolism, rendering these cells vulnerable to injury. Successful treatment reverses IgG glycosylation to patterns comparable to those in patients with nonrenal SLE. These data lay a strong foundation for larger translational studies evaluating the potential of IgG glycosylation as a predictive and pharmacodynamic biomarker for LN. This work also supports a need for the development of approaches to control the aberrant glycosylation of self-targeting IgG in patients with LN as a mechanism to minimize podocytopathy.

Much remains unknown regarding T follicular helper 17 (TFH17) cells commonly found in autoimmune patients. We previously showed that (and here ask why) egress of gut segmented filamentous bacteria (SFB)-induced TFH cells from Peyer's patches (PP) to systemic sites promotes arthritis. We found splenic TFH17 cells are gut derived. Functional analyses using fate-mapping mice revealed a c-Maf-dependent and SFB-induced TH17-to-TFH cell reprogramming that dominantly occurs in PPs. Unlike conventional TFH cells, TH17-derived TFH cells are highly migratory and atypically concentrated in the dark zone of germinal centers (GCs). Compared to conventional TFH cells, TH17-derived TFH cells express higher levels of TFH-associated functional molecules and more robustly conjugate with B cells. Gain- and loss-of-function studies demonstrated their dominance in promoting GC B cells and arthritis. Notably, murine gut TH17-derived TFH signatures exist in rheumatoid arthritis patients. Thus, gut T cell plasticity generates atypical, potent TFH cells promoting systemic autoimmunity.

Tripartite motif-containing 21 (TRIM21) is a cytoplasmic protein with E3 ubiquitin ligase activity. Although autoantibodies against TRIM21 are frequently detected in patients with systemic lupus erythematosus (SLE), its role in disease pathogenesis remains unclear. Here we demonstrate that TRIM21 directly interacts with the stimulator of interferon genes (STING) to regulate type I interferon (IFN) production. In both induced and spontaneous murine models of lupus, TRIM21 deficiency exacerbated lupus-like pathology and heightened IFN production after STING activation. By contrast, TRIM21 overexpression attenuated autoimmunity in lupus-prone mice. Mechanistically, TRIM21 binds to STING and promotes its degradation via the ubiquitin-proteasome pathway. In patients with SLE, TRIM21 expression levels inversely correlated with STING expression, type I IFN levels and autoantibody titers. These findings suggest that targeting the TRIM21-STING axis may offer a therapeutic strategy to reduce type I IFN production in SLE.

Systemic lupus erythematous is a clinically and pathogenetically heterogeneous disease that has long challenged researchers and clinicians aiming to improve its treatment. Advances over the past 75 years have revealed a number of key immune mechanisms that drive clinical manifestations, paving the way for the development of therapies that go beyond broad immunosuppression to improve clinical efficacy and reduce side effects. These include approaches aimed at specific immune pathways, and emerging efforts to restore immune homeostasis, such as chimeric antigen receptor T cell therapies to eliminate pathogenic B cells, low-dose interleukin 2 or regulatory T cell therapies. Although hopes for durable remissions or cure are rising, major obstacles remain owing to the complex nature of the disease. In this Review, we discuss emerging therapeutic strategies designed to address these challenges.

OBJECTIVE: Interleukin-17-producing CD4+ Th17 cells contribute to the pathogenesis of autoimmune diseases, including crescentic glomerulonephritis. Although ADAM9 has been reported to contribute to organ inflammation, the mechanism remains poorly understood. The goal of the current study was to investigate how ADAM9 alters T cell metabolism to promote the generation of Th17 cell differentiation.

METHODS: We induced antiglomerular basement membrane (anti-GBM) glomerulonephritis in Adam9+/+ and Adam9-/- mice using sheep anti-GBM IgG and compared disease severity. Glycolysis in Th17 cells was measured using a Seahorse XFp Extracellular Flux Analyzer (Agilent Technologies, Inc), and metabolomic analysis was conducted on Th17 cells from both Adam9+/+ and Adam9-/- mice. We measured the GLUT1 expression in Th17 cells from Adam9+/+ and Adam9-/- mice and insulin-like growth factor 1 (IGF-1)-treated Th17 cells. Finally, we assessed the protease activity of ADAM9 on IGF-binding protein 4 (IGFBP4).

RESULTS: Mice deficient in ADAM9 had limited numbers of kidney-infiltrating CD4+ T cells and suffered reduced kidney damage and inflammation following the injection of sheep anti-GBM IgG. ADAM9 deficiency led to decreased GLUT1 expression and glycolysis in Th17 cells. Mechanistically, we found that ADAM9 cleaved IGFBP4 and enabled the release of IGF-1, which enhanced the expression of GLUT1 and promoted glycolysis.

CONCLUSION: By cleaving IGFBP4, ADAM9 releases IGF-1, which in turn upregulates GLUT1 expression and promotes glycolysis in Th17 cells. These findings suggest that targeting ADAM9 or blocking IGF-1 should provide a therapeutic strategy for autoimmune diseases.

Pemphigus vulgaris is a B cell-mediated autoimmune disease characterized by autoantibodies targeting desmoglein-3 (Dsg3), a critical adhesion molecule in epithelial tissues. Current treatments rely on broad immunosuppression, highlighting the need for more targeted therapeutic approaches in pemphigus vulgaris and other autoantibody-driven disorders. We engineered a therapeutic fusion protein consisting of the pathogenic domains of Dsg3 linked to either human immunoglobulin G1 (IgG1) or mouse IgG2a (Dsg3-Fc). In vitro, Dsg3-Fc selectively eliminated Dsg3-autoreactive B cells. In vivo, Dsg3-Fc effectively depleted human B cells expressing patient-derived anti-Dsg3 B cell receptors, even in the presence of circulating autoantibodies. Moreover, Dsg3-Fc inhibited both disease initiation and progression in a polyclonal, active pemphigus vulgaris model in immunocompetent mice. In addition, Dsg3-Fc rapidly neutralized pathogenic autoantibodies without inducing systemic toxicity. These findings demonstrate that targeting pathogenic B cells and neutralizing autoantibodies through autoantigen-Fc fusion proteins may represent a promising therapeutic strategy for pemphigus vulgaris and potentially other autoantibody-mediated diseases without the need for global immunosuppression.

OBJECTIVE: Upregulation of Interleukin 23 (IL-23) in the serum and kidneys of patients with lupus nephritis (LN) has been demonstrated, but its effect on podocytes remains unknown. We hypothesized that IL-23 contributes to podocyte injury and that targeted deletion of IL-23R in podocytes of lupus-prone mice can prevent the development of glomerulonephritis.

METHODS: Kidney biopsies were immunostained for IL-23R. In vitro experiments were conducted using a human podocyte cell line and primary murine podocytes. Human podocytes stimulated with IL-23 underwent bulk-RNA sequencing. The expression of IL-23R, structure and motility of podocytes were assessed. Podocytes isolated from B6 wild type mice injected with a minicircle (MC) encoding IL-23 were studied. To assess the role of IL-23R in the development of nephritis, we generated podocyte-specific Il23r deficient MRL/lpr lupus-prone mice.

RESULTS: IL-23R was highly expressed in the glomeruli of patients with LN. IL-23R expression was also upregulated in human podocytes and primary podocytes isolated from B6 mice after IL-23 stimulation. Human podocytes stimulated with IL-23 showed decreased expression of synaptopodin and remodeling of the actin cytoskeleton. IL-23 MC-administered mice exhibited a significant increase in the expression of IL-23R and phosphorylated STAT3 (pSTAT3) in podocytes. Finally, MRL/lpr.Podo-Cre+ Il23rfl/fl mice showed decreased clinical and histologic features of LN.

CONCLUSION: IL-23R expression is increased in podocytes from mice and humans with systemic lupus erythematosus. IL-23 signaling disrupts the cytoskeleton in podocytes and increases their mobility leading to the development of glomerulonephritis. Podocyte-specific deletion of Il23r in lupus-prone mice abrogates the development of LN.

OBJECTIVE: T cell receptor αβ+ (TCRαβ+) CD4- CD8- double-negative (DN) T cells are expanded in lupus-prone mice and patients with systemic lupus erythematosus (SLE), produce interleukin-17 (IL-17), and contribute to disease pathogenesis. However, it is not known whether there is functional heterogeneity within this population. This study aimed to determine whether a subset of DN T cells produces IL-10 and whether the ratio of IL-17+/IL-10+ DN T cells correlates with disease activity.

METHODS: Flow cytometry was used to analyze DN T cells in lupus-prone MRL/lpr mice and patients with SLE. IL-17+ and IL-10+ DN T cells were identified and quantified in the peripheral blood and lymphoid organs. Correlations between the IL-17+/IL-10+ DN T cell ratio and clinical parameters, including the SLE Disease Activity Index (SLEDAI) and proteinuria, were examined.

RESULTS: IL-17+ DN T cells were increased in lupus-prone mice, whereas IL-10+ DN T cells decreased in aging MRL/lpr mice. In patients with SLE (n = 67), the IL-17+/IL-10+ DN T cell ratio was associated with higher SLEDAI scores and was elevated in those with proteinuria. A longitudinal analysis of patients with SLE similarly showed a positive correlation between SLEDAI scores and the IL-17+/IL-10+ DN T cell ratio.

CONCLUSION: The observation of nonoverlapping IL-10+ and IL-17+ DN T cells suggests heterogeneity within the DN T cells in both lupus-prone mice and patients. The IL-17+/IL-10+ DN T cell ratio may serve as a biomarker to monitor disease activity in patients with SLE.

OBJECTIVES: Systemic lupus erythematosus (SLE) is a complex autoimmune disease. Significant morbidity and early mortality necessitate early intervention. This study harnessed SLE-associated immune dysregulation to create a Lupus Classification Risk Index (LCRII) and Lupus Disease Activity Immune Index (LDAII) that identified individuals at risk for SLE classification and disease activity.

METHODS: The LCRII was developed from 84 military personnel who developed classified SLE (≥4 American College of Rheumatology criteria) versus matched healthy controls, which was confirmed in 56 lupus blood relatives who developed SLE versus 154 matched unaffected relatives and 77 unrelated controls. The LDAII was informed by SLE patient visits with low (n=132) or active (n=179) disease and 48 matched controls. Data from blood samples assessed for circulating SLE-associated autoantibody specificities and soluble immune mediators informed the LCRII and LDAII. Random forest modelling guided the selection of informative analytes.

RESULTS: An LCRII informed by 32 or 17 log-transformed/standardised mediators, weighted by their correlation to SLE-associated autoantibodies, differentiated pre-SLE individuals before reaching disease classification (area under the curve (AUC) ≥0.79, p<0.0001; effect size ≥1.1), even before the appearance of clinical criteria (AUC ≥0.74, p<0.0001; effect size ≥0.9). The LCRII-32, LCRII-17 and select mediators, MCP-3/CCL7, TNFRII, stem cell factor (SCF), IL-1α, IP-10/CXCL10 and TGF-β differentiated renal and serositis classification criteria (p<0.05). An LDAII informed by 26 or 13 log-transformed/standardised mediators, weighted by their correlation to SLE-associated autoantibodies or disease activity (hybrid Systemic Lupus Erythematosus Disease Activity Index; hSLEDAI), differentiated SLE patients with low (hSLEDAI <4) or active (hSLEDAI ≥4) disease (AUC >0.6, p ≤0.002, effect size ≥0.4), including clinical/serologic active versus quiescent disease (AUC ≥0.7, p<0.0001, effect size ≥0.6). The LDAII-26, LDAII-13 and select mediators MCP-1/CCL2, TNFRII, SCF, IL-2Rα, IL-10 and TGF-β differentiated renal and serositis manifestations.

CONCLUSIONS: We have conceptualised two immune mediator-informed indexes, the LCRII that predicts SLE from months to years before clinical presentation, and the LDAII that analogously predicts active disease in SLE to distinguish patients who would benefit from early intervention.