Publications

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.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by widespread organ involvement including the kidney. Calcium/calmodulin-dependent protein kinase IV (CaMK4) has been shown to conrol immune cell nad podocyte function. To address the effect of genetic podocyte-specific CaMK4 deficiency on systemic autoimmunity and kidney pathology in lupus-prone mice we generated B6.lpr.Camk4flox..podocincre mice. Although podocyte-specific CaMK4 deletion in the lupus-prone Br.lpr mice did not affect systemic autoimmune response parameters, it led to significant improvement of kidney pathology and clinical outcomes. Specifically, B6.lpr.Camk4flox..podocincre mice exhibited reduced glomerular pathology, characterized by less mesangial cell proliferation and diminished immune complex deposition, accompanied by decreased levels of albuminuria and improved creatinine levels. CaMK4 deficiency in podocytes averted the deposition of immune complexes in the kidney. Interestingly, we found increased deposition of immune complexes in the liver. We conclude that CaMK4 expression in podocytes is central to the development of LN and its targeted deletion in podocytes prevents its development without affecting systemic autoimmunity while immune complexes appear to be re-directed from the kidney to the liver.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by immune dysregulation and widespread inflammation. Natural killer (NK) cells, essential for immune surveillance, exhibit profound dysfunction in SLE, including impaired cytotoxicity and cytokine production. However, the mechanisms underlying these abnormalities remain poorly understood. This study investigates how the accumulation of dysfunctional mitochondria due to defective mitophagy contributes to NK cell impairment in SLE and explores strategies to restore their function.

METHODS: Mitochondrial structure and function in NK cells from SLE patients (n=104) and healthy controls (n=104) were assessed using flow cytometry, transmission electron microscopy, and proteomics. Mitophagy-related gene expression was quantified by RT-qPCR. The effects of Urolithin A, a mitophagy activator, and hydroxychloroquine (HCQ) on mitochondrial recycling and NK cell function were evaluated in vitro .

RESULTS: SLE NK cells exhibited accumulation of enlarged, dysfunctional mitochondria, impaired lysosomal acidification, and increased cytosolic mitochondrial DNA leakage, consistent with defective mitophagy. Proteomic and transcriptional analyses revealed downregulation of key mitophagy-related genes. These abnormalities were associated with diminished NK cell effector functions, including reduced degranulation and cytokine production. In vitro , treatment with Urolithin A enhanced mitophagy, improved mitochondrial and lysosomal function, and restored NK cell effector responses. HCQ was also associated with partial recovery of mitochondrial recycling and NK cell function.

CONCLUSION: These findings identify mitochondrial dysfunction and impaired mitophagy as major contributors to NK cell abnormalities in SLE. By uncovering a novel immunometabolic mechanism, this offers new insight into SLE pathogenesis and highlights potential therapeutic strategies targeting mitochondrial quality control.

Systemic lupus erythematosus (SLE) is characterized by dysfunctional regulatory T cells (Tregs). We previously showed that protein phosphatase 2A (PP2A) plays a critical role in maintaining the suppressive function of Tregs. Here, we analyzed phosphoproteomics and metabolomics data from PP2A-wild type and PP2A-deficient Tregs and demonstrated that PP2A regulates Treg function through the pentose phosphate pathway (PPP). Furthermore, we proved that the PPP metabolite gluconolactone (GDL) enhances in vitro induced (i)Treg differentiation and function by promoting forkhead box protein 3 and phosphorylated signal transducer and activator of transcription 5 expression and inhibits T helper 17 (TH17) differentiation in murine cells. In short-term imiquimod-induced autoimmunity in mice, treatment with GDL alleviates inflammation by inhibiting TH17 cells. GDL promotes Tregs function and alleviates skin lesions in MRL.lpr lupus-prone mice in vivo. It also promotes Tregs differentiation and function in ex vivo experiments using cells from patients with SLE. Last, in patients suffering from cutaneous lupus erythematosus, topical application of a GDL-containing cream controlled skin inflammation and improved the clinical and histologic appearance of the skin lesions within 2 weeks. Together, we have identified GDL as a PPP metabolite and showed mechanistically that it restores immune regulation in vitro and in vivo by inducing Treg suppressive function and inhibiting TH17 cells. GDL should be considered as a treatment approach for inflammatory and autoimmune diseases.

Protein phosphatases play a critical role in maintaining immune homeostasis by regulating various signaling pathways involved in immune cell activation, differentiation, and function. In the context of systemic autoimmune diseases, dysregulation of phosphatase activity contributes to aberrant immune responses, leading to chronic inflammation and tissue damage. This review explores the role of key phosphatases from the protein serine/threonine phosphatase and protein tyrosine phosphatase families that are implicated in systemic autoimmunity. We discuss their diverse roles in immune cell subsets, the mechanisms by which their dysregulation drives autoimmune pathogenesis, and the therapeutic potential of targeting these enzymes.

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.

INTRODUCTION: Antibody-mediated rejection (ABMR) after allogeneic kidney transplantation is a substantial clinical problem for which there are no specific treatments. High endothelial venules (HEV) are specialized veins which are normally present only in lymph nodes (LN) facilitating immune cell entry. Here, we show that kidneys undergoing rejection develop HEV-like structures derived from host cells.

METHODS: We developed a nano-delivery system targeting HEVs to simultaneously deliver therapeutics to draining LN and kidney allografts.

RESULTS: Using this system, we preferentially delivered IL-21 neutralizing antibody (NP-HEV[αIL21]) to draining LN and kidney allografts resulting in improved graft function and recipient survival. The NP-HEV[αIL21] system also decreased alloreactive B cell responses, donor-specific antibody production, and ABMR-like lesions in kidney grafts.

CONCLUSIONS: Our study provides a therapeutic strategy to selectively target distinct effector sites to attenuate B-cell alloimmunity while limiting effects of broad systemic immunosuppression in kidney transplantation.

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.