Predictors of a weak antibody response to COVID-19 mRNA vaccine in systemic lupus erythematosus.

OBJECTIVES: To characterize the antibody response to COVID-19 mRNA vaccination in patients with Systemic Lupus Erythematosus (SLE) and identify predictors of poor response. METHODS: SLE patients who are followed at the Beth Israel Deaconess Medical Center Lupus Cohort (BID-LC) were enrolled. SARS-CoV-2 IgG Spike antibody was measured in patients who received two doses of either the BNT162b2 (Pfizer-BioNTech) or the mRNA-1273 (Moderna) COVID-19 vaccine (n = 62). We defined non-responders as patients with an IgG Spike antibody titer less than two-fold (< 2) the index value of the test and responders as patients with antibody levels greater or equal to two-fold (≥ 2). A web-based survey was used to collect information regarding immunosuppressive medication use and SLE flares after vaccination. RESULTS: In our cohort of lupus patients, 76% were vaccine responders. The use of two or more immunosuppressive drugs was associated with being a non-responder (Odds Ratio 5.26; 95% CI 1.23-22.34, p = 0.02). Both Belimumab use and higher Prednisone dose were associated with vaccine non-response (p = 0.04 and p = 0.04). The non-responder group had higher mean levels of serum IL-18 than the responder group (p = 0.04) as well as lower C3 levels (p = 0.01). Lupus flares and breakthrough infections were uncommon post-vaccination. CONCLUSIONS: Immunosuppressive medications have a negative impact on vaccine humoral response in SLE individuals. We observed a trend towards vaccine no-response in BNT162b2 recipients and a relationship between IL-18 and impaired antibody response that merits further investigation.

Phosphofructokinase P fine-tunes T regulatory cell metabolism, function, and stability in systemic autoimmunity.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by defective regulatory T (Treg) cells. Here, we demonstrate that a T cell-specific deletion of calcium/calmodulin-dependent protein kinase 4 (CaMK4) improves disease in B6.lpr lupus-prone mice and expands Treg cells. Mechanistically, CaMK4 phosphorylates the glycolysis rate-limiting enzyme 6-phosphofructokinase, platelet type (PFKP) and promotes aerobic glycolysis, while its end product fructose-1,6-biphosphate suppresses oxidative metabolism. In Treg cells, a CRISPR-Cas9-enabled Pfkp deletion recapitulated the metabolism of Camk4-/- Treg cells and improved their function and stability in vitro and in vivo. In SLE CD4+ T cells, PFKP enzymatic activity correlated with SLE disease activity and pharmacologic inhibition of CaMK4-normalized PFKP activity, leading to enhanced Treg cell function. In conclusion, we provide molecular insights in the defective metabolism and function of Treg cells in SLE and identify PFKP as a target to fine-tune Treg cell metabolism and thereby restore their function.

The deacetylase SIRT2 contributes to autoimmune disease pathogenesis by modulating IL-17A and IL-2 transcription.

Aberrant IL-17A expression together with reduced IL-2 production by effector CD4+ T cells contributes to the pathogenesis of systemic lupus erythematosus (SLE). Here, we report that Sirtuin 2 (SIRT2), a member of the family of NAD+-dependent histone deacetylases, suppresses IL-2 production by CD4+ T cells while promoting their differentiation into Th17 cells. Mechanistically, we show that SIRT2 is responsible for the deacetylation of p70S6K, activation of the mTORC1/HIF-1α/RORγt pathway and induction of Th17-cell differentiation. Additionally, SIRT2 was shown to be responsible for the deacetylation of c-Jun and histones at the Il-2 gene, resulting in decreased IL-2 production. We found that the transcription factor inducible cAMP early repressor (ICER), which is overexpressed in T cells from people with SLE and lupus-prone mice, bound directly to the Sirt2 promoter and promoted its transcription. AK-7, a SIRT2 inhibitor, limited the ability of adoptively transferred antigen-specific CD4+ T cells to cause autoimmune encephalomyelitis in mice and limited disease in lupus-prone MRL/lpr mice. Finally, CD4+ T cells from SLE patients exhibited increased expression of SIRT2, and pharmacological inhibition of SIRT2 in primary CD4+ T cells from patients with SLE attenuated the ability of these cells to differentiate into Th17 cells and promoted the generation of IL-2-producing T cells. Collectively, these results suggest that SIRT2-mediated deacetylation is essential in the aberrant expression of IL-17A and IL-2 and that SIRT2 may be a promising molecular target for new SLE therapies.

Role of Glutaminase 2 in Promoting CD4+ T Cell Production of Interleukin-2 by Supporting Antioxidant Defense in Systemic Lupus Erythematosus.

OBJECTIVE: Glutaminase (GLS) isoenzymes GLS1 and GLS2 catalyze the first step of glutaminolysis. GLS1 is requisite for Th17 cell differentiation, and its inhibition suppresses autoimmune disease in animals, but the function of GLS2 is not known. The aim of this study was to investigate the role of GLS2 in CD4+ T cell function and systemic lupus erythematosus (SLE) pathogenesis. METHODS: We measured reactive oxygen species (ROS) levels, lipid peroxidation, and mitochondrial mass and polarization by flow cytometry, interleukin-2 (IL-2) production by a dual luciferase assay, and CpG DNA methylation of Il2 by a real-time polymerase chain reaction system. The impact of the overexpression of wild-type GLS1, wild-type GLS2, or mutated GLS2 at the PDZ domain-binding motif in CD4+ T cells was examined. Furthermore, GLS2 expression in CD4+ T cells from lupus-prone mice and patients with SLE was analyzed by Western blotting. RESULTS: GLS2, but not GLS1, reduced ROS levels and lipid peroxidation and restored mitochondrial function in T cells. GLS2 promoted IL-2 production through the demethylation of the Il2 promoter. Mutation of the PDZ domain-binding motif abated the ability of GLS2 to regulate IL-2 and ROS levels. In lupus-prone mice and patients with SLE, the expression of GLS2 was decreased in CD4+ T cells. Finally, GLS2 overexpression corrected ROS levels and restored IL-2 production by CD4+ T cells from lupus-prone mice and SLE patients. CONCLUSION: Our findings suggest that GLS2 has a crucial role in IL-2 production by CD4+ T cells by supporting antioxidant defense, and they offer a new approach to correcting IL-2 production by T cells in SLE.

Glutathione peroxidase 4-regulated neutrophil ferroptosis induces systemic autoimmunity.

The linkage between neutrophil death and the development of autoimmunity has not been thoroughly explored. Here, we show that neutrophils from either lupus-prone mice or patients with systemic lupus erythematosus (SLE) undergo ferroptosis. Mechanistically, autoantibodies and interferon-α present in the serum induce neutrophil ferroptosis through enhanced binding of the transcriptional repressor CREMα to the glutathione peroxidase 4 (Gpx4, the key ferroptosis regulator) promoter, which leads to suppressed expression of Gpx4 and subsequent elevation of lipid-reactive oxygen species. Moreover, the findings that mice with neutrophil-specific Gpx4 haploinsufficiency recapitulate key clinical features of human SLE, including autoantibodies, neutropenia, skin lesions and proteinuria, and that the treatment with a specific ferroptosis inhibitor significantly ameliorates disease severity in lupus-prone mice reveal the role of neutrophil ferroptosis in lupus pathogenesis. Together, our data demonstrate that neutrophil ferroptosis is an important driver of neutropenia in SLE and heavily contributes to disease manifestations.

Estrogen-Induced hsa-miR-10b-5p Is Elevated in T Cells From Patients With Systemic Lupus Erythematosus and Down-Regulates Serine/Arginine-Rich Splicing Factor 1.

OBJECTIVE: Autoimmune diseases affect women disproportionately more than men. Estrogen is implicated in immune cell dysfunction, yet its precise molecular roles are not fully known. We recently identified new roles for serine/arginine-rich splicing factor 1 (SRSF1) in T cell function and autoimmunity. SRSF1 levels are decreased in T cells from patients with systemic lupus erythematosus (SLE) and are associated with active disease and comorbidity. However, the molecular mechanisms that control SRSF1 expression are unknown. Srsf1 messenger RNA (mRNA) has a long 3'-untranslated region (3'-UTR), suggesting posttranscriptional control. This study was undertaken to investigate the role of estrogen and posttranscriptional mechanisms of SRSF1 regulation in T cells and SLE. METHODS: In silico bioinformatics analysis of Srsf1-3'-UTR revealed multiple microRNA (miRNA; miR)-binding sites. Additional screening and literature searches narrowed down hsa-miR-10b-5p for further study. Peripheral blood T cells from healthy individuals and SLE patients were evaluated for mRNA and miRNA expression by quantitative reverse transcription-polymerase chain reaction, and SRSF1 protein levels were assessed by immunoblotting. T cells were cultured with ß-estradiol, and transient transfections were used to overexpress miRNAs. Luciferase assays were used to measure 3'-UTR activity. RESULTS: We demonstrated that estrogen increased hsa-miR-10b-5p expression in human T cells, and hsa-miR-10b-5p down-regulated SRSF1 protein expression. Mechanistically, hsa-mir-10b-5p regulated SRSF1 posttranscriptionally via control of its 3'-UTR activity. Importantly, hsa-miR-10b-5p expression levels were elevated in T cells from healthy women compared to healthy men and also elevated in T cells from SLE patients. CONCLUSION: We identified a previously unrecognized molecular link between estrogen and gene regulation in immune cells, with potential relevance to systemic autoimmune disease.

ADAM9 enhances Th17 cell differentiation and autoimmunity by activating TGF-ß1.

The a disintegrin and metalloproteinase (ADAM) family of proteinases alter the extracellular environment and are involved in the development of T cells and autoimmunity. The role of ADAM family members in Th17 cell differentiation is unknown. We identified ADAM9 to be specifically expressed and to promote Th17 differentiation. Mechanistically, we found that ADAM9 cleaved the latency-associated peptide to produce bioactive transforming growth factor ß1, which promoted SMAD2/3 phosphorylation and activation. A transcription factor inducible cAMP early repressor was found to bind directly to the ADAM9 promoter and to promote its transcription. Adam9-deficient mice displayed mitigated experimental autoimmune encephalomyelitis, and transfer of Adam9-deficient myelin oligodendrocyte globulin-specific T cells into Rag1-/- mice failed to induce disease. At the translational level, an increased abundance of ADAM9 levels was observed in CD4+ T cells from patients with systemic lupus erythematosus, and ADAM9 gene deletion in lupus primary CD4+ T cells clearly attenuated their ability to differentiate into Th17 cells. These findings revealed that ADAM9 as a proteinase provides Th17 cells with an ability to activate transforming growth factor ß1 and accelerates its differentiation, resulting in aberrant autoimmunity.

Aberrantly glycosylated IgG elicits pathogenic signaling in podocytes and signifies lupus nephritis.

Lupus nephritis (LN) is a serious complication occurring in 50% of patients with systemic lupus erythematosus (SLE) for which there is a lack of biomarkers, a lack of specific medications, and a lack of a clear understanding of its pathogenesis. The expression of calcium/calmodulin kinase IV (CaMK4) is increased in podocytes of patients with LN and lupus-prone mice, and its podocyte-targeted inhibition averts the development of nephritis in mice. Nephrin is a key podocyte molecule essential for the maintenance of the glomerular slit diaphragm. Here, we show that the presence of fucose on N-glycans of IgG induces, whereas the presence of galactose ameliorates, podocyte injury through CaMK4 expression. Mechanistically, CaMK4 phosphorylates NF-κB, upregulates the transcriptional repressor SNAIL, and limits the expression of nephrin. In addition, we demonstrate that increased expression of CaMK4 in biopsy specimens and in urine podocytes from people with LN is linked to active kidney disease. Our data shed light on the role of IgG glycosylation in the development of podocyte injury and propose the development of "liquid kidney biopsy" approaches to diagnose LN.

Splicing factor SRSF1 limits IFN-γ production via RhoH and ameliorates experimental nephritis.

OBJECTIVE: CD4 T helper 1 (Th1) cells producing IFN-γ contribute to inflammatory responses in the pathogenesis of SLE and lupus nephritis. Moreover, elevated serum type II IFN levels precede the appearance of type I IFNs and autoantibodies in patient years before clinical diagnosis. However, the molecules and mechanisms that control this inflammatory response in SLE remain unclear. Serine/arginine-rich splicing factor 1 (SRSF1) is decreased in T cells from SLE patients, and restrains T cell hyperactivity and systemic autoimmunity. Our objective here was to evaluate the role of SRSF1 in IFN-γ production, Th1 differentiation and experimental nephritis. METHODS: T cell-conditional Srsf1-knockout mice were used to study nephrotoxic serum-induced nephritis and evaluate IFN-γ production and Th1 differentiation by flow cytometry. RNA sequencing was used to assess transcriptomics profiles. RhoH was silenced by siRNA transfections in human T cells by electroporation. RhoH and SRSF1 protein levels were assessed by immunoblots. RESULTS: Deletion of Srsf1 in T cells led to increased Th1 differentiation and exacerbated nephrotoxic serum nephritis. The expression levels of RhoH are decreased in Srsf1-deficient T cells, and silencing RhoH in human T cells leads to increased production of IFN-γ. Furthermore, RhoH expression was decreased and directly correlated with SRSF1 in T cells from SLE patients. CONCLUSION: Our study uncovers a previously unrecognized role of SRSF1 in restraining IFN-γ production and Th1 differentiation through the control of RhoH. Reduced expression of SRSF1 may contribute to pathogenesis of autoimmune-related nephritis through these molecular mechanisms.

Systemic lupus erythematosus favors the generation of IL-17 producing double negative T cells.

Mature double negative (DN) T cells are a population of αß T cells that lack CD4 and CD8 coreceptors and contribute to systemic lupus erythematosus (SLE). The splenic marginal zone macrophages (MZMs) are important for establishing immune tolerance, and loss of their number or function contributes to the progression of SLE. Here we show that loss of MZMs impairs the tolerogenic clearance of apoptotic cells and alters the serum cytokine profile, which in turn provokes the generation of DN T cells from self-reactive CD8+ T cells. Increased Ki67 expression, narrowed TCR V-beta repertoire usage and diluted T-cell receptor excision circles confirm that DN T cells from lupus-prone mice and patients with SLE undergo clonal proliferation and expansion in a self-antigen dependent manner, which supports the shared mechanisms for their generation. Collectively, our results provide a link between the loss of MZMs and the expansion of DN T cells, and indicate possible strategies to prevent the development of SLE.

Splicing factor SRSF1 controls T cell homeostasis and its decreased levels are linked to lymphopenia in systemic lupus erythematosus.

OBJECTIVE: Lymphopenia is a frequent clinical manifestation and risk factor for infections in SLE, but the underlying mechanisms are not fully understood. We previously identified novel roles for the RNA-binding protein serine arginine-rich splicing factor 1 (SRSF1) in the control of genes involved in signalling and cytokine production in human T cells. SRSF1 is decreased in T cells from patients with SLE and associates with severe disease. Because SRSF1 controls the expression of apoptosis-related genes, we hypothesized that SRSF1 controls T cell homeostasis and, when reduced, leads to lymphopenia. METHODS: We evaluated SRSF1 expression in T cells from SLE patients by immunoblots and analysed its correlation with clinical parameters. T cell conditional Srsf1 knockout mice were used to evaluate lymphoid cells and apoptosis by flow cytometry. Quantitative PCR and immunoblots were used to assess Bcl-xL mRNA and protein expression. SRSF1 overexpression was performed by transient transfections by electroporation. RESULTS: We found that low SRSF1 levels correlated with lymphopenia in SLE patients. Selective deletion of Srsf1 in T cells in mice led to T cell lymphopenia, with increased apoptosis and decreased expression of the anti-apoptotic Bcl-xL. Lower SRSF1 expression correlated with low Bcl-xL levels in T cells and lower Bcl-xL levels associated with lymphopenia in SLE patients. Importantly, overexpression of SRSF1 rescued survival of T cells from patients with SLE. CONCLUSION: Our studies uncovered a previously unrecognized role for SRSF1 in the control of T cell homeostasis and its reduced expression as a molecular defect that contributes to lymphopenia in systemic autoimmunity.

The CD38/NAD/SIRTUIN1/EZH2 Axis Mitigates Cytotoxic CD8 T Cell Function and Identifies Patients with SLE Prone to Infections.

Patients with systemic lupus erythematosus (SLE) suffer frequent infections that account for significant morbidity and mortality. T cell cytotoxic responses are decreased in patients with SLE, yet the responsible molecular events are largely unknown. We find an expanded CD8CD38high T cell subset in a subgroup of patients with increased rates of infections. CD8CD38high T cells from healthy subjects and patients with SLE display decreased cytotoxic capacity, degranulation, and expression of granzymes A and B and perforin. The key cytotoxicity-related transcription factors T-bet, RUNX3, and EOMES are decreased in CD8CD38high T cells. CD38 leads to increased acetylated EZH2 through inhibition of the deacetylase Sirtuin1. Acetylated EZH2 represses RUNX3 expression, whereas inhibition of EZH2 restores CD8 T cell cytotoxic responses. We propose that high levels of CD38 lead to decreased CD8 T cell-mediated cytotoxicity and increased propensity to infections in patients with SLE, a process that can be reversed pharmacologically.