Unraveling transcriptomic signatures and dysregulated pathways in systemic lupus erythematosus across disease states.

OBJECTIVES: This study aims to elucidate the transcriptomic signatures and dysregulated pathways in patients with Systemic Lupus Erythematosus (SLE), with a particular focus on those persisting during disease remission. METHODS: We conducted bulk RNA-sequencing of peripheral blood mononuclear cells (PBMCs) from a well-defined cohort comprising 26 remission patients meeting the Low Lupus Disease Activity State (LLDAS) criteria, 76 patients experiencing disease flares, and 15 healthy controls. To elucidate immune signature changes associated with varying disease states, we performed extensive analyses, including the identification of differentially expressed genes and pathways, as well as the construction of protein-protein interaction networks. RESULTS: Several transcriptomic features recovered during remission compared to the active disease state, including down-regulation of plasma and cell cycle signatures, as well as up-regulation of lymphocytes. However, specific innate immune response signatures, such as the interferon (IFN) signature, and gene modules involved in chromatin structure modification, persisted across different disease states. Drug repurposing analysis revealed certain drug classes that can target these persistent signatures, potentially preventing disease relapse. CONCLUSION: Our comprehensive transcriptomic study revealed gene expression signatures for SLE in both active and remission states. The discovery of gene expression modules persisting in the remission stage may shed light on the underlying mechanisms of vulnerability to relapse in these patients, providing valuable insights for their treatment.

Transcriptomic Features of systemic lupus erythematosus Patients in Flare and Changes during Acute In-Hospital Treatment.

OBJECTIVES: Systemic lupus erythematosus (SLE) is a complex autoimmune disease with varying symptoms and multi-organ damage. Relapse-remission cycles often persist for many patients for years with the current treatment. Improved understanding of molecular changes caused by SLE flare and intensive treatment may result in more targeted therapies. METHODS: RNA-sequencing was performed on peripheral blood mononuclear cells (PBMCs) from 65 SLE patients in flare, collected both before (SLE1) and after (SLE2) in-hospital treatment, along with 15 healthy controls (HC). Differentially expressed genes (DEGs) were identified among the three groups. Enriched functions and key molecular signatures of the DEGs were analyzed and scored to elucidate the transcriptomic changes during treatment. RESULTS: Few upregulated genes in SLE1 vs HC were affected by treatment (SLE2 vs SLE1), mostly functional in interferon signalling (IFN), plasmablasts, and neutrophils. IFN and plasmablast signatures were repressed, but the neutrophil signature remained unchanged or enhanced by treatment. The IFN and neutrophil scores together stratified the SLE samples. IFN scores correlated well with leukopenia, while neutrophil scores reflected relative cell compositions but not cell counts. CONCLUSIONS: In-hospital treatment significantly relieved SLE symptoms with expression changes of a small subset of genes. Notably, IFN signature changes matched SLE flare and improvement, while enhanced neutrophil signature upon treatment suggested the involvement of low-density granulocytes (LDG) in disease development.

Human germline heterozygous gain-of-function STAT6 variants cause severe allergic disease.

STAT6 (signal transducer and activator of transcription 6) is a transcription factor that plays a central role in the pathophysiology of allergic inflammation. We have identified 16 patients from 10 families spanning three continents with a profound phenotype of early-life onset allergic immune dysregulation, widespread treatment-resistant atopic dermatitis, hypereosinophilia with esosinophilic gastrointestinal disease, asthma, elevated serum IgE, IgE-mediated food allergies, and anaphylaxis. The cases were either sporadic (seven kindreds) or followed an autosomal dominant inheritance pattern (three kindreds). All patients carried monoallelic rare variants in STAT6 and functional studies established their gain-of-function (GOF) phenotype with sustained STAT6 phosphorylation, increased STAT6 target gene expression, and TH2 skewing. Precision treatment with the anti-IL-4Rα antibody, dupilumab, was highly effective improving both clinical manifestations and immunological biomarkers. This study identifies heterozygous GOF variants in STAT6 as a novel autosomal dominant allergic disorder. We anticipate that our discovery of multiple kindreds with germline STAT6 GOF variants will facilitate the recognition of more affected individuals and the full definition of this new primary atopic disorder.

Targeted Gene Sanger Sequencing Should Remain the First-Tier Genetic Test for Children Suspected to Have the Five Common X-Linked Inborn Errors of Immunity.

To address inborn errors of immunity (IEI) which were underdiagnosed in resource-limited regions, our centre developed and offered free genetic testing for the most common IEI by Sanger sequencing (SS) since 2001. With the establishment of The Asian Primary Immunodeficiency (APID) Network in 2009, the awareness and definitive diagnosis of IEI were further improved with collaboration among centres caring for IEI patients from East and Southeast Asia. We also started to use whole exome sequencing (WES) for undiagnosed cases and further extended our collaboration with centres from South Asia and Africa. With the increased use of Next Generation Sequencing (NGS), we have shifted our diagnostic practice from SS to WES. However, SS was still one of the key diagnostic tools for IEI for the past two decades. Our centre has performed 2,024 IEI SS genetic tests, with in-house protocol designed specifically for 84 genes, in 1,376 patients with 744 identified to have disease-causing mutations (54.1%). The high diagnostic rate after just one round of targeted gene SS for each of the 5 common IEI (X-linked agammaglobulinemia (XLA) 77.4%, Wiskott-Aldrich syndrome (WAS) 69.2%, X-linked chronic granulomatous disease (XCGD) 59.5%, X-linked severe combined immunodeficiency (XSCID) 51.1%, and X-linked hyper-IgM syndrome (HIGM1) 58.1%) demonstrated targeted gene SS should remain the first-tier genetic test for the 5 common X-linked IEI.

Phenomic Analysis of Chronic Granulomatous Disease Reveals More Severe Integumentary Infections in X-Linked Compared With Autosomal Recessive Chronic Granulomatous Disease.

Background: Chronic granulomatous disease (CGD) is an inborn error of immunity (IEI), characterised by recurrent bacterial and fungal infections. It is inherited either in an X-linked (XL) or autosomal recessive (AR) mode. Phenome refers to the entire set of phenotypes expressed, and its study allows us to generate new knowledge of the disease. The objective of the study is to reveal the phenomic differences between XL and AR-CGD by using Human Phenotype Ontology (HPO) terms. Methods: We collected data on 117 patients with genetically diagnosed CGD from Asia and Africa referred to the Asian Primary Immunodeficiency Network (APID network). Only 90 patients with sufficient clinical information were included for phenomic analysis. We used HPO terms to describe all phenotypes manifested in the patients. Results: XL-CGD patients had a lower age of onset, referral, clinical diagnosis, and genetic diagnosis compared with AR-CGD patients. The integument and central nervous system were more frequently affected in XL-CGD patients. Regarding HPO terms, perianal abscess, cutaneous abscess, and elevated hepatic transaminase were correlated with XL-CGD. A higher percentage of XL-CGD patients presented with BCGitis/BCGosis as their first manifestation. Among our CGD patients, lung was the most frequently infected organ, with gastrointestinal system and skin ranking second and third, respectively. Aspergillus species, Mycobacterium bovis, and Mycobacteirum tuberculosis were the most frequent pathogens to be found. Conclusion: Phenomic analysis confirmed that XL-CGD patients have more recurrent and aggressive infections compared with AR-CGD patients. Various phenotypic differences listed out can be used as clinical handles to distinguish XL or AR-CGD based on clinical features.

Excessive deubiquitination of NLRP3-R779C variant contributes to very-early-onset inflammatory bowel disease development.

BACKGROUND: Very-early-onset inflammatory bowel disease (VEOIBD) is a chronic inflammatory disease of the gastrointestinal tract occurring during infancy or early childhood. NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome has emerged as a crucial regulator of intestinal homeostasis; however, whether NLRP3 variants may modify VEOIBD risk is unknown. OBJECTIVE: We sought to investigate whether and how a rare NLRP3 variant, found in 3 patients with gastrointestinal symptoms, contributes to VEOIBD development. METHODS: Whole-exome sequencing and bioinformatic analysis were performed to screen disease-associated NLRP3 variants from a cohort of children with VEOIBD. Inflammasome activation was determined in reconstituted HEK293T human embryonic kidney cells with NLRP3 inflammasome components, doxycycline-inducible NLRP3 macrophages, as well as PBMCs and biopsies from patients with NLRP3 variants. Pathogenesis of the variants was determined using a dextran sulfate sodium-induced acute colitis model. RESULTS: We identified a dominant gain-of-function missense variant of NLRP3, encoded by rs772009059 (R779C), in 3 patients with gastrointestinal symptoms. Functional analysis revealed that R779C increased NLRP3 inflammasome activation and pyroptosis in macrophages. This was mediated by enhanced deubiquitination of NLRP3 via binding with deubiquitinases BRCC3 and JOSD2, which are highly expressed in myeloid cells. In a dextran sulfate sodium-induced acute colitis model, NLRP3-R779C in hematopoietic cells resulted in more severe colitis, which can be ameliorated via knockdown of BRCC3 or JOSD2. CONCLUSIONS: BRCC3 and JOSD2 mediate NLRP3-R779C deubiquitination, which promotes NLRP3 inflammasome activation and the risk of developing VEOIBD.

RU486 Metabolite Inhibits CCN1/Cyr61 Secretion by MDA-MB-231-Endothelial Adhesion.

Successful adhesion of circulating tumor cells (CTCs) to microvascular endothelium of distant metastatic tissue is the key starting step of metastatic cascade that could be effectively chemoprevented as we demonstrated previously. Here, we hypothesize that the hetero-adhesion may produce secretory biomarkers that may be important for both premetastatic diagnosis and chemoprevention. We show that co-incubation of triple-negative breast cancer (TNBC) cell line MDA-MB-231 with human pulmonary microvascular endothelial monolayers (HPMEC) secretes Cyr61 (CCN1), primarily from MDA-MB-231. However, addition of metapristone (RU486 metabolite) to the co-incubation system inhibits Cyr61 secretion probably via the Cyr61/integrin αvß1 signaling pathway without significant cytotoxicity on both MDA-MB-231 and HPMEC. Transfection of MDA-MB-231 with Cyr61-related recombinant plasmid or siRNA enhances or reduces Cyr61 expression, accordingly. The transfection significantly changes hetero-adhesion and migration of MDA-MB-231, and the changed bioactivities by overexpressed CYR61 could be antagonized by metapristone in vitro. Moreover, the circulating MDA-MB-231 develops lung metastasis in mice, which could be effectively prevented by oral metapristone without significant toxicity. The present study, for the first time, demonstrates that co-incubation of MDA-MB-231 with HPMEC secrets CYR61 probably via the CYR61/integrin αvß1 signaling pathway to promote adhesion-invasion of TNBC (early metastatic step). Metapristone, by interfering the adhesion-invasion process, prevents metastasis from happening.

Promising Compounds From Murraya exotica for Cancer Metastasis Chemoprevention.

Most of the present anticancer drugs are highly cytotoxic and focus mainly on killing tumor cells rather than slowing the progress of cancer metastasis. Evidence has been reported that bridges the mechanisms of inflammation and tumor invasion. Therefore, we evaluated the potency in cancer metastasis chemoprevention of compounds and a coumarin extracted from Murraya exotica, which is known for its anti-inflammation bioactivity. By carrying out experiments in vitro, we found the root extracts more efficient than the leaf extracts in restraining cell migration of MDA-MB-231 cells, while leaf extracts presented slightly stronger inhibition of tumor cell adhesion at low concentrations. In addition, compared to root extracts, a novel coumarin identified previously from root extracts showed equal inhibition on cancer cell adhesion and less inhibition on cell migration. All extracts used in this study presented low cytotoxicity in vitro. Through comparison of the contents of leaf and root extracts from M exotica, several compounds are considered promising against cancer metastasis. This study evaluates the worth of further development of M exotica to find its effect on cancer metastasis chemoprevention.

Pharmacoproteomic analysis reveals that metapristone (RU486 metabolite) intervenes E-cadherin and vimentin to realize cancer metastasis chemoprevention.

Metapristone is the most predominant biological active metabolite of mifepristone, and being developed as a novel cancer metastasis chemopreventive agent by us. Despite its prominent metastasis chemopreventive effect, the underlying mechanism remains elusive. Our study, for the first time, demonstrated that metapristone had the ability to prevent breast cancer cells from migration, invasion, and interfere with their adhesion to endothelial cells. To explore the underlying mechanism of metapristone, we employed the iTRAQ technique to assess the effect of metapristone on MDA-MB-231 cells. In total, 5,145 proteins were identified, of which, 311 proteins showed significant differences in metapristone-treated cells compared to the control group (P-value < 0.05). Bioinformatic analysis showed many differentially expressed proteins (DEPs) functionally associated with post-translational modification, chaperones, translation, transcription, replication, signal transduction, etc. Importantly, many of the DEPs, such as E-cadherin, vimentin, TGF-ß receptor I/II, smad2/3, ß-catenin, caveolin, and dystroglycan were associated with TGF-ß and Wnt signaling pathways, which were also linked to epithelial-to-mesenchymal transition (EMT) process. Further validation of the epithelial marker "E-caderin" and mesenchymal marker "vimetin" were carried out using immunoblot and immunofluorescence. These results have revealed a novel mechanism that metapristone-mediated metastasis chemoprevention is through intervening the EMT-related signaling pathways.

Thirty-day rat toxicity study reveals reversible liver toxicity of mifepristone (RU486) and metapristone.

OBJECTIVE: Mifepristone (RU486) is an oral first-line contraceptive used by hundreds of millions of women, and recently it was tested for anticancer activity in both genders worldwide. We are developing metapristone (the N-monodemethyl RU486) as a potential metastasis chemopreventive. The present acute and 30-d subacute toxicity study aimed at examining and compared in parallel the potential toxicity of the two drugs. METHODS: The single-dose acute toxicity and 30-d subacute toxicity studies were conducted in mice and rats, respectively, by gavaging metapristone or mifepristone at various doses. Blood samples and organs were collected for blood chemistry, hematology and histology analyses. RESULTS: Oral mifepristone (3000 mg/kg) caused 30% and 40% death in female and male mice, respectively, within 15 h post-dosing. In comparison, the same dose of metapristone produced 30% acute death in males only. Thirty-day oral administration of the two drugs to rats (12.5, 50 and 200 mg/kg/day) caused reversible hepatotoxicity that only occurred at 200 mg/kg/day group, evidenced by the elevated liver enzyme activity and liver organ weight. CONCLUSION: The present study, for the first time, reveals reversible hepatotoxicity in rats caused by the 30-d consecutive administration at the high dose, and warns the potential hepatotoxicity caused by long-term administrations of high doses of mifepristone or metapristone in clinical trials but not by the acute single abortion doses.

Systems pharmacology of mifepristone (RU486) reveals its 47 hub targets and network: comprehensive analysis and pharmacological focus on FAK-Src-Paxillin complex.

Mifepristone (RU486), a synthetic steroid compound used as an abortifacient drug, has received considerable attention to its anticancer activity recently. To explore the possibility of using mifepristone as a cancer metastasis chemopreventive, we performed a systems pharmacology analysis of mifepristone-related molecules in the present study. Data were collected by using Natural Language Processing (NLP) and 513 mifepristone-related genes were dug out and classified functionally using a gene ontology (GO) hierarchy, followed by KEGG pathway enrichment analysis. Potential signal pathways and targets involved in cancer were obtained by integrative network analysis. Total thirty-three proteins were involved in focal adhesion-the key signaling pathway associated with cancer metastasis. Molecular and cellular assays further demonstrated that mifepristone had the ability to prevent breast cancer cells from migration and interfere with their adhesion to endothelial cells. Moreover, mifepristone inhibited the expression of focal adhesion kinase (FAK), paxillin, and the formation of FAK/Src/Paxillin complex, which are correlated with cell adhesion and migration. This study set a good example to identify chemotherapeutic potential seamlessly from systems pharmacology to cellular pharmacology, and the revealed hub genes may be the promising targets for cancer metastasis chemoprevention.