Mitotic MTH1 inhibitor TH1579 induces PD-L1 expression and inflammatory response through the cGAS-STING pathway.

The mitotic MTH1 inhibitor TH1579 is a dual inhibitor that inhibits mitosis and incorporation of oxidative DNA damage and leads to cancer-specific cell death. The response to immune checkpoint inhibitor (ICI) treatment is often augmented by DNA damaging agents through the cGAS-STING pathway. This study investigates whether TH1579 can improve the efficacy of immune checkpoint blockades through its immunomodulatory properties. Various human and murine cancer cell lines were treated with mitotic MTH1i TH1579, and the expression of PD-L1 and T-cell infiltration-related chemokines was analysed by flow cytometry and real-time qPCR. Syngeneic mouse models were established to examine the combined effect of TH1579 and PD-L1 blockade. In our investigation, we found that TH1579 upregulates PD-L1 expression at both the protein and mRNA levels in human cancer cell lines. However, in murine cell lines, the increase was less pronounced. An in vivo experiment in a syngeneic mouse melanoma model showed that TH1579 treatment significantly increased the efficacy of atezolizumab, an anti-PD-L1 antibody, compared to vehicle or atezolizumab monotherapy. Furthermore, TH1579 exhibited immune-modulatory properties, elevating cytokines such as IFN-ß and chemokines including CCL5 and CXCL10, in a cGAS-STING pathway-dependent manner. In conclusion, TH1579 has the potential to improve ICI treatment by modulating immune checkpoint-related proteins and pathways.

Disentangling the riddle of systemic lupus erythematosus with antiphospholipid syndrome: blood transcriptome analysis reveals a less-pronounced IFN-signature and distinct molecular profiles in venous versus arterial events.

INTRODUCTION: Systemic lupus erythematosus with antiphospholipid syndrome (SLE-APS) represents a challenging SLE endotype whose molecular basis remains unknown. METHODS: We analysed whole-blood RNA-sequencing data from 299 patients with SLE (108 SLE-antiphospholipid antibodies (aPL)-positive, including 67 SLE-APS; 191 SLE-aPL-negative) and 72 matched healthy controls (HC). Pathway enrichment analysis, unsupervised weighted gene coexpression network analysis and machine learning were applied to distinguish disease endotypes. RESULTS: Patients with SLE-APS demonstrated upregulated type I and II interferon (IFN) pathways compared with HC. Using a 100-gene random forests model, we achieved a cross-validated accuracy of 75.6% in distinguishing these two states. Additionally, the comparison between SLE-APS and SLE-aPL-negative revealed 227 differentially expressed genes, indicating downregulation of IFN-α and IFN-γ signatures, coupled with dysregulation of the complement cascade, B-cell activation and neutrophil degranulation. Unsupervised analysis of SLE transcriptome identified 21 gene modules, with SLE-APS strongly linked to upregulation of the 'neutrophilic/myeloid' module. Within SLE-APS, venous thromboses positively correlated with 'neutrophilic/myeloid' and 'B cell' modules, while arterial thromboses were associated with dysregulation of 'DNA damage response (DDR)' and 'metabolism' modules. Anticardiolipin and anti-ß2GPI positivity-irrespective of APS status-were associated with the 'neutrophilic/myeloid' and 'protein-binding' module, respectively. CONCLUSIONS: There is a hierarchical upregulation and-likely-dependence on IFN in SLE with the highest IFN signature observed in SLE-aPL-negative patients. Venous thrombotic events are associated with neutrophils and B cells while arterial events with DDR and impaired metabolism. This may account for their differential requirements for anticoagulation and provide rationale for the potential use of mTOR inhibitors such as sirolimus and the direct fIIa inhibitor dabigatran in SLE-APS.

Impaired serotonin synthesis in hippocampus of murine lupus represents an early neuropsychiatric event.

BACKGROUND: Despite significant progress in understanding the mechanisms underlying hippocampal involvement in neuropsychiatric systemic lupus erythematosus (NPSLE), our understanding of how neuroinflammation affects the brain neurotransmitter systems is limited. To date, few studies have investigated the role of neurotransmitters in pathogenesis of NPSLE with contradictory results. METHODS: Hippocampal tissue from NZB/W-F1 lupus-prone mice and age-matched control strains were dissected in both pre-nephritic (3-month-old) and nephritic (6-month-old) stages. High-Performance Liquid Chromatography (HPLC) was used to evaluate the level of serotonin (5-HT), dopamine (DA), and their metabolites 5-HIAA and DOPAC, respectively, in mouse hippocampi. RESULTS: Lupus mice exhibit decreased levels of serotonin at the early stages of the disease, along with intact levels of its metabolite 5-HIAA. The 5-HT turnover ratio (5-HIAA/5-HT ratio) was increased in the hippocampus of lupus mice at pre-nephritic stage suggesting that low hippocampal serotonin levels in lupus are attributed to decreased serotonin synthesis. Both DA and DOPAC levels remained unaffected in lupus hippocampus at both early and late stages. CONCLUSION: Impaired hippocampal serotonin synthesis in the hippocampus of lupus-prone mice represents an early neuropsychiatric event. These findings may have important implications for the use of symptomatic therapy in diffuse NPSLE.

Transcriptomic and proteomic profiling reveals distinct pathogenic features of peripheral non-classical monocytes in systemic lupus erythematosus.

Peripheral blood monocytes propagate inflammation in systemic lupus erythematosus (SLE). Three major populations of monocytes have been recognized namely classical (CM), intermediate (IM) and non-classical monocytes (NCM). Herein, we performed a comprehensive transcriptomic, proteomic and functional characterization of the three peripheral monocytic subsets from active SLE patients and healthy individuals. Our data demonstrate extensive molecular disruptions in circulating SLE NCM, characterized by enhanced inflammatory features such as deregulated DNA repair, cell cycle and heightened IFN signaling combined with differentiation and developmental cues. Enhanced DNA damage, elevated expression of p53, G0 arrest of cell cycle and increased autophagy stress the differentiation potential of NCM in SLE. This immunogenic profile is associated with an activated macrophage phenotype of NCM exhibiting M1 characteristics in the circulation, fueling the inflammatory response. Together, these findings identify circulating SLE NCM as a pathogenic cell type in the disease that could represent an additional therapeutic target.

A network-based approach reveals long non-coding RNAs associated with disease activity in lupus nephritis: key pathways for flare and potential biomarkers to be used as liquid biopsies.

Objective: A blood-based biomarker is needed to assess lupus nephritis (LN) disease activity, minimizing the need for invasive kidney biopsies. Long non-coding RNAs (lncRNAs) are known to regulate gene expression, appear to be stable in human plasma, and can serve as non-invasive biomarkers. Methods: Transcriptomic data of whole blood samples from 74 LN patients and 20 healthy subjects (HC) were analyzed to identify differentially expressed (DE) lncRNAs associated with quiescent disease and flares. Weighted gene co-expression network analysis (WGCNA) was performed to uncover lncRNAs with a central role (hub lncRNAs) in regulating key biological processes that drive LN disease activity. The association of hub lncRNAs with disease activity was validated using RT-qPCR on an independent cohort of 15 LN patients and 9 HC. cis- and trans-targets of validated lncRNAs were explored in silico to examine potential mechanisms of their action. Results: There were 444 DE lncRNAs associated with quiescent disease and 6 DE lncRNAs associated with flares (FDR <0.05). WGCNA highlighted IFN signaling and B-cell activity/adaptive immunity as the most significant processes contributing to nephritis activity. Four disease-activity-associated lncRNAs, namely, NRIR, KLHDC7B-DT, MIR600HG, and FAM30A, were detected as hub genes and validated in an independent cohort. NRIR and KLHDC7B-DT emerged as potential key regulators of IFN-mediated processes. Network analysis suggests that FAM30A and MIR600HG are likely to play a central role in the regulation of B-cells in LN through cis-regulation effects and a competing endogenous RNA mechanism affecting immunoglobulin gene expression and the IFN-λ pathway. Conclusions: The expression of lncRNAs NRIR, KLHDC7B-DT, FAM30A, and MIR600HG were associated with disease activity and could be further explored as blood-based biomarkers and potential liquid biopsy on LN.

Down-regulation of human long non-coding RNA LINC01187 is associated with nephropathies.

Chronic kidney diseases affect a substantial percentage of the adult population worldwide. This observation emphasizes the need for novel insights into the molecular mechanisms that control the onset and progression of renal diseases. Recent advances in genomics have uncovered a previously unanticipated link between the non-coding genome and human kidney diseases. Here we screened and analysed long non-coding RNAs (lncRNAs) previously identified in mouse kidneys by genome-wide transcriptomic analysis, for conservation in humans and differential expression in renal tissue from healthy and diseased individuals. Our data suggest that LINC01187 is strongly down-regulated in human kidney tissues of patients with diabetic nephropathy and rapidly progressive glomerulonephritis, as well as in murine models of kidney diseases, including unilateral ureteral obstruction, nephrotoxic serum-induced glomerulonephritis and ischemia/reperfusion. Interestingly, LINC01187 overexpression in human kidney cells in vitro inhibits cell death indicating an anti-apoptotic function. Collectively, these data suggest a negative association of LINC01187 expression with renal diseases implying a potential protective role.

Return of the Tbx5; lineage-tracing reveals ventricular cardiomyocyte-like precursors in the injured adult mammalian heart.

The single curative measure for heart failure patients is a heart transplantation, which is limited due to a shortage of donors, the need for immunosuppression and economic costs. Therefore, there is an urgent unmet need for identifying cell populations capable of cardiac regeneration that we will be able to trace and monitor. Injury to the adult mammalian cardiac muscle, often leads to a heart attack through the irreversible loss of a large number of cardiomyocytes, due to an idle regenerative capability. Recent reports in zebrafish indicate that Tbx5a is a vital transcription factor for cardiomyocyte regeneration. Preclinical data underscore the cardioprotective role of Tbx5 upon heart failure. Data from our earlier murine developmental studies have identified a prominent unipotent Tbx5-expressing embryonic cardiac precursor cell population able to form cardiomyocytes, in vivo, in vitro and ex vivo. Using a developmental approach to an adult heart injury model and by employing a lineage-tracing mouse model as well as the use of single-cell RNA-seq technology, we identify a Tbx5-expressing ventricular cardiomyocyte-like precursor population, in the injured adult mammalian heart. The transcriptional profile of that precursor cell population is closer to that of neonatal than embryonic cardiomyocyte precursors. Tbx5, a cardinal cardiac development transcription factor, lies in the center of a ventricular adult precursor cell population, which seems to be affected by neurohormonal spatiotemporal cues. The identification of a Tbx5-specific cardiomyocyte precursor-like cell population, which is capable of dedifferentiating and potentially deploying a cardiomyocyte regenerative program, provides a clear target cell population for translationally-relevant heart interventional studies.

Microglia activation in the presence of intact blood-brain barrier and disruption of hippocampal neurogenesis via IL-6 and IL-18 mediate early diffuse neuropsychiatric lupus.

INTRODUCTION: Inflammatory mediators are detected in the cerebrospinal fluid of systemic lupus erythematosus patients with central nervous system involvement (NPSLE), yet the underlying cellular and molecular mechanisms leading to neuropsychiatric disease remain elusive. METHODS: We performed a comprehensive phenotyping of NZB/W-F1 lupus-prone mice including tests for depression, anxiety and cognition. Immunofluorescence, flow cytometry, RNA-sequencing, qPCR, cytokine quantification and blood-brain barrier (BBB) permeability assays were applied in hippocampal tissue obtained in both prenephritic (3-month-old) and nephritic (6-month-old) lupus mice and matched control strains. Healthy adult hippocampal neural stem cells (hiNSCs) were exposed ex vivo to exogenous inflammatory cytokines to assess their effects on proliferation and apoptosis. RESULTS: At the prenephritic stage, BBB is intact yet mice exhibit hippocampus-related behavioural deficits recapitulating the human diffuse neuropsychiatric disease. This phenotype is accounted by disrupted hippocampal neurogenesis with hiNSCs exhibiting increased proliferation combined with decreased differentiation and increased apoptosis in combination with microglia activation and increased secretion of proinflammatory cytokines and chemokines. Among these cytokines, IL-6 and IL-18 directly induce apoptosis of adult hiNSCs ex vivo. During the nephritic stage, BBB becomes disrupted which facilitates immune components of peripheral blood, particularly B-cells, to penetrate into the hippocampus further augmenting inflammation with locally increased levels of IL-6, IL-12, IL-18 and IL-23. Of note, an interferon gene signature was observed only at nephritic-stage. CONCLUSION: An intact BBB with microglial activation disrupting the formation of new neurons within the hippocampus represent early events in NPSLE. Disturbances of the BBB and interferon signature are evident later in the course of the disease.

Antiviral Innate Immune Responses in Autoimmunity: Receptors, Pathways, and Therapeutic Targeting.

Innate immune receptors sense nucleic acids derived from viral pathogens or self-constituents and initiate an immune response, which involves, among other things, the secretion of cytokines including interferon (IFN) and the activation of IFN-stimulated genes (ISGs). This robust and well-coordinated immune response is mediated by the innate immune cells and is critical to preserving and restoring homeostasis. Like an antiviral response, during an autoimmune disease, aberrations of immune tolerance promote inflammatory responses to self-components, such as nucleic acids and immune complexes (ICs), leading to the secretion of cytokines, inflammation, and tissue damage. The aberrant immune response within the inflammatory milieu of the autoimmune diseases may lead to defective viral responses, predispose to autoimmunity, or precipitate a flare of an existing autoimmune disease. Herein, we review the literature on the crosstalk between innate antiviral immune responses and autoimmune responses and discuss the pitfalls and challenges regarding the therapeutic targeting of the mechanisms involved.

ATR-mediated DNA damage responses underlie aberrant B cell activity in systemic lupus erythematosus.

B cells orchestrate autoimmune responses in patients with systemic lupus erythematosus (SLE), but broad-based B cell-directed therapies show only modest efficacy while blunting humoral immune responses to vaccines and inducing immunosuppression. Development of more effective therapies targeting pathogenic clones is a currently unmet need. Here, we demonstrate enhanced activation of the ATR/Chk1 pathway of the DNA damage response (DDR) in B cells of patients with active SLE disease. Treatment of B cells with type I IFN, a key driver of immunity in SLE, induced expression of ATR via binding of interferon regulatory factor 1 to its gene promoter. Pharmacologic targeting of ATR in B cells, via a specific inhibitor (VE-822), attenuated their immunogenic profile, including proinflammatory cytokine secretion, plasmablast formation, and antibody production. Together, these findings identify the ATR-mediated DDR axis as the orchestrator of the type I IFN-mediated B cell responses in SLE and as a potential novel therapeutic target.

Cross-species transcriptome analysis for early detection and specific therapeutic targeting of human lupus nephritis.

OBJECTIVES: Patients with lupus nephritis (LN) are in urgent need for early diagnosis and therapeutic interventions targeting aberrant molecular pathways enriched in affected kidneys. METHODS: We used mRNA-sequencing in effector (spleen) and target (kidneys, brain) tissues from lupus and control mice at sequential time points, and in the blood from 367 individuals (261 systemic lupus erythematosus (SLE) patients and 106 healthy individuals). Comparative cross-tissue and cross-species analyses were performed. The human dataset was split into training and validation sets and machine learning was applied to build LN predictive models. RESULTS: In murine SLE, we defined a kidney-specific molecular signature, as well as a molecular signature that underlies transition from preclinical to overt disease and encompasses pathways linked to metabolism, innate immune system and neutrophil degranulation. The murine kidney transcriptome partially mirrors the blood transcriptome of patients with LN with 11 key transcription factors regulating the cross-species active LN molecular signature. Integrated protein-to-protein interaction and drug prediction analyses identified the kinases TRRAP, AKT2, CDK16 and SCYL1 as putative targets of these factors and capable of reversing the LN signature. Using murine kidney-specific genes as disease predictors and machine-learning training of the human RNA-sequencing dataset, we developed and validated a peripheral blood-based algorithm that discriminates LN patients from normal individuals (based on 18 genes) and non-LN SLE patients (based on 20 genes) with excellent sensitivity and specificity (area under the curve range from 0.80 to 0.99). CONCLUSIONS: Machine-learning analysis of a large whole blood RNA-sequencing dataset of SLE patients using human orthologs of mouse kidney-specific genes can be used for early, non-invasive diagnosis and therapeutic targeting of LN. The kidney-specific gene predictors may facilitate prevention and early intervention trials.

The Neurodevelopmental Disorders Associated Gene Rnf113a Regulates Survival and Differentiation Properties of Neural Stem Cells.

RNF113A (Ring Finger Protein 113A) is genetically associated with autism spectrum disorders and X-linked trichothiodystrophy (TTD) syndrome. Loss-of-function mutations in human RNF113A are causally linked to TTD, which is characterized by abnormal development of the central nervous system (CNS) and mental retardation. How the loss of RNF113A activity affects brain development is not known. Here we identify Rnf113a1 as a critical regulator of cell death and neurogenesis during mouse brain development. Rnf113a1 gene exhibits widespread expression in the embryonic CNS. Knockdown studies in embryonic cortical neural stem/progenitor cells (NSCs) and the mouse cortex suggest that Rnf113a1 controls the survival, proliferation, and differentiation properties of progenitor cells. Importantly, Rnf113a1 deficiency triggers cell apoptosis via a combined action on essential regulators of cell survival, including p53, Nupr1, and Rad51. Collectively, these observations establish Rnf113a1 as a regulatory factor in CNS development and provide insights into its role in neurodevelopmental defects associated with TTD and autism.

Nuclear receptor NR5A2 negatively regulates cell proliferation and tumor growth in nervous system malignancies.

Nervous system malignancies are characterized by rapid progression and poor survival rates. These clinical observations underscore the need for novel therapeutic insights and pharmacological targets. To this end, here, we identify the orphan nuclear receptor NR5A2/LRH1 as a negative regulator of cancer cell proliferation and promising pharmacological target for nervous system-related tumors. In particular, clinical data from publicly available databases suggest that high expression levels of NR5A2 are associated with favorable prognosis in patients with glioblastoma and neuroblastoma tumors. Consistently, we experimentally show that NR5A2 is sufficient to strongly suppress proliferation of both human and mouse glioblastoma and neuroblastoma cells without inducing apoptosis. Moreover, short hairpin RNA-mediated knockdown of the basal expression levels of NR5A2 in glioblastoma cells promotes their cell cycle progression. The antiproliferative effect of NR5A2 is mediated by the transcriptional induction of negative regulators of the cell cycle, CDKN1A (encoding for p21cip1), CDKN1B (encoding for p27kip1) and Prox1 Interestingly, two well-established agonists of NR5A2, dilauroyl phosphatidylcholine (DLPC) and diundecanoyl phosphatidylcholine, are able to mimic the antiproliferative action of NR5A2 in human glioblastoma cells via the induction of the same critical genes. Most importantly, treatment with DLPC inhibits glioblastoma tumor growth in vivo in heterotopic and orthotopic xenograft mouse models. These data indicate a tumor suppressor role of NR5A2 in the nervous system and render this nuclear receptor a potential pharmacological target for the treatment of nervous tissue-related tumors.

Patrolling human SLE haematopoietic progenitors demonstrate enhanced extramedullary colonisation; implications for peripheral tissue injury.

Systemic lupus erythematosus (SLE) is an autoimmune disease where bone-marrow-derived haematopoietic cells have a key role in its pathogenesis with accumulating evidence suggesting an aberrant function of haematopoietic stem/progenitor cells (HSPCs). We examined whether patrolling HSPCs differ from bone-marrow HSPCs both in SLE and healthy individuals, and how they participate in peripheral tissue injury. By employing next-generation RNA sequencing, the transcriptomes of CD34+ HSPCs deriving from the bone marrow and those patrolling the bloodstream of both healthy and individuals with SLE were compared. Patrolling SLE and Healthy human HSPC kinetics were examined through their inoculation into humanised mice. Patrolling and bone-marrow HSPCs have distinct molecular signatures, while patrolling SLE HSPCs showed an enhanced extramedullary gene expression profile. Non-mobilised, SLE-derived circulating HSPCs demonstrated altered homing capacities. Xenotransplantation of circulating HSPCs in humanised mice showed that human peripheral blood HSPCs possess the ability for extramedullary organ colonisation to the kidneys. Circulating and bone marrow-derived HSPCs are distinct in steady and diseased states. Patrolling SLE CD34+ HSPCs are able to home at extramedullary sites such as the spleen and kidneys, potentially participating in peripheral tissue injury.

DNA Damage Response in the Adaptive Arm of the Immune System: Implications for Autoimmunity.

In complex environments, cells have developed molecular responses to confront threats against the genome and achieve the maintenance of genomic stability assuring the transfer of undamaged DNA to their progeny. DNA damage response (DDR) mechanisms may be activated upon genotoxic or environmental agents, such as cytotoxic drugs or ultraviolet (UV) light, and during physiological processes requiring DNA transactions, to restore DNA alterations that may cause cellular malfunction and affect viability. In addition to the DDR, multicellular organisms have evolved specialized immune cells to respond and defend against infections. Both adaptive and innate immune cells are subjected to DDR processes, either as a prerequisite to the immune response, or as a result of random endogenous and exogenous insults. Aberrant DDR activities have been extensively studied in the immune cells of the innate arm, but not in adaptive immune cells. Here, we discuss how the aberrant DDR may lead to autoimmunity, with emphasis on the adaptive immune cells and the potential of therapeutic targeting.