StarGazer: A Hybrid Intelligence Platform for Drug Target Prioritization and Digital Drug Repositioning Using Streamlit.

Target prioritization is essential for drug discovery and repositioning. Applying computational methods to analyze and process multi-omics data to find new drug targets is a practical approach for achieving this. Despite an increasing number of methods for generating datasets such as genomics, phenomics, and proteomics, attempts to integrate and mine such datasets remain limited in scope. Developing hybrid intelligence solutions that combine human intelligence in the scientific domain and disease biology with the ability to mine multiple databases simultaneously may help augment drug target discovery and identify novel drug-indication associations. We believe that integrating different data sources using a singular numerical scoring system in a hybrid intelligent framework could help to bridge these different omics layers and facilitate rapid drug target prioritization for studies in drug discovery, development or repositioning. Herein, we describe our prototype of the StarGazer pipeline which combines multi-source, multi-omics data with a novel target prioritization scoring system in an interactive Python-based Streamlit dashboard. StarGazer displays target prioritization scores for genes associated with 1844 phenotypic traits, and is available via https://github.com/AstraZeneca/StarGazer.

Wiskott-Aldrich syndrome protein restricts cGAS/STING activation by dsDNA immune complexes.

Dysregulated sensing of self-nucleic acid is a leading cause of autoimmunity in multifactorial and monogenic diseases. Mutations in Wiskott-Aldrich syndrome protein (WASp), a key regulator of cytoskeletal dynamics in immune cells, cause autoimmune manifestations and increased production of type I IFNs by innate cells. Here we show that immune complexes of self-DNA and autoantibodies (DNA-ICs) contribute to elevated IFN levels via activation of the cGAS/STING pathway of cytosolic sensing. Mechanistically, lack of endosomal F-actin nucleation by WASp caused a delay in endolysosomal maturation and prolonged the transit time of ingested DNA-ICs. Stalling in maturation-defective organelles facilitated leakage of DNA-ICs into the cytosol, promoting activation of the TBK1/STING pathway. Genetic deletion of STING and STING and cGAS chemical inhibitors abolished IFN production and rescued systemic activation of IFN-stimulated genes in vivo. These data unveil the contribution of cytosolic self-nucleic acid sensing in WAS and underscore the importance of WASp-mediated endosomal actin remodeling in preventing innate activation.

Frontline Science: Kindlin-3 is essential for patrolling and phagocytosis functions of nonclassical monocytes during metastatic cancer surveillance.

Nonclassical monocytes maintain vascular homeostasis by patrolling the vascular endothelium, responding to inflammatory signals, and scavenging cellular debris. Nonclassical monocytes also prevent metastatic tumor cells from seeding new tissues, but whether the patrolling function of nonclassical monocytes is required for this process is unknown. To answer this question, we utilized an inducible-knockout mouse that exhibits loss of the integrin-adaptor protein Kindlin-3 specifically in nonclassical monocytes. We show that Kindlin-3-deficient nonclassical monocytes are unable to patrol the vascular endothelium in either the lungs or periphery. We also find that Kindlin-3-deficient nonclassical monocytes cannot firmly adhere to, and instead "slip" along, the vascular endothelium. Loss of patrolling activity by nonclassical monocytes was phenocopied by ablation of LFA-1, an integrin-binding partner of Kindlin-3. When B16F10 murine melanoma tumor cells were introduced into Kindlin-3-deficient mice, nonclassical monocytes showed defective patrolling towards tumor cells and failure to ingest tumor particles in vivo. Consequently, we observed a significant, 4-fold increase in lung tumor metastases in mice possessing Kindlin-3-deficient nonclassical monocytes. Thus, we conclude that the patrolling function of nonclassical monocytes is mediated by Kindlin-3 and essential for these cells to maintain vascular endothelial homeostasis and prevent tumor metastasis to the lung.

Engineering Caveolae-Targeted Lipid Nanoparticles To Deliver mRNA to the Lungs.

Efficacious use of therapeutic gene delivery via nanoparticles is hampered by the challenges associated with targeted delivery to tissues of interest. Systemic administration of lipid nanoparticle (LNP)-encapsulated mRNA leads to a protein expressed predominantly in the liver and spleen. Here, LNP encapsulating mRNA was covalently conjugated to an antibody, specifically binding plasmalemma vesicle-associated protein (PV1) as a means to target lung tissue. Systemic administration of PV1-targeted LNPs demonstrated significantly increased delivery of mRNA to the lungs and a 40-fold improvement in protein expression in the lungs, compared with control LNPs. We also investigated the effect of LNP size to determine optimal tissue distribution and transfection. Larger-size PV1-targeted LNPs not only have the elasticity to target the PV1 expressed in the caveolae but also enable robust mRNA expression in the lungs. Targeted delivery of mRNA to the lungs is a promising approach in the treatment of lung diseases.

Antibody Fragment F(ab')2 Targeting Caveolae-Associated Protein PV1 for Selective Kidney Targeting and Retention.

Targeted strategies to deliver and retain drugs to kidneys are needed to improve drug accumulation and efficacy in a myriad of kidney diseases. These drug delivery systems show potential for improving the therapeutic windows of drugs acting in the kidney. Biodistribution of antibody-based therapeutics in vivo is governed by several factors including binding affinity, size, and valency. Investigations of how the biophysical and biochemical properties of biologics enable them to overcome biological barriers and reach kidneys are therefore of interest. Although renal accumulation of antibody fragments in cancer diagnostics and treatment has been observed, reports on effective delivery of antibody fragments to the kidneys remain scarce. Previously, we demonstrated that targeting plasmalemma vesicle-associated protein (PV1), a caveolae-associated protein, can promote accumulation of antibodies in both the lungs and the kidneys. Here, by fine-tuning the binding affinity of an antibody toward PV1, we observe that the anti-PV1 antibody with reduced binding affinity lost the capability for kidney targeting while retaining the lung targeting activity, suggesting that binding affinity is a critical factor for kidney targeting of the anti-PV1 antibody. We next use the antibody fragment F(ab')2 targeting PV1 to assess the dual effects of rapid kidney filtration and PV1 targeting on kidney-selective targeting. Ex vivo fluorescence imaging results demonstrated that after rapidly accumulating in kidneys at 4 h, PV1-targeted F(ab')2 was continually retained in the kidney at 24 h, whereas the isotype control F(ab')2 underwent urinary elimination with significantly reduced signaling in the kidney. Confocal imaging studies confirmed the localization of PV1-targeted F(ab')2 in the kidney. In addition, the monovalent antibody fragment (Fab-C4) lost the capability for kidney homing, indicating that the binding avidity of anti-PV1 F(ab')2 is important for kidney targeting. Our findings suggest that PV1-targeted F(ab')2 might be useful as a drug carrier for renal targeting and highlight the importance of affinity optimization for tissue targeting antibodies.

Low-density granulocytes activate T cells and demonstrate a non-suppressive role in systemic lupus erythematosus.

OBJECTIVES: The presence of proinflammatory low-density granulocytes (LDG) has been demonstrated in autoimmune and infectious diseases. Recently, regulatory neutrophilic polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) were identified in systemic lupus erythematosus (SLE). Because LDG and PMN-MDSC share a similar phenotype with contrasting functional effects, we explored these cells in a cohort of patients with SLE. METHODS: LDG and normal-density granulocytes (NDG) were isolated from fresh blood of healthy donors (HD) and patients with SLE. Associations between LDG and clinical manifestations were analysed. Multicolor flow cytometry and confocal imaging were performed to immunophenotype the cells. The ability of LDG and NDG to suppress T cell function and induce cytokine production was quantified. RESULTS: LDG prevalence was elevated in SLE versus HD, associated with the interferon (IFN) 21-gene signature and disease activity. Also, the LDG-to-lymphocyte ratio associated better with SLE disease activity index than neutrophil-to-lymphocyte ratio. SLE LDG exhibited significantly heightened surface expression of various activation markers and also of lectin-like oxidised low-density lipoprotein receptor-1, previously described to be associated with PMN-MDSC. Supernatants from SLE LDG did not restrict HD CD4+ T cell proliferation in an arginase-dependent manner, suggesting LDG are not immunosuppressive. SLE LDG supernatants induced proinflammatory cytokine production (IFN gamma, tumour necrosis factor alpha and lymphotoxin alpha) from CD4+ T cells. CONCLUSIONS: Based on our results, SLE LDG display an activated phenotype, exert proinflammatory effects on T cells and do not exhibit MDSC function. These results support the concept that LDG represent a distinct proinflammatory subset in SLE with pathogenic potential, at least in part, through their ability to activate type 1 helper responses.

Targeted drug delivery via caveolae-associated protein PV1 improves lung fibrosis.

Systemic administration of bio-therapeutics can result in only a fraction of drug reaching targeted tissues, with the majority of drug being distributed to tissues irrelevant to the drug's site of action. Targeted delivery to specific organs may allow for greater accumulation, better efficacy, and improved safety. We investigated how targeting plasmalemma vesicle-associated protein (PV1), a protein found in the endothelial caveolae of lungs and kidneys, can promote accumulation in these organs. Using ex vivo fluorescence imaging, we show that intravenously administered αPV1 antibodies localize to mouse lungs and kidneys. In a bleomycin-induced idiopathic pulmonary fibrosis (IPF) mouse model, αPV1 conjugated to Prostaglandin E2 (PGE2), a known anti-fibrotic agent, significantly reduced collagen content and fibrosis whereas a non-targeted PGE2 antibody conjugate failed to slow fibrosis progression. Our results demonstrate that PV1 targeting can be utilized to deliver therapeutics to lungs and this approach is potentially applicable for various lung diseases.

Characterisation of anifrolumab, a fully human anti-interferon receptor antagonist antibody for the treatment of systemic lupus erythematosus.

OBJECTIVE: We investigated the mechanistic and pharmacological properties of anifrolumab, a fully human, effector-null, anti-type I interferon (IFN) alpha receptor 1 (IFNAR1) monoclonal antibody in development for SLE. METHODS: IFNAR1 surface expression and internalisation on human monocytes before and after exposure to anifrolumab were assessed using confocal microscopy and flow cytometry. The effects of anifrolumab on type I IFN pathway activation were assessed using signal transducer and activator of transcription 1 (STAT1) phosphorylation, IFN-stimulated response element-luciferase reporter cell assays and type I IFN gene signature induction. The ability of anifrolumab to inhibit plasmacytoid dendritic cell (pDC) function and plasma cell differentiation was assessed by flow cytometry and ELISA. Effector-null properties of anifrolumab were assessed in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays with B cells. RESULTS: Anifrolumab reduced cell surface IFNAR1 by eliciting IFNAR1 internalisation. Anifrolumab blocked type I IFN-dependent STAT1 phosphorylation and IFN-dependent signalling induced by recombinant and pDC-derived type I IFNs and serum of patients with SLE. Anifrolumab suppressed type I IFN production by blocking the type I IFN autoamplification loop and inhibited proinflammatory cytokine induction and the upregulation of costimulatory molecules on stimulated pDCs. Blockade of IFNAR1 suppressed plasma cell differentiation in pDC/B cell co-cultures. Anifrolumab did not exhibit CDC or ADCC activity. CONCLUSIONS: Anifrolumab potently inhibits type I IFN-dependent signalling, including the type I IFN autoamplification loop, and is a promising therapeutic for patients with SLE and other diseases that exhibit chronic dysfunctional type I IFN signalling.

Galectin-9 inhibits TLR7-mediated autoimmunity in murine lupus models.

Uncontrolled secretion of type I IFN, as the result of endosomal TLR (i.e., TLR7 and TLR9) signaling in plasmacytoid DCs (pDCs), and abnormal production of autoantibodies by B cells are critical for systemic lupus erythematosus (SLE) pathogenesis. The importance of galectin-9 (Gal-9) in regulating various autoimmune diseases, including lupus, has been demonstrated. However, the precise mechanism by which Gal-9 mediates this effect remains unclear. Here, using spontaneous murine models of lupus (i.e., BXSB/MpJ and NZB/W F1 mice), we demonstrate that administration of Gal-9 results in reduced TLR7-mediated autoimmune manifestations. While investigating the mechanism underlying this phenomenon, we observed that Gal-9 inhibits the phenotypic maturation of pDCs and B cells and abrogates their ability to mount cytokine responses to TLR7/TLR9 ligands. Importantly, immunocomplex-mediated (IC-mediated) and neutrophil extracellular trap-mediated (NET-mediated) pDC activation was inhibited by Gal-9. Additionally, the mTOR/p70S6K pathway, which is recruited by both pDCs and B cells for TLR-mediated IFN secretion and autoantibody generation, respectively, was attenuated. Gal-9 was found to exert its inhibitory effect on both the cells by interacting with CD44.

Deleting an Nr4a1 Super-Enhancer Subdomain Ablates Ly6Clow Monocytes while Preserving Macrophage Gene Function.

Mononuclear phagocytes are a heterogeneous family that occupy all tissues and assume numerous roles to support tissue function and systemic homeostasis. Our ability to dissect the roles of individual subsets is limited by a lack of technologies that ablate gene function within specific mononuclear phagocyte sub-populations. Using Nr4a1-dependent Ly6Clow monocytes, we present a proof-of-principle approach that addresses these limitations. Combining ChIP-seq and molecular approaches we identified a single, conserved, sub-domain within the Nr4a1 enhancer that was essential for Ly6Clow monocyte development. Mice lacking this enhancer lacked Ly6Clow monocytes but retained Nr4a1 gene expression in macrophages during steady state and in response to LPS. Because Nr4a1 regulates inflammatory gene expression and differentiation of Ly6Clow monocytes, decoupling these processes allows Ly6Clow monocytes to be studied independently.

In vivo Imaging of Tumor and Immune Cell Interactions in the Lung.

Immunotherapy has demonstrated great therapeutic potential by activating the immune system to fight cancer. However, little is known about the specific dynamics of interactions that occur between tumor and immune cells. In this protocol we describe a novel method to visualize the interaction of tumor and immune cells in the lung of live mice, which can be applied to other organs. In this protocol fluorescent-labeled tumor cells are transferred to recipient mice expressing fluorescently tagged immune cells. Tumor-immune cell interactions in the lung are then imaged by confocal or two photon microscopy. Analysis of tumor interactions with immune cells using this protocol should aid in a better understanding of the importance of these interactions and their role in developing immunotherapies.

Quantification of Tumor Material Uptake.

Extracellular tumor material including exosomes, microvesicles and apoptotic tumor debris may help cancers invade new organs. Enhancing the removal of extracellular tumor material by immune cells represents a novel immunotherapy approach for preventing cancer metastasis. This protocol quantifies the uptake and removal of extracellular tumor material from circulation and tissues by immune cells. In this assay fluorescent tumor cells are transferred into mice, and then immune cells are quantified by either flow cytometry or imaging cytometry for their uptake of tumor material.

Transcription factor Nr4a1 couples sympathetic and inflammatory cues in CNS-recruited macrophages to limit neuroinflammation.

The molecular mechanisms that link the sympathetic stress response and inflammation remain obscure. Here we found that the transcription factor Nr4a1 regulated the production of norepinephrine (NE) in macrophages and thereby limited experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Lack of Nr4a1 in myeloid cells led to enhanced NE production, accelerated infiltration of leukocytes into the central nervous system (CNS) and disease exacerbation in vivo. In contrast, myeloid-specific deletion of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, protected mice against EAE. Furthermore, we found that Nr4a1 repressed autocrine NE production in macrophages by recruiting the corepressor CoREST to the Th promoter. Our data reveal a new role for macrophages in neuroinflammation and identify Nr4a1 as a key regulator of catecholamine production by macrophages.

Patrolling monocytes control tumor metastasis to the lung.

The immune system plays an important role in regulating tumor growth and metastasis. Classical monocytes promote tumorigenesis and cancer metastasis, but how nonclassical "patrolling" monocytes (PMo) interact with tumors is unknown. Here we show that PMo are enriched in the microvasculature of the lung and reduce tumor metastasis to lung in multiple mouse metastatic tumor models. Nr4a1-deficient mice, which specifically lack PMo, showed increased lung metastasis in vivo. Transfer of Nr4a1-proficient PMo into Nr4a1-deficient mice prevented tumor invasion in the lung. PMo established early interactions with metastasizing tumor cells, scavenged tumor material from the lung vasculature, and promoted natural killer cell recruitment and activation. Thus, PMo contribute to cancer immunosurveillance and may be targets for cancer immunotherapy.

The transcription factor NR4A1 is essential for the development of a novel macrophage subset in the thymus.

Tissue macrophages function to maintain homeostasis and regulate immune responses. While tissue macrophages derive from one of a small number of progenitor programs, the transcriptional requirements for site-specific macrophage subset development are more complex. We have identified a new tissue macrophage subset in the thymus and have discovered that its development is dependent on transcription factor NR4A1. Functionally, we find that NR4A1-dependent macrophages are critically important for clearance of apoptotic thymocytes. These macrophages are largely reduced or absent in mice lacking NR4A1, and Nr4a1-deficient mice have impaired thymocyte engulfment and clearance. Thus, NR4A1 functions as a master transcription factor for the development of this novel thymus-specific macrophage subset.

Nonclassical patrolling monocyte function in the vasculature.

Nonclassical patrolling monocytes are characterized by their unique ability to actively patrol the vascular endothelium under homeostatic and inflammatory conditions. Patrolling monocyte subsets (CX3CR1(high)Ly6C(-) in mouse and CX3CR1(high)CD14(dim)CD16(+) in humans) are distinct from the classical monocyte subsets (CCR2(high)Ly6C(+) in mouse and CCR2(high)CD14(+)CD16(-) in humans) and exhibit unique functions in the vasculature and inflammatory disease. Patrolling monocytes function in several disease settings to remove damaged cells and debris from the vasculature and have been associated with wound healing and the resolution of inflammation in damaged tissues. This review highlights the unique functions of these patrolling monocytes in the vasculature and during inflammation.

NR4A nuclear receptors in immunity and atherosclerosis.

PURPOSE OF REVIEW: To understand chronic inflammatory diseases such as atherosclerosis, we require in-depth knowledge on immune-cell differentiation, function of specific immune-cell subsets and endothelial cell-mediated extravasation. In this review, we summarize a number of very recent observations on the pivotal function of NR4A nuclear receptors in immunity and atherosclerosis. RECENT FINDINGS: NR4A nuclear receptors are involved in negative selection of thymocytes, Treg differentiation and the development of Ly6C monocytes. Nur77 and Nurr1 attenuate atherosclerosis in mice whereas NOR-1 aggravates vascular lesion formation. SUMMARY: These exciting, novel insights on the function of NR4A nuclear receptors in immunity, vascular cells and atherosclerosis will initiate a plethora of studies to understand the underlying molecular mechanisms, which will culminate in the identification of novel NR4A targets to modulate chronic inflammatory disease.

Increased cholesterol content in gammadelta (γδ) T lymphocytes differentially regulates their activation.

Gammadelta (γδ) T lymphocytes respond quickly upon antigen encounter to produce a cytokine response. In this study, we sought to understand how functions of γδ T cells are differentially regulated compared to αß T cells. We found that cholesterol, an integral component of the plasma membrane and a regulator of TCR signaling, is increased in γδ T cells compared to αß T cells, and modulates their function. Higher levels of activation markers, and increased lipid raft content in γδ cells suggest that γδ T cells are more activated. Cholesterol depletion effectively decreased lipid raft formation and activation of γδ T cells, indicating that increased cholesterol content contributes to the hyper-activated phenotype of γδ T cells, possibly through enhanced clustering of TCR signals in lipid rafts. TCR stimulation assays and western blotting revealed that instead of a lower TCR threshold, enhanced TCR signaling through ERK1/2 activation is likely the cause for high cholesterol-induced rapid activation and proliferation in γδ T cells. Our data indicate that cholesterol metabolism is differentially regulated in γδ T cells. The high intracellular cholesterol content leads to enhanced TCR signaling and increases activation and proliferation of γδ T cells.

Nr4a1-dependent Ly6C(low) monocytes monitor endothelial cells and orchestrate their disposal.

The functions of Nr4a1-dependent Ly6C(low) monocytes remain enigmatic. We show that they are enriched within capillaries and scavenge microparticles from their lumenal side in a steady state. In the kidney cortex, perturbation of homeostasis by a TLR7-dependent nucleic acid "danger" signal, which may signify viral infection or local cell death, triggers Gαi-dependent intravascular retention of Ly6C(low) monocytes by the endothelium. Then, monocytes recruit neutrophils in a TLR7-dependent manner to mediate focal necrosis of endothelial cells, whereas the monocytes remove cellular debris. Prevention of Ly6C(low) monocyte development, crawling, or retention in Nr4a1(-/-), Itgal(-/-), and Tlr7(host-/-BM+/+) and Cx3cr1(-/-) mice, respectively, abolished neutrophil recruitment and endothelial killing. Prevention of neutrophil recruitment in Tlr7(host+/+BM-/-) mice or by neutrophil depletion also abolished endothelial cell necrosis. Therefore, Ly6C(low) monocytes are intravascular housekeepers that orchestrate the necrosis by neutrophils of endothelial cells that signal a local threat sensed via TLR7 followed by the in situ phagocytosis of cellular debris.