IL-37 requires the receptors IL-18Rα and IL-1R8 (SIGIRR) to carry out its multifaceted anti-inflammatory program upon innate signal transduction.

Interleukin 37 (IL-37) and IL-1R8 (SIGIRR or TIR8) are anti-inflammatory orphan members of the IL-1 ligand family and IL-1 receptor family, respectively. Here we demonstrate formation and function of the endogenous ligand-receptor complex IL-37-IL-1R8-IL-18Rα. The tripartite complex assembled rapidly on the surface of peripheral blood mononuclear cells upon stimulation with lipopolysaccharide. Silencing of IL-1R8 or IL-18Rα impaired the anti-inflammatory activity of IL-37. Whereas mice with transgenic expression of IL-37 (IL-37tg mice) with intact IL-1R8 were protected from endotoxemia, IL-1R8-deficient IL-37tg mice were not. Proteomic and transcriptomic investigations revealed that IL-37 used IL-1R8 to harness the anti-inflammatory properties of the signaling molecules Mer, PTEN, STAT3 and p62(dok) and to inhibit the kinases Fyn and TAK1 and the transcription factor NF-κB, as well as mitogen-activated protein kinases. Furthermore, IL-37-IL-1R8 exerted a pseudo-starvational effect on the metabolic checkpoint kinase mTOR. IL-37 thus bound to IL-18Rα and exploited IL-1R8 to activate a multifaceted intracellular anti-inflammatory program.

A single-cell transcriptome atlas of the adult human retina.

The retina is a specialized neural tissue that senses light and initiates image processing. Although the functional organization of specific retina cells has been well studied, the molecular profile of many cell types remains unclear in humans. To comprehensively profile the human retina, we performed single-cell RNA sequencing on 20,009 cells from three donors and compiled a reference transcriptome atlas. Using unsupervised clustering analysis, we identified 18 transcriptionally distinct cell populations representing all known neural retinal cells: rod photoreceptors, cone photoreceptors, Müller glia, bipolar cells, amacrine cells, retinal ganglion cells, horizontal cells, astrocytes, and microglia. Our data captured molecular profiles for healthy and putative early degenerating rod photoreceptors, and revealed the loss of MALAT1 expression with longer post-mortem time, which potentially suggested a novel role of MALAT1 in rod photoreceptor degeneration. We have demonstrated the use of this retina transcriptome atlas to benchmark pluripotent stem cell-derived cone photoreceptors and an adult Müller glia cell line. This work provides an important reference with unprecedented insights into the transcriptional landscape of human retinal cells, which is fundamental to understanding retinal biology and disease.

Renal participation of myeloperoxidase in antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis.

Myeloperoxidase (MPO) is an important neutrophil lysosomal enzyme, a major autoantigen, and a potential mediator of tissue injury in MPO-ANCA-associated vasculitis (MPO-AAV) and glomerulonephritis. Here we examined MPO deposition in kidney biopsies from 47 patients with MPO-AAV. Leukocyte accumulation and fibrin deposition consistent with cell-mediated immunity was a major feature. Tubulointerstitial macrophage, CD4+ and CD8+ T-cell, and neutrophil numbers correlated with low presenting eGFR. MPO was not detected in kidneys from patients with minimal change or thin basement membrane disease, but was prominent in glomerular, periglomerular, and tubulointerstitial regions in MPO-AAV. Extracellular MPO released from leukocytes was pronounced in all MPO-AAV patients. Similar numbers of neutrophils and macrophages expressed MPO in the kidneys, but colocalization studies identified neutrophils as the major source of extracellular MPO. Extraleukocyte MPO was prominent in neutrophil extracellular traps in the majority of patients; most of which had traps in half or more glomeruli. These traps were associated with more neutrophils and more MPO within glomeruli. Glomerular MPO-containing macrophages generated extracellular trap-like structures. MPO also localized to endothelial cells and podocytes. The presence of the most active glomerular lesions (both segmental necrosis and cellular crescents) correlated with intraglomerular CD4+ cells and MPO+ macrophages. Thus, cellular and extracellular MPO may cause glomerular and interstitial injury.

GILZ overexpression inhibits endothelial cell adhesive function through regulation of NF-κB and MAPK activity.

Glucocorticoid-induced leucine zipper (GILZ) is an anti-inflammatory protein first identified in T lymphocytes. We recently observed that GILZ is highly expressed in synovial endothelial cells in rheumatoid arthritis. However, the function of GILZ in endothelial cells is unknown. To investigate the actions of GILZ in this cell type, we induced GILZ expression in HUVECs via transient transfection. GILZ overexpression significantly reduced the capacity of TNF-stimulated HUVECs to support leukocyte rolling, adhesion, and transmigration. These effects were associated with decreased expression of E-selectin, ICAM-1, CCL2, CXCL8, and IL-6. Experiments in a human microvascular endothelial cell line demonstrated that TNF-inducible NF-κB activity was significantly inhibited by overexpression of GILZ. Exogenous GILZ inhibited TNF-induced NF-κB p65 DNA binding, although this occurred in the absence of an effect on p65 nuclear translocation, indicating that the mechanism of action of exogenous GILZ in endothelial cells differs from that reported in other cell types. GILZ overexpression also inhibited TNF-induced activation of p38, ERK, and JNK MAPKs, as well as increased expression of the MAPK inhibitory phosphatase, MKP-1. In contrast, silencing endogenous GILZ in glucocorticoid-treated HUVECs did not alter their capacity to support leukocyte interactions. These data demonstrate that exogenous GILZ exerts inhibitory effects on endothelial cell adhesive function via a novel pathway involving modulation of NF-κB p65 DNA binding and MAPK activity. Induction of GILZ expression in endothelial cells may represent a novel therapeutic modality with the potential to inhibit inflammatory leukocyte recruitment.

Trop2 regulates motility and lamellipodia formation in cultured fetal lung fibroblasts.

Proliferation and migration of fibroblasts are vital for fetal lung development. However, the regulatory mechanisms are poorly understood. We have previously shown that TROP2 gene expression is closely associated with fetal lung cell proliferation in vivo and that TROP2 knockdown decreases proliferation of fetal lung fibroblasts in culture. We hypothesized that the Trop2 protein also regulates the morphology and motility of fetal lung fibroblasts. Fibroblasts isolated from fetal rat lungs (gestational age embryonic day 19) adopted a myofibroblast-like morphology in culture. Trop2 protein was localized to lamellipodia. TROP2 siRNA significantly decreased: TROP2 mRNA levels by 77%, the proportion of cells containing Trop2 protein by 70%, and cell proliferation by 50%. TROP2 siRNA also decreased the degree of motility as determined by the number of gridlines that cells moved across (2.2 ± 0.2 vs. 3.2 ± 0.2; P < 0.001). TROP2 knockdown altered cell morphology, causing a notable absence of lamellipodia and abnormal localization of components of the cell migration apparatus, and it reduced phosphorylated ERK1 and ERK2 levels. In contrast, TROP2 overexpression significantly increased: TROP2 mRNA levels by 40-fold, cell proliferation by 40%, the proportion of cells that were motile by 20%, and the number of gridlines that cells moved across (2.1 ± 0.2 vs. 1.6 ± 0.1; P < 0.001). Our data suggest that Trop2 regulates cell proliferation and motility and that it does so by regulating the ERK pathway and several critical components of the cell migration apparatus.

The oncogene Trop2 regulates fetal lung cell proliferation.

The factors regulating growth of the developing lung are poorly understood, although the degree of fetal lung expansion is critical. The oncogene Trop2 (trophoblast antigen 2) is upregulated during accelerated fetal lung growth, and we hypothesized that it may regulate normal fetal lung growth. We investigated Trop2 expression in the fetal and neonatal sheep lung during accelerated and delayed lung growth induced by alterations in fetal lung expansion, as well as in response to glucocorticoids. Trop2 expression was measured using real-time PCR and localized spatially using in situ hybridization and immunofluorescence. During normal lung development, Trop2 expression was higher at 90 days gestational age (GA; 4.0 ± 0.8) than at 128 days GA (1.0 ± 0.1), decreased to 0.5 ± 0.1 at 142 days GA (full term ∼147 days GA), and was positively correlated to lung cell proliferation rates (r = 0.953, P < 0.005). Trop2 expression was regulated by fetal lung expansion, but not by glucocorticoids. It was increased nearly threefold by 36 h of increased fetal lung expansion (P < 0.05) and was reduced to ∼55% of control levels by reduced fetal lung expansion (P < 0.05). Trop2 expression was associated with lung cell proliferation during normal and altered lung growth, and the TROP2 protein colocalized with Ki-67-positive cells in the fetal lung. TROP2 was predominantly localized to fibroblasts and type II alveolar epithelial cells. Trop2 small interfering RNA decreased Trop2 expression by ∼75% in cultured fetal rat lung fibroblasts and decreased their proliferation by ∼50%. Cell viability was not affected. This study demonstrates that TROP2 regulates lung cell proliferation during development.

Drug discovery using induced pluripotent stem cell models of neurodegenerative and ocular diseases.

The revolution of induced pluripotent stem cell (iPSC) technology provides a platform for development of cell therapy, disease modeling and drug discovery. Recent technological advances now allow us to reprogram a patient's somatic cells into induced pluripotent stem cells (iPSCs). Together with methods to differentiate these iPSCs into disease-relevant cell types, we are now able to model disease in vitro using iPSCs. Importantly, this represents a robust in vitro platform using patient-specific cells, providing opportunity for personalized precision medicine. Here we provide a review of advances using iPSC for drug development, and discuss the potential and limitations of iPSCs for drug discovery in neurodegenerative and ocular diseases. Emerging technologies that can facilitate the search for new drugs by assessment using in vitro disease models will also be discussed, including organoid differentiation, organ-on-chip, direct reprogramming and humanized animal models.

Development of a pipeline for automated, high-throughput analysis of paraspeckle proteins reveals specific roles for importin α proteins.

We developed a large-scale, unbiased analysis method to measure how functional variations in importin (IMP) α2, IMPα4 and IMPα6 each influence PSPC1 and SFPQ nuclear accumulation and their localization to paraspeckles. This addresses the hypothesis that individual IMP protein activities determine cargo nuclear access to influence cell fate outcomes. We previously demonstrated that modulating IMPα2 levels alters paraspeckle protein 1 (PSPC1) nuclear accumulation and affects its localization into a subnuclear domain that affects RNA metabolism and cell survival, the paraspeckle. An automated, high throughput, image analysis pipeline with customisable outputs was created using Imaris software coupled with Python and R scripts; this allowed non-subjective identification of nuclear foci, nuclei and cells. HeLa cells transfected to express exogenous full-length and transport-deficient IMPs were examined using SFPQ and PSPC1 as paraspeckle markers. Thousands of cells and >100,000 nuclear foci were analysed in samples with modulated IMPα functionality. This analysis scale enabled discrimination of significant differences between samples where paraspeckles inherently display broad biological variability. The relative abundance of paraspeckle cargo protein(s) and individual IMPs each influenced nuclear foci numbers and size. This method provides a generalizable high throughput analysis platform for investigating how regulated nuclear protein transport controls cellular activities.

Activated Renal Dendritic Cells Cross Present Intrarenal Antigens After Ischemia-Reperfusion Injury.

BACKGROUND: The healthy kidney contains an extensive population of renal mononuclear phagocytes (RMPs), with substantial phenotypic and functional diversity. However, how this diverse population is affected by ischemia-reperfusion injury (IRI), an obligate part of renal transplantation, is not yet well understood. The aim of this study was to characterize the phenotypic and functional alterations in RMPs induced by IRI. METHODS: Renal mononuclear phagocytes were studied 24 and 72 hours after 30 minutes of renal ischemia or sham operation. Kidneys were digested and the phenotypes of renal leukocyte populations were analyzed via flow cytometry. Multiphoton microscopy was used to image renal dendritic cells (DCs) in vivo using CD11c reporter mice. The capacity of renal DCs to present antigen was examined by assessment of proliferation of ovalbumin-specific T cells in rat insulin promoter-membrane-bound ovalbumin transgenic mice after sham or IRI procedures. RESULTS: Ischemia-reperfusion injury induced influx of monocytes, DCs, macrophages, and neutrophils into the kidney. Classification of RMP subpopulations based on CD11b/CD11c expression demonstrated that the RMPs that increased in response to IRI were predominantly newly recruited monocyte-derived inflammatory DCs. In vivo multiphoton imaging of CD11c-EYFP mice revealed that intrarenal DCs exhibited increased number and activity of dendrites in the postischemic period. Ischemia-reperfusion injury also promoted DC-dependent cross-presentation of renal antigens to CD8 T cells in the draining lymph node. CONCLUSIONS: In response to renal IRI, RMP populations are skewed toward those derived from inflammatory monocyte precursors. In addition, renal DCs undergo functional activation, increasing their capacity to activate antigen-specific CD8 T cells in renal draining lymph nodes.

Homodimerization attenuates the anti-inflammatory activity of interleukin-37.

Dysregulation of the inflammatory response underlies numerous diseases. Although most interleukin-1 family cytokines are proinflammatory, human interleukin-37 (IL-37) is a powerful, broad-spectrum inhibitor of inflammation and immunity. We determined the crystal structure of IL-37 to establish the anti-inflammatory mechanism of this key cytokine in view of developing IL-37-based therapies. We found that two ß-trefoil fold IL-37 molecules form a head-to-head dimer that is stable in solution. IL-37 variants mutated to convert the cytokine into an obligate monomer were up to 13-fold more effective than the dimer in suppressing proinflammatory events both in primary human blood cells and in vivo in murine endotoxic shock. Therapeutic exploitation of the powerful anti-inflammatory properties of monomeric IL-37 may prove beneficial in treating a wide range of inflammatory and autoimmune disorders.

Quantitative Analysis of the Microtubule Interaction of Rabies Virus P3 Protein: Roles in Immune Evasion and Pathogenesis.

Although microtubules (MTs) are known to have important roles in intracellular transport of many viruses, a number of reports suggest that specific viral MT-associated proteins (MAPs) target MTs to subvert distinct MT-dependent cellular processes. The precise functional importance of these interactions and their roles in pathogenesis, however, remain largely unresolved. To assess the association with disease of the rabies virus (RABV) MAP, P3, we quantitatively compared the phenotypes of P3 from a pathogenic RABV strain, Nishigahara (Ni) and a non-pathogenic Ni-derivative strain, Ni-CE. Using confocal/live-cell imaging and dSTORM super-resolution microscopy to quantify protein interactions with the MT network and with individual MT filaments, we found that the interaction by Ni-CE-P3 is significantly impaired compared with Ni-P3. This correlated with an impaired capacity to effect association of the transcription factor STAT1 with MTs and to antagonize interferon (IFN)/STAT1-dependent antiviral signaling. Importantly, we identified a single mutation in Ni-CE-P3 that is sufficient to inhibit MT-association and IFN-antagonist function of Ni-P3, and showed that this mutation alone attenuates the pathogenicity of RABV. These data provide evidence that the viral protein-MT interface has important roles in pathogenesis, suggesting that this interface could provide targets for vaccine/antiviral drug development.

Enriched retinal ganglion cells derived from human embryonic stem cells.

Optic neuropathies are characterised by a loss of retinal ganglion cells (RGCs) that lead to vision impairment. Development of cell therapy requires a better understanding of the signals that direct stem cells into RGCs. Human embryonic stem cells (hESCs) represent an unlimited cellular source for generation of human RGCs in vitro. In this study, we present a 45-day protocol that utilises magnetic activated cell sorting to generate enriched population of RGCs via stepwise retinal differentiation using hESCs. We performed an extensive characterization of these stem cell-derived RGCs by examining the gene and protein expressions of a panel of neural/RGC markers. Furthermore, whole transcriptome analysis demonstrated similarity of the hESC-derived RGCs to human adult RGCs. The enriched hESC-RGCs possess long axons, functional electrophysiological profiles and axonal transport of mitochondria, suggestive of maturity. In summary, this RGC differentiation protocol can generate an enriched population of functional RGCs from hESCs, allowing future studies on disease modeling of optic neuropathies and development of cell therapies.

Amnion cell-mediated immune modulation following bleomycin challenge: controlling the regulatory T cell response.

INTRODUCTION: The immunomodulatory properties of human amnion epithelial cells (hAECs) have been previously described in several disease models. We previously reported on the ability of hAECs to influence macrophage phenotype and chemotaxis. In this study, we aim to elucidate the contribution of regulatory T cells (Tregs) to macrophage polarisation and downstream effects on inflammation and fibrosis in a bleomycin model of lung injury. METHODS: Either CD45(+)/FoxP3(+) Tregs or CD45(+)/FoxP3 (-) non-Tregs were adoptively transferred into Rag1 (-/-) mice immediately prior to bleomycin challenge. Four million hAECs were administered 24 hours later. Outcomes were measured 7 or 14 days later. RESULTS: Mitigation of lung inflammation and fibrosis was observed only in animals that received both hAECs and Tregs. hAEC treatment also induced the maturation of non-Tregs into FoxP3-expressing Tregs. This event was found to be transforming growth factor-beta (TGFß)-dependent. Furthermore, polarisation of macrophages from M1 to M2 occurred only in animals that received hAECs and Tregs. CONCLUSIONS: This study provides the first evidence that Tregs are required for hAEC-mediated macrophage polarisation and consequential mitigation of bleomycin-induced lung injury. Uncovering the interactions between hAECs, macrophages, and T-cell subsets is central to understanding the mechanisms by which hAECs elicit lung repair.

Glucocorticoid-induced leucine zipper (GILZ) regulates testicular FOXO1 activity and spermatogonial stem cell (SSC) function.

Spermatogonia stem cell (SSC) self-renewal and differentiation are tightly regulated processes that ensure a continued production of mature sperm throughout male adulthood. In the present study, we investigated the role of glucocorticoid-induced leucine zipper (GILZ) in maintenance of the male germline and spermatogenesis. GILZ was detectable in germ cells of wild type mice on the day of birth, suggesting a role for GILZ in prospermatogonia and SSC pool formation. Gilz KO mice were generated and adult males were azoospermic and sterile. During the first wave of spermatogenesis in Gilz KO mice, spermatogenesis arrested part way through pachytene of meiosis I. Subsequent waves resulted in a progressive depletion of germ cells through apoptosis to ultimately produce a Sertoli cell-only phenotype. Further, in contrast to wild type littermates, PLZF(+) cells were detected in the peri-luminal region of Gilz KO mice at day 6 post-natal, suggesting a defect in prospermatogonia migration in the absence of GILZ. At age 30 days, transient accumulation of PLZF(+) cells in a subset of tubules and severely compromised spermatogenesis were observed in Gilz KO mice, consistent with defective SSC differentiation. GILZ deficiency was associated with an increase in FOXO1 transcriptional activity, which leads to activation of a selective set of FOXO1 target genes, including a pro-apoptotic protein, BIM. On the other hand, no evidence of a heightened immune response was observed. Together, these results suggest that GILZ suppresses FOXO1 nuclear translocation, promotes SSC differentiation over self-renewal, and favours germ cell survival through inhibition of BIM-dependent pro-apoptotic signals. These findings provide a mechanism for the effects of GILZ on spermatogenesis and strengthen the case for GILZ being a critical molecule in the regulation of male fertility.

Multiphoton imaging reveals a new leukocyte recruitment paradigm in the glomerulus.

In contrast with many capillary beds, the glomerulus readily supports leukocyte recruitment. However, little is known regarding the actions of leukocytes following their recruitment to glomeruli. We used multiphoton confocal microscopy to examine leukocyte behavior in the glomerular microvasculature. In normal glomeruli, neutrophils and monocytes were retained in capillaries for several minutes, remaining static or migrating intravascularly. Induction of glomerular inflammation resulted in an increase in the duration of retention of static and migratory leukocytes. In response to immune complex deposition, both static and migratory neutrophils generated oxidants in inflamed glomeruli via a Mac-1-dependent mechanism. Our results describe a new paradigm for glomerular inflammation, suggesting that the major effect of acute inflammation is to increase the duration of leukocyte retention in the glomerulus. Moreover, these findings describe a previously unknown form of multicellular intravascular patrolling that involves both monocytes and neutrophils, which may underlie the susceptibility of the glomerulus to inflammation.

Generation of human female reproductive tract epithelium from human embryonic stem cells.

BACKGROUND: Recent studies have identified stem/progenitor cells in human and mouse uterine epithelium, which are postulated to be responsible for tissue regeneration and proliferative disorders of human endometrium. These progenitor cells are thought to be derived from Müllerian duct (MD), the primordial female reproductive tract (FRT). METHODOLOGY/PRINCIPAL FINDINGS: We have developed a model of human reproductive tract development in which inductive neonatal mouse uterine mesenchyme (nMUM) is recombined with green fluorescent protein (GFP)-tagged human embryonic stem cells (hESCs); GFP-hESC (ENVY). We demonstrate for the first time that hESCs can be differentiated into cells with a human FRT epithelial cell phenotype. hESC derived FRT epithelial cells emerged from cultures containing MIXL1(+) mesendodermal precursors, paralleling events occurring during normal organogenesis. Following transplantation, nMUM treated embryoid bodies (EBs) generated epithelial structures with a typical MD phenotype that expressed the MD markers PAX2, HOXA10. Functionally, the hESCs derived FRT epithelium responded to exogenous estrogen by proliferating and secreting uterine-specific glycodelin A (GdA). CONCLUSIONS/SIGNIFICANCE: These data show nMUM can induce differentiation of hESC to form the FRT epithelium. This may provide a model to study early developmental events of the human FRT.

The generation of live offspring from vitrified oocytes.

Oocyte cryopreservation is extremely beneficial for assisted reproductive technologies, the treatment of infertility and biotechnology and offers a viable alternative to embryo freezing and ovarian grafting approaches for the generation of embryonic stem cells and live offspring. It also offers the potential to store oocytes to rescue endangered species by somatic cell nuclear transfer and for the generation of embryonic stem cells to study development in these species. We vitrified mouse oocytes using a range of concentrations of trehalose (0 to 0.3 M) and demonstrated that 0.1 and 0.3 M trehalose had similar developmental rates, which were significantly different to the 0.2 M cohort (P<0.05). As mitochondria are important for fertilisation outcome, we observed that the clustering and distribution of mitochondria of the 0.2 M cohort were more affected by vitifrication than the other groups. Nevertheless, all 3 cohorts were able to develop to blastocyst, following in vitro fertilisation, although developmental rates were better for the 0.1 and 0.3 M cohorts than the 0.2 M cohort (P<0.05). Whilst blastocysts gave rise to embryonic stem-like cells, it was apparent from immunocytochemistry and RT-PCR that these cells did not demonstrate true pluripotency and exhibited abnormal karyotypes. However, they gave rise to teratomas following injection into SCID mice and differentiated into cells of each of the germinal layers following in vitro differentiation. The transfer of 2-cell embryos from the 0.1 and 0.3 M cohorts resulted in the birth of live offspring that had normal karyotypes (9/10). When 2-cell embryos from vitrified oocytes underwent vitrification, and were thawed and transferred, live offspring were obtained that exhibited normal karyotypes, with the exception of one offspring who was larger and died at 7 months. We conclude that these studies highlight the importance of the endometrial environment for the maintenance of genetic stability and thus the propagation of specific genetic traits.