Stabilization of myeloid-derived HIFs promotes vascular regeneration in retinal ischemia.

The retinal vasculature is tightly organized in a structure that provides for the high metabolic demand of neurons while minimizing interference with incident light. The adverse impact of retinal vascular insufficiency is mitigated by adaptive vascular regeneration but exacerbated by pathological neovascularization. Aberrant growth of neovessels in the retina is responsible for impairment of sight in common blinding disorders including retinopathy of prematurity, proliferative diabetic retinopathy, and age-related macular degeneration. Myeloid cells are key players in this process, with diverse roles that can either promote or protect against ocular neovascularization. We have previously demonstrated that myeloid-derived VEGF, HIF1, and HIF2 are not essential for pathological retinal neovascularization. Here, however, we show by cell-specific depletion of Vhl in a mouse model of retinal ischemia (oxygen-induced retinopathy, OIR) that myeloid-derived HIFs promote VEGF and bFGF expression and enhance vascular regeneration in association with improved density and organization of the astrocytic network.

MAP4K4 Inhibition Promotes Survival of Human Stem Cell-Derived Cardiomyocytes and Reduces Infarct Size In Vivo.

Heart disease is a paramount cause of global death and disability. Although cardiomyocyte death plays a causal role and its suppression would be logical, no clinical counter-measures target the responsible intracellular pathways. Therapeutic progress has been hampered by lack of preclinical human validation. Mitogen-activated protein kinase kinase kinase kinase-4 (MAP4K4) is activated in failing human hearts and relevant rodent models. Using human induced-pluripotent-stem-cell-derived cardiomyocytes (hiPSC-CMs) and MAP4K4 gene silencing, we demonstrate that death induced by oxidative stress requires MAP4K4. Consequently, we devised a small-molecule inhibitor, DMX-5804, that rescues cell survival, mitochondrial function, and calcium cycling in hiPSC-CMs. As proof of principle that drug discovery in hiPSC-CMs may predict efficacy in vivo, DMX-5804 reduces ischemia-reperfusion injury in mice by more than 50%. We implicate MAP4K4 as a well-posed target toward suppressing human cardiac cell death and highlight the utility of hiPSC-CMs in drug discovery to enhance cardiomyocyte survival.

Use of bioreactors for culturing human retinal organoids improves photoreceptor yields.

BACKGROUND: The use of human pluripotent stem cell-derived retinal cells for cell therapy strategies and disease modelling relies on the ability to obtain healthy and organised retinal tissue in sufficient quantities. Generating such tissue is a lengthy process, often taking over 6 months of cell culture, and current approaches do not always generate large quantities of the major retinal cell types required. METHODS: We adapted our previously described differentiation protocol to investigate the use of stirred-tank bioreactors. We used immunohistochemistry, flow cytometry and electron microscopy to characterise retinal organoids grown in standard and bioreactor culture conditions. RESULTS: Our analysis revealed that the use of bioreactors results in improved laminar stratification as well as an increase in the yield of photoreceptor cells bearing cilia and nascent outer-segment-like structures. CONCLUSIONS: Bioreactors represent a promising platform for scaling up the manufacture of retinal cells for use in disease modelling, drug screening and cell transplantation studies.

Clinical Remission of Sight-Threatening Non-Infectious Uveitis Is Characterized by an Upregulation of Peripheral T-Regulatory Cell Polarized Towards T-bet and TIGIT.

Background: Non-infectious uveitis can cause chronic relapsing and remitting ocular inflammation, which may require high dose systemic immunosuppression to prevent severe sight loss. It has been classically described as an autoimmune disease, mediated by pro-inflammatory Th1 and Th17 T-cell subsets. Studies suggest that natural immunosuppressive CD4+CD25+FoxP3+ T-regulatory cells (Tregs) are involved in resolution of inflammation and may be involved in the maintenance of clinical remission. Objective: To investigate whether there is a peripheral blood immunoregulatory phenotype associated with clinical remission of sight-threatening non-infectious uveitis by comparing peripheral blood levels of Treg, Th1, and Th17, and associated DNA methylation and cytokine levels in patients with active uveitic disease, control subjects and patients (with previously active disease) in clinical remission induced by immunosuppressive drugs. Methods: Isolated peripheral blood mononuclear cells (PBMC) from peripheral blood samples from prospectively recruited subjects were analyzed by flow cytometry for CD3, CD4, FoxP3, TIGIT, T-bet, and related orphan receptor γt. Epigenetic DNA methylation levels of FOXP3 Treg-specific demethylated region (TSDR), FOXP3 promoter, TBX21, RORC2, and TIGIT loci were determined in cryopreserved PBMC using a next-generation sequencing approach. Related cytokines were measured in blood sera. Functional suppressive capacity of Treg was assessed using T-cell proliferation assays. Results: Fifty patients with uveitis (intermediate, posterior, and panuveitis) and 10 control subjects were recruited. The frequency of CD4+CD25+FoxP3+ Treg, TIGIT+ Treg, and T-bet+ Treg and the ratio of Treg to Th1 were significantly higher in remission patients compared with patients with active uveitic disease; and TIGIT+ Tregs were a significant predictor of clinical remission. Treg from patients in clinical remission demonstrated a high level of in vitro suppressive function compared with Treg from control subjects and from patients with untreated active disease. PBMC from patients in clinical remission had significantly lower methylation levels at the FOXP3 TSDR, FOXP3 promoter, and TIGIT loci and higher levels at RORC loci than those with active disease. Clinical remission was also associated with significantly higher serum levels of transforming growth factor ß and IL-10, which positively correlated with Treg levels, and lower serum levels of IFNγ, IL-17A, and IL-22 compared with patients with active disease. Conclusion: Clinical remission of sight-threatening non-infectious uveitis has an immunoregulatory phenotype characterized by upregulation of peripheral Treg, polarized toward T-bet and TIGIT. These findings may assist with individualized therapy of uveitis, by informing whether drug therapy has induced phenotypically stable Treg associated with long-term clinical remission.

Late neuroprogenitors contribute to normal retinal vascular development in a Hif2a-dependent manner.

In the adult central nervous system, endothelial and neuronal cells engage in tight cross-talk as key components of the so-called neurovascular unit. Impairment of this important relationship adversely affects tissue homeostasis, as observed in neurodegenerative conditions including Alzheimer's and Parkinson's disease. In development, the influence of neuroprogenitor cells on angiogenesis is poorly understood. Here, we show in mouse that these cells interact intimately with the growing retinal vascular network, and we identify a novel regulatory mechanism of vasculature development mediated by hypoxia-inducible factor 2a (Hif2a). By Cre-lox gene excision, we show that Hif2a in retinal neuroprogenitor cells upregulates the expression of the pro-angiogenic mediators vascular endothelial growth factor and erythropoietin, whereas it locally downregulates the angiogenesis inhibitor endostatin. Importantly, absence of Hif2a in retinal neuroprogenitor cells causes a marked reduction of proliferating endothelial cells at the angiogenic front. This results in delayed retinal vascular development, fewer major retinal vessels and reduced density of the peripheral deep retinal vascular plexus. Our findings demonstrate that retinal neuroprogenitor cells are a crucial component of the developing neurovascular unit.

Prevention of Photoreceptor Cell Loss in a Cln6nclf Mouse Model of Batten Disease Requires CLN6 Gene Transfer to Bipolar Cells.

The neuronal ceroid lipofuscinoses (NCLs) are inherited lysosomal storage disorders characterized by general neurodegeneration and premature death. Sight loss is also a major symptom in NCLs, severely affecting the quality of life of patients, but it is not targeted effectively by brain-directed therapies. Here we set out to explore the therapeutic potential of an ocular gene therapy to treat sight loss in NCL due to a deficiency in the transmembrane protein CLN6. We found that, although Cln6nclf mice presented mainly with photoreceptor degeneration, supplementation of CLN6 in photoreceptors was not beneficial. Because the level of CLN6 is low in photoreceptors but high in bipolar cells (retinal interneurons that are only lost in Cln6-deficient mice at late disease stages), we explored the therapeutic effects of delivering CLN6 to bipolar cells using adeno-associated virus (AAV) serotype 7m8. Bipolar cell-specific expression of CLN6 slowed significantly the loss of photoreceptor function and photoreceptor cells. This study shows that the deficiency of a gene normally expressed in bipolar cells can cause the loss of photoreceptors and that this can be prevented by bipolar cell-directed treatment.

Assessment of AAV Vector Tropisms for Mouse and Human Pluripotent Stem Cell-Derived RPE and Photoreceptor Cells.

Adeno-associated viral vectors are showing great promise as gene therapy vectors for a wide range of retinal disorders. To date, evaluation of therapeutic approaches has depended almost exclusively on the use of animal models. With recent advances in human stem cell technology, stem cell-derived retina now offers the possibility to assess efficacy in human organoids in vitro. Here we test six adeno-associated virus (AAV) serotypes [AAV2/2, AAV2/9, AAV2/8, AAV2/8T(Y733F), AAV2/5, and ShH10] to determine their efficiency in transducing mouse and human pluripotent stem cell-derived retinal pigment epithelium (RPE) and photoreceptor cells in vitro. All the serotypes tested were capable of transducing RPE and photoreceptor cells in vitro. AAV ShH10 and AAV2/5 are the most efficient vectors at transducing both mouse and human RPE, while AAV2/8 and ShH10 achieved similarly robust transduction of human embryonic stem cell-derived cone photoreceptors. Furthermore, we show that human embryonic stem cell-derived photoreceptors can be used to establish promoter specificity in human cells in vitro. The results of this study will aid capsid selection and vector design for preclinical evaluation of gene therapy approaches, such as gene editing, that require the use of human cells and tissues.

Activation of podocyte Notch mediates early Wt1 glomerulopathy.

The Wilms' tumor suppressor gene, WT1, encodes a zinc finger protein that regulates podocyte development and is highly expressed in mature podocytes. Mutations in the WT1 gene are associated with the development of renal failure due to the formation of scar tissue within glomeruli, the mechanisms of which are poorly understood. Here, we used a tamoxifen-based CRE-LoxP system to induce deletion of Wt1 in adult mice to investigate the mechanisms underlying evolution of glomerulosclerosis. Podocyte apoptosis was evident as early as the fourth day post-induction and increased during disease progression, supporting a role for Wt1 in mature podocyte survival. Podocyte Notch activation was evident at disease onset with upregulation of Notch1 and its transcriptional targets, including Nrarp. There was repression of podocyte FoxC2 and upregulation of Hey2 supporting a role for a Wt1/FoxC2/Notch transcriptional network in mature podocyte injury. The expression of cleaved Notch1 and HES1 proteins in podocytes of mutant mice was confirmed in early disease. Furthermore, induction of podocyte HES1 expression was associated with upregulation of genes implicated in epithelial mesenchymal transition, thereby suggesting that HES1 mediates podocyte EMT. Lastly, early pharmacological inhibition of Notch signaling ameliorated glomerular scarring and albuminuria. Thus, loss of Wt1 in mature podocytes modulates podocyte Notch activation, which could mediate early events in WT1-related glomerulosclerosis.

Transplanted Donor- or Stem Cell-Derived Cone Photoreceptors Can Both Integrate and Undergo Material Transfer in an Environment-Dependent Manner.

Human vision relies heavily upon cone photoreceptors, and their loss results in permanent visual impairment. Transplantation of healthy photoreceptors can restore visual function in models of inherited blindness, a process previously understood to arise by donor cell integration within the host retina. However, we and others recently demonstrated that donor rod photoreceptors engage in material transfer with host photoreceptors, leading to the host cells acquiring proteins otherwise expressed only by donor cells. We sought to determine whether stem cell- and donor-derived cones undergo integration and/or material transfer. We find that material transfer accounts for a significant proportion of rescued cells following cone transplantation into non-degenerative hosts. Strikingly, however, substantial numbers of cones integrated into the Nrl-/- and Prph2rd2/rd2, but not Nrl-/-;RPE65R91W/R91W, murine models of retinal degeneration. This confirms the occurrence of photoreceptor integration in certain models of retinal degeneration and demonstrates the importance of the host environment in determining transplantation outcome.

Recapitulation of Human Retinal Development from Human Pluripotent Stem Cells Generates Transplantable Populations of Cone Photoreceptors.

Transplantation of rod photoreceptors, derived either from neonatal retinae or pluripotent stem cells (PSCs), can restore rod-mediated visual function in murine models of inherited blindness. However, humans depend more upon cone photoreceptors that are required for daylight, color, and high-acuity vision. Indeed, macular retinopathies involving loss of cones are leading causes of blindness. An essential step for developing stem cell-based therapies for maculopathies is the ability to generate transplantable human cones from renewable sources. Here, we report a modified 2D/3D protocol for generating hPSC-derived neural retinal vesicles with well-formed ONL-like structures containing cones and rods bearing inner segments and connecting cilia, nascent outer segments, and presynaptic structures. This differentiation system recapitulates human photoreceptor development, allowing the isolation and transplantation of a pure population of stage-matched cones. Purified human long/medium cones survive and become incorporated within the adult mouse retina, supporting the potential of photoreceptor transplantation for treating retinal degeneration.

Differentiation and Transplantation of Embryonic Stem Cell-Derived Cone Photoreceptors into a Mouse Model of End-Stage Retinal Degeneration.

The loss of cone photoreceptors that mediate daylight vision represents a leading cause of blindness, for which cell replacement by transplantation offers a promising treatment strategy. Here, we characterize cone differentiation in retinas derived from mouse embryonic stem cells (mESCs). Similar to in vivo development, a temporal pattern of progenitor marker expression is followed by the differentiation of early thyroid hormone receptor ß2-positive precursors and, subsequently, photoreceptors exhibiting cone-specific phototransduction-related proteins. We establish that stage-specific inhibition of the Notch pathway increases cone cell differentiation, while retinoic acid signaling regulates cone maturation, comparable with their actions in vivo. MESC-derived cones can be isolated in large numbers and transplanted into adult mouse eyes, showing capacity to survive and mature in the subretinal space of Aipl1-/- mice, a model of end-stage retinal degeneration. Together, this work identifies a robust, renewable cell source for cone replacement by purified cell suspension transplantation.

Egress of sperm autoantigen from seminiferous tubules maintains systemic tolerance.

Autoimmune responses to meiotic germ cell antigens (MGCA) that are expressed on sperm and testis occur in human infertility and after vasectomy. Many MGCA are also expressed as cancer/testis antigens (CTA) in human cancers, but the tolerance status of MGCA has not been investigated. MGCA are considered to be uniformly immunogenic and nontolerogenic, and the prevailing view posits that MGCA are sequestered behind the Sertoli cell barrier in seminiferous tubules. Here, we have shown that only some murine MGCA are sequestered. Nonsequestered MCGA (NS-MGCA) egressed from normal tubules, as evidenced by their ability to interact with systemically injected antibodies and form localized immune complexes outside the Sertoli cell barrier. NS-MGCA derived from cell fragments that were discarded by spermatids during spermiation. They egressed as cargo in residual bodies and maintained Treg-dependent physiological tolerance. In contrast, sequestered MGCA (S-MGCA) were undetectable in residual bodies and were nontolerogenic. Unlike postvasectomy autoantibodies, which have been shown to mainly target S-MGCA, autoantibodies produced by normal mice with transient Treg depletion that developed autoimmune orchitis exclusively targeted NS-MGCA. We conclude that spermiation, a physiological checkpoint in spermatogenesis, determines the egress and tolerogenicity of MGCA. Our findings will affect target antigen selection in testis and sperm autoimmunity and the immune responses to CTA in male cancer patients.

Hypoxia inducible factors are dispensable for myeloid cell migration into the inflamed mouse eye.

Hypoxia inducible factors (HIFs) are ubiquitously expressed transcription factors important for cell homeostasis during dynamic oxygen levels. Myeloid specific HIFs are crucial for aspects of myeloid cell function, including their ability to migrate into inflamed tissues during autoimmune disease. This contrasts with the concept that accumulation of myeloid cells at ischemic and hypoxic sites results from a lack of chemotactic responsiveness. Here we seek to address the role of HIFs in myeloid trafficking during inflammation in a mouse model of human uveitis. We show using mice with myeloid-specific Cre-deletion of HIFs that myeloid HIFs are dispensable for leukocyte migration into the inflamed eye. Myeloid-specific deletion of Hif1a, Epas1, or both together, had no impact on the number of myeloid cells migrating into the eye. Additionally, stabilization of HIF pathways via deletion of Vhl in myeloid cells had no impact on myeloid trafficking into the inflamed eye. Finally, we chemically induce hypoxemia via hemolytic anemia resulting in HIF stabilization within circulating leukocytes to demonstrate the dispensable role of HIFs in myeloid cell migration into the inflamed eye. These data suggest, contrary to previous reports, that HIF pathways in myeloid cells during inflammation and hypoxia are dispensable for myeloid cell tissue trafficking.

Flow cytometric analysis of inflammatory and resident myeloid populations in mouse ocular inflammatory models.

Myeloid cells make a pivotal contribution to tissue homeostasis during inflammation. Both tissue-specific resident populations and infiltrating myeloid cells can cause tissue injury through aberrant activation and/or dysregulated activity. Reliable identification and quantification of myeloid cells within diseased tissues is important to understand pathological inflammatory processes. Flow cytometry is a valuable technique for leukocyte analysis, but a standardized flow cytometric method for myeloid cell populations in the eye is lacking. Here, we validate a reproducible flow cytometry gating approach to characterize myeloid cells in several commonly used models of ocular inflammation. We profile and quantify myeloid subsets across these models, and highlight the value of this strategy in identifying phenotypic differences using Ccr2-deficient mice. This method will aid standardization in the field and facilitate future investigations into the roles of myeloid cells during ocular inflammation.

The HSP70 co-chaperone DNAJC14 targets misfolded pendrin for unconventional protein secretion.

Mutations in SLC26A4, which encodes pendrin, are responsible for hearing loss with an enlarged vestibular aqueduct and Pendred syndrome. The most prevalent mutation in East Asia is p.H723R (His723Arg), which leads to defects in protein folding and cell-surface expression. Here we show that H723R-pendrin can be rescued to the cell surface by an HSP70 co-chaperone DNAJC14-dependent unconventional trafficking pathway. Blockade of ER-to-Golgi transport or activation of ER stress signals induced Golgi-independent cell-surface expression of H723R-pendrin and restored its cell-surface Cl(-)/HCO3(-) exchange activity. Proteomic and short interfering RNA screenings with subsequent molecular analyses showed that Hsc70 and DNAJC14 are required for the unconventional trafficking of H723R-pendrin. Moreover, DNAJC14 upregulation was able to induce the unconventional cell-surface expression of H723R-pendrin. These results indicate that Hsc70 and DNAJC14 play central roles in ER stress-associated unconventional protein secretion and are potential therapeutic targets for diseases such as Pendred syndrome, which arise from transport defects of misfolded proteins.

PD-1 Expression and Cytokine Secretion Profiles of Mycobacterium tuberculosis-Specific CD4+ T-Cell Subsets; Potential Correlates of Containment in HIV-TB Co-Infection.

HIV co-infection is an important risk factor for tuberculosis (TB) providing a powerful model in which to dissect out defective, protective and dysfunctional Mycobacterium tuberculosis (MTB)-specific immune responses. To identify the changes induced by HIV co-infection we compared MTB-specific CD4+ responses in subjects with active TB and latent TB infection (LTBI), with and without HIV co-infection. CD4+ T-cell subsets producing interferon-gamma (IFN-γ), interleukin-2 (IL-2) and tumour necrosis factor-alpha (TNF-α) and expressing CD279 (PD-1) were measured using polychromatic flow-cytometry. HIV-TB co-infection was consistently and independently associated with a reduced frequency of CD4+ IFN-γ and IL-2-dual secreting T-cells and the proportion correlated inversely with HIV viral load (VL). The impact of HIV co-infection on this key MTB-specific T-cell subset identifies them as a potential correlate of mycobacterial immune containment. The percentage of MTB-specific IFN-γ-secreting T-cell subsets that expressed PD-1 was increased in active TB with HIV co-infection and correlated with VL. This identifies a novel correlate of dysregulated immunity to MTB, which may in part explain the paucity of inflammatory response in the face of mycobacterial dissemination that characterizes active TB with HIV co-infection.

Cardiac-Restricted IGF-1Ea Overexpression Reduces the Early Accumulation of Inflammatory Myeloid Cells and Mediates Expression of Extracellular Matrix Remodelling Genes after Myocardial Infarction.

Strategies to limit damage and improve repair after myocardial infarct remain a major therapeutic goal in cardiology. Our previous studies have shown that constitutive expression of a locally acting insulin-like growth factor-1 Ea (IGF-1Ea) propeptide promotes functional restoration after cardiac injury associated with decreased scar formation. In the current study, we investigated the underlying molecular and cellular mechanisms behind the enhanced functional recovery. We observed improved cardiac function in mice overexpressing cardiac-specific IGF-1Ea as early as day 7 after myocardial infarction. Analysis of gene transcription revealed that supplemental IGF-1Ea regulated expression of key metalloproteinases (MMP-2 and MMP-9), their inhibitors (TIMP-1 and TIMP-2), and collagen types (Col 1α1 and Col 1α3) in the first week after injury. Infiltration of inflammatory cells, which direct the remodelling process, was also altered; in particular there was a notable reduction in inflammatory Ly6C+ monocytes at day 3 and an increase in anti-inflammatory CD206+ macrophages at day 7. Taken together, these results indicate that the IGF-1Ea transgene shifts the balance of innate immune cell populations early after infarction, favouring a reduction in inflammatory myeloid cells. This correlates with reduced extracellular matrix remodelling and changes in collagen composition that may confer enhanced scar elasticity and improved cardiac function.

Differences in antigen-specific CD4+ responses to opportunistic infections in HIV infection.

HIV-infected individuals with severe immunodeficiency are at risk of opportunistic infection (OI). Tuberculosis (TB) may occur without substantial immune suppression suggesting an early and sustained adverse impact of HIV on Mycobacterium tuberculosis (MTB)-specific cell mediated immunity (CMI). This prospective observational cohort study aimed to observe differences in OI-specific and MTB-specific CMI that might underlie this. Using polychromatic flow cytometry, we compared CD4+ responses to MTB, cytomegalovirus (CMV), Epstein-Barr virus (EBV) and Candida albicans in individuals with and without HIV infection. MTB-specific CD4+ T-cells were more polyfunctional than virus specific (CMV/EBV) CD4+ T-cells which predominantly secreted IFN-gamma (IFN-γ) only. There was a reduced frequency of IFN-γ and IL-2 (IL-2)-dual-MTB-specific cells in HIV-infected individuals, which was not apparent for the other pathogens. MTB-specific cells were less differentiated especially compared with CMV-specific cells. CD127 expression was relatively less frequent on MTB-specific cells in HIV co-infection. MTB-specific CD4+ T-cells PD-1 expression was infrequent in contrast to EBV-specific CD4+ T-cells. The variation in the inherent quality of these CD4+ T-cell responses and impact of HIV co-infection may contribute to the timing of co-infectious diseases in HIV infection.

Monocyte/Macrophage-derived IGF-1 Orchestrates Murine Skeletal Muscle Regeneration and Modulates Autocrine Polarization.

Insulin-like growth factor 1 (IGF-1) is a potent enhancer of tissue regeneration, and its overexpression in muscle injury leads to hastened resolution of the inflammatory phase. Here, we show that monocytes/macrophages constitute an important initial source of IGF-1 in muscle injury, as conditional deletion of the IGF-1 gene specifically in mouse myeloid cells (ϕIGF-1 CKO) blocked the normal surge of local IGF-1 in damaged muscle and significantly compromised regeneration. In injured muscle, Ly6C+ monocytes/macrophages and CD206+ macrophages expressed equivalent IGF-1 levels, which were transiently upregulated during transition from the inflammation to repair. In injured ϕIGF-1 CKO mouse muscle, accumulation of CD206+ macrophages was impaired, while an increase in Ly6C+ monocytes/macrophages was favored. Transcriptional profiling uncovered inflammatory skewing in ϕIGF-1 CKO macrophages, which failed to fully induce a reparative gene program in vitro or in vivo, revealing a novel autocrine role for IGF-1 in modulating murine macrophage phenotypes. These data establish local macrophage-derived IGF-1 as a key factor in inflammation resolution and macrophage polarization during muscle regeneration.