B Cells and IL-21-Producing Follicular Helper T Cells Cooperate to Determine the Dynamic Alterations of Premetastatic Tumor Draining Lymph Nodes of Breast Cancer.

Metastasis is the major cause of cancer-related death, and lymph node is the most common site of metastasis in breast cancer. However, the alterations that happen in tumor-draining lymph nodes (TDLNs) to form a premetastatic microenvironment are largely unknown. Here, we first report the dynamic changes in size and immune status of TDLNs before metastasis in breast cancer. With the progression of tumor, the TDLN is first enlarged and immune-activated at early stage that contains specific antitumor immunity against metastasis. The TDLN is then contracted and immunosuppressed at late stage before finally getting metastasized. Mechanistically, B and follicular helper T (Tfh) cells parallelly expand and contract to determine the size of TDLN. The activation status and specific antitumor immunity of CD8+ T cells in the TDLN are determined by interleukin-21 (IL-21) produced by Tfh cells, thus showing parallel changes. The turn from activated enlargement to suppressed contraction is due to the spontaneous contraction of germinal centers mediated by follicular regulatory T cells. On the basis of the B-Tfh-IL-21-CD8+ T cell axis, we prove that targeting the axis could activate TDLNs to resist metastasis. Together, our findings identify the dynamic alterations and regulatory mechanisms of premetastatic TDLNs of breast cancer and provide new strategies to inhibit lymph node metastasis.

Angiogenic and Fibrogenic Dual-effect of Gremlin1 on Proliferative Diabetic Retinopathy.

Ocular angiogenic diseases, such as proliferative diabetic retinopathy (PDR), are often characterized by pathological new vessels and fibrosis formation. Anti-vascular endothelial growth factor (VEGF) therapy, despite of its efficiency to inhibit new vessels, has limitations, including drug resistance and retinal fibrosis. Here, we identified that Gremlin1, a novel angiogenesis and fibrosis inducer, was secreted from Müller glial cells, and its expression increased in the vitreous fluid from patients with PDR. Mechanistically, Gremlin1 triggered angiogenesis by promoting endothelial-mesenchymal transition via the EGFR/RhoA/ROCK pathway. In addition, Gremlin1 activated microglia to present profibrotic and fibrogenic properties. Further, anti-Gremlin1 antibody inhibited ocular angiogenesis and microglia fibrosis in mouse models. Collectively, Gremlin1 could be a potential therapeutic target in the treatment of ocular angiogenic diseases.

Epigenetic regulation in the commitment of progenitor cells during retinal development and regeneration.

Retinal development is initiated by multipotent retinal progenitor cells, which undergo several rounds of cell divisions and subsequently terminal differentiation. Retinal regeneration is usually considered as the recapitulation of retinal development, which share common mechanisms underlying the cell cycle re-entry of adult retinal stem cells and the differentiation of retinal neurons. However, how proliferative retinal progenitor cells perform a precise transition to postmitotic retinal cell types during the process of development and regeneration remains elusive. It is proposed that both the intrinsic and extrinsic programming are involved in the transcriptional regulation of the spatio-temporal fate commitment. Epigenetic modifications and the regulatory mechanisms at both DNA and chromatin levels are also postulated to play an important role in the timing of differentiation of specific retinal cells. In the present review, we have summarized recent knowledge of epigenetic regulation that underlies the commitment of retinal progenitor cells in the settings of retinal development and regeneration.

Single-Cell Transcriptomic Profiling of Human Retinal Organoids Revealed a Role of IGF1-PHLDA1 Axis in Photoreceptor Precursor Specification.

Purpose: Cone and rod photoreceptors in the retina convert light to electrical signals which are transmitted to the visual cortex of the brain. Abnormal photoreceptor development and degeneration results in blindness. So far, the mechanism that controls photoreceptor specification and its subsequent fate bifurcation remain elusive. Methods: To trace and enrich the human photoreceptor lineage, we first engineered H9 human embryonic stem cell (hESC) reporter line by fusing EGFP to endogenous BLIMP1 using CRISPR/CAS9 gene-editing technology, and then used the cell line to generate 3D retinal organoids. Following EGFP-based cell sorting, single-cell RNA-sequencing was conducted via 10x Genomics Chromium system, and the data were analyzed using Seurat. Immunofluorescence combined with lentivirus-mediated knockdown and overexpression experiments were used as validation approaches. Results: Single-cell transcriptomic profiling revealed that retinal progenitor cells were temporally programmed to differentiate to cone and rod sequentially. We identified PHLDA1 as a novel regulator of photoreceptor specification. PHLDA1 mediated the effects of IGF1 through IGF1R, and inhibited AKT phosphorylation during photoreceptor development. Conclusions: Our data established a transcriptomic cell atlas of the human photoreceptor lineage, and identified IGF1-PHLDA1 axis to regulate human photoreceptor development.

The Role of Adaptive Immunity in Diabetic Retinopathy.

Diabetic retinopathy (DR) is currently one of the common causes of vision loss in working-age adults. It is clinically diagnosed and classified according to the vascular changes in the fundus. However, the activation of immune cells occurs before these vascular changes become detectable. These, together with molecular studies and the positive clinical outcomes of anti-inflammatory treatment, highlight the pivotal involvement of the immune system. The role of innate immunity in DR pathophysiology has been studied in depth, but the contribution of adaptive immunity remains largely elusive. This review aims to summarize our current understanding of the activation mechanism of adaptive immunity in DR microenvironments and to discuss the relationship between adaptive immunity and local vascular units or innate immunity, which opens new avenues for clinical applications in DR treatment.

Somatostatin signalling promotes the differentiation of rod photoreceptors in human pluripotent stem cell-derived retinal organoid.

OBJECTIVES: Stem cell-derived photoreceptor replacement therapy is a promising strategy for the treatment of retinal degenerative disease. The development of 3D retinal organoids has permitted the production of photoreceptors. However, there is no strategy to enrich a specific photoreceptor subtype due to inadequate knowledge of the molecular mechanism underlying the photoreceptor fate determination. Hence, our aim is to explore the uncharacterized function of somatostatin signalling in human pluripotent stem cell-derived photoreceptor differentiation. MATERIALS AND METHODS: 3D retinal organoids were achieved from human embryonic stem cell. The published single-cell RNA-sequencing datasets of human retinal development were utilized to further investigate the transcriptional regulation of photoreceptor differentiation. The assays of immunofluorescence staining, lentivirus transfection, real-time quantitative polymerase chain reaction and western blotting were performed. RESULTS: We identified that the somatostatin receptor 2 (SSTR2)-mediated signalling was essential for rod photoreceptor differentiation at the precursor stage. The addition of the cognate ligand somatostatin in human 3D retinal organoids promoted rod photoreceptor differentiation and inhibited cone photoreceptor production. Furthermore, we found that the genesis of rod photoreceptors was modulated by endogenous somatostatin specifically secreted by developing retinal ganglion cells. CONCLUSIONS: Our study identified SSTR2 signalling as a novel extrinsic regulator for rod photoreceptor fate determination in photoreceptor precursors, which expands the repertoire of functional signalling pathways in photoreceptor development and sheds light on the optimization of the photoreceptor enrichment strategy.

Single-Cell Transcriptomics Reveals Novel Role of Microglia in Fibrovascular Membrane of Proliferative Diabetic Retinopathy.

Vitreous fibrovascular membranes (FVMs), the hallmark of proliferative diabetic retinopathy (PDR), cause retinal hemorrhage, detachment, and eventually blindness. However, little is known about the pathophysiology of FVM. In this study, we used single-cell RNA sequencing on surgically harvested PDR-FVMs and generated a comprehensive cell atlas of FVM. Eight cellular compositions were identified, with microglia as the major cell population. We identified a GPNMB+ subpopulation of microglia, which presented both profibrotic and fibrogenic properties. Pseudotime analysis further revealed the profibrotic microglia was uniquely differentiated from retina-resident microglia and expanded in the PDR setting. Ligand-receptor interactions between the profibrotic microglia and cytokines upregulated in PDR vitreous implicated the involvement of several pathways, including CCR5, IFNGR1, and CD44 signaling, in the microglial activation within the PDR microenvironment. Collectively, our description of the novel microglia phenotypes in PDR-FVM may offer new insight into the cellular and molecular mechanism underlying the pathogenesis of DR, as well as potential signaling pathways amenable to disease-specific intervention.

Identification of a novel GPR143 mutation in a large Chinese family with isolated foveal hypoplasia.

BACKGROUND: Pathogenic variants of G-protein coupled receptor 143 (GPR143) gene often leads to ocular albinism type I (OA1) characterized by nystagmus, iris and fundus hypopigmentation, and foveal hypoplasia. In this study, we identified a novel hemizygous nonsense mutation in GPR143 that caused an atypical manifestation of OA1. CASE PRESENTATION: We reported a large Chinese family in which all affected individuals are afflicted with poor visual acuity and foveal hypoplasia without signs of nystagmus. Fundus examination of patients showed an absent foveal reflex and mild hypopigmentation. The fourth grade of foveal hypoplasia and the reduced area of blocked fluorescence at foveal region was detected in OCT. OCTA imaging showed the absence of foveal avascular zone. In addition, the amplitude of multifocal ERG was reduced in the central ring. Gene sequencing results revealed a novel hemizygous mutation (c.939G > A) in GPR143 gene, which triggered p.W313X. However, no iris depigmentation and nystagmus were observed among both patients and carriers. CONCLUSIONS: In this study, we reported a novel nonsense mutation of GPR143 in a large family with poor visual acuity and isolated foveal hypoplasia without nystagmus, which further expanded the genetic mutation spectrum of GPR143.

Transcriptome profiling of the low-salinity stress responses in the gills of the juvenile Pseudopleuronectes yokohamae.

Salinity is an important environmental factor that affects the life cycle of fish, including their growth, development and reproduction. The marbled flounder, Pseudopleuronectes yokohamae, is an important economic resource and serves as a good model to investigate osmoregulation, as it can adapt to a wide range of salinity levels. However, the lack of genomic resources for this species has hampered the understanding of the mechanisms underlying its salinity tolerance. In this study, RNA-Seq analysis was conducted to identify genes related to salinity adaptation and osmotic regulation in the gill tissue of marbled flounder exposed to different concentrations of environmental salinity (6 and 30 ppt). After de novo assembly, 19,265 genes were annotated by the Nr database. A comparison of expression between the two salinity groups revealed 673 differentially expressed genes, of which 180 were upregulated and 493 were downregulated in the low salinity group relative to the high salinity group. The related molecular biological processes were explored from several important perspectives, and potential functions were determined by enrichment analyses, including those of metabolites in ion transportation, energy metabolism and protein synthesis, and immune responses. This study is the first transcriptomic study conducted on marbled flounder, and it revealed many novel sequences for further biological analyses. In addition, the candidate genes identified in the gene expression analysis provided insights into responses to salinity change and molecular mechanisms underlying osmoregulation in the gills of marbled flounder.

Comparative transcriptomic analysis reveals the gene expression profiles in the liver and spleen of Japanese pufferfish (Takifugu rubripes) in response to Vibrio harveyi infection.

Japanese pufferfish (Takifugu rubripes) is one of the main marine aquatic fish species cultured in Asia due to its high nutritional value. In recent years, disease caused by Vibrio harveyi infections have led to serious mortality in Japanese pufferfish industry. To understand the complex molecular mechanisms between V. harveyi and Japanese pufferfish, we performed a transcriptome analysis of liver and spleen samples from Japanese pufferfish at 1 and 2 day post-infection. Between-group comparisons revealed 922 genes that were significantly differentially expressed. The altered genes emphasized the function in several immune related pathways including MAPK signaling pathway, JAK-STAT signaling pathway, toll-like receptor signaling pathway, cytokine-cytokine receptor interaction and lysosomal pathway. The data generated in this study provided insight into the responses of Japanese pufferfish against V. harveyi at the transcriptome level, promoting our comprehensive understanding of immune responses for aquatic animal against V. harveyi.

Molecular characterization and expression analysis of galectins in Japanese pufferfish (Takifugu rubripes) in response to Vibrio harveyi infection.

Galectins are a family of proteins with conserved carbohydrate recognition domains (CRDs) that bind to specific glycans, including the glycans on the surface of pathogens, and therefore play a role in cytokine secretion, cell activation, migration, adhesion and apoptosis. Currently, galectins have been extensively studied in mammalian species but rarely studied in teleost fish species. In this study, a total of 12 galectin genes were characterized to understand the molecular mechanisms of galectin function in Japanese pufferfish (Takifugu rubripes). Phylogenetic analyses and syntenic analyses confirmed their correct annotation and suggested the strongest relationships to tetraodon. Furthermore, expression analyses were conducted in healthy tissues of Japanese pufferfish and after infection with Vibrio harveyi in the intestine, liver and spleen. The results showed that galectin genes were widely expressed in all examined tissues; however, most of the galectin genes were highly expressed in mucosal tissues (skin, gill and intestine). Moreover, majority of the galectin genes were significantly regulated after V. harveyi infection in the intestine, liver and spleen, suggesting that galectins were involved in the immune response to V. harveyi infection in Japanese pufferfish. This study established the foundation for future studies of galectin gene functions.

CRB2 mutation causes autosomal recessive retinitis pigmentosa.

Retinitis pigmentosa (RP), the most common form of inherited retinal dystrophies, exhibits significant genetic heterogeneity. The crumbs homolog 2 (CRB2) protein, together with CRB1 and CRB3, belongs to the Crumbs family. Given that CRB1 mutations account for 4% of RP cases, the role of CRB2 mutations in RP etiology has long been hypothesized but never confirmed. Herein, we report the identification of CRB2 as a novel RP causative gene in a Chinese consanguineous family and have analyzed its pathogenic effects. Comprehensive ophthalmic and systemic evaluations confirmed the clinical diagnosis of the two patients in this family as RP. WES revealed a homozygous missense mutation, CRB2 p.R1249G, to segregate the RP phenotype, which was highly conserved among multiple species. In vitro cellular study revealed that this mutation not only interrupted the stability of the transcribed CRB2 mRNA and the encoded CRB2 protein, but also interfered with the wild type CRB2 mRNA/protein and decreased their expression. This mutation was also shown to trigger epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE) cells, thus impairing regular RPE phagocytosis and induce RPE degeneration and apoptosis. Thus, we conclude that CRB2 p.R1249G mutation causes RP via accelerating EMT, dysfunction and loss of RPE cells, and establish CRB2 as a novel Crumbs family member associated with non-syndromic RP. We provide important hints for understanding of CRB2 defects and retinopathy, and for the involvement of EMT of RPE cells in RP pathogenesis.

Molecular characterization and expression analysis of the interleukin 1b gene in Pacific cod (Gadus macrocephalus).

Interleukin 1b (IL-1b) is a member of the cytokine family that serves as major mediators for early pro-inflammatory responses. During artificial breeding of Pacific cod (Gadus macrocephalus), outbreaks of NNV in the early development stages could lead to high mortality. Recent research reported that IL-1b participated in the host-virus interaction against NNV infection, however, no IL-1b gene has been identified from Pacific cod so far. In this study, we described the identification and characterization of a IL-1b from Pacific cod transcriptome at both transcript and protein level. Sequence alignment and phylogenetic analysis were conducted to determine and confirm their evolutionary relationship. Expression analysis revealed that IL-1b was expressed in all tested tissues and the western blot analysis confirmed the same expression profiles using purified recombinant IL-1b protein. In addition, significant up-regulation was detected in IL-1b transcripts during early development stages at 30dph and 50dph. After challenged with poly I:C, IL-1b was significantly induced after 24 h. Taken together, these results strongly suggested that IL-1b might play an essential role in innate defense mechanism of Pacific cod.