CRB1-associated retinal degeneration is dependent on bacterial translocation from the gut.

The Crumbs homolog 1 (CRB1) gene is associated with retinal degeneration, most commonly Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP). Here, we demonstrate that murine retinas bearing the Rd8 mutation of Crb1 are characterized by the presence of intralesional bacteria. While normal CRB1 expression was enriched in the apical junctional complexes of retinal pigment epithelium and colonic enterocytes, Crb1 mutations dampened its expression at both sites. Consequent impairment of the outer blood retinal barrier and colonic intestinal epithelial barrier in Rd8 mice led to the translocation of intestinal bacteria from the lower gastrointestinal (GI) tract to the retina, resulting in secondary retinal degeneration. Either the depletion of bacteria systemically or the reintroduction of normal Crb1 expression colonically rescued Rd8-mutation-associated retinal degeneration without reversing the retinal barrier breach. Our data elucidate the pathogenesis of Crb1-mutation-associated retinal degenerations and suggest that antimicrobial agents have the potential to treat this devastating blinding disease.

Metagenomic Sequencing Analysis Identifies Cross-Cohort Gut Microbial Signatures Associated With Age-Related Macular Degeneration.

Purpose: Alterations in the gut microbiota have been associated with age-related macular degeneration (AMD). However, the dysbiosis shared by different ethnicity and geographic groups, which may associate with the disease pathogenesis, remain underexplored. Here, we characterized dysbiosis of the gut microbiota in patients with AMD from Chinese and Swiss cohorts and identified cross-cohort signatures associated with AMD. Methods: Shotgun metagenomic sequencing was performed on fecal samples from 30 patients with AMD and 30 healthy subjects. Published datasets with 138 samples from Swiss patients with AMD and healthy subjects were re-analyzed. Comprehensive taxonomic profiling was conducted by matching to the RefSeq genome database, metagenome-assembled genome (MAG) database, and Gut Virome Database (GVD). Functional profiling was performed by reconstruction of the MetaCyc pathways. Results: The α-diversity of the gut microbiota was decreased in patients with AMD according to taxonomic profiles generated using MAG but not RefSeq database as reference. The Firmicutes/Bacteroidetes ratio was also decreased in patients with AMD. Among AMD-associated bacteria shared between Chinese and Swiss cohorts, Ruminococcus callidus, Lactobacillus gasseri, and Prevotellaceae (f) uSGB 2135 were enriched in patients with AMD, whereas Bacteroidaceae (f) uSGB 1825 was depleted in patients with AMD and was negatively associated with hemorrhage size. Bacteroidaceae was one of the major hosts of phages associated with AMD. Three degradation pathways were reduced in AMD. Conclusions: These results demonstrated that dysbiosis of the gut microbiota was associated with AMD. We identified cross-cohort gut microbial signatures involving bacteria, viruses, and metabolic pathways, which potentially serve as promising targets for the prevention or treatment of AMD.

Epigenetic drug screen identified IOX1 as an inhibitor of Th17-mediated inflammation through targeting TET2.

BACKGROUND: Targeting helper T cells, especially Th17 cells, has become a plausible therapy for many autoimmune diseases. METHODS: Using an in vitro culture system, we screened an epigenetics compound library for inhibitors of IFN-γ and IL-17 expression in murine Th1 and Th17 cultures. FINDINGS: This identified IOX1 as an effective suppressor of IL-17 expression in both murine and human CD4+ T cells. Furthermore, we found that IOX1 suppresses Il17a expression directly by targeting TET2 activity on its promoter in Th17 cells. Using established pre-clinical models of intraocular inflammation, treatment with IOX1 in vivo reduced the migration/infiltration of Th17 cells into the site of inflammation and tissue damage. INTERPRETATION: These results provide evidence of the strong potential for IOX1 as a viable therapy for inflammatory diseases, in particular of the eye. FUNDING: This study was supported by the National Key Research and Development Program of China 2021YFA1101200 (2021YFA1101204) to LW and XW; the National Natural Science Foundation of China 81900844 to XH and 82171041 to LW; the China Postdoctoral Science Foundation 2021M700776 and the Scientific Research Project of Guangdong Provincial Bureau of Traditional Chinese Medicine 20221373 to YZ; and the National Institute for Health Research (NIHR) Biomedical Research Centre at Moorfields Eye Hospital NHS (National Health Service) Foundation Trust and University College London Institute of Ophthalmology, UK (DAC, LPS, PJPL, MS, ADD and RWJL). The views expressed are those of the authors and not necessarily those of the NIHR or the UK's Department of Health and Social Care.

Metagenomic Profiling of the Ocular Surface Microbiome in Patients After Allogeneic Hematopoietic Stem Cell Transplantation.

PURPOSE: To investigate the characteristics of the ocular surface microbiome in patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and the associations between the microbial dysbiosis and chronic ocular graft-versus-host disease (oGVHD). DESIGN: Prospective cohort study. METHODS: Ocular surface samples from 48 healthy subjects and 76 patients after allo-HSCT, including 50 patients with chronic oGVHD and 26 patients without oGVHD, were collected. Species-level composition of the ocular surface microbiome was surveyed via metagenomic shotgun sequencing. OGVHD was diagnosed and graded according to the International Chronic Ocular GVHD Consensus Group criteria. RESULTS: The α-diversity of the microbiota was significantly decreased in patients after allo-HSCT. Nevertheless, we detected more types of viral species in the allo-HSCT group than the healthy group, especially anelloviruses. The mismatch of donor-recipient sex was only negatively associated with the α-diversity in male but not female recipients. Moreover, the microbiome of patients with oGVHD was distinct from patients without oGVHD. Gordonia bronchialis and Pseudomonas parafulva were enriched in patients with oGVHD and positively associated with International Chronic Ocular GVHD score. CONCLUSIONS: This study suggests that the ocular surface microbiome after allo-HSCT is characterized by a loss of diversity. Furthermore, the microbial dysbiosis at the ocular surface is associated with the status and severity of chronic oGVHD. These results lay the groundwork for future investigations of the potential microbial mechanism for oGVHD.

Identification of an intraocular microbiota.

The current dogma in ophthalmology and vision research presumes the intraocular environment to be sterile. However, recent evidence of intestinal bacterial translocation into the bloodstream and many other internal organs including the eyes, found in healthy and diseased animal models, suggests that the intraocular cavity may also be inhabited by a microbial community. Here, we tested intraocular samples from over 1000 human eyes. Using quantitative PCR, negative staining transmission electron microscopy, direct culture, and high-throughput sequencing technologies, we demonstrated the presence of intraocular bacteria. The possibility that the microbiome from these low-biomass communities could be a contamination from other tissues and reagents was carefully evaluated and excluded. We also provide preliminary evidence that a disease-specific microbial signature characterized the intraocular environment of patients with age-related macular degeneration and glaucoma, suggesting that either spontaneous or pathogenic bacterial translocation may be associated with these common sight-threatening conditions. Furthermore, we revealed the presence of an intraocular microbiome in normal eyes from non-human mammals and demonstrated that this varied across species (rat, rabbit, pig, and macaque) and was established after birth. These findings represent the first-ever evidence of intraocular microbiota in humans.

COVID-19 immune features revealed by a large-scale single-cell transcriptome atlas.

A dysfunctional immune response in coronavirus disease 2019 (COVID-19) patients is a recurrent theme impacting symptoms and mortality, yet a detailed understanding of pertinent immune cells is not complete. We applied single-cell RNA sequencing to 284 samples from 196 COVID-19 patients and controls and created a comprehensive immune landscape with 1.46 million cells. The large dataset enabled us to identify that different peripheral immune subtype changes are associated with distinct clinical features, including age, sex, severity, and disease stages of COVID-19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was found in diverse epithelial and immune cell types, accompanied by dramatic transcriptomic changes within virus-positive cells. Systemic upregulation of S100A8/A9, mainly by megakaryocytes and monocytes in the peripheral blood, may contribute to the cytokine storms frequently observed in severe patients. Our data provide a rich resource for understanding the pathogenesis of and developing effective therapeutic strategies for COVID-19.

Geographic Difference Shaped Human Ocular Surface Metagenome of Young Han Chinese From Beijing, Wenzhou, and Guangzhou Cities.

Purpose: Microbial ecosystems interact with the human body and affect human health. The microbial community on the ocular surface remains an underexplored territory despite its importance as the first line of defense barrier that protects the eye and ultimately sight. We investigated how age and sex affected human ocular surface microbiome, and in the present study wanted to understand how geographic difference shaped the microbiome in the ocular surface. Methods: We collected conjunctival specimens of 172 eyes from 86 healthy volunteers living in three Chinese cities, namely, Guangzhou, Wenzhou, and Beijing. Using the direct metagenomic shotgun sequencing approach, we characterized how geographic difference affected the human ocular microbiome. Results: We surveyed the taxonomic composition and metabolic function of the microbiota on human ocular surface. We showed that the ocular surface microbiota was composed of bacteria, viruses, and fungi. A geographical difference in both composition and function of the conjunctival microbiome suggests that the environment people lived in shapes their conjunctival microbiome, especially the dominate species. Conclusions: Our study provides a reference catalog of the healthy conjunctival metagenome and raises a concern for environmental influences on the ocular surface microbiome.

CSMD: a computational subtraction-based microbiome discovery pipeline for species-level characterization of clinical metagenomic samples.

MOTIVATION: Microbiome analyses of clinical samples with low microbial biomass are challenging because of the very small quantities of microbial DNA relative to the human host, ubiquitous contaminating DNA in sequencing experiments and the large and rapidly growing microbial reference databases. RESULTS: We present computational subtraction-based microbiome discovery (CSMD), a bioinformatics pipeline specifically developed to generate accurate species-level microbiome profiles for clinical samples with low microbial loads. CSMD applies strategies for the maximal elimination of host sequences with minimal loss of microbial signal and effectively detects microorganisms present in the sample with minimal false positives using a stepwise convergent solution. CSMD was benchmarked in a comparative evaluation with other classic tools on previously published well-characterized datasets. It showed higher sensitivity and specificity in host sequence removal and higher specificity in microbial identification, which led to more accurate abundance estimation. All these features are integrated into a free and easy-to-use tool. Additionally, CSMD applied to cell-free plasma DNA showed that microbial diversity within these samples is substantially broader than previously believed. AVAILABILITY AND IMPLEMENTATION: CSMD is freely available at https://github.com/liuyu8721/csmd. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Inhibition of Experimental Age-Related Macular Degeneration by ZQMT in Mice.

BACKGROUND: Age-related macular degeneration (AMD) is a progressive and irreversible eye disease. The anti-vascular endothelial growth factor (VEGF) therapy has revolutionized the treatment of neovascular AMD. However, the expense for such treatment is quite high. METHODS: We used a traditional Chinese medicine ZQMT as an alternative therapeutic regimen for AMD. We employed two in vivo animal models mimicking dry and wet AMD respectively to assess the therapeutic efficacy of ZQMT on treating AMD-related retinopathy. AMD-related retinopathy in Crb1rd8 mice was evaluated from week 1 to 8 by fundus photography. Laser-induced choroidal neovascularization (CNV) was evaluated by fluorescein angiography and histopathology. RESULTS: ZQMT increased CX3CR1 expression in murine CD4+ T cells either cultured in vitro or directly isolated from animals treated with ZQMT. We also performed both in vitro and in vivo studies to confirm that ZQMT has no apparent toxic effects. ZQMT alleviated AMD-related retinopathy in both Crb1rd8 and CNV models. Depletion of CCL2 and CX3CR1 in Crb1rd8 mice abolished the efficacy of ZQMT, suggesting that CCL2 and/or CX3CR1 may underlie the mechanisms of ZQMT in treating AMD-related retinopathy in mice. CONCLUSION: In summary, our study supports the protective roles of a traditional Chinese medicine ZQMT in AMD.

Association of IGFN1 variant with polypoidal choroidal vasculopathy.

BACKGROUND: Polypoidal choroidal vasculopathy (PCV) and neovascular age-related macular degeneration (nAMD) share a similar phenotype but are different in their clinical manifestations, responses to treatment and prognosis. Whether PCV is a subtype of AMD or a distinct entity from nAMD remains unknown. Therefore, we performed a whole-exome sequencing based association analysis to compare the genetic architecture of PCV and nAMD in Han Chinese. METHODS: Whole-exome sequencing analysis was performed on 21 nAMD cases, 20 PCV cases and 20 healthy controls. As a follow-up validation, 145 nAMD cases, 160 PCV cases and 193 controls were genotyped using the Sequenom MassARRAY platform (Sequenom, San Diego, CA, USA). RESULTS: A novel variant, c.6196A>G in the IGFN1 gene, was significantly associated with only PCV (combined p = 7.1 × 10-11 , odds ratio = 9.44), but not with nAMD (combined p = 0.683, odds ratio = 1.30). The minor allele G conferred an increased risk of PCV. CONCLUSIONS: The findings of the present study indicate that, although some of the susceptibility loci are shared between PCV and nAMD, a unique genetic signature may decide the pathogenesis of PCV.

Identification of a novel compound targeting the nuclear export of influenza A virus nucleoprotein.

Although antiviral drugs are available for the treatment of influenza infection, it is an urgent requirement to develop new antiviral drugs regarding the emergence of drug-resistant viruses. The nucleoprotein (NP) is conserved among all influenza A viruses (IAVs) and has no cellular equivalent. Therefore, NP is an ideal target for the development of new IAV inhibitors. In this study, we identified a novel anti-influenza compound, ZBMD-1, from a library of 20,000 compounds using cell-based influenza A infection assays. We found that ZBMD-1 inhibited the replication of H1N1 and H3N2 influenza A virus strains in vitro, with an IC50 ranging from 0.41-1.14 µM. Furthermore, ZBMD-1 inhibited the polymerase activity and specifically impaired the nuclear export of NP. Further investigation indicated that ZBMD-1 binds to the nuclear export signal 3 (NES3) domain and the dimer interface of the NP pocket. ZBMD-1 also protected mice that were challenged with lethal doses of A/PR/8/1934 (H1N1) virus, effectively relieving lung histopathology changes, as well as strongly inhibiting the expression of pro-inflammatory cytokines/chemokines, without inducing toxicity effects in mice. These results suggest that ZBMD-1 is a promising anti-influenza compound which can be further investigated as a useful strategy against IAVs in the future.

The Influence of Age and Sex on Ocular Surface Microbiota in Healthy Adults.

Purpose: A growing body of evidence suggests that the microbiome of the ocular surface confers potent immunoregulatory functions and has a key role in the physiologic maintenance of healthy eyes and in the pathogenesis of ocular diseases. Although the microbiome is known to be affected by age and sex, the influence of these factors on ocular surface microbiota in healthy adults remains largely unknown. Methods: Ocular surface microbiome samples were obtained from the inferior bulbar conjunctiva of 48 young and 42 old adults at Zhongshan Ophthalmic Center. Using metagenomic shotgun sequencing, we characterized the sex- and age-differences in conjunctival microbiome profiles of healthy adults. Results: Male and female groups differed only in the ß diversity of bacterial communities, while there were significant differences in bacterial composition, metabolic functions, and the abundance of antibiotic resistance genes between young and old adult groups. Conclusions: Our findings suggest that age and sex collectively shape the conjunctival microbiome, and may change the immune homeostasis of the ocular surface through alterations of its commensal microbiome.

Host Protein Moloney Leukemia Virus 10 (MOV10) Acts as a Restriction Factor of Influenza A Virus by Inhibiting the Nuclear Import of the Viral Nucleoprotein.

UNLABELLED: The viral ribonucleoprotein (vRNP) complex of influenza A viruses (IAVs) contains an RNA-dependent RNA polymerase complex (RdRp) and nucleoprotein (NP) and is the functional unit for viral RNA transcription and replication. The vRNP complex is an important determinant of virus pathogenicity and host adaptation, implying that its function can be affected by host factors. In our study, we identified host protein Moloney leukemia virus 10 (MOV10) as an inhibitor of IAV replication, since depletion of MOV10 resulted in a significant increase in virus yield. MOV10 inhibited the polymerase activity in a minigenome system through RNA-mediated interaction with the NP subunit of vRNP complex. Importantly, we found that the interaction between MOV10 and NP prevented the binding of NP to importin-α, resulting in the retention of NP in the cytoplasm. Both the binding of MOV10 to NP and its inhibitory effect on polymerase activity were independent of its helicase activity. These results suggest that MOV10 acts as an anti-influenza virus factor through specifically inhibiting the nuclear transportation of NP and subsequently inhibiting the function of the vRNP complex. IMPORTANCE: The interaction between the influenza virus vRNP complex and host factors is a major determinant of viral tropism and pathogenicity. Our study identified MOV10 as a novel host restriction factor for the influenza virus life cycle since it inhibited the viral growth rate. Conversely, importin-α has been shown as a determinant for influenza tropism and a positive regulator for viral polymerase activity in mammalian cells but not in avian cells. MOV10 disrupted the interaction between NP and importin-α, suggesting that MOV10 could also be an important host factor for influenza virus transmission and pathogenicity. Importantly, as an interferon (IFN)-inducible protein, MOV10 exerted a novel mechanism for IFNs to inhibit the replication of influenza viruses. Furthermore, our study potentially provides a new drug design strategy, the use of molecules that mimic the antiviral mechanism of MOV10.

RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs.

Human immunodeficiency virus type 1 (HIV-1) exploits multiple host factors during its replication. The REV/RRE-dependent nuclear export of unspliced/partially spliced viral transcripts needs the assistance of host proteins. Recent studies have shown that MOV10 overexpression inhibited HIV-1 replication at various steps. However, the endogenous MOV10 was required in certain step(s) of HIV-1 replication. In this report, we found that MOV10 potently enhances the nuclear export of viral mRNAs and subsequently increases the expression of Gag protein and other late products through affecting the Rev/RRE axis. The co-immunoprecipitation analysis indicated that MOV10 interacts with Rev in an RNA-independent manner. The DEAG-box of MOV10 was required for the enhancement of Rev/RRE-dependent nuclear export and the DEAG-box mutant showed a dominant-negative activity. Our data propose that HIV-1 utilizes the anti-viral factor MOV10 to function as a co-factor of Rev and demonstrate the complicated effects of MOV10 on HIV-1 life cycle.