Purpose:Uveitis (inflammation inside the eye) is a disabling manifestation of the post-Ebola syndrome that affects 10% to 35% of individuals who survive the infection. Post-Ebola uveitis presents with diverse clinical features but frequently involves the posterior segment of the eye, where the retinal pigment epithelium plays a key role in directing immune responses. Our previous work shows that this epithelium is relatively susceptible to infection with Zaire ebolavirus (EBOV), the strain responsible for most Ebola outbreaks. In addition to production roles, viral proteins may act to alter the molecular responses of host cells. Methods:We investigated the activity of EBOV viral protein 24 (VP24) in human retinal pigment epithelial cells. An EBOV VP24 expression plasmid was constructed in-house. Multiple primary cell isolates were lipofectamine-transfected, first with VP24 or control expression plasmids and then with polyinosinic-polycytidylic acid (poly I:C) to simulate viral RNA. A type I interferon (IFN) response to transfection was confirmed by an IFN-β enzyme-linked immunosorbent assay. Cellular immune responses after 4- and 24-h exposures to poly I:C were characterized by reverse transcription-quantitative polymerase chain reaction. Results:Multidimensional scaling, drawing on 19 immune response-related gene transcripts, covering antiviral, immunomodulatory, and proinflammatory molecules, demonstrated changes in gene expression profiles following transfection. Analysis of individual cell isolates showed a range of changes, including upregulation and downregulation of different gene transcripts across the two investigated time points. Conclusions:Our findings suggest VP24 elicits variable immune responses from human retinal pigment epithelial cells, potentially contributing to the variation in clinical presentations of uveitis in Ebola survivors.

Purpose:Ocular toxoplasmosis, caused by infection with Toxoplasma gondii, is characterized by retinal necrosis and reactive intraocular inflammation. Müller glial cells are a principal retinal host cell population for T. gondii. The goal of this research was to delineate potential involvements of Müller glial cells in ocular toxoplasmosis at a molecular level. Design:Laboratory-based study. Samples:Human retinal Müller glial cells infected with T. gondii plus noninfected cells. Methods:Monolayers of Müller glial cells isolated from human retina (6 donor eye pairs) were infected for 24 hours with GT1 or GPHT strain T. gondii tachyzoites (multiplicity of infection of 5), or incubated in parallel without infection. Total RNA and small RNA were extracted from cell monolayers, sequenced on the Illumina NovaSeq 6000 and NextSeq 550 platforms, respectively, and aligned to GRCh38. Transcriptomic responses to infection with each strain were compared for differential expression (false discovery rate <5% and twofold change). These data were interrogated for enrichment of Reactome and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and Gene Ontology in InnateDB; putative transcription factor binding sites in HOMER; and potential microRNA-mRNA interactions in multiMiR. Main Outcome Measures:Total and small RNA transcriptomes. Results:6.3% of total RNA and 2.4% of small RNA changed in GT1-infected cells (582 upregulated and 210 downregulated transcripts and 20 upregulated microRNAs) versus 4.3% of total RNA and 1.5% of small RNA in GPHT-infected cells (400 upregulated and 137 downregulated transcripts and 12 upregulated microRNAs). Seventy-six transcripts and 4 microRNAs were different between strains; most were increased by both, but GT1 induced higher levels than GPHT. Enriched pathways and ontologies were dominated by DNA replication and intracellular metabolic activities, and the immune response for GT1 and GPHT. Seven of 8 transcription factor binding sites were shared for GT1 and GPHT infections, all overexpressed, including sites for p65/RELA and E2F family members. Across the strains, miR-18a-5p was the most connected microRNA in predicted mRNA target networks. Conclusions:This work demonstrates that human retinal Müller glial cells shift to a proliferative and inflammatory phenotype when infected with T. gondii tachyzoites, consistent with a central role in the characteristic pathology of ocular toxoplasmosis. Financial Disclosures:Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Aim: Severe dengue is correlated with a decrease in the circulating complement regulator, factor H (FH) and prior work has shown that dengue virus (DENV) infection induces FH mRNA but not FH protein release. Here, the mechanisms of this phenomenon were defined. Methods: HEK293 cells were infected with DENV-2 and changes in FH mRNA and protein were analyzed by real time reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and fluorescent microscopy. Additionally, cells were stimulated with size fractionated supernatants from DENV-infected cells, supernatant containing DENV non-structural protein-1 (NS1) without virus particles, and infections performed with or without the toll-like receptor-4 (TLR4) antagonist, TAK-242. Mass spectrometry was used to define the protein content of the fractionated supernatant, and treatment of cells with sialidase or heparinase was used to define cell-associated FH protein. Results: DENV-infection induced full-length FH mRNA and cell-associated FH protein. Microscopy demonstrated membrane and intracellular-associated FH with a cytoskeletal and perinuclear localisation, in both DENV positive and uninfected neighboring bystander cells. Fractionation of cultured supernatant from DENV-infected cells demonstrated that secreted factors > 50 (kilodaltons) kDa induced FH mRNA and this could be blocked with TAK-242 but was not simulated by the TLR4 agonist, DENV NS1. Mass spectrometry detected DENV envelope, membrane and NS1, complement component 5 (C5), and complement FB, and indicated a > 20-fold increase in C4, inter-alpha-trypsin inhibitor heavy chain H2 (ITIH2), and alpha-2-macroglobulin in the > 50 kDa fraction from DENV-infected compared with conditioned media from uninfected cells. Sialic acid levels were unchanged and cleavage did not affect release of FH from DENV-infected compared to uninfected cells. In contrast, sulphated glycosaminoglycans (GAGs) were reduced in the cultured supernatant and cell lysates following DENV-infection, and heparinase cleavage released significantly more FH from DENV-compared with uninfected cells. Conclusions: Following DENV-infection, secreted molecules induce FH that remains intracellular and with increased binding to cell surface heparan sulphate. The mediators of induction of FH mRNA act in trans and via TLR4 but this is not likely to be via DENV NS1. The retention of FH in the local environment of the infected cell could benefit the virus by negating local complement killing of cells, and/or benefit the host by inhibition of heparan sulphate-mediated DENV infection to restrict viral spread.

Intercellular adhesion molecule 1 (ICAM-1) is a central cell adhesion molecule for retinal transendothelial migration of the leukocytes in non-infectious posterior uveitis. Inhibiting ICAM1 gene transcription reduces induction of ICAM-1 in inflamed retinal endothelium. Based on published literature implicating transcription factor ETS-1 as an activator of ICAM1 gene transcription, we investigated the effect of ETS-1 blockade on ICAM-1 levels in cytokine-stimulated human retinal endothelial cells. We first examined ICAM1 and ETS1 transcript expression in human retinal endothelial cells exposed to tumor necrosis factor-alpha (TNF-α) or interleukin-1beta (IL-1β). ICAM1 and ETS1 transcripts were increased in parallel in primary human retinal endothelial cell isolates (n = 5) after a 4-hour stimulation with TNF-α or IL-1β (p ≤ 0.012 and ≤ 0.032, respectively). We then assessed the effect of ETS-1 blockade by small interfering (si)RNA on cellular ICAM1 transcript and membrane-bound ICAM-1 protein. ETS1 transcript was reduced by greater than 90% in cytokine-stimulated and non-stimulated human retinal endothelial cell monolayers following a 48-hour treatment with two ETS-1-targeted siRNA, in comparison to negative control non-targeted siRNA (p ≤ 0.0002). The ETS-1 blockade did not reduce ICAM1 transcript expression nor levels of membrane-bound ICAM-1 protein, rather it increased both for a majority of siRNA-treatment and cytokine-stimulation conditions (p ≤ 0.018 and ≤ 0.004, respectively). These unexpected findings indicate that ETS-1 blockade increases ICAM-1 transcript and protein levels in human retinal endothelial cells. Thus ETS-1-targeting would be expected to promote rather than inhibit retinal transendothelial migration of leukocytes in non-infectious posterior uveitis.

Uveitis, or intraocular inflammation, is a potentially blinding condition that mostly affects the working-age population. The cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1β, play a role in the pathogenesis of non-infectious uveitis and have been linked to the breakdown of the inner blood-retinal barrier, composed mainly of retinal endothelial cells, leading to macular oedema and vascular leakage. However, the effects of TNF-α and IL-1β on human retinal endothelial function are not fully understood. In this work, we investigated the impact of TNF-α and IL-1β on several aspects of human retinal endothelial cell biology. Through a real-time biosensor, the impact of TNF-α and IL-1β on formation of a retinal endothelial cell barrier was analyzed. Changes in junctional components were assessed via RT-qPCR and immunolabelling. Cell survival, necrosis and apoptosis were appraised via cell proliferation and flow cytometric studies. Tumor necrosis factor-α and IL-1β impaired the electrical resistance of the retinal endothelial cell barrier, while the addition of a potentially barrier-impairing cytokine, IL-6, did not enhance the effect of TNF-α and IL-1β. Level of the gene transcript encoding zonula occludens (ZO)-1 was diminished, while ZO-1 protein configuration was changed by TNF-α and IL-1β. Both cytokines affected human retinal endothelial cell proliferation and viability, while only TNF-α increased rates of necrosis. These results indicate that TNF-α and IL-1β are important drivers of retinal endothelial dysfunction in non-infectious uveitis, suggesting that targeting these cytokines is critical when treating complications of uveitis, such as macular oedema and vascular leakage.

As a key host protein involved in cellular infection by the severe acute respiratory syndrome coronavirus (SARS-CoV-2), angiotensin converting enzyme (ACE)2 is an ideal target for antiviral drugs. Manipulation of transcription provides opportunity for graduated blockade that preserves physiological functions. We sought to develop a model system for evaluating manipulation of ACE2 gene transcription using human retinal pigment epithelium. Retinal pigment epithelial cell isolates were prepared from human posterior eyecups (n = 11 individual isolates). The cells expressed ACE2 transcript and protein, and expression was not induced by hypoxia mimetic dimethyloxaloylglycine, or inflammatory cytokine IL-1β. ACE2 gene transcription factors were predicted in silico and cross-referenced with the human retinal pigment epithelial cell transcriptome, and five candidate transcription factors were identified: ETS proto-oncogene 1 transcription factor (ETS1), nuclear factor I C (NFIC), nuclear receptor subfamily 2 group C member 1 (NR2C1), TEA domain transcription factor 1 (TEAD1), and zinc finger protein 384 (ZNF384). The candidates were individually targeted in cells by transfection with small interfering (si)RNA. Knockdowns reduced mean cellular expression of all the transcription factors in comparison to expression in cells transfected with control non-targeted siRNA. Mean cellular ACE2 transcript was reduced under the condition of NR2C1 knockdown, but not for ETS1, NFIC, TEAD1, and ZNF384 knockdowns. Our findings build on previous work demonstrating the potential for drugging gene transcription. Importantly, we show the value of human retinal pigment epithelium as a system for evaluating ACE2 transcriptional blockade, a possible approach for treating SARS-CoV-2 infection. Brief Research Report.

Interleukin (IL)-6 is an inflammatory cytokine present in the eye during non-infectious uveitis, where it contributes to the progression of inflammation. There are two major IL-6 signaling pathways: classic signaling and trans-signaling. Classic signaling requires cellular expression of the IL-6 receptor (IL-6R), which exists in membrane-bound (mIL-6R) and soluble (sIL-6R) forms. Prevailing dogma is that vascular endothelial cells do not produce IL-6R, relying on trans-signaling during inflammation. However, the literature is inconsistent, including with respect to human retinal endothelial cells. We examined IL-6R transcript and protein expression in multiple primary human retinal endothelial cell isolates, and assessed the effect of IL-6 on the transcellular electrical resistance of monolayers. Using reverse transcription-polymerase chain reaction, IL-6R, mIL-6R and sIL-6R transcripts were amplified in 6 primary human retinal endothelial isolates. Flow cytometry on 5 primary human retinal endothelial cell isolates under non-permeabilizing conditions and following permeabilization demonstrated intracellular stores of IL-6R and the presence of mIL-6R. When measured in real-time, transcellular electrical resistance of an expanded human retinal endothelial cell isolate, also shown to express IL-6R, decreased significantly on treatment with recombinant IL-6 in comparison to non-treated cells across 5 independent experiments. Our findings indicate that human retinal endothelial cells produce IL-6R transcript and functional IL-6R protein. The potential for classic signaling in human retinal endothelial cells has implications for the development of therapeutics targeted against IL-6-mediated pathology in non-infectious uveitis.

Aboriginal and Torres Strait Islander People (respectfully referred to as Indigenous Australians herein) are disparately burdened by many infectious and chronic diseases relative to Australians with European genetic ancestry. Some of these diseases are described in other populations to be influenced by the inherited profile of complement genes. These include complement factor B, H, I and complement factor H-related (CFHR) genes that can contribute to a polygenic complotype. Here the focus is on the combined deletion of CFHR1 and 3 to form a common haplotype (CFHR3–1Δ). The prevalence of CFHR3–1Δ is high in people with Nigerian and African American genetic ancestry and correlates to a higher frequency and severity of systemic lupus erythematosus (SLE) but a lower prevalence of age-related macular degeneration (AMD) and IgA-nephropathy (IgAN). This pattern of disease is similarly observed among Indigenous Australian communities. Additionally, the CFHR3–1Δ complotype is also associated with increased susceptibility to infection with pathogens, such as Neisseria meningitidis and Streptococcus pyogenes, which also have high incidences in Indigenous Australian communities. The prevalence of these diseases, while likely influenced by social, political, environmental and biological factors, including variants in other components of the complement system, may also be suggestive of the CFHR3–1Δ haplotype in Indigenous Australians. These data highlight a need to define the Indigenous Australian complotypes, which may lead to the discovery of new risk factors for common diseases and progress towards precision medicines for treating complement-associated diseases in Indigenous and non-Indigenous populations. Herein, the disease profiles suggestive of a common complement CFHR3–1Δ control haplotype are examined.

The interaction between leukocytes and cytokine-activated retinal endothelium is an initiating step in non-infectious uveitis involving the posterior eye, mediated by cell adhesion molecules. However, because cell adhesion molecules are required for immune surveillance, therapeutic interventions would ideally be employed indirectly. Using 28 primary human retinal endothelial cell isolates, this study sought to identify transcription factor targets for reducing levels of the key retinal endothelial cell adhesion molecule, intercellular adhesion molecule (ICAM)-1, and limiting leukocyte binding to the retinal endothelium. Five candidate transcription factors—C2CD4B, EGR3, FOSB, IRF1, and JUNB—were identified by differential expression analysis of a transcriptome generated from IL-1β- or TNF-α-stimulated human retinal endothelial cells, interpreted in the context of the published literature. Further filtering involved molecular studies: of the five candidates, C2CD4B and IRF1 consistently demonstrated extended induction in IL-1β- or TNF-α-activated retinal endothelial cells and demonstrated a significant decrease in both ICAM-1 transcript and ICAM-1 membrane-bound protein expression by cytokine-activated retinal endothelial cells following treatment with small interfering RNA. RNA interference of C2CD4B or IRF1 significantly reduced leukocyte binding in a majority of human retinal endothelial cell isolates stimulated by IL-1β or TNF-α. Our observations suggest that the transcription factors C2CD4B and IRF1 may be potential drug targets for limiting leukocyte–retinal endothelial cell interactions in non-infectious uveitis involving the posterior eye.

Uveitis is a common manifestation of post-Ebola syndrome, associated with persistence of Ebola virus (EBOV; Zaire ebolavirus ) inside the eye. The iris and retinal pigment epithelia are key components of the blood-ocular barriers, but have the capacity to act as hosts for microorganisms. We investigated the ability of EBOV to productively infect these cell populations. Donor-matched human iris and retinal pigment epithelial isolates (n = 5) were infected with EBOV at a multiplicity of infection of 1 for up to 72 hours. Parallel cultures were infected with Reston virus (RESTV; Reston ebolavirus ) or Zika virus (ZIKV), or held uninfected under the same conditions. Viral transcript expression by RT-qPCR on total cellular RNA, cytoimmunofluorescence, and assays of 50% tissue culture infected dose of culture supernatant showed that both iris and retinal pigment epithelial isolates were permissive to infection, and supported replication and release of EBOV, as well as RESTV and ZIKV. However, in comparison to cells isolated from iris, those from retina demonstrated obvious EBOV-induced cytopathic effect, had higher intracellular EBOV nucleoprotein transcript, expressed intracellular EBOV protein more widely, and released EBOV at higher titer. Comparable results were obtained for isolates infected with RESTV and ZIKV. Consistent with observations of retinal pigment epithelial scars in Ebola survivors, our results suggest that an early event in post-Ebola uveitis is infection of the retinal pigment epithelium. Relative susceptibility of retinal pigment epithelial cells to infection with RESTV and ZIKV, as well as EBOV, implies this phenomenon may relate to a cell-specific attribute, such as high phagocytic activity.

Retinal endothelial cell activation is a central event in non-infectious posterior uveitis. There is recent interest in long non-coding (lnc)RNA-targeted therapeutics for retinal diseases. We aimed to identify human retinal endothelial cell lncRNAs that might be involved in activation. Eleven candidate lncRNAs were identified: GAS5, KCNQ1OT1, LINC00294, MALAT1, MEG3, MIR155HG, NEAT1, NORAD, OIP5-AS1, SENCR, TUG1. Expression was assessed by RT-PCR in human retinal endothelial cells, at baseline and following activation with interleukin (IL)-1β and tumor necrosis factor (TNF)-α. IL-1β significantly upregulated MEG3 and SENCR at 4 and 24 hours; LINC00294, NORAD, OIP5-AS1 and TUG1 at 24 hours; and MIR155HG at 4, 24 and 48 hours; but downregulated GAS5 at 24 and 48 hours. TNF-α significantly upregulated KCNQ1OT1, LINC00294, MEG3, NORAD and SENCR at 4 hours; SENCR and TUG1 at 24 hours; and MIR155HG at all time points. Future studies involving manipulation of MIR155HG may be warranted to explore potential therapeutic applications for non-infectious posterior uveitis.

Purpose: Molecular profiling of human retinal endothelial cells provides opportunities to understand the roles of this cell population in maintenance of the blood-ocular barrier, and its involvements in diverse retinal vasculopathies. We aimed to generate a transcriptome of human retinal endothelial cells in the unstimulated state, and following treatment with inflammatory cytokines linked to cell dysfunction. Methods: Endothelial cells were isolated from retinae of five human cadaveric donors, and treated for 60 minutes and 24 hours with interleukin-1β or tumor necrosis factor-α, or exposed to medium alone for the same intervals. Expression of intercellular adhesion molecule-1 was measured by RT-qPCR to confirm cytokine-induced activation of the cells. RNA was sequenced on the Illumina NovaSeq 6000 platform. Reads were aligned to the human GRCh38 genome, and reads that aligned to Ensembl-annotated genes were counted. Quality control of sequencing was performed with FastQC, and sequences were classified by Kraken. Results: A human retinal endothelial cell RNA-sequencing dataset with mean of 99% reads aligned to the human genome was produced as raw RNA sequence data (FASTQ files) and processed read data (XLSX files). Multidimensional scaling analysis showed a strong donor effect, which was readily controlled by ComBat. Conclusions: Our dataset may be useful for human retinal endothelial cell transcriptomic assemblies, functional gene annotating and/or gene expression and enrichment analyses, as well as cross-dataset harmonization. Translational Relevance: The molecular profile of the human retinal endothelium is a source of candidate biologic targets for retinal vasculopathies.

Prevalence of dengue retinopathy varies across epidemics, with the disease linked to circulation of dengue virus serotype 1 (DENV-1). The retinal pigment epithelium has been implicated in the pathology. We investigated infectivity, molecular response, and barrier function of epithelial cells inoculated with DENV strains from different outbreaks in Singapore. Monolayers of human retinal pigment epithelial cells (multiple primary cell isolates and the ARPE-19 cell line) were inoculated with six DENV strains, at multiplicity of infection of 10; uninfected and recombinant strain-infected controls were included where relevant. Infectivity and cell response were assessed primarily by RT-qPCR on total cellular RNA, and barrier function was evaluated as electrical resistance across monolayers. Higher viral RNA loads were measured in human retinal pigment epithelial cells infected with DENV-1 strains from the 2005 Singapore epidemic, when retinopathy was prevalent, versus DENV-1 strains from the 2007 Singapore epidemic, when retinopathy was not observed. Type I interferon (IFN) transcripts (IFN-β and multiple IFN-stimulated genes) were up-regulated, and impact on barrier function was more pronounced, for cells infected with DENV-1 strains from the 2005 versus the 2007 Singapore epidemics. Aside from serotype, strain of DENV may determine the potential to induce retinal pathology. Identification of molecular markers of disease-associated DENV strains may provide insights into the pathogenesis of dengue retinopathy.

During recent Zika epidemics, adults infected with Zika virus (ZIKV) have developed organ-specific inflammatory complications. The most serious Zika-associated inflammatory eye disease is uveitis, which is commonly anterior in type, affecting both eyes and responding to corticosteroid eye drops. Mechanisms of Zika-associated anterior uveitis are unknown, but ZIKV has been identified in the aqueous humor of affected individuals. The iris pigment epithelium is a target cell population in viral anterior uveitis, and it acts to maintain immune privilege within the anterior eye. Interactions between ZIKV and human iris pigment epithelial cells were investigated with infectivity assays and RNA-sequencing. Primary cell isolates were prepared from eyes of 20 cadaveric donors, and infected for 24 hours with PRVABC59 strain ZIKV or incubated uninfected as control. Cytoimmunofluorescence, RT-qPCR on total cellular RNA, and focus-forming assays of culture supernatant showed cell isolates were permissive to infection, and supported replication and release of infectious ZIKV. To explore molecular responses of cell isolates to ZIKV infection at the whole transcriptome level, RNA was sequenced on the Illumina NextSeq 500 platform, and results were aligned to the human GRCh38 genome. Multidimensional scaling showed clear separation between transcriptomes of infected and uninfected cell isolates. Differential expression analysis indicated a vigorous molecular response of the cell to ZIKV: 7,935 genes were differentially expressed between ZIKV-infected and uninfected cells (FDR < 0.05), and 99% of 613 genes that changed at least two-fold were up-regulated. Reactome and KEGG pathway and Gene Ontology enrichment analyses indicated strong activation of viral recognition and defense, in addition to biosynthesis processes. A CHAT network included 6275 molecular nodes and 24 contextual hubs in the cell response to ZIKV infection. Receptor-interacting serine/threonine kinase 1 (RIPK1) was the most significantly connected contextual hub. Correlation of gene expression with read counts assigned to the ZIKV genome identified a negative correlation between interferon signaling and viral load across isolates. This work represents the first investigation of mechanisms of Zika-associated anterior uveitis using an in vitro human cell model. The results suggest the iris pigment epithelium mounts a molecular response that limits intraocular pathology in most individuals.

Ocular toxoplasmosis is the commonest clinical manifestation of infection with obligate intracellular parasite, Toxoplasma gondii. Active ocular toxoplasmosis is characterized by replication of T. gondii tachyzoites in the retina, with reactive inflammation. The multifunctional retinal pigment epithelium is a key target cell population for T. gondii. Since the global gene expression profile is germane to understanding molecular involvements of retinal pigment epithelial cells in ocular toxoplasmosis, we performed RNA-Sequencing (RNA-Seq) of human cells following infection with T. gondii tachyzoites. Primary cell isolates from eyes of cadaveric donors (n = 3), and the ARPE-19 human retinal pigment epithelial cell line, were infected for 24 h with GT-1 strain T. gondii tachyzoites (multiplicity of infection = 5) or incubated uninfected as control. Total and small RNA were extracted from cells and sequenced on the Illumina NextSeq 500 platform; results were aligned to the human hg19 reference sequence. Multidimensional scaling showed good separation between transcriptomes of infected and uninfected primary cell isolates, which were compared in edgeR software. This differential expression analysis revealed a sizeable response in the total RNA transcriptome- with significantly differentially expressed genes totaling 7,234 (28.9% of assigned transcripts)-but very limited changes in the small RNA transcriptome -totaling 30 (0.35% of assigned transcripts) and including 8 microRNA. Gene ontology and pathway enrichment analyses of differentially expressed total RNA in CAMERA software, identified a strong immunologic transcriptomic signature. We conducted RT-qPCR for 26 immune response-related protein-coding and long non-coding transcripts in epithelial cell isolates from different cadaveric donors (n = 3), extracted by a different isolation protocol but similarly infected with T. gondii, to confirm immunological activity of infected cells. For microRNA, increases in miR-146b and miR-212 were detected by RT-qPCR in 2 and 3 of these independent cell isolates. Biological network analysis in the InnateDB platform, including 735 annotated differentially expressed genes plus 2,046 first-order interactors, identified 10 contextural hubs and 5 subnetworks in the transcriptomic immune response of cells to T. gondii. Our observations provide a solid base for future studies of molecular and cellular interactions between T. gondii and the human retinal pigment epithelium to illuminate mechanisms of ocular toxoplasmosis.

OBJECTIVE:Toxoplasmosis may follow consumption of undercooked meat containing Toxoplasma gondii cysts. Lamb is considered to pose the highest risk for contamination across meats. Red meat is often served undercooked, yet there are no current data on T. gondii contamination of Australian sourced and retailed lamb. We sought to address this gap in public health knowledge.METHODS:Lamb mincemeat was purchased at the supermarket counter three times weekly for six months. T. gondii was detected by real-time polymerase chain reaction (PCR) of DNA extracted from the meat following homogenisation. Purchases were also tested for common foodborne bacterial pathogens.RESULTS:Conservative interpretation of PCR testing (i.e. parasite DNA detected in three of four tests) gave a probability of 43% (95% confidence interval, 32%-54%) that lamb mincemeat was contaminated with T. gondii. None of the purchases were contaminated with Campylobacter jejuni, Salmonella species or S. enterica serovar Typhimurium, indicating sanitary meat processing.CONCLUSIONS:Australian lamb is commonly contaminated with T. gondii. Future studies should be directed at testing a range of red meats and meat cuts. Implications for public health: Consuming undercooked Australian lamb has potential to result in toxoplasmosis. There may be value in health education around this risk.

PURPOSE. Retinal damage in ocular toxoplasmosis reflects Toxoplasma gondii-induced cell lysis and reactive inflammation. Human retinal histopathology demonstrates the presence of neutrophils, but activities of this leukocyte subset are unstudied. We conducted in vitro experiments to evaluate roles for neutrophils as retinal taxis for T. gondii and as contributors to the inflammation.METHODS. Human neutrophils were isolated from peripheral blood. Migration to diseaserelevant chemokines was evaluated in transwells, seeded with human retinal endothelial cells for some assays, using neutrophils infected with GT-1 strain T. gondii tachyzoites. Neutrophils were cocultured with T. gondii-infected ARPE-19 and primary human retinal pigment epithelial cells, and production of reactive oxygen species (ROS) was estimated by dihydroethidium reaction. Proteins produced by T. gondii-infected ARPF.-19 cells were profiled by immunoarray, and candidate neutrophil-activating proteins were targeted with specific blocking antibody in coculture assays.RESULTS. Infection with T. gondii arrested neutrophil migration across retinal endothelium regardless of the presence of CXCL8. Migration to CXCL1, CXCL2, and CXCL8 also was significantly inhibited in infected neutrophils. Neutrophils generated more ROS when cocultured with infected versus uninfected ARPE-19 cells and three of four primary retinal pigment epithelial cell isolates. Infected ARPE-19 cells augmented the synthesis of 12 neutrophil-activating proteins also expressed by primary retinal pigment epithelial cells. Antibody blockade of granulocyte-macrophage colony-stimulating factor, interleukin-6 (1L-6) and 11,18 significantly reduced ROS production by neutrophils cocultured with T. gondii-infected ARPE-19 cells.CONCLUSIONS. Our findings support involvement of neutrophils in retinal inflammation, but not parasite transport, in the setting of ocular toxoplasmosis.

Retinal infection with Toxoplasma gondii—ocular toxoplasmosis—is a common cause of vision impairment worldwide. Pathology combines parasite-induced retinal cell death and reactive intraocular inflammation. Müller glial cells, which represent the supporting cell population of the retina, are relatively susceptible to infection with T. gondii. We investigated expression of long non-coding RNAs (lncRNAs) with immunologic regulatory activity in Müller cells infected with virulent T. gondii strains—GT1 (haplogroup 1, type I) and GPHT (haplogroup 6). We first confirmed expression of 33 lncRNA in primary cell isolates. MIO-M1 human retinal Müller cell monolayers were infected with T. gondii tachyzoites (multiplicity of infection = 5) and harvested at 4, 12, 24, and 36 h post-infection, with infection being tracked by the expression of parasite surface antigen 1 (SAG1). Significant fold-changes were observed for 31 lncRNAs at one or more time intervals. Similar changes between strains were measured for BANCR, CYTOR, FOXD3-AS1, GAS5, GSTT1-AS1, LINC-ROR, LUCAT1, MALAT1, MIR22HG, MIR143HG, PVT1, RMRP, SNHG15, and SOCS2-AS1. Changes differing between strains were measured for APTR, FIRRE, HOTAIR, HOXD-AS1, KCNQ1OT1, LINC00968, LINC01105, lnc-SGK1, MEG3, MHRT, MIAT, MIR17HG, MIR155HG, NEAT1, NeST, NRON, and PACER. Our findings suggest roles for lncRNAs in regulating retinal Müller cell immune responses to T. gondii, and encourage future studies on lncRNA as biomarkers and/or drug targets in ocular toxoplasmosis.