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Human leucocyte antigen B*27 (HLA-B*27) is strongly associated with inflammatory diseases of the spine and pelvis (for example, ankylosing spondylitis (AS)) and the eye (that is, acute anterior uveitis (AAU))1. How HLA-B*27 facilitates disease remains unknown, but one possible mechanism could involve presentation of pathogenic peptides to CD8+ T cells. Here we isolated orphan T cell receptors (TCRs) expressing a disease-associated public β-chain variable region–complementary-determining region 3β (BV9–CDR3β) motif2,3,4 from blood and synovial fluid T cells from individuals with AS and from the eye in individuals with AAU. These TCRs showed consistent α-chain variable region (AV21) chain pairing and were clonally expanded in the joint and eye. We used HLA-B*27:05 yeast display peptide libraries to identify shared self-peptides and microbial peptides that activated the AS- and AAU-derived TCRs. Structural analysis revealed that TCR cross-reactivity for peptide–MHC was rooted in a shared binding motif present in both self-antigens and microbial antigens that engages the BV9–CDR3β TCRs. These findings support the hypothesis that microbial antigens and self-antigens could play a pathogenic role in HLA-B*27-associated disease.

Uveitis is a heterogeneous collection of diseases. We tested the hypothesis that despite the diversity of uveitides, there could be common mechanisms shared by multiple subtypes, and that evidence of these common mechanisms may be detected as gene expression profiles in whole blood.Cohort study.Ninety subjects with uveitis including axial spondyloarthritis (n = 17), sarcoidosis (n = 13), inflammatory bowel disease (n = 12), tubulointerstitial nephritis with uveitis (n = 10), or idiopathic uveitis (n = 38) as well as 18 healthy controls were enrolled, predominantly at Oregon HealthScience University. RNA-Seq data generated from peripheral, whole blood identified 19,859 unique transcripts. We analyzed gene expression pathways via Kyoto Encyclopedia of Genes and Genomes and Gene Ontology (GO). We validated our list of upregulated genes by comparison to a previously published study on peripheral blood gene expression among 50 subjects with diverse forms of uveitis.Both the Kyoto Encyclopedia of Genes and Genomes and GO analysis identified multiple shared pathways or GO terms with a P value of.0001. Almost all pathways related to the immune response and/or response to an infection. A total of 119 individual transcripts were upregulated by at least 1.5-fold and false discovery rate.05, and 61 were downregulated by similar criteria. Comparing mRNA from our study with a false discovery rate.05 and the prior report, we identified 10 common gene transcripts: ICAM1, IL15RA, IL15, IRF1, IL10RB, GSK3A, TYK2, MEF2A, MEF2B, and MEF2D.Many forms of uveitis share overlapping mechanisms. These data support the concept that a single therapeutic approach could benefit diverse forms of this disease.

Purpose To identify molecular features that distinguish individuals with shared clinical features of granulomatous uveitis. Design Cross-sectional observational study. Participants Four eyes from patients with active granulomatous uveitis. Methods We performed single-cell RNA sequencing with antigen-receptor sequence analysis to obtain an unbiased gene expression survey of ocular immune cells and to identify clonally expanded lymphocytes. Main Outcomes Measures For each inflamed eye, we measured the proportion of distinct immune cell types, the amount of B- or T-cell clonal expansion, and the transcriptional profile of T and B cells. Results Each individual showed robust clonal expansion arising from a single T- or B-cell lineage, suggesting distinct, antigen-driven pathogenic processes in each patient. This variability in clonal expansion was mirrored by individual variability in CD4 T-cell populations, whereas ocular CD8 T cells and B cells were more similar transcriptionally among patients. Finally, ocular B cells displayed evidence of class switching and plasmablast differentiation within the ocular microenvironment, providing additional support for antigen-driven immune responses in granulomatous uveitis. Conclusions Collectively, our study identified both conserved and individualized features of granulomatous uveitis, illuminating parallel pathophysiologic mechanisms and suggesting that future personalized therapeutic approaches may be warranted.

PURPOSE: To test the hypothesis that idiopathic uveitis can be categorized into subtypes based on gene expression from blood. DESIGN: Case control study METHODS: We applied RNA-Seq to peripheral blood from patients with uveitis associated with one of four systemic diseases, including axial spondyloarthritis (n=17), sarcoidosis (n=13), inflammatory bowel disease (n=12), tubulo-interstitial nephritis with uveitis (n=10), or idiopathic uveitis (n=38) as well as 18 healthy controls evaluated predominantly at Oregon Health & Science University. A high dimensional negative binomial regression model implemented in the edgeR R package compared each disease group against the controls. The 20 most distinctive genes for each diagnosis were extracted. Out of 80 genes, there were 75 unique genes. A classification algorithm was developed by fitting a gradient boosting tree with 5-fold cross-validation. mRNA from subjects with idiopathic uveitis was analyzed to see if any fit clinically and by gene expression pattern with one of the diagnosable entities. RESULTS: For uveitis associated with a diagnosable systemic disease, gene expression profiling achieved an overall accuracy of 85% (balanced average of sensitivity plus specificity, p-value < 0.001). Although the majority of patients with idiopathic uveitis presumably have none of these four associated systemic diseases, gene expression profiles helped to reclassify 11 of 38 subjects. CONCLUSIONS: Peripheral blood gene expression profiling is a potential adjunct in accurate differential diagnosis of the cause of uveitis. Validation of these results and characterization of the gene expression profile from additional discrete diagnoses could enhance the value of these observations.

Metagenomic next-generation sequencing is a powerful method for pathogen detection that combines advanced genome sequencing technology with cutting-edge bioinformatics to analyze microbial populations. Metagenomic next-generation sequencing has the potential to identify uncommon, unculturable, and even previously unidentified pathogens from a clinical isolate. Of particular interest to ophthalmology, this robust data extraction can occur from very small volume clinical samples. Here we discuss the opportunities and limitations of this technique and their current and future application to ophthalmic diagnostics.

Background: Cytomegalovirus retinitis (CMVR) is the primary cause of AIDS-related blindness and remains a substantial cause of blindness in resource-limited settings. Retinal detachment (RD) occurs in approximately 1/3 of patients with CMV retinitis. There is limited capacity to treat retinal detachment in resource-limited settings, and retinal detachment commonly results in permanent profound vision loss. Prophylactic argon laser demarcation was widely used in the pre-HAART era in high income countries, and we here describe our experience with this safe, low-cost intervention to prevent blindness from CMV retinitis-related retinal detachment in a resource-limited setting, in the current era where effective antiretroviral therapy is available. Methods: A non-randomized retrospective observational cohort study was conducted over 4 years in patients on cART (combined antiretroviral therapy) and with inactive AIDS-related CMVR involving at least 25% of the retina, and who were treated with prophylactic argon laser demarcation (LD). The main outcomes were retinal detachment and loss of vision.Results: In a 45 eyes from 38 patients with inactive CMVR on cART for HIV/AIDS who underwent prophylactic laser demarcation, there were no retinal detachments and no loss of vision.Conclusion: In this series of patients at risk for CMVR-related retinal detachment who were treated with prophylactic laser demarcation, retinal detachment was prevented and visual acuity was maintained. Prophylactic argon laser demarcation is a safe, effective, and a realistically affordable intervention to reduce blindness in patients with AIDS-related CMV retinitis.

Macrophages undergo programmatic changes with age, leading to altered cytokine polarization and immune dysfunction, shifting these critical immune cells from protective sentinels to disease promoters. The molecular mechanisms underlying macrophage inflammaging are poorly understood. Using an unbiased RNA sequencing (RNA-seq) approach, we identified Mir146b as a microRNA whose expression progressively and unidirectionally declined with age in thioglycollate-elicited murine macrophages. Mir146b deficiency led to altered macrophage cytokine expression and reduced mitochondrial metabolic activity, two hallmarks of cellular aging. Single-cell RNA-seq identified patterns of altered inflammation and interferon gamma signaling in Mir146b-deficient macrophages. Identification of Mir146b as a potential regulator of macrophage aging provides novel insights into immune dysfunction associated with aging.

The tissue-specific drivers of neurosarcoidosis remain poorly defined. To identify cerebrospinal fluid (CSF) specific, antigen-driven T and B cell responses, we performed single-cell RNA sequencing of CSF and blood cells from neurosarcoid participants coupled to T and B cell receptor sequencing. In contrast to pulmonary sarcoidosis, which is driven by CD4 T cells, we found CD8 T cell clonal expansion enriched in the neurosarcoid CSF. These CSF-enriched CD8 T cells were composed of two subsets with differential expression of EBI2, CXCR3, and CXCR4. Lastly, our data suggest that IFNγ signaling may distinguish neurosarcoidosis from other neurological disorders.

Purpose: To describe an uncommon presentation of ocular infection caused by human herpes simplex virus 2 (HSV-2).Methods: Case report.Results: A 32-year-old female with no prior history of mucocuta...

Ocular complications are common in uveitis and may eventually require surgical management. These complications include cataracts, uveitic glaucoma, epiretinal membranes, band keratopathy, and retinal detachments, among many others. Perioperative surgical planning and inflammatory control are paramount in these complex cases to reduce the risk of postoperative complications such as persistent inflammation and/or cystoid macular edema, hypotony, ocular hypertension, and band keratopathy. This chapter discusses the special considerations and management steps to improve surgical outcomes in uveitic eyes.

Herpes simplex virus 1 (HSV-1) is a nearly ubiquitous human pathogen, remaining dormant in its human host the majority of the time. The interaction between HSV-1 and the immune system represents a complicated balance of power that allows the virus to persist in the host for a lifetime. However, disruptions in the immune system can activate the virus with the potential to cause devastating infections in the central nervous system (CNS). We present a patient who suffered three consecutive yearly HSV-1 CNS episodes (encephalitis, seizure, and retinitis), each within days of his influenza vaccination. We highlight subtle immunologic defects in this patient that may have allowed unchecked viral replication and resultant disease manifestations, as well as the potential role of influenza vaccine in tipping this balance in favor of HSV-1.

We report a high-throughput application of multispectral imaging flow cytometry (MIFC) for analyzing the expression and localization of both RNA and protein molecules in a heterogeneous population of cells. The approach was developed using polyadenylated nuclear (PAN) RNA, an abundant, noncoding RNA expressed by Kaposi's sarcoma–associated herpesvirus (KSHV) during the lytic phase of infection. High levels of PAN RNA are, in part, dependent on its interaction with poly(A)-binding protein C1 (PABPC1), which relocalizes from the cytoplasm to the nucleus of lytically infected cells. We quantitatively tracked the cytoplasmic to nuclear translocation of PABPC1 and examined how this translocation relates to the expression and localization of viral RNA and protein molecules in KSHV-infected cells. This high-throughput approach will be useful for other systems in which changes in subcellular localization of RNA and protein molecules need to be monitored simultaneously.

Kaposi sarcoma-associated herpesvirus (KSHV; also known as HHV8) is the causative agent of two B cell tumors, multicentric Castleman disease (MCD) and primary effusion lymphoma (PEL). However, little is known about the nature of the specific B cell subtype(s) most susceptible to infection. Identifying these cells would provide direct insight into KSHV transmission and virus-induced transformation. To identify this subset and to determine whether infection alters its cellular phenotype, we exposed human tonsillar cells to KSHV and characterized infected cells using high-throughput multispectral imaging flow cytometry (MIFC). Stable expression of the virally encoded latency-associated nuclear antigen (LANA), a marker of latent KSHV infection, was observed predominantly in cells expressing the l light chain of the B cell receptor. These LANA+ B cells proliferated and exhibited similarities to the cells characteristic of MCD (IgMl-expressing plasmablasts), including blasting morphology with elevated expression of Ki67, variable expression of CD27, and high levels of IgM and IL-6 receptor. Furthermore, the proportion of infected cells showing a blasting phenotype increased upon addition of exogenous IL-6. Our data lead us to propose that oral transmission of KSHV involves the latent infection of a subset of tonsillar IgMl-expressing B cells, which then proliferate as they acquire the plasmablast phenotype characteristic of MCD.

To clarify the role and regulation of eosinophils, we subjected several key eosinophil-related genetically engineered mice to a chronic model of allergic airway inflammation aiming to identify results that were independent of the genetic targeting strategy. In particular, mice with defects in eosinophil development (Δdbl-GATA) and eosinophil recruitment [mice deficient in CCR3 (CCR3 knockout) and mice deficient in both eotaxin-1 and eotaxin-2 (eotaxin-1/2 double knockout)] were subjected to Aspergillus fumigatus -induced allergic airway inflammation. Allergen-induced eosinophil recruitment into the airway was abolished by 98%, 94%, and 99% in eotaxin-1/2 double knockout, CCR3 knockout, and Δdbl-GATA mice, respectively. Importantly, allergen-induced type II T helper lymphocyte cytokine production was impaired in the lungs of eosinophil- and CCR3-deficient mice. The absence of eosinophils correlated with reduction in allergen-induced mucus production. Notably, by using global transcript expression profile analysis, a large subset (29%) of allergen-induced genes was eosinophil- and CCR3-dependent; pathways downstream from eosinophils were identified, including in situ activation of coagulation in the lung. In summary, we present multiple lines of independent evidence that eosinophils via CCR3 have a central role in chronic allergic airway disease.

IL-13 overexpression in the lung induces inflammatory and remodeling responses that are prominent features of asthma. Whereas most studies have concentrated on the development of IL-13-induced disease, far fewer studies have focused on the reversibility of IL-13-induced pathologies. This is particularly important because current asthma therapy appears to be poor at reversing lung remodeling. In this manuscript, we used an externally regulatable transgenic system that targets expression of IL-13 to the lung with the aim of characterizing the reversibility process. After 4 wk of doxycycline (dox) exposure, IL-13 expression resulted in mixed inflammatory cell infiltration, mucus cell metaplasia, lung fibrosis, and airspace enlargement (emphysema). After withdrawal of dox, IL-13 protein levels were profoundly reduced by 7 d and below baseline by 14 d. During this time frame, the level of lung eosinophils returned to near normal, whereas macrophages, lymphocytes, and neutrophils remained markedly elevated. IL-13-induced mucus cell metaplasia significantly decreased (91%) 3 wk after withdrawal of dox, showing strong correlation with reduced eosinophil levels. In contrast, IL-13-induced lung fibrosis did not significantly decline 4 wk after dox withdrawal. Importantly, IL-13-induced emphysema persisted, but modestly declined 4 wk after dox. Examination of transcript expression profiles identified a subset of genes that remained increased weeks after transgene expression was no longer detected. Notably, numerous IL-13-induced cytokines and enzymes were reversible (IL-6 and cathepsins), whereas others were sustained (CCL6 and chitinases) after IL-13 withdrawal, respectively. Thus, several hallmark features of IL-13-induced lung pathology persist and are dissociated from eosinophilia after IL-13 overexpression ceases.

The expression of distinct chemokines within the asthmatic lung suggests that specific regulatory mechanisms may mediate various stages of asthmatic disease. Global transcript expression profiling was used to define the spectrum and kinetics of chemokine involvement in an experimental murine model of asthma. Seventeen chemokines were induced in the lungs of allergen-inoculated mice, as compared with saline-treated mice. Two (CXCL13 and CCL9) of the 17 identified chemokines have not previously been associated with allergic airway disease. Seven (7 of 17; CCL2, CCL7, CCL9, CCL11, CXCL1, CXCL5, CXCL10) of the allergen-induced chemokines were induced early after allergen challenge and remained induced throughout the experimental period. Three chemokines (CXCL2, CCL3, and CCL17) were induced only during the early phase of the inflammatory response after the initial allergen challenge, while seven chemokines (CCL6, CCL8, CCL12, CCL22, CXCL9, CXCL12, and CXCL13) were increased only after a second allergen exposure. Unexpectedly, expression of only three chemokines, CCL11, CCL17, and CCL22, was STAT6 dependent, and many of the identified chemokines were overexpressed in STAT6-deficient mice, providing an explanation for the enhanced neutrophilic inflammation seen in these mice. Notably, IFN-γ and STAT1 were shown to contribute to the induction of two STAT6-independent chemokines, CXCL9 and CXCL10. Taken together, these results show that only a select panel of chemokines (those targeting Th2 cells and eosinophils) is positively regulated by STAT6; instead, many of the allergen-induced chemokines are negatively regulated by STAT6. Collectively, we demonstrate that allergen-induced inflammation involves coordinate regulation by STAT1, STAT6, and IFN-γ.