Transcriptome network analysis implicates CX3CR1-positive type 3 dendritic cells in non-infectious uveitis.

Background: Type I interferons (IFNs) promote the expansion of subsets of CD1c+ conventional dendritic cells (CD1c+ DCs), but the molecular basis of CD1c+ DCs involvement in conditions not associated without elevated type I IFNs remains unclear. Methods: We analyzed CD1c+ DCs from two cohorts of non-infectious uveitis patients and healthy donors using RNA-sequencing followed by high-dimensional flow cytometry to characterize the CD1c+ DC populations. Results: We report that the CD1c+ DCs pool from patients with non-infectious uveitis is skewed toward a gene module with the chemokine receptor CX3CR1 as the key hub gene. We confirmed these results in an independent case-control cohort and show that the disease-associated gene module is not mediated by type I IFNs. An analysis of peripheral blood using flow cytometry revealed that CX3CR1+ DC3s were diminished, whereas CX3CR1- DC3s were not. Stimulated CX3CR1+ DC3s secrete high levels of inflammatory cytokines, including TNF-alpha, and CX3CR1+ DC3 like cells can be detected in inflamed eyes of patients. Conclusions: These results show that CX3CR1+ DC3s are implicated in non-infectious uveitis and can secrete proinflammatory mediators implicated in its pathophysiology. Funding: The presented work is supported by UitZicht (project number #2014-4, #2019-10, and #2021-4). The funders had no role in the design, execution, interpretation, or writing of the study.

A Network of Serum Proteins Predict the Need for Systemic Immunomodulatory Therapy at Diagnosis in Noninfectious Uveitis.

Purpose: Early identification of patients with noninfectious uveitis requiring steroid-sparing immunomodulatory therapy (IMT) is currently lacking in objective molecular biomarkers. We evaluated the proteomic signature of patients at the onset of disease and associated proteomic clusters with the need for IMT during the course of the disease. Design: Multicenter cohort study. Participants: Two hundred thirty treatment-free patients with active noninfectious uveitis. Methods: We used aptamer-based proteomics (n = 1305 proteins) and a bioinformatic pipeline as a molecular stratification tool to define the serum protein network of a Dutch discovery cohort (n = 78) of patients and healthy control participants and independently validated our results in another Dutch cohort (n = 111) and a United States cohort (n = 67). Multivariate Cox analysis was used to assess the relationship between the protein network and IMT use. Main Outcome Measures: Serum protein levels and use of IMT. Results: Network-based analyses revealed a tightly coexpressed serum cluster (n = 85 proteins) whose concentration was consistently low in healthy control participants (n = 26), but varied among patients with noninfectious uveitis (n = 52). Patients with high levels of the serum cluster at disease onset showed a significantly increased need for IMT during follow-up, independent of anatomic location of uveitis (hazard ratio, 3.42; 95% confidence interval, 1.22-9.5; P = 0.019). The enrichment of neutrophil-associated proteins in the protein cluster led to our finding that the neutrophil count could serve as a clinical proxy for this proteomic signature (correlation: r = 0.57, P = 0.006). In an independent Dutch cohort (n = 111), we confirmed that patients with relatively high neutrophil count at diagnosis (> 5.2 × 109/L) had a significantly increased chance of requiring IMT during follow-up (hazard ratio, 3.2; 95% confidence interval, 1.5-6.8; P = 0.002). We validated these findings in a third cohort of 67 United States patients. Conclusions: A serum protein signature correlating with neutrophil levels was highly predictive for IMT use in noninfectious uveitis. We developed a routinely available tool that may serve as a novel objective biomarker to aid in clinical decision-making for noninfectious uveitis.

Tissue-Resident Memory CD8+ T Cells From Skin Differentiate Psoriatic Arthritis From Psoriasis.

OBJECTIVE: To compare immune cell phenotype and function in psoriatic arthritis (PsA) versus psoriasis in order to better understand the pathogenesis of PsA. METHODS: In-depth immunophenotyping of different T cell and dendritic cell subsets was performed in patients with PsA, psoriasis, or axial spondyloarthritis and healthy controls. Subsequently, we analyzed cells from peripheral blood, synovial fluid (SF), and skin biopsy specimens using flow cytometry, along with high-throughput transcriptome analyses and functional assays on the specific cell populations that appeared to differentiate PsA from psoriasis. RESULTS: Compared to healthy controls, the peripheral blood of patients with PsA was characterized by an increase in regulatory CD4+ T cells and interleukin-17A (IL-17A) and IL-22 coproducing CD8+ T cells. One population specifically differentiated PsA from psoriasis: i.e., CD8+CCR10+ T cells were enriched in PsA. CD8+CCR10+ T cells expressed high levels of DNAX accessory molecule 1 and were effector memory cells that coexpressed skin-homing receptors CCR4 and cutaneous lymphocyte antigen. CD8+CCR10+ T cells were detected under inflammatory and homeostatic conditions in skin, but were not enriched in SF. Gene profiling further revealed that CD8+CCR10+ T cells expressed GATA3, FOXP3, and core transcriptional signature of tissue-resident memory T cells, including CD103. Specific genes, including RORC, IFNAR1, and ERAP1, were up-regulated in PsA compared to psoriasis. CD8+CCR10+ T cells were endowed with a Tc2/22-like cytokine profile, lacked cytotoxic potential, and displayed overall regulatory function. CONCLUSION: Tissue-resident memory CD8+ T cells derived from the skin are enhanced in the circulation of patients with PsA compared to patients with psoriasis alone. This may indicate that aberrances in cutaneous tissue homeostasis contribute to arthritis development.

A pan-inflammatory microRNA-cluster is associated with orbital non-Hodgkin lymphoma and idiopathic orbital inflammation.

Non-Hodgkin orbital lymphoma (NHOL) and idiopathic orbital inflammation (IOI) are common orbital conditions with largely unknown pathophysiology that can be difficult to diagnose. In this study we aim to identify serum miRNAs associated with NHOL and IOI. We performed OpenArray® miRNA profiling in 33 patients and controls. Differentially expressed miRNAs were technically validated across technology platforms and replicated in an additional cohort of 32 patients and controls. We identified and independently validated a serum miRNA profile of NHOL that was remarkably similar to IOI and characterized by an increased expression of a cluster of eight miRNAs. Pathway enrichment analysis indicated that the miRNA-cluster is associated with immune-mediated pathways, which we supported by demonstrating the elevated expression of this cluster in serum of patients with other inflammatory conditions. The cluster contained miR-148a, a key driver of B-cell tolerance, and miR-365 that correlated with serum IgG and IgM concentrations. In addition, miR-29a and miR-223 were associated with blood lymphocyte and neutrophil populations, respectively. NHOL and IOI are characterized by an abnormal serum miRNA-cluster associated with immune pathway activation and linked to B cell and neutrophil dysfunction.

Aqueous Humor Analysis Identifies Higher Branched Chain Amino Acid Metabolism as a Marker for Human Leukocyte Antigen-B27 Acute Anterior Uveitis and Disease Activity.

PURPOSE: Human leukocyte antigen-B27 (HLA-B27)-positive acute anterior uveitis (AAU) has a higher recurrence rate and shows more anterior chamber cell infiltration compared with HLA-B27-negative patients, suggesting distinct etiologies of these clinically overlapping conditions. To advance our understanding of the biology of AAU, we characterized the metabolic profile of aqueous humor (AqH) of patients with HLA-B27-associated AAU (B27-AAU) and noninfectious idiopathic AAU (idiopathic AAU). DESIGN: Experimental laboratory study. METHODS: AqH samples from 2 independent cohorts totaling 30 patients with B27-AAU, 16 patients with idiopathic AAU, and 20 patients with cataracts underwent 2 individual rounds of direct infusion mass spectrometry. Features predicted by direct infusion mass spectrometry that facilitated maximum separation between the disease groups in regression models were validated by liquid chromatography/tandem mass spectrometry-based quantification with appropriate standards. RESULTS: Partial least square-discriminant analysis revealed metabolite profiles that were able to separate patients with B27-AAU from those with iodiopathic AAU. Pathway enrichment analysis, based on metabolites on which separation of the groups in the partial least square-discriminant analysis model was based, demonstrated the involvement of branched-chain amino acid biosynthesis, ascorbate and aldarate metabolism, the tricarboxylic acid cycle, and glycolysis-diverting pathways (eg, serine biosynthesis) across all investigated cohorts. Notably, the metabolite ketoleucine was elevated in B27-AAU across all 3 runs and moderately-but robustly-correlated with anterior chamber cell count (correlation coefficient range 0.41-0.81). CONCLUSIONS: These results illustrate metabolic heterogeneity between HLA-B27-positive and HLA-B27-negative AAU, including an increase of branched-chain amino acid biosynthesis, that reflects disease activity in AAU.

High-Dimensional Profiling Reveals Heterogeneity of the Th17 Subset and Its Association With Systemic Immunomodulatory Treatment in Non-infectious Uveitis.

Background: Non-infectious uveitis (NIU) is a severe intra ocular inflammation, which frequently requires prompt systemic immunosuppressive therapy (IMT) to halt the development of vision-threatening complications. IMT is considered when NIU cannot be treated with corticosteroids alone, which is unpredictable in advance. Previous studies have linked blood cell subsets to glucocorticoid sensitivity, which suggests that the composition of blood leukocytes may early identify patients that will require IMT. Objective: To map the blood leukocyte composition of NIU and identify cell subsets that stratify patients that required IMT during follow-up. Methods: We performed controlled flow cytometry experiments measuring a total of 37 protein markers in the blood of 30 IMT free patients with active non-infectious anterior, intermediate, and posterior uveitis, and compared these to 15 age and sex matched healthy controls. Results from manual gating were validated by automatic unsupervised gating using FlowSOM. Results: Patients with uveitis displayed lower relative frequencies of Natural Killer cells and higher relative frequencies of memory T cells, in particular the CCR6+ lineages. These results were confirmed by automatic gating by unsupervised clustering using FlowSOM. We observed considerable heterogeneity in memory T cell subsets and abundance of CXCR3-CCR6+ (Th17) cells between the uveitis subtypes. Importantly, regardless of the uveitis subtype, patients that eventually required IMT in the course of the study follow-up exhibited increased CCR6+ T cell abundance before commencing therapy. Conclusion: High-dimensional immunoprofiling in NIU patients shows that clinically distinct forms of human NIU exhibit shared as well as unique immune cell perturbations in the peripheral blood and link CCR6+ T cell abundance to systemic immunomodulatory treatment.

A Disease-Associated MicroRNA Cluster Links Inflammatory Pathways and an Altered Composition of Leukocyte Subsets to Noninfectious Uveitis.

Purpose: The cause of noninfectious uveitis (NIU) is poorly understood but is considered to be mediated by a complex interplay between genetic, environmental, and-relatively unexplored-epigenetic factors. MicroRNAs (miRNAs) are noncoding small RNAs that are important epigenetic regulators implicated in pathologic signaling. Therefore, we mapped the circulating miRNA-ome of NIU patients and studied miRNA perturbations within the broader context of the immune system. Methods: We designed a strategy to robustly identify changes in the miRNA profiles of two independent cohorts totaling 54 untreated patients with active and eye-restricted disease and 26 age-matched controls. High-resolution miRNA-ome data were obtained by TaqMan OpenArray technology and subsequent RT-qPCR. Flow cytometry data, and proteomic data spanning the cellular immune system, were used to map the uveitis-miRNA signature to changes in the composition of specific leukocyte subsets in blood. Results: Using stringent selection criteria, we identified and independently validated an miRNA cluster that is associated with NIU. Pathway enrichment analysis for genes targeted by this cluster revealed significant enrichment for the PI3K/Akt, MAPK, FOXO, and VEGF signaling pathways, and photoreceptor development. In addition, unsupervised multidomain analyses linked the presence of the uveitis-associated miRNA cluster to a different composition of leukocyte subsets, more specifically, CD16+CD11c+HLA-DR- cells. Conclusions: Together, this study identified a unique miRNA cluster associated with NIU that was related to changes in leukocyte subsets demonstrating systemic changes in epigenetic regulation underlying NIU.

Prevalence and characteristics of ocular pain in non-infectious uveitis: a quality of life study.

BACKGROUND/AIM: To survey the frequency, character, severity and impact of ocular pain on quality of life in adult patients with non-infectious uveitis (NIU). METHODS: This patient-requested cross-sectional survey study describes the results of three self-administered questionnaires (the National Eye Institute Visual Function Questionnaire, the 36-Item Short Form Health Survey (SF-36) and the McGill Pain Questionnaire Dutch Language Version) from 147 patients with NIUs from a university-based tertiary referral centre in Utrecht. RESULTS: The mean Visual Function Questionnaire (VFQ) Ocular Pain Score of all patients with NIU was 72 (±24), which is significantly lower than an ocular disease-free reference group (90±15, P<0.0001), indicating more ocular pain. This was true for all types of NIU, regardless of the localisation: although Ocular Pain Scores were lower in patients with anterior uveitis (AU) compared with patients with non-AU (mean 62 (±24) vs 74 (±24), P=0.04), patients with non-AU still scored substantially lower than the reference group that had no ocular history (P<0.0001). Patients with NIU also scored significantly lower on all other VFQ subscales as well as on the SF-36 subscales 'Role Limitations due to physical problems', 'Vitality', 'General health' and 'Bodily Pain' compared with controls. The VFQ Ocular Pain subscale correlated with other quality of life subscales (both VFQ-25 and SF-36), indicating a relationship between pain and quality of life. CONCLUSION: This study shows that ocular pain is highly prevalent in patients with NIU, regardless of the localisation. Furthermore, ocular pain has an impact on quality of life.

Aberrant leukocyte telomere length in Birdshot Uveitis.

PURPOSE: Birdshot Uveitis (BU) is an archetypical chronic inflammatory eye disease, with poor visual prognosis, that provides an excellent model for studying chronic inflammation. BU typically affects patients in the fifth decade of life. This suggests that it may represent an age-related chronic inflammatory disease, which has been linked to increased erosion of telomere length of leukocytes. METHODS: To study this in detail, we exploited a sensitive standardized quantitative real-time polymerase chain reaction to determine the peripheral blood leukocyte telomere length (LTL) in 91 genotyped Dutch BU patients and 150 unaffected Dutch controls. RESULTS: Although LTL erosion rates were very similar between BU patients and healthy controls, we observed that BU patients displayed longer LTL, with a median of log (LTL) = 4.87 (= 74131 base pair) compared to 4.31 (= 20417 base pair) in unaffected controls (P<0.0001). The cause underpinning the difference in LTL could not be explained by clinical parameters, immune cell-subtype distribution, nor genetic predisposition based upon the computed weighted genetic risk score of genotyped validated variants in TERC, TERT, NAF1, OBFC1 and RTEL1. CONCLUSIONS: These findings suggest that BU is accompanied by significantly longer LTL.

Systemic inflammatory immune signatures in a patient with CRB1 linked retinal dystrophy.

BACKGROUND: In this report we describe, for the first time, the activation of the peripheral immune compartment in a patient with a CRB1 linked retinal degenerative disease, masquerading as intermediate uveitis. METHODS: To monitor the immune system during systemic immunosuppressive treatment, given for the initial diagnosis of intermediate uveitis, blood samples were taken before and during therapy, for analysis of peripheral blood mononuclear cell-subsets and circulating immune mediators. RESULTS: The levels of various pro-inflammatory immune mediators (including MIF, TSLP, CCL2/MCP-1, CXCL9, CXCL10, IFN-ß, IL-6, IL-17, IL-21, IL-22, and IL-23) were elevated in serum at the first time point, and decreased under immunosuppressive treatment. In parallel, the frequency of activated (CD86+) CD1c+ myeloid dendritic cells in blood was proportional to the central foveal thickness measured by optical coherence tomography. CONCLUSIONS: These observations challenge the current view on the distinct pathophysiology of retinal degenerative and retinal inflammatory conditions in this patient.

Potential Predictors of Poor Visual Outcome in Human Leukocyte Antigen-B27-Associated Uveitis.

PURPOSE: To identify potential predictors of permanent vision loss in patients with human leukocyte antigen (HLA)-B27-associated uveitis in a tertiary referral center. DESIGN: Retrospective case-control study. METHODS: The charts of 212 patients (338 eyes) with HLA-B27-associated uveitis that visited the University Medical Center Utrecht with a follow-up of at least 6 months were retrospectively studied. Clinical features at presentation and during follow-up were compared to final visual outcome in quiescent state. Eyes with (sub-) normal vision (>20/50) were compared with visually impaired (≤20/50) and blind (≤5/50, or a visual field of <10 degrees) eyes, using survival analysis. A multivariate Cox proportional hazards analysis was performed to analyze potential predictors for permanent vision loss. RESULTS: Median follow-up was 10.4 years (range, 0.5-44.7 years). During follow-up 226 eyes (66%) experienced vision loss up to 20/50, but most recovered. Twenty patients (9%) became permanently visually impaired or blind in at least 1 eye because of uveitis, after a median of 9.7 years (range, 0-20.9 years). The main cause was secondary glaucoma or related to glaucoma surgery (12/22 eyes, 55%). Survival analysis showed, after adjustment for age and sex, an ocular pressure of >21 mm Hg, hypotony, and panuveitis to be potential predictors at presentation, and the development of secondary glaucoma or hypotony to be predictors of blindness or visual impairment during follow-up. CONCLUSIONS: The long-term visual prognosis of HLA-B27-associated uveitis is relatively good, but the true incidence of permanent vision loss is probably still underestimated. Our findings highlight the importance of proper control of intraocular pressure.

Characteristics of childhood uveitis leading to visual impairment and blindness in the Netherlands.

PURPOSE: To investigate the clinical characteristics of childhood uveitis leading to visual impairment or blindness. METHODS: In this descriptive study, we reviewed data from the medical records of 58 children with visual impairment or blindness due to childhood uveitis, which were seen at an institute for visually impaired patients (Bartiméus) between January 1981 and December 2012, in a retrospective, cross-sectional manner. RESULTS: Thirty-two of the 58 children (55%) were visually impaired and 26 (45%) were legally blind. Uveitis was posterior in 76% of all cases. Infectious uveitis represented 74% of all cases, of which 86% was congenital. Five patients (9%) had uveitis related to a systemic disease, and ten patients (17%) had idiopathic uveitis. There was a decrease in infectious causes over the last decades (p = 0.04) and an increase in idiopathic uveitis (p < 0.01), but the rate of children with posterior uveitis remained constant. There was an overall decrease in the number of children with uveitis referred to Bartiméus. The number of ocular complications at the time of intake was higher in children with acquired disease compared with congenital diseases (p < 0.01), as it was in children with non-infectious uveitis compared with infectious uveitis (p = 0.04). Most comorbidities that were noted were seen in children with infectious uveitis. CONCLUSION: Most patients suffering from visual impairment or blindness due to childhood uveitis had posterior and/or infectious uveitis, mostly congenital. There is a shift in causes which shows a decrease in infectious causes and an increase in idiopathic causes.