Profiling the peripheral immune response to ex vivo TNF stimulation in untreated juvenile idiopathic arthritis using single cell RNA sequencing.

BACKGROUND: Juvenile Idiopathic Arthritis (JIA) is an autoimmune disease with a heterogenous clinical presentation and unpredictable response to available therapies. This personalized transcriptomics study sought proof-of-concept for single-cell RNA sequencing to characterize patient-specific immune profiles. METHODS: Whole blood samples from six untreated children, newly diagnosed with JIA, and two healthy controls were cultured for 24 h with or without ex vivo TNF stimulation and subjected to scRNAseq to examine cellular populations and transcript expression in PBMCs. A novel analytical pipeline, scPool, was developed wherein cells are first pooled into pseudocells prior to expression analysis, facilitating variance partitioning of the effects of TNF stimulus, JIA disease status, and individual donor. RESULTS: Seventeen robust immune cell-types were identified, the abundance of which was significantly affected by TNF stimulus, which resulted in notable elevation of memory CD8 + T-cells and NK56 cells, but down-regulation of naïve B-cell proportions. Memory CD8 + and CD4 + T-cells were also both reduced in the JIA cases relative to two controls. Significant differential expression responses to TNF stimulus were also characterized, with monocytes showing more transcriptional shifts than T-lymphocyte subsets, while the B-cell response was more limited. We also show that donor variability exceeds the small degree of possible intrinsic differentiation between JIA and control profiles. An incidental finding of interest was association of HLA-DQA2 and HLA-DRB5 expression with JIA status. CONCLUSIONS: These results support the development of personalized immune-profiling combined with ex-vivo immune stimulation for evaluation of patient-specific modes of immune cell activity in autoimmune rheumatic disease.

Immunometabolic Analysis of Synovial Fluid from Juvenile Idiopathic Arthritis Patients.

Juvenile idiopathic arthritis (JIA) is an inflammatory rheumatic disorder. Polymorphonuclear neutrophils (PMNs) are present in JIA synovial fluid (SF), but with variable frequency. SF PMNs in JIA were previously shown to display high exocytic but low phagocytic and immunoregulatory activities. To further assess whether the degree of SF neutrophilia associated with altered immune responses in JIA, we collected SF and blood from 16 adolescent JIA patients. SF and blood leukocytes were analyzed by flow cytometry. SF and plasma were used for immune mediator quantification and metabolomics. Healthy donor blood T cells were cultured in SF to evaluate its immunoregulatory activities. PMN and T cell frequencies were bimodal in JIA SF, delineating PMN high/T cell low (PMNHigh) and PMN low/T cell high (PMNLow) samples. Proinflammatory mediators were increased in SF compared with plasma across patients, and pro- and anti-inflammatory mediators were further elevated in PMNHigh SF. Compared to blood, SF PMNs showed increased exocytosis and programmed death-1/programmed death ligand-1 expression, and SF PMNs and monocytes/macrophages had increased surface-bound arginase-1. SPADE analysis revealed SF monocyte/macrophage subpopulations coexpressing programmed death-1 and programmed death ligand-1, with higher expression in PMNHigh SF. Healthy donor T cells showed reduced coreceptor expression when stimulated in PMNHigh versus PMNLow SF. However, amino acid metabolites related to the arginase-1 and IDO-1 pathways did not differ between the two groups. Hence, PMN predominance in the SF of a subset of JIA patients is associated with elevated immune mediator concentration and may alter SF monocyte/macrophage phenotype and T cell activation, without altering immunoregulatory amino acids.

Temporomandibular Joint Acoustic Emissions in Children With Juvenile Idiopathic Arthritis Differ From Those in Healthy Children.

PURPOSE: Articulation of the temporomandibular joint (TMJ) generates sounds with specific characteristics known as joint acoustic emissions (AEs). The purpose of this project was to determine if AEs as described by the joint health score (JHS) in children with juvenile idiopathic arthritis (JIA) differ from AEs in healthy children. METHODS: The investigators implemented a cross-sectional study with age- and sex-matched controls to compare AEs from 4 groups: (1) healthy subjects without TMJ sounds, (2) healthy subjects with TMJ sounds, (3) subjects with JIA without TMJ sounds, and (4) subjects with TMJ sounds. Predictor variables were JIA status (ie JIA/healthy) and joint sounds (present/absent). The outcome variable was AEs. Subjects wore a specialized headset and performed specific jaw movements that generated AEs. AEs were recorded and analyzed using an aggregated decision tree classification model that calculates a JHS for each group. JHSs were compared using a receiver operating characteristic curve and classification accuracies. The study team used a 2-tailed unpaired t-test to determine if score distributions were different. Significance was P < .05. RESULTS: A total of 51 subjects (102 TMJs; 37 females) with an average age of 13.1 years (range, 7 to 18) participated. Children with JIA and TMJ sounds had AEs with large repetitive clicks. Children with JIA without sounds had smaller repetitive clicks. Healthy children had grinding sounds with lower amplitude. The receiver operating characteristic curve had a classification accuracy of 71.6%. This accuracy compares against the gold standard clinical assessment for placing these patients into their groups (JIA vs healthy). JHSs of children with TMJ sounds and children with JIA and TMJ sounds were statistically significant (P < .0001). CONCLUSION: In our sample, the AE of TMJs in healthy children may be different than that in children with JIA. Assessment of an AE is a promising and noninvasive technique to determine involvement of TMJs in children with JIA.

Impact of the Season of Birth on the Development of Juvenile Idiopathic Arthritis in the United States: A Nationwide Registry-based Study.

OBJECTIVE: Autoimmune disorders result from the interplay of genetic and environmental factors. Many autoimmune disorders are associated with specific seasons of birth, implicating a role for environmental determinants in their etiopathology. We investigated if there is an association between the season of birth and the development of juvenile idiopathic arthritis (JIA). METHODS: Birth data from 10,913 children with JIA enrolled at 62 Childhood Arthritis and Rheumatology Research Alliance Registry sites was compared with 109,066,226 US births from the same period using a chi-square goodness-of-fit test. Season of birth of the JIA cohort was compared to the US population estimate using a 2-sided 1-sample test for a binomial proportion and corrected for multiple comparisons. Secondary analysis was performed for JIA categories, age of onset, and month of birth. RESULTS: A greater proportion of children with JIA were born in winter (January-March) compared to the US general population (25.72% vs 24.08%; corrected P < 0.0001). This observation was also true after stratifying for age of onset (≤ or > 6 yrs). When analyzed by the month of birth, a greater proportion of children with JIA were born in January compared to the US population (9.44% vs 8.13%; corrected P < 0.0001). CONCLUSION: Relative to the general population, children with JIA are more often born in the winter, and specifically in the month of January. These observations support the hypothesis that seasonal variations in exposures during the gestational and/or early postnatal periods may contribute to development of JIA.

Knee Acoustic Emissions as a Digital Biomarker of Disease Status in Juvenile Idiopathic Arthritis.

In this paper, we quantify the joint acoustic emissions (JAEs) from the knees of children with juvenile idiopathic arthritis (JIA) and support their use as a novel biomarker of the disease. JIA is the most common rheumatic disease of childhood; it has a highly variable presentation, and few reliable biomarkers which makes diagnosis and personalization of care difficult. The knee is the most commonly affected joint with hallmark synovitis and inflammation that can extend to damage the underlying cartilage and bone. During movement of the knee, internal friction creates JAEs that can be non-invasively measured. We hypothesize that these JAEs contain clinically relevant information that could be used for the diagnosis and personalization of treatment of JIA. In this study, we record and compare the JAEs from 25 patients with JIA-10 of whom were recorded a second time 3-6 months later-and 18 healthy age- and sex-matched controls. We compute signal features from each of those record cycles of flexion/extension and train a logistic regression classification model. The model classified each cycle as having JIA or being healthy with 84.4% accuracy using leave-one-subject-out cross validation (LOSO-CV). When assessing the full JAE recording of a subject (which contained at least 8 cycles of flexion/extension), a majority vote of the cycle labels accurately classified the subjects as having JIA or being healthy 100% of the time. Using the output probabilities of a JIA class as a basis for a joint health score and test it on the follow-up patient recordings. In all 10 of our 6-week follow-up recordings, the score accurately tracked with successful treatment of the condition. Our proposed JAE-based classification model of JIA presents a compelling case for incorporating this novel joint health assessment technique into the clinical work-up and monitoring of JIA.