CERS1 is a Biomarker of Staphylococcus aureus Abundance and Atopic Dermatitis Severity.

BACKGROUND: Atopic dermatitis (AD) is an inflammatory skin condition characterized by widely variable cutaneous Staphylococcus aureus abundance that contributes to disease severity and rapidly responds to type 2 immune blockade (i.e., dupilumab). The molecular mechanisms regulating S. aureus levels between AD subjects remain poorly understood. OBJECTIVE: To investigate host genes that may be predictive of S. aureus abundance and correspond with AD severity. METHODS: Data derived from the NIH/NIAID-funded (NCT03389893 [ADRN-09]) randomized, double-blind, and placebo-controlled multicenter study of dupilumab in adults (n=71 subjects) with moderate-severe AD. Bulk RNA-sequencing of skin biopsies (n=57 lesional, 55 non-lesional) was compared to epidermal S. aureus abundance, lipidomics, and AD clinical measures. RESULTS: S. aureus abundance and ceramide synthase 1 (CERS1) expression positively correlated at baseline across both non-lesional (r=0.29, p=0.030) and lesional (r=0.41, p=0.0015) skin. Lesional CERS1 expression also positively correlated with AD severity (i.e., SCORing AD [SCORAD] r=0.44, p=0.0006) and skin barrier dysfunction (transepidermal water loss area under the curve [TEWL AUC] r=0.31, p=0.025) at baseline. CERS1 expression (forms C18:0-sphingolipids) was negatively associated with elongation of very long chain fatty acids (ELOVL6; C16:0→C18:0) expression and corresponded with a shorter chain length sphingolipid composition. Dupilumab rapidly reduced CERS1 expression (day 7) and ablated the relationship with S. aureus abundance and ELOVL6 expression by day 21. CONCLUSION: CERS1 is a unique molecular biomarker of S. aureus abundance and AD severity that may contribute to dysfunctional skin barrier and shorter chain sphingolipid composition through fatty acid sequestration as a maladaptive compensatory response to reduced ELOVL6.

Automated multi-scale computational pathotyping (AMSCP) of inflamed synovial tissue.

Rheumatoid arthritis (RA) is a complex immune-mediated inflammatory disorder in which patients suffer from inflammatory-erosive arthritis. Recent advances on histopathology heterogeneity of RA synovial tissue revealed three distinct phenotypes based on cellular composition (pauci-immune, diffuse and lymphoid), suggesting that distinct etiologies warrant specific targeted therapy which motivates a need for cost effective phenotyping tools in preclinical and clinical settings. To this end, we developed an automated multi-scale computational pathotyping (AMSCP) pipeline for both human and mouse synovial tissue with two distinct components that can be leveraged together or independently: (1) segmentation of different tissue types to characterize tissue-level changes, and (2) cell type classification within each tissue compartment that assesses change across disease states. Here, we demonstrate the efficacy, efficiency, and robustness of the AMSCP pipeline as well as the ability to discover novel phenotypes. Taken together, we find AMSCP to be a valuable cost-effective method for both pre-clinical and clinical research.

High-throughput micro-CT analysis identifies sex-dependent biomarkers of erosive arthritis in TNF-Tg mice and differential response to anti-TNF therapy.

BACKGROUND: Development of reliable disease activity biomarkers is critical for diagnostics, prognostics, and novel drug development. Although computed tomography (CT) is the gold-standard for quantification of bone erosions, there are no consensus approaches or rationales for utilization of specific outcome measures of erosive arthritis in complex joints. In the case of preclinical models, such as sexually dimorphic tumor necrosis factor transgenic (TNF-Tg) mice, disease severity is routinely quantified in the ankle through manual segmentation of the talus or small regions of adjacent bones primarily due to the ease in measurement. Herein, we sought to determine the particular hindpaw bones that represent reliable biomarkers of sex-dependent disease progression to guide future investigation and analysis. METHODS: Hindpaw micro-CT was performed on wild-type (n = 4 male, n = 4 female) and TNF-Tg (n = 4 male, n = 7 female) mice at monthly intervals from 2-5 (females) and 2-8-months (males) of age, since female TNF-Tg mice exhibit early mortality from cardiopulmonary disease at approximately 5-6-months. Further, 8-month-old WT (n = 4) and TNF-Tg males treated with anti-TNF monoclonal antibodies (n = 5) or IgG placebo isotype controls (n = 6) for 6-weeks were imaged with micro-CT every 3-weeks. For image analysis, we utilized our recently developed high-throughput and semi-automated segmentation strategy in Amira software. Synovial and osteoclast histology of ankle joints was quantified using Visiopharm. RESULTS: First, we demonstrated that the accuracy of automated segmentation, determined through analysis of ~9000 individual bones by a single user, was comparable in wild-type and TNF-Tg hindpaws before correction (79.2±8.9% vs 80.1±5.1%, p = 0.52). Compared to other bone compartments, the tarsal region demonstrated a sudden, specific, and significant bone volume reduction in female TNF-Tg mice, but not in males, by 5-months (4-months 4.3± 0.22 vs 5-months 3.4± 0.62 mm3, p<0.05). Specifically, the cuboid showed significantly reduced bone volumes at early timepoints compared to other tarsals (i.e., 4-months: Cuboid -24.1±7.2% vs Talus -9.0±5.9% of 2-month baseline). Additional bones localized to the anterolateral region of the ankle also exhibited dramatic erosions in the tarsal region of females, coinciding with increased synovitis and osteoclasts. In TNF-Tg male mice with severe arthritis, the talus and calcaneus exhibited the most sensitive response to anti-TNF therapy measured by effect size of bone volume change over treatment period. CONCLUSIONS: We demonstrated that sexually dimorphic changes in arthritic hindpaws of TNF-Tg mice are bone-specific, where the cuboid serves as a reliable early biomarker of erosive arthritis in female mice. Adoption of automated segmentation approaches in pre-clinical or clinical models has potential to translate quantitative biomarkers to monitor bone erosions in disease and evaluate therapeutic efficacy.

Atopic dermatitis and IgE-mediated food allergy: Common biologic targets for therapy and prevention.

OBJECTIVE: To highlight common mechanistic targets for the treatment of atopic dermatitis (AD) and IgE-mediated food allergy (IgE-FA) with potential to be effective for both diseases and prevent atopic progression. DATA SOURCES: Data sources were PubMed searches or National Clinical Trials (NCT)-registered clinical trials related to AD, IgE-FA, and other atopic conditions, especially focused on the pediatric population. STUDY SELECTIONS: Human seminal studies and/or articles published in the past decade were emphasized with reference to preclinical models when relevant. NCT-registered clinical trials were filtered by inclusion of pediatric subjects younger than 18 years with special focus on children younger than 12 years as a critical period when AD and IgE-FA diseases may often be concurrent. RESULTS: AD and IgE-FA share several pathophysiologic features, including epithelial barrier dysfunction, innate and adaptive immune abnormalities, and microbial dysbiosis, which may be critical for the clinical progression between these diseases. Revolutionary advances in targeted biologic therapies have shown the benefit of inhibiting type 2 immune responses, using dupilumab (anti-interleukin-4Rα) or omalizumab (anti-IgE), to potentially reduce symptom burden for both diseases in pediatric populations. Although the potential for biologics to promote disease remission (AD) or sustained unresponsiveness (IgE-FA) remains unclear, the refinement of biomarkers to predict infants at risk for atopic disorders provides promise for prevention through timely intervention. CONCLUSION: AD and IgE-FA exhibit common features that may be leveraged to develop biologic therapeutic strategies to treat both conditions and even prevent atopic progression. Future studies should be designed with consistent age stratification in the pediatric population and standardized regimens of adjuvant oral immunotherapy or dose escalation (IgE-FA) to improve cross-study interpretation.

Distinct mast cell subpopulations within and around lymphatic vessels regulate lymph flow and progression of inflammatory-erosive arthritis in TNF-transgenic mice.

Objective: Inflammatory-erosive arthritis is exacerbated by dysfunction of joint-draining popliteal lymphatic vessels (PLVs). Synovial mast cells are known to be pro-inflammatory in rheumatoid arthritis (RA). In other settings they have anti-inflammatory and tissue reparative effects. Herein, we elucidate the role of mast cells on PLV function and inflammatory-erosive arthritis in tumor necrosis factor transgenic (TNF-tg) mice that exhibit defects in PLVs commensurate with disease progression. Methods: Whole mount immunofluorescent microscopy, toluidine blue stained histology, scanning electron microscopy, and in silico bioinformatics were performed to phenotype and quantify PLV mast cells. Ankle bone volumes were assessed by µCT, while corresponding histology quantified synovitis and osteoclasts. Near-infrared indocyanine green imaging measured lymphatic clearance as an outcome of PLV draining function. Effects of genetic MC depletion were assessed via comparison of 4.5-month-old WT, TNF-tg, MC deficient KitW-sh/W-sh (cKit-/-), and TNF-tg x cKit-/- mice. Pharmacological inhibition of mast cells was assessed by treating TNF-tg mice with placebo or cromolyn sodium (3.15mg/kg/day) for 3-weeks. Results: PLVs are surrounded by MCT+/MCPT1+/MCPT4+ mast cells whose numbers are increased 2.8-fold in TNF-tg mice. The percentage of peri-vascular degranulating mast cells was inversely correlated with ICG clearance. A population of MCT+/MCPT1-/MCPT4- mast cells were embedded within the PLV structure. In silico single-cell RNA-seq (scRNAseq) analyses identified a population of PLV-associated mast cells (marker genes: Mcpt4, Cma1, Cpa3, Tpsb2, Kit, Fcer1a & Gata2) with enhanced TGFß-related signaling that are phenotypically distinct from known MC subsets in the Mouse Cell Atlas. cKit-/- mice have greater lymphatic defects than TNF-tg mice with exacerbation of lymphatic dysfunction and inflammatory-erosive arthritis in TNF-tg x cKit-/- vs. TNF-Tg mice. Cromolyn sodium therapy stabilized PLV mast cells, increased TNF-induced bone loss, synovitis, and osteoclasts, and decreased ICG clearance. Conclusions: Mast cells are required for normal lymphatic function. Genetic ablation and pharmacological inhibition of mast cells exacerbates TNF-induced inflammatory-erosive arthritis with decreased lymphatic clearance. Together, these findings support an inflammatory role of activated/degranulated peri-PLV mast cells during arthritic progression, and a homeostatic role of intra-PLV mast cells, in which loss of the latter dominantly exacerbates arthritis secondary to defects in joint-draining lymphatics, warranting investigation into specific cellular mechanisms.

Multi-omics analysis identifies IgG2b class-switching with ALCAM-CD6 co-stimulation in joint-draining lymph nodes during advanced inflammatory-erosive arthritis.

Introduction: Defective lymphatic drainage and translocation of B-cells in inflamed (Bin) joint-draining lymph node sinuses are pathogenic phenomena in patients with severe rheumatoid arthritis (RA). However, the molecular mechanisms underlying this lymphatic dysfunction remain poorly understood. Herein, we utilized multi-omic spatial and single-cell transcriptomics to evaluate altered cellular composition (including lymphatic endothelial cells, macrophages, B-cells, and T-cells) in the joint-draining lymph node sinuses and their associated phenotypic changes and cell-cell interactions during RA development using the tumor necrosis factor transgenic (TNF-Tg) mouse model. Methods: Popliteal lymph nodes (PLNs) from wild-type (n=10) and TNF-Tg male mice with "Early" (5 to 6-months of age; n=6) and "Advanced" (>8-months of age; n=12) arthritis were harvested and processed for spatial transcriptomics. Single-cell RNA sequencing (scRNAseq) was performed in PLNs from the TNF-Tg cohorts (n=6 PLNs pooled/cohort). PLN histopathology and ELISPOT along with ankle histology and micro-CT were evaluated. Histopathology of human lymph nodes and synovia was performed for clinical correlation. Results: Advanced PLN sinuses exhibited an increased Ighg2b/Ighm expression ratio (Early 0.5 ± 0.1 vs Advanced 1.4 ± 0.5 counts/counts; p<0.001) that significantly correlated with reduced talus bone volumes in the afferent ankle (R2 = 0.54, p<0.001). Integration of single-cell and spatial transcriptomics revealed the increased IgG2b+ plasma cells localized in MARCO+ peri-follicular medullary sinuses. A concomitant decreased Fth1 expression (Early 2.5 ± 0.74 vs Advanced 1.0 ± 0.50 counts, p<0.001) within Advanced PLN sinuses was associated with accumulation of iron-laden Prussian blue positive macrophages in lymph nodes and synovium of Advanced TNF-Tg mice, and further validated in RA clinical samples. T-cells were increased 8-fold in Advanced PLNs, and bioinformatic pathway assessment identified the interaction between ALCAM+ macrophages and CD6+ T-cells as a plausible co-stimulatory mechanism to promote IgG2b class-switching. Discussion: Collectively, these data support a model of flare in chronic TNF-induced arthritis in which loss of lymphatic flow through affected joint-draining lymph nodes facilitates the interaction between effluxing macrophages and T-cells via ALCAM-CD6 co-stimulation, initiating IgG2b class-switching and plasma cell differentiation of the expanded Bin population. Future work is warranted to investigate immunoglobulin clonality and potential autoimmune consequences, as well as the efficacy of anti-CD6 therapy to prevent these pathogenic events.

Implementation of automated behavior metrics to evaluate voluntary wheel running effects on inflammatory-erosive arthritis and interstitial lung disease in TNF-Tg mice.

BACKGROUND: Although treatment options and algorithms for rheumatoid arthritis (RA) have improved remarkably in recent decades, there continues to be no definitive cure or pharmacologic intervention with reliable long-term efficacy. For this reason, the combination of medications and healthy lifestyle modifications are essential for controlling joint disease, and extra-articular manifestations of RA, such as interstitial lung disease (ILD) and other lung pathologies, which greatly impact morbidity and mortality. Generally, exercise has been deemed beneficial in RA patients, and both patients and clinicians are motivated to incorporate effective non-pharmacologic interventions. However, there are limited evidence-based and specific exercise regimens available to support engagement in such activities for RA patients. Here, we provided the continuous opportunity for exercise to mice and implemented automated recording and quantification of wheel running behavior. This allowed us to describe the associated effects on the progression of inflammatory-erosive arthritis and ILD in the tumor necrosis factor transgenic (TNF-Tg) mouse model of RA. METHODS: Wild-type (WT; males, n=9; females, n=9) and TNF-Tg (males, n=12; females, n=14) mice were singly housed with free access to a running wheel starting at 2 months until 5 to 5.5 months of age. Measures of running included distance, rate, length, and number of run bouts, which were derived from continuously recorded data streams collected automatically and in real-time. In vivo lung, ankle, and knee micro-computed tomography (micro-CT), along with terminal micro-CT and histology were performed to examine the association of running behaviors and disease progression relative to sedentary controls. RESULTS: TNF-Tg males and females exhibited significantly reduced running distance, rate, length, and number of run bouts compared to WT counterparts by 5 months of age (p<0.0001). Compared to sedentary controls, running males and females showed increased aerated lung volumes (p<0.05) that were positively correlated with running distance and rate in female mice (WT: Distance, ρ=0.705/rate, ρ=0.693 (p<0.01); TNF-Tg: ρ=0.380 (p=0.06)/ρ=0.403 (p<0.05)). Talus bone volumes were significantly reduced in running versus sedentary males and negatively correlated with running distance and rate in TNF-Tg mice (male: ρ=-903/ρ=-0.865; female: ρ=-0.614/ρ=-0.594 (p<0.001)). Histopathology validated the lung and ankle micro-CT findings. CONCLUSIONS: Implementation of automated wheel running behavior metrics allows for evaluation of longitudinal activity modifications hands-off and in real-time to relate with biomarkers of disease severity. Through such analysis, we determined that wheel running activity increases aerated lung volumes, but exacerbates inflammatory-erosive arthritis in TNF-Tg mice. To the end of a clinically relevant model, additional functional assessment of these outcomes and studies of pain behavior are warranted.

Near-Infrared Imaging of Indocyanine Green Identifies Novel Routes of Lymphatic Drainage from Metacarpophalangeal Joints in Healthy Human Hands.

Background: Collecting lymphatic vessel (CLV) dysfunction has been implicated in various diseases, including rheumatoid arthritis (RA). RA patients with active hand arthritis exhibit significantly reduced lymphatic clearance of the web spaces adjacent to the metacarpophalangeal (MCP) joints and a reduction in total and basilic-associated CLVs on the dorsal surface of the hand by near-infrared (NIR) imaging of indocyanine green (ICG). In this pilot study, we assessed direct lymphatic drainage from MCP joints and aimed to visualize the total lymphatic anatomy using novel dual-agent relaxation contrast magnetic resonance lymphography (DARC-MRL) in the upper extremity of healthy human subjects. Methods and Results: Two healthy male subjects >18 years old participated in the study. We performed NIR imaging along with conventional- or DARC-MRL following intradermal web space and intra-articular MCP joint injections. ICG (NIR) or gadolinium (Gd) (MRL) was administered to visualize the CLV anatomy of the upper extremity. Web space draining CLVs were associated with the cephalic side of the antecubital fossa, while MCP draining CLVs were localized to the basilic side of the forearm by near-infrared indocyanine green imaging. The DARC-MRL methods used in this study did not adequately nullify the contrast in the blood vessels, and limited Gd-filled CLVs were identified. Conclusion: MCP joints predominantly drain into basilic CLVs in the forearm, which may explain the reduction in basilic-associated CLVs in the hands of RA patients. Current DARC-MRL techniques show limited identification of healthy lymphatic structures, and further refinement in this technique is necessary. Clinical trial registration number: NCT04046146.