Erosive Inflammatory Arthritis Research Articles

Androgen Exhibits a Protective Role Against Focal Erosions in Murine TNF-induced Inflammatory Arthritis.

Rheumatoid arthritis (RA) is characterized by erosive pathology associated with joint inflammation and a sexual dimorphism with increased prevalence in females. Here, we aim to determine whether androgen is protective against inflammatory-erosive disease in TNF-transgenic (TNF-Tg) mice. Wild-type (WT) and TNF-Tg male mice underwent sham (WT, n = 3; TNF-Tg, n = 7) or orchiectomy (WT, n = 3; TNF-Tg, n = 7) surgery at 1 month old to remove androgen production confirmed by serum testosterone concentration. Cohorts of orchiectomized TNF-Tg males were treated with either 5ɑ-dihydrotestosterone (.025 mg/day) (n = 3) or placebo (n = 3) via subcutaneous pellet insertion. Weekly clinical measures, along with mid-hindpaw bone volumes and ankle histology at 3 months old were evaluated for all groups. Orchiectomies in TNF-Tg males significantly decreased serum testosterone (P < .05), weight gain (P < .001), and mid-hindpaw bone volumes (P < .05) in comparison to sham TNF-Tg mice. The cuboid bone also had increased synovitis by histology with the loss of androgen (P < .05). Treatment of orchiectomized TNF-Tg males with 5ɑ-dihydrotestosterone protected against the changes in weight gain (P < .01) and bone erosion (P < .05) associated with decreased osteoclast number in the cuboid (P < .01). In the TNF-Tg model of chronic inflammatory arthritis, androgen is protective in erosive disease. The loss of endogenous androgen significantly accelerated the progression of inflammatory-erosive arthritis in male TNF-Tg mice to a similar severity as age-matched female mice. In addition, treatment with exogenous androgen prevented this observed bone loss in orchiectomized TNF-Tg males. Overall, androgen delays and limits bone erosion even in the presence of active inflammation and future studies are warranted to elucidate the associated mechanisms.

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.

DNA hypomethylation ameliorates erosive inflammatory arthritis by modulating interferon regulatory factor-8

Epigenetic regulation plays a crucial role in the pathogenesis of autoimmune diseases such as inflammatory arthritis. DNA hypomethylating agents, such as decitabine (DAC), have been shown to dampen inflammation and restore immune homeostasis. In the present study, we demonstrate that DAC elicits potent anti-inflammatory effects and attenuates disease symptoms in several animal models of arthritis. Transcriptomic and epigenomic profiling show that DAC-mediated hypomethylation regulates a wide range of cell types in arthritis, altering the differentiation trajectories of anti-inflammatory macrophage populations, regulatory T cells, and tissue-protective synovial fibroblasts (SFs). Mechanistically, DAC-mediated demethylation of intragenic 5'-Cytosine phosphate Guanine-3' (CpG) islands of the transcription factor Irf8 (interferon regulatory factor 8) induced its re-expression and promoted its repressor activity. As a result, DAC restored joint homeostasis by resetting the transcriptomic signature of negative regulators of inflammation in synovial macrophages (MerTK, Trem2, and Cx3cr1), TREGs (Foxp3), and SFs (Pdpn and Fapα). In conclusion, we found that Irf8 is necessary for the inhibitory effect of DAC in murine arthritis and that direct expression of Irf8 is sufficient to significantly mitigate arthritis.

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

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. 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. 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 TGFβ-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. 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.

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. 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. 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. 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

BackgroundAlthough 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.MethodsWild-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.ResultsTNF-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.ConclusionsImplementation 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.

Persistent popliteal lymphatic muscle cell coverage defects despite amelioration of arthritis and recovery of popliteal lymphatic vessel function in TNF-Tg mice following anti-TNF therapy

While rheumatoid arthritis patients and tumor necrosis factor transgenic (TNF-Tg) mice with inflammatory-erosive arthritis display lymphatic drainage deficits, the mechanisms responsible remain unknown. As ultrastructural studies of joint-draining popliteal lymphatic vessels (PLVs) in TNF-Tg mice revealed evidence of lymphatic muscle cell (LMC) damage, we aimed to evaluate PLV-LMC coverage in TNF-Tg mice. We tested the hypothesis that alpha smooth muscle actin (αSMA)+ PLV-LMC coverage decreases with severe inflammatory-erosive arthritis, and is recovered by anti-TNF therapy facilitated by increased PLV-LMC turnover during amelioration of joint disease. TNF-Tg mice with established disease received anti-TNF monoclonal antibody (mAb) or placebo IgG isotype control mAb therapy (n = 5) for 6-weeks, while wild-type (WT) littermates (n = 8) received vehicle (PBS). Bromodeoxyuridine (BrdU) was also administered daily during the treatment period to monitor PLV-LMC turnover. Effective anti-TNF therapy was confirmed by longitudinal assessment of popliteal lymph node (PLN) volume via ultrasound, PLV contraction frequency via near-infrared imaging of indocyanine green, and ankle bone volumes via micro-computed tomography (micro-CT). Terminal knee micro-CT, and ankle and knee histology were also performed. PLVs were immunostained for αSMA and BrdU to evaluate PLV-LMC coverage and turnover, respectively, via whole-mount fluorescent microscopy. Anti-TNF therapy reduced PLN volume, increased talus and patella bone volumes, and reduced tarsal and knee synovial areas compared to placebo treated TNF-Tg mice (p < 0.05), as expected. Anti-TNF therapy also increased PLV contraction frequency at 3-weeks (from 0.81 ± 1.0 to 3.2 ± 2.0 contractions per minute, p < 0.05). However, both anti-TNF and placebo treated TNF-Tg mice exhibited significantly reduced αSMA+ PLV-LMC coverage compared to WT (p < 0.05). There was no correlation of αSMA+ PLV-LMC coverage restoration with amelioration of inflammatory-erosive arthritis. Similarly, there was no difference in PLV-LMC turnover measured by BrdU labeling between WT, TNF-Tg placebo, and TNF-Tg anti-TNF groups with an average of < 1% BrdU+ PLV-LMCs incorporated per week. Taken together these results demonstrate that PLV-LMC turnover in adult mice is limited, and that recovery of PLV function during amelioration of inflammatory-erosive arthritis occurs without restoration of αSMA+ LMC coverage. Future studies are warranted to investigate the direct and indirect effects of chronic TNF exposure, and the role of proximal inflammatory cells on PLV contractility.

Single-cell transcriptomics of popliteal lymphatic vessels and peripheral veins reveals altered lymphatic muscle and immune cell populations in the TNF-Tg arthritis model

BackgroundLymphatic dysfunction exists in tumor necrosis factor transgenic (TNF-Tg) mice and rheumatoid arthritis (RA) patients. While joint-draining TNF-Tg popliteal lymphatic vessels (PLVs) have deficits in contractility during end-stage arthritis, the nature of lymphatic muscle cells (LMCs) and their TNF-altered transcriptome remain unknown. Thus, we performed single-cell RNA-sequencing (scRNAseq) on TNF-Tg LMCs in PLVs efferent to inflamed joints versus wild-type (WT) controls.MethodsSingle-cell suspensions of PLVs were sorted for smooth muscle cells (SMCs), which was validated by Cspg4-Cre;tdTomato reporter gene expression. Single-cell RNA-seq was performed on a 10x Genomics platform and analyzed using the Seurat R package. Uniform Manifold Approximation and Projections (UMAPs) and Ingenuity Pathway Analysis software were used to assess cell clusters and functional genomics in WT vs. TNF-Tg populations.ResultsFluorescent imaging of Cspg4-Cre;tdTomato vessels demonstrated dim PLVs and strong reporter gene expression in the adjacent superficial saphenous vein, which was corroborated by flow cytometry of LMCs and vascular smooth muscle cells (VSMCs) from these vessels. Due to their unique morphology, these populations could also be readily detected by scatter analysis of cells from non-fluorescent mice. Bioinformatics analysis of flow sorted WT and TNF-Tg cells identified 20 unique cell clusters that together were 22.4% LMCs, 15.0% VSMCs, and 62.6% non-muscle cells of 8879 total cells. LMCs and M2-macrophages were decreased, while inflammatory monocytes were increased in TNF-Tg lower limb vasculature. SMC populations were defined by Cald1, Tpm1, and Pdgfrb expression and were enriched in myofibroblast-like gene expression. TNF-Tg LMCs exhibited enhanced functional genomics associated with cell death, phagocyte recruitment, and joint inflammation. Among the most prominent TNF-induced genes in SMCs were Mmp3, Cxcl12, and Ccl19, and the most downregulated genes were Zbtb16, Galnt15, and Apod.ConclusionsSingle-cell RNA-seq can be used to investigate functional genomics of lower limb vasculature in mice. Our findings confirm the inflammatory transcriptome of TNF-Tg vessels and altered gene expression in SMC populations. This study further supports a potential role of mesenchymal stromal cells in inflammatory-erosive arthritis pathogenesis, and warrants future studies to define the effects of this TNF-altered transcriptome on PLV function and joint homeostasis.

O01 Post-transplant lymphoproliferative disorder (PTLD) in a patient with rheumatoid arthritis

O01 Post-transplant lymphoproliferative disorder (PTLD) in a patient with rheumatoid arthritis

INOS dependent and independent phases of lymph node expansion in mice with TNF-induced inflammatory-erosive arthritis

IntroductionA pivotal effect of lymphatic vessel (LV) function in joint homeostasis was identified in the tumor necrosis factor-transgenic (TNF-Tg) mouse model of rheumatoid arthritis (RA). Specifically, loss of LV contractions is associated with progressive synovitis and erosions. Furthermore, draining lymph node expansion is a biomarker of arthritic progression, and both macrophages and lymphatic endothelial cells express inducible nitric oxide synthase (iNOS), which disrupts LV contraction and transport of immune cells to the draining lymph nodes. Therefore, to directly assess these relationships, we tested the hypothesis that TNF-Tg mice with global genetic ablation of iNOS (iNOS−/−) will show delayed draining lymph node expansion, maintained LV contractions, and decreased synovitis and erosions.MethodiNOS−/−× TNF-Tg female and male mice, and control littermates (iNOS−/−, TNF-Tg, and WT), were examined with (1) ultrasound to determine popliteal lymph node (PLN) volume and (2) near-infrared imaging (NIR) to assess popliteal LV contraction frequency, and differences between genotypes were assessed at 3, 4, 5, and 6 months of age. Knees and PLN were harvested at 4 months in females and 6 months in males, to assess synovitis, bone erosions, and cellular accumulation in PLN sinuses via histology.ResultsInitially, an increase in PLN volume was observed for both female and male iNOS−/−× TNF-Tg and TNF-Tg compared to their WT and iNOS−/− counterparts at 2 and 3 months, respectively. Subsequently, TNF-Tg PLNs continue to increase in volume, while iNOS−/−× TNF-Tg did not increase in volume from the initial timepoints. WT and iNOS−/− PLN volume was unchanged throughout the experiment. LV contraction frequency was increased at 4 months in females and 5 months in males, in the iNOS−/−× TNF-Tg mice compared to the TNF-Tg. Synovitis and erosions were moderately reduced in iNOS−/−× TNF-Tg versus TNF-Tg knees in females, while no differences in knee pathology were observed in males.ConclusionsGenetic iNOS ablation maintains draining lymph node volume and LV function during TNF-induced inflammatory arthritis and is associated with moderately decreased joint inflammation and damage.

New Insights for RANKL as a Proinflammatory Modulator in Modeled Inflammatory Arthritis

Receptor activator of nuclear factor-κB ligand (RANKL), a member of the Tumor Necrosis Factor (TNF) superfamily, constitutes the master regulator of osteoclast formation and bone resorption, whereas its involvement in inflammatory diseases remains unclear. Here, we used the human TNF transgenic mouse model of erosive inflammatory arthritis to determine if the progression of inflammation is affected by either genetic inactivation or overexpression of RANKL in transgenic mouse models. TNF-mediated inflammatory arthritis was significantly attenuated in the absence of functional RANKL. Notably, TNF overexpression could not compensate for RANKL-mediated osteopetrosis, but promoted osteoclastogenesis between the pannus and bone interface, suggesting RANKL-independent mechanisms of osteoclastogenesis in inflamed joints. On the other hand, simultaneous overexpression of RANKL and TNF in double transgenic mice accelerated disease onset and led to severe arthritis characterized by significantly elevated clinical and histological scores as shown by aggressive pannus formation, extended bone resorption, and massive accumulation of inflammatory cells, mainly of myeloid origin. RANKL and TNF cooperated not only in local bone loss identified in the inflamed calcaneous bone, but also systemically in distal femurs as shown by microCT analysis. Proteomic analysis in inflamed ankles from double transgenic mice overexpressing human TNF and RANKL showed an abundance of proteins involved in osteoclastogenesis, pro-inflammatory processes, gene expression regulation, and cell proliferation, while proteins participating in basic metabolic processes were downregulated compared to TNF and RANKL single transgenic mice. Collectively, these results suggest that RANKL modulates modeled inflammatory arthritis not only as a mediator of osteoclastogenesis and bone resorption but also as a disease modifier affecting inflammation and immune activation.

Targeting lymphatic function as a novel therapeutic intervention for rheumatoid arthritis.

Although clinical outcomes for patients with rheumatoid arthritis (RA) have greatly improved with the use of biologic and conventional DMARDs, approximately 40% of patients do not achieve primary clinical outcomes in randomized trials, and only a small proportion achieve lasting remission. Over the past decade, studies in murine models point to the critical role of the lymphatic system in the pathogenesis and therapy of inflammatory-erosive arthritis, presumably by the removal of catabolic factors, cytokines and inflammatory cells from the inflamed synovium. Murine studies demonstrate that lymphatic drainage increases at the onset of inflammatory-erosive arthritis but, as inflammation progresses to a more chronic phase, lymphatic clearance declines and both structural and cellular changes are observed in the draining lymph node. Specifically, chronic damage to the lymphatic vessel from persistent inflammation results in loss of lymphatic vessel contraction followed by lymph node collapse, reduced lymphatic drainage, and ultimately severe synovitis and joint erosion. Notably, clinical pilot studies in patients with RA report lymph node changes following treatment, and thus draining lymphatic vessels and nodes could represent a potential biomarker of arthritis activity and response to therapy. Most importantly, targeting lymphatics represents an innovative strategy for therapeutic intervention for RA.

Brief Report: Treatment of Tumor Necrosis Factor-Transgenic Mice With Anti-Tumor Necrosis Factor Restores Lymphatic Contractions, Repairs Lymphatic Vessels, and May Increase Monocyte/Macrophage Egress.

Recent studies have demonstrated that there is an inverse relationship between lymphatic egress and inflammatory arthritis in affected joints. As a model, tumor necrosis factor (TNF)-transgenic mice develop advanced arthritis following draining lymph node (LN) collapse, and loss of lymphatic contractions downstream of inflamed joints. It is unknown if these lymphatic deficits are reversible. This study was undertaken to test the hypothesis that anti-TNF therapy reduces advanced erosive inflammatory arthritis, associated with restoration of lymphatic contractions, repair of damaged lymphatic vessels, and evidence of increased monocyte egress. TNF-transgenic mice with advanced arthritis and collapsed popliteal LNs were treated with anti-TNF monoclonal antibody (10 mg/kg weekly) or placebo for 6 weeks, and effects on knee synovitis, lymphatic vessel ultrastructure and function, and popliteal LN cellularity were assessed by ultrasound, histology, transmission electron microscopy (TEM), near-infrared indocyanine green imaging, and flow cytometry. Anti-TNF therapy significantly decreased synovitis (∼5-fold; P < 0.05 versus placebo), restored lymphatic contractions, and significantly increased the number of popliteal LN monocyte/macrophages (∼2-fold; P < 0.05 versus placebo). TEM demonstrated large activated macrophages attached to damaged lymphatic endothelium in mice with early arthritis, extensively damaged lymphatic vessels in placebo-treated mice with advanced arthritis, and rolling leukocytes in repaired lymphatic vessels in mice responsive to anti-TNF therapy. These findings support the concept that anti-TNF therapy ameliorates erosive inflammatory arthritis, in part via restoration of lymphatic vessel contractions and potential enhancement of inflammatory cell egress.

Lymphatic imaging to assess rheumatoid flare: mechanistic insights and biomarker potential.

Proliferation of draining lymphatic vessels coupled with dynamic changes in lymph node volume and flow are characteristic features in rheumatoid arthritis (RA). Furthermore, impaired lymph egress from inflamed synovium is associated with joint flare in murine models of inflammatory-erosive arthritis. Unfortunately, advances towards a greater understanding of lymphatic changes in RA pathogenesis have been slow due to the absence of outcome measures to quantify lymphatic function in vivo. While lymphoscintigraphy is the current standard to assess lymphedema and sentinel lymph nodes in cancer patients, its sensitivity and specificity are inadequate to study lymphatics in RA. The emergence of high-resolution MRI, power Doppler ultrasound, and near-infrared imaging that permits real-time quantification of lymphatic function in animal models has been a major advance, and these techniques have produced a new paradigm of altered lymphatic function that underlies both acute arthritic flare and chronic inflammation. In acute flare, lymphatic drainage increases several fold, whereas no lymphatic contractions are detected in lymph vessels draining chronic arthritic joints. Moreover, these outcomes are now being adapted to study lymphatics in RA towards the development of novel biomarkers of arthritic flare and the discovery of new therapeutic targets. In particular, interventions that directly increase lymphatic egress from diseased joints by opening collateral lymphatic vessels, and that restore lymphatic vessel contractions, provide novel therapeutic approaches with potential for minimal toxicity and immunosuppression. To summarize the origins of this field, recent advances, and future directions, we herein review: current knowledge of lymphatics in RA based on classic literature; new in-vivo imaging modalities that have elucidated how lymphatics modulate acute versus chronic joint inflammation in murine models; and how these preclinical outcome measures are being translated to study lymphatic function in RA inflammation and how effective RA therapies alter lymphatic flow and lymph nodes draining flaring joints. Trial registration: ClinicalTrials.gov NCT02680067. Registered 7 December 2015; ClinicalTrials.gov NCT01098201. Registered 30 March 2010; and ClinicalTrials.gov NCT01083563. Registered 8 March 2010.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1092-0) contains supplementary material, which is available to authorized users.

TNF signals are dispensable for the generation of CD23+ CD21/35-high CD1d-high B cells in inflamed lymph nodes

Tumor necrosis factor (TNF) is a key cytokine in rheumatoid arthritis (RA) pathogenesis, as underscored by the clinical effectiveness of TNF antagonists. While several of TNF’s key targets in RA are well understood, its many pleiotropic effects remain to be elucidated. TNF-transgenic mice develop inflammatory-erosive arthritis associated with disruption of draining lymph node histology and function, and accumulation of B cells with unique phenotypic and functional features consistent with contribution to pathogenesis (B cells in inflamed nodes, Bin). Bin cell induction depends on the inflamed microenvironment, but the specific signals are unknown. Using anti-TNF treatment and TNF-receptor-deficient mice, here we show that Bin cells are induced and maintained independently of B cell-intrinsic TNF signals.

In vivo quantification of lymph viscosity and pressure in lymphatic vessels and draining lymph nodes of arthritic joints in mice

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease with episodic flares. In TNF-Tg mice, a model of inflammatory-erosive arthritis, the popliteal lymph node (PLN) enlarges during the pre-arthritic 'expanding' phase, and then 'collapses' with adjacent knee flare associated with the loss of the intrinsic lymphatic pulse. As the mechanisms responsible are unknown, we developed in vivo methods to quantify lymph viscosity and pressure in mice with wild-type (WT), expanding and collapsed PLN. While no differences in viscosity were detected via multiphoton fluorescence recovery after photobleaching (MP-FRAP) of injected FITC-BSA, a 32.6% decrease in lymph speed was observed in vessels afferent to collapsed PLN (P<0.05). Direct measurement of intra-lymph node pressure (LNP) demonstrated a decrease in expanding PLN versus WT pressure (3.41±0.43 vs. 6.86±0.56cmH2O; P<0.01), which dramatically increased to 9.92±1.79cmH2O in collapsed PLN. Lymphatic pumping pressure (LPP), measured indirectly by slowly releasing a pressurized cuff occluding indocyanine green (ICG), demonstrated an increase in vessels afferent to expanding PLN versus WT (18.76±2.34 vs. 11.04±1.47cmH2O; P<0.01), which dropped to 2.61±0.72cmH2O (P<0.001) after PLN collapse. Herein, we document the first in vivo measurements of murine lymph viscosity and lymphatic pressure, and provide evidence to support the hypothesis that lymphangiogenesis and lymphatic transport are compensatory mechanisms to prevent synovitis via increased drainage of inflamed joints. Furthermore, the decrease in lymphatic flow and loss of LPP during PLN collapse are consistent with decreased drainage from the joint during arthritic flare, and validate these biomarkers of RA progression and possibly other chronic inflammatory conditions.

Bone fragility beyond strength and mineral density: Raman spectroscopy predicts femoral fracture toughness in a murine model of rheumatoid arthritis

Clinical prediction of bone fracture risk primarily relies on measures of bone mineral density (BMD). BMD is strongly correlated with bone strength, but strength is independent of fracture toughness, which refers to the bone's resistance to crack initiation and propagation. In that sense, fracture toughness is more relevant to assessing fragility-related fracture risk, independent of trauma. We hypothesized that bone biochemistry, determined by Raman spectroscopy, predicts bone fracture toughness better than BMD. This hypothesis was tested in tumor necrosis factor-transgenic mice (TNF-tg), which develop inflammatory-erosive arthritis and osteoporosis. The left femurs of TNF-tg and wild type (WT) littermates were measured with Raman spectroscopy and micro-computed tomography. Fracture toughness was assessed by cutting a sharp notch into the anterior surface of the femoral mid-diaphysis and propagating the crack under 3 point bending. Femoral fracture toughness of TNF-tg mice was significantly reduced compared to WT controls (p=0.04). A Raman spectrum-based prediction model of fracture toughness was generated by partial least squares regression (PLSR). Raman spectrum PLSR analysis produced strong predictions of fracture toughness, while BMD was not significantly correlated and produced very weak predictions. Raman spectral components associated with mineralization quality and bone collagen were strongly leveraged in predicting fracture toughness, reiterating the limitations of mineralization density alone.

Mechanisms of bone fragility in a mouse model of glucocorticoid‐treated rheumatoid arthritis: Implications for insufficiency fracture risk

Glucocorticoid (GC) therapy is associated with increased risk of fracture in patients with rheumatoid arthritis (RA). To elucidate the cause of this increased risk, we examined the effects of chronic erosive inflammatory arthritis and GC treatment on bone quality, structure, and biomechanical properties in a murine model. Mice with established arthritis and expressing human tumor necrosis factor α (TNFα) transgene (Tg) and their wild-type (WT) littermates were continually treated with GC (prednisolone 5 mg/kg/day via subcutaneous controlled-release pellet) or placebo for 14, 28, or 42 days. Microstructure, biomechanical properties, chemical composition, and morphology of the tibiae and lumbar vertebral bodies were assessed by micro-computed tomography, biomechanical testing, Raman spectroscopy, and histology, respectively. Serum markers of bone turnover were also determined. TNF-Tg and GC treatment additively decreased mechanical strength and stiffness in both the tibiae and the vertebral bodies. GC treatment in the TNF-Tg mice increased the ductility of tibiae under torsional loading. These changes were associated with significant alterations in the biochemical and structural composition of the mineral and organic components of the bone matrix, a decrease in osteoblast activity and bone formation, and an increase in osteoclast activity. Our findings indicate that the concomitant decrease in bone strength and increase in bone ductility associated with chronic inflammation and GC therapy, coupled with the significant changes in the bone quality and structure, may increase the susceptibility of the bone to failure under low-energy loading. This may explain the mechanism of symptomatic insufficiency fractures in patients with RA receiving GC therapy who do not have radiographic manifestations of fracture.

Diagnostic radiographique d’un rhumatisme des mains et des poignets

Hand and wrist are the most frequent target of a large pannel of arthritis. The radiological diagnostic is often difficult at an early stage. The first radiological sign to be used is the presence of marginal bone erosions which is the hallmark of erosive inflammatory arthritis such as rheumatoid arthritis and psoriatic arthritis and, less commonly, gout. Marginal erosions occur at sites where intra-articular bone is not protected by an overlying hyaline cartilage. Presence of bone proliferation in association with marginal erosion or at enthesitic sites are the hallmark of spondylarthropathies, mainly psoriatic arthritis at the hand and wrist since it is lacking in rheumatoïd arthritis. Type of joint space narrowing is another important sign since diffuse joint space narrowing is very suggestive of inflammatory arthritis while focal joint space narrowing is suggestive of osteoarthritis and metabolic disorders. In the absence of marginal erosions, the radiological appearance is often suggestive of a degenerative disease with osteophytes and subchondral sclerosis. In such cases, distribution of the abnormal joints is a key sign since osteoarthritis predominates in the interphalangeal joints while CCPD arthropathy and hemochromatosis arthropathy are more marked in the metacarpophalangeal joints.

Validation of 3-dimensional ultrasound versus magnetic resonance imaging quantification of popliteal lymph node volume as a biomarker of erosive inflammatory arthritis in mice

Validation of 3-dimensional ultrasound versus magnetic resonance imaging quantification of popliteal lymph node volume as a biomarker of erosive inflammatory arthritis in mice