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.

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.

The Enigmas of Lymphatic Muscle Cells: Where Do They Come From, How Are They Maintained, and Can They Regenerate?

Lymphatic muscle cell (LMC) contractility and coverage of collecting lymphatic vessels (CLVs) are integral to effective lymphatic drainage and tissue homeostasis. In fact, defects in lymphatic contractility have been identified in various conditions, including rheumatoid arthritis, inflammatory bowel disease, and obesity. However, the fundamental role of LMCs in these pathologic processes is limited, primarily due to the difficulty in directly investigating the enigmatic nature of this poorly characterized cell type. LMCs are a unique cell type that exhibit dual tonic and phasic contractility with hybrid structural features of both vascular smooth muscle cells (VSMCs) and cardiac myocytes. While advances have been made in recent years to better understand the biochemistry and function of LMCs, central questions regarding their origins, investiture into CLVs, and homeostasis remain unanswered. To summarize these discoveries, unexplained experimental results, and critical future directions, here we provide a focused review of current knowledge and open questions related to LMC progenitor cells, recruitment, maintenance, and regeneration. We also highlight the high-priority research goal of identifying LMC-specific genes towards genetic conditional- inducible in vivo gain and loss of function studies. While our interest in LMCs has been focused on understanding lymphatic dysfunction in an arthritic flare, these concepts are integral to the broader field of lymphatic biology, and have important potential for clinical translation through targeted therapeutics to control lymphatic contractility and drainage.

Dendritic cell-specific transmembrane protein is required for synovitis and bone resorption in inflammatory arthritis.

Objective: Dendritic Cell-Specific Transmembrane Protein (DC-STAMP) is essential for the formation of fully functional multinucleated osteoclasts. DC-STAMP deficient mice, under physiological conditions, exhibit osteopetrosis and develop systemic autoimmunity with age. However, the function of DC-STAMP in inflammation is currently unknown. We examined whether genetic ablation of DC-STAMP attenuates synovitis and bone erosion in TNF transgenic (Tg) and K/BxN serum-induced murine rheumatoid arthritis. Methods: We evaluated arthritis onset in Tg(hTNF) mice lacking DC-STAMP and 50:50 chimeric mice by visual examination, measurement of ankle width, micro-CT-scan analysis and quantitation of the area occupied by osteoclasts in bone sections. To further investigate the cellular and molecular events modulated by DC-STAMP, we measured serum cytokines, determined changes in cytokine mRNA expression by monocytes activated with IL4 or LPS/IFNγ and enumerated immune cells in inflamed mouse joints. Results: Synovitis, bone loss and matrix destruction are markedly reduced in Dcstamp-/-;Tg(hTNF) mice. These mice had significantly lower CCL2 and murine TNF serum levels and exhibited impaired monocyte joint migration compared to Tg(hTNF) mice. The reduced arthritic severity in Dcstamp deficient mice was associated with compromised monocyte chemotaxis, cytokine production, and M2 polarization. Conclusion: These results reveal that DC-STAMP modulates both bone resorption and inflammation and may serve as an activity biomarker and therapeutic target in inflammatory arthritis and metabolic bone disease.

Extracellular sulfatase-2 is overexpressed in rheumatoid arthritis and mediates the TNF-α-induced inflammatory activation of synovial fibroblasts.

Extracellular sulfatase-2 (Sulf-2) influences receptor-ligand binding and subsequent signaling by chemokines and growth factors, yet Sulf-2 remains unexplored in inflammatory cytokine signaling in the context of rheumatoid arthritis (RA). In the present study, we characterized Sulf-2 expression in RA and investigated its potential role in TNF-α-induced synovial inflammation using primary human RA synovial fibroblasts (RASFs). Sulf-2 expression was significantly higher in serum and synovial tissues from patients with RA and in synovium and serum from hTNFtg mice. RNA sequencing analysis of TNF-α-stimulated RASFs showed that Sulf-2 siRNA modulated ~2500 genes compared to scrambled siRNA. Ingenuity Pathway Analysis of RNA sequencing data identified Sulf-2 as a primary target in fibroblasts and macrophages in RA. Western blot, ELISA, and qRT‒PCR analyses confirmed that Sulf-2 knockdown reduced the TNF-α-induced expression of ICAM1, VCAM1, CAD11, PDPN, CCL5, CX3CL1, CXCL10, and CXCL11. Signaling studies identified the protein kinase C-delta (PKCδ) and c-Jun N-terminal kinase (JNK) pathways as key in the TNF-α-mediated induction of proteins related to cellular adhesion and invasion. Knockdown of Sulf-2 abrogated TNF-α-induced RASF proliferation. Sulf-2 knockdown with siRNA and inhibition by OKN-007 suppressed the TNF-α-induced phosphorylation of PKCδ and JNK, thereby suppressing the nuclear translocation and DNA binding activity of the transcription factors AP-1 and NF-κBp65 in human RASFs. Interestingly, Sulf-2 expression positively correlated with the expression of TNF receptor 1, and coimmunoprecipitation assays demonstrated the binding of these two proteins, suggesting they exhibit crosstalk in TNF-α signaling. This study identified a novel role of Sulf-2 in TNF-α signaling and the activation of RA synoviocytes, providing the rationale for evaluating the therapeutic targeting of Sulf-2 in preclinical models of RA.

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.

BACKGROUND: Lymphatic 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. METHODS: Single-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. RESULTS: Fluorescent 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. CONCLUSIONS: Single-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.

A high-throughput semi-automated bone segmentation workflow for murine hindpaw micro-CT datasets.

INTRODUCTION: Micro-computed tomography (µCT) is a valuable imaging modality for longitudinal quantification of bone volumes to identify disease or treatment effects for a broad range of conditions that affect bone health. Complex structures, such as the hindpaw with up to 31 distinct bones in mice, have considerable analytic potential, but quantification is often limited to a single bone volume metric due to the intensive effort of manual segmentation. Herein, we introduce a high-throughput, user-friendly, and semi-automated method for segmentation of murine hindpaw µCT datasets. METHODS: In vivo µCT was performed on male (n = 4; 2-8-months) and female (n = 4; 2-5-months) C57BL/6 mice longitudinally each month. Additional 9.5-month-old male C57BL/6 hindpaws (n = 6 hindpaws) were imaged by ex vivo µCT to investigate the effects of resolution and integration time on analysis outcomes. The DICOMs were exported to Amira software for the watershed-based segmentation, and watershed markers were generated automatically at approximately 80% accuracy before user correction. The semi-automated segmentation method utilizes the original data, binary mask, and bone-specific markers that expand to the full volume of the bone using watershed algorithms. RESULTS: Compared to the conventional manual segmentation using Scanco software, the semi-automated approach produced similar raw bone volumes. The semi-automated segmentation also demonstrated a significant reduction in segmentation time for both experienced and novice users compared to standard manual segmentation. ICCs between experienced and novice users were >0.9 (excellent reliability) for all but 4 bones. DISCUSSION: The described semi-automated segmentation approach provides remarkable reliability and throughput advantages. Adoption of the semi-automated segmentation approach will provide standardization and reliability of bone volume measures across experienced and novice users and between institutions. The application of this model provides a considerable strategic advantage to accelerate various research opportunities in pre-clinical bone and joint analysis towards clinical translation.

Ex vivo Demonstration of Functional Deficiencies in Popliteal Lymphatic Vessels From TNF-Transgenic Mice With Inflammatory Arthritis.

Background: Recent studies demonstrated lymphangiogenesis and expansion of draining lymph nodes during chronic inflammatory arthritis, and lymphatic dysfunction associated with collapse of draining lymph nodes in rheumatoid arthritis (RA) patients and TNF-transgenic (TNF-Tg) mice experiencing arthritic flare. As the intrinsic differences between lymphatic vessels afferent to healthy, expanding, and collapsed draining lymph nodes are unknown, we characterized the ex vivo behavior of popliteal lymphatic vessels (PLVs) from WT and TNF-Tg mice. We also interrogated the mechanisms of lymphatic dysfunction through inhibition of nitric oxide synthase (NOS). Methods: Popliteal lymph nodes (PLNs) in TNF-Tg mice were phenotyped as Expanding or Collapsed by in vivo ultrasound and age-matched to WT littermate controls. The PLVs were harvested and cannulated for ex vivo functional analysis over a relatively wide range of hydrostatic pressures (0.5-10 cmH2O) to quantify the end diastolic diameter (EDD), tone, amplitude (AMP), ejection fraction (EF), contraction frequency (FREQ), and fractional pump flow (FPF) with or without NOS inhibitors Data were analyzed using repeated measures two-way ANOVA with Bonferroni's post hoc test. Results: Real time videos of the cannulated PLVs demonstrated the predicted phenotypes of robust vs. weak contractions of the WT vs. TNF-Tg PLV, respectively. Quantitative analyses confirmed that TNF-Tg PLVs had significantly decreased AMP, EF, and FPF vs. WT (p < 0.05). EF and FPF were recovered by NOS inhibition, while the reduction in AMP was NOS independent. No differences in EDD, tone, or FREQ were observed between WT and TNF-Tg PLVs, nor between Expanding vs. Collapsed PLVs. Conclusion: These findings support the concept that chronic inflammatory arthritis leads to NOS dependent and independent draining lymphatic vessel dysfunction that exacerbates disease, and may trigger arthritic flare due to decreased egress of inflammatory cells and soluble factors from affected joints.

Characterization of the temporal, cell-specific and interferon-inducible patterns of indoleamine 2,3 dioxygenase 1 (IDO1) expression in the human placenta across gestation.

INTRODUCTION: The human placenta performs multiple functions necessary for successful pregnancy, but the metabolic pathways and molecular mechanisms responsible for regulating placental development and functions remain incompletely understood. Catabolism of the essential amino acid tryptophan has numerous critical roles in normal physiology, including inflammation. The kynurenine pathway, which accounts for ∼90% of tryptophan breakdown, is mediated by indoleamine 2,3 dioxygenase 1 (IDO1) in the placenta. In pregnant mice, alterations of IDO1 activity or expression result in fetal resorption and a preeclampsia-like phenotype. Decreased IDO1 expression at the maternal-fetal interface has also been linked to preeclampsia, in utero growth restriction and recurrent miscarriage in humans. These collective observations suggest essential role(s) for IDO1 in maintaining healthy pregnancy. Despite these important roles, the precise temporal, cell-specific and inflammatory cytokine-mediated patterns of IDO1 expression in the human placenta have not been thoroughly characterized across gestation. METHODS: Western blot and whole mount immunofluorescence (WMIF) were utilized to characterize and quantify basal and interferon (IFN)-inducible IDO1 expression in 1st trimester (7-13 weeks), 2nd trimester (14-22 weeks) and term (39-41 weeks) placental villi. RESULTS: IDO1 expression is activated in the human placenta between the 13th and 14th weeks of pregnancy, increases through the 2nd trimester and remains elevated at term. Constitutive IDO1 expression is restricted to placental endothelial cells. Interestingly, different types of IFNs have distinct effects on IDO1 expression in the human placenta. DISCUSSION: Our collective results are consistent with potential role(s) for IDO1 in the regulation of vascular functions in placental villi.

TNF-Polarized Macrophages Produce Insulin-like 6 Peptide to Stimulate Bone Formation in Rheumatoid Arthritis in Mice.

The risk of osteoporosis is increased in rheumatoid arthritis (RA). Anti-tumor necrosis factor (TNF) therapy has markedly improved the outcomes of RA patients but does not improve osteoporosis in some reports. This could be a combined result of disease severity and other therapeutic agents, such as glucocorticoids that accelerate osteoporosis progression. We evaluated the effects of anti-TNF therapy on osteoporosis in an animal model of RA and explored the possible mechanisms involved. Six-week-old TNF transgenic (TNF-Tg) mice with early stage erosive arthritis were treated with TNF antibody (Ab) or control immunoglobulin (IgG) weekly for 4 weeks. We found that TNF Ab completely blocked the development of erosive arthritis in TNF-Tg mice, but only slightly increased vertebral bone mass, associated with reduction in parameters of both bone resorption and formation. Similarly, TNF Ab slightly increased trabecular bone mass in tibias of 8-month-old TNF-Tg mice with advanced erosive arthritis. Interestingly, TNFα increased osteoblast differentiation from mouse bone marrow stromal cells (BMSCs) containing large number of macrophages but not from pure mesenchymal progenitor cells (MPCs). TNFα-polarized macrophages (TPMs) did not express iNos and Arginase 1, typical markers of inflammatory and resident macrophages. Interestingly, TPMs stimulated osteoblast differentiation, unlike resident and inflammatory macrophages polarized by IL-4 and interferon-λ, respectively. RNA-seq analysis indicated that TPMs produced several anabolic factors, including Jagged1 and insulin like 6 (INSL6). Importantly, inhibition of either Jagged1 or INSL6 blocked TNFα-induced osteoblast differentiation. Furthermore, INSL6 Ab significantly decreased the expansion of TNF-induced MPCs in BMSCs, and anti-TNF Ab reduced INSL6 expression by macrophages in vitro and in TNF-Tg mice in vivo. We conclude that TPMs produce INSL6 to stimulate bone formation and anti-TNF Ab blocks not only enhanced bone resorption but also the anabolic effect of TPMs on bone, limiting its effect to increase bone mass in this model of RA. © 2021 American Society for Bone and Mineral Research (ASBMR).

Humanized Mice Exhibit Exacerbated Abscess Formation and Osteolysis During the Establishment of Implant-Associated Staphylococcus aureus Osteomyelitis.

Staphylococcus aureus is the predominant pathogen causing osteomyelitis. Unfortunately, no immunotherapy exists to treat these very challenging and costly infections despite decades of research, and numerous vaccine failures in clinical trials. This lack of success can partially be attributed to an overreliance on murine models where the immune correlates of protection often diverge from that of humans. Moreover, S. aureus secretes numerous immunotoxins with unique tropism to human leukocytes, which compromises the targeting of immune cells in murine models. To study the response of human immune cells during chronic S. aureus bone infections, we engrafted non-obese diabetic (NOD)-scid IL2Rγnull (NSG) mice with human hematopoietic stem cells (huNSG) and analyzed protection in an established model of implant-associated osteomyelitis. The results showed that huNSG mice have increases in weight loss, osteolysis, bacterial dissemination to internal organs, and numbers of Staphylococcal abscess communities (SACs), during the establishment of implant-associated MRSA osteomyelitis compared to NSG controls (p < 0.05). Flow cytometry and immunohistochemistry demonstrated greater human T cell numbers in infected versus uninfected huNSG mice (p < 0.05), and that T-bet+ human T cells clustered around the SACs, suggesting S. aureus-mediated activation and proliferation of human T cells in the infected bone. Collectively, these proof-of-concept studies underscore the utility of huNSG mice for studying an aggressive form of S. aureus osteomyelitis, which is more akin to that seen in humans. We have also established an experimental system to investigate the contribution of specific human T cells in controlling S. aureus infection and dissemination.

Distinct vasculotropic versus osteotropic features of S. agalactiae versus S. aureus implant-associated bone infection in mice.

Osteomyelitis is a devastating complication of orthopaedic surgery and commonly caused by Staphylococcus aureus (S. aureus) and Group B Streptococcus (GBS, S. agalactiae). Clinically, S. aureus osteomyelitis is associated with local inflammation, abscesses, aggressive osteolysis, and septic implant loosening. In contrast, S. agalactiae orthopaedic infections generally involve soft tissue, with acute life-threatening vascular spread. While preclinical models that recapitulate the clinical features of S. aureus bone infection have proven useful for research, no animal models of S. agalactiae osteomyelitis exist. Here, we compared the pathology caused by these bacteria in an established murine model of implant-associated osteomyelitis. In vitro scanning electron microscopy and CFU quantification confirmed similar implant inocula for both pathogens (~105 CFU/pin). Assessment of mice at 14 days post-infection demonstrated increased S. aureus virulence, as S. agalactiae infected mice had significantly greater body weight, and fewer CFU on the implant and in bone and adjacent soft tissue (p < 0.05). X-ray, µCT, and histologic analyses showed that S. agalactiae induced significantly less osteolysis and implant loosening, and fewer large TRAP+ osteoclasts than S. aureus without inducing intraosseous abscess formation. Most notably, transmission electron microscopy revealed that although both bacteria are capable of digesting cortical bone, S. agalactiae have a predilection for colonizing blood vessels embedded within cortical bone while S. aureus primarily colonizes the osteocyte lacuno-canalicular network. This study establishes the first quantitative animal model of S. agalactiae osteomyelitis, and demonstrates a vasculotropic mode of S. agalactiae infection, in contrast to the osteotropic behavior of S. aureus osteomyelitis.

Lineage tracing reveals evidence of a popliteal lymphatic muscle progenitor cell that is distinct from skeletal and vascular muscle progenitors.

Loss of popliteal lymphatic vessel (PLV) contractions, which is associated with damage to lymphatic muscle cells (LMCs), is a biomarker of disease progression in mice with inflammatory arthritis. Currently, the nature of LMC progenitors has yet to be formally described. Thus, we aimed to characterize the progenitors of PLV-LMCs during murine development, towards rational therapies that target their proliferation, recruitment, and differentiation onto PLVs. Since LMCs have been described as a hybrid phenotype of striated and vascular smooth muscle cells (VSMCs), we performed lineage tracing studies in mice to further clarify this enigma by investigating LMC progenitor contribution to PLVs in neonatal mice. PLVs from Cre-tdTomato reporter mice specific for progenitors of skeletal myocytes (Pax7+ and MyoD+) and VSMCs (Prrx1+ and NG2+) were analyzed via whole mount immunofluorescent microscopy. The results showed that PLV-LMCs do not derive from skeletal muscle progenitors. Rather, PLV-LMCs originate from Pax7-/MyoD-/Prrx1+/NG2+ progenitors similar to VSMCs prior to postnatal day 10 (P10), and from a previously unknown Pax7-/MyoD-/Prrx1+/NG2- muscle progenitor pathway during development after P10. Future studies of these LMC progenitors during maintenance and repair of PLVs, along with their function in other lymphatic beds, are warranted.

Altered Lymphatic Vessel Anatomy and Markedly Diminished Lymph Clearance in Affected Hands of Patients With Active Rheumatoid Arthritis.

OBJECTIVE: To assess differences between lymphatic function in the affected hands of rheumatoid arthritis (RA) patients with active synovitis and that of healthy controls, using indocyanine green (ICG) dye and near-infrared (NIR) imaging. METHODS: NIR imaging of the hands of 8 patients with active RA and 13 healthy controls was performed following web space injection of 0.1 ml of 100 µM ICG. The percentage of ICG retention in the web spaces was determined by NIR imaging at baseline and at 7 days (±1 day) after the initial injections; image analysis provided contraction frequency. ICG+ lymphatic vessel (LV) length and branching architecture were assessed. RESULTS: Retention of ICG in RA hands was higher compared to controls (P < 0.01). The average contraction frequency of ICG+ LVs in RA patients and in controls did not differ (mean ± SD 0.53 ± 0.39 contractions/minute versus 0.51 ± 0.35 contractions/minute). Total ICG+ LV length in RA hands was lower compared to healthy controls (58.3 ± 15.0 cm versus 71.4 ± 16.1 cm; P < 0.001), concomitant with a decrease in the number of ICG+ basilic LVs in the hands of RA patients (P < 0.05). CONCLUSION: Lymphatic drainage in the hands of RA patients with active disease was reduced compared to controls. This reduction was associated with a decrease in total length of ICG+ LVs on the dorsal surface of the hands, which continued to contract at a similar rate to that observed in controls. These findings provide a plausible mechanism for exacerbation of synovitis and joint damage, specifically the accumulation and retention of inflammatory cells and catabolic factors in RA joints due to impaired efferent lymphatic flow. NIR/ICG imaging of RA hands is feasible and warrants formal investigation as a primary outcome measure for arthritis disease severity and/or persistence in future clinical trials.