Objective
Understanding the pathogenesis of systemic sclerosis (SSc) is confounded by considerable disease heterogeneity. Animal models of SSc that recapitulate distinct subsets of disease at the ...molecular level have not been delineated. We applied interspecies comparative analysis of genomic data from multiple mouse models of SSc and patients with SSc to determine which animal models best reflect the SSc intrinsic molecular subsets.
Methods
Gene expression measured in skin from mice with sclerodermatous graft‐versus‐host disease (GVHD), bleomycin‐induced fibrosis, Tsk1/+ or Tsk2/+ mice was mapped to human orthologs and compared to SSc skin biopsy–derived gene expression. Transforming growth factor β (TGFβ) activation was assessed using a responsive signature in mice, and tumor necrosis factor receptor superfamily member 12A (TNFRSF12A) expression was measured in SSc patient and mouse skin.
Results
Gene expression in skin from mice with sclerodermatous GVHD and bleomycin‐induced fibrosis corresponded to that in SSc patients in the inflammatory molecular subset. In contrast, Tsk2/+ mice showed gene expression corresponding to the fibroproliferative SSc subset. Enrichment of a TGFβ‐responsive signature was observed in both Tsk2/+ mice and mice with bleomycin‐induced skin fibrosis. Expression of TNFRSF12A (the TWEAK receptor/fibroblast growth factor–inducible 14) was elevated in skin from patients with fibroproliferative SSc and the skin of Tsk2/+ mice.
Conclusion
This study reveals similarities in cutaneous gene expression between distinct mouse models of SSc and specific molecular subsets of the disease. Different pathways underlie the intrinsic subsets including TGFβ, interleukin‐13 (IL‐13), and IL‐4. We identify a novel target, Tnfrsf12a, with elevated expression in skin from patients with fibroproliferative SSc and Tsk2/+ mice. These findings will inform mechanistic and translational preclinical studies in SSc.
Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection among all infants worldwide and remains a significant cause of morbidity and mortality. To address this ...unmet medical need, MK‐1654, a half‐life extended RSV neutralizing monoclonal antibody, is in clinical development for the prevention of RSV disease in infants. This was a phase I, randomized, placebo‐controlled, single‐site, double‐blind trial of MK‐1654 in 44 healthy Japanese adults. The safety, tolerability, pharmacokinetics, antidrug antibodies (ADAs), and serum neutralizing antibody (SNA) titers against RSV were evaluated for 1 year after a single intramuscular (i.m.) or intravenous (i.v.) dose of MK‐1654 or placebo in five groups (100 mg i.m., 300 mg i.m., 300 mg i.v., 1000 mg i.v., or placebo). MK‐1654 was generally well‐tolerated in Japanese adults. There were no serious drug‐related adverse events (AEs) reported in any MK‐1654 recipient and no discontinuations due to any AEs in the study. The half‐life of MK‐1654 ranged from 76 to 91 days across dosing groups. Estimated bioavailability was 86% for 100 mg i.m. and 77% for 300 mg i.m. One participant out of 33 (3.0%) developed detectable ADA with no apparent associated AEs. The RSV SNA titers increased in a dose‐dependent manner among participants who received MK‐1654. These data support the development of MK‐1654 for use in Japanese infants.
As the only cell capable of bone resorption, the osteoclast is a central mediator of skeletal homeostasis and disease. To efficiently degrade mineralized tissue, these multinucleated giant cells ...secrete acid into a resorption lacuna formed between their apical membrane and the bone surface. For each proton pumped into this extracellular compartment, one bicarbonate ion remains in the cytoplasm. To prevent alkalinization of the cytoplasm, a basolateral bicarbonate/chloride exchanger provides egress for intracellular bicarbonate. However, the identity of this exchanger is unknown. Here, we report that the bicarbonate/chloride exchanger, solute carrier family 4, anion exchanger, member 2 (SLC4A2), is up-regulated during osteoclast differentiation. Suppression of Slc4a2 expression by RNA interference inhibits the ability of RAW cells, a mouse macrophage cell line, to differentiate into osteoclasts and resorb mineralized matrix in vitro. Accordingly, Slc4a2-deficient mice fail to remodel the primary, cartilaginous skeletal anlagen. Abnormal multinucleated giant cells are present in the bone marrow of Slc4a2-deficient mice. Though these cells express the osteoclast markers CD68, cathepsin K, and NFATc1, compared with their wild-type (WT) counterparts they are larger, fail to express tartrate-resistant acid phosphatase (TRAP) activity, and display a propensity to undergo apoptosis. In vitro Slc4a2-deficient osteoclasts are unable to resorb mineralized tissue and cannot form an acidified, extracellular resorption compartment. These data highlight SLC4A2 as a critical mediator of osteoclast differentiation and function in vitro and in vivo.
Osteochondromas are common benign osteocartilaginous tumors in children and adolescents characterized by cartilage-capped bony projections on the surface of bones. These tumors often cause pain, ...deformity, fracture, and musculoskeletal dysfunction, and they occasionally undergo malignant transformation. The pathogenesis of osteochondromas remains poorly understood. Here, we demonstrate that nuclear factor of activated T cells c1 and c2 (NFATc1 and NFATc2) suppress osteochondromagenesis through individual and combinatorial mechanisms. In mice, conditional deletion of NFATc1 in mesenchymal limb progenitors, Scleraxis-expressing (Scx-expressing) tendoligamentous cells, or postnatally in
-expressing cells resulted in osteochondroma formation at entheses, the insertion sites of ligaments and tendons onto bone. Combinatorial deletion of NFATc1 and NFATc2 gave rise to larger and more numerous osteochondromas in inverse proportion to gene dosage. A population of entheseal NFATc1- and Aggrecan-expressing cells was identified as the osteochondroma precursor, previously believed to be growth plate derived or perichondrium derived. Mechanistically, we show that NFATc1 restricts the proliferation and chondrogenesis of osteochondroma precursors. In contrast, NFATc2 preferentially inhibits chondrocyte hypertrophy and osteogenesis. Together, our findings identify and characterize a mechanism of osteochondroma formation and suggest that regulating NFAT activity is a new therapeutic approach for skeletal diseases characterized by defective or exaggerated osteochondral growth.
Neutralizing mAbs can prevent communicable viral diseases. MK-1654 is a respiratory syncytial virus (RSV) F glycoprotein neutralizing monoclonal antibody (mAb) under development to prevent RSV ...infection in infants. Development and validation of methods to predict efficacious doses of neutralizing antibodies across patient populations exposed to a time-varying force of infection (i.e., seasonal variation) are necessary.
Five decades of clinical trial literature were leveraged to build a model-based meta-analysis (MBMA) describing the relationship between RSV serum neutralizing activity (SNA) and clinical endpoints. The MBMA was validated by backward translation to animal challenge experiments and forward translation to predict results of a recent RSV mAb trial. MBMA predictions were evaluated against a human trial of 70 participants who received either placebo or one of four dose-levels of MK-1654 and were challenged with RSV NCT04086472. The MBMA was used to perform clinical trial simulations and predict efficacy of MK-1654 in the infant target population.
The MBMA established a quantitative relationship between RSV SNA and clinical endpoints. This relationship was quantitatively consistent with animal model challenge experiments and results of a recently published clinical trial. Additionally, SNA elicited by increasing doses of MK-1654 in humans reduced RSV symptomatic infection rates with a quantitative relationship that approximated the MBMA. The MBMA indicated a high probability that a single dose of ≥ 75 mg of MK-1654 will result in prophylactic efficacy (> 75% for 5 months) in infants.
An MBMA approach can predict efficacy of neutralizing antibodies against RSV and potentially other respiratory pathogens.
Bone remodeling requires osteoclasts to generate and maintain an acidified resorption compartment between the apical membrane and the bone surface to solubilize hydroxyapatite crystals within the ...bone matrix. This acidification process requires (i) apical proton secretion by a vacuolar H ⁺-ATPase, (ii) actin cytoskeleton reorganization into a podosome belt that forms a gasket to restrict lacunar acid leakage, and (iii) basolateral chloride uptake and bicarbonate extrusion by an anion exchanger to provide Cl ⁻ permissive for apical acid secretion while preventing cytoplasmic alkalinization. Here we show that osteoclast-targeted deletion in mice of solute carrier family 4 anion exchanger member 2 (Slc4a2) results in osteopetrosis. We further demonstrate a previously unrecognized consequence of SLC4A2 loss of function in the osteoclast: dysregulation of calpain-dependent podosome disassembly, leading to abnormal actin belt formation, cell spreading, and migration. Rescue of SLC4A2-deficient osteoclasts with functionally defined mutants of SLC4A2 indicates regulation of actin cytoskeletal reorganization by anion-exchange activity and intracellular pH, independent of SLC4A2’s long N-terminal cytoplasmic domain. These data suggest that maintenance of intracellular pH in osteoclasts through anion exchange regulates the actin superstructures required for bone resorption.
Tissue remodeling with fibrosis is a predominant pathophysiological mechanism of many human diseases. Systemic sclerosis is a rare, often lethal, disorder of unknown etiology manifested by dermal ...fibrosis (scleroderma) and excessive connective tissue deposition in internal organs. Currently, there are no available antifibrotic therapeutics, a reflection of our lack of understanding of this process. Animal models of scleroderma are useful tools to dissect the transcription factors and cytokines that govern fibrosis. A disproportionate increase of type 2 cytokines, like TGF-β and IL-4, more than type 1 cytokines, like IFN-γ, is thought to underlie the pathogenesis of scleroderma. In this study, we show that mice deficient in the transcription factor T-box expressed in T cells (T-bet), a master regulator of type 1 immunity, display increased sensitivity to bleomycin-induced dermal sclerosis. Despite the well-established role of T-bet in adaptive immunity, we also show that RAG2⁻/⁻ mice, which lack T and B cells, are vulnerable to bleomycin-induced scleroderma and that RAG2/T-bet double-deficient mice maintain the increased sensitivity to bleomycin observed in T-bet⁻/⁻ mice. Furthermore, overexpression of T-bet in T cells does not affect the induction of skin sclerosis in this model. Lastly, we show that IL-13 is the profibrotic cytokine regulated by T-bet in this model. Together, we conclude that T-bet serves as a repressor of dermal sclerosis through an IL-13-dependent pathway in innate immune cells. T-bet, and its transcriptional network, represent an attractive target for the treatment of systemic sclerosis and other fibrosing disorders.
Abstract Osteoclasts are specialized secretory cells of the myeloid lineage important for normal skeletal homeostasis as well as pathologic conditions of bone including osteoporosis, inflammatory ...arthritis and cancer metastasis. Differentiation of these multinucleated giant cells from precursors is controlled by the cytokine RANKL, which through its receptor RANK initiates a signaling cascade culminating in the activation of transcriptional regulators which induce the expression of the bone degradation machinery. The transcription factor nuclear factor of activated T-cells c1 (NFATc1) is the master regulator of this process and in its absence osteoclast differentiation is aborted both in vitro and in vivo. Differential mRNA expression analysis by microarray is used to identify genes of potential physiologic relevance across nearly all biologic systems. We compared the gene expression profile of murine wild-type and NFATc1-deficient osteoclast precursors stimulated with RANKL and identified that the majority of the known genes important for osteoclastic bone resorption require NFATc1 for induction. Here, five novel RANKL-induced, NFATc1-dependent transcripts in the osteoclast are described: Nhedc2 , Rhoc , Serpind1 , Adcy3 and Rab38 . Despite reasonable hypotheses for the importance of these molecules in the bone resorption pathway and their dramatic induction during differentiation, the analysis of mice with mutations in these genes failed to reveal a function in osteoclast biology. Compared to littermate controls, none of these mutants demonstrated a skeletal phenotype in vivo or alterations in osteoclast differentiation or function in vitro. These data highlight the need for rigorous validation studies to complement expression profiling results before functional importance can be assigned to highly regulated genes in any biologic process.
The fundamental mechanisms that drive the pathogenesis of systemic sclerosis (SSc) remain elusive, despite over 50 years of investigation. Here, we review recent progress in the understanding of the ...immunopathogenesis of SSc. In particular, we consider interleukin-13 (IL13), and its upstream and downstream pathways, as an example of an immune system-derived mediator involved in fibrotic and vascular pathology. Emerging results linking pattern-recognition receptors and interferon pathways to SSc are also stressed. We discuss genetic data linking the immune system to SSc risk and efforts to apply animal models to subsets of patients recently resolved by gene expression profiling. These developments will help build a context for better understanding of previous observations and design of the next generation of studies that may eventually lead to effective treatment.
In the original version of this Article, financial support was not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to include support from the National Football ...League Players Association.
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