Understanding the Transcriptomic Landscape to Drive New Innovations in Musculoskeletal Regenerative Medicine.

PURPOSE OF REVIEW: RNA-sequencing (RNA-seq) is a novel and highly sought-after tool in the field of musculoskeletal regenerative medicine. The technology is being used to better understand pathological processes, as well as elucidate mechanisms governing development and regeneration. It has allowed in-depth characterization of stem cell populations and discovery of molecular mechanisms that regulate stem cell development, maintenance, and differentiation in a way that was not possible with previous technology. This review introduces RNA-seq technology and how it has paved the way for advances in musculoskeletal regenerative medicine. RECENT FINDINGS: Recent studies in regenerative medicine have utilized RNA-seq to decipher mechanisms of pathophysiology and identify novel targets for regenerative medicine. The technology has also advanced stem cell biology through in-depth characterization of stem cells, identifying differentiation trajectories and optimizing cell culture conditions. It has also provided new knowledge that has led to improved growth factor use and scaffold design for musculoskeletal regenerative medicine. This article reviews recent studies utilizing RNA-seq in the field of musculoskeletal regenerative medicine. It demonstrates how transcriptomic analysis can be used to provide insights that can aid in formulating a regenerative strategy.

Deletion of c-FLIP from CD11bhi Macrophages Prevents Development of Bleomycin-induced Lung Fibrosis.

Idiopathic pulmonary fibrosis is a progressive lung disease with complex pathophysiology and fatal prognosis. Macrophages (MΦ) contribute to the development of lung fibrosis; however, the underlying mechanisms and specific MΦ subsets involved remain unclear. During lung injury, two subsets of lung MΦ coexist: Siglec-Fhi resident alveolar MΦ and a mixed population of CD11bhi MΦ that primarily mature from immigrating monocytes. Using a novel inducible transgenic system driven by a fragment of the human CD68 promoter, we targeted deletion of the antiapoptotic protein cellular FADD-like IL-1ß-converting enzyme-inhibitory protein (c-FLIP) to CD11bhi MΦ. Upon loss of c-FLIP, CD11bhi MΦ became susceptible to cell death. Using this system, we were able to show that eliminating CD11bhi MΦ present 7-14 days after bleomycin injury was sufficient to protect mice from fibrosis. RNA-seq analysis of lung MΦ present during this time showed that CD11bhi MΦ, but not Siglec-Fhi MΦ, expressed high levels of profibrotic chemokines and growth factors. Human MΦ from patients with idiopathic pulmonary fibrosis expressed many of the same profibrotic chemokines identified in murine CD11bhi MΦ. Elimination of monocyte-derived MΦ may help in the treatment of fibrosis. We identify c-FLIP and the associated extrinsic cell death program as a potential pathway through which these profibrotic MΦ may be pharmacologically targeted.

Phagocytosis of microparticles by alveolar macrophages during acute lung injury requires MerTK.

Microparticles are a newly recognized class of mediators in the pathophysiology of lung inflammation and injury, but little is known about the factors that regulate their accumulation and clearance. The primary objective of our study was to determine whether alveolar macrophages engulf microparticles and to elucidate the mechanisms by which this occurs. Alveolar microparticles were quantified in bronchoalveolar fluid of mice with lung injury induced by LPS and hydrochloric acid. Microparticle numbers were greatest at the peak of inflammation and declined as inflammation resolved. Isolated, fluorescently labeled particles were placed in culture with macrophages to evaluate ingestion in the presence of endocytosis inhibitors. Ingestion was blocked with cytochalasin D and wortmannin, consistent with a phagocytic process. In separate experiments, mice were treated intratracheally with labeled microparticles, and their uptake was assessed though microscopy and flow cytometry. Resident alveolar macrophages, not recruited macrophages, were the primary cell-ingesting microparticles in the alveolus during lung injury. In vitro, microparticles promoted inflammatory signaling in LPS primed epithelial cells, signifying the importance of microparticle clearance in resolving lung injury. Microparticles were found to have phosphatidylserine exposed on their surfaces. Accordingly, we measured expression of phosphatidylserine receptors on macrophages and found high expression of MerTK and Axl in the resident macrophage population. Endocytosis of microparticles was markedly reduced in MerTK-deficient macrophages in vitro and in vivo. In conclusion, microparticles are released during acute lung injury and peak in number at the height of inflammation. Resident alveolar macrophages efficiently clear these microparticles through MerTK-mediated phagocytosis.

Three Unique Interstitial Macrophages in the Murine Lung at Steady State.

The current paradigm in macrophage biology is that some tissues mainly contain macrophages from embryonic origin, such as microglia in the brain, whereas other tissues contain postnatal-derived macrophages, such as the gut. However, in the lung and in other organs, such as the skin, there are both embryonic and postnatal-derived macrophages. In this study, we demonstrate in the steady-state lung that the mononuclear phagocyte system is comprised of three newly identified interstitial macrophages (IMs), alveolar macrophages, dendritic cells, and few extravascular monocytes. We focused on similarities and differences between the three IM subtypes, specifically, their phenotype, location, transcriptional signature, phagocytic capacity, turnover, and lack of survival dependency on fractalkine receptor, CX3CR1. Pulmonary IMs were located in the bronchial interstitium but not the alveolar interstitium. At the transcriptional level, all three IMs displayed a macrophage signature and phenotype. All IMs expressed MER proto-oncogene, tyrosine kinase, CD64, CD11b, and CX3CR1, and were further distinguished by differences in cell surface protein expression of CD206, Lyve-1, CD11c, CCR2, and MHC class II, along with the absence of Ly6C, Ly6G, and Siglec F. Most intriguingly, in addition to the lung, similar phenotypic populations of IMs were observed in other nonlymphoid organs, perhaps highlighting conserved functions throughout the body. These findings promote future research to track four distinct pulmonary macrophages and decipher the division of labor that exists between them.

Cell Origin Dictates Programming of Resident versus Recruited Macrophages during Acute Lung Injury.

Two populations of alveolar macrophages (AMs) coexist in the inflamed lung: resident AMs that arise during embryogenesis, and recruited AMs that originate postnatally from circulating monocytes. The objective of this study was to determine whether origin or environment dictates the transcriptional, metabolic, and functional programming of these two ontologically distinct populations over the time course of acute inflammation. RNA sequencing demonstrated marked transcriptional differences between resident and recruited AMs affecting three main areas: proliferation, inflammatory signaling, and metabolism. Functional assays and metabolomic studies confirmed these differences and demonstrated that resident AMs proliferate locally and are governed by increased tricarboxylic acid cycle and amino acid metabolism. Conversely, recruited AMs produce inflammatory cytokines in association with increased glycolytic and arginine metabolism. Collectively, the data show that even though they coexist in the same environment, inflammatory macrophage subsets have distinct immunometabolic programs and perform specialized functions during inflammation that are associated with their cellular origin.

Pioglitazone restores phagocyte mitochondrial oxidants and bactericidal capacity in chronic granulomatous disease.

BACKGROUND: Deficient production of reactive oxygen species (ROS) by the phagocyte nicotinamide adenine dinucleotide (NADPH) oxidase in patients with chronic granulomatous disease (CGD) results in susceptibility to certain pathogens secondary to impaired oxidative killing and mobilization of other phagocyte defenses. Peroxisome proliferator-activated receptor (PPAR) γ agonists, including pioglitazone, approved for type 2 diabetes therapy alter cellular metabolism and can heighten ROS production. It was hypothesized that pioglitazone treatment of gp91(phox-/-) mice, a murine model of human CGD, would enhance phagocyte oxidant production and killing of Staphylococcus aureus, a significant pathogen in patients with this disorder. OBJECTIVES: We sought to determine whether pioglitazone treatment of gp91(phox-/-) mice enhanced phagocyte oxidant production and host defense. METHODS: Wild-type and gp91(phox-/-) mice were treated with the PPARγ agonist pioglitazone, and phagocyte ROS and killing of S aureus were investigated. RESULTS: As demonstrated by 3 different ROS-sensing probes, short-term treatment of gp91(phox-/-) mice with pioglitazone enhanced stimulated ROS production in neutrophils and monocytes from blood and neutrophils and inflammatory macrophages recruited to tissues. Mitochondria were identified as the source of ROS. Findings were replicated in human monocytes from patients with CGD after ex vivo pioglitazone treatment. Importantly, although mitochondrial (mt)ROS were deficient in gp91(phox-/-) phagocytes, their restoration with treatment significantly enabled killing of S aureus both ex vivo and in vivo. CONCLUSIONS: Together, the data support the hypothesis that signaling from the NADPH oxidase under normal circumstances governs phagocyte mtROS production and that such signaling is lacking in the absence of a functioning phagocyte oxidase. PPARγ agonism appears to bypass the need for the NADPH oxidase for enhanced mtROS production and partially restores host defense in CGD.

Polyelectrolyte Complex Hydrogels with Controlled Mechanics Affect Mesenchymal Stem Cell Differentiation Relevant to Growth Plate Injuries.

The growth plate is a complex cartilage structure in long bones that mediates growth in children. When injured, the formation of a "bony bar" can occur which impedes normal growth and can cause angular deformities or growth arrest. Current treatments for growth plate injuries are limited and result in poor patient outcomes, necessitating research toward novel treatments that can prevent bony bar formation and stimulate cartilage regeneration. This study investigates alginate-chitosan polyelectrolyte complex (PEC) hydrogels as an injectable biomaterial system to prevent bony bar formation. Biomaterial properties including stiffness and degradation are quantified, and the effect that material properties have on mesenchymal stem cell (MSC) fate is quantified in vitro. Specifically, this study aims to elucidate the effectiveness of biomaterial-based control over the differentiation behavior of MSCs toward osteogenic or chondrogenic lineages using biochemical metabolite assays and quantitative real time PCR. Further, the PEC hydrogels are employed in a rat growth plate injury model to determine their effectiveness in preventing bony bar formation in vivo. Results indicate that hydrogel composition and material properties affect the differentiation tendency of MSCs in vitro, and the PEC hydrogels show promise as an injectable biomaterial for growth plate injuries.

Analysis of Physeal Fractures from the United States National Trauma Data Bank.

BACKGROUND: Pediatric long-bone physeal fractures can lead to growth deformities. Previous studies have reported that physeal fractures make up 18-30% of total fractures. This study aimed to characterize physeal fractures with respect to sex, age, anatomic location, and Salter-Harris (SH) classification from a current multicenter national database. METHODS: A retrospective cohort study was performed using the 2016 United States National Trauma Data Bank (NTDB). Patients ≤ 18 years of age with a fracture of the humerus, radius, ulna, femur, tibia, or fibula were included. RESULTS: The NTDB captured 132,018 patients and 58,015 total fractures. Physeal fractures made up 5.7% (3291) of all long-bone fractures, with males accounting for 71.0% (2338). Lower extremity physeal injuries comprised 58.6% (1929) of all physeal fractures. The most common site of physeal injury was the tibia comprising 31.8% (1047), 73.9% (774) of which were distal tibia fractures. Physeal fractures were greatest at 11 years of age for females and 14 years of age for males. Most fractures were SH Type II fractures. DISCUSSION AND CONCLUSIONS: Our analysis indicates that 5.7% of pediatric long-bone fractures involved the physis, with the distal tibia being the most common. These findings suggest a lower incidence of physeal fractures than previous studies and warrant further investigation.

The heterogeneous mechanical properties of adolescent growth plate cartilage: A study in rabbit.

The growth plate is a cartilaginous tissue that functions to lengthen bones in children. When fractured, however, the growth plate can lose this critical function. Our understanding of growth plate fracture and mechanobiology is currently hindered by sparse information on the growth plate's microscale spatial gradients in mechanical properties. In this study, we performed microindentation across the proximal tibia growth plate of 9-week-old New Zealand White rabbits (n = 15) to characterize spatial variations in mechanical properties using linear elastic and nonlinear poroelastic material models. Mean indentation results for Hertz reduced modulus ranged from 380 to 690 kPa, with a peak in the upper hypertrophic zone and significant differences (p < 0.05) between neighboring zones. Using a subset of these animals (n = 7), we characterized zonal structure and extracellular matrix content of the growth plate through confocal fluorescent microscopy and Raman spectroscopy mapping. Comparison between mechanical properties and matrix content across the growth plate showed that proteoglycan content correlated with compressive modulus. This study is the first to measure poroelastic mechanical properties from microindentation across growth plate cartilage and to discern differing mechanical properties between the upper and lower hypertrophic zones. This latter finding may explain the location of typical growth plate fractures. The spatial variation in our reported mechanical properties emphasize the heterogeneous structure of the growth plate which is important to inform future regenerative implant design and mechanobiological models.

Polyamine import and accumulation causes immunomodulation in macrophages engulfing apoptotic cells.

Phagocytosis of apoptotic cells, termed efferocytosis, is critical for tissue homeostasis and drives anti-inflammatory programming in engulfing macrophages. Here, we assess metabolites in naive and inflammatory macrophages following engulfment of multiple cellular and non-cellular targets. Efferocytosis leads to increases in the arginine-derived polyamines, spermidine and spermine, in vitro and in vivo. Surprisingly, polyamine accumulation after efferocytosis does not arise from retention of apoptotic cell metabolites or de novo synthesis but from enhanced polyamine import that is dependent on Rac1, actin, and PI3 kinase. Blocking polyamine import prevents efferocytosis from suppressing macrophage interleukin (IL)-1ß or IL-6. This identifies efferocytosis as a trigger for polyamine import and accumulation, and imported polyamines as mediators of efferocytosis-induced immune reprogramming.

Association of Sputum Neutrophil Extracellular Trap Subsets With IgA Anti-Citrullinated Protein Antibodies in Subjects at Risk for Rheumatoid Arthritis.

OBJECTIVE: Mechanisms leading to anti-citrullinated protein antibody (ACPA) generation in rheumatoid arthritis (RA) are hypothesized to originate in the lung. We undertook this study to understand associations between neutrophil extracellular trap (NET) formation in the lung and local ACPA generation in subjects at risk of developing RA. METHODS: Induced sputum was collected from 49 subjects at risk of developing RA, 12 patients with RA, and 18 controls. Sputum neutrophils were tested for ex vivo NET formation, and sputum-induced NET formation of control neutrophils was measured using immunofluorescence imaging. Sputum macrophages were tested for ex vivo endocytosis of apoptotic and opsonized cells. Levels of ACPA, NET remnants, and inflammatory proteins were quantified in sputum supernatant. RESULTS: Spontaneous citrullinated histone H3 (Cit-H3)-expressing NET formation was higher in sputum neutrophils from at-risk subjects and RA patients compared to controls (median 12%, 22%, and 0%, respectively; P < 0.01). In at-risk subjects, sputum IgA ACPA correlated with the percentage of neutrophils that underwent Cit-H3+ NET formation (r = 0.49, P = 0.002) and levels of Cit-H3+ NET remnants (r = 0.70, P < 0.001). Reduced endocytic capacity of sputum macrophages was found in at-risk subjects and RA patients compared to controls. Using a mediation model, we found that sputum inflammatory proteins were associated with sputum IgA ACPA through a pathway mediated by Cit-H3+ NET remnants. Sputum-induced Cit-H3+ NET formation also correlated with sputum levels of interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor in at-risk subjects, suggesting a causal relationship. CONCLUSION: These data support a potential mechanism for mucosal ACPA generation in subjects at risk of developing RA, whereby inflammation leads to increased citrullinated protein-expressing NETs that promote local ACPA generation.

Air-Inflation of Murine Lungs with Vascular Perfusion-Fixation.

Lung histology is often used to investigate the contributions provided by airspace cells during lung homeostasis and disease pathogenesis. However, commonly used instillation-based fixation methods can displace airspace cells and mucus into terminal airways and can alter tissue morphology. In comparison, vascular perfusion-fixation techniques are superior at preserving the location and morphology of cells within airspaces and the mucosal lining. However, if positive airway pressure is not simultaneously applied, regions of the lungs may collapse and capillaries may bulge into the alveolar spaces, leading to distortion of the lung anatomy. Herein, we describe an inexpensive method for air-inflation during vascular perfusion-fixation to preserve the morphology and location of airway and alveolar cells and interstitium in murine lungs for downstream histologic studies. Constant air pressure is delivered to the lungs via the trachea from a sealed, air-filled chamber that maintains pressure via an adjustable liquid column while fixative is perfused through the right ventricle.

Immunocytochemical analysis of syntaxin-1 in rat circumvallate taste buds.

Mammalian buds contain a variety of morphological taste cell types, but the type III taste cell is the only cell type that has synapses onto nerve processes. We hypothesize that taste cell synapses utilize the SNARE protein machinery syntaxin, SNAP-25, and synaptobrevin, as is used by synapses in the central nervous system (CNS) for Ca2+-dependent exocytosis. Previous studies have shown that taste cells with synapses display SNAP-25- and synaptobrevin-2-like immunoreactivity (LIR) (Yang et al. [2000a] J Comp Neurol 424:205-215, [2004] J Comp Neurol 471:59-71). In the present study we investigated the presynaptic membrane protein, syntaxin-1, in circumvallate taste buds of the rat. Our results indicate that diffuse cytoplasmic and punctate syntaxin-1-LIR are present in different subsets of taste cells. Diffuse, cytoplasmic syntaxin-1-LIR is present in type III cells while punctate syntaxin-1-LIR is present in type II cells. The punctate syntaxin-1-LIR is believed to be associated with Golgi bodies. All of the synapses associated with syntaxin-1-LIR taste cells are from type III cells onto nerve processes. These results support the proposition that taste cell synapses use classical SNARE machinery such as syntaxin-1 for neurotransmitter release in rat circumvallate taste buds.

Qualitative and quantitative differences between taste buds of the rat and mouse.

BACKGROUND: Numerous electrophysiological, ultrastructural, and immunocytochemical studies on rodent taste buds have been carried out on rat taste buds. In recent years, however, the mouse has become the species of choice for molecular and other studies on sensory transduction in taste buds. Do rat and mouse taste buds have the same cell types, sensory transduction markers and synaptic proteins? In the present study we have used antisera directed against PLCbeta2, alpha-gustducin, serotonin (5-HT), PGP 9.5 and synaptobrevin-2 to determine the percentages of taste cells expressing these markers in taste buds in both rodent species. We also determined the numbers of taste cells in the taste buds as well as taste bud volume. RESULTS: There are significant differences (p < 0.05) between mouse and rat taste buds in the percentages of taste cells displaying immunoreactivity for all five markers. Rat taste buds display significantly more immunoreactivity than mice for PLCbeta2 (31.8% vs 19.6%), alpha-gustducin (18% vs 14.6%), and synaptobrevin-2 (31.2% vs 26.3%). Mice, however, have more cells that display immunoreactivity to 5-HT (15.9% vs 13.7%) and PGP 9.5 (14.3% vs 9.4%). Mouse taste buds contain an average of 85.8 taste cells vs 68.4 taste cells in rat taste buds. The average volume of a mouse taste bud (42,000 microm3) is smaller than a rat taste bud (64,200 microm3). The numerical density of taste cells in mouse circumvallate taste buds (2.1 cells/1000 microm3) is significantly higher than that in the rat (1.2 cells/1000 microm3). CONCLUSION: These results suggest that rats and mice differ significantly in the percentages of taste cells expressing signaling molecules. We speculate that these observed dissimilarities may reflect differences in their gustatory processing.

Induction of TGF-ß1 synthesis by macrophages in response to apoptotic cells requires activation of the scavenger receptor CD36.

BACKGROUND/OBJECTIVE: Phosphatidylserine (PS) exposed on apoptotic cells has been shown to stimulate production of transforming growth factor-ß (TGF-ß) and promote anti-inflammatory responses. However, the PS receptor(s) responsible for this induction has not been clearly determined. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, using RAWTßRII cells in which a truncated dominant negative TGF-ß receptor II was stably transfected in order to avoid auto-feedback induction of TGF-ß, we show that TGF-ß1 synthesis is initiated via activation of the scavenger receptor, CD36. The response requires exposure of PS on the apoptotic cell surface and was absent in macrophages lacking CD36. Direct activation of CD36 with an anti-CD36 antibody initiated TGF-ß1 production, and signaling pathways involving both Lyn kinase and ERK1/2 were shown to participate in CD36-driven TGF-ß1 expression. CONCLUSION/SIGNIFICANCE: Since CD36 has been previously implicated in activation of secreted latent TGF-ß, the present study indicates its role in the multiple steps to generation of this important biological mediator.