Faculty Bibliography

Atherosclerosis is a chronic inflammatory disease, and developing therapies to promote its regression is an important clinical goal. We previously established that atherosclerosis regression is characterized by an overall decrease in plaque macrophages and enrichment in markers of alternatively activated M2 macrophages. We have now investigated the origin and functional requirement for M2 macrophages in regression in normolipidemic mice that received transplants of atherosclerotic aortic segments. We compared plaque regression in WT normolipidemic recipients and those deficient in chemokine receptors necessary to recruit inflammatory Ly6Chi (Ccr2-/- or Cx3cr1-/-) or patrolling Ly6Clo (Ccr5-/-) monocytes. Atherosclerotic plaques transplanted into WT or Ccr5-/- recipients showed reduced macrophage content and increased M2 markers consistent with plaque regression, whereas plaques transplanted into Ccr2-/- or Cx3cr1-/- recipients lacked this regression signature. The requirement of recipient Ly6Chi monocyte recruitment was confirmed in cell trafficking studies. Fate-mapping and single-cell RNA sequencing studies also showed that M2-like macrophages were derived from newly recruited monocytes. Furthermore, we used recipient mice deficient in STAT6 to demonstrate a requirement for this critical component of M2 polarization in atherosclerosis regression. Collectively, these results suggest that continued recruitment of Ly6Chi inflammatory monocytes and their STAT6-dependent polarization to the M2 state are required for resolution of atherosclerotic inflammation and plaque regression.

Rotavirus, a leading cause of severe gastroenteritis and diarrhoea in young children, accounts for around 215,000 deaths annually worldwide. Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-kappaB signalling, raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa. The mechanism by which enteric viruses are sensed and restricted in vivo, especially by NOD-like receptor (NLR) inflammasomes, is largely unknown. Here we uncover and mechanistically characterize the NLR Nlrp9b that is specifically expressed in intestinal epithelial cells and restricts rotavirus infection. Our data show that, via RNA helicase Dhx9, Nlrp9b recognizes short double-stranded RNA stretches and forms inflammasome complexes with the adaptor proteins Asc and caspase-1 to promote the maturation of interleukin (Il)-18 and gasdermin D (Gsdmd)-induced pyroptosis. Conditional depletion of Nlrp9b or other inflammasome components in the intestine in vivo resulted in enhanced susceptibility of mice to rotavirus replication. Our study highlights an important innate immune signalling pathway that functions in intestinal epithelial cells and may present useful targets in the modulation of host defences against viral pathogens.

Abnormalities of the endosomal-lysosomal network (ELN) are a signature feature of Alzheimer's disease (AD). These include the earliest known cytopathology that is specific to AD and that affects endosomes and induces the progressive failure of lysosomes, each of which are directly linked by distinct mechanisms to neurodegeneration. The origins of ELN dysfunction and beta-amyloidogenesis closely overlap, which reflects their common genetic basis, the established early involvement of endosomes and lysosomes in amyloid precursor protein (APP) processing and clearance, and the pathologic effect of certain APP metabolites on ELN functions. Genes that promote beta-amyloidogenesis in AD (APP, PSEN1/2, and APOE4) have primary effects on ELN function. The importance of primary ELN dysfunction to pathogenesis is underscored by the mutations in more than 35 ELN-related genes that, thus far, are known to cause familial neurodegenerative diseases even though different pathogenic proteins may be involved. In this article, I discuss growing evidence that implicates AD gene-driven ELN disruptions as not only the antecedent pathobiology that underlies beta-amyloidogenesis but also as the essential partner with APP and its metabolites that drive the development of AD, including tauopathy, synaptic dysfunction, and neurodegeneration. The striking amelioration of diverse deficits in animal AD models by remediating ELN dysfunction further supports a need to integrate APP and ELN relationships, including the role of amyloid-beta, into a broader conceptual framework of how AD arises, progresses, and may be effectively therapeutically targeted.-Nixon, R. A. Amyloid precursor protein and endosomal-lysosomal dysfunction in Alzheimer's disease: inseparable partners in a multifactorial disease.

Macrophages perform critical functions in both innate immunity and cholesterol metabolism. Here, we report that activation of Toll-like receptor 4 (TLR4) in macrophages causes lanosterol, the first sterol intermediate in the cholesterol biosynthetic pathway, to accumulate. This effect is due to type I interferon (IFN)-dependent histone deacetylase 1 (HDAC1) transcriptional repression of lanosterol-14alpha-demethylase, the gene product of Cyp51A1. Lanosterol accumulation in macrophages, because of either treatment with ketoconazole or induced conditional disruption of Cyp51A1 in mouse macrophages in vitro, decreases IFNbeta-mediated signal transducer and activator of transcription (STAT)1-STAT2 activation and IFNbeta-stimulated gene expression. These effects translate into increased survival to endotoxemic shock by reducing cytokine secretion. In addition, lanosterol accumulation increases membrane fluidity and ROS production, thus potentiating phagocytosis and the ability to kill bacteria. This improves resistance of mice to Listeria monocytogenes infection by increasing bacterial clearance in the spleen and liver. Overall, our data indicate that lanosterol is an endogenous selective regulator of macrophage immunity.

Adenosine A2a receptor (A2aR) stimulation promotes the synthesis of collagens I and III, and we have recently demonstrated that there is crosstalk between the A2aR and WNT/beta-catenin signaling pathway. In in vitro studies, A2aR signaling for collagen III expression was mediated by WNT/beta-catenin signaling in human dermal fibroblasts; we further verified whether the crosstalk between A2aR and Wnt/beta-catenin signaling was involved in diffuse dermal fibrosis in vivo. Wnt-signaling reporter mice (Tcf/Lef:H2B-GFP) were challenged with bleomycin and treated with the selective A2aR antagonist istradefylline (KW6002) or vehicle. Dermal fibrosis was quantitated and nuclear translocation of beta-catenin in fibroblasts was assessed by double-staining for Green fluorescent protein or dephosphorylated beta-catenin or beta-catenin phosphorylated at Ser552, and vimentin. KW6002 significantly reduced skin thickness, skinfold thickness, breaking tension, dermal hydroxyproline content, myofibroblast accumulation, and collagen alignment in bleomycin-induced dermal fibrosis. Also, there was increased expression of Tcf/Lef:H2B-GFP reporter in bleomycin-induced dermal fibrosis, an effect that was diminished by treatment with KW6002. Moreover, KW6002 significantly inhibited nuclear translocation of Tcf/Lef:H2B-GFP reporter, as well as dephosphorylated beta-catenin and beta-catenin phosphorylated at Ser552. Our work supports the hypothesis that pharmacologic blockade of A2aR inhibits the WNT/beta-catenin signaling pathway, contributing to its capacity to inhibit bleomycin-induced dermal fibrosis.

Importance: The notion that systemic isotretinoin taken within 6 to 12 months of cutaneous surgery contributes to abnormal scarring or delayed wound healing is widely taught and practiced; however, it is based on 3 small case series from the mid-1980s. Objective: To evaluate the body of literature to provide evidence-based recommendations regarding the safety of procedural interventions performed either concurrently with, or immediately following the cessation of systemic isotretinoin therapy. Evidence Review: A panel of national experts in pediatric dermatology, procedural/cosmetic dermatology, plastic surgery, scars, wound healing, acne, and isotretinoin was convened. A systematic PubMed review of English-language articles published from 1982 to 2017 was performed using the following search terms: isotretinoin, 13-cis-retinoic acid, Accutane, retinoids, acitretin, surgery, surgical, laser, ablative laser, nonablative laser, laser hair removal, chemical peel, dermabrasion, wound healing, safety, scarring, hypertrophic scar, and keloid. Evidence was graded, and expert consensus was obtained. Findings: Thirty-two relevant publications reported 1485 procedures. There was insufficient evidence to support delaying manual dermabrasion, superficial chemical peels, cutaneous surgery, laser hair removal, and fractional ablative and nonablative laser procedures for patients currently receiving or having recently completed isotretinoin therapy. Based on the available literature, mechanical dermabrasion and fully ablative laser are not recommended in the setting of systemic isotretinoin treatment. Conclusions and Relevance: Physicians and patients may have an evidence-based discussion regarding the known risk of cutaneous surgical procedures in the setting of systemic isotretinoin therapy. For some patients and some conditions, an informed decision may lead to earlier and potentially more effective interventions.

INTRODUCTION: According to a recent study from the Center of Disease Control and Prevention, 1 in every 10 Americans aged 12 and older reported chronic use of antidepressants. Chronic use of serotonin re-uptake inhibitors (SSRI) has been linked to impaired bone mineral accrual during skeletal development and osteoporosis [1,2]. We investigated the effect of fluoxetine, the most commonly prescribed SSRI in the U.S., on the complex program of bone regeneration in two disparate models of fracture repair in mice, followed by a thorough assessment of the in vitro mineralization capacity of primary osteoprogenitor cells (OPCs). We hypothesized that fluoxetine exerts a negative effect on osteoblast proliferation and differentiation during the process of fracture repair, resulting in a less mineralized and weaker bony callus. METHODS: Twelve week-old C57BL/6J mice were used following the IACUC guidelines at our institution. Fluoxetine was delivered in the drinking water at 10 mg/kg/day dose during the 3 weeks before surgeries to simulate chronic SSRI use [3]. Bone fracture repair through endochondral ossification was analyzed using a well-established femur fracture model stabilized with an intramedullary rod. Fracture callus was examined at 14 and 28 days. Intramembranous ossification was analyzed using a 1-mm monocortical tibial defect model. Here, injuries were allowed to heal for 7 or 14 days. Samples were subjected to microCT analysis, histomorphometry, TRAP and ALP histochemistry and immunolabeling for osteocalcin and runx2. A set of fractured femurs at d28 was subjected to 4-point biomechanical bending tests. All mice were continuously treated with fluoxetine during the repair period, except for a group of mice in which we aimed at understanding how discontinuation of the SSRI at the time of fracture would affect fracture healing (Fig. 1A). For the in vitro studies, bone marrow stromal cells were cultured in growth media alone or in presence of 5, 10 or 20 microM fluoxetine, with and without serotonin. Cell proliferation was measured using a BrdU colorimetric assay and apoptotic cells were detected by TUNEL labeling. bMSCs were also cultured in osteogenic differentiation media alone or with the aforementioned fluoxetine concentrations. Mineralization activity was analyzed by alizarin red staining and ALP activity and the expression of osteogenic markers was evaluated by qRT-PCR. An additional set of in vitro experiments was carried out with serotonin supplementation at 50mM in growth media or osteogenic media. Cell proliferation and osteogenic differentiation were examined. Student's t test with Holm-Sidak correction were used to quantify differences described in this study. Error bars represent standard deviation. An asterisk symbol (*) denotes a p value of less than 0.05. RESULTS: Fluoxetine-treated mice developed a normal cartilaginous callus at 14 days after fracture. At 28 days, the fluoxetine-treated animals demonstrated a significantly smaller and biomechanically weaker bony callus (Fig. 1B). In order to further dissect the mechanism that resulted in a smaller osseous regenerate, we studied the healing process of monocortical tibial defects as an intramembranous model of bone healing, which confirmed a direct effect of fluoxetine on osteoblast differentiation and mineralization. In vitro studies established that fluoxetine treatment decreases osteogenic differentiation and mineralization and that this effect is serotonin-independent. Finally, in a translational approach, we tested whether cessation of the medication would result in restoration of the regenerative potential. Interestingly, histologic and microCT analysis revealed non-union formation in these animals with fibrous tissue interposition within the callus (Fig. 1). DISCUSSION: In summary, our current study shows that chronic fluoxetine treatment negatively affects bone healing by inhibiting proliferation, osteoblast differentiation and mineralization. Data from this study and others provide strong evidence that chronic SSRI use leads to osteoporosis, which is associated with an increased fracture risk. In a translation arm of our study, we aimed at studying the effect of fluoxetine cessation at the time of fracture. In this group, we surprisingly encountered the consistent formation of non-unions with persistent fibrous tissue interposition. Further studies are now focusing at understanding this intriguing finding. (Figure Presented)

INTRODUCTION: Many adult tissues harbor stem cells, which theoretically could be employed for injury repair [1, 2], but the origins of these cells, and the factors that influence their developmental potency, are poorly understood. The skeleton contains tissue-specific stem cells, which are responsible for maintaining bone mass and for regenerating new bone following injury. By genetic cell lineage labeling we established that adult skeletal stem cells come from two embryonic lineages, the mesoderm and the neural crest [3]. Although both populations give rise to cartilage and bone, they are not functionally equivalent: Neural crest (NC)-derived skeletal progenitor cells are more osteogenic, and exhibit robust plasticity in bone grafting assays compared to mesoderm-derived skeletal stem cells [3]. The embryonic origin, however, is not the only attribute that differs between cells from the cranial versus appendicular skeleton. The embryonic Hox code is responsible for positional patterning during development, and our data show that the Hox code still serves as positional memory during adult bone regeneration. The goal of this study was to further elucidate and understand the molecular basis for this remarkable plasticity of the NC-derived, Hox-negative progenitor cell compared to the rather committed mesoderm-derived, Hox-positive progenitor cell. METHODS: The periosteum from four different skeletal sites (F-frontal bone, H-hyoid bone, P-parietal bone, T-tibia) was carefully collected into RNAlater, snap frozen in liquid nitrogen, then RNA isolation was carried out. RNA obtained was inspected on Bioanalyzer and samples with RIN superior to 8 were then subjected to RNA sequencing utilizing high output, paired-end reads using the Illumina HiSeq 2500 System. Three replicates from each skeletal location were tested. Bioinformatic analysis was performed with TopHat (version 2.0.9), Cufflinks (version 2.2.0) and Htseq (version 0.6.1.p.1). The expression of Hox genes with the most relevant expression levels detected by RNAseq was validated by qRT-PCR. RESULTS SECTION: Utilizing RNA sequencing we first set out to test whether the embryonic Hox code continues to be expressed in adult cells, and if the Hox expression pattern matches that of the embryo. We harvested periosteum from four distinct locations, each representing a unique signature of embryonic Hox code (positive/negative) and embryonic origin (neural crest/mesoderm). The periosteum from the tibia (MD, Hox-pos), hyoid (NC, Hox-pos), frontal (NC, Hox-neg) and parietal bone (MD, Hox-neg) was isolated, followed by standard RNA preparation. We then used RNAseq to identify the transcriptome of these four cell origins. First we hypothesized that the embryonic Hox status of periosteal cell is maintained into adulthood. Analysis of the RNAseq data confirmed this hypothesis: Parietal and frontal bones showed insignificant amounts of Hox transcripts, while the tibia and hyoid showed relatively high expression levels (Fig. 1A). Next, we aimed at understanding whether periosteal progenitor cells can be distinguished by their embryonic origin or by their Hox code expression. A first suggestions that the Hox code is a more defining characteristic than the embryonic origin was shown by the hierarchical cluster analysis, in which the progenitors from the tibia and hyoid and the progenitors from the frontal and parietal bone clustered together according to their Hox expression profile (Fig. 1B). We then confirmed this distinction according to the Hox code by plotting the results in an MA plot. Here, every dot represents one gene, genes with an adjusted p value less than 0.01 are shown in red. Comparison of neural crest (frontal and hyoid) and mesoderm (parietal and tibia) derived progenitors revealed a paucity of differentially expressed genes, while comparing Hox-negative (frontal and parietal) and Hox-positive (hyoid and tibia) progenitors resulted in an abundance of differentially expressed genes (Fig. 1C). This analysis suggests that it is the Hox status that characterizes and distinguishes skeletal progenitor cells more accurately than the embryonic origin. DISCUSSION: In previous experiments, we made an unexpected discovery: skeletal stem cells come in at least two "flavors". Using a genetic cell lineage labeling strategy, we identified a mesodermderived population that is responsible for remodeling and repair in long bones, and a second population derived from the NC that remodels and repairs craniofacial bones [3]. These results established for the first time that NC- and mesoderm-derived bones heal through the selective recruitment of skeletal stem/progenitor cells from their own embryonic origins. However, this selective 'flavor' has to somehow be imprinted into the progenitor cell. The Hox code represent a mechanism crucial for embryonic patterning, and therefore we sought to investigate whether the positional memory, defined by the Hox expression pattern, persists into adulthood, and if so, if it represents the mechanism by which adult progenitor cells can be characterized. Our gene transcription analysis confirmed our hypothesis that it is in fact the Hox code that distinguishes progenitor cells more effectively than the embryonic origin. The finding that progenitor cells maintain positional memory throughout their life, and that this identity is associated with a unique transcriptional profile may have significant clinical implications. If bones preferentially heal using cells that share the same positional origin (Hox code), then reparative strategies may have to take this variable into account in order to be maximally effective. (Figure Presented)

INTRODUCTION: All tissues are affected by aging, but diseases that weaken the skeleton constitute the most prevalent chronic impairment in the United States. Although skeletal diseases and conditions are seldom fatal, they can significantly compromise function and diminish quality of life. Perhaps most importantly, age-related changes in skeletal health can be traced back to a decline in both the number and function of osteoprogenitor cells (OPCs). However, the cause for the decline in both the number and function of OPCs is not well understood. Chronic inflammation in the elderly (inflamm-aging) is thought to be a major contributor to this decline in the regenerative capacity of many tissues, including the skeleton. In contrast to a well-balanced inflammatory response after trauma, which is crucial for successful bone repair, chronic unbalanced elevation of pro-inflammatory cytokines has been shown to inhibit regeneration in a variety of tissues. We hypothesize that inflamm-aging is the major cause for the decline in OPC number and dysfunction in elderly patients and that this decline in OPC number and dysfunction can be halted by treatment with an anti-inflammatory drug. METHODS: Young, 12 week-old and aged, 52 week-old C57BL/6J were used following the IACUC guidelines at our institution. Aged animals were randomly distributed into a no-treatment (n=5) and a treatment group (n=5). Animals in the no-treatment group received regular drinking water, while animals in the treatment group received sodium salicylate water (12mg/day) for 8 weeks. The inflammatory status of young and aged untreated and treated mice was assessed using a multiplex platform screening for multiple pro- and anti-inflammatory cytokines, and utilizing qRT-PCR for IL-1, IL-6, NF-kappaB, TNF-alpha. FACS analysis using the LepR as a marker for osteogenic precursor cells was employed to identify the effect of chronic low-level inflammation on progenitor cell number. In addition, bMSCs were harvested from femurs and tibia from young and aged untreated and treated mice and cultured in growth media and osteogenic media. Cell proliferation and osteogenic differentiation (qRT-PCR for col 1, runx2, osx and oc, and alizarin red and alkaline phosphatase staining) were assessed in vitro. Results are presented in the form of mean +/- standard deviation, with N equal to the number of samples analyzed. Two-tailed Student's t-tests were used to determine significant differences between data sets that are normally distributed. For non-normally distributed data sets, Mann-Whitney U test was used. Significance was attained at p < 0.05 and all statistical analyses were performed with Graphpad Prism software (GraphPad Software, San Diego, California). RESULTS: First, we set out to identify age-related chronic inflammation in mice. We analyzed blood by multiplex analysis and tibial and femoral bone marrow by qRT-PCR for pro-inflammatory markers. Both analyses revealed an increase in pro-inflammatory and a decrease in anti-inflammatory cytokines in aged animals, confirming the presence of inflamm-aging (Fig. 1A). Next, we aimed at understanding how aging effects osteoprogenitor cell number using flow cytometry. We harvested cells from young and aged mice, removed red blood cells, and then stained with antibodies to CD31, CD45, Ter-119 and LepR. Cell sorting was performed and CD31^-CD45^-Ter^-119^-LepR+ cells were isolated and quantified. Flow cytometry analysis revealed that 0.38% of cells from 12 week old mice were LepR⁺ osteoprogenitor cells, confirming the findings published by Zhou et al. Analysis of the cells from 52 week-old mice revealed a significant decrease in number to 0.017% of bone marrow cells (Fig. 1B). Next, we had to establish that an 8-week course of sodium salicylate successfully represses chronic inflammation. In response to NSAID treatment, the expression level of IL-10 significantly increased above the level of the juvenile animals, while the NF-kappaB, TNFalpha and Cox-2 levels returned to baseline (Fig. 1A). This experiment served as a proof-of-principle that mice exhibit inflamm-aging and that this inflammatory state can be suppressed by administration of an NSAID. Having established that our dosing of the systemic NSAID suppresses chronic ageinduced inflammation, we assessed whether OPC frequency changes as a result of suppressed inflamm-aging. Bone marrow from 3 month-old, 12 month-old and 12 monthold NSAID-treated animals was subjected to flow cytometry. OPC frequency declined with aging, however, after an 8 week course of NSAID treatment, we noticed a two-fold increase in LepR⁺ OPCs within the bone marrow (Fig. 1B). We then aimed at testing whether NSAID treatment of aged mice resulted in a restoration of the osteogenic potential of this OPC population. Quantitative RT-PCR of the bone marrow of sodium salicylate-treated mice showed an increase in osteogenic gene expression (osx, oc and alkaline phosphatase) compared to untreated aged mice. The expression level of osx reached that of young, 3 month-old mice, while oc and ALP expression levels were significantly higher than those of juvenile animals (Fig. 1C). In order to further characterize this increase in osteogenic potential, we harvested MSCs from young and aged treated and untreated animals, plated them in vitro and then subjected them to osteogenic differentiation media. Mineralization assays and expression analysis of osx, oc and ALP showed decreased osteogenesis of aged cells, while treatment with sodium salicylate recovered this decline and resulted in restoration of the osteogenic potential (Fig. 1D). DISCUSSION: These experiments demonstrate for the first time that age-related chronic inflammation is responsible for the decreased proliferative and osteogenic potential of aged OPCs and that this process is reversible by anti-inflammatory treatment. The findings from this study may have a profound translational impact: If we could restore the regenerative potential of the aged skeleton by treating age-related inflammation, then theoretically, we may have a tool at hand to improve the healing process of osteoporotic fracture patients. (Figure Presented)