Immune Cell and Adipose Gene Expression Reprogramming in Juvenile Monkeys Born to Health and Weight-Diverse Mothers

Over 93 million Americans are obese and 66 million suffer from metabolic disease. Roughly 40% of obese people do not have metabolic abnormalities (metabolically health obese [MHO]), while approximately 15% of lean do (metabolically unhealthy lean [MUL]). African green monkeys (AGMs) demonstrate naturally occurring obesity and metabolic syndrome (MetS) without diet manipulation, and MetS criteria are heritable. Age-matched maternal AGMs were classified by adjusted MetS criteria ([n=44]; waist >40cm, fasting glucose (FG) >100 mg/dL, SBP/DBP >135/85mmHg, and HDL-c <50mg/dL) and classified as metabolically healthy lean (MHL), MHO, MUL or metabolically unhealthy obese (MUO). Age, weight and sex-matched pre-pubertal juvenile offspring from these mothers were additionally selected (n=9-11/group; ages=1.1-3.4years) for evaluation. We assessed monocyte subtypes by flow cytometry, and subcutaneous adipose gene expression patterns by RNAseq. Non-classical monocytes were increased in obese and unhealthy mothers (MHO p=0.02, MUL p=0.003, MUO p=0.00002) compared to MHL. MUL and MUO juvenile offspring also had more non-classical monocytes compared to MHL (p=0.05 and p=0.07). Monocyte chemoattractant protein-1 (MCP)-1 was measured in plasma and found to be elevated in MUO juveniles (p=0.02). Patterns of increased cytokine and extracellular matrix gene expression were seen in MUL and MUO juveniles’ adipose (6-7/group), mirroring obese and unhealthy mothers’ adipose gene expression patterns. Maternal health and obesity influence offspring immune cells and adipose gene expression prior to weight gain and metabolic disease onset. Our data underscore maternal monocyte and adipose profiles as inherited phenotypes that present prior to adipose expansion and may be targets to improve intergenerational health trajectories.

Reverse transcription PCR-based detection of matrix and hemagglutinin-neuraminidase genes among Avian orthoavulavirus 1 clinical isolates in the Philippines, 1991-2017

Newcastle disease (ND) is a highly infectious disease that affects devastatingly the avian population worldwide. It is caused by Avian orthoavulavirus 1 (AOAV-1), or better known as Newcastle disease virus belonging to phylum Negarnaviricota, class Monjiviricetes, order Mononegavirales and family Paramyxoviridae. This virus consists of six principal structural proteins namely: the nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN) and the large protein RNA dependent RNA polymerase (L).. The present study aimed to molecularly detect the M and HN gene segments of the AOAV-1 field isolates from clinical cases in the Philippines from 1991 through 2017. RT-PCR amplification and sequence analyses using primers NDV-For4359 and NDV-Rev4788 which anneal to the matrix gene and primers NDV-For6369 and NDV-Rev6598 targeting the HN genes, identified all isolates to be AOAV-1. Determining the different genes of the virus would greatly help scientists and researchers to accurately identify the viral isolates in order to improve epidemiological studies and surveillance of the disease in the country.

The extracellular matrix gene, Svep1, orchestrates airway patterning and the transition from lung branching morphogenesis to alveolar maturation in the mouse

Proper lung development and function requires two independent but interrelated processes: branching morphogenesis to form the airway tree, and alveolar cell differentiation for peripheral gas exchange. The disruption of either branching or differentiation results in severe respiratory deficiencies and often in neonatal death. The molecular mechanisms that control branching patterns and the transition to alveolar differentiation are not completely understood. Here we report on the in vitro and in vivo characterization of the lungs of mouse embryos lacking a functional Svep1 gene. Our data demonstrate that the SVEP1 extracellular matrix protein is critical for the process of transitioning from branching to alveolar maturation. Svep1-/- embryos on a C57BL/6J genetic background are characterized by hypoplastic lungs and a disorganized increase in distal airway tips which disrupts airway architecture and lobe shape. The lungs of Svep1 knockout embryos also have defects in alveolar differentiation. In vitro lung explant experiments demonstrated that SVEP1 normally inhibits branching morphogenesis and that treatment with a SVEP1 peptide can rescue the branching defects observed in Svep1 knockouts. Our findings reveal for the first time that Svep1 is essential for constructing the basic airway architecture and for the transition from lung branching to alveolar differentiation. Our results suggest therapeutic strategies to enhance lung development in patients with life-threatening respiratory disorders such as the lung hypoplasia and prematurity observed in neonates with congenital diaphragmatic hernia (CDH).

Null cyp1b1 Activity in Zebrafish Leads to Variable Craniofacial Defects Associated with Altered Expression of Extracellular Matrix and Lipid Metabolism Genes

CYP1B1 loss of function (LoF) is the main known genetic alteration present in recessive primary congenital glaucoma (PCG), an infrequent disease characterized by delayed embryonic development of the ocular iridocorneal angle; however, the underlying molecular mechanisms are poorly understood. To model CYP1B1 LoF underlying PCG, we developed a cyp1b1 knockout (KO) zebrafish line using CRISPR/Cas9 genome editing. This line carries the c.535_667del frameshift mutation that results in the 72% mRNA reduction with the residual mRNA predicted to produce an inactive truncated protein (p.(His179Glyfs*6)). Microphthalmia and jaw maldevelopment were observed in 23% of F0 somatic mosaic mutant larvae (144 hpf). These early phenotypes were not detected in cyp1b1-KO F3 larvae (144 hpf), but 27% of adult (four months) zebrafish exhibited uni- or bilateral craniofacial alterations, indicating the existence of incomplete penetrance and variable expressivity. These phenotypes increased to 86% in the adult offspring of inbred progenitors with craniofacial defects. No glaucoma-related phenotypes were observed in cyp1b1 mutants. Transcriptomic analyses of the offspring (seven dpf) of cyp1b1-KO progenitors with adult-onset craniofacial defects revealed functionally enriched differentially expressed genes related to extracellular matrix and cell adhesion, cell growth and proliferation, lipid metabolism (retinoids, steroids and fatty acids and oxidation–reduction processes that include several cytochrome P450 genes) and inflammation. In summary, this study shows the complexity of the phenotypes and molecular pathways associated with cyp1b1 LoF, with species dependency, and provides evidence for the dysregulation of extracellular matrix gene expression as one of the mechanisms underlying the pathogenicity associated with cyp1b1 disruption.

Null cyp1b1 Activity in Zebrafish Leads to Variable Craniofacial Defects Associated with Altered Expression of Extracellular Matrix and Lipid Metabolism Genes

CYP1B1 loss-of-function (LoF) is the main known genetic alteration present in recessive primary congenital glaucoma (PCG), an infrequent disease characterized by delayed embryonic development of the ocular iridocorneal angle and caused by poorly understood molecular mechanisms. To model CYP1B1 LoF underlying PCG, we developed a cyp1b1 knockout (KO) zebrafish line using CRISPR/Cas9 genome editing. This line carries the c.535_667del frameshift mutation that results in a 72% mRNA reduction with residual mRNA predicted to produce an inactive truncated protein (p.(His179Glyfs*6)). Craniofacial defects and jaw maldevelopment were observed in 23% of somatic mosaic F0 crispant larvae (144 hpf). These early phenotypes were not detected in KO F3 larvae (144 hpf) but 27% of adult fishes (4 months) showed uni or bilateral craniofacial alterations, indicating the existence of incomplete penetrance and variable expressivity. These phenotypes increased to 86% in the adult offspring of inbred progenitors with craniofacial defects. No glaucoma-related phenotypes were observed in the cyp1b1 mutants. Transcriptomic analyses of the offspring (7dpf) of KO cyp1b1 progenitors with adult-onset craniofacial defects revealed that differentially expressed genes were functionally enriched in groups related with extracellular matrix and cell adhesion, cell growth and proliferation, lipid metabolism (retinoids, steroids, and fatty acids, and oxidation-reduction processes which included several cytochrome P450 genes) and inflammation. In summary, this study shows the complexity of phenotypes and molecular pathways associated with cyp1b1 LoF, with species-dependency, and provides evidence for dysregulation of extracellular matrix gene expression as one of the mechanisms underlaying pathogenicity associated with cyp1b1 disruption.

OP0248 DEVELOPMENT OF A 3D SKIN MICROTISSUE MODEL FOR FIBROTIC DISEASES

Background:Traditional preclinical approaches, such as two-dimensional cell culture and animal models, are often inadequate to mimic the pathophysiological features of complex diseases such as systemic sclerosis (SSc). Human specific targets, such as the recently described pro-fibrotic long non coding RNA (lncRNA) H19X1, are becoming increasingly relevant in preclinical research, creating the need of new strategies and tools in translational medicine. The employment of novel three-dimensional (3D) culture systems, where multiple cell types are included, is filling an important gap left by the traditional preclinical methods.Objectives:To develop an easy to produce 3D fibrotic skin microtissues model for translational proof of concept studies.Methods:Two thousand five hundred dermal fibroblasts isolated from skin of SSc patients were seeded in ultra-low attachment 96-well plates. Fibroblast were let to aggregate into spheres for 48h. Two thousand five hundred primary normal human keratinocytes were added to the culture and let to layer onto the fibroblast spheres for 72h. H19X silencing experiments were used as proof of concept studies. H19X silencing with antisense oligonucleotides or transfections with a scrambled control were performed in fibroblasts prior to the sphere formation for 24h. TGFβ (10 ng/ml) was added to microtissue to exacerbate the fibrotic phenotype. Haematoxylin eosin staining as well as immunohistochemistry staining for vimentin and cytokeratin 10 was performed. Skin microtissues were processed for RNA and protein isolation. Pro-collagen Iα1 and fibronectin were quantified in the supernatants with ELISA.Results:The microtissues presented a core of SSc fibroblast as revealed by vimentin staining and an external layer of keratinocytes as revealed by cytokeratin 10 staining, mimicking the human skin architecture. Gene expression analysis following TGFβ stimulation displayed induced expression of extracellular matrix gene COL1A1 (p=0.044) and the myofibroblast marker ACTA2 (p=0.018), indicating that the microtissues were able to develop a fibrotic response. Microtissues, where H19X was silenced, displayed reduced gene expression of COL1A1 and ACTA2 after TGFβ stimulation (COL1A1 p=0.007, ACTA2 p=0.045). Additionally, H19X silencing led to lower levels of αSMA protein expression (p=0.009) and pro-collagen1α1 secretion (p=0.039) in the supernatant of the microtissue cultures as revealed by Western Blot and ELISA, respectively. FN1 expression and fibronectin protein levels were not significantly reduced in the microtissues after H19X silencing.Conclusion:We were able to produce a 3D microtissue resembling skin architecture that can respond to fibrotic stimuli. Knockdown experiments of pro-fibrotic lncRNA H19X confirmed the potential of the model as screening platform for novel pro-fibrotic effectors. A future aim will be to optimize the model for high-throughput automated screening platforms.References:[1]Pachera, E., et al. (2020). “Long noncoding RNA H19X is a key mediator of TGF-β–driven fibrosis.” The Journal of Clinical Investigation 130(9): 4888-4905.Disclosure of Interests:Elena Pachera: None declared, Gabriela Kania: None declared, Astrid Juengel: None declared, Maurizio Calcagni Speakers bureau: Arthrex, Consultant of: Medartis, Arthrex, SilkBiomaterials, Grant/research support from: Medartis, Oliver Distler Speakers bureau: Actelion, Bayer, Boehringer Ingelheim, Medscape, Novartis, Roche, Consultant of: Abbvie, Actelion, Acceleron Pharma, Amgen, AnaMar, Arxx Therapeutics, Bayer, Baecon Discovery, Blade Therapeutics, Boehringer, CSL Behring, ChemomAb, Corpuspharma, Curzion Pharmaceuticals, Ergonex, Galapagos NV, GSK, Glenmark Pharmaceuticals, Inventiva, Italfarmaco, iQvia, -Kymera, Medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Roche, Sanofi, UCB, Grant/research support from: Abbvie, Actelion, Bayer, Boehringer Ingelheim, Kymera Therapeutics, Mitsubishi Tanabe

Polycystin-2 Is Required for Chondrocyte Mechanotransduction and Traffics to the Primary Cilium in Response to Mechanical Stimulation

Primary cilia and associated intraflagellar transport are essential for skeletal development, joint homeostasis, and the response to mechanical stimuli, although the mechanisms remain unclear. Polycystin-2 (PC2) is a member of the transient receptor potential polycystic (TRPP) family of cation channels, and together with Polycystin-1 (PC1), it has been implicated in cilia-mediated mechanotransduction in epithelial cells. The current study investigates the effect of mechanical stimulation on the localization of ciliary polycystins in chondrocytes and tests the hypothesis that they are required in chondrocyte mechanosignaling. Isolated chondrocytes were subjected to mechanical stimulation in the form of uniaxial cyclic tensile strain (CTS) in order to examine the effects on PC2 ciliary localization and matrix gene expression. In the absence of strain, PC2 localizes to the chondrocyte ciliary membrane and neither PC1 nor PC2 are required for ciliogenesis. Cartilage matrix gene expression (Acan, Col2a) is increased in response to 10% CTS. This response is inhibited by siRNA-mediated loss of PC1 or PC2 expression. PC2 ciliary localization requires PC1 and is increased in response to CTS. Increased PC2 cilia trafficking is dependent on the activation of transient receptor potential cation channel subfamily V member 4 (TRPV4) activation. Together, these findings demonstrate for the first time that polycystins are required for chondrocyte mechanotransduction and highlight the mechanosensitive cilia trafficking of PC2 as an important component of cilia-mediated mechanotransduction.