Mechanisms of GH action on the skeleton: role of SOCS2

Determining the mechanisms by which growth hormone (GH) enhances bone growth and development has proven difficult. GH can act either systemically via the stimulation of liver insulin like growth factor (IGF)-1, or locally via activation of the GH receptor (GHR). Furthermore, the local actions of GH may be IGF-1 dependent (indirect) or independent (direct). Suppressor of cytokine signalling 2 (SOCS2) has been identified as an important regulator of GH signalling via the JAK/STAT pathway. The SOCS2 knockout (Socs2-/-) mouse is characterised by its overgrowth phenotype despite no elevation in systemic GH and IGF-1 levels. It therefore offers a valid and novel model to investigate the local effects of enhanced GH signalling on the skeleton. The work presented in this thesis investigates the Socs2-/- mouse model to better understand the actions of local GH on longitudinal bone growth and bone accrual. Ex vivo metatarsal organ cultures, osteoblast cultures, and in vivo approaches are used to unravel the mechanisms of GH action on the skeleton. This thesis also explores the potential of SOCS2 as primary mediator of inflammatory induced bone loss through the utilisation of the dextran sulphate sodium (DSS) model of colitis. Embryonic and postnatal ex vivo metatarsal organ cultures are used to study the mechanism of GH action on longitudinal bone growth. Specifically, the present work highlights that enhanced linear growth in the absence of SOCS2 is associated with an increase in the GH regulated proteins, IGF-2 and IGF binding protein 3 (IGFBP3), but not IGF-1. This indicates that IGF-1 may not be essential for mediating GH action on bone growth. Completion of an in depth analysis of the bone phenotype of juvenile and adult, male and female Socs2-/- mice reveals an anabolic phenotype consistent with increased GH signalling. Male Socs2-/- mice are shown to have a greater enhancement of cortical parameters compared to females, resulting in increased bone strength. Investigation of the mechanisms behind the enhanced bone accrual in Socs2-/- mice identifies SOCS2 as the primary SOCS protein regulating GH signalling in primary osteoblasts. The JAK/STAT pathway is confirmed as the key signalling pathway targeted by SOCS2. Despite this enhanced signalling there is little evidence presented in this thesis to suggest that GH actions on osteoblasts and ultimately bone mass are mediated through increased Igf1 expression. GH treatment is shown to be anabolic to bone of young juvenile Socs2-/- mice, but not WT mice. This increase in bone mass is associated with increase bone p-STAT5 signalling, but no increase in Igf1 levels indicating that GH may have IGF-1 independent effects in the Socs2-/- mouse model. GH treatment of young mice also reveals an age and sex specific effect of GH action where GH does not stimulate growth until approximately 3 weeks of age. From 3 weeks of age, WT female mice show increased growth in response to GH, but males do not. The increased growth is associated with increased p-STAT5 signalling and increased bone area. This thesis also confirms SOCS2 a critical mediator of bone loss associated with inflammation. The present results show that deteriorated trabecular bone health in colitic mice is associated with elevated Socs2 expression in bone. Furthermore, despite similar levels of gut inflammation observed in Socs2-/- mice with DSS induced colitis these mice are partly protected from poor bone health. The work described herein has used the Socs2-/- mouse model to strengthen our understanding of the actions of local GH on skeletal growth and development. It also provides compelling evidence for the importance of SOCS2 as a mediator of bone loss in cases of inflammatory bowel disease

Selective Myeloid Depletion of Galectin-3 Offers Protection Against Acute and Chronic Lung Injury

Rationale: Galectin-3 (Gal-3) is an immune regulator and an important driver of fibrosis in chronic lung injury, however, its role in acute lung injury (ALI) remains unknown. Previous work has shown that global deletion of galectin-3 reduces collagen deposition in a bleomycin-induced pulmonary fibrosis model (MacKinnon et al., Am. J. Respir. Crit. Care Med., 2012, 185, 537–46). An inhaled Gal-3 inhibitor, GB0139, is undergoing Phase II clinical development for idiopathic pulmonary fibrosis (IPF). This work aims to elucidate the role of Gal-3 in the myeloid and mesenchymal compartment on the development of acute and chronic lung injury. Methods:LgalS3(fl/fl) mice were generated and crossed with mice expressing the myeloid (LysM) and mesenchymal (Pdgfrb) cre drivers to yield LysM-cre (+/-) /LgalS3 (fl/fl) and Pdgfrb-cre (+/-) /LgalS3 (fl/fl) mice. The response to acute (bleomycin or LPS) or chronic (bleomycin) lung injury was compared to globally deficient Gal-3 (−/−) mice. Results: Myeloid depletion of Gal-3 led to a significant reduction in Gal-3 expression in alveolar macrophages and neutrophils and a reduction in neutrophil recruitment into the interstitium but not into the alveolar space. The reduction in interstitial neutrophils corelated with decreased levels of pulmonary inflammation following acute bleomycin and LPS administration. In addition, myeloid deletion decreased Gal-3 levels in bronchoalveolar lavage (BAL) and reduced lung fibrosis induced by chronic bleomycin. In contrast, no differences in BAL Gal-3 levels or fibrosis were observed in Pdgfrb-cre (+/-) /LgalS3 (fl/fl) mice. Conclusions: Myeloid cell derived Galectin-3 drives acute and chronic lung inflammation and supports direct targeting of galectin-3 as an attractive new therapy for lung inflammation

Suppressor of cytokine signaling 2 (SOCS2) deletion protects bone health of mice with DSS induced inflammatory bowel disease.

Individuals with inflammatory bowel disease (IBD) often present with poor bone health. The development of targeted therapies for this bone loss requires a fuller understanding of the underlying cellular mechanisms. Although bone loss in IBD is multifactorial the altered sensitivity and secretion of growth hormone (GH) and insulin-like growth factor-1 (IGF-1) in IBD is understood to be a critical contributing mechanism. The expression of suppressor of cytokine signaling 2 (SOCS2), a well-established negative regulator of GH signaling, is stimulated by pro-inflammatory cytokines. Therefore, it is likely that SOCS2 expression represents a critical mediator through which pro-inflammatory cytokines inhibit GH/IGF-1 signaling and decrease bone quality in IBD. Utilising the DSS model of colitis we have revealed that endogenously elevated GH function in the Socs2−/− mouse protects the skeleton from osteopenia. Micro-computed tomography assessment of DSS treated wild-type mice revealed a worsened trabecular architecture compared to control mice. Specifically, DSS treated WT mice had significantly decreased bone volume (BV/TV) (41%; p<0.05), trabecular thickness (16%; p<0.05), trabecular number (30%; p<0.05), and a resulting increase in trabecular separation (19%; <0.05). In comparison, the trabecular bone of Socs2 deficient mice was partially protected from the adverse effects of DSS. The reduction in a number of parameters including BV/TV (21%; p<0.05) was less, and no changes were observed in trabecular thickness or separation. This protected phenotype was unlikely to be a consequence of improved mucosal health in the DSS treated Socs2−/− mice but rather a result of unregulated GH signaling directly on bone. These studies indicate that the absence of SOCS2 is protective against bone loss typical of IBD. This study also provides an improved understanding of the relative effects of GH/IGF-1 on bone health in experimental colitis, information that is essential before these drugs are explored as bone protective agents in children and adults with IBD

A versatile nuclei extraction protocol for single nucleus sequencing in non-model species – optimization in various Atlantic salmon tissues

The use of single cell sequencing technologies has exploded over recent years, and is now commonly used in many non-model species. Sequencing nuclei instead of whole cells has become increasingly popular, as it does not require the processing of samples immediately after collection. Here we present a highly effective nucleus isolation protocol that outperforms previously available method in challenging samples in a non-model specie. This protocol can be successfully applied to extract nuclei from a variety of tissues and species

Single cell RNA sequencing and lineage tracing confirm mesenchyme to epithelial transformation (MET) contributes to repair of the endometrium at menstruation

The human endometrium experiences repetitive cycles of tissue wounding characterised by piecemeal shedding of the surface epithelium and rapid restoration of tissue homeostasis. In this study, we used a mouse model of endometrial repair and three transgenic lines of mice to investigate whether epithelial cells that become incorporated into the newly formed luminal epithelium have their origins in one or more of the mesenchymal cell types present in the stromal compartment of the endometrium. Using scRNAseq, we identified a novel population of PDGFRb + mesenchymal stromal cells that developed a unique transcriptomic signature in response to endometrial breakdown/repair. These cells expressed genes usually considered specific to epithelial cells and in silico trajectory analysis suggested they were stromal fibroblasts in transition to becoming epithelial cells. To confirm our hypothesis we used a lineage tracing strategy to compare the fate of stromal fibroblasts (PDGFRa+) and stromal perivascular cells (NG2/CSPG4+). We demonstrated that stromal fibroblasts can undergo a mesenchyme to epithelial transformation and become incorporated into the re-epithelialised luminal surface of the repaired tissue. This study is the first to discover a novel population of wound-responsive, plastic endometrial stromal fibroblasts that contribute to the rapid restoration of an intact luminal epithelium during endometrial repair. These findings form a platform for comparisons both to endometrial pathologies which involve a fibrotic response (Asherman’s syndrome, endometriosis) as well as other mucosal tissues which have a variable response to wounding

Single cell transcriptomics of Atlantic salmon (Salmo salar L.) liver reveals cellular heterogeneity and immunological responses to challenge by Aeromonas salmonicida

Copyright © 2022 Taylor, Ruiz Daniels, Dobie, Naseer, Clark, Henderson, Boudinot, Martin and Macqueen.Peer reviewedPublisher PD

Design and manufacture of an optimised side-shifted PPM 2 EMAT array for use in mobile robotic localisation

Guided wave Electro Magnetic Acoustic Transducers (EMATs) offer an elegant method for structural inspection and localisation relative to geometric features, such as welds. This paper presents a Lorentz force EMAT construction framework, where a numerical model has been developed for optimising Printed Circuit Board (PCB) coil parameters as well as a methodology for optimising magnet array parameters to a user’s needs. This framework was validated experimentally to show its effectiveness through comparison to an industry built EMAT. The framework was then used to design and manufacture a Side-Shifted Unidirectional Periodic Permanent Magnet (PPM) EMAT for use on a mobile robotic system, which uses guided waves for ranging to build internal maps of a given subject, identifying welded sections, defects and other structural elements. The unidirectional transducer setup was shown to operate in simulation and was then manufactured to compare to the bidirectional transmitter and two-receiver configurations on a localisation system. The unidirectional setup was shown to have clear benefits over the bidirectional setup for mapping an unknown environment using guided waves as there were no dead spots of mapping where signal direction could not be interpreted. Additionally, overall package size was significantly reduced, which in turn allows more measurements to be taken within confined spaces and increases robotic crawler mobility

Single-cell RNA sequencing redefines the mesenchymal cell landscape of mouse endometrium

The endometrium is a dynamic tissue that exhibits remarkable resilience to repeated episodes of differentiation, breakdown, regeneration, and remodeling. Endometrial physiology relies on a complex interplay between the stromal and epithelial compartments with the former containing a mixture of fibroblasts, vascular, and immune cells. There is evidence for rare populations of putative mesenchymal progenitor cells located in the perivascular niche of human endometrium, but the existence of an equivalent cell population in mouse is unclear. We used the Pdgfrb‐BAC‐eGFP transgenic reporter mouse in combination with bulk and single‐cell RNA sequencing to redefine the endometrial mesenchyme. In contrast to previous reports we show that CD146 is expressed in both PDGFRβ + perivascular cells and CD31 + endothelial cells. Bulk RNAseq revealed cells in the perivascular niche which express the high levels of Pdgfrb as well as genes previously identified in pericytes and/or vascular smooth muscle cells (Acta2, Myh11, Olfr78, Cspg4, Rgs4, Rgs5, Kcnj8, and Abcc9). scRNA‐seq identified five subpopulations of cells including closely related pericytes/vascular smooth muscle cells and three subpopulations of fibroblasts. All three fibroblast populations were PDGFRα+/CD34 + but were distinct in their expression of Ngfr/Spon2/Angptl7 (F1), Cxcl14/Smoc2/Rgs2 (F2), and Clec3b/Col14a1/Mmp3 (F3), with potential functions in the regulation of immune responses, response to wounding, and organization of extracellular matrix, respectively. Immunohistochemistry was used to investigate the spatial distribution of these populations revealing F1/NGFR + cells in most abundance beside epithelial cells. We provide the first definitive analysis of mesenchymal cells in the adult mouse endometrium identifying five subpopulations providing a platform for comparisons between mesenchymal cells in endometrium and other adult tissues which are prone to fibrosis

Mapping the cellular landscape of Atlantic salmon head kidney by single cell and single nucleus transcriptomics

Single-cell transcriptomics is the current gold standard for global gene expression profiling, not only in mammals and model species, but also in non-model fish species. This is a rapidly expanding field, creating a deeper understanding of tissue heterogeneity and the distinct functions of individual cells, making it possible to explore the complexities of immunology and gene expression on a highly resolved level. In this study, we compared two single cell transcriptomic approaches to investigate cellular heterogeneity within the head kidney of healthy farmed Atlantic salmon (Salmo salar). We compared 14,149 cell transcriptomes assayed by single cell RNA-seq (scRNA-seq) with 18,067 nuclei transcriptomes captured by single nucleus RNA-Seq (snRNA-seq). Both approaches detected eight major cell populations in common: granulocytes, heamatopoietic stem cells, erythrocytes, mononuclear phagocytes, thrombocytes, B cells, NK-like cells, and T cells. Four additional cell types, endothelial, epithelial, interrenal, and mesenchymal cells, were detected in the snRNA-seq dataset, but appeared to be lost during preparation of the single cell suspension submitted for scRNA-seq library generation. We identified additional heterogeneity and subpopulations within the B cells, T cells, and endothelial cells, and revealed developmental trajectories of heamatopoietic stem cells into differentiated granulocyte and mononuclear phagocyte populations. Gene expression profiles of B cell subtypes revealed distinct IgM and IgT-skewed resting B cell lineages and provided insights into the regulation of B cell lymphopoiesis. The analysis revealed eleven T cell sub-populations, displaying a level of T cell heterogeneity in salmon head kidney comparable to that observed in mammals, including distinct subsets of cd4/cd8-negative T cells, such as tcrγ positive, progenitor-like, and cytotoxic cells. Although snRNA-seq and scRNA-seq were both useful to resolve cell type-specific expression in the Atlantic salmon head kidney, the snRNA-seq pipeline was overall more robust in identifying several cell types and subpopulations. While scRNA-seq displayed higher levels of ribosomal and mitochondrial genes, snRNA-seq captured more transcription factor genes. However, only scRNA-seq-generated data was useful for cell trajectory inference within the myeloid lineage. In conclusion, this study systematically outlines the relative merits of scRNA-seq and snRNA-seq in Atlantic salmon, enhances understanding of teleost immune cell lineages, and provides a comprehensive list of markers for identifying major cell populations in the head kidney with significant immune relevance.</p

Mapping the cellular landscape of Atlantic salmon head kidney by single cell and single nucleus transcriptomics

The study was funded by grants from the Research Council Norway (ID:302191), the University of Edinburgh's Data Driven Innovation Initiative (Scottish Funding Council Beacon ‘Building Back Better’ Call), and the Biotechnology and Biological Sciences Research Council, including the institutional strategic programme grants BBS/E/D/10002071, BBS/E/RL/230001C, BBS/E/D/20002174, BBS/E/RL/230002B, and the responsive mode grants BB/W005859/1 and BB/W008564/1. NH is supported by a Wellcome Trust Senior Research Fellowship in Clinical Science (ref. 219542/Z/19/Z). UG is supported by the Research Council of Norway (ID:274635).Peer reviewe