Craig Lawless

The mammalian retina contains high amounts of metals/metalloid-selenium. Their dyshomeostases are associated with certain retinal diseases. We carried out this bioinformatics study to identify the relationships between putative retinal metal/selenium binding proteins, their molecular functions, and biological processes. Identification of putative mouse metal/selenium binding proteins was based on known binding motifs, domains, patterns, and profiles. Annotations were obtained from Uniprot keywords ‘metal binding’, ‘metal ion co-factors’, ‘selenium proteins’. Protein functions were estimated by associative frequency with key words in UniProt annotations. The raw data of five mouse proteomics PRIDE datasets (available to date) were downloaded and processed with Mascot against the mouse taxa of Uniprot (SwissProt/Trembl) and MaxQuant (version 1.6.10.43) for qualitative and quantitative datasets, respectively. Clinically relevant variants were evaluated using archives and aggregated information in ClinVar. The 438 proteins common to all the retina proteomics datasets were used to identify over-represented Gene Ontology categories. The putative mouse retinal metal/metalloid binding proteins identified are mainly involved in: (1) metabolic processes (enzymes), (2) homeostasis, (3) transport (vesicle mediated, transmembrane, along microtubules), (4) cellular localization, (5) regulation of signalling and exocytosis, (6) organelle organization, (7) (de)phosphorylation, and (8) complex assembly. Twenty-one proteins were identified as involved in response to light stimulus and/or visual system development. An association of metal ion binding proteins rhodopsin, photoreceptor specific nuclear receptor, calcium binding protein 4 with disease-related mutations in inherited retinal conditions was identified, where the mutations affected an area within or in close proximity to the metal binding site or domain. These findings suggest a functional role for the putative metal/metalloid binding site in retinal proteins in certain retinal disorders.

Pulmonary fibrosis is an increasing and major cause of death worldwide. Understanding the cellular and molecular mechanisms underlying the pathophysiology of lung fibrosis may lead to urgently needed diagnostic and prognostic strategies for the disease. SOX9 is a core transcription factor that has been associated with fibrotic disease, however its role and regulation in acute lung injury and/or fibrosis have not been fully defined. In this study we apply a hypothesis based approach to uncover unique SOX9-protein signatures associated with both acute lung injury and fibrotic progression. Using in vivo models of lung injury in the presence or absence of SOX9, our study shows SOX9 is essential to the damage associated response of alveolar epithelial cells from an early time-point in lung injury. In parallel, as disease progresses, SOX9 is responsible for regulating tissue damaging ECM production from pro-fibrotic fibroblasts. In determining the in vivo role of SOX9 we identified secreted ECM components downstream of SOX9 as markers of acute lung injury and fibrosis. To underscore the translational potential of our SOX9-regulated markers, we analysed serum samples from acute COVID19, post COVID19 and idiopathic pulmonary fibrosis (IPF) patient cohorts. Our hypothesis driven SOX9-panels showed significant capability in all cohorts at identifying patients who had poor disease outcomes. This study shows that SOX9 is functionally critical to disease in acute lung injury and pulmonary fibrosis and its regulated pathways have diagnostic, prognostic and therapeutic potential in both COVID19 and IPF disease.

Daily rhythms in mammalian behaviour and physiology are generated by a multi-oscillator circadian system entrained through environmental cues (e.g. light and feeding). The presence of tissue niche-dependent physiological time cues has been proposed, allowing tissues the ability of circadian phase adjustment based on local signals. However, to date, such stimuli have remained elusive. Here we show that daily patterns of mechanical loading and associated osmotic challenge within physiological ranges reset circadian clock phase and amplitude in cartilage and intervertebral disc tissues in vivo and in tissue explant cultures. Hyperosmolarity (but not hypo-osmolarity) resets clocks in young and ageing skeletal tissues and induce genome-wide expression of rhythmic genes in cells. Mechanistically, RNAseq and biochemical analysis revealed the PLD2-mTORC2-AKT-GSK3β axis as a convergent pathway for both in vivo loading and hyperosmolarity-induced clock changes. These results reveal diurnal patterns of mechanical loading and consequent daily oscillations in osmolarity as a bona fide tissue niche-specific time cue to maintain skeletal circadian rhythms in sync.

Chemokine-driven leukocyte recruitment is a key component of the immune response and of various diseases. Therapeutically targeting the chemokine system in inflammatory disease has been unsuccessful, which has been attributed to redundancy. We investigated why chemokines instead have specific, specialized functions, as demonstrated by multiple studies. We analyzed the expression of genes encoding chemokines and their receptors across species, tissues, and diseases. This analysis revealed complex expression patterns such that genes encoding multiple chemokines that mediated recruitment of the same leukocyte type were expressed in the same context, such as the genes encoding the CXCR3 ligands CXCL9, CXCL10, and CXCL11. Through biophysical approaches, we showed that these chemokines differentially interacted with extracellular matrix glycosaminoglycans (ECM GAGs), which was enhanced by sulfation of specific GAGs. Last, in vivo approaches demonstrated that GAG binding was critical for the CXCL9-dependent recruitment of specific T cell subsets but not of others, irrespective of CXCR3 expression. Our data demonstrate that interactions with ECM GAGs regulated whether chemokines were presented on cell surfaces or remained more soluble, thereby affecting chemokine availability and ensuring specificity of chemokine action. Our findings provide a mechanistic understanding of chemokine-mediated immune cell recruitment and identify strategies to target specific chemokines during inflammatory disease.

Desmosomes are multiprotein adhesion complexes that link intermediate filaments to the plasma membrane, ensuring the mechanical integrity of cells across tissues, but how they participate in the wider signalling network to exert their full function is unclear. To investigate this we carried out multiplexed protein proximity mapping using biotinylation (BioID). The combined interactomes of the essential desmosomal proteins desmocollin 2a, plakoglobin and plakophilin 2a (Pkp2a) in Madin-Darby canine kidney epithelial cells were mapped and their differences and commonalities characterised as desmosome matured from Ca ²⁺ -dependence to the mature, Ca ²⁺ -independent, hyperadhesive state, which predominates in tissues. Results suggest that individual desmosomal proteins have distinct roles in connecting to cellular signalling pathways and that these roles alter substantially when cells change their adhesion state. The data provide further support for a dualistic concept of desmosomes in which the properties of Pkp2a differ from those of the other, more stable proteins. This body of data provides an invaluable resource for analysis of desmosome function.

Collagen is a key structural component of multicellular organisms and is arranged in a highly organised manner. In structural tissues such as tendons, collagen forms bundles of parallel fibres between cells, which appear within a 24 hour window between E13.5 and E14.5 during mouse embryonic development. Current models assume that the organised structure of collagen requires direct cellular control, whereby cells actively lay down collagen fibrils from cell surfaces. However, such models appear incompatible with the time- and length-scales of fibril formation. We propose a phase-transition model to account for the rapid development of ordered fibrils in embryonic tendon, reducing reliance on active cellular processes. We develop phase-field crystal simulations of collagen fibrillogenesis in domains derived from electron micrographs of inter-cellular spaces in embryonic tendon and compare results qualitatively and quantitatively to observed patterns of fibril formation. To test the prediction of this phase-transition model that free protomeric collagen should exist in the intercellular spaces prior to the formation of observable fibrils, we use laser-capture microdissection, coupled with mass spectrometry, which demonstrates steadily increasing free collagen in intercellular spaces up to E13.5, followed by a rapid reduction of free collagen that coincides with the appearance of less soluble collagen fibrils. The model and measurements together provide evidence for extracellular self-assembly of collagen fibrils in embryonic mouse tendon, supporting an additional mechanism for rapid collagen fibril formation during embryonic development.

Chemokine driven leukocyte recruitment is a key component of the immune response and is central to a wide range of diseases. However, there has yet to be a clinically successful therapeutic approach that targets the chemokine system during inflammatory disease; possibly due to the supposed redundancy of the chemokine system. A range of recent studies have demonstrated that the chemokine system is in fact based on specificity of function. Here we have generated a resource to analyse chemokine gene (ligand and receptor) expression across different species, tissues and diseases; revealing complex expression patterns whereby multiple chemokine ligands that mediate recruitment of the same leukocyte type are expressed in the same context, e.g. the CXCR3 ligands CXCL9, 10 and 11. We use biophysical approaches to show that CXCL9, 10 and 11 have very different interactions with extracellular matrix glycosaminoglycans (GAGs) which is exacerbated by specific GAG sulphation. Finally, in vivo approaches demonstrate that GAG-binding is critical for CXCL9 driven recruitment of specific T cell subsets (e.g. CD4 ⁺ ) but not others (e.g. CD8 ⁺ ), independent of CXCR3 expression. Our data demonstrate that chemokine expression is complex and that multiple ligands are likely needed for robust leukocyte recruitment across tissues and diseases. We also demonstrate that ECM GAGs facilitate decoding of these complex chemokine signals so that they are either primarily presented on GAG-coated cell surfaces or remain more soluble. Our findings represent a new mechanistic understanding of chemokine mediated immune cell recruitment and identify novel avenues to target specific chemokines during inflammatory disease.

Usual interstitial pneumonia (UIP) is a histological pattern characteristic of idiopathic pulmonary fibrosis (IPF). The UIP pattern is patchy with histologically normal lung adjacent to dense fibrotic tissue. At this interface, fibroblastic foci (FF) are present and are sites where myofibroblasts and extracellular matrix (ECM) accumulate. Utilizing laser capture microdissection-coupled mass spectrometry, we interrogated the FF, adjacent mature scar, and adjacent alveoli in 6 fibrotic (UIP/IPF) specimens plus 6 nonfibrotic alveolar specimens as controls. The data were subjected to qualitative and quantitative analysis and histologically validated. We found that the fibrotic alveoli protein signature is defined by immune deregulation as the strongest category. The fibrotic mature scar classified as end-stage fibrosis whereas the FF contained an overabundance of a distinctive ECM compared with the nonfibrotic control. Furthermore, FF were positive for both TGFB1 and TGFB3 , whereas the aberrant basaloid cell lining of FF was predominantly positive for TGFB2 . In conclusion, spatial proteomics demonstrated distinct protein compositions in the histologically defined regions of UIP/IPF tissue. These data revealed that FF are the main site of collagen biosynthesis and that the adjacent alveoli are abnormal. This essential information will inform future mechanistic studies on fibrosis progression.