Biology subjects

Dodd, R. J.

Publications and source records attributed to Dodd, R. J..

4 recordsLinked to original sources

bioRxiv2025

The hyaluronan receptor CD44 drives COVID-19 severity through its regulation of neutrophil migration

The novel respiratory disease COVID-19 caused by the coronavirus SARS-CoV-2 continues to be a public health emergency worldwide, and there is a need for more effective therapy for patients. The relationship between the extracellular matrix and the host immune response to infection is severely understudied. Deposition of the polysaccharide hyaluronan (HA) into the lungs is associated with more severe COVID-19 disease outcomes. HA is a major component of the extracellular matrix in connective tissues and is abundant in many parts of the body, including cartilage, skin, brain, and vitreous body. CD44 is the primary receptor for HA and is found on almost all immune cells in the lung. Known functions of CD44 include mediation of immune cell migration, activation, and differentiation. We hypothesized that increased HA deposition during COVID-19 increases CD44-mediated immune cell infiltration into lungs and results in more severe pathology. Here, we report that in mice infected with a mouse-adapted strain of SARS-CoV-2, treatment with a combination of two anti-CD44 monoclonal antibodies confers a significant survival benefit and reduces weight loss and clinical score of the mice on Day 4 post infection. We show that anti-CD44 treatment decreases many key cytokines and chemokines in the bronchoalveolar lavage fluid on Day 4. With flow cytometry, we show that anti-CD44 reduces the numbers of neutrophils in infected lungs. We also show through immunofluorescence that treatment with anti-CD44 antibodies reduces colocalization of HA and CD45 in lung sections, indicating that HAs interaction with immune cells contributes to pathology. Our findings demonstrate that disruption of HA-receptor interactions is a way to prevent inflammatory pathology in pulmonary infection.

immunology↗

bioRxiv2024

Extracellular matrix phenotyping by imaging mass cytometry defines distinct cellular matrix environments associated with allergic airway inflammation.

The extracellular matrix (ECM) forms the scaffold in which cells reside and interact. The composition of this scaffold guides the development of local immune responses and tissue function. With the advent of multiplexed spatial imaging methodologies, investigating the intricacies of cellular spatial organisation are more accessible than ever. However, the relationship between cellular organisation and ECM composition has been broadly overlooked. Using imaging mass cytometry, we investigated the association between cellular niches and their surrounding matrix environment during allergic airway inflammation in two commonly used mouse strains. By first classifying cells according to their canonical intracellular markers and then by developing a novel analysis pipeline to independently characterise a cells ECM environment, we integrated analysis of both intracellular and extracellular data. Applying this methodology to three distinct tissue regions we reveal disparate and restricted responses. Recruited neutrophils were dispersed within the alveolar parenchyma, alongside a loss of alveolar type I cells and an expansion of alveolar type II cells. This activated parenchyma was associated with increased proximity to hyaluronan and chondroitin sulphate. In contrast, infiltrating CD11b+ and MHCII+ cells accumulated in the adventitial cuff and aligned with an expansion of the subepithelial layer. This expanded subepithelial region was enriched for closely interacting stromal and CD11b+ immune cells which overlaid regions enriched for type-I and type-III collagen. The cell-cell and cell-matrix interactions identified here will provide a greater understanding of the mechanisms and regulation of allergic disease progression across different inbred mouse strains and provide specific pathways to target aspects of remodelling during allergic pathology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/623782v2_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@dcaf56org.highwire.dtl.DTLVardef@7b5d7forg.highwire.dtl.DTLVardef@1376d4eorg.highwire.dtl.DTLVardef@1e95801_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

The structural organisation of pentraxin-3 and its interactions with heavy chains of inter-alpha-inhibitor regulate crosslinking of the hyaluronan matrix

Pentraxin-3 (PTX3) is an octameric protein, comprised of eight identical protomers, that has diverse functions in reproductive biology, innate immunity and cancer. PTX3 interacts with the large polysaccharide hyaluronan (HA) to which heavy chains (HCs) of the inter--inhibitor (II) family of proteoglycans are covalently attached, playing a key role in the (non-covalent) crosslinking of HC*HA complexes. These interactions stabilise the cumulus matrix, essential for ovulation and fertilisation in mammals, and are also implicated in the formation of pathogenic matrices in the context of viral lung infections. To better understand the physiological and pathological roles of PTX3 we have analysed how its quaternary structure underpins HA crosslinking via its interactions with HCs. A combination of X-ray crystallography, cryo-electron microscopy (cryo-EM) and AlphaFold predictive modelling revealed that the C-terminal pentraxin domains of the PTX3 octamer are arranged in a central cube, with two long extensions on either side, each formed from four protomers assembled into tetrameric coiled-coil regions, essentially as described by (Noone et al., 2022; doi:10.1073/pnas.2208144119). From crystallography and cryo-EM data, we identified a network of inter-protomer salt bridges that facilitate the assembly of the octamer. Small angle X-ray scattering (SAXS) validated our model for the octameric protein, including the analysis of two PTX3 constructs: a tetrameric Half-PTX3 and a construct missing the 24 N-terminal residues ({Delta}1-24-PTX3). SAXS determined a length of [~]520 [A] for PTX3 and, combined with 3D variability analysis of cryo-EM data, defined the flexibility of the N-terminal extensions. Biophysical analyses revealed that the prototypical heavy chain HC1 does not interact with PTX3 at pH 7.4, consistent with our previous studies showing that, at this pH, PTX3 only associates with HC*HA complexes if they are formed in its presence. However, PTX3 binds to HC1 at acidic pH, and can also be incorporated into pre-formed HC*HA complexes under these conditions. This provides a novel mechanism for the regulation of PTX3-mediated HA crosslinking (e.g., during inflammation), likely mediated by a pH-dependent conformational change in HC1. The PTX3 octamer was found to associate simultaneously with up to eight HC1 molecules and, thus, has the potential to form a major crosslinking node within HC*HA matrices, i.e., where the physical and biochemical properties of resulting matrices could be tuned by the HC/PTX3 composition.

biochemistry↗

bioRxiv2024

Chemical modification of hyaluronan oligosaccharides differentially modulates hyaluronan- hyaladherin interactions.

The glycosaminoglycan hyaluronan (HA) is a ubiquitous, non-sulphated polysaccharide with diverse biological roles mediated through its interactions with HA-binding proteins (HABPs). Most HABPs belong to the Link module superfamily, including the major HA receptor, CD44, and secreted protein TSG-6, which catalyzes the covalent transfer of Heavy Chains (HC) from inter-a-inhibitor (IaI) onto HA. The structures of the HA-binding domains (HABD) of CD44 (HABD_CD44) and TSG-6 (Link_TSG6) have been determined and their interactions with HA extensively characterized. The mechanisms of binding are different, with Link_TSG6 interacting with HA primarily via ionic and CH-{pi} interactions, whereas HABD_CD44 binds solely via hydrogen bonds and van der Waals forces. Here we exploit these differences to generate HA oligosaccharides, chemically modified at their reducing ends, that bind specifically and differentially to these target HABPs. Hexasaccharides (HA6AN) modified with 2- or 3-aminobenzoic acid or 2-amino-4-methoxybenzoic acid (HA6-2AA, HA6-3AA, HA6-2A4MBA, respectively) had increased affinities for Link_TSG6 compared to unmodified HA6AN. These modifications did not increase the affinity for CD44_HABD. A model of HA6-2AA (derived from the solution dynamic 3D structure of HA4-2AA) was docked into the Link_TSG6 structure, providing evidence that the 2AA-carboxyl forms a salt bridge with Arginine-81. These modeling results informed a 2nd series of chemical modifications for HA oligosaccharides, which again showed differential binding to the two proteins. Several modifications to HA4 and HA6 were found to convert the oligosaccharide into substrates for HC-transfer, whereas unmodified HA4 and HA6 are not. This study has generated valuable research tools to further understand HA biology.

biochemistry↗