Tara E. Sutherland

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 is 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 in-bred mouse strains. We have integrated both intracellular and extracellular analysis by first classifying cells according to their canonical marker expression and then utilising a novel pipeline to independently characterise their ECM environment. Applying this methodology across three distinct lung tissue regions we reveal region-specific and spatially constrained allergic inflammatory responses. In our model of allergic airway inflammation, 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 specifically in BALB/c mice. This region was around the airway-adjacent pulmonary artery and aligned with an expansion of the subepithelial airway region. 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 detailed map of cell and matrix interactions generated here provides a critical resource for understanding ECM contribution to specific lung niches during allergic disease. It reveals strain-dependent differences in inflammation and remodelling and generates novel hypotheses for targeting aspects of remodelling during allergic airway pathology.

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 HA’s 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.

Chitinase-like proteins (CLPs) are established biomarkers of inflammation and airway remodeling in asthma, yet their direct contribution toward disease pathogenesis is unknown. In a mouse model of type 2/type 17 airway inflammation induced by house dust mite, ragweed, and Aspergillus fumigatus (DRA) allergens, we demonstrate that murine CLPs Ym1 ( Chil3 ) and Ym2 ( Chil4 ) exert distinct and potent effects on airway extracellular matrix (ECM) composition during chronic lung pathology. Using both Ym1-knockout ( Chil3 ^−/− ) and Ym2-knockdown ( Chil4 ^KD/KD ) mice, we found that these CLPs have fundamental roles in airway remodeling that are independent of interleukin-13 (IL-13) and IL-17A signaling pathways. Antibody-mediated inhibition of CLPs after pathology has developed reverses airway remodeling independently of chronic inflammation. However, this inhibition is not sufficient to reduce airway hyperresponsiveness (AHR) in allergic animals. Instead, the absence of CLPs increases epithelial damage and leads to a loss of bronchial epithelial integrity. This work disentangles chronic IL-13 and IL-17A signaling from the development of allergic airway pathology and reveals CLPs as orchestrators of airway remodeling that can produce both protective and adverse outcomes.

Spatial proteomics technologies now profile cells and the extracellular matrix (ECM) together in situ . Yet analysis tools remain cell-centric, despite the ECM playing an essential role in health and disease. Here we present Mantpy, a frame-work that represents the ECM, and its interface with cells, as spatial graphs. Mantpy builds ECM graphs directly from matrix markers and links them with cell graphs for joint cell–ECM analysis, supporting graph statistics, explainable graph deep learning and visualisation. Mantpy extends from a single ECM marker to multiplexed cell–ECM panels. It recovers layered tissue architecture in the intestine from collagen IV alone, quantifies infection-associated ECM remodelling in mouse liver, and relates cells to the surrounding matrix in mouse lung, where cellular context recovers synthetically corrupted matrix labels. Released with ECM-inclusive datasets and interoperating with the scverse ecosystem and pytorch geometric. Mantpy brings the matrix into spatial analysis alongside the cell.

Chitinase-like proteins (CLPs) are of wide interest due to their significant roles during both biological and pathological processes. Human CLPs such as YKL-40 have been suggested as biomarkers of disease severity in many conditions. Murine CLPs include Brp39, Ym1, and Ym2 and these are similarly upregulated in multiple mouse models of pathology. Investigation of these molecules, particularly Ym1 and Ym2, is plagued by complexity in the genomic locus due to recent gene duplication events in the C57BL/6 strain. Using a novel CRISPR-Cas9 targeting approach involving CB6 mixed background embryos, we generated a Ym1 deficient mouse. Validation using flow cytometry, ELISA, and immunofluorescence confirmed no expression of mature Ym1 protein with no alteration in the expression of related chitinases/CLP genes including Chia and Chil4. This new transgenic mouse line will be key for investigating CLP functions and the genetic approach utilised may provide a useful strategy for other genes which show differences between inbred mouse strains.

The intestine is a site of diverse functions including digestion, nutrient absorption, immune surveillance, and microbial symbiosis. Intestinal microRNAs (miRNAs) are detectable in faeces and regulate barrier integrity, host-microbe interactions and the immune response, potentially offering valuable non-invasive tools to study intestinal health. However, current experimental methods are suboptimal and heterogeneity in study design limits the utility of faecal miRNA data. Here, we develop an optimised protocol for faecal miRNA detection and report a reproducible murine faecal miRNA profile in healthy mice. We use this pipeline to study faecal miRNAs during infection with the gastrointestinal helminth, Trichuris muris, revealing roles for miRNAs in fibrosis and wound healing. Intestinal fibrosis was confirmed in vivo using Hyperion® imaging mass cytometry, demonstrating the efficacy of this approach. Further applications of this optimised pipeline to study host-microbe interactions and intestinal disease will enable the generation of hypotheses and therapeutic strategies in diverse contexts.

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

A consistent feature of lung injury is a rapid and sustained accumulation of hyaluronan (HA). The rodent gut‐dwelling nematode Nippostrongylus brasiliensis (Nb) induces tissue damage as it migrates through the lungs. Type 2 immune responses are essential for the repair of the lungs, hence Nb infection is a well‐established model to study immune‐mediated lung repair. We found that Nb infection was associated with increased HA in the lung, which peaked at d7 post‐infection (p.i.). Deposition of HA in the alveolar epithelium correlated with regions of damaged tissue and the type 2 immune response, which is characterized by eosinophilia and increased type 2 cytokines such as IL‐13. Consistent with the accumulation of HA, we observed increased expression of the major synthase Has2, alongside decreased expression of Hyal1, Hyal2, and Tmem2, which can degrade existing HA. Expression of Tsg6 was also increased and correlated with the presence of inter‐α‐inhibitor heavy chain–HA complexes (HC·HA) at d7 p.i. Using IL‐13‐deficient mice, we found that the accumulation of HA during Nb infection was IL‐13 dependent. Our data thus provide further evidence that IL‐13 is a modulator of the HA matrix during lung challenge and links IL‐13‐mediated HA regulation to tissue repair pathways.

Chitinase-like proteins (CLPs) are biomarkers of inflammation and airway remodelling in asthma, yet their direct contribution towards disease pathogenesis is unknown. Using a mouse model of allergen-induced type 2/type 17 airway inflammation we sought to directly investigate the role of the murine CLPs Ym1 and Ym2 during chronic lung pathology. Data demonstrated distinct chronic inflammatory roles for Ym2, IL-13, and IL-17a signalling pathways. Notably, only CLPs were key for initiating the pathogenic accumulation and re-organisation of the pulmonary extracellular matrix (ECM) environment. Furthermore, inhibition of CLPs after chronic pathology developed, reversed airway remodelling independently of chronic inflammation. These studies disentangle chronic IL-13 and IL-17a signalling from the development of allergic airway remodelling and instead highlight a central role for CLPs, which provides new avenues to therapeutically target aberrant ECM accumulation.