Biology subjects

Hegarty, L. M.

Publications and source records attributed to Hegarty, L. M..

4 recordsLinked to original sources

bioRxiv2026

Fixation matters: duration in fixative prior to immunofluorescent analysis directly impacts macrophage visualisation in epithelial tissues

Macrophages are now recognised as key players in a range of tissues and biological processes, responding to injury and infection, and facilitating development and regeneration. As the importance of macrophage crosstalk within these processes has been revealed, so too has the significance of studying the spatial positioning of macrophages within the tissue of interest. As such, immunofluorescent microscopy-based analysis is becoming an increasingly attractive technique for immunology research. While tissue fixation preserves the tissue architecture and immobilises target antigens, prolonged fixation can negatively impact protein recognition. We report that prolonged exposure to a paraformaldehyde-based fixative profoundly impacts detection of cell surface markers that define macrophage subsets in the mouse submandibular gland, in contrast to epithelial cell markers which appear more robust. We find that this that this is not exclusive to the salivary gland, and similar effects are seen in the pancreas and kidney. Importantly, a short duration of fixation allowed the detection of macrophage subsets in both mouse and human tissue without compromising the detection of other markers. Adoption of a short fixation approach enables accurate detection of a wide range of cell types in tissues, and facilitates exploration of spatial positioning and cell proximity by immunofluorescent microscopy analysis.

immunology↗

bioRxiv2025

Tissue resident colonic macrophages persist through acute inflammation and adapt to aid tissue repair

Macrophages are crucial for the maintenance of intestinal homeostasis, are considered key proinflammatory effector cells during intestinal inflammation and are implicated in tissue repair following injury or inflammation. Whether these roles are attributed to distinct subsets of macrophages or if macrophages retain a degree of plasticity in the intestine remains poorly understood. Here, through a combination of single cell RNA sequencing, lineage-tracing and immunofluorescence imaging, we define three major subpopulations of murine, colonic macrophages on the basis of CD11c and CD163 expression. These macrophages occupy discrete anatomical niches and display distinct replenishment kinetics. They all accumulate during acute colitis and Cx3cr1-based fate mapping shows that they persist through to inflammation resolution. Moreover, marked transcriptional differences exist between the macrophages present in health and their counterparts in the post-inflammation environment, demonstrating that inflammation leads to transcriptional rewiring of the resident macrophages in a subset-specific manner. Intriguingly, there were minimal transcriptional changes between long-lived macrophages and their recently differentiated counterparts, indicating the environment exerted a greater influence than ontogeny or the time of residency on their functional states in resolution.

immunology↗

bioRxiv2025

DYNAMIC CHANGES TO THE INTESTINAL ENVIRONMENT OCCUR THROUGHOUT RECOVERY FROM EXPERIMENTAL ISCHAEMIC STROKE

Stroke survivors experience a plethora of complications during recovery, including gastrointestinal symptoms. Intestinal dysfunction is reported to occur rapidly following stroke in both humans and animal models and alterations such as reduced barrier integrity, lymphocyte loss, and an altered microbiota have been suggested to contribute to poor neurological outcomes. Despite the persistence of gastrointestinal symptoms in many stroke survivors, how the intestinal environment changes over the course of stroke recovery remains poorly understood. Here, we use an experimental model of ischemic stroke to profile the gastrointestinal tract over a three month period of recovery.We have shown that experimental stroke leads to structural alterations to the colon, impaired transit times and an altered bacterial community composition. No impairments to barrier function were detected at any time point and transit times recover within 2 weeks post stroke. In contrast, structural and bacterial community alterations remain up to 3 months post stroke and are accompanied by abnormalities that develop only during chronic recovery, such as altered antibody coating of bacteria. These results suggest that the gastrointestinal system is dynamically altered over the course of experimental stroke recovery and that certain defects persist chronically after stroke.

immunology↗

bioRxiv2022

CSF1R-dependent macrophages in the salivary gland are essential for epithelial regeneration following radiation-induced injury

The salivary glands often become damaged in individuals receiving radiotherapy for head and neck cancer, resulting in xerostomia, or chronic dry mouth. This leads to detrimental effects on their health and quality of life, for which there is no regenerative therapy. Macrophages are the predominant cell type in the salivary glands and are attractive therapeutic targets due to their unrivalled capacity to drive tissue repair and regeneration. Yet, the nature and role of macrophages in salivary gland homeostasis and whether or not they contribute to tissue repair/regeneration following injury is not well understood. Here, we have used single cell RNA-seq, multi-parameter flow cytometry and fluorescence microscopy to map the heterogeneity of the salivary gland macrophage compartment throughout development and following radiation-induced injury. We show that there are highly dynamic changes in the composition of the salivary gland macrophage compartment with age, in part due to changes in the ontogeny of these cells, determined using a suite of complementary fate mapping systems. A combination of mutant mice and antibody blockade demonstrates that salivary gland macrophages are dependent on CSF1, but not IL-34 or GM-CSF, for their development and maintenance. Finally, using an in vivo model of radiation-induced salivary gland injury combined with a novel Mafb-specific depletion system, we demonstrate an essential role for macrophages. Without macrophages the clearance of cells with DNA damage, and effective tissue repair following such injury, is severely comprised. Our data, therefore, indicate a strong case for exploring the therapeutic potential of manipulating macrophages in order to promote tissue repair and thus minimise salivary gland dysfunction after radiotherapy.

immunology↗