Biology subjects

Balic, A.

Publications and source records attributed to Balic, A..

7 recordsLinked to original sources

bioRxiv2026

CSF1R-dependent macrophages control B cell development and function in the chicken immune system.

Acquired immunity in mammals depends upon capture and presentation of antigens by specialised macrophage populations in splenic marginal zone and lymph node sinuses and follicular dendritic cells (FDC) within germinal centres. Cells referred to as FDC in chickens express CSF1R, the receptor for macrophage colony-stimulating factor (CSF1) and IL34. We utilised single cell RNA-seq on CSF1R+ cells from chicken spleen to identify monocytes and two distinct populations of macrophages. TIMD4/C1Q/MAFB+ macrophages were enriched for expression of genes involved in iron metabolism. A MARCO/VSIG4+ population expressed SPIC, a transcription factor associated with red pulp macrophages in mammals but also expressed receptors (CR2) and trophic factors (TNFSF13, CXCL13) associated with mammalian FDC. SPIC+ cells were located within follicles in spleen, caecal tonsil and bursa. We generated a CSF1R knockout in the chicken germ line. Mutant birds lack macrophages in the embryo. They were indistinguishable from wild type at hatch and behaved and fed normally but from day 5-6 post hatch they failed to thrive. Loss of CSF1R function in hatchlings led to monocytopenia and granulocytosis and the loss of macrophage subpopulations in lymphoid organs. Consistent with their expression of B cell trophic factors, the loss of follicular macrophages in the bursa was associated with involution and severe B cell deficiency in the circulation and spleen. In summary, lymphoid tissues of chickens contain specialised macrophage populations with distinct expression profiles. The details of regulation by CSF1R, specialised functions and underlying transcriptional regulation are quite different between birds and mammals.

immunology↗

bioRxiv2024

A single-nucleus census of immune and non-immune cell types for the major immune organ systems of chicken.

In the avian host, comprehensively cataloging immune cell types, their transcriptome profiles, and varying molecular responses to pathogen challenges are necessary steps toward a better understanding of the interplay between genetics and disease resilience. We present a first nuclei atlas of immune cell types derived from the three main immune organs of layer chickens, including spleen, bursa, and thymus. In bursa we also present, an accounting of cell type activation with the bacterial toxin lipopolysaccharide (LPS). Our analysis includes 36,370 total nuclei and 16, 12, and 12 transcriptionally distinct clusters for spleen, bursa, and thymus, respectively. We discover nuclei molecular profiles that uniquely distinguish states of the transcriptome within cell type that could serve as new means to characterize avian immune subtypes. We further subcluster refined immune cell type classifications, specifically highlighting the transcriptomic diversity of B and T cell subtypes. In the bursa, inferred intercellular communication and signaling pathway enrichment analyses across immune and non-immune cell types demonstrate the unappreciated complexity of the B cell repertoire in a model mimicking systemic bacterial infection. This census of all cell types in both primary and one major secondary avian immune organ system, although preliminary, provides a first review of how nuclei transcribe numerous genes, known and unknown, a critical prerequisite for the study avian immunogenetics by cell type.

immunology↗

bioRxiv2024

Trigeminal ganglion and tooth innervation modifications following genetic and pharmacological Nogo-A inhibition

Nogo-A is a major regulator of neural development and regeneration, but its role in tooth innervation remains largely unknown. Neurons from trigeminal ganglia support teeth homeostasis and regeneration, and disorders of their function could have significant pathophysiological consequences. In this study, we show that Nogo-A is expressed in the trigeminal ganglia and in the neurons innervating the teeth, and that its deletion affects both the number and patterning of neurons in teeth. In organotypic cultures, Nogo-A blocking antibodies affect the trigeminal ganglia-derived neuronal outgrowths and allow premature innervation of tooth germs. RNA sequencing analysis revealed that Nogo-A deletion induces alterations linked to functions at synapses and interference with neurotrophin signalling during the differentiation and maturation of trigeminal neurons. Taken together, these results reveal for the first time the importance of Nogo-A as a major regulator of tooth innervation and point to its potential as a clinical therapeutic target.

neuroscience↗

bioRxiv2024

Infectious bronchitis virus vaccination, but not the presence of XCR1, is correlated with large differences in chicken caecal microbiota

The chicken immune system and microbiota play vital roles in maintaining gut homeostasis and protecting against pathogens. In mammals, XCR1+ conventional dendritic cells (cDCs) are located in the gut-draining lymph nodes and play a major role in gut homeostasis. These cDCs sample antigens in the gut luminal contents and limit the inflammatory response to gut commensal microbes by generating appropriate regulatory and effector T-cell responses. We hypothesised that these cells play similar roles in sustaining gut homeostasis in chickens, and that chickens lacking XCR1 were likely to contain a dysbiotic caecal microbiota. Here we compare the caecal microbiota of chickens that were either heterozygous or homozygous XCR1 knockouts, that had or had not been vaccinated for infectious bronchitis virus (IBV). We used short-read (Illumina) and long-read (PacBio HiFi) metagenomic sequencing to reconstruct 670 high-quality, strain-level metagenome assembled genomes. We found no significant differences between alpha-diversity or the abundance of specific microbial taxa between genotypes. However, IBV vaccination was found to correlate with significant differences in the richness and beta-diversity of the microbiota, and to the abundance of forty bacterial genera. In conclusion, we found that a lack of XCR1 was not correlated with significant changes in the chicken microbiota, but IBV vaccination was. Data SummaryThe raw sequencing data for this project, as well as primary assemblies, putative genome bins and species-level MAGs, are available in the European Nucleotide Archive under project PRJEB64517. Strain-level MAGs are available through Edinburgh DataShare (https://doi.org/10.7488/ds/7678). Impact statementChickens play a vital role in global food systems, with 74 billion chickens killed for meat and 1.6 trillion chicken eggs produced in 2021 alone. The gut microbiota plays a vital role in the health and nutrition of the chicken, contributing to gut homeostasis and the production of nutrients that can be absorbed and used by the host bird. The chicken gut microbiota represents an excellent target to improve pathogen resistance and nutrition, as farmed chickens are not exposed to a maternal hen and thereby develop a low-diversity gut microbiota that negatively impacts their immune development and gut health. In order to develop such microbiota interventions, we first need to understand the fundamental biology behind immune-microbiota interactions. While it is well known that the chicken gut microbiota plays an important role in gut homeostasis, the mechanisms behind this phenomenon are poorly understood. In mammals, XCR1+ conventional dendritic cells have been shown to play a role in maintaining gut homeostasis. In this study we compare the gut microbiota of heterozygous and homozygous XCR1 knockout chickens that did or did not receive infectious bronchitis virus vaccination. We found that the gut microbiota of heterozygous and homozygous XCR1 knockout chickens did not differ significantly, but IBV vaccination did significantly correlate with differences in the microbiota composition.

microbiology↗

bioRxiv2023

Single-cell transcriptomics analysis reveals extracellular matrix remodelling in carious human dental pulp

The carious lesion is a bacteria caused destruction of tooth mineralized matrices marked by concurrent tissue reparative and immune responses in the dental pulp. While major molecular players in tooth pulp decay have been uncovered, a detailed map of the molecular and cellular landscape of the diseased pulp is still missing. Here we used single-cell RNA sequencing analysis, to generate a comprehensive single-cell atlas of the carious human dental pulp tissue. Our data demonstrated modifications in various cell clusters of the carious pulp, such as immune cells, mesenchymal stem cells (MSC) and fibroblasts, when compared to the healthy dental pulp. These changes include upregulation of genes encoding extracellular matrix (ECM) components and the enrichment of the fibroblast cluster with myofibroblasts. Assessment of the Fibronectin fibres mechanical strain showed a significant tension reduction in the carious human pulp, compared to the healthy one. Collectively, the present data demonstrate molecular, cellular and biomechanical alterations in the carious pulp tissue, indicative of extensive ECM remodelling and reminiscent of fibrosis observed in other organs.

cell biology↗

bioRxiv2022

Nogo-A regulates the fate of human dental pulp stem cells towards osteogenic, adipogenic, and neurogenic differentiation

Human teeth are highly innervated organs that contain a variety of mesenchymal stem cell populations that could be used for cell-based regenerative therapies. Specific molecules are often used in these treatments to favorably modulate stem cells function and fate. Nogo-A, a key regulator of neuronal growth and differentiation, is already used in clinical tissue regeneration trials. While the functions of Nogo-A in neuronal tissues are extensively explored, its role in teeth still remains unknown. In this work, we first immunohistochemically analyzed the distribution of Nogo-A protein in the dental pulp of human teeth. Nogo-A is localized in a variety of cellular and structural components of the dental pulp, including odontoblasts, fibroblasts, neurons and vessels. We also cross-examined Nogo expression in the various pulp cell clusters in a single cell RNA sequencing dataset of human dental pulp, which showed high levels of expression in all cell clusters, including that of stem cells. We then assessed the role of Nogo-A on the fate of human dental pulp stem cells and their differentiation capacity in vitro. Using immunostaining, Alizarin Red S and Oil Red O staining we showed that Nogo-A delayed the differentiation of cultured dental pulp stem cells towards the osteogenic, adipogenic and neurogenic lineages, while addition of the blocking anti-Nogo-A antibody had opposite effects. These results were further confirmed by qRT-PCR, which demonstrated overexpression of genes involved in osteogenic (RUNX2, ALP, SP7/OSX), adipogenic (PPAR-{gamma}2, LPL) and neurogenic (DCX, TUBB3, NEFL) differentiation in presence of the anti-Nogo-A antibody. Conversely, the osteogenic and adipogenic genes were downregulated by Nogo-A. Taken together, our results show that the functions of Nogo-A are not restricted to neuronal cells, but are extended to other cell populations, including dental pulp stem cells. We show that Nogo-A regulates their fates towards osteogenic, adipogenic and neurogenic differentiation, thus indicating its potential use in the clinics.

cell biology↗

bioRxiv2018

Characterisation of subpopulations of chicken mononuclear phagocytes that express TIM4 and the macrophage colony-stimulating factor receptor (CSF1R)

The phosphatidylserine receptor, TIM4, encoded by TIMD4, mediates the phagocytic uptake of apoptotic cells. We applied anti-chicken TIM4 monoclonal antibodies, in combination with CSF1R reporter transgenes to dissect the function of TIM4 in chick (Gallus gallus). During development in ovo, TIM4 was present on the large majority of macrophages but expression became more heterogeneous post-hatch. Blood monocytes expressed KUL01, class II MHC and CSF1R-mApple uniformly. Around 50% of monocytes were positive for surface TIM4. They also expressed many other monocyte-specific transcripts at a higher level than TIM4- monocytes. In liver, highly-phagocytic TIM4hi cells shared many transcripts with mammalian Kupffer cells and were associated with uptake of apoptotic cells. Although they expressed CSF1R mRNA, Kupffer cells did not express the CSF1R-mApple transgene, suggesting that additional CSF1R transcriptional regulatory elements are required by these cells. By contrast, CSF1R-mApple was detected in liver TIM4lo and TIM4- cells which were not phagocytic and were more abundant than Kupffer cells. These cells expressed CSF1R, alongside high levels of FLT3, MHCII, XCR1 and other markers associated with conventional dendritic cells (cDC) in mice. In bursa, TIM4 was present on the cell surface of two populations. Like Kupffer cells, bursal TIM4hi phagocytes co-expressed many receptors involved in apoptotic cell recognition. TIM4lo cells appear to be a sub-population of bursal B cells. In overview, TIM4 is associated with phagocytes that eliminate apoptotic cells in the chick. In the liver, TIM4 and CSF1R reporters distinguished Kupffer cells from an abundant population of DC-like cells.

immunology↗