Biology subjects

Butcher, S. K.

Publications and source records attributed to Butcher, S. K..

3 recordsLinked to original sources

bioRxiv2026

Alternative promoters used during myeloid differentiation and upon activation change the gene products available for innate immune programs.

Macrophages are innate immune cells present in most tissues of the body, whose molecular programs are determined by their ontogeny and environment. From the earliest stages of embryonic development, macrophages are recruited into developing tissues where they support organogenesis with trophic factors such as WNT, VEGF and PDGF. While macrophage subsets have been described in different tissues at single cell resolution, little is known about transcript isoforms and proteoforms that underpin their differentiation and function. Here we assessed enhancer, promoter, transcript and proteomic variation as pluripotent stem cells differentiate to macrophages, identifying over 200 previously uncharacterised genes and over 20,000 new mRNA isoforms, updating our current understanding of the human genome, its regulation and potential output. Newly discovered myeloid-expressed transcripts and proteins were enriched for motifs associated with secreted proteins, and these included previously uncharacterised isoforms of growth factors, in which we predict N-terminal changes impact on their location and function. Activation of primary adult monocytes and monocyte-derived macrophages was also characterised by the expression of diverse transcript isoforms, largely arising from alternate transcription initiation sites and predicted to impact on the acute response to bacterial or fungal stimuli. Understanding the full spectrum of gene products expressed by these cells further extends our understanding of the phenotypic plasticity and trophic potential of macrophages in human development and may lead to the discovery of new clinical targets for tissue engineering or immune-related studies. Graphical AbstractIn this manuscript, Berrocal-Rubio and colleagues examined the differentiation of human macrophages using an iPSC model of tissue macrophage biology. Combining long-read sequencing technology with promoter profiling identified over 17, 700 genes implicated in pluripotency-myeloid specification. 7% of transcripts profiled from previously characterised genes were predicted to encode new proteins, and a further 3% of transcripts were derived from genes newly discovered in this project. They confirmed that a high proportion of these alternate transcripts were detectable in primary monocytes but also discovered that activation of primary monocytes led to further alternate promoter usage, with the potential to further diversify the innate immune responses to a broad set of pathogens. The newly described macrophage genes and transcripts encoded proteins enriched for motifs associated with secreted peptides. These data suggest that alternate transcription of macrophage genes leads to new effectors of innate immune function, that include a substantially expanded number of growth factors or secreted proteins. Created in BioRender. Berrocal, M. (2025) https://BioRender.com/d0n47le O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/703142v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@454cf0org.highwire.dtl.DTLVardef@1be28e8org.highwire.dtl.DTLVardef@16fb2e8org.highwire.dtl.DTLVardef@4aca30_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2024

Efficient generation of human dendritic cells from iPSC by introducing a feeder-free expansion step for hematopoietic progenitors

Dendritic cells (DCs) are rare innate immune cells that are essential regulators of anti-tumour, anti-viral and vaccine responses by the adaptive immune system. Conventional dendritic cells, particularly the cDC1 subset, are most desired for DC-based immunotherapies, however, it can be difficult to isolate sufficient numbers of primary cells from patients. The most common alternate sources of DC are ex vivo, such as monocyte-derived or DC expanded from cord blood hematopoietic progenitors. Induced pluripotent stem cells (iPSC) offer a promising solution, providing an opportunity for in vitro generating DCs that are suitable for patient-derived or off-the-shelf batch-manufactured cells. Here, we developed an in vitro protocol designed to maximise the yield of iPSC-derived DC progenitors, with the specific goal of generating DC1-like cells. The iPSC-DCs subsets generated by our method could be partitioned by cell surface phenotypes of cDC1, cDC2 and DC3, but they were most transcriptionally similar to monocyte-derived DC (MoDC). Stimulated iPSC-DCs generated pro-inflammatory cytokines, expressed migratory chemokine receptors including CCR7 which indicates capacity to traffic through lymphatic endothelium, and upregulated co-stimulatory molecules, indicating their potential for productive interactions with T-cells. This method offers a promising step towards an expandable source of allogeneic human dendritic cells for future applications.

cell biology↗

bioRxiv2018

Endotoxin Tolerance Induced by Different TLR Ligands

Endotoxin tolerance is a long-recognised property of macrophages that leads to an altered response to repeated or chronic exposure to Toll-like receptor (TLR) ligands. The physiological role of tolerance is to limit the potential damage to host tissue that may otherwise result from prolonged production of pro-inflammatory cytokines. Endotoxin tolerance is induced by all TLRs tested to date, however, tolerance induced by the TLR4 ligand lipopolysaccharide (LPS) is by far the best studied. LPS tolerance involves a global transcriptional shift from a pro-inflammatory response toward one characterised by the expression of anti-inflammatory and pro-resolution factors. Although largely reversible, LPS-tolerance leads to a hybrid macrophage activation state that is pro-inflammatory in nature but possesses distinct regulatory anti-inflammatory features. Remarkably, a comparative transcriptomic analysis of tolerance induced by different TLR ligands has not previously been reported. Here we describe the transcriptomic profiles of mouse macrophages tolerised with ligands for TLR2, TLR3, TLR4 and TLR 9. While we identified TLR-specific transcriptional profiles in macrophages tolerised with each ligand, tolerance induced by TLR4 was the most comprehensive state, such that each gene tolerised by any of the TLRs tested was also found to be tolerised by TLR4. Pro-inflammatory cytokines are not universally supressed in all tolerant cells but distinct patterns of cytokine expression distinguished TLR-specific tolerance. Analysis of gene regulatory regions revealed specific DNA sequence motifs associated with distinct states of TLR tolerance, implicating previously identified as well as novel transcriptional regulators of tolerance in macrophages. These data provide a basis for the future exploitation of TLR-specific tolerant states to achieve therapeutic re-programming of the innate immune response.

immunology↗