Biology subjects

Locksley, R. M.

Publications and source records attributed to Locksley, R. M..

15 recordsLinked to original sources

bioRxiv2026

RasGRP1 promotes efficient intestinal tuft cell differentiation

Intestinal stem and progenitor cells balance proliferation and lineage commitment to maintain epithelial homeostasis and adapt to environmental challenges. Tuft cells are a rare chemosensory lineage that rapidly expands in response to the cytokine IL-13 during helminth infection, yet how epithelial progenitor states influence this response remains incompletely understood. Using genetic mouse models, intestinal organoids, and single-cell approaches, we identify the Ras guanine nucleotide exchange factor RasGRP1 as an epithelial-intrinsic promoter of efficient tuft cell differentiation. Rasgrp1 is expressed in rare crypt epithelial cells distinct from Lgr5-positive stem cells. Once cells entered the tuft cell differentiation trajectory, Rasgrp1 deficiency has little impact on tuft cell maturation, including expression of the Pou2f3 tuft cell transcription factor. Instead, RasGRP1 acts at the progenitor stage, where it restrains EGFR-driven proliferation and supports a transcriptionally heterogeneous transit-amplifying state, including a subset of cells expressing Pou2f3, that is largely lost upon Rasgrp1 deficiency. Consistent with this model, EGFR inhibition restores tuft cell numbers in Rasgrp1-deficient organoids and cooperates with IL-13 to enhance tuft cell differentiation. Rasgrp1-deficient mice exhibit delayed tuft cell hyperplasia and inefficient helminth clearance. Conversely, constitutive epithelial RasGRP1 overexpression, uncoupled from its endogenous regulation, drives spontaneous tuft cell hyperplasia and accelerates parasite clearance, but alters the balance of intestinal epithelial lineages. Together, our findings identify RasGRP1 as a cell-intrinsic regulator that restrains progenitor proliferation and promotes responsiveness to IL-13, thereby coupling growth factor signaling, cell cycle control, and immune-driven epithelial lineage specification.

cell biology↗

bioRxiv2026

Mite-induced migratory skin-like CD103+ ILC2s establish lung residency and impact immunity

Tissue-resident immune cells are critical for lung homeostasis and protection against inflammation. Here, we describe a novel population of migratory inflammatory CD103+ skin ILC2s (skILC2s) that accumulate in the lung vasculature following transient infestation with ubiquitous Demodex mites, hair follicle commensals that drive local skILC2 proliferation and entry into blood. With time, ex-skILC2s become tissue resident in the lung, occupy previously described immune cell adventitial niches and respond earlier to lung perturbations than endogenous lung ST2+ ILC2s to alter the trajectories of a subsequent immune response. Thus, like transiting ex-gut ILC2s described after small intestinal infection by helminths and protists, ex-skILC2s migrate post-birth in response to pathobionts to change the inflammatory milieu of the lung, revealing a common paradigm by which internal mucosal responses become shaped by ILC2s from barrier epithelia colonized post-birth. HighlightsO_LIDemodex mites infestation drives local expansion of CD103+ skin ILC2s that egress and enter the circulation C_LIO_LIMigratory CD103+ skin-like ILC2s infiltrate into lung, adapt to the local environment and establish long-term residency C_LIO_LIPrior Demodex mite exposure re-organizes the lung ILC2 landscape C_LIO_LIThe distinct cytokine and receptor repertoire of ex-skILC2 alters lung immunity C_LI

immunology↗

bioRxiv2026

Circulating inflammatory ILC2s as a biomarker of gastric cancer progression

Gastric cancer is frequently diagnosed at an advanced stage, contributing to poor patient outcomes and highlighting the need for blood-based biomarkers that can detect disease earlier and track progression. We previously showed that a tuft cell-IL25-type 2 innate lymphoid cell (ILC2) circuit promotes gastric metaplasia and gastric tumour development, but whether this pathway gives rise to a circulating ILC2 population remained unknown. Here, we show that IL25-responsive inflammatory ILC2s (iILC2s) emerge in the blood across gastric disease progression and are selectively associated with gastric tumour contexts. Using multiple mouse models spanning SPEM, early gastric adenoma, and advanced gastric cancer, we found that circulating ILC2s increased progressively with disease severity, with the dominant increase occurring in the IL17RB+ iILC2 subset. This response was not observed in pancreatic or ovarian tumour models, indicating that circulating iILC2 mobilisation is not a general consequence of tumour burden. Instead, mobilisation was linked to gastric disease and accompanied by increased ILC2 accumulation in distal mucosal tissues. Genetic tuft cell ablation or therapeutic blockade of IL25 or IL13 significantly reduced circulating iILC2s, demonstrating that this response depends on the tuft cell-IL25 circuit. In vivo, mobilised iILC2s were associated with enhanced growth of distal tumour implants, supporting a functional contribution beyond the primary gastric site. Translationally, circulating ILC2s were elevated in patients with gastric inflammatory disease and gastric cancer, with the IL25-responsive subset predominating. Circulating ILC2s also showed strong discriminatory power between gastric cancer patients and healthy controls in ROC analysis, supporting their potential utility as a blood-based biomarker. Together, these findings identify circulating iILC2s as a systemic hallmark of gastric tuft cell-ILC2 signalling and establish their mechanistic and translational relevance in gastric cancer.

cancer biology↗

bioRxiv2025

IL-25-induced memory ILC2s mediate long-term small intestinal adaptation

The adaptation of intestinal helminths to vertebrates evolved strategies to attenuate host tissue damage to support reproductive needs of parasites necessary to disseminate offspring to the environment. Helminths initiate the IL-25-mediated tuft cell-ILC2 circuit that enhances barrier protection of the host although viable parasites can target and limit the pathway. We used IL-25 to create small intestinal adaptation marked by anatomic, cell compositional and immunologic changes that persisted months after induction. Small intestinal adaptation was associated with heightened resistance to barrier pathogens, including in the lung, and sustained by transcriptionally and epigenetically modified, tissue-resident, memory-effector ILC2s distinct from those described by innate training; epithelial stem cells remained unaltered. Despite requiring IL-25 for induction, memory ILC2s maintained an activated state in the absence of multiple alarmins and supported mucosal resilience while avoiding adverse sensitization to chronic inflammation, revealing a pathway for deploying innate immune cells to coordinate a distributed mucosal defense.

immunology↗

bioRxiv2024

Telocytes link epithelial nutrient sensing with amplification of the ILC2-tuft cell circuit

Group 2 innate lymphocytes (ILC2s) are prevalent in small intestine but engagement of type 2 immunity during basal processes are incompletely described. Thymic stromal lymphopoietin (TSLP), a cytokine implicated in ILC2 activation, was constitutively expressed in villus telocytes and crypt-associated trophocytes, specialized fibroblasts that sustain epithelial identity. Feeding increased TSLP and induced ILC2 type 2 cytokines that were attenuated by deletion of TSLP in PDGFR+ stromal cells or TSLP receptor on ILC2s. Mouse and human telocytes expressed receptors for glucagon-like peptide-2 (GLP-2), which is released by enteroendocrine cells (EECs) after eating. GLP-2 induced intestinal TSLP, TSLP-dependent ILC2 cytokine production, and tuft cell hyperplasia. The telocyte-alarmin relay couples EEC nutrient detection with amplification of a tuft cell chemosensory circuit that diversifies surveillance of ingested cargo. One-Sentence SummaryIntestinal telocyte TSLP relays signals from enteroendocrine cells to ILC2s to amplify the tuft cell circuit in response to feeding.

immunology↗

bioRxiv2023

Epithelial zonation along the mouse and human small intestine defines five discrete metabolic domains

A key aspect of nutrient absorption is the exquisite division of labor across the length of the small intestine, with individual classes of micronutrients taken up at different positions. For millennia, the small intestine was thought to comprise three segments with indefinite borders: the duodenum, jejunum, and ileum. By examining fine-scale longitudinal segmentation of the mouse and human small intestines, we identified transcriptional signatures and upstream regulatory factors that define five domains of nutrient absorption, distinct from the three traditional sections. Spatially restricted expression programs were most prominent in nutrient-absorbing enterocytes but initially arose in intestinal stem cells residing in three regional populations. While a core signature was maintained across mice and humans with different diets and environments, domain properties were influenced by dietary changes. We established the functions of Ppar- and Cdx1 in patterning lipid metabolism in distal domains and generated a predictive model of additional transcription factors that direct domain identity. Molecular domain identity can be detected with machine learning, representing the first systematic method to computationally identify specific intestinal regions in mice. These findings provide a foundational framework for the identity and control of longitudinal zonation of absorption along the proximal:distal small intestinal axis.

genomics↗

bioRxiv2023

Structural characterization of ligand binding and pH-specific enzymatic activity of mouse Acidic Mammalian Chitinase

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.

biochemistry↗

bioRxiv2023

Group 2 innate lymphoid cells constrain type 3/17 lymphocytes in shared stromal niches to restrict liver fibrosis

Group 2 innate lymphoid cells (ILC2s) cooperate with adaptive Th2 cells as key organizers of tissue type 2 immune responses, while a spectrum of innate and adaptive lymphocytes coordinate early type 3/17 immunity. Both type 2 and type 3/17 lymphocyte associated cytokines are linked to tissue fibrosis, but how their dynamic and spatial topographies may direct beneficial or pathologic organ remodelling is unclear. Here we used volumetric imaging in models of liver fibrosis, finding accumulation of periportal and fibrotic tract IL-5+ lymphocytes, predominantly ILC2s, in close proximity to expanded type 3/17 lymphocytes and IL-33high niche fibroblasts. Ablation of IL-5+ lymphocytes worsened carbon tetrachloride-and bile duct ligation-induced liver fibrosis with increased niche IL-17A+ type 3/17 lymphocytes, predominantly {gamma}{delta} T cells. In contrast, concurrent ablation of IL-5+ and IL-17A+ lymphocytes reduced this progressive liver fibrosis, suggesting a cross-regulation of type 2 and type 3 lymphocytes at specialized fibroblast niches that tunes hepatic fibrosis.

immunology↗

bioRxiv2022

IL-13-programmed airway tuft cells produce PGE2, which promotes CFTR-dependent mucociliary function

Chronic type 2 (T2) inflammatory diseases of the respiratory tract are characterized by mucus overproduction and disordered mucociliary function, which are largely attributed to the effects of IL-13 on common epithelial cell types (mucus secretory and ciliated cells). The role of rare cells in airway T2 inflammation is less clear, though tuft cells have been shown to be critical in the initiation of T2 immunity in the intestine. Using bulk and single cell RNA sequencing of airway epithelium and mouse modeling, we find that IL-13 expands and programs airway tuft cells towards eicosanoid metabolism, and that tuft cell deficiency leads to a reduction in airway prostaglandin E2 (PGE2) concentration. Allergic airway epithelia bear a signature of prostaglandin E2 activation, and PGE2 activation leads to CFTR-dependent ion and fluid secretion and accelerated mucociliary transport. Together these data reveal a role for tuft cells in regulating epithelial mucociliary function in the allergic airway.

immunology↗

bioRxiv2022

Inhibition of the tuft cell/ILC2 axis reduces gastric tumor development in mice

Although gastric cancer is a leading cause of cancer-related deaths, systemic treatment strategies remain scarce. Here we explore a metabolite-triggered circuit between epithelial tuft cells and innate lymphoid type 2 cells (ILC2) that is evolutionarily optimized for intestinal remodeling in response to helminth infection. We demonstrate that tuft cell-derived interleukin 25 (IL25) acts as an alarmin on ILC2s to induce the release of IL13 as a growth factor for tuft cells, and propose that this model drives early metaplastic remodeling and gastric tumor formation. Genetic ablation of tuft cells, ILC2s or antibody-mediated neutralization of IL13 or IL25 reduces the growth of established tumors. Thus, the tuft cell/ILC2 axis provides an opportunity to therapeutically inhibit preneoplastic lesions and early-stage gastric cancer through repurposing of antibody-mediated therapies. One-Sentence SummaryTuft cells and type 2 innate lymphoid cells offer a new therapeutic target in gastric disease.

cancer biology↗

bioRxiv2021

Alveolar macrophages strictly rely on GM-CSF from alveolar epithelial type 2 cells before and after birth

Programs defining tissue-resident macrophage identity depend on local environmental cues. For alveolar macrophages (AMs), these signals are provided by immune and non-immune cells, and include GM-CSF (CSF2). However, evidence to functionally link components of this intercellular crosstalk remains scarce. We thus developed new transgenic mice to profile pulmonary GM-CSF expression, which we detected in both immune cells, including group 2 innate lymphoid cells and {gamma}{delta} T cells, as well as AT2s. AMs were unaffected by constitutive deletion of hematopoietic Csf2 and basophil depletion. Instead, AT2 lineage-specific constitutive and inducible Csf2 deletion revealed the non-redundant function of AT2-derived GM-CSF in instructing AM fate, establishing the postnatal AM compartment, and maintaining AMs in adult lungs. This AT2-AM relationship begins during embryogenesis, where nascent AT2s timely induce GM-CSF expression to support the proliferation and differentiation of fetal monocytes contemporaneously seeding the tissue, and persists into adulthood, when epithelial GM-CSF remains restricted to AT2s.

immunology↗

bioRxiv2021

Type 2 innate immunity regulates hair follicle homeostasis to control Demodex pathosymbionts

Allergic skin diseases are common, but basal roles for type 2 immunity in cutaneous homeostasis are incompletely understood. Here, we show that skin group 2 innate lymphoid cells (ILC2s) are the predominant resident cells that secretes IL-13, which attenuates epithelial cell proliferation during anagen throughout the hair follicle (HF), including in HF stem cells. Although HF are normal in the absence of type 2 immunity, colonization with the commensal mite, Demodex musculi, results in epithelial proliferation and aberrant hair follicle morphology accompanied by loss of stable commensalism with massive infestation and expansion of inflammatory ILC2s and other immune cells. Topical anti-parasitic agents, but not broad-spectrum antibiotics, reversed the phenotype. Commensal Demodex colonization of mammalian hair follicles is ubiquitous, including in humans, revealing an unanticipated role for ILC2s and type 2 immunity for skin homeostasis in the normal environment. One Sentence SummaryDynamic regulation of skin physiology by Type 2 innate immunity regulates healthy commensalism by parasitic Demodex mites.

immunology↗

bioRxiv2019

Dissecting the chitinolytic activity of mammalian chitinases

Chitin is an abundant polysaccharide used by a large range of organisms for structural rigidity and water repulsion. As such, the insoluble crystalline structure of chitin poses significant challenges for enzymatic degradation. Vertebrates do not produce chitin, but do express chitin degrading enzymes. Acidic mammalian chitinase, the primary enzyme involved in the degradation of environmental chitin in mammalian lungs, is a processive glycosyl hydrolase that may be able to make multiple hydrolysis events for each binding event. Mutations to acidic mammalian chitinase have been associated with asthma, and genetic deletion of the enzyme in mice results in significantly increased morbidity and mortality with age. We initially set out to reverse this phenotype by engineering hyperactive acidic mammalian chitinase variants. Using a directed evolution screening approach using commercial fluorogenic substrates, we identified mutations with consistent increases in activity. To determine whether the activity increases observed with oligomeric substrates were consistent with more biologically relevant chitin substrates, we developed new assays to quantify chitinase activity with colloidal crystalline chitin, and identified a high throughput fluorogenic assay that gives sufficient signal to noise advantages to quantify changes to activity due to the addition or removal of a chitin binding domain to the enzyme. We show that the activity increasing mutations derived from our directed evolution screen were lost when crystalline substrates were used. In contrast, naturally occurring gain-of-function mutations gave similar results with oligomeric and crystalline substrates. We also show that the activity differences between acidic mammalian chitinase and chitotriosidase are reduced in the context of crystalline substrate, suggesting that previously reported activity differences with oligomeric substrates may have been largely driven by differential substrate specificity for the oligomers. These results highlight the need for assays against more physiological substrates when engineering complex metabolic enzymes, and provide a new approach that may be broadly applicable to engineering glycosyl hydrolases.

biochemistry↗

bioRxiv2018

Skin inflammation driven by differentiation of quiescent tissue-resident ILCs into a spectrum of pathogenic effectors

Psoriasis pathology is driven by the type 3 cytokines IL-17 and Il-22, but little is understood about the dynamics that initiate alterations in tissue homeostasis. Here, we use mouse models, single-cell RNA-seq (scRNA-seq), computational inference and cell lineage mapping to show that psoriasis induction reconfigures the functionality of skin-resident ILCs to initiate disease. Tissue-resident ILCs amplified an initial IL-23 trigger and were sufficient, without circulatory ILCs, to drive pathology, indicating that ILC tissue remodeling initiates psoriasis. Skin ILCs expressed type 2 cytokines IL-5 and IL-13 in steady state, but were epigenetically poised to become ILC3-like cells. ScRNA-seq profiles of ILCs from psoriatic and naive skin of wild type (WT) and Rag1-/- mice form a dense continuum, consistent with this model of fluid ILC states. We inferred biological \"topics\" underlying these states and their relative importance in each cell with a generative model of latent Dirichlet allocation, showing that ILCs from untreated skin span a spectrum of states, including a naive/quiescent-like state and one expressing the Cd74 and Il13 but little Il5. Upon disease induction, this spectrum shifts, giving rise to a greater proportion of classical Il5- and Il13- expressing \"ILC2s\" and a new, mixed ILC2/ILC3-like subset, expressing Il13, Il17, and Il22. Using these key topics, we related the cells through transitions, revealing a quiescence-ILC2-ILC3s state trajectory. We demonstrated this plasticity in vivo, combining an IL-5 fate mouse with IL-17A and IL-22 reporters, validating the transition of IL-5-producing ILC2s to IL-22- and IL-17A-producing cells during disease initiation. Thus, steady-state skin ILCs are actively repressed and cued for a plastic, type 2 response, which, upon induction, morphs into a type 3 response that drives psoriasis. This suggests a general model where specific immune activities are primed in healthy tissue, dynamically adapt to provocations, and left unchecked, drive pathological remodeling.

immunology↗

bioRxiv2018

Detection of succinate by intestinal tuft cells triggers a type 2 innate immune circuit

Initiation of immune responses requires innate immune sensing, but immune detection of the helminths, protists, and allergens that stimulate type 2 immunity remains poorly understood. In the small intestine, type 2 immune responses are regulated by a tuft cell-ILC2 signaling circuit. Tuft cells express components of a canonical taste transduction pathway, including the membrane channel TRPM5, but the ligands and receptors that activate tuft cells in the small intestine are unknown. Here we identify succinate as the first ligand that activates intestinal tuft cells to initiate type 2 immune responses. Using mRNA-Seq on tuft cells from different tissues, we show that all tuft cells express the intracellular taste transduction pathway, but expression of upstream receptors is tissue-specific. In the small intestine, tuft cells express the succinate receptor SUCNR1. Remarkably, providing succinate in drinking water is sufficient to induce a multifaceted type 2 immune response in the murine small intestine, involving all known components of the tuft-ILC2 circuit. The helminth Nippostrongylus brasiliensis secretes succinate as a metabolite, and sensing of both succinate and N. brasiliensis requires tuft cells and TRPM5, suggesting a novel paradigm in which type 2 immunity monitors microbial metabolism. Manipulation of succinate sensing may have therapeutic benefit in numerous intestinal diseases.

immunology↗