Biology subjects

Smyth, D. J.

Publications and source records attributed to Smyth, D. J..

9 recordsLinked to original sources

bioRxiv2026

Evolutionary adaptation of helminth immunomodulators to their hosts

Background: Parasitic nematodes have evolved secreted effector proteins which interact with host molecules to modulate the host immune response. These interactions can be highly specific and are under strong selective pressure. Here, we investigate the host-pathogen interaction between Heligmosomoides bakeri and its natural host Mus musculus, and between H. polygyrus and its natural host Apodemus sylvaticus. We focus on the Alarmin Release Inhibitor family (previously called HpARI) produced by heligmosomid parasites, which interact with IL-33 and heparan sulphate. Results: Using comparative genomics, we identified duplications and diversifications of the ARI family including multiple copies of ARI1 and conserved ARI3 homologues in both H. bakeri and H. polygyrus. We identify a new family member, HpolARI4, encoded by H. polygyrus, as well as a more distant homologue, HmixARI, encoded by Heligmosomum mixtum, a parasite of the bank vole (Myodes glareolus). Our functional assays identify that HpolARI4 preferentially blocks A. sylvaticus IL-33 but has little activity against M. musculus IL-33, whereas HbakARI2 is highly specific for M. musculus IL-33. We furthermore show differential interaction with host heparan sulphate, with HbakARI2 and HpolARI4 showing high affinity binding, whereas HbakARI3 shows no binding. We further test this family against non-host human IL-33 and show that functional suppression can be achieved by HbakARI3 in vitro and in vivo. Conclusion: By integrating genomic data, structural insights, and functional assays, we reveal how the expansion and diversification of the ARI family drive host-specific IL-33 modulation, providing a molecular model for nematode-host co-evolution.

immunology↗

bioRxiv2025

Transcriptomic and proteomic analysis show minimal role formast cell ST2 in primary Heligmosomoides polygyrus bakeriinfection

The IL-33/ST2 pathway is important as part of the type 2 immune response against helminth infections. Mast cells express the highest levels of the IL-33 receptor subunit ST2 of any immune cell, and mast cells can mediate type 2 immune inflammation, however the role of IL-33-driven mast cell responses in helminth infection is poorly understood. We sought to determine the role of mast cell ST2 expression during Heligmosomoides polygyrus bakeri (Hpb) infection by generating mast cell conditional ST2 knockout (MCPT5Cre x ST2f/f, cKO) mice. These mice have normal frequencies of mast cells at steady state, but show specific and strong (albeit incomplete) knockdown of ST2 expression on mast cells. On Hpb infection, faecal egg and adult worm burden were similar between cKO and littermate controls, as were mast cell degranulation markers, serum IgE and goblet cell hyperplasia. Therefore, we conclude that mast cell ST2 does not play a dominant role in Hpb infection. To further investigate the immune response to infection in cKO and littermate controls, transcriptomic and proteomic changes were assessed in duodenal tissues in infected versus naive mice in cKO and control mice. Minimal transcriptomic and proteomic changes were seen between genotypes, whereas substantial changes were seen between naive and infected mice, regardless of genotype. Hpb infection induced local increases at the transcript and protein level for mast cell proteases (MCPT1 and MCPT2), resistin-like molecules (RELM and RELM{beta}), and markers such as the phospholipase PLA2G4C and the pore-forming protein gasdermin C. Bulk proteomic analysis was also searched against the Hpb genome to identify Hpb proteins present in the duodenal tissues. A list of 60 Hpb proteins of interest were identified in infected duodenal samples, of which 18 contain a signal peptide and are present in the excretory/secretory products of Hpb (HES) (likely secretory products including immunomodulatory proteins); 28 proteins are present in HES but do not contain a signal peptide (likely excretory products); and 14 proteins are not present in HES (likely proteins present in the remnants of Hpb within the duodenum). This work thus provides datasets for changes in the mouse intestine due to Hpb infection, at both the transcript and protein level, as well as a dataset of Hpb proteins detectable in the mouse duodenum at day 14 of infection.

immunology↗

bioRxiv2025

HpBoRB, a helminth-derived CCP domain protein which binds RELMβ

Helminth infections persist by influencing host immunity through the release of immunomodulatory proteins which prevent immune ejection. The intestinal nematode Heligmosomoides polygyrus bakeri (Hpb) secretes multiple families of immunomodulatory proteins, many of which are composed of consecutive Complement Control Protein (CCP) domains. We hypothesized that further CCP domain proteins are secreted by the parasite to interact with the host. We identified an unusually large number of CCP domain-containing proteins in the genome of Hpb, and cloned a range of these for screening in an Avidity-based Extracellular Interaction Screening (AVEXIS) assay, focussing on interactions with host immune proteins. This screen confirmed the binding of known immunomodulators (HpBARI, TGM1) for their targets (ST2, TGFBR2) and identified a new interaction between a 2 CCP domain Hpb protein and mouse resistin-like molecule beta (RELM{beta}), a host protein demonstrated to have anti-helminth properties. This protein was named Binder of RELM{beta} (HpBoRB). This interaction was confirmed in ELISA, competition assays, size exclusion chromatography and surface plasmon resonance experiments, identifying a subnanomolar affinity interaction between HpBoRB and RELM{beta}. These data may indicate that Hpb interferes with the potent anti-helminth host protein RELM{beta} and adds to our knowledge of the host-parasite interactions mediated by Hpb secreted proteins.

immunology↗

bioRxiv2024

Vaccination against helminth IL-33-modulators permits immune-mediated parasite ejection

The murine intestinal nematode Heligmosomoides polygyrus bakeri powerfully modulates the host immune response. This is achieved in part through the HpARI family (HpARI1/2/3), which act on IL-33, and the HpBARI family (HpBARI and HpBARI_Hom2), which act on ST2. Here, we find that this immunomodulation is evident only in the first week of infection, with abrogation of ST2 detection and systemic suppression of IL-33-dependent responses. Vaccination with individual HpARI or HpBARI family members raised antibody responses which could block these proteins immunomodulatory activities. During infection, vaccination could release the host from immunosuppression: HpARI2 vaccination resulted in much increased ILC2 and Th2 immunity, with heightened serum IL-4 and IL-5 responses, but did not abrogate ST2 suppression. In contrast, a HpBARI+HpBARI_Hom2 vaccination cocktail resulted in abrogation of ST2 suppression, and again increased Th2 immunity and serum cytokine responses. Either of the HpARI2 or the HpBARI cocktail vaccinations provided significant protection against subsequent H. polygyrus bakeri infection. We therefore show a proof of principle that vaccination with immunomodulatory proteins can protect the host against infection, and can be used as a tool for blocking the effects of specific parasite-derived proteins.

immunology↗

bioRxiv2023

The helminth TGF-beta mimic TGM4 is a modular ligand that binds CD44, CD49d and TGF-beta receptors to preferentially target myeloid cells

The murine helminth parasite Heligmosomoides polygyrus expresses a family of modular proteins which, replicating the functional activity of the immunomodulatory cytokine TGF-{beta}, have been named TGM (TGF-{beta} Mimic). Multiple domains bind to different receptors, including TGF-{beta} receptors T{beta}RI (ALK5) and T{beta}RII through domains 1-3, and prototypic family member TGM1 binds the cell surface co-receptor CD44 through domains 4-5. This allows TGM1 to induce T lymphocyte Foxp3 expression, characteristic of regulatory (Treg) cells, and to activate a range of TGF-{beta}-responsive cell types. In contrast, a related protein, TGM4, targets a much more restricted cell repertoire, primarily acting on myeloid cells, with less potent effects on T cells and lacking activity on other TGF-{beta}-responsive cell types. TGM4 binds avidly to myeloid cells by flow cytometry, and can outcompete TGM1 for cell binding. Analysis of receptor binding in comparison to TGM1 reveals a 10-fold higher affinity than TGM1 for TGF{beta}R-I (T{beta}RI), but a 100-fold lower affinity for T{beta}RII through Domain 3. Consequently, TGM4 is more dependent on co-receptor binding; in addition to CD44, TGM4 also engages CD49d (Itga4) through Domains 1-3, as well as CD206 and Neuropilin-1 through Domains 4 and 5. TGM4 was found to effectively modulate macrophage populations, inhibiting lipopolysaccharide-driven inflammatory cytokine production and boosting interleukin (IL)-4-stimulated responses such as Arginase-1 in vitro and in vivo. These results reveal that the modular nature of TGMs has allowed the fine tuning of the binding affinities of the T{beta}R- and co-receptor binding domains to establish cell specificity for TGF-{beta} signalling in a manner that cannot be attained by the mammalian cytokine.

immunology↗

bioRxiv2022

A helminth mimic of TGF-β, TGM, enhances regenerative cutaneous wound healing and modulates immune cell recruitment and activation

Intestinal helminth parasites express excretory/secretory (ES) molecules, which modulate the type-2 immune response including anti-inflammatory and tissue repair pathways. TGF-{beta} mimic (TGM), an ES molecule secreted by Heligmosomoides polygyrus (Hp), binds TGF-{beta} receptors yet lacks structural homology to TGF-{beta} and exhibits distinct receptor interactions. We demonstrate TGM treatment enhanced wound healing and tissue regeneration in an in vivo wound biopsy model. TGM, in a 1.5% carboxymethylcellulose solution, was topically administered beneath a Tegaderm layer. Through histological analysis, increased restoration of normal tissue structure in the wound beds of TGM-treated mice was observed during mid- to late-stage wound healing. These observations included accelerated re-epithelialization and hair follicle regeneration, without increased scarring. Flow cytometric and gene expression analysis showed differential expansion of myeloid populations at different stages of wound healing. This included enhanced early accumulation and persistence of macrophages in TGM-treated wounds during the initial inflammatory phase. Additionally, the percentage of alternatively activated (M2) macrophages expressing CD206 was reduced with TGM treatment during early and mid-stage wound healing. scRNAseq analysis of TGM-treated wounds indicate upregulation of multiple wound healing-associated genes without expression of CD206 within macrophage subsets. Experiments with truncated TGM constructs revealed that the TGF{beta}-R binding domain was essential in enhancing the wound healing response. In summary, TGM can accelerate skin wound healing and pro-restorative maturation through its interaction with the TGF-{beta} receptor and stimulate the recruitment and reprogramming of specific macrophage subsets. This study indicates a role for TGM as a potential novel therapeutic option for enhanced wound healing. One-Sentence SummaryA helminth-derived protein leads to rapid wound closure, skin regeneration, and reprogramming of macrophage activation through TGF-{beta}R binding.

immunology↗

bioRxiv2019

Single-cell transcriptomics identifies an effectorness gradient shaping the response of CD4+ T cells to cytokines

Naive CD4+ T cells coordinate the immune response by acquiring an effector phenotype in response to cytokines. However, the cytokine responses in memory T cells remain largely understudied. We used quantitative proteomics, bulk RNA-seq and single-cell RNA-seq of over 40,000 human naive and memory CD4+ T cells to generate a detailed map of cytokine-regulated gene expression programs. We demonstrated that cytokine response differs substantially between naive and memory T cells and showed that memory cells are unable to differentiate into the Th2 phenotype. Moreover, memory T cells acquire a Th17-like phenotype in response to iTreg polarization. At the single-cell level, we demonstrated that T cells form a continuum which progresses from naive to effector memory T cells. This continuum is accompanied by a gradual increase in the expression levels of chemokines and cytokines and thus represents an effectorness gradient. Finally, we found that T cell cytokine responses are determined by where the cells lie in the effectorness gradient and identified genes whose expression is controlled by cytokines in an effectorness-dependent manner. Our results shed light on the heterogeneity of T cells and their responses to cytokines, provide insight into immune disease inflammation and could inform drug development.

immunology↗

bioRxiv2019

Immune disease variants modulate gene expression in regulatory CD4+ T cells and inform drug targets

Identifying cellular functions dysregulated by disease associated variants could implicate novel pathways for drug targeting or modulation in cell therapies. Variants associated with immune diseases point towards the role of CD4+ regulatory T cells (Tregs), cell type critical for immune homeostasis. To translate the effects of immune disease alleles on Treg function we mapped genetic regulation (QTL) of gene expression and chromatin activity in Tregs. We identified 123 loci where Treg QTLs colocalized with immune disease variants. These colocalizations indicated that dysregulation of key Treg pathways, including cell activation via CD28 and IL-2 signalling mediated by STAT5A contribute to the shared pathobiology of immune diseases. Finally, using disease GWAS signals colocalizing with Treg QTLs, we identified 34 known drug targets and 270 targets with drug tractability evidence. Our study is the first in-depth characterization of immune disease variant effects on Treg gene expression modulation and dysregulation of Treg function.

genetics↗

bioRxiv2017

Cells With Treg-Specific FOXP3 Demethylation But Low CD25 Are Prevalent In Autoimmunity

Identification of alterations in the cellular composition of the human immune system is key to understanding the autoimmune process. Recently, a subset of FOXP3+ cells with low CD25 expression was found to be increased in peripheral blood from systemic lupus erythematosus (SLE) patients, although its functional significance remains controversial. Here we find in comparisons with healthy donors that the frequency of FOXP3+ cells within CD127lowCD25low CD4+ T cells (here defined as CD25lowFOXP3+ T cells) is increased in patients affected by autoimmune disease of varying severity, from combined immunodeficiency with active autoimmunity, SLE to type 1 diabetes. We show that CD25lowFOXP3+ T cells share phenotypic features resembling conventional CD127lowCD25highFOXP3+ Tregs, including demethylation of the Treg-specific epigenetic control region in FOXP3 that is highly enriched in HELIOS+ cells, and lack of IL-2 production. As compared to conventional Tregs, more CD25lowFOXP3+HELIOS+ T cells are in cell cycle (33.0% vs 20.7% Ki-67+; P = 1.3 x 10-9) and express the late-stage inhibitory receptor PD-1 (67.2% vs 35.5%; P = 4.0 x 10-18), while having reduced expression of the early-stage inhibitory receptor CTLA-4, as well as other Treg markers, such as FOXP3 and CD15s. The number of CD25lowFOXP3+ T cells are highly correlated (P = 1.2 x 10-19) with the proportion of CD25highFOXP3+ T cells in cell cycle (Ki-67+). These findings suggest that CD25lowFOXP3+ T cells represent a subset of Tregs that are derived from CD25highFOXP3+ T cells, and are a peripheral marker of recent Treg expansion in response to an autoimmune reaction in tissues.\n\nHighlights- FOXP3+ compartment within CD127lowCD25low T cells is expanded in autoimmune patients.\n\n- Increased numbers of CD25lowFOXP3+ T cells are a circulating marker of autoimmunity.\n\n- CD25lowFOXP3+ HELIOS+ T cells are fully demethylated at the FOXP3 TSDR.\n\n- CD25lowFOXP3+ T cells could represent a terminal stage of regulatory T cells.

immunology↗