Biology subjects

Mincham, K. T.

Publications and source records attributed to Mincham, K. T..

8 recordsLinked to original sources

bioRxiv2023

Mapping lung hematopoietic progenitors: Developmental kinetics and response to Influenza A viral infection

The bone marrow is a specialised niche responsible for the maintenance of hematopoietic stem and progenitor cells during homeostasis and inflammation. Recent studies however have extended this essential role to the extramedullary and extravascular lung microenvironment. Here, we provide further evidence for a reservoir of hematopoietic stem and progenitor cells within the lung from embryonic day 18.5 until adulthood. These lung progenitors display distinct microenvironment-specific developmental kinetics compared to their bone marrow counterparts, exemplified by a rapid shift from a common myeloid to megakaryocyte-erythrocyte progenitor dominated niche with increasing age. In adult mice, Influenza A viral infection results in a transient reduction in multipotent progenitors within the lungs, with a parallel increase in downstream granulocyte-macrophage progenitors and dendritic cell populations associated with acute viral infections. Our findings suggest lung hematopoietic progenitors play a role in re-establishing immunological homeostasis in the respiratory mucosa, which may have significant clinical implications for maintaining pulmonary health following inflammatory perturbation.

immunology↗

bioRxiv2023

CXCL17 binds efficaciously to glycosaminoglycans with the potential to modulate chemokine signalling

CXCL17 is a mucosally secreted protein, and the most recently identified human chemokine, an assignment based on protein fold prediction and chemotactic activity for leukocytes. However, these credentials have been the subject of much recent discussion and no experimental evidence has been presented regarding the definitive structure of CXCL17. In this study, we evaluated the structural and chemoattractant credentials of CXCL17 to better characterise this molecule, and gain deeper insights into its functional role as a glycosaminoglycan (GAG) binding protein. In the absence of structural information, in silico modelling techniques assessed the likelihood of CXCL17 adopting a chemokine-fold. Recombinant CXCL17 was synthesized in mammalian and prokaryotic systems. Modified Boyden chamber and real-time chemotaxis assays assessed the ability of CXCL17 to promote chemotaxis of murine splenocytes, human neutrophils and CXCR1-transfectants. The efficacy of CXCL17 binding to GAGs was quantified with solid-phase assays and bio-layer interferometry techniques. All modelling efforts failed to support classification of CXCL17 as a chemokine based on its predicted conformation. Recombinant CXCL17 was observed to dimerize as a function of concentration, a characteristic of several chemokines. Contrary to a previous report, CXCL17 was not chemotactic for murine splenocytes, although it was a low-potency chemoattractant for human neutrophils at micromolar concentrations, several orders of magnitude higher than those required for CXCL8. As anticipated due to its highly basic nature, CXCL17 bound to GAGs robustly, with key C-terminal motifs implicated in this process. While inactive via CXCR1, CXCL17 was found to inhibit CXCR1-mediated chemotaxis of transfectants to CXCL8 in a dose-dependent manner. In summary, despite finding little evidence for chemokine-like structure and function, CXCL17 readily bound GAGs, and could modulate chemotactic responses to another chemokine in vitro. We postulate that such modulation is a consequence of superior GAG-binding, and that C-terminal fragments of CXCL17 may serve as prototypic inhibitors of chemokine function.

immunology↗

bioRxiv2022

Full Spectrum Flow Cytometry for High-Dimensional Immunophenotyping of Murine Innate Lymphoid Cells

Purpose and appropriate sample typesThis 25-parameter, 22-colour full spectrum flow cytometry panel was designed and optimised for the comprehensive enumeration and functional characterisation of innate lymphoid cell (ILC) subsets in mouse tissues (Table 1). The panel presented here allows the discrimination of ILC progenitors (ILCP), ILC1, ILC2, NCR+ ILC3, NCR- ILC3, CCR6+ lymphoid tissue-inducer (LTi)-like ILC3 and mature natural killer (NK) cell populations. Further characterisation of ILC and NK cell functional profiles in response to stimulation is provided by the inclusion of subset-specific cytokine markers, and proliferation markers. Development and optimisation of this panel was performed on freshly isolated cells from adult BALB/c lungs and small intestine lamina propria, and ex vivo stimulation with phorbol 12-myrisate 13-acetate, ionomycin and pro-ILC activating cytokines. O_TBL View this table: org.highwire.dtl.DTLVardef@1b1415forg.highwire.dtl.DTLVardef@3adc6forg.highwire.dtl.DTLVardef@5e0aeaorg.highwire.dtl.DTLVardef@1e9e61org.highwire.dtl.DTLVardef@2ff463_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONSummary Table C_TABLECAPTION C_TBL Ethical compliance statementAll mouse experiments were performed in accordance with the recommendations in the Guide for the Use of Laboratory Animals of Imperial College London, with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. All animal procedures and care conformed strictly to the UK Home Office Guidelines under the Animals (Scientific Procedures) Act 1986, and the protocols were approved by the Home Office of Great Britain.

cell biology↗

bioRxiv2021

Metabolic dysfunction induced by high-fat diet modulates hematopoietic stem and myeloid progenitor cells in brown adipose tissue of mice

Brown adipose tissue (BAT) may be an important metabolic regulator of whole-body glucose. While important roles have been ascribed to macrophages in regulating metabolic functions in BAT, little known is known of the roles of other immune cells subsets, particularly dendritic cells (DCs). Eating a high fat diet may compromise the development of hematopoietic stem and progenitor cells (HSPC) - which give rise to DCs - in bone marrow, with less known of its effects in BAT. We have previously demonstrated that ongoing exposure to low-dose ultraviolet radiation (UVR) significantly reduced the whitening effect of eating a high-fat diet upon interscapular (i)BAT of mice. Here, we examined whether this observation may be linked to changes in the phenotype of HSPC and myeloid-derived immune cells in iBAT and bone marrow of mice using 12-colour flow cytometry. Many HSPC subsets declined in both iBAT and bone marrow with increasing metabolic dysfunction. Conversely, with rising adiposity and metabolic dysfunction, conventional (c)DCs increased in both of these tissues. When compared to low-fat diet, consumption of high-fat diet significantly reduced proportions of myeloid, common myeloid and megakaryocyte-erythrocyte progenitors in iBAT, and short-term hematopoietic stem cells in bone marrow. In mice fed a high-fat diet, exposure to low-dose UVR significantly reduced proportions of cDCs in iBAT, independently of nitric oxide release from irradiated skin (blocked using the scavenger, cPTIO), but did not significantly modify HSPC subsets in either tissue. Further studies are needed to determine whether changes in these cell populations contribute towards metabolic dysfunction.

cell biology↗

bioRxiv2021

Protection against neonatal respiratory viral infection via maternal treatment during pregnancy with the benign immune training agent OM-85

ObjectivesIncomplete maturation of immune regulatory functions at birth are antecedent to the heightened risk for severe respiratory infections during infancy. Our forerunner animal model studies demonstrated that maternal treatment with the benign microbial-derived immune modulating agent OM-85 during pregnancy promotes accelerated maturation of immune regulatory networks in the developing fetal bone marrow. Here, we aimed to establish proof-of-concept that this would enhance resilience to severe early life respiratory viral infection during the neonatal period. MethodsPregnant BALB/c mice were treated orally with OM-85 during gestation and their offspring infected intranasally with a mouse-adapted rhinovirus (vMC0) at postnatal day 2. We then assessed clinical course, lung viral titres and lung immune parameters to determine whether offspring from OM-85 treated mothers demonstrate enhanced immune protection against neonatal vMC0 infection. ResultsOffspring from OM-85 treated mothers display enhanced capacity to clear an otherwise lethal respiratory viral infection during the neonatal period, with a concomitant reduction in the exaggerated nature of the ensuing immune response. These treatment effects were associated with accelerated postnatal myeloid cell seeding of neonatal lungs and enhanced expression of microbial sensing receptors in lung tissues, coupled in particular with enhanced capacity to rapidly expand and maintain networks of lung dendritic cells expressing function-associated markers crucial for maintenance of local immune homeostasis in the face of pathogen challenge. ConclusionMaternal OM-85 treatment may represent a novel therapeutic strategy to reduce the burden, and potential long-term sequlae, of severe neonatal respiratory viral infection by accelerating development of innate immune competence.

immunology↗

bioRxiv2020

High-Dimensional Immunophenotyping of Murine T-cell and B-cell Subsets

Purpose and appropriate sample typesThis 19-parameter, 18-colour flow cytometry panel was designed and optimised to enable the comprehensive and simultaneous immunophenotyping of distinct T-cell and B-cell subsets within murine lymphoid tissues (Table 1). Cellular populations identified by employing this OMIP include 4 major subsets of B-cells (memory, activated, plasma cells and plasmablasts) and 7 major subsets of CD4+ T-cells (naive, central memory, effector memory, helper, regulatory, follicular helper and follicular regulatory). Staining was performed on freshly isolated splenocytes from 21-day-old neonatal BALB/c mice, however due to the omission of mouse strain-specific markers, this OMIP can be implemented across a range of murine models where in-depth immunophenotyping of the diverse repertoire of T-cell and B-cell populations localised within lymphoid tissues is required. O_TBL View this table: org.highwire.dtl.DTLVardef@1b488fdorg.highwire.dtl.DTLVardef@18dd356org.highwire.dtl.DTLVardef@2dce91org.highwire.dtl.DTLVardef@154cc27org.highwire.dtl.DTLVardef@27237c_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONSummary Table C_TABLECAPTION C_TBL

cell biology↗

bioRxiv2019

Transplacentally mediated immune training to accelerate fetal immune competence

We recently reported that the offspring of mice treated during pregnancy with the microbial-derived immunomodulator OM-85 manifest striking resistance postnatally to allergic airways inflammation, and localised the potential treatment target to the fetal cDC progenitor compartment which expands to increase the pool of precursors available at birth, enabling accelerated postnatal seeding of the lung mucosal cDC network required for establishment of immunological homeostasis in the airways. Here, we profile maternal OM-85 treatment-associated transcriptomic signatures in fetal bone marrow, and identify a series of immunometabolic pathways which provide essential metabolites for accelerated myelopoiesis, that are hallmarks of classical "immune training". In addition, the cDC progenitor compartment displayed treatment-associated activation of the XBP1-ERN1 signalling axis which has previously been shown to be essential for tissue survival of cDC, particularly within the lung microenvironment. Our forerunner studies indicate uniquely rapid turnover of airway mucosal cDCs at baseline, with further large-scale upregulation of population dynamics during aeroallergen and/or pathogen challenge. XBP1-ERN1 signalling plays a key role in mitigation of ER stress-associated toxicity which frequently accompanies DC hyper-activation during intense immunoinflammatory responses, and we suggest that enhanced capacity for XBP1-ERN1-dependent cDC survival within the airway mucosal tissue microenvironment may be a crucial element of the OM-85-mediated transplacental "innate immune training" process which results in enhanced resistance to airway inflammatory disease during the high-risk early postnatal period.

immunology↗

bioRxiv2017

Transplacental protection against asthma by maternal treatment with a bacterial-derived immunomodulatory agent

Studies in European and US farming populations have documented major reductions in asthma prevalence in offspring of mothers exposed to microbial breakdown products present in farm dusts and unprocessed foods. This was associated with enhancement of innate immune competence in the offspring. We sought to (i) identify a safe therapeutic that would reproduce these immunomodulatory effects in a murine model, (ii) elucidate underlying mechanism(s)-of-action, and (iii) develop a scientific rationale for progressing this approach to human trials. We demonstrate in mice that maternal treatment during pregnancy with the microbial-derived immunomodulator OM85, which has been used clinically in adults and children in Europe for >30 years for bolstering resistance to infection-associated airways inflammation, markedly reduces the susceptibility of the offspring of treated mothers to development of experimental atopic asthma. We identify bone marrow precursors of the dendritic cell populations responsible for airway mucosal immune surveillance as the primary targets for the asthma-protective effects of maternal OM85 treatment in the offspring.

immunology↗