Biology subjects

Pingen, M.

Publications and source records attributed to Pingen, M..

11 recordsLinked to original sources

bioRxiv2025

Chemokine receptor expression defines a trajectory from monocytes to mature macrophages.

CCR1, CCR2 and CCR5 direct recruitment of monocytes and macrophages in inflammation. However, the discrete role for each receptor in monocyte/macrophage biology remains poorly understood, with previous reports citing receptor redundancy. Using transcriptomic approaches to examine inflammatory chemokine receptor expression on lung interstitial macrophage populations, we demonstrate that interstitial macrophages can be divided into three distinct subsets, each of which express specific patterns of chemokine receptors, and that there are dynamic changes in chemokine receptor expression as macrophages differentiate from monocytes in the lung. Furthermore, macrophages expressing different combinations of chemokine receptors are transcriptionally distinct, suggesting non-redundant functions for CCR1, 2 and 5. Finally, we examined changes in macrophage chemokine receptor expression in vitro after treatment with varied TLR ligands, and show that CCR1 is specifically increased in response to bacterial but not viral ligands. Our data provide compelling evidence that macrophage chemokine receptor expression is not redundant, but specific and malleable in response to discrete inflammatory stimuli.

immunology↗

bioRxiv2025

Inflammatory chemokine receptors CCR1, CCR2, CCR3 and CCR5 are essential for an optimal T cell response to influenza.

Inflammatory chemokine receptors CCR1/2/3/5 (iCCRs) play an important role in the recruitment of immune cells involved in innate immune functions and orchestrating the adaptive immune response. Here we utilise an influenza A virus (IAV) challenge to investigate the combinatorial roles of the iCCRs in the anti-IAV immune response. We did not observe any gross differences in infection-driven pathology in the absence of iCCRs. Despite iCCR deletion resulting in decreased migration of monocytes, migratory macrophages and B cells to lungs during acute IAV infection, no differences in dendritic cell numbers were observed. Whilst the total number of T cells was similar in lungs of iCCR-deficient mice, the number of IAV-specific CD4 but not CD8 T cells in the lung was strongly reduced in the absence of iCCRs. Furthermore, fewer CD4, but not CD8, T cells produced IFN-{gamma}. This CD4 T cell phenotype persisted into the memory stage of infection, with fewer IAV-specific and IFN-{gamma}+ CD4 but not CD8 T cells at 29 days post infection. In conclusion, despite having no impact on dendritic cell migration between the lung and the draining lymph node, iCCR deletion is associated with an altered CD4 T cell response to IAV infection.

immunology↗

bioRxiv2024

Lung structural cell dynamics are altered by influenza virus infection experience leading to rapid immune protection following viral re-challenge

Lung structural cells, including epithelial cells and fibroblasts, form barriers against pathogens and trigger immune responses following infections such as influenza A virus. This response leads to the recruitment of innate and adaptive immune cells required for viral clearance. Some of these recruited cells remain within the lung following infection and contribute to enhanced viral control following subsequent infections. There is growing evidence that structural cells can also display long-term changes following infection or insults. Here we investigate long-term changes to mouse lung epithelial cells, fibroblasts, and endothelial cells following influenza virus infection and find that all three cell types maintain an imprint of the infection, particularly in genes associated with communication with T cells. Lung epithelial cells from IAV-infected mice display functional changes by more rapidly controlling influenza virus than cells from naive animals. This rapid anti-viral response and increased expression of molecules required to communicate with T cells demonstrates sustained and enhanced functions following infection. These data suggest lung structural cells could be effective targets for vaccines to boost durable protective immunity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/604410v5_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@2774e0org.highwire.dtl.DTLVardef@6a39e6org.highwire.dtl.DTLVardef@1ff5863org.highwire.dtl.DTLVardef@103df12_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LILung epithelial cells, fibroblasts, and blood endothelial cells maintain an inflammatory imprint of influenza A virus (IAV) infection for at least 40 days post-infection. C_LIO_LIIn vivo re-infection leads to a more spatially restricted anti-viral response compared to primary IAV-infected animals. C_LIO_LIT cells are not required for enhanced viral control early after re-infection in vivo C_LIO_LIEx vivo lung epithelial cells from IAV-infected mice more rapidly control IAV than cells from naive animals in the absence of immune cells. C_LI

immunology↗

bioRxiv2023

Disruption of maternal ACKR3 has profound effects on embryos and offspring.

ACKR3, scavenges and degrades the stem cell recruiting chemokine CXCL12 which is essential for proper embryonic, and in particular hematopoietic, development. Here we demonstrate strong expression of ACKR3 on trophoblasts. Using a pharmacological blocker we demonstrate that ACKR3 is essential for preventing movement of CXCL12 from the mother to the embryo with elevated plasma CXCL12 levels being detected in embryos from ACKR3-blocker treated mothers. Mice born to mothers treated with the blocker are lighter and shorter than those born to vehicle-treated mothers and, in addition, display profound anaemia associated with a markedly reduced bone marrow hematopoietic stem cell population. Importantly, whilst the hematopoietic abnormalities are corrected as mice age, our studies reveal a postnatal window during which offspring of ACKR3 blocker treated mice are unable to mount effective inflammatory responses to inflammatory/infectious stimuli. Overall, these data demonstrate that ACKR3 is essential for preventing CXCL12 transfer from mother to embryo and for ensuring properly regulated CXCL12 control over the development of the hematopoietic system.

developmental biology↗

bioRxiv2023

Triphasic production of IFNγ by innate and adaptive lymphocytes following influenza A virus infection

Interferon gamma (IFN{gamma}) is a potent antiviral cytokine that can be produced by many innate and adaptive immune cells during infection. Currently, our understanding of which cells produce IFN{gamma} and where they are located at different stages of an infection are limited. We have used reporter mice to investigate in vivo expression of IFN{gamma} in the lung and secondary lymphoid organs during and following influenza A virus (IAV) infection. We observed a triphasic production of IFN{gamma} expression. Unconventional T cells and innate lymphoid cells, particularly NK cells, were the dominant producers of early IFN{gamma}, while CD4 and CD8 T cells were the main producers by day 10 post-infection. Following viral clearance, some memory CD4 and CD8 T cells continued to produce IFN{gamma} in the lungs and draining lymph node. Interestingly, IFN{gamma} production by lymph node Natural Killer (NK), NKT and innate lymphoid 1 cells also continued to be above naive levels, suggesting memory-like phenotypes for these cells. Analysis of the localisation of IFN{gamma}+ memory CD4 and CD8 T cells demonstrated that cytokine+ T cells were located near airways and in the lung parenchyma. Following a second IAV challenge, lung IAV specific CD8 T cells rapidly increased their expression of IFN{gamma} while CD4 T cells in the draining lymph node increased their IFN{gamma} response. Together, these data suggest that IFN{gamma} production fluctuates based on cellular source and location, both of which could impact subsequent immune responses.

immunology↗

bioRxiv2021

Mosquito saliva sialokinin-dependent enhancement of arbovirus infection through endothelial barrier leakage

Viruses transmitted by Aedes mosquitoes constitute an increasingly important global health burden. Defining common determinants of host susceptibility to this large group of heterogenous pathogens are key for informing the rational design of new pan-viral medicines. Infection of the vertebrate host with these viruses is enhanced by the presence of mosquito saliva, a complex mixture of salivary gland-derived factors and microbiota. We show that enhancement of infection by saliva was dependent on vascular function and was independent of most anti-saliva immune responses, including to salivary microbiota. Instead, the Aedes gene product sialokinin mediated enhancement of virus infection through a rapid reduction in endothelial barrier integrity. Sialokinin is unique within the insect world as having vertebrate-like tachykinin sequence and is absent from non-vector competent Anopheles mosquitoes, whose saliva was not pro-viral and did not induce similar vascular permeability. Therapeutic strategies targeting sialokinin have potential to limit disease severity following infection with Aedes mosquito-borne viruses.

microbiology↗

bioRxiv2020

A novel antiviral formulation inhibits a range of enveloped viruses.

Some free fatty acids derived from milk and vegetable oils are known to have potent antiviral and antibacterial properties. However, therapeutic applications of short to medium chain fatty acids are limited by physical characteristics such as immiscibility in aqueous solutions. We evaluated a novel proprietary formulation based on an emulsion of short chain caprylic acid, ViroSAL, for its ability to inhibit a range of viral infections in vitro and in vivo. In vitro, ViroSAL inhibited the enveloped viruses Epstein-Barr, measles, herpes simplex, Zika and orf parapoxvirus, together with Ebola, Lassa, vesicular stomatitis and SARS-CoV-1 pseudoviruses, in a concentration- and time-dependent manner. Evaluation of the components of ViroSAL revealed that caprylic acid was the main antiviral component; however, the ViroSAL formulation significantly inhibited viral entry compared with caprylic acid alone. In vivo, ViroSAL significantly inhibited Zika and Semliki Forest Virus replication in mice following the inoculation of these viruses into mosquito bite sites. In agreement with studies investigating other free fatty acids, ViroSAL had no effect on norovirus, a non-enveloped virus, indicating that its mechanism of action may be via surfactant disruption of the viral envelope. We have identified a novel antiviral formulation that is of great interest for prevention and/or treatment of a broad range of enveloped viruses.

microbiology↗

bioRxiv2019

Chemokine receptors CCR1 and ACKR2 coordinate mammary gland development

Macrophages are key regulators of developmental processes, including those involved in mammary gland development. We previously demonstrated that the atypical chemokine receptor, ACKR2, contributes to control of ductal epithelial branching in the developing mammary gland by regulating macrophage dynamics. ACKR2 is a chemokine-scavenging receptor, which mediates its effects through collaboration with inflammatory chemokine receptors (iCCRs). Here we reveal that ACKR2, and the iCCR CCR1, reciprocally regulate branching morphogenesis in the mammary gland, whereby stromal ACKR2 modulates levels of the shared ligand CCL7 to control the movement of a key population of CCR1-expressing macrophages to the ductal epithelium. In addition estrogen, which is essential for ductal elongation during puberty, upregulates CCR1 expression on macrophages. The age at which girls develop breasts is decreasing, which raises the risk of diseases including breast cancer. This study presents a previously unknown mechanism controlling the rate of mammary gland development during puberty and highlights potential therapeutic targets. SummaryIn the mammary gland during puberty, availability of the chemokine CCL7 is controlled by a scavenging receptor ACKR2 and provides a key signal to macrophages which have the receptor CCR1. Together, this controls the timing of development.

immunology↗

bioRxiv2019

A novel methodology for defining stromal expression of atypical chemokine receptors in vivo

Analysis of chemokine receptor, and atypical chemokine receptor, expression is frequently hampered by the lack of availability of high-quality antibodies and the species-specificity of those that are available. We have previously described methodology utilising Alexa-Fluor labelled chemokine ligands as versatile reagents to detect receptor expression. Previously this has been limited to haematopoietic cells and methodology for assessing expression of receptors on stromal cells has been lacking. Amongst chemokine receptors the ones most frequently expressed on stromal cells belong to the atypical chemokine receptor subfamily. These receptors do not signal in the classic sense in response to ligand but scavenge their ligands and degrade them and thus sculpt in vivo chemokine gradients. Here we demonstrate the ability to use either intratracheal, or intravenous, Alexa-Fluor labelled chemokine administration to detect stromal cell populations expressing the atypical chemokine receptor ACKR2. Using this methodology we demonstrate, for the first time, expression of ACKR2 on blood endothelial cells. This observation sets the lung aside from other tissues in which ACKR2 is exclusively expressed on lymphatic endothelial cells. In summary therefore we described a novel method for the in situ labelling of atypical chemokine receptor expressing cells appropriate for subsequent flow cytometric analysis. We propose that this methodology will work in a range of species and for a range of receptors and therefore will have significant versatility

immunology↗

bioRxiv2019

Site-directed M2 proton channel inhibitors enable synergistic combination therapy for rimantadine-resistant pandemic influenza

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable.\n\nRational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic \"swine\" H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance.

microbiology↗

bioRxiv2019

Pan-viral protection against arboviruses by targeting inoculation site-based skin macrophages

Arthropod-borne viruses (arboviruses) are important human pathogens for which there are no specific antiviral medicines. The large number of genetically-distinct arbovirus species, coupled with the unpredictable nature of their outbreaks, has made developing virus-specific anti-viral medicines challenging. Instead, we have defined and targeted a key aspect of the host innate immune response to virus at the arthropod bite that is common to all arbovirus infections, potentially circumventing the need for virus-specific therapies at this site. Using mouse models and human skin explants, we identify innate immune responses by dermal macrophages in the skin as a key determinant of disease severity. Post-exposure treatment of the inoculation site by a topical innate immune agonist significantly suppressed both the local and subsequent systemic course of infection and improved clinical outcome in mice to infection with a variety of arboviruses from the Alphavirus, Flavivirus and Orthobunyavirus genuses. In the absence of treatment, anti-viral interferon expression to virus in the skin was restricted to dermal dendritic cells. In contrast, targeting the more populous skin-resident macrophages with an immune agonist elicited protective responses in key cellular targets of virus that otherwise replicated virus to high levels. By defining and targeting a key aspect of the innate immune response to virus at the mosquito bite site, we have shown that it is possible to improve outcome to infection by targeting pathways activated at the site of inoculation, and thereby identified a putative new strategy for limiting disease following infection with a variety of genetically-distinct arboviruses.\n\nOne-sentence summaryWe demonstrate that activation of innate immune responses to arbovirus at the mosquito bite is a limiting factor for preventing efficient systemic dissemination of virus and that therapeutic targeting of skin-resident macrophages can have defining inhibitory effects on the later systemic course.

immunology↗