Enhanced CCR2 expression by ACKR2-deficient NK cells increases tumoricidal cell therapy efficacy.

Chemokines regulate leukocyte navigation to inflamed sites and specific tissue locales and may therefore be useful for ensuring accurate homing of cell therapeutic products. We, and others, have shown that atypical chemokine receptor 2 (ACKR2), deficient mice (ACKR2-/-) are protected from metastasis development in cell line and spontaneous mouse models. We have shown that this relates to enhanced CCR2 expression on ACKR2-/- NK cells allowing them to home more effectively to CCR2 ligand expressing metastatic deposits. Here we demonstrate that the metastatic-suppression phenotype in ACKR2-/- mice is not a direct effect of the absence of ACKR2. Instead, enhanced NK cell CCR2 expression is caused by passenger-mutations that originate from creation of the ACKR2-/- mouse strain in 129 embryonic stem cells. We further demonstrate that simple selection of CCR2+ NK cells enriches for a population of cells with enhanced anti-metastatic capabilities. Given the widespread expression of CCR2 ligands by tumors, our study highlights CCR2 as a potentially important contributor to NK cell tumoricidal cell therapy.

Disruption of placental ACKR3 impairs growth and hematopoietic development of offspring.

ACKR3 scavenges and degrades the stem cell recruiting chemokine CXCL12, which is essential for proper embryonic and, in particular, haematopoietic development. Here, we demonstrate strong expression of ACKR3 on trophoblasts. Using a maternally administered pharmacological blocker and Cre-mediated genetic approaches, we demonstrate that trophoblast 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 haematopoietic stem cell population. Importantly, although the haematopoietic 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 haematopoietic system.

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

Interferon gamma (IFNγ) 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 IFNγ and where they are located at different stages of an infection is limited. We have used reporter mice to investigate in vivo expression of Ifnγ mRNA in the lung and secondary lymphoid organs during and following influenza A virus (IAV) infection. We observed a triphasic production of Ifnγ expression. Unconventional T cells and innate lymphoid cells, particularly NK cells, were the dominant producers of early Ifnγ, 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 express Ifnγ in the lungs and draining lymph node. Interestingly, Ifnγ production by lymph node natural killer (NK), NKT, and innate lymphoid type 1 cells also continued to be above naïve levels, suggesting memory-like phenotypes for these cells. Analysis of the localization of Ifnγ+ 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 Ifnγ while CD4 T cells in the draining lymph node increased their Ifnγ response. Together, these data suggest that Ifnγ production fluctuates based on cellular source and location, both of which could impact subsequent immune responses.

Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity.

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of neurons, astrocytes, oligodendrocytes, and microglia. Due to increasing efforts to replace and reduce animal use, a variety of in vitro cell culture systems have been developed to explore innate cell properties, which allow the development of therapeutics for CNS infections and pathologies. Whilst certain research questions can be addressed by human-based cell culture systems, such as (induced) pluripotent stem cells, working with human cells has its own limitations with regard to availability, costs, and ethics. Here, we describe a unique protocol for isolating and culturing cells from embryonic mouse brains. The resulting mixed neural cell cultures mimic several cell populations and interactions found in the brain in vivo. Compared to current equivalent methods, this protocol more closely mimics the characteristics of the brain and also garners more cells, thus allowing for more experimental conditions to be investigated from one pregnant mouse. Further, the protocol is relatively easy and highly reproducible. These cultures have been optimized for use at various scales, including 96-well based high throughput screens, 24-well microscopy analysis, and 6-well cultures for flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. This culture method is a powerful tool to investigate infection and immunity within the context of some of the complexity of the CNS with the convenience of in vitro methods.

Analysis of combinatorial chemokine receptor expression dynamics using multi-receptor reporter mice.

Inflammatory chemokines and their receptors are central to the development of inflammatory/immune pathologies. The apparent complexity of this system, coupled with lack of appropriate in vivo models, has limited our understanding of how chemokines orchestrate inflammatory responses and has hampered attempts at targeting this system in inflammatory disease. Novel approaches are therefore needed to provide crucial biological, and therapeutic, insights into the chemokine-chemokine receptor family. Here, we report the generation of transgenic multi-chemokine receptor reporter mice in which spectrally distinct fluorescent reporters mark expression of CCRs 1, 2, 3, and 5, key receptors for myeloid cell recruitment in inflammation. Analysis of these animals has allowed us to define, for the first time, individual and combinatorial receptor expression patterns on myeloid cells in resting and inflamed conditions. Our results demonstrate that chemokine receptor expression is highly specific, and more selective than previously anticipated.

Mosquito saliva enhances virus infection through sialokinin-dependent vascular leakage.

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

Exploration of immunological responses underpinning severe fever with thrombocytopenia syndrome virus infection reveals IL-6 as a therapeutic target in an immunocompromised mouse model.

Dabie bandavirus (previously severe fever with thrombocytopenia syndrome virus; SFTSV), is an emerging tick-borne bunyavirus responsible for severe fever with thrombocytopenia syndrome (SFTS), a disease with high case fatality that is characterized by high fever, thrombocytopenia, and potentially lethal hemorrhagic manifestations. Currently, neither effective therapeutic strategies nor approved vaccines exist for SFTS. Therefore, there remains a pressing need to better understand the pathogenesis of the disease and to identify therapeutic strategies to ameliorate SFTS outcomes. Using a type I interferon (IFN)-deficient mouse model, we investigated the viral tropism, disease kinetics, and the role of the virulence factor nonstructural protein (NSs) in SFTS. Ly6C+ MHCII+ cells in the lymphatic tissues were identified as an important target cell for SFTSV. Advanced SFTS was characterized by significant migration of inflammatory leukocytes, notably neutrophils, into the lymph node and spleen, however, these cells were not required to orchestrate the disease phenotype. The development of SFTS was associated with significant upregulation of proinflammatory cytokines, including high levels of IFN-γ and IL-6 in the serum, lymph node, and spleen. Humoral immunity generated by inoculation with delNSs SFTSV was 100% protective. Importantly, NSs was critical to the inhibition of the host IFNɣ response or downstream IFN-stimulated gene production and allowed for the establishment of severe disease. Finally, therapeutic but not prophylactic use of anti-IL-6 antibodies significantly increased the survival of mice following SFTSV infection and, therefore, this treatment modality presents a novel therapeutic strategy for treating severe SFTS.

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. Rational 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.

Lipid-specific IgMs induce antiviral responses in the CNS: implications for progressive multifocal leukoencephalopathy in multiple sclerosis.

Progressive multi-focal leukoencephalopathy (PML) is a potentially fatal encephalitis caused by JC polyomavirus (JCV). PML principally affects people with a compromised immune system, such as patients with multiple sclerosis (MS) receiving treatment with natalizumab. However, intrathecal synthesis of lipid-reactive IgM in MS patients is associated with a markedly lower incidence of natalizumab-associated PML compared to those without this antibody repertoire. Here we demonstrate that a subset of lipid-reactive human and murine IgMs induce a functional anti-viral response that inhibits replication of encephalitic Alpha and Orthobunyaviruses in multi-cellular central nervous system cultures. These lipid-specific IgMs trigger microglia to produce IFN-ß in a cGAS-STING-dependent manner, which induces an IFN-α/ß-receptor 1-dependent antiviral response in glia and neurons. These data identify lipid-reactive IgM as a mediator of anti-viral activity in the nervous system and provide a rational explanation why intrathecal synthesis of lipid-reactive IgM correlates with a reduced incidence of iatrogenic PML in MS.

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 severe acute respiratory syndrome coronavirus 1 (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 surfactant disruption of the viral envelope. We have identified a novel antiviral formulation that is of great interest for the prevention and/or treatment of a broad range of enveloped viruses, particularly those of the skin and mucosal surfaces.