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.

Host Inflammatory Response to Mosquito Bites Enhances the Severity of Arbovirus Infection.

Aedes aegypti mosquitoes are responsible for transmitting many medically important viruses such as those that cause Zika and dengue. The inoculation of viruses into mosquito bite sites is an important and common stage of all mosquito-borne virus infections. We show, using Semliki Forest virus and Bunyamwera virus, that these viruses use this inflammatory niche to aid their replication and dissemination in vivo. Mosquito bites were characterized by an edema that retained virus at the inoculation site and an inflammatory influx of neutrophils that coordinated a localized innate immune program that inadvertently facilitated virus infection by encouraging the entry and infection of virus-permissive myeloid cells. Neutrophil depletion and therapeutic blockade of inflammasome activity suppressed inflammation and abrogated the ability of the bite to promote infection. This study identifies facets of mosquito bite inflammation that are important determinants of the subsequent systemic course and clinical outcome of virus infection.

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.

Chemokine receptors coordinately regulate macrophage dynamics and mammary gland development.

Macrophages are key regulators of developmental processes, including those involved in mammary gland development. We have previously demonstrated that the atypical chemokine receptor ACKR2 contributes to the control of ductal epithelial branching in the developing mammary gland by regulating macrophage dynamics. ACKR2 is a chemokine-scavenging receptor that mediates its effects through collaboration with inflammatory chemokine receptors (iCCRs). Here, we reveal reciprocal regulation of 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, oestrogen, 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.

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.

Placental chemokine compartmentalisation: A novel mammalian molecular control mechanism.

Atypical chemokine receptor 2 (ACKR2) is a chemokine-scavenging receptor. ACKR2-/-embryos display a reduction in size of a novel, to our knowledge, embryonic skin macrophage population referred to as 'intermediate' cells. CC chemokine receptor 2 (CCR2)-/-embryos display an identical phenotype, indicating that these cells require CCR2 to enable them to populate embryonic skin. Further analysis revealed that ACKR2-/-embryos have higher circulating concentrations of the CCR2 ligand, CC ligand 2 (CCL2); thus, ACKR2 regulates intraembryonic CCL2 levels. We show that ACKR2 is strongly expressed by trophoblasts and that it blocks movement of inflammatory chemokines, such as CCL2, from the maternal decidua into the embryonic circulation. We propose that trophoblastic ACKR2 is responsible for ensuring chemokine compartmentalisation on the maternal decidua, without which chemokines enter the embryonic circulation, disrupting gradients essential for directed intraembryonic cell migration. Overall, therefore, we describe a novel, to our knowledge, molecular mechanism whereby maternal decidual chemokines can function in a compartmentalised fashion without interfering with intraembryonic leukocyte migration. These data suggest similar functions for other atypical chemokine receptors in the placenta and indicate that defects in such receptors may have unanticipated developmental consequences.

Analysis of lung stromal expression of the atypical chemokine receptor ACKR2 reveals unanticipated expression in murine blood endothelial cells.

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 utilizing Alexa-Fluor-labeled chemokine ligands as versatile reagents to detect receptor expression. Previously this has been limited to hematopoietic cells and methodology for assessing expression of receptors on stromal cells has been lacking. Among 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-labeled 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 and suggest unique roles for ACKR2 in the pulmonary environment.

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.

The Atypical Chemokine Receptor Ackr2 Constrains NK Cell Migratory Activity and Promotes Metastasis.

Chemokines have been shown to be essential players in a range of cancer contexts. In this study, we demonstrate that mice deficient in the atypical chemokine receptor Ackr2 display impaired development of metastasis in vivo in both cell line and spontaneous models. Further analysis reveals that this relates to increased expression of the chemokine receptor CCR2, specifically by KLRG1+ NK cells from the Ackr2-/- mice. This leads to increased recruitment of KLRG1+ NK cells to CCL2-expressing tumors and enhanced tumor killing. Together, these data indicate that Ackr2 limits the expression of CCR2 on NK cells and restricts their tumoricidal activity. Our data have important implications for our understanding of the roles for chemokines in the metastatic process and highlight Ackr2 and CCR2 as potentially manipulable therapeutic targets in metastasis.

Infection with the frequently transmitted HIV-1 M41L variant has no influence on selection of tenofovir resistance.

OBJECTIVES: In ∼10% of newly diagnosed HIV-1 patients, drug-resistant viral variants are detected. In such transmitted HIV-1 variants, the thymidine analogue mutation (TAM) M41L is frequently observed as a single resistance mutation and these viral variants often belong to phylogenetic transmission clusters. The presence of at least three TAMs, in particular patterns with M41L/L210W, impairs the efficacy of the extensively used drug tenofovir. We investigated whether the presence of a single M41L mutation at baseline influences the selection of resistance to tenofovir and emtricitabine in vitro and in vivo. METHODS: The impact of M41L on the development of drug resistance to tenofovir and emtricitabine was determined by extensive in vitro selection experiments and investigation of the virological outcome of patients on a first-line regimen. RESULTS: The presence of a single M41L mutation did not influence the selected mutational profile or the genetic barrier to resistance to tenofovir and/or emtricitabine during long-term in vitro selection experiments. In vivo, virological outcome of first-line regimens containing tenofovir and emtricitabine was comparable between patients diagnosed with HIV-1 harbouring M41L (n=17, 16 were part of one transmission cluster) and WT virus (n=248). CONCLUSIONS: Detection of a single M41L reverse transcriptase mutation at baseline did not influence the development of resistance in vitro or virological outcome on tenofovir-containing regimens in patients belonging to a large transmission cluster. Our results indicate that a high genetic barrier regimen may not be required when patients are diagnosed with HIV variants containing a single M41L mutation in reverse transcriptase.