Single-cell proteomics and transcriptomics capture eosinophil development and identify the role of IL-5 in their lineage transit amplification.

The activities, ontogeny, and mechanisms of lineage expansion of eosinophils are less well resolved than those of other immune cells, despite the use of biological therapies targeting the eosinophilia-promoting cytokine interleukin (IL)-5 or its receptor, IL-5Rα. We combined single-cell proteomics and transcriptomics and generated transgenic IL-5Rα reporter mice to revisit eosinophilopoiesis. We reconciled human and murine eosinophilopoiesis and provided extensive cell-surface immunophenotyping and transcriptomes at different stages along the continuum of eosinophil maturation. We used these resources to show that IL-5 promoted eosinophil-lineage expansion via transit amplification, while its deletion or neutralization did not compromise eosinophil maturation. Informed from our resources, we also showed that interferon response factor-8, considered an essential promoter of myelopoiesis, was not intrinsically required for eosinophilopoiesis. This work hence provides resources, methods, and insights for understanding eosinophil ontogeny, the effects of current precision therapeutics, and the regulation of eosinophil development and numbers in health and disease.

Validation of Calcein Violet as a New Marker of Semen Membrane Integrity in Domestic Animals.

Many fluorochromes routinely used in semen quality analysis emit in the green and red channels, limiting their possible combination for multiple parameter analysis. The use of fluorophores emitting in different light channels broadens the possibilities of combination to expand the range of simultaneously evaluated criteria. This is of great interest in cases of small ejaculated volumes, such as those naturally occurring in roosters, small dog breeds and drones (Apis mellifera). The purpose of this experiment is to establish Calcein Violet (CaV), a blue fluorochrome, as a marker of viability and acrosomal integrity in domestic animals in order to free the red and green channels. SYBR®14/Propidium Iodide (PI) was used as reference dye, heat-treated samples as negative controls, serial staining combination for validation and epifluorescence microscopy for observation. Dead spermatozoa marked in red with PI showed no blue fluorescence either from the head or the tail. Live spermatozoa showed a decreasing blue emission from head to tail when single stained with CaV. Unreacted acrosomes showed intense blue fluorescence irrespective of plasma membrane integrity. This needs to be further confirmed for species with small and difficult to observe heads. Establishment of CaV as a marker of membrane integrity by fluorescence microscopy is a decisive first step towards further technical development and use with flow cytometry.

Parasitic worms affect virus coinfection: a mechanistic overview.

Helminths are parasitic worms that coevolve with their host, usually resulting in long-term persistence through modulating host immunity. The multifarious mechanisms altering the immune system induced by helminths have significant implications on the control of coinfecting pathogens such as viruses. Here, we explore the recent literature to highlight the main immune alterations and mechanisms that affect the control of viral coinfection. Insights from these mechanisms are valuable in the understanding of clinical observations in helminth-prevalent areas and in the design of new therapeutic and vaccination strategies to control viral diseases.

Wildebeest-Derived Malignant Catarrhal Fever: A Bovine Peripheral T Cell Lymphoma Caused by Cross-Species Transmission of Alcelaphine Gammaherpesvirus 1.

Gammaherpesviruses (γHVs) include viruses that can induce lymphoproliferative diseases and tumors. These viruses can persist in the long term in the absence of any pathological manifestation in their natural host. Alcelaphine gammaherpesvirus 1 (AlHV-1) belongs to the genus Macavirus and asymptomatically infects its natural host, the wildebeest (Connochaetes spp.). However, when transmitted to several susceptible species belonging to the order Artiodactyla, AlHV-1 is responsible for the induction of a lethal lymphoproliferative disease, named wildebeest-derived malignant catarrhal fever (WD-MCF). Understanding the pathogenic mechanisms responsible for the induction of WD-MCF is important to better control the risks of transmission and disease development in susceptible species. The aim of this review is to synthesize the current knowledge on WD-MCF with a particular focus on the mechanisms by which AlHV-1 induces the disease. We discuss the potential mechanisms of pathogenesis from viral entry into the host to the maintenance of viral genomes in infected CD8+ T lymphocytes, and we present current hypotheses to explain how AlHV-1 infection induces a peripheral T cell lymphoma-like disease.

A Vaccine Targeting Ovine Herpesvirus 2 Glycoprotein B Protects against Sheep-Associated Malignant Catarrhal Fever.

Malignant catarrhal fever (MCF) is a complex and often fatal disease of ungulates. Effective vaccines are needed to avoid MCF outbreaks and mitigate losses. This study aimed to evaluate a sheep-associated MCF (SA-MCF) vaccine candidate targeting ovine herpesvirus 2 (OvHV-2) glycoprotein B (gB). Rabbits were used as a laboratory animal model to test the safety, immunogenicity, and protective efficacy of a chimeric virus consisting of a recombinant, non-pathogenic strain of alcelaphine herpesvirus-1 encoding OvHV-2 ORF8 to express gB (AlHV-1∆ORF73/OvHV-2-ORF8). Viral-vectored immunizations were performed by using the AlHV-1∆ORF73/OvHV-2-ORF8 chimera alone or as a DNA prime (OvHV-2-ORF8)-virus boost regimen. The viral vector was inoculated by intravenous or intramuscular routes and the DNA was delivered by intradermal shots using a gene gun. The vaccine candidates were deemed safe as no clinical signs were observed following any of the immunizations. Anti-OvHV-2 gB antibodies with neutralizing activity were induced by all immunogens. At three weeks post-final immunization, all animals were challenged intranasally with a lethal dose of OvHV-2. MCF protection rates ranging from 66.7% to 71.4% were observed in vaccinated rabbits, while all mock-vaccinated animals developed the disease. The significant protective efficacy obtained with the vaccine platforms tested in this study encourages further trials in relevant livestock species, such as cattle and bison.

Elp3-mediated codon-dependent translation promotes mTORC2 activation and regulates macrophage polarization.

Macrophage polarization is a process whereby macrophages acquire distinct effector states (M1 or M2) to carry out multiple and sometimes opposite functions. We show here that translational reprogramming occurs during macrophage polarization and that this relies on the Elongator complex subunit Elp3, an enzyme that modifies the wobble uridine base U34 in cytosolic tRNAs. Elp3 expression is downregulated by classical M1-activating signals in myeloid cells, where it limits the production of pro-inflammatory cytokines via FoxO1 phosphorylation, and attenuates experimental colitis in mice. In contrast, alternative M2-activating signals upregulate Elp3 expression through a PI3K- and STAT6-dependent signaling pathway. The metabolic reprogramming linked to M2 macrophage polarization relies on Elp3 and the translation of multiple candidates, including the mitochondrial ribosome large subunit proteins Mrpl3, Mrpl13, and Mrpl47. By promoting translation of its activator Ric8b in a codon-dependent manner, Elp3 also regulates mTORC2 activation. Elp3 expression in myeloid cells further promotes Wnt-driven tumor initiation in the intestine by maintaining a pool of tumor-associated macrophages exhibiting M2 features. Collectively, our data establish a functional link between tRNA modifications, mTORC2 activation, and macrophage polarization.

Laboratory modelling of urban flooding.

This paper presents two datasets obtained from laboratory experiments of urban flooding in a street network performed at the University of Liège. The experimental model represents a part of a synthetic urban district that consists of three inlets, three outlets and several three- and four- branches crossroads. The following experimental data was produced: (i) dataset 1: time-series of flow depths at model inlets and time-series of discharges at model outlets for a two-branch junction model, a two-branch bifurcation model and a district model. The datasets were generated by varying the upstream and downstream boundary conditions, i.e. flooding conditions; (ii) dataset 2 includes the same data type as dataset 1 complemented by 2D surface velocity measured using the non-intrusive LSPIV technique for eight urban form configurations in the district model. The collected data enable improving the understanding of the effect of urban forms on the urban flood processes. These two datasets are valuable for validating and improving numerical or analytical models of urban flooding and may contribute to flood risk management and flood-resilient urban design.

Development of a Specific Anti-capsid Antibody- and Magnetic Bead-Based Immunoassay to Detect Human Norovirus Particles in Stool Samples and Spiked Mussels via Flow Cytometry.

Human noroviruses impose a considerable health burden globally. Here, a flow cytometry approach designed for their detection in biological waste and food samples was developed using antibody-coated magnetic beads. Antipeptide antibodies against murine norovirus and various human norovirus genotypes were generated for capture and coated onto magnetic beads. A flow cytometry assay was then implemented to detect bead-bound human norovirus GI.3 in patient stool samples and in norovirus-spiked mussel digestive tissues. The detection limit for stool samples was 105 gc/mL, thus bettering detection limits of commercially available norovirus diagnosis quick kits of 100-fold; the detection limit in spiked mussels however was ten-fold higher than in stool samples. Further assays showed a decrease in fluorescence intensity for heat- or UV-inactivated virus particles. Overall, we demonstrate the application of a flow cytometry approach for direct detection of small non-enveloped virus particles such as noroviruses. An adaptation of the technology to routine diagnostics has the potential to contribute a rapid and sensitive tool to norovirus outbreak investigations. Further improvements to the method, notably decreasing the detection limit of the approach, may allow the analysis of naturally contaminated food and environmental samples.

Il4ra-independent vaginal eosinophil accumulation following helminth infection exacerbates epithelial ulcerative pathology of HSV-2 infection.

How helminths influence the pathogenesis of sexually transmitted viral infections is not comprehensively understood. Here, we show that an acute helminth infection (Nippostrongylus brasiliensis [Nb]) induced a type 2 immune profile in the female genital tract (FGT). This leads to heightened epithelial ulceration and pathology in subsequent herpes simplex virus (HSV)-2 infection. This was IL-5-dependent but IL-4 receptor alpha (Il4ra) independent, associated with increased FGT eosinophils, raised vaginal IL-33, and enhanced epithelial necrosis. Vaginal eosinophil accumulation was promoted by IL-33 induction following targeted vaginal epithelium damage from a papain challenge. Inhibition of IL-33 protected against Nb-exacerbated HSV-2 pathology. Eosinophil depletion reduced IL-33 release and HSV-2 ulceration in Nb-infected mice. These findings demonstrate that Nb-initiated FGT eosinophil recruitment promotes an eosinophil, IL-33, and IL-5 inflammatory circuit that enhances vaginal epithelial necrosis and pathology following HSV-2 infection. These findings identify a mechanistic framework as to how helminth infections can exacerbate viral-induced vaginal pathology.

Nappe oscillations on free-overfall structures, data from laboratory experiments.

This paper presents a dataset obtained from fifty-two laboratory experiments of nappe oscillations on free overfall structures. Data were collected on two complementary experimental setups, each consisting of a linear weir model. The dataset covers test configurations involving varied geometric parameters (i.e. weir crest shape, weir width, fall height and nappe confinement) and inflow discharges. The following experimental data were produced: assessment of nappe oscillation occurrence and associated frequencies. The later measurements were performed using characterization techniques (image and sound analysis) developed for this research. Reuse of the collected data will support efforts to improve the understanding of the physical processes underpinning nappe oscillation and to validate numerical modelling of the phenomenon.

A gammaherpesvirus licenses CD8 T cells to protect the host from pneumovirus-induced immunopathologies.

Human respiratory syncytial virus (RSV) is a pneumovirus that causes severe infections in infants worldwide. Despite intensive research, safe and effective vaccines against RSV have remained elusive. The main reason is that RSV infection of children previously immunized with formalin-inactivated-RSV vaccines has been associated with exacerbated pathology, a phenomenon called RSV vaccine-enhanced respiratory disease. In parallel, despite the high RSV prevalence, only a minor proportion of children develop severe diseases. Interestingly, variation in the immune responses against RSV or following RSV vaccination could be linked with differences of exposure to microbes during childhood. Gammaherpesviruses (γHVs), such as the Epstein-Barr virus, are persistent viruses that deeply influence the immune system of their host and could therefore affect the development of pneumovirus-induced immunopathologies for the long term. Here, we showed that a previous ɣHV infection protects against both pneumovirus vaccine-enhanced disease and pneumovirus primary infection and that CD8 T cells are essential for this protection. These observations shed a new light on the understanding of pneumovirus-induced diseases and open new perspectives for the development of vaccine strategies.

Alcelaphine herpesvirus 1 genes A7 and A8 regulate viral spread and are essential for malignant catarrhal fever.

Alcelaphine herpesvirus 1 (AlHV-1) is a gammaherpesvirus that is carried asymptomatically by wildebeest. Upon cross-species transmission to other ruminants, including domestic cattle, AlHV-1 induces malignant catarrhal fever (MCF), which is a fatal lymphoproliferative disease resulting from proliferation and uncontrolled activation of latently infected CD8+ T cells. Two laboratory strains of AlHV-1 are used commonly in research: C500, which is pathogenic, and WC11, which has been attenuated by long-term maintenance in cell culture. The published genome sequence of a WC11 seed stock from a German laboratory revealed the deletion of two major regions. The sequence of a WC11 seed stock used in our laboratory also bears these deletions and, in addition, the duplication of an internal sequence in the terminal region. The larger of the two deletions has resulted in the absence of gene A7 and a large portion of gene A8. These genes are positional orthologs of the Epstein-Barr virus genes encoding envelope glycoproteins gp42 and gp350, respectively, which are involved in viral propagation and switching of cell tropism. To investigate the degree to which the absence of A7 and A8 participates in WC11 attenuation, recombinant viruses lacking these individual functions were generated in C500. Using bovine nasal turbinate and embryonic lung cell lines, increased cell-free viral propagation and impaired syncytia formation were observed in the absence of A7, whereas cell-free viral spread was inhibited in the absence of A8. Therefore, A7 appears to be involved in cell-to-cell viral spread, and A8 in viral cell-free propagation. Finally, infection of rabbits with either mutant did not induce the signs of MCF or the expansion of infected CD8+ T cells. These results demonstrate that A7 and A8 are both essential for regulating viral spread and suggest that AlHV-1 requires both genes to efficiently spread in vivo and reach CD8+ T lymphocytes and induce MCF.

Pre-conception maternal helminth infection transfers via nursing long-lasting cellular immunity against helminths to offspring.

Maternal immune transfer is the most significant source of protection from early-life infection, but whether maternal transfer of immunity by nursing permanently alters offspring immunity is poorly understood. Here, we identify maternal immune imprinting of offspring nursed by mothers who had a pre-conception helminth infection. Nursing of pups by helminth-exposed mothers transferred protective cellular immunity to these offspring against helminth infection. Enhanced control of infection was not dependent on maternal antibody. Protection associated with systemic development of protective type 2 immunity in T helper 2 (TH2) impaired IL-4Rα-/- offspring. This maternally acquired immunity was maintained into maturity and required transfer (via nursing) to the offspring of maternally derived TH2-competent CD4 T cells. Our data therefore reveal that maternal exposure to a globally prevalent source of infection before pregnancy provides long-term nursing-acquired immune benefits to offspring mediated by maternally derived pathogen-experienced lymphocytes.

Flow and detailed 3D morphodynamic data from laboratory experiments of fluvial dike breaching.

This paper presents a dataset obtained from fifty four laboratory experiments of the breaching of fluvial dikes due to flow overtopping. Data were collected on two complementary experimental setups, each consisting of a main channel representing the river, an erodible lateral dike and a floodplain. The dataset covers seven test series, involving varying hydraulic boundary conditions (e.g. inflow discharge, downstream boundary conditions), main channel dimensions, as well as bottom and dike material. The following experimental data were produced: time series of water levels in the main channel, time series of flow discharges in the main channel and through the breach, as well as high resolution 3D reconstructions of the breach geometry during its expansion. The latter measurements were performed using a novel non-intrusive laser profilometry technique developed for this research. Reuse of the collected data will support efforts to improve our understanding of the physical processes underpinning fluvial dike breaching. It will also enable benchmarking the accuracy of conceptual or detailed numerical models for the prediction of dike breaching, which is central to flood risk management.

Recruitment of hepatic macrophages from monocytes is independent of IL-4Rα but is associated with ablation of resident macrophages in schistosomiasis.

Alternatively activated Mφs (AAMφ) accumulate in hepatic granulomas during schistosomiasis and have been suggested to originate in the bone marrow. What is less understood is how these Mφ responses are regulated after S. mansoni infection. Here, we investigated the role of IL-4 receptor α-chain (IL-4Rα)-signalling in the dynamics of liver Mφ responses. We observed that IL-4Rα signalling was dispensable for the recruitment of Ly6Chi monocytes and for their conversion into F4/80hi CD64hi CD11bhi Mφ. Moreover, while IL-4Rα provided an AAMφ phenotype to liver F4/80hi CD64hi CD11bhi Mφ that was associated with regulation of granuloma formation, it was dispensable for host survival. Resident F4/80hi CD64hi CD11blo Mφ did not upregulate the AAMφ signature gene Ym1. Rather, resident Mφ nearly disappeared by week 8 after infection and artificial ablation of resident Mφ in CD169DTR mice did not affect the response to S. mansoni infection. Interestingly, ablation of CD169+ cells in naive mice resulted in the accumulation of F4/80hi CD64hi CD11bhi Mφ, which was amplified when ablation occurred during schistosomiasis. Altogether, our results suggest the ablation of resident KCs after S. mansoni infection to be associated with the recruitment and accumulation of F4/80hi CD64hi CD11bhi Mφ with lyz2-dependent IL-4Rα contributing to the regulation of granuloma inflammation but being dispensable for host survival.

Maternal helminth infections and the shaping of offspring immunity.

Helminth infections leave a long-lasting immunological footprint on their hosts. Clinical studies have provided first evidence that maternal helminth infections can result in an altered immune profile in their offspring which can potentially shape how they respond to conditions throughout life. This can relate to changes in offspring induction of immune responses against other diseases. However, whether these changes result in actual changes in offspring ability to control disease is unclear. Our understanding of which immune mechanisms are altered and how they are changed is limited. In this review, we highlight what we know from human and mouse studies about this important context of helminth exposure. Moreover, we discuss how mechanisms such as antibody transfer, antigen exposure, maternal cell uptake, chimerism and epigenetics are all likely to be functional contributors to the striking changes that are seen in offspring born or nursed by helminth exposed mothers.

Helminth-induced IL-4 expands bystander memory CD8+ T cells for early control of viral infection.

Infection with parasitic helminths can imprint the immune system to modulate bystander inflammatory processes. Bystander or virtual memory CD8+ T cells (TVM) are non-conventional T cells displaying memory properties that can be generated through responsiveness to interleukin (IL)-4. However, it is not clear if helminth-induced type 2 immunity functionally affects the TVM compartment. Here, we show that helminths expand CD44hiCD62LhiCXCR3hiCD49dlo TVM cells through direct IL-4 signaling in CD8+ T cells. Importantly, helminth-mediated conditioning of TVM cells provided enhanced control of acute respiratory infection with the murid gammaherpesvirus 4 (MuHV-4). This enhanced control of MuHV-4 infection could further be explained by an increase in antigen-specific CD8+ T cell effector responses in the lung and was directly dependent on IL-4 signaling. These results demonstrate that IL-4 during helminth infection can non-specifically condition CD8+ T cells, leading to a subsequently raised antigen-specific CD8+ T cell activation that enhances control of viral infection.