Search

Peer reviewed: True, EROS (essential for reactive oxygen species) protein is indispensable for expression of gp91phox, the catalytic core of the phagocyte NADPH oxidase. EROS deficiency in humans is a novel cause of the severe immunodeficiency, chronic granulomatous disease, but its mechanism of action was unknown until now. We elucidate the role of EROS, showing it acts at the earliest stages of gp91phox maturation. It binds the immature 58 kDa gp91phox directly, preventing gp91phox degradation and allowing glycosylation via the oligosaccharyltransferase machinery and the incorporation of the heme prosthetic groups essential for catalysis. EROS also regulates the purine receptors P2X7 and P2X1 through direct interactions, and P2X7 is almost absent in EROS-deficient mouse and human primary cells. Accordingly, lack of murine EROS results in markedly abnormal P2X7 signalling, inflammasome activation, and T cell responses. The loss of both ROS and P2X7 signalling leads to resistance to influenza infection in mice. Our work identifies EROS as a highly selective chaperone for key proteins in innate and adaptive immunity and a rheostat for immunity to infection. It has profound implications for our understanding of immune physiology, ROS dysregulation, and possibly gene therapy.

Visceral leishmaniasis is a deadly infectious disease caused by Leishmania donovani, a kinetoplastid parasite for which no licensed vaccine is available. To identify potential vaccine candidates, we systematically identified genes encoding putative cell surface and secreted proteins essential for parasite viability and host infection. We identified a protein encoded by

Trypanosoma vivaxis a unicellular hemoparasite, and a principal cause of animal African trypanosomiasis (AAT), a vector-borne and potentially fatal livestock disease across sub-Saharan Africa. Previously, we identified diverseT.vivax-specific genes that were predicted to encode cell surface proteins. Here, we examine the immune responses of naturally and experimentally infected hosts to these unique parasite antigens, to identify immunogens that could become vaccine candidates. Immunoprofiling of host serum shows that one particular family (Fam34) elicits a consistent IgG antibody response. This gene family, which we now callVivaxin, encodes at least 124 transmembrane glycoproteins that display quite distinct expression profiles and patterns of genetic variation. We focused on one gene (viv-β8) that encodes one particularly immunogenic vivaxin protein and which is highly expressed during infections but displays minimal polymorphism across the parasite population. Vaccination of mice with VIVβ8 adjuvanted with Quil-A elicits a strong, balanced immune response and delays parasite proliferation in some animals but, ultimately, it does not prevent disease. Although VIVβ8 is localized across the cell body and flagellar membrane, live immunostaining indicates that VIVβ8 is largely inaccessible to antibody in vivo. However, our phylogenetic analysis shows that vivaxin includes other antigens shown recently to induce immunity againstT.vivax. Thus, the introduction of vivaxin represents an important advance in our understanding of theT.vivaxcell surface. Besides being a source of proven and promising vaccine antigens, the gene family is clearly an important component of the parasite glycocalyx, with potential to influence host-parasite interactions.

Anti-TNFα and anti-IL-23 antibodies are highly effective therapies for Crohn’s disease or ulcerative colitis in a proportion of patients. V56B2 is a novel bispecific domain antibody in which a llama-derived IL-23p19-specific domain antibody, humanised and engineered for intestinal protease resistance, V900, was combined with a previously-described TNFα-specific domain antibody, V565. V56B2 contains a central protease-labile linker to create a single molecule for oral administration. Incubation of V56B2 with trypsin or human faecal supernatant resulted in a complete separation of the V565 and V900 monomers without loss of neutralising potency. Following oral administration of V900 and V565 in mice, high levels of each domain antibody were detected in the faeces, demonstrating stability in the intestinal milieu. In ex vivo cultures of colonic biopsies from IBD patients, treatment with V565 or V900 inhibited tissue phosphoprotein levels and with a combination of the two, inhibition was even greater. These results support further development of V56B2 as an oral therapy for IBD with improved safety and efficacy in a greater proportion of patients as well as greater convenience for patients compared with traditional monoclonal antibody therapies.

Summary Signalling lymphocyte activation molecule family member 9 (SLAMF9) is an orphan receptor of the CD2/SLAM family of leucocyte surface proteins. Examination of SLAMF9 expression and function indicates that SLAMF9 promotes inflammation by specialized subsets of antigen‐presenting cells. Within healthy liver and circulating mouse peripheral blood mononuclear cells, SLAMF9 is expressed on CD11b+, Ly6C−, CD11clow, F4/80low, MHC‐II+, CX3CR1+ mononuclear phagocytes as well as plasmacytoid dendritic cells. In addition, SLAMF9 can be found on peritoneal B1 cells and small (F4/80low), but not large (F4/80high), peritoneal macrophages. Upon systemic challenge with Salmonella enterica Typhimurium, Slamf9−/− mice were impaired in their ability to clear the infection from the liver. In humans, SLAMF9 is up‐regulated upon differentiation of monocytes into macrophages, and lipopolysaccharide stimulation of PMA‐differentiated, SLAMF9 knockdown THP‐1 cells showed an essential role of SLAMF9 in production of granulocyte–macrophage colony‐stimulating factor, tumour necrosis factor‐α, and interleukin‐1β. Taken together, these data implicate SLAMF9 in the initiation of inflammation and clearance of bacterial infection., Signalling lymphocyte activation molecule family member 9 (SLAMF9) is a cell surface receptor found on specialized subsets of mononuclear phagocytes. Deficiencies in SLAMF9 expression lead to altered pro‐inflammatory cytokine production in human cells and reduced capacity to clear Salmonella infection in mice.

The discovery of a Salmonella-targeting phage from the waterways of the United Kingdom provided an opportunity to address the mechanism by which Chi-like bacteriophage (phage) engages with bacterial flagellae. The long tail fibre seen on Chi-like phages has been proposed to assist the phage particle in docking to a host cell flagellum, but the identity of the protein that generates this fibre was unknown. We present the results from genome sequencing of this phage, YSD1, confirming its close relationship to the original Chi phage and suggesting candidate proteins to form the tail structure. Immunogold labelling in electron micrographs revealed that YSD1_22 forms the main shaft of the tail tube, while YSD1_25 forms the distal part contributing to the tail spike complex. The long curling tail fibre is formed by the protein YSD1_29, and treatment of phage with the antibodies that bind YSD1_29 inhibits phage infection of Salmonella. The host range for YSD1 across Salmonella serovars is broad, but not comprehensive, being limited by antigenic features of the flagellin subunits that make up the Salmonella flagellum, with which YSD1_29 engages to initiate infection.

SummaryInterferon induced transmembrane protein 3 (IFITM3) is an important viral restriction factor in viral pathogenesis that also exhibits poorly understood immune regulatory functions. Here, using human and mouse models, we demonstrate that IFITM3 regulates MyD88-dependent TLR-mediated cytokine production following dendritic cell exposure to cytomegalovirus (CMV), and this process limits viral pathogenesisin vivo. IFITM3 also restricted pro-inflammatory (IL-6) cytokine production in response to influenza. IFITM3 bound to and promoted ubiquitination and proteasomal degradation of the reticulon 4 isoform Nogo-B. We reveal that Nogo-B mediates TLR-dependent pro-inflammatory cytokine production and promotes viral pathogenesisin vivo, and this process involved alteration of TLR dynamics. The anti-inflammatory function of IFITM3 was intrinsically linked to its ability to regulate Nogo-B. Thus, we uncover Nogo-B as an unappreciated driver of viral pathogenesis and highlight a novel immune regulatory pathway where IFITM3 fine-tunes TLR responsiveness of myeloid cells to viral stimulation.

Experimental mouse models are central to basic biomedical research; however, variability exists across genetically identical mice and mouse facilities making comparisons difficult. Whether specific indigenous gut bacteria drive immunophenotypic variability in mouse models of human disease remains poorly understood. We performed a large-scale experiment using 579 genetically identical laboratory mice from a single animal facility, designed to identify the causes of disease variability in the widely used dextran sulphate sodium mouse model of inflammatory bowel disease. Commonly used treatment endpoint measures-weight loss and intestinal pathology-showed limited correlation and varied across mouse lineages. Analysis of the gut microbiome, coupled with machine learning and targeted anaerobic culturing, identified and isolated two previously undescribed species, Duncaniella muricolitica and Alistipes okayasuensis, and demonstrated that they exert dominant effects in the dextran sulphate sodium model leading to variable treatment endpoint measures. We show that the identified gut microbial species are common, but not ubiquitous, in mouse facilities around the world, and suggest that researchers monitor for these species to provide experimental design opportunities for improved mouse models of human intestinal diseases.

Trypanosomes are protozoan parasites that cause infectious diseases including human African trypanosomiasis (sleeping sickness), and nagana in economically-important livestock animals1,2. An effective vaccine against trypanosomes would be an important control tool, but the parasite has evolved sophisticated immunoprotective mechanisms including antigenic variation3 that present an apparently insurmountable barrier to vaccination. Here we show using a systematic genome-led vaccinology approach4 and a murine model of Trypanosoma vivax infection5 that protective invariant subunit vaccine antigens can be identified. Vaccination with a single recombinant protein comprising the extracellular region of a conserved cell surface protein localised to the flagellum membrane termed “invariant flagellum antigen from T. vivax” (IFX) induced long-lasting protection. Immunity was passively transferred with immune serum, and recombinant monoclonal antibodies to IFX could induce sterile protection and revealed multiple mechanisms of antibody-mediated immunity, including a major role for complement. Our discovery identifies a vaccine candidate for an important parasitic disease that has constrained the socioeconomic development of sub-Saharan African countries6 and challenges long-held views that vaccinating against trypanosome infections cannot be achieved.

The mammalian gut microbiota consists of hundreds of anaerobic bacterial species that shape intestinal homeostasis and influence host immune responses. Although the causal roles of specific human gut bacterial species in health and disease are emerging, the role of indigenous gut bacteria in driving immunophenotypic variability in mouse models of human disease remains poorly understood. We performed a large-scale experiment using 579 laboratory mice designed to identify and validate the causes of disease variability in the widely used dextran sulphate sodium (DSS) mouse model of inflammatory bowel disease. Using microbiome analysis, coupled with machine learning and targeted anaerobic culturing, we identified and isolated the novel gut pathobiont species Duncaniella muricolitica and Alistipes okayasuensis and fulfilled Koch’s postulates in mice to show that each pathobiont exerts dominant effects in the DSS model leading to variable treatment responses. We show these pathobiont species are common, but not ubiquitous, in mouse facilities around the world, raising experimental design opportunities for improved mouse models of human intestinal diseases.

In spite of growing evidence supporting the occurrence of complex interactions between Schistosoma and gut bacteria in mice and humans, no data is yet available on whether worm-mediated changes in microbiota composition are dependent on the baseline gut microbial profile of the vertebrate host. In addition, the impact of such changes on the susceptibility to, and pathophysiology of, schistosomiasis remains largely unexplored. In this study, mice colonized with gut microbial populations from a human donor (HMA mice), as well as microbiota-wild type (WT) animals, were infected with Schistosoma mansoni, and alterations of their gut microbial profiles at 50 days post-infection were compared to those occurring in uninfected HMA and WT rodents, respectively. Significantly higher worm and egg burdens, together with increased specific antibody responses to parasite antigens, were observed in HMA compared to WT mice. These differences were associated to extensive dissimilarities between the gut microbial profiles of each HMA and WT groups of mice at baseline; in particular, the gut microbiota of HMA animals was characterized by low microbial alpha diversity and expanded Proteobacteria, as well as by the absence of putative immunomodulatory bacteria (e.g. Lactobacillus). Furthermore, differences in infection-associated changes in gut microbiota composition were observed between HMA and WT mice. Altogether, our findings support the hypothesis that susceptibility to S.mansoni infection in mice is partially dependent on the composition of the host baseline microbiota. Moreover, this study highlights the applicability of HMA mouse models to address key biological questions on host-parasite-microbiota relationships in human helminthiases.

Trypanosomes are protozoan parasites that cause infectious diseases, including African trypanosomiasis (sleeping sickness) in humans and nagana in economically important livestock1,2. An effective vaccine against trypanosomes would be an important control tool, but the parasite has evolved sophisticated immunoprotective mechanisms—including antigenic variation3—that present an apparently insurmountable barrier to vaccination. Here we show, using a systematic genome-led vaccinology approach and a mouse model of Trypanosoma vivax infection4, that protective invariant subunit vaccine antigens can be identified. Vaccination with a single recombinant protein comprising the extracellular region of a conserved cell-surface protein that is localized to the flagellum membrane (which we term ‘invariant flagellum antigen from T. vivax’) induced long-lasting protection. Immunity was passively transferred with immune serum, and recombinant monoclonal antibodies to this protein could induce sterile protection and revealed several mechanisms of antibody-mediated immunity, including a major role for complement. Our discovery identifies a vaccine candidate for an important parasitic disease that has constrained socioeconomic development in countries in sub-Saharan Africa5, and provides evidence that highly protective vaccines against trypanosome infections can be achieved. Systemic genome-led vaccinology and a mouse model of Trypanosoma vivax infection identify protective invariant subunit vaccine antigens, and demonstrate the possibility of generating effective vaccines that induce long-lasting protection against trypanosome infections.

FBXO7 encodes an F box containing protein that interacts with multiple partners to facilitate numerous cellular processes and has a canonical role as part of an SCF E3 ubiquitin ligase complex. Mutation of FBXO7 is responsible for an early onset Parkinsonian pyramidal syndrome and genome-wide association studies have linked variants in FBXO7 to erythroid traits. A putative orthologue in Drosophila, nutcracker, has been shown to regulate the proteasome, and deficiency of nutcracker results in male infertility. Therefore, we reasoned that modulating Fbxo7 levels in a murine model could provide insights into the role of this protein in mammals. We used a targeted gene trap model which retained 4 - 16 % residual gene expression and assessed the sensitivity of phenotypic traits to gene dosage. Fbxo7 hypomorphs showed regenerative anaemia associated with a shorter erythrocyte half-life, and male mice were infertile. Alterations to T cell phenotypes were also observed, which intriguingly were both T cell intrinsic and extrinsic. Hypomorphic mice were also sensitive to infection with Salmonella, succumbing to a normally sublethal challenge. Despite these phenotypes, Fbxo7 hypomorphs were produced at a normal Mendelian ratio with a normal lifespan and no evidence of neurological symptoms. These data suggest that erythrocyte survival, T cell development and spermatogenesis are particularly sensitive to Fbxo7 gene dosage.

Salmonella Typhimurium is a major diarrheal pathogen and associated with invasive nontyphoid Salmonella (iNTS) disease in vulnerable populations. We present the first characterization of iNTS organisms in Southeast Asia and describe a different evolutionary trajectory from that of organisms causing iNTS in sub-Saharan Africa. In Vietnam, the globally distributed monophasic variant of Salmonella Typhimurium, the serovar I:4,[5],12:i:− ST34 clone, has reacquired a phase 2 flagellum and gained a multidrug-resistant plasmid to become associated with iNTS disease in HIV-infected patients. We document distinct communities of S. Typhimurium and I:4,[5],12:i:− in animals and humans in Vietnam, despite the greater mixing of these host populations here. These data highlight the importance of whole-genome sequencing surveillance in a One Health context in understanding the evolution and spread of resistant bacterial infections., Nontyphoidal Salmonella (NTS), particularly Salmonella enterica serovar Typhimurium, is among the leading etiologic agents of bacterial enterocolitis globally and a well-characterized cause of invasive disease (iNTS) in sub-Saharan Africa. In contrast, S. Typhimurium is poorly defined in Southeast Asia, a known hot spot for zoonotic disease with a recently described burden of iNTS disease. Here, we aimed to add insight into the epidemiology and potential impact of zoonotic transfer and antimicrobial resistance (AMR) in S. Typhimurium associated with iNTS and enterocolitis in Vietnam. We performed whole-genome sequencing and phylogenetic reconstruction on 85 human (enterocolitis, carriage, and iNTS) and 113 animal S. Typhimurium isolates isolated in Vietnam. We found limited evidence for the zoonotic transmission of S. Typhimurium. However, we describe a chain of events where a pandemic monophasic variant of S. Typhimurium (serovar I:4,[5],12:i:− sequence type 34 [ST34]) has been introduced into Vietnam, reacquired a phase 2 flagellum, and acquired an IncHI2 multidrug-resistant plasmid. Notably, these novel biphasic ST34 S. Typhimurium variants were significantly associated with iNTS in Vietnamese HIV-infected patients. Our study represents the first characterization of novel iNTS organisms isolated outside sub-Saharan Africa and outlines a new pathway for the emergence of alternative Salmonella variants into susceptible human populations.

TNFα is an important cytokine in inflammatory bowel disease. V565 is a novel anti-TNFα domain antibody developed for oral administration in IBD patients, derived from a llama domain antibody and engineered to enhance intestinal protease resistance. V565 activity was evaluated in TNFα-TNFα receptor-binding ELISAs as well as TNFα responsive cellular assays and demonstrated neutralisation of both soluble and membrane TNFα with potencies similar to those of adalimumab. Although sensitive to pepsin, V565 retained activity after lengthy incubations with trypsin, chymotrypsin, and pancreatin, as well as mouse small intestinal and human ileal and faecal supernatants. In orally dosed naïve and DSS colitis mice, high V565 concentrations were observed in intestinal contents and faeces and immunostaining revealed V565 localisation in mouse colon tissue. V565 was detected by ELISA in post-dose serum of colitis mice, but not naïve mice, demonstrating penetration of disrupted epithelium. In an ex vivo human IBD tissue culture model, V565 inhibition of tissue phosphoprotein levels and production of inflammatory cytokine biomarkers was similar to infliximab, demonstrating efficacy when present at the disease site. Taken together, results of these studies provide confidence that oral V565 dosing will be therapeutic in IBD patients where the mucosal epithelial barrier is compromised.

The interferon-inducible transmembrane (IFITM) family of proteins has been shown to restrict a broad range of viruses in vitro and in vivo by halting progress through the late endosomal pathway. Further, single nucleotide polymorphisms (SNPs) in its sequence have been linked with risk of developing severe influenza virus infections in humans. The number of viruses restricted by this host protein has continued to grow since it was first demonstrated as playing an antiviral role; all of which enter cells via the endosomal pathway. We therefore sought to test the limits of antimicrobial restriction by Ifitm3 using a knockout mouse model. We showed that Ifitm3 does not impact on the restriction or pathogenesis of bacterial (Salmonella typhimurium, Citrobacter rodentium, Mycobacterium tuberculosis) or protozoan (Plasmodium berghei) pathogens, despite in vitro evidence. However, Ifitm3 is capable of restricting respiratory syncytial virus (RSV) in vivo either through directly restricting RSV cell infection, or by exerting a previously uncharacterised function controlling disease pathogenesis. This represents the first demonstration of a virus that enters directly through the plasma membrane, without the need for the endosomal pathway, being restricted by the IFITM family; therefore further defining the role of these antiviral proteins.

Failure to maintain a normal in vivo erythrocyte half-life results in the development of hemolytic anemia. Half-life is affected by numerous factors, including energy balance, electrolyte gradients, reactive oxygen species, and membrane plasticity. The heterotrimeric AMP-activated protein kinase (AMPK) is an evolutionarily conserved serine/threonine kinase that acts as a critical regulator of cellular energy balance. Previous roles for the alpha 1 and gamma 1 subunits in the control of erythrocyte survival have been reported. In the work described here, we studied the role of the beta 1 subunit in erythrocytes and observed microcytic anemia with compensatory extramedullary hematopoiesis together with splenomegaly and increased osmotic resistance., Highlights • Prkab1tm1b/tm1b mice were generated and phenotyped. • Prkab1tm1b/tm1b mice presented with microcytic anemia. • Erythrocytes lacking Prkab1 have a variable morphologic appearance. • Prkab1-deficient erythrocytes have increased osmotic resistance. • The importance of the beta 1 isoform in erythrocyte integrity is discussed.

Background Reactive oxygen species (ROS), generated via the phagocyte NADPH oxidase cytochrome b558, are essential for effective immune responses to common and serious pathogens. The phagocyte NADPH oxidase is a multisubunit protein complex and deficiency of either the membrane bound or cytoplasmic components leads to chronic granulomatous disease, a serious and often fatal illness characterised by recurrent infections and autoimmunity. Moreover, abnormal generation of ROS has been implicated in the pathogenesis of multigenic autoimmune diseases such as systemic lupus erythematosus. Eros (essential for reactive oxygen species), encoded by bc017643 , is a novel transmembrane protein that is highly expressed in the immune system and highly conserved in evolution but has no previously identified function. Eros is an orthologue of the plant protein Ycf4, necessary for expression of proteins of the photosynthetic photosystem 1 complex, an NADPH oxio-reductase complex. We elucidated its role in infection in mice. Methods ROS are essential for host defence against the serious bacterial pathogen Salmonella enterica serovar Typhimurium. We screened individual knockout mice (Wellcome Trust Knockout mouse project) for susceptibility to salmonella infection. Having identified mice deficient in Eros as being highly susceptible to salmonella, we used ex-vivo approaches including reactive oxygen burst assays and western blot, to characterise their defect further. Findings We found that Eros was essential for host defence to infection. Eros was crucial for generating reactive oxygen species through regulation of the essential NADPH oxidase components, gp91 and p22. Eros-deficient mice expressed almost no gp91 and p22 in neutrophils and macrophages secondary to accelerated degradation in the absence of Eros. As a result Eros-deficient mice died rapidly after infection with salmonella or listeria. Eros also regulated the ROS-dependent formation of neutrophil extracellular traps and melanoma metastases. Interpretation We have found a a key role for Eros in regulating host defence. The finding that Eros-deficient mice lack gp91 and p22 at the protein, though not mRNA, level shows how these key components of the reatcive oxygen burst are protected from degradation and furthers our understanding of reactive oxygen burst biology. Eros is highly conserved between mouse and man so it is likely that it also has a crucial role in human immunity. Funding Wellcome Trust, Academy of Medical Sciences starter grant.