Katherine Harcourt

Epithelial IL-22RA1-Mediated Fucosylation Promotes Intestinal Colonization Resistance to an Opportunistic Pathogen

Summary Our intestinal microbiota harbors a diverse microbial community, often containing opportunistic bacteria with virulence potential. However, mutualistic host-microbial interactions prevent disease by opportunistic pathogens through poorly understood mechanisms. We show that the epithelial interleukin-22 receptor IL-22RA1 protects against lethal Citrobacter rodentium infection and chemical-induced colitis by promoting colonization resistance against an intestinal opportunistic bacterium, Enterococcus faecalis. Susceptibility of Il22ra1−/− mice to C. rodentium was associated with preferential expansion and epithelial translocation of pathogenic E. faecalis during severe microbial dysbiosis and was ameloriated with antibiotics active against E. faecalis. RNA sequencing analyses of primary colonic organoids showed that IL-22RA1 signaling promotes intestinal fucosylation via induction of the fucosyltransferase Fut2. Additionally, administration of fucosylated oligosaccharides to C. rodentium-challenged Il22ra1−/− mice attenuated infection and promoted E. faecalis colonization resistance by restoring the diversity of anaerobic commensal symbionts. These results support a model whereby IL-22RA1 enhances host-microbiota mutualism to limit detrimental overcolonization by opportunistic pathogens.

Signatures of Adaptation in Human Invasive Salmonella Typhimurium ST313 Populations from Sub-Saharan Africa

Two lineages of Salmonella enterica serovar Typhimurium (S. Typhimurium) of multi-locus sequence type ST313 have been linked with the emergence of invasive Salmonella disease across sub-Saharan Africa. The expansion of these lineages has a temporal association with the HIV pandemic and antibiotic usage. We analysed the whole genome sequence of 129 ST313 isolates representative of the two lineages and found evidence of lineage-specific genome degradation, with some similarities to that observed in S. Typhi. Individual ST313 S. Typhimurium isolates exhibit a distinct metabolic signature and modified enteropathogenesis in both a murine and cattle model of colitis, compared to S. Typhimurium outside of the ST313 lineages. These data define phenotypes that distinguish ST313 isolates from other S. Typhimurium and may represent adaptation to a distinct pathogenesis and lifestyle linked to an-immuno-compromised human population.

Defining the Range of Pathogens Susceptible to Ifitm3 Restriction Using a Knockout Mouse Model

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.

Ubiquitin Specific Protease 21 Is Dispensable for Normal Development, Hematopoiesis and Lymphocyte Differentiation

USP21 is a ubiquitin specific protease that catalyzes protein deubiquitination, however the identification of its physiological substrates remains challenging. USP21 is known to deubiquitinate transcription factor GATA3 and death-domain kinase RIPK1 in vitro, however the in vivo settings where this regulation plays a biologically significant role remain unknown. In order to determine whether USP21 is an essential and non-redundant regulator of GATA3 or RIPK1 activity in vivo, we characterized Usp21-deficient mice, focusing on mouse viability and development, hematopoietic stem cell function, and lymphocyte differentiation. The Usp21-knockout mice were found to be viable and fertile, with no significant dysmorphology, in contrast to the GATA3 and RIPK1 knockout lines that exhibit embryonic or perinatal lethality. Loss of USP21 also had no effect on hematopoietic stem cell function, lymphocyte development, or the responses of antigen presenting cells to TLR and TNFR stimulation. GATA3 levels in hematopoietic stem cells or T lymphocytes remained unchanged. We observed that aged Usp21-knockout mice exhibited spontaneous T cell activation, however this was not linked to altered GATA3 levels in the affected cells. The contrast in the phenotype of the Usp21-knockout line with the previously characterized GATA3 and RIPK1 knockout mice strongly indicates that USP21 is redundant for the regulation of GATA3 and RIPK1 activity during mouse development, in hematopoietic stem cells, and in lymphocyte differentiation. The Usp21-deficient mouse line characterized in this study may serve as a useful tool for the future characterization of USP21 physiological functions.

Eros is a novel transmembrane protein that controls the phagocyte respiratory burst and is essential for innate immunity

The phagocyte respiratory burst is crucial for innate immunity. The transfer of electrons to oxygen is mediated by a membrane-bound heterodimer, comprising gp91phox and p22phox subunits. Deficiency of either subunit leads to severe immunodeficiency. We describe Eros (essential for reactive oxygen species), a protein encoded by the previously undefined mouse gene bc017643, and show that it is essential for host defense via the phagocyte NAPDH oxidase. Eros is required for expression of the NADPH oxidase components, gp91phox and p22phox. Consequently, Eros-deficient mice quickly succumb to infection. Eros also contributes to the formation of neutrophil extracellular traps (NETS) and impacts on the immune response to melanoma metastases. Eros is an ortholog of the plant protein Ycf4, which is necessary for expression of proteins of the photosynthetic photosystem 1 complex, itself also an NADPH oxio-reductase. We thus describe the key role of the previously uncharacterized protein Eros in host defense.

The antiviral restriction factor IFN-induced transmembrane protein 3 prevents cytokine-driven CMV pathogenesis

The antiviral restriction factor IFN-induced transmembrane protein 3 (IFITM3) inhibits cell entry of a number of viruses, and genetic diversity within IFITM3 determines susceptibility to viral disease in humans. Here, we used the murine CMV (MCMV) model of infection to determine that IFITM3 limits herpesvirus-associated pathogenesis without directly preventing virus replication. Instead, IFITM3 promoted antiviral cellular immunity through the restriction of virus-induced lymphopenia, apoptosis-independent NK cell death, and loss of T cells. Viral disease in Ifitm3–/– mice was accompanied by elevated production of cytokines, most notably IL-6. IFITM3 inhibited IL-6 production by myeloid cells in response to replicating and nonreplicating virus as well as following stimulation with the TLR ligands Poly(I:C) and CpG. Although IL-6 promoted virus-specific T cell responses, uncontrolled IL-6 expression in Ifitm3–/– mice triggered the loss of NK cells and subsequently impaired control of MCMV replication. Thus, IFITM3 represents a checkpoint regulator of antiviral immunity that controls cytokine production to restrict viral pathogenesis. These data suggest the utility of cytokine-targeting strategies in the treatment of virus-infected individuals with impaired IFITM3 activity.

Preclinical Development of a Novel, Orally-Administered Anti-Tumour Necrosis Factor Domain Antibody for the Treatment of Inflammatory Bowel Disease

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 AMP-activated protein kinase beta 1 subunit modulates erythrocyte integrity.

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.

Interferon lambda is required for interferon gamma-expressing NK cell responses but does not afford antiviral protection during acute and persistent murine cytomegalovirus infection

Interferon lambda (IFNλ) is a group of cytokines that belong to the IL-10 family. They exhibit antiviral activities against certain viruses during infection of the liver and mucosal tissues. Here we report that IFNλ restricts in vitro replication of the β-herpesvirus murine cytomegalovirus (mCMV). However, IFNλR1-deficient (Ifnλr1-/-) mice were not preferentially susceptible to mCMV infection in vivo during acute infection after systemic or mucosal challenge, or during virus persistence in the mucosa. Instead, our studies revealed that IFNλ influences NK cell responses during mCMV infection. Ifnλr1-/- mice exhibited defective development of conventional interferon-gamma (IFNγ)-expressing NK cells in the spleen during mCMV infection whereas accumulation of granzyme B-expressing NK cells was unaltered. In vitro, development of splenic IFNγ+ NK cells following stimulation with IL-12 or, to a lesser extent, IL-18 was abrogated by IFNλR1-deficiency. Thus, IFNλ regulates NK cell responses during mCMV infection and restricts virus replication in vitro but is redundant in the control of acute and persistent mCMV replication within mucosal and non-mucosal tissues.

New Variant of Multidrug-Resistant Salmonella enterica Serovar Typhimurium Associated with Invasive Disease in Immunocompromised Patients in Vietnam

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. ABSTRACT 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. IMPORTANCE 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.