Toxoplasma gondii‐infected natural killer cells display a hypermotility phenotype in vivo

Toxoplasma gondii is a highly prevalent intracellular protozoan parasite that causes severe disease in congenitally infected or immunocompromised hosts. T. gondii is capable of invading immune cells and it has been suggested that the parasite harnesses the migratory pathways of these cells to spread through the body. Although in vitro evidence suggests that the parasite further enhances its spread by inducing a hypermotility phenotype in parasitized immune cells, in vivo evidence for this phenomenon is scarce. Here we use a physiologically relevant oral model of T. gondii infection, in conjunction with two‐photon laser scanning microscopy, to address this issue. We found that a small proportion of natural killer (NK) cells in mesenteric lymph nodes contained parasites. Compared with uninfected ‘bystander’ NK cells, these infected NK cells showed faster, more directed and more persistent migratory behavior. Consistent with this, infected NK cells showed impaired spreading and clustering of the integrin, LFA‐1, when exposed to plated ligands. Our results provide the first evidence for a hypermigratory phenotype in T. gondii‐infected NK cells in vivo, providing an anatomical context for understanding how the parasite manipulates immune cell motility to spread through the host

A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF-β– and retinoic acid–dependent mechanism

Foxp3+ regulatory T (T reg) cells play a key role in controlling immune pathological re actions. Many develop their regulatory activity in the thymus, but there is also evidence for development of Foxp3+ T reg cells from naive precursors in the periphery. Recent studies have shown that transforming growth factor (TGF)-β can promote T reg cell development in culture, but little is known about the cellular and molecular mechanisms that mediate this pathway under more physiological conditions. Here, we show that after antigen activation in the intestine, naive T cells acquire expression of Foxp3. Moreover, we identify a population of CD103+ mesenteric lymph node dendritic cells (DCs) that induce the devel opment of Foxp3+ T reg cells. Importantly, promotion of T reg cell responses by CD103+ DCs is dependent on TGF-β and the dietary metabolite, retinoic acid (RA). These results newly identify RA as a cofactor in T reg cell generation, providing a mechanism via which functionally specialized gut-associated lymphoid tissue DCs can extend the repertoire of T reg cells focused on the intestine

Essential role for CD103 in the T cell–mediated regulation of experimental colitis

The integrin CD103 is highly expressed at mucosal sites, but its role in mucosal immune regulation remains poorly understood. We have analyzed the functional role of CD103 in intestinal immune regulation using the T cell transfer model of colitis. Our results show no mandatory role for CD103 expression on T cells for either the development or CD4+CD25+ regulatory T (T reg) cell–mediated control of colitis. However, wild-type CD4+CD25+ T cells were unable to prevent colitis in immune-deficient recipients lacking CD103, demonstrating a nonredundant functional role for CD103 on host cells in T reg cell–mediated intestinal immune regulation. Non–T cell expression of CD103 is restricted primarily to CD11chighMHC class IIhigh dendritic cells (DCs). This DC population is present at a high frequency in the gut-associated lymphoid tissue and appears to mediate a distinct functional role. Thus, CD103+ DCs, but not their CD103− counterparts, promoted expression of the gut-homing receptor CCR9 on T cells. Conversely, CD103− DCs promoted the differentiation of IFN-γ–producing T cells. Collectively, these data suggest that CD103+ and CD103− DCs represent functionally distinct subsets and that CD103 expression on DCs influences the balance between effector and regulatory T cell activity in the intestine

Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans

A functionally distinct subset of CD103+ dendritic cells (DCs) has recently been identified in murine mesenteric lymph nodes (MLN) that induces enhanced FoxP3+ T cell differentiation, retinoic acid receptor signaling, and gut-homing receptor (CCR9 and α4β7) expression in responding T cells. We show that this function is specific to small intestinal lamina propria (SI-LP) and MLN CD103+ DCs. CD103+ SI-LP DCs appeared to derive from circulating DC precursors that continually seed the SI-LP. BrdU pulse-chase experiments suggested that most CD103+ DCs do not derive from a CD103− SI-LP DC intermediate. The majority of CD103+ MLN DCs appear to represent a tissue-derived migratory population that plays a central role in presenting orally derived soluble antigen to CD8+ and CD4+ T cells. In contrast, most CD103− MLN DCs appear to derive from blood precursors, and these cells could proliferate within the MLN and present systemic soluble antigen. Critically, CD103+ DCs with similar phenotype and functional properties were present in human MLN, and their selective ability to induce CCR9 was maintained by CD103+ MLN DCs isolated from SB Crohn's patients. Thus, small intestinal CD103+ DCs represent a potential novel target for regulating human intestinal inflammatory responses

A novel <i>in vitro</i> model of the small intestinal epithelium in co-culture with ‘gut-like’ dendritic cells

Abstract Cross-talk between dendritic cells (DCs) and the intestinal epithelium is important in the decision to mount a protective immune response to a pathogen or to regulate potentially damaging responses to food antigens and the microbiota. Failures in this decision-making process contribute to the development of intestinal inflammation, making the molecular signals that pass between DCs and intestinal epithelial cells potential therapeutic targets. Until now, in vitro models with sufficient complexity to understand these interactions have been lacking. Here, we outline the development of a co-culture model of in vitro differentiated ‘gut-like’ DCs with small intestinal organoids (enteroids). Sequential exposure of murine bone marrow progenitors to Flt3L, granulocyte macrophage colony-stimulating factor (GM-CSF) and all-trans-retinoic acid (RA) resulted in the generation of a distinct population of conventional DCs expressing CD11b+SIRPα+CD103+/− (cDC2) exhibiting retinaldehyde dehydrogenase (RALDH) activity. These ‘gut-like’ DCs extended transepithelial dendrites across the intact epithelium of enteroids. ‘Gut-like’ DC in co-culture with enteroids can be utilized to define how epithelial cells and cDCs communicate in the intestine under a variety of different physiological conditions, including exposure to different nutrients, natural products, components of the microbiota, or pathogens. Surprisingly, we found that co-culture with enteroids resulted in a loss of RALDH activity in ‘gut-like’ DCs. Continued provision of GM-CSF and RA during co-culture was required to oppose putative negative signals from the enteroid epithelium. Our data contribute to a growing understanding of how intestinal cDCs assess environmental conditions to ensure appropriate activation of the immune response.</jats:p

Bioengineering commensal bacteria-derived outer membrane vesicles for delivery of biologics to the gastrointestinal and respiratory tract

Gram-negative bacteria naturally produce and secrete nanosized outer membrane vesicles (OMVs). In the human gastrointestinal tract, OMVs produced by commensal Gram-negative bacteria can mediate interactions amongst host cells (including between epithelial cells and immune cells) and maintain microbial homeostasis. This OMV-mediated pathway for host-microbe interactions could be exploited to deliver biologically active proteins to the body. To test this we engineered the Gram-negative bacterium Bacteroides thetaiotaomicron (Bt), a prominent member of the intestinal microbiota of all animals, to incorporate bacteria-, virus- and human-derived proteins into its OMVs. We then used the engineered Bt OMVs to deliver these proteins to the respiratory and gastrointestinal (GI)-tract to protect against infection, tissue inflammation and injury. Our findings demonstrate the ability to express and package both Salmonella enterica ser. Typhimurium-derived vaccine antigens and influenza A virus (IAV)-derived vaccine antigens within or on the outer membrane of Bt OMVs. These antigens were in a form capable of eliciting antigen-specific immune and antibody responses in both mucosal tissues and systemically. Furthermore, immunisation with OMVs containing the core stalk region of the IAV H5 hemagglutinin from an H5N1 strain induced heterotypic protection in mice to a 10-fold lethal dose of an unrelated subtype (H1N1) of IAV. We also showed that OMVs could express the human therapeutic protein, keratinocyte growth factor-2 (KGF-2), in a stable form that, when delivered orally, reduced disease severity and promoted intestinal epithelial repair and recovery in animals administered colitis-inducing dextran sodium sulfate. Collectively, our data demonstrates the utility and effectiveness of using Bt OMVs as a mucosal biologics and drug delivery platform technology

Non‐canonical autophagy functions of ATG16L1 in epithelial cells limit lethal infection by influenza A virus

Influenza A virus (IAV) and SARS-CoV-2 (COVID-19) cause pandemic infections where cytokine storm syndrome and lung inflammation lead to high mortality. Given the high social and economic cost of respiratory viruses, there is an urgent need to understand how the airways defend against virus infection. Here we use mice lacking the WD and linker domains of ATG16L1 to demonstrate that ATG16L1-dependent targeting of LC3 to single-membrane, non-autophagosome compartments - referred to as non-canonical autophagy - protects mice from lethal IAV infection. Mice with systemic loss of non-canonical autophagy are exquisitely sensitive to low-pathogenicity IAV where extensive viral replication throughout the lungs, coupled with cytokine amplification mediated by plasmacytoid dendritic cells, leads to fulminant pneumonia, lung inflammation and high mortality. IAV was controlled within epithelial barriers where non-canonical autophagy reduced IAV fusion with endosomes and activation of interferon signalling. Conditional mouse models and ex vivo analysis showed that protection against IAV infection of lung was independent of phagocytes and other leucocytes. This establishes non-canonical autophagy in airway epithelial cells as a novel innate defence that restricts IAV infection and lethal inflammation at respiratory surfaces

The WD and linker domains of ATG16L1 required for non-canonical autophagy limit lethal influenza A virus infection at epithelial surfaces

Phagocytosis and autophagy represent two evolutionarily ancient pathways that provide an important defense against infection by delivering pathogens to lysosomes for degradation. Phagocytosis and autophagy are linked by non-canonical autophagy pathways that conjugate LC3 to endo-lysosome compartments to facilitate phagosome maturation and lysosome fusion. A role for non-canonical autophagy in host defence is implied from cellular studies in vitro, but critically, these studies have rarely been extended to infection of model organisms with intact epithelial barriers and complex immune systems. To address this, we developed a mouse model with specific loss of non-canonical autophagy by removing the WD and linker domain of ATG16L1 required for recruitment of LC3 to endo-lysosome compartments. The mice retain the coiled-coiled domain of ATG16L1 required for conventional autophagy and maintain tissue and immunological homeostasis. Mice with systemic loss of non-canonical autophagy are exquisitely sensitive to low-pathogenicity murine-adapted influenza A virus leading to extensive viral replication throughout the lungs, cytokine dysregulation, fulminant pneumonia and lung inflammation leading to high mortality associated with virulent strains. Conditional mouse models and ex vivo analysis showed that protection against IAV infection of lung required non-canonical autophagy within epithelial barriers but was independent of phagocytes and other leukocytes. This establishes non-canonical autophagy pathways in epithelial cells as a novel innate defence mechanism that can restrict IAV infection at mucosal surfaces

Stem cell‐derived enteroid cultures as a tool for dissecting host‐parasite interactions in the small intestinal epithelium.

Toxoplasma gondii and Cryptosporidium spp. can cause devastating pathological effects in humans and livestock, and in particular to young or immunocompromised individuals. The current treatment plans for these enteric parasites are limited due to long drug courses, severe side effects, or simply a lack of efficacy. The study of the early interactions between the parasites and the site of infection in the small intestinal epithelium has been thwarted by the lack of accessible, physiologically relevant, and species-specific models. Increasingly, 3D stem cell-derived enteroid models are being refined and developed into sophisticated models of infectious disease. In this review we shall illustrate the use of enteroids to spearhead research into enteric parasitic infections, bridging the gap between cell line cultures and in vivo experiments

Cardiac and vascular structure and function parameters do not improve with alternate nightly home hemodialysis: An interventional cohort study

Background: Nightly extended hours hemodialysis may improve left ventricular hypertrophy and function and endothelial function but presents problems of sustainability and increased cost. The effect of alternate nightly home hemodialysis (NHD) on cardiovascular structure and function is not known