Purinergic signaling: a common path in the macrophage response against Mycobacterium tuberculosis and Toxoplasma gondii.

Immune responses are essential for the protection of the host against external dangers or infections and are normally efficient in the clearance of invading microbes. However, some intracellular pathogens have developed strategies to replicate and survive within host cells resulting in latent infection associated with strong inflammation. This excessive response can cause cell and tissue damage and lead to the release of the intracellular content, in particular the nucleotide pool, into the extracellular space. Over the last decade, new studies have implicated metabolites from the purinergic pathway in shaping the host immune response against intracellular pathogens and proved their importance in the outcome of the infection. This review aims to summarize how the immune system employs the purinergic system either to fight the pathogen, or to control collateral tissue damage. This will be achieved by focusing on the macrophage response against two intracellular pathogens, the human etiologic agent of tuberculosis, Mycobacterium tuberculosis and the protozoan parasite, Toxoplasma gondii

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

An open-format enteroid culture system for interrogation of interactions between Toxoplasma gondii and the intestinal epithelium.

When transmitted through the oral route, Toxoplasma gondii first interacts with its host at the small intestinal epithelium. This interaction is crucial to controlling initial invasion and replication, as well as shaping the quality of the systemic immune response. It is therefore an attractive target for the design of novel vaccines and adjuvants. However, due to a lack of tractable infection models, we understand surprisingly little about the molecular pathways that govern this interaction. The in vitro culture of small intestinal epithelium as 3D enteroids shows great promise for modeling the epithelial response to infection. However, the enclosed luminal space makes the application of infectious agents to the apical epithelial surface challenging. Here, we have developed three novel enteroid-based techniques for modeling T. gondii infection. In particular, we have adapted enteroid culture protocols to generate collagen-supported epithelial sheets with an exposed apical surface. These cultures retain epithelial polarization, and the presence of fully differentiated epithelial cell populations. They are susceptible to infection with, and support replication of, T. gondii. Using quantitative label-free mass spectrometry, we show that T. gondii infection of the enteroid epithelium is associated with up-regulation of proteins associated with cholesterol metabolism, extracellular exosomes, intermicrovillar adhesion, and cell junctions. Inhibition of host cholesterol and isoprenoid biosynthesis with Atorvastatin resulted in a reduction in parasite load only at higher doses, indicating that de novo synthesis may support, but is not required for, parasite replication. These novel models therefore offer tractable tools for investigating how interactions between T. gondii and the host intestinal epithelium influence the course of infection

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

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