Cleaved CD95L perturbs in vitro macrophages responses to Toxoplasma gondii.

Toxoplasma gondii infects approximately 1-2 billion people, and manipulation of the macrophage response is critical to host and parasite survival. A cleaved (cl)-CD95L form can promote cellular migration and we have previously shown that cl-CD95L aggravates inflammation and pathology in systemic lupus erythematosus (SLE). Findings have shown that CD95L is upregulated during human infection, therefore we examined the effect of cl-CD95L on the macrophage response to T. gondii. . We find that cl-CD95L promotes parasite replication in macrophages, associated with increased arginase-1 levels, mediated by signal transducer and activator of transcription (STAT)6. Inhibition of both arginase-1 and STAT6 reversed the effects of cl-CD95L. Phospho-kinase array showed that cl-CD95L alters Janus Kinases (JAK)/STAT, mammalian target of rapamycin (mTOR), and Src kinase signals. By triggering changes in JAK/STAT cl-CD95L may limit anti-parasite effectors.

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.

Proteomic Profiling of Enteroid Cultures Skewed toward Development of Specific Epithelial Lineages.

Recently, 3D small intestinal organoids (enteroids) have been developed from cultures of intestinal stem cells which differentiate in vitro to generate all the differentiated epithelial cell types associated with the intestine and mimic the structural properties of the intestine observed in vivo. Small-molecule drug treatment can skew organoid epithelial cell differentiation toward particular lineages, and these skewed enteroids may provide useful tools to study specific epithelial cell populations, such as goblet and Paneth cells. However, the extent to which differentiated epithelial cell populations in these skewed enteroids represent their in vivo counterparts is not fully understood. This study utilises label-free quantitative proteomics to determine whether skewing murine enteroid cultures toward the goblet or Paneth cell lineages results in changes in abundance of proteins associated with these cell lineages in vivo. Here, proteomics data confirms that skewed enteroids recapitulate important features of the in vivo gut environment, demonstrating that they can serve as useful models for the investigation of normal and disease processes in the intestine. Furthermore, comparison of mass spectrometry data with histology data contained within the Human Protein Atlas identifies putative novel markers for goblet and Paneth cells.

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.

Monophasic expression of FliC by Salmonella 4,[5],12:i:- DT193 does not alter its pathogenicity during infection of porcine intestinal epithelial cells.

Non-typhoidal serotypes of Salmonella enterica remain important food-borne pathogens worldwide and the frequent emergence of epidemic strains in food-producing animals is a risk to public health. In recent years, Salmonella 4,[5],12:i:- isolates, expressing only phase 1 (FliC) of the two flagellar antigens, have emerged and increased in prevalence worldwide. In Europe, the majority of 4,[5],12:i:- isolates belong to phage types DT193 and DT120 of Salmonella Typhimurium and pigs have been identified as the reservoir species. In this study we investigated the ability of pig-derived monophasic (4,[5],12:i:-) and biphasic DT193 isolates to invade a porcine intestinal epithelial cell line (IPEC-1) and activate TLR-5, IL-8 and caspases. We found that the 4,[5],12:i:- isolates exhibited comparable adhesion and invasion to that of the virulent S. Typhimurium isolate 4/74, suggesting that these strains could be capable of colonizing the small intestine of pigs in vivo. Infection with 4,[5],12:i:- and biphasic DT193 isolates resulted in approximately the same level of TLR-5 (a flagellin receptor) and IL-8 (a proinflammatory chemokine) mRNA upregulation. The monophasic variants also elicited similar levels of caspase activation and cytotoxicity to the phase-variable DT193 isolates. These findings suggest that failure of 4,[5],12:i:- DT193 isolates to express a second phase of flagellar antigen (FljB) is unlikely to hamper their pathogenicity during colonization of the porcine intestinal tract.

Infection-induced regulation of natural killer cells by macrophages and collagen at the lymph node subcapsular sinus.

Infection leads to heightened activation of natural killer (NK) cells, a process that likely involves direct cell-to-cell contact, but how this occurs in vivo is poorly understood. We have used two-photon laser-scanning microscopy in conjunction with Toxoplasma gondii mouse infection models to address this question. We found that after infection, NK cells accumulated in the subcapsular region of the lymph node, where they formed low-motility contacts with collagen fibers and CD169(+) macrophages. We provide evidence that interactions with collagen regulate NK cell migration, whereas CD169(+) macrophages increase the activation state of NK cells. Interestingly, a subset of CD169(+) macrophages that coexpress the inflammatory monocyte marker Ly6C had the most potent ability to activate NK cells. Our data reveal pathways through which NK cell migration and function are regulated after infection and identify an important accessory cell population for activation of NK cell responses in lymph nodes.

Regulatory T cells suppress systemic and mucosal immune activation to control intestinal inflammation.

The gastrointestinal (GI) tract is the main interface where the body encounters exogenous antigens. It is crucial that the local response here is tightly regulated to avoid an immune reaction against dietary antigens and commensal flora while still mounting an efficient defense against pathogens. Faults in establishing intestinal tolerance can lead to disease, inducing local and often also systemic inflammation. Studies in human as well as in animal models suggest a role for regulatory T cells (Tregs) in maintaining intestinal homeostasis. Transfer of Tregs can not only prevent the development of colitis in animal models but also cure established disease, acting both systemically and at the site of inflammation. In this review, we discuss the major regulatory pathways, including transforming growth factor-beta (TGF-beta), interleukin-10 (IL-10), and cytotoxic T-lymphocyte antigen-4 (CTLA-4), and their role in Treg-mediated control of systemic and mucosal responses. In addition, we give an overview of the known mechanisms of lymphocyte migration to the intestine and discuss how CD103 expression can influence the balance between regulatory and effector T cells. Further understanding of the factors that control the activity of Tregs in different immune compartments may facilitate the design of strategies to target regulation in a tissue-specific way.