Modulation of lytic molecules restrain serial killing in γδ T lymphocytes.

γδ T cells play a pivotal role in protection against various types of infections and tumours, from early childhood on and throughout life. They consist of several subsets characterised by adaptive and innate-like functions, with Vγ9Vδ2 being the largest subset in human peripheral blood. Although these cells show signs of cytotoxicity, their modus operandi remains poorly understood. Here we explore, using live single-cell imaging, the cytotoxic functions of γδ T cells upon interactions with tumour target cells with high temporal and spatial resolution. While γδ T cell killing is dominated by degranulation, the availability of lytic molecules appears tightly regulated in time and space. In particular, the limited co-occurrence of granzyme B and perforin restrains serial killing of tumour cells by γδ T cells. Thus, our data provide new insights into the cytotoxic arsenal and functions of γδ T cells, which may guide the development of more efficient γδ T cell based adoptive immunotherapies.

The Lysosomal Calcium Channel TRPML1 Maintains Mitochondrial Fitness in NK Cells through Interorganelle Cross-Talk.

Cytotoxic lymphocytes eliminate cancer cells through the release of lytic granules, a specialized form of secretory lysosomes. This compartment is part of the pleomorphic endolysosomal system and is distinguished by its highly dynamic Ca2+ signaling machinery. Several transient receptor potential (TRP) calcium channels play essential roles in endolysosomal Ca2+ signaling and ensure the proper function of these organelles. In this study, we examined the role of TRPML1 (TRP cation channel, mucolipin subfamily, member 1) in regulating the homeostasis of secretory lysosomes and their cross-talk with mitochondria in human NK cells. We found that genetic deletion of TRPML1, which localizes to lysosomes in NK cells, led to mitochondrial fragmentation with evidence of collapsed mitochondrial cristae. Consequently, TRPML1-/- NK92 (NK92ML1-/-) displayed loss of mitochondrial membrane potential, increased reactive oxygen species stress, reduced ATP production, and compromised respiratory capacity. Using sensitive organelle-specific probes, we observed that mitochondria in NK92ML1-/- cells exhibited evidence of Ca2+ overload. Moreover, pharmacological activation of the TRPML1 channel in primary NK cells resulted in upregulation of LC3-II, whereas genetic deletion impeded autophagic flux and increased accumulation of dysfunctional mitochondria. Thus, TRPML1 impacts autophagy and clearance of damaged mitochondria. Taken together, these results suggest that an intimate interorganelle communication in NK cells is orchestrated by the lysosomal Ca2+ channel TRPML1.

Trickle infection with Heligmosomoides polygyrus results in decreased worm burdens but increased intestinal inflammation and scarring.

Introduction: Intestinal roundworms cause chronic debilitating disease in animals, including humans. Traditional experimental models of these types of infection use a large single-dose infection. However, in natural settings, hosts are exposed to parasites on a regular basis and when mice are exposed to frequent, smaller doses of Heligmosomoides polygyrus, the parasites are cleared more quickly. Whether this more effective host response has any negative consequences for the host is not known. Results: Using a trickle model of infection, we found that worm clearance was associated with known resistance-related host responses: increased granuloma and tuft cell numbers, increased levels of granuloma IgG and decreased intestinal transit time, as well as higher serum IgE levels. However, we found that the improved worm clearance was also associated with an inflammatory phenotype in and around the granuloma, increased smooth muscle hypertrophy/hyperplasia, and elevated levels of Adamts gene expression. Discussion: To our knowledge, we are the first to identify the involvement of this protein family of matrix metalloproteinases (MMPs) in host responses to helminth infections. Our results highlight the delicate balance between parasite clearance and host tissue damage, which both contribute to host pathology. When continually exposed to parasitic worms, improved clearance comes at a cost.

Increases in helminth-induced IL-21 protein levels disappear upon sample freezing.

IL-21 is a much studied cytokine that has been implicated in the regulation of TH1, TH2, TH17 and regulatory immune responses; its signalling is a promising therapeutic target for autoimmune, inflammatory and infectious diseases. Despite its biological importance, measuring IL-21 reliably has proved difficult. ELISAs are commonly used to measure cytokines in various biological samples. However, results obtained are only as good as the quality of the sample. Here, we show that when using fresh samples, a significant increase in IL-21 was measured in the intestinal homogenate of mice infected with the intestinal worm Heligmosomoides polygyrus. This difference disappeared when samples were frozen in either liquid nitrogen for two days or at -80 °C for three weeks, with levels in both naïve and infected animals decreasing. This was not observed for the IL-13 cytokine, where freezing had no impact on levels measured. Our study highlights the importance of sample storage to measuring biomarkers. Since modulating IL-21 signalling is such an important potential therapeutic avenue, accurately measuring the levels of this cytokine is key to assessing its role in various research models and clinical settings.

Toxoplasma gondii: One Organism, Multiple Models.

Toxoplasma gondii is an intensely studied protozoan parasite. It is also used as a model organism to research additional clinically relevant human and veterinary parasites due to ease of in vitro culture and genetic manipulation. Recently, it has been developed as a model of inflammatory bowel disease, due to their similar pathologies. However, researchers vary widely in how they use T. gondii, which makes study comparisons and interpretation difficult. The aim of this review is to provide researchers with a tool to: (i) determine the appropriateness of the different T. gondii models to their research, (ii) interpret results from the wide range of study conditions, and (iii) consider new advances in technology which could improve or refine their experimental setup.

Host-Imposed Copper Poisoning Impacts Fungal Micronutrient Acquisition during Systemic Candida albicans Infections.

Nutritional immunity is a process whereby an infected host manipulates essential micronutrients to defend against an invading pathogen. We reveal a dynamic aspect of nutritional immunity during infection that involves copper assimilation. Using a combination of laser ablation inductively coupled mass spectrometry (LA-ICP MS) and metal mapping, immunohistochemistry, and gene expression profiling from infected tissues, we show that readjustments in hepatic, splenic and renal copper homeostasis accompany disseminated Candida albicans infections in the mouse model. Localized host-imposed copper poisoning manifests itself as a transient increase in copper early in the kidney infection. Changes in renal copper are detected by the fungus, as revealed by gene expression profiling and fungal virulence studies. The fungus responds by differentially regulating the Crp1 copper efflux pump (higher expression during early infection and down-regulation late in infection) and the Ctr1 copper importer (lower expression during early infection, and subsequent up-regulation late in infection) to maintain copper homeostasis during disease progression. Both Crp1 and Ctr1 are required for full fungal virulence. Importantly, copper homeostasis influences other virulence traits-metabolic flexibility and oxidative stress resistance. Our study highlights the importance of copper homeostasis for host defence and fungal virulence during systemic disease.

A novel renal epithelial cell in vitro assay to assess Candida albicans virulence.

Candida albicans, an opportunistic fungal pathogen, can cause severe systemic infections in susceptible patient groups. Systemic candidiasis is mainly studied in the mouse intravenous challenge model, where progressive infection correlates with increased early renal chemokine levels. To develop a new in vitro assay to assess C. albicans virulence, which reflects the events occurring in the murine infection model, renal M-1 cortical collecting duct epithelial cells were evaluated as the early producers of cytokines in response to C. albicans. We show that renal epithelial cells respond only to live C. albicans cells capable of forming hyphae, producing chemokines KC and MIP-2, with levels correlating with epithelial cell damage. By assaying epithelial cell responses to strains of known virulence in the murine intravenous challenge model we demonstrate that renal epithelial cells can discriminate between virulent and attenuated strains. This simple, novel assay is a useful initial screen for altered virulence of C. albicans mutants or clinical isolates in vitro and provides an alternative to the mouse systemic infection model.

The contribution of mouse models to our understanding of systemic candidiasis.

Some Candida species are common commensals, which can become opportunistic pathogens in susceptible hosts. In severely ill patients, Candida species, particularly Candida albicans, can cause life-threatening systemic infections. These infections are difficult to diagnose, as symptoms are similar to those of systemic bacterial infections. These difficulties can lead to delays in initiation in antifungal therapy, which contributes to the high mortality rates (> 40%) associated with these infections. In order to investigate systemic Candida infection, mouse models have been developed that mimic human disease, the most common being the intravenous infection model and the gastrointestinal colonization and dissemination model. This review discusses the two models and the contributions that they have made to our understanding of fungal virulence, host response to infection and the development of novel antifungal therapies and diagnostics.