Method for simultaneous tracking of thousands of unlabeled cells within a transparent 3D matrix.

Three-dimensional tracking of cells is one of the most powerful methods to investigate multicellular phenomena, such as ontogenesis, tumor formation or wound healing. However, 3D tracking in a biological environment usually requires fluorescent labeling of the cells and elaborate equipment, such as automated light sheet or confocal microscopy. Here we present a simple method for 3D tracking large numbers of unlabeled cells in a collagen matrix. Using a small lensless imaging setup, consisting of an LED and a photo sensor only, we were able to simultaneously track ~3000 human neutrophil granulocytes in a collagen droplet within an unusually large field of view (>50 mm2) at a time resolution of 4 seconds and a spatial resolution of ~1.5 µm in xy- and ~30 µm in z-direction. The setup, which is small enough to fit into any conventional incubator, was used to investigate chemotaxis towards interleukin-8 (IL-8 or CXCL8) and N-formylmethionyl-leucyl-phenylalanine (fMLP). The influence of varying stiffness and pore size of the embedding collagen matrix could also be quantified. Furthermore, we demonstrate our setup to be capable of telling apart healthy neutrophils from those where a condition of inflammation was (I) induced by exposure to lipopolysaccharide (LPS) and (II) caused by a pre-existing asthma condition. Over the course of our experiments we have tracked more than 420.000 cells. The large cell numbers increase statistical relevance to not only quantify cellular behavior in research, but to make it suitable for future diagnostic applications, too.

KIR3DS1 directs NK cell-mediated protection against human adenovirus infections.

Human adenoviruses (HAdVs) are a major cause for disease in children, in particular after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Currently, effective therapies for HAdV infections in immunocompromised hosts are lacking. To decipher immune recognition of HAdV infection and determine new targets for immune-mediated control, we used an HAdV infection 3D organoid system, based on primary human intestinal epithelial cells. HLA-F, the functional ligand for the activating NK cell receptor KIR3DS1, was strongly up-regulated and enabled enhanced killing of HAdV5-infected cells in organoids by KIR3DS1+ NK cells. In contrast, HLA-A and HLA-B were significantly down-regulated in HAdV5-infected organoids in response to adenoviral E3/glycoprotein19K, consistent with evasion from CD8+ T cells. Immunogenetic analyses in a pediatric allo-HSCT cohort showed a reduced risk to develop severe HAdV disease and faster clearance of HAdV viremia in children receiving KIR3DS1+/HLA-Bw4+ donor cells compared with children receiving non­KIR3DS1+/HLA-Bw4+ cells. These findings identify the KIR3DS1/HLA-F axis as a new target for immunotherapeutic strategies against severe HAdV disease.

Enhanced Glycolysis Is Required for Antileishmanial Functions of Neutrophils Upon Infection With Leishmania donovani.

Visceral leishmaniasis (VL) is a fatal parasitic disease if untreated. Treatment options of VL diminish due to emerging drug resistance. Although the principal host cells for the multiplication of Leishmania are macrophages, neutrophils are the first cells infected with the parasites rapidly after parasite inoculation. Leishmania can survive in neutrophils despite the potent antimicrobial effector functions of neutrophils that can eliminate the parasites. Recently, the growing field of immunometabolism provided strong evidence for the therapeutic potential in targeting metabolic processes as a means of controlling immune effector functions. Therefore, the understanding of the immunometabolic profile of neutrophils during Leishmania infection could provide new promising targets for host-directed therapies against VL. To our knowledge, this is the first study addressing the bioenergetics profile of L. donovani-infected primary human neutrophils. Transcriptome analysis of L. donovani-infected neutrophils revealed an early significant upregulation of several glycolytic enzymes. Extracellular flux analysis showed that glycolysis and glycolytic capacity were upregulated in L. donovani-infected neutrophils at 6 h post infection. An increased glucose uptake and accumulation of glycolytic end products were further signs for an elevated glycolytic metabolism in L. donovani-infected neutrophils. At the same time point, oxidative phosphorylation provided NADPH for the oxidative burst but did not contribute to ATP production. Inhibition of glycolysis with 2-DG significantly reduced the survival of L. donovani promastigotes in neutrophils and in culture. However, this reduction was due to a direct antileishmanial effect of 2-DG and not a consequence of enhanced antileishmanial activity of neutrophils. To further address the impact of glucose metabolism during the first days of infection in vivo, we treated C57BL/6 mice with 2-DG prior to infection with L. donovani and assessed the parasite load one day and seven days post infection. Our results show, that seven days post-infection the parasite load of 2-DG treated animals was significantly higher than in mock treated animals. This data indicates that glycolysis serves as major energy source for antimicrobial effector functions against L. donovani. Inhibition of glycolysis abrogates important neutrophil effector functions that are necessary the initial control of Leishmania infection.

Cytokines and metabolic regulation: A framework of bidirectional influences affecting Leishmania infection.

Leishmania, a protozoan parasite inflicting the complex of diseases called Leishmaniases, resides and replicates as amastigotes within mammalian macrophages. As macrophages are metabolically highly active and can generate free radicals that can destroy this parasite, Leishmania also devise strategies to modulate the host cell metabolism. However, the metabolic changes can also be influenced by the anti-leishmanial immune response mediated by cytokines. This bidirectional, dynamic and complex metabolic coupling established between Leishmania and its host is the result of a long co-evolutionary process. Due to the continuous alterations imposed by the host microenvironment, such metabolic coupling continues to be dynamically regulated. The constant pursuit and competition for nutrients in the host-Leishmania duet alter the host metabolic pathways with major consequences for its nutritional reserves, eventually affecting the phenotype and functionality of the host cell. Altered phenotype and functions of macrophages are particularly relevant to immune cells, as perturbed metabolic fluxes can crucially affect the activation, differentiation, and functions of host immune cells. All these changes can deterministically direct the outcome of an infection. Cytokines and metabolic fluxes can bidirectionally influence each other through molecular sensors and regulators to dictate the final infection outcome. Our studies along with those from others have now identified the metabolic nodes that can be targeted for therapy.

An Attempt to Polarize Human Neutrophils Toward N1 and N2 Phenotypes in vitro.

Neutrophils act as the first line of defense against invading pathogens. Although traditionally considered in context of their antimicrobial effector functions, the importance of tumor-associated neutrophils (TANs) in the development of cancer has become increasingly clear during the last decade. With regard to their high plasticity, neutrophils were shown to acquire an anti-tumorigenic N1 or a pro-tumorigenic N2 phenotype. Despite the urgent need to get a comprehensive understanding of the interaction of TANs with their tumor microenvironment, most studies still rely on murine tumor models. Here we present for the first time a polarization attempt to generate N1 and N2 neutrophils from primary human neutrophils in vitro. Our results underscore that N1-polarized neutrophils have a pro-inflammatory phenotype characterized among others by a higher level of intercellular adhesion molecule (ICAM)-1 and high secretion of interferon (IFN)γ-induced protein 10 (IP-10)/C-X-C motif chemokine 10 (CXCL10) and tumor necrosis factor (TNF). Further, we demonstrate that neutrophils incubated under a tumor-mimicking in vitro environment show a high cell surface expression of C-X-C motif chemokine receptor 2 (CXCR2) and secrete high levels of interleukin (IL)-8. These findings suggest that it is feasible to polarize blood-derived primary human neutrophils toward N1- and N2-like phenotypes in vitro. Further, we hypothesized that the presence of anti-inflammatory neutrophil phenotype is not a phenomenon limited to cancer but also occurs when neutrophils are infected with intracellular pathogens. Indeed, our findings indicate that N2-polarized neutrophils exert a markedly decreased capacity to kill the protozoan parasite Leishmania donovani and therefore permit parasite persistence.

Type-1 interferons prolong the lifespan of neutrophils by interfering with members of the apoptotic cascade.

Polymorphonuclear Neutrophils (PMNs) are metabolically highly active phagocytes, present in abundant numbers in the circulation. These active cells take the onus of clearing invading pathogens by crowding at inflammatory sites in huge numbers. Though PMNs are extremely short living and die upon spontaneous apoptosis, extended lifespan has been observed among those cells arrive at the inflammation sites or tackle intracellular infections or face any microbial challenges. The delay/inhibition of spontaneous apoptosis of these short-living cells at the inflammatory core rather helps in combating pathogens. Like many candidates, type-1 interferons (type-1 IFNs) is a group of cytokines predominant at the inflammation site. Although there are some isolated reports, a systematic study is still lacking which addresses the impact of the predominant type of interferon on the spontaneous apoptosis of neutrophils. Here in, we have observed that exposure of these IFNs (IFN-ß, IFN-α & IFN-ω etc) on human neutrophils prevents the degradation of the Bfl1, an important anti-apoptotic partner in the apoptotic cascade. Treatment showed a significant reduction in the release of cytochrome-C in the cytosol, a critical regulator in the intrinsic apoptotic pathway. We also noticed a reduction in the conversion of procaspase -3 to active caspase-3, a crucial executioner caspase towards initiation of apoptosis. Taken together our results show that exposure to interferon interferes with apoptotic pathways of neutrophils and thereby delay its spontaneous apoptosis. These findings would help us further deciphering specific roles if these inflammatory agents are causing any immune-metabolomic changes on PMNs at the inflammatory and infection core.