Lack of strong innate immune reactivity renders macrophages alone unable to control productive Varicella-Zoster Virus infection in an isogenic human iPSC-derived neuronal co-culture model.

peer reviewedWith Varicella-Zoster Virus (VZV) being an exclusive human pathogen, human induced pluripotent stem cell (hiPSC)-derived neural cell culture models are an emerging tool to investigate VZV neuro-immune interactions. Using a compartmentalized hiPSC-derived neuronal model allowing axonal VZV infection, we previously demonstrated that paracrine interferon (IFN)-α2 signalling is required to activate a broad spectrum of interferon-stimulated genes able to counteract a productive VZV infection in hiPSC-neurons. In this new study, we now investigated whether innate immune signalling by VZV-challenged macrophages was able to orchestrate an antiviral immune response in VZV-infected hiPSC-neurons. In order to establish an isogenic hiPSC-neuron/hiPSC-macrophage co-culture model, hiPSC-macrophages were generated and characterised for phenotype, gene expression, cytokine production and phagocytic capacity. Even though immunological competence of hiPSC-macrophages was shown following stimulation with the poly(dA:dT) or treatment with IFN-α2, hiPSC-macrophages in co-culture with VZV-infected hiPSC-neurons were unable to mount an antiviral immune response capable of suppressing a productive neuronal VZV infection. Subsequently, a comprehensive RNA-Seq analysis confirmed the lack of strong immune responsiveness by hiPSC-neurons and hiPSC-macrophages upon, respectively, VZV infection or challenge. This may suggest the need of other cell types, like T-cells or other innate immune cells, to (co-)orchestrate an efficient antiviral immune response against VZV-infected neurons

Varicella-zoster virus recapitulates its immune evasive behaviour in matured hiPSC-derived neurospheroids

peer reviewedVaricella-zoster virus (VZV) encephalitis and meningitis are potential central nervous system (CNS) complications following primary VZV infection or reactivation. With Type-I interferon (IFN) signalling being an important first line cellular defence mechanism against VZV infection by the peripheral tissues, we here investigated the triggering of innate immune responses in a human neurallike environment. For this, we established and characterised 5-month matured hiPSC-derived neurospheroids (NSPHs) containing neurons and astrocytes. Subsequently, NSPHs were infected with reporter strains of VZV (VZV eGFP-ORF23) or Sendai virus (SeV eGFP), with the latter serving as an immune-activating positive control. Live cell and immunocytochemical analyses demonstrated VZV eGFP-ORF23 infection throughout the NSPHs, while SeV eGFP infection was limited to the outer NSPH border. Next, NanoString digital transcriptomics revealed that SeV eGFP-infected NSPHs activated a clear Type-I IFN response, Frontiers in Immunology frontiersin.org 0

Activation of Interferon-Stimulated Genes following Varicella-Zoster Virus Infection in a Human iPSC-Derived Neuronal In Vitro Model Depends on Exogenous Interferon-\u3b1

Varicella-zoster virus (VZV) infection of neuronal cells and the activation of cell-intrinsic antiviral responses upon infection are still poorly understood mainly due to the scarcity of suitable human in vitro models that are available to study VZV. We developed a compartmentalized human-induced pluripotent stem cell (hiPSC)-derived neuronal culture model that allows axonal VZV infection of the neurons, thereby mimicking the natural route of infection. Using this model, we showed that hiPSC-neurons do not mount an effective interferon-mediated antiviral response following VZV infection. Indeed, in contrast to infection with Sendai virus, VZV infection of the hiPSC-neurons does not result in the upregulation of interferon-stimulated genes (ISGs) that have direct antiviral functions. Furthermore, the hiPSC-neurons do not produce interferon-α (IFNα), a major cytokine that is involved in the innate antiviral response, even upon its stimulation with strong synthetic inducers. In contrast, we showed that exogenous IFNα effectively limits VZV spread in the neuronal cell body compartment and demonstrated that ISGs are efficiently upregulated in these VZV-infected neuronal cultures that are treated with IFNα. Thus, whereas the cultured hiPSC neurons seem to be poor IFNα producers, they are good IFNα responders. This could suggest an important role for other cells such as satellite glial cells or macrophages to produce IFNα for VZV infection control

Necroptosis signalling promotes inflammation, airway remodelling and emphysema in COPD

Rationale: Necroptosis, mediated by RIPK3 and MLKL, is a form of regulated necrosis that can drive tissue inflammation and destruction, however its contribution to COPD pathogenesis is poorly understood. Objectives: To determine the role of necroptosis in COPD. Methods: Levels of RIPK3, MLKL and activated phospho-MLKL were measured in lung tissues of COPD patients and non-COPD controls. Necroptosis-related mRNA and proteins and cell death were examined in the lungs and pulmonary macrophages of mice with cigarette smoke (CS)-induced experimental COPD. The responses of Ripk3- and Mlkl-deficient (-/-) mice to CS exposure were compared to wild-type mice. Combined inhibition of apoptosis (pan-caspase inhibitor qVD-OPh) and necroptosis (Mlkl-/- mice) was assessed. Measurements and main results: Protein levels of MLKL and pMLKL but not RIPK3 were increased in lung tissues of COPD patients compared to never smokers or smoker non-COPD controls. Necroptosis-related mRNA and protein levels were increased in lung tissue and macrophages in CS-exposed mice/experimental COPD. Ripk3 or Mlkl deletion prevented airway inflammation in response to acute CS-exposure. Ripk3 deficiency reduced airway inflammation and remodelling and development of emphysematous pathology following chronic CS-exposure. Mlkl deletion and qVD-OPh treatment reduced chronic CS-induced airway inflammation, but only Mlkl deletion prevented airway remodelling and emphysema. Ripk3 or Mlkl deletion and qVD-OPh treatment reduced CS-induced lung cell death. Conclusions: Necroptosis is induced by CS exposure and increased in COPD patient lungs and experimental COPD. Inhibiting necroptosis attenuates CS-induced airway inflammation, airway remodelling and emphysema. Targeted inhibition of necroptosis is a potential therapeutic strategy in COPD

Luminescent human iPSC-derived neurospheroids enable modeling of neurotoxicity after oxygen-glucose deprivation

Despite the considerable impact of stroke on both the individual and on society, a neuroprotective therapy for stroke patients is missing. This is partially due to the current lack of a physiologically relevant human in vitro stroke model. To address this problem, we have developed a luminescent human iPSC-derived neurospheroid model that enables real-time read-out of neural viability after ischemia-like conditions. We subjected 1- and 4-week-old neurospheroids, generated from iPSC-derived neural stem cells, to 6 h of oxygen-glucose deprivation (OGD) and measured neurospheroid luminescence. For both, we detected a decrease in luminescent signal due to ensuing neurotoxicity, as confirmed by conventional LDH assay and flow cytometric viability analysis. Remarkably, 1-week-old, but not 4-week-old neurospheroids recovered from OGD-induced injury, as evidenced by their reduced but overall increasing luminescence over time. This underscores the need for more mature neurospheroids, more faithfully recapitulating the in vivo situation. Furthermore, treatment of oxygen- and glucose-deprived neurospheroids with the pan-caspase inhibitor Z-VAD-FMK did not increase overall neural survival, despite its successful attenuation of apoptosis, in a human-based 3D environment. Nevertheless, owing to its three-dimensional organization and real-time viability reporting potential, the luminescent neurospheroids may become readily adopted in high-throughput screens aimed at identification of new therapeutic agents to treat acute ischemic stroke patients