Varicella-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 neural-like 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 (VZVeGFP-ORF23) or Sendai virus (SeVeGFP), with the latter serving as an immune-activating positive control. Live cell and immunocytochemical analyses demonstrated VZVeGFP-ORF23 infection throughout the NSPHs, while SeVeGFP infection was limited to the outer NSPH border. Next, NanoString digital transcriptomics revealed that SeVeGFP-infected NSPHs activated a clear Type-I IFN response, while this was not the case in VZVeGFP-ORF23-infected NSPHs. Moreover, the latter displayed a strong suppression of genes related to IFN signalling and antigen presentation, as further demonstrated by suppression of IL-6 and CXCL10 production, failure to upregulate Type-I IFN activated anti-viral proteins (Mx1, IFIT2 and ISG15), as well as reduced expression of CD74, a key-protein in the MHC class II antigen presentation pathway. Finally, even though VZVeGFP-ORF23-infection seems to be immunologically ignored in NSPHs, its presence does result in the formation of stress granules upon long-term infection, as well as disruption of cellular integrity within the infected NSPHs. Concluding, in this study we demonstrate that 5-month matured hiPSC-derived NSPHs display functional innate immune reactivity towards SeV infection, and have the capacity to recapitulate the strong immune evasive behaviour towards VZV.

With 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 (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.

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.

Rationale: Necroptosis, mediated by RIPK3 (receptor-interacting protein kinase 3) and MLKL (mixed lineage kinase domain-like), is a form of regulated necrosis that can drive tissue inflammation and destruction; however, its contribution to chronic obstructive pulmonary disease (COPD) pathogenesis is poorly understood. Objectives: To determine the role of necroptosis in COPD. Methods: Total and active (phosphorylated) RIPK3 and MLKL were measured in the lung tissue of patients with COPD and control subjects without COPD. Necroptosis-related mRNA and proteins as well as cell death were examined in lungs and pulmonary macrophages of mice with cigarette smoke (CS)-induced experimental COPD. The responses of Ripk3-/- and Mlkl-/- mice to acute and chronic CS exposure were compared with those of wild-type mice. The combined inhibition of apoptosis (with the pan-caspase inhibitor quinoline-Val-Asp-difluorophenoxymethylketone [qVD-OPh]) and necroptosis (with deletion of Mlkl in mice) was assessed. Measurements and Main Results: The total MLKL protein in the epithelium and macrophages and the pRIPK3 and pMLKL in lung tissue were increased in patients with severe COPD compared with never-smokers or smoker control subjects without COPD. Necroptosis-related mRNA and protein levels were increased in the lungs and macrophages in CS-exposed mice and experimental COPD. Ripk3 or Mlkl deletion prevented airway inflammation upon acute CS exposure. Ripk3 deficiency reduced airway inflammation and remodeling as well as the development of emphysematous pathology after chronic CS exposure. Mlkl deletion and qVD-OPh treatment reduced chronic CS-induced airway inflammation, but only Mlkl deletion prevented airway remodeling and emphysema. Ripk3 or Mlkl deletion and qVD-OPh treatment reduced CS-induced lung-cell death. Conclusions: Necroptosis is induced by CS exposure and is increased in the lungs of patients with COPD and in experimental COPD. Inhibiting necroptosis attenuates CS-induced airway inflammation, airway remodeling, and emphysema. Targeted inhibition of necroptosis is a potential therapeutic strategy in COPD.

Background: Necroptosis is an inflammatory type of regulated cell death, mediated by receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). COPD involves airway inflammation and loss of alveolar tissue (emphysema), as a consequence of cigarette smoke (CS) inhalation. There is evidence that CS-induced necroptosis contributes to the pathogenesis of COPD. Objectives: We investigated the presence of necroptotic markers in human COPD. To assess the contribution of necroptosis to pulmonary inflammation, airway remodelling and emphysema, we chronically exposed RIPK3- and MLKL-deficient mice to CS. Methods: Protein expression levels of RIPK3 and (phosphorylated) MLKL in lungs of never smokers, smokers without airflow obstruction and COPD patients (GOLD II to IV) were evaluated by immunohistochemistry (IHC) and immunoblotting. Wild-type, RIPK3- and MLKL-deficient mice were exposed to CS for 8 weeks. BALF was collected and total and differential leukocytes were enumerated, and histology of lung tissue was assessed. Results: IHC of human lungs revealed positive staining for RIPK3 and MLKL, primarily in airway epithelial cells. Quantification demonstrated increased expression of MLKL but not RIPK3 in airway epithelium of COPD patients, compared to smokers and never smokers. In addition, phosphorylation of MLKL was increased in lung tissue of patients with severe COPD. RIPK3- and MLKL-deficient mice were protected against inflammation in experimental COPD, and both strains showed protection against CS-induced airway remodelling and emphysema, compared to wild-type controls. Conclusions: We provide evidence that necroptosis contributes to the pathogenesis of chronic disease features of COPD.

Background: Necroptosis is an inflammatory type of regulated cell death, mediated by receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). COPD involves airway inflammation and loss of alveolar tissue (emphysema), as a consequence of cigarette smoke (CS) inhalation. There is evidence that CS-induced necroptosis contributes to the pathogenesis of COPD. Objectives: We investigated the presence of necroptotic markers in human COPD. To assess the contribution of necroptosis to pulmonary inflammation, airway remodelling and emphysema, we chronically exposed RIPK3- and MLKL-deficient mice to CS. Methods: Protein expression levels of RIPK3 and (phosphorylated) MLKL in lungs of never smokers, smokers without airflow obstruction and COPD patients (GOLD II to IV) were evaluated by immunohistochemistry (IHC) and immunoblotting. Wild-type, RIPK3- and MLKL-deficient mice were exposed to CS for 8 weeks. BALF was collected and total and differential leukocytes were enumerated, and histology of lung tissue was assessed. Results: IHC of human lungs revealed positive staining for RIPK3 and MLKL, primarily in airway epithelial cells. Quantification demonstrated increased expression of MLKL but not RIPK3 in airway epithelium of COPD patients, compared to smokers and never smokers. In addition, phosphorylation of MLKL was increased in lung tissue of patients with severe COPD. RIPK3- and MLKL-deficient mice were protected against inflammation in experimental COPD, and both strains showed protection against CS-induced airway remodelling and emphysema, compared to wild-type controls. Conclusions: We provide evidence that necroptosis contributes to the pathogenesis of chronic disease features of COPD.