Live-Cell Insights Newsletter: Vol. 1, 2020 Featured publications

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Virology

Exacerbated Apoptosis of Cells Infected with Infectious Bursal Disease Virus upon Exposure to Interferon Alpha

Deciphering a mechanism for IBVD-induced immunosuppression

Infectious bursal disease virus (IBDV) is a highly contagious viral infection of avian species that is characterized by high mortality rates, giving rise to the immunosuppressive disease IBD in domestic chickens, which is of concern for the commercial poultry industry. Infection with IBDV results in loss of B-lymphocytes, which produce IgM antibody, also affecting macrophages and monocytes, as well as the destruction of the avian bursa of Fabricious (lymphoid organ, site of B cell development in birds). There is also damage to secondary organs. Overall, these combined alterations result immunosuppression in survivors, making them vulnerable to secondary infection, as well as non-responsive to subsequent vaccination to other pathogens.  

It has been suggested that this immunosuppression occurs through massive apoptosis of B cells and the development of a cytokine storm, which ensues as proinflammatory factors damage tissues, with elevation of IFN-γ and T-cell immunosuppression. Despite research in this area, the underlying mechanism of this immune suppression had remained a mystery.

This paper by Cugas-Gaona et al. from the Centro Nacional de Biotecnología-CSIC in Madrid expands our understanding of the underlying the immunosupression and pathogenesis arising IBVD infection, providing new mechanistic insight into the apoptosis of IBDV infected cells exposed to type 1 interferon (IFN).

Key findings include:

• During IBDV infection, cells treated with hIFN-α had increased PKR phosphorylation and upregulated gene expression of IFN-β. A gradual increase in PSRP cleavage was observed by Western blot analysis, and IncuCyte® Live-Cell analysis was utilized to confirm the corresponding increase in apoptosis over time.

• IBDV infected HeLa cells were treated with hIFN-α, triggering marked apoptotic death. This correlated with PKR phosphorylation and upregulated gene expression of the IFN-β.  IncuCyte® Caspase3/7 reagent was used to monitor the apoptosis during the 24 hr. infection period.  Blocking of viral dsRNA replication in IBDV-infected HeLa cells was performed with the polymerase inhibitor, 7-deaza-2’-C-methyladeosine (7DMA) in cells treated with or without IFN.  Cells treated with 7DMA showed reduced apoptosis, with a slight reduction in those not treated with IFN, demonstrating that the replication/trans-cription of viral RNA was necessary for apoptosis of cells infected with IBDV and treated with IFN.

• IBDV infection was studied in chicken DF-1 cells, which also underwent apoptosis with chicken IFN-α treatment.  IncuCyte® assessment of apoptosis was performed, revealing that signs of infection occurred earlier in human HeLa cells as compared to chicken DF-1 cells, but IFN treatment also resulted in enhanced apoptosis. This indicated similar trends for both avian and mammalian models of infection.

• This study demonstrated that cells infected with IBDV undergo marked apoptosis when exposed to IFN-α.

• PKR, TNF-α, and NF-κB are important proteins in the apoptotic response to IFN-α during IBVD infection, and their downregulation reduced apoptosis.

• During IBDV infection, the IBDV viral genomic dsRNA aids in triggering apoptosis.

Read the full paper in Journal of Virology, March 2018.

VIROLOGY

Macrophage Migration Inhibitory Factor Enhances Influenza-Associated Mortality in Mice

A possible new target to bolster host immunity and reduce viral disease

Despite advances in influenza vaccination, the efficacy remains sub-optimal due to acute respiratory inflammation and persistent mortality from influenza.   A first line of defense is the activation of innate immune responses in the lung mucosa, which elicit the recruitment and activation of immune cells, and production of cytokines.  Antiviral proteins may curb viral replication, most importantly, the interferon (IFN) family of proteins. Additionally, this inflammatory response must be balanced by an anti-inflammatory response to prevent tissue damage and the resulting Acute Respiratory Distress Syndrome (ARDS).

Macrophage migration inhibitory factor (MIF) serves to regulate the innate immune response through a variety of means, including apoptosis inhibition, activation of macrophages, and stimulation of NLRP3 inflammasome. The role of MIF in influenza infection has not been defined.  This study by Smith and colleagues, in a joint effort between the University of Michigan and Yale School of Medicine, attempts to further define the role of MIF using a mouse model of influenza using influenza virus (IAV) strain PR8.

Key findings include:

• MIF-deficient mice that have influenza viral infection have less inflammation, a lower viral load, and reduced mortality compared to WT controls.

• Cytotoxicity assays, along with apoptosis assays using IncuCyte live-cell imaging and analysis, IncuCyte Caspase-3/7, and Annexin-V, were employed to assess IAV viral replication in GFP-labeled bronchoalveolar lavage (BAL) cells from infected mice for 24 hrs.

• Tg mice that overexpress Mif in alveolar epithelia have more inflammation, viral load, and higher mortality

• Blocking MIF using antibodies during influenza infection of WT mice resulted in increased survival.

• Prior to influenza viral infection, the lungs from MIF deficient mice had reduced levels of a mitophagy protein, parkin., which acts to regulate antiviral signaling responses. This protein caused negative regulation of antiviral signaling prior to infection, Lung cells from the MIF deficient mice also showed enhanced levels of antiviral typeI/III IFN in air-spaces post infection as compared to WT control animals.

• In vitro, human lung epithelial cultures treated with recombinant human MIF displayed an increased number of influenza virus-infected cells.

• The authors conclude that MIF impairs the antiviral immune response of the host in the tested models, augmenting the inflammatory process during influenza infection.

Read the full paper in JCI Insight, July 2019.

Immunology

IFN-β Improves Sepsis-Related Alveolar Macrophage Dysfunction and Postseptic Acute Respiratory Distress Syndrome–Related Mortality

The macrophage gets some help from an old friend

Acute respiratory distress syndrome (ARDS) is an inflammatory condition of the lungs that may progress to respiratory failure.  ARDS is a critical complication of medical conditions such as sepsis, pneumonia, aspiration, and viral infections, and has a high mortality rate. Fluid from small blood vessels in the lungs leaks into the alveoli, causing the characteristic shortness of breath, There may also be alterations in the levels of surfactant and pulmonary fibrosis. Vital oxygen is unable to enter the bloodstream and reach the organs, resulting in organ failure. 

ARDS is most commonly associated with sepsis, which is characterized by disruption of immune cell populations and dysregulation between the pro- and anti-inflammatory balance of the immune system.  Despite much research, the underlying alterations to innate immunity within the lung associated with this condition are not well understood.

In this international, collaborative study by Hiruma et al., the therapeutic effect of IFN-β administration during post-septic Acute Respiratory Distress Syndrome (ARDS) was investigated in a “two-hit” model of cecal ligation and puncture (CLP) mouse model of sepsis followed by infection with Pseudomonas aeruginosa to establish bacterial pneumonia.  The authors proposed a mechanism for the lung injury associated with sepsis and ARDS, and the therapeutic action of IFN-β administration.

Key findings include:

• Post-septic ARDS was established in the CLP mouse model, followed by a second hit pneumonia infection with Pseudomonas aerugenosa., Mice were then administered IFN-β, while controls were not. The sepsis was associated with increases in alveolar TNF-α and IL-10. IncuCyte® pHrodo Green E. coli Bioparticles were used to assess the phagocytic capacity of alveolar macrophages using IncuCyte® live-cell analysis. A reduction of phagocytosis by alveolar macrophages and secretion of KC (CXCL1 chemokine) was observed following sepsis, which was consistent with reprogramming of the innate immune system. It was restored by administration of IFN-β.

• Ex vivo exposure of the alveolar macrophages to TNF-α and IL-10 also resulted in impaired cytokine release.

• The KC responses of alveolar macrophages from the test condition of sepsis followed by pneumonia were lessened as compared to that of either sepsis or pneumonia only, and there were fewer neutrophils in to lung alveoli, despite more in the lungs overall. There was also increased injury, as well as a reduction in bacterial clearance that was associated with an increase in mortality

• Administration of IFN-β reversed an impaired alveolar macrophage cytokine secretion by TNF-α and IL-10 in vitro. In vivo, there was improvement of bacterial clearance and recruitment of neutrophils to the alveoli, with an improvement in survival and lung epithelial permeability, as well as reduced lung injury.

• These results uncovered a “incomplete alveolar neutrophil recruitment” as a mechanism for ARDS following sepsis, along with the finding that IFN-β restored this function to improve survival in this mouse model of sepsis.

Read the full paper in American Journal of Respiratory Cell and Molecular Biology, July 2018.

Oncology Therapeutics

Characterizing Cellular Responses During Oncolytic Maraba Virus Infection

New insights on the attenuation of oncolytic viral propagation

Exploiting the tumor killing properties of oncolytic viruses continues to be of interest as a possible treatment strategy for a variety of cancers. Maraba virus has emerged as a promising candidate due to the virus’ high propensity for malignant cell killing and lower toxicity in normal cell lines.  To further develop this agent as a possible therapeutic, more information is needed on how this virus interacts with tumors and the immune system, as well as a greater understanding of how the virus exploits host translational mechanisms for the production of viral proteins.

In this international, collaborative study by Hassanzadeh et al., the authors utilized an interferon-sensitive mutant Maraba virus (MG1), to better characterize how the virus is propagated, and discern host/virus interactions, focusing on the regulation of translation, and the attenuation of Marba virus propagation.

Key findings include:

• Two regulators of translation were found in infected cells that regulate protein synthesis: 4E-binding protein (4E-BP1) and Eukaryotic Initiation Factor 2 (eIF2α).

• In particular, eIF2α was indicated in the repression of host translation in MG1-infected cells, while still permitting MG1 protein synthesis.

• eIF2α phosphorylation was found to play a negative during MG1 infection. IncuCyte® live cell imaging was used to monitor the rate of viral propagation through confluency of GFP signal in WT and S51 MEFs. IncuCyte® Cytotox Red reagent was used to monitor the infection of mock- or infected cells over the time course of infection. MG1 virus showed a higher rate of infection in S51A MEFs compare to WT MEFs.

• eIF2α was found to aid expression of the anti-apoptotic Bcl-xL protein and MG1 during viral infections.

• Another translation factor, Eukaryotic translation initiation factor 5B (eIF5β), was also found to aid expression of the anti-apoptotic Bcl-xL protein and MG1 during infection with virus in an eIF5B knockdown study. IncuCyte® was used to observe the effect of knocking down eIF5B on cell viability and rate of infection with MG1. A reduction in eIF5B limited MG1 viral propagation in host cells, enhancing cell survival during infection.

• Overall, the upregulation of Bcl-xL conferred a survival benefit for the host cell, and allowed propagation of the virus. The inhibition of eIF5B indirectly results in down-regulation of Bcl-xL, which also indirectly attenuates viral propagation.

Read the full paper in International Journal of Molecular Science, January 2018.

Immunotherapy

Blocking Expression of Inhibitory Receptor NKG2A Overcomes Tumor Resistance to NK Cells

A new approach to overcome tumor resistance

The development of tumor resistance is a key impediment to immunotherapy using natural killer (NK) cells, which have the ability to detect transformed cells and eliminate them through the interaction of activating/inhibitory receptors on the NK with their targets.  The inhibitory receptor CD94/NK group 2 member A (NKG2A) binds to the polymorphic HLA Class 1 molecule (HLA-E), which presents peptides from other Class I HLA.  NKG2A ligation with HLA-E and associated peptides causes NKG2A to transduce an inhibitory signal, resulting in the suppression of NK activities such as the secretion of cytokines and cytotoxic activities. HLA-E overexpression is found in several tumor types and is associated with poor prognosis. Cells expressing NKG2A+ are found in a variety of cancers, suggesting suppression on NK activity.

In this study by Kamiya et al. from the National University of Singapore and MedSix Therapeutics, the authors examined HLAE expression in over 10,000 tumor samples, with additional analysis for association with KLRC1 (NKG2A) and KLRD1 (CD94).  The purpose was to generate NK cells that were not inhibited by HLA-E.  The group designed protein expression blockers (PEBLS) that halt the transport of NKG2A to the cell surface of NK cells, and assessed the effects of NKG2A downregulation on tumor resistance to NK killing via HLA-E.

Key findings include:

• NG2A was removed from the surface of NK cells through use of a construct of single chain variable fragments derived from an anati-NG2A monoclonal antibody (anti-NKG2A PEBLS), which prevented the protein surface expression of NKG2A and its co-receptor CD94 (NKG2Anull NK cells).

• IncuCyte® proliferation assays were used as part of the functional long-term assessment of cytotoxicity of PEBL-transduced NK cells (NKG2Anull NK cells) and controls against K562 leukemia cells transduced with mCherry. Downregulation of NKG2A resulted in an increase of NK cytoxicity against tumor cells, which expressed HLA-E and HLA-G (GpHLA-E). This response was more robust than that generated by use of an anti-NKG2A antibody. NK proliferation was not affected.

• IncuCyte® was used in the long-term assessment of cytotoxicity of PEBL-transduced NK and controls against GpHLA-E+ transduced target cell lines and U2OS-GpHLA-E spheroids. Again, superior killing was demonstrated. Similar results were found in spheroid tumors of U2OS-mCherry co-cultured with NK cells.

• These results were further confirmed in xenograft models of immunodeficient mice, where NKG2A^nul NK cells displayed enhanced killing of tumors expressing HLA-E as compared to NKG2A + cells.

• The downregulation of NKG2A therefore increases the anti-tumor activity of NK.

Read the full paper in Journal of Clinical Investigation, March 2019.

Immunotherapy

PAK4 Inhibition Improves PD-1 Blockade Immunotherapy

A novel strategy to overcome immune checkpoint inhibition

Blocking programmed cell death protein 1 (PD-1) continues to be of intense interest as an immunotherapy approach for the enhanced efficacy of anti-tumor T cells. However, patients without pre-existing immune cell infiltration of their tumors may be resistant to this therapeutic approach. It has been suggested that cancer-cell intrinsic mechanisms may play a role in the exclusion of T cells from the tumors, in particular alterations in the WNT/β-catenin signaling pathway.

In this multi-institutional study by Abril-Rodriquez et al., the authors undertook a transcriptional analysis of tumor biopsies with patients with advanced melanoma who had been treated with anti-PD-1.  They identified an actionable drug target, p21-activated kinase 4 (PAK4), which could be inhibited in combination with immune checkpoint inhibition with the end goal of overcoming tumor resistance by increasing infiltration of T cells and dendritic cells.

Key findings include:

• PAK4 expression was elevated in tumor biopsies from patients that were not responsive to anti-PD-1 treatment who also had low infiltration of T cells and dendritic cells, suggesting it may be a driver to treatment resistance.

• The expression of PAK4 in melanoma tumor biopsies correlated with activation of the WNT/β-catenin signaling pathway, and PAK4 regulated WNT signaling in vitro.

• Studies were undertaken to evaluate the possible synergy between PAK4 inhibition and PD-1 blockade. The IncuCyte® Live-Cell Analysis System was used to measure cell confluency in MC38 PAK4 knockouts (mouse colon adenocarcinoma) and controls subjected to TNF treatment. Proliferation was decreased as compared to untreated controls: C38 WT (41%), MC38PAK4 KO6.9 (95%), and MC38 PAK4KO6.10 (74%). This indicated that the MC MC38 PAK4 knockout clones were more also more sensitive to TNF and contributed to the observed phenotype, which was consistent with previous reports.

• The combined use of anti-PD-1 and PAK4 inhibitor KPT-9274 demonstrated a more robust anti-tumor response than treatment with PD-1 only, suggesting synergy.

• In the in vivo mouse models using C57BL/6 mice with B1PAK4KO or B16 WT tumors, PAK4 inhibition with a murine anti-PD-1 antibody resulted in decreased tumor growth.

Read the full paper in Nature, December 2019.