ABSTRACT
Breast cancer is the most common form of cancer and the second cancer-causing death in females. Although remission rates are high if detected early, survival rates drop substantially when breast cancer becomes metastatic. The most common sites of metastatic breast cancer are bone, liver and lung. Respiratory viral infections inflict illnesses on countless people. The latest pandemic caused by the respiratory virus, SARS-CoV-2, has infected more than 600 million worldwide, with documented COVID-related death upward of 1 million in the United States alone. Respiratory viral infections result in increased inflammation with immune cell influx and expansion to facilitate viral clearance. Prior studies have shown that inflammation, including through neutrophils, can contribute to dormant cancer cells reawakening and outgrowth. Moreover, inhibition of IL6 has been shown to decrease breast cancer lung metastasis in mouse models. However, how respiratory viral infections contribute to breast cancer lung metastasis remains to be unraveled. Using MMTV/PyMT and MMTV/NEU mouse models of breast cancer lung metastasis and influenza A virus as a model respiratory virus, we demonstrated that acute influenza infection and the accompanying inflammation and immune cell influx awakens and dramatically increased proliferation and expansion of dormant disseminated cancer cells (DCC) in the lungs. Acute influenza infection leads to immune influx and expansion, including neutrophils and macrophages, with increased proportion of MHCII+ macrophages in early time points, and a sustained decrease in CD206+ macrophages starting 6 days post-infection until 28 days after the initial infection. Additionally, we observed a sustained accumulation of CD4+ T cells around expanding tumor cells for as long as 28 days after the infection. Notably, neutrophil depletion or IL6 knockout reversed the flu-induced dormant cell expansion in the lung. Finally, awakened DCC exhibited downregulation of vimentin immunoreactivity, suggesting a role for phenotypic plasticity in DCC outgrowth following viral infection. In conclusion, we show that respiratory viral infections awaken and increase proliferation of dormant breast cancer cells in the lung, and that depletion of neutrophils or blocking IL6 reverses influenza-induced dormant cell awakening and proliferation.
ABSTRACT
Background: We previously screened 10 human lung and upper airway cell lines expressing variable levels of endogenous ACE2/TMPRSS2. We found that H522 human lung adenocarcinoma cells supported SARS-CoV-2 replication independent of ACE2, whereas the ACE2 positive cell lines were not permissive to infection. Type I/III interferons (IFNs) potently restrict SARS-CoV-2 replication through the actions of hundreds of interferon-stimulated genes (ISGs) that are upregulated upon IFN signaling. Here we report that a number of ACE2 positive airway cell lines are unable to support SARS-CoV-2 replication due to basal activation of the cGAS-STING DNA sensing pathway and subsequent upregulation of IFNs and ISGs which restrict SARS-CoV-2 replication. Method(s): SARS-CoV-2 WT strain 2019-nCoV/USA-WA1/2020 viral replication was detected through analysis of cell associated RNA. RNA sequencing was used to study the basal level of genes in the type-I IFN pathway in the 10 cell lines, which was further validated by western blotting and qRT-PCR. A panel of 5 cell lines, with varying expression levels of ACE2 and TMPRSS2, were pre-treated with Ruxolitinib, a JAK1/2 inhibitor. A siRNA-mediated screen was used to determine the molecular basis of basally high expression of ISGs in cell lines. CRISPR knockout of IFN-alpha receptor and cGAS-STING pathway components was conducted in parallel Results: Here we show that higher basal levels of IFN pathway activity underlie the inability of ACE2+ cell lines to support virus replication. Importantly, this IFN-induced block can be overcome by chemical inhibition and genetic disruption of the IFN signaling pathway or by ACE2 overexpression, suggesting that one or more saturable ISGs underlie the lack of permissivity of these cells. Ruxolitinib treatment increased SARS-CoV-2 RNA levels by nearly 3 logs in OE21 and SCC25. Furthermore, the baseline activation of the STING-cGAS pathway accounts for the high ISG levels and genetic disruption of the cGAS-STING pathway enhances levels by nearly 2 and 3 logs of virus replication in the two separate ACE2+ cell line models respectively. Conclusion(s): Our findings demonstrate that cGAS-STING-dependent activation of IFN-mediated innate immunity underlies the inability of ACE2+ airway cell lines to support SARS-CoV-2 replication. Our study highlights that in addition to ACE2, basal activation of cGAS-STING pathway, IFNs and ISGs may play a key role in defining SARS-CoV-2 cellular tropism and may explain the complex SARS-CoV- 2 pathogenesis in vivo.
ABSTRACT
Butyrylcholinesterase (BChE), a hepatic enzyme produced by the liver is affected by and influences a variety of inflammatory, infectious and metabolic dysfunction processes. Considering that COVID-19 is a multisystem disorder related to conditions influenced by BChE, the potential interrelation of the two is reviewed. BChE is altered in a variety of infectious diseases, and serves as a prognostic marker in both infections and in non-infectious diseases. Closely related to acetylcholinesterase (AChE), BChE plays a role in modulating inflammation via the cholinergic system. It forms part of the signaling pathway linking the immune system, nervous system and the endocrine system. COVID-19 progresses to a stage of unregulated inflammation in a subset of subjects. Cholinergic dysfunction could be potentially responsible for a march to cytokine storm. BChE could influence the course of COVID-19 by acting through the brain-immune-endocrine axis via cholinergic transmission, as well as affecting factors predicting adverse outcomes of COVID-19 (obesity, insulin resistance, coronary artery disease, type 2 diabetes mellitus). Interestingly, variant forms of the enzyme with impaired hydrolytic activity are reported in endogamous ethnic populations. It would be instructive to study the effect of COVID-19 in these natural human knock-out equivalents. Biomed Rev 2021;32: 37-46.
ABSTRACT
Introduction: Management of acute respiratory distress including COVID-19 pneumonia involves O2 supplementation, which is lifesaving, but causes severe hyperoxic acute lung injury (HALI). AT2 cells are the most affected cell type in hyperoxia (HO). NADPH oxidase (NOX) is a major source of reactive oxygen species (ROS) in HO. NOX4, the only functionally active NOX present in mitochondria, and primarily produces H2O2 as well as mtROS has been shown to be involved in several human pathologies. Not much is known about NOX4-induced mitochondrial injury in HALI. The current study aims to determine the role of AT2 epithelial cell NOX4 in HALI and the impact of HO on the modulation of mtROS and mitochondrial dynamics in HALI. Methods: Nox4-/-Spc-Cre animals were generated using tamoxifen induction and the knockdown was validated. The Nox4- /-Spc-Cre knockout (KO) and wild type (WT) mice were exposed to room air (NO) or 95% O2 (HO) for 66h to study the structural and functional changes in the lung. Transmission Electron Microscopy (TEM) was used to study the HO-induced changes in mitochondria. Isolated primary AT2 and/ mouse lung epithelial (MLE) cell line was investigated for mtROS, mt dynamics and apoptosis. Mitochondrial injury was assessed in Nox4 WT and Nox4 silenced cells. Results: C57BL/6J WT animals subjected to HO for 66h showed increased expression of NOX4, determining the role of NOX4 in HALI. The H&E staining demonstrated significant HALI in Nox4 WT animals exposed to HO compared to Nox4 KO as determined by increased infiltration of neutrophils, alveolar wall thickening and presence of proteinaceous debris in the alveolar space. Further, increased BAL cell count and protein levels, increased AT2 cell death and elevation of the proinflammatory cytokine IL- 6 and the chemokine KC was seen in WT animals compared to Nox4 KO. Analysis of lung tissues by TEM showed mitochondrial swelling, cristae damage and mitophagy in AT2 cells due to HO. Changes in mt injury markers were also observed. HO-induced NOX4 increase in primary AT2/ MLE-12 cells resulted in increased mtROS production and apoptosis, which was reduced with Nox4 siRNA silencing. Conclusion: This study suggests that the HO induced NOX4 expression in mouse lung, and deletion of Nox4 gene in AT2 cells reduced mtROS production and apoptosis and protected the lungs from severe hyperoxic lung injury. These results suggest NOX4 as a potential target for the treatment of HALI.
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Background and Aims: Type 2 diabetes (T2D) is a major risk factor for developing severe infectious disease, such as COVID-19. The endocrine and immune system closely interact following viral infection, which is deregulated in T2D. Previously, we showed in humans and mice that viral infection causes transient insulin resistance, which can lead to permanent loss of glycemic control in subjects with pre-diabetes. How changes in systemic glycemia benefit the antiviral response, and how this derails in T2D is mostly unknown. Methods: Mice were infected with virulent strains of cytomegalovirus or lymphocytic choriomeningitis virus. Glucose-, insulin- and pyruvate-tolerance tests and hyperinsulinemic euglycemic clamping were used to determine the metabolic state of animals. Conditional knock-out models were used to measure the impact of cytokines on metabolism of specific organs. Dietinduced obesity models were used to determine the impact of hyperglycemia on the antiviral response. Results: Severe viral infection causes pancreatic β-cell hyperfunctionality following their stimulation with the cytokine IFNγ by local T cells. Virus-induced hyperinsulinemia impaired glucose release by the liver and promoted induction of fasting metabolism, because of reduced hepatic glycogenolysis, causing relative, transient hypoglycemia (RHG). RHG was beneficial to the antiviral response by promoting the release of antiviral cytokines by endothelial cells, which impaired viral replication. Obese mice failed to induce fastng metabolsim, resulting in lower antiviral cytokines, higher viral titers and increased pathology. Conclusions: Metabolic adaptations following infection are of major importance for optimal control of viral replication. In context of T2D, these changes cannot be accomplished, thus leading to more frequent and severe infections.
ABSTRACT
Vaccines prevent 4-5 million deaths a year making them the principal tool of medical intervention worldwide. Nucleoside-modified mRNA was developed over 15 years ago and has become the darling of the COVID-19 pandemic with the first 2 FDA approved vaccines based on it. These vaccines show greater than 90% efficacy and outstanding safety in clinical use. The mechanism for the outstanding immune response induction are the prolonged production of antigen leading to continuous loading of germinal centers and the adjuvant effect of the LNPs, which selectively stimulate T follicular helper cells that drive germinal center responses. Vaccine against many pathogens, including HIV, HCV, HSV2, CMV, universal influenza, coronavirus variants, pancoronavirus, nipah, norovirus, malaria, TB, and many others are currently in development. Nucleoside-modified mRNA is also being developed for therapeutic protein delivery. Finally, nucleoside-modified mRNA-LNPs are being developed and used for gene therapy. Cas9 knockout to treat transthyretin amyloidosis has shown success in phase 1 trials. We have developed the ability to target specific cells and organs, including lung, brain, heart, CD4+ cells, all T cells, and bone marrow stem cells, with LNPs allowing specific delivery of gene editing and insertion systems to treat diseases such as sickle cell anemia. Nucleoside-modified mRNA will have an enormous potential in the development of new medical therapies.
ABSTRACT
As solid organ transplant (SOT) recipients receive therapeutic immunosuppression that compromises their immune response to infections and vaccines, they have a high risk of developing severe COVID-19 and an increased risk of COVID-19-related death. The constant immunosuppression may result in reduction of efficiency of immunotherapy. Thus, a therapy is required that enables efficient viral clearance against SARS-CoV-2 whilst simultaneously maintaining immunosuppressive treatment in transplant patients to prevent transplant rejection. Here, we propose adoptive transfer of SARS-CoV-2-specific T-cells rendered resistant to the common immunosuppressant Tacrolimus to optimize performance in immunosuppressed patients. By using a GMP-compatible, vector-free CRISPR-Cas9-based, gene-editing approach, we knocked out the cell-intrinsic adaptor protein FKBP12, which is required for the immunosuppressive function of Tacrolimus, and generated Tacrolimus-resistant SARS-CoV-2-reactive T-cell products (TCPs) from the blood of SARS-CoV-2 convalescent donors. Functional and phenotypical characterization of these products in depth, including single cell CITE- and TCR sequencing analyses, showed that the gene modification did not impact the functional potency of the Tacrolimus-resistant SARS-CoV-2-specific TCPs compared to unmodified SARS-CoV-2-specific TCPs, but confirmed resistance to Tacrolimus and sensitivity to alternative immunosuppressive drugs from the same class (safety switch). Based on the promising results, we aim to clinically validate this approach in transplant recipients. Our strategy has the potential to prevent or ameliorate severe COVID-19 in the SOT setting whilst preventing allogeneic organ rejection. Our platform technology allows targeting of different SARS-CoV-2 variants and other viruses, thus multiplying its potential therapeutic use.
ABSTRACT
The proceedings contain 593 papers. The topics discussed include: association of transthyretin VAL122ILE variant with incident heart failure and mortality among Black Americans: insights from the regards study;vitamin d modulates histone modifications governing the natriuretic peptide receptor-a gene;cell to cell communication through entanglement and superconductivity improving left ventricular function in an uncoupled state;association of serum lipid levels with COVID-19 infection, severity and mortality;impaired glucose tolerance in guanylyl cyclase/natriuretic peptide receptor-a gene-knockout and gene-duplication mutant mice;meta-analysis of randomized vs observational studies of the effects of invasive therapy in patients with non-ST-elevation myocardial infarction and chronic kidney disease;a retrospective analysis of mortality in adult patients with acute coronary syndrome and cardiogenic shock requiring temporary mechanical circulatory support;and mitochondrial myopathy mimicking Guillain-Barre syndrome in a 21-year-old graduate student.
ABSTRACT
VITT is an immune-based complication of adenoviral-based vaccines used to immunize against SARS_CoV2. The antibodies in VITT have been described as directed at the platelet-specific chemokine PF4 (CXCL4). While the clinical course and target chemokine in VITT has much in common with the better-known thrombocytopenic/prothrombotic disorder, heparin-induced thrombocytopenia (HIT), which involves antibodies directed against PF4 bound to the polyanion heparin, the specific loci where VITT and PF4/polyanion HIT antibodies bind appear to differ in studies using alanine-scanning mutations of PF4 (Nature, 2021. DOI: 10.1038/s41586-021-03744-4). The VITT antigenic site localizes to a heparin-binding domain. Unlike the dominant HIT locus, the VITT locus is conserved not only between human and mouse PF4, but also between PF4 and the related platelet-specific chemokine NAP2 (CXCL7). NAP2 is also expressed and stored in platelet alpha-granules and is present in equimolar concentrations to PF4. Unlike PF4, NAP2 avidly binds the chemokine receptor CXCR2 and strongly activates neutrophils. We now show that antibodies from patients who developed VITT after both AstraZeneca (AZ) or Johnson and Johnson (JJ) adenoviral vaccines, unlike HIT antibodies, recognize mouse PF4 (Figure 1A). More importantly, both AZ and JJ VITT antibodies bound NAP2, while none of the HIT antibodies tested bound PF4 or NAP2 in the absence of heparin (Figure 1A). These results are consistent with the alanine-scanning studies that distinguish the HIT and VITT binding sites. Dynamic light scattering (DLS) showed that NAP2 and PF4 bind to the adenoviral vectors, including Ad5 and the AZ vector ChAdOx5, which leads to expression of SARS_CoV2 spike protein. ChAdOx2 vaccine and CsCl 2-purified ChAdOx2 bound to both proteins, but form larger complexes with NAP2 than with PF4 even at lower concentrations of this chemokine (Figure 1C). Removal of anti-PF4 antibodies by hPF4-Sepharose abrogated PF4-dependent binding, but did not significantly reduce binding to NAP2 (not shown), indicating that VITT plasma contains discrete pools of anti-PF4 and anti-NAP2 antibodies that may have distinct functional properties. Sandwich ELISA (not shown) and Western blot analysis of purified VITT IgG demonstrates the presence of hPF4-IgG and NAP2-IgG immune complexes in purified patient's IgG (Figure 2A). Functional studies show that both PF4 and NAP2 can activate platelets in the presence of VITT antibodies. Anti-PF4-depleted VITT IgG fraction retains the ability to activate platelets in the presence of NAP2 (Figure 2B). Thus, unlike HIT, VITT appears to target a shared antigenic site on the related chemokines PF4 and NAP2. This raises the question as to whether NAP2, as one the most abundant platelet chemokines released from activated platelets, is involved in the initiation and propagation of the immunothrombotic response. Additional studies are needed to see whether NAP2, which can potently and specifically activate neutrophils via CXCLR2, contributes to the specific thromboinflammatory phenotype seen in VITT. We propose using FcgammaRIIA+ mice that concurrently express human PF4 and NAP2 and specific knockout of each chemokine, available in our group, to further understand the pathogenesis of VITT and its thrombocytopenic/ prothrombotic phenotype. [Formula presented] Disclosures: Padmanabhan: Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees. Cines: Dova: Consultancy;Rigel: Consultancy;Treeline: Consultancy;Arch Oncol: Consultancy;Jannsen: Consultancy;Taventa: Consultancy;Principia: Other: Data Safety Monitoring Board.