Lung Epithelial Cell Transcriptional Regulation as a Factor in COVID-19 Associated Coagulopathies

SARS-CoV-2 has rapidly become a global pandemic. In addition to the acute pulmonary symptoms of COVID-19 (the disease associated with SARS-CoV-2 infection), pulmonary and distal coagulopathies have caused morbidity and mortality in many patients. Currently, the molecular pathogenesis underlying COVID-19 associated coagulopathies are unknown. Identifying the molecular basis of how SARS-CoV-2 drives coagulation is essential to mitigating short and long term thrombotic risks of sick and recovered COVID-19 patients. We aimed to perform coagulation focused transcriptome analysis of in vitro infected primary respiratory epithelial cells, patient derived bronchial alveolar lavage (BALF) cells, and circulating immune cells during SARS-CoV-2 infection. Our objective was to identify transcription mediated signaling networks driving coagulopathies associated with COVID-19. We analyzed recently published experimentally and clinically derived bulk or single cell RNA sequencing datasets of SARS-CoV-2 infection to identify changes in transcriptional regulation of blood coagulation. We also confirmed that the transcriptional expression of a key coagulation regulator was recapitulated at the protein level. We specifically focused our analysis on lung tissue expressed genes regulating the extrinsic coagulation cascade and the plasminogen activation system. Analyzing transcriptomic data of in vitro infected normal human bronchial epithelial (NHBE) cells and patient derived BALF samples revealed that SARS-CoV-2 infection induces the extrinsic blood coagulation cascade and suppresses the plasminogen activation system. We also performed in vitro SARS-CoV-2 infection experiments on primary human lung epithelial cells to confirm that transcriptional upregulation of Tissue Factor, the extrinsic coagulation cascade master regulator, manifested at the protein level. Further, infection of NHBEs with influenza A virus (IAV) did not drive key regulators of blood coagulation in a similar manner as SARS-CoV-2. Additionally, peripheral blood mononuclear cells (PBMCs) did not differentially express genes regulating the extrinsic coagulation cascade or plasminogen activation system during SARS-CoV-2 infection, suggesting that they are not directly inducing coagulopathy through these pathways. The hyper-activation of the extrinsic blood coagulation cascade and the suppression of the plasminogen activation system in SARS-CoV-2 infected epithelial cells may drive diverse coagulopathies in the lung and distal organ systems. Understanding how hosts drive such transcriptional changes with SARS-CoV-2 infection may enable the design of host-directed therapeutic strategies to treat COVID-19 and other coronaviruses inducing hyper-coagulation

The lung, the niche, and the microbe: Exploring the lung microbiome in cancer and immunity

The lung is a complex and unique organ system whose biology is strongly influenced by environmental exposure, oxygen abundance, connection to extrapulmonary systems via a dense capillary network, and an array of immune cells that reside in the tissue at steady state. The lung also harbors a low biomass community of commensal microorganisms that are dynamic during both health and disease with the capacity to modulate regulatory immune responses during diseases such as cancer. Lung cancer is the third most common cancer worldwide with the highest mortality rate amongst cancers due to the difficulty of an early diagnosis. This review discusses the current body of work addressing the interactions between the lung microbiota and the immune system, and how these two components of the pulmonary system are linked to lung cancer development and outcomes. Bringing in lessons from broader studies examining the effects of the gut microbiota on cancer outcomes, we highlight many challenges and gaps in this nascent field

Surviving Deadly Lung Infections: Innate Host Tolerance Mechanisms in the Pulmonary System

Much research on infectious diseases focuses on clearing the pathogen through the use of antimicrobial drugs, the immune response, or a combination of both. Rapid clearance of pathogens allows for a quick return to a healthy state and increased survival. Pathogen-targeted approaches to combating infection have inherent limitations, including their pathogen-specific nature, the potential for antimicrobial resistance, and poor vaccine efficacy, among others. Another way to survive an infection is to tolerate the alterations to homeostasis that occur during a disease state through a process called host tolerance or resilience, which is independent from pathogen burden. Alterations in homeostasis during infection are numerous and include tissue damage, increased inflammation, metabolic changes, temperature changes, and changes in respiration. Given its importance and sensitivity, the lung is a good system for understanding host tolerance to infectious disease. Pneumonia is the leading cause of death for children under five worldwide. One reason for this is because when the pulmonary system is altered dramatically it greatly impacts the overall health and survival of a patient. Targeting host pathways involved in maintenance of pulmonary host tolerance during infection could provide an alternative therapeutic avenue that may be broadly applicable across a variety of pathologies. In this review, we will summarize recent findings on tolerance to host lung infection. We will focus on the involvement of innate immune responses in tolerance and how an initial viral lung infection may alter tolerance mechanisms in leukocytic, epithelial, and endothelial compartments to a subsequent bacterial infection. By understanding tolerance mechanisms in the lung we can better address treatment options for deadly pulmonary infections

Rapid Pathogen-Induced Apoptosis: A Mechanism Used by Dendritic Cells to Limit Intracellular Replication of Legionella pneumophila

Dendritic cells (DCs) are specialized phagocytes that internalize exogenous antigens and microbes at peripheral sites, and then migrate to lymphatic organs to display foreign peptides to naïve T cells. There are several examples where DCs have been shown to be more efficient at restricting the intracellular replication of pathogens compared to macrophages, a property that could prevent DCs from enhancing pathogen dissemination. To understand DC responses to pathogens, we investigated the mechanisms by which mouse DCs are able to restrict replication of the intracellular pathogen Legionella pneumophila. We show that both DCs and macrophages have the ability to interfere with L. pneumophila replication through a cell death pathway mediated by caspase-1 and Naip5. L. pneumophila that avoided Naip5-dependent responses, however, showed robust replication in macrophages but remained unable to replicate in DCs. Apoptotic cell death mediated by caspase-3 was found to occur much earlier in DCs following infection by L. pneumophila compared to macrophages infected similarly. Eliminating the pro-apoptotic proteins Bax and Bak or overproducing the anti-apoptotic protein Bcl-2 were both found to restore L. pneumophila replication in DCs. Thus, DCs have a microbial response pathway that rapidly activates apoptosis to limit pathogen replication

Both TLR2 and TRIF Contribute to Interferon-β Production during Listeria Infection

Synthesis of interferon-β (IFN-β) is an innate response to cytoplasmic infection with bacterial pathogens. Our recent studies showed that Listeria monocytogenes limits immune detection and IFN-β synthesis via deacetylation of its peptidoglycan, which renders the bacterium resistant to lysozyme degradation. Here, we examined signaling requirements for the massive IFN-β production resulting from the infection of murine macrophages with a mutant strain of L. monocytogenes, ΔpgdA, which is unable to modify its peptidoglycan. We report the identification of unconventional signaling pathways to the IFN-β gene, requiring TLR2 and bacterial internalization. Induction of IFN-β was independent of the Mal/TIRAP adaptor protein but required TRIF and the transcription factors IRF3 and IRF7. These pathways were stimulated to a lesser degree by wild-type L. monocytogenes. They operated in both resident and inflammatory macrophages derived from the peritoneal cavity, but not in bone marrow-derived macrophages. The novelty of our findings thus lies in the first description of TLR2 and TRIF as two critical components leading to the induction of the IFN-β gene and in uncovering that individual macrophage populations adopt different strategies to link pathogen recognition signals to IFN-β gene expression

Coinfection With Influenza A Virus and Klebsiella oxytoca: An Underrecognized Impact on Host Resistance and Tolerance to Pulmonary Infections

Pneumonia is a world health problem and a leading cause of death, particularly affecting children and the elderly (1, 2). Bacterial pneumonia following infection with influenza A virus (IAV) is associated with increased morbidity and mortality but the mechanisms behind this phenomenon are not yet well-defined (3). Host resistance and tolerance are two processes essential for host survival during infection. Resistance is the host's ability to clear a pathogen while tolerance is the host's ability to overcome the impact of the pathogen as well as the host response to infection (4–8). Some studies have shown that IAV infection suppresses the immune response, leading to overwhelming bacterial loads (9–13). Other studies have shown that some IAV/bacterial coinfections cause alterations in tolerance mechanisms such as tissue resilience (14–16). In a recent analysis of nasopharyngeal swabs from patients hospitalized during the 2013–2014 influenza season, we have found that a significant proportion of IAV-infected patients were also colonized with Klebsiella oxytoca, a gram-negative bacteria known to be an opportunistic pathogen in a variety of diseases (17). Mice that were infected with K. oxytoca following IAV infection demonstrated decreased survival and significant weight loss when compared to mice infected with either single pathogen. Using this model, we found that IAV/K. oxytoca coinfection of the lung is characterized by an exaggerated inflammatory immune response. We observed early inflammatory cytokine and chemokine production, which in turn resulted in massive infiltration of neutrophils and inflammatory monocytes. Despite this swift response, the pulmonary pathogen burden in coinfected mice was similar to singly-infected animals, albeit with a slight delay in bacterial clearance. In addition, during coinfection we observed a shift in pulmonary macrophages toward an inflammatory and away from a tissue reparative phenotype. Interestingly, there was only a small increase in tissue damage in coinfected lungs as compared to either single infection. Our results indicate that during pulmonary coinfection a combination of seemingly modest defects in both host resistance and tolerance may act synergistically to cause worsened outcomes for the host. Given the prevalence of K. oxytoca detected in human IAV patients, these dysfunctional tolerance and resistance mechanisms may play an important role in the response of patients to IAV

A novel series of positive modulators of the AMPA receptor : discovery and structure based hit-to-lead studies

Starting from an HTS derived hit 1, application of biostructural data facilitated rapid optimization to lead 22, a novel AMPA receptor modulator. This is the first demonstration of how structure based drug design can be exploited in an optimization program for a glutamate receptor

Expression of Natural Killer Receptor Alleles at Different Ly49 Loci Occurs Independently and Is Regulated by Major Histocompatibility Complex Class I Molecules

Ly49 receptor genes are expressed by subsets of natural killer (NK) cells in an overlapping fashion, accounting for the capacity of NK subsets to attack host cells that have selectively downregulated self–major histocompatibility complex (MHC) class I molecules. It was shown previously that most NK cells express only one or the other allele of a given Ly49 gene, while a smaller population expresses both alleles. However, the methods used to detect monoallelic and biallelic cells were nonquantitative. Here, new allele-specific antibodies were used to provide the first quantitative examination of biallelic and monoallelic expression of Ly49A and Ly49G2. The results demonstrate conclusively that most Ly49A+ and Ly49G2+ NK cells express the corresponding gene in a monoallelic fashion, with a smaller subset expressing both alleles. Unexpectedly, biallelic Ly49A+ NK cells were more numerous than predicted by completely independent allelic expression, suggesting some heterogeneity among NK progenitors in the potential to express a given Ly49 gene. The data also show that cells expressing one allele of Ly49G2 may express Ly49A from the same or opposite chromosome with equal likelihood, indicating that the expressed allele is chosen independently for different Ly49 genes. Finally, the data demonstrate that biallelic expression of Ly49A or Ly49G2 occurs least frequently in mice that express ligands for these receptors (H-2d mice), and most frequently in class I–deficient mice. Thus, biallelic expression of Ly49 genes is regulated by interactions of NK cell progenitors with MHC class I molecules

Structure based evolution of a novel series of positive modulators of the AMPA receptor

Starting from compound 1, we utilized biostructural data to successfully evolve an existing series into a new chemotyope with a promising overall profile, exemplified by