The Inovirus Pf4 Triggers Antiviral Responses and Disrupts the Proliferation of Airway Basal Epithelial Cells.

BACKGROUND: The inovirus Pf4 is a lysogenic bacteriophage of Pseudomonas aeruginosa (Pa). People with Cystic Fibrosis (pwCF) experience chronic airway infection with Pa and a significant proportion have high numbers of Pf4 in their airway secretions. Given the known severe damage in the airways of Pa-infected pwCF, we hypothesized a high Pf4 burden can affect airway healing and inflammatory responses. In the airway, basal epithelial cells (BCs) are a multipotent stem cell population critical to epithelium homeostasis and repair. We sought to investigate the transcriptional responses of BCs under conditions that emulate infection with Pa and exposure to high Pf4 burden. METHODS: Primary BCs isolated from pwCF and wild-type (WT) donors were cultured in vitro and exposed to Pf4 or bacterial Lipopolysaccharide (LPS) followed by transcriptomic and functional assays. RESULTS: We found that BCs internalized Pf4 and this elicits a strong antiviral response as well as neutrophil chemokine production. Further, we found that BCs that take up Pf4 demonstrate defective migration and proliferation. CONCLUSIONS: Our findings are highly suggestive of Pf4 playing a role in the pathogenicity of Pa in the airways. These findings provide additional evidence for the ability of inoviruses to interact with mammalian cells and disrupt cell function.

Recommendations for the Equitable and Widespread Implementation of Liquid Biopsy for Cancer Care.

Liquid biopsies-tests that detect circulating tumor cellular components in the bloodstream-have the potential to transform cancer by reducing health inequities in screening, diagnostics, and monitoring. Today, liquid biopsies are being used to guide treatment choices for patients and monitor for cancer recurrence, and promising work in multi-cancer early detection is ongoing. However, without awareness of the barriers to adoption of this new technology and a willingness to build mitigation efforts into the implementation of widespread liquid biopsy testing, the communities that could most benefit may be the last to access and use them. In this work, we review the challenges likely to affect the accessibility of liquid biopsies in both the general population and underserved populations, and recommend specific actions to facilitate equitable access for all patients.

Bacterial outer membrane vesicles bound to bacteriophages modulate neutrophil responses to bacterial infection.

Pseudomonas aeruginosa is a major human pathogen, particularly effective at colonizing the airways of patients with cystic fibrosis. Bacteriophages are highly abundant at infection sites, but their impact on mammalian immunity remains unclear. We previously showed that Pf4, a temperate filamentous bacteriophage produced by P. aeruginosa, modifies the innate immune response to P. aeruginosa infections via TLR3 signaling, but the underlying mechanisms remained unclear. Notably, Pf4 is a single-stranded DNA and lysogenic phage, and its production does not typically result in lysis of its bacterial host. We identified previously that internalization of Pf4 by human or murine immune cells triggers maladaptive viral pattern recognition receptors and resulted in bacterial persistence based on the presence of phage RNA. We report now that Pf4 phage dampens inflammatory responses to bacterial endotoxin and that this is mediated in part via bacterial vesicles attached to phage particles. Outer membrane vesicles (OMVs) are produced by Gram-negative bacteria and play a key role in host pathogen interaction. Recently, evidence has emerged that OMVs differentially package small RNAs. In this study, we show that Pf4 are decorated with OMVs that remain affixed to Pf4 despite of purification steps. These phages are endocytosed by human cells and delivered to endosomal vesicles. We demonstrate that short RNAs within the OMVs form hairpin structures that trigger TLR3-dependent type I interferon production and antagonize production of antibacterial cytokines and chemokines. In particular, Pf4 phages inhibit CXCL5, preventing efficient neutrophil chemotaxis in response to endotoxin. Moreover, blocking IFNAR or TLR3 signaling abrogates the effect of Pf4 bound to OMVs on macrophage activation. In a murine acute pneumonia model, mice treated with Pf4 associated with OMVs show significantly less neutrophil infiltration in BAL fluid than mice treated with purified Pf4. These changes in macrophage phenotype are functionally relevant: conditioned media from cells exposed to Pf4 decorated with OMVs are significantly less effective at inducing neutrophil migration in vitro and in vivo. These results suggest that Pf4 phages alter innate immunity to bacterial endotoxin and OMVs, potentially dampening inflammation at sites of bacterial colonization or infection.

Monocytes use protrusive forces to generate migration paths in viscoelastic collagen-based extracellular matrices.

Circulating monocytes are recruited to the tumor microenvironment, where they can differentiate into macrophages that mediate tumor progression. To reach the tumor microenvironment, monocytes must first extravasate and migrate through the type-1 collagen rich stromal matrix. The viscoelastic stromal matrix around tumors not only stiffens relative to normal stromal matrix, but often exhibits enhanced viscous characteristics, as indicated by a higher loss tangent or faster stress relaxation rate. Here, we studied how changes in matrix stiffness and viscoelasticity, impact the three-dimensional migration of monocytes through stromal-like matrices. Interpenetrating networks of type-1 collagen and alginate, which enable independent tunability of stiffness and stress relaxation over physiologically relevant ranges, were used as confining matrices for three-dimensional culture of monocytes. Increased stiffness and faster stress relaxation independently enhanced the 3D migration of monocytes. Migrating monocytes have an ellipsoidal or rounded wedge-like morphology, reminiscent of amoeboid migration, with accumulation of actin at the trailing edge. Matrix adhesions and Rho-mediated contractility were dispensable for monocyte migration in 3D, but migration did require actin polymerization and myosin contractility. Mechanistic studies indicate that actin polymerization at the leading edge generates protrusive forces that open a path for the monocytes to migrate through in the confining viscoelastic matrices. Taken together, our findings implicate matrix stiffness and stress relaxation as key mediators of monocyte migration and reveal how monocytes use pushing forces at the leading edge mediated by actin polymerization to generate migration paths in confining viscoelastic matrices. Significance Statement: Cell migration is essential for numerous biological processes in health and disease, including for immune cell trafficking. Monocyte immune cells migrate through extracellular matrix to the tumor microenvironment where they can play a role in regulating cancer progression. Increased extracellular matrix (ECM) stiffness and viscoelasticity have been implicated in cancer progression, but the impact of these changes in the ECM on monocyte migration remains unknown. Here, we find that increased ECM stiffness and viscoelasticity promote monocyte migration. Interestingly, we reveal a previously undescribed adhesion-independent mode of migration whereby monocytes generate a path to migrate through pushing forces at the leading edge. These findings help elucidate how changes in the tumor microenvironment impact monocyte trafficking and thereby disease progression.

Biochemical, biophysical, and immunological characterization of respiratory secretions in severe SARS-CoV-2 infections.

Thick, viscous respiratory secretions are a major pathogenic feature of COVID-19, but the composition and physical properties of these secretions are poorly understood. We characterized the composition and rheological properties (i.e., resistance to flow) of respiratory secretions collected from intubated COVID-19 patients. We found the percentages of solids and protein content were greatly elevated in COVID-19 compared with heathy control samples and closely resembled levels seen in cystic fibrosis, a genetic disease known for thick, tenacious respiratory secretions. DNA and hyaluronan (HA) were major components of respiratory secretions in COVID-19 and were likewise abundant in cadaveric lung tissues from these patients. COVID-19 secretions exhibited heterogeneous rheological behaviors, with thicker samples showing increased sensitivity to DNase and hyaluronidase treatment. In histologic sections from these same patients, we observed increased accumulation of HA and the hyaladherin versican but reduced tumor necrosis factor-stimulated gene-6 staining, consistent with the inflammatory nature of these secretions. Finally, we observed diminished type I interferon and enhanced inflammatory cytokines in these secretions. Overall, our studies indicated that increases in HA and DNA in COVID-19 respiratory secretion samples correlated with enhanced inflammatory burden and suggested that DNA and HA may be viable therapeutic targets in COVID-19 infection.

Filamentous bacteriophage delays healing of Pseudomonas-infected wounds.

Chronic wounds infected by Pseudomonas aeruginosa (Pa) are characterized by disease progression and increased mortality. We reveal Pf, a bacteriophage produced by Pa that delays healing of chronically infected wounds in human subjects and animal models of disease. Interestingly, impairment of wound closure by Pf is independent of its effects on Pa pathogenesis. Rather, Pf impedes keratinocyte migration, which is essential for wound healing, through direct inhibition of CXCL1 signaling. In support of these findings, a prospective cohort study of 36 human patients with chronic Pa wound infections reveals that wounds infected with Pf-positive strains of Pa are more likely to progress in size compared with wounds infected with Pf-negative strains. Together, these data implicate Pf phage in the delayed wound healing associated with Pa infection through direct manipulation of mammalian cells. These findings suggest Pf may have potential as a biomarker and therapeutic target in chronic wounds.

Biochemical, Biophysical, and Immunological Characterization of Respiratory Secretions in Severe SARS-CoV-2 (COVID-19) Infections.

Thick, viscous respiratory secretions are a major pathogenic feature of COVID-19 disease, but the composition and physical properties of these secretions are poorly understood. We characterized the composition and rheological properties (i.e. resistance to flow) of respiratory secretions collected from intubated COVID-19 patients. We find the percent solids and protein content are greatly elevated in COVID-19 compared to heathy control samples and closely resemble levels seen in cystic fibrosis, a genetic disease known for thick, tenacious respiratory secretions. DNA and hyaluronan (HA) are major components of respiratory secretions in COVID-19 and are likewise abundant in cadaveric lung tissues from these patients. COVID-19 secretions exhibit heterogeneous rheological behaviors with thicker samples showing increased sensitivity to DNase and hyaluronidase treatment. In histologic sections from these same patients, we observe increased accumulation of HA and the hyaladherin versican but reduced tumor necrosis factorâ€"stimulated gene-6 (TSG6) staining, consistent with the inflammatory nature of these secretions. Finally, we observed diminished type I interferon and enhanced inflammatory cytokines in these secretions. Overall, our studies indicate that increases in HA and DNA in COVID-19 respiratory secretion samples correlate with enhanced inflammatory burden and suggest that DNA and HA may be viable therapeutic targets in COVID-19 infection.

Bacteriophages and the Immune System.

Bacteriophages-viruses that infect bacteria-are abundant within our bodies, but their significance to human health is only beginning to be explored. Here, we synthesize what is currently known about our phageome and its interactions with the immune system. We first review how phages indirectly affect immunity via bacterial expression of phage-encoded proteins. We next review how phages directly influence innate immunity and bacterial clearance. Finally, we discuss adaptive immunity against phages and its implications for phage/bacterial interactions. In light of these data, we propose that our microbiome can be understood as an interconnected network of bacteria, bacteriophages, and human cells and that the stability of these tri-kingdom interactions may be important for maintaining our immunologic and metabolic health. Conversely, the disruption of this balance, through exposure to exogenous phages, microbial dysbiosis, or immune dysregulation, may contribute to disease.

The phosphorylation state of both hERG and KvLQT1 mediates protein-protein interactions between these complementary cardiac potassium channel alpha subunits.

KvLQT1 and hERG are the α-subunits of the voltage-gated K+ channels which carry the cardiac repolarizing currents IKs and IKr, respectively. These currents function in vivo with some redundancy to maintain appropriate action potential durations (APDs) in cardiomyocytes. As such, protein-protein interactions between hERG and KvLQT1 may be important in normal cardiac electrophysiology, as well as in arrhythmia and sudden cardiac death. Previous phenomenological observations of functional, mutual downregulation between these complementary repolarizing currents in transgenic rabbit models and human cell culture motivate our investigations into protein-protein interactions between hERG and KvLQT1. Previous data suggest that a dynamic, physical interaction between hERG and KvLQT1 modulates the respective currents. However, the mechanism by which hERG-KvLQT1 interactions are regulated is still poorly understood. Phosphorylation is proposed to play a role since modifying the phosphorylation state of each protein has been shown to alter channel kinetics, and both hERG and KvLQT1 are targets of the Ser/Thr protein kinase PKA, activated by elevated intracellular cAMP. In this work, quantitative apFRET analyses of phosphonull and phosphomimetic hERG and KvLQT1 mutants indicate that unphosphorylated hERG does not interact with KvLQT1, suggesting that hERG phosphorylation is important for wild-type proteins to interact. For proteins already potentially interacting, phosphorylation of KvLQT1 appears to be the driving factor abrogating hERG-KvLQT1 interaction. This work increases our knowledge about hERG-KvLQT1 interactions, which may contribute to the efforts to elucidate mechanisms that underlie many types of arrhythmias, and also further characterizes novel protein-protein interactions between two distinct potassium channel families.

Pf Bacteriophage and Their Impact on Pseudomonas Virulence, Mammalian Immunity, and Chronic Infections.

Pf bacteriophage are temperate phages that infect the bacterium Pseudomonas aeruginosa, a major cause of chronic lung infections in cystic fibrosis (CF) and other settings. Pf and other temperate phages have evolved complex, mutualistic relationships with their bacterial hosts that impact both bacterial phenotypes and chronic infection. We and others have reported that Pf phages are a virulence factor that promote the pathogenesis of P. aeruginosa infections in animal models and are associated with worse skin and lung infections in humans. Here we review the biology of Pf phage and what is known about its contributions to pathogenesis and clinical disease. First, we review the structure, genetics, and epidemiology of Pf phage. Next, we address the diverse and surprising ways that Pf phages contribute to P. aeruginosa phenotypes including effects on biofilm formation, antibiotic resistance, and motility. Then, we cover data indicating that Pf phages suppress mammalian immunity at sites of bacterial infection. Finally, we discuss recent literature implicating Pf in chronic P. aeruginosa infections in CF and other settings. Together, these reports suggest that Pf bacteriophage have direct effects on P. aeruginosa infections and that temperate phages are an exciting frontier in microbiology, immunology, and human health.

Bacteriophage trigger antiviral immunity and prevent clearance of bacterial infection.

Bacteriophage are abundant at sites of bacterial infection, but their effects on mammalian hosts are unclear. We have identified pathogenic roles for filamentous Pf bacteriophage produced by Pseudomonas aeruginosa (Pa) in suppression of immunity against bacterial infection. Pf promote Pa wound infection in mice and are associated with chronic human Pa wound infections. Murine and human leukocytes endocytose Pf, and internalization of this single-stranded DNA virus results in phage RNA production. This triggers Toll-like receptor 3 (TLR3)- and TIR domain-containing adapter-inducing interferon-ß (TRIF)-dependent type I interferon production, inhibition of tumor necrosis factor (TNF), and the suppression of phagocytosis. Conversely, immunization of mice against Pf prevents Pa wound infection. Thus, Pf triggers maladaptive innate viral pattern-recognition responses, which impair bacterial clearance. Vaccination against phage virions represents a potential strategy to prevent bacterial infection.