Inflammatory profiles in sputum and blood of people with TB with and without HIV coinfection.

Although tuberculosis (TB) remains a major killer among infectious diseases and the leading cause of death for people with HIV, drivers of immunopathology, particularly at the site of infection in the lungs remain incompletely understood. To fill this gap, we compared cytokine profiles in paired plasma and sputum samples collected from adults with pulmonary TB with and without HIV. We found that people with pulmonary TB with HIV had significantly higher markers of inflammation in both plasma and sputum than those without HIV; these differences were present despite a similar extent of radiographic involvement. We also found that the strength and direction of correlations between biomarkers in the blood and lung compartments differed by HIV status and people with HIV had more positive correlations than those without HIV. Future studies can further explore these differences in inflammation by HIV status across the blood and lung compartments and seek to establish how these profiles may be associated with long-term outcomes and lung health after completion of TB treatment.

Why Certain Repurposed Drugs Are Unlikely to Be Effective Antivirals to Treat SARS-CoV-2 Infections.

Most repurposed drugs have proved ineffective for treating COVID-19. We evaluated median effective and toxic concentrations (EC50, CC50) of 49 drugs, mostly from previous clinical trials, in Vero cells. Ratios of reported unbound peak plasma concentrations, (Cmax)/EC50, were used to predict the potential in vivo efficacy. The 20 drugs with the highest ratios were retested in human Calu-3 and Caco-2 cells, and their CC50 was determined in an expanded panel of cell lines. Many of the 20 drugs with the highest ratios were inactive in human Calu-3 and Caco-2 cells. Antivirals effective in controlled clinical trials had unbound Cmax/EC50 ≥ 6.8 in Calu-3 or Caco-2 cells. EC50 of nucleoside analogs were cell dependent. This approach and earlier availability of more relevant cultures could have reduced the number of unwarranted clinical trials.

Transcriptional reprogramming of infiltrating neutrophils drives lung pathology in severe COVID-19 despite low viral load.

Troubling disparities in COVID-19-associated mortality emerged early, with nearly 70% of deaths confined to Black/African American (AA) patients in some areas. However, targeted studies on this vulnerable population are scarce. Here, we applied multiomics single-cell analyses of immune profiles from matching airways and blood samples of Black/AA patients during acute SARS-CoV-2 infection. Transcriptional reprogramming of infiltrating IFITM2+/S100A12+ mature neutrophils, likely recruited via the IL-8/CXCR2 axis, leads to persistent and self-sustaining pulmonary neutrophilia with advanced features of acute respiratory distress syndrome (ARDS) despite low viral load in the airways. In addition, exacerbated neutrophil production of IL-8, IL-1ß, IL-6, and CCL3/4, along with elevated levels of neutrophil elastase and myeloperoxidase, were the hallmarks of transcriptionally active and pathogenic airway neutrophilia. Although our analysis was limited to Black/AA patients and was not designed as a comparative study across different ethnicities, we present an unprecedented in-depth analysis of the immunopathology that leads to acute respiratory distress syndrome in a well-defined patient population disproportionally affected by severe COVID-19.

Baricitinib attenuates the proinflammatory phase of COVID-19 driven by lung-infiltrating monocytes.

SARS-CoV-2-infected subjects are generally asymptomatic during initial viral replication but may suffer severe immunopathology after the virus has receded and monocytes have infiltrated the airways. In bronchoalveolar lavage fluid from severe COVID-19 patients, monocytes express mRNA encoding inflammatory mediators and contain SARS-CoV-2 transcripts. We leverage a human small airway model of infection and inflammation, whereby primary blood monocytes transmigrate across SARS-CoV-2-infected lung epithelium to characterize viral burden, gene expression, and inflammatory mediator secretion by epithelial cells and monocytes. In this model, lung-infiltrating monocytes acquire SARS-CoV-2 from the epithelium and upregulate expression and secretion of inflammatory mediators, mirroring in vivo data. Combined use of baricitinib (Janus kinase inhibitor) and remdesivir (nucleoside analog) enhances antiviral signaling and viral clearance by SARS-CoV-2-positive monocytes while decreasing secretion of proneutrophilic mediators associated with acute respiratory distress syndrome. These findings highlight the role of lung-infiltrating monocytes in COVID-19 pathogenesis and their importance as a therapeutic target.

Macrophage PD-1 associates with neutrophilia and reduced bacterial killing in early cystic fibrosis airway disease.

BACKGROUND: Macrophages are the major resident immune cells in human airways coordinating responses to infection and injury. In cystic fibrosis (CF), neutrophils are recruited to the airways shortly after birth, and actively exocytose damaging enzymes prior to chronic infection, suggesting a potential defect in macrophage immunomodulatory function. Signaling through the exhaustion marker programmed death protein 1 (PD-1) controls macrophage function in cancer, sepsis, and airway infection. Therefore, we sought to identify potential associations between macrophage PD-1 and markers of airway disease in children with CF. METHODS: Blood and bronchoalveolar lavage fluid (BALF) were collected from 45 children with CF aged 3 to 62 months and structural lung damage was quantified by computed tomography. The phenotype of airway leukocytes was assessed by flow cytometry, while the release of enzymes and immunomodulatory mediators by molecular assays. RESULTS: Airway macrophage PD-1 expression correlated positively with structural lung damage, neutrophilic inflammation, and infection. Interestingly, even in the absence of detectable infection, macrophage PD-1 expression was elevated and correlated with neutrophilic inflammation. In an in vitro model mimicking leukocyte recruitment into CF airways, soluble mediators derived from recruited neutrophils directly induced PD-1 expression on recruited monocytes/macrophages, suggesting a causal link between neutrophilic inflammation and macrophage PD-1 expression in CF. Finally, blockade of PD-1 in a short-term culture of CF BALF leukocytes resulted in improved pathogen clearance. CONCLUSION: Taken together, these findings suggest that in early CF lung disease, PD-1 upregulation associates with airway macrophage exhaustion, neutrophil takeover, infection, and structural damage.

Neutrophil-derived extracellular vesicles promote feed-forward inflammasome signaling in cystic fibrosis airways.

Cystic fibrosis (CF) airways feature high extracellular levels of the IL-1 family of proinflammatory mediators. These mediators are cleavage products of caspase-1, the final protease in the inflammasome cascade. Due to the proven chronic presence of reprogrammed neutrophils in the CF airway lumen, understanding inflammasome signaling in these cells is of great importance to understand how disease is perpetuated in this milieu. Here, we hypothesized that CF airway neutrophils contribute to chronic inflammation, in part, via the packaging of inflammasome-inducing signals in extracellular vesicles (EVs). We confirmed that CF airway fluid is enriched in IL-1α, IL-1ß, and IL-18, and that CF airway neutrophils up-regulate the activating receptor IL-1R1. Meanwhile, down-modulatory signals such as IL-1R2 and IL-1RA are unchanged. Active caspase-1 itself is present in CF airway fluid EVs, with neutrophil-derived EVs being most enriched. Using a transmigration model of CF airway inflammation, we show that CF airway fluid EVs are necessary and sufficient to induce primary granule exocytosis by naïve neutrophils (hallmark of reprogramming) and concomitantly activate caspase-1 and IL-1ß production by these cells and that the addition of triple-combination highly effective CFTR modulator therapy does not abrogate these effects. Finally, EVs from activated neutrophils can deliver active caspase-1 to primary tracheal epithelial cells and induce their release of IL-1α. These findings support the existence of a feed-forward inflammatory process by which reprogrammed CF airway neutrophils bypass 2-step control of inflammasome activation in neighboring cells (naïve neutrophils and epithelial cells) via the transfer of bioactive EVs.

Inactivation of SARS-CoV-2 and COVID-19 Patient Samples for Contemporary Immunology and Metabolomics Studies.

Due to the severity of COVID-19 disease, the U.S. Centers for Disease Control and Prevention and World Health Organization recommend that manipulation of active viral cultures of SARS-CoV-2 and respiratory secretions from COVID-19 patients be performed in biosafety level (BSL)3 laboratories. Therefore, it is imperative to develop viral inactivation procedures that permit samples to be transferred to lower containment levels (BSL2), while maintaining the fidelity of complex downstream assays to expedite the development of medical countermeasures. In this study, we demonstrate optimal conditions for complete viral inactivation following fixation of infected cells with commonly used reagents for flow cytometry, UVC inactivation in sera and respiratory secretions for protein and Ab detection, heat inactivation following cDNA amplification for droplet-based single-cell mRNA sequencing, and extraction with an organic solvent for metabolomic studies. Thus, we provide a suite of viral inactivation protocols for downstream contemporary assays that facilitate sample transfer to BSL2, providing a conceptual framework for rapid initiation of high-fidelity research as the COVID-19 pandemic continues.

Mass production of human airway-like neutrophils via transmigration in an organotypic model of human airways.

Neutrophils are difficult to study, particularly in tissues, due to their short half-life and propensity for activation. We describe an organotypic airway model that uses patient airway fluid to enable the transmigration of blood neutrophils to acquire an airway-like phenotype in order to better understand their contribution to airway diseases. In particular, we showcase how conditioned neutrophils modulate their bacteria-killing abilities. For complete details on the use and execution of this protocol, please refer to Margaroli et al. (2021).

Transcriptional firing represses bactericidal activity in cystic fibrosis airway neutrophils.

Neutrophils are often considered terminally differentiated and poised for bacterial killing. In chronic diseases such as cystic fibrosis (CF), an unexplained paradox pits massive neutrophil presence against prolonged bacterial infections. Here, we show that neutrophils recruited to CF airways in vivo and in an in vitro transmigration model display rapid and broad transcriptional firing, leading to an upregulation of anabolic genes and a downregulation of antimicrobial genes. Newly transcribed RNAs are mirrored by the appearance of corresponding proteins, confirming active translation in these cells. Treatment by the RNA polymerase II and III inhibitor α-amanitin restores the expression of key antimicrobial genes and increases the bactericidal capacity of CF airway neutrophils in vitro and in short-term sputum cultures ex vivo. Broadly, our findings show that neutrophil plasticity is regulated at the site of inflammation via RNA and protein synthesis, leading to adaptations that affect their canonical functions (i.e., bacterial clearance).

Immunomodulation in Cystic Fibrosis: Why and How?

Cystic fibrosis (CF) lung disease is characterized by unconventional mechanisms of inflammation, implicating a chronic immune response dominated by innate immune cells. Historically, therapeutic development has focused on the mutated cystic fibrosis transmembrane conductance regulator (CFTR), leading to the discovery of small molecules aiming at modulating and potentiating the presence and activity of CFTR at the plasma membrane. However, treatment burden sustained by CF patients, side effects of current medications, and recent advances in other therapeutic areas have highlighted the need to develop novel disease targeting of the inflammatory component driving CF lung damage. Furthermore, current issues with standard treatment emphasize the need for directed lung therapies that could minimize systemic side effects. Here, we summarize current treatment used to target immune cells in the lungs, and highlight potential benefits and caveats of novel therapeutic strategies.

Immunogenicity of RNA Replicons Encoding HIV Env Immunogens Designed for Self-Assembly into Nanoparticles.

RNA replicons are a promising platform technology for vaccines. To evaluate the potential of lipid nanoparticle-formulated replicons for delivery of HIV immunogens, we designed and tested an alphavirus replicon expressing a self-assembling protein nanoparticle immunogen, the glycoprotein 120 (gp120) germline-targeting engineered outer domain (eOD-GT8) 60-mer. The eOD-GT8 immunogen is a germline-targeting antigen designed to prime human B cells capable of evolving toward VRC01-class broadly neutralizing antibodies. Replicon RNA was encapsulated with high efficiency in 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-based lipid nanoparticles, which provided effective delivery in the muscle and expression of luciferase lasting ∼30 days in normal mice, contrasting with very brief and low levels of expression obtained by delivery of equivalent modified mRNA (modRNA). eOD-GT8 60-mer-encoding replicons elicited high titers of gp120-specific antibodies following a single injection in mice, and increased levels of antigen-specific germinal center B cells compared with protein immunization. Immunization of transgenic mice expressing human inferred-germline VRC01 heavy chain B cell receptors that are the targets of the eOD antigen led to priming of B cells and somatic hypermutation consistent with VRC01-class antibody development. Altogether, these data suggest replicon delivery of Env immunogens may be a promising avenue for HIV vaccine development.

In vitro evolution of enhanced RNA replicons for immunotherapy.

Self-replicating (replicon) RNA is a promising new platform for gene therapy, but applications are still limited by short persistence of expression in most cell types and low levels of transgene expression in vivo. To address these shortcomings, we developed an in vitro evolution strategy and identified six mutations in nonstructural proteins (nsPs) of Venezuelan equine encephalitis (VEE) replicon that promoted subgenome expression in cells. Two mutations in nsP2 and nsP3 enhanced transgene expression, while three mutations in nsP3 regulated this expression. Replicons containing the most effective mutation combinations showed enhanced duration and cargo gene expression in vivo. In comparison to wildtype replicon, mutants expressing IL-2 injected into murine B16F10 melanoma showed 5.5-fold increase in intratumoral IL-2 and 2.1-fold increase in infiltrating CD8 T cells, resulting in significantly slowed tumor growth. Thus, these mutant replicons may be useful for improving RNA therapeutics for vaccination, cancer immunotherapy, and gene therapy.

Neutrophil Dysfunction in the Airways of Children with Acute Respiratory Failure Due to Lower Respiratory Tract Viral and Bacterial Coinfections.

Neutrophils are recruited to the airways of patients with acute respiratory distress syndrome (ARDS) where they acquire an activated pro-survival phenotype with an enhanced respiratory burst thought to contribute to ARDS pathophysiology. Our in vitro model enables blood neutrophil transepithelial migration into cell-free tracheal aspirate fluid from patients to recapitulate the primary airway neutrophil phenotype observed in vivo. Neutrophils transmigrated through our model toward airway fluid from children with lower respiratory viral infections coinfected with bacteria had elevated levels of neutrophil activation markers but paradoxically exhibited an inability to kill bacteria and a defective respiratory burst compared with children without bacterial coinfection. The airway fluid from children with bacterial coinfections had higher levels of neutrophil elastase activity, as well as myeloperoxidase levels compared to children without bacterial coinfection. Neutrophils transmigrated into the aspirate fluid from children with bacterial coinfection showed decreased respiratory burst and killing activity against H. influenzae and S. aureus compared to those transmigrated into the aspirate fluid from children without bacterial coinfection. Use of a novel transmigration model recapitulates this pathological phenotype in vitro that would otherwise be impossible in a patient, opening avenues for future mechanistic and therapeutic research.

Activated PMN Exosomes: Pathogenic Entities Causing Matrix Destruction and Disease in the Lung.

Here, we describe a novel pathogenic entity, the activated PMN (polymorphonuclear leukocyte, i.e., neutrophil)-derived exosome. These CD63+/CD66b+ nanovesicles acquire surface-bound neutrophil elastase (NE) during PMN degranulation, NE being oriented in a configuration resistant to α1-antitrypsin (α1AT). These exosomes bind and degrade extracellular matrix (ECM) via the integrin Mac-1 and NE, respectively, causing the hallmarks of chronic obstructive pulmonary disease (COPD). Due to both ECM targeting and α1AT resistance, exosomal NE is far more potent than free NE. Importantly, such PMN-derived exosomes exist in clinical specimens from subjects with COPD but not healthy controls and are capable of transferring a COPD-like phenotype from humans to mice in an NE-driven manner. Similar findings were observed for another neutrophil-driven disease of ECM remodeling (bronchopulmonary dysplasia [BPD]). These findings reveal an unappreciated role for exosomes in the pathogenesis of disorders of ECM homeostasis such as COPD and BPD, providing a critical mechanism for proteolytic damage.

Small-molecule-based regulation of RNA-delivered circuits in mammalian cells.

Synthetic mRNA is an attractive vehicle for gene therapies because of its transient nature and improved safety profile over DNA. However, unlike DNA, broadly applicable methods to control expression from mRNA are lacking. Here we describe a platform for small-molecule-based regulation of expression from modified RNA (modRNA) and self-replicating RNA (replicon) delivered to mammalian cells. Specifically, we engineer small-molecule-responsive RNA binding proteins to control expression of proteins from RNA-encoded genetic circuits. Coupled with specific modRNA dosages or engineered elements from a replicon, including a subgenomic promoter library, we demonstrate the capability to externally regulate the timing and level of protein expression. These control mechanisms facilitate the construction of ON, OFF, and two-output switches, with potential therapeutic applications such as inducible cancer immunotherapies. These circuits, along with other synthetic networks that can be developed using these tools, will expand the utility of synthetic mRNA as a therapeutic modality.