Joseph S. Bednash

The acute respiratory distress syndrome is a heterogenous syndrome characterized by the rapid development of respiratory failure. Nearly 40% of patients who develop ARDS will die, and there is growing interest in identification of biomarkers to identify patients at risk of death and/or inform treatment decisions. Most prior work on biomarkers in ARDS has focused on the plasma compartment, but there is concern that circulating biomarkers may not reflect alveolar pathobiology. The anti-inflammatory microRNA-146a has been shown to be upregulated in inflammatory cells in human bronchoalveolar lavage fluid, but it is not known if these levels correspond with outcomes. We measured miR-146a expression by digital droplet PCR in human biospecimens from four different cohorts of patients with respiratory failure requiring mechanical ventilation – two plasma cohorts, one bronchoalveolar lavage cohort, and one heat moisture exchange (HME) filter fluid cohort. We found that miR-146a was detectible in plasma, bronchoalveolar lavage fluid, and HME fluid. However, only when measured in the alveolar space, was miR-146a expression significantly lower in older adults and those who died. It did not correlate with outcomes when measured in plasma. To our knowledge, this is the first report that nucleotides can be measured in HME fluid and builds upon expanding literature that circulating biomarkers may not reflect complex biology of the alveolar microenvironment during ARDS.

Acute Respiratory Distress Syndrome (ARDS), a heterogeneous syndrome of hypoxic respiratory failure secondary to dysregulated pulmonary inflammation, is caused by diverse insults. This heterogeneity presents challenges for mechanistic and therapeutic research, as evidenced by conflicting results from trials of n−3 polyunsaturated fatty acid (PUFA) supplementation for ARDS. PUFAs and downstream oxylipins are important to pulmonary inflammation but are not well defined in ARDS. We hypothesized that differences in fatty acid metabolism, as measured by levels of n−3 and n−6 PUFAs and oxylipins, are associated with differences in ARDS outcomes, inflammation, and causes. To test this, PUFAs/oxylipins were measured by LC MS/MS in plasma samples from 90 patients with ARDS. Pro‐inflammatory cytokines and chemokines IL‐6 and IL‐8 were measured by ELISA. Multivariable linear regressions modeled the relationship between PUFAs/oxylipins, inflammation, and ARDS mortality, severity, and cause. Multiple n−3 and n−6 PUFA‐derived oxylipins were decreased in severe ARDS. We did not detect differences in PUFAs/oxylipins by mortality. PUFAs/oxylipins varied by cause of ARDS, especially between patients with sepsis and those with trauma. Furthermore, specific oxylipins were associated with IL‐6 and IL‐8. As we observed that oxylipins varied by disease severity and underlying cause, these metabolites may function as biomarkers and suggest dysregulated mechanisms of lung repair. Furthermore, while oxylipins are derived from PUFAs, differences in PUFAs did not directly correlate with changes in oxylipins, suggesting altered lipid metabolism as a mechanism. Further consideration of differences in lipid metabolism in ARDS could identify subgroups with differential responses to n−3 PUFA supplementation or other therapies.

Aminoacyl‐ tRNA Synthetases ( aaRS ) are important regulators of cytokine signaling. Multiple cytoplasmic aaRS family members have been observed to be secreted in response to various stimuli to modulate downstream responses, however, agonist‐induced cellular release of aaRS from mitochondria has not been described. In particular, TNFα is a potent mediator of aaRS release. BEAS ‐ 2B cells were utilized to study the release of mitochondrial Aspartyl‐ tRNA Synthetase ( DARS2 ) in response to various cytokines. The role of DARS2 in paracrine signaling was evaluated using adoptive media transfer from BEAS ‐ 2B to recipient THP1 cells. To identify pathways governing DARS2 secretion, blocking antibodies chemical inhibitors and siRNA technology was employed. Herein, we describe DARS2 as the first mitochondrial aaRS released in response to TNFα from airway epithelia. Once secreted, DARS2 binds to macrophages, is internalized, thereby inducing an M1 ‐like phenotype in recipient macrophages. DARS2 release from airway epithelia is in part, TNFα ‐receptor 1 dependent, and requires the endosomal sorting complex required for extracellular transport.

Severe COVID‐19 is often complicated by hypoxemic respiratory failure and acute respiratory distress syndrome (ARDS). Mechanisms governing lung injury and repair in ARDS remain poorly understood. We hypothesized that plasma proteomics may uncover protein biomarkers correlated with COVID‐19 ARDS severity. We analyzed the plasma proteome from 32 patients with ARDS and COVID‐19 using an aptamer‐based platform of 7289 proteins, and correlated protein measurements with sequential organ failure assessment (SOFA) scores at days 1 and 7 of ICU admission. We identified 184 differentially abundant proteins correlated with SOFA at day 1 and 46 proteins at day 7. In a longitudinal analysis, we correlated dynamic changes in protein abundance and SOFA between days 1 and 7 and identified 40 significant proteins. Pathway analysis of significant proteins identified increased ephrin signaling and acute phase response signaling correlated with increased SOFA scores between days 1 and 7, while pathways related to pulmonary fibrosis signaling and wound healing had a negative correlation. These findings suggest that persistent inflammation may drive disease severity, while repair processes correlate with improvements in organ dysfunction. This approach is generalizable to future ARDS cohorts for identification of biomarkers and disease mechanisms as we strive towards targeted therapies in ARDS.

Pseudomonas aeruginosa (P.a.) is a gram-negative, opportunistic bacterium abundantly present in the environment. Often P.a. infections cause severe pneumonia, if left untreated. Surprisingly, up to 30% of patients admitted to the hospital for community- acquired pneumonia develop adverse cardiovascular complications such as myocardial infarction, arrhythmia, left ventricular dysfunction, and heart failure. However, the underlying mechanism of infection-mediated cardiac dysfunction is not yet known. Recently, we demonstrated that P.a. infection of the lungs led to severe cardiac electrical abnormalities and left ventricular dysfunction with limited P.a. dissemination to the heart tissue. To understand the mechanism of cardiac dysfunction during P.a. infection, we utilized both in vitro and in vivo models. Our results revealed that inflammatory cytokines contribute but are not solely responsible for severe contractile dysfunction in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Instead, exposure of hiPSC-CMs with conditioned media from P.a. infected human monocyte-derived macrophages (hMDMs) was sufficient to cause severe contractile dysfunction and arrhythmia in hiPSC-CMs. Specifically, exosomes released from infected hMDMs and bacterial outer membrane vesicles (OMVs) are the major drivers of cardiomyocyte contractile dysfunction. By using LC-MS/MS, we identified bacterial proteins, including toxins that are packaged in the exosomes and OMVs, which are responsible for contractile dysfunction. Furthermore, we demonstrated that systemic delivery of bacterial OMVs to mice caused severe cardiac dysfunction, mimicking the natural bacterial infection. In summary, we conclude that OMVs released during infection enter circulation and drive cardiac dysfunction.

BACKGROUND: Evidence suggests that COVID-19 predisposes to cardiovascular diseases (CVDs). While monocytes/macrophages play a central role in the immunopathogenesis of atherosclerosis, less is known about their immunopathogenic mechanisms that lead to CVDs during COVID-19. Natural killer (NK) cells, which play an intermediary role during pathologies like atherosclerosis, are dysregulated during COVID-19. Here, we sought to investigate altered immune cells and their associations with CVD risk during severe COVID-19. METHODS: We measured plasma biomarkers of CVDs and determined phenotypes of circulating immune subsets using spectral flow cytometry. We compared these between patients with severe COVID-19 (severe, n=31), those who recovered from severe COVID-19 (recovered, n=29), and SARS-CoV-2–uninfected controls (controls, n=17). In vivo observations were supported using in vitro assays to highlight possible mechanistic links between dysregulated immune subsets and biomarkers during and after COVID-19. We performed multidimensional analyses of published single-cell transcriptome data of monocytes and NK cells during severe COVID-19 to substantiate in vivo findings. RESULTS: During severe COVID-19, we observed alterations in cardiometabolic biomarkers including oxidized–low-density lipoprotein, which showed decreased levels in severe and recovered groups. Severe patients exhibited dysregulated monocyte subsets, including increased frequencies of proinflammatory intermediate monocytes (also observed in the recovered) and decreased nonclassical monocytes. All identified NK-cell subsets in the severe COVID-19 group displayed increased expression of activation and tissue-resident markers, such as CD69 (cluster of differentiation 69). We observed significant correlations between altered immune subsets and plasma oxidized–low-density lipoprotein levels. In vitro assays revealed increased uptake of oxidized–low-density lipoprotein into monocyte-derived macrophages in the presence of NK cells activated by plasma of patients with severe COVID-19. Transcriptome analyses confirmed enriched proinflammatory responses and lipid dysregulation associated with epigenetic modifications in monocytes and NK cells during severe COVID-19. CONCLUSIONS: Our study provides new insights into the involvement of monocytes and NK cells in the increased CVD risk observed during and after COVID-19.

Introduction: The acute respiratory distress syndrome (ARDS) is a common complication of severe COVID-19 and contributes to patient morbidity and mortality. ARDS is a heterogeneous syndrome caused by various insults, and results in acute hypoxemic respiratory failure. Patients with ARDS from COVID-19 may represent a subgroup of ARDS patients with distinct molecular profiles that drive disease outcomes. Here, we hypothesized that longitudinal transcriptomic analysis may identify distinct dynamic pathobiological pathways during COVID-19 ARDS.Methods: We identified a patient cohort from an existing ICU biorepository and established three groups for comparison: 1) patients with COVID-19 ARDS that survived hospitalization (COVID survivors, n = 4), 2) patients with COVID-19 ARDS that did not survive hospitalization (COVID non-survivors, n = 5), and 3) patients with ARDS from other causes as a control group (ARDS controls, n = 4). RNA was isolated from peripheral blood mononuclear cells (PBMCs) at 4 time points (Days 1, 3, 7, and 10 following ICU admission) and analyzed by bulk RNA sequencing.Results: We first compared transcriptomes between groups at individual timepoints and observed significant heterogeneity in differentially expressed genes (DEGs). Next, we utilized the likelihood ratio test to identify genes that exhibit different patterns of change over time between the 3 groups and identified 341 DEGs across time, including hemoglobin subunit alpha 2 (HBA1, HBA2), hemoglobin subunit beta (HBB), von Willebrand factor C and EGF domains (VWCE), and carbonic anhydrase 1 (CA1), which all demonstrated persistent upregulation in the COVID non-survivors compared to COVID survivors. Of the 341 DEGs, 314 demonstrated a similar pattern of persistent increased gene expression in COVID non-survivors compared to survivors, associated with canonical pathways of iron homeostasis signaling, erythrocyte interaction with oxygen and carbon dioxide, erythropoietin signaling, heme biosynthesis, metabolism of porphyrins, and iron uptake and transport.Discussion: These findings describe significant differences in gene regulation during patient ICU course between survivors and non-survivors of COVID-19 ARDS. We identified multiple pathways that suggest heme and red blood cell metabolism contribute to disease outcomes. This approach is generalizable to larger cohorts and supports an approach of longitudinal sampling in ARDS molecular profiling studies, which may identify novel targetable pathways of injury and resolution.

Clinical data demonstrate an increased predisposition to cardiovascular disease (CVD) following severe COVID-19 infection. This may be driven by a dysregulated immune response associated with severe disease. Monocytes and vascular tissue resident macrophages play a critical role in atherosclerosis, the main pathology leading to ischemic CVD. Natural killer (NK) cells are a heterogenous group of cells that are critical during viral pathogenesis and are known to be dysregulated during severe COVID-19 infection. Their role in atherosclerotic cardiovascular disease has recently been described. However, the contribution of their altered phenotypes to atherogenesis following severe COVID-19 infection is unknown. We demonstrate for the first time that during and after severe COVID-19, circulating proinflammatory monocytes and activated NK cells act synergistically to increase uptake of oxidized low-density lipoprotein (Ox-LDL) into vascular tissue with subsequent foam cell generation leading to atherogenesis despite recovery from acute infection. Our data provide new insights, revealing the roles of monocytes/macrophages, and NK cells in COVID-19-related atherogenesis.

The impact of pre-existing common cold coronavirus (CCCoV) antibodies (Abs) on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune responses and pathogenesis remains poorly defined. We evaluated these associations in a cohort of hospitalized patients with COVID-19 and respiratory failure of varying severity. Patients with respiratory failure from other causes (non-COVID-19) were evaluated as controls. We demonstrated a positive correlation between levels of CCCoV and SARS-CoV-2 Abs using CCCoV and SARS-CoV-2 N and S protein peptide-specific ELISA. Consistent with the above, moderately increased levels of CCCoV-specific Abs in non-COVID-19 vs. COVID-19 patients suggest potential protective effects. Further, higher SARS-CoV-2 N protein-specific and CCCoV Ab levels were observed among surviving vs. non-surviving COVID-19 positive patients. However, the highest SARS-CoV-2 N and S protein-specific IgG and IgA Ab levels were noted in the patients with the most severe clinical disease. Finally, advanced age, cancer and immunosuppression were associated with significantly higher mortality and reduced SARS-CoV-2 and CCCoV Ab levels. Thus, our data highlight that sufficient SARS-CoV-2 N protein-specific Ab responses improve clinical outcomes in severely ill COVID-19 patients. We also confirmed that pre-existing CCCoV-specific Abs do not inhibit the SARS-CoV-2 Ab response and may further reduce the prevalence and/or severity of COVID-19.