Carmen Mikacenic

Candida albicans ( Calb ) in the lower respiratory tract (LRT) is considered a rare cause of pneumonia, yet its frequent recovery from LRT secretions of mechanically ventilated patients associates with poor clinical outcomes. To determine whether Calb contributes to lung injury, we conducted a translational investigation spanning two independent, prospective human cohorts, murine models of lung injury, and in vitro assays. In critically ill patients, Calb was the most abundant fungus in LRT specimens, and its detection associated with increased markers of lung injury, prolonged mechanical ventilation, and increased mortality. In a murine model, intratracheal Calb was sufficient to disrupt the air-blood barrier and recruit neutrophils, effects markedly attenuated with heat-killed Calb . Neutrophil depletion led to uncontrolled fungal growth, systemic dissemination and mortality, with surviving mice exhibiting worsened barrier disruption, demonstrating that neutrophils support pathogen control while lung injury is driven by the live organism. Calb induced lung epithelial cytotoxicity as well as barrier disruption in human alveolar epithelial cells at air liquid interface. Yeast-locked mutants without hyphal morphogenesis demonstrated attenuated barrier disruption in human alveolar cells and diminished lung injury in mice, despite higher fungal burden. In human cohort data, LRT microbiome profiles with high Calb abundance and codominant bacterial pathogens predicted worse mortality, and mouse models confirmed that Calb amplifies lung barrier disruption when followed by Pseudomonas aeruginosa inoculation, an effect requiring hyphal morphogenesis. These findings establish that LRT Calb causes direct air-blood barrier disruption through hyphal morphogenesis and amplifies bacterial lung injury, challenging the prevailing view of Candida as an innocent respiratory bystander.

Background Acute respiratory failure (ARF) is common, exhibits variable outcomes, and arises from diverse causes that incompletely reflect underlying biology. Although the pandemic accelerated molecular investigation of ARF in COVID-19, whether these biologic signals generalize to ARF in hospital populations of varying etiology and severity remains unknown. Methods We analyzed aptamer-based plasma proteomics collected within 48 hours of admission across two multihospital studies of varying etiology and severity, including individuals aged ≥14 years with acute viral or respiratory illness, stratified by COVID-19 and non-COVID-19 status (N = 1,096 total across four cohorts). The primary outcome was critical ARF, defined as advanced respiratory support or death by day 7. We identified proteins reproducibly associated with critical ARF in all cohorts, then contextualized protein associations with pathway analysis. Additionally, we developed proteomics-based models for two clinical purposes: predicting critical ARF and identifying heterogeneity of treatment response. Results We identify 9 proteins associated with critical ARF across cohorts (FDR < 0.05). Seven proteins related to innate immunity, oxidative stress, and barrier disruption are higher in critical ARF, while two proteins linked to immune signaling are lower. Pathway analysis identifies dysregulated inflammatory and fibrotic signaling pathways. Protein-based models improve discrimination of critical ARF beyond clinical variables in non-COVID-19 cohorts. A causal forests model finds elevated IL1RL1 (soluble ST2) is associated with greater benefit from imatinib. Conclusions A proteomic signature linked to ARF-related outcomes is reproducible across four cohorts with a breadth of etiologies and severity. The proteins may inform design of novel treatments and enriched clinical trials.

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.

Macrophage activation syndrome (MAS) is driven by a hyperinflammatory response characterized by aberrant activation of lymphocytes and phagocytes. While monocytes and macrophages are thought to be important in MAS pathogenesis, their role remains poorly understood. We used bulk and single-cell RNA sequencing (RNA-Seq) on sorted monocytes from children with MAS and healthy controls to identify transcriptional changes during MAS. We defined a MAS signature in classical monocytes that correlated with ferritin and was elevated in monocytes from systemic lupus erythematosus and COVID-19 patients. We also identified a subset of classical monocytes with high levels of interferon-stimulated genes (ISGs) that expanded during MAS. Surprisingly, the transcriptional signature of these cells was driven by type I IFNs, rather than IFNγ. Consistent with this finding, we detected increased levels of circulating IFNβ during MAS, suggesting that IFNβ plays an unrecognized role in driving MAS monocyte responses. We also identified a MAS-associated CD8 ⁺ T cell population with a distinctive transcriptional signature. We used cell-cell communication algorithms to predict increased immunoregulatory interactions between monocytes and T cells during MAS. Together, these results provide new evidence for a role for type I IFN during MAS and identify a unique CD8 ⁺ T cell population that may contribute to MAS pathophysiology.

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.

CONTEXT: COVID-19 has been associated with features of a cytokine storm syndrome with some patients sharing features with the hyperinflammatory disorder, secondary hemophagocytic lymphohistiocytosis (sHLH). HYPOTHESIS: We hypothesized that proteins associated with sHLH from other causes will be associated with COVID-sHLH and that subjects with fatal COVID-sHLH would have defects in immune-related pathways. METHODS AND MODELS: We identified two cohorts of adult patients presenting with COVID-19 at two tertiary care hospitals in Seattle, Washington in 2020 and 2021. In this observational study, we assessed clinical laboratory values and plasma proteomics. Subjects identified as having sHLH (ferritin > 1000 plus cytopenias in two or more lineages [WBC < 5000 odds ratio [OR] ANC (absolute neutrophil count) < 1000, hemoglobin < 9 or hematocrit < 27, platelets < 100,000], and elevated transaminases [either AST (aspartate aminotransferase) or ALT (alanine aminotransferase) > 30] OR subjects with a ferritin > 3000) were compared with those with COVID-19 without sHLH. We identified 264 patients with COVID-19 of whom 24 met our sHLH definition. Eight patients who died of COVID-sHLH underwent genomic sequencing to identify variants in immune-related genes. RESULTS: Nine percent of enrolled COVID-19 subjects met our defined criteria for sHLH (n = 24/264). Using broad serum proteomic approaches (O-link and SomaScan), we identified three proteins increased in subjects with COVID-19-associated sHLH (soluble PD-L1 [sPD-L1], tumor necrosis factor-R1, and interleukin [IL]-18BP, p < 0.05 for O-link and false discovery rate < 0.05 for SomaScan), supporting a role for proteins previously associated with other forms of sHLH (IL-18BP and soluble tumor necrosis factor receptor 1). We also identified candidate proteins and pathways associated with COVID-sHLH, including sPD-L1 and the syntaxin pathway. We detected pathogenic variants in DOCK8 and TMPRSS15 in deceased individuals with COVID-sHLH, further suggesting that alterations in immune-related processes may contribute to hyperinflammation and fatal outcomes in COVID-19. INTERPRETATIONS AND CONCLUSIONS: Proteins increased in COVID-19-associated sHLH, such as sPD-L1, and pathways, such as the syntaxin pathway, suggest important roles for the immune response in driving sHLH in the context of COVID-19.

The relationship between the PD-L1 (Programmed Death-Ligand 1)/PD-1 pathway, lung inflammation, and clinical outcomes in acute respiratory distress syndrome (ARDS) is poorly understood. We sought to determine whether PD-L1/PD-1 in the lung or blood is associated with ARDS and associated severity. We measured soluble PD-L1 (sPD-L1) in plasma and lower respiratory tract samples (ARDS1 [n = 59] and ARDS2 [n = 78]) or plasma samples alone (ARDS3 [n = 149]) collected from subjects with ARDS and tested for associations with mortality using multiple regression. We used mass cytometry to measure PD-L1/PD-1 expression and intracellular cytokine staining in cells isolated from BAL fluid (n = 18) and blood (n = 16) from critically ill subjects with or without ARDS enrolled from a fourth cohort. Higher plasma concentrations of sPD-L1 were associated with mortality in ARDS1, ARDS2, and ARDS3. In contrast, higher concentrations of sPD-L1 in the lung were either not associated with mortality (ARDS2) or were associated with survival (ARDS1). Alveolar PD-1POS T cells had more intracellular cytokine staining than PD-1NEG T cells. Subjects without ARDS had a higher ratio of PD-L1POS alveolar macrophages to PD-1POS T cells than subjects with ARDS. We conclude that sPD-L1 may have divergent cellular sources and/or functions in the alveolar versus blood compartments, given distinct associations with mortality. Alveolar leukocyte subsets defined by PD-L1 or PD-1 cell-surface expression have distinct cytokine secretion profiles, and the relative proportions of these subsets are associated with ARDS.