A no-Wnt situation for alveolar macrophage self-renewal.

Alveolar macrophages (AMs) are central to defense against respiratory pathogens. Impediments in restoring AMs after infection increase the risk for superinfection, which is associated with significant morbidity and mortality worldwide. In this issue of Immunity, Zhu et al. report a Wnt-ß-catenin-HIF-1α axis in AMs that promotes an inflammatory phenotype while restricting proliferation and self-renewal.

In Vivo Evolution of a Klebsiella pneumoniae Capsule Defect With wcaJ Mutation Promotes Complement-Mediated Opsonophagocytosis During Recurrent Infection.

BACKGROUND: Klebsiella pneumoniae carbapenemase-producing K pneumoniae (KPC-Kp) bloodstream infections are associated with high mortality. We studied clinical bloodstream KPC-Kp isolates to investigate mechanisms of resistance to complement, a key host defense against bloodstream infection. METHODS: We tested growth of KPC-Kp isolates in human serum. In serial isolates from a single patient, we performed whole genome sequencing and tested for complement resistance and binding by mixing study, direct enzyme-linked immunosorbent assay, flow cytometry, and electron microscopy. We utilized an isogenic deletion mutant in phagocytosis assays and an acute lung infection model. RESULTS: We found serum resistance in 16 of 59 (27%) KPC-Kp clinical bloodstream isolates. In 5 genetically related bloodstream isolates from a single patient, we noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ. Disruption of wcaJ was associated with decreased polysaccharide capsule, resistance to complement-mediated killing, and surprisingly, increased binding of complement proteins. Furthermore, an isogenic wcaJ deletion mutant exhibited increased opsonophagocytosis in vitro and impaired in vivo control in the lung after airspace macrophage depletion in mice. CONCLUSIONS: Loss of function in wcaJ led to increased complement resistance, complement binding, and opsonophagocytosis, which may promote KPC-Kp persistence by enabling coexistence of increased bloodstream fitness and reduced tissue virulence.

Lung Epithelium Releases Growth Differentiation Factor 15 in Response to Pathogen-mediated Injury.

GDF15 (growth differentiation factor 15) is a stress cytokine with several proposed roles, including support of stress erythropoiesis. Higher circulating GDF15 levels are prognostic of mortality during acute respiratory distress syndrome, but the cellular sources and downstream effects of GDF15 during pathogen-mediated lung injury are unclear. We quantified GDF15 in lower respiratory tract biospecimens and plasma from patients with acute respiratory failure. Publicly available data from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were reanalyzed. We used mouse models of hemorrhagic acute lung injury mediated by Pseudomonas aeruginosa exoproducts in wild-type mice and mice genetically deficient for Gdf15 or its putative receptor, Gfral. In critically ill humans, plasma levels of GDF15 correlated with lower respiratory tract levels and were higher in nonsurvivors. SARS-CoV-2 infection induced GDF15 expression in human lung epithelium, and lower respiratory tract GDF15 levels were higher in coronavirus disease (COVID-19) nonsurvivors. In mice, intratracheal P. aeruginosa type II secretion system exoproducts were sufficient to induce airspace and plasma release of GDF15, which was attenuated with epithelial-specific deletion of Gdf15. Mice with global Gdf15 deficiency had decreased airspace hemorrhage, an attenuated cytokine profile, and an altered lung transcriptional profile during injury induced by P. aeruginosa type II secretion system exoproducts, which was not recapitulated in mice deficient for Gfral. Airspace GDF15 reconstitution did not significantly modulate key lung cytokine levels but increased circulating erythrocyte counts. Lung epithelium releases GDF15 during pathogen injury, which is associated with plasma levels in humans and mice and can increase erythrocyte counts in mice, suggesting a novel lung-blood communication pathway.

Serum metabolomic signatures of fatty acid oxidation defects differentiate host-response subphenotypes of acute respiratory distress syndrome.

BACKGROUND: Fatty acid oxidation (FAO) defects have been implicated in experimental models of acute lung injury and associated with poor outcomes in critical illness. In this study, we examined acylcarnitine profiles and 3-methylhistidine as markers of FAO defects and skeletal muscle catabolism, respectively, in patients with acute respiratory failure. We determined whether these metabolites were associated with host-response ARDS subphenotypes, inflammatory biomarkers, and clinical outcomes in acute respiratory failure. METHODS: In a nested case-control cohort study, we performed targeted analysis of serum metabolites of patients intubated for airway protection (airway controls), Class 1 (hypoinflammatory), and Class 2 (hyperinflammatory) ARDS patients (N = 50 per group) during early initiation of mechanical ventilation. Relative amounts were quantified by liquid chromatography high resolution mass spectrometry using isotope-labeled standards and analyzed with plasma biomarkers and clinical data. RESULTS: Of the acylcarnitines analyzed, octanoylcarnitine levels were twofold increased in Class 2 ARDS relative to Class 1 ARDS or airway controls (P = 0.0004 and < 0.0001, respectively) and was positively associated with Class 2 by quantile g-computation analysis (P = 0.004). In addition, acetylcarnitine and 3-methylhistidine were increased in Class 2 relative to Class 1 and positively correlated with inflammatory biomarkers. In all patients within the study with acute respiratory failure, increased 3-methylhistidine was observed in non-survivors at 30 days (P = 0.0018), while octanoylcarnitine was increased in patients requiring vasopressor support but not in non-survivors (P = 0.0001 and P = 0.28, respectively). CONCLUSIONS: This study demonstrates that increased levels of acetylcarnitine, octanoylcarnitine, and 3-methylhistidine distinguish Class 2 from Class 1 ARDS patients and airway controls. Octanoylcarnitine and 3-methylhistidine were associated with poor outcomes in patients with acute respiratory failure across the cohort independent of etiology or host-response subphenotype. These findings suggest a role for serum metabolites as biomarkers in ARDS and poor outcomes in critically ill patients early in the clinical course.

Update on the Features and Measurements of Experimental Acute Lung Injury in Animals: An Official American Thoracic Society Workshop Report.

Advancements in methods, technology, and our understanding of the pathobiology of lung injury have created the need to update the definition of experimental acute lung injury (ALI). We queried 50 participants with expertise in ALI and acute respiratory distress syndrome using a Delphi method composed of a series of electronic surveys and a virtual workshop. We propose that ALI presents as a "multidimensional entity" characterized by four "domains" that reflect the key pathophysiologic features and underlying biology of human acute respiratory distress syndrome. These domains are 1) histological evidence of tissue injury, 2) alteration of the alveolar-capillary barrier, 3) presence of an inflammatory response, and 4) physiologic dysfunction. For each domain, we present "relevant measurements," defined as those proposed by at least 30% of respondents. We propose that experimental ALI encompasses a continuum of models ranging from those focusing on gaining specific mechanistic insights to those primarily concerned with preclinical testing of novel therapeutics or interventions. We suggest that mechanistic studies may justifiably focus on a single domain of lung injury, but models must document alterations of at least three of the four domains to qualify as "experimental ALI." Finally, we propose that a time criterion defining "acute" in ALI remains relevant, but the actual time may vary based on the specific model and the aspect of injury being modeled. The continuum concept of ALI increases the flexibility and applicability of the definition to multiple models while increasing the likelihood of translating preclinical findings to critically ill patients.

Racial Diversity Among American Cardiologists: Implications for the Past, Present, and Future.

In the United States, race-based disparities in cardiovascular disease care have proven to be pervasive, deadly, and expensive. African American/Black, Hispanic/Latinx, and Native/Indigenous American individuals are at an increased risk of cardiovascular disease and are less likely to receive high-quality, evidence-based medical care as compared with their White American counterparts. Although the United States population is diverse, the cardiovascular workforce that provides its much-needed care lacks diversity. The available data show that care provided by physicians from racially diverse backgrounds is associated with better quality, both for minoritized patients and for majority patients. Not only is cardiovascular workforce diversity associated with improvements in health care quality, but racial diversity among academic teams and research scientists is linked with research quality. We outline documented barriers to achieving workforce diversity and suggest evidence-based strategies to overcome these barriers. Key strategies to enhance racial diversity in cardiology include improving recruitment and retention of racially diverse members of the cardiology workforce and focusing on cardiovascular health equity for patients. This review draws attention to academic institutions, but the implications should be considered relevant for nonacademic and community settings as well.

IL-37-induced activation of glycogen synthase kinase 3ß promotes IL-1R8/Sigirr phosphorylation, internalization, and degradation in lung epithelial cells.

Interleukin (IL)-37 diminishes a variety of inflammatory responses through ligation to its receptor IL-1R8/Sigirr. Sigirr is a Toll like receptor/IL-1R family member. We have shown that Sigirr is not stable in response to IL-37 treatment. IL-37-induced Sigirr degradation is mediated by the ubiquitin-proteasome system, and the process is reversed by a deubiquitinase, USP13. However, the molecular mechanisms by which USP13 regulates Sigirr stability have not been revealed. In this study, we investigate the roles of glycogen synthesis kinase 3ß (GSK3ß) in Sigirr phosphorylation and stability. IL-37 stimulation induced Sigirr phosphorylation and degradation, as well as activation of GSK3ß. Inhibition of GSK3ß attenuated IL-37-induced Sigirr phosphorylation, while exogenous expressed GSK3ß promoted Sigirr phosphorylation at threonine (T)372 residue. Sigirr association with GSK3ß was detected. Amino acid residues 51-101 in GSK3ß were identified as the Sigirr binding domain. These data indicate that GSK3ß mediates IL-37-induced threonine phosphorylation of Sigirr. Further, we investigated the role of GSK3ß-mediated phosphorylation of Sigirr in Sigirr degradation. Inhibition of GSK3ß attenuated IL-37-induced Sigirr degradation, while T372 mutant of Sigirr was resistant to IL-37-mediated degradation. Furthermore, inhibition of Sigirr phosphorylation prevented Sigirr internalization and association with USP13, suggesting GSK3ß promotes Sigirr degradation through disrupting Sigirr association with USP13.

SCFFBXO17 E3 ligase modulates inflammation by regulating proteasomal degradation of glycogen synthase kinase-3ß in lung epithelia.

Glycogen synthase kinase-3ß (GSK3ß) has diverse biological roles including effects on cellular differentiation, migration, and inflammation. GSK3ß phosphorylates proteins to generate phosphodegrons necessary for recognition by Skp1/Cullin-1/F-box (SCF) E3 ubiquitin ligases leading to subsequent proteasomal degradation of these substrates. However, little is known regarding how GSK3ß protein stability itself is regulated and how its stability may influence inflammation. Here we show that GSK3ß is degraded by the ubiquitin-proteasome pathway in murine lung epithelial cells through lysine 183 as an acceptor site for K48 polyubiquitination. We have identified FBXO17 as an F-box protein subunit that recognizes and mediates GSK3ß polyubiquitination. Both endogenous and ectopically expressed FBXO17 associate with GSK3ß, and its overexpression leads to decreased protein levels of GSK3ß. Silencing FBXO17 gene expression increased the half-life of GSK3ß in cells. Furthermore, overexpression of FBXO17 inhibits agonist-induced release of keratinocyte-derived cytokine (KC) and interleukin-6 (IL-6) production by cells. Thus, the SCFFBXO17 E3 ubiquitin ligase complex negatively regulates inflammation by targeting GSK3ß in lung epithelia.

FBXO17 promotes cell proliferation through activation of Akt in lung adenocarcinoma cells.

BACKGROUND: The ubiquitin-proteasome pathway, mediated in part, by ubiquitin E3 ligases, is critical in regulating cellular processes such as cell proliferation, apoptosis, and migration. FBXO17 was recently identified as an F-box protein that targets glycogen synthase kinase-3ß to the E3 ubiquitin ligase protein complex for polyubiquitination and proteasomal degradation. Here, we identified that in several lung adenocarcinoma cell lines, FBXO17 cellular protein was detected at relatively high levels, as was expression in a subset of lung cancers. Hence, we investigated the effects of FBXO17 on cell proliferation. METHODS: Single cell RNA sequencing analysis was performed on a resection of a non-small cell lung carcinoma tumor to examine FBXO17 expression. Multiple lung cancer cell lines were immunoblotted, and The Cancer Genome Atlas was analyzed to determine if FBXO17 expression was amplified in a subset of lung cancers. A549 cells were transfected with empty vector or FBXO17-V5 plasmid and immunoblotted for Akt pathway mediators including PDK1, ERK1/2, ribosomal protein S6, and CREB. Cell proliferation and viability were analyzed by trypan blue exclusion, BrdU incorporation and an MTS-based fluorometric assay. Studies were also performed after transfecting with sifbxo17. Samples were used in an RNA microarray analysis to evaluate pathways affected by reduced FBXO17 gene expression. RESULTS: We observed that overexpression of FBXO17 increased A549 cell proliferation coupled with Akt activation. Ectopically expressed FBXO17 also increased ERK1/2 kinase activation and increased phosphorylation of RPS6, a downstream target of mTOR. We also observed an increased number of cells in S-phase and increased metabolic activity of lung epithelial cells expressing FBXO17. FBXO17 knockdown reduced Akt Ser 473 phosphorylation approaching statistical significance with no effect on Thr 308. However, ERK1/2 phosphorylation, cellular metabolic activity, and overall cell numbers were reduced. When we analyzed RNA profiles of A549 cells with reduced FBXO17 expression, we observed downregulation of several genes associated with cell proliferation and metabolism. CONCLUSIONS: These data support a role for FBXO17 abundance, when left unchecked, in regulating cell proliferation and survival through modulation of Akt and ERK kinase activation. The data raise a potential role for the F-box subunit in modulating tumorigenesis.

Post-translational modification of the interferon-gamma receptor alters its stability and signaling.

The IFN gamma receptor 1 (IFNGR1) binds IFN-γ and activates gene transcription pathways crucial for controlling bacterial and viral infections. Although decreases in IFNGR1 surface levels have been demonstrated to inhibit IFN-γ signaling, little is known regarding the molecular mechanisms controlling receptor stability. Here, we show in epithelial and monocytic cell lines that IFNGR1 displays K48 polyubiquitination, is proteasomally degraded, and harbors three ubiquitin acceptor sites at K277, K279, and K285. Inhibition of glycogen synthase kinase 3 beta (GSK3ß) destabilized IFNGR1 while overexpression of GSK3ß increased receptor stability. We identified critical serine and threonine residues juxtaposed to ubiquitin acceptor sites that impacted IFNGR1 stability. In CRISPR-Cas9 IFNGR1 generated knockout cell lines, cellular expression of IFNGR1 plasmids encoding ubiquitin acceptor site mutations demonstrated significantly impaired STAT1 phosphorylation and decreased STAT1-dependent gene induction. Thus, IFNGR1 undergoes rapid site-specific polyubiquitination, a process modulated by GSK3ß. Ubiquitination appears to be necessary for efficient IFNGR1-dependent gamma gene induction and represents a relatively uncharacterized regulatory mechanism for this receptor.