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

Abstract

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.

Keywords: GDF15; GFRAL; acute respiratory failure; cytokines; stress erythropoiesis.

Figure 1.

Higher plasma GDF15 (growth differentiation factor 15) levels are associated with worse clinical outcomes and lower respiratory tract dysbiosis and positively correlate with lower respiratory tract GDF15 levels. (A–C) Plasma GDF15 levels from critically ill patients (n = 103) with acute respiratory failure by clinical group: patients with risk factors for (ARFA), or a diagnosis of, acute respiratory distress syndrome (ARDS) and airway controls ventilated for non-pulmonary indications (A). (B) Host response phenotypes as classified by a four-variable model consisting of angiopoietin-2, soluble TNF receptor-1, procalcitonin, and bicarbonate and (C) survival status at 30 days. (D) Plasma GDF15 levels are displayed for a subset of patients (n = 45) separated by Dirichlet multinomial model clusters of bacterial composition. Clusters had significant differences in bacterial abundance and α-diversity characterized as low (n = 14), intermediate (n = 19), and high (n = 12) α-diversity. The low-diversity cluster, characterized by a high abundance of typical pathogenic bacteria indicative of lower respiratory tract infection or dysbiosis, showed significantly higher plasma GDF15 levels. (E) Endotracheal aspirate (ETA) GDF15 levels are presented stratified by clinical classification of ARDS, at-risk for ARDS, or airway control mechanically ventilated for non-pulmonary illness (n = 110). Range displayed with box plot displaying median and IQR. P values represent differences by Kruskal-Wallis test with Dunn’s post hoc test using Benjamini-Hochberg correction for multiple comparisons. (F) Scatter plot of plasma and ETA GDF15 levels normalized to total protein level (n = 44, Spearman ρ displayed). Each point represents a single patient, and the P value represents significance by Wilcoxon rank sum test unless otherwise stated.

Figure 2.

Mice release GDF15 during lung injury induced by Pseudomonas aeruginosa type II secretion system exoproducts. Wild-type (WT) C57Bl/6J mice were intratracheally inoculated with vehicle (n = 6) or the supernatant (SN) of P. aeruginosa grown in culture. Parent P. aeruginosa (P. aeruginosa cell-free SN [PA SN], n = 8) and a genetically modified strain (ΔxcpQ SN, n = 6) deficient for the type II secretion system chaperone protein xcpQ were used. Necropsy was performed 20 hours after inoculation. (A) Gross appearance of BAL fluid, (B) BAL protein concentrations, (C) plasma GDF15 levels (vehicle group, n = 5), and (D) BAL GDF15 levels. (E) Murine lung epithelial cells (MLE12) were exposed in vitro to vehicle or PA SN. Cell lysates were collected 4 hours after exposure, and Gdf15 transcripts (E) and protein (F) were measured. (G) Gdf15^fl/fl mice (“control”) or epithelial-specific knockout (KO) Gdf15^fl/fl;SFTPC-Cre (“epithelial KO”) mice were exposed to PA SN (n = 9 control and n = 14 epithelial KO), and BAL (G) and plasma (H) GDF15 was measured. In H, plasma GDF15 concentration was normalized to control to compare across experiments. Each tube or point represents an individual mouse, and the group median is displayed. Groups were compared using a Mann-Whitney test.

Figure 3.

Human lung epithelium releases GDF15 in response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. (A) Human lung alveolar epithelial cells infected with SARS-CoV-2 (n = 3) reveal increased levels of GDF15 by label-free quantification (LFQ) compared with control sham infection (n = 3) (30). Each point represents the median protein expression of upregulated proteins. GDF15 is red, whereas all other proteins are black. Box plot displays the median and IQR. (B) Publicly available single-cell RNA sequencing data from BAL fluid collected from patients infected with severe coronavirus disease (COVID-19) pneumonia (n = 6) or moderate COVID-19 pneumonia (n = 3) and healthy controls (n = 3; from Gene Expression Omnibus database accession GSE145926). Cells identified as lung epithelium by cluster analysis display significantly increased GDF15 RNA levels (P < 6.9 × 10⁻¹²⁰ for severe COVID compared with healthy controls). (C) Correlogram of associations of ETA GDF15 levels (46 patients with 76 total time points on sampling Days 1, 5, and 10) with host inflammatory response biomarkers during COVID-19 pneumonia requiring mechanical ventilation. All ETA protein levels are normalized to total protein. The number in the circle displays the Spearman correlation. The direction and magnitude of correlation are displayed by color, nonsignificant correlations are blank, and P values are adjusted for multiple comparisons by Benjamini-Hochberg correction with significance set to P < 0.05. (D) Box-and-whisker plots showing ETA GDF15 levels stratified by sampling day (n = 29 on Day 1, n = 27 on Day 5, and n = 20 on Day 10; total n = 46) and comparing nonsurvivors (dark gray) and survivors (light green). Boxes represent the IQRs, horizontal lines represent the medians, and vertical lines extend from minimum to maximum values. Dashed lines connect the median values for each group at each sampling day to show longitudinal trends in GDF15 levels. Ang-2 = angiopoietin-2; ARFA = at risk for ARDS; C19 = coronavirus disease; LFQ = label-free quantification; Procal = procalcitonin; RAGE = soluble receptor for advanced glycation end-products; ST2 = suppression of tumorigenicity-2 (also known as IL-1 receptor ligand-1 or IL-33 receptor); sTNFR1 = soluble TNF receptor-1.

Figure 4.

Global deficiency of Gdf15 is marked by an altered airspace cytokine profile during lung injury induced by P. aeruginosa exoproducts. In two experiments, WT (n = 19) and Gdf15^−/− mice (n = 16) were intratracheally inoculated with PA SN, and BAL fluid and plasma were collected 20 hours after infection. (A) Gross images of BAL hemorrhage from each experiment. (B) BAL absorbance (optical density [OD]) at 540 nm (OD₅₄₀) normalized to the WT median value to compare across experiments, (C) BAL IgM normalized to the WT median value to compare across experiments, (D) TNF-α, (E) BAL TNF-α, (F) plasma granulocyte colony–stimulating factor, (G) BAL granulocyte colony–stimulating factor, (H) BAL polymorphonuclear leukocytes counts normalized to the WT median value to compare across experiments, and (I) BAL neutrophil elastase activity in WT (n = 24) and Gdf15^−/− mice (n = 21) normalized to the WT median to compare across experiments. Each tube or point represents a single mouse. Groups were compared using a Mann-Whitney test. Nonsignificant comparisons are not displayed. G-CSF = granulocyte colony–stimulating factor. PMN = polymorphonuclear leukocyte.

Figure 5.

Global genetic deficiency of Gfral does not recapitulate the lung phenotype seen in Gdf15^−/− mice during lung injury induced by P. aeruginosa exoproducts. WT (n = 7) and mice globally deficient for GFRAL (Gfral^−/−; n = 6) were intratracheally inoculated with PA SN. (A) Gross images of BAL hemorrhage. (B) BAL OD₅₄₀ showing increased hemorrhage in Gfral^−/− mice. (C) BAL cellularity. (D) Polymorphonuclear cell counts in BAL assessed by manual differential count. (E) BAL total protein by bicinchoninic acid assay. (F–I) The indicated proteins were measured by ELISA in BAL or plasma. (J) BAL neutrophil elastase activity (OD₄₀₀ at 24 h) and (K) BAL TNF-α. Each tube or point represents a single mouse. Groups were compared using a Mann-Whitney test. P values are displayed for all comparisons.

Figure 6.

Global genetic deficiency of Gdf15 alters the lung transcriptional profile during lung injury elicited by intratracheal instillation of P. aeruginosa exoproducts. WT mice (n = 5) and mice globally deficient for GDF15 (Gdf15^−/−; n = 5) were intratracheally inoculated with PA SN, and RNA was collected 20 hours after infection. (A) Volcano plot of normalized RNA counts with annotations showing 3,005 differentially expressed genes with adjusted P value <0.05. (B) Heat map of top differentially expressed genes after regularized log transformation of RNA counts and removal of pseudogenes clustered by row with genotype by column. (C and D) Dot plots of overrepresentation analysis for Gene Ontology molecular functions (P < 0.01, q < 0.05) in the 20 most upregulated (C) and downregulated (D) gene sets using all genes with statistically significant differential RNA counts (adjusted P < 0.05). (E) Gene set enrichment analysis of Hallmark pathways (adjusted P < 0.05) of all transcripts from control and Gdf15^−/− mice. (F and G) CXCL10 was quantified by ELISA in lung homogenate (F) and BAL fluid (G). Each dot represents a single mouse. Groups were compared using a Mann-Whitney test.

Figure 7.

Intratracheal recombinant murine GDF15 at the time of instillation of P. aeruginosa exoproducts increases plasma levels of GDF15 and circulating erythrocyte counts in mice globally deficient for Gdf15. In two experiments, WT and Gdf15^−/− mice were intratracheally administered recombinant murine GDF15 (rmGDF15) or an equivalent volume of vehicle (n = 11 WT with vehicle, n = 9 Gdf15^−/− mice with vehicle, and n = 12 Gdf15^−/− mice with rmGDF15) at the time of intratracheal inoculation with PA SN. Note that two mice were removed from the Gdf15^−/− vehicle group and one mouse was removed from the Gdf15^−/− rmGDF15 group because of an absence of lung injury observed at necropsy. BAL fluid, plasma, and lung tissue were collected 20 hours after infection. (A) BAL GDF15. (B) CXCL10 levels in homogenate of lavaged left lung (adjusted to total protein in homogenate) after normalization to the median of the Gdf15^−/− vehicle group. (C) BAL CXCL10 levels after normalization to the median of the Gdf15^−/− group. (D) BAL TNF-α levels after normalization. (E) Plasma GDF15. (F–H) Hemavet counts of plasma neutrophils (F), monocytes (G), and erythrocytes (H) after normalization. Each point represents a single mouse. Gdf15^−/− vehicle and Gdf15^−/− rmGDF15 groups were compared using a Mann-Whitney test. WT is displayed as reference. All P values for statistical comparisons are displayed.