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Transcriptional firing represses bactericidal activity in cystic fibrosis airway neutrophils

Margaroli, Camilla, Moncada-Giraldo, Diego, Gulick, Dalia Arafat, Dobosh, Brian, Giacalone, Vincent D., Forrest, Osric A., Sun, Fangxu, Gu, Chunhui, Gaggar, Amit, Kissick, Haydn, Wu, Ronghu, Gibson, Greg, Tirouvanziam, Rabindra·2021·Cell Reports Medicine
DOI 10.1016/j.xcrm.2021.100239PMC PMC8080108
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Figure 1 GRIM neutrophils show an increase in RNA content in the cystic fibrosis lung in vivo and in vitro (A) Blood (red) and sputum (blue) neutrophils from patients with CF (N = 7) were analyzed by flow cytometry. (B) Airway neutrophils displayed a characteristic loss of the phagocytic receptor CD16 and release of primary granules, measured by surface CD63. Increased total RNA content compared to their matched counterpart was measured by flow cytometry. (C) Blood neutrophils (red, N = 5) were transmigrated in vitro to sputum supernatant from patients with CF (CFASN, blue) or the chemoattractant leukotriene B4 (LTB4) as a transmigration control (gray). (D and E) Flow cytometry analysis revealed an increase in RNA content in CFASN airway neutrophils compared to matched blood neutrophils, which was confirmed by quantification by bioanalyzer (E). (F) Genes that were commonly upregulated or downregulated both in vivo and in vitro were used to conduct a Gene Ontology (GO) term RNA analysis. Top 20 pathways for the upregulated and downregulated genes are shown; significance of enriched pathways was established using the false discovery rate (FDR) q < 5%. Results are shown as median and interquartile range. Statistical analysis was performed by Wilcoxon matched-pairs signed rank test; ∗∗p < 0.01, n.s., not significant.
Figure 1 GRIM neutrophils show an increase in RNA content in the cystic fibrosis lung in vivo and in vitro (A) Blood (red) and sputum (blue) neutrophils from patients with CF (N = 7) were analyzed by flow cytometry. (B) Airway neutrophils displayed a characteristic loss of the phagocytic receptor CD16 and release of primary granules, measured by surface CD63. Increased total RNA content compared to their matched counterpart was measured by flow cytometry. (C) Blood neutrophils (red, N = 5) were transmigrated in vitro to sputum supernatant from patients with CF (CFASN, blue) or the chemoattractant leukotriene B4 (LTB4) as a transmigration control (gray). (D and E) Flow cytometry analysis revealed an increase in RNA content in CFASN airway neutrophils compared to matched blood neutrophils, which was confirmed by quantification by bioanalyzer (E). (F) Genes that were commonly upregulated or downregulated both in vivo and in vitro were used to conduct a Gene Ontology (GO) term RNA analysis. Top 20 pathways for the upregulated and downregulated genes are shown; significance of enriched pathways was established using the false discovery rate (FDR) q < 5%. Results are shown as median and interquartile range. Statistical analysis was performed by Wilcoxon matched-pairs signed rank test; ∗∗p < 0.01, n.s., not significant.CC BY · PubMed Central
Figure 2 The CF airway microenvironment imparts distinct transcriptional signatures onto GRIM neutrophils (A) Principal component analysis (PCA) of the transcriptome from neutrophils used in the in vitro transmigration model showed a distinct profile for each condition (blood, LTB4-transmigrated, and CFASN-transmigrated; N = 5 independent experiments). (B) Primary, secondary, and tertiary granule effector proteins were downregulated (gray) in the CFASN group compared to their blood counterpart, while respective structural granule proteins were upregulated (black). (C) Gene set enrichment analysis (GSEA) showed enriched glycolysis and mTOR metabolic pathways in the CFASN neutrophils compared to LTB4, as well as changes in the immunological profile, reflecting the acquisition of myeloid-derived suppressor cell (MDSC) function. (D) Of the genes uniquely upregulated or downregulated compared to the blood, those that were significantly different (log2 fold change greater or less than 2 or −2, and p < 0.01) between CFASN and LTB4 were analyzed by GSEA using the hallmark (H), reactome (R), and KEGG (K) datasets. Top 10 pathways (FDR < 5%) are shown for the upregulated and downregulated gene sets.
Figure 2 The CF airway microenvironment imparts distinct transcriptional signatures onto GRIM neutrophils (A) Principal component analysis (PCA) of the transcriptome from neutrophils used in the in vitro transmigration model showed a distinct profile for each condition (blood, LTB4-transmigrated, and CFASN-transmigrated; N = 5 independent experiments). (B) Primary, secondary, and tertiary granule effector proteins were downregulated (gray) in the CFASN group compared to their blood counterpart, while respective structural granule proteins were upregulated (black). (C) Gene set enrichment analysis (GSEA) showed enriched glycolysis and mTOR metabolic pathways in the CFASN neutrophils compared to LTB4, as well as changes in the immunological profile, reflecting the acquisition of myeloid-derived suppressor cell (MDSC) function. (D) Of the genes uniquely upregulated or downregulated compared to the blood, those that were significantly different (log2 fold change greater or less than 2 or −2, and p < 0.01) between CFASN and LTB4 were analyzed by GSEA using the hallmark (H), reactome (R), and KEGG (K) datasets. Top 10 pathways (FDR < 5%) are shown for the upregulated and downregulated gene sets.CC BY · PubMed Central
Figure 3 Transcriptional and functional adaptation of neutrophils to the CF airway microenvironment occurs over time Blood neutrophils (red) were transmigrated in vitro toward CFASN and collected at 1, 2, 4, and 6 h post-transmigration (N = 5 independent experiments). (A and B) Surface CD63 expression was quantified by flow cytometry (A), and RNA content by bioanalyzer (B). (C) The transcriptional profile obtained by RNA sequencing for each time point was compared to blood (N = 3 subjects). Significantly different genes are defined as log2 greater than 2 or less than −2 for upregulated genes (left) and dowregulated genes (right), respectively, and indicated in Venn diagram form to show the overlap between time points. (D) Pathway analysis for downregulated genes by GSEA (FDR q < 5%) shows reduced expression of granule effector proteins (reactome “neutrophil degranulation” pathway) over time relative to blood. Dot plots indicate the number of genes in the overlap between the experimental gene set and the reactome neutrophil degranulation gene set, while the level of significance of pathway enrichment at each time point is indicated by color. Data are shown as median and interquartile range.
Figure 3 Transcriptional and functional adaptation of neutrophils to the CF airway microenvironment occurs over time Blood neutrophils (red) were transmigrated in vitro toward CFASN and collected at 1, 2, 4, and 6 h post-transmigration (N = 5 independent experiments). (A and B) Surface CD63 expression was quantified by flow cytometry (A), and RNA content by bioanalyzer (B). (C) The transcriptional profile obtained by RNA sequencing for each time point was compared to blood (N = 3 subjects). Significantly different genes are defined as log2 greater than 2 or less than −2 for upregulated genes (left) and dowregulated genes (right), respectively, and indicated in Venn diagram form to show the overlap between time points. (D) Pathway analysis for downregulated genes by GSEA (FDR q < 5%) shows reduced expression of granule effector proteins (reactome “neutrophil degranulation” pathway) over time relative to blood. Dot plots indicate the number of genes in the overlap between the experimental gene set and the reactome neutrophil degranulation gene set, while the level of significance of pathway enrichment at each time point is indicated by color. Data are shown as median and interquartile range.CC BY · PubMed Central
Figure 4 Functional adaptation to the CF airway microenvironment depends upon de novo transcription Blood neutrophils (red) were transmigrated in vitro for 2 h toward CFASN and subsequently incubated for 8 h with or without the transcriptional blocker α-amanitin (N = 3 independent experiments). (A–D) CFASN transmigrated neutrophils treated with α-amanitin showed reduced release of primary granules (A), concomitant with lower presence of NE in the extracellular milieu (B), reduced secretion of extracellular vesicles (EVs) (C), and increased killing of Pseudomonas aeruginosa (D). (E and F) Short-term treatment of primary CF sputum neutrophils with α-amanitin (N = 3 independent experiments) led to similar patterns of reduced exocytosis (E) and increased killing of P. aeruginosa (F). Data are shown as median and interquartile range and analyzed using the Wilcoxon matched-pairs signed rank test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 4 Functional adaptation to the CF airway microenvironment depends upon de novo transcription Blood neutrophils (red) were transmigrated in vitro for 2 h toward CFASN and subsequently incubated for 8 h with or without the transcriptional blocker α-amanitin (N = 3 independent experiments). (A–D) CFASN transmigrated neutrophils treated with α-amanitin showed reduced release of primary granules (A), concomitant with lower presence of NE in the extracellular milieu (B), reduced secretion of extracellular vesicles (EVs) (C), and increased killing of Pseudomonas aeruginosa (D). (E and F) Short-term treatment of primary CF sputum neutrophils with α-amanitin (N = 3 independent experiments) led to similar patterns of reduced exocytosis (E) and increased killing of P. aeruginosa (F). Data are shown as median and interquartile range and analyzed using the Wilcoxon matched-pairs signed rank test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.CC BY · PubMed Central

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Metadata

Authors
Margaroli, C., Moncada-Giraldo, D., Gulick, D. A., Dobosh, B., Giacalone, V. D., Forrest, O. A., Sun, F., Gu, C., Gaggar, A., Kissick, H., Wu, R., Gibson, G., & Tirouvanziam, R.
ORCID
Venue
Cell Reports Medicine
Publisher
Elsevier BV
Published
2021-04
Volume
2
Issue
4
Pages
100239
ISSN
2666-3791
DOI
10.1016/j.xcrm.2021.100239
PMC
PMC8080108
Cited by
38
References
3
Language
en
Licence
Funders
  • National Institutes of Health
  • National Heart, Lung, and Blood Institute (R01HL126603)
  • Cystic Fibrosis Foundation (ROWE19R0, TIROUV15A0)
  • Emory University
  • Children's Healthcare of Atlanta

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