Research ArticleFUNGAL INFECTIONS

Microhemorrhage-associated tissue iron enhances the risk for Aspergillus fumigatus invasion in a mouse model of airway transplantation

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Science Translational Medicine  21 Feb 2018:
Vol. 10, Issue 429, eaag2616
DOI: 10.1126/scitranslmed.aag2616
  • Fig. 1 Alloimmune-mediated rejection causes microhemorrhage in airway transplants.

    Comparison of allotransplants and syntransplants (n = 3 to 6 per group) by day posttransplant for (A) mean blood perfusion units measured by Doppler flowmetry and (B) fluorescein isothiocyanate (FITC)–conjugated lectin perfusion. a.u., arbitrary units. (B) Microbeads (yellow arrows) are extravasated (red) or colocalized (yellow) with microvascular FITC-lectin staining (green). Magnification, ×10. Scale bar, 100 μm. (C) Mean percent area perfused in the tracheal transplant model. (D) Microbead density area, measured by the percentage of the tracheal transplant with extravasated fluorescent microbeads. Data are means ± SEM and analyzed by Student’s t test. (E) Transmission electron micrograph of transplanted mouse trachea depicts vascular intimal layer (dashed red ellipse) with erythrocyte collections (yellow asterisks). The white arrow indicates an extravasating erythrocyte. Magnification, ×4500. (F) Tissue oxygen tension (PO2, mmHg) by day posttransplant in allotransplants and syntransplants (n = 3 to 6 per group) measured by Oxyprobe. Data are means ± SEM and analyzed by Student’s t test.

  • Fig. 2 Acute rejection induces tissue hemorrhage and dysregulates iron metabolism, increasing allograft iron content.

    (A) Iron concentration measured by inductively coupled plasma mass spectroscopy. Data are means ± SEM of graft iron content in parts per billion (ppb) per milligram of dry weight of trachea (n = 9) shown as a function of day posttransplant and analyzed by Student’s t test. (B) Representative serial sections of day 12 allotransplant mouse trachea stained with Perl’s Prussian blue stain for ferric iron and Turnbull’s stain for ferrous iron. Turnbull stain positive control is an untransplanted graft treated with ferrous sulfate topical solution. Magnification, ×20. Scale bar, 100 μm. (C) Representative immunofluorescent staining of day 12 mouse tracheal allotransplant (n = 3) with anti–light-chain ferritin antibody (red) compared to untransplanted graft (control). Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Magnification, ×20. Scale bar, 100 μm. (D) Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) for host iron metabolism genes in allotransplants (n = 3 to 9) on days 6 to 12 posttransplant compared to untransplanted grafts (control). Data are means ± SEM and analyzed by multiple t tests with the Holm-Sidak method to correct for multiple comparisons. (E) RT-qPCR fold change differences for hepcidin and natural resistance–associated macrophage protein 1 (nRAMP1) in day 12 mouse tracheal allotransplants and syntransplants (n = 5) relative to untransplanted trachea *P = 0.0079, **P = 0.04 analyzed by nonparametric Mann-Whitney U test.

  • Fig. 3 Iron increases A. fumigatus metabolism and affects its growth direction in culture.

    (A) Comparative A. fumigatus metabolism [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay] in RPMI culture medium (control) or in the presence of hemoglobin (HgB) or iron dextran (n = 32 wells) analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. (B) Growth area (in square millimeters) of A. fumigatus WT and ΔsreA, ΔhapX, and ΔsreAcccA mutants on iron-sufficient (Fe+) and iron-deficient (Fe) agar plates (n = 3) analyzed by Student’s t test. (C) Relative difference in growth area of A. fumigatus WT and mutant strains in wells containing iron dextran supplemented or unsupplemented RPMI culture medium (n = 3) analyzed by Student’s t test. (D) Representative A. fumigatus cultures comparing hyphal growth morphology in WT and ΔsreA mutant exposed to iron dextran and phosphate-buffered saline (PBS). Growth morphology (yellow line) is shown relative to the iron dextran well (24 hours). The convex pattern denotes positive tropism for iron; the concave pattern denotes negative tropism. (E) Representative light microscopy image of WT A. fumigatus hyphal growth (18 hours). Magnification, ×5. Scale bar, 50 μm. The inset depicts hyphal tips with wide-angle (>45°) branching toward the iron dextran–containing well (n = 3).

  • Fig. 4 Tissue iron promotes A. fumigatus invasion in the orthotopic tracheal transplant mouse model.

    (A) A. fumigatus invasion in mouse tracheal allotransplants (n = 4 to 9 per group) on days 6 to 12 posttransplant graded using a 0-to-4 histologic scale. Data are means ±SEM and analyzed by nonparametric Mann-Whitney U test. (B) Perl’s Prussian blue staining of the iron area (in square micrometers) in mouse tracheal allotransplants (n = 4 to 9 per group) on days 6 to 12 posttransplant. Data are means ± SEM analyzed by nonparametric Mann-Whitney U test. (C) Invasion by A. fumigatus of day 8 allotransplants (n = 8 to 9 per group) after treatment by intraperitoneal injections of PBS (control), DFO (deferoxamine), or DFX (deferasirox), analyzed by nonparametric Mann-Whitney U test. (D) Invasion by A. fumigatus of day 8 syntransplants (n = 5 to 9 per group) from hemochromatosis knockout (Hfe−/−) donor mice and WT donor mice (control). (E) Invasion by A. fumigatus of day 8 allotransplants (n = 5 to 9 per group) from Hfe−/− and WT donor mice, analyzed by nonparametric Mann-Whitney U test. (F) Invasion of day 12 allotransplants (n = 5 to 6 per group) by A. fumigatus hapX, sreA, and sreA/cccA mutants or WT A. fumigatus, analyzed by nonparametric Mann-Whitney U test.

  • Fig. 5 Exogenous iron stimulates A. fumigatus invasion in syntransplants.

    (A) Representative Grocott’s methenamine silver staining of mouse tracheal syntransplants treated with vehicle (control, left) or FeSO4 topical solution reveals deep invasion by A. fumigatus (red arrows) in the FeSO4 solution–treated graft. Magnification, ×20. Scale bar, 100 μm. (B) Invasion by A. fumigatus of day 8 syntransplants (n = 6 to 11 per group) treated with topical FeSO4 solution or vehicle control. Data are means ± SEM analyzed by nonparametric Mann-Whitney U test. (C) Representative Prussian blue staining of syntransplants (n = 4 per group) treated with FeSO4 topical solution (top) or vehicle control (bottom). Black arrowheads depict tissue iron deposits (blue). Magnification, ×20. Scale bar, 100 μm. (D) Doppler flowmetry perfusion studies comparing blood perfusion units between syntransplants (n = 4 per group) treated with FeSO4 solution or vehicle control, analyzed by Student’s t test. (E) Graft tissue oxygen tension (PO2, mmHg) comparing vehicle control or FeSO4 solution–treated syngrafts (n = 4 per group). Analyzed by Student’s t test. TL, tracheal lumen; C, cartilage ring.

  • Fig. 6 Acute rejection is characterized by a proinflammatory macrophage phenotype.

    (A) Mean number of macrophages (F4/80+ cells per trachea) in day 12 mouse tracheal allotransplants and syntransplants (n = 3 per group) treated with FeSO4 solution or vehicle control. Data are means ± SEM analyzed by two-way ANOVA with Tukey’s multiple comparison test. (B) Representative immunohistochemistry with F4/80 antibody staining of mouse tracheal sections from day 12 allotransplants and syntransplants (n = 3 per group) treated with FeSO4 solution or vehicle control. Magnification, ×40. Scale bar, 50 μm. (C) Representative flow cytometry analysis of mouse tracheal transplants comparing day 12 syntransplants and allotransplants gated on cell surface CD11b and F4/80 expression. (D) Representative flow cytometry analysis of mouse tracheal transplants comparing day 12 syntransplants and allotransplants re-gated on the expression of CD206. M1 denotes classically activated macrophages, and M2 denotes alternatively activated macrophages.

  • Fig. 7 Iron is increased in allograft airways from lung transplant patients.

    (A) Schematic diagram depicting biopsy strategy for human lung transplant patients, comparing paired samples from nontransplanted (recipient, black) and transplanted (donor, red) airways. (B) Proportion of cells staining positive for iron in biopsy samples from lung transplant patients (n = 5) comparing recipient and transplanted airways. Data are means ± SEM analyzed by Wilcoxon matched-pairs signed rank test. (C) Representative Prussian blue staining of a recipient airway biopsy and (D) a transplant airway biopsy, depicting iron-laden (blue) macrophages. Magnification, ×40. Scale bar, 50 μm.

  • Table 1 Iron studies in human lung transplant patients.

    CF, cystic fibrosis; IPF, idiopathic pulmonary fibrosis; ND, not detected; BAL, bronchoalveolar fluid.

    Age (years)DiagnosisTime
    posttransplant
    (weeks)
    # Cells iron stain positive/biopsyBAL iron
    content
    (μg/liter)
    InfectionRejection grade
    TransplantRecipient
    27CF58221431.4Pseudomonas
    aeruginosa
    A0
    53IPF11814929353.4NDA0
    35CF1065434430.5NDA1
    67IPF2334740279.3P. aeruginosaA2
    21CF812723550.9Enterobacter
    cloacae
    A1

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/429/eaag2616/DC1

    Supplementary materials and methods

    Table S1. RT-qPCR genes and primers.

    Fig. S1. Iron content, as measured by ICP-MS, in day 12 syntransplants and allotransplants.

    Fig. S2. RT-qPCR of host iron metabolism genes in day 12 syntransplants and allotransplants.

    Fig. S3. Schematic of culture plate for growth morphology and positive tropism studies.

    Fig. S4. Culture plates showing positive tropism.

    Fig. S5. Experimental design of animal studies.

    Fig. S6. Semiquantitative histopathological scale for grading fungal invasion and burden.

    Fig. S7. Fungal burden by iron groups in the orthotopic tracheal transplantation model.

    Fig. S8. Increased fungal invasion in allotransplants treated with FeSO4 topical solution.

    Fig. S9. Alloimmune-mediated microvascular ischemia and iron overload do not decrease immune effector cell numbers in the orthotopic tracheal transplantation model.

    Fig. S10. Schematic diagram illustrating how acute allograft rejection induces transplant iron overload, promoting an invasive A. fumigatus phenotype.

  • Supplementary Material for:

    Microhemorrhage-associated tissue iron enhances the risk for Aspergillus fumigatus invasion in a mouse model of airway transplantation

    Joe L. Hsu, Olga V. Manouvakhova, Karl V. Clemons, Mohammed Inayathullah, Allen B. Tu, Raymond A. Sobel, Amy Tian, Hasan Nazik, Venkata R. Pothineni, Shravani Pasupneti, Xinguo Jiang, Gundeep S. Dhillon, Harmeet Bedi, Jayakumar Rajadas, Hubertus Haas, Laure Aurelian, David A. Stevens, Mark R. Nicolls*

    *Corresponding author. Email: mnicolls{at}stanford.edu

    Published 21 February 2018, Sci. Transl. Med. 10, eaag2616 (2018)
    DOI: 10.1126/scitranslmed.aag2616

    This PDF file includes:

    • Materials and Methods
    • Table S1. RT-qPCR genes and primers.
    • Fig. S1. Iron content, as measured by ICP-MS, in day 12 syntransplants and allotransplants.
    • Fig. S2. RT-qPCR of host iron metabolism genes in day 12 syntransplants and allotransplants.
    • Fig. S3. Schematic of culture plate for growth morphology and positive tropism studies.
    • Fig. S4. Culture plates showing positive tropism.
    • Fig. S5. Experimental design of animal studies.
    • Fig. S6. Semiquantitative histopathological scale for grading fungal invasion and burden.
    • Fig. S7. Fungal burden by iron groups in the orthotopic tracheal transplantation model.
    • Fig. S8. Increased fungal invasion in allotransplants treated with FeSO4 topical solution.
    • Fig. S9. Alloimmune-mediated microvascular ischemia and iron overload do not decrease immune effector cell numbers in the orthotopic tracheal transplantation model.
    • Fig. S10. Schematic diagram illustrating how acute allograft rejection induces transplant iron overload, promoting an invasive A. fumigatus phenotype.

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