Research ArticleVascular Biology

Methicillin-resistant Staphylococcus aureus causes sustained collecting lymphatic vessel dysfunction

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Science Translational Medicine  17 Jan 2018:
Vol. 10, Issue 424, eaam7964
DOI: 10.1126/scitranslmed.aam7964
  • Fig. 1 Collecting popliteal lymphatic vessels exhibit diminished contraction and lymph velocity after MRSA infection.

    C57BL/6 mice were infected subcutaneously in the hindlimb with 2 × 106 to 4 × 106 colony-forming units (CFU) of MRSA for 4 days or left uninfected. (A) Representative sequential images from intravital microscopy of PLVs perfused with FITC-dextran (2 million molecular weight). Scale bar, 50 μm (see movies S1 and S2). (B) Representative traces from PLV wall measurements show lymphatic diameter and contraction over time (n = 8 to 11, each group). (C) Ejection fraction, indicating strength of lymphatic vessel contraction, in uninfected mice and mice infected with WT MRSA after 4 days (n = 8 to 11, each group). (D) Frequency indicates the counts per minute (cpm) of PLV contractions in uninfected mice and mice infected with WT MRSA after 4 days (n = 7 to 10, each group). (E) Top: Representative depth versus time image of the optical coherence tomography intensity signal (grayscale) from a fixed transverse location that is used to identify the upper and lower boundaries of PLVs in uninfected animals and during active infection (day 4). LV, lymphatic vessel. Instantaneous lymph velocity (color) in the PLV in uninfected animals and during active infection (day 4) is overlaid on structural OCT depth scan. Bottom: Representative trace of instantaneous velocity of lymph flow averaged over the PLV cross-sectional area in uninfected animals and during active infection (day 4). (F) Time-averaged mean velocity of lymph flow in uninfected animals and during active infection (n = 3 to 11 measurements in each group). For (C), (D), and (F), statistical analysis was performed using Student’s unpaired two-sided t test. Error bars show SEM. *P < 0.05.

  • Fig. 2 CLV dysfunction persists after clearance of MRSA.

    (A) Infectious burden measured by colony formation assay in the draining PLN (left) at the indicated time points (n = 6 to 14, all groups). Infectious burden in the skin and underlying muscle tissue (right) from the site of infection at the indicated time points (n = 4 to 5, all groups). (B) Gene expression array of mouse inflammatory cytokines and receptors comparing tissue from the site of infection collected at day 4 (left) and day 60 (right) after MRSA infection. ΔΔCT was calculated with normalization of raw data to housekeeping genes. The graph plots normalized gene expression levels (log10) from control (uninfected) skin and muscle tissue (n = 2) on the x axis versus normalized gene expression levels (log10) of day 4 (n = 2) or day 60 (n = 2) post-infection skin and muscle tissue on the y axis. Circles above the upper gray line identify significantly up-regulated genes (P < 0.05); the blue line and lower gray line represent the boundary for genes unchanged and significantly down-regulated, respectively. Individual samples were loaded into each qPCR array plate and normalized to skin from uninfected mice. (C) Ejection fraction (n = 3 to 8) and (D) frequency (n = 3 to 10) show the strength and number, respectively, of PLV contractions over time. Each time point represents a different cohort of mice; days 1, 2, 4, 30, 60, and 120 were measured. (E) Representative depth versus time image of the depth-resolved optical coherence tomography intensity from a fixed transverse location that is used to identify the upper and lower boundaries of PLVs in uninfected animals and after clearance of infection (day 35). Instantaneous lymph velocity (color) in the lymphatic vessel in uninfected animals and after clearance of infection (day 35) is overlaid on structural OCT depth scan. (F) Time-averaged mean velocity of lymph in uninfected animals and after clearance of infection (day 35; n = 11 to 17 measurements in each group). Statistical analysis was performed using Student’s unpaired two-sided t test. Error bars show SEM. *P < 0.05.

  • Fig. 3 Inhibition of iNOS is not sufficient to restore CLV contraction during MRSA infection.

    (A) Representative sections of control skin (from uninfected mice) and skin 4 days after MRSA infection. Lymphatic vessels were identified with anti–LYVE-1 antibody (red) and nuclei with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Anti-iNOS antibody (green) identified iNOS-positive cells; n = 4. Scale bar, 300 μm. (B) Serial sections to the images in (A) were stained with anti–Gr-1 antibody (blue), anti-iNOS antibody (green), and anti-Ly6G antibody (red); n = 4. Scale bar, 50 μm. (C) Sterile PBS (50 μl) or LTA (50 μl) suspended in sterile PBS was injected into the hindlimb of C57BL/6 and C57BL/6-iNOS−/− mice. On day 4 after injection, the ejection fraction shows the strength of PLV contraction from the indicated concentrations of LTA (n = 3 to 4, each group). (D) C57BL/6 mice were injected subcutaneously in the hindlimb with PBS (uninfected) or infected subcutaneously in the hindlimb with 2 × 106 to 4 × 106 CFU of WT MRSA or MRSA deficient in nitric oxide production (ΔsaNOS). C57BL/6-iNOS−/− mice were infected subcutaneously in the hindlimb with 2 × 106 to 4 × 106 CFU of WT MRSA or ΔsaNOS MRSA. Ejection fraction 4 days after infection shows the strength of PLV contraction among respective groups (n = 8 to 11, each group). For (C) and (D), one-way analysis of variance (ANOVA) comparison with Fisher’s least significant difference post hoc analysis was used to determine significance. Error bars show SEM. *P < 0.05.

  • Fig. 4 Infection-induced CLV dysfunction is associated with decreased LMC coverage.

    (A) Representative immunohistochemical images of uninfected mouse hindlimb and mouse hindlimb 36 hours after infection stained with αSMA (crimson, arrowhead) and TUNEL (brown); n = 4 mice per group. Scale bar, 40 μm. (B) Representative intravital image from segment of PLV of an αSMAPDsRed/C57BL/6 mouse interstitially injected with FITC-dextran; n = 4 mice per group. Arrowhead indicates lymphatic vessel. Arrow points to blood vessel. Scale bar, 100 μm. (C) Representative intravital image from PLV segments of αSMAPDsRed/C57BL/6 mice on the indicated days. Scale bar, 100 μm. (D) Quantification of αSMA-positive cells per 100 μm of PLV; n = 4 to 8 mice per group. (E) Representative image from PLV segments of C57BL/6 mice, stained with anti-αSMA, on the indicated days. Scale bar, 100 μm. (F) Computer-automated quantification of % αSMA-positive area of PLV; n = 3 to 4 mice for each group. (G) Representative image from a segment of PLV stained with anti-αSMA (red) 260 days after infection and corresponding control (contralateral) PLV. Scale bar, 25 μm. Arrowheads indicate adjacent blood vessel (posterior tibial artery). (H) Computer-automated quantification of % αSMA coverage of PLV 260 days after infection or corresponding age-matched control (contralateral) popliteal lymphatic vessel (n = 5, both groups). For (D) and (F), statistical analysis was performed using one-way ANOVA comparison with Fisher’s least significant difference post hoc analysis. For (H), statistical analysis was performed using Student’s unpaired two-sided t test. Error bars show SEM. *P < 0.05.

  • Fig. 5 MRSA protein causes muscle cell death.

    (A) Cell viability [relative luciferase units (RLU)] analysis of hSMCs after exposure for 24 hours to control medium (TSB), MRSA-conditioned medium, or MRSA-conditioned medium incubated for 18 to 24 hours at 37°C with trypsin; n = 3 independent experiments. (B) Graph depicting LDH release from hSMCs (left y axis) and cell viability (RLU, right y axis) after exposure for 6 hours to control medium (TSB), MRSA-conditioned medium, or doxycycline; n = 3 independent experiments. (C) Mouse LMC viability analysis (RLU) after exposure for 6 hours to control medium (TSB) or (WT) MRSA-conditioned medium; n = 3 independent experiments. For (A), one-way ANOVA comparison with Fisher’s least significant difference post hoc analysis was performed. For (B), statistical analysis was performed using multiple t tests for comparators indicated. For (C), statistical analysis was performed using Student’s unpaired two-sided t test compared to the control group. Error bars show SEM. *P < 0.05. N.D., not detected.

  • Fig. 6 Effect of MRSA toxins on LMCs and CLV function.

    (A) Identification by mass spectrometry of the relative abundance of proteins (area) from <100-kDa fraction of MRSA-conditioned medium. (B) Heat map of the most highly expressed toxins, as identified by mass spectrometry; also depicted is the corresponding toxin expression level in the absence of agr. (C) Graph depicting LDH release from LMCs (left y axis) and cell viability (RLU, right y axis) after incubation for 24 hours with normal growth medium only (−), α-hemolysin (1.0 μg/ml), δ-hemolysin (100 μg/ml), or PSMα1 (100 μg/ml). Statistical analysis was performed using multiple t tests relative to untreated control for cytolysis and cell viability. *P < 0.05; n = 3 independent experiments. (D) Cell viability analysis of murine LMCs (mLMCs) after exposure for 24 hours to control medium (TSB), MRSA-conditioned medium, or conditioned medium of isogenic MRSA mutants, as indicated; n = 3 independent experiments. *P < 0.05 relative to control medium. hla, α-hemolysin; hld, δ-hemolysin. (E) Lymphatic ejection fraction (n = 4 to 5 mice for each group) and (F) frequency (n = 5 to 6 mice for each group) of lymphatic vessel contraction was measured in mice infected with WT or the indicated mutant MRSA strains. (G) Computer-automated quantification of % αSMA-positive area of PLV in animals 30 days after infection with WT or the indicated mutant MRSA strain; n = 3 to 4 mice for each group. For (D) to (G), statistical analysis was performed by one-way ANOVA comparison with Fisher’s least significance difference post hoc analysis. Error bars show SEM. *P < 0.05.

  • Fig. 7 MRSA virulence proteins cause LMC death and diminished CLV function.

    (A) mLMC viability analysis after exposure for 24 hours to control medium (TSB), agr mutant–conditioned medium, or MRSA-conditioned medium; n = 3 independent experiments. (B) LDH release from LMCs incubated with conditioned medium from WT or agr mutant MRSA; n = 3 independent experiments. (C) Computer-automated quantification of % αSMA-positive area of PLV in uninfected animals and animals 30 days after agr mutant MRSA infection; n = 3 to 4 mice for each group. (D) Representative traces from lymphatic vessel wall measurements show lymphatic diameter and contraction over time in uninfected mice and mice infected with WT or agr mutant MRSA for 30 days. (E) Ejection fraction shows the strength of lymphatic vessel contraction in uninfected mice and mice infected with WT or agr mutant MRSA for 30 days; n = 8 to 17. (F) Frequency indicates the cpm of lymphatic vessel contractions in uninfected mice and mice infected with WT or agr mutant MRSA for 30 days; n = 8 to 15. For (E) and (F), statistical analysis was performed by one-way ANOVA comparison with Fisher’s least significance difference post hoc analysis. Error bars show SEM. *P < 0.05.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/424/eaam7964/DC1

    Materials and Methods

    Fig. S1. MRSA infection leads to acute inflammation and CLV dilation.

    Fig. S2. CLV function remains impaired after the clearance of MRSA.

    Fig. S3. Attenuation of host inflammation is not sufficient to restore CLV contraction during MRSA infection.

    Fig. S4. Effect of MRSA infection on the blood vasculature.

    Fig. S5. Characterization of LMCs, vascular muscle cells, and LECs.

    Fig. S6. Identification of apoptotic cells in skin abscesses caused by MRSA infection.

    Fig. S7. MRSA toxins contribute to reduced hSMC and LEC viability.

    Movie S1. Representative lymphatic contraction of afferent CLV to the PLN in uninfected mouse.

    Movie S2. Representative lymphatic contraction of afferent CLV to the PLN in MRSA-infected (CA-MRSA USA300 JE2 strain) mouse 4 days after infection.

    Movie S3. Representative lymphatic contraction of afferent CLV to the PLN in MRSA-infected (CA-MRSA USA300 strain) mouse 30 days after infection.

    Movie S4. Representative lymphatic contraction of popliteal lymphatic vessel afferent CLV to the PLN in agr mutant–infected mouse 30 days after infection.

    Table S1. List of proteins identified by mass spectrometry of supernatant from WT CA-MRSA USA300 JE2 strain (provided as an Excel file).

    Table S2. Primary data (provided as an Excel file).

    Reference (59)

  • Supplementary Material for:

    Methicillin-resistant Staphylococcus aureus causes sustained collecting lymphatic vessel dysfunction

    Dennis Jones, Eelco F. J. Meijer, Cedric Blatter, Shan Liao, Ethel R. Pereira, Echoe M. Bouta, Keehoon Jung, Shan Min Chin, Peigen Huang, Lance L. Munn, Benjamin J. Vakoc, Michael Otto, Timothy P. Padera*

    *Corresponding author. Email: tpadera{at}steele.mgh.harvard.edu

    Published 17 January 2018, Sci. Transl. Med. 10, eaam7964 (2018)
    DOI: 10.1126/scitranslmed.aam7964

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. MRSA infection leads to acute inflammation and CLV dilation.
    • Fig. S2. CLV function remains impaired after the clearance of MRSA.
    • Fig. S3. Attenuation of host inflammation is not sufficient to restore CLV contraction during MRSA infection.
    • Fig. S4. Effect of MRSA infection on the blood vasculature.
    • Fig. S5. Characterization of LMCs, vascular muscle cells, and LECs.
    • Fig. S6. Identification of apoptotic cells in skin abscesses caused by MRSA infection.
    • Fig. S7. MRSA toxins contribute to reduced hSMC and LEC viability.
    • Legends for movies S1 to S4
    • Legends for tables S1 and S2
    • Reference (59)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Representative lymphatic contraction of afferent CLV to the PLN in uninfected mouse.
    • Movie S2 (.avi format). Representative lymphatic contraction of afferent CLV to the PLN in MRSA-infected (CA-MRSA USA300 JE2 strain) mouse 4 days after infection.
    • Movie S3 (.avi format). Representative lymphatic contraction of afferent CLV to the PLN in MRSA-infected (CA-MRSA USA300 strain) mouse 30 days after infection.
    • Movie S4 (.avi format). Representative lymphatic contraction of popliteal lymphatic vessel afferent CLV to the PLN in agr mutant–infected mouse 30 days after infection.
    • Table S1. List of proteins identified by mass spectrometry of supernatant from WT CA-MRSA USA300 JE2 strain (provided as an Excel file).
    • Table S2. Primary data (provided as an Excel file).

    [Download Tables S1 and S2]

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