Research ArticleINNATE IMMUNITY

Skin infections are eliminated by cooperation of the fibrinolytic and innate immune systems

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Science Immunology  22 Sep 2017:
Vol. 2, Issue 15, eaan2725
DOI: 10.1126/sciimmunol.aan2725
  • Fig. 1 NFATc2-deficient mice do not eliminate skin C. albicans infections.

    (A) Lesions in WT and NFATc2-deficient mice at the indicated time points after C. albicans hyphae injection in the deep derma. WT animals (top) undergo ulceration and C. albicans elimination, whereas NFATc2-deficient mice (bottom) develop a persistent abscess. (B) Kaplan-Meier curve showing the percentage of WT and NFATc2-deficient mice undergoing ulceration after C. albicans administration at the indicated time points; n = 10; log-rank test. Results are representative of at least six independent experiments. (C) Hematoxylin and eosin staining of WT (top) and NFATc2-deficient (bottom) mouse skin lesions at the indicated time points after C. albicans infections. Larger magnification of selected areas of the same section are shown to evidence granulocyte recruitment (left). PicroSirius Red staining is also shown to evidence collagen depositions (right, red deposits) and periodic acid–Schiff (PAS) staining to evidence Candida. See also fig. S2 for higher magnifications. Representative histological sections of four independent experiments are shown; see also fig. S3 (A and B). (D) WT and RAG-2–deficient mice respond similarly to primary skin infections with C. albicans. The Kaplan-Meier curve shows the percentage of WT and RAG-2–deficient mice undergoing ulceration after C. albicans administration at the indicated time points; n = 18 per group. (E) Digital image analysis quantification of collagen staining. Five fields from two sections (24 hours after infection) of two independent experiments were analyzed. The analyzed fields covered the entire sections excluding the skin. Means and SEM are depicted. n = 5; statistical significance, two-tailed t test.

  • Fig. 2 Fibroblast activation in WT and NFATc2-deficient mice.

    (A) Histology of lesions induced by C. albicans hyphae in WT and NFATc2-deficient mice 48 hours after infections. Fibroblasts surrounding granulocytes are highlighted with black arrows. (B) Representative images of α-SMA immunohistochemical staining in skin sections of WT and NFATc2-deficient mice 6 and 8 days after C. albicans infection. PicroSirius Red staining is also shown to evidence collagen capsule. Representative histological sections of three independent experiments are shown; see also fig. S7.

  • Fig. 3 The TGF-β pathway is activated in the skin after C. albicans infection and is required to contain the infection.

    (A) Western analysis of SMAD2/3 phosphorylation at the indicated time points after infection of WT and NFATc2-deficient mice. Data were quantified and normalized on β-actin. Data are representative of three independent experiments. nt, untreated mice. (B) Representative images of p-SMAD2,3 immunohistochemical staining in skin sections 24 hours after C. albicans infection. p-SMAD2,3–positive cells are brown. The experiment was repeated twice with similar results; see also fig. S8. (C) Abscess formation in WT and NFATc2-deficient mice in the presence or not of the TGF-β inhibitor SB-431542. TGF-β inhibitor was administered ip (50 μg per mouse) for 3 days starting 1 day before C. albicans infection; the day of the infection was also coadministered locally with the hyphae. Note that the abscess is more diffused when the TGF-β pathway is inhibited. Two independent experiments with eight animals per group were performed. (D) Visualization of C. albicans (purple staining, brown arrows) by PAS staining in WT and NFATc2-deficient mouse skin lesions 24 to 48 hours after C. albicans infections in the presence of TGF-β inhibitor. PicroSirius Red staining of selected areas is also shown to evidence collagen depositions (arrows). Note that the collagen capsule is disorganized if the animals are treated with the TGF-β inhibitor and C. albicans can exit the abscess (see fig. S6 for higher magnifications). Representative histological sections of three independent experiments are shown; see also fig. S10.

  • Fig. 4 IFN-γ antagonizes TGF-β in vivo.

    (A) qRT-PCR analysis of IFN-γ mRNA in C. albicans–infected tissues of WT and NFATc2-deficient mice at the indicated time points after infection; each dot represents a different mouse. Means and SEM are depicted. Statistical significance was determined with a two-way ANOVA. ns, not statistically significant. (B) IFN-γ immunohistochemical staining (brown cells) in skin sections 24 hours after C. albicans infection. Representative histological sections from two independent experiments are shown; see also fig. S11. (C) Left: Kaplan-Meier curve showing the percentage of WT and NFATc2-deficient mice undergoing ulceration after C. albicans administration in the presence or not of IFN-γ at the indicated time points; n (WT) = 11, n (NFATc2) = 10, n (WT + rIFN-γ) = 5, n (NFATc2 + rIFN-γ) = 5. rIFN-γ, recombinant IFN-γ. Right: Kaplan-Meier curve showing the percentage of WT and IFN-γ–deficient mice undergoing ulceration after C. albicans administration in the presence or not of the indicated antibodies at the indicated time points; n (WT + isotype control) = 8, n (WT) = 12, n (WT + α–IFN-γ) = 10, n (IFN-γ–deficient) = 14; log-rank test. (D) Hematoxylin and eosin staining of WT and NFATc2-deficient mouse skin lesions 48 hours after C. albicans infections in the presence or not of the indicated stimuli. Larger magnification of PicroSirius Red staining of selected areas is also shown to evidence collagen depositions. The collagen capsule is loose in NFATc2-deficient mice if they are treated with rIFN-γ (1 μg per mouse). In contrast, the collagen capsule becomes thick in WT animals if IFN-γ is neutralized by an anti–IFN-γ antibody (50 μg per mouse). (E) Western blot analysis of SMAD2/3 phosphorylation in WT animals treated with C. albicans hyphae or C. albicans hyphae and rIFN-γ. Western blot analysis was performed 6 hours after C. albicans infection. Data were quantified and normalized on β-actin. Data are representative of two independent experiments. (F) Western blot analysis of SMAD2/3 phosphorylation from the infected skin of WT animals treated with C. albicans hyphae, C. albicans hyphae and the IFN-γ neutralizing antibody, or C. albicans hyphae and the isotype control antibody. Western blot analysis was performed 24 hours after C. albicans infection. Data were quantified and normalized on β-actin. Data are representative of three independent experiments. (G) In vitro WT and NFATc2-deficient skin fibroblast proliferation in the presence or not of the indicated stimuli (TGF-β, 3 ng/ml; IFN-γ, 150 U/ml). Each dot represents a different sample. (H) α-SMA immunohistochemical staining in skin sections of IFN-γ–deficient mice 7 days after C. albicans infection. PicroSirius Red staining is also shown to evidence collagen capsule.

  • Fig. 5 IFN-γ activates the fibrinolytic system.

    (A) Increase of active plasmin levels in WT and NFATc2-deficient mice at the indicated time points after C. albicans infection. Each dot represents a single mouse. Means and SEM are depicted; a two-way ANOVA was used for statistics. The experiment was repeated twice with similar results. (B) Hematoxylin and eosin staining of WT mouse skin lesions 48 hours after C. albicans infections in the presence of PAI-1 (0.65 μg per mouse) and NFATc2-deficient mice skin lesions. Larger magnification of PicroSirius Red staining of selected areas is also shown to evidence collagen depositions. Representative histological sections from two independent experiments are shown; see also fig. S13. (C) Kaplan-Meier curves showing the percentage of mice undergoing ulceration after C. albicans administration. Where indicated, mice were treated with PAI-1 (0.65 μg per mouse, coadministered with C. albicans). n (WT) = 10, n (WT + PAI-1) = 24; log-rank test. (D) Representative images of α-SMA immunohistochemical staining in skin sections of NFATc2-deficient and PAI-1–treated WT animals 8 and 7 days after C. albicans infection. (E) Left: Western blot analysis of tPA and PAI-1 levels measured in WT and NFATc2-deficient animals at the indicated hours after C. albicans infection. Data are representative of three independent experiments. Middle and right: Western blot analysis of tPA and PAI-1 levels measured in WT and NFATc2-deficient animals treated with an anti–IFN-γ blocking antibody and with rIFN-γ, respectively, at the time of C. albicans infection. The analysis was performed at the indicated time points. Data are representative of two independent experiments. (F) Left: Increase of active plasmin levels in NFATc2-deficient mice at the indicated time points after C. albicans infection in the presence or not of rIFN-γ. Right: Increase of active plasmin levels in WT mice at the indicated time points after C. albicans infection in the presence or not of anti–IFN-γ blocking antibody. Each dot represents a single mouse. Means and SEM are depicted; a two-way ANOVA was used for statistics. (G) qRT-PCR analysis of IFN-γ mRNA in C. albicans–infected tissues before and 6 hours after the infection in WT animals depleted or not of NK cells. Each dot represents a single mouse. Means and SEM are depicted; a two-way ANOVA was used for statistics. (H) Kaplan-Meier curve showing the percentage of WT (n = 9), NK cell–depleted WT (n = 8), and NFATc2-deficient mice (n = 10) undergoing ulceration after C. albicans administration at the indicated time points. Log-rank test was used. (I) IFN-γ immunohistochemical staining in skin sections (brown cells) of WT mice treated or not with anti-asialo GM to eliminate NK cells (NK-depleted) and infected with C. albicans. Note that IFN-γ+ cells are strongly reduced in NK-depleted mice. (J) Kaplan-Meier curve showing the percentage of NFATc2-deficient mice (n = 10), NFATc2-deficient mice reconstituted with activated IFN-γ–sufficient NK cells (n = 10) or IFN-γ–deficient NK cells (n = 10), and WT animals (n = 8) undergoing ulceration at the indicated time points after C. albicans administration. Log-rank test was used.

  • Fig. 6 DCs are required for NK cell activation.

    (A) IFN-γ immunohistochemical staining in skin sections (brown cells) of DOG mice and DC-depleted DOG mice (DOG + DT) after C. albicans infection. Representative histological sections from two independent experiments are shown; see also fig. S19. (B) Kaplan-Meier curve showing the percentage of DC-sufficient (n = 22) and DC-depleted (n = 32) DOG mice (DOG + DT) undergoing ulceration after C. albicans administration at the indicated time points; log-rank test. (C) Absolute numbers of IFN-γ+ NK cells at the draining lymph nodes of WT and NFATc2-deficient mice 4 hours after C. albicans infections. Where indicated, mice were cotreated with C. albicans and rIL-2. Each symbol represents a different mouse. Means and SDM are depicted; a two-way ANOVA test was used for statistics. (D) Absolute numbers of IFN-γ+ NK cells at the draining lymph nodes of DOG mice treated or not with DT 4 hours after C. albicans infections. Each symbol represents a different mouse. Means and SDM are depicted; a two-way ANOVA test was used for statistics. (E) IL-2 released in the supernatants by WT or NFATc2-deficient BMDCs and BM macrophages (MF) before and after C. albicans exposure (MOI, 0.05). IL-2 released by DCs–NK cell and macrophages–NK cell cocultures is also shown. Each dot represents a different sample. (F) Immature or C. albicans–activated WT and NFATc2-deficient BMDCs were cultured with NK cells for 18 hours. Where indicated, IL-2 was blocked using the S4B6 anti–IL-2 antibody (α–IL-2), or rIL-2 was added to the cultures. Levels of IFN-γ in the supernatant were then quantified by ELISA. Each dot represents a different sample. Representative data of two independent experiments are shown. KO, knockout. (G) Absolute numbers of IL-2+ DCs at the draining lymph nodes of WT and NFATc2-deficient mice 4 hours after C. albicans infections. Each symbol represents a different mouse. Means and SDM are depicted; a two-way ANOVA test was used for statistics. (H) Kaplan-Meier curves showing the percentage of NFATc2-deficient mice undergoing ulceration after C. albicans administration in the presence or not of the indicated stimuli and at the indicated time points; n (Nfatc2−/−) = 10, n (Nfatc2−/− + IL-2) = 8, n (Nfatc2−/− + IL-2 + α–IFN-γ) = 10; log-rank test. (I) Kaplan-Meier curves showing the percentage of NFATc2-deficient mice reconstituted with DCs of the indicated genotype undergoing ulceration after C. albicans administration. Where indicated, NK cells were depleted. n = 12 per group; log-rank test.

  • Fig. 7 Innate immune and fibrinolytic systems cooperate also during bacterial infections.

    (A) Kaplan-Meier curve showing the percentage of WT (n = 6) and IFN-γ–deficient mice (n = 10) undergoing ulceration after S. aureus administration at the indicated time points; log-rank test. Representative data of two independent experiments. (B) Hematoxylin and eosin staining of WT and IFN-γ–deficient mice skin lesions 48 hours after S. aureus infection. PicroSirius Red staining is also shown to evidence collagen depositions. (C) Western blot analysis of SMAD2/3 phosphorylation at the indicated time points after S. aureus infection of WT and IFN-γ–deficient mice. Data were quantified and normalized on β-actin. Data are representative of three independent experiments. (D) Western blot analysis of tPA and PAI-1 levels measured in WT and IFN-γ–deficient animals at the indicated hours after S. aureus infection.

  • Fig. 8 NFAT activation in innate immune cells dictates the sterilization of C. albicans skin infection.

    Schematic of C. albicans skin infection containment and elimination. Distinct phases can be identified in the inflammatory process that takes place after C. albicans skin infections. During the very early phases (1), granulocytes are recruited and form an abscess around the invading microorganisms to avoid infection spreading. The containment of the infection is ensured by fibroblasts that, once activated by TGF-β, proliferate and deposit collagen around the abscess to form a capsule. Later, IFN-γ, produced by NK cells activated in the draining lymph nodes (dLN), antagonizes TGF-β and thus avoids excessive fibroblast activation and excessive collagen deposition and also avoids the differentiation of fibroblasts into myofibroblasts (2). Last, IFN-γ ensures the activation of the fibrinolytic system and the consequent activation of metalloproteinases by plasmin (3). During the elimination phase, proteinases digest the collagen capsule and induce skin ulceration for the microbial expulsion out of the skin (3). NFATc2 activation in DCs after C. albicans exposure leads to IL-2 production. In turn, IL-2 is required to elicit IFN-γ release by NK cells (2). In the absence of NFATc2, IFN-γ is not produced in sufficient amount to counteract the TGF-β pathway (2 Nfatc2−/−) and to induce the activation of the fibrinolytic system (3 Nfatc2−/−); therefore, fibroblasts are hyperactive, deposit excessive collagen, and differentiate in myofibroblasts (3 Nfatc2−/−). This leads to the formation of a thick capsule that prevents skin ulceration and microbial expulsion out of the skin.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/15/eaan2725/DC1

    Fig. S1. Expression of NFATc1, NFATc2, and NFATc3 in immune cells.

    Fig. S2. Magnifications of the selected areas shown in Fig. 1C.

    Fig. S3. Additional histological images of the abscess after C. albicans infection.

    Fig. S4. Histology of the abscess 1 month after infection of NFATc2-deficient mice.

    Fig. S5. Granulocyte and monocyte recruitment at the infection site of WT and NFATc2-deficient mice.

    Fig. S6. Visualization of C. albicans at the infection site 6 to 8 days after infection.

    Fig. S7. Additional histological images of α-SMA staining in skin sections of WT and NFATc2-deficient mice.

    Fig. S8. Additional histological images of p-SMAD2,3 staining.

    Fig. S9. Magnifications of the selected areas shown in Fig. 3D.

    Fig. S10. Additional histological images of the abscesses after C. albicans infection in the presence of a TGF-β inhibitor.

    Fig. S11. Additional histological images of IFN-γ staining.

    Fig. S12. IFN-γ induces capsule digestion.

    Fig. S13. Additional histological images of the abscesses after C. albicans infection in the presence of PAI-1.

    Fig. S14. Inhibition of plasmin or MMP-3 interferes with C. albicans elimination.

    Fig. S15. IFN-γ mRNA is up-regulated in the infected skin of RAG-2–deficient mice.

    Fig. S16. DCs are necessary for the activation of NK cells in the presence of C. albicans in vitro.

    Fig. S17. DC depletion after DT injection in DOG mice.

    Fig. S18. Granulocyte and monocyte recruitment at the infection site of DOG mice treated or not with DT to deplete DCs.

    Fig. S19. Additional histological images of IFN-γ staining in the infected skin of DOG mice.

    Fig. S20. IL-12 production by BMDCs after C. albicans stimulation.

    Fig. S21. Original Western blots shown in Fig. 3A.

    Fig. S22. Original Western blots shown in Fig. 4 (E and F).

    Fig. S23. Original Western blots shown in Fig. 5E.

    Fig. S24. Original Western blots shown in Fig. 7C.

    Fig. S25. Original Western blots shown in Fig. 7D.

    Fig. S26. Representative isotype control stainings for immunohistochemical analyses.

    Fig. S27. Gating strategies used in cytofluorimetric analyses.

    Fig. S28. Representative isotype control stainings for cytofluorimetric analyses.

    Raw data for Figs. 1 (B, D, and E), 4 (A, C, and G), 5 (A, C, F, G, H, and J), 6 (B to I), and 7A; and figs. S1 (A and B), S5, S14, S15, S16, S17A, S18, and S20 (Microsoft Excel format).

  • Supplementary Materials

    Supplementary Material for:

    Skin infections are eliminated by cooperation of the fibrinolytic and innate immune systems

    William Santus, Simona Barresi, Francesca Mingozzi, Achille Broggi, Ivan Orlandi, Giulia Stamerra, Marina Vai, Alessandra M. Martorana, Alessandra Polissi, Julia R. Köhler, Ningning Liu, Ivan Zanoni,* Francesca Granucci*

    *Corresponding authors. Email: francesca.granucci{at}unimib.it (F.G.); ivan.zanoni{at}childrens.harvard.edu (I.Z.)

    Published 22 September 2017, Sci. Immunol. 2, eaan2725 (2017)
    DOI: 10.1126/sciimmunol.aan2725

    This PDF file includes:

    • Fig. S1. Expression of NFATc1, NFATc2, and NFATc3 in immune cells.
    • Fig. S2. Magnifications of the selected areas shown in Fig. 1C.
    • Fig. S3. Additional histological images of the abscess after C. albicans infection.
    • Fig. S4. Histology of the abscess 1 month after infection of NFATc2-deficient mice.
    • Fig. S5. Granulocyte and monocyte recruitment at the infection site of WT and NFATc2-deficient mice.
    • Fig. S6. Visualization of C. albicans at the infection site 6 to 8 days after infection.
    • Fig. S7. Additional histological images of α-SMA staining in skin sections of WT and NFATc2-deficient mice.
    • Fig. S8. Additional histological images of p-SMAD2,3 staining.
    • Fig. S9. Magnifications of the selected areas shown in Fig. 3D.
    • Fig. S10. Additional histological images of the abscesses after C. albicans infection in the presence of a TGF-β inhibitor.
    • Fig. S11. Additional histological images of IFN-γ staining.
    • Fig. S12. IFN-γ induces capsule digestion.
    • Fig. S13. Additional histological images of the abscesses after C. albicans infection in the presence of PAI-1.
    • Fig. S14. Inhibition of plasmin or MMP-3 interferes with C. albicans elimination.
    • Fig. S15. IFN-γ mRNA is up-regulated in the infected skin of RAG-2–deficient mice.
    • Fig. S16. DCs are necessary for the activation of NK cells in the presence of C. albicans in vitro.
    • Fig. S17. DC depletion after DT injection in DOG mice.
    • Fig. S18. Granulocyte and monocyte recruitment at the infection site of DOG mice treated or not with DT to deplete DCs.
    • Fig. S19. Additional histological images of IFN-γ staining in the infected skin of DOG mice.
    • Fig. S20. IL-12 production by BMDCs after C. albicans stimulation.
    • Fig. S21. Original Western blots shown in Fig. 3A.
    • Fig. S22. Original Western blots shown in Fig. 4 (E and F).
    • Fig. S23. Original Western blots shown in Fig. 5E.
    • Fig. S24. Original Western blots shown in Fig. 7C.
    • Fig. S25. Original Western blots shown in Fig. 7D.
    • Fig. S26. Representative isotype control stainings for immunohistochemical analyses.
    • Fig. S27. Gating strategies used in cytofluorimetric analyses.
    • Fig. S28. Representative isotype control stainings for cytofluorimetric analyses.

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    Other Supplementary Material for this manuscript includes the following:

    • Raw data for Figs. 1 (B, D, and E), 4 (A, C, and G), 5 (A, C, F, G, H, and J), 6 (B to I), and 7A; and figs. S1 (A and B), S5, S14, S15, S16, S17A, S18, and S20 (Microsoft Excel format).

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