Research ArticleInfectious Disease

CXCR1-mediated neutrophil degranulation and fungal killing promote Candida clearance and host survival

See allHide authors and affiliations

Science Translational Medicine  20 Jan 2016:
Vol. 8, Issue 322, pp. 322ra10
DOI: 10.1126/scitranslmed.aac7718
  • Fig. 1. Systemic candidiasis induces the expression of Cxcr1 and its ligand Cxcl5.

    (A) Cxcr1 is induced in Cxcr1+/+ kidneys at days 1, 4, and 7 after infection. P = 0.0023, P = 0.0012, and P = 0.0022 for Cxcr1 expression at days 1, 4, and 7 versus day 0, respectively (n = 5 to 8; two independent experiments; Mann-Whitney test). (B) Cxcl5 is induced in Cxcr1+/+ kidneys at days 1, 4, and 7 after infection. P = 0.01, P = 0.0016, and P = 0.0004 for Cxcl5 expression at days 1, 4, and 7 versus day 0, respectively (n = 5 to 8; two independent experiments; t test). (C) Cxcl5 is significantly induced at the protein level in Cxcr1+/+ kidneys after infection (n = 6 to 8; two independent experiments). **P = 0.0012; Mann-Whitney test; ***P = 0.0003; t test with Welch’s correction. Data represent means ± SEM.

  • Fig. 2. Systemic candidiasis results in increased mortality in Cxcr1−/− mice due to enhanced fungal proliferation in the kidney and renal failure.

    (A) Schematic showing the generation of Cxcr1−/− mice by homologous recombination. WT, wild-type. (B) Mortality rates of Cxcr1+/+ and Cxcr1−/− mice after intravenous challenge with Candida (n = 25; summary data of three independent experiments). P = 0.015; log-rank (Mantel-Cox) test. (C) Fungal burden in the kidneys of Cxcr1+/+ and Cxcr1−/− mice at days 4 (P = 0.0135; n = 14 to 15; three independent experiments; t test with Welch’s correction) and 7 (P = 0.025; n = 30 to 31; six independent experiments; Mann-Whitney test) after infection. (D) Renal function is significantly compromised in Cxcr1−/− mice after infection. Shown are summary data of serum blood urea nitrogen (BUN) (P = 0.0477; Mann-Whitney test) and creatinine (P = 0.0403; Mann-Whitney test) at day 7 after infection (n = 17 to 18; three independent experiments). (E) Histopathology. Representative periodic acid–Schiff (PAS) staining of Cxcr1+/+ and Cxcr1−/− kidney sections at day 4 after infection. Original magnification, ×20; Scale bars, 500 μm (n = 15; three independent experiments). All quantitative data represent means ± SEM.

  • Fig. 3. Neutrophils are the major Cxcr1-expressing immune cells in the kidney during systemic candidiasis, whereas Cxcl5 is evenly expressed among various immune cell types.

    (A and B) Relative expression of Cxcr1 (A) and Cxcl5 (B) in resident macrophages (day 0) and in neutrophils, monocytes, inflammatory macrophages, and T cells (day 4 after infection) FACS-sorted from Cxcr1+/+ kidneys (n = 3 to 6; two independent experiments). ****P < 0.0001 for neutrophil Cxcr1 expression relative to other immune cells by one-way analysis of variance (ANOVA) with Bonferroni correction. Data represent means ± SEM.

  • Fig. 4. Cxcr1 does not mediate neutrophil trafficking in the kidney during systemic candidiasis.

    (A and B) Accumulation of neutrophils in Cxcr1+/+ and Cxcr1−/− kidneys at days 1, 4, and 7 after infection. (A) Number of neutrophils. (B) Percent of neutrophils within total CD45+ leukocytes (n = 6 to 15; two to four independent experiments). (C) Induction of neutrophil-targeted CXC chemokines in Cxcr1+/+ and Cxcr1−/− kidneys after Candida infection (n = 6 to 8; two independent experiments). (D and E) Ratio of CD45.1+Cxcr1+/+ and CD45.2+Cxcr1−/− neutrophils in the blood before infection and in the blood and kidney at days 1 (D) and 4 (E) after Candida infection of mixed bone marrow radiation chimeras (n = 6; two independent experiments). Data represent means ± SEM.

  • Fig. 5. Cxcr1 deficiency impairs the killing capacity of kidney neutrophils against Candida and results in decreased hyphal damage in the infected kidney.

    (A) Representative PAS staining of Cxcr1+/+ and Cxcr1−/− kidney sections (day 4 after infection) showing degraded hyphal elements within Cxcr1+/+ neutrophil abscesses but intact hyphae within Cxcr1−/− neutrophil abscesses. Original magnification, ×400. Scale bars, 100 μm. (B)Candida hyphal length within Cxcr1+/+ and Cxcr1−/− renal neutrophil abscesses at day 4 after infection (n = 452 to 466 randomly selected hyphal element measurements obtained from 12 to 13 kidneys per genotype; summary data from three independent experiments). ****P < 0.0001; Mann-Whitney test. (C) Cxcr1−/− neutrophils MACS-sorted from the kidney at day 4 after infection have impaired ability to damage opsonized (***P = 0.0009) and unopsonized (**P = 0.0034) Candida hyphae ex vivo (n = 8; four independent experiments; t test). All quantitative data represent means ± SEM.

  • Fig. 6. Cxcr1−/− neutrophils exhibit cell-intrinsic defects in degranulation and non-oxidative Candida killing but no impaired survival, maturation, or phagocytosis.

    (A) Neutrophil survival. Percentage of live, apoptotic, and dead neutrophils in the kidney at day 4 after infection determined by annexin V and 7-AAD FACS staining (n = 8; two independent experiments). (B) Neutrophil maturation. Mean fluorescence intensity of the maturation markers Ly6G, CD11b, and CD18 expressed on the surface of kidney neutrophils at day 4 after infection (n = 4 to 18; two to five independent experiments). (C) Neutrophil phagocytosis. Cxcr1+/+ and Cxcr1−/− neutrophils isolated from uninfected bone marrows have similar capacity to internalize dTomato-expressing C. albicans (n = 4; two independent experiments). (D) Neutrophil oxidative burst. Mean fluorescence intensity of rhodamine 123 measured by FACS in Cxcr1+/+ and Cxcr1−/− neutrophils MACS-sorted from the kidney at day 4 after infection with or without additional ex vivo stimulation with PMA (phorbol 12-myristate 13-acetate) (n = 4; two independent experiments). (E) Neutrophil degranulation. β-Methylumbelliferone (MUB) amount measured as a marker of release of granule β-glucuronidase in Cxcr1+/+ and Cxcr1−/− neutrophils MACS-sorted from the kidney at day 4 after infection (P = 0.004; n = 13; four independent experiments; Mann-Whitney test). (F) Neutrophil MPO content. Mean fluorescence intensity of MPO assessed by intracellular FACS in Cxcr1+/+ and Cxcr1−/− kidney neutrophils at day 4 after infection (P = 0.0202; n = 15; four independent experiments; t test). (G) Neutrophil MPO release. Release of granule MPO from Cxcr1+/+ and Cxcr1−/− neutrophils MACS-sorted from the kidney at day 4 after infection after ex vivo infection with opsonized (P = 0.0308; t test) or unopsonized (P = 0.0384; Mann-Whitney test) Candida hyphae (n = 17; four independent experiments). (H to J) Cxcr1−/− neutrophils isolated from uninfected bone marrows exhibit defects in Candida hyphal killing (P = 0.0032; n = 12; six independent experiments; t test with Welch’s correction for opsonized hyphae; and P = 0.0021; n = 9; five independent experiments; t test with Welch’s correction for unopsonized hyphae) (H), degranulation capacity measured as β-glucuronidase release (P = 0.0033; n = 6; three independent experiments; t test) (I), and intracellular MPO content (P = 0.0207; n = 8; four independent experiments; Mann-Whitney test) (J). Data represent means ± SEM.

  • Fig. 7. The mutant CXCR1-T276 allele is associated with impaired fungal killing and degranulation in human neutrophils.

    (A) Healthy donors carrying the CXCR1-T276 allele have similar absolute neutrophil counts with CXCR1-WT donors (n = 5 to 11). (B to E) Neutrophils of healthy donors carrying the CXCR1-T276 allele exhibit impaired ability to damage opsonized and unopsonized Candida yeast (B) (P = 0.0067 for opsonized and P = 0.0332 for unopsonized yeast; n = 5 to 6; five independent experiments; t test with Welch’s correction) and hyphae (C) (P = 0.0023 for opsonized and P = 0.0012 for unopsonized hyphae; n = 6 to 7; five independent experiments; Mann-Whitney test) ex vivo, defective degranulation as measured by release of β-glucuronidase (P = 0.0381; n = 4 to 6; three independent experiments; Mann-Whitney test) (D), and decreased intracellular MPO content (P = 0.0012; n = 5 to 6; three independent experiments; t test) (E).

  • Table 1. Association of the mutant CXCR1-T276 allele with development of disseminated candidiasis after candidemia in patients of mixed European descent (n = 153).

    ANC, absolute neutrophil count.

    VariableUnivariate analysis
    P value
    Multivariate analysis
    OR (95% CI)
    CXCR1-T276 allele (CG + GG)0.04553.95 (1.20–13.03)
    Male sex0.5264
    Immunocompromised state0.2882
    Hematopoietic stem cell transplantation0.5845*
    Solid organ transplantation0.01024.03 (1.46–11.17)
    Active malignancy0.3656
    Solid tumor0.5967
    Leukemia1.0000*
    Lymphoma0.3124*
    Chemotherapy within the past 3 months0.6836
    Neutropenia (ANC < 500 cells/mm3)0.4837
    Surgery within the past 30 days0.102
    Receipt of total parenteral nutrition0.08913.19 (1.19–8.21)
    Dialysis-dependent1.0000*
    Acute renal failure0.2652
    Liver disease0.1551
    Intensive care unit admission within the past 14 days0.5278

    *Fisher’s exact test.

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/8/322/322ra10/DC1

      Methods

      Fig. S1. Cxcr1 deficiency does not result in developmental renal defects.

      Fig. S2. Cxcl5 promotes dose-dependent Cxcr1-mediated calcium flux in mouse neutrophils.

      Fig. S3. Cxcl1 and Cxcl2 do not promote significant Cxcr1-dependent calcium flux in mouse neutrophils.

      Fig. S4. Systemic candidiasis results in increased renal tissue injury in Cxcr1−/− mice.

      Fig. S5. Cxcr1 deficiency does not impair fungal clearance in the liver, spleen, or brain after Candida infection.

      Fig. S6. Cxcr1 deficiency does not result in histological abnormalities in the liver, brain, or spleen after Candida infection.

      Fig. S7. Gating strategy for FACS sorting of hematopoietic cells from kidney.

      Fig. S8. Kidney neutrophils express Cxcr1 and Cxcl5 at steady state and after systemic candidiasis.

      Fig. S9. Cxcr1 deficiency does not affect the production of neutrophils in the bone marrow after Candida infection.

      Fig. S10. Cxcr1 deficiency does not affect the accumulation of neutrophils in the blood after Candida infection.

      Fig. S11. Cxcr1 deficiency does not impair the induction of Cxcl5 in the Candida-infected kidney.

      Fig. S12. Cxcr1 deficiency does not impair the accumulation of mononuclear phagocytes in the kidney after Candida infection.

      Fig. S13. Cxcr1 does not impair the induction of proinflammatory cytokines and chemokines in the kidney after Candida infection.

      Fig. S14. Cxcr2 is expressed on blood and kidney neutrophils of Cxcr1+/+ and Cxcr1−/− mice at steady state and during systemic candidiasis.

      Fig. S15. A selective Cxcr2 antagonist does not decrease neutrophil accumulation in the Candida-infected kidney of Cxcr1−/− mice.

      Fig. S16. Cxcr1 deficiency results in a cell-intrinsic defect in killing of Candida yeast forms.

      Fig. S17. Cxcr1 deficiency does not impair the formation of neutrophil extracellular traps ex vivo.

      Fig. S18. Cxcr1 deficiency does not impair the accumulation of NK cells or the induction of Il23a and Gmcsf in the kidney after Candida infection.

      Fig. S19. Cxcr1 deficiency does not impair the production of reactive oxygen species by bone marrow neutrophils at steady state.

      Table S1. Demographic and clinical characteristics of the candidemic and control subjects enrolled in the present study.

      Table S2. Association of the mutant CXCR1-T276 allele with susceptibility to systemic candidiasis in subjects of mixed European descent (n = 304).

      Table S3. Association of the mutant CXCR1-T276 allele with development of persistent fungemia in candidemic patients of mixed European descent (n = 153).

      Data File 1. Excel file with all data values in tabular format.

      References (3235)

    • Supplementary Material for:

      CXCR1-mediated neutrophil degranulation and fungal killing promote Candida clearance and host survival

      Muthulekha Swamydas, Ji-Liang Gao, Timothy J. Break, Melissa D. Johnson, Martin Jaeger, Carlos A. Rodriguez, Jean K. Lim, Nathaniel M. Green, Amanda L. Collar, Brett G. Fischer, Chyi-Chia Richard Lee, John R. Perfect, Barbara D. Alexander, Bart-Jan Kullberg, Mihai G. Netea, Philip M. Murphy, Michail S. Lionakis*

      *Corresponding author. E-mail: lionakism{at}niaid.nih.gov

      Published 20 January 2016, Sci. Transl. Med. 8, 322ra10 (2016)
      DOI: 10.1126/scitranslmed.aac7718

      This PDF file includes:

      • Methods
      • Fig. S1. Cxcr1 deficiency does not result in developmental renal defects.
      • Fig. S2. Cxcl5 promotes dose-dependent Cxcr1-mediated calcium flux in mouse neutrophils.
      • Fig. S3. Cxcl1 and Cxcl2 do not promote significant Cxcr1-dependent calcium flux in mouse neutrophils.
      • Fig. S4. Systemic candidiasis results in increased renal tissue injury in Cxcr1−/− mice.
      • Fig. S5. Cxcr1 deficiency does not impair fungal clearance in the liver, spleen, or brain after Candida infection.
      • Fig. S6. Cxcr1 deficiency does not result in histological abnormalities in the liver, brain, or spleen after Candida infection.
      • Fig. S7. Gating strategy for FACS sorting of hematopoietic cells from kidney.
      • Fig. S8. Kidney neutrophils express Cxcr1 and Cxcl5 at steady state and after systemic candidiasis.
      • Fig. S9. Cxcr1 deficiency does not affect the production of neutrophils in the bone marrow after Candida infection.
      • Fig. S10. Cxcr1 deficiency does not affect the accumulation of neutrophils in the blood after Candida infection.
      • Fig. S11. Cxcr1 deficiency does not impair the induction of Cxcl5 in the Candida-infected kidney.
      • Fig. S12. Cxcr1 deficiency does not impair the accumulation of mononuclear phagocytes in the kidney after Candida infection.
      • Fig. S13. Cxcr1 does not impair the induction of proinflammatory cytokines and chemokines in the kidney after Candida infection.
      • Fig. S14. Cxcr2 is expressed on blood and kidney neutrophils of Cxcr1+/+ and Cxcr1−/− mice at steady state and during systemic candidiasis.
      • Fig. S15. A selective Cxcr2 antagonist does not decrease neutrophil accumulation in the Candida-infected kidney of Cxcr1−/− mice.
      • Fig. S16. Cxcr1 deficiency results in a cell-intrinsic defect in killing of Candida yeast forms.
      • Fig. S17. Cxcr1 deficiency does not impair the formation of neutrophil extracellular traps ex vivo.
      • Fig. S18. Cxcr1 deficiency does not impair the accumulation of NK cells or the induction of Il23a and Gmcsf in the kidney after Candida infection.
      • Fig. S19. Cxcr1 deficiency does not impair the production of reactive oxygen species by bone marrow neutrophils at steady state.
      • Table S1. Demographic and clinical characteristics of the candidemic and control subjects enrolled in the present study.
      • Table S2. Association of the mutant CXCR1-T276 allele with susceptibility to systemic candidiasis in subjects of mixed European descent (n = 304).
      • Table S3. Association of the mutant CXCR1-T276 allele with development of persistent fungemia in candidemic patients of mixed European descent (n = 153).
      • References (3235)

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Data File 1. Excel file with all data values in tabular format.

    Navigate This Article