Research ArticleNutrition

A high-salt diet compromises antibacterial neutrophil responses through hormonal perturbation

See allHide authors and affiliations

Science Translational Medicine  25 Mar 2020:
Vol. 12, Issue 536, eaay3850
DOI: 10.1126/scitranslmed.aay3850
  • Fig. 1 HSD exacerbates pyelonephritis in mice.

    Mice were fed low-salt diet (LSD), NSD, or HSD for 1 week and infected with UPEC. Eighteen hours after infection, kidneys were analyzed. (A) Quantification of E. coli colony-forming units in kidney homogenates. (B) Representative microscopy pictures of kidney sections infected with E. coli expressing GFP. Dim green, autofluorescence; bright green, E. coli GFP stained with anti-GFP Alexa Fluor 488 antibody; red, CD11b-APC; blue, CD45-V421. Yellow rectangles indicate areas with visible UPEC. (C to E) Flow cytometric quantification of kidney dendritic cells (viable CD45+, MHCII+, and CD11c+) (C), inflammatory monocytes (viable CD45+, Ly6G, and Ly6C+) (D), and neutrophils (viable CD45+, Ly6Cint, and Ly6G+) (E) 18 hours after infection in mice fed NSD or HSD for 1 week. (F) Flow cytometric quantification of reactive oxygen species (ROS) production by kidney neutrophils using the DCFDA dye. (G) Flow cytometric quantification of inducible nitric oxide synthase (iNOS) expression by kidney neutrophils. (H) FACS plot of intracellular E. coli contained in kidney neutrophils. The numbers indicate percentages of neutrophils containing E. coli and geometric mean fluorescence intensities (MFIs). SSC, side scatter. (I) Quantification of bacterial viability in neutrophils isolated from infected kidneys based on gentamicin protection assay and phagocytosis. Live and dead bacteria were measured by flow cytometry (E. coli MFI). Live bacteria were measured by colony-forming units. Both measures were normalized to the average of the NSD group, and normalized live bacterial counts were divided by normalized total bacterial counts. MFI0, MFI of unstained sample; AU, arbitrary units. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA (A) and unpaired Student’s t test (C to I). Bar graphs indicate means and SEM. The experiments were performed at least two times with seven mice per group. Graphs depict pooled experiments.

  • Fig. 2 HSD-induced intrarenal effects.

    Mice were fed NSD or HSD for 1 week, and kidneys were analyzed. (A) RT-PCR quantification of Nfat5 expression relative to Gapdh and control [kidney cortex (Cor) NSD] in kidneys of uninfected mice fed NSD or HSD for 1 week. OMed, outer medulla; IMed, inner medulla. (B) Atomic absorption spectroscopy quantification of sodium content in kidneys of uninfected mice. (C) Flow cytometric quantification of sodium (Asante NaTRIUM Green-2) in kidney neutrophils 18 hours after infection in mice fed NSD or HSD for 1 week. (D) Quantification of bacterial viability in bone marrow neutrophils exposed in vitro to +40 mM NaCl gentamicin protection assay and phagocytosis combined. *P < 0.05 by unpaired Student’s t test (A to D). Bar graphs indicate means and SEM. The experiments were performed at least twice with 5 (A and D), 17 (i.e., kidneys from 17 mice were pooled for one measurement) (B), and 3 (C) mice per group. Graphs depict pooled experiments.

  • Fig. 3 HSD-induced systemic effects.

    Mice were fed NSD or HSD for 1 week, and spleen, bone marrow blood, and kidneys were analyzed. (A) RT-PCR quantification of spleen Nfat5 expression relative to Gapdh and control (NSD) under homeostatic conditions in mice. (B) Volcano plot showing differentially regulated genes in neutrophils isolated from the bone marrow of NSD- or HSD-fed uninfected mice and heatmap showing expression of selected genes in bone marrow neutrophils from HSD-fed mice. GC, glucocorticoid. (C) ELISA quantification of serum corticosterone in uninfected mice fed NSD or HSD for 1 week. (D) INTEGRA clinical analyzer quantification of urea content in kidneys of uninfected mice fed NSD or HSD for 1 week. (E) In silico prediction of glucocorticoid response elements in Nfat5 promoter. ARE, androgen response element; GRE, glucocorticoid response element; PRE, progesterone response element; Ar, androgen receptor; Nr3c1, glucocorticoid receptor; Nr3c2, mineralocorticoid receptor; Pgr, progesterone receptor. (F) RT-PCR quantification of spleen Nfat5, Tsc22d3 (TSC22 domain family protein 3), Per1 (period circadian protein homolog 1), and Sgk1 (serum and glucocorticoid-regulated kinase 1) expression relative to Gapdh and control (NSD + veh) in mice injected subcutaneously for 1 week daily with 60 μg of dexamethasone (dex) or PBS. *P < 0.05 and **P < 0.01 by unpaired Student’s t test. Bar graphs indicate means and SEM. The experiments were performed at least twice with four (A) or five (B to F) mice per group. Graphs depict pooled experiments.

  • Fig. 4 Mechanism of the HSD-mediated glucocorticoid increase.

    Mice were fed NSD or HSD for 1 week, and blood and adrenal glands were analyzed. (A to C) ELISA quantification of serum corticosterone at 9 a.m. and 6 p.m. (A), ACTH (B), and serum angiotensin II (C) in uninfected mice fed HSD and NSD for 1 week. (D) RT-PCR quantification of adrenal gland Cyp11b2 (aldosterone synthase gene) expression in uninfected mice fed HSD and NSD for 1 week. (E) ELISA quantification of serum aldosterone in uninfected mice fed HSD and NSD for 1 week. (F) RT-PCR quantification of adrenal gland Cyp11b2 expression in mice fed HSD and implanted with angiotensin II (AngII) osmotic minipumps (1.25 ng/min per gram) for 1 week. (G and H) ELISA quantification of serum aldosterone (G) and serum corticosterone (H) in mice fed HSD and implanted with angiotensin II osmotic minipumps (1.25 ng/min per gram) for 1 week. *P < 0.05, **P < 0.01, and ***P < 0.001 by unpaired Student’s t test (A to H). Bar graphs indicate means and SEM. The experiments were performed at least twice with five (A to C and E) and six (D and F to H) mice per group. Graphs depict pooled experiments.

  • Fig. 5 Neither HSD-induced aldosterone decrease nor glucocorticoid-induced urea, Nfat5, or glucosuria exacerbate pyelonephritis.

    Mice were fed NSD or HSD for 1 week and infected with UPEC. Eighteen hours after infection, kidneys were analyzed. Additional treatments are indicated below. (A) Quantification of kidney colony-forming units 18 hours after infection in mice treated for 1 week with 50 mg/kg per day of spironolactone (spiro) or 50 μg/kg per day of aldosterone. (B) Flow cytometric quantification of GFP+ UPEC phagocytosis by bone marrow neutrophils exposed to urea concentrations found in the OMed of mice fed NSD (+60 mM) or HSD (+80 mM). (C) In vitro bacterial viability in bone marrow neutrophils exposed to an additional 60 or 80 mM urea. Gentamicin protection assay and phagocytosis combined. (D) Flow cytometry quantification of dead bone marrow neutrophils after exposure to 60 and 80 mM urea for 3 hours in vitro. (E) Flow cytometry quantification of apoptotic and necrotic bone marrow neutrophils after exposure to 600 or 1200 mM urea for 3 hours in vitro. (F to H) Mice have been exposed to HSD for 1 week and injected intraperitoneally with vehicle or 80 mg/kg per day of arginase inhibitor N-ω-hydroxy-l-norarginine (NOHA). (F) Quantification of colony-forming units in kidney homogenates 18 hours after infection. (G) Flow cytometric quantification of kidney neutrophils (viable CD45+, Ly6Cint, and Ly6G+). (H) Quantification of bacterial viability in neutrophils isolated from infected kidneys based on gentamicin protection assay and phagocytosis. (I to K) Mice were exposed to NSD or HSD for 1 week and injected subcutaneously twice with 2 mg/kg of unspecific (Neg1) or Nfat5-targeting (iNFAT5) antisense oligonucleotides. (I) Quantification of kidney colony-forming units 18 hours after infection. (J) Flow cytometric quantification of kidney neutrophils. (K) Quantification of bacterial viability in neutrophils isolated from infected kidneys based on gentamicin protection assay and phagocytosis. (L) Absorbance quantification (wavelength, 570 nm) of UPEC growth in urine collected from mice fed NSD or HSD for 1 week. *P < 0.05 and ***P < 0.001 by one-way ANOVA (A, E, and I to K) and unpaired Student’s t test (B to D, F to H, and L). Bar graphs indicate means and SEM. The experiments were performed at least twice with five (A and L), four (B to D), three (E), six (F to H), and seven (I to K) mice per group. Graphs depict pooled experiments.

  • Fig. 6 HSD-induced hyperglucocorticoidism suppresses the defense against pyelonephritis and listeriosis.

    Mice were fed NSD or HSD for 1 week and analyzed or infected with UPEC or L. monocytogenes and analyzed. Additional treatments are indicated below. (A) Quantification of in vitro bacterial viability in bone marrow neutrophils exposed to different dexamethasone concentrations. Gentamicin protection assay and phagocytosis combined. (B) Quantification of kidney colony-forming units 18 hours after infection in mice injected subcutaneously for 1 week daily with 60 μg of dexamethasone or vehicle. (C) Quantification of bacterial viability in neutrophils isolated from infected kidneys based on gentamicin protection assay and phagocytosis combined. (D) Quantification of kidney colony-forming units 18 hours after infection in mice injected subcutaneously for 1 week daily with 1 mg of mifepristone (mif) or vehicle. (E) Quantification of bacterial viability in neutrophils isolated from infected kidneys based on gentamicin protection assay and phagocytosis combined. (F) Quantification of in vitro bacterial viability in blood neutrophils from uninfected mice fed NSD or HSD for 1 week. Gentamicin protection assay and phagocytosis combined. (G) Gene set enrichment analysis (GSEA) plot for gene ontology (GO) biological process phagosome maturation in neutrophils isolated from the bone marrow of NSD- or HSD-fed uninfected mice. NES, normalized enrichment score; FDR, false discovery rate. (H) Quantification of in vitro phagosomal pH in blood neutrophils from uninfected mice fed NSD or HSD for 1 week. (I) Quantification of spleen colony forming units 3 days after L. monocytogenes infection [5 × 104 per mouse intraperitoneally (i.p.)]. Mice were fed NSD or HSD for 1 week. (J) Quantification of bacterial viability in neutrophils isolated from infected spleens based on gentamicin protection assay and phagocytosis combined. (K) Quantification of spleen colony-forming units 3 days after L. monocytogenes infection (5 × 104 per mouse i.p.). Mice were fed HSD and injected subcutaneously for 1 week with 1 mg of mifepristone daily. (L) Quantification of bacterial viability in neutrophils isolated from infected spleens based on gentamicin protection assay and phagocytosis combined. *P < 0.05, **P < 0.01, and ***P < 0.001 by unpaired Student’s t test (A to L). Bar graphs indicate means and SEM. The experiments were performed at least twice with five (A and H to L), seven (B to E), and three (F) mice per group. Graphs depict pooled experiments.

  • Fig. 7 An HSD causes hyperglucocorticoidism and worsens ex vivo antibacterial neutrophil function in humans.

    Analysis of urine and plasma values of 10 healthy human volunteers that consumed 6 g/day of NaCl for 1 week in addition to their normal diet. Quantification of sodium content in morning urine (A), aldosterone (B), and corticosterone (C) in plasma collected in the evening, numbers of blood neutrophils (live, CD16+, and CD15+ cells). (D) Quantification of in vitro bacterial viability in blood neutrophils. (E) Gentamicin protection assay and phagocytosis combined. *P < 0.05 paired Student’s t test. Dots indicate each of the 10 individual human volunteers.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/536/eaay3850/DC1

    Fig. S1. Influence of different salt concentrations on UPEC growth and measurement of urine excretion.

    Fig. S2. No influence of an HSD on intrarenal cytokine production and neutrophil numbers.

    Fig. S3. Sodium staining in kidney neutrophils.

    Fig. S4. High salt effect on macrophages and T cells.

    Fig. S5. HSD effect on steroid biosynthesis and urea effect on Nfat5.

    Fig. S6. Effect of urea on macrophage function.

    Fig. S7. Antisense oligonucleotide–mediated NFAT5 knockdown in the kidney.

    Fig. S8. An HSD does not cause major changes in serum and urine glucose concentrations.

    Fig. S9. Effect of dexamethasone on macrophage function.

    Fig. S10. Measuring intraphagosomal pH.

    Fig. S11. An HSD impairs the defense against Listeria in the liver.

    Fig. S12. Epidemiologic correlation between human salt intake and UTI incidence in various countries.

    Fig. S13. Mechanism of HSD-mediated glucocorticoid increase and pyelonephritis/listeriosis exacerbation.

    Fig. S14. Validation of the method to measure UPEC phagocytosis.

    Table S1. GO analysis of genes down-regulated in bone marrow neutrophils upon HSD exposure.

    Data file S1. Primary data.

  • The PDF file includes:

    • Fig. S1. Influence of different salt concentrations on UPEC growth and measurement of urine excretion.
    • Fig. S2. No influence of an HSD on intrarenal cytokine production and neutrophil numbers.
    • Fig. S3. Sodium staining in kidney neutrophils.
    • Fig. S4. High salt effect on macrophages and T cells.
    • Fig. S5. HSD effect on steroid biosynthesis and urea effect on Nfat5.
    • Fig. S6. Effect of urea on macrophage function.
    • Fig. S7. Antisense oligonucleatide-mediated NFAT5 knockdown in the kidney.
    • Fig. S8. An HSD does not cause major changes in serum and urine glucose concentrations.
    • Fig. S9. Effect of dexamethasone on macrophage function.
    • Fig. S10. Measuring intraphagosomal pH.
    • Fig. S11. An HSD impairs the defense against Listeria in the liver.
    • Fig. S12. Epidemiologic correlation between human salt intake and UTI incidence in various countries.
    • Fig. S13. Mechanism of HSD-mediated glucocorticoid increase and pyelonephritis/listeriosis exacerbation.
    • Fig. S14. Validation of the method to measure UPEC phagocytosis.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). GO analysis of genes down-regulated in bone marrow neutrophils upon HSD exposure.
    • Data file S1 (Microsoft Excel format). Primary data.

Stay Connected to Science Translational Medicine

Navigate This Article