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Sodium chloride is an ionic checkpoint for human TH2 cells and shapes the atopic skin microenvironment

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Science Translational Medicine  20 Feb 2019:
Vol. 11, Issue 480, eaau0683
DOI: 10.1126/scitranslmed.aau0683
  • Fig. 1 NaCl enhances TH2 and suppresses TH1 cell responses in memory T cells.

    (A to D) Human memory TH cells were sorted from fresh peripheral blood mononuclear cells (PBMCs) as CD4+CD14CD45RA T cells by flow cytometry and stimulated for a total culture period of 5 days in the presence (high) or absence (low) of additional 50 mM NaCl with anti-CD3 and anti-CD28 monoclonal antibody (mAb) for 48 hours. (A) Intracellular staining and flow cytometry [fluorescence-activated cell sorter (FACS)] on day 5 after phorbol 12-myristate 13-acetate (PMA) and ionomycin restimulation. FACS staining of an individual experiment (left) and cumulative data is shown, with each circle representing one donor (IL-17, n = 18; IL-4, n = 17; IL-13, n = 11; IFN-γ, n = 22). (B) ELISA of cell culture supernatants analyzed on day 5 (n = 3). (C) Flow cytometry and ELISA of T cells stimulated as in (A) and (B) and treated in IL-4 or IL-12 cytokine microenvironments (FACS panels are representative of three experiments; ELISA, n = 3). P = 0.007 (IL-4) and P = 0.003 (IFN-γ), Kruskal-Wallis test. Dunn’s multiple comparisons post hoc test was performed for comparisons between IL-4 and high NaCl. (D) IL-12RB expression as shown by FACS. One representative experiment and cumulative data (n = 5) are shown. (E) Skin CD3+ T cells obtained after abdominoplasties were sorted and characterized by flow cytometry according to fig. S4 and stimulated and analyzed as in (A) (n = 3). Paired Student’s t tests were used for comparisons between two groups. n.s., not significant.

  • Fig. 2 NaCl induces the transcriptional activation of TH2 and suppression of TH1 programs.

    (A) Human memory T cells were stimulated with anti-CD3 and anti-CD28 mAb for 48 hours of a 5-day culture period in the presence (high) or absence (low) of additional 50 mM NaCl before intracellular staining for transcription factors on day 5. Representative flow cytometry analyses (left) and cumulative data (right) are shown, with each circle indicating one donor and experiment (GATA-3, n = 4; T-bet, n = 3). MFI, mean fluorescence intensity. (B) T cell clones were generated from CD4+ CD45RA memory T cells during a 14-day culture period with irradiated allogeneic feeder cells and phytohemagglutinin and were restimulated and analyzed as in (A). T cell clones were randomly selected from growing cultures for restimulation experiments. Each circle represents an individual T cell clone (GATA-3, n = 14; T-bet, n = 24; ROR-γt, n = 24). Paired Student’s t test was used for comparison between two groups (A and B). (C) Memory T cells were stimulated as in (A) and (B) in low or high NaCl conditions or in the presence of recombinant polarizing cytokines in low NaCl conditions for 5 days before restimulation for 30 min with IL-4, IL-12, and IL-6. Phosphorylation of STAT molecules was assessed after intracellular staining and flow cytometry (n = 3). P = 0.025 (p-STAT6), P = 0.004 (p-STAT4), and P = 0.01 (p-STAT3), Kruskal-Wallis test.

  • Fig. 3 NaCl reprograms distinct TH cell subsets to acquire TH2 properties.

    (A) Polarized human memory TH cell subsets were sorted ex vivo according to the differential expression of chemokine receptor surface markers and restimulated in the presence (high) or absence (low) of additional 50 mM NaCl with CD3 and CD28 mAb for 48 hours of a 5-day culture period. Cytokine expression was determined by intracellular cytokine staining and flow cytometry. Paired Student’s t test was used for comparison between two groups. (B) Random T cell clones that were generated from memory T cells were stimulated as in (A), and their cytokine coexpression pattern was measured in the presence of low versus high NaCl concentrations. Every circle represents a unique T cell clone. (C) Individual T cell clones that were generated from memory T cells were selected on the basis of being negative for IL-4 or IL-17 expression. They were restimulated in the presence of low versus high NaCl concentrations for 5 days before intracellular staining. Two individual clones from one experiment and blood donor (n = 3) are shown. (D) Intracellular cytokine straining of human CD4+ CD45RA memory T cells after stimulation as in (A). The same T cell cultures were then split and restimulated for another 5 days in low and high NaCl conditions before intracellular cytokine staining and flow cytometric analysis following PMA and ionomycin stimulation on day 10 (n = 4). Paired Student’s t test was used for comparison between two groups. (E) Heatmap with average CpG methylation as measured by deep sequencing of bisulfite PCR amplicons for indicated regions (p, promoter; I, intron; e, exon; numbers in brackets indicate amplicons for different subregions of the respective genes; D1 to D3, donors 1 to 3; comparison of matched low versus high NaCl conditions for D1 to D3 by Student’s t test with false discovery rate–adjusted P values).

  • Fig. 4 NaCl promotes TH2 cell differentiation from naïve T cell precursors in the absence of polarizing cytokines.

    (A) Cytokine expression was determined by flow cytometry after PMA and ionomycin restimulation for 5 hours. Left: Cytokine expression in one individual blood donor. Right: Cumulative data of all donors. (B) Supernatants from the cultures in (A) were tested by ELISA and normalized to cell numbers by counting beads. (C) Transcription factor expression was determined by flow cytometry after treatment as in (A) and (B). Each circle represents a separate blood donor. Paired Student’s t test was used for comparison between two groups. gMFI, geometric MFI.

  • Fig. 5 SGK-1 and NFAT5 mechanistically link salt signaling with the TH2 program.

    (A) qRT-PCR of memory TH cells. Paired Student’s t test was used for comparison between two groups. A.U., arbitrary units. (B) Cytokine production in supernatants was determined by ELISA after stimulation in low- or high-salt conditions for 5 days and shRNA-mediated silencing of SGK1 or NFAT5 (n = 3 to 6). The control conditions indicated as (−) contain scrambled shRNA. For IL-4 and IFN-γ, P ≤ 0.05 (mixed-effects analysis for multiple comparisons). Unpaired Student’s t test was used for comparison between two groups. (C) Intracellular staining and flow cytometry of the master transcription factor GATA-3 (P ≤ 0.05, Kruskal-Wallis test) and T-bet (P = 0.12, Kruskal-Wallis test) after stimulation in low- or high-salt conditions for 5 days and shRNA-mediated silencing of NFAT5 and SGK1. Dunn’s post hoc test was not significant (n.s.) for comparison between low NaCl conditions with scrambled shRNA and high NaCl conditions with shRNA for NFAT5 or SGK1. *P ≤ 0.05, Dunn’s post hoc test for comparison with low NaCl conditions containing scrambled shRNA.

  • Fig. 6 The skin of atopic dermatitis patients has elevated sodium content.

    (A) NAA of 4-mm skin punch biopsies. The punch biopsies were taken from lesional and nonlesional skin of atopic dermatitis patients and of healthy controls (patients undergoing plastic surgery) (table S2, patient information). The concentration of sodium is shown (mean ± SEM). The right panel illustrates the clinical presentation of a representative patient with atopic dermatitis. (B) NAA from lesional and nonlesional skin of psoriasis patients. Right: Clinical presentation of a representative patient with psoriasis. (C) Representative spectrum of gamma quanta emission. The spectrum has been recorded after a thermal neutron irradiation with 4.3 × 1016 cm−2 and a counting time of 30 min 1 day after the end of exposure. Unpaired Student’s t test was used for comparison between two groups.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/11/480/eaau0683/DC1

    Materials and Methods

    Fig. S1. The viability of human T cells is preserved over a wide range of NaCl concentrations.

    Fig. S2. TH17 cell signature molecules are up-regulated upon stimulation of memory TH cells with NaCl.

    Fig. S3. NaCl increases memory TH2 cell responses independently of exogenous or autocrine IL-4.

    Fig. S4. NaCl induces up-regulation of IL-4, IL-17, as well as IL-4 and IL-17 double-positive T cells.

    Fig. S5. NaCl enhances TH2 and suppresses TH1 cell responses in TH cell clones.

    Fig. S6. Comparison of different osmolytes demonstrates NaCl to be the most potent inducer of IL-4 and suppressor of IFN-γ in memory T cells.

    Fig. S7. Memory CD8 T cells show stable IL-4 and IFN-γ expression upon stimulation with NaCl but up-regulate IL-17.

    Fig. S8. Skin T cells are distinct from blood T cells and differ in their expression of the tissue residency markers CD69 and CD103.

    Fig. S9. NaCl induces the up-regulation of the skin-homing chemokine receptor CCR8, which enriches for TH2-associated cytokines.

    Fig. S10. Transcriptome-wide enrichment of a TH2 cell signature by NaCl.

    Fig. S11. NaCl induces TH2 cell polarization independently of exogenous or autocrine IL-4.

    Fig. S12. TH17-polarizing cytokine conditions abrogate the TH2-promoting effect of NaCl.

    Fig. S13. Mouse T cells differentiate into TH2 cells in response to NaCl.

    Fig. S14. SGK-1 is a downstream target of NFAT5.

    Fig. S15. GATA-3 and T-bet are not regulated by NFAT5 and SGK-1 in low NaCl conditions.

    Fig. S16. Model.

    Table S1. Selected regions and primer sequences used for DNA methylation analyses.

    Table S2. Patient information.

    Data file S1. Primary data.

    References (5054)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. The viability of human T cells is preserved over a wide range of NaCl concentrations.
    • Fig. S2. TH17 cell signature molecules are up-regulated upon stimulation of memory TH cells with NaCl.
    • Fig. S3. NaCl increases memory TH2 cell responses independently of exogenous or autocrine IL-4.
    • Fig. S4. NaCl induces up-regulation of IL-4, IL-17, as well as IL-4 and IL-17 double-positive T cells.
    • Fig. S5. NaCl enhances TH2 and suppresses TH1 cell responses in TH cell clones.
    • Fig. S6. Comparison of different osmolytes demonstrates NaCl to be the most potent inducer of IL-4 and suppressor of IFN-γ in memory T cells.
    • Fig. S7. Memory CD8 T cells show stable IL-4 and IFN-γ expression upon stimulation with NaCl but up-regulate IL-17.
    • Fig. S8. Skin T cells are distinct from blood T cells and differ in their expression of the tissue residency markers CD69 and CD103.
    • Fig. S9. NaCl induces the up-regulation of the skin-homing chemokine receptor CCR8, which enriches for TH2-associated cytokines.
    • Fig. S10. Transcriptome-wide enrichment of a TH2 cell signature by NaCl.
    • Fig. S11. NaCl induces TH2 cell polarization independently of exogenous or autocrine IL-4.
    • Fig. S12. TH17-polarizing cytokine conditions abrogate the TH2-promoting effect of NaCl.
    • Fig. S13. Mouse T cells differentiate into TH2 cells in response to NaCl.
    • Fig. S14. SGK-1 is a downstream target of NFAT5.
    • Fig. S15. GATA-3 and T-bet are not regulated by NFAT5 and SGK-1 in low NaCl conditions.
    • Fig. S16. Model.
    • Table S1. Selected regions and primer sequences used for DNA methylation analyses.
    • Table S2. Patient information.
    • References (5054)

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    Other Supplementary Material for this manuscript includes the following:

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