Research ArticleAutoimmunity

Kidney tissue hypoxia dictates T cell–mediated injury in murine lupus nephritis

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Science Translational Medicine  08 Apr 2020:
Vol. 12, Issue 538, eaay1620
DOI: 10.1126/scitranslmed.aay1620
  • Fig. 1 Renal-infiltrating T cells are located in areas of hypoxia.

    (A and B) Gene Set Enrichment Analysis plots comparing gene signatures of renal-infiltrating CD4+ and CD8+ T cells, to splenic to CD4+ and CD8+ T cells, respectively, on the basis of the hypoxia signatures generated by comparing triple PHD (prolyl-4-hydroxylase domain proteins)–knockout (TKO) CD4+ to wild-type (WT) CD4+ T cells, and comparing VHL (Von-Hippel Lindau) tumor suppressor knockout (KO) CD8+ T cells to lymphocytic choriomenigitis virus–specific P14 T cell receptor transgenic CD8+ T cells taken from virally infected mice. (C) Representative confocal microscopy of lymphocytic aggregates of CD4+ (blue) and CD8+ T cells (magenta) with HIF-1α nuclear staining (red) located in regions of hypoxia (pimonidazole, green). (D) Summary of nuclear HIF-1α and pimonidazole staining and combined staining of CD4+ and CD8+ T cells within renal lymphocytic aggregates. (E and F) Representative data and summary of pimonidazole and HIF-1α staining of activated (CD44hi) splenic versus renal CD4+ and CD8+ T cells isolated from kidneys of 16- to 18-week-old MRL/lpr mice (n = 4 and 8, respectively). MFI, mean fluorescence intensity. (G and H) Representative data of pimonidazole and HIF-1α staining in kidney versus spleen CD4+ (G) and CD8+ T cells (H) as in (E) and (F). Representative of three experiments, n = 4 to 8 animals per group. Data shown are means ± SD; statistical analysis by two-tailed paired t test (E and F). **P < 0.01 and ***P < 0.001.

  • Fig. 2 Renal T cells survive in hypoxia through Bnip3 alternative splicing mediated by HIF-1–dependent PDK2.

    (A and B) Representative data and summary of PDK2 (A) and BNIP3 (B) expression in activated (CD44hi) splenic versus renal CD4+ and CD8+ T cells isolated from kidneys of 16- to 18-week-old MRL/lpr mice (n = 6 and 7, respectively). (C and D) Quantification of two forms of Bnip3 transcripts, full length (Bnip3FL) and exon 3 deleted (Bnip3Δex3) in CD4+ (C) and CD8+ T cells (D) (n = 8). (E) Binding of HIF-1α to Bnip3 and Pdk2 promoter regions, as determined by chromatin immunoprecipitation (ChIP) and real-time quantitative PCR (qPCR). IgG, immunoglobulin G. (F to H) Mean fluorescence intensity of HIF-1α (F), BNIP3 (G), and PDK2 (H) in TH1-activated CD4+ T cells transduced with empty vector (EV) or two different constructs of Hif1α knockdowns after 2 days in hypoxic cultures. (I) Ratio of Bnip3FL and Bnip3Δex3 mRNAs in TH1-activated CD4+ T cells transduced with either EV or knockdown constructs targeting Hif1a, Pdk2, Bnip3FL, or Bnip3Δex3 1 day after adding DMOG. (J) Percentage of MitoTracker Deep Redlo annexin V+ cells of TH1-activated CD4+ T cells transduced with knockdown vectors after 3 days of hypoxic culture. Data shown are means ± SD; statistical analysis by two-tailed paired t test (A to E) and unpaired t test (F to J). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 3 T cell effector function in hypoxia is mediated by HIF-1–regulated proline metabolism facilitating glycolysis.

    (A) RNA-sequencing gene expression (mean) of splenic versus renal CD8+ and CD4+ T cells isolated from kidneys of 14-week-old MRL/lpr mice. (B) PRODH expression in activated (CD44hi) splenic versus renal CD4+ and CD8+ T cells isolated from kidneys of 16- to 18-week-old MRL/lpr mice (n = 7). (C) Mxi1 mRNA in TH1-activated CD4+ T cells transduced with either EV or knockdown constructs targeting Hif1a 1 day after adding DMOG. (D to F) Mean fluorescence intensity of PRODH in TH1-activated CD4+ T cells transduced with EV, or two different Hif1a knockdown constructs (D), two different Mxi1 knockdown constructs (E), or Myc knockdown constructs (F) after 2 days of hypoxic culture. (G and H) Percentage of IFN-γ+ cells of the live TH1-activated CD4+ T cells, and activated CD8+ T cells, transduced with the different knockdown vectors after 3 days (for CD4+ T cells) or 1 day (for CD8+ T cells) of hypoxia culture. (I) Mean fluorescence intensity of granzyme B in live activated CD8+ T cells transduced with the different knockdown vectors after 1 day of hypoxia culture. (J and K) Baseline OCR (J) and extracellular acidification rate (ECAR) (K) of control-, DMOG-, and DMOG- and THFA-treated TH1-activated CD4+ T cells. (L and M) Baseline OCR (L) and ECAR (M) of control-, DMOG-, and DMOG- and THFA-treated CD8+ T cells. (N and O) NADH/NAD+ ratio of control-, THFA-, DMOG-, and DMOG- and THFA-treated TH1-activated CD4+ T cells (N) and CD8+ T cells (O). Data shown are means ± SD; statistical analysis by two-tailed paired t test (B) and unpaired t test (C to O). ns = P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 4 Selective HIF-1 blockade eliminates renal injury in murine lupus nephritis.

    (A) Numbers of renal-infiltrating CD4+ and CD8+ T cells isolated from 16-week-old male MRL/lpr mice after 4 weeks of treatment with either PX-478 or PBS (n = 11 and 10, respectively). (B) Semiquantitative urine dipstick analysis for proteinuria from 16-week-old male MRL/lpr mice treated with PX-478 or PBS. (C) Pathological scores of the 16-week-old male MRL/lpr mice treated with PX-478 or PBS, as assessed by the NIH activity index. (D to F) Representative glomerular (D), perivascular aggregates near the corticomedullary junction (E), and immunofluorescence staining of glomerular IgG2a (F) from the same mice after 4 weeks of treatment with PX-478 and PBS. Representative of three experiments, n = 10 to 11 animals per group. (G to I) Representative pimonidazole staining of kidney sections from 16-week-old MRL/lpr mice treated with PBS (G) or PX-478 (H), and quantification of pimonidazole-positive cortical tubular cells (I). Reference line indicates pimonidazole-positive renal tubular cells in control Fas-intact MRL+/+ mice. n = 5, 6, and 4, respectively. (J) Kaplan-Meier survival curve of MRL/lpr mice treated with PBS or PX-478 (n = 10 animals each group). Data shown are means ± SD; statistical analysis by two-tailed t test (A to C and I) and log-rank test (J). ns = P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 5 Genetic ablation of HIF-1α in T cells eliminates infiltrating T cells in lupus nephritis, reverses hypoxia, and prolongs survival.

    (A) Numbers of renal-infiltrating CD4+ and CD8+ T cells isolated from 6-month-old Cd4cre.Hif1afl/fl and Hif1afl/fl B6.Sle1.Yaa male mice (n = 12 and 8, respectively). (B) Semiquantitative urine dipstick analysis for proteinuria the same mice as (A). (C) Pathological scores of the same mice using the NIH activity index for total NIH activity index (n = 16 and 12, respectively). (D to F) Representative glomerular (D), perivascular aggregates near the corticomedullary junction (E) and immunofluorescence staining of glomerular IgG2c (F) from the same mice. (G to I) Representative pimonidazole (Hypoxyprobe) staining of kidney sections and quantification of pimonidazole-positive renal tubular cells in the cortex. Reference line represents the pimonidazole staining in renal cortex of control B6 mice, n = 6, 6, and 4, respectively. (J) Kaplan-Meier survival curve of Cd4cre.Hif1afl/fl and Hif1afl/fl B6.Sle1.Yaa male mice, n = 9 animals each group. Data shown are means ± SD; statistical analysis by two-tailed t test (A to C) and log-rank test (J) ns = P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 6 Hypoxia-regulated T cell survival and effector pathways are up-regulated in human lupus nephritis.

    (A to C) Immunohistochemistry staining of IDH1 (brown) + CD3 (red) (A), PDK2 (brown) (B), and PRODH (brown) (C) in human lupus nephritis biopsy sample. (D to F) Immunohistochemistry staining of IDH1 (red) + CD8 (brown) (D), PDK2 (brown) (E), and PRODH (brown) (F) in inflamed human tonsil. (G to I) Quantification of IDH1 (G), PDK2 (H), and PRODH-positive (I) staining in dense lymphocytic aggregates of lupus nephritis and T cell zone in inflamed tonsils. (J to L) Merged immunofluorescence staining images of CD4, CD8, 4′,6-diamidino-2-phenylindole (DAPI) with either IDH1 (J), PDK2 (K), or PRODH (L).

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/538/eaay1620/DC1

    Materials and Methods

    Fig. S1. Gating strategy for renal-infiltrating CD4+ and CD8+ T cells.

    Fig. S2. Renal-infiltrating CD4+ and CD8+ T cells are functionally active and phenotypically distinct.

    Fig. S3. Reversal of tissue damage and hypoxia after T cell depletion in lupus nephritis.

    Fig. S4. Validation of hypoxic signatures in renal-infiltrating CD4+ and CD8+ T cells using a published transcriptome dataset.

    Fig. S5. Illustration used to define renal pimonidazole staining.

    Fig. S6. Regions of hypoxia extend to the renal cortex in murine lupus nephritis.

    Fig. S7. Effect of hypoxia on T cell populations in lupus nephritis.

    Fig. S8. Predominant HIF-1α but not HIF-2α expression in renal-infiltrating T cells.

    Fig. S9. T cell depletion reverses renal cortical hypoxia.

    Fig. S10. HIF-1–regulated survival pathway in renal-infiltrating T cells from B6.Sle1.Yaa lupus-prone mice.

    Fig. S11. HIF-1–controlled survival pathways are up-regulated in hypoxia and pseudohypoxia T cell cultures.

    Fig. S12. Alternative splicing of BNIP3 regulated by PDK2 promotes T cell survival in hypoxia.

    Fig. S13. HIF-1–dependent T cell survival in renal hypoxia mediated by PDK2-driven alternative splicing of BNIP3.

    Fig. S14. IDH1 and PRODH are up-regulated in renal-infiltrating T cells.

    Fig. S15. PRODH is up-regulated in hypoxia and pseudohypoxia T cell cultures.

    Fig. S16. Changes in mitochondrial function and glycolysis upon blocking proline metabolism in pseudohypoxia.

    Fig. S17. HIF-1–controlled proline metabolism facilitates glycolysis.

    Fig. S18. Validation of phenotypic analyses of renal-infiltrating CD4+ and CD8+ T cells using a published transcriptome dataset.

    Fig. S19. Cellular adaptation in hypoxia is perturbed after selective HIF-1 blockade.

    Fig. S20. Selective HIF-1 blockade is therapeutic in murine lupus nephritis.

    Fig. S21. Phenotypic analyses of the nephritic lupus-prone mice treated with a HIF-1 antagonist.

    Fig. S22. HIF-1 pharmacological blockade reduces autoantibody production and abolishes GC formation in murine lupus.

    Fig. S23. Genetic ablation of HIF-1α in T cells reduces glycolytic capacity and tubulointerstitial infiltrations in lupus nephritis.

    Fig. S24. Hypoxia-regulated T cell survival and effector transcripts in human lupus nephritis.

    Fig. S25. Hypoxia-regulated proteins in noninflamed regions of human lupus nephritis.

    Table S1. Up-regulated pathways in renal-infiltrating T cells in murine lupus nephritis.

    Table S2. Targeted sequences for shRNA design.

    Table S3. Primers for qPCR.

    Data file S1. Summary of the transcriptome data of kidney versus spleen T cells with respective fold changes and statistics (provided as separate Excel file).

    References (6265)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Gating strategy for renal-infiltrating CD4+ and CD8+ T cells.
    • Fig. S2. Renal-infiltrating CD4+ and CD8+ T cells are functionally active and phenotypically distinct.
    • Fig. S3. Reversal of tissue damage and hypoxia after T cell depletion in lupus nephritis.
    • Fig. S4. Validation of hypoxic signatures in renal-infiltrating CD4+ and CD8+ T cells using a published transcriptome dataset.
    • Fig. S5. Illustration used to define renal pimonidazole staining.
    • Fig. S6. Regions of hypoxia extend to the renal cortex in murine lupus nephritis.
    • Fig. S7. Effect of hypoxia on T cell populations in lupus nephritis.
    • Fig. S8. Predominant HIF-1α but not HIF-2α expression in renal-infiltrating T cells.
    • Fig. S9. T cell depletion reverses renal cortical hypoxia.
    • Fig. S10. HIF-1–regulated survival pathway in renal-infiltrating T cells from B6.Sle1.Yaa lupus-prone mice.
    • Fig. S11. HIF-1–controlled survival pathways are up-regulated in hypoxia and pseudohypoxia T cell cultures.
    • Fig. S12. Alternative splicing of BNIP3 regulated by PDK2 promotes T cell survival in hypoxia.
    • Fig. S13. HIF-1–dependent T cell survival in renal hypoxia mediated by PDK2-driven alternative splicing of BNIP3.
    • Fig. S14. IDH1 and PRODH are up-regulated in renal-infiltrating T cells.
    • Fig. S15. PRODH is up-regulated in hypoxia and pseudohypoxia T cell cultures.
    • Fig. S16. Changes in mitochondrial function and glycolysis upon blocking proline metabolism in pseudohypoxia.
    • Fig. S17. HIF-1–controlled proline metabolism facilitates glycolysis.
    • Fig. S18. Validation of phenotypic analyses of renal-infiltrating CD4+ and CD8+ T cells using a published transcriptome dataset.
    • Fig. S19. Cellular adaptation in hypoxia is perturbed after selective HIF-1 blockade.
    • Fig. S20. Selective HIF-1 blockade is therapeutic in murine lupus nephritis.
    • Fig. S21. Phenotypic analyses of the nephritic lupus-prone mice treated with a HIF-1 antagonist.
    • Fig. S22. HIF-1 pharmacological blockade reduces autoantibody production and abolishes GC formation in murine lupus.
    • Fig. S23. Genetic ablation of HIF-1α in T cells reduces glycolytic capacity and tubulointerstitial infiltrations in lupus nephritis.
    • Fig. S24. Hypoxia-regulated T cell survival and effector transcripts in human lupus nephritis.
    • Fig. S25. Hypoxia-regulated proteins in noninflamed regions of human lupus nephritis.
    • Table S1. Up-regulated pathways in renal-infiltrating T cells in murine lupus nephritis.
    • Table S2. Targeted sequences for shRNA design.
    • Table S3. Primers for qPCR.
    • Legend for data file S1
    • References (6265)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1. Summary of the transcriptome data of kidney versus spleen T cells with respective fold changes and statistics (provided as separate Excel file).

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