Research ArticleBARRIER DYSFUNCTION

The antiprotease SPINK7 serves as an inhibitory checkpoint for esophageal epithelial inflammatory responses

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Science Translational Medicine  06 Jun 2018:
Vol. 10, Issue 444, eaap9736
DOI: 10.1126/scitranslmed.aap9736
  • Fig. 1 Expression of SPINK5 and SPINK7 genes in EoE and their sequence analysis.

    (A) FPKM values for SPINK family members determined from RNA-seq of esophageal biopsies [n = 6 control patients having 0 eosinophils per high-power field (control) and n = 10 patients with active EoE]. Data are means ± SD. P value was calculated by t test (unpaired, two-tailed). (B) Phylogenetic distribution of kazal domains of SPINK5 and SPINK7. A molecular dendrogram of kazal domains was drawn by using the ClustalΩ program set at the default parameters. (C) Immunofluorescence staining of esophageal biopsy sections for SPINK5 (magenta) and SPINK7 (cyan) with 4′,6-diamidino-2-phenylindole (DAPI)–stained nuclei (blue); representative images of four sections from different control patients and four sections from different patients with EoE are shown. The white dashed line separates the epithelium from the lamina propria. (D) Normalized SPINK7 mRNA expression values (n = 50 control patients and n = 83 patients with EoE). Each point represents an individual patient. Statistical significance was calculated according to the Mann-Whitney U test. (E) FPKM values of SPINK5, SPINK7, and CCL26 in differentiated EPC2 cells that were either untreated or treated with IL-13 (100 ng/ml) every other day for 7 days. P value was calculated by t test (unpaired, two-tailed) (F) SPINK7 mRNA expression from esophageal biopsies from 14 healthy controls, 22 patients with inactive EoE, and 19 patients with active EoE. P value was calculated by t test (unpaired, two-tailed). (G) Correlation of SPINK5 and SPINK7 expression with other EoE transcriptome genes as assessed by the EoE diagnostic panel (17) (n = 85 control and EoE patients) using Spearman correlations.

  • Fig. 2 Loss of SPINK5 and SPINK7 affects epithelial barrier function and architecture.

    (A) Hematoxylin and eosin (H&E)–stained sections of either NSC-treated, SPINK5-silenced, or SPINK7-silenced EPC2 cells and primary esophageal epithelial cells after ALI differentiation (day 14). Arrows point to the noncellular areas that were formed. (B) TEER (ohm/cm2) measurement from NSC-treated, SPINK7-silenced, and SPINK5-silenced EPC2 cells at day 7 of ALI differentiation. Data are the means ± SD from three independent experiments performed in triplicate. P value was calculated by t test (unpaired, two-tailed). Quantitative polymerase chain reaction (PCR) analysis of SPINK5 expression (C) or SPINK7 expression (D) of control (NSC), SPINK7-silenced, and SPINK5-silenced EPC2 cells that were grown for 14 days in ALI culture. Data are the means ± SD of three independent experiments performed in triplicate. P value was calculated by t test (unpaired, two-tailed). (E) A heatmap representing the fold change of 17 EDC genes that are significantly (P < 0.05) altered by SPINK7 depletion in EPC2 cells after ALI differentiation (day 14) from three independent experiments. (F) FLG mRNA expression in NSC-treated or SPINK7-silenced EPC2 or primary esophageal epithelial cells obtained from control patients after ALI differentiation. Data are means ± SD. P value was calculated by t test (unpaired, two-tailed). (G) FLG protein expression in NSC-treated or SPINK7-silenced EPC2 cells after ALI differentiation was assessed by Western blot. (H) Representative images of coimmunofluorescence of FLG (green), E-cadherin (magenta), and nuclear DAPI stain (blue) in NSC-treated or SPINK7-silenced EPC2 cells after ALI differentiation. (I) Representative electron microscopy images of NSC-treated or SPINK7-silenced EPC2 cells after ALI differentiation (day 14) from three independent experiments. Arrows depict microplicae at the epithelial junctions. The dashed arrows depict the absence of microplicae at epithelial junctions. (J) Representative immunostained sections of E-cadherin (cyan and green) and DAPI (blue) of NSC-treated or SPINK7-silenced EPC2 cells grown with high Ca2+ (top) or after 14 days of ALI differentiations (bottom) from three independent experiments. (K) Representative immunostained sections of DAPI (blue) and DSG1 (magenta) of NSC-treated or SPINK7-silenced EPC2 cells after 14 days of ALI differentiation from three independent experiments. (L) Reconstituted three-dimensional confocal images of NSC-treated or SPINK7-silenced EPC2 cells after ALI differentiation (day 14) stained with DAPI (blue), E-cadherin (green), and DSG1 (magenta). Movie presentations of the data are available in the Supplementary Materials (movies S1 and S2). Images were processed by Imaris software.

  • Fig. 3 Loss of SPINK7 induces esophageal mucosa transcriptome centered on inflammation.

    (A) Venn diagram depicting the number of genes differentially expressed (DE) as identified by RNA-seq of patients with EoE as compared to control (fold change > |2|, P < 0.05, FPKM > 1) [the EoE transcriptome has 1658 DE genes (9)] and in SPINK7 gene silencing as compared to NSC treatment in EPC2 cells differentiated in ALI cultures for 14 days (SPINK7 silencing has 270 DE genes). Genes overlapping between these two data sets were identified (97 genes). Gene ontology (GO) analyses of the SPINK7-EoE overlap gene set depicting human diseases or cell biology are presented according to the P values (−log10) for pathway discovery. (B) Venn diagram depicting the number of genes DE as identified by RNA-seq of EPC2 cells after ALI differentiation after IL-13 stimulation compared to untreated cells (IL-13 was added starting from day 7 of the ALI cultures and was added three times per week in a concentration of 100 ng/ml) (fold change > |2|, P < 0.05, FPKM > 1) [the IL-13 transcriptome has 1161 DE genes (9)] and in SPINK7 gene silencing as compared to NSC treatment in EPC2 cells differentiated in ALI cultures for 14 days (SPINK7 silencing has DE 270 genes). Genes overlapping between these two data sets were identified (119 genes). GO analyses of the SPINK7–IL-13 overlap gene set depicting human diseases, cell biology, or localization is presented according to the P values (−log10) for pathway discovery. (C) Left: Venn diagram depicting the number of genes that overlap among EoE, SPINK7 gene silencing, and IL-13 transcriptomes. Expression of the overlapping genes (47 genes) is represented by a heatmap (right) according to their fold change in SPINK7 silencing as compared to NSC treatment, EoE as compared to control, or cells treated with IL-13 as compared to cells treated with vehicle (9).

  • Fig. 4 Loss of SPINK7 induces cytokine release.

    (A) Heatmap of expression of cytokines and chemokines derived from supernatants of NSC-treated or SPINK7-silenced EPC2 cells after ALI differentiation (day 14) that were altered [all indicated cytokines and chemokines were expressed at a concentration >1 pg/ml, fold change (FC) > |2|, P < 0.05]. Data are presented as the mean fold change of SPINK7 compared to NSC of three independent experiments performed in duplicate or triplicate. Blue, down-regulated cytokines after SPINK7 silencing compared to NSC; yellow, up-regulated cytokines after SPINK7 silencing compared to NSC. The label “CXCL1/2/3” represents the collective detection of CXCL1, CXCL2, and CXCL3. (B) IL-8 protein in supernatants from NSC-treated or SPINK7-silenced EPC2 cells after ALI differentiation (day 14). Each point represents one data point from three independent experiments performed in triplicate. (C) A representative experiment of TSLP release from EPC2 cells that were grown in high-calcium media for 64 hours and then stimulated for 8 hours with the indicated concentrations of polyinosinic-polycytidylic acid (polyI:C). Cell supernatants were assessed for TSLP levels from three independent experiments. Data are the means ± SD of a representative experiment. All P values were calculated by t test (unpaired, two-tailed).

  • Fig. 5 SPINK7 CRISPR/Cas9 KO cells phenocopy SPINK7-silenced cells and reveal induction of an EMT marker and increased MMP proteolytic activity.

    (A) A representative TEER (ohm/cm2) measurement from SPINK7 CRISPR/Cas9 KO and control EPC2 cells at day 9 of ALI differentiation from three independent experiments performed in six replicates. Data are means ± SD. (B) FLG mRNA expression in control or SPINK7-KO EPC2 cells after ALI differentiation. Data are the means ± SD from four independent experiments performed in six replicates. (C) Quantification of uPA activity in supernatants derived from differentiated SPINK7 KO and control EPC2 cells that were loaded with 1 active unit (AU) of purified uPA. uPA activity in the supernatants is calculated as AUs according to standard dilutions of uPA. The dashed line represents the amount of uPA that was added to the supernatants. Data are the means ± SD of a representative experiment from three independent experiments performed in six replicates. (D) Representative data of TSLP release from EPC2 cells that were grown in high-calcium media for 64 hours and then stimulated for 8 hours with the indicated concentrations of polyI:C. Cell supernatants were assessed for TSLP concentrations from three independent experiments. Data are means ± SD. (E) VIM mRNA expression in control or SPINK7-KO EPC2 cells after ALI differentiation. Data are the means ± SD from four independent experiments. (F) Representative immunostained sections of DAPI (blue) and vimentin (VIM; magenta) of control NSC-treated or SPINK7-silenced EPC2 after 14 days of ALI differentiation from three independent experiments. Quantification of MMPs (G) or MMP9 (H) proteolytic activity in supernatants derived from NSC-treated or SPINK7-silenced EPC2 differentiated cells. MMP activity in the supernatants is calculated according to standard dilutions of MMP9. Data are the means ± SD of three independent experiments performed in triplicate. (I) GO analyses of the CRISPR/Cas9 SPINK7 KO differentiated cells compared to CRISPR/Cas9 control cells. Numbers presented are the P values (−log10). All P values were calculated by t test (unpaired, two-tailed) except GO analysis, which uses analysis of variance (ANOVA) test.

  • Fig. 6 SPINK7 regulates eosinophil function by regulating the uPA/uPAR pathway in the esophagus.

    (A) Quantification of uPA activity in supernatants derived from NSC-treated or SPINK7-silenced EPC2 cells during ALI differentiation (days 7 to 9). uPA activity in the supernatants is calculated as AUs according to standard dilutions of uPA. Data are the means ± SD of three independent experiments performed in triplicate. P values were calculated by t test (unpaired, two-tailed). (B) FPKM values of uPA in the esophagus of patients with EoE (n = 8) and controls (n = 5). Data are means ± SD. P values were calculated by t test (unpaired, two-tailed). (C) Analysis of the uPA proteolytic activity (AU) in esophageal biopsies from patients with EoE and control individuals (n = 6 for each cohort). Data are means ± SD. P values were calculated by t test (unpaired, two-tailed). (D) Analysis of the pan protease activity in esophageal biopsies from patients with EoE and control individuals (n = 6 for each cohort). (E) The expression of the D1 domain of uPAR was quantified [according to the mean fluorescence intensity (MFI) using flow cytometry] on the cell surface of eosinophils that were incubated with supernatants from control or SPINK7 KO differentiated cells. (F) The expression of the D1 domain of uPAR was quantified (MFI using flow cytometry) on the cell surface of eosinophils (7AADlow, CD45+, CD11B+, SIGLEC8+) derived from either blood or esophageal biopsies from patients with EoE (n = 8 individuals). Each point represents a single patient. P value was calculated by t test (paired, two-tailed). IgG1, immunoglobulin G1. (G) Using flow cytometry, expression of D1 and D2D3 domains of uPAR was quantified (according to the MFI) on the cell surface of eosinophils derived from esophageal biopsies of additional six patients with EoE. Each point represents a single patient. P value was calculated by t test (paired, two-tailed). (H) Supernatants derived from either SPINK7 KO cells, control cells, or purified eosinophils treated with uPA (10 nM) or a combination of A23187 (10 μM) and phorbol 12-myristate 13-acetate (50 nM) (Ion + PMA), which induced the release of EPX from the eosinophils purified from blood of three healthy donors. Data are means ± SD. P value was calculated by t test (unpaired, two-tailed).

  • Fig. 7 A1AT administration ameliorates the effects caused by loss of SPINK7.

    (A) Quantification of trypsin-like activity in supernatants derived from NSC-treated or SPINK7-silenced EPC2 cells that were treated with A1AT or vehicle after ALI differentiation. Data are the means ± SD of three independent experiments. (B) Electrical resistance measurements of NSC-treated or SPINK7-silenced EPC2 cells that were treated with A1AT or a vehicle during ALI differentiation. Data are means ± SD. (C) H&E staining of NSC-treated or SPINK7-silenced EPC2 cells that were treated with A1AT or a vehicle after ALI differentiation (day 14). (D) Coimmunofluorescence staining of E-cadherin (magenta) and FLG (green) of NSC-treated or SPINK7-silenced EPC2 cells that were treated with A1AT or vehicle after ALI differentiation. P values were calculated by t test (unpaired, two-tailed).

  • Fig. 8 Epistasis between genetic variants in TSLP and PLAU contributes to EoE susceptibility.

    (A) Genetic interaction between TSLP (rs2289277) and SNPs in 68 atopy-related genes. Logistic regression analyses with interaction term were performed using 725 cases and 412 controls. Each circle represents a single SNP, which is colored according to its locus. Covariates include age, sex, and three principal components. Multiple testing threshold, P < 0.00036 is shown as the upper dashed line, whereas P < 0.05 is shown as the lower dashed line. The PLAU gene is denoted with a rectangle. (B) Forest plot evaluating the combinatorial effects of the presence of at least one minor allele at TSLP (rs2289277-G) and PLAU (rs2459449-T) and polymorphic sequences are shown.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/444/eaap9736/DC1

    Materials and Methods

    Fig. S1. Differential sequence and expression of SPINK5 and SPINK7.

    Fig. S2. SPINK5 and SPINK7 are esophageal epithelial differentiation markers.

    Fig. S3. Analysis of the SPINK7 transcriptome reveals a differentiation defect.

    Fig. S4. Loss of SPINK7 induces intercellular dilated spaces and barrier impairment.

    Fig. S5. Analysis of the SPINK7 transcriptome reveals overlaps with the EoE and IL-13–induced transcriptomes.

    Fig. S6. SPINK7 gene silencing induces cytokine release.

    Fig. S7. Generation of SPINK7 KO EPC2 cells.

    Fig. S8. uPA administration does not alter barrier integrity or promote overexpression of EMT markers in the ALI culture system.

    Fig. S9. uPAR expression on immune cells and the effect of uPA on eosinophils.

    Fig. S10. A summary of the effect of barrier gene deficiency on SPINK7 expression and a model of SPINK7 pathway in EoE.

    Movie S1. Immunofluorescence of junctional proteins of differentiated control EPC2 cells.

    Movie S2. Immunofluorescence of junctional proteins of differentiated SPINK7-silenced EPC2 cells.

    Table S1. Correlation between the expression of SPINK5 or SPINK7 with EoE signature genes.

    Table S2. SPINK7 transcriptome.

    Table S3. Differentiation transcriptome.

    Table S4. CRISPR/Cas9 SPINK7 KO transcriptome.

    References (4046)

  • Supplementary Material for:

    The antiprotease SPINK7 serves as an inhibitory checkpoint for esophageal epithelial inflammatory responses

    Nurit P. Azouz, Mario A. Ynga-Durand, Julie M. Caldwell, Ayushi Jain, Mark Rochman, Demetria M. Fischesser, Leanne M. Ray, Mary C. Bedard, Melissa K. Mingler, Carmy Forney, Matthew Eilerman, Jonathan T. Kuhl, Hua He, Jocelyn M. Biagini Myers, Vincent A. Mukkada, Philip E. Putnam, Gurjit K. Khurana Hershey, Leah C. Kottyan, Ting Wen, Lisa J. Martin, Marc E. Rothenberg*

    *Corresponding author. Email: rothenberg{at}cchmc.org

    Published 6 June 2018, Sci. Transl. Med. 10, eaap9736 (2018)
    DOI: 10.1126/scitranslmed.aap9736

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Differential sequence and expression of SPINK5 and SPINK7.
    • Fig. S2. SPINK5 and SPINK7 are esophageal epithelial differentiation markers.
    • Fig. S3. Analysis of the SPINK7 transcriptome reveals a differentiation defect.
    • Fig. S4. Loss of SPINK7 induces intercellular dilated spaces and barrier impairment.
    • Fig. S5. Analysis of the SPINK7 transcriptome reveals overlaps with the EoE and IL-13–induced transcriptomes.
    • Fig. S6. SPINK7 gene silencing induces cytokine release.
    • Fig. S7. Generation of SPINK7 KO EPC2 cells.
    • Fig. S8. uPA administration does not alter barrier integrity or promote overexpression of EMT markers in the ALI culture system.
    • Fig. S9. uPAR expression on immune cells and the effect of uPA on eosinophils.
    • Fig. S10. A summary of the effect of barrier gene deficiency on SPINK7 expression and a model of SPINK7 pathway in EoE.
    • Legends for movies S1 and S2
    • Legends for tables S1 to S4
    • References (4046)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Immunofluorescence of junctional proteins of differentiated control EPC2 cells.
    • Movie S2 (.avi format). Immunofluorescence of junctional proteins of differentiated SPINK7-silenced EPC2 cells.
    • Table S1 (Microsoft Excel format). Correlation between the expression of SPINK5 or SPINK7 with EoE signature genes.
    • Table S2 (Microsoft Excel format). SPINK7 transcriptome.
    • Table S3 (Microsoft Excel format). Differentiation transcriptome.
    • Table S4 (Microsoft Excel format). CRISPR/Cas9 SPINK7 KO transcriptome.

    [Download Movie S1]

    [Download Movie S2]

    [Download Tables S1 to S4]

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