Research ArticleInflammation

An anti-inflammatory eicosanoid switch mediates the suppression of type-2 inflammation by helminth larval products

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Science Translational Medicine  22 Apr 2020:
Vol. 12, Issue 540, eaay0605
DOI: 10.1126/scitranslmed.aay0605
  • Fig. 1 Topical treatment with Hpb larval extract (HpbE) modulates type-2 airway inflammation in mice.

    (A) Experimental model of house dust mite (HDM)–induced allergic airway inflammation and intranasal (i.n.) treatment with HpbE. (B) BALF cell counts in mice sensitized (1 μg) and challenged (10 μg) with HDM ± intranasal treatment with HpbE (5 μg), 48 hours after the last challenge and treatment. (C) Representative hematoxylin and eosin (H&E)– or periodic acid–Schiff (PAS)–stained lung tissue from mice sensitized to HDM ± treatment with HpbE. Scale bars, 100 μm. (D) Concentrations of 15-HETE (LC-MS/MS) or IL-5, IL-6, eotaxin, and RANTES (Bioplex) in BALF from mice sensitized to HDM ± treatment with HpbE. Results are pooled from two independent experiments (B and D) or representative of stainings performed for two independent experiments (C). Results are presented as means ± SEM; n = 3 to 9 (naïve) or n = 5 to 17 (treated) per group. Statistical significance was determined by Kruskal-Wallis test followed by Dunn’s multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 2 HpbE-treated macrophages produce less LTs and modulate allergic airway inflammation via PGE2.

    (A) Eicosanoids (LC-MS/MS) produced by mouse bone marrow–derived macrophages (BMDMs) after treatment with Hpb larval extract (HpbE) (24 hours) and stimulation with A23187 (10 min). (B) Relative gene expression of AA-metabolizing enzymes [quantitative polymerase chain reaction (qPCR)] in mouse BMDM treated with HpbE. (C) Heat map showing major PUFA metabolites (LC-MS/MS) produced by human monocyte-derived macrophages (MDMs) ± treatment with HpbE followed by A23187 (10 min). (D) Amounts of major bioactive AA metabolites (LC-MS/MS) produced by human MDM ± treatment with HpbE +A23187 (10 min). (E) Relative gene expression of eicosanoid pathway proteins (qPCR) in human MDM ± treatment with HpbE. (F) Frequencies of total leukocytes and total numbers of eosinophils in BALF of mice sensitized to HDM + intranasal transfer of HpbE-conditioned or untreated BMDM (MΦ) [wild type (wt) or Ptges−/−], 18 hours after the last challenge and transfer. Dashed lines indicate eosinophil prevalence in HDM-sensitized mice. (G) Representative H&E-stained lung tissue from mice sensitized to HDM ± intranasal transfer of untreated or HpbE-conditioned BMDM (wt or Ptges−/−). Scale bars, 100 μm. Data are presented as means ± SEM; n = 8 BMDM from C57BL/6 mice (A and B); n = 10 to 15 MDM from healthy human blood donors (C to E); n = 7 to 9 mice per group (F and G). Statistical significance was determined by Wilcoxon test (A to E) or Mann-Whitney test (F). *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 3 HpbE induces type-2–suppressive cytokines and prevents M2 polarization.

    (A) Amounts of IL-10 and IL-1β (ELISA) produced by human MDM (n = 14 to 15) ± treatment with HpbE. (B) Amounts of TNF-α, IL-6, IL-12p70, IL-18, IL-27, IL-33, and CCL17/TARC (Bioplex) produced by human MDM (n = 3) after treatment with HpbE. (C) Amounts of IL-10 and IL-1β (Bioplex) produced by mouse BMDM (n = 8) ± treatment with HpbE. (D) Gene expression of M2 markers (qPCR) in human MDM (n = 9 to 10) ± treatment with HpbE. (E) Gene expression of M2 markers (qPCR) in mouse BMDM (n = 5) ± treatment with HpbE. Data are presented as means ± SEM. Statistical significance was determined by Wilcoxon test. **P < 0.01; ***P < 0.001. n.d., not detectable.

  • Fig. 4 HpbE modulates eicosanoid profiles and chemotaxis of granulocytes in a human setting of type-2 inflammation.

    (A) Heat map showing major PUFA metabolites (LC-MS/MS) produced by mixed human granulocytes ± treatment with HpbE (24 hours) followed by A23187 (10 min). (B) Amounts of major eicosanoids (LC-MS/MS) produced by mixed human granulocytes ± treatment with HpbE (24 hours) + A23187 (10 min). (C) Amounts of cysLTs [enzyme immunoassay (EIA), validated by LC-MS/MS] produced by purified human eosinophils ± treatment with HpbE (24 hours) + A23187 (10 min). (D) Relative gene expression of AA-metabolizing enzymes (qPCR) in mixed human granulocytes ± treatment with HpbE. (E) Expression of LT synthetic enzymes (LTC4S and LTA4H) (flow cytometry) in human eosinophils ± treatment with HpbE. (F) Chemotaxis of granulocytes from patients with AERD toward nasal polyp secretions ± treatment with HpbE/fluticasone propionate (FP)/montelukast (MK). Dashed line depicts basal migration. (G) Surface expression of chemotactic receptors (CCR3 and CRTH2) (flow cytometry) in human eosinophils ± treatment with HpbE. (H) Chemotaxis of mixed human granulocytes ± pretreatment with conditioned medium from MDM (±HpbE, ±COX inhibitor indomethacin). Dashed line depicts basal migration. Data are pooled from at least three independent experiments and presented as means ± SEM; n = 6 to 9 mixed granulocytes or purified eosinophils from human blood donors. Statistical significance was determined by Wilcoxon test (two groups) or Friedman test (four groups). *P < 0.05; **P < 0.01.

  • Fig. 5 Induction of type-2–suppressive macrophages by HpbE is mediated via HIF-1α, p38 MAPK, and COX.

    (A) Representative immunofluorescence staining of HIF-1α, COX-2, 4′,6-diamidino-2-phenylindole (DAPI) (cell nuclei), and F4/80 in BMDM ± treatment with HpbE. (B) Eicosanoid production (LC-MS/MS) in BMDM (wt or HIF-1αfloxed/floxedxLysMCre) ± treatment with HpbE (24 hours) + A23187 (10 min). (C) Amounts of IL-6, TNF-α, IL-1β, or IL-10 (Bioplex) in BMDM (wt or HIF-1αfloxed/floxedxLysMCre) ± treatment with HpbE. (D) Gene expression of M2 markers (qPCR) in BMDM (wt or HIF-1αfloxed/floxedxLysMCre) ± treatment with HpbE. (E) Protein amounts of phospho-p38, total p38, COX-2, or β-actin (Western blot) in MDM ± treatment with HpbE. Left, representative blots for n = 3 blood donors; right, quantification for n = 5 to 9 donors. (F and G) Amounts of IL-10 or IL-1β (ELISA) in MDM ± treatment with HpbE ± inhibitors of p38 (VX-702), COX (indomethacin), or HIF-1α (acriflavine). (H) Fold change of eicosanoid enzymes in MDM treated with HpbE ± inhibitors of p38 (VX-702), COX (indomethacin), or HIF-1α (acriflavine). Dotted lines indicate expression in untreated cells. Data are pooled from at least two independent experiments and presented as means ± SEM; n = 5 to 9. Statistical significance was determined by two-way ANOVA (A to D), Wilcoxon test (E), or Friedman test (F to H). *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 6 GDH is a major immunoregulatory factor in HpbE.

    (A and B) Amounts of prostanoids (EIA) or IL-10 and IL-1β (ELISA) in human MDM (A) or chemotaxis of human PMNs (B) ± treatment with HpbE or heat-inactivated HpbE (HpbE 90°C). (C) Amounts of IL-10 (ELISA) in human MDM ± treatment with HpbE ± pretreatment with proteinase K (prot K). (D) Size exclusion chromatogram for fractionation of HpbE. (E) Amounts of TXB2 (EIA) or IL-10 (ELISA) in MDM ± treatment with HpbE fractions. (F) Summary of results from MS identification of proteins in active fractions of HpbE. (G) Amounts of PGE2 (EIA) or IL-10 (ELISA) in MDM ± treatment with HpbE ± inhibitor of GDH (GDHi; bithionol, 20 μM). (H) Amounts of PGE2 or total COX metabolites (LC-MS/MS) in MDM ± treatment with HpbE ± inhibitor of GDH (bithionol, 20 or 100 μM). (I) Amounts of PGE2 (EIA) or IL-10 (ELISA) in MDM ± treatment with HpbE ± different dilutions of a monoclonal antibody (clone 4F8) against Hpb GDH. Data are pooled from at least two independent experiments and presented as means ± SEM for MDM from n = 2 to 10 healthy human blood donors. Statistical significance was determined by Friedman test. *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 7 Recombinant Hpb GDH induces anti-inflammatory mediators in human macrophages and suppresses allergic airway inflammation in mice.

    (A) Amounts of eicosanoids (LC-MS/MS/EIA) or IL-10 (ELISA) in human MDM ± treatment with recombinant Hpb GDH (5 μg/ml) (24 hours) ± A23187 (10 min). (B) BALF cell counts in mice sensitized (1 μg) and challenged with HDM (10 μg) ± intranasal treatment with Hpb GDH (10 μg), 48 hours after the last challenge and treatment. (C) Representative H&E- or PAS-stained lung tissue from mice sensitized to HDM ± treatment with Hpb GDH. Scale bars, 100 μm. Results are pooled from at least two independent experiments (A and B) or representative of stainings performed for two independent experiments (C). Results are presented as means ± SEM for MDM from n = 7 to 9 healthy human blood donors or n = 9 to 10 mice per group. Statistical significance was determined by Friedman test (A) or Kruskal-Wallis test followed by Dunn’s multiple comparison test (B). *P = 0.05; **P = 0.01; ***P < 0.001.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/540/eaay0605/DC1

    Supplementary Materials and Methods

    Fig. S1. PUFA metabolites produced by human MDM in response to treatment with HpbE and impact of HpbE-treated Ptgs2−/− BMDM on eosinophilic airway inflammation.

    Fig. S2. HpbE has stronger eicosanoid-modulatory effects than glucocorticosteroids.

    Fig. S3. HpbE has a distinct potential to induce type-2–suppressive mediators compared to other helminth products or helminth-associated bacteria.

    Fig. S4. Viability and LTA4H expression in human eosinophils and neutrophils and PUFA metabolites produced by human PMNs in response to treatment with HpbE.

    Fig. S5. HpbE induces a regulatory eicosanoid and cytokine profile in mixed and isolated CD14+ human PBMCs.

    Fig. S6. Effect of COX-2, NF-κB, PI3K, PTEN, or PKA inhibition on HpbE-driven modulation of cytokines and eicosanoid pathways.

    Fig. S7. Effect of neutralizing antibodies against PRRs (TLR2 and dectin-1/2) or IL-1β on HpbE-driven modulation of eicosanoids and IL-10 in human MDM.

    Fig. S8. Bithionol does not affect cell viability and L3 stage HpbE shows a higher GDH activity as compared to L4 or L5 extracts.

    Fig. S9. Newly generated monoclonal antibodies recognize Hpb GDH, clone 4F8 reduces HpbE-induced PGE2 and IL-10 production, and bithionol partially inhibits activity of recombinant Hpb GDH.

    Fig. S10. Sequence of Hpb GDH is distinct from human GDH and contains potential predicted glycosylation sites.

    Table S1. LC-MS/MS panel of PUFAs and PUFA metabolites.

    Table S2. Proteins present in active fractions of HpbE identified by MS.

    Table S3. Primer sequences for qPCR.

    Table S4. Reagents and resources.

    Data file S1. Primary data.

    References (5963)

  • The PDF file includes:

    • Supplementary Materials and Methods
    • Fig. S1. PUFA metabolites produced by human MDM in response to treatment with HpbE and impact of HpbE-treated Ptgs2−/− BMDM on eosinophilic airway inflammation.
    • Fig. S2. HpbE has stronger eicosanoid-modulatory effects than glucocorticosteroids.
    • Fig. S3. HpbE has a distinct potential to induce type-2–suppressive mediators compared to other helminth products or helminth-associated bacteria.
    • Fig. S4. Viability and LTA4H expression in human eosinophils and neutrophils and PUFA metabolites produced by human PMNs in response to treatment with HpbE.
    • Fig. S5. HpbE induces a regulatory eicosanoid and cytokine profile in mixed and isolated CD14+ human PBMCs.
    • Fig. S6. Effect of COX-2, NF-κB, PI3K, PTEN, or PKA inhibition on HpbE-driven modulation of cytokines and eicosanoid pathways.
    • Fig. S7. Effect of neutralizing antibodies against PRRs (TLR2 and dectin-1/2) or IL-1β on HpbE-driven modulation of eicosanoids and IL-10 in human MDM.
    • Fig. S8. Bithionol does not affect cell viability and L3 stage HpbE shows a higher GDH activity as compared to L4 or L5 extracts.
    • Fig. S9. Newly generated monoclonal antibodies recognize Hpb GDH, clone 4F8 reduces HpbE-induced PGE2 and IL-10 production, and bithionol partially inhibits activity of recombinant Hpb GDH.
    • Fig. S10. Sequence of Hpb GDH is distinct from human GDH and contains potential predicted glycosylation sites.
    • Legends for tables S1 and S2
    • Table S3. Primer sequences for qPCR.
    • Table S4. Reagents and resources.
    • References (5963)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). LC-MS/MS panel of PUFAs and PUFA metabolites.
    • Table S2 (Microsoft Excel format). Proteins present in active fractions of HpbE identified by MS.
    • Data file S1 (Microsoft Excel format). Primary data.

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