The Role of Fucoidans Isolated from the Sporophylls of Undaria pinnatifida against Particulate-Matter-Induced Allergic Airway Inflammation: Evidence of the Attenuation of Oxidative Stress and Inflammatory Responses.

Balb/c mouse lung tissue section, PM-exacerbated allergic airway inflammation model

Female Balb/c mice, 20-25 g, 7-8 weeks old (OrientBio, Sungnam, Korea); n = 7 per group across seven groups

Balb/c mouse lung homogenate, PM-exacerbated allergic airway inflammation model

Female Balb/c mice, 20-25 g, 7-8 weeks old (OrientBio, Sungnam, Korea); n = 7 per group across seven groups

Balb/c mouse serum, PM-exacerbated allergic airway inflammation model

Female Balb/c mice, 20-25 g, 7-8 weeks old (OrientBio, Sungnam, Korea); n = 7 per group across seven groups

Balb/c mouse cohort, PM-exacerbated allergic airway inflammation model

Female Balb/c mice, 20-25 g, 7-8 weeks old (OrientBio, Sungnam, Korea); n = 7 per group across seven groups; airway responsiveness measured 24 h after the final PM challenge with nebulised methacholine at 0, 12.5, 25 and 50 mg/mL

Balb/c mouse bronchoalveolar lavage fluid, PM-exacerbated allergic airway inflammation model

Female Balb/c mice, 20-25 g, 7-8 weeks old (OrientBio, Sungnam, Korea); n = 7 per group across seven groups

Balb/c mouse trachea tissue section, PM-exacerbated allergic airway inflammation model

Female Balb/c mice, 20-25 g, 7-8 weeks old (OrientBio, Sungnam, Korea); n = 7 per group across seven groups

MDA positive cell percentage in lung sections (immunohistochemistry)

Malondialdehyde adducts stained by immunohistochemistry on 3 um lung sections and quantified as the MDA positive cell percentage (Figure 1A-H). PM exposure as well as OVA significantly enhanced the MDA positive cell percentage in the PM only, OVA only and OVA + PM groups relative to the healthy control (p < 0.0005), and fucoidans dose-dependently attenuated it relative to OVA + PM. The paper reports this endpoint as images plus a bar chart with no absolute percentages, so no magnitude is recorded; the two directions it states are in response_comparisons without a change value.

Exposure conditions

Outputs

Evidence

Malondialdehyde level in lung homogenate (thiobarbituric acid assay)

MDA measured in lung homogenate supernatant by the thiobarbituric acid reaction read at 532 nm against an MDA standard curve (Figure 1I). PM and OVA each raised lung MDA by about 88-90% over the healthy control (p < 0.05), and fucoidan lowered the PM-exacerbated level dose-dependently, by 51.2% at 100 mg/kg and by 70.4% at 400 mg/kg. The paper states that a high dose of fucoidan was more or similarly effective to prednisone, the positive control. All stated percentages are in response_comparisons; the paper gives no absolute MDA concentrations.

Exposure conditions

Outputs

Evidence

Malondialdehyde level in serum (lipid peroxidation assay kit)

Serum MDA measured with a colorimetric lipid peroxidation assay kit at the micromolar level against a standard curve (Figure 1J). PM exposure raised serum MDA by 74.6% in both the PM only and the OVA + PM groups relative to the healthy control (both p < 0.005), and fucoidan attenuated the PM-exacerbated level dose-dependently, by 44.8% at 100 mg/kg and by 78.0% at 400 mg/kg (both p < 0.0005). The paper gives no absolute serum MDA concentrations, so the percentages live in response_comparisons.

Exposure conditions

Outputs

Evidence

8-OHdG level in serum (DNA/RNA oxidative damage ELISA)

Serum 8-hydroxy-2'-deoxyguanosine measured as the 8-OHdG-acetylcholinesterase conjugate with a DNA/RNA oxidative damage ELISA kit (Figure 1K). 8-OHdG rose significantly over the healthy control in OVA only (81.4%), PM only (122.2%) and OVA + PM (116.8%), all p < 0.005. Fucoidans reduced serum 8-OHdG by 11.8% at both doses but the reduction was not significant, whereas prednisone reduced it by 25.7% (p < 0.05). The authors read this contrast as fucoidans and prednisone attenuating PM-induced oxidative stress through different signalling pathways. No absolute 8-OHdG concentrations are reported.

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Outputs

Evidence

Airway resistance (Penh) under methacholine challenge

Enhanced pause (Penh) measured by whole-body plethysmography 24 h after the final PM challenge, with nebulised methacholine at 0, 12.5, 25 and 50 mg/mL and readings averaged over 3 min (Figure 2A). At the high methacholine concentration (50 mg/mL) the PM only, OVA only and OVA + PM groups all showed significantly increased airway hyper-responsiveness over the healthy control (p < 0.0005); PM exacerbated OVA-induced hyper-responsiveness by 20.7% (p < 0.05); and high-dose fucoidans attenuated the PM-exacerbated hyper-responsiveness by 43.0% (p < 0.0005), similarly to prednisone. Penh is a dimensionless plethysmography index rather than a spirometric volume or a measured resistance, and the paper reports it only as figure curves, so no magnitude is recorded in the output records.

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Evidence

Inflammatory cell differential in BALF (Diff Quick)

Differential counts of macrophages, eosinophils, neutrophils, lymphocytes and basophils on methanol-fixed BALF smears stained with a Diff Quick kit (Figure 2B). High-dose fucoidans significantly attenuated the PM-exacerbated infiltration of eosinophils, neutrophils and lymphocytes (p < 0.05), and attenuated neutrophil infiltration more effectively than prednisone did; the macrophage and basophil counts fell dose-dependently but not significantly. The paper reports the counts as figure bars with no numbers, so no magnitude is recorded. The H&E inflammatory activity grade of trachea and lung sections (Figure 3O, 3P) corroborates the same key event but has no assay class in this schema; its result is quoted here as the second evidence item.

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Evidence

IL-4, IL-17a and IL-33 in BALF (ELISA)

IL-4, IL-17a and IL-33 measured by ELISA in BALF supernatant (Figure 12D-F). IL-4 was significantly increased in OVA + PM over the healthy control (p < 0.05) and high-dose fucoidans attenuated it (p < 0.05); IL-17a was appreciably increased in OVA + PM and attenuated by high-dose fucoidan without reaching significance; IL-33 was significantly increased in OVA + PM and attenuated by high-dose fucoidan. BALFSputumOutput has no IL-4, IL-17a or IL-33 slot and the paper reports the concentrations only as figure bars, so these output records carry their group and condition with no magnitude.

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Evidence

PAS positive cell percentage in trachea sections

Periodic acid-Schiff staining of trachea sections, counterstained with 0.5% methyl green, quantified as the PAS positive cell percentage in ImageJ v1.46 (Figure 10). Goblet cell hyperplasia and mucus secretion increased by 287.5% in OVA + PM relative to the healthy control (p < 0.0005); the PM only and OVA only groups also showed elevated PAS positive cells, but the highest proportion was in OVA + PM. Fucoidan treatments attenuated the PAS positive cell proportion by 9.1% at 100 mg/kg and by 36.7% at 400 mg/kg (p < 0.005). The paper gives no absolute percentages, so the stated changes live in response_comparisons.

Exposure conditions

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Evidence

PAS positive cell percentage in lung sections

Periodic acid-Schiff staining of lung sections quantified as the PAS positive cell percentage in ImageJ v1.46 (Figure 10J-P). Mucus secretion and goblet cell hyperplasia increased by 378.0% in OVA + PM relative to the healthy control (p < 0.0005), and PM induced PAS positive cells in the lungs by 49.0% over OVA alone (p < 0.005). Fucoidan treatments reduced the number of PAS positive cells by 35.2% at 100 mg/kg (p < 0.005) and by 66.8% at 400 mg/kg (p < 0.0005). The paper separately reports, without quantification, that PM exposure enhanced mucus secretion with enlarged submucosal mucous glands and that fucoidans dose-dependently reversed both (Figure 11); there is no submucosal-gland slot in this schema, so that observation is recorded here and in the goblet-to-mucin key event relationship rather than as a measurement.

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Outputs

Evidence

Congo red positive eosinophils in trachea sections

Eosinophils stained with Congo red on trachea sections and counted per site in ImageJ v1.46 (Figure 4A-G, 4O). PM increased eosinophil infiltration in the trachea of OVA-sensitised mice by 500.0% over the healthy control (p < 0.0005), and OVA + PM exceeded OVA alone by 56.4% (p < 0.005). Fucoidan treatments attenuated the PM-exacerbated infiltration dose-dependently, by 52.0% at 100 mg/kg and by 72.0% at 400 mg/kg. The paper reports eosinophil counts only as figure bars, so no magnitude is recorded.

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Outputs

Evidence

Congo red positive eosinophils in lung sections

Eosinophils stained with Congo red on lung sections and counted per site in ImageJ v1.46 (Figure 4H-N, 4P). The highest infiltration was in OVA + PM: 566.6% above the healthy control (p < 0.0005) and 74.0% above OVA only (p < 0.005). Fucoidan attenuated eosinophil infiltration dose-dependently, by 45.0% at 100 mg/kg and by 65.0% at 400 mg/kg, and the high dose was as effective as prednisone. No absolute counts are reported.

Exposure conditions

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Evidence

Gr-1+ granulocytes in trachea sections (immunohistochemistry)

Gr-1+ granulocytes detected by immunohistochemistry on trachea sections and counted per site in ImageJ v1.46 (Figure 5A-G, 5O). OVA + PM raised the granulocyte influx by 200.0% over the healthy control (p < 0.0005) and PM exacerbated Gr-1+ infiltration by 52.5% over OVA alone. Fucoidan reduced the influx by 36.6% at 100 mg/kg and 53.3% at 400 mg/kg, and prednisone by 55.5%. No absolute counts are reported.

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Outputs

Evidence

Gr-1+ granulocytes in lung sections (immunohistochemistry)

Gr-1+ granulocytes detected by immunohistochemistry on lung sections and counted per site in ImageJ v1.46 (Figure 5H-N, 5P). Granulocyte infiltration rose 247.8% in OVA + PM over the healthy control, and PM exacerbated OVA-induced granulocyte infiltration by 113.3% over OVA alone (p < 0.0005). High-dose fucoidans reduced granulocyte infiltration by 66.4% relative to OVA + PM (p < 0.0005) and were more effective than prednisone. No absolute counts are reported.

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Outputs

Evidence

F4/80+ macrophages in trachea sections (immunohistochemistry)

F4/80+ macrophages detected by immunohistochemistry on trachea sections and counted per site in ImageJ v1.46 (Figure 6A-G, 6O). OVA + PM showed clearly enhanced macrophage infiltration, +250.0% over the healthy control (p < 0.0005). Fucoidans significantly reduced macrophage infiltration relative to OVA + PM, by 34.7% at 100 mg/kg (p < 0.05) and 45.7% at 400 mg/kg (p < 0.0005). No absolute counts are reported.

Exposure conditions

Outputs

Evidence

F4/80+ macrophages in lung sections (immunohistochemistry)

F4/80+ macrophages detected by immunohistochemistry on lung sections and counted per site in ImageJ v1.46 (Figure 6H-N, 6P). Macrophage infiltration rose 515.3% in PM + OVA over the healthy control (p < 0.0005), and PM exacerbated macrophage infiltration by 58.4% over OVA alone. Both fucoidan doses significantly attenuated the PM-exacerbated infiltration, by 74.3% and 81.2% respectively (both p < 0.0005), and the high dose was more or similarly effective to prednisone. No absolute counts are reported.

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Evidence

CD4+ T lymphocytes in trachea sections (immunohistochemistry)

CD4+ T cells detected by immunohistochemistry on trachea sections and counted per site in ImageJ v1.46 (Figure 7A-G, 7O). CD4+ T cells increased by 64.1% in OVA + PM over the healthy control (p < 0.005), and fell dose-dependently with fucoidan treatment, by 15.4% at 100 mg/kg and by 56.4% at 400 mg/kg (p < 0.05). No absolute counts are reported.

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Evidence

CD4+ T lymphocytes in lung sections (immunohistochemistry)

CD4+ T cells detected by immunohistochemistry on lung sections and counted per site in ImageJ v1.46 (Figure 7H-N, 7P). CD4+ T cell infiltration rose 197.8% in OVA + PM over the healthy control. The paper also reports that PM exacerbated OVA-induced CD4+ T cell infiltration by 24.5% over OVA alone at p < 0.09 - which does not meet the paper's own 0.05 significance criterion, so that comparison is recorded as not significant. High-dose fucoidan reduced CD4+ T cell infiltration by 59.1% relative to OVA + PM (p < 0.0005), similarly to prednisone. No absolute counts are reported.

Exposure conditions

Outputs

Evidence

Activated mast cells in trachea sections (toluidine blue)

Mast cells stained with 0.5% toluidine blue on trachea sections and counted per site in ImageJ v1.46 (Figure 9A-H). Activated mast cells increased by 123.5% in OVA + PM over the healthy control (p < 0.0005) and fell dose-dependently with fucoidan treatment, by 28.9% at 100 mg/kg and by 50.0% at 400 mg/kg (p < 0.005). No absolute counts are reported.

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Outputs

Evidence

Degranulated mast cells in trachea sections (toluidine blue)

Degranulated mast cells counted per site on toluidine-blue-stained trachea sections in ImageJ v1.46 (Figure 9I). OVA + PM showed the highest degranulation of all groups, 133.6% above OVA alone. Fucoidan treatments reversed the PM-induced degranulation by 51.9% at 100 mg/kg and by 76.7% at 400 mg/kg (p < 0.005), and the high dose was as effective as prednisone. No absolute counts are reported.

Exposure conditions

Outputs

Evidence

Total IgE in serum (ELISA)

Total serum IgE measured by ELISA (Figure 8A). Total IgE increased by 280.1% in OVA + PM over the healthy control (p < 0.05), and PM augmented the serum IgE level of OVA-sensitised mice by 68.3% over OVA alone (p < 0.05). High-dose fucoidan treatment attenuated the PM-exacerbated level considerably, by 33.2% (p < 0.005), though the paper notes fucoidans were not as effective as prednisone. No absolute IgE concentrations are reported.

Exposure conditions

Outputs

Evidence

OVA-specific IgE in serum (ELISA)

OVA-specific IgE measured by ELISA in serum (Figure 8B). The OVA-specific IgE level was markedly increased in OVA + PM over the healthy control (p < 0.0005) and 18.90% above the OVA only group (p < 0.05). A high dose of fucoidans significantly attenuated the OVA-specific IgE level by 56.3% relative to OVA + PM (p < 0.005) and was more effective than prednisone. No absolute concentrations are reported.

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Evidence

OVA-specific IgG1 in serum (ELISA)

OVA-specific IgG1 measured by ELISA on serum diluted 1:2000 (Figure 8C). OVA + PM moderately increased the OVA-specific IgG1 level compared with OVA only, and high-dose fucoidan treatment attenuated it significantly, by 90.0% relative to OVA + PM (p < 0.05), similarly to prednisone. No absolute concentrations are reported.

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Outputs

Evidence

OVA-specific IgG2a in serum (ELISA)

OVA-specific IgG2a measured by ELISA in serum (Figure 8D). This is the one immunoglobulin endpoint fucoidans did not change: the paper states plainly that fucoidans did not affect the OVA-specific IgG2a level. It is recorded here so that the negative result travels with the positive ones rather than being dropped. No absolute concentrations and no percent changes are reported for this endpoint, so it has no response_comparisons entry.

Exposure conditions

Outputs

Evidence

IL-4, IL-17a and IL-33 in serum (ELISA)

IL-4, IL-17a and IL-33 measured by ELISA in serum (Figure 12A-C). IL-4 was significantly increased in OVA + PM over the healthy control (p < 0.005) and attenuated by high-dose fucoidans (p < 0.05); IL-17a was appreciably increased in OVA + PM and attenuated by high-dose fucoidan without reaching significance; serum IL-33 was the one cytokine fucoidans did not attenuate. The paper reports no absolute concentrations, so these output records carry no magnitude.

Exposure conditions

Outputs

Evidence

IL-4, IL-17a and IL-33 in lung homogenate (ELISA)

IL-4, IL-17a and IL-33 measured by ELISA in lung homogenate supernatant (Figure 12G-I). IL-4 was significantly increased in OVA + PM over the healthy control (p < 0.05) and attenuated by high-dose fucoidans (p < 0.05); IL-17a was appreciably increased and attenuated without reaching significance; IL-33 was significantly increased in the lungs of OVA + PM mice and attenuated by high-dose fucoidan, which the authors read as contributing to the attenuated Th2 response. No absolute concentrations are reported.

Exposure conditions

Outputs

Evidence

MDA positive cell percentage in lung, OVA + PM vs healthy control

PM exposure as well as OVA significantly enhanced the MDA positive cell percentage in the lungs relative to the healthy control. Reported significance threshold: p < 0.0005. The paper states no magnitude for this change, so change_type and change_value are omitted.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

MDA positive cell percentage in lung, OVA + PM + fucoidans 400 mg/kg vs OVA + PM

Fucoidans dose-dependently attenuated the MDA positive cell percentage in the lungs compared with OVA + PM. The paper reports no significance threshold for this comparison. The paper states no magnitude for this change, so change_type and change_value are omitted.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Lung MDA, PM only vs healthy control

Lung MDA increased by 88.4% in the PM only group relative to the healthy control. Reported significance threshold: p < 0.05.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Lung MDA, OVA only vs healthy control

Lung MDA increased by 88.6% in the OVA only group relative to the healthy control. Reported significance threshold: p < 0.05.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Lung MDA, OVA + PM vs healthy control

Lung MDA increased by 89.5% in the OVA + PM group relative to the healthy control. Reported significance threshold: p < 0.05.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Lung MDA, OVA + PM + fucoidans 100 mg/kg vs OVA + PM

Lung MDA fell by 51.2% with low-dose (100 mg/kg) fucoidan treatment relative to OVA + PM. The paper reports no significance threshold for this comparison. The paper reports the threshold p < 0.05 for the high-dose reduction only.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Lung MDA, OVA + PM + fucoidans 400 mg/kg vs OVA + PM

Lung MDA fell by 70.4% with high-dose (400 mg/kg) fucoidan treatment relative to OVA + PM. Reported significance threshold: p < 0.05.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Serum MDA, PM only vs healthy control

Serum MDA increased by 74.6% in the PM only group relative to the healthy control. Reported significance threshold: p < 0.005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Serum MDA, OVA + PM vs healthy control

Serum MDA increased by 74.6% in the OVA + PM group relative to the healthy control - the same percentage the paper reports for PM only. Reported significance threshold: p < 0.005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Serum MDA, OVA + PM + fucoidans 100 mg/kg vs OVA + PM

Serum MDA fell by 44.8% with low-dose (100 mg/kg) fucoidan treatment relative to OVA + PM. Reported significance threshold: p < 0.0005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Serum MDA, OVA + PM + fucoidans 400 mg/kg vs OVA + PM

Serum MDA fell by 78.0% with high-dose (400 mg/kg) fucoidan treatment relative to OVA + PM. Reported significance threshold: p < 0.0005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Serum 8-OHdG, OVA only vs healthy control

Serum 8-OHdG increased by 81.4% in the OVA only group relative to the healthy control. Reported significance threshold: p < 0.005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Serum 8-OHdG, PM only vs healthy control

Serum 8-OHdG increased by 122.2% in the PM only group relative to the healthy control. Reported significance threshold: p < 0.005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Serum 8-OHdG, OVA + PM vs healthy control

Serum 8-OHdG increased by 116.8% in the OVA + PM group relative to the healthy control. Reported significance threshold: p < 0.005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Serum 8-OHdG, OVA + PM + fucoidans 100 mg/kg vs OVA + PM

Serum 8-OHdG fell by 11.8% with low-dose fucoidan treatment relative to OVA + PM. The paper states explicitly that this reduction was not significant.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Serum 8-OHdG, OVA + PM + fucoidans 400 mg/kg vs OVA + PM

Serum 8-OHdG fell by 11.8% with high-dose fucoidan treatment relative to OVA + PM - the paper reports the same percentage for both fucoidan doses and states explicitly that the reduction was not significant.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Serum 8-OHdG, OVA + PM + prednisone vs OVA + PM

Serum 8-OHdG fell by 25.7% with prednisone relative to OVA + PM. Reported significance threshold: p < 0.05. The authors contrast this with the non-significant fucoidan effect on the same marker and infer that fucoidans and prednisone attenuate PM-induced oxidative stress through different signalling pathways.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Airway hyper-responsiveness (Penh), OVA + PM vs OVA only

PM exacerbated OVA-induced airway hyper-responsiveness by 20.7%. Reported significance threshold: p < 0.05. Measured at the high methacholine concentration (50 mg/mL).

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Airway hyper-responsiveness (Penh), OVA + PM + fucoidans 400 mg/kg vs OVA + PM

High-dose fucoidans markedly attenuated the PM-exacerbated airway hyper-responsiveness by 43.0% at 50 mg/mL methacholine. Reported significance threshold: p < 0.0005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

BALF eosinophil infiltration, OVA + PM + fucoidans 400 mg/kg vs OVA + PM

High-dose fucoidans significantly attenuated the PM-exacerbated infiltration of eosinophils in BALF. Reported significance threshold: p < 0.05. The paper reports the BALF differential as figure bars with no counts or percentages, so change_type and change_value are omitted.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

BALF neutrophil infiltration, OVA + PM + fucoidans 400 mg/kg vs OVA + PM

High-dose fucoidans significantly attenuated the PM-exacerbated infiltration of neutrophils in BALF. Reported significance threshold: p < 0.05. The paper reports the BALF differential as figure bars with no counts or percentages, so change_type and change_value are omitted.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

BALF lymphocyte infiltration, OVA + PM + fucoidans 400 mg/kg vs OVA + PM

High-dose fucoidans significantly attenuated the PM-exacerbated infiltration of lymphocytes in BALF. Reported significance threshold: p < 0.05. The paper reports the BALF differential as figure bars with no counts or percentages, so change_type and change_value are omitted.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

BALF macrophage count, OVA + PM + fucoidans 400 mg/kg vs OVA + PM

Fucoidans dose-dependently attenuated the PM-exacerbated macrophage count in BALF, but the paper states explicitly that this reduction was not significant. No magnitude is reported.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Trachea eosinophil infiltration, OVA + PM vs healthy control

PM increased eosinophil infiltration in the trachea of OVA-sensitised mice by 500.0% relative to the healthy control. Reported significance threshold: p < 0.0005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Trachea eosinophil infiltration, OVA + PM vs OVA only

OVA + PM exacerbated eosinophil infiltration in the trachea by 56.4% relative to the OVA only group. Reported significance threshold: p < 0.005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Trachea eosinophil infiltration, OVA + PM + fucoidans 100 mg/kg vs OVA + PM

Low-dose fucoidan treatment suppressed PM-exacerbated eosinophil infiltration in the trachea by 52.0% relative to OVA + PM. The paper reports no significance threshold for this comparison.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Trachea eosinophil infiltration, OVA + PM + fucoidans 400 mg/kg vs OVA + PM

High-dose fucoidan treatment suppressed PM-exacerbated eosinophil infiltration in the trachea by 72.0% relative to OVA + PM. The paper reports no significance threshold for this comparison.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Lung eosinophil infiltration, OVA + PM vs healthy control

Eosinophil infiltration in the lungs was 566.6% above the healthy control in the OVA + PM group. Reported significance threshold: p < 0.0005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Lung eosinophil infiltration, OVA + PM vs OVA only

Eosinophil infiltration in the lungs was 74.0% above the OVA only group in the OVA + PM group. Reported significance threshold: p < 0.005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Lung eosinophil infiltration, OVA + PM + fucoidans 100 mg/kg vs OVA + PM

Low-dose fucoidan treatment suppressed eosinophil infiltration in the lungs by 45.0% relative to OVA + PM. The paper reports no significance threshold for this comparison.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Lung eosinophil infiltration, OVA + PM + fucoidans 400 mg/kg vs OVA + PM

High-dose fucoidan treatment suppressed eosinophil infiltration in the lungs by 65.0% relative to OVA + PM, as effectively as prednisone. The paper reports no significance threshold for this comparison.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Trachea Gr-1+ granulocyte influx, OVA + PM vs healthy control

The OVA + PM group enhanced the influx of granulocytes in the trachea by 200.0% relative to the healthy control. Reported significance threshold: p < 0.0005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Trachea Gr-1+ granulocyte influx, OVA + PM vs OVA only

PM exacerbated Gr-1+ cell infiltration by 52.5% in the trachea of OVA-sensitised mice relative to OVA alone. The paper reports no significance threshold for this comparison.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Trachea Gr-1+ granulocyte influx, OVA + PM + fucoidans 100 mg/kg vs OVA + PM

Low-dose fucoidan treatment reduced the PM-exacerbated influx of Gr-1+ cells in the trachea by 36.6% relative to OVA + PM. The paper reports no significance threshold for this comparison.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Trachea Gr-1+ granulocyte influx, OVA + PM + fucoidans 400 mg/kg vs OVA + PM

High-dose fucoidan treatment reduced the PM-exacerbated influx of Gr-1+ cells in the trachea by 53.3% relative to OVA + PM. The paper reports no significance threshold for this comparison.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Trachea Gr-1+ granulocyte influx, OVA + PM + prednisone vs OVA + PM

Prednisone attenuated the granulocyte influx in the trachea by 55.5% relative to OVA + PM. The paper reports no significance threshold for this comparison.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Lung Gr-1+ granulocyte infiltration, OVA + PM vs healthy control

Granulocyte infiltration in the lungs increased by 247.8% in OVA + PM relative to the healthy control. The paper reports no significance threshold for this comparison.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Lung Gr-1+ granulocyte infiltration, OVA + PM vs OVA only

PM exacerbated OVA-induced granulocyte infiltration in the lungs by 113.3% relative to OVA alone. Reported significance threshold: p < 0.0005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Lung Gr-1+ granulocyte infiltration, OVA + PM + fucoidans 400 mg/kg vs OVA + PM

High-dose fucoidan treatment significantly reduced granulocyte infiltration in the lung by 66.4% relative to OVA + PM, and the paper states fucoidans were more effective than prednisone on this endpoint. Reported significance threshold: p < 0.0005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Trachea F4/80+ macrophage infiltration, OVA + PM vs healthy control

The OVA + PM group showed clearly enhanced macrophage infiltration in the trachea, +250.0% relative to the healthy control. Reported significance threshold: p < 0.0005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Trachea F4/80+ macrophage infiltration, OVA + PM + fucoidans 100 mg/kg vs OVA + PM

Low-dose fucoidan treatment significantly reduced macrophage infiltration in the trachea by 34.7% relative to OVA + PM. Reported significance threshold: p < 0.05.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Trachea F4/80+ macrophage infiltration, OVA + PM + fucoidans 400 mg/kg vs OVA + PM

High-dose fucoidan treatment significantly reduced macrophage infiltration in the trachea by 45.7% relative to OVA + PM. Reported significance threshold: p < 0.0005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Lung F4/80+ macrophage infiltration, OVA + PM vs healthy control

F4/80+ macrophage infiltration in the lungs of PM + OVA mice increased by 515.3% relative to the healthy control. Reported significance threshold: p < 0.0005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Lung F4/80+ macrophage infiltration, OVA + PM vs OVA only

PM exacerbated macrophage infiltration in the lungs of OVA-sensitised mice by 58.4% relative to OVA alone. Reported significance threshold: p < 0.005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Lung F4/80+ macrophage infiltration, OVA + PM + fucoidans 100 mg/kg vs OVA + PM

Low-dose fucoidan treatment significantly attenuated PM-exacerbated macrophage infiltration in the lungs by 74.3% relative to OVA + PM. Reported significance threshold: p < 0.0005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Lung F4/80+ macrophage infiltration, OVA + PM + fucoidans 400 mg/kg vs OVA + PM

High-dose fucoidan treatment significantly attenuated PM-exacerbated macrophage infiltration in the lungs by 81.2% relative to OVA + PM. Reported significance threshold: p < 0.0005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Trachea CD4+ T cell infiltration, OVA + PM vs healthy control

CD4+ T cells in the trachea increased by 64.1% in OVA + PM relative to the healthy control. Reported significance threshold: p < 0.005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Trachea CD4+ T cell infiltration, OVA + PM + fucoidans 100 mg/kg vs OVA + PM

Low-dose fucoidan treatment reduced CD4+ T cell infiltration in the trachea by 15.4% relative to OVA + PM. The paper reports no significance threshold for this comparison.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Trachea CD4+ T cell infiltration, OVA + PM + fucoidans 400 mg/kg vs OVA + PM

High-dose fucoidan treatment reduced CD4+ T cell infiltration in the trachea by 56.4% relative to OVA + PM. Reported significance threshold: p < 0.05.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Lung CD4+ T cell infiltration, OVA + PM vs healthy control

CD4+ T cell infiltration in the lungs increased by 197.8% in OVA + PM relative to the healthy control. The paper reports no significance threshold for this comparison.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Lung CD4+ T cell infiltration, OVA + PM vs OVA only

PM exacerbated OVA-induced CD4+ T cell infiltration in the lungs by 24.5% relative to OVA alone, but the paper reports this at p < 0.09 - above the p < 0.05 criterion it sets in its own statistics section, so this change is NOT statistically significant.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Lung CD4+ T cell infiltration, OVA + PM + fucoidans 400 mg/kg vs OVA + PM

High-dose fucoidan treatment significantly reduced CD4+ T cell infiltration in the lungs by 59.1% relative to OVA + PM. Reported significance threshold: p < 0.0005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Activated mast cells in trachea, OVA + PM vs healthy control

Activated mast cells in the trachea increased by 123.5% in OVA + PM relative to the healthy control. Reported significance threshold: p < 0.0005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Activated mast cells in trachea, OVA + PM + fucoidans 100 mg/kg vs OVA + PM

Low-dose fucoidan treatment reduced mast cell activation by 28.9% relative to OVA + PM. The paper reports no significance threshold for this comparison.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Activated mast cells in trachea, OVA + PM + fucoidans 400 mg/kg vs OVA + PM

High-dose fucoidan treatment reduced mast cell activation by 50.0% relative to OVA + PM. Reported significance threshold: p < 0.005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Degranulated mast cells in trachea, OVA + PM vs OVA only

PM exacerbated OVA-induced mast cell degranulation by 133.6% relative to OVA alone. The paper reports no significance threshold for this comparison.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Degranulated mast cells in trachea, OVA + PM + fucoidans 100 mg/kg vs OVA + PM

Low-dose fucoidan treatment reversed the PM-induced degranulation of mast cells in the trachea by 51.9% relative to OVA + PM. The paper reports no significance threshold for this comparison.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Degranulated mast cells in trachea, OVA + PM + fucoidans 400 mg/kg vs OVA + PM

High-dose fucoidan treatment reversed the PM-induced degranulation of mast cells in the trachea by 76.7% relative to OVA + PM, as effectively as prednisone. Reported significance threshold: p < 0.005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Total serum IgE, OVA + PM vs healthy control

Total serum IgE increased by 280.1% in OVA + PM relative to the healthy control. Reported significance threshold: p < 0.05.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Total serum IgE, OVA + PM vs OVA only

PM augmented the serum IgE level of OVA-sensitised mice by 68.3% relative to OVA alone. Reported significance threshold: p < 0.05.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Total serum IgE, OVA + PM + fucoidans 400 mg/kg vs OVA + PM

High-dose fucoidan treatment attenuated the PM-exacerbated serum IgE level by 33.2% relative to OVA + PM, though the paper states fucoidans were not as effective as prednisone. Reported significance threshold: p < 0.005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

OVA-specific serum IgE, OVA + PM vs OVA only

The OVA-specific IgE level was 18.90% higher in OVA + PM than in the OVA only group. Reported significance threshold: p < 0.05.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

OVA-specific serum IgE, OVA + PM + fucoidans 400 mg/kg vs OVA + PM

High-dose fucoidan treatment significantly attenuated the OVA-specific IgE level by 56.3% relative to OVA + PM and was more effective than prednisone. Reported significance threshold: p < 0.005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

OVA-specific serum IgG1, OVA + PM + fucoidans 400 mg/kg vs OVA + PM

High-dose fucoidan treatment attenuated the OVA-specific IgG1 level significantly, by 90.0% relative to OVA + PM, similarly to prednisone. Reported significance threshold: p < 0.05.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Trachea PAS positive cell proportion, OVA + PM vs healthy control

PAS staining of the trachea showed a 287.5% increase in goblet cell hyperplasia and mucus secretion in OVA + PM relative to the healthy control. Reported significance threshold: p < 0.0005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Trachea PAS positive cell proportion, OVA + PM + fucoidans 100 mg/kg vs OVA + PM

Low-dose fucoidan treatment attenuated the PAS positive cell proportion in the trachea by 9.1% relative to OVA + PM. The paper reports no significance threshold for this comparison. The paper reports the threshold p < 0.005 for the high-dose reduction only.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Trachea PAS positive cell proportion, OVA + PM + fucoidans 400 mg/kg vs OVA + PM

High-dose fucoidan treatment attenuated the PAS positive cell proportion in the trachea by 36.7% relative to OVA + PM. Reported significance threshold: p < 0.005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Lung PAS positive cell proportion, OVA + PM vs healthy control

Mucus secretion and goblet cell hyperplasia in the lungs increased by 378.0% in OVA + PM relative to the healthy control. Reported significance threshold: p < 0.0005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Lung PAS positive cell proportion, OVA + PM vs OVA only

PM clearly induced PAS positive cells in the lungs, by 49.0% relative to OVA alone. Reported significance threshold: p < 0.005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Lung PAS positive cell proportion, OVA + PM + fucoidans 100 mg/kg vs OVA + PM

Low-dose fucoidan treatment reduced the number of PAS positive cells in the lungs by 35.2% relative to OVA + PM. Reported significance threshold: p < 0.005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Lung PAS positive cell proportion, OVA + PM + fucoidans 400 mg/kg vs OVA + PM

High-dose fucoidan treatment reduced the number of PAS positive cells in the lungs by 66.8% relative to OVA + PM. Reported significance threshold: p < 0.0005.

Statistical test: one-way analysis of variance followed by Tukey's multiple comparison test

Increased oxidative stress leads to airway inflammation

The paper's framing of its own exposure arm: PM inhalation triggers oxidative stress, which drives the inflammation that then sustains airway injury. The oxidative markers (lung and serum MDA, serum 8-OHdG) and the inflammatory endpoints (BALF differential, eosinophil, granulocyte, macrophage and CD4+ T cell infiltration) rose together in the same animals, and oral fucoidans lowered both. The causal step itself is argued from the literature rather than tested here - there is no antioxidant-only arm that would separate the oxidative stress from the inflammation - so the support is recorded as moderate.

Airway inflammation is accompanied by increased systemic inflammation

The same exposure that raised airway inflammatory endpoints also raised serum total IgE, OVA-specific IgE and IgG1, serum IL-4 and serum IL-17a, and high-dose fucoidans lowered both sets together. The paper measures the airway and systemic compartments in parallel without testing which drives which, so this is recorded as an association with weak support rather than a causal step.

Airway inflammation leads to goblet cell hyperplasia

The paper states its own causal reading explicitly: the goblet cell hyperplasia and increased mucus secretion seen in PM-exposed mice are taken to be a consequence of the PM-exacerbated Th2/Th17 response and macrophage infiltration. Both sides were measured in the same animals - the inflammatory cell infiltration by BALF differential and immunohistochemistry, the hyperplasia by PAS staining of trachea and lung - and both fell together under fucoidan treatment.

Goblet cell hyperplasia leads to mucin hypersecretion

PAS staining measured goblet cell hyperplasia and mucus secretion as one endpoint in both trachea and lung, and the paper separately reports that PM exposure enhanced mucus secretion with enlarged submucosal mucous glands, which fucoidans dose-dependently reversed (Figure 11). The submucosal gland observation is the paper's only separate evidence for the secretory side and is qualitative, with no score or magnitude, so this relationship is recorded with weak support.

Mucin hypersecretion leads to decreased lung function

The adverse-outcome end of the pathway. Airway hyper-responsiveness measured as Penh rose in the PM only, OVA only and OVA + PM groups and fell by 43.0% with high-dose fucoidans, alongside the fall in PAS positive cells; the paper argues the mechanism from the clinical literature - mucus plugs obstructing the airways and mediators inducing bronchoconstriction - rather than testing it, so support is weak.

Airway inflammation leads to decreased lung function

Eosinophil influx is the specific inflammatory step the paper places upstream of airway hyper-responsiveness, and both were measured here: Congo red eosinophil counts rose 500.0% in the trachea and 566.6% in the lungs of OVA + PM mice, while Penh rose significantly over the healthy control in the same groups and fell 43.0% under high-dose fucoidans. The link is argued from the pattern and the literature rather than from an intervention that separates the two, so support is moderate.

PM-exacerbated mouse model of allergic airway inflammation

Mice (n = 7 per group) randomly divided into seven groups: healthy control, PM only, OVA only, OVA + PM, OVA + PM + fucoidans (100 mg/kg), OVA + PM + fucoidans (400 mg/kg), and OVA + PM + prednisone (5 mg/kg). Mice in the OVA induction groups were sensitised with OVA (10 ug) dissolved in 200 uL saline and 2 mg Al(OH)3 by intraperitoneal injection. Two weeks after sensitisation, mice in groups other than the healthy control and OVA only groups were nebulised with PM (5 mg/m3, 30 min/day) daily for 7 consecutive days. Together with PM exposure the PM-exposed mice were orally gavaged with either a low dose (100 mg/kg) or high dose (400 mg/kg) of fucoidans, or prednisone (5 mg/kg), suspended in saline; other groups were orally administered saline. The protocol follows a previously described model with minor modifications. Fucoidans were supplied by Haewon Biotech Co., Ltd. (Seoul, Korea). PM was certified reference material (CRM) No. 28 urban aerosol from the National Institute for Environmental Studies (NIES), Japan, the majority of which was <2.5 um in diameter. Animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Institutional Ethical Committee of Jeju National University.

Trachea and lung fixation, embedding and sectioning

Mice were sacrificed one day after the final exposure to PM (day 22) and lung and trachea sections were fixed in 10% formalin. After fixation, samples were embedded in paraffin and sectioned at 3 um onto coated glasses.

Lung tissue homogenisation for lipid peroxidation and cytokine assays

Lung tissues homogenised using a tacoPrep bead beater (GeneReach Biotechnology Co., Taichung City, Taiwan) in RIPA buffer containing protease inhibitors (2 mM Na3VO4, 1 mM PMSF, 10 ug/mL aprotinin, 10 ug/mL leupeptin). The homogenate was centrifuged at 3000x g for 10 min at 4 C and the supernatant used for assay; protein concentration in the supernatant was measured by the Bradford protein assay method.

Serum separation from cardiac-puncture blood

Blood was collected from anaesthetised mice on day 22 via cardiac puncture using a heparinised syringe. Serum was separated from blood cells by centrifuging the collected blood at 12,000 rpm for 10 min at 4 C.

Bronchoalveolar lavage fluid collection

1 mL of DPBS was instilled into the lungs (intra-tracheal) using a cannula and BALF gently collected. For differential cell counts the collected BALF was centrifuged at 300x g for 5 min at 4 C and the supernatant separated for cytokine analysis; for the cytokine ELISAs reported in the Methods the collected BALF was centrifuged at 12,000 rpm for 10 min at 4 C and the supernatant analysed.

Thiobarbituric acid assay for lipid peroxidation in lung homogenate

An equal amount of protein from the lung homogenate supernatant was mixed with 10% (w/v) trichloroacetic acid and incubated for 10 min at 4 C. After centrifuging at 2000x g for 15 min at 4 C the resulting supernatant was reacted with an equal volume of 0.67% (w/v) thiobarbituric acid in a heating block for 10 min. The supernatant was separated by centrifuging at 2000x g for 15 min at 4 C and its absorbance measured at 532 nm using a microplate reader. The malondialdehyde (MDA) content in the samples was measured with the predetermined MDA standard curve.

Equipment: tacoPrep bead beater (GeneReach Biotechnology Co., Taichung City, Taiwan); Microplate reader, absorbance at 532 nm

Lipid peroxidation assay kit for serum malondialdehyde

The MDA level in mouse serum was measured using a lipid peroxidation assay kit following the manufacturer's instructions. MDA activity was measured at the micromolar level based on the standard curve using a colorimetric assay.

DNA/RNA oxidative damage ELISA for serum 8-OHdG

Serum 8-OHdG-acetylcholinesterase conjugate (DNA/RNA oxidative damage tracer) measured with the DNA/RNA oxidative damage ELISA kit (Cayman Chemicals, Ann Arbor, MI, USA) following the manufacturer's recommendations.

Whole-body plethysmography with methacholine challenge

After 24 h of the final challenge with PM the mice were nebulised with different concentrations of methacholine (0, 12.5, 25, 50 mg/mL) for 10 min in a whole body chamber. Immediately after each nebulisation the enhanced pause (Penh) was measured in a plethysmography chamber (Allmedicus, Anyang, Korea). The readings were averaged for a 3 min period.

Diff Quick differential cell count on BALF smears

The collected BALF was centrifuged (300x g, 5 min, at 4 C) and the supernatant separated for cytokine analysis. The cells were fixed in methanol and smears prepared on glass slides. The smears were stained with a Diff Quick staining kit (Sysmex Corporation, Kobe, Japan) and the cell compositions of macrophages, eosinophils, neutrophils, lymphocytes and basophils determined using a microscope with an Olympus DP-72 microscope camera (Olympus, Tokyo, Japan).

Haematoxylin and eosin staining with inflammation grading

Tissue sections were stained with haematoxylin and eosin (H&E) and observed under a light microscope (Olympus, Tokyo, Japan) for histopathological examinations. Inflammation in trachea and lung sections was scored as follows: 0, normal; 1, few inflammatory cells infiltrated; 2, observed inflammatory cells one cell layer deep; 3, observed inflammatory cells 2-4 cell layers deep; and 4, infiltrated inflammatory cells of more than four cell layers deep.

Congo red staining for eosinophil infiltration

Infiltration of eosinophils in the lung and trachea tissue sections was confirmed by Congo red staining. Deparaffinised tissues were immersed in Congo red working solution for 30 min, washed in running tap water and counterstained with 0.5% methyl green. Stained sections were observed under a microscope and representative areas captured using an Olympus DP-72 microscope camera (Olympus, Tokyo, Japan). The number of Congo red stained eosinophils per site from at least three sites of each section (3 sections were taken from each mouse) was quantified using ImageJ (v1.46) software.

Toluidine blue staining for mast cell count and degranulation

Mast cell infiltration in the trachea was evaluated using toluidine blue staining. Deparaffinised trachea sections were immersed in 0.5% toluidine blue solution and counterstained with 0.5% methyl green. Stained sections were observed under a microscope and representative areas captured using an Olympus DP-72 microscope camera (Olympus, Tokyo, Japan). The number of mast cells per site and the number of degranulated mast cells per site from at least three sites of each section (3 sections were taken from each mouse) were quantified using ImageJ (v1.46) software.

Periodic acid-Schiff staining for goblet cells and mucus secretion

Trachea and lung sections were stained with periodic acid-Schiff (PAS) and counterstained with 0.5% methyl green to evaluate goblet cell metaplasia and mucus secretion. The PAS-positive cell percentage from at least three sites of each section (3 sections were taken from each mouse) was quantified using ImageJ (v1.46) software.

Immunohistochemistry for malondialdehyde adducts

Trachea and lung sections on glass slides were de-paraffinised in a xylene series and rehydrated in an ethanol series. Nonspecific binding was blocked by immersing tissues in 0.3% hydrogen peroxide with rabbit or horse serum for 30 min. Following blocking, tissues were incubated with MDA antibody (1:400) overnight at 4 C. After washing, tissues were incubated with biotinylated anti-rat IgG or anti-rabbit IgG for 45 min followed by treatment with an avidin-biotin peroxidase complex using the Vectastain Elite ABC kit (Vector Inc., Burlingame, CA, USA). HRP binding sites were detected with 3,3'-diaminobenzidine (DAB, Vector Inc.). Images were captured using an Olympus DP-72 microscope camera (Olympus, Tokyo, Japan). The number of positive cells per site from at least three sites of each section (3 sections were taken from each mouse) was acquired with ImageJ (v1.46) software.

Immunohistochemistry for Gr-1+ granulocytes

As for the MDA immunohistochemistry protocol, with mouse Gr-1 antibody (1:200) as the overnight primary antibody; the paper states the secondary step as a disjunction - biotinylated anti-rat IgG or anti-rabbit IgG for 45 min - without saying which goes with which primary, so no assignment is made here.

Immunohistochemistry for F4/80+ macrophages

As for the MDA immunohistochemistry protocol, with purified anti-mouse F4/80 antibody (1:200) as the overnight primary antibody; the paper states the secondary step as a disjunction - biotinylated anti-rat IgG or anti-rabbit IgG for 45 min - without saying which goes with which primary, so no assignment is made here.

Immunohistochemistry for CD4+ T lymphocytes

As for the MDA immunohistochemistry protocol, with anti-CD4 antibody (1:400) as the overnight primary antibody; the paper states the secondary step as a disjunction - biotinylated anti-rat IgG or anti-rabbit IgG for 45 min - without saying which goes with which primary, so no assignment is made here.

ELISA for total serum IgE

Total IgE in serum measured with an ELISA kit (Bethyl Lab, Montgomery, TX, USA) following the manufacturer's recommendations.

ELISA for OVA-specific serum IgE

OVA-specific IgE in serum measured with an ELISA kit (BioLegend, San Diego, CA, USA) following the manufacturer's recommendations.

ELISA for OVA-specific serum IgG1

OVA-specific IgG1 measured with an ELISA kit (Cayman Chemicals, Ann Arbor, MI, USA) on serum diluted 1:2000, following the manufacturer's recommendations.

ELISA for OVA-specific serum IgG2a

OVA-specific IgG2a measured with an ELISA kit (BioLegend, San Diego, CA, USA) following the manufacturer's recommendations.

ELISA for IL-4, IL-17a and IL-33 in serum, BALF and lung homogenate

IL-4 and IL-17a measured with ELISA kits from BioLegend (San Diego, CA, USA) and IL-33 with the Mouse IL-33 ELISA kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), each following the manufacturer's recommendations. The same three kits were applied to serum, to BALF supernatant and to lung homogenate supernatant.