Additional file 1 of White blood cells and chronic rhinosinusitis: a Mendelian randomization study

Additional file 1: Figure S1. F-statistics of IV SNPs used in MR Analysis. Figure S2. A SNP effect on neutrophil count and CRS. Two-sample MR analyses performed using GWAS summary statistics for neutrophil count (exposure trait) and CRS (outcome trait). Primary analysis was performed using inverse-variance weighted two-sample MR. B SNP effect on lymphocyte count and CRS. Two-sample MR analyses performed using GWAS summary statistics for lymphocyte count (exposure trait) and CRS (outcome trait). Primary analysis was performed using inverse-variance weighted two-sample MR. C SNP effect on monocyte count and CRS. Two-sample MR analyses performed using GWAS summary statistics for monocyte count (exposure trait) and CRS (outcome trait). Primary analysis was performed using inverse-variance weighted two-sample MR. D SNP effect on basophil count and CRS. Two-sample MR analyses performed using GWAS summary statistics for basophil count (exposure trait) and CRS (outcome trait). Primary analysis was performed using inverse-variance weighted two-sample MR. Figure S3. A SNP effect on neutrophil count and CRS. Two-sample MR analysis performed using GWAS summary statistics for neutrophil count (exposure trait) and CRS (outcome trait). Inverse variance weighted MR results: OR 1.04, 95% CI (0.94, 1.15), p=0.49. B SNP effect on lymphocyte count and CRS. Two-sample MR analysis performed using GWAS summary statistics for lymphocyte count (exposure trait) and CRS (outcome trait). Inverse variance weighted MR results: OR 1.08, 95% CI (0.97, 1.20), p=0.17. C SNP effect on monocyte count and CRS. Two-sample MR analysis performed using GWAS summary statistics for monocyte count (exposure trait) and CRS (outcome trait). Inverse variance weighted MR results: OR 1.12, 95% CI (1.04, 1.20), p=0.002. D SNP effect on basophil count and CRS. Two-sample MR analysis performed using GWAS summary statistics for basophil count (exposure trait) and CRS (outcome trait). Inverse variance weighted MR results: OR 1.12, 95% CI (0.97, 1.29), p=0.12.Figure S4. Funnel plots for white blood cell types

Additional file 2 of White blood cells and chronic rhinosinusitis: a Mendelian randomization study

Additional file 2: Table S1. Egger intercept test

B Cells Play Key Roles in Th2-Type Airway Immune Responses in Mice Exposed to Natural Airborne Allergens

<div><p>Humans are frequently exposed to various airborne allergens. In addition to producing antibodies, B cells participate in immune responses via various mechanisms. The roles of B cells in allergic airway inflammation and asthma have been controversial. We examined the functional importance of B cells in a mouse model of asthma, in which mice were exposed repeatedly to common airborne allergens. Naïve wild-type BALB/c mice or B cell-deficient JH^−/− mice were exposed intranasally to a cocktail of allergen extracts, including <i>Alternaria</i>, <i>Aspergillus</i>, and house dust mite, every other day for two weeks. Ovalbumin was included in the cocktail to monitor the T cell immune response. Airway inflammation, lung pathology, and airway reactivity were analyzed. The airway exposure of naïve wild type mice to airborne allergens induced robust eosinophilic airway inflammation, increased the levels of Th2 cytokines and chemokines in the lung, and increased the reactivity to inhaled methacholine. These pathological changes and immune responses were attenuated in B cell-deficient JH^−/− mice. The allergen-induced expansion of CD4⁺ T cells was impaired in the lungs and draining lymph nodes of JH^−/− mice. Furthermore, lymphocytes from JH^−/− mice failed to produce Th2 cytokines in response to ovalbumin re-stimulation <i>in vitro</i>. Our results suggest that B cells are required for the optimal development of Th2-type immune responses and airway inflammation when exposed to common airborne allergens. The therapeutic targeting of B cells may be beneficial to treat asthma in certain patients.</p></div

Airway reactivity is reduced in JH^-/- mice.

<p>WT or JH^-/- mice were intranasally exposed to OAAH or PBS for 2 weeks, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121660#pone.0121660.g001" target="_blank">Fig. 1A</a>. (A) Airway reactivity to inhaled methacholine was assessed by whole-body plethysmography, as described in the Methods section. Results are presented as the percentage of baseline (i.e. before PBS or methacholine challenge) and as mean±SEM (n = 4 and 6 in PBS and OAAH, respectively). *, p<0.05, between WT mice and JH^-/- mice exposed to OAAH. (B) Lung sections were stained with the periodic acid-Schiff stain. Original magnification, 160×. Scale bars, 50 μm. Data are representative of two independent experiments.</p

Th2-type cellular and humoral immune responses are decreased in JH^-/- mice.

<p>WT or JH^-/- mice were intranasally exposed to OAAH or PBS for 2 weeks, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121660#pone.0121660.g001" target="_blank">Fig. 1A</a>. (A) Cytokine levels in lung homogenates were determined by ELISA. (B) Blood was collected from these mice 24 hours after the last allergen exposure. The antibody levels in the plasma were analyzed by ELISA. (C) Single-cell suspensions of the lung were stained with anti-CD3 and anti-CD4 antibodies and analyzed by flow cytometry. Cell numbers were calculated based on cell counts and percentages of CD3⁺ and CD4⁺ cells. Results are presented as mean±SEM (n = 5 mice per group). *, p<0.05; **, p<0.01, between the groups indicated by horizontal lines. Data are representative of two independent experiments.</p

Th2-type responses in draining lymph nodes are attenuated in JH^-/- mice.

<p>WT or JH-^/- mice were intranasally exposed to OAAH for 2 weeks, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121660#pone.0121660.g001" target="_blank">Fig. 1A</a>. (A) Mediastinal lymph nodes (MLNs) were harvested, and cells were stained with anti-CD3 and anti-CD4 antibodies and analyzed by flow cytometry. Results are presented as mean±SEM (n = 5 mice per group). *, p<0.05; **, p<0.01. Data are representative of two independent experiments. (B) MLN cells were cultured with medium alone or with OVA (100 μg/ml) in 96-well tissue culture plates for 5 days. The levels of cytokines in the culture supernatants were analyzed by ELISA. *; p<0.05 between the groups indicated by horizontal lines. Data (mean±range, n = 2) are representative of three independent experiments.</p

Allergen exposure-induced airway inflammation is attenuated in B cell-deficient JH^-/- mice.

<p>(A) The experimental protocol is shown. Wild-type (WT) or JH^-/- mice were intranasally exposed to a cocktail of allergens (OAAH; ovalbumin, <i>Alternaria</i>, <i>Aspergillus</i>, house dust mite) on days 0, 2, 4, 7, 9, 11, and 14, and they were analyzed on day 15. (B) Cell differentials in BAL fluids were counted and presented as mean±standard error of the mean (SEM; n = 4 and 6 in PBS and OAAH, respectively). **, p<0.01, between WT BALB/c and JH^-/- mice exposed to OAAH. (C) Lung sections were stained with hematoxylin and eosin stains (upper and middle panels) or with the anti-mouse MBP antibody (lower panels). Original magnification, 160×. Scale bars, 50 μm. Data are representative of two independent experiments. (D) Chemokine levels in BAL fluids were determined by ELISA. Results are presented as mean±SEM (n = 4 and 6 in PBS and OAAH, respectively).</p

Eos secrete a soluble factor(s) to enhance HMCL proliferation.

<p>MV and exosomes were isolated from BM Eos SN. S1 and S2 are the MV-free and exosome-free supernatants, respectively. The various fractions were tested for their ability to stimulate HMCL proliferation as assessed by [3H]TdR-incorporation assay. Results are representative of 3 independent experiments. * <i>p</i><0.01; ** <i>p</i><0.001; n.s., not significant.</p

Eosinophils and PCs are found in close proximity in the human bone marrow.

<p>BM biopsies were obtained from normal donors (<b>A, C</b>) and MM patients (<b>B, D</b>) and stained with H&E (<b>A, B</b>) or using immunofluorescence (<b>C, D</b>) for selective visualization of PCs and Eos. In immunofluorescence staining, PCs were stained with anti-CD138 mAb (green) and Eos were stained with chromotrope 2R (red). Autofluorescent red blood cells are shown in yellow in these overlaid images. Images are representative of 5 normal donor and 10 MM patient BM biopsies. (<b>E</b>) Quantitation of Eos across 6 random fields from each immunofluorescence-stained sample dividing Eos into 3 categories: 1) Eos in direct contact with PCs; 2) Eos within a 3-cell distance of PCs; and 3) Eos more than a 3-cell distance away from the closest PC. Samples #1-5 are BM biopsies from normal donors. Samples #6–8 are from patients with MGUS. Samples #10–12 are from patients with SMM. Samples #9 and 13–15 are from patients with MM. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070554#pone.0070554.s003" target="_blank">Table S1</a> for more detail.</p

Proliferation of primary CD138+ MM cells is enhanced by treatment with Eos SN.

<p>Proliferation of the CD138+ cells upon treatment with Eos SN or CM was assessed by BrdU labeling. The figure shows representative flow cytometric analysis of the BrdU staining of 2 Eos-responsive (Patient 1 and 2) and 1 Eos-nonresponsive (Patient 3) samples. A total of 6 samples were analyzed.</p