Percent positivity of IL-17R and IL-22R expression on HBE cells gated on AQP3- versus AQP3+ cells.

<p>*denotes p<0.05 compared to AQP3- gates. n = 3 donors, Mann-Whitney comparison.</p><p>Primary HBE cells from 3 separate donors were stained anti-AQP3, anti-IL22R, anti-IL-17RA, or anti-IL-17RC. Cells were gates on AQP3 and the percentage that stained for IL-17RA, IL-17RC, or IL22R are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020333#pone-0020333-t001" target="_blank">Table 1</a> as mean percentages ± SEM.</p

Cigarette smoke extract enhances Th17 differentiation <i>in vivo</i>.

<p>(A) OTII mice were given 50 µg OVA in 50 µL HBSS or CSE intratracheally on day 0, day 7 and sacrificed on day 14. Single cell suspensions harvested from lung digest and mediastinal lymph nodes were re-stimulated with PMA and ionomycin in the presence of Golgi-plug for 5h, intracellular cytokines were analyzed by FACS (A). Gene expression in lung tissue was analyzed by real-time RT-PCR (B, n = 5 per group, * denotes p<0.05 compared to OVA alone).</p

Th17 cytokines induce MMP expression in HBE cells.

<p>(A) Primary HBE cells were stimulated with media or a combination of 10/-50 ng/ml IL-22 and 50 ng/ml of IL-17A for 48 hours (n = 3 donors per condition). RNA was harvested and subjected to hybridization on Hu-133 arrays for RNA sequencing as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020333#s4" target="_blank">Methods</a>. Transcript abundance or hybridization intensity was measured on normalized data in Qseq software (Lasergene). Scatter plot analysis of normalized transcript abundance for Affymetrix (x-axis) and RNA-seq (y-axis) are shown. (B) Relative gene expression of MMPs and TIMP2 from RNA-seq data. (C) Up-regulation of <i>Ccl2</i> by IL-17 and IL-22 combination treatment in primary HBE cells as measured by RNAseq. (D) CCL2, MMP3 and MMP12 gene expression were analyzed by real-time RT-PCR.</p

Requirement of IL-17RA for emphysema.

<p>Representative histological sections of mouse lungs stained with hematoxylin and eosin after six months of exposure to air (sham exposed) (A,B) or cigarette smoke (C,D) demonstrate C57BL/6 mice develop alveolar enlargement (C) as opposed to IL-17RA−/− mice (D). (E) Enlargement of airspaces was quantified using the mean linear intercept method. This method determines the parenchymal surface area relative to lung volume. An increase in Lm represents an increase in the alveolar space as seen in the WT smoking mice versus sham air. This increase in Lm was not seen in the IL-17A−/− mice over sham air controls. All lung tissues were inflated to 25cm water pressure with neutral buffered formalin. Original magnification: x 50 of representative sections.</p

image_4.JPG

<p>Using the CRISPR/Cas9 gene-editing technology, we recently produced a number of rabbits with mutations in immune function genes, including FOXN1, PRKDC, RAG1, RAG2, and IL2RG. Seven founder knockout rabbits (F0) and three male IL2RG null (−/y) F1 animals demonstrated severe combined immunodeficiency (SCID), characterized by absence or pronounced hypoplasia of the thymus and splenic white pulp, and absence of immature and mature T and B-lymphocytes in peripheral blood. Complete blood count analysis showed severe leukopenia and lymphocytopenia accompanied by severe neutrophilia. Without prophylactic antibiotics, the SCID rabbits universally succumbed to lung infections following weaning. Pathology examination revealed severe heterophilic bronchopneumonia caused by Bordetella bronchiseptica in several animals, but a consistent feature of lung lesions in all animals was a severe interstitial pneumonia caused by Pneumocystis oryctolagi, as confirmed by histological examination and PCR analysis of Pneumocystis genes. The results of this study suggest that these SCID rabbits could serve as a useful model for human SCID to investigate the disease pathogenesis and the development of gene and drug therapies.</p

image_3.JPEG

<p>Using the CRISPR/Cas9 gene-editing technology, we recently produced a number of rabbits with mutations in immune function genes, including FOXN1, PRKDC, RAG1, RAG2, and IL2RG. Seven founder knockout rabbits (F0) and three male IL2RG null (−/y) F1 animals demonstrated severe combined immunodeficiency (SCID), characterized by absence or pronounced hypoplasia of the thymus and splenic white pulp, and absence of immature and mature T and B-lymphocytes in peripheral blood. Complete blood count analysis showed severe leukopenia and lymphocytopenia accompanied by severe neutrophilia. Without prophylactic antibiotics, the SCID rabbits universally succumbed to lung infections following weaning. Pathology examination revealed severe heterophilic bronchopneumonia caused by Bordetella bronchiseptica in several animals, but a consistent feature of lung lesions in all animals was a severe interstitial pneumonia caused by Pneumocystis oryctolagi, as confirmed by histological examination and PCR analysis of Pneumocystis genes. The results of this study suggest that these SCID rabbits could serve as a useful model for human SCID to investigate the disease pathogenesis and the development of gene and drug therapies.</p

table_1.DOCX

<p>Using the CRISPR/Cas9 gene-editing technology, we recently produced a number of rabbits with mutations in immune function genes, including FOXN1, PRKDC, RAG1, RAG2, and IL2RG. Seven founder knockout rabbits (F0) and three male IL2RG null (−/y) F1 animals demonstrated severe combined immunodeficiency (SCID), characterized by absence or pronounced hypoplasia of the thymus and splenic white pulp, and absence of immature and mature T and B-lymphocytes in peripheral blood. Complete blood count analysis showed severe leukopenia and lymphocytopenia accompanied by severe neutrophilia. Without prophylactic antibiotics, the SCID rabbits universally succumbed to lung infections following weaning. Pathology examination revealed severe heterophilic bronchopneumonia caused by Bordetella bronchiseptica in several animals, but a consistent feature of lung lesions in all animals was a severe interstitial pneumonia caused by Pneumocystis oryctolagi, as confirmed by histological examination and PCR analysis of Pneumocystis genes. The results of this study suggest that these SCID rabbits could serve as a useful model for human SCID to investigate the disease pathogenesis and the development of gene and drug therapies.</p

image_2.JPEG

<p>Using the CRISPR/Cas9 gene-editing technology, we recently produced a number of rabbits with mutations in immune function genes, including FOXN1, PRKDC, RAG1, RAG2, and IL2RG. Seven founder knockout rabbits (F0) and three male IL2RG null (−/y) F1 animals demonstrated severe combined immunodeficiency (SCID), characterized by absence or pronounced hypoplasia of the thymus and splenic white pulp, and absence of immature and mature T and B-lymphocytes in peripheral blood. Complete blood count analysis showed severe leukopenia and lymphocytopenia accompanied by severe neutrophilia. Without prophylactic antibiotics, the SCID rabbits universally succumbed to lung infections following weaning. Pathology examination revealed severe heterophilic bronchopneumonia caused by Bordetella bronchiseptica in several animals, but a consistent feature of lung lesions in all animals was a severe interstitial pneumonia caused by Pneumocystis oryctolagi, as confirmed by histological examination and PCR analysis of Pneumocystis genes. The results of this study suggest that these SCID rabbits could serve as a useful model for human SCID to investigate the disease pathogenesis and the development of gene and drug therapies.</p

image_1.JPEG

<p>Using the CRISPR/Cas9 gene-editing technology, we recently produced a number of rabbits with mutations in immune function genes, including FOXN1, PRKDC, RAG1, RAG2, and IL2RG. Seven founder knockout rabbits (F0) and three male IL2RG null (−/y) F1 animals demonstrated severe combined immunodeficiency (SCID), characterized by absence or pronounced hypoplasia of the thymus and splenic white pulp, and absence of immature and mature T and B-lymphocytes in peripheral blood. Complete blood count analysis showed severe leukopenia and lymphocytopenia accompanied by severe neutrophilia. Without prophylactic antibiotics, the SCID rabbits universally succumbed to lung infections following weaning. Pathology examination revealed severe heterophilic bronchopneumonia caused by Bordetella bronchiseptica in several animals, but a consistent feature of lung lesions in all animals was a severe interstitial pneumonia caused by Pneumocystis oryctolagi, as confirmed by histological examination and PCR analysis of Pneumocystis genes. The results of this study suggest that these SCID rabbits could serve as a useful model for human SCID to investigate the disease pathogenesis and the development of gene and drug therapies.</p

β-Glucan plus LPS promote neutrophilic inflammation in OVA-induced allergic inflammation.

<p>(A) Timeline for OVA i.p. sensitizations (25 μg in 1 mg alum) and i.t. challenges (10 μg) with the combination of OVA, β-glucan (1x10⁷ particles) and LPS (100 ng), as indicated. (B) Airway inflammation in challenged C57BL/6 mice showing the number of total leukocytes, eosinophils (Siglec-F^hiGr-1^loCD11c^lo), neutrophils (Gr-1^hiCD11b^hiF4/80^lo), inflammatory macrophage/monocytes (F4/80^hiCD11b^hiGr-1^lo) and T cells (CD3^hiCD4^hi). (C) Total serum IgE levels in challenged mice, as indicated. (<i>D</i>) Pulmonary cytokine concentrations in BALF of challenged C57BL/6 mice, as indicated. (E) Numbers of recruited neutrophils in the BALF of challenged C57BL/6 wild type, Dectin-1^-/- and TLR-4^-/- mice, as indicated. (F) H&E and PAS stains of formalin fixed lung sections (left) from C57BL/6 wild type mice, challenged as indicated. Scale bars represent 100 μm (H&E) and 50 μm (PAS). Quantification (right) of mucus producing goblet cell area in PAS stained sections. (G) Airway resistance (R) in intubated C57BL/6 wild type mice exposed to increasing doses of nebulised methacholine (MCh), as indicated. *p<0.05, n.s., not significant. Shown are the mean ± SEM of pooled data from at least 2 independent experiments (n = 14–16 mice/group).</p