Involvement of IL-17RB⁺<i>i</i>NKT cells in the development of RSV-induced AHR.

<p>(A) Schematic showing the protocol for RSV-induced AHR. Mice were i.n. administered with RSV (10⁶ pfu) or PBS alone as a control 4 times at 10-day intervals. Mice were i.p. immunized with rec Gs/alum (50 µg/2 mg) 4 d after first RSV infections. Three days after the last RSV administration, mice were exposed i.n. to rec Gs and were measured 1 d later. (B) Development of RSV-induced AHR in BALB/c, but not in <i>Jα18</i>^−/− or <i>Il17rb</i>^−/− mice. Changes in R_L are depicted. The RSV-infected, rec Gs immunized, BALB/c mice had a greatly increased AHR compared to the other three groups. Results are expressed as the mean ± SEM. * <i>p</i><0.05 and ** <i>p</i><0.01. (C, D) Total and differential cell counts (C) and cytokines (D) in BAL fluid. BAL fluid was collected 24 h after challenge of the mice depicted in (B) with intranasal rec Gs. Results are expressed as the mean ± SEM. * <i>p</i><0.05 and ** <i>p</i><0.01. (E) Histological examination of lung tissues by H&E and PAS staining. RSV infected, rec Gs immunized, BALB/c, <i>Jα18</i>^−/− or <i>IL17rb</i>^−/−, mice were compared with control BALB/c mice (rec Gs alone). Bars indicate 100 µm. (F) AHR development after cell transfer of spleen IL-17RB⁺<i>i</i>NKT cells into <i>Jα18</i>^−/− mice. Indicated cell numbers of sorted IL-17RB⁺, IL-17RB⁻<i>i</i>NKT cells or total <i>i</i>NKT cells from spleen, or PBS control, were i.v. transferred into rec-Gs/alum-sensitized <i>Jα18</i>^−/− mice 24 h before RSV treatment (on the day 9, 19, and 29), and then challenged with rec Gs (24 h) and measurement of lung resistance (48 h). Each group of IL-17RB⁺<i>i</i>NKT cell-transferred mice was compared to other three groups. * <i>p</i><0.05, ** <i>p</i><0.01 calculated by Kruskal Wallis test. The results represent one out of four experiments with five mice in each group.</p

Function of <i>i</i>NKT cell subtypes in the spleen.

<p>(A) Global gene expression profiles in <i>i</i>NKT subtypes in the thymus and spleen. Tree view representation of clustering analysis among the four <i>i</i>NKT subtypes in thymus and spleen from B6 and BALB/c. The values represent coefficients between the indicated panels. <i>r</i>²>0.95 in red, 0.85<<i>r</i>²<0.95 in orange, and <i>r</i>²<0.85 in blue. One representative experiment of three is shown. (B) Plasticity and stability of <i>i</i>NKT subtypes. The four <i>i</i>NKT cell subtypes in the thymus were sorted and each subtype (5×10⁵) was i.v. transferred into independent <i>Jα18</i>^−/− mice (<i>n</i> = 3). 10 d after transfer, α-GalCer/CD1d dimer⁺ TCRβ⁺ cells in spleen were analyzed by FACS for the expression of IL-17RB and CD4. Representative data from three experiments are shown. (C–F) In vitro cytokine production by splenic <i>i</i>NKT cell subtypes (red, CD4⁻ IL-17RB⁺; orange, CD4⁺ IL-17RB⁺; blue, CD4⁻ IL-17RB⁻; green, CD4⁺ IL-17RB⁻). Sorted splenic <i>i</i>NKT subtypes (5×10⁴ cells/100 µL) were co-cultured with BM-DCs (5×10³/100 µL) for 48 h in the presence of α-GalCer (100 ng/µL) (C), IL-12 (10 ng/µL) (D), IL-23 (10 ng/µL) (E), and IL-25 (10 ng/µL) (F). Levels of IFN-γ, IL-4, IL-9, IL-10, IL-13, IL-17A, and IL-22 in the supernatants were analyzed by ELISA or CBA. Data are mean ± SD of triplicate wells. One representative experiment of three is shown.</p

<i>i</i>NKT cell subtypes in the periphery.

<p>(A–C) FACS profile of peripheral <i>i</i>NKT cells in B6 mice. α-GalCer/CD1d dimer⁺ TCRβ⁺<i>i</i>NKT cells (A) and <i>i</i>NKT subtypes based on the expression of CD44 and NK1.1 (B) or CD4 and IL-17RB (C) in spleen, liver, BM, lung, inguinal LN, and mesenteric LN from B6 or <i>Il17rb</i>^−/− mice. Numbers indicate percentage of total mononuclear cells (A) and <i>i</i>NKT cells (B, C). (D) Number of cells of each <i>i</i>NKT subtype based on the expression of IL-17RB and CD4 in thymus and periphery of B6 and BALB/c mice. Cell numbers were calculated based on the results from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001255#pbio-1001255-g004" target="_blank">Figures 4A, 4C</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001255#pbio.1001255.s008" target="_blank">S8A</a>, and S8B. IL-17RB⁺<i>i</i>NKT cells were mainly localized in spleen, lung, inguinal LN, and mesenteric LN, whereas hardly any were observed in liver and BM. One representative experiment of three is shown.</p

Involvement of <i>E4bp4</i> in cytokine production by CD4⁺ IL-17RB⁺<i>i</i>NKT cells in response to IL-25.

<p>(A, B) Quantitative analysis of <i>Rorc</i> (A) and <i>E4bp4</i> (B) in <i>i</i>NKT cell subtypes after cytokine treatment. Sorted <i>i</i>NKT cell subtypes (5×10⁴/100 µL) from thymus (left) or spleen (right) were co-cultured with BM-DCs (5×10³/100 µL) in the presence or absence of IL-23 (10 ng/ml) or IL-25 (10 ng/ml) for 24 h. The <i>i</i>NKT cell subtypes were then sorted again and analyzed for expression of the indicated genes by quantitative real-time PCR. The data are representative of three independent experiments (mean ± SEM). (C, D) Cytokine production by CD4⁺ IL-17RB⁺<i>i</i>NKT cells in response to IL-25. Sorted CD4⁺ IL-17RB⁺<i>i</i>NKT cells (5×10⁴/100 µL) from thymus (C) or spleen (D) of B6 or <i>E4bp4</i>^−/− mice were co-cultured with BM-DCs (5×10³/100 µL) in the presence of IL-25 (10 ng/ml) for 48 h and then the levels of the indicated cytokines in the tissue culture media were analyzed. <i>i</i>NKT cells from B6 were compared to those from <i>E4bp4</i>^−/− mice. ** <i>p</i><0.01 calculated by <i>t</i> test. The data are representative of three independent experiments (mean ± SD).</p

Function of <i>i</i>NKT cells in the spleen and liver from <i>Il17rb</i>^−/− and <i>Il15</i>^L117P mice.

<p>(A) FACS profile of spleen and liver mononuclear cells in WT, <i>Il17rb</i>^−/− and <i>Il15</i>^L117P mice on a B6 background. Numbers are percentage of gated cells. α-GalCer/CD1d dimer⁺<i>i</i>NKT cells and α-GalCer/CD1d dimer⁻ NK1.1⁺ NK cells were slightly decreased in <i>Il17rb</i>^−/− mice and markedly reduced in <i>Il15</i>^L117P mice. (B) IL-17RB expression in spleen and liver <i>i</i>NKT cells of WT B6, <i>Il17rb</i>^−/− and <i>Il15</i>^L117P mice. Shaded profiles in the histograms indicate the background staining with isotype matched control mAb. (C, D) In vitro cytokine production by spleen <i>i</i>NKT cells from <i>Il17rb</i>^−/− and <i>Il15</i>^L117P mice (C) and by liver <i>i</i>NKT cells from <i>Il17rb</i>^−/− mice (D). Sorted <i>i</i>NKT cells (5×10⁴/100 µL) from spleen and liver of WT B6 and <i>Il17rb</i>^−/− mice were co-cultured with BM-DCs (5×10³/100 µL) for 48 h in the presence of the indicated doses of α-GalCer. The <i>Il17rb</i>^−/−<i>i</i>NKT cells produced IFN-γ at levels equivalent to WT, while T_H2 and T_H17 cytokine production, except for IL-4, were severely impaired. (E) <i>i</i>NKT cell-dependent cytokine production in WT B6 and <i>Il17rb</i>^−/− mice in vivo. α-GalCer (2 µg) was i.v. injected and the levels of cytokines in serum were analyzed at the indicated time points. The serum IFN-γ levels were similar in both mice, whereas production of T_H2 and T_H17 cytokines, except for IL-4, was significantly reduced in the <i>Il17rb</i>^−/− mice. Cytokines were measured by ELISA or a cytometric bead array system at the indicated time points. Data are mean ± SDs from three mice and repeated three times with similar results.</p

Differential gene expression and cytokine production among thymic <i>i</i>NKT cell subtypes from B6 mice.

<p>(A, B, D, H) Quantitative PCR analysis of thymic <i>i</i>NKT subtypes. Thymic <i>i</i>NKT cells further divided into four subtypes based on the expression of CD4 and IL-17RB (red, CD4⁻ IL-17RB⁺; orange, CD4⁺ IL-17RB⁺; blue, CD4⁻ IL-17RB⁻; green, CD4⁺ IL-17RB⁻). One representative out of three experiments is shown (mean ± SEM). (A) The purity of the sorted cells was confirmed by the relative <i>Il17rb</i> and <i>Cd4</i> mRNA expression levels in the respective subtypes. <i>Il2rb</i> ( = <i>Cd122</i>) expression was restricted to CD4⁻ and CD4⁺, IL-17RB⁻<i>i</i>NKT cells. (B) Expression of T_H1/T_H2/T_H17 related genes. T_H1 related: <i>Ifng</i>, <i>Tbx21</i> and <i>Stat4</i>, T_H2 related; <i>Il4</i> and <i>Gata3</i>, and T_H17 related: <i>Il17a</i>, <i>Il22</i> and <i>Rorc</i> transcripts were analyzed. (D) Expression of cytokine receptor genes. Receptor for IL-12, IL-23, and IL-25 were analyzed. The component chains of the various receptors are IL-12 receptor: IL-12Rβ2/IL-12Rβ1; IL-23 receptor: IL-23R/IL-12Rβ1; IL-25 receptor: IL-17RB/IL-17RA. (H) Expression of chemokine receptor genes. <i>Ccr4</i>, <i>Ccr6</i>, <i>Ccr7</i>, <i>Cxcr3</i>, and <i>Cxcr6</i>. (C, E, F, G) In vitro cytokine production by thymic <i>i</i>NKT cell subtypes (red, CD4⁻ IL-17RB⁺; orange, CD4⁺ IL-17RB⁺; blue, CD4⁻ IL-17RB⁻; green, CD4⁺ IL-17RB⁻). Sorted thymic <i>i</i>NKT subtypes (5×10⁴ cells/100 µL) were co-cultured with BM-DCs (5×10³/100 µL) for 48 h in the presence of α-GalCer (100 ng/µL) (C), IL-12 (10 ng/µL) (E), IL-23 (10 ng/µL) (F), or IL-25 (10 ng/µL) (G). Levels of IFN-γ, IL-4, IL-9, IL-10, IL-13, IL-17A, and IL-22 were analyzed. The data are representative of three independent experiments (mean ± SEM).</p

Profile of <i>i</i>NKT cells in the thymus of <i>Il17rb</i>^−/− and <i>Il15</i>^L117P mice.

<p>(A, B) FACS profiles of thymus (A) and enriched thymic <i>i</i>NKT cells (B) in WT, <i>Il17rb</i>^−/− and <i>Il15</i>^L117P mice on a B6 background. (A) α-GalCer/CD1d dimer⁺<i>i</i>NKT cells were slightly decreased in <i>Il17rb</i>^−/− mice and markedly reduced in <i>Il15</i>^L117P mice. (B) There was a loss of the NK1.1⁻ population in <i>Il17rb</i>^−/− thymic <i>i</i>NKT cells, while <i>Il15</i>^L117P thymic <i>i</i>NKT cells showed impairment of the NK1.1⁺ population. (C) IL-17RB and CD122 expression by thymic <i>i</i>NKT cell populations of B6 mice. IL-17RB and CD122 expression in CD4⁻ and CD4⁺ of CD44^lo NK1.1⁻ (Stage 1), CD44^lo NK1.1⁺ (Stage 2), and CD44^hi NK1.1⁺ (Stage 3) populations were analyzed. IL-17RB expression was observed in Stages 1/2, while CD122 expression was in the Stage 3 cells. (D, E) Profiles of thymic <i>i</i>NKT cells in B6 and <i>Il15</i>^L117P mice showing expression of IL-17RB and CD4 (D) and further divided into CD44 and NK1.1 subpopulations (E). The percentage of IL-17RB⁺<i>i</i>NKT cells was increased due to the loss of expansion of IL17RB⁻<i>i</i>NKT cells in <i>Il15</i>^L117P mice. CD4⁻ and CD4⁺, IL-17RB⁺<i>i</i>NKT cells were almost all Stage 1 and Stage 2 in both WT B6 and <i>Il15</i>^L117P mice. On the other hand, the majority of CD4⁻ and CD4⁺, IL-17RB⁻<i>i</i>NKT cells were Stage 3 in both WT B6 and <i>Il15</i>^L117P mice. Loss of expansion of CD4⁻ and CD4⁺, IL-17RB⁻<i>i</i>NKT cells was also observed in <i>Il15</i>^L117P mice. (F, G) Percentage (F) and cell number (G) of the total <i>i</i>NKT cells and the four subtypes (i.e. IL-17RB^+/− and CD4^+/−) in B6, <i>Il17rb</i>^−/− and <i>Il15</i>^L117P mice based on their CD44 and NK1.1 expression patterns. The number of CD4⁻ and CD4⁺, IL-17RB⁻<i>i</i>NKT cells was significantly decreased especially in Stage 3 in <i>Il15</i>^L117P mice compared to WT B6 mice. By contrast, CD4⁻ and CD4⁺, IL-17RB⁺<i>i</i>NKT cells in <i>Il15</i>^L117P mice were present in numbers comparable to WT. Results are representative of those from three independent experiments. (H, I) Development of <i>i</i>NKT subtypes in Stages 1 and 2. Stage 1 and 2 cells in the four <i>i</i>NKT subtypes (i.e. IL-17RB^+/− and CD4^+/−) from WT B6 mice were sorted and cocultured with dGuo treated 15 dpc FT lobes from <i>Jα18</i>^−/− mice (1,000 cells/well). 10 d after culture, cells were recovered and analyzed the surface expression pattern in CD44 versus NK1.1 (H) and CD4 versus IL-17RB (I). IL-17RB⁻ precursors gave rise through Stage 2 to Stage 3 cells with IL-17RB⁻, while IL-17RB⁺ subtypes gave rise to Stage 2 cells with IL-17RB⁺. Results are representative of those from three independent experiments.</p

Medullary regions are severely affected in Foxn1-Stat3-CKO mice.

<p>(A) Macroscopy of the thymus derived from Stat3-flox/flox (Stat3f/f) and Foxn1Cre::Stat3f/f mice at 7 weeks of age. (B) Flowcytometric profiles of developing thymocytes derived from Stat3f/f and Foxn1Cre::Stat3f/f mice, at 7 weeks of age. (C) Cryostat sections of thymic tissue from Stat3f/f and Foxn1Cre::Stat3f/f mice (7 weeks of age) were stained with the mTEC specific antibody, ER-TR5 (red) and counterstained with DAPI (blue). Scale bars: 400 μm. (D) Cryostat sections of neonatal thymic tissue from Stat3f/f and Foxn1Cre::Stat3f/f mice were stained with antibody directed to K14 (green) and counterstained with DAPI (blue) Scale bars: 100 μm. (E) Total thymic cellularity of control (containing Cre-f/+ and f/f) and Foxn1Cre::Stat3f/f (Cre-f/f) mice at indicated ages. Bar stands for the average value of each experimental group. ns denotes a non-significant difference (P>0.1) in Student’s t test. (F) Changes in the proportional area of medullary regions in thymus tissue sections of control (containing Cre-f/+ and f/f, n = 8, 6, 7, 3 for neo, 3, 6, 12 week, respectively) and Foxn1Cre::Stat3f/f (Cre-f/f, n = 5, 4, 6, 5 for neo, 3, 6, 12 week, respectively) mice in the first 12 weeks of life. The area occupied by mTECs in thymus was quantitatively measured in sections stained with K14 antibody using Axiovision4 software (Carl Zeiss). Error bar stands for the standard deviation. ns denotes a non-significant difference (P>0.1) in Student’s t test. **;P<0.005, ***;P<0.0005. (G) Representative flow cytometric profiles showing frequencies of major TEC populations from 12 weeks old Stat3f/f and Foxn1Cre::Stat3f/f mice. EpCAM⁺CD45^- fraction represents whole TEC population, and UEA1 vs Ly51 profile was displayed for the cells gated on EpCAM⁺CD45^- fraction, where UEA1^highLy51^low and UEA1^lowLy51^high fraction were defined as mTEC and cTEC population, respectively. (H) Ratio of mTEC vs cTECs in flow cytometric analysis of control (containing Cre-f/+ and f/f, n = 4) and Foxn1Cre::Stat3f/f mice (n = 5) at 12 weeks of age is shown.</p

Maturation of mTECs was not affected in Foxn1-Stat3-CKO.

<p>Cryostat sections of the thymus were stained with antibodies directed to K14 (green) and UEA1 (red) (A), and with antibodies to ERTR5 (red) and AIRE (green) (B). Sections were counterstained with DAPI (blue). Scale bars: 400 μm.</p

Medullary regions are severely affected while cTECs were normally regenerated in K5-Cre-Stat3-CKO mice.

<p>(A) Gross anatomical analysis of the thymus derived from 7 week old K5Cre::Stat3f/+ and K5Cre::Stat3f/f mice. Scale bars: 1 mm. (B) CD4 and CD8 cell surface expression on thymocytes derived from 7 week old Stat3f/f and K5Cre::Stat3f/f mice. (C) Cryostat sections of thymic tissues derived from either K5Cre::Stat3f/+ or K5Cre::Stat3f/f mice at 7 weeks of age. The tissues were stained with anti-K8 (red) and anti-K14 (green) antibody and counterstained with DAPI (blue). Scale bars: 400 μm. (D) Experimental design for data presented in panels (E) and (F). K5-Cre::Stat3-f/f mice were lethally irradiated and then rescued by hematopoietic stem cell transplantation (wild type C57BL/6). After 4 weeks, mice were sacrificed and the thymus was examined. (E) Gross anatomical analysis of the grafted thymus 4 weeks after hematopoietic stem cell transplantation. (F) Cryostat sections of the thymus were stained with anti-K14 antibody (green) and counterstained with DAPI (blue). Scale bars: 400 μm.</p