Effects of Mycobacteria Major Secretion Protein, Ag85B, on Allergic Inflammation in the Lung

<div><p>Many epidemiological studies have suggested that the recent increase in prevalence and severity of allergic diseases such as asthma is inversely correlated with <i>Mycobacterium bovis</i> bacillus Calmette Guerin (BCG) vaccination. However, the underlying mechanisms by which mycobacterial components suppress allergic diseases are not yet fully understood. Here we showed the inhibitory mechanisms for development of allergic airway inflammation by using highly purified recombinant Ag85B (rAg85B), which is one of the major protein antigens secreted from <i>M. tuberculosis</i>. Ag85B is thought to be a single immunogenic protein that can elicit a strong Th1-type immune response in hosts infected with mycobacteria, including individuals vaccinated with BCG. Administration of rAg85B showed a strong inhibitory effect on the development of allergic airway inflammation with induction of Th1-response and IL-17and IL-22 production. Both cytokines induced by rAg85B were involved in the induction of Th17-related cytokine-production innate immune cells in the lung. Administration of neutralizing antibodies to IL-17 or IL-22 in rAg85B-treated mice revealed that IL-17 induced the infiltration of neutrophils in BAL fluid and that allergen-induced bronchial eosinophilia was inhibited by IL-22. Furthermore, enhancement of the expression of genes associated with tissue homeostasis and wound healing was observed in bronchial tissues after rAg85B administration in a Th17-related cytokine dependent manner. The results of this study provide evidence for the potential usefulness of rAg85B as a novel approach for anti-allergic effect and tissue repair other than the role as a conventional TB vaccine.</p></div

Innate immune cells that secrete Th17-related cytokines are induced by rAg85B administration in BAL fluid.

<p>OVA-immunized (i.p., day0 and 14) and sensitized (5% aerosolized-OVA, day21 to 25) BALB/c mice were challenged with PBS or rAg85B protein (i.p. (100 µg; days 0 and 14) and i.n. (20 µg; days 21, 23, and 25)). At 24 h after the last OVA sensitization, BAL fluid from naïve or OVA sensitized BALB/c mice treated with PBS or rAg85B, were harvested. BAL cells were stimulated with ionomycin and PMA for 5 h, and with brefeldin A added in the last 3 h. Flow cytometry of BAL cells from PBS-treated (upper) and rAg85B protein-treated (lower) OVA-sensitized mice stained with anti-CD3, anti-CD4, anti-CD8, anti-Gr-1, anti-γδ TCR, anti-NKp46, anti-CD11c, anti-CD127 (IL-7R) and Lineage specific marker (CD3, CD19, Gr-1, CD11b, CD11c). Numbers in quadrants indicate percent of cells in each (<i>A</i>). Intracellular IL-17 and IL-22 staining in indicated cells by flow cytometry (dot plots) and absolute numbers of those cell populations (side graphs) in the BAL fluid (<i>B, C, D, E, F, G</i>). Data are representative of at least two independent experiments (**P<0.01 compared with OVA control. error bars, s.d.; n = 6 mice).</p

Functions of rAg85B in allergic inflammation.

<p>Experimental design used to investigate the effects of rAg85B on OVA-induced allergic lung inflammation (<i>A</i>). BALB/c mice were intraperitoneally immunized with OVA on days 0 and 14. On days 21 to 25 after the first immunization, mice were exposed to aerosolized 5% OVA for 20 min. Three hours prior to OVA inhalation, the mice were i.p. (100 µg; days 0 and 14) and i.n. (20 µg; days 21, 23, and 25) administered rAg85B. One day after the last challenge, the BAL cells were counted (<i>B</i>) and OVA-specific serum IgE concentrations were determined by ELISA (<i>C</i>). Flow cytometry of BAL cells from naïve or OVA sensitized BALB/c mice treated with PBS or rAg85B, stained with anti-Gr-1 and anti-Siglec-F. Numbers adjacent to outlined area indicate percent of eosinophils (Gr-1^dull, Siglec-F⁺), and neutrophils (Gr-1⁺, Siglec-F^neg) (<i>D</i>). Formalin-fixed tissue sections were stained with hematoxylin and eosin to visualized cell recruitment (upper row, scale bar, 100 mm), Masson's trichrome (center row, scale bar, 100 mm), and α-smooth muscle actin (lower row, scale bar, 50 mm). Numbers in quadrants indicate the score scale from 0 to 5 in each. (<i>E</i>). Data are representative of at least three independent experiments. (*P<0.05, **P<0.01 compared with OVA control. error bars, s.d.; n = 6 mice).</p

Administration of rAg85B resulted in the reduction of Th2 cytokine and chemokine levels and in the enhancement of Th1 and Th17 cytokine levels in BAL fluid.

<p>OVA-immunized (i.p., day0 and 14) and sensitized (5% aerosolized-OVA, day21 to 25) BALB/c mice were challenged with PBS or rAg85B protein (i.p. (100 µg; days 0 and 14) and i.n. (20 µg; days 21, 23, and 25)). At 24 h after the last OVA sensitization, BAL fluid from naïve or OVA sensitized BALB/c mice treated with PBS or rAg85B, were harvested. Levels of cytokines in the BAL fluid were measured directly by ELISA. Data are representative of at least three independent experiments (*P<0.05, **P<0.01 compared with OVA control. error bars, s.d.; n = 6 mice).</p

Effects of rAg85B to Toll-Like and NOD-Like Receptor.

<p>The results are provided as optical density values (650 nm).</p><p>The values represent the means and standard deviations of three screenings.</p><p>TLR/NLR ligand screening were performed by InvivoGen, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106807#s2" target="_blank">Methods</a>.</p><p>Effects of rAg85B to Toll-Like and NOD-Like Receptor.</p

Administaration of rAg85B induced immune deviation from a Th2-response towards a Th1, Th17-related response in OVA-stimulated LN cells.

<p>OVA-immunized (i.p., day0 and 14) and sensitized (5% aerosolized-OVA, day21 to 25) BALB/c mice were challenged with PBS or rAg85B protein (i.p. (100 µg; days 0 and 14) and i.n. (20 µg; days 21, 23, and 25)). At 24 h after the last OVA sensitization, mediastinal lymph nodes (MLNs) from naïve or OVA sensitized BALB/c mice treated with PBS or rAg85B, were harvested. MLNs were cultured with OVA (50 µg/ml), and cytokines in the culture supernatant were determined 48 h after incubation by ELISA. Data are representative of at least three independent experiments (*P<0.05, **P<0.01 compared with OVA control. error bars, s.d.; n = 6 mice).</p

Schematic illustration of the proposed effects of rAg85B in a mouse model of allergic inflammation.

<p>IFN-γ and IL-17-producing Th cells are induced in regional lymph nodes by rAg85B challenge, however, Th17 cells do not enter the lung unlike Th1 cells. Th17-related cytokine-secreting cells in lungs from rAg85B-administered mice are innate immune cells including γδT cells, IL-7R⁺ Lin⁻ cells, CD3⁻ NKp46⁺ cells and CD11c⁺ cells. IL-17 and IL-22 induced by rAg85B in an allergic environment have crucial roles in not only anti-allergic effects but also regulation of tissue homeostatic reactions.</p

Ag85B administration promotes Th17-related innate responses in the lung.

<p>OVA-sensitized BALB/c mice (5% aerosolized-OVA, day21 to 25) were challenged intranasally with PBS (control), rAg85B (rAg85B + isotype control), rAg85B plus neutralizing IL-17 (rAg85B + IL-17Ab) or IL-22 (rAg85B + IL-22Ab), or a combination of both antibodies (rAg85B +IL-17/22Ab) on days 21, 23, and 25. The isotype-matched control antibody was treated with the same time course as neutralization Ab i.n. administration. Lungs from naïve or OVA sensitized BALB/c mice treated with PBS or rAg85B with/without neutralization Ab were sampled one day after the last challenge for histological analysis and quantification of mRNA levels. Lung sections were stained with hematoxylin and eosin (left row, scale bar, 100 mm), Masson's trichrome (center row, scale bar, 100 mm), α-smooth muscle actin (right row, scale bar, 50 mm). Numbers in quadrants indicate the score scale from 0 to 5 in each (<i>A</i>). Real-time RT-PCR was performed for the indicated molecules expression on RNA isolated from individual mice lungs (<i>B</i>). Data are representative of at least two independent experiments (*P<0.05, **P<0.01 compared with rAg85B+isotype control challenged group. error bars, s.d.; n = 6 mice).</p

IFN-γ and IL-17-producing CD4-negative cell subsets proliferated in BAL fluid after rAg85B administration.

<p>OVA-immunized (i.p., day0 and 14) and sensitized (5% aerosolized-OVA, day21 to 25) BALB/c mice were challenged with PBS or rAg85B protein (i.p. (100 µg; days 0 and 14) and i.n. (20 µg; days 21, 23, and 25)). At 24 h after the last OVA sensitization, BAL fluid from naïve or OVA sensitized BALB/c mice treated with PBS or rAg85B, were harvested. BAL cells were stimulated with ionomycin and PMA for 5 h, and with brefeldin A added in the last 3 h. Flow cytometry of stimulated BAL cells from PBS-treated (upper) and rAg85B protein-treated (lower) OVA-sensitized mice stained with specific antibodies indicated marker. Numbers in quadrants indicate percent of cells in each (<i>A</i>). Absolute numbers of various cell populations (above graphs) in BAL fluid (<i>B</i>, <i>C</i>). Data are representative of three independent experiments (**P<0.01 compared with OVA control. error bars, s.d.; n = 6 mice).</p

IFN-γ and IL-17-producing CD4 T cell subsets proliferated in lymph nodes after rAg85B administration.

<p>OVA-immunized (i.p., day0 and 14) and sensitized (5% aerosolized-OVA, day21 to 25) BALB/c mice were challenged with PBS or rAg85B protein (i.p. (100 µg; days 0 and 14) and i.n. (20 µg; days 21, 23, and 25)). At 24 h after the last OVA sensitization, mediastinal lymph nodes (MLNs) from naïve or OVA sensitized BALB/c mice treated with PBS or rAg85B, were harvested. MLNs were stimulated with ionomycin and PMA for 5 h, and with brefeldin A added in the last 3 h. Flow cytometry of stimulated MLNs from naïve (upper), PBS-treated (middle) and rAg85B protein-treated (lower) OVA-sensitized mice stained with specific antibodies indicated marker. Numbers in quadrants indicate percent of cells in each (<i>A</i>). Absolute numbers of various cell populations (above graphs) in lymph nodes (<i>B</i>, <i>C</i>). Data are representative of three independent experiments (*P<0.05, **P<0.01 compared with OVA control. error bars, s.d.; n = 6 mice).</p