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Ag85B DNA vaccine suppresses airway inflammation in a murine model of asthma.

Wu J, Xu J, Cai C, Gao X, Li L, Zhong N - Respir. Res. (2009)

Bottom Line: IL-4 production was significantly decreased in the BAL fluid (32.0 +/- 7.6 pg/ml vs. 130.8 +/- 32.6 pg/ml, p < 0.01) and in the splenocyte supernatant (5.1 +/- 1.6 pg/ml vs. 10.1 +/- 2.3 pg/ml, p < 0.05) in the pMG-Ag85B group compared with the OVA-sensitized control group, while IFN-gamma production was increased in the BAL fluid (137.9 +/- 25.6 pg/ml vs. 68.4 +/- 15.3 pg/ml, p < 0.05) and in the splenocyte supernatant (20.1 +/- 5.4 pg/ml vs. 11.3 +/- 3.2 pg/ml, p < 0.05).In a murine model of asthma induced by OVA, intranasal immunization with pMG-Ag85B significantly reduced allergic airway inflammation with less eosinophil infiltration.This protective effect was associated with decreased IL-4 and increased IFN-gamma production in the BAL fluid and in the supernatant of cultured splenocytes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Respiratory Disease, Peking University First Hospital, Beijing, PR China. wjxst@hotmail.com

ABSTRACT

Background: In allergic asthma, Th2 lymphocytes are believed to play important roles in orchestrating airway eosinophilia and inflammation. Resetting the Th1/Th2 imbalance may have a therapeutic role in asthma. The mycobacterium tuberculosis 30-kilodalton major secretory protein (antigen 85B, Ag85B) can protect animals from M. tuberculosis infection by inducing a Th1-dominant response.

Methods: In this study, the Ag85B gene was cloned into pMG plasmids to yield the pMG-Ag85B plasmid. The expression of Ag85B gene in murine bronchial epithelia cells was detected by Western blotting and immunohistochemical staining after intranasal immunization with reconstructed pMG-Ag85B plasmids. The protective effect of pMG-Ag85B plasmids immunization in airway inflammation was evaluated by histological examination and bronchoalveolar lavage (BAL). IL-4 and IFN-gamma levels in the BAL and supernatant from splenocyte culture were determined using ELISA kits.

Results: The Ag85B gene was successfully expressed in murine bronchial epithelia cells by intranasal immunization with reconstructed pMG-Ag85B plasmids. Using a murine model of asthma induced by ovalbumin (OVA), pMG-Ag85B immunization significantly inhibited cellular infiltration across the airway epithelium with a 37% decrease in the total number of cells (9.6 +/- 2.6 x 10(5)/ml vs. 15.2 +/- 3.0 x 10(5)/ml, p < 0.05) and a 74% decrease in the number of eosinophils (1.4 +/- 0.2 x 10(5)/ml vs. 5.4 +/- 1.1 x 10(5)/ml, p < 0.01) compared with the OVA-sensitized control group. There was no difference in the number of neutrophils in BAL fluid between the pMG-Ag85B group, the OVA-sensitized control group and the empty pMG group. IL-4 production was significantly decreased in the BAL fluid (32.0 +/- 7.6 pg/ml vs. 130.8 +/- 32.6 pg/ml, p < 0.01) and in the splenocyte supernatant (5.1 +/- 1.6 pg/ml vs. 10.1 +/- 2.3 pg/ml, p < 0.05) in the pMG-Ag85B group compared with the OVA-sensitized control group, while IFN-gamma production was increased in the BAL fluid (137.9 +/- 25.6 pg/ml vs. 68.4 +/- 15.3 pg/ml, p < 0.05) and in the splenocyte supernatant (20.1 +/- 5.4 pg/ml vs. 11.3 +/- 3.2 pg/ml, p < 0.05).

Conclusion: In a murine model of asthma induced by OVA, intranasal immunization with pMG-Ag85B significantly reduced allergic airway inflammation with less eosinophil infiltration. This protective effect was associated with decreased IL-4 and increased IFN-gamma production in the BAL fluid and in the supernatant of cultured splenocytes.

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Immunization with pMG-Ag85B inhibited inflammatory cell infiltration in the lung. A. Timing of the sensitization, immunization and challenge (NS = normal saline). B. Lung tissue was taken 24 hours after the last OVA challenge. H&E staining of lung sections from the normal (upper left), OVA (upper right), empty pMG (lower left) and pMG-Ag85B (lower right) groups. n = 6 mice per group. C, D. BAL fluid was collected 24 hours after the last OVA challenge. The total number of cells (C) and number of eosinophils (D) were counted. Values are means ± SD for seven animals. *P < 0.05, **P < 0.01, for the pMG-Ag85B group versus the OVA-sensitized control group; +P < 0.05, ++P < 0.01, for the pMG-Ag85B group versus the pMG group (unpaired two-sided Student's t-test).
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Figure 2: Immunization with pMG-Ag85B inhibited inflammatory cell infiltration in the lung. A. Timing of the sensitization, immunization and challenge (NS = normal saline). B. Lung tissue was taken 24 hours after the last OVA challenge. H&E staining of lung sections from the normal (upper left), OVA (upper right), empty pMG (lower left) and pMG-Ag85B (lower right) groups. n = 6 mice per group. C, D. BAL fluid was collected 24 hours after the last OVA challenge. The total number of cells (C) and number of eosinophils (D) were counted. Values are means ± SD for seven animals. *P < 0.05, **P < 0.01, for the pMG-Ag85B group versus the OVA-sensitized control group; +P < 0.05, ++P < 0.01, for the pMG-Ag85B group versus the pMG group (unpaired two-sided Student's t-test).

Mentions: Since Ag85B was successfully expressed in vivo, we wondered whether Ag85B could protect mice from the development of asthma. We used the OVA sensitization/challenge asthma model. In this model, mice were intraperitoneally injected with high doses of OVA protein once a week for 3 weeks, rested for 4 weeks, and then challenged with OVA through the airway. These mice developed serious inflammation in the lung compared with the saline-treated mice, mimicking the pathological process of asthma. In this study, during the resting phase, mice were intranasally immunized twice with pMG-Ag85B plasmid DNA, empty pMG or saline. All mice were then challenged with 1% OVA through the airway except the saline group and lung inflammation was examined 24 hours later (Fig. 2A). In the OVA-sensitized control group, histological examination revealed shedding of the airway epithelium and swelling of the bronchiolar wall with cellular infiltration, particularly in the parabronchiolar and perivascular area (Fig. 2B, upper right). However, pMG-Ag85B immunization greatly inhibited cellular infiltration across the whole area (Fig. 2B, lower right). No inflammation was observed in the saline group (Fig. 2B, upper left). Consistent with the histological data, the total number of cells and the number of eosinophils in the BAL fluid was significantly increased in the OVA-sensitized control group compared with the saline group (Fig. 2C&2D). In the pMG-Ag85B group, OVA-induced inflammation was suppressed with a 37% decrease in the total number of cells (9.6 ± 2.6 × 105/ml vs. 15.2 ± 3.0 × 105/ml, p < 0.05) and a 74% decrease in the number of eosinophils (1.4 ± 0.2 × 105/ml vs. 5.4 ± 1.1 × 105/ml, p < 0.01) compared with the OVA-sensitized control group. There were no significant differences in the total number of cells or number of eosinophils between the empty pMG group and the OVA-sensitized control group (Fig. 2C&2D). There was no significant difference in the number of neutrophils in BAL fluid between the pMG-Ag85B group, the OVA-sensitized control group and the empty pMG group (pMG-Ag85B: 2.2 ± 0.4 × 105; OVA-sensitized control: 2.5 ± 0.5 × 105 cells; pMG: 2.3 ± 0.5 × 105; p > 0.05), while the number of neutrophils in BAL fluid was significantly increased in the three test groups compared with that in the normal control group (normal control group: 0.8 ± 0.1 × 105 cells; all p < 0.05)


Ag85B DNA vaccine suppresses airway inflammation in a murine model of asthma.

Wu J, Xu J, Cai C, Gao X, Li L, Zhong N - Respir. Res. (2009)

Immunization with pMG-Ag85B inhibited inflammatory cell infiltration in the lung. A. Timing of the sensitization, immunization and challenge (NS = normal saline). B. Lung tissue was taken 24 hours after the last OVA challenge. H&E staining of lung sections from the normal (upper left), OVA (upper right), empty pMG (lower left) and pMG-Ag85B (lower right) groups. n = 6 mice per group. C, D. BAL fluid was collected 24 hours after the last OVA challenge. The total number of cells (C) and number of eosinophils (D) were counted. Values are means ± SD for seven animals. *P < 0.05, **P < 0.01, for the pMG-Ag85B group versus the OVA-sensitized control group; +P < 0.05, ++P < 0.01, for the pMG-Ag85B group versus the pMG group (unpaired two-sided Student's t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Immunization with pMG-Ag85B inhibited inflammatory cell infiltration in the lung. A. Timing of the sensitization, immunization and challenge (NS = normal saline). B. Lung tissue was taken 24 hours after the last OVA challenge. H&E staining of lung sections from the normal (upper left), OVA (upper right), empty pMG (lower left) and pMG-Ag85B (lower right) groups. n = 6 mice per group. C, D. BAL fluid was collected 24 hours after the last OVA challenge. The total number of cells (C) and number of eosinophils (D) were counted. Values are means ± SD for seven animals. *P < 0.05, **P < 0.01, for the pMG-Ag85B group versus the OVA-sensitized control group; +P < 0.05, ++P < 0.01, for the pMG-Ag85B group versus the pMG group (unpaired two-sided Student's t-test).
Mentions: Since Ag85B was successfully expressed in vivo, we wondered whether Ag85B could protect mice from the development of asthma. We used the OVA sensitization/challenge asthma model. In this model, mice were intraperitoneally injected with high doses of OVA protein once a week for 3 weeks, rested for 4 weeks, and then challenged with OVA through the airway. These mice developed serious inflammation in the lung compared with the saline-treated mice, mimicking the pathological process of asthma. In this study, during the resting phase, mice were intranasally immunized twice with pMG-Ag85B plasmid DNA, empty pMG or saline. All mice were then challenged with 1% OVA through the airway except the saline group and lung inflammation was examined 24 hours later (Fig. 2A). In the OVA-sensitized control group, histological examination revealed shedding of the airway epithelium and swelling of the bronchiolar wall with cellular infiltration, particularly in the parabronchiolar and perivascular area (Fig. 2B, upper right). However, pMG-Ag85B immunization greatly inhibited cellular infiltration across the whole area (Fig. 2B, lower right). No inflammation was observed in the saline group (Fig. 2B, upper left). Consistent with the histological data, the total number of cells and the number of eosinophils in the BAL fluid was significantly increased in the OVA-sensitized control group compared with the saline group (Fig. 2C&2D). In the pMG-Ag85B group, OVA-induced inflammation was suppressed with a 37% decrease in the total number of cells (9.6 ± 2.6 × 105/ml vs. 15.2 ± 3.0 × 105/ml, p < 0.05) and a 74% decrease in the number of eosinophils (1.4 ± 0.2 × 105/ml vs. 5.4 ± 1.1 × 105/ml, p < 0.01) compared with the OVA-sensitized control group. There were no significant differences in the total number of cells or number of eosinophils between the empty pMG group and the OVA-sensitized control group (Fig. 2C&2D). There was no significant difference in the number of neutrophils in BAL fluid between the pMG-Ag85B group, the OVA-sensitized control group and the empty pMG group (pMG-Ag85B: 2.2 ± 0.4 × 105; OVA-sensitized control: 2.5 ± 0.5 × 105 cells; pMG: 2.3 ± 0.5 × 105; p > 0.05), while the number of neutrophils in BAL fluid was significantly increased in the three test groups compared with that in the normal control group (normal control group: 0.8 ± 0.1 × 105 cells; all p < 0.05)

Bottom Line: IL-4 production was significantly decreased in the BAL fluid (32.0 +/- 7.6 pg/ml vs. 130.8 +/- 32.6 pg/ml, p < 0.01) and in the splenocyte supernatant (5.1 +/- 1.6 pg/ml vs. 10.1 +/- 2.3 pg/ml, p < 0.05) in the pMG-Ag85B group compared with the OVA-sensitized control group, while IFN-gamma production was increased in the BAL fluid (137.9 +/- 25.6 pg/ml vs. 68.4 +/- 15.3 pg/ml, p < 0.05) and in the splenocyte supernatant (20.1 +/- 5.4 pg/ml vs. 11.3 +/- 3.2 pg/ml, p < 0.05).In a murine model of asthma induced by OVA, intranasal immunization with pMG-Ag85B significantly reduced allergic airway inflammation with less eosinophil infiltration.This protective effect was associated with decreased IL-4 and increased IFN-gamma production in the BAL fluid and in the supernatant of cultured splenocytes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Respiratory Disease, Peking University First Hospital, Beijing, PR China. wjxst@hotmail.com

ABSTRACT

Background: In allergic asthma, Th2 lymphocytes are believed to play important roles in orchestrating airway eosinophilia and inflammation. Resetting the Th1/Th2 imbalance may have a therapeutic role in asthma. The mycobacterium tuberculosis 30-kilodalton major secretory protein (antigen 85B, Ag85B) can protect animals from M. tuberculosis infection by inducing a Th1-dominant response.

Methods: In this study, the Ag85B gene was cloned into pMG plasmids to yield the pMG-Ag85B plasmid. The expression of Ag85B gene in murine bronchial epithelia cells was detected by Western blotting and immunohistochemical staining after intranasal immunization with reconstructed pMG-Ag85B plasmids. The protective effect of pMG-Ag85B plasmids immunization in airway inflammation was evaluated by histological examination and bronchoalveolar lavage (BAL). IL-4 and IFN-gamma levels in the BAL and supernatant from splenocyte culture were determined using ELISA kits.

Results: The Ag85B gene was successfully expressed in murine bronchial epithelia cells by intranasal immunization with reconstructed pMG-Ag85B plasmids. Using a murine model of asthma induced by ovalbumin (OVA), pMG-Ag85B immunization significantly inhibited cellular infiltration across the airway epithelium with a 37% decrease in the total number of cells (9.6 +/- 2.6 x 10(5)/ml vs. 15.2 +/- 3.0 x 10(5)/ml, p < 0.05) and a 74% decrease in the number of eosinophils (1.4 +/- 0.2 x 10(5)/ml vs. 5.4 +/- 1.1 x 10(5)/ml, p < 0.01) compared with the OVA-sensitized control group. There was no difference in the number of neutrophils in BAL fluid between the pMG-Ag85B group, the OVA-sensitized control group and the empty pMG group. IL-4 production was significantly decreased in the BAL fluid (32.0 +/- 7.6 pg/ml vs. 130.8 +/- 32.6 pg/ml, p < 0.01) and in the splenocyte supernatant (5.1 +/- 1.6 pg/ml vs. 10.1 +/- 2.3 pg/ml, p < 0.05) in the pMG-Ag85B group compared with the OVA-sensitized control group, while IFN-gamma production was increased in the BAL fluid (137.9 +/- 25.6 pg/ml vs. 68.4 +/- 15.3 pg/ml, p < 0.05) and in the splenocyte supernatant (20.1 +/- 5.4 pg/ml vs. 11.3 +/- 3.2 pg/ml, p < 0.05).

Conclusion: In a murine model of asthma induced by OVA, intranasal immunization with pMG-Ag85B significantly reduced allergic airway inflammation with less eosinophil infiltration. This protective effect was associated with decreased IL-4 and increased IFN-gamma production in the BAL fluid and in the supernatant of cultured splenocytes.

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Related in: MedlinePlus