Mycobacterium tuberculosis-Specific Antigen Rv3619c Effectively Alleviates Allergic Asthma in Mice

Abstract

Despite significant advances, asthma remains a cause of premature death, and current treatments are suboptimal. Antigen-specific Th2 cells and their cytokines are primary mediators of the pathophysiological changes seen in asthma. Studies in animal models have shown that mycobacteria can suppress the asthma phenotype by alteration of the Th1/Th2 cytokines ratio. In this study, utilizing a Th1 delivery system to modulate the allergic airway inflammation in a Th2-driven model of asthma, we evaluated the efficacy of immunization with Mycobacterium tuberculosis-specific antigen Rv3619c, either alone or in combination with low dose dexamethasone. The rv3619c gene was cloned in an expression plasmid pGES-TH-1, expressed in Escherichia coli, and the recombinant protein Rv3619c was purified to homogeneity using affinity chromatography. Mice were immunized with the recombinant protein emulsified in Freund's Incomplete Adjuvant (IFA) alone and in combination with low dose dexamethasone, and then challenged with ovalbumin (OVA). Airway inflammation was assessed by quantifying airway cytology, histological changes and Th2 cytokine (IL-5) secretion from splenocytes. OVA-specific IgE, IgG and IgG1 from sera was assessed, as well as pERK1/2 expression in the lung tissue. Immunization with recombinant Rv3619c alone inhibited the OVA-induced increase in total cell counts, eosinophil airway cell infiltration in BAL fluid, perivascular and peribronchial inflammation and fibrosis, and goblet cell hyper/metaplasia. In addition, Rv3619c/IFA inhibited the OVA-induced IL-5 in spleen cells, OVA-specific IgE, IgG, and IgG1 levels in sera, and pERK1/2 expression in lung tissue. Immunization with Rv3619c/IFA in combination with low dose dexamethasone resulted in an enhanced effect on some but not all the asthma features. Taken together, this study demonstrates that immunization with Rv3619c/IFA, alone or in combination with dexamethasone, may be an effective treatment strategy for the prevention of asthma.

Keywords: Mycobacterium tuberculosis; Rv3619c; Th1; Th2; asthma; hygiene hypothesis; inflammation; ovalbumin.

Figure 1

Immunization protocol with Rv3619c/IFA in ovalbumin (OVA)-challenged murine model of asthma. 6–8 weeks ok female balb/c mice (n = 10) were immunized intraperitonealy on day 0 with either 2 µg of Rv3619c emulsified with Freund’s Incomplete Adjuvant (IFA) and boosted with protein only on days 14 and 28 [Rv3619c/IFA and Rv3619c/IFA+ dexamethasone (DEX) (0.5 mg) groups]. All other groups (n = 10) were immunized with PBS emulsified with IFA on day 0 and PBS only on days 14 and 28. All mice (except PBS group mice) were immunized intraperitoneally with OVA/AluG on day 42. Mice were then challenged intranasally with OVA for four consecutive days (days 52 to 55). The PBS group mice were challeneged intranasally with PBS on the same days. Mice in Rv3619c/IFA/dexamethasone (DEX) (0.5 mg), dexamethasone (DEX) (0.5 mg), and dexamethasone (DEX) (3 mg) groups were treated with either dexamethasone an hour prior to intranasal challenge. All mice were sacrificed on day 56 and Bronchoalveolar lavage fluid (BALF) lung tissue, blood and spleens were collected.

Figure 2

Effect of Rv3619c/Freund’s Incomplete Adjuvant (IFA) alone and in combination with low dose dexamethasone (0.5 mg) treatment on total (A) and differential (B) cells counts in ovalbumin (OVA)-challenged mice. Immunization of mice with Rv3619c/IFA alone or in combination with dexamethasone (DEX) (0.5 mg) treatment significantly inhibited OVA-induced BAL total cell counts and eosinophilia. Data are expressed as mean ± SEM (n = 10). ^#P < 0.05 versus time-matched PBS-challenged mice. *P < 0.05 versus time-matched ovalbumin-challenged mice.

Figure 3

A representative low-magnification light photomicrographs (A) and score (B) display H&E staining of whole lung samples from PBS-challenged/vehicle, ovalbumin (OVA)-challenged/vehicle, OVA-challenged/Rv3619c/IFA, OVA-challenged/dexamethasone (DEX) (3 mg), OVA-challenged/Rv3619c/IFA/dexamethasone (DEX) (0.5 mg), and OVA-challenged/dexamethasone (DEX) (0.5 mg). OVA-challenged/vehicle treated mice had marked peribronchial and perivascular inflammatory cell infiltrations compared with PBS-challenged/vehicle treated mice. Immunization with Rv3619c/IFA alone resulted in a significant reduction in the peribronchial and perivascular inflammatory cell infiltration compared to OVA-challenged/vehicle treated mice and was comparable with high dose dexamethasone (3 mg). Immunization with Rv3619c/IFA in combination with dexamethasone (0.5 mg) resulted in a greater inhibitory effect on the inflammatory response compared to immunization with Rv3619c/IFA alone. B = bronchioles, BV = blood vessels, (➞) = marked peribronchial and perivascular inflammatory cell infiltrations. Data are expressed as mean ± SEM (n = 10). ^#P < 0.05 versus time-matched PBS-challenged mice. *P < 0.05 versus time-matched OVA-challenged mice. **P < 0.05 versus Rv3619c/IFA and dexamethasone (0.5 mg).

Figure 4

A representative low-magnification light photomicrographs (A) and score (B) display Masson’s Trichrome staining of whole lung samples from PBS-challenged/vehicle, ovalbumin (OVA)-challenged/vehicle, OVA-challenged/Rv3619c/IFA, OVA-challenged/dexamethasone (DEX) (3 mg), OVA-challenged/Rv3619c/IFA/dexamethasone (DEX) (0.5 mg), and OVA-challenged/dexamethasone (DEX) (0.5 mg). OVA-challenged/vehicle treated mice had marked perivascular and peribronchial fibrosis compared with PBS-challenged mice. Immunization with Rv3619c/IFA alone resulted in a significant reduction in the perivascular and peribronchial fibrosis compared to OVA-challenged/vehicle treated mice and was comparable to the high dose dexamethasone (3 mg). Immunization with Rv3619c/IFA in combination with dexamethasone (0.5 mg) treatment did have any greater effect on the perivascular and peribronchial fibrosis compared to Rv3619c/IFA alone. B = bronchioles, BV = blood vessels, (➞) = marked peribronchial and perivascular fibrosis. Data are expressed as mean ± SEM (n = 10). ^#P < 0.05 versus time-matched PBS-challenged mice. *P < 0.05 versus time-matched OVA-challenged mice.

Figure 5

A representative low-magnification light photomicrographs (A) and score (B) display PAS staining of whole lung samples from PBS-challenged/vehicle, ovalbumin (OVA)-challenged/vehicle, OVA-challenged/Rv3619c/Freund’s Incomplete Adjuvant (IFA), OVA-challenged/dexamethasone (DEX) (3 mg), OVA-challenged/Rv3619c/IFA/dexamethasone (DEX) (0.5 mg), and OVA-challenged/dexamethasone (DEX) (0.5 mg). OVA-challenged/vehicle treated mice had marked bronchial mucus production and goblet cell hyper/metaplasia compared with PBS-challenged mice. Immunization with Rv3619c/IFA alone resulted in a significant reduction in the bronchial mucus production and goblet cell hyper/metaplasia compared to OVA-challenged/vehicle treated mice and was somewhat comparable to the high dose dexamethasone (3 mg). Immunization with Rv3619c/IFA in combination with dexamethasone (0.5 mg) did have any greater effects on the bronchial mucus production and goblet cell hyper/metaplasia compared when compared to Rv3619c/IFA alone. B = bronchioles, BV = blood vessels, (➞) = marked bronchial mucus production and goblet cell hyper/metaplasia. Data are expressed as mean ± SEM (n = 10). ^#P < 0.05 versus time-matched PBS-challenged mice. *P < 0.05 versus time-matched OVA-challenged mice.

Figure 6

Effect of Rv3619c/Freund’s Incomplete Adjuvant (IFA) alone and in combination with low dose dexamethasone (DEX) (0.5 mg) treatment on Th2 cytokine (IL-5) secretion from spleen cells of ovalbumin (OVA)-challenged mice. Immunization of mice with Rv3619c/IFA showed a significant inhibition of secretion of Th2 cytokine (IL-5). Immunization with Rv3619c/IFA in combination with dexamethasone (0.5 mg) treatment did have any greater effects on the IL-5 levels. Data are expressed as mean ± SEM (n = 10). ^#P < 0.05 versus time-matched PBS-challenged mice. *P < 0.05 versus time-matched OVA-challenged mice.

Figure 7

Effect of Rv3619c/Freund’s Incomplete Adjuvant (IFA) alone and in combination with low dose dexamethasone (DEX) (0.5 mg) treatment on sera levels of ovalbumin (OVA)-specific IgE (A) IgG (B) and IgG1 (C) from OVA-challenged mice. Immunization with Rv3619c/IFA alone resulted in significant reduction in the sera levels of OVA-specific IgE, IgG, and IgG1. Immunization with Rv3619c/IFA alone or in combination with dexamethasone (0.5 mg) treatment did have any greater effects on the OVA-specific IgE, IgG, and IgG1. Data are expressed as mean ± SEM (n = 10). ^#P < 0.05 versus time-matched PBS-challenged mice. *P < 0.05 versus time-matched OVA-challenged mice. **P < 0.05 versus Rv3619c/IFA and dexamethasone (0.5 mg).

Figure 8

A representative low-magnification light photomicrographs (A) and score (B) display pERK1/2 fluorescence of whole lung samples from PBS-challenged/vehicle, ovalbumin (OVA)-challenged/vehicle, OVA-challenged/Rv3619c/Freund’s Incomplete Adjuvant (IFA), OVA-challenged/dexamethasone (DEX) (3 mg), OVA-challenged/Rv3619c/IFA/dexamethasone (DEX) (0.5 mg), and OVA-challenged/dexamethasone (DEX) (0.5 mg). OVA-challenged/vehicle treated mice had marked increase in pERK1/2 signals compared with PBS-challenged mice. Immunization with Rv3619c/IFA alone resulted in a significant reduction in the pERK1/2 signals compared to OVA-challenged/vehicle treated mice and was comparable to the high dose dexamethasone (3 mg). Immunization of mice with Rv3619c/IFA in combination with dexamethasone (0.5 mg) did not result in any significant enhancement of the effects of Rv3619c/IFA alone. Data are expressed as mean ± SEM (n = 5). ^#P < 0.05 versus time-matched PBS-challenged mice. *P < 0.05 versus time-matched OVA-challenged mice.