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. 2011 Apr;164(1):80-9.
doi: 10.1111/j.1365-2249.2010.04313.x. Epub 2011 Jan 14.

Induction of β-defensins by l-isoleucine as novel immunotherapy in experimental murine tuberculosis

C E Rivas-Santiago  1 , B Rivas-Santiago, D A León, J Castañeda-Delgado, R Hernández Pando
Affiliations

Induction of β-defensins by l-isoleucine as novel immunotherapy in experimental murine tuberculosis

C E Rivas-Santiago et al. Clin Exp Immunol. 2011 Apr.

Abstract

Tuberculosis is a worldwide health problem, and multidrug-resistant (MDR) and extensively multidrug-resistant (XMDR) strains are rapidly emerging and threatening the control of this disease. These problems motivate the search for new treatment strategies. One potential strategy is immunotherapy using cationic anti-microbial peptides. The capacity of l-isoleucine to induce beta-defensin expression and its potential therapeutic efficiency were studied in a mouse model of progressive pulmonary tuberculosis. BALB/c mice were infected with Mycobacterium tuberculosis strain H37Rv or with a MDR clinical isolate by the intratracheal route. After 60 days of infection, when disease was in its progressive phase, mice were treated with 250 µg of intratracheal l-isoleucine every 48 h. Bacillary loads were determined by colony-forming units, protein and cytokine gene expression were determined by immunohistochemistry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), respectively, and tissue damage was quantified by automated morphometry. Administration of l-isoleucine induced a significant increase of beta-defensins 3 and 4 which was associated with decreased bacillary loads and tissue damage. This was seen in animals infected with the antibiotic-sensitive strain H37Rv and with the MDR clinical isolate. Thus, induction of beta-defensins might be a potential therapy that can aid in the control of this significant infectious disease.

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Figures

Fig. 1
Fig. 1
Kinetics of human β-defensin 2 gene expression in the lung epithelial cell line A549 after stimulation with different concentrations of l-isoleucine. (a) A549 cells were stimulated with l-isoleucine using the indicated concentrations. At the indicated time-points the cells were collected, the RNA was isolated and the number of mRNA copies of β-defensin was quantified by real-time reverse transcription–polymerase chain reaction (RT–PCR). All values are mean ± standard deviation of five different experiments. Asterisks represent statistical significance when compared with the control non-stimulated cells. (b) Immunocytochemistry confirmed protein production in the condition in which the cells showed the highest β-defensin expression (25 µg/ml for 18 h), while non-stimulated cells do not show immunostaining (inset). (c) In comparison with l-isoleucine, A549 cells stimulated with 50 µg of d-isoleucine during 48 h did not induce human β-defensin 2 gene expression. Asterisks represent statistical significance when compared with the control non-stimulated cells.
Fig. 2
Fig. 2
Effect on the β-defensin gene expression in non-infected mice after the administration of l-isoleucine. Groups of BALB/c mice received the indicated amount of l-isoleucine by the intratracheal route, and were euthanized at different time-points and their lungs were used to isolate total mRNA to determine the expression of murine β-defensin 3 (a) and 4 (b). In comparison with l-isoleucine, administration of 250 µg of d-isoleucine or the vehicle saline solution (control group) did not induce gene expression of β-defensin 3 (c) or β-defensin 4 (d). All values are mean ± standard deviation of five mice from three independent experiments. Asterisks represent statistical significance when compared with the control group that received only the vehicle (P < 0·005).
Fig. 3
Fig. 3
Effect of l-isoleucine administration during advanced disease in the lungs of mice infected with drug-sensitive H37Rv strain. (a) l-isoleucine administration (white symbols) starting 60 days after infection increased mBD3 and mBD4 (b) gene expression when compared with control mice (black symbols). (c) l-isoleucine treatment also decreased pulmonary bacterial loads and (d) the area of pneumonia. (e) In comparison with the control group, l-isoleucine treatment increased the expression of interferon-γ and tumour necrosis factor-α (f). Each point corresponds to the mean and standard deviation of five mice group in one representative experiment. Asterisks represent statistical significance when compared to the control non-treated group (*P < 0·05; **P < 0·01).
Fig. 4
Fig. 4
Effect of l-isoleucine administration during advanced disease in the lungs of mice infected with a multidrug-resistant strain. (a) l-isoleucine administration (white symbols) starting 60 days after infection increased mBD3 and (b) mBD4 gene expression when compared with control mice (black symbols). (c) l-isoleucine treatment also decreased pulmonary bacilli burdens, and after 60 days of treatment the lung surface affected by pneumonia (d) when compared with the control group. l-isoleucine treatment increased the expression of interferon-γ (e) and tumour necrosis factor-α (f). Each point corresponds to the mean and standard deviation of five mice group in one representative experiment. Asterisks represent statistical significance when compared to the control non-treated group (*P < 0·05, **P < 0·01).
Fig. 5
Fig. 5
Representative lung histopathology and immunohistochemistry after 2 months of treatment with l-isoleucine and in a control non-treated mouse. (a) Very low expression of mBD3 in the bronchial epithelium (arrows) in the lung of control mouse after 4 months of infection with H37Rv strain. (b) In contrast, there is strong mBD3 immunostaining in the bronchial epithelium (arrows) and in some macrophages in the lung of the mouse infected with strain H37Rv and treated with l-isoleucine. (c) The control animal shows extensive pneumonia after 4 months of infection with drug-sensitive strain H37Rv. (d) In comparison, the l-isoleucine treated mouse shows less lung surface area affected by pneumonia (arrow). (e) Representative micrograph showing extensive pneumonia after 4 months of infection with the multidrug-resistant clinical isolate, while the L-isoleucine-treated mouse shows less lung consolidation (f).
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