Indoleamine-2,3-dioxygenase activity induces neutrophil apoptosis

Introduction

Influenza-related mortality is often caused by secondary bacterial pneumonia. We have previously shown that the tryptophan-catabolizing enzyme indoleamine-2,3-dioxygenase (IDO) critically impairs host defense against secondary bacterial pneumonia [1]. Since inhibition of IDO resulted in increased neutrophil numbers during primary viral infection, we hypothesized that tryptophan degradation and/or the generation of downstream metabolites induces neutrophil apoptosis. In the present study we aimed to investigate the impact of IDO-mediated tryptophan metabolism on neutrophil apoptosis in vitro and in vivo.

Methods

Freshly isolated neutrophils were cultured in the presence or absence of tryptophan, kynurenine and 3-hydroxy-anthranilic acid. Apoptosis was identified by annexin V/propidium iodine staining (%, mean ± SD). To confirm our in vitro data, transgenic mice that conditionally express IDO in the airway epithelium upon doxycycline (dox) treatment and control mice were challenged with LPS (1 μg) or Klebsiella pneumoniae (10⁴ colony-forming units) intranasally and sacrificed after 24 hours to count neutrophils in bronchoalveolar lavage fluid (total number, mean ± SD). Statistical analysis was performed by Student's t test or Mann-Whitney U test where appropriate. P < 0.05 was considered significant.

Results

Both kynurenine and 3-hydroxy-anthranilic acid enhanced apoptosis in freshly isolated neutrophils (60.3 ± 8.7% and 45.5 ± 1.7% respectively vs. 33.5 ± 8.1% under control conditions, both P < 0.05), which was reversed by adding tryptophan. Conditional transgenic mice, which showed marked expression of IDO in the pulmonary compartment, had reduced neutrophil numbers in bronchoalveolar lavage fluid after challenge with K. pneumoniae (3.36 ± 1.92 × 10⁵ vs. 12.1 ± 9.0 × 10⁵ in dox-treated littermates, P < 0.05) and LPS (1.88 ± 1.22 × 10⁵ vs. 5.21 ± 3.81 × 10⁵ in control-treated transgenic mice, P < 0.05), which was associated with active caspase-3 staining in dox-treated mice, but not in control mice.

Conclusions

Neutrophils undergo apoptosis in presence of kynurenine or 3-hydroxy-anthranilic acid and the absence of tryptophan. Pulmonary IDO expression, as occurs during influenza infection, enhances neutrophil apoptosis in vivo and may impair host defense against secondary bacterial infections.