In this study, to our best knowledge, we report for the first time that 13-S-HODE, a linoleic acid metabolite, causes mitochondrial dysfunction and bronchial epithelial injury. Although much is known about leukotrienes in asthma12
, much less attention has been given to other lipid metabolites. We studied 13-S-HODE because of increasing evidence of the role of mitochondrial dysfunction in asthma7,8,9
and high concentrations of 13-S-HODE are found in reticulocytes during degeneration of mitochondria of reticulocytes16
. On the other hand, mitochondrial dysfunction seems to be crucial in the genesis of epithelial injury and asthma pathogenesis in mice7,8,9
. Similarly dysfunctional mitochondria have been found in human asthmatic bronchial epithelia30
. Transfer of mitochondria from stem cells to alveolar epithelial cells reversed acute lung injury in sepsis, indicating the crucial role that mitochondrial health in lung diseases17
. In this context, understanding the effects of 13-S-HODE on airway epithelium is essential because we found its levels to be high in the airway secretions and extracellular fluids. Also it is practically difficult to reduce the levels of 13-S-hydroxyoctadecadienoic acid (13-S-HODE) as there are many sources for its synthesis13,14,15
We showed how exogenous induction of 13-S-HODE can lead to mitochondrial dysfunction in cultured human bronchial epithelium. We found that 13-S-HODE leads to mitochondrial structural alterations such as loss of cristae, swelling and increase in intramitochondrial calcium. As increase in calcium levels activate calpain we measured calpain activity not only in mitochondria but also in cytosol. Increase in cytosolic calpain activity with 13-S-HODE administration indicates that 13-S-HODE might have increased the levels of intracellular calcium. Increase in mitochondrial calpain activity with 13-S-HODE is interesting in the context of mitochondrial structural alterations as calpain 10 overexpression is known to cause mitochondrial fragmentation and apoptosis in neurons31
. Calpain 10, mitochondrial dominant calpain, is one of the recently reported candidate molecules in other chronic inflammatory diseases such as diabetes mellitus19,31
though there is no such report in asthma. Similarly we also found that increased calpain 10 activity in allergized lungs and importantly it was found to be increased in bronchial epithelia of allergized lungs.
Next we wanted to determine the effect of 13-S-HODE in in vivo
conditions. To determine this, we instilled 13-S-HODE intranasally mimicking the increase in its levels in asthmatic conditions. 13-S-HODE not only causes mitochondrial dysfunction and injury in bronchial epithelia but also causes similar features in naïve mouse. These features were associated with increase in the levels of cytokines such as IL-25 and IL-33 which are the indicators of epithelial cell stress5,6
. These cytokines are known to have proinflammatory properties as they induce the polarization of Th2 cells5,6
. Evidently we found an increase in the levels of IL-4. In addition, we found an increase in the levels of IL-17 in C57BL/6 mice though IL-17 was not upregulated in BALB/c mouse with 13-S-HODE administration (data not shown). These 13-S-HODE induced features were associated with severe difficulty in breathing. To understand the reasons for this difficulty in breathing, we determined the lung function. We found a significant reduction in elastance and increase in compliance. In addition, 13-S-HODE administration to allergic mice leads to worsen the airway hyperresponsiveness (data not shown). 13-S-HODE induced features in mouse such as airway neutrophilia, reduction in elastance and increase in epithelial stress related cytokines and IL-17 levels are interesting and these features are the indicators of severe asthma and emphysema20,21,22,32
. We found an increase in 13-S-HODE in airways of human asthmatics, and 13-S-HODE neutralization attenuates asthma mimicking features in mice, so 13-S-HODE could be targeted for asthma therapeutics. Increases in the levels of IL-17 cytokines, airway neutrophilia are also the features of steroid resistant conditions. Our preliminary results indicate that steroid administration to 13-S-HODE given allergic mice leads to no reduction in airway neutrophilia though it reduced airway eosinophilia (data not shown). However, the involvement of 13-S-HODE in emphysema and steroid resistance requires further detailed investigation.
13-S-HODE administration also leads to alter calcium homeostasis both in bronchial epithelia and mouse lungs. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) array profiles of 13-S-HODE–instilled lung showed a significant upregulation of the genes involved in calcium-dependent mitochondrial transporters (aralar
) and carnitine shuttle from cytosol to mitochondria (carnitine palmitoyltransferase 1b
and solute carrier slc25a20
) (Supplementary Table 1
) though 13-S-HODE did not affect various tested OXPHOS genes except cox5a, atp5a1,
(Supplementary Table 2
). Furthermore, we were able to show, by using genetic and pharmacological strategies, that the effects of 13-S-HODE were due to extracellular activation of TRPV1 channels, resulting in calcium influx and calpain activation. Though TRP receptors are well studied in airway in the aspect of neuronal cells scarce studies are available in non-neural tissues. Earlier study indicated that TRPV1 has no effect in asthma pathogenesis33
. This may be true in C57BL/6 mice as we found no upregulation of TRPV1 in C57BL/6 mouse whereas we found that TRPV1 levels were upregulated in allergic BALB/c mouse. In support to our findings, differential distribution of TRPV1 has been reported in airway dorsal root ganglion neurons as BALB/c mice have more capsaicin-sensitive receptors (TRPV1) than do those of C57BL/6 mice34
. This difference also could be one of the reasons that asthma mimicking features were not alleviated in C57BL/6 mice in an earlier study33
. However, the reasons for increased TRPV1 expression in allergized lungs are not clear. Transport of TRPV1 to the cell membrane by nerve growth factor has been reported in neurons, and nerve growth factor levels are increased in allergic inflammatory conditions35,36
. Accordingly, we found its expression on the surface of the bronchial epithelium in allergized mice. Although the contribution of TRPV1-mediated cell death to the pathogenesis of diseases such as diabetes mellitus and neurodegenerative diseases is known31
, such effects have not been studied in allergic airway inflammation. Although we did not focus on other types of vanilloid receptors, one cannot rule out the possibility of their roles in some 13-S-HODE–mediated effects. Together, these data indicated that 13-S-HODE concentrations were increased in extracellular fluids of asthmatic patients and extracellular receptor-mediated effects of 13-S-HODE are relevant to asthma pathogenesis. Thus, extracellular 13-S-HODE present in airways may activate TRPV1 present in the bronchial epithelial membrane to alter intracellular calcium homeostasis, thereby resulting in increased mitochondrial calcium and initiation of apoptosis (). It is also possible that 13-S-HODE may have receptor independent effects that need further investigations.
In conclusion, our findings indicate a novel role of extracellular 13-S-HODE, a lipid metabolite derived from linoleic acid, in allergic responses and airway epithelial injury. We further identify TRPV1 as one of its downstream targets. Since 13-S-HODE leads to key features of steroid refractory conditions such as neutrophilia and increased in IL-17 cytokines, 13-S-HODE should be explored further as a potential therapeutic target.