(506) Antiviral response and ROS signaling triggers Warburg Effect and stimulates Pseudomonas aeruginosa (Pa) biofilm growth

Background: Bacterial infections in the airways are a hallmark of cystic fibrosis (CF). In CF adults, these infections lead to lung function impairment and become chronic and dominated by Pseudomonas aeruginosa (Pa). Clinical reports show that respiratory viral infections worsen
Pa infection and lung function. Moreover, using an in vitro model of CF epithelium, we have seen that infection with disparate respiratory viruses enhances Pa biofilm growth through the antiviral response (Hendricks M, et al. PNAS. 2016). A feature of respiratory viral infections is the increase in reactive oxygen species (ROS), functioning both as antimicrobial and as signaling molecules involved in metabolic regulation. It is well established that ROS can lead to the stabilization of Hypoxia-inducible factor 1 alpha (HIF-1α), which induces the transcription of genes involved in glycolysis, such as Hexokinase-2 (HK2), rate limiting enzyme of Warburg Effect (WE).

Methods: Using an in vitro model of CF epithelium, we used ROS scavengers during respiratory syncytial virus (RSV) infection or under polyI:C stimulation, to test the effect of ROS in the apical secretion of stimulatory factors for Pa biofilm growth.

Results: We observed that apical secretion from cells overexpressing HK2 enhances Pa biofilm growth. Based on this, we asked whether ROS triggered during the antiviral response induces HK2 and apical secretion of L-lactate and if this enhances Pa biofilm growth. Using ROS scavengers during respiratory syncytial virus (RSV) infection, we tested the effect of ROS in the apical secretion of stimulatory factors for Pa biofilm growth. We observed induction of Pa biofilm when grown in apical secretions from RSV infected CFBE cells, while we observed a loss of Pa biofilm when grown in apical secretions from RSV infected CFBE41o- cells with ROS scavenging. We then induced ROS with polyinosinic-polycytidylic acid (polyI:C), analog of double-stranded RNA that mimics viral infection and triggers the antiviral response. We observed that while polyI:C enhanced Pa biofilm, scavenging ROS led to the loss of Pa biofilm growth. This data suggests that ROS induced by RSV infection and the antiviral response mediates the release of Pa biofilm stimulatory factors. We next measured HK2 expression by RT-qPCR and apical L-lactate release in RSV infection, polyI:C or IFN-β treatment. We observed upregulation of HK2 by RSV infection and by the antiviral response. Likewise, we detected higher L-lactate secretion in CM from RSV infected or IFN-β treated CFBE41o- cells compared to untreated control, suggesting that the antiviral response increases HK2 expression and apical release of L-lactate. We further scavenged ROS in RSV infection and measured HK2 and L-lactate secretion to test the role of ROS in inducing WE. We observed decrease in both HK2 abundance and apical L-lactate secretion, suggesting that RSV infection induces HK2 and apical secretion of L-lactate through ROS signaling. Finally, to assess of the effect of L-lactate on Pa biofilm growth, we treated CFBE41o- cells with IFN-β and oxamate, inhibitor of LDHA, enzyme that converts pyruvate to L-lactate. Under oxamate treatment, we observed a reduction in Pa biofilm growth compared to IFN-β only, suggesting that L-lactate stimulates Pa biofilm growth.

Conclusions: Overall, this data suggests that ROS triggered by antiviral signaling initiates metabolic reprogramming, as a mechanism of promoting Pa biofilm during co-infection.

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