Interferon signaling drives epithelial metabolic reprogramming to promote secondary bacterial infection

Grace P. Carreno-Florez; Brian R. Kocak; Matthew R. Hendricks; Jeffrey A. Melvin; Katrina B. Mar; Jessica Kosanovich; Rachel L. Cumberland; Greg M. Delgoffe; Sruti Shiva; Kerry M. Empey; John W. Schoggins; Jennifer M. Bomberger

doi:10.1371/journal.ppat.1011719

Abstract

Clinical studies report that viral infections promote acute or chronic bacterial infections at multiple host sites. These viral-bacterial co-infections are widely linked to more severe clinical outcomes. In experimental models in vitro and in vivo, virus-induced interferon responses can augment host susceptibility to secondary bacterial infection. Here, we used a cell-based screen to assess 389 interferon-stimulated genes (ISGs) for their ability to induce chronic Pseudomonas aeruginosa infection. We identified and validated five ISGs that were sufficient to promote bacterial infection. Furthermore, we dissected the mechanism of action of hexokinase 2 (HK2), a gene involved in the induction of aerobic glycolysis, commonly known as the Warburg effect. We report that HK2 upregulation mediates the induction of Warburg effect and secretion of L-lactate, which enhances chronic P. aeruginosa infection. These findings elucidate how the antiviral immune response renders the host susceptible to secondary bacterial infection, revealing potential strategies for viral-bacterial co-infection treatment.

Author summary

Viral infections can make the host susceptible to secondary bacterial infections. Increasing evidence indicates that viral-bacterial co-infections cause worse clinical manifestations and lead to life-threatening acute or chronic bacterial infections. The host immune response against a viral infection promotes secondary bacterial infection; however, our understanding of how the antiviral response favors a subsequent bacterial infection is limited. Here, we aimed to assess the role of a suite of genes induced by the antiviral response, known as interferon-stimulated genes (ISGs), in the enhancement of Pseudomonas aeruginosa infection, which is frequently found in viral-bacterial co-infections in the airways of immunocompromised individuals. We found that the ISG hexokinase 2 (HK2), which drives metabolic reprogramming and mediates the secretion of the metabolite lactate by the respiratory epithelium, promotes chronic P. aeruginosa infection. These findings improve our understanding of how the antiviral response changes the host mucosal environment, enabling a subsequent bacterial infection.

Citation: Carreno-Florez GP, Kocak BR, Hendricks MR, Melvin JA, Mar KB, Kosanovich J, et al. (2023) Interferon signaling drives epithelial metabolic reprogramming to promote secondary bacterial infection. PLoS Pathog 19(11): e1011719. https://doi.org/10.1371/journal.ppat.1011719

Editor: Vincent T. Lee, University of Maryland, UNITED STATES

Received: July 13, 2023; Accepted: September 28, 2023; Published: November 8, 2023

Copyright: © 2023 Carreno-Florez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files. In addition, all data underlying the findings are available from Dryad with the identifier doi:10.5061/dryad.djh9w0w5v [66].

Funding: This work was supported by a Fulbright-ICETEX Pasaporte a la Ciencia fellowship to GPCF; the National Institutes of Health (NIH) grant T32AI49820 and Cystic Fibrosis Foundation (CFF) grant MELVIN15F0 to JAM; the NIH grant T32AI060525 to MRH; and NIH grants R01HL123771 and R33HL137077 and CFF grants BOMBER18G0 and RDP BOMBER19R0 to JMB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Increasing evidence shows that viral infections render the host susceptible to secondary bacterial infection in multiple host sites. The clinical manifestations of viral infections vary from mild, with symptoms that resolve within days, to more severe in cases that require hospitalization. Viral-bacterial co-infections are often linked to worsened outcomes, leading to extended stays in intensive care units, with prolonged and more severe clinical symptoms [1,2]

Viral infections predispose to the acquisition of acute or chronic bacterial infections. They are associated with multiple diseases in the airways [3–9], the auditory cavity [10], and the urinary [11] and gastrointestinal tracts [12–15]. Moreover, they have been linked to severe conditions such as meningitis and sepsis [11]. Clinical studies have shown that influenza virus and respiratory syncytial virus (RSV) predispose the airways to infection by Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Staphylococcus aureus in children and adults [4–6,8,16,17] and these co-infections are associated with more severe symptoms [9,18,19]. Moreover, in adults and those suffering from chronic lung disease, RSV has been shown to predispose to chronic Pseudomonas aeruginosa infection [3,7,9,18,19]. While clinical studies examining viral-bacterial co-infections have become more common, there remains limited research about the mechanisms by which viral infections predispose to secondary bacterial infection and, in particular, how the antiviral immune response promotes co-infection.

Previous work by our laboratory and others has linked a preceding viral infection and subsequent antiviral immune responses to an increased likelihood of secondary bacterial infection [20–23]. Studies in an in vivo model of airway infection have shown that the antiviral response to different respiratory viruses impairs the innate immune control of Streptococcus pneumoniae and leads to an increased bacterial burden in the nasal cavity [20]. Similarly, we have previously shown that the antiviral immune response enhances chronic Pseudomonas aeruginosa infection in an in vitro model that closely mimics the respiratory tract [22,23]. Although viral-bacterial co-infection models have been used to show that antiviral interferon signaling is detrimental to the clearance of bacterial infections, the underlying mechanisms remain unknown.

Viral infections trigger an antiviral response that reshapes the host intracellular and extracellular environment. This antiviral response involves a signaling cascade that drives increased expression of hundreds of interferon-stimulated genes (ISGs), some of which have antiviral effector functions [24,25]. The development and implementation of high-throughput ISG screens have allowed the identification of the antiviral functions of hundreds of ISGs by multiple viruses [26,27]; however, their role in viral-bacterial co-infections has yet to be tested.

To define mechanisms by which antiviral interferon signaling promotes bacterial infections, specifically through the induction of ISGs, we implemented a lentivirus-based ISG screen to identify ISGs that promote chronic P. aeruginosa infection in the respiratory tract. We identified 5 hit ISGs and further dissected the mechanism by which one hit, hexokinase 2 (HK2), stimulates P. aeruginosa biofilm formation. HK2 encodes one of the first rate-limiting enzymes in glycolysis. The enzyme HK2 is predominantly upregulated in cells undergoing aerobic glycolysis, also known as the Warburg effect (WE). This process is linked to a higher demand for glucose to supply mitochondrial respiration and shunts the excess glucose toward the synthesis of L-lactate, which is ultimately secreted and thus accessible in the extracellular milieu [28–31]. Our results suggest that the antiviral interferon response is the key driver of this metabolic reprogramming that supports secondary bacterial infections and thus contributes to transkingdom interactions.

Results

Identification of ISGs that promote P. aeruginosa biofilm growth

We have previously demonstrated that respiratory syncytial virus (RSV) infection increases P. aeruginosa biofilm growth through IFN signaling [22]. To define mechanisms by which antiviral interferon signaling enhances P. aeruginosa biofilm growth, we hypothesized that respiratory epithelial cells secrete biofilm-stimulatory factors. To test this hypothesis, human ΔF508/ΔF508 cystic fibrosis airway epithelial cells (CFBE41o-, hereafter called CF AECs) were grown as a polarized monolayer and stimulated with IFN-β (1000 IU/mL). The secretions from the apical compartment were collected and inoculated with GFP-tagged P. aeruginosa. After allowing biofilm biogenesis, we imaged and quantified biofilm biomass via fluorescence microscopy. We observed an increase in P. aeruginosa biofilm biomass grown in the apical secretions of IFN-β-stimulated CF AECs compared to the unstimulated control (Fig 1A). To determine whether specific ISGs induce P. aeruginosa biofilm formation, we screened in duplicate a library of 389 ISGs using a previously described cell-based lentiviral system [26]. The lentiviral vector co-expresses an individual ISG and the red fluorescent protein TagRFP (Fig 1B). To test the transduction efficiency and validate our screening method, we transduced CF AECs with 2 genes that positively regulate the expression of ISGs (IRF1 and IRF9) and 4 ISGs with known functions (RSAD2, CH25H, IFITM3, and SLC25A28). We selected the lowest lentivirus dose yielding 80% RFP/ISG-expressing+ cells (Fig 1C). The ISG screen included P. aeruginosa grown in minimal essential medium (MEM) as a negative control and inoculation of P. aeruginosa in the apical secretions of cells with lentiviral overexpression of the firefly luciferase (Fluc) gene as a control of transduction (S1 Fig). CF AECs were transduced with the lentiviruses expressing each ISG for 72 h. We incubated GFP-producing P. aeruginosa in apical secretions from each ISG and assessed biofilm growth. Using fluorescence microscopy, we imaged, quantified, and averaged 6 different fields of GFP-expressing P. aeruginosa biofilms grown in apical secretions for each ISG tested. From 2 biological screening replicates, we identified a set of ISGs inducing the highest biofilm biomass production compared to the global mean (Fig 1D). We then calculated a z-score based on the biomass obtained from each condition, and z-scores above 2 were considered to indicate significance. Conditions with a z-score of 2 in both screens were considered hits. We identified 6 hit ISGs that significantly enhanced P. aeruginosa biofilm growth: TNF Receptor Superfamily Member 10a (TNRFSF10A, z score = 2.7), PML Nuclear Body Scaffold (PML, z score = 2.1), Apolipoprotein B MRNA Editing Enzyme Catalytic Subunit 3A (APOBEC3A, z score = 3.1), MYD88 Innate Immune Signal Transduction Adaptor (MYD88, z score = 3.6), EH Domain Containing 4 (EHD4, z score = 2.6) and Hexokinase 2 (HK2, z score = 3) (Fig 1E).

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Fig 1. Identification of ISGs that promote P. aeruginosa biofilm growth.

(A) Biofilm biomass of GFP-expressing P. aeruginosa grown in apical secretions from IFN-β-stimulated CF AECs determined using a static abiotic biofilm assay. Representative fields of 3 independent replicates (left) and quantification of biofilm biomass using Nikon NIS-Elements from images acquired at 20x (right). (B) Diagram of ISG overexpression screen and biofilm biomass quantification created with Biorender.com. (C) Transduction efficiency using different lentivirus doses, with 50 uL of lentiviral particles as the lowest dose reaching 80% transduction. (D) Average of P. aeruginosa biofilm biomass per ISG in 2 independent screens represented as a dot plot, highlighting ISGs with the highest biomass increase (green dots) measured under abiotic conditions. (E) PCA of screen 1 and 2 highlighting 6 ISGs with a Z-score ≥2 (in green). The 6 ISGs hits are TFRSF10A, PML, APOBEC3A, MYD88, EHD4 and HK2. For all experiments n ≥ 3. Data are presented as mean ± SEM. ****p < 0.0001.

https://doi.org/10.1371/journal.ppat.1011719.g001

Verification of TNFRSF10A, PML, APOBEC3A, MYD88, EHD4 and HK2 as ISG hits enhancing P. aeruginosa biofilm growth

With the identification of TNFRSF10A, PML, APOBEC3A, MYD88, EHD4, and HK2 as ISGs that promote a significant increase of P. aeruginosa biofilm formation, we tested whether these 6 ISG hits were induced by IFN or respiratory viral infection. We used qRT-PCR to assess their expression in CF AECs stimulated with IFN-β or infected with two different respiratory viruses, RSV or human rhinovirus (hRV14). We observed upregulation of the 6 ISGs during IFN-β stimulation (Fig 2A) and during infection with either of the two respiratory viruses relevant to chronic lung disease patients (Fig 2B and 2C), indicating that their upregulation is mediated by IFN signaling.

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Fig 2. Confirmation of selected hit ISGs that stimulate P. aeruginosa biofilm formation.

(A-C) mRNA fold change of TNFRSF10A, PML, MYD88, EHD4 and HK2 in IFN-β-stimulated (A), RSV-infected (B) or hRV-infected (C) CF AECs measured by quantitative RT-PCR (qRT-PCR). (D) Biofilm biomass of GFP-expressing P. aeruginosa grown in apical secretions from CF AECs overexpressing GFP (transfection control), TNFRSF10A, PML, MYD88, EHD4 or HK2 using liposome-mediated transfection and measured under static abiotic conditions. Biofilm biomass quantified with Nikon NIS-Elements from images acquired at 20x. For all experiments n ≥ 3. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

https://doi.org/10.1371/journal.ppat.1011719.g002

We then implemented a secondary validation for the 6 ISG hits. We adapted an overexpression platform using liposome-mediated transfection of plasmid encoding each ISG hit in CF AECs. After transfection, we collected apical secretions to grow GFP-expressing P. aeruginosa biofilms for 6 h. We then imaged and quantified biofilm biomass by fluorescence microscopy. We observed induction of P. aeruginosa biofilm formation on CF AECs overexpressing TNFRSF10A, PML, MYD88, EHD4, and HK2 (Fig 2D). We detected cytotoxicity triggered by APOBEC3A overexpression and did not further pursue this ISG hit in subsequent experiments. These results suggest that TNFRSF10A, PML, MYD88, EHD4, and HK2 are induced during IFN signaling and that their upregulation provokes the secretion of molecule(s) that stimulate P. aeruginosa biofilm formation.

RSV infection induces hexokinase 2 (HK2) through the antiviral response and triggers the Warburg effect

We next investigated the mechanism by which the hit gene HK2 promoted P. aeruginosa biofilm formation. HK2 was prioritized because of the robust biofilm induction in the primary screen (z score = 3) and secondary validation (Figs 1D and 2D). HK2 encodes the enzyme hexokinase 2 (HK2), one of the four hexokinase isoforms found in mammalian tissues. Hexokinases are responsible for the first rate-limiting step in glycolysis. HK1 is constitutively produced in most cell type and leads glycolysis toward the synthesis of pyruvate and its subsequent transport to the mitochondria, where it supports respiration and oxidative phosphorylation. In addition, cells with high glucose flux [30,31] also express HK2, which leads the excess of pyruvate toward the synthesis of L-lactate through a process known as aerobic glycolysis or Warburg effect (WE) [28,32] (Fig 3A). To determine if antiviral immune signaling triggers the WE, we stimulated CF AECs with IFN-β and measured the glycolytic status. We measured glucose consumption and oxygen consumption rate (OCR) to investigate how glycolysis supplies mitochondrial respiration. We observed increased glucose consumption under IFN-β stimulation (Fig 3B), in accordance with higher glycolytic flux commonly found in cells undergoing the WE. We also observed a similar OCR (Fig 3C) and basal respiration (S2A Fig) during IFN-β stimulation, indicating that glycolysis is feeding mitochondrial respiration. We additionally assessed glycolytic function during RSV infection. Likewise, we found increased glucose consumption (Fig 3B) and a similar trend in OCR and basal respiration (S3A and S3B Fig). Glucose consumption was also increased during hRV14 infection (S4A Fig). These results suggest that antiviral IFN signaling leads to higher glycolytic flux, which supports the WE and mitochondrial respiration.

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Fig 3. RSV infection induces HK2 expression and triggers the Warburg effect in bronchial epithelial cells through the antiviral response.

(A) Diagram of glycolysis depicting the fate of pyruvate toward oxidative phosphorylation or toward aerobic glycolysis (WE) created with Biorender.com. (B) Glucose consumption in the basolateral medium of IFN-β-stimulated or RSV-infected CF AECs measured by colorimetric assay. (C) OCR in CFBE41o- cells during IFN-β stimulation determined using Seahorse assay. (D) L-lactate concentration measured in apical secretions from CF AECs during IFN-β stimulation or RSV infection measured by colorimetric assay. (E) ECAR in IFN-β-stimulated CFBE41o- cells determined using Seahorse assay. Lung tissue and bronchoalveolar lavage fluid (BALF) were collected from BALB/cJ neonatal mice infected with line 19F RSV (5x10⁵ pfu/g) 3 and 7 days post-infection. (F) mRNA expression of Ifn-B, Inf-L, and HK2, expressed as fold change, was measured in lung tissue. (G) Quantification of the L-lactate concentration in BALF determined using colorimetric assay. For all experiments n ≥ 3. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

https://doi.org/10.1371/journal.ppat.1011719.g003

We next measured the secretion of L-lactate and extracellular acidification rate (ECAR) to determine if IFN signaling is also driving glycolysis toward Warburg metabolism. We found higher lactate levels in secretions from IFN-β-stimulated CF AECs than in the unstimulated control (Fig 3D). We observed similar results in secretions from cells infected with RSV (Figs 3D and S3E) and hRV14 (S4B Fig), We also found an increased ECAR and enhanced basal glycolysis in cells under IFN stimulation (Figs 3E and S2B) and during RSV infection (S3C and S3D Fig), as measured by Seahorse mitochondrial stress analysis. To examine if HK2 upregulation was sufficient to cause this metabolic shift, we overexpressed HK2 in AECs and examined lactate secretion. We observed that HK2 overexpression induces L-lactate secretion, suggesting that HK2 upregulation is sufficient to promote lactate secretion (S5 Fig). Overall, these data suggest that viral-induced IFN signaling leads to an increase in glucose consumption and apical L-lactate secretion in bronchial epithelial cells, consistent with IFN-induced HK2 reprogramming the metabolic state of the respiratory epithelium to the WE.

Next, we wanted to determine if our in vitro observations of antiviral IFN signaling inducing the WE could be recapitulated in an in vivo model. We infected neonatal BALB/cJ mice with 5x10⁵ pfu/g line 19F RSV [33] and collected lung tissue and bronchoalveolar lavage fluid (BALF) at 3 and 7 days post-infection (dpi). We found increased expression of Ifn-B, Ifn-L, and HK2 at both time points compared to that in the uninfected control, which is consistent with the induction of the antiviral response along with the upregulation of the glycolytic gene HK2 (Fig 3F). We next measured L-lactate secretion in BALF collected at 3 and 7 dpi. We found increased levels of L-lactate in RSV-infected mice compared to that in the controls (Fig 3G). These results suggest that RSV infection induces Warburg metabolism in vivo. Taken together, these findings demonstrate that the IFN signaling induced during a viral infection triggers the WE.

Lactate secreted by the CF bronchial epithelium stimulates P. aeruginosa biofilm growth

After observing that HK2 upregulation by IFN signaling triggers the WE both in vitro and in vivo, we examined the impact of epithelial metabolic reprogramming on P. aeruginosa biofilm biogenesis. To this end, we used pharmacological inhibitors of the WE during IFN-β stimulation or HK2 overexpression. We used 2-deoxy-glucose (2-DG), an analogue of glucose, to inhibit HK2 function and sodium oxamate (NaOx), an analogue of pyruvate, to inhibit the conversion of pyruvate to L-lactate. We treated CF AECs with either 2-DG (10 mM) or NaOx (50 mM), collected apical secretions, and inoculated P. aeruginosa into those secretions for biofilm biomass quantification. We observed that secretions from IFN-β-stimulated CF AECs enhanced P. aeruginosa biofilm formation compared to that of the unstimulated control, with a reduction in P. aeruginosa biofilm biomass when IFN-β-stimulated CF AECs were treated with 2-DG or NaOx (Fig 4A). We further used HK2-overexpression to determine whether HK2 induction alone is sufficient for inducing P. aeruginosa biofilm formation. We observed that while apical secretions from HK2-overexpressing CF AECs enhanced P. aeruginosa biofilm biomass, the presence of each of these glycolytic inhibitors led to a decrease in P. aeruginosa biofilm biomass to untreated levels (Fig 4B). These findings suggest that inhibiting the synthesis of L-lactate and its apical secretion impairs P. aeruginosa biofilm growth.

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Fig 4. L-lactate stimulates P. aeruginosa biofilm growth.

(A, B) Biofilm biomass of GFP-expressing P. aeruginosa grown in apical secretions from IFN-β-stimulated CF AECs (A) or grown in apical secretions from HK2-overexpressing CF AECs (B) treated with 2-DG or NaOx and assessed by a static abiotic biofilm assay. HK2 overexpression was induced through liposome-mediated transfection of a plasmid encoding HK2. Representative fields of 3 independent replicates (left) and quantification of biofilm biomass using Nikon NIS-Elements with images acquired at 20x (right). (C) Biofilm biomass of GFP-producing P. aeruginosa co-cultured with CFBE41o- cells under constant flow with L-lactate supplementation assessed under biotic conditions. Biofilms (green) and nuclei stained with Hoechst (blue) were imaged by live-cell microscopy (left), and biofilm biomass was quantified by Nikon NIS-Elements (right). For all experiments n ≥ 3. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

https://doi.org/10.1371/journal.ppat.1011719.g004

Finally, we wanted to determine whether growing P. aeruginosa in the presence of L-lactate is sufficient to induce biofilm formation. To this end, we used a live-cell imaging system in which CF AECs are co-cultured with GFP-producing P. aeruginosa for 6 h under a constant flow of medium supplemented with L-lactate (10 mM) [22,34,35]. We used L-lactate, as it is the isomeric form synthesized in mammalian cells and applied the concentration released during RSV infection. We next imaged biofilms grown on CF AECs by fluorescence microscopy and quantified the biofilm biomass. We observed enhanced P. aeruginosa biofilm formation in medium supplemented with L-lactate compared to that in the untreated control (Fig 4C), suggesting that lactate secreted by the airway epithelium enhances P. aeruginosa biofilm formation. Overall, these findings are consistent with the conclusion that IFN signaling stimulates the expression of the ISG HK2, which drives metabolic reprogramming and the WE, stimulates apical L-lactate secretion, and thereby enhances P. aeruginosa biofilm growth.

Discussion

The expression of ISGs during viral infection leads to changes in the intracellular environment, thus regulating the host-virus crosstalk. Although the functions of many ISGs have been studied in the context of viral infections, little is known about the functions of virus-induced ISGs in the setting of viral-bacterial co-infections, in which IFN signaling is known to potentiate secondary bacterial infections. Here, we screened 389 ISGs previously described to regulate multiple viral infections [26], and we identified 5 hit ISGs, namely, TNRFSF10A, PML, MYD88, EHD4 and HK2, that robustly promoted P. aeruginosa biofilm growth. Moreover, we dissected the mechanism of HK2 in enhancing P. aeruginosa biofilm growth, showing that it instigates aerobic glycolysis and the apical secretion of L-lactate, which thereby potentiates P. aeruginosa biofilm growth. Taken together, our findings support a model in which antiviral signaling mediates metabolic reprogramming in the airway epithelium, which stimulates P. aeruginosa biofilm biogenesis. Finally, this study demonstrates that applying high-throughput based ISG screens, previously used to screen single viral [26] or bacterial [36] infections offers a valuable tool to study viral-bacterial co-infections, furthering our understanding of transkingdom interactions during co-infection.

Although ISGs are canonically referred to as antiviral effectors, the upregulation of HK2 mainly has a proviral role among respiratory viral infections [37–40]. Multiple viruses enhance glucose metabolism in the host, even in the presence of oxygen to supply mitochondrial respiration, thus resembling the WE widely studied in cancer, also known as aerobic glycolysis. Although the main role of aerobic glycolysis appears to be the rapid generation of ATP, it also funnels metabolites to other pathways including the pentose phosphate pathway (PPP) for the synthesis of nucleosides, fatty acids and antioxidant molecules and the hexosamine biosynthetic pathway (HBP) that supports the generation of metabolites for protein and lipid glycosylation [28,29,32]. As intracellular parasites, viruses rely on the host cell machinery and resources for viral replication; thus, it is not surprising that aerobic glycolysis plays a key role in the success of viral infection. HK2 encodes the first rate-limiting enzyme of aerobic glycolysis, and it is upregulated during infections caused by multiple respiratory viruses including RSV, influenza virus, human metapneumovirus (hMPV) and human rhinovirus (hRV) [37–41]. Interestingly, HK2 has a proviral function in RSV, influenza virus, hMPV and adenovirus 5 infection [37–41] but exerts an antiviral function in hRV-C infection [41], revealing some virus-specific roles. Our results show for the first time that the induction of HK2 is the result of the host antiviral immune program, i.e., the IFN response.

A hallmark feature of the WE is the secretion of lactate in the extracellular environment, and recently, the role of lactate in host-pathogen interactions is increasingly appreciated after long being considered a waste product of glucose metabolism. Interestingly, it was recently proposed that lactate produced during virus-induced aerobic glycolysis inhibits RIG-I signaling, thus suppressing RIG-I-mediated IFN synthesis and allowing viral replication [42]. This hypothesis suggests that HK2-driven aerobic glycolysis supports viral infections by providing energy and biomolecules for replication, as well as by providing lactate as a metabolite to evade host defense.

Lactate exists as two enantiomers, L- and D-lactate. While L-lactate is the main form found in vertebrates as a byproduct of either anaerobic or aerobic glycolysis, D-lactate is mainly synthesized by bacteria under anaerobic conditions [43]. It is well documented that L-lactate is present at high levels at sites of inflammation [44,45]. And notably, clinical studies of acute or chronic infection and inflammation in the lungs have shown a predominance of the L-lactate form, suggesting that the host, specifically innate immune cells and the epithelium, is the main source of this glycolytic metabolite [41–45].

Although the role of L-lactate in viral-bacterial co-infections is unknown, increasing evidence from research on acute and chronic infections in the airways suggests an association between viral infection, increased L-lactate levels, and secondary bacterial infection. Clinical evidence from airway samples and studies on in vitro models of the respiratory epithelium have shown that viral infections increase the concentration of L-lactate [44]. In addition, some clinical studies have linked respiratory viral infections and higher levels of L-lactate with episodes of pulmonary exacerbation, characterized by worsened lung symptoms, including worsened bacterial infection [44,46]. Here, using a model of viral-bacterial co-infection in the airways, we demonstrate that the antiviral IFN response promotes secondary P. aeruginosa infection through the upregulation of the ISG HK2, which is required for the WE and apical L-lactate secretion. Overall, these findings suggest that P. aeruginosa uses lactate secreted from the airway epithelium during the antiviral response to promote biofilm formation.

Multiple bacteria that infect the airways, including P. aeruginosa, Neisseria meningitidis and Haemophilus influenzae, use host-derived lactate for colonization and persistence [47–55]. In studies of the CF airways, it has been found that lactate in CF sputum and synthetic CF sputum medium (SCFM) is among a diverse group of carbon sources used by P. aeruginosa, highlighting that P. aeruginosa can respond to the nutritional cues of the CF lung environment [50,51]. Growing a clinical isolate of P. aeruginosa in medium supplemented with lactate led to a genetic profile associated with the biofilm lifestyle, inducing the upregulation of genes involved in phenazine production and downregulation of genes involved in swarming motility [52]. Interestingly, in our study we observed that L-lactate stimulates P. aeruginosa biofilm biogenesis. Future work will examine the underlying mechanism by which P. aeruginosa responds to L-lactate to induce biofilm. In summary, the host-derived L-lactate produced during the antiviral immune response plays a key role in promoting secondary bacterial infection.

A majority of the research on viral-bacterial co-infections has focused on how the antiviral response renders the host susceptible to secondary bacterial infections. However, fewer studies point out that having a bacterial infection first can also predispose the host to a secondary viral infection [56–59]. Recent studies suggest that S. pneumoniae colonization promotes the infectivity and transmission of influenza [59]. In addition, previous work from our laboratory demonstrated that P. aeruginosa secretes at least one virulence factor that reduces viral antigen presentation and recognition of influenza-infected respiratory epithelial cells [60]. However, more studies are necessary to determine if immunometabolic regulation is part of a broadly applicable mechanism by which bacterial infections can predispose or impact the severity of viral infections.

While IFN signaling is known to be detrimental to the host defense against a variety of extracellular pathogens, the mechanisms underlying this dysfunction remain poorly understood. The studies herein have identified the downstream ISGs TNRFSF10A, PML, MYD88, EHD4, and HK2 that potentiate secondary bacterial infection. The function of these ISGs are in different cellular functions, including cell death and survival [61], nuclear bodies structure [62], inflammatory signaling [63], endocytic trafficking [64] and glucose metabolism [65]. While no apparent overlap in cellular functions suggest synergy of these ISGs in promoting biofilm, further studies are required to identify whether they act synergistically or through independent mechanisms to promote biofilm. Here, we uncovered the molecular mechanism by which hexokinase 2 mediates biofilm formation due to metabolic reprogramming in the host. By elucidating the role of specific pathways downstream of the antiviral IFN response that promote secondary bacterial infection, we might identify new host targets with therapeutic potential to combat the poor outcomes observed, particularly in chronic lung disease patients.

Materials and methods

Ethics statement

In vivo assays strictly followed the recommendations of the NIH Guide for the Care and Use of Laboratory Animals [56] and were approved by the University of Pittsburgh Institutional Animal Care and Use Committee (IACUC) (protocol number 14023340). Neonatal BALB/cJ mice were handled according to IACUC guidelines, and all efforts were made to minimize animal suffering. Mice were housed at the University of Pittsburgh Division of Laboratory Animal Resources.

Cell lines, viruses, and bacterial strains

We used the immortalized human CF airway epithelial cell line CFBE41o- (referred to herein as CF AECs) collected from explanted lungs of CF patients, following the protocol approved by the Institutional Review Board at the University of Pittsburgh [35]. CFBE41o- cells were seeded on Transwell filters and grown at air-liquid interface for 7–10 days or until well polarized or differentiated. For viral infections, CF AECs were apically infected with purified RSV A2 strain (MOI = 1) resuspended in minimal essential medium (MEM) without phenol red for 72 h at 37°C and with 5% CO₂. For bacterial infections, GFP-expressing Pseudomonas aeruginosa strain PAO1 was used as previously described [22].

Glycolytic inhibition experiments

2-Deoxy-glucose (2-DG, Sigma D8375) and sodium oxamate (NaOx, Sigma 02751) were purchased from Sigma Aldrich. For glycolytic inhibition during IFN-β stimulation, either 10 mM 2-DG or 50 mM NaOx was added to CF AECs basolaterally for 2 h at 37°C and with 5% CO₂. The basolateral medium was replaced with medium supplemented with IFN-β (1000 IU/mL, R&D 8499-IF-010/CF), MEM without phenol red was added apically, and apical secretions were collected after 18 h for static abiotic biofilm formation experiments. For glycolytic inhibition during HK2 overexpression, 48 h post-transfection, cells were treated with either 10 mM of 2-DG or 50 mM NaOx as previously described, and MEM without phenol was added apically. Apical secretions were collected 72 h post-transfection.

ISG screen

The ISG library and the generation of the lentiviral particles were previously described [26]. Briefly, a library of 389 ISG was compiled based on microarrays of IFN-treated cells, and each ISG was inserted in a bicistronic lentivirus co-expressing the ISG and the red fluorescent protein TagRFP. Transduction efficiency was visually assessed as the percentage of cells that were RFP+. CF cells were spinoculated with equal volumes of lentivirus-containing supernatants in a one gene per well format at 800 xg for 45 minutes at 37°C. Lentivirus was removed at 6 hours post-transduction. ISG-conditioned supernatants were subsequently collected 48 hours post-transduction and maintained at 4°C. Each ISG-conditioned supernatant was added to a glass-bottomed 48 well plate and inoculated with PAO1 (OD₆₀₀ = 0.01) for 6 h. Biofilms formed were imaged at 20x, and the biofilm biomass was quantified with a Nikon Ti inverted fluorescence microscope using Nikon Imaging Software (NIS) Elements AR 4.6. The ISG screen included PAO1 grown in MEM as negative control, PAO1 grown in MEM supernatant from cells overexpressing the Fluc gene, and PAO1 grown in supernatant from cells without lentiviral transduction, as transduction controls.

Abiotic biofilm assay

GFP-expressing PAO1 (OD of the culture normalized to 0.05) was inoculated in apical secretions of CF AECs supplemented with 0.4% L-arginine in glass-bottomed dishes (MatTek Corporation), and biofilm formation was measured after 6 h of growth at 37°C and with 5% CO₂. Z-stack images were recorded from 6–10 random fields per dish using a Nikon Ti-inverted fluorescence microscope. The area (μm²) and biofilm volume (μm³) of each Z-stack was measured using NIS Elements AR 4.6, and the biofilm biomass was determined by dividing the biomass volume/area (μm³/μm²).

Mitochondrial function and aerobic glycolysis assessment

The OCR and ECAR were assessed using the Seahorse platform (Agilent) following 18 h of IFN-β stimulation or 24 h of RSV infection (MOI = 1). The OCR (pmol/minute) and ECAR (mpH/minute) were measured in basal conditions (medium alone) and after the sequential addition of oligomycin, FCCP, 2-DG, and the combination of rotenone and antimycin between 90–120 minutes. Glucose consumption (Abcam, ab65333) and lactate secretion (Abcam, ab65330) were measured after 18 h of IFN-β stimulation or 72 h of RSV infection using colorimetric assays. Glucose consumption was determined by subtracting the glucose concentration in basolateral medium from the glucose concentration in the feeding medium. For lactate secretion, MEM without phenol red was added apically for 18 h during IFN-β stimulation or for the last 24 h of RSV infection and was collected to determine the extracellular lactate concentration.

Biotic biofilm imaging

CFBE410- cells under the constant flow of medium with 10 mM L-lactate (Sigma Aldrich, L7022) were inoculated with PAO1, and biofilm growth was recorded through a previously described live-cell imaging system [22, 35]

Quantitative real-time PCR

RNA was extracted from CF AECs or neonatal BALB/cJ mice using a Qiagen RNA extraction kit (RNeasy Mini Kit, 74106) following the manufacturer instructions. PCR assays were run in a BioRad CFX connect system, and RNA expression was determined using the 2^-ΔΔCT method.

In vivo RSV infection

In vivo RSV assays strictly followed the recommendations of the NIH Guide for the Care and Use of Laboratory Animals [56] and were approved by the University of Pittsburgh Institutional Animal Care and Use Committee (IACUC) (protocol number 14023340). Neonatal BALB/cJ mice were infected intranasally with 5 × 10⁵ pfu/g body weight RSV line 19 or an equal volume of PBS, as previously described [22, 33]. Bronchoalveolar lavage fluid was collected to measure L-lactate levels, and lungs were harvested to quantify RNA expression.

Statistical analysis

GraphPad Prism version 6.0 (GraphPad) was used for statistical analysis. Means were compared using Student’s t test or, for multiple comparisons, ANOVA with Tukey’s post hoc test. p < 0.05 was considered significant.

Supporting information

S1 Fig. ISG screen controls for P. aeruginosa biofilm growth.

Representative images of GFP-producing P. aeruginosa biofilms under abiotic conditions grown in MEM (negative control), secretions from cells overexpressing FLUC (transduction control), and secretions from cells transfected with an ISG that resulted in low biofilm formation induction (SLC16A1) and an ISG that resulted in high biofilm formation induction (CPT1A). 20x images captured with Nikon NIS-Elements.

https://doi.org/10.1371/journal.ppat.1011719.s001

(TIF)

S2 Fig. IFN-β signaling triggers non-mitochondrial glucose consumption.

Measurement of (A) basal respiration and (B) basal glycolysis in CFBE41o- stimulated with IFN-β (1000 IU/mL) using a Seahorse assay. For all experiments n ≥ 3. Data are presented as mean ± SEM. *p < 0.05.

https://doi.org/10.1371/journal.ppat.1011719.s002

(TIF)

S3 Fig. RSV induces the expression of the ISG HK2 and triggers the Warburg effect in bronchial epithelial cells.

(A) OCR, (B) basal respiration, (C) ECAR, and (D) basal glycolysis measured in RSV-infected CFBE41o- cells using a Seahorse assay. (E) L-lactate concentration in apical secretions from primary CF AECs during RSV infection measured by colorimetric assay. For all experiments n ≥ 3. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01.

https://doi.org/10.1371/journal.ppat.1011719.s003

(TIF)

S4 Fig. hRV14 infection induces glucose consumption and apical L-lactate secretion in CF AECs.

Measurement of (A) glucose consumption in growth medium and (B) L-lactate concentration in apical secretions of CF AECs infected with hRV14 (MOI 0.1) for 72 h using a colorimetric assay. For all experiments n ≥ 3. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01.

https://doi.org/10.1371/journal.ppat.1011719.s004

(TIF)

S5 Fig. HK2-overexpression in CF AECs enhances L-lactate synthesis and apical secretion.

Colorimetric measurement of L-lactate concentration from apical secretions of HK2-overexpressing CF AECs, 72 h post-transfection. For all experiments n ≥ 3. Data are presented as mean ± SEM. ***p < 0.001.

https://doi.org/10.1371/journal.ppat.1011719.s005

(TIFF)

Acknowledgments

We thank the Airway Biology Core at the University of Pittsburgh for providing primary airway epithelial cells and Anthony Richardson, Paula Zamora, and Alexis Duray for the helpful comments.

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Figures

Abstract

Author summary

Introduction

Results

Identification of ISGs that promote P. aeruginosa biofilm growth

Verification of TNFRSF10A, PML, APOBEC3A, MYD88, EHD4 and HK2 as ISG hits enhancing P. aeruginosa biofilm growth

RSV infection induces hexokinase 2 (HK2) through the antiviral response and triggers the Warburg effect

Lactate secreted by the CF bronchial epithelium stimulates P. aeruginosa biofilm growth

Discussion

Materials and methods

Ethics statement

Cell lines, viruses, and bacterial strains

Glycolytic inhibition experiments

ISG screen

Abiotic biofilm assay

Mitochondrial function and aerobic glycolysis assessment

Biotic biofilm imaging

Quantitative real-time PCR

In vivo RSV infection

Statistical analysis

Supporting information

S1 Fig. ISG screen controls for P. aeruginosa biofilm growth.

S2 Fig. IFN-β signaling triggers non-mitochondrial glucose consumption.

S3 Fig. RSV induces the expression of the ISG HK2 and triggers the Warburg effect in bronchial epithelial cells.

S4 Fig. hRV14 infection induces glucose consumption and apical L-lactate secretion in CF AECs.

S5 Fig. HK2-overexpression in CF AECs enhances L-lactate synthesis and apical secretion.

Acknowledgments

References