THP-1 cells were plated in 6-well plates at a density of 0.5 × 106 per well. They were differentiated with phorbol myristate acetate (5 nM) for 48 h and cultured in complete RPMI medium for 72 h. Cells were infected with IAV WSN at a multiplicity of infection (MOI) of 1, 2, or 3 for 2 h. Cells were then washed with phosphatebuffered saline (PBS) and cultured in complete RPMI medium for 24 h. The cell-free supernatant was collected for ELISA. For drug-therapy experiments, differentiated THP-1 cells were treated with MCC950 (1 µM, Adipogen), anakinra (0.5 µg/mL, Kineret™), or vehicle control (PBS) for 1 h. Immediately after the drug treatment, cells were infected with IAV WSN (MOI 2) for 2 h. The infected cells were washed with PBS and cultured in complete RPMI medium containing MCC950 (1 µM), anakinra (0.5 µg/mL), or vehicle control for 24 h. The cell-free supernatant was collected for ELISA. ELISA was done for IL-1β (eBioscience, San Diego, CA, USA) and Caspase-1 (R&D Systems, Minneapolis, MN, USA).

# Cell Imaging

Human THP-1 monocytes were plated on sterilized 18CIR-1 coverglasses in a 12-well plate at a density of 0.25–0.5 million cells per well and differentiated. Cells were then treated with an MOI of 2 of IAV for 24 h and probed for active caspase-1 by means of FAM-YVAD-FMK (FAM-FLICA caspase-1 assay kit #97, ImmunoChemistry, Bloomington, MN, USA) according to the manufacturer's instructions. Nuclei were labeled with Hoechst 33342, and then cells were fixed in 2.7% paraformaldehyde for 5 min at room temperature. Images were acquired by means of a Nikon A1R laser scanning confocal microscope.

#### *In Vivo* Influenza Virus Infection

Juvenile (4-week-old) mice were anesthetized with isoflurane and infected intratracheally with WSN [12.5 plaque forming units (PFU) in 50 μL PBS] or an equal volume of PBS.

#### Inflammasome Inhibition *In Vivo*

MCC950 (Adipogen) reconstituted in sterile PBS was administered intraperitoneally in juvenile mice at a dose of 10 mg/kg daily beginning on day 3 postinfection (p.i.) until tissue harvest, death, or recovery. Anakinra (Kineret™) was administered intraperitoneally in juvenile mice at a dose of 100 mg/kg on day 3 p.i. until tissue harvest, death, or recovery.

#### Bronchoalveolar Lavage Fluid (BALF) Harvest

A 20-gauge angiocatheter was ligated into the trachea, and the lungs were lavaged twice with sterile PBS (700 µL). The lavage fluid was centrifuged at 1,000 *g* for 10 min. The pellet was resuspended, and the cells were counted using the Invitrogen Countess Automated Cell Counter (Invitrogen, Grand Island, NY, USA). Protein levels in the supernatant were measured by Bradford Assay (BioRad), and cytokine levels were measured using ELISA. Interleukin (IL)-18 was measured using the mouse IL-18 ELISA Kit (MBL International Corporation, Woburn, MA, USA) according to the manufacturer's instructions. Interleukin-6 (IL-6) was measured using the mouse IL-6 Ready-Set-GO ELISA Kits (eBioscience, San Diego, CA, USA). Interferon (IFN)-α was measured using the mouse IFN Alpha ELISA Kit (PBL Assay Science, Piscataway, NJ, USA).

#### Wet-to-Dry Weight Ratios

Mice were anesthetized and lungs were surgically removed *en bloc.* Lungs were weighed in a tared container. The lungs were then dried at 45°C in a Speed-Vac SC100 evaporator (Thermo Scientific, Waltham, MA, USA) until a constant weight was obtained, and the wet-to-dry weight ratio was calculated.

#### Histology

Mice were anesthetized and lungs were perfused *via* the right ventricle with 10 mL HBSS with calcium and magnesium. A 22-gauge angiocatheter was sutured into the trachea, heart and lungs were removed en bloc, and then lungs were inflated with 0.7 mL of 4% paraformaldehyde at a pressure not exceeding 16 cm H2O. Tissue was fixed in 4% paraformaldehyde overnight at 4°C, then processed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Images were acquired by means of a TissueGnostics automated slide imaging system (TissueGnostics, Vienna, Austria).

#### Lung Harvest and Homogenization

For plaque assay, lungs were homogenized in PBS (20 µL/mg lung). For western blot, lungs were homogenized in RIPA buffer with protease inhibitor (20 mM Tris–HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, Roche complete ULTRA Tablet). Homogenized lungs were centrifuged at 1,000 *g*. The supernatant was frozen at 80°C.

#### Western Blot

The presence of indicated proteins in lung homogenates from day 7 p.i. was assessed by western blotting using the following antibodies: NLRP3 (Adipogen), Caspase-1 (14F468) (Santa Cruz sc-56036), ASC (Adipogen), IL-18 (Biovision, 5180R-10), and Actin (Santa Cruz).

#### Flow Cytometry for Intracellular Staining of NLRP3 Inflammasome Components

Mice were anesthetized and lungs were perfused *via* the right ventricle with 10 mL HBSS with Ca2+ and Mg2+. The lung lobes were removed and inflated with enzyme solution (5 mL of 0.2 mg/mL DNase I and 2 mg/mL Collagenase D in HBSS with Ca2+ and Mg2+) using a 30G needle. The tissue was minced and then processed in GentleMACS dissociator (Miltenyi) according to the manufacturer's instructions. Processed lungs were passed through a 40 µm cell strainer, and red blood cells were lysed with BD Pharm Lyse (BD Biosciences, San Jose, CA, USA). Remaining cells were counted with a Countess Cell Counter (Invitrogen, Grand Island, NY, USA). CD45 microbeads were added, and cells were eluted according to the Miltenyi manufacturer's instructions. Cells were stained with viability dye Aqua (Invitrogen) and stained with a mixture of fluorochromeconjugated antibodies (see **Table 1** for lists of fluorochromes, antibodies, manufacturers, and clones). Data were acquired on a BD LSR II flow cytometer using BD FACSDiva software (BD Biosciences), and data analyses were performed with FlowJo software (TreeStar, Ashland, OR, USA). Cell populations were identified using sequential gating strategy, and the percentage of cells in the live/singlets gate was multiplied by the number of live cells to obtain an absolute live-cell count. The expression of activation markers is presented as median fluorescence intensity (MFI).

#### Plaque Assay

Confluent monolayers of MDCK cells were infected with stock virus or lung homogenate serially diluted in 1% bovine serum albumin Dulbecco's Modified Eagle Medium (DMEM) for 2 h at 37°C. Plates were washed with PBS and an overlay of 50% 2× Replacement Media (2× DMEM, 0.12 M NaHCO3, 2% Penn– Strep, and 1% HEPES), 50% avecil (2.35%), and *N*-acetyl trypsin (1.5 µg/mL) remained on the cells for 72 h at 37°C. Overlay was removed, and the monolayers were then stained with Naphthalene Blue-Black and plaques counted.

#### Statistical Analysis

Data are expressed as means ± SD. Differences between two groups were assessed by using a Student's *t*-test. Differences between three or more groups were assessed using one-way analysis of variance with a Bonferroni multiple comparisons test. Values of *P* < 0.05 were considered to be significant. The log rank test was used in the analysis of the Kaplan–Meier curve. All analyses were performed using GraphPad Prism software version 6.0 for Windows (GraphPad Software, San Diego, CA, USA).

# RESULTS

# MCC950 and Anakinra Decrease NLRP3 Inflammasome Activity *In Vitro* in Macrophages Infected with IAV

MCC950 is a small molecule inhibitor of the NLRP3 inflammasome. Although its exact mechanism of action is unknown, it has been shown to be specific to NLRP3 and to prevent the activation of caspase-1 and the maturation and secretion of IL-1β and IL-18 in response to multiple NLRP3 inflammasome stimuli (14–16). Anakinra is a synthetic version of the naturally occurring IL-1β receptor antagonist. It prevents the downstream signaling of the IL-1β receptor. We tested the ability of MCC950 and anakinra to inhibit the NLRP3 inflammasome *in vitro.*

To determine the IAV inoculation dose necessary for NLRP3 inflammasome activation, we infected THP-1 cells (a human monocyte cell line derived from a 1-year-old patient) with IAV at an MOI of 1, 2, and 3 for 24 h. As shown in **Figures 1A,B**, there was a dose dependent increase in caspase-1 and IL-1β levels in the supernatant from IAV-infected macrophage cells. Based on these results we chose to test the ability of MCC950 and anakinra to inhibit NLRP3 inflammasome activation in response IAV at an MOI of 2.

THP-1 cells were pretreated with MCC950 (1 µM) or anakinra (0.5 µg/mL) and then infected with IAV (A/WSN/2009) at an MOI of 2. As shown in **Figures 1C,D**, cells infected with IAV had a robust increase in caspase-1 and IL-1β in the supernatant. In contrast, when the cells were treated with the NLRP3 inhibitor, MCC950, or the IL-1β receptor antagonist, anakinra, detection of caspase-1 and IL-1β during IAV infection was greatly reduced. Caspase-1 activation was also assessed using a specific fluorescent probe, FAM-YVAD-FMK (17). Cells infected with IAV showed robust caspase-1 activation following treatment with IAV, with caspase-1 forming aggregates throughout the cytoplasm. However, caspase-1 activation was severely reduced in cells treated with MCC950 prior to IAV infection. No caspase-1 activation was observed in uninfected THP-1 cells (**Figure 1E**).

#### MCC950 Improves Survival of Juvenile Mice Infected with IAV

The induction of IL-1β by IAV has been shown to be NLRP3 inflammasome dependent (8–10). We assessed the ability of MCC950 to prevent NLRP3 inflammasome activation and alter the young host's inflammatory response to IAV infection. Juvenile mice were infected with IAV [A/WSN/2009 12.5 PFU intratracheal (i.t.)] to achieve infection of the lower respiratory tract. Starting on day 3 p.i., we administered MCC950 [10 mg/ kg intraperitoneal (i.p.), once daily (q.d.)] or an equal volume of vehicle control (i.p., q.d.) until recovery or death. We observed that the median survival of IAV-infected, vehicle-treated juvenile mice was day 11 p.i, with only 18% of PBS-treated mice surviving infection (**Figure 2A**). In contrast, 75% of IAV-infected, MCC950-treated juvenile mice were alive on day 11 p.i. IAV infection is typically associated with significant weight loss, which was observed in both IAV-infected, PBS-treated and IAVinfected, MCC950-treated mice. Importantly, the majority of the IAV-infected, MCC950-treated mice began to regain weight between days 8 and 9 p.i. (**Figure 2B**). Surviving IAV-infected, MCC950-treated juvenile mice exhibited coat ruffling, febrile shaking, and mild lethargy, but the majority of animals recovered. At 7 days p.i., indices of lung injury were not different between IAV-infected, PBS-treated and IAV-infected, MCC950-treated mice. Both groups displayed elevated levels of cellular infiltration (**Figure 2C**) and protein leakage (**Figure 2D**) into the BALF, and both groups had increased wet-to-dry weight ratios (**Figure 2E**). In accordance with this, histological examination of IAV-infected, PBS-treated and IAV-infected, MCC950-treated mice on day 7 p.i. demonstrated a similar degree of lung injury at this time point (**Figures 2F,G**).

### MCC950 Inhibits the NLRP3 Inflammasome in Juvenile Lungs

We next sought to compare NLRP3 inflammasome activation in IAV-infected, PBS-treated and IAV-infected, MCC950-treated juvenile mice. IL-18 was elevated in the BALF from IAVinfected, PBS-treated mice, but was significantly attenuated in IAV-infected, MCC950-treated mice (**Figure 3A**). Importantly, the IAV-induced increase in NLRP3 protein expression observed in PBS-treated mice was absent in MCC950-treated mice (**Figure 3B**). Additional western blot analysis of inflammasome components in lung homogenates showed a similar increase in ASC in response to IAV infection in both PBS-treated and MCC950-treated mice (**Figure 3C**). Mature caspase-1 was increased in the BALF from IAV-infected, PBS-treated mice, compared to uninfected controls (**Figure 3D**). In contrast, caspase-1 secretion was inhibited in IAV-infected, MCC950-treated mice (**Figure 3D**). IL-6 and tumor necrosis-α (TNF-α), which are inflammatory cytokines that are not dependent on NLRP3 inflammasome activation, were not different between the two treatment groups (**Figures 3E,F**).

#### Figure 1 | Continued

MCC950 and anakinra inhibit influenza A virus (IAV)-induced NOD-like receptor protein 3 inflammasome activation in THP-1 macrophages. Differentiated human THP-1 macrophages were infected with IAV (WSN) at a multiplicity of infection (MOI) of 1, 2, or 3 for 2 h. Infected cells were cultured for 24 h, and supernatant was evaluated by ELISA for (A) caspase-1 or (B) interleukin-1β (IL-1β). Differentiated human THP-1 macrophages were treated with MCC950, anakinra, or vehicle control. Cells were either infected with IAV (WSN, MOI 2) for 2 h or sham infected and treated with drug therapy alone. Cells were then washed and cultured in media containing MCC950, anakinra, or vehicle control. Supernatant was collected 24 h after IAV infection and evaluated by ELISA for (C) caspase-1 or (D) IL-1β. # indicates significant elevation over all other conditions. (E) MCC590-treated or vehicle-treated cells were fixed 24 h following IAV infection (WSN MOI 2) and fluorescently labeled to show active caspase-1 (green) and nuclei (blue). Scale bars, 10 µm.

#### MCC950 Does Not Prevent Monocyte Recruitment to the Lungs but Does Inhibit NLRP3 Inflammasome Activation in Alveolar Macrophages

Macrophages are a main source of NLRP3 inflammasome activation during IAV infection (18). To investigate the impact of MCC950 treatment on NLRP3 inflammasome activation in these cells, we isolated alveolar macrophages (CD45+, CD64+, CD11c+, Siglec F+) and monocyte-derived cells (CD45+, CD11b+, Ly6C+, CD64+) from the lungs of IAV-infected, PBStreated and IAV-infected, MCC950-treated mice using 10-color flow cytometry (19). In addition, we assessed the expression levels of NLRP3, caspase-1, and IL-1β with intracellular staining.

were infected with IAV (WSN 12.5 plaque forming unit intratracheal) and treated with MCC950 (10 mg/kg intraperitoneal daily) or phosphate-buffered saline (PBS) control beginning on day 3 postinfection (p.i.). Bronchoalveolar lavage fluid (BALF) or whole lungs were collected from IAV-infected, MCC950-treated mice and IAV-infected, PBS-treated mice 7 days p.i. (A) Interleukin-18 (IL-18) in BALF as measured by ELISA. (B,C) NLRP3 and ASC in lung homogenates as measured by Western blot. (D) Caspase-1 in BALF as measured by ELISA. (E) Interleukin-6 (IL-6) and (F) tumor necrosis-α (TNF-α) in BALF as measured by ELISA. \**p* < 0.05, \*\**p* < 0.01, \*\*\*\**p* < 0.0001.

IAV infection caused an influx of monocyte-derived cells into the lungs. When analyzing all CD45+ cells, a similar number of alveolar macrophages (**Figure 4A**) and monocyte-derived cells (data not shown) were found in both treatment groups on day 7 p.i. To determine if MCC950 inhibited the expression of NLRP3 in these cells, we examined the median fluorescence intensity (MFI) of the inflammasome components, NLRP3 and caspase-1, and the product of its activation, IL-1β. All components of the NLRP3 inflammasome measured were significantly elevated in the alveolar macrophages of IAV-infected mice compared to uninfected controls (data not shown and **Figures 4B–D**). IAVinfected, MCC950-treated mice had significantly decreased levels of NLRP3 and IL-1β in alveolar macrophages (**Figures 4C,D**). These results are consistent with our finding that IAV-infected, MCC950-treated mice had decreased levels of NLRP3 in homogenized lungs as measured by Western blot, and IL-18 in BALF as measured by ELISA (see **Figure 3**).

# MCC950 Does Not Impact Type I Interferon Production or Viral Clearance in Juvenile IAV Infection

Interferon-α (IFN-α) is a type I interferon secreted in response to viral infection to control viral replication and prevent propagation of the infection to neighboring cells. Mice infected with IAV, as well as mice treated with MCC950, had an increase in IFN-α levels on day 7 p.i. (**Figure 5A**). Consistent with this, viral

Figure 4 | MCC950 treatment decreases NOD-like receptor protein 3 (NLRP3) inflammasome activation in alveolar macrophages in juvenile mice infected with influenza A virus (IAV). Juvenile mice were infected with IAV (WSN 12.5 plaque forming unit intratracheal) and treated with MCC950 (10 mg/kg intraperitoneal daily) or phosphate-buffered saline (PBS) control beginning on day 3 postinfection (p.i.). Lungs were harvested 7 days p.i. and evaluated by flow cytometry for NLRP3 inflammasome activation using intracellular staining. (A) Number of alveolar macrophages in lung homogenates. (B–D) Median fluorescence intensity (MFI) of caspase-1, interleukin-1β (IL-1β), and NLRP3 in alveolar macrophages. \**p* < 0.05, \*\**p* < 0.01.

\*\**p* < 0.01.

titers in the lung homogenates of IAV-infected, PBS-treated and IAV-infected, MCC950-treated mice as measured by plaque assay were equal (**Figure 5B**).

#### Anakinra Does Not Protect Juvenile Mice from IAV Infection

NOD-like receptor protein 3-dependent production of IL-1β and IL-18 may have downstream consequences with regard to IAVinduced inflammation and disease. IL-1β and IL-18 bind their cell-surface receptors (IL-1R and IL-18R, respectively) expressed on a range of cell types to induce potent NF-κB-dependent secondary cytokine production (20, 21). Importantly, lack of IL-1R resulted in reduced lung immunopathology following H1N1 infection, suggesting that IL-1R signaling may increase damage to the lung (22). Anakinra competes for the IL-1 receptor and blocks the actions of IL-1β. We investigated the impact of anakinra treatment on survival in IAV-infected juvenile mice. Juvenile mice were infected with IAV (A/WSN/2009 12.5 PFU i.t.) and anakinra (100 mg/kg i.p.) or an equal volume of vehicle control was administered i.p., q.d. beginning on day 3 p.i. and continuing until recovery or death. There was no statistically significant difference in the survival of IAV-infected mice treated with anakinra compared to those given vehicle control (**Figure 6A**). Initiating anakinra therapy on day 2 p.i. or day 4 p.i. also did not improve survival (data not shown). Measurement of protein leakage, IL-18, and IL-6 in BALF failed to show a difference between anakinra and control-treated mice on day 7 p.i. (**Figures 6B–D**). IFN-α secretion was not impacted by anakinra therapy, either, and viral titers from lung homogenates were equal in IAV-infected anakinra-treated mice, and IAV-infected PBS control-treated mice (**Figures 6E,F**). Finally, a similar degree of lung injury was seen on histological examination of IAV-infected, PBS-treated and IAV-infected, anakinra-treated mice on day 7 p.i. (**Figure 6G**).

#### DISCUSSION

The host response to IAV can exacerbate the morbidity associated with IAV infection (4, 23, 24). It is well established that the NLRP3 inflammasome is a major component of the host response to IAV (6). It is activated by the influenza M2 proton channel and results in the production of the potent inflammatory cytokines, IL-1β and IL-18 (7). While pathogen clearance and host survival depend on adequate activation of the innate immune system, an excessive inflammatory response to infection can be harmful to the young host. The NLRP3 inflammasome is protective in lethal mouse models of IAV infection (9–11). Loss of NLRP3, ASC, or caspase-1 in mice leads to decreased IL-1β and IL-18 secretion and increases mortality from IAV. Alternatively, excessive inflammasome activation may decrease survival by exacerbating the lung injury seen in lethal IAV infection (6). Therefore, inflammasome signaling must be tightly controlled to promote eradication of the virus while limiting collateral damage to the host. In severe IAV infection, this balance is not achieved, making the NLRP3 inflammasome an attractive therapeutic target. Early modulation of its activity may not only limit the production of injurious inflammatory cytokines, but also prevent the pyroptotic cell death and the ensuing tissue destruction caused by activated caspase-1. Thus, the identification of small molecule inhibitors of the NLRP3 inflammasome offers considerable therapeutic promise. To establish the optimal degree of NLRP3 inflammasome activation during IAV infection, we sought to modulate NLRP3 inflammasome signaling, with the goal of protecting juvenile mice from IAV-induced lung injury. We investigated the efficacy of MCC950, a potent inhibitor of NLRP3, as well as anakinra, a known inhibitor of the IL-1β pathway *via* IL-1 receptor.

MCC950 has been shown to be a specific NLRP3 inhibitor and to be protective in multiple models of injurious NLRP3 inflammasome activation (14–16). It can be given by oral, intravenous, and i.p. routes and is effective at doses ranging from 4 to 20 mg/kg in mouse models of autoimmune disease (experimental autoimmune encephalitis) (14), diseases of constitutive NLRP3 activation (cryopyrin-associated periodic syndrome) (14), and disorders in which NLRP3 has been shown to play an important role [including cardiac infarction (25) and non-alcoholic steatohepatitis (26)]. *In vitro* treatment of IAV-infected THP-1 macrophages with MCC950 confirmed that IAV-induced NLRP3 inflammasome

activation is effectively inhibited with this molecule. Interestingly, treatment of IAV-infected cells with the IL-1β receptor antagonist, anakinra, did not just block downstream signaling from the IL-1β receptor. It also appeared to inhibit NLRP3 inflammasome activation, as demonstrated by decreased caspase-1 and IL-1β in the supernatant from IAV-infected, anakinra-treated cells. Although anakinra classically targets IL-1β signaling by blocking the interaction of IL-1β with its receptor, it has also been shown to bind to and inhibit caspase-1, which likely explains our finding of NLRP3 inflammasome inhibition in IAV-infected cells treated with anakinra (27, 28). Consequently, we hypothesized that both MCC950 and anakinra had the potential to protect juvenile mice from IAV-induced NLRP3 inflammasome activation and lung injury.

Juvenile mice treated with MCC950 beginning 3 days p.i. were protected from IAV-induced mortality. We chose this timing for the initiation of therapy because this likely corresponds to when children infected with IAV develop symptoms and seek medical attention. The protection from IAV-induced mortality was associated with a decreased amount of NLRP3 in the lung homogenates, and decreased IL-18 levels in the BALF, from IAV-infected, MCC950-treated mice compared to IAV-infected, vehicle-treated mice, indicating that inhibition of the NLRP3 inflammasome was achieved. However, this protection from IAV-induced mortality was not associated with a decrease in traditional markers of lung injury, including cellular infiltration and protein leakage into the alveolar space. Since MCC950 therapy did not prevent IAV-induced lung injury on day 7 p.i., this suggests that MCC950 treatment improved survival by either halting disease progression or enhancing recovery. When CD45+ cells from IAV-infected mice were examined with intracellular staining, the greatest impact of NLRP3 inflammasome inhibition was found in alveolar macrophages. In these cells, MCC950 treatment decreased NLRP3 and IL-1β levels. As alveolar macrophages can promote alveolar epithelial cell repair (24), the switch from an inflammatory to an anti-inflammatory phenotype in alveolar macrophages may play an important role in recovery from IAV. We reason that inhibiting the NLRP3 inflammasome in this cell population may have played a key role in the beneficial effects of MCC950 therapy.

Other groups have shown varying degrees of protection from IAV (12) or the IAV virulence factor PB1-F2 (29) using MCC950 in murine models of adult IAV. Tate et al. were able to delay death from two different strains of IAV (A/PR/8/34 and HKx31) by a few days with MCC950 (5 mg/kg intranasal) treatment. Notably, timing of initiation of MCC950 therapy was important in their model, with early administration of the drug on day 1 p.i. harmful, and late administration after day 3 beneficial. This could be consistent with our proposal that NLRP3 inflammasome inhibition is important for recovery from IAV infection, rather than prevention of IAV-induced lung injury. However, evaluation of lung injury was not performed in their study. They were able to demonstrate prevention of immune cell infiltration into the lungs and decreased cytokine production, including IL-1β, IL-18, TNF-α, and IL-6, in BALF and serum. In our model of juvenile IAV infection, we did not see the same inhibition of immune cell recruitment to the lungs with MCC950 therapy (10 mg/kg i.p.). We also did not see the same suppression of IL-6 or TNF-α production. However, we were not surprised by this finding because these cytokines are not dependent on NLRP3 inflammasome activation. Importantly, we did see evidence of NLRP3 inflammasome inhibition in the resident alveolar macrophages, and decreased IL-18 in BALF, which may have contributed to the increased survival we found in mice treated with MCC950. Differences in our models may explain the disparate findings regarding cellular recruitment and cytokine suppression, highlighting the importance of the microenvironment when modulating the immune response to a pathogen. We administered the drug i.p. instead of intranasal to avoid repeated exposure to anesthesia, but these two delivery methods could result in different concentrations of the drug in the alveolar space. In addition, the half-life of the drug may be altered by delivery method, potentially limiting its efficacy. Perhaps more importantly, we used juvenile mice, which may have a propensity for worse disease (4, 30, 31). Innate immune signaling has been shown to be vary widely depending on age, making age-relevant models critical when studying inflammatory diseases (32–38).

Genetic deletion of NLRP3 inflammasome components leads to worse outcomes in IAV infection (9–11). This argues that abolishment of NLRP3 inflammasome signaling during IAV infection is harmful and that early NLRP3 inflammasome activation is necessary for controlling the infection and viral clearance. The finding from Tate et al. that early inhibition of NLRP3 with MCC950 increases mortality from IAV infection is consistent with this (12). In contrast, late inhibition was protective, which supports our finding that MCC950 treatment beginning 3 days p.i. improved survival in juvenile mice infected with IAV. The sensitivity of the outcome of NLRP3 modulation to timing and degree of inhibition is not unique to IAV infection, but a common theme in inflammatory responses to pathogens, where inadequate inflammation impairs pathogen clearance, but excessive inflammation causes collateral tissue damage and enhanced injury. This emphasizes the need for careful characterization of optimal treatment strategies in clinically relevant, age appropriate, models.

In contrast to our results from MCC950 treatment, anakinra treatment of IAV-infected juvenile mice did not show protection from IAV-induced mortality or lung injury. Despite achieving NLRP3 inflammasome suppression *in vitro*, anakinra therapy did not effectively decrease NLRP3 inflammasome activation or IL-18 secretion into the alveolar space in our *in vivo* model of juvenile IAV infection. Therefore, it was not surprising that we could not demonstrate protection from IAV with anakinra treatment. Instead, it suggests that once daily dosing with i.p. delivery was not sufficient to achieve caspase-1 inhibition. Alternatively, it argues that isolated IL-1 receptor antagonism is insufficient to protect juvenile mice from IAV infection because it leaves IL-18 signaling intact. There is one report of anakinra therapy (100 μg/mouse, intravenous, daily from day 2 to 6 p.i.) improving survival in IAV infection in adult mice (A/PR/8/34) (39), but only mortality was evaluated. Differences in our model, especially the age of the mice, may explain the discrepancy in our findings.

Influenza A virus is a source of significant morbidity and mortality in children, but current therapies are limited to early antiviral treatment and supportive care. There is considerable need for new strategies to improve outcomes in pediatric IAV infection, and the use of juvenile models to test these strategies is critical. Targeting the NLRP3 inflammasome may be beneficial in juvenile IAV infection and does not appear to impact viral clearance. Better understanding of how NLRP3 inflammasome inhibition improves mortality in juvenile IAV infection, and identification of the optimal timing and method of NLRP3 inflammasome inhibition in juvenile IAV infection, deserve further study.

#### ETHICS STATEMENT

This study was carried out in accordance with United States federal guidelines and was approved by The Institutional Animal Care and Use Committee at Northwestern University.

# AUTHOR CONTRIBUTIONS

BC contributed to all aspects of this manuscript including experimental design, conduction of experiments, interpretation of data, and manuscript preparation. NR, YC, JD, and DS contributed to the conduction of the experiments, interpretation of data, and manuscript preparation. KS and CK contributed to experimental design, conduction of experiments, interpretation of data, and manuscript preparation. KR contributed to experimental design, interpretation of data, and manuscript preparation.

#### ACKNOWLEDGMENTS

Histology services were provided by the Northwestern University Research Histology and Phenotyping Laboratory, which is supported by NCI P30-CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center. Imaging work was performed at the Northwestern University Center for Advanced Microscopy, generously supported by NCI CCSG P30-CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center. Flow cytometry was supported by the Northwestern University—Flow Cytometry Core Facility (Cancer Center Support Grant NCI CA060553).

#### REFERENCES

#### FUNDING

This work was supported by the Pediatric Critical Care and Trauma Scientist Development Program (NICHD K12HD047349), the American Lung Association, the American Thoracic Society, the Respiratory Health Association, and the National Institutes of Health (NHLBI R01HL128194 and 2P01HL071643-11A1).

persist throughout the first month of life. *Clin Immunol* (2009) 133:228–37. doi:10.1016/j.clim.2009.07.003

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Coates, Staricha, Ravindran, Koch, Cheng, Davis, Shumaker and Ridge. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Inflammatory Responses Regulating Alveolar Ion Transport during Pulmonary Infections

*Christin Peteranderl 1 \*, Jacob I. Sznajder2 , Susanne Herold1 and Emilia Lecuona2*

*1Department of Internal Medicine II, University of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Giessen, Germany, 2Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA*

The respiratory epithelium is lined by a tightly balanced fluid layer that allows normal O2 and CO2 exchange and maintains surface tension and host defense. To maintain alveolar fluid homeostasis, both the integrity of the alveolar–capillary barrier and the expression of epithelial ion channels and pumps are necessary to establish a vectorial ion gradient. However, during pulmonary infection, auto- and/or paracrine-acting mediators induce pathophysiological changes of the alveolar–capillary barrier, altered expression of epithelial Na,K-ATPase and of epithelial ion channels including epithelial sodium channel and cystic fibrosis membrane conductance regulator, leading to the accumulation of edema and impaired alveolar fluid clearance. These mediators include classical pro-inflammatory cytokines such as TGF-β, TNF-α, interferons, or IL-1β that are released upon bacterial challenge with *Streptococcus pneumoniae*, *Klebsiella pneumoniae*, or *Mycoplasma pneumoniae* as well as in viral infection with influenza A virus, pathogenic coronaviruses, or respiratory syncytial virus. Moreover, the pro-apoptotic mediator TNF-related apoptosisinducing ligand, extracellular nucleotides, or reactive oxygen species impair epithelial ion channel expression and function. Interestingly, during bacterial infection, alterations of ion transport function may serve as an additional feedback loop on the respiratory inflammatory profile, further aggravating disease progression. These changes lead to edema formation and impair edema clearance which results in suboptimal gas exchange causing hypoxemia and hypercapnia. Recent preclinical studies suggest that modulation of the alveolar–capillary fluid homeostasis could represent novel therapeutic approaches to improve outcomes in infection-induced lung injury.

Keywords: ion channel, ion pumps, edema, cytokines, Na-K-ATPase, cystic fibrosis membrane conductance regulator, epithelial sodium channel, lung injury

*Edited by:*

*Rudolf Lucas, Augusta University, USA*

#### *Reviewed by:*

*Sigrid A. Langhans, Alfred I. duPont Hospital for Children, USA Jean-francois Pittet, University of Alabama, USA*

*\*Correspondence:*

*Christin Peteranderl<EMAIL_ADDRESS>uni-giessen.de*

#### *Specialty section:*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology*

*Received: 23 February 2017 Accepted: 31 March 2017 Published: 18 April 2017*

#### *Citation:*

*Peteranderl C, Sznajder JI, Herold S and Lecuona E (2017) Inflammatory Responses Regulating Alveolar Ion Transport during Pulmonary Infections. Front.

# INTRODUCTION

The major task of the respiratory tract is the exchange between inhaled atmospheric oxygen and carbon dioxide carried by the bloodstream, which is ensured by a thin but large surface area formed by type I and type II alveolar epithelial cells. Both the upper and the lower respiratory epithelia are lined by a thin (0.2 µM) aqueous layer (1), referred to as airway surface liquid (ASL) and alveolar lining fluid (AFL), respectively. This fluidic component serves—in concerted action with surfactant, mucus, and ciliary beat—to reduce alveolar surface tension and prevent atelectasis as well as to defend against invading pathogens. To maintain the composition of the ASL and AFL and to prevent alveolar flooding, lung fluid homeostasis is tightly controlled by the expression and activity of ion channels and pumps. These channels and pumps establish an osmotic gradient between airspace and interstitium, driving paracellular or aquaporin- (AQP3, 4, and 5) (2) mediated fluid movement across the respiratory epithelium. Among these, the apical amiloride-sensitive epithelial sodium channel (ENaC) and the amiloride-insensitive cyclic nucleotide-gated cation channel (CNG) acting together with the basolaterally located Na,K-ATPase (NKA) promote transcellular sodium transport (3), which is accompanied in the alveolar epithelium by chloride uptake from the apical cystic fibrosis membrane conductance regulator (CFTR) (4). However, in the airway, CFTR promotes chloride secretion to regulate mucus density (5). In addition, Ca2+-activated ion channels (CaCC) promote apical chloride secretion, further supported by basolateral chloride uptake *via* Na+/K+/2Cl− cotransporters (NKCC) (6) as well as potassium ion channels such as Kv7.1, contributing to cellular membrane potential and buildup of an electrochemical gradient necessary for apical chloride secretion (7). Additional factors influencing fluid homeostasis are epithelial (im)permeability established by tight junction proteins as well as endothelial integrity limiting the extravasation of fluid from the blood vessels driven by changes in the capillary hydrostatic pressure (8, 9).

Pulmonary infections commonly disturb ion and thus fluid homeostasis, resulting in abnormal changes of ASL, AFL, and alveolar edema formation. Both viral and bacterial pathogens are common causative agents for acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS), which are characterized by a widespread inflammation within the lungs, extensive flooding of the alveolar airspace with protein-rich exudate fluid and impaired gas exchange leading to respiratory failure and resulting in mortality rates of 40–58% (10, 11). Additionally, sepsis resulting from primary infections at other sites is often complicated by the development of severe lung injury during the onset of bacteremia, resulting in lung failure and accounting for as many as half of all cases of ARDS (12). Although some of the pathogen-derived effects on ion transport during lung injury have been reported to be caused directly by the pathogen–host cell interaction (13), accumulating evidence suggests that autoand paracrine mediators of local and/or systemic inflammatory responses mounted upon pathogen recognition and replication induce—among other pathophysiological changes—impaired ion transport and alveolar fluid clearance (AFC), resulting in edema formation and persistence. Importantly, mortality in ARDS patients has repeatedly been found to correlate with persistence of alveolar edema (11, 14).

In this review, we will highlight advances in the understanding of how inflammatory responses in pulmonary infection affect ion transport, including common patterns and unique pathways activated by different respiratory pathogens, and how these mechanisms might be modulated to improve the outcomes of ARDS patients.

# MEDIATORS MODULATING ION AND FLUID HOMEOSTASIS

There are numerous reports showing that pulmonary infection leads to loss of barrier integrity and edema accumulation as well as the role of distinct mediators on impairing ion channel or transporter function on the alveolar, bronchial, and gut epithelia. However, there have been few studies showing how infectious agents modulate soluble signaling molecules that affect ion and fluid homeostasis. Several reports from the last decade have reestablished an important role for soluble, inflammatory mediators in the progression of ARDS. For example, Lee et al. demonstrated that exposure of human ATII cells to pulmonary edema fluid derived from ARDS patients alone was sufficient to downregulate the ion channels and pumps involved in AFC, including ENaC, the NKA, and CFTR (15). Concomitantly, it was established that viral or bacterial lung infections lead to edema accumulation and impair clearance *via* the induction of paracrine factors. For example, influenza A virus (IAV) has been shown to increase apical potassium secretion by upregulation of the apical potassium channel KCNN4 by a paracrine signaling event, thus disturbing the osmotic gradient necessary for edema clearance (16). Similarly, *Pseudomonas aeruginosa* evokes a strong inflammatory response and lung edema accumulation related with the modulation of ENaC subunit expression (17, 18). In the next paragraphs, we will provide an overview on interconnections of mediators released in pulmonary infection and their effects on ion and fluid homeostasis (**Figure 1**).

#### Interferon

Once cells detect pathogens by their specific and specialized pattern recognition receptors, they produce interferons (IFN), which can be detected—if not actively suppressed by a given pathogen—in most pulmonary infection scenarios. Effects of IFN on fluid homeostasis seem to be mostly limited to gamma IFN (IFN-γ), which have been attributed a modulatory role in both innate and adaptive immunity (19, 20). IFN-γ has been reported to decrease sodium transport at levels as low as 10 U/ml (21). Moreover, IFN-γ can also directly decrease chloride currents along the bronchial epithelium by downregulating CFTR due to a posttranscriptional modulation of CFTR messenger RNA (mRNA) stability and thus half-life (21–23). In contrast, both class I IFN, IFN-α, and IFN-β that are usually implicated in mounting a direct cellular pathogen-restrictive response do not modulate CFTR mRNA or protein abundance (22). IFN-α appears to negatively impact NKA cell membrane protein abundance during IAV infection *via* activating the metabolic sensor AMP-kinase (AMPK) (24). However, to date,

can be downregulated by reactive oxygen and nitrogen species (RONS) and ATP, transforming growth factor beta (TGF-β) or interleukin-1 beta (IL-1β) upon *Streptococcus pneumoniae* and influenza A virus (IAV) infection. Sodium is secreted at the basolateral side by the Na,K-ATPase (NKA), which is modulated in lipopolysaccharide (LPS)-induced lung injury as well as upon *Mycoplasma pulmonis*, IAV, coronavirus (CoV), or adenovirus challenge. RONS, interferon-alpha (IFN-α), and TNF-related apoptosis-inducing ligand (TRAIL) lead to a decrease in NKA abundance or activity. In parallel, chloride is taken up (alveolar epithelium) or secreted (airway) by the cystic fibrosis membrane conductance regulator (CFTR) and secreted by apical Ca2+-activated ion channels (CaCC), supported by basolateral potassium channels (not shown) and Na+/K+/2Cl− cotransporters (NKCC). While extracellular ATP enhances chloride secretion by CaCC, CFTR action is reduced by IFN-γ and interleukin-8 (IL-8) in CoV, IAV, respiratory syncytial virus (RSV), or *Mycoplasma pneumoniae* infection.

there is no data supporting whether this effect of IFN-α on ion transport is a generalized response during pulmonary infections.

# Tumor Necrosis Factor Alpha (TNF-**α**)

Tumor necrosis factor alpha is a classical cytokine produced upon local or systemic inflammation, regulating differential processes such as proliferation and differentiation of immune cells as well as cell death (25–27). After initial conflicting studies, it has by now become clear that it plays a dichotomic role in lung fluid reabsorption (28). On one hand, TNF-α ligation to its receptor TNF receptor 1 (TNFR1, also named CD120a or p55) inhibits ENaC activity both *in vitro* and *in vivo via* a PKC-dependent mechanism (29). On the other hand, a distinct lectin-like domain of TNF different from the receptor-binding domain, which can be mimicked by the 17-amino acid circular TIP peptide (30), has been reported to increase edema reabsorption in rat bacterial pneumonia (31). Application of the TIP peptide has been demonstrated to elevate ENaC expression and open probability (32) resulting in enhanced AFC in *P. aeruginosa*-treated rats *in vivo* (31) and has furthermore been reported to increase NKA activity (33). In addition to its direct effects on ion channels and pumps of the alveolar epithelium, the TNF-α/TNFR1 interaction also modulates the integrity of the alveolar barrier, as it increases endothelial expression of chemoattractants and adhesion molecules including the interleukin-8 (IL-8; formerly called neutrophil chemotactic factor)/IL-8 receptor 2 axis, the intercellular adhesion molecule-1, platelet endothelial cell adhesion molecule-1, and vascular adhesion molecule-1, and thus promotes excessive recruitment of mononuclear phagocytes and neutrophils during lung inflammation (30, 34, 35). Importantly, besides cellular transmigration itself, neutrophil-derived proteases and neutrophil extracellular traps are central drivers of both endothelial and epithelial injury (36).

# Interleukin-1 Beta (IL-1**β**)

Interleukin-1 beta is one of the most commonly found cytokines in pulmonary edema and bronchoalveolar lavage fluids in experimental and human ARDS (37, 38) and is, for example, induced during *Klebsiella pneumoniae* bacterial pneumonia (39–41). It is mainly produced by macrophages and, similarly to TNF-α, has a major impact on cell proliferation, differentiation, and cell death. In pulmonary inflammation, IL-1β increases lung barrier permeability in *in vitro* and *in vivo* models of ARDS (41, 42) and may contribute to alveolar edema in lung injury models by impairing fluid reabsorption from the lungs. This can in part be attributed to decreased sodium absorption due to a decrease in αENaC expression and trafficking to the apical membrane of ATII cells (43). In addition, IL-1β in *Streptococcus pneumonia* infection (44)—and also TNF-α and IFN-γ (45)—can influence ion transport processes *via* activation of the pro-coagulant factors (46). Thrombin in particular has been demonstrated to impair AFC by increasing the PKC-ζ-dependent endocytosis of the alveolar NKA (47).

#### Interleukin-8

Interleukin-8 is a chemotactic factor that correlates with neutrophil accumulation in distal airspaces of patients with ARDS and is a predictor of mortality (48–50). IL-8 is secreted by bronchial epithelial cells and can be induced by *Mycoplasma pneumoniae* antigen or live *M. pneumoniae* (51) as well as by severe acute respiratory syndrome coronavirus spike protein or respiratory syncytial virus infection (52, 53). The rate of AFC is impaired by high levels of IL-8 and is significantly lower in patients who have a pulmonary edema fluid concentration of IL-8 above 4,000 pg/ml (54). Mechanistically, IL-8 inhibits beta-2 adrenergic receptor (β2AR) agonist-stimulated fluid transport across rat and human alveolar epithelia. This inhibition is mediated by a PI3Kdependent desensitization and downregulation of the β2AR from the cell membrane associated with an inhibition of cyclic AMP generation normally observed in response to β2AR agonist stimulation (54).

# Transforming Growth Factor Beta (TGF-**β**)

The cytokine TGF-β is a critical factor for the development of ARDS. Besides its established role in dampening inflammatory responses (55), e.g., by driving macrophages toward an antiinflammatory phenotype (56), it increases alveolar epithelial permeability to promote edema formation upon lipopolysaccharide (LPS) stimulation (57). Furthermore, TGF-β has been shown to inhibit amiloride-sensitive sodium transport by an ERK1/2-dependent inhibition of the αENaC subunit promoter activity, decreasing αENaC mRNA and protein expression (58). In addition, Peters et al. (59) demonstrated that TGF-β leads to the subsequent activation of phospholipase D1, phosphatidylinositol-4-phosphate 5-kinase 1α, and NADPH oxidase 4 (Nox4). Nox4 activation results in the production of reactive oxygen species (ROS) that in turn reduce cell surface stability of the αβγENaC complex and thus promote edema fluid accumulation. Moreover, TGF-β decreases NKA β1 subunit expression, resulting in decreased NKA activity in lung epithelial cells (60, 61). In further support of a role for TGF-β in lung injury, TGF-β levels are increased in lung fluids from patients with ALI/ ARDS (62) and in murine models of *Streptococcus pneumoniae* and IAV infection (63, 64). Of note, TGF-β has been proposed to further aggravate edema formation in IAV infection by increasing epithelial cell death, causing a disruption of epithelial barrier integrity (64). Moreover, it has been implicated in the upregulation of cellular adhesins which increase host susceptibility to bacterial co-infections (65) posing a major risk for increased viral pneumonia-associated morbidity and mortality during influenza epidemics (66).

# TNF-Related Apoptosis-Inducing Ligand (TRAIL)

The principal role of TRAIL, highly released by lung macrophages upon viral infection, is to drive infected cells into apoptosis to limit pathogen spread. TRAIL has been reported to be produced especially during viral respiratory infections, including IAV-, adenovirus-, and paramyxovirus infection, and cell sensitivity to TRAIL-induced apoptosis is enhanced in infected cells by increased TRAIL-receptor expression (67, 68). However, this process also affects alveolar epithelial barrier integrity leading to edema accumulation (67, 69). Moreover, TRAIL signaling leads to NKA downregulation in IAV infection in non-infected neighboring alveolar epithelial cells mediated by AMPK (24). Accordingly, TRAIL signaling reduces AFC and promotes edema formation. In addition, TRAIL release upon IAV infection further favors bacterial superinfection with *S. pneumoniae*, aggravating lung injury (70).

#### Nucleotides

During acute infection, extracellular nucleotides often serve as danger signals involved in recognition and control of pathogens by promoting the recruitment of inflammatory cells, stimulating pro-inflammatory cytokines, and increasing the production of ROS or nitric oxide (NO) (71, 72). Extracellular ATP, which can be released from the airway epithelia and is produced by endothelial cells upon acute inflammation, binds to P2 purinergic receptors to promote a calcium signaling-dependent stimulation of CaCC and a decreased open probability of ENaC (73, 74). Moreover, extracellular adenosine, produced from ATP by hydrolysis *via* the ecto-5′-nucleotidase CD73, is increased in bronchoalveolar lavage fluid of IAV-infected mice, and genetic deletion of the A1-adenosine-receptor is protective (75). However, CD73 is only to a limited extent involved in the progression of lung injury and has no effect on pulmonary edema formation (76).

#### Reactive Oxygen and Nitrogen Species (RONS)

Reactive oxygen and nitrogen species have been shown to be involved in the development of epithelial injury in pathologic situations, including LPS-/sepsis-induced lung injury as well as viral pneumonia, in which RONS are produced in large quantities by alveolar phagocytes (77). Studies in rabbit and piglet lungs further elucidated that RONS affect AFC and edema persistence by inhibiting both the activity of ENaC and alveolar epithelial NKA (78, 79).

# EFFECTS OF ION CHANGES ON CYTOKINE PRODUCTION

To add to the complexity of airway and alveolar fluid regulation, it has been suggested that not only ion channels, pumps, and transporters are modulated by signaling factors released upon pulmonary infection but also changes in ion transport influence the respiratory inflammatory response. For example, the transporter NKCC1—which plays a critical role in basolateral ion transport—can affect the severity of pneumonia and sepsis and consequently severity of lung injury, by regulating the ability of the alveolar–capillary barrier to modulate neutrophil infiltration into the air spaces of the lung (80). Lack of NKCC1 in a mouse model of pneumonia infection with *K. pneumonia* or LPS resulted in increased numbers of neutrophils in the lavage fluid, decreased bacteremia, and importantly mortality. It has, therefore, been suggested that the activity of NKCC1 contributes to edema formation and decreased neutrophil migration into the lung air spaces, probably contributing to reduce bacterial killing and the subsequent development of severe sepsis (81–83). Similarly, mutations of CFTR can amplify lung inflammation by upregulating pro-inflammatory responses caused by an increase in cytokine production upon NFκB activation in lung epithelial cells (84). Lack of functional neutrophilic CFTR in a model of LPS-induced lung inflammation contributes to inflammatory imbalance with NFκB translocation and a reduction of antiinflammatory cytokines such as IL-10, favoring the increase in lung vascular permeability (85). Also ion imbalances in response to expression of viral ion channels or viroporins, has been recognized as potential pathogen recognition pathway that favors inflammasome activation and the release of IL-1β, TNF, and IL-6, which might contribute to the limitation of virus spreading (86, 87).

## THERAPEUTIC MODULATION OF THE ALVEOLAR–CAPILLARY FLUID BALANCE DURING PULMONARY INFECTION

As stated above, pulmonary infections—especially in severe cases—can lead to lung edema accumulation and impaired edema clearance. Lung edema results in impaired oxygenation and organ dysfunction which if not resolved leads to high mortality of patients with ARDS (11, 14). Current treatment options for infection-induced ARDS include antivirals and antibiotics. However, there is increased antibiotic resistance—reported for pathogens such as *K. pneumoniae*, *Escherichia coli*, *Staphylococcus aureus* and *P. aeruginosa* (82, 83, 88)—or lack of readily available treatment options for some acute emerging agents such as zoonotic influenza viruses or middle east respiratory syndrome coronavirus (89–91). Current approaches to treat ARDS patients include low tidal volume mechanical ventilation, positive end expiratory pressure, fluid management, and extracorporeal membrane oxygenation as measures to primarily improve oxygenation (92). Interestingly, lung-protective ventilation strategies have not only been reported to reduce mortality by 22% in patients with ARDS but also to diminish the number of neutrophils and the concentration of pro-inflammatory cytokines released in patient lavage fluids.

Novel approaches targeting host mediators known to promote lung edema formation and impair clearance such as studies on TIP peptide [see Tumor Necrosis Factor Alpha (TNF-α) above] administration in ARDS are being studied. Initial reports showed that AP301, a synthetic peptide mimicking TIP, induces ENaC activity in type II alveolar epithelial cells from dogs, pigs, and rats (93) and improves lung function in a porcine lung injury model (94). A subsequent phase II clinical trial with AP301 in ventilated ARDS patients resulted in improved AFC and oxygenation of these patients (95). Also, mesenchymal stem cells, which have been reported to improve epithelial barrier integrity in human

#### REFERENCES

AEC II treated with a cytokine mix composed of a combination of IL-1β, TNFα, and IFNγ (96), are currently tested for safety and efficacy in phase II trials (clinical trial identifiers NCT02097641, NCT01775774, NCT02112500). Studies on β2-agonists, which had been previously shown to improve vectorial sodium transport and edema clearance (97, 98), did not improve ARDS outcomes (99, 100), possibly due to an enhanced inflammatory response driven by lung macrophages (101). Further treatment options targeting para- or autocrine signaling events affecting AFC in preclinical models include glucocorticoids that suppress inflammation and upregulate both NKA (102) and ENaC (103, 104), neutralizing antibodies directed against virus-specific release of macrophage TRAIL that improve NKA expression as well as AFC in IAV-infected mice (24) and nitric oxide synthase inhibitors aminoguanidine or N(omega)-monomethyl-l-arginine (l-NMMA) that protect against pulmonary edema in LPS-induced lung injury as well as in IAV infection (77, 105).

### CONCLUSION

Pathogen-induced lung injury but also sepsis can lead to widespread respiratory inflammation that favors accumulation of lung edema leading to multiorgan dysfunction and poor outcomes. Recent advances in the development of novel treatment strategies targeting respiratory ion homeostasis show encouraging results, identifying them as promising candidates to improve AFC in ALI which could potentially improve the survival of patients with ARDS.

# AUTHOR CONTRIBUTIONS

CP, SH, JS, and EL have performed bibliographic research and drafted the manuscript.

# FUNDING

This work was supported by the German Research Foundation (SFB-TR84 B2, SFB1021 C05, KFO309 P2/P8, EXC147), by the German Center for Lung Research (DZL), by the German Center for Infection Research (DZIF), and by the National Institutes of Health (R37-HL48129 and HL071643).

epithelial cells.  Results from the ALIVE study. *Intensive Care Med* (2004) 30:51–61. doi:10.1007/s00134-003-2136-x

outcomes in acute respiratory distress syndrome (BALTI-2): a multicentre, randomised controlled trial. *Lancet* (2012) 379:229–35. doi:10.1016/ S0140-6736(11)61623-1

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Peteranderl, Sznajder, Herold and Lecuona. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# The Role of Transient Receptor Potential Vanilloid 4 in Pulmonary Inflammatory Diseases

*Rachel G. Scheraga, Brian D. Southern, Lisa M. Grove and Mitchell A. Olman\**

*Cleveland Clinic, Department of Pathobiology, Lerner Research Institute, Cleveland, OH, USA*

Ion channels/pumps are essential regulators of organ homeostasis and disease. In the present review, we discuss the role of the mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), in cytokine secretion and pulmonary inflammatory diseases such as asthma, cystic fibrosis (CF), and acute lung injury/acute respiratory distress syndrome (ARDS). TRPV4 has been shown to play a role in lung diseases associated with lung parenchymal stretch or stiffness. TRPV4 indirectly mediates hypotonicity-induced smooth muscle contraction and airway remodeling in asthma. Further, the literature suggests that in CF TRPV4 may improve ciliary beat frequency enhancing mucociliary clearance, while at the same time increasing pro-inflammatory cytokine secretion/lung tissue injury. Currently it is understood that the role of TRPV4 in immune cell function and associated lung tissue injury/ARDS may depend on the injury stimulus. Uncovering the downstream mechanisms of TRPV4 action in pulmonary inflammatory diseases is likely important to understanding disease pathogenesis and may lead to novel therapeutics.

Keywords: transient receptor potential vanilloid 4, ion channels, asthma, pulmonary vascular disease, acute respiratory distress syndrome

### INTRODUCTION

Ion channels and pumps play multiple important roles in cell homeostasis (1). They function to allow passive, agonist-induced, or voltage-dependent flux of specific ions in and out of the cell (1, 2). Dysregulation of channel function and/or expression can lead to organ dysfunction and disease (1–3). Recent studies have shown that a transient receptor potential (TRP) channel family member, transient receptor potential vanilloid 4 (TRPV4), is implicated in inflammatory lung diseases such as asthma, cystic fibrosis (CF), acute lung injury/acute respiratory distress syndrome (ARDS), and pulmonary fibrosis (4–10). In fact, these studies show that TRPV4 can regulate inflammatory cytokines that play key roles in orchestrating lung tissue homeostasis and inflammatory lung disease (4, 7, 10–14). Dysregulation of cytokines leads to alterations in cell–cell interactions, lung tissue remodeling, and repair (15). Regulating cytokine secretion through the modulation of ion channels such as TRPV4 may mediate inflammatory lung diseases. Therefore, TRPV4 may be a potential target for lung disease pathogenesis (16). This review summarizes and integrates the data from our laboratory and others to further the understanding of the TRPV4–cytokine interaction in pulmonary inflammation.

#### *Edited by:*

*Rudolf Lucas, Augusta University, USA*

#### *Reviewed by: Osama F. Harraz,*

*University of Vermont, USA Alexander Dietrich, Ludwig-Maximilians-Universität München, Germany André Dagenais, Institute of Clinical Research de Montreal (IRCM), Canada*

#### *\*Correspondence:*

*Mitchell A. Olman<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology*

*Received: 03 February 2017 Accepted: 12 April 2017 Published: 04 May 2017*

#### *Citation:*

*Scheraga RG, Southern BD, Grove LM and Olman MA (2017) The Role of Transient Receptor Potential Vanilloid 4 in Pulmonary Inflammatory Diseases. Front. Immunol. 8:503. doi: 10.3389/fimmu.2017.00503*

# THE TRPV4 CHANNEL

Intracellular calcium is tightly regulated in a spatiotemporal manner through a system of ion channels and membrane pumps (17). One such channel is TRPV4, a transmembrane (TM) cation channel of the TRP superfamily (18). TRPV4 is an 871 amino acid protein that has 6 TM domains, an ion pore located between TM5 and 6, an NH2 terminal intracellular sequence with several ankyrin-type repeats, and a COOH-terminal intracellular tail (19, 20). Both the NH2 and COOH termini interact with signal kinases, other molecules [e.g., nitric oxide (NO)], and scaffolding proteins (21). The intracellular tails contain several activity-modifying phosphorylation sites. TRPV4 is sensitized and activated by both chemical [5,6-epoxyeicosatrienoic acid (EET) and 4 alpha-phorbol 12,13-didecanoate (4-αPDD)] and physical stimuli (temperature 27–35°C, membrane stretch, and hypotonicity) (22–25). TRPV4 is ubiquitously expressed in many cell types in the respiratory system. In the setting of pulmonary inflammation, TRPV4 has been found to be highly expressed and upregulated in airway smooth muscle, vascular endothelial cells, alveolar epithelial cells, and immune cells such as macrophages and neutrophils (12, 16, 21, 26–28). TRPV4 has been implicated in the pathogenesis of asthma, CF, and sterile and infectionassociated ARDS (4–10, 29).

### THE ROLE OF TRPV4 IN INFLAMMATORY LUNG DISEASES

#### Asthma

Asthma is a chronic lung disease characterized by airway inflammation and remodeling, excess bronchial secretions, and smooth muscle hypertrophy and contraction leading to airway narrowing (bronchoconstriction). Recent work shows that TRPV4 mediates airway wall thickness, goblet cell recruitment, collagen expression, fibrotic airway remodeling, and increased expression of transforming growth factor-β (TGF-β) in a house dust mite (*Dermatophagoides farinae*) mouse model of asthma (30). The authors also show that TRPV4 mediates TGF-βdependent myofibroblast differentiation *in vitro* through the ras homolog gene family member A (RhoA), p38, and PI3Kα (30). *In vitro* exposure of airway smooth muscle or tracheal rings to hypotonic solutions causes smooth muscle cell contraction, and some asthmatic patients are hypersensitive to this stimulus. To that end, it has been found that small nucleotide polymorphisms in the G allele in the coding region and 3′ flanking region of the TRPV4 gene, as first identified in COPD, are associated with a greater reduction in pulmonary function after hypotonic saline administration (8, 31). Interestingly, the calcium and contractile response of smooth muscle cells to hypotonic saline involves interactions between the cysteinyl leukotriene pathway and TRPV4 (12, 32). These findings suggest that downregulation of TRPV4 may be a therapeutic target in some etiologies and genetic variants of asthma. Of note, different TRPV4 activation stimuli beyond hypotonicity utilize different pathways for TRPV4 activation. For example, hypotonicity induces TRPV4 activation through phospholipase A2 (PLA2)/P450 epoxygenase-dependent generation of EETs, while heat and 4αPDD are PLA2/P450-independent (25). Further study of the mode of TRPV4 activation in individual diseases would support disease-specific, pathway-targeted therapy. While asthma is an inflammatory disease, there is no current evidence linking Th2 type cytokines and TRPV4 in the pathogenesis of asthma. Hence, this is an avenue for future studies.

# Cystic Fibrosis

Cystic fibrosis is characterized by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a membranebased chloride channel, which initially causes dehydration of the airway surface liquid thereby increasing susceptibility to bacterial and fungal infections (e.g., *Pseudomonas*, *Staphylococcus*, *Burkholderia*, atypical mycobacterium) (33). TRPV4 interacts with CFTR on several levels. TRPV4-dependent calcium influx in response to hypotonicity is reduced in human CF epithelial cells (34). Furthermore, other hypotonicity-induced TRPV4 chemical activators (5,6-, 8,9-, 11,12-, and 14,15-EET) and their metabolites (5,6 DHET) have been measured in the sputum of CF patients (10). Although the current consensus suggests that dehydration of airway mucous is the predominant cause of impaired mucociliary clearance in CF, recent considerations have been put forth to increase ciliary function or ciliary beat frequency (CBF) as a means to improve mucociliary clearance (35, 36). Concordantly, TRPV4-deleted tracheal epithelial cells have decreased CBF in response to ATP, 4αPDD, and temperature, whereas CBF in response to hyperviscosity was similar in wild-type (WT) and TRPV4 deleted cells. These data suggest that TRPV4 agonism might increase CBF; however, the effects on CF prognosis remain to be determined (37).

The pathogenesis of CF is also characterized by cytokinemediated airway inflammation. Recently, both cytokines/ chemokines and lipid mediators secreted from epithelial cells have been identified as key components in the inflammatory process. In this regard, TRPV4 activation induces epithelial cell secretion of pro-inflammatory cytokines/chemokines and active lipid mediators (e.g., IL-8, cytosolic PLA2, prostaglandin E2, NF-κB, AA, etc.) in response to lipopolysaccharide (LPS) (10). Secretion of IL-8/KC, in both bronchial epithelial cells and in intact mice lungs in response to TPRV4 activation, was increased upon inhibition of CFTR (10). These data demonstrate that TRPV4 has pleotropic effects on CF pathogenesis. Further study of the individual molecular pathways downstream of TRPV4 in CF may identify selectivity in the TRPV4 responses that can then be marshaled for therapeutic intent.

# ACUTE LUNG INJURY/ARDS

Acute respiratory distress syndrome is a syndrome characterized by patchy lung inflammation along with cytokine release leading to alveolar space edema, exudate, and collapse. The pathogenesis of ARDS is complex; it is characterized by endothelial and alveolar epithelial injury followed by recruitment and accumulation of inflammatory cells in the injured alveolus (38). ARDS is a consequence of non-infectious (trauma, hemorrhage, lung ventilator stretch) or infectious (sepsis, pneumonia) causes (39). As the biological processes that underlie the lung injury and their molecular drivers are not fully understood, medical therapy directed at the lung inflammatory response has yet to successfully modify the course of ARDS. Experimental animal and patient studies demonstrate the lung injury and resolution phases of ARDS are mediated through a complex orchestration of cytokines/chemokines (e.g., IL-1β, TNFα, IL-8, IL-6, and IL-10) (40–44). Studies show that both sterile (e.g., ventilator-induced stretch) and infectious [e.g., intra-tracheal (IT) LPS] triggers of ARDS result in stiffening (reduced compliance) of the lung tissue (45, 46).

The role of TRPV4 in ARDS is context/etiology-dependent. It has been shown that TRPV4 mediates the lung injury response to a sterile stimulus *in vivo* [i.e., hydrochloric acid (HCl)], as assessed by inflammatory cell influx, lung vascular permeability (wet/dry ratio, Evans blue dye extravasation, and total protein), lung histopathology and physiology, and pro-inflammatory cytokine levels (IL-1β, VEGF, KC, G-CSF, MCP-1, RANTES, MIP-2, and IL-6) (7, 14). Protection from the acute lung injury response to IT HCl was noted in mice that lack TRPV4 (TRPV4 KO), or in mice that were treated with three different small molecule inhibitors of TRPV4 (7, 14). Importantly, two of these inhibitors (GSK2220691 and GSK2337429A) show efficacy when administered 30 min after IT HCl (7). Thus, these inhibitors show promise as a novel and exciting therapeutic/preventative approach for acute lung injury (7). *In vitro* stimulation of human and murine neutrophils (with platelet-activating factor or LPS) induced TRPV4-dependent calcium influx, reactive oxygen species (ROS) production, adhesion chemotaxis, and Rac activation (14). Taken together, these data suggest that neutrophils possess the capacity to mediate acute lung injury in a TRPV4-dependent manner. Whether the *in vivo* lung injury response to HCl is solely dependent on neutrophil TRPV4, as opposed to TRPV4 in other cell types, remains to be determined. In addition to TRPV4's effect on the cytokine/ inflammatory changes in ARDS, TRPV4 actions can induce lung endothelial barrier dysfunction *in vitro* and *in vivo*, as well as cause disruption of alveolar type I epithelial cells leading to lung vascular leak and alveolar edema (9, 29). These findings are the rationale for a clinical trial of TRPV4 antagonists in high venous pressure-induced pulmonary edema (https://clinicaltrials.gov).

#### TRPV4 AND MACROPHAGE FUNCTION IN LUNG INJURY

A similar TRPV4-dependent lung injury response has been demonstrated in macrophages in high volume ventilator-induced lung injury (6, 47). Mice lacking TRPV4 (TRPV4 KO) had less vascular leak, pulmonary edema (wet/dry ratio, filtration coefficient), and NO production in response to high volumes (peak inflation pressure 35 cm H2O) when compared to WT controls. TRPV4 also seemed to partially mediate the increase in injury due to the combined effects of high volume ventilation and induced hyperthermia (40°C). Analysis of alveolar macrophages after high volume ventilation revealed that TRPV4 KO macrophages had less production of NO and ROS than those from WT mice. As in the HCl model, pretreatment with a non-selective TRP inhibitor (ruthenium red) prevented the increase in vascular permeability from combined high volume ventilation/hyperthermia in WT mice (48). Adoptive transfer of WT macrophages to TRPV4 KO mice reestablished the lung injury seen in WT mice. These data suggest that macrophage-specific TRPV4 acts as a mechanical and temperature sensor to initiate/mediate the acute lung injury induced by high volume ventilation (47).

Our laboratory is studying the role of TRPV4 in macrophage function during infection-associated lung injury. Alveolar macrophages are known to be effector cells in bacterial and particle clearance, and in the injury/repair process (49). We chose to explore the role of the calcium ion channel, TRPV4, in macrophage phagocytosis, as intracellular calcium is known to be required for the phagocytic process, and because TRPV4 plays a role in force-dependent cytoskeletal changes in other systems/ cell types (7–9, 29, 47, 50, 51). Studies show that the process of phagocytosis in macrophages requires integration of the signals from macrophage surface receptors, pathogens, and the extracellular matrix (52–54). However, the effects of matrix stiffness on the macrophage phenotypic response or its signal transduction pathways have yet to be fully elucidated.

We recently published the novel observation that TRPV4 integrates the LPS and matrix stiffness signals to control macrophage function, which promotes host defense and resolution from lung injury (4). After demonstrating that TRPV4 is expressed and functionally active in murine bone marrow-derived macrophages, we studied the macrophage response to LPS on matrices of varying physiological-range stiffnesses. We demonstrated that TRPV4 mediates LPS-stimulated macrophage phagocytosis of both opsonized particles (IgG-coated latex beads) and non-opsonized particles (*Escherichia coli*) *in vitro*. Matrix stiffness in the range seen in inflamed or fibrotic lung (>25 kPa) augmented the LPS phagocytic response by 151 ± 3% (4). Inhibition of TRPV4 by siRNA or pharmacologic inhibitors completely abrogated both the LPS effect, as well as the matrix stiffness effect, on phagocytosis. These data indicate that both the LPS and stiffness effect on macrophage phagocytosis are TRPV4 dependent (4).

As TRPV4 is required for macrophage phagocytosis *in vitro* in a stiffness-dependent manner, we next sought to examine the role of TRPV4 on macrophage phagocytosis after intratracheally (IT) administered LPS *in vivo*. Despite the influx of neutrophils, alveolar macrophages were the predominant cell type that phagocytosed IT administered IgG-coated beads following IT LPS (24 h) in WT mice (4). As seen *in vitro*, the *in vivo* enhancement effect of IT LPS on alveolar macrophage phagocytosis was lost upon deletion of TRPV4 (TRPV4 KO mice) (**Figure 1**) (4). This effect is not explained by a difference in macrophage recruitment. Concordant with the *in vitro* data, our *in vivo* data demonstrate that LPSinduced alveolar macrophage phagocytosis is TRPV4 dependent.

Studies suggest that macrophage-released cytokines modulate bacterial clearance and the lung injury/repair process, in the context of injury-related stiffened matrix (52–55). Recognizing the complexity of tissue responses to individual cytokines/ chemokines, we chose to focus initially on IL-1β and IL-10, as they are well-known key mediators of lung injury/resolution (56–58). TRPV4 also modulates the LPS signal for cytokine production. Specifically, IL-1β secretion was decreased by half,

and IL-10 secretion increased approximately twofold in WT alveolar macrophages compared with TRPV4 KO macrophages in response to LPS. Such a profile would predict that TRPV4 mediates a net inflammation-suppressive response to LPS. Interestingly, this TRPV4 modulation of the LPS signal required a matrix stiffness in the range of injured or fibrotic lung (≥25 kPa). As illustrated in the schematic model, macrophage TRPV4 is sensitized by a stiff matrix (as seen in ARDS) to modulate the infectious (LPS—experimental surrogate for Gram-negative bacterial lung infection) signal toward an anti-inflammatory macrophage phenotype (**Figure 1**).

Collectively, our data demonstrate that TRPV4 responds to extracellular matrix stiffness, thereby altering the LPS signal to mediate macrophage phagocytosis and cytokine production (4). Despite the limitations in extrapolating our simplified experimental system to *in vivo* lung injury, the data point to TRPV4 as an important mechanosensor that mediates macrophage function differently in lung homeostasis, and in the context of pulmonary infection-induced inflammation. We speculate that under basal conditions, the resident lung macrophage response to LPS is modified (less phagocytic, more IL-1β) as a consequence of low lung tissue stiffness (i.e., 1–3 kPa) thereby enhancing recruitment of professional bactericidal cells (neutrophils) (55). After an acute inflammatory or infectious insult, a separate population of monocytes is recruited from the bone marrow to populate both interstitial and injured alveolar compartment in the context of denuded, exposed interstitial matrix (40). There are two overlapping phases of ARDS. During the initial injury phase (days 1–10), lung tissue is predominantly edematous and exudative, while during the fibroproliferative phase (days 7–28), there is increased deposition of interstitial and alveolar type I and III collagen (40). Both phases of ARDS (fibroproliferative > acute) exhibit clear evidence of increased stiffness at the whole organ level (40, 46, 59), but, limited mechanical data are available at the cellular level of resolution. A recent study shows that lung alveolar vessel wall stiffness is increased >10-fold (3 versus 43 kPa) after IT LPS (48 h) in mice compared to controls, as measured by atomic force microscopy, well within the range examined in our study (>8–25 kPa) (45, 46). We further speculate that, after injury, the macrophage phagocytic response to LPS is upregulated along with a cytokine profile that promotes resolution in a TRPV4 dependent manner as a consequence of tissue stiffening. Such a scenario would support tissue stiffness, TRPV4-dependent shift in the macrophage phenotype that is commensurate with the appropriate phase of the injury/repair process.

Thus, our findings suggest that TRPV4 regulates a feed-forward mechanism of phagocytosis in activated lung tissue macrophages when they interact with stiffened infection/injury-associated lung matrix. This concept is further supported by the observation that surfactant protein B-deficient mice have altered alveolar macrophage shape and function in association with increased alveolar surface tension (60). The macrophage activation phenotypes (M1/M2 classification) are well established *in vitro*. The classically activated M1 macrophage phenotype, induced by INFγ, TNFα, and LPS, exhibits inflammatory/bactericidal properties. In contrast, the alternatively activated M2 macrophage phenotype, induced by IL-4 and IL-13, exhibits tissue repair/ fibrotic properties (49, 55). Data are emerging that the *in vivo* macrophage phenotypes are more heterogeneous and plastic than the *in vitro* derived M1/M2 classification. Our published cytokine data (↑IL-1β, ↓IL-10) with inhibition of TRPV4 indeed suggests that TRPV4 mediates polarization toward M1-like phenotype (4, 61, 62). However, a complex array of cytokines contributes to the pathogenesis of ARDS, and targeting individual cytokines has not been shown to alter the disease process, indicating the net inflammatory balance is important (41–44, 63).

Our findings regarding the role of TRPV4 in downregulating the pro-inflammatory, bacterial clearance-inducing LPS signal are opposite to those in neutrophils in response to sterile inflammation, or in macrophages upon stretch-induced tissue injury. Lung injury is dependent on cytokine production and inflammatory cell influx in response to activation of pattern recognition receptors by damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). There are multiple known ligand–receptor interactions and intracellular signaling pathways that are both DAMP/PAMP-receptor specific and overlapping. We speculate that differences in the interaction of TRPV4 signals with infectious PAMP signals versus sterile tissue injury DAMP signals might explain the differences between our infectious model and the sterile lung injury model. Defining the specific molecular pathways and interactions in individual injury models is a fruitful avenue of research that may lead to novel therapeutic targets.

#### Table 1 | *In vitro* and *in vivo* studies of the role of transient receptor potential vanilloid 4 (TRPV4) in inflammatory pulmonary diseases.

*This table is only a partial representation of the literature, given the focused nature of the mini review. We apologize for any work omitted from this review. We summarize the cited literature on the role of TRPV4 in asthma, CF, acute lung injury/ARDS, and pulmonary fibrosis.*

#### SUMMARY

In summary, ion channels are important in the pathogenesis of inflammatory lung diseases, and the ion channel TRPV4 plays a specific role in mediating lung diseases associated with parenchymal stretch and inflammation or infection. The data reviewed in this work on the role of TRPV4 in pulmonary inflammatory diseases are summarized in **Table 1**. TRPV4 activation and its downstream signaling pathways differ in response to varying stimuli, cell types, and contexts. In asthma, TRPV4 mediates hypotonicity-induced airway hyperresponsiveness, but not release of Th2 cytokines (12, 32). In CF, TRPV4 appears to play important, yet paradoxical, roles in CBF/mucociliary clearance and epithelial cell pro-inflammatory cytokine (IL-8/KC) secretion (35, 36). TRPV4 may also play different roles in ARDS depending on the underlying etiology (4, 7, 14, 48). We, and others, have shown that macrophage and neutrophil TRPV4 regulate

#### REFERENCES

pro-inflammatory cytokine secretion. Lastly, in pulmonary fibrosis, TRPV4 has been shown to mediate the mechanosensing that drives myofibroblast differentiation and experimental lung fibrosis in mice (5). Collectively, TRPV4 is shown to play a novel role in modulating cytokine secretion and pulmonary inflammation and therefore may be involved in the pathogenesis of many respiratory diseases.

#### AUTHOR CONTRIBUTIONS

RS, BS, LG, and MO reviewed the literature and wrote the paper.

#### FUNDING

This work was supported by NIH grants (HL-133380) to RS and (HL-085324 and HL-119792) to MO and Genentech Research Award to BS.

during ventilator-induced lung injury in isolated mouse lungs. *Am J Physiol Lung Cell Mol Physiol* (2007) 293:L923–32. doi:10.1152/ajplung.00221.2007

TRPV4 in lung epithelium: potential involvement in cystic fibrosis. *Am J Physiol Lung Cell Mol Physiol* (2016) 311(3):L664. doi:10.1152/ajplung.00442.2015

*Dermatophagoides farinae*-induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation. *FASEB J* (2017) 31(4):1556–70. doi:10.1096/fj.201601045R

of TRPV4. *Am J Respir Cell Mol Biol* (2008) 38(4):386–92. doi:10.1165/ rcmb.2007-0192OC

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Scheraga, Southern, Grove and Olman. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

*Paulina Gwoz´ dzin´ska 1 , Benno A. Buchbinder 1 , Konstantin Mayer 1 , Susanne Herold 1 , Rory E. Morty 1,2, Werner Seeger 1,2 and István Vadász 1 \**

*1Department of Internal Medicine, Justus Liebig University, Universities of Giessen and Marburg Lung Center, German Center for Lung Research, Giessen, Germany, 2Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany*

#### *Edited by:*

*Rudolf Lucas, Augusta University, USA*

# *Reviewed by:*

*Aaron Hanukoglu, Tel Aviv University, Israel Juerg Hamacher, Lindenhof Hospital, Switzerland*

#### *\*Correspondence:*

*István Vadász<EMAIL_ADDRESS>uni-giessen.de*

#### *Specialty section:*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology*

*Received: 26 February 2017 Accepted: 04 May 2017 Published: 23 May 2017*

#### *Citation:*

*Gwoździńska P, Buchbinder BA, Mayer K, Herold S, Morty RE, Seeger W and Vadász I (2017) Hypercapnia Impairs ENaC Cell Surface Stability by Promoting Phosphorylation, Polyubiquitination and Endocytosis of β-ENaC in a Human Alveolar Epithelial Cell Line. Front. Immunol. 8:591. doi: 10.3389/fimmu.2017.00591*

Acute lung injury is associated with formation of pulmonary edema leading to impaired gas exchange. Patients with acute respiratory distress syndrome (ARDS) require mechanical ventilation to improve oxygenation; however, the use of relatively low tidal volumes (to minimize further injury of the lung) often leads to further accumulation of carbon dioxide (hypercapnia). Hypercapnia has been shown to impair alveolar fluid clearance (AFC), thereby causing retention of pulmonary edema, and may lead to worse outcomes; however, the underlying molecular mechanisms remain incompletely understood. AFC is critically dependent on the epithelial sodium channel (ENaC), which drives the vectorial transport of Na+ across the alveolar epithelium. Thus, in the current study, we investigated the mechanisms by which hypercapnia effects ENaC cell surface stability in alveolar epithelial cells (AECs). Elevated CO2 levels led to polyubiquitination of β-ENaC and subsequent endocytosis of the α/β-ENaC complex in AECs, which were prevented by silencing the E3 ubiquitin ligase, Nedd4-2. Hypercapnia-induced ubiquitination and cell surface retrieval of ENaC were critically dependent on phosphorylation of the Thr615 residue of β-ENaC, which was mediated by the extracellular signalregulated kinase (ERK)1/2. Furthermore, activation of ERK1/2 led to subsequent activation of AMP-activated protein kinase (AMPK) and c-Jun N-terminal kinase (JNK)1/2 that in turn phosphorylated Nedd4-2 at the Thr899 residue. Importantly, mutation of Thr899 to Ala markedly inhibited the CO2-induced polyubiquitination of β-ENaC and restored cell surface stability of the ENaC complex, highlighting the critical role of Nedd4-2 phosphorylation status in targeting ENaC. Collectively, our data suggest that elevated CO2 levels promote activation of the ERK/AMPK/JNK axis in a human AEC line, in which ERK1/2 phosphorylates β-ENaC whereas JNK mediates phosphorylation of Nedd4-2, thereby facilitating the channel–ligase interaction. The hypercapnia-induced ENaC dysfunction may contribute to impaired alveolar edema clearance and thus, interfering with these molecular mechanisms may improve alveolar fluid balance and lead to better outcomes in patients with ARDS.

Keywords: carbon dioxide, epithelial sodium channel, sodium transport, ubiquitination, alveolar fluid clearance, alveolar epithelium, mitogen-activated protein kinase signaling

# INTRODUCTION

Carbon dioxide (CO2) is formed as a by-product of cellular respiration and is eliminated from the body during breathing (1). In respiratory disorders that are associated with alveolar hypoventilation, retention of CO2 is often detected, which leads to elevated CO2 concentrations in the blood, also known as hypercapnia (2). For example, patients with severe acute respiratory distress syndrome (ARDS) frequently present with hypercapnia, which may be enhanced due to mechanical ventilation with low tidal volumes to minimize further ventilatorinduced injuries to the lung (3). It is increasingly evident that the alveolar epithelium is capable of sensing of elevated CO2 levels, which initiate specific signaling signatures and alter the function of alveolar epithelial and other cells (2, 4). While some of these effects are anti-inflammatory, which may be beneficial in the context of excessive inflammation, others impair innate immunity, mitochondrial function, cellular repair, and alveolar epithelial barrier function, which are clearly detrimental in the setting of ARDS (5–10).

A fully functional alveolar epithelial barrier is crucial for maintaining optimal fluid balance and gas exchange in the lung (11, 12). In order to keep the alveolar space "dry," excess alveolar liquid is reabsorbed from the air space into the interstitium by a well-characterized active sodium transport process in which Na+ enters the alveolar epithelial type I and type II cells through the apically located epithelial sodium channel (ENaC) and is subsequently pumped out basolaterally by the Na,K-ATPase. This creates a Na+ gradient, which drives paracellular movement of water leading to its clearance from the alveolar space (11, 12). Importantly, it has been clearly demonstrated that in most patients with ARDS alveolar fluid clearance (AFC) is impaired and that those patients with ARDS and impaired AFC the mortality is significantly higher than in ARDS patients with normal AFC (13).

Other than in the alveolar epithelium, ENaC molecules are expressed in the apical surface of various tight epithelia including kidney, colon, and respiratory airways where ENaC is located along the entire length of motile cilia and regulate osmolarity of the periciliary fluid (14). ENaC usually consists of three subunits (α [or δ, depending on the species, tissue, and cell type], β, and γ) (15–17). A functional ENaC complex requires at least one α- or δ-subunit, whereas the β- and γ-subunits are necessary for proper trafficking and activity of the channel (15, 16, 18). In line with this notion, mice lacking α-ENaC are unable to clear lung fluid from the alveoli and die nearly immediately after birth (19). The significance of β-ENaC in the regulation of channel activity has been highlighted in transgenic mice overexpressing this subunit in the lung (20). In these animals, an increase in alveolar epithelial Na+ uptake due to β-ENaC overexpression, probably by promoting trafficking of ENaC to the cell surface and enhancing channel activity, leads to lung dehydration and causes a CF-like phenotype. Cell surface abundance of ENaC is modified by the E3 ubiquitin ligase Nedd4-2, which by interaction with the PY motif, located at the C-termini of each subunit of the channel, promotes ubiquitination and subsequent clathrin*-*mediated endocytosis of the channel (21–23). Nedd4-2−/− mice have enhanced ENaC expression and function; however, this genetic manipulation has lethal consequences (24). In contrast, overexpression of the ligase causes a decrease in ENaC density at the plasma membrane (PM) and reduces Na+ transport (25).

Previous studies have proposed the involvement of phosphorylation in the mechanisms regulating Nedd4-2 binding to ENaC (26, 27). For example, extracellular signal-regulated kinase (ERK), c*-*Jun N*-*terminal kinase (JNK), and recently also the cellular energy sensor, AMP-activated protein kinase (AMPK), have been described as potential modulators of the ENaC/Nedd4-2 interaction by phosphorylating the E3 ligase or the target (28–32). We have previously described that hypercapnia markedly impairs AFC and initiates a specific signaling pattern in the alveolar epithelium, including rapid activation of ERK, AMPK, and JNK and subsequent downregulation of the Na,K-ATPase (7, 8, 33). Considering the pivotal role of ENaC in AFR and that several kinases, which have previously been suggested to alter ENaC/ Nedd4-2 interaction, are activated by elevated CO2 levels, in the current study we sought to determine whether ENaC is effected by hypercapnia and provide evidence that excess CO2 initiates ERK-mediated β-ENaC phosphorylation and AMPK/JNKdependent activation of Nedd4-2 leading to an enhancement of β-ENaC polyubiquitination and, thus, to endocytosis of the ENaC complex form the cell surface. Since ENaC activity is essential for optimal lung fluid balance, the hypercapnia-induced alterations in ENaC cell surface stability may cause further aggravation of lung injury.

#### MATERIALS AND METHODS

#### Cell Culture

Human epithelial A549 cells (ATCC, CCL 185) were grown in DMEM supplemented with 10% fetal bovine serum and 100 U/ml penicillin, 100 µg/ml streptomycin as previously described (8). Experiments were performed on subconfluent monolayers of cells. Cells were incubated in a humidified atmosphere of 5% CO2/95% air at 37°C.

# CO2 Exposure

A549 cells were treated with 40 or 120 mmHg CO2 (normocapnia and hypercapnia, respectively). Before each experiment, fresh solutions were prepared with DMEM-Ham's F-12 medium and Tris base. The buffering capacity of the experimental media was modified by changing the initial pH using Tris base to obtain a pH of 7.4 at 40 and 120 mmHg CO2 (8). The desired CO2 concentrations and pH levels were obtained by equilibrating the experimental media overnight in a humidified chamber from BioSpherix Ltd. (NY, USA). The C-Chamber's atmosphere was controlled with a PRO-CO2 Carbon Dioxide controller (Biospherix Ltd.). In the chamber, cells were treated with a pCO2 of 40 or 120 mmHg while keeping 21% O2 balanced with N2. Before and after CO2 exposure, pH, pCO2, and pO2 levels in the media were measured using a Rapidlab blood gas analyzer (Siemens, Erlangen, Germany).

# Plasmids, Constructs, Site-Directed Mutagenesis, Antibodies, and Inhibitors

pEYFP-C1-expressing α-ENaC was constructed by PCR amplifying α-ENaC gene using as a template pTNT-α-ENaC and oligonucleotide primers α-ENaC forward 5′-GAATTCAATGG-AGGGGAACAAGCTGGAGG-3′ and α-ENaC reverse 5′-GGATCCCTTGTCATCGTCATCCTTGTAATCGGGCCCC CCCAGAGGAC-3′. The resulting amplicon was digested with *Eco*RI/BamH1 and ligated to the multiple cloning site (MCS) of pEYFP-C1 plasmid. The pEYFP-C1 vector contained the epitopetag eYFP at the N-terminus and a FLAG-tag at the C-terminus. Thus, anti-GFP or anti-FLAG antibodies recognize the α-ENaC construct at a predicted size of 118 kDa, 1,073 amino acids [α-ENaC (90 kDa) plus YFP (27 kDa) and FLAG (1 kDa)]. pcDNA3.1V5/His expressing β-ENaC was constructed by PCR amplifying β-ENaC gene using as a template cDNA transcribed from total mRNA isolated from A549 cells and oligonucleotide primers β-ENaC forward 5′-CTCGGATCCACATGCACGTGAAGAAGTACCT-3′ and β-ENaC reverse 5′-GCACTCGAGGATGGCATCACCCT-CACTGT-3′. The resulting amplicon was digested with Xho1/ BamH1 and ligated to MCS of pcDNA3.1V5/His plasmid. Finally, *E. coli* DH5α were transformed using the constructed plasmid. Anti-V5 antibodies recognize the β-ENaC construct at a predicted size of 96 kDa, 872 amino acids [β-ENaC plus V5 at the C-terminus (1 kDa)]. pCMV-HA-C-expressing γ-ENaC was constructed by PCR amplifying γ-ENaC gene using as a template pTNT-γENaC and oligonucleotide primers γ-ENaC forward 5′-AGGCCCGAATTCATGGCACCCGGAGAGAAGAT-3′ and γ-ENaC reverse 5′-GTAGCCGGTACCGAGCTCATC-CAGCATCTGGG-3′. The resulting amplicon was digested and ligated to MCS of pCMV-HA-C plasmid. The pCMV-HA-C vector contained the epitope-tag myc at the N-terminus and an HA-tag at the C-terminus. Anti-HA antibodies recognize γ-ENaC at a predicted size of 97 kDa, 881 amino acids [γ-ENaC plus myc (1 kDa) and HA (1 kDa)]. pRK5-HA-ubiquitin was a gift from Ted Dawson [Addgene 17608 (34)] and the pCI HA NEDD4L plasmid was a gift from Joan Massague [Addgene 27000 (35)]. Site-directed mutagenesis was used to perform point mutation of T899A in human Nedd4-2 using Quick Change Mutagenesis Kit from Stratagene (La Jolla, CA, USA) in accordance to the manufacturer's instructions. The primer sequences were as follows: Nedd4-2 forward: 5′-ACTGCAGTTTGTCGCAGGGACATCG-CGAG-3′, Nedd4-2 reverse: 5′ CTCGCGATGTCCCTGCGAC-AAACTGCAGT-3′. Immunoblot analysis of epitope-tagged ENaC expressed in A549 cells were performed with a mouse anti-GFP antibody from Roche (Basel, Switzerland), a mouse anti-HA antibody (Covance, Princeton, NJ, USA), a mouse anti-V5 antibody and a mouse antibody against transferrin receptor used as loading control of biotinylated proteins from Invitrogen (Waltham, MA,

USA; Figure S1 in Supplementary Material). A rabbit antibody directed against β-actin was used as loading control of cytoplasmic ENaC and was purchased from Sigma Aldrich (Saint Louis, MO, USA). The inhibitor of AMPK, Compound C, was from Merck Millipore (Darmstadt, Germany). The inhibitor of MEK, U0126 was from Promega (Fitchburg, WI, USA). siRNA against AMPK-α1 and Nedd4-2 and scrambled siRNA control were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

#### Transient Transfection

A549 cells were transiently transfected with eYFP-α-ENaC, β-ENaC-V5, HA-ubiquitin, HA-Nedd4-2 wild type, or mutant by using nucleofection, as previously described (36). Briefly, cells were resuspended in 100 µl of the nucleofection solution SF (Lonza, Cologne, Germany), and 4–6 µg of DNA was added. Cells were placed in a cuvette and pulsed with the specified cell-type nucleofector program. After 10 min of incubation time, cells were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. In some studies, 24 h before nucleofection with ENaC plasmids, cells were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen, Waltham, MA, USA) according to the instructions of the manufacturer. Experiments were performed 48 h later.

# Cell Surface Biotinylation

A549 cells were labeled for 20 min using 1 mg/ml EZ-Link *N*HS-SS-biotin (Pierce Biotechnology, Waltham, MA, USA) and lysed in lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EGTA, 10% glycerol, 1% TritonX100). Surface proteins were pulled down with streptavidin-agarose beads from Pierce Biotechnology (Waltham, MA, USA) and analyzed by SDS-PAGE and immunoblot, as described previously (8).

# Ubiquitination Studies

A549 cells were transfected with ENaC plasmids (2 µg of each) and HA-ubiquitin (3 µg). In some studies, cells were cotransfected with a plasmid coding HA-Nedd4-2 (wild type or mutant T899A, 2 µg) or siRNAs (against AMPK-α1, Nedd4-2, or scrambled). Cells were exposed to 40 mmHg CO2 (Ctrl) or to 120 mmHg CO2 (CO2) for 15 or 30 min and lysed on ice in lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EGTA, 10% glycerol, 1% TritonX100), containing a protease inhibitor cocktail from Roche. After lysing the samples, proteins were resolved in 8% polyacrylamide gel, transferred to nitrocellulose membrane (Optitran; Schleicher & Schuell, Dassel, Germany) using a semidry apparatus from Bio-Rad (Hercules, Berkeley, CA, USA). Membranes were blocked in 5% fat-free dried milk powder and immunoblotted with anti-GFP or anti-V5 to detect α- or β-ENaC, respectively. Films were overexposed to detect ENaC ubiquitin conjugates.

# Phosphorylation Experiments

Phosphorylation studies of ERK1/2, AMPK-α1, and c-Jun were performed using antibodies from Cell Signaling (Danvers, MA, USA). The anti-phospho-β-ENaC (T615) antibody was from Abcam (Cambridge, UK). A549 cells were treated with normal or elevated CO2 concentrations (40 or 120 mmHg, respectively) for the desired times, and then were washed with PBS twice and were lysed on ice in lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EGTA, 10% glycerol, 1% TritonX100). Samples having the same amount of protein were resuspended in Laemmli sample buffer and boiled for 10 min at 98°C and immunoblotted with specific antibodies.

#### Statistics

Data are presented as mean ± SEM and were analyzed using one-way analysis of variance (ANOVA) followed by a multiple comparison with the Dunnet test. *p* values of less than 0.05 were considered significant. GraphPad prism 6 (GraphPad software, San Diego, CA, USA) was used for the analysis and presentation of data.

#### RESULTS

# Acute Exposure to Elevated CO2 Levels Leads to ENaC Endocytosis by Promoting Polyubiquitination of **β**-ENaC

To test whether high CO2 levels promote endocytosis of ENaC, A549 cells were co-transfected with plasmids encoding the human α- and β-subunit of ENaC and the PM abundance of these proteins was measured after exposure of cells to physiological (pCO2 40 mmHg; normocapnia) or elevated (pCO2 120 mmHg; hypercapnia) CO2 concentrations at a pHe of 7.4 for 30 min. Exposure of cells to elevated CO2 levels decreased α- and β-ENaC cell surface abundance by approximately 60% (**Figure 1A**), whereas the total protein level remained unaffected (**Figure 1B**). To determine whether elevated CO2 levels lead to ubiquitination of either ENaC subunit, A549 cells were co-transfected with α- or β-ENaC and ubiquitin containing HA-tag (HA-Ub) and exposed the cells to 40 or 120 mmHg CO2 for 15 min. Whereas no ubiquitination of α-ENaC in response to hypercapnia was

evident (**Figure 1C**), a marked increase in β-ENaC ubiquitination in total cell lysates was observed when cells were treated with elevated CO2 and immunoblotted with an antibody against V5 (**Figure 1D**). We detected a "polyubiquitin smear" above the molecular size of ENaC suggesting the presence of β-ENaC ubiquitin conjugates. This observation suggested that β-ENaC is a substrate of CO2-induced ubiquitination.

#### Nedd4-2 Mediates the Hypercapnia-Induced ENaC Polyubiquitination and Endocytosis

In subsequent studies, we silenced the endogenous Nedd4-2 with a specific siRNA to study whether Nedd4-2 mediates β-ENaC polyubiquitination during hypercapnia. Of note, the elevated CO2-induced ENaC ubiquitination was prevented by Nedd4-2 silencing (**Figure 2A**). To further test whether Nedd4-2 silencing altered ENaC PM stability, we measured cell surface ENaC abundance in A549 cells co-transfected with a scrambled siRNA (si-Scr.) or siRNA against Nedd4-2. Importantly, cells exposed to increased CO2 concentrations treated with siRNA targeting Nedd4-2 had increased ENaC α- and β-subunit density at the PM (**Figure 2B**). Thus, upon hypercapnia, Nedd4-2 targets β-ENaC, leading to polyubiquitination of the β-subunit of the channel, which results in decreased abundance of the α/β-ENaC complex at the cell surface.

## Hypercapnia Induces ERK1/2-Dependent Phosphorylation of **β**-ENaC at Thr615 and Downregulates Surface Abundance of the Channel by Facilitating **β**-ENaC Polyubiquitination and Endocytosis of the **α**/**β**-ENaC Complex

In agreement with a previous report describing increased ERK 1/2 activity in alveolar epithelial cells (AECs) exposed to hypercapnia (33), we found a rapid and transient phosphorylation of ERK1/2 in A549 cells exposed to elevated CO2 (**Figure 3A**). Moreover, ERK1/2 activation was paralleled by phosphorylation of β-ENaC at the Thr 615 residue (**Figure 3B**). To test whether ERK-dependent β-ENaC phosphorylation was sufficient to promote polyubiquitination of β-ENaC in response to high CO2, we co-transfected A549 cells with β-ENaC and HA-ubiquitin. Cells were pre-treated with the MEK (upstream of ERK) inhibitor U0126 and exposed to elevated CO2 concentrations for 30 min. Inhibition of ERK prevented phosphorylation and polyubiquitination of the ENaC β-subunit (**Figure 3C**). To further prove that the hypercapnia-induced ENaC cell surface retrieval is dependent on ERK1/2, A549 cells were co-transfected with ENaC plasmids, and exposed to normo- or hypercapnia as described above and observed that inhibition of ERK1/2 markedly increased the number of the ENaC molecules at the cell surface upon hypercapnia exposure (**Figure 3D**). Together, these data indicate that the hypercapnia-induced ERK1/2 activation promotes ENaC internalization by phosphorylation-dependent ubiquitination of the β-subunit of the channel.

### JNK1/2-Dependent Nedd4-2 Phosphorylation at Thr899 Facilitates **β**-ENaC Polyubiquitination and Endocytosis

Because JNK activation has been implicated in the CO2-induced signaling pattern in AEC, which led to inhibition of the Na, K-ATPase (7), we next investigated the effects of JNK phosphorylation in AEC exposed to hypercapnia on ENaC. Activity of JNK1/2 was assessed by phosphorylation of c-Jun, a downstream target of JNK1/2. In line with the previously published data, we observed a rapid and time-dependent JNK activation induced by hypercapnia, which returned to baseline within 30 min of exposure to elevated CO2 levels (**Figure 4A**). To further investigate whether increased activity of Nedd4-2 is crucial to decrease hypercapnia-induced ENaC cell surface abundance, we next mutated a single amino acid in the catalytic domain of Nedd4-2 (T899A). The Thr899 residue within the HECT domain of the E3 ligase has previously been reported to be involved in the Nedd4-2-mediated ubiquitination of α-ENaC (37). A549 cells were co-transfected with HA-Ub and HA-Nedd4-2 wild type (WT) or HA-Nedd4-2 mutant (T899) constructs and exposed to 40 or 120 mmHg CO2 for 30 min. Of note, we found that phosphorylation of Thr899 played a central role in the hypercapniainduced ubiquitination of β-ENaC, as in A549 cells expressing the Nedd4-2 in which the Thr899 has been mutated to an alanine (T899A), which cannot be phosphorylated, the level of β-ENaC polyubiquitination significantly decreased (**Figure 4B**). To further investigate whether this decrease in the ubiquitination of β-ENaC due to the lack of phosphorylation at the Thr899 residue of Nedd4-2 correlated with an increase in ENaC cell surface stability, cell surface biotinylation studies were performed. Importantly, overexpression of the Nedd4-2 mutant (T899A) also prevented endocytosis of the α/β-ENaC complex during hypercapnia (**Figure 4C**). Moreover and further confirming the central role of Nedd4-2 phosphorylation at Thr899 in the ubiquitination and subsequent endocytosis of ENaC, we observed increased levels of ENaC proteins at the cell surface after overexpression of the Nedd4-2 T899A mutant. Finally and in line with the above described findings, pretreatment of A549 cells with the potent and specific JNK inhibitor, SP600125, also fully prevented the hypercapnia-induced endocytosis of ENaC (**Figure 4D**).

## Hypercapnia Induces ENaC Endocytosis by ERK1/2-Dependent AMPK-**α**1 Activation

AMP-activated protein kinase, which has been shown to activate Nedd4-2 and inhibit ENaC (29), has also been described as one of the central mediators of the hypercapnia-induced alveolar epithelial dysfunction and a downstream target of ERK upon CO2 exposure (8, 33). Furthermore, we have previously observed that AMPK activates JNK1/2 in AEC when exposed to elevated CO2 levels (7). In line with these previously published observations, we measured a rapid and transient phosphorylation of AMPK-α1 in A549 cells exposed to hypercapnia (**Figure 5A**), which was dependent on activation of ERK (**Figure 5B**) and

upstream of JNK (Figure S2 in Supplementary Material). To determine whether activation of AMPK was necessary for the hypercapnia-induced polyubiquitination of β-ENaC, A549 cells were co-transfected with β-ENaC, HA-ubiquitin, and a specific siRNA against AMPK-α1 (or a scrambled siRNA) and were exposed to normal or elevated CO2 levels and observed a significant decrease in polyubiquitination of β-ENaC after CO2 exposure (**Figure 5C**). As a second approach, endogenous AMPK was inhibited by compound C after co-transfection of A549 cells with β-ENaC and HA-ubiquitin. Similar to our data that we obtained with AMPK silencing, exposure of the cells to hypercapnia in the presence of the inhibitor markedly

decreased the hypercapnia-induced β-ENaC polyubiquitination (**Figure 5D**). To further confirm the role of AMPK-α1 in the CO2-induced downregulation of ENaC, we transfected A549 cells with α- and β-ENaC and exposed to elevated CO2 levels for 30 min in the presence or absence of the above mentioned siRNA against AMPK-α1 (**Figure 5E**) or compound C (Figure S3 in Supplementary Material) and observed that silencing or inhibition of AMPK stabilized ENaC proteins at the PM upon hypercapnia. Taken together, these latter studies suggest that AMPK by activation of JNK and subsequent phosphorylation of Nedd4-2 plays a central role in the hypercapnia-induced ubiquitination and endocytosis of ENaC.

# DISCUSSION

In the present study, we show that elevated CO2 levels initiate a specific signaling pattern leading to ubiquitination-mediated retrieval of ENaC from the PM, thereby reducing cell surface abundance of the channel in a human AEC line. Hypercapnia is associated with a number of acute and chronic pulmonary diseases; however, it is not evident to what extent and by which mechanisms these elevated levels of CO2 may further impact on disease states. While hypercapnia and the associated acidosis have been shown to have anti-inflammatory effects, which might be advantageous at sites of excessive inflammation, recently, it has been clearly demonstrated that by impairing innate immunity, cellular repair, and alveolar epithelial function, elevated CO2 may play a role in the pathogenesis of ARDS and COPD (2, 5, 9, 10, 38). Furthermore, it is increasingly evident that patients with ARDS and COPD who present with hypercapnia have worse outcomes (3, 39, 40).

A major function of the alveolar epithelium is the clearance of excess alveolar fluid, thereby promoting effective gas exchange. This clearance is mediated by the concerted action of various sodium transporters, among which the apically located ENaC

and the basolateral Na,K-ATPase have been identified as key players. Indeed, we have previously shown that the Na,K-ATPase is downregulated by hypercapnia; however, a potential regulation of ENaC by carbon dioxide has not been previously investigated. Various factors have been shown to affect ENaC cell surface abundance and function, including interleukin-1β, interleukin-4, transforming growth factor-β, LPS, or hypoxia (41–45), which similar to hypercapnia are often observed in patients with respiratory failure. As reducing hypercapnia without further damaging the lung is challenging, a better understanding of the molecular patterns initiated by elevated CO2 levels may help us to interfere with the deleterious signals, thereby rescuing or at least not further aggravating lung damage.

Ubiquitination is a posttranslational modification that regulates trafficking and stability of proteins (46). Numerous studies described that depending on the stimulus ENaC subunits may undergo multimono- or polyubiquitination leading to channel retrieval from the cell surface or degradation of ENaC (47–49). It is also well documented that the phosphorylation status of target molecules and the E3 ubiquitin ligase often play a pivotal role in the initiation of ubiquitination (50, 51). Previous findings established the significance of mitogen-activated protein kinase (MAPK) in the hypercapnia-induced impairment of AFC (7, 33). Moreover, it has also been described that ERK and JNK, two prominent members of the MAPK family, may alter the phosphorylation status of ENaC and the E3 ligase of the channel, Nedd4-2, respectively (31, 37).

Thus, we first investigated whether elevated CO2 concentrations affect ENaC cell surface stability by a mechanism involving ubiquitination of the channel and whether the MAPK pathway is involved in the hypercapnia-induced signaling events. Of note, a remarkable and rapid increase in polyubiquitination of β-ENaC and a significant reduction of the cell surface abundance of the α/β-ENAC complex were observed in AEC exposed to hypercapnia, as early as 30 min after CO2 exposure, suggesting that ENaC function is probably sensitive to changes in CO2 levels. In contrast, in the first half an hour after CO2 exposure, total intracellular levels of ENaC remained unchanged, suggesting that CO2 influenced

amount of both proteins were determined by immunoblotting. Graph represents the p-AMPK-α1/AMPK-α1 ratio. Representative immunoblots of p-AMPK-α1, p-ERK1/2 and total level of AMPK-α1 and ERK1/2 are shown. Values are expressed as mean ± SEM (*n* = 3; \**p* < 0.05; \*\*\**p* < 0.001). (C) A549 cells were co-transfected with β-ENaC, HA-ubiquitin, and siRNA targeting AMPK-α1 or a scrambled siRNA and were treated with 40 or 120 mmHg CO2 for 30 min. β-ENaC polyubiquitinated isoforms were determined with anti-V5 antibody. Representative immunoblots of β-ENaC, AMPK-α1, and β-actin are shown. (D) A549 cells were co-transfected with β-ENaC and HA-ubiquitin and were treated with 40 mmHg CO2 or 120 mmHg CO2 for 30 min at a pHe of 7.4 in the presence or absence of compound C (20 µM, 30 min pretreatment). Total ubiquitinated β-ENaC was detected as described above. Representative immunoblots of β-ENaC, p-AMPK-α1, and total AMPK-α1 are shown. (E) Cells were co-transfected with α- and β-ENaC and siRNA targeting AMPK-α1 or a scrambled siRNA and exposed to 40 mmHg CO2 or 120 mmHg CO2 for 30 min at a pHe of 7.4. Biotinylated ENaC proteins were detected by immunoblotting. Representative western blots of αand β-ENaC at the plasma membrane (PM) and total protein abundance of ENaC, p-AMPK-α1, and AMPK-α1 are shown. Bars present mean ± SEM (*n* = 3; \**p* < 0.05; \*\**p* < 0.01).

the trafficking of the channel rather than protein degradation. Furthermore, no significant changes in the ubiquitination status of α-ENaC have been detected upon hypercapnic treatment, highlighting and further confirming the central regulatory role of β-ENaC in the trafficking of the channel (52).

Our data demonstrate that elevated CO2 levels cause a rapid and time-dependent ERK1/2 activation followed by phosphorylation of β-ENaC at the Thr615 residue. Moreover, genetic inhibition of Nedd4-2, the E3 ubiquitin ligase that drives ubiquitination of the various ENaC subunits (49), by a specific siRNA reduced β-ENaC polyubiquitination and prevented the hypercapnia-induced redistribution of α- and β-ENaC from the PM to the intracellular store, indicating a central role for Nedd4-2 in ENaC ubiquitination and endocytosis in AEC exposed to hypercapnia. Indeed, ERK1/2 has previously been described as a negative regulator of ENaC. For example, Eaton et al. showed that protein kinase C-δ drives ERK activation leading to ENaC internalization (30). Another study established that the ERK-mediated ENaC downregulation is promoted by phosphorylation β- and γ-ENaC, resulting in enhancement of Nedd4-2/ENaC interaction and thus, decreased Na+ transport (31). Therefore, hypercapnia by enhancing ERK activity promotes phosphorylation of the ENaC β-subunit, which may increase the affinity of the E3 ubiquitin ligase to ENaC.

We have previously shown that JNK is also implicated in CO2 responses and that phosphorylation of the kinase is required for the CO2-induced inhibition of the Na,K-ATPase in the alveolar epithelium (7). The significance of JNK in cellular adaptation to stress has been shown by several studies (53). Of note, the possible role of JNK in modulating Nedd4-2 activity and ENaC current has been reported in polarized kidney epithelial cells (28). Remarkably, this study also showed that the Thr899 residue in the HECT (homologous to the E6-AP carboxyl terminus) domain of Nedd4-2 may be phosphorylated by JNK1, which was required for ubiquitination of α-ENaC (28). To assess the potential involvement of JNK-mediated Nedd4-2 phosphorylation in the hypercapnia-induced downregulation of ENaC, we mutated Thr899 to Ala to prevent phosphorylation of the E3 ligase at this residue. Importantly, this point mutation largely prevented the CO2-induced polyubiquitination of β-ENaC although activation of JNK was evident and stabilized α- and β-ENaC at the cell surface. Thus, our data together with the previously published literature suggest that phosphorylation of Nedd4-2 by JNK at the Thr899 residue is critical for the hypercapnia-induced ubiquitination of β–ENaC, which drives endocytosis of the ENaC complex from the PM in AEC.

We have previously shown that AMPK, a cellular metabolic sensor that inhibits several ion transporters including the cystic fibrosis transmembrane conductance regulator, Na,K-ATPase, and ENaC, is rapidly activated by hypercapnia (8, 29, 54). Regarding the regulation of ENaC, it has been shown that chemical stimulation of AMPK by 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside inhibited ENaC activity in lung epithelial cells (55). Moreover, enhanced abundance of ENaC channels at the cell surface was reported in the distal airways in AMPK-α1−/− mice (28). Interestingly, AMPK has also been reported to regulate Nedd4-2 activity (26, 29). In the current study, treatment of AEC with a specific siRNA against AMPK-α1 or an AMPK-α inhibitor, compound C markedly decreased CO2-induced β-ENaC polyubiquitination and endocytosis of α-, and β-ENaC, which is consistent with previous findings showing that in human embryonic kidney cells, AMPK activation promoted Nedd4-2/ENaC association (26). Furthermore, and in line with a previously published study (7), we also show that in the context of hypercapnia, AMPK is an upstream regulator of JNK. Thus, it is probable that the AMPK-regulated effects of CO2 on Nedd4-2 and ENaC are indirect and mediated by JNK. Moreover, although AMPK is an early element of the CO2-induced signaling pattern, its activation appears to be downstream of ERK upon hypercapnic exposure. This is of particular importance as ERK appears two have a dual role in the hypercapnia-induced downregulation of ENaC. On the one hand, it rapidly phosphorylates the β-subunit of the channel and by activating AMPK and JNK, it indirectly promotes phosphorylation of the E3 ligase Nedd4-2 as well. Of note, both of these phosphorylation events seem to be critically required for the CO2-induced ubiquitination and subsequent endocytosis of ENaC, probably by enhancing the association of the E3 ligase and the target molecule.

Our study has some clear limitations. Although we show a rapid activation of ERK and a subsequent phosphorylation of β-ENaC at the Thr615 residue, which is a known target of ERK, we have not investigated the potential rescue of β-ENaC ubiquitination or trafficking of the ENaC complex after preventing phosphorylation at this specific site. A mutation of this residue will be necessary to definitely prove that ERK-promoted phosphorylation of β-ENaC at this residue drives the downregulation of the channel upon hypercapnia. Moreover, the current study was performed exclusively in AECs and further *in vivo* investigations will be necessary to establish the role of the hypercapnia-induced signaling events identified in the current manuscript in ENaC-driven AFC and alveolar epithelial barrier dysfunction in an animal model of hypercapnic acute lung injury.

Taken together, our study shows for the first time that upon exposure to elevated CO2 levels, ENaC cell surface abundance is rapidly downregulated in a human AEC line by a specific, CO2-induced and ERK-, AMPK-, and JNK-mediated signaling pathway, which promotes phosphorylation of both β-ENaC and Nedd4-2, leading to ubiquitination of β-ENaC and subsequent internalization of the α/β-ENaC complex. This novel signaling pathway may contribute to the persistence of alveolar edema and thus, interfering with these molecular mechanisms may improve alveolar fluid balance and lead to better outcomes in patients with ARDS and hypercapnia.

# AUTHOR CONTRIBUTIONS

Conception or design of the work: PG and IV; acquisition, analysis, or interpretation of data: PG, BB, KM, SH, RM, WS, and IV; drafting the work: PG and IV; revising it critically for important intellectual content: PG, BB, KM, SH, RM, WS, and IV. All the authors approved the final version of the manuscript and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

# ACKNOWLEDGMENTS

The authors thank Mrs. Miriam Wessendorf (Universities of Giessen and Marburg Lung Center) for her excellent technical assistance.

#### FUNDING

This work was supported by grants from the Excellence Cluster "Cardio Pulmonary System" (ECCPS), the German Center for Lung Research (DZL), the Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz (LOEWE) of the

#### REFERENCES

Hessen State Ministry of Higher Education, Research and the Arts, and the Deutsche Forschungsgemeinschaft (Clinical Research Unit KFO309/1) (for KM, SH, RM, WS, and IV). IV was supported by a Clinical Scientist Career Program grant from the ECCPS.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://journal.frontiersin.org/article/10.3389/fimmu.2017.00591/ full#supplementary-material.

phosphorylation. *J Biol Chem* (2002) 277(16):13539–47. doi:10.1074/jbc. M111717200

signaling pathway. *J Biol Chem* (2005) 280(19):18579–89. doi:10.1074/jbc. M410561200

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Gwoździńska, Buchbinder, Mayer, Herold, Morty, Seeger and Vadász. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

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# TnF lectin-like Domain restores epithelial sodium channel Function in Frameshift Mutants associated with Pseudohypoaldosteronism Type 1B

*Anita Willam1,2\*, Mohammed Aufy 1 , Susan Tzotzos 2 , Dina El-Malazi1 , Franziska Poser 1 Alina Wagner 1 , Birgit Unterköfler 1 , Didja Gurmani 1 , David Martan1 , Shahid Muhammad Iqbal 1 , Bernhard Fischer 2 , Hendrik Fischer 2 , Helmut Pietschmann2 Istvan Czikora3 , Rudolf Lucas 3 , Rosa Lemmens-Gruber 1 and Waheed Shabbir 1,2*

*1Department of Pharmacology and Toxicology, University of Vienna, Vienna, Austria, 2APEPTICO GmbH, Vienna, Austria, 3 Vascular Biology Center, Medical College of Georgia, Augusta University, Augusta, GA, United States*

Previous *in vitro* studies have indicated that tumor necrosis factor (TNF) activates amiloride-sensitive epithelial sodium channel (ENaC) current through its lectin-like (TIP) domain, since cyclic peptides mimicking the TIP domain (e.g., solnatide), showed ENaCactivating properties. In the current study, the effects of TNF and solnatide on individual ENaC subunits or ENaC carrying mutated glycosylation sites in the α-ENaC subunit were compared, revealing a similar mode of action for TNF and solnatide and corroborating the previous assumption that the lectin-like domain of TNF is the relevant molecular structure for ENaC activation. Accordingly, TNF enhanced ENaC current by increasing open probability of the glycosylated channel, position N511 in the α-ENaC subunit being identified as the most important glycosylation site. TNF significantly increased Na+ current through ENaC comprising only the pore forming subunits α or δ, was less active in ENaC comprising only β-subunits, and showed no effect on ENaC comprising γ-subunits. TNF did not increase the membrane abundance of ENaC subunits to the extent observed with solnatide. Since the α-subunit is believed to play a prominent role in the ENaC current activating effect of TNF and TIP, we investigated whether TNF and solnatide can enhance αβγ-ENaC current in α-ENaC loss-of-function frameshift mutants. The efficacy of solnatide has been already proven in pathological conditions involving ENaC in phase II clinical trials. The frameshift mutations αI68fs, αT169fs, αP197fs, αE272fs, αF435fs, αR438fs, αY447fs, αR448fs, αS452fs, and αT482fs have been reported to cause pseudohypoaldosteronism type 1B (PHA1B), a rare, life-threatening, salt-wasting disease, which hitherto has been treated only symptomatically. In a heterologous expression system, all frameshift mutants showed significantly reduced amiloride-sensitive whole-cell current compared to wild type αβγ-ENaC, whereas membrane abundance varied between mutants. Solnatide restored function in α-ENaC frameshift mutants to current density levels of wild type ENaC or higher despite their lacking a binding site for solnatide, previously located to the region between TM2 and the C-terminus of

#### *Edited by:*

*Jixin Zhong, Case Western Reserve University, United States*

#### *Reviewed by:*

*Rui Li, Montreal Neurological Institute and Hospital, Canada Qingfeng Chen, University of Texas Southwestern Medical Center, United States Shanzhong Gong, University of Texas at Austin, United States*

#### *\*Correspondence:*

*Anita Willam<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology*

*Received: 27 February 2017 Accepted: 08 May 2017 Published: 29 May 2017*

#### *Citation:*

*Willam A, Aufy M, Tzotzos S, El-Malazi D, Poser F, Wagner A, Unterköfler B, Gurmani D, Martan D, Iqbal SM, Fischer B, Fischer H, Pietschmann H, Czikora I, Lucas R, Lemmens-Gruber R and Shabbir W (2017) TNF Lectin-Like Domain Restores Epithelial Sodium Channel Function in Frameshift Mutants Associated with Pseudohypoaldosteronism Type 1B. Front. Immunol. 8:601. doi: 10.3389/fimmu.2017.00601*

the α-subunit. TNF similarly restored current density to wild type levels in the mutant αR448fs. Activation of βγ-ENaC may contribute to this moderate current enhancement, but whatever the mechanism, experimental data indicate that solnatide could be a new strategy to treat PHA1B.

Keywords: lectin-like domain of tumor necrosis factor, TIP peptides, solnatide (AP301), amiloride-sensitive epithelial sodium channel, pseudohypoaldosteronism type 1B

#### INTRODUCTION

Tumor necrosis factor (TNF) is a mammalian inflammatory cytokine, which exerts a plethora of effects primarily aimed at defending the host against invading pathogens. Apart from mediating its activities through cross-linking with specific receptors on the surface of mammalian cells (1), TNF participates in innate immune functions through a lectin-like (TIP) domain, spatially distinct from the TNF-receptor binding site (2–4). The lectin-like domain of TNF recognizes and interacts with specific oligosaccharide moieties, in particular *N*,*N*′-diacetylchitobiose (5). TNF is crucially involved in the control of *Trypanosoma brucei brucei* and *T. cruzei* infections, through the trypanolytic effect triggered by interaction of its lectin-like domain with the N-linked *N*,*N*′-diacetylchitobiose core of the variant surface glycoproteins (VSG) of these organisms (1, 2, 6–14). Another effect of the TNF TIP domain observed in early work was the amiloride-sensitive increase in membrane conductance in microvascular endothelial cells (MVECs) (4) and alveolar epithelial cells (15), an effect which we now know is due to activation of the amiloride-sensitive epithelial sodium channel (ENaC) (16). The potential physiological role of the lectin-like domain of TNF in resolution of alveolar edema has been demonstrated in various rodent models of flooded lungs (15, 17, 18). Furthermore, transgenic mice expressing a mutated TNF lectin-like domain are more prone to develop lung edema than their wild-type (WT) counterparts when challenged with the bacterial toxin pneumolysin (PLY) (19).

The synthetic, cyclic, 17-residue peptide, solnatide, mimics the lectin-like domain (TIP) of human TNF (2). Like TNF, TIP peptide can influence regulation of alveolar fluid balance. Solnatide has been shown to activate fluid reabsorption in *in situ* and *in vivo* flooded rat lung models (18) and a mouse version the TIP peptide, mTIP, decreased pulmonary edema in isolated, endotoxin-injured rabbit lung (20). Moreover, solnatide, instilled intratracheally into rats prior to lung transplantation, significantly improved lung function, indicating its use as a potential therapy for ischemia reperfusion injury associated with lung transplantation (21). Inhalation of nebulized solnatide in a porcine bronchoalveolar lavage (BAL) model of acute lung injury (ALI) resulted in an increased PaO2/FiO2 ratio and reduced extravascular lung water index (EVLWI) (22). More recently, solnatide demonstrated profound therapeutic activity in a rat model of pulmonary edema induced by acute hypobaric hypoxia and exercise (23).