Chronic respiratory diseases are some of the leading causes of illness and death globally [1
]. Chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and asthma can all be fatal diseases that affect lung function by increasing inflammation and decreasing mucus clearance. Inflammation is a natural biological response, and at a local site of damage or infection, it is an important mechanism for the immune system to clear harmful stimuli. However, excessive inflammation, like that which occurs during sepsis, can damage delicate organ structures, such as those found in the respiratory system. Prolonged inflammation and corresponding damage of critical organ systems can be lethal [2
Millions of people suffer globally from chronic respiratory diseases, including estimates of 65 million with COPD, 300 million with asthma, and 70,000 with CF [1
]. Approximately 3 million people die each year from COPD [1
]. Regrettably, it is estimated that 250,000 deaths are attributed to asthma each year, many of which could have been prevented with sufficient medical intervention [9
]. Life expectancy of a patient with CF was only 5 years in the 1960s, and while the quality and length of life have improved dramatically since then, CF remains a debilitating and ultimately fatal disease [6
]. As these diseases continue to have such an aggressive impact on human mortality, it is critical to progress research into the causes, prevention, and new treatments of these diseases.
Searching for novel treatments for these diseases requires exploring new drug candidates with bioactivity that can combat inflammation and mucus accumulation. The marine environment provides a diverse source of natural products with potential to treat human ailments. The marine dinoflagellate Karenia brevis
is one such source of bioactive compounds. This unicellular alga produces a number of bioactive compounds with therapeutic potential, including the neurotoxic brevetoxins (PbTxs), hemibrevetoxin B, brevisin, brevisamide, tamulamides A and B, and brevenal [10
]. Brevenal (Figure 1
) was the first natural nontoxic ligand described that displaces PbTxs from binding to voltage-sensitive sodium channels [16
]. In cell models, brevenal has been found to antagonize PbTx-induced elevations in intracellular calcium levels [17
] and reduce cell death in the presence of highly toxic concentrations of PbTxs [18
]. In vivo models have shown that brevenal can attenuate PbTx-induced bronchoconstriction and increase tracheal mucosal velocity in sheep [19
]. The ability of brevenal to increase tracheal mucosal velocity and mucocilliary clearance has led to the patenting of brevenal as a treatment for COPD, cystic fibrosis, and asthma, as well as attempts by Silurian Pharmaceuticals to begin Phase I clinical testing for the treatment of cystic fibrosis.
During the in vivo investigation of the antagonistic effects of brevenal against brevetoxins, it was discovered that brevenal alone could also attenuate the effects of other inflammatory agents. For example, brevenal has been shown to attenuate neutrophil elastase-induced bronchoconstriction and decrease neutrophil recruitment into the lung [20
]. These results indicate anti-inflammatory effects not typically seen with traditional lung clearing pharmaceuticals [20
]. Brevenal has also been found to attenuate PbTx-induced activity and sodium influx in, but not activation of, mast cells, a key immune cell that coordinates allergic responses [24
]. While brevenal shows promise as a potential therapeutic for lung disease, the mechanism by which brevenal can attenuate inflammation remains unclear.
Secondary to airway restriction, chronic respiratory diseases are often compounded by the effects of inflammation. An excessive inflammatory response can cause serious damage to lung tissues, decreasing quality of life and increasing debilitating symptoms associated with COPD, asthma, and CF. As such, ideal drug candidates for chronic respiratory disease will have a dual effect: Combat the root cause of disease (e.g., bronchoconstriction or mucus accumulation) and simultaneously reduce damaging inflammation. The purpose of our study was to examine the effects of brevenal on pro- and anti-inflammatory cytokine production from lung epithelial cells and immune cells, as an additive mechanism to its influence on airway restriction. Macrophages were used for this study because of their role in coordinating inflammatory responses, both in the lung and systemically. We further examined the effects of brevenal on phenotypic markers of macrophage activation to determine the mechanisms by which brevenal exerts an anti-inflammatory response, thereby demonstrating its utility for treating chronic respiratory diseases.
Chronic respiratory diseases are a leading cause of death globally, and include COPD, CF, and asthma [1
]. These diseases are exacerbated by inflammation, which can inhibit lung function and cause lung damage. Ideal novel treatments for chronic respiratory diseases dually address mucus clearance and inflammation. Brevenal, a natural product isolated from marine algae, has been shown to increase mucociliary clearance in animal models of chronic respiratory disease [19
]. Brevenal also demonstrated the potential for anti-inflammatory action not typically seen with traditional lung clearing pharmaceuticals [20
]. However, these previous studies were unable to determine the mechanism of action by which brevenal reduced lung inflammation.
The purpose of our study was to examine the anti-inflammatory effects of brevenal in the lung, and to determine potential mechanisms of action of this putative respiratory therapeutic. Native lung epithelial cells and cell lines (such as A549 cells) are known to produce cytokines and chemokines in response to inflammatory stimuli that recruit inflammatory cells to the site of infection or damage. IL-8 is a proinflammatory chemokine that attracts neutrophils to sites where an immune response has been initiated, where they then propagate the inflammatory reaction [27
]. Our results indicate that brevenal can reduce inflammatory cytokine secretion from lung epithelial cells after exposure to an inflammatory stimulus, thereby potentially reducing an exacerbation of inflammation. These results are similar to what has been found with corticosteroid treatment, where budesonide reduced IL-8 secretion from bronchial epithelial cells [28
Additionally, our studies examined brevenal’s anti-inflammatory action in macrophages. Macrophages respond to harmful stimuli and cytokines to propagate immune responses by amplifying chemical signals, making them pivotal in the initial immune response [29
]. TNFα is a proinflammatory cytokine that initiates the innate immune response by influencing other cells to produce more proinflammatory cytokines. Inflammatory diseases (e.g., sepsis and rheumatoid arthritis) are often characterized by excessive production of TNFα [30
]. In addition to TNFα, we measured macrophage production of IL-1β, a potent proinflammatory cytokine associated with immunopathologies [31
], IL-6, a pleiotropic cytokine with pro- and anti-inflammatory influences [32
], and IL-10, an anti-inflammatory cytokine that suppresses the immune response and prevents tissue damage [33
]. Our data indicate that brevenal acts as an anti-inflammatory agent due to its ability to reduce proinflammatory cytokine secretion and not affect production of pleiotropic or anti-inflammatory cytokines. One would not expect nor desire a potential lung therapeutic to have a severe effect on the ability of immune sentinel cells to respond to stimuli and produce cytokines. We suggest that brevenal would have a modest but significant physiological effect in only partially reducing inflammatory responses. Key to this suggestion is that brevenal affects the most potent inflammatory cytokines (i.e., IL-1β and TNFα), which are particularly problematic in inflammatory diseases [32
]. However, brevenal does not unilaterally inhibit secretion of all cytokines. IL-6 is considered pleiotropic and immunomodulatory because it can have multiple effects, including stimulating the immune response to trauma and activating lymphocytes [32
]. Indeed, other anti-inflammatory drugs, such as corticosteroids, have been found to inhibit an entire suite of cytokines, from the initial phase inflammatory mediators IL-1 β and TNFα, to immunomodulatory interleukins (including IL-6), to even the anti-inflammatory IL-10 [34
]. Our study found that brevenal reduced inflammatory cytokine secretion by 20%–35%. The corticosteroid budesonide has been found to reduce TNFα secretion from macrophages in vitro by approximately 40% at a similar dose of 1 nM [35
]. Additionally, it has been found that serum levels of IL-1β and TNFα are only 45%–50% lower in healthy patients than those hospitalized with sepsis [36
]. As such, we suggest that our results would translate in vivo to a measured yet significant physiological effect to reduce damaging inflammation in lung disease, without compromising the necessary healing response of the immune system. However, further studies would need to be completed to fully demonstrate such an effect.
Our results also indicate a biphasic response to brevenal in cytokine secretion, where lower doses are more effective than higher doses. While the mechanisms behind this effect are not completely clear at this time, such biphasic responses are not uncommon in pharmaceutical agents. Indeed, marine-derived compounds have previously been shown to have biphasic responses, including other compounds from the same algal source as brevenal [37
]. Further studies would be needed to determine the exact reason for such biphasic effects, though it could be due to a development of tolerance by the cells at higher doses of brevenal. Regardless, brevenal is primarily advocated as therapeutic for lung diseases in which mucocilliary clearance is the target. Any anti-inflammatory effects would be secondary and synergistic to the primary effect of increasing tracheal mucosal velocity and mucocilliary clearance. Indeed, it has been suggested that general anti-inflammatory effects are a leading reason why brevenal is believed to be superior over current CF treatments [22
]. As a treatment for lung clearance, however, brevenal has been stated to be ineffective at doses below 0.1 nM [39
], making doses below this less physiologically relevant for either use.
Brevenal’s ability to reduce proinflammatory cytokine secretion led us to investigate its effect on macrophage cell surface receptor expression as a possible mechanism for these observations. First, we examined TLR4 expression because it binds LPS and initiates the innate immune response [40
]. However, our results do not indicate that brevenal alters TLR4 expression or the ability of macrophages to respond to bacterial infection via the expression of this receptor. As brevenal therefore must act through another mechanism, we examined the ability of brevenal to alter activation states of macrophages. The M1 phenotype is the classical activation pathway associated with response to infection and production of proinflammatory cytokines [41
]. Here, we show that brevenal treatment reduced the M1 phenotype marker (CD86) in macrophages, demonstrating a mechanism for suppression of inflammatory activation. The M2 phenotype is indicative of the alternative activation pathway associated with wound and tissue healing [41
]. Brevenal reduced the M2 phenotype marker (CD206) and also reduced cell size and complexity of macrophages.
Interestingly, our results only extend to the single cell population of RAW 264.7 cells, as brevenal did not have an effect on activation states or size and complexity in aggregated cells. The mechanisms behind this finding are not immediately clear, but aggregation alone could certainly have confounding effects. It could be that brevenal cannot overrule an already activated macrophage, and therefore has greater utility as a prophylactic than as a rescue therapeutic for lung inflammation. If patients are already using brevenal to increase mucocilliary clearance, then it could have an added synergistic effect of a modest reduction in pulmonary inflammation. Such an effect would be ideal to maintain the ability of cells to respond to infection. Additionally, we found that brevenal reduced the percentage of cells in the aggregated population at low doses. As such, brevenal could have an effect on the ability of cells to aggregate, but further studies would be needed to confirm this hypothesis.
Overall, our results suggest that brevenal treatment elicits an anti-inflammatory response in epithelial cells and macrophages in addition to improving mucociliary clearance shown in previous studies. The mechanism of its immunomodulatory effect is through decreased activation of macrophages in general, putting them in a so-called quiet state, rather than shifting phenotype. This mechanism of action is ideal for reducing lung inflammation, especially in patients with chronic respiratory diseases where inflammation can be severely damaging and potentially lethal. These findings support further investigation on the utility of brevenal as a dual-action therapeutic for life threatening pulmonary diseases, including COPD, asthma, and CF.
4. Materials and Methods
4.1. Cell Culture
Human alveolar epithelial cells (A549, CCL-185), murine alveolar macrophage cells (MH-S, CRL-2019), and murine macrophage-like cells (RAW 264.7, CRL-2278) were utilized in this study (ATCC, Manassas, VA, USA). A549 cells were cultured in F12K medium, MH-S in RPMI 1640 medium, and RAW 264.7 in DMEM with Glutamax (Thermo Fisher Scientific, Waltham, MA, USA). All media contained 10% FBS and penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA).
4.2. Brevenal Treatment
Cells were treated with brevenal acquired from Dr. Daniel Baden, extracted from K. brevis
cultures as previously described [16
]. A certificate of purity was obtained, demonstrating purity of greater than 95%, as determined by high pressure liquid chromatography analysis (single peak elution from C-18 reverse phase column, UV detection at 215 nm and 290 nm, mobile phase 89% methanol and 11% water). Doses of treatment ranged from 0.1 pM to 100 nM. The choice of doses was based on toxicity studies and previous studies and efficacy of brevenal as a treatment for increasing tracheal mucosal velocity and mucocilliary clearance, as any anti-inflammatory effect would be additive to its use as a CF therapy. Previous studies found efficacy of brevenal from 1 ng/mL (1.52 nM equivalent) to 50 µg/mL (76.22 µM equivalent) [20
]. Additionally, brevenal has been found to be ineffective below 1 ng/mL [39
]. Previous studies with corticosteroids have used similar ranges of 0.1 nM to 100 nM [28
4.3. Cytotoxicity Assays
Cytotoxicity assays were conducted for each cell line as previously described [38
]. Briefly, cells were seeded into 96-well plates, incubated at 37 °C for 24 h, and treated with brevenal, positive control (PbTx-2), or negative control of absolute ethanol (Fisher Scientific, Waltham, MA, USA). Brevenal and PbTx-2 were extracted from K. brevis
cultures grown by colleagues at UNCW as described previously [16
]. Cells were treated with a nuclear stain, Hoechst 33342 (Invitrogen Life Technologies, Waltham, MA, USA) for 24 h. Fluorescence was measured using the DAPI filter on an Image Xpress Micro (Molecular Devices, LLC; San Jose, CA, USA). Four images per well were acquired and analyzed with MetaXpress Image Acquisition and Analysis v220.127.116.11 software (Molecular Devices, LLC; San Jose, CA, USA).
4.4. Quantification of Pro- and Anti-Inflammatory Cytokines
A549, MH-S, and RAW 264.7 cells were seeded in 6-well plates, treated with brevenal [0-100 nM], and incubated at 37 °C for 24 h before stimulation with 400 ng/mL lipopolysaccharide (LPS) (Sigma-Aldrich; St. Louis, MO, USA). Supernatant was collected and stored at −20 °C until enzyme linked immunosorbent assays (ELISAs) were performed to measure cytokine production, using methods described previously [44
]. For murine lines (RAW 264.7 and MH-S), IL-1β, IL-10, and TNFα DuoSet®
ELISA kits (R&D Systems, Inc.; Minneapolis, MN, USA) were used to detect cytokines production with brevenal and LPS treatment. Antibodies were used for the detection of murine IL-6 (BD PharmingenTM
, San Diego, CA, USA). For human A549 cells, human IL-8 and TNFα, DuoSet®
ELISA kits (R&D Systems, Inc.; Minneapolis, MN, USA) were used. Absorbance was read at 450 nm using a FlexStation III plate reader (Molecular Devices, LLC; San Jose, CA, USA).
4.5. Flow Cytometry Assessment for Toll-Like Receptor 4 (TLR4), Mannose Receptor (CD206), and CD86 Receptor Expression
RAW 264.7 macrophages were seeded in 6-well plates and incubated at 37 °C until cells adhered and grew to confluence before being treated with ethanol (negative control), brevenal, or brevenal and LPS. Antibody staining and flow cytometry experiments were initiated following a 24 h incubation with treatment at 37 °C. Cells were harvested, centrifuged, and media were removed. The cells were stained on ice for 1 hr with 2 μg/mL (TLR4) or 4 μg/mL (CD206 or CD86) fluorescent antibody solution. Unbound antibody was removed and cells were resuspended in cold PBS prior to analysis on a BD FACSCelestaTM flow cytometer (BD Biosciences; San Jose, CA, USA).
Expression of Toll-like receptor 4 (TLR4 or CD284/MD-2) was measured using phycoerythrin (PE)-conjugated rat anti-mouse antibodies directed against CD284/MD-2 (MTS510; BD Pharmingen™, San Diego, CA, USA). Expression of mannose receptors (CD206) was measured using Alexa Fluor® 488-conjugated rabbit anti-mouse antibodies directed against CD206 (clone EPR6828(B); abcam, Cambridge, MA, USA). Expression of CD86 receptors was measured using allophycocyanin (APC)-conjugated rat anti-mouse antibodies directed against CD86 (clone GL-1; abcam, Cambridge, MA, USA).
4.6. Statistical Analyses
Cell viability was determined by image analysis of Hoechst 33342 nuclear dye. Cell counts were averaged from four images per well. Cytotoxicity was calculated as the percentage of viable cells in treatment wells compared to control wells. Cytotoxicity calculations from 96-well images were automated by SAS v9.1.3 software (SAS Institute Inc., Cary, NC, USA) and non-linear regression curve-fit was performed in GraphPad Prism v4.03 (GraphPad Software, San Diego, CA, USA). Data points are averages of three replicate 96-well plate experiments.
Cytokine concentration was measured by absorbance values fit to a standard curve. IL-8 was quantified in pg/mL and IL-1β, TNFα, IL-6, and IL-10 were quantified in ng/mL. Values for each treatment were averaged and standard deviation and standard error were calculated. One-way ANOVAs were performed using JMP Pro v14.0 with post hoc analysis to determine statistical significance (p < 0.05) (SAS Institute Inc., Cary, NC, USA).
For flow cytometry experiments, the mean values for fluorescence intensity and/or forward scatter and side scatter were recorded for each sample and averages were calculated within treatments. Standard deviation and standard error were calculated, and one-way ANOVAs were performed using JMP Pro v14.0 with post hoc analysis, including Tukey’s all pairs tests, to determine statistical significance (p < 0.05) (SAS Institute Inc., Cary, NC, USA).