Time-dependent effect of clarithromycin on pro-inflammatory cytokines in CRS*

Background : The purpose of this study was to assess the time-effect of clarithromycin on the inflammatory response in chronic rhinosinusitis (CRS), to further explore the use of macrolides in cell culture as a model for CRS, and its action on the immune system. Methodology : The time effect of clarithromycin on several cytokines was examined for IL-1β, IL-4, IL-5, IL-8 and GM-CSF. Samples prior and post-incubation were assessed, as well as samples collected 24h following removal of clarithromycin to determine if any immunomodulatory effect persisted. Cytokines were quantified using ProcartaPlex TM assays. Results : Of the 5 cytokines assessed, only IL-1β and IL-8 production were significantly inhibited at 4h. Increased levels of IL-4 were observed at 72 hours of incubation and returned to near baseline levels after its removal. IL-8 showed the most time-dependent relationship with clarithromycin. No differences between the expression of IL-5 and GM-CSF were found. Conclusions : The present work suggests a specific and dose-dependent impact of clarithromycin on the inflammatory response in CRS. Moreover, the immunomodulatory effects of clarithromycin on the cytokines IL-4 and IL-8 varied depending on length of exposure to clarithromycin. Further studies to further establish the relationship between length of exposure and cytokine expression, and with additional “actors” in CRS pathophysiology should be considered. This may enable us in the future to determine appropriate duration of macrolide therapy in patients with CRS. in-vitro


Introduction
Despite its prevalence and impact on health, chronic rhinosinusitis aetiology remains incompletely understood (1) . Diagnosis is primarily based on clinical symptoms, with endoscopy or computerized tomography (CT) scans used to assist diagnosis by showing presence of mucosal changes and nasal polyps.
This allows a division into phenotypes -CRSwNP and CRSsNP -, which frequently guides diagnosis, prognosis, and treatment.
However, this classification does not reflect its heterogeneity in clinical presentation, pathology and therapeutic response (1,2) .
The pathophysiological mechanisms underlying CRS involve cellular infiltrate of neutrophils, macrophages and proinflamma-tory cytokines associated with helper T cell type 1, 2 and 17 (3) .
More, research has been taken to analyse the cytokine signatures associated with Th1, Th2 and Th17 inflammation to help us subtype according to endotype rather than phenotype (4) . A more robust expression of these cytokine markers could serve as an aid for guiding therapeutic decisions in the future.
Recently, it has been suggested that several macrolides, such as erythromycin, azithromycin and clarithromycin, are effective for the treatment of respiratory diseases including CRS. Their action consists of inhibiting or killing pathogens and down-regulate pro-inflammatory mechanisms (5,6) . While long-term low dose macrolide therapy has been considered an option in the ma-nagement of CRS, the evidence base for this remains uncertain and is the subject of an ongoing major trial (7) . The National Institute for Health and Care Excellence (NICE) has no clear guidance on dosage, duration of therapy or the patient groups most likely to benefit, based on an international consensus statement published in 2016 (8) . Systematic reviews published in recent years have concluded that macrolides may be effective in improving endoscopic and CT scores in CRS patients compared to baseline, but that effectiveness is likely to depend on appropriate patient selection (9)(10)(11) . The mechanism of their anti-inflammatory effect in CRS is an area of active research, with much of our understanding based on studies of relatively small sample sizes. A 2019 systematic review of 22 randomised controlled trials identified that, in CRS patients, macrolides may downregulate expression of pro-inflammatory cytokines IL-1β, IL-6, TNF-alpha and IL-8, amongst others (12) , and that a decrease in Th2 cytokines were reported more frequently than a decrease in Th1. There, authors recommended further research to explore the exact mechanisms underlying the immunomodulatory effects of macrolides to optimise usage and identify appropriate patient groups who may benefit.
Evidence examining the relationship between time dependent macrolide exposure or concentration dependent macrolide exposure and cytokine responses in CRS is extremely limited.
Two studies examining cytokine expression in mice following macrolide therapy suggested that different exposure times to macrolides may result in different changes in cytokine expression (13,14) . Duration of macrolide therapy may be a factor in responsiveness to macrolide therapy in CRS, but further studies are required to establish whether variation in expression of cytokines implicated in CRS occurs depending on duration of macrolide exposure.
While macrolides may be effective in CRS, lack of understanding and guidance on appropriate antibiotic usage has resulted in unnecessary antibiotic prescribing. This raises concerns as this treatment can have long-term consequences by promoting antimicrobial resistance to antibiotics, with one study demonstrating an increase in proportion of macrolide resistant streptococci following macrolide therapy (5,15) . This risk appears to be greater in long term therapy compared to short term therapy (16) . Antimicrobial resistance is considered a significant threat to patients' safety in Europe (17) and promotion of appropriate antibiotic usage is part of the UK's 5-year antimicrobial resistance strategy (18) . Other concerns with macrolide usage include the risk of adverse effects, such as Clostridium difficile colitis and effects on cardiac conduction (19) .
A better understanding of macrolides involved in CRS, such as their mechanisms of action and time dependent effects, could enable us to predict patient responses to macrolide therapy and enable a more personalised treatment (20,21) . For instance, it will help us understand whether high doses in the short-term are more beneficial than lower doses in the long-term (5,22) .
In this study, clarithromycin was selected rather than erythromycin, since two reviews have raised concerns about its cardiac toxicity, especially in patients presenting a long QT interval (23,24) .
Moreover, clarithromycin was selected rather than other macrolides due to its characteristic of therapeutic serum concentrations and high tissue concentrations (25) , and has been described as having an immunomodulatory effect on respiratory diseases and also CRS (26)(27)(28) , through the inhibition of neutrophilic inflammation and macrophage activation (12) .
In summary, clarithromycin was preferred since erythromycin has poor tolerability, previous randomized controlled trial revealed poor efficacy with azithromycin administration and roxithromycin has limited availability in the UK. Clarithromycin, on the other hand, is readily available in the UK with a reasonable sideeffect profile (25) and it is currently recommended as an option by EPOS and ENT-UK rhinosinusitis commissioning guidelines for the treatment of CRS patients in a secondary care setting (26)(27)(28) .
Therefore, the aim of this work was to explore and compare the anti-inflammatory effect of clarithromycin in vivo on different inflammatory mediators, with direct correlation on different phenotypes of CRS.

Ethics statement
All experiments were evaluated and approved by East Midlands -Leicester Central Research Ethics Committee. Due to methodological issues, the A549 cell line rather than patient samples were used to obtain the final results (see details below). All patient tissue samples initially used were disposed of according to principles of the Human Tissue Act 2004.

Cell culture
The cell line used in this work was A549 adenocarcinoma human alveolar basal epithelial cell line, which is used nowadays for both basic research and drug discovery. It was deemed as a good replacement for epithelial cells culture from CRS patients due to its similarity to the nasal mucosal tissue. The line was

Cell cultures
The trypan blue dye exclusion test was performed on the culture supernatants after incubation. There was no significant difference in cell viability between the treatment and control specimens (data not shown).

Effects of clarithromycin on in-vitro cytokine and chemokine expression
Clarithromycin in the dose used in these experiments did not exhibit significant effects on cell viability (results not shown).

Cell counts and viability
All cells freshly isolated, for tissue culture or cryopreservation were counted and assessed for viability, based on trypan blue dye exclusion. A small volume (10μl) of the sample was transferred to a well of a round bottom 96 well plate (Nunc) and   (29) .

Cell culture and clarithromycin treatment
To generate epithelial cell media, A549 cells, at an initial count of 0.05x106 cells, were grown in 24-well culture plates (Nunc) until confluent. Before treatment, supernatants were harvested from each well and stored at -80°C, to form our baseline samples.
After incubation with 0.064mg/ml clarithromycin solution for 4, low or below the detection threshold in each group and we were unable to analyse the potential immunomodulatory effect of clarithromycin.

Discussion
Our results demonstrate reduced expression of IL-1β after incubation with clarithromycin, although only significant at 4 hours.
In cell sample groups which had been exposed to clarithromycin

Interpretations
Macrolides are important therapeutic options in the treatment of many chronic inflammatory diseases due to their immunomodulatory effects, and therefore they can be clinically effective in CRS (12) . However, little is known about how macrolides affect specific pathophysiological features of CRS. In this study, the anti-inflammatory activity of clarithromycin and its immunomodulatory function was investigated, as well as, to assess the duration of its effects. Therefore, this macrolide was administered for 4, 12, 24, 48 and 72 hours, and mediators' levels were measured before and after incubation, and after a washout period of 24h.
The goal of this study was to evaluate the action of clarithromycin on the selected cytokine levels -GM-CSF, IL-1β, -4, -5 and -8. Previously, Courcey et al. (30) performed a similar experiment using nasal epithelial cells that were stimulated in order to assess cytokine concentrations. The study design is identical to the one adopted on this project, so their protocol was tested.
Unfortunately, we were unable to obtain primary sinonasal epithelial cells in culture and an immortalised respiratory cell line was privileged to carry out this work. Currently, there are no immortalized nasal cell lines from either "normal" or patients suffering from CRS. As such, we investigated the possibility of using currently available cell lines that could mimic the upper respiratory cells. Previous studies established primary nasal fibroblast cultures, while others used immortalized lower respiratory epithelial cell lines, such as A549 or BEAS-2B given the similarities between the upper and lower airways (31) . The A549 adenocarcinoma human alveolar basal epithelial cell line is used nowadays for both basic research and drug discovery, and was widely available at our laboratory. Given its similarity to the nasal mucosal tissue (31) , it was privileged to act as a replacement of epithelial cells culture from CRS patients.
Literature research was performed for this study to select the mediators of interest -IL-1β, -4, -5, -8 and GM-CSF. Recent studies have clearly demonstrated that in addition to antibacterial effects, macrolides may also have anti-inflammatory effects. Previous research has shown that reduction of IL-1β may be a potential mechanism of macrolides (32) . Our results also demonstrate reduced expression of IL-1β after incubation with clarithromycin, although only significant at 4 hours. IL-1β is associated with neutrophilic CRS and clarithromycin may impair production or secretion of these cytokines. This may result in reduced neutrophil accumulation in the sinus mucosa, thereby reducing the inflammation underlying CRS. This may be of particular use in Asian CRSwNP populations, with research suggesting a tendency towards neutrophilic inflammation in Chinese patients (33) . Multiple studies in Asian populations have described patients with neutrophilia as difficult to treat despite treatment with endoscopic sinus surgery and poor response to corticosteroid treatment compared to patients with predominantly eosinophilic inflammation (34)(35)(36) . Macrolide therapy may be an alternative or adjunctive treatment option in these patient groups.
In cell sample groups which had been exposed to clarithromycin Existing research suggests that macrolides may reduce levels of IL-4 (12) . While incubation with clarithromycin for 4 hours demonstrated a reduction in IL-4, this was not statistically significant, and incubation for other times demonstrated an increase in IL-4 following addition of clarithromycin, significant after incubation for 72 hours (p<0.01). However further research with larger sample sizes should be conducted to determine whether the trend observed at 24 and 48 hours is significant. As IL-4 has been associated with epithelial barrier dysfunction in CRS (37) , and epithelial barrier dysfunction has been implicated as a pathological mechanism in CRS, IL-4 is an appealing drug target. If future studies find that IL-4 expression is significantly increased Similarly, exposure to clarithromycin for 12 hours also reduced IL-8 levels, although not significantly. Concentrations of IL-8 were further reduced following removal of clarithromycin, and this was significant (p<0.01). This may suggest that incubation with clarithromycin for 12 hours is sufficient for the initial reduction to persist. After 24, 48 and 72 hours of exposure to clarithromycin, IL-8 levels appeared to increase, and removal reduced IL-8 concentrations close to baseline levels. The reduction following removal was significant at 24 hours, and the increase in IL-8 and reduction following removal was also significant at 48 and 72 hours (p<0.01). These results support Shinkai et al. 's research examining macrolide antibiotics in COPD (38) . They found that IL-8 levels decreased over 6 hours and then increased at 12-72 hours after exposure to clarithromycin and found similar results with azithromycin at 24 and 48 hours, although in our study incubation for 12 hours was associated with an initial reduction in IL-8. This could be due to a severe depletion of Il-4 and IL-8, which on its turn increases Th1/Th2 ratios, favouring the expression of these cytokines during a prolonged exposi-  (12) , and therefore, future studies examining IL-5 in this cell line may need to consider stimulation of the A549 cells with TNF-a or Interferon-γ (42) . Other cell lines might also prove to be more adequate in such conditions than the one used in this study.
Many of the samples tested for GM-CSF yielded undetectable results. Previous studies examining GM-CSF in lung cancer and inflammation in airway epithelium have detected little or no GM-CSF in resting A549 cells (43,44) . Future studies examining GM-CSF and time dependent clarithromycin exposure using A549 cells as a model for CRS will need to stimulate the cells with IL-1β and use more samples to provide meaningful results (45) .
While GM-CSF was detected in a small number of samples, these levels were low and differences between groups were not found to be statistically significant.
One previous study has considered the relationship between time dependent exposure to macrolides and GM-CSF levels while examining how macrolide antibiotics modulate ERK phosphorylation and cytokine production in patients with chronic obstructive pulmonary disease (COPD) (38) . These researchers found that clarithromycin increased GM-CSF at 48 hours. This is an unexpected result, given that other research has proposed that clarithromycin may reduce levels of GM-CSF (46) , suggesting time dependent exposure may have a role. Examining the expression of GM-CSF in response to different time lengths of clarithromycin exposure warrants further research. GM-CSF is proved to be elevated in asthma (12) . Given that CRS is thought to share some of the pathophysiological mechanisms of asthma in the upper airways and that GM-CSF is elevated during symptomatic exacerbations in patients with CRS (47) , more research based on the recommendations from this study may provide useful information which could be used to guide management.
Clarithromycin in the doses used in these experiments did not exhibit significant effects on cell viability. These findings outline a specific and dose-dependent impact of clarithromycin on the inflammatory response in CRS.

Generalisability
This study showed changes in the cytokine expression profile associated with exposure to clarithromycin in one particular cell line -A459. At the outset our underlying assumption was that different patients will have different patterns of cytokine expression and different changes in cytokine expression profile when exposed to clarithromycin. Further studies on samples of upper respiratory nasal mucosa from different individuals may produce a different pattern of cytokine change, so unnoticeable cytokines in our study should not automatically be excluded from further studies.
The results which have emerged, combined with suggestions from previous research examining macrolides in other airways diseases warrant further investigations into the time dependent effects of macrolide antibiotics in CRS. This is an area which has been little investigated but may enable development of appropriate guidance for macrolide prescribing in CRS patients.

Conclusions
This study demonstrates that the effect of clarithromycin exposure on the cytokines measured here varies over time. Several interesting patterns have emerged in this study. While some cytokines presented no real change, or no obvious pattern of change; others showed apparent trends which were not statistically significant. Lastly, others displayed significant changes.
Regarding the lasting effect of clarithromycin, we could note two different patterns: a reversal of the clarithromycin effect or a prolongation of the effect. This could indicate that macrolides may have a long-lasting effect on immune mediators beyond the time that therapy is completed, as well as showing a timedependent effect.
Determining appropriate duration and dose of macrolide therapy is essential to enable clinicians to balance risks with minimal treatment duration for persistent anti-inflammatory effect. As existing research proposes that mechanisms of actions of different macrolides may vary between each other (20) , studies examining other time dependent immunomodulatory effects on these cytokines with other macrolide antibiotics should also be conducted. This would explore whether the effects observed are exclusive to clarithromycin or also present in other macrolides.