key: cord-0954495-31zlp1fm authors: Pollock, Jennifer; Chalmers, James D. title: The Immunomodulatory Effects of Macrolide Antibiotics in Respiratory Disease date: 2021-11-03 journal: Pulm Pharmacol Ther DOI: 10.1016/j.pupt.2021.102095 sha: 7144a9aa32522933b297a6cc194b22746a7a8c87 doc_id: 954495 cord_uid: 31zlp1fm Macrolide antibiotics are well known for their antibacterial properties, but extensive research in the context of inflammatory lung disease has revealed that they also have powerful immunomodulatory properties. It has been demonstrated that these drugs are therapeutically beneficial in various lung diseases, with evidence they significantly reduce exacerbations in patients with COPD, asthma, bronchiectasis and cystic fibrosis. The efficacy demonstrated in patients infected with macrolide tolerant organisms such as Pseudomonas aeruginosa supports the concept that their efficacy is at least partly related to immunomodulatory rather than antibacterial effects. Inconsistent data and an incomplete understanding of their mechanisms of action hampers the use of macrolide antibiotics as immunomodulatory therapies. Macrolides recently demonstrated no clinically relevant immunomodulatory effects in the context of COVID-19 infection. This review provides an overview of macrolide antibiotics and discusses their immunomodulatory effects and mechanisms of action in the context of inflammatory lung disease. Macrolide antibiotics are a group of natural products produced by the genus Streptomyces 1 . They contain a macrocyclic lactone ring and are classified as 14-, 15-or 16-membered based on the number of carbon atoms within this structure 1 (Table 1) . Macrolides are bacteriostatic, predominantly against Gram-positive bacteria, as they competitively bind the bacterial 50S ribosomal subunit thus reducing protein synthesis and preventing replication 2 . Erythromycin (ERY) 14 The immunomodulatory properties of macrolides were first described in 1987 by Kudoh and colleagues in a study of diffuse panbronchiolitis (DPB) 7 , a rare, severe and progressive inflammatory lung disease driving irreversible lung damage 8 (Table 2) . They found that long-term daily treatment with 400-600mg of ERY suppressed DPB symptoms and increased the patients life expectancy. Indeed, it is estimated that the 10-year mortality rate of DPB reduced from 90% to 10% after ERY became standard therapy. It was initially believed this was simply due to the antibiotic activity of the macrolides, but this was challenged by the observation that DPB patients are typically infected with the macrolide-resistant Gram-negative pathogen Pseudomonas aeruginosa, and that patients experienced clinical benefits despite serum levels of ERY being substantially below the antibacterial threshold 9 . This unanticipated discovery led to the theory that low-dose macrolide therapy might have immunomodulatory properties beyond its antibacterial actions. Since then, clinical and experimental research has aimed to decipher these effects and reveal the potential mechanisms of action. However, there is much controversy regarding these effects and the underlying mechanisms have yet to be completely defined. J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f It is widely reported that long-term treatment with macrolide antibiotics, particularly 14-and 15membered macrolides, improve clinical outcomes in several respiratory diseases (Table 2) including COPD, BE and CF which are primarily regarded as neutrophil-driven disorders. There are some apparently contradictory results between clinical trials with some trials reporting improvements in symptoms, lung function and QoL, with others reporting no clinical benefit in these areas (Table 2 ). However, differences in dosing and/or duration of treatment, as well as the use of different macrolides between studies, could explain these discrepancies. These trials, however, all claim to have used doses below the reported antibacterial threshold ( Table 2 ). The magnitude of effect in these studies is remarkably similar (30-60% reduction in exacerbation frequency) despite diversity in pathophysiology between these conditions, suggesting a degree of shared biology. The results of randomized trials in these diseases have been extensively reviewed elsewhere and so are not discussed here in detail 9, 30 . Nevertheless, several aspects of these trials support the view that macrolides have effects beyond traditional antimicrobial effects. In bronchiectasis, an individual patient data meta-analysis of 3 randomized trials showed an overall reduction in exacerbations of 51% along with improvements in symptoms. In responder analysis, the group of patients with the greatest response were patients chronically infected with Pseudomonas aeruginosa (rate ratio 0·36 (0·18-0·72)), an organism that is not traditionally considered susceptible to macrolide antibiotics 28 . Similarly, in CF, randomized trials of macrolides in patients chronically infected with P. aeruginosa were clearly positive with improvements in FEV1 and prolonged time to first exacerbation (0.65; 95% CI, 0.44-0.95; P =.03) 18 while no improvements in lung function in patients without P. aeruginosa were observed, although exacerbations were still reduced 19 . In asthma, a disease not typically associated with chronic bacterial infection, the efficacy of AZM in reducing asthma exacerbations further supports an immunomodulatory effect. In the AMAZES trial of 420 patients with asthma 10 , exacerbations were reduced by 41% (0·59 [95% CI 0·47-0·74]) overall with similar results between eosinophilic and non-eosinophilic asthma (eosinophilic asthma rate ratio (0·66 (0·47-0·93)) vs non-eosinophilic (0·52 (0·29-0·94))). Together, these data support the idea that macrolides are having effects greater than would be expected from bacterial J o u r n a l P r e -p r o o f Clinical studies investigating the long-term effects of macrolides in chronic lung diseases have reported adverse events including hearing impairment and gastrointestinal complications. Furthermore, while rare, cardiotoxicity has been associated with macrolide therapy 9 . Macrolides can prolong the cardiac QT interval and inhibit metabolism of proarrhythmogenic drugs, leading to syncope and sudden death. Prolongation of the QT interval was reported in a number of COVID-19 trials of AZM and led to participants being withdrawn from the study 41, 42 . Extra care must therefore be taken when prescribing macrolides, and a baseline electrocardiogram and strict surveillance for drug-drug interactions has been recommended to prevent severe cardiac events 46 . Another major concern is the rising prevalence of antimicrobial resistance (AMR). A recent metaanalysis shows significant association between long-term macrolide therapy and AMR 47 . Similarly, a retrospective study reported AMR in pneumococcal species in CF patients 4 years after macrolide treatment 48 . Therefore, a major question should be whether long-term low-dose macrolide therapy risks increasing antibiotic resistance long term. In summary from a clinical perspective, it is perceived that macrolides have therapeutic activity extending beyond their role as antibiotics. By understanding the basis for this unexpected finding, it might be possible to improve or enhance the non-antibiotic functions of macrolides, apply them to other inflammatory diseases, avoid side effects, and limit AMR. J o u r n a l P r e -p r o o f Clinical studies report that macrolides reduce immune cell infiltration into the lungs of asthmatics and BE patients 49, 50 . Reduced lung leukocyte counts have also been reported in murine models of P. aeruginosa endobronchial infection and pulmonary fibrosis following administration of AZM and 14-membered macrolides, respectively, compared with untreated controls 51, 52 . It is suggested that macrolides attenuate leukocyte migration into the lung by reducing chemokine and adhesion molecule production by airway epithelial cells. It is unclear why this does not result in similar harmful effects as CXCR2 or potent leukotriene B4 antagonism, but may be because macrolides do not entirely inhibit neutrophil migration into the lung, or that macrolides have less of a direct impact on neutrophils and alter migration of other leukocytes also. by ERY treatment, likely because of reduced adhesion molecule expression and chemokine secretion by cultured epithelial cells 53 . ERY blocked the release of key neutrophil chemoattractants, CXCL8 and IL-6 53 . Similarly, CAM decreased CXCL8 and IL-6 secretion by a human epithelial cell line in vitro 54 . Clinical trials report decreased levels of CXCL8 in human airways in response to macrolide treatment 49, 50 , and murine models of sepsis treated with macrolides show decreased inflammatory cytokine levels, including IL-6, in the airways and blood 55 . Additionally, AZM, CAM and ROX were reported to inhibit in vitro spontaneous production of soluble mediators including CXCL8 and IL-6 in COPD sputum samples 56 . In this study, ERY was described as "very weakly active" and no statistically significant changes in inflammatory molecule secretion were observed. It has also been reported that AZM does not reduce expression of CXCL1 and CXCL2, the murine chemokines involved in neutrophil chemotaxis 57 , although it is important to note that this study used cultured epithelial cells from wildtype and CF murine models. Thus, although there are some inconsistencies between studies and variation between macrolides, it appears that macrolides can, in some circumstances, reduce chemoattractant production. Macrolides are also reported to influence integrin expression. ERY inhibited the release of soluble ICAM-1, an integrin adhesion molecule, from human bronchial epithelial cells in vitro 53 One study found that macrolides did not alter the induction of other adhesion molecule genes, such as selectins, in mice 52 , despite these genes also being regulated by Nuclear Factor kappa-lightchain-enhancer of activated B cells (NF-κB) similar to integrins 60 . The reasons for these apparent discrepancies are not yet clear but may be due to these molecules not being key to transepithelial migration 61 . J o u r n a l P r e -p r o o f Multiple potential effects of macrolide antibiotics on macrophage function have been described (summarised in Figure 1 ). Prolonged neutrophil lifespan caused by delayed apoptosis is thought to be prominent in many chronic diseases 79, 80 . It is thought that macrolides shorten neutrophil lifespan by inducing apoptosis, which may be one possible explanation for the reduced neutrophil numbers seen in clinical trials of macrolides 49, 50 and the observed therapeutic benefits. Initial in vitro data showed that 14-and 15-membered macrolides decrease neutrophil survival and increase apoptosis 69 . A variety of different techniques were used in this study to confirm these results, including western blot, transmission electron microscopy and cell viability assays, highlighting a compelling case for macrolide-induced neutrophil apoptosis. Moreover, others report increased neutrophil apoptosis in healthy human volunteers following three-day treatment with AZM 73 . This study used microscopy to identify apoptosis-associated morphological changes in neutrophils and revealed prolonged effects of AZM, with increased levels of apoptotic cells being detected 28 days after the last administration of the drug. In addition, increased apoptosis of isolated blood neutrophils has been reported in calves, pigs and mice following macrolide treatment 81, 82, 83 . These studies strongly support the idea that macrolides induce neutrophil apoptosis. Importantly, the benefits of neutrophil apoptosis may go deeper than simply reducing neutrophil lifespan. Promoting neutrophil apoptosis reduces the likelihood of cells undergoing necrosis and releasing inflammatory mediators into the local lung environment. In turn, enhanced neutrophil apoptosis alongside the macrolide-induced enhanced efferocytic capacity of macrophages (Section 3.1.2) may contribute to the beneficial effects of macrolides, given that efferocytosis promotes an anti-inflammatory environment 84 . J o u r n a l P r e -p r o o f Macrolides are reported to modulate NETosis, the process where web-like structures composed of chromatin, histones and granule proteins are released to entrap bacteria. NET release can cause significant tissue damage in lung disease and is therefore being investigated as a potential therapeutic target 85 . Bystrzycka and colleagues (2017) demonstrated that AZM can suppress human neutrophil production of NETs induced by phorbol 12-myristate 13-acetate (PMA) 74 . Moreover, ERY suppressed NET release from human and murine neutrophils exposed in vitro to cigarette smoke, a known trigger of NETosis and an inflammatory stimulus in COPD, and reduced the number of NETs in the bronchoalveolar fluid of cigarette smoke-exposed mice 77 . In addition, a recent observational, multicohort study investigating the role of NETs in BE disease severity, and the potential of macrolide antibiotics to reduce NETs, showed that long-term low-dose AZM therapy significantly reduced NETs in sputum of both bronchiectasis and asthmatic patients, highlighting macrolides as a potential therapy for these diseases 86 . Over the course of one year, UK-based BE patients with active Pseudomonas infection were treated with 250mg of AZM thrice weekly, and asthmatic patients enrolled in the AMAZES study 10 were treated with 500mg AZM thrice weekly. Analysis of sputum samples obtained at baseline and following therapy showed that AZM significantly reduced NET concentration compared with either a matched cohort not receiving macrolide therapy or those receiving placebo. Of note, in comparison to asthmatics with neutrophil dominant inflammation, those with eosinophil-dominant inflammation (characterised by >3% sputum eosinophils) saw no significant reduction in NET concentration following macrolide therapy, highlighting the overall effect seen was driven by a marked reduction in NET concentration in neutrophilic disease patients. Some studies, however, report conflicting findings regarding the effect of macrolides on NETs 75, 76 . AZM and CAM alone were reported to induce NET formation in vitro 75 . This study also found that neutrophils from patients with Helicobacter Differences in study design, including neutrophil isolation methods and NETosis detection, in addition to differences in disease plus the concentrations/antibiotics used could all account for differences in results. Furthermore, NETosis is a heterogeneous process and macrolides may differentially affect these distinct NETosis pathways. Therefore, understanding the exact role macrolides play is difficult, and more research is needed to decipher exactly how macrolides influence NETosis. The oxidative burst defines intracellular ROS production and is vital for efficient killing of ingested pathogens. Early in vitro evidence suggested that macrolides might impair the oxidative burst 78 . However, out of the macrolides tested, including ROX, AZM, ERY, SPM, JM and OLE, only ROX had this activity. Recent data using human neutrophils reported macrolides alone did not affect ROS production in vitro, but cells treated with the highest concentration of AZM did significantly inhibit the production of ROS 74 . Other studies report macrolides having no effect on neutrophil ROS 87 or having differential effects between different stimuli ex vivo 73 Macrolides seemingly polarise DCs to a tolerogenic phenotype but there are conflicting reports. Three papers have reported that macrolides shift human and murine DCs towards a tolerogenic phenotype in vitro 89 J o u r n a l P r e -p r o o f Macrolide-treated mBMDCs and hMDCs are reported to have enhanced IL-10 and decreased inflammatory cytokine expression including IL-6 and IL-12, providing further evidence that macrolides drive a tolerogenic DC phenotype 89, 90, 91 . In one study, AZM and CAM both significantly increased IL-10 production from mBMDCs but only CAM significantly reduced inflammatory IL-6 production 92 . Another study reported that AZM treatment decreased levels of both pro-inflammatory and anti-inflammatory cytokines 91 . Differences in experimental design could again account for these discrepancies but collectively data suggest macrolides increase antiinflammatory cytokine production in vitro, suggesting they induce a tolerogenic DC phenotype. However, their impact on primary DCs in vivo has yet to be investigated. J o u r n a l P r e -p r o o f Macrolides reportedly modulate T-cell function both directly and indirectly (Figure 3 ). Research relating to macrolides and B-cell function is lacking, with the exception of a preliminary human study indicating that in vivo antibody production was unaffected by macrolides 93 . Since it was noted that macrolides reduce lymphocyte numbers in the lung of DPB patients in vivo 95 , much research has examined if macrolides increase T-cell apoptosis. Macrolides increase apoptosis of activated T-cells and a Jurkat T-cell line in vitro 96, 97 , but this only occurred at high macrolide concentrations, i.e. ≥100ug/ml. It is possible, given that macrolides accumulate within cells over time, that these immunomodulatory effects may only be seen at high concentrations. However, it is reported that these concentrations are well above those found in human tissues 98 . Other in vitro studies similarly found lower doses of AZM and CAM did not induce T-cell apoptosis 91, 98 . Also, while some studies looked at lymphocytes as a single population (and showed macrolides increased apoptosis) 96 , others looked specifically at CD4+ T cells and found no effect of macrolides on apoptosis 91, 97, 98 . Therefore, low-dose macrolide therapy might increase apoptosis of specific T-cell subsets, highlighted by decreased CD8+ T-cells numbers and unchanged CD4+ T-cell numbers in macrolide-treated patients 95 . J o u r n a l P r e -p r o o f In vitro studies found human CD4+ T-cell proliferation was significantly inhibited by low-dose AZM therapy 91, 94 . However, some report that macrolide-induced inhibition of T-cell proliferative responses was only apparent at high macrolide concentrations 91, 97, 98 , similar to effects seen for apoptosis. This suggests the decrease in T-cell numbers seen in macrolide-treated patients may be a consequence of the reduction in pro-inflammatory cytokines involved in recruitment and proliferation, as previously discussed, rather than a direct effect of macrolides on T-cell proliferation and/or apoptosis. Lastly, research suggests macrolides directly suppress T-cell cytokine production. AZM decreased IL-17 production by human and murine Th17 cells in a dose-dependent manner highlighting a direct inhibitory effect of macrolides on T-cell cytokine production 97, 98, 99 . This is interesting given that Th17 responses are particularly important in providing protection from bacterial infections, a common feature of many inflammatory lung diseases, but also contributes to clinical exacerbations. Therefore, reduced IL-17 may, on the one hand, contribute to chronic bacterial infections by hampering vital immune responses but on the other, and in line with clinical data (Table 2) , may benefit the patient by reducing exacerbations. Therefore, while macrolides likely affect aspects of adaptive immunity, open questions remain. While evidence for macrolides inducing tolerogenic DCs seems largely consistent, inconclusive and conflicting studies on T-cell function highlight a need for further research. J o u r n a l P r e -p r o o f likely have a different mechanism of mTOR modulation than Rapamycin, possibly by directly binding mTOR without the need for a co-factor. Phosphorylation by Akt can stimulate or inhibit different target proteins. Therefore, macrolideinduced immunomodulatory effects may be the result of differential effects on different proteins in the pathway i.e. Akt activation may cause some proteins to be upregulated and others to be downregulated to ultimately give rise to the effects seen. J o u r n a l P r e -p r o o f 5.2 NF-κB and AP-1 Initial research found that various 14-membered macrolides inhibit NF-κB activation in bronchial epithelial cells and peripheral blood mononuclear cells 100, 101, 102 . As NF-κB governs chemokine and cytokine expression, it was the proposed mechanism for macrolide-induced cytokine attenuation and further hinted that inhibition of these transcription factors may cause other antiinflammatory effects. Macrolide treatment was found to specifically affect inhibitor of nuclear factor kappa B (IκB) proteins 81,103 ( Figure 4 ). Using human tracheal cells, AZM treatment inhibited IκB-α degradation 103 . Likewise, TUL significantly decreased phosphorylated IκB-α levels in LPS-stimulated bovine neutrophils, therefore decreasing IκB-α degradation 81 . This suggests macrolides may inhibit IKK, the enzyme responsible for the phosphorylation and breakdown of IκB-α ( Figure 4) . Therefore, it is likely macrolides have inhibitory effects on NF-κB. J o u r n a l P r e -p r o o f In summary, macrolides possess immunomodulatory properties that likely contribute to their observed efficacy in the treatment of inflammatory respiratory diseases. Their efficacy in clinical scenarios where antibiotic effects are less likely, such as eosinophilic asthma, BE and CF with Pseudomonas, further hint that immunomodulatory effects are a key mechanism behind their therapeutic action. However, much controversy exists surrounding a definitive mechanism of action for these drugs and whether antimicrobial effects are possibly a significant driver of the clinical benefits of macrolide therapy. Including the ability of macrolides to accumulate within tissues to concentrations above the antibacterial threshold, the intimate relationship between infection and host immune response alludes to the idea that targeting infection through antibacterial mechanisms may inadvertently reduce the inflammatory processes implicated in disease, and those supposedly dampened by macrolides. Multiple other findings further hint at the potential importance of antimicrobial effects. One of which is the discovery that macrolideresistant pathogens, namely Pseudomonas, may be macrolide-sensitive in the context of the lung. This was highlighted by evidence showing that P. aeruginosa harbours increased susceptibility to macrolides when cultured in bronchoalveolar lavage fluid compared to standard cell culture media 105 , findings potentially more representative of the in vivo effects occurring in the macrolidetreated lung. Also, evidence highlights Pseudomonas isolates from CF patients acquire resistance to macrolides 106 , a process that would not occur in naturally macrolide-resistant organisms. This data, alongside observations that chronically infected Pseudomonas patients often show greater clinical benefit when treated with macrolides 18 (data previously overviewed in Section 1.3), highlights a compelling case for additional antibiotic effects. While outwith the scope of this review, macrolides reportedly possess additional antiviral effects 107 and these antiviral effects/improvements in host viral defense may similarly contribute to the therapeutic effects of macrolides given that a common clinical outcome of long-term macrolide therapy is reduced exacerbations, which are often of viral origin. Therefore, it may be more accurate to look at the immune modulation provided by macrolides as an interplay between improved host response to infection, dampening of dysregulated inflammation and the targeted elimination of several relevant and susceptible airway pathogens. Regardless, the evidence throughout this review shows macrolides possess more than just antimicrobial properties. As such, macrolides may have potential in diseases where immune response dysregulation contributes to disease pathogenesis, such as in cancer and RA. This is supported by clinical data showing cancer patients, including lung cancer patients, respond better to cancer therapy when given in combination with CAM 108 , and an improvement in RA symptoms after macrolide therapy 109, 110 . However, as macrolide therapy comes with disadvantages (Section 1.5), it must be carefully evaluated whether the advantages of macrolides outweigh the safety concerns and future research is needed to answer questions regarding long-term safety of macrolides. Finally, by further understanding the immunological pathways targeted by macrolides and the magnitude to which their immunomodulatory effects drive clinical outcomes opposed to their antimicrobial effects, the development of non-antibiotic macrolide-like drugs could see the benefits of macrolide therapy optimised and the drawbacks addressed. In conclusion, macrolide antibiotics have shown potent immunomodulatory properties in aspects of innate and adaptive immunity and these effects likely contribute to their therapeutic benefit in the context of inflammatory respiratory diseases alongside their well-established antimicrobial properties. 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Macrolide structures (Table 1) were created using ChemSpider, Royal Society of Chemistry (2021).