Cells of the innate immune system detect danger signals via so called “Pattern recognition receptors” (PRRs). A multi-protein complex termed “inflammasome” is among the most important platforms integrating signalling by different PRRs and is mainly activated by molecules of the NOD-like receptor (NLR) family. Once assembled, inflammasome formation leads to the activation of inflammatory caspases and the subsequent secretion of the pro-inflammatory cytokines IL-1 and IL-18. Apart from the canonical activation by NLRs, recent research has outlined the importance of a non-canonical inflammasome activation by a direct sensing of LPS through the inflammatory caspases-4/5/11. The supreme importance of the inflammasome in the regulation of inflammation is underlined by the contribution of its deregulation in the pathogenesis of wide-spread diseases such as diabetes mellitus, COPD and asthma.
Macrolides are a group of antibiotics mainly used in the treatment of respiratory diseases. In addition to their anti-infective effect, they are known to exhibit a broad range of anti-inflammatory properties, which are thought to contribute to their favourable clinical effects. The utilization of macrolides in low-dose regimen is increasingly discussed for the long-term treatment of inflammatory diseases with a bacteriological component, particularly COPD. As deregulated inflammasome signalling has been shown to be involved in many of these diseases, the aim of this study was to analyse the impact of macrolides on this signalling platform.
First, we measured the impact of the most important macrolide, azithromycin, on cytokine production of LPS-or flagellin-stimulated human monocytes. We noted that IL-1ß production was specifically inhibited in LPS-stimulated monocytes, whereas the production of other pro-inflammatory cytokines (IL-6, IL-8, TNF-α) were unaffected. To determine whether this effect was class specific within macrolides we performed the same experiment with clarithromycin and roxithromycin, two additional representatives of this antibiotic class. In contrast to azithromycin, however, these compounds were not able to alter cytokine secretion of monocytes. As pharmakokinetic studies have established a much higher intracellular accumulation of azithromycin as compared to other compounds of this class, we hypothesized that the intracellular concentration might be responsible for the diverging effects. Indeed, interference with its Ca++-dependent uptake (by Ca++-depletion and L-type Ca++-channel blockade) entirely abolished its cytokine-modulatory properties. Since the 8 effect of azithromycin was restricted to LPS-stimulated monocytes, the interference with non-canonical inflammasome mediated sensing of intracellular LPS was feasible. Furthermore, we detected cellular LPS-internalisation, thus supporting the possibility of an impact on intracellular sensing. Indeed, we observed down-regulation of caspase-4 expression by azithromycin treatment in LPS-stimulated monocytes. On the other hand, there was no measurable impact on NF-κB activation, which was in line with the lacking effect on other pro-inflammatory cytokines. To expand our observation to a more complex pathophysiological setting, we established a murine LPS sepsis model and showed that azithromycin treatment specifically dampened IL-1ß levels when compared to controls.
The results of this work establish a so far undescribed impact of azithromycin on the non-canonical inflammasome, which might be helpful to improve strategies in the treatment of inflammasome-mediated diseases.