Snježana Čužić

Azithromycin and Clarithromycin Inhibit Lipopolysaccharide-Induced Murine Pulmonary Neutrophilia Mainly through Effects on Macrophage-Derived Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-1β

Macrolide antibiotics possess immunomodulatory/anti-inflammatory properties. These properties are considered fundamental for the efficacy of macrolide antibiotics in the treatment of chronic inflammatory diseases like diffuse panbronchiolitis and cystic fibrosis. However, the molecular mechanisms and cellular targets of anti-inflammatory/immunomodulatory macrolide activity are still not fully understood. To describe anti-inflammatory effects of macrolides in more detail and to identify potential biomarkers of their activity, we have investigated the influence of azithromycin and clarithromycin on the inflammatory cascade leading to neutrophil infiltration into lungs after intranasal lipopolysaccharide challenge in mice. Azithromycin and clarithromycin pretreatment reduced total cell and neutrophil numbers in bronchoalveolar lavage fluid and myeloperoxidase concentration in lung tissue. In addition, concentrations of several inflammatory mediators, including CCL2, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-1β (IL-1β), tumor necrosis factor α, and sE-selectin in lung homogenates were decreased after macrolide treatment. Inhibition of cytokine production observed in vivo was also corroborated in vitro in lipopolysaccharide-stimulated monocytes/macrophages, but not in an epithelial cell line. In summary, results presented in this article confirm that macrolides can suppress neutrophil-dominated pulmonary inflammation and suggest that the effect is mediated through inhibition of GM-CSF and IL-1β production by alveolar macrophages. Besides GM-CSF and IL-1β, CCL2 and sE-selectin are also identified as potential biomarkers of macrolide anti-inflammatory activity in the lungs.

Azithromycin drives in vitro GM-CSF/IL-4-induced differentiation of human blood monocytes toward dendritic-like cells with regulatory properties.

Azithromycin, a macrolide antibacterial, has been shown to modify the phenotype of macrophages. We have investigated whether azithromycin in vitro is able to modulate the differentiation of human blood monocytes to DCs. iA‐DCs appear to have a unique phenotype, characterized by increased granularity, adherence, and a surface molecule expression profile similar to that of MDCs, namely, CD1a–CD14–CD71+CD209high, as well as high CD86 and HLA‐DR expression. The iA‐DC phenotype is associated with increased IL‐6 and IL‐10 release, increased CCL2 and CCL18 expression and release, and M‐CSF expression, as well as reduced CCL17 expression and release. Upon maturation with LPS, A‐DCs and MDCs exhibit decreased expression of HLA‐DR and costimulatory molecules, CD40 and CD83, as well as an increase in IL‐10 and a decrease in CCL17 and CXCL11 secretion. These modulated responses of iA‐DCs were associated with the ability to reduce a MLR, together with enhanced phagocytic and efferocytotic properties. Azithromycin, added 2 h before activation of iDCs with LPS, enhanced IL‐10 release and inhibited IL‐6, IL‐12p40, CXCL10, CXCL11, and CCL22 release. In conclusion, azithromycin modulates the differentiation of blood monocyte‐derived DCs to form iA‐DCs with a distinct phenotype similar to that of iMDCs, accompanied by enhanced phagocytic and efferocytic capabilities. It also modifies LPS‐induced DC maturation by decreasing surface molecule expression required for T cell activation, increasing IL‐10 production, and inducing MLR‐reducing properties.

Azithromycin inhibits macrophage interleukin-1β production through inhibition of activator protein-1 in lipopolysaccharide-induced murine pulmonary neutrophilia☆

Macrolide antibiotics, including azithromycin, also possess anti-inflammatory properties. However, the molecular mechanism(s) of activity as well as the target cells for their action have not been unambiguously identified as yet. In this study, the effects of azithromycin on lipopolysaccharide (LPS)-induced pulmonary neutrophilia were investigated in mice. Using immunohistochemistry, mRNA and specific protein assays, we confirmed that azithromycin ameliorates LPS-induced pulmonary neutrophilia by inhibiting interleukin-1β (IL-1β) expression and production selectively in alveolar macrophages as well as in LPS-stimulated J774.2 macrophage-derived cells in vitro. Inhibition by azithromycin of neutrophilia and IL-1β was accompanied by prevention of nuclear expression of activator protein-1 (AP-1) in both alveolar macrophages and J774.2 cells. The macrolide did not alter nuclear factor kappa B (NF-κB) or extracellular signal-regulated kinase 1/2 (ERK1/2) expression, activation or localization in LPS-stimulated lungs or in J774.2 cells. In conclusion, we have shown that inhibition of LPS-induced pulmonary neutrophilia and IL-1β concentrations in lung tissue following azithromycin treatment is mediated through effects on alveolar macrophages. In addition, we have shown for the first time, in an in vivo model, that azithromycin inhibits AP-1 activation in alveolar macrophages, an action confirmed on J774.2 cells in vitro.

Differential evaluation of excisional non-occluded wound healing in db/db mice.

The full-thickness wound in the genetically diabetic (db/db) mouse is a commonly used model of impaired wound healing. We investigated delayed healing of non-occluded, excisional, full-thickness, dermal wounds in db/db mice in comparison to their normal littermate controls and refined methods for monitoring skin wound re-epithelialization, contraction, granulation tissue formation, and inflammation. We have confirmed with a computer-assisted planimetry method the results of previous studies showing that healing of non-occluded full excision wounds in db/db mice does not occur by contraction as much as in healthy mice. In addition, we have developed separate histological methods for the assessment of re-epithelialization, contraction, granulation tissue (mature, immature, fibrosis), and inflammation (lipogranulomas, secondary, nonspecific). Using a new approach to histological assessment, we have shown that wound closure in db/db mice is delayed owing to: (1) delayed granulation tissue maturation; (2) ‘‘laced,’’ widely distributed granulation tissue around fat lobules; and (3) obstruction by lipogranulomas, whereas the rate of re-epithelialization seems to be the same as in C57Bl/6 mice. This methodology should permit a more precise differentiation of effects of novel therapeutic agents on the wound healing process in db/db mice.

Claudin-3 and Clara Cell 10 kDa Protein as Early Signals of Cigarette Smoke–Induced Epithelial Injury along Alveolar Ducts

Smoking-associated chronic obstructive pulmonary disease is characterized by inflammation, changes affecting small airways, and development of emphysema. Various short- and long-term models have been introduced to investigate these processes. The aim of the present study was to identify markers of early epithelial injury/adaptation in a short-term animal model of cigarette smoke exposure. Initially, male BALB/c mice were exposed to smoke from one to five cigarettes and lung changes were assessed 4 and 24 hr after smoking cessation. Subsequently, animals were exposed to smoke from five cigarettes for 2 consecutive days and lungs investigated daily until the seventh postexposure day. Lung homogenates cytokines were determined, bronchioloalveolar fluid cells were counted, and lung tissue was analyzed by immunohistochemistry. Exposure to smoke from a single cigarette induced slight pulmonary neutrophilia. Smoke from two cigarettes additionally induced de novo expression of tight junction protein, claudin-3, by alveolar duct (AD) epithelial cells. Further increases in smoke exposure induced epithelial changes in airway progenitor regions. During the recovery period, the severity/frequency of epithelial reactions slowly decreased, coinciding with the switch from acute to a chronic inflammatory reaction. Claudin-3 and Clara cell 10 kDa protein were identified as possible markers of early tobacco smoke–induced epithelial injury along ADs.

Fluorescently labeled macrolides as a tool for monitoring cellular and tissue distribution of azithromycin

Exceptional therapeutic effects of macrolides in treating various infections and inflammatory conditions can be significantly contributed to their unique pharmacokinetic properties. Macrolides accumulate in cells and tissues, with concentrations usually 10 to more than 100 times higher of those measured in plasma. Intracellular distribution of macrolides has so far been examined using extensive subcellular fractionation techniques, radiolabeled compounds and conventional pharmacokinetic methods. In this study we evaluated four fluorescently labeled macrolides on their applicability to monitor azithromycin distribution in vitro and in vivo. 9-Deoxo-9a-{3-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]propyl}-9a-aza-9a-homoerythromycin A (9a-NBD-azithromycin) was selected as a compound with most similar cellular pharmacokinetics to azithromycin. 9a-NBD-azithromycin demonstrated antimicrobial properties comparable to azithromycin, displayed the same biological activity profile in LPS-stimulated J774A.1 murine macrophage cells and, even though it accumulated in cells almost 50% more than azithromycin, it showed same rate of retention. Identical to azithromycin, 9a-NBD-azithromycin was localized in lysosomes of J774A.1 cells. Two hours after 9a-NBD-azithromycin was administered intraperitonally to mice, a strong fluorescent signal was located in kidneys and liver and slightly weaker in the spleen. In kidneys, the signal was concentrated in tubuli, and glomeruli were negative. Patchy florescence in hepatocytes supports lysosomal cellular localization. Weaker staining of white pulp compared to red pulp of spleen is in agreement with lower accumulation of azithromycin in lymphocytes compared to other cell types present. We conclude that 9a-NBD-azithromycin can be used as a fluorescent analog of azithromycin to visualize its distribution in in vitro systems, and is also suitable for in vivo studies.