Mihailo Banjanac

Azithromycin distinctively modulates classical activation of human monocytes in vitro

BACKGROUND AND PURPOSE Azithromycin has been reported to modify activation of macrophages towards the M2 phenotype. Here, we have sought to identify the mechanisms underlying this modulatory effect of azithromycin on human monocytes, classically activated in vitro.

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.

Intensity of macrolide anti-inflammatory activity in J774A.1 cells positively correlates with cellular accumulation and phospholipidosis

Some macrolide antibiotics were reported to inhibit interleukin-6 (IL6) and prostaglandin-E2 (PGE(2)) production by bacterial lipopolysaccharide (LPS) stimulated J774A.1 cells. Macrolides are also known to accumulate in cells and some were proven inducers of phospholipidosis. In the present study, with a set of 18 mainly 14- and 15-membered macrolides, we have investigated whether these macrolide induced phenomena in J774A.1 cells are connected. In LPS-stimulated J774A.1 cells, the extent of inhibition of proinflammatory markers (IL6 and PGE(2)) by macrolides significantly correlated with their extent of accumulation in cells, as well as with the induction of phospholipidosis, and cytotoxic effects in prolonged culture (with correlation coefficients (R) ranging from 0.78 to 0.93). The effects observed were related to macrolide binding to phospholipids (CHI IAM), number of positively charged centres, and were inversely proportional to the number of hydrogen bond donors. Similar interdependence of effects was obtained with chloroquine and amiodarone, whereas for dexamethasone and indomethacin these effects were not linked. The observed macrolide induced phenomena in J774A.1 cells were reversible and elimination of the macrolides from the culture media prevented phospholipidosis and the development of cytotoxicity in long-term cultures. Based on comparison with known clinical data, we conclude that LPS-stimulated J774A.1 cells in presented experimental setup are not a representative cellular model for the evaluation of macrolide anti-inflammatory potential in clinical trials. Nevertheless, our study shows that, at least in in vitro models, binding to biological membranes may be the crucial factor of macrolide mechanism of action.

Impairment of lysosomal functions by azithromycin and chloroquine contributes to anti-inflammatory phenotype.

Azithromycin and chloroquine have been shown to exhibit anti-inflammatory activities in a number of cellular systems, but the mechanisms of these activities have still not been clarified unequivocally. Since both drugs are cationic, accumulate in acidic cellular compartments and bind to phospholipids with a consequent increase in lysosomal pH and induce phospholipidosis, we examined the relevance of these common properties to their anti-inflammatory activities. We compared also these effects with effects of concanamycin A, compound which inhibits acidification of lysosomes. All three compounds increased lysosomal pH, accumulation of autophagic vacuoles and ubiquitinated proteins and impaired recycling of TLR4 receptor with consequences in downstream signaling in LPS-stimulated J774A.1 cells. Azithromycin and chloroquine additionally inhibited arachidonic acid release and prostaglandin E2 synthesis. Therefore, impairment of lysosomal functions by azithromycin and chloroquine deregulate TLR4 recycling and signaling and phospholipases activation and lead to anti-inflammatory phenotype in LPS-stimulated J774A.1 cells.

Anti-inflammatory mechanism of action of azithromycin in LPS-stimulated J774A.1 cells

Azithromycin is a macrolide antibiotic with well-described anti-inflammatory properties which can be attributed, at least partially, to its action on macrophages. We have previously shown, with 18 different macrolide molecules, that IL-6 and PGE₂ inhibition correlates with macrolide accumulation, as well as with their binding to phospholipids in J774A.1 cells. The present study was performed in order to substantiate the hypothesis that biological membranes are a target for macrolide anti-inflammatory activity. By analyzing the effect of azithromycin on overall eicosanoid production, we found that in LPS-stimulated J774A.1 cells, azithromycin, like indomethacin, inhibited the synthesis of all eicosanoids produced downstream of COX. Upstream of COX, azithromycin inhibited arachidonic acid release in the same way as a cPLA₂ inhibitor, while indomethacin had no effect. Further comparison revealed that in LPS-stimulated J774A.1 cells, the cPLA₂ inhibitor showed the same profile of inhibition as azithromycin in inhibiting PGE₂, IL-6, IL-12p40 and arachidonic acid release. Therefore, we propose that the anti-inflammatory activity of azithromycin in this model may be due to interactions with cPLA₂, causing inadequate translocation of the enzyme or disturbing physical interactions with its substrates.

Macrolactonolides: a novel class of anti- inflammatory compounds

A new concept in design of safe glucocorticoid therapy was introduced by conjugating potent glucocorticoid steroids with macrolides (macrolactonolides). These compounds were synthesized from various steroid 17β-carboxylic acids and 9a-N-(3-aminoalkyl) derivatives of 9-deokso-9a-aza-9a-homoeritromicin A and 3-descladinosyl-9-deokso-9a-aza-9a-homoeritromicin A using stable alkyl chain. Combining property of macrolides to preferentially accumulate in immune cells, especially in phagocyte cells, with anti-inflammatory activity of classic steroids, we designed molecules which showed good anti-inflammatory activity in ovalbumin (OVA) induced asthma in rats. The synthesis, in vitro and in vivo anti-inflammatory activity of this novel class of compounds are described.

Macrolones Are a Novel Class of Macrolide Antibiotics Active against Key Resistant Respiratory Pathogens In Vitro and In Vivo

ABSTRACT As we face an alarming increase in bacterial resistance to current antibacterial chemotherapeutics, expanding the available therapeutic arsenal in the fight against resistant bacterial pathogens causing respiratory tract infections is of high importance. The antibacterial potency of macrolones, a novel class of macrolide antibiotics, against key respiratory pathogens was evaluated in vitro and in vivo. MIC values against Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, and Haemophilus influenzae strains sensitive to macrolide antibiotics and with defined macrolide resistance mechanisms were determined. The propensity of macrolones to induce the expression of inducible erm genes was tested by the triple-disk method and incubation in the presence of subinhibitory concentrations of compounds. In vivo efficacy was assessed in a murine model of S. pneumoniae-induced pneumonia, and pharmacokinetic (PK) profiles in mice were determined. The in vitro antibacterial profiles of macrolones were superior to those of marketed macrolide antibiotics, including the ketolide telithromycin, and the compounds did not induce the expression of inducible erm genes. They acted as typical protein synthesis inhibitors in an Escherichia coli transcription/translation assay. Macrolones were characterized by low to moderate systemic clearance, a large volume of distribution, a long half-life, and low oral bioavailability. They were highly efficacious in a murine model of pneumonia after intraperitoneal application even against an S. pneumoniae strain with constitutive resistance to macrolide-lincosamide-streptogramin B antibiotics. Macrolones are the class of macrolide antibiotics with an outstanding antibacterial profile and reasonable PK parameters resulting in good in vivo efficacy.