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Previous experiments from our laboratory have demonstrated that the antibiotic ciprofloxacin is substrate for a MRP-like transporter in J774 macrophages and that a prolonged exposure of these cells to ciprofloxacin selects for resistant cells, in which ciprofloxacin accumulation is strongly reduced. These resistant cells overexpress mostly MRP4 and, to a lesser extent, MRP2. NSAIDs, like diclofenac and indomethacin, were described as preferential MRP4 inhibitors.
In the present study, we have studied the potential competition between ciprofloxacin and NSAIDs (diclofenac and indomethacin) for a common efflux pathway in wild-type and ciprofloxacin-resistant macrophages.
After having characterised NSAIDs accumulation in J774 macrophages, we have studied competition between ciprofloxacin and NSAIDs in wild-type and resistant cells.
These competition studies showed that:
(1) indomethacin can increase ciprofloxacin accumulation to a same level as gemfibrozil (a MRP non specific inhibitor) both in wild-type and resistant cells. This higher accumulation is related to a slower efflux.
(2) indomethacin accumulation is lower in resistant cells that in wild-type cells but is restored in the presence of ciprofloxacin.
(3) diclofenac increases ciprofloxacin accumulation in both wild-type and resistant cells but to a lower extent than indomethacin.
(4) diclofenac accumulation is similar in wild-type and ciprofloxacin-resistant cells, increased by gemfibrozil but not by ciprofloxacin.
These results suggest that (a) the two NSAIDs can inhibit the transporter responsible for ciprofloxacin efflux in macrophages (probably MRP4), (b) indomethacin is a substrate for this transporter while diclofenac seems to be substrate for another MRP-related transporter.
Ongoing studies using MDCK cells overexpressing selectively different MRP try to identify the diclofenac transporter and to confirm the MRP4 implication in indomethacin and ciprofloxacin transport. Les travaux antérieurs du laboratoire ont montré que la ciprofloxacine était substrat d'un transporteur d'efflux de la famille des MRP dans les macrophages J774 et que l'exposition chronique de ces cellules à la ciprofloxacine sélectionnait des cellules résistantes dans lesquelles l'accumulation de ciprofloxacine est très faible. Ces cellules résistantes surexpriment surtout la MRP4 mais aussi la MRP2. Les anti-inflammatoires, notamment le diclofenac et l'indométacine, sont décrits comme des inhibiteurs préférentiels de MRP4. Dans ce travail, nous avons voulu étudier la compétition potentielle entre la ciprofloxacine et les AINS (diclofenac et indométacine) pour le transporteur d'efflux dans des macrophages J774 sauvages et résistants mais aussi dans des cellules MDCK sauvages et transfectées sélectivement par diverses MRP.
Après avoir caractérisé l'accumulation des AINS dans les macrophages en fonction du temps et de la concentration, nous avons étudié la compétition entre la ciprofloxacine et les AINS pour un même transporteur d'efflux dans des macrophages J774.
Ces études de compétition ont montré que :
1) l'indométacine est capable d'augmenter le niveau d'accumulation de la ciprofloxacine à un même niveau que le gemfibrozil, un inhibiteur non spécifique des MRP, dans les cellules sauvages et résistantes. Cette accumulation accrue est liée à un efflux ralenti.
2) l'accumulation de l'indométacine est plus faible dans les cellules résistantes que dans les cellules sauvages, et est restaurée en présence de ciprofloxacine.
3) le diclofenac augmente à un moindre niveau l'accumulation de ciprofloxacine dans les 2 types cellulaires.
4) l'accumulation de diclofenac est similaire dans les 2 types cellulaires, non influencée par la ciprofloxacine mais augmentée par le gemfibrozil.
Ces données suggèrent que les 2 AINS peuvent inhiber le transporteur responsable de l'efflux de la ciprofloxacine dans les macrophages (probablement MRP4), que l'indométacine est elle-même substrat de ce transporteur alors que le diclofenac serait substrat d'un autre transporteur MRP.
Des études sont en cours sur des cellules MDCK surexprimant sélectivement diverses MRP pour tâcher d'identifier le transporteur du diclofenac et de confirmer l'implication de la MRP4 dans le transport de l'indométacine et de la ciprofloxacine.

Macrophages --- Ciprofloxacin --- Fluoroquinolones --- Anti-Inflammatory Agents, Non-Steroidal --- Drug Interactions

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Background : Airway inflammation is of pivotal importance in cystic fibrosis (DF) lung disease. We have previously demonstrated in F508del-CF mice a lung inflammation characterized by macrophage infiltration which reduced after azithromycin treatment. The present study aimed at characterizing phenotype of macrophages isolated from wild-type or F508del-CF (CF) mice and assessing the effects of azithromycin on these immune cells.
Methods : Macrophages purified from lung and peritoneal cavity were stimulated either with lipopolysaccarides from Pseudomonas aeruginosa plus interferon-γ or with interleukin-(IL)-4 plus IL-13, either in the presence or in the absence of 1mg/l azithromycin. Macrophage phenotypes were investigated in primary culture 6h after stimulation by different M1 (pro-inflammatory) and M2 (anti-inflammatory) markers at the mRNA level.
Results : A marked macrophage infiltration was observed in both lungs and peritoneal cavity suggesting a systemic macrophage accumulation in CF mice. Non-stimulated and stimulated CF peritoneal macrophages overexpressed M1 (IL1-β and TNF-α) and M2 (Arginase-1, IL-10 and A20) markers as compared to wild-type peritoneal cells. Similar pattern of responses to M1 and M2 stimuli were observed in CF alveolar macrophages. These findings indicated that the immune responses are exacerbated in CF macrophages. Curiously, in non-stimulated CD alveolar macrophages the expression of M1 and M2 mediators was down-regulated. Moreover, we found that azithromycin reduced M1 macrophages activation in CF and wild-type alveolar macrophage.
Conclusions : Our findings further support the concept that overall macrophage immune responses are altered in CF and that azithromycin particularly downregulates M1 differentiation

Cystic Fibrosis --- Phenotype --- Azithromycin --- Macrophages --- Mice --- Cystic Fibrosis Transmembrane Conductance Regulator