Cigarette Smoke Extract Induced Disfuction Of Macrophages And The Invovled Mechanism

Cigarette smoke (CS) is associated with increased risk of different disorders in hunman. Immunological dysfunction especially in macrophages is one of important reasons in the initiation, progression and exacerbation of smoke-related pulmonary illnesses. Our research focused on cigarette smoke induced ruction damage of macrophage and involved mechanism.In the first part, we examined toxicological effects of cigarette smoke extract (CSE) on mouse Ana-1macrophages and tried to elucidate the involved mechanism. The results showed CSE induced cell apoptosis accompanied by increased releasing of lactate dehydrogenase (LDH), mitochondrial injury and oxidative stress. It also inhibited anti-apoptosis protein Bcl-2expression and promoted pro-apoptosis protein Bax and Bad expressions. Moreover, low-dose CSE (<15%) or short-time treatment (<12h) increased nuclear NF-κB levels of macrophages; On the contrary, high-dose CSE (>15%) or long-time treatment (>12h) decreased it. These observations were in correspondence with changes of intracellular ROS level and antioxidant enzymes’activity. Furthermore, pretreatment with10μM of NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) for1h significantly enhanced macrophage apoptosis. Taken together, these data implied that mitochondrial dysfunction and oxidative stress played important roles in the injury of Ana-1cells caused by CSE, which was related to NF-κB pathway; an anti-apoptotic program played a dominant role at low doses/short-term exposure to CSE, whereas a pro-apoptotic program was initiated at high doses/long-term exposure.In the second part, we investigated the effect of CSE and CS on M2polarization of macrophages.After Ana-1cells treated with lower concentration of CSE (<4%), the intracellular ROS levels, the NO production and phagocytic ability were significantly declined. CSE also promoted the level of surface molecules CD163and decreased the level of MHC-II of Ana-1cells. Besides, the mRNA expressions of IL-12p40, iNOS and TNF-a were down-regulated and the expressions of IL-10, IL-6, TGF-β1and TGF-P2were up-regulated by CSE. Furthermore, Western blotting analysis showed that CSE enhanced the levels of p-JAK2and p-STAT3, and meantime promoted the level of NF-KBp50both in cytoplasm and nuclear; however, CSE promoted NF-KBp65level in cytoplasm and inhibited it in nuclear. When treated Ana-1cells with p-STAT3inhibitor WP1066along with CSE, macrophages down-regulated IL-10expression induced by CSE and up-regulated CSE-induced IL-12expression.In order to confirm the above results, experiments on mouse peritoneal macrophages, mouse macrophages from bronchoalveolar lavage (BAL) and mouse bone marrow-derived macrophages were carried. The results showed CSE enhanced the levels of CD163, down-regulated pro inflammatory cytokines and up-regulated anti-inflammatory cytokines in the three primary macrophages. This is in according with the results of Ana-1cells.Furthermore, the results were verified by the animal experiment. After BALB/c mice were used to undergo smoking for6days, BAL macrophages were collected and CD163marker was detected. The results showed the percentage of CD163positive macrophages in BAL from smoking mice is significantly higher than that of control mice.In a conclusion, cigarette smoke may induce macrophage M2-polarization and the involved mechanism may be corporately mediated via JAK2/STAT3activation and NF-κB inhibition. This may partially account for the initiation, progression and deterioration of smoking-related disorders.