Influence of Mycoplasma cynos infection on the expression of inflammatory response genes in canine macrophage cell line DH82

Mycoplasma cynos je edina mikoplazma, za katero so doslej dokazali povezavo z razvojem bolezni dihal pri psih. Bila je večkrat izolirana iz obolelih psov, predvsem tistih, živečih v psarnah. Domneva se, da je, tako kot druge mikoplazme, ki naseljujejo dihala, lahko povezana s številnimi bolezenskimi zapleti ter, da je v klinični obravnavi teh stanj pogosto spregledana. Vzrok je verjetno v tem, da je relativno slabo proučena, predvsem je slabo poznana povezava med bakterijo in celicami gostitelja. V okviru magistrske naloge smo z uporabo metode PCR v realnem času, preverili vpliv okužbe trajne pasje celične linije DH82 z M. cynos na izražanje genov za citokine IL-1beta, IL-6, IL-8 in za transkripcijski dejavnik NF-kapaB1. Ugotovili smo, da se okužba z M. cynos odraža v postopnem zviševanju relativnih sprememb v izražanju gena za IL-1beta do 12 h po okužbi, v postopnem zniževanju izražanja gena za IL-6 do 12 h po okužbi, v postopnem zviševanju izražanja gena za IL-8 do 24 h in nato padcu izražanja po 48 h ter v relativno majhnem spreminjanju izražanja gena za NF-kapaB1 do 6 h po okužbi. Okužba ima tudi vpliv na morfološke spremembe celic DH82. Po 24 h po okužbi se te kažejo v obliki vakuolizacije celic, po 48 h pa še v lizi posameznih celic DH82. Pri teh dveh časovnih točkah je opazen tudi padec viabilnosti celic, in sicer na približno 87 % po 24 h in na 70 % po 48 h. Opazili smo tudi padec števila M. cynos v supernatantih celic DH82 v prvih 3 h po okužbi. V supernatantih celic DH82 nismo zaznali spremenjenih koncentracij dušikovega oksida (NO).Mycoplasma cynos is the only species of mycoplasma that has been proven to be associated with the development of respiratory disease in dogs. It was isolated several times from the diseased dogs, mainly those living in kennels. Like other mycoplasmas that inhabit the respiratory system, it is believed that M. cynos is associated with many health complications and that it is often overlooked in clinical treatments. The reason may be in the lack of studies, especially in terms of the relationship between the bacterium and its host cells. Using quantitative PCR, we analysed the influence of M. cynos infection on the expression of genes for cytokines IL-1beta, IL-6, IL-8 and the transcription factor NF-kappaB1 in the immortalised dog cell line DH82. We came to a conclusion that the infection results in a gradual increase in IL-1beta gene expression until 12 h after the infection, in a gradual decrease in IL-6 gene expression until 12 h after the infection, in a gradual increase in IL-8 gene expression until 24 h and then a decrease in 48 h after the infection, and in relatively minor variation of NF-kappaB1 gene expression until 6 h after the infection. M. cynos infection also has an influence on the morphology of DH82 cells, which is manifested in increased vacuolisation 24 h following the infection and in the lysis of individual DH82 cells after 48 h. During this time, an apparent drop in cell viability can also be observed. The viability dropped to 87% after 24 h and to 70% after 48 h after the infection. We also observed a drop in M. cynos concentrations in DH82 cell supernatants in the first 3 h after the infection. We were unable to detect any changes in nitric oxide (NO) concentrations in DH82 cell supernatants, after the infection with M. cynos