Comparative inflammatory cytokines gene expression in culture of human macrophages infected with Leishmania tropica and L. major parasites
Comparative inflammatory cytokines gene expression in culture of human macrophages infected with Leishmania tropica and L. major parasites
Fatemeh Darzi,1,*Mahmoud Nateghi Rostami,2Roshanak Davoudian,3
1. Department of Parasitology, Pasteur Institute of Iran 2. Department of Parasitology, Pasteur Institute of Iran 3. Department of Parasitology, Pasteur Institute of Iran
Introduction: Human cutaneous leishmaniasis is mainly caused by two species Leishmania tropica and L. major in Old World. The type of skin lesion, its duration, severity and appearance is different between Anthroponotic Cutaneous Leishmaniasis (ACL) due to L. tropica and Zoonotic CL (ZCL) due to L. major. Since Pro- and anti-inflammatory cytokines significantly contribute in severity and outcome of CL lesion, this experiment aimed to compare the gene expression changes of these cytokines in culture of human macrophages infected with L. major and L. tropica parasites.
Methods: Peripheral blood mononuclear cells were isolated from a healthy individual without the history of CL from non-endemic region and macrophages were prepared by culture of adherent monocytes in 6well flat-bottomed plates for 6 days in 37 °C incubator with 5% CO2. Macrophages (3x10^5/well) were stimulated with either live L. major or live L. tropica promastigotes (Parasite:MQ ratio of 10:1) or unstimulated. At three time points T1=4, T2=10 and T3=24 hours after stimulation, RNAs were extracted and gene expression of TNF, IL-1B, IL- 6, TGF-B, and IL-10 were measured by relative real-time RT-PCR method using ΔΔCt calculation.
Results: Significant upregulation of TNF, TGF-B and IL-1B genes was shown in stimulated macrophages. The expression of IL-1B in macrophages stimulated with L. major was significantly higher than that of L.tropica. In L. tropica stimulated macrophages the expression of TNF was 93.05 fold higher in T2=10 hr compared to T1=4 hr and significantly decreased after 24 hr. In L. major stimulated macrophages the expression of TGF-B was 213.78 fold higher in T2=10 hr compared to T1=4 and significantly decreased after 24 hr, but in L. tropica stimulated macrophages TGF-B expression was significantly higher in T3=24 hr culture.
Conclusion: This study suggested alterations in pro-inflammatory cytokine IL1B and anti-inflammatory cytokine TGF-B associated with L. major vs. L. tropica infection in vitro. Changes in pro-pro- and anti-inflammatory cytokines seems to play a role in differences occur between severity/appearance of ACL and ZCL lesions.