Comparative in vitro elicitation of reactive oxygen/nitrogen species and cytokines in Chlamydia pneumoniae and Chlamydia trachomatis infected human monocytes.

Comparative in vitro elicitation of reactive oxygen/nitrogen species and cytokines in Chlamydia pneumoniae and Chlamydia trachomatis infected human monocytes. Marangoni A.1, Bergamini C.2, Fato R.2, Cavallini C.3, Foschi C 1, Russo I.1, Nardini P.1, Cevenini R.1 1Section of Microbiology-DESOS, 2Dipartimento di Biochimica “G. Moruzzi”, and 3Cardiovascular Department, University of Bologna, Bologna, Italy INTRODUCTION An increasing number of in vitro studies suggests that chlamydial species can infect immune cells, at least at low level. These infections may alter immune cell function, promote chlamydial persistence in the host and contribute to the progression of several chronic inflammatory diseases. The limited infection that occurs can affect antigen processing, cytokine production and susceptibility to apoptosis. Regarding that, some authors reported a copious release of reactive oxygen species in Chlamydia pneumoniae-infected macrophages and others the induction of various interleukins and TNF- in blood mononuclear cells after the infection. The aim of this study was to elucidate in vitro infection characteristics, evaluate reactive oxygen (ROS) and reactive nitrogen species (RNS) production and cytokine release in human blood monocytes infected by human pathogens C. pneumoniae (CP) and C. trachomatis (CT). METHODS Human monocytes were isolated from healthy volunteer peripheral blood by centrifugation over Ficoll-Paque and cultured in RPMI 1640. Fresh isolated monocytes were seeded on glass coverlips in 24 well-plates and infected with viable CP and CT elementary bodies (infectivity ratio: 5 EBs/cell). At various time, infectivity rate was evaluated by recultivating disrupted monocytes in LL-CMK2. Infection by CT and CP was carried out as control in the same cellular line. Infected cells were fixed and stained with a fluorescence-labelled MAb to the LPS of Chlamydia (Meridian Diagnostics) at 24, 48, 72 and 96 hours post infection. The number of inclusion was determined by fluorescence microscopy. In order to evaluate the production of ROS and RNS, monocytes were infected with viable CT and CP EBs in the presence of 10µM DCFDA or 5µM DAF, respectively ROS and RNS fluorescent probes. Phorbol and LPS treatment was used as positive control, for ROS and RNS respectively. Cell fluorescence increase was measured with a spectrofluorometer (Wallac Victor multilabel counter). Cytokine production was evaluated by measuring mRNA expression of IFNs and TNF-α at different times after infection: CT and CP-infected monocytes were lysed by adding Trizol (Invitrogen) and total RNA was isolated (RNeasy Micro Kit, Qiagen). cDNA was synthesized and a Real-time RT-PCR was performed in a Lightcycler system (Roche Diagnostics), with the SYBR Green fast start kit (Lyghtcycler® FastStart DNA MasterPLUS SYBR Green I). Primers used in the reaction mixture to assess IFN-α, IFN-β, IFN-γ and TNF-α levels were from SuperArray (SABiosciences Corporation). Fluorescence was measured at the end of each extension step and samples were run in duplicate, using for calculations the average threshold cycle (Ct). A positive control was included by stimulating monocytes with 1 µg/ml LPS. RESULTS Infectivity rate of CP-infected monocytes was 12% at 24 hours after the infection, 2% at 48 hours and 0% at 72 and 96 hours, in comparison with control infected permissive LL-CMK2 cells. On the contrary, we were not able to recover infectious CT at any time after the infection. At 3 hours after infection, CT-infected monocytes were able to produce higher level of both ROS and RNS, in comparison with untreated control and CP-infected monocytes (P=0.024). At later time points there were not significant differences between CT and CP-stimulated monocytes. The expression of IFN-α, IFN-β, IFN-γ was only slightly increased at 4 and 6 hours after infection in CT-infected monocytes, compared to negative control. On t...