Rac1 Regulates the NLRP3 Inflammasome Which Mediates IL-1beta Production in Chlamydophila pneumoniae Infected Human Mononuclear Cells

Chlamydophila pneumoniae causes acute respiratory tract infections and has been associated with development of asthma and atherosclerosis. The production of IL-1β, a key mediator of acute and chronic inflammation, is regulated on a transcriptional level and additionally on a posttranslational level by inflammasomes. In the present study we show that C. pneumoniae-infected human mononuclear cells produce IL-1β protein depending on an inflammasome consisting of NLRP3, the adapter protein ASC and caspase-1. We further found that the small GTPase Rac1 is activated in C. pneumoniae-infected cells. Importantly, studies with specific inhibitors as well as siRNA show that Rac1 regulates inflammasome activation in C. pneumoniae-infected cells. In conclusion, C. pneumoniae infection of mononuclear cells stimulates IL-1β production dependent on a NLRP3 inflammasome-mediated processing of proIL-1β which is controlled by Rac1

Role of Rac1 in the production of IL-1β in <i>C. pneumoniae</i>-infected cells.

<p>(A) PBMCs were incubated with different concentrations of the Rac1 inhibitor NSC23766 for 30 min and subsequently infected with <i>C. pneumoniae</i> (MOI 3), or (C) cells were first infected with <i>C. pneumoniae</i> (MOI 3) and NSC23766 was added 2.5 h post-infection. After incubating 16 hrs production of IL-1β was quantified by ELISA. Total RNA was harvested for quantification of chlamydial 16s rRNA production (B, D) using real-time PCR as indicated in the Materials and Methods section. (E) THP-1 cells were transfected with control siRNA or siRNA specific for Rac1. After 48 h, cells were infected with <i>C. pneumoniae</i> (MOI 3) for 16 hrs and knock down of Rac1 was assessed by reverse transcription PCR. (F) HEp-2 reinfection assay in which siRNA-transfected THP-1 cells infected with <i>C. pneumoniae</i> (MOI 0.5; 72 h) were harvested and inoculated onto monolayers of HEp-2 cells. Infected cells were then stained for Chlamydia 48 h p.i. and clamydial inclusions were counted. Data shown are representative for at least three (A–D) or two (E, F) experiments performed in duplicates.</p

<i>C. pneumoniae</i> stimulates production of mature IL-1β in human PBMCs.

<p>(A) Human PBMCs were infected with different MOI of <i>C. pneumoniae</i> for 16 hrs and production of IL-1β was determined by ELISA. (B) Human PBMCs were infected with <i>C. pneumoniae</i> (MOI 3) for different time intervals and amounts of mature IL-1β (17 kDA) in the cell supernatant was visualized by Western Blot. The western blot is representative of three independent experiments. Results obtained from ELISAs represent mean ± SD of three independent experiments.</p

Caspase-1, ASC and NLRP3 mediate IL-1β production in <i>C. pneumoniae</i>-infected cells.

<p>(A) THP-1 monocytes were infected with <i>C. pneumoniae</i> (MOI 3) for different time intervalls. Cell lysates were harvested and assayed for procaspase-1 and caspase-1 p20. (B, C) PBMCs were transfected with control siRNA or siRNA specific for caspase-1. After 48 h, cells were infected with <i>C. pneumoniae</i> (MOI 3) for 16 hrs. Expression of caspase-1 was examined by reverse transcription PCR, and supernatants were subjected to IL-1β ELISA. (D–G) PBMCs were transfected with control siRNA or siRNA specific for ASC (D, E) or NLRP3 (F, G). After 48 h, cells were infected with <i>C. pneumoniae</i> (MOI 3), expression of ASC (D) and NLRP3 (F) was examined by reverse transcription PCR, and supernatants were subjected to IL-1β ELISA (E, G). (H) Cells were transfected with siRNA as indicated and, after 48 h, infected with <i>C. pneumoniae</i> (MOI 3). Cell lysates were assayed for procaspase-1 and caspase-1 p20. (I) Mouse BMMs obtained from wildtype and Nlrp3−/− mice were infected with <i>C. pneumoniae</i> (MOI 3) for 16 hrs. Production of mIL-1β was quantified by ELISA. Western Blots are representative for at least three independent experiments. Results obtained from ELISAs represent mean ± SD of three independent experiments.</p

Rac1 controls IL-1β production at a posttranscriptional level in <i>C. pneumoniae</i>-infected cells.

<p>PBMCs were transfected with control siRNA or siRNA specific for Rac1. After 48 h, cells were infected with <i>C. pneumoniae</i> (MOI 3) for 16 hrs and knock down of Rac1 was assessed by reverse transcription PCR (A). Cell supernatants were subjected to IL-1β ELISA (B), and levels of pro-IL-1β mRNA were analyzed by Q-PCR (C). (D) THP-1 cells were incubated with the indicated concentrations of NSC23766 for 30 min and afterwards infected with <i>C. pneumoniae</i> (MOI 3) for 8 h. Cell lysates were assayed for pro-caspase-1 and caspase-1 p20 by Western blot. The western Blot is representative of three independent experiments. (E, F) THP-1 cells seeded on coverslips were treated or not treated with NSC23766, and infected with <i>C. pneumoniae</i> for 20 h. Bacteria (red) and ASC (green) were visualized by confocal laser scanning microscopy using specific antibodies. The arrowheads point to ASC foci. Images are representative of three independent experiments (original magnification 63×).</p