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

Image-based multiplex immune profiling of cancer tissues: translational implications. A report of the International Immuno-oncology Biomarker Working Group on Breast Cancer

Recent advances in the field of immuno-oncology have brought transformative changes in the management of cancer patients. The immune profile of tumours has been found to have key value in predicting disease prognosis and treatment response in various cancers. Multiplex immunohistochemistry and immunofluorescence have emerged as potent tools for the simultaneous detection of multiple protein biomarkers in a single tissue section, thereby expanding opportunities for molecular and immune profiling while preserving tissue samples. By establishing the phenotype of individual tumour cells when distributed within a mixed cell population, the identification of clinically relevant biomarkers with high-throughput multiplex immunophenotyping of tumour samples has great potential to guide appropriate treatment choices. Moreover, the emergence of novel multi-marker imaging approaches can now provide unprecedented insights into the tumour microenvironment, including the potential interplay between various cell types. However, there are significant challenges to widespread integration of these technologies in daily research and clinical practice. This review addresses the challenges and potential solutions within a structured framework of action from a regulatory and clinical trial perspective. New developments within the field of immunophenotyping using multiplexed tissue imaging platforms and associated digital pathology are also described, with a specific focus on translational implications across different subtypes of cancer

Zellautonome angeborene Immunantwort in humanen Endothelzellen auf die Infektion mit Chlamydophila pneumoniae

Wirtszellen verfügen über bisher unzureichend verstandene zellautonome Immunmechanismen zur Abwehr von intrazellulären Bakterien. In dieser Arbeit wurden zwei Abwehrmechanismen charakterisiert, die in Endothelzellen und Makrophagen Infektionen durch C. pneumoniae bekämpfen. Es konnte gezeigt werden, dass C. pneumoniae über einen MAVS-abhängigen Signalweg in humanen Endothelzellen erkannt wird. Diese Erkennung aktiviert die Transkriptionsfaktoren IRF3 und IRF7 und nachfolgend eine IRF3/7-abhängige Typ I-IFN-Produktion. Typ I-IFN bewirken auto- und parakrin eine Kontrolle der intrazellulären Infektion mit C. pneumoniae. Zum anderen wurde gezeigt, dass das mitochondriale Molekül NLRX1 eine zellautonome Abwehr gegen C. pneumoniae in Endothelzellen und in Makrophagen vermittelt. Diese NLRX1-abhängige intrazelluläre Abwehr ist unabhängig von verschiedenen, bisher mit NLRX1 in Verbindung gebrachten Signalwegen. Die Ergebnisse zeigen somit zum ersten Mal, dass NLRX1 eine zellautonome Abwehr gegen intrazelluläre Bakterien vermittelt. Daraus gewonnene Erkenntnisse sowie die Ergebnisse zukünftiger Arbeiten zur Klärung der NLRX1- und MAVS-aktivierenden chlamydialen Moleküle und den durch Typ-I-IFN-abhängigen intrazellulären Abwehrmechanismen könnten bei der Erforschung neuartiger antibakterieller Therapien hilfreich sein. Diese ist angesichts der weltweiten signifikanten Zunahme von mehrfach-resistenten Infektionserregern, unbedingt notwendig.The cell autonomous defense mechanisms against intracellular bacteria in host cells are so far insufficiently understood. In the present work two defense mechanisms involved in the elimination of C. pneumoniae in endothelial cells and in macrophages were characterized. It could be shown that C. pneumoniae is recognized by a MAVS-dependent signaling pathway in human endothelial cells. This recognition activates the transcription factors IRF3 and IRF7 and subsequently an IRF3/7-dependent type I-IFN production. Type-I-IFNs induce an auto- and paracrine control mechanism against the intracellular infection with C. pneumoniae. Additionally it could be shown for the first time that the mitochondrial NLR molecule NLRX1 mediates a cell autonomous defense mechanism against C. pneumoniae and most likely other intracellular bacteria in human endothelial cells and murine macrophages. This NLRX1-dependent intracellular defense mechanism is independent of the different mechanisms which were so far linked to NLRX1. The outcome of this work and future studies to identify chlamydial molecules responsible for the activation of the MAVS- and NLRX1-dependent signaling pathways as well as the effector mechanisms responsible for Type-I-IFN-dependent control of intracellular chlamydial replication could be very helpful in the development of novel antibacterial therapies

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

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

<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