Non-Apoptotic Toxicity of <em>Pseudomonas aeruginosa</em> toward Murine Cells

<div><p>Although <em>P. aeruginosa</em> is especially dangerous in cystic fibrosis (CF), there is no consensus as to how it kills representative cell types that are of key importance in the lung. This study concerns the acute toxicity of the sequenced strain, PAO1, toward a murine macrophage cell line (RAW 264.7). Toxicity requires brief contact with the target cell, but is then delayed for more than 12 h. None of the classical toxic effectors of this organism is required and cell death occurs without phagocytosis or acute perturbation of the actin cytoskeleton. Apoptosis is not required for toxicity toward either RAW 264.7 cells or for alveolar macrophages. Transcriptional profiling shows that encounter between PAO1 and RAW 264.7 cells elicits an early inflammatory response, followed by growth arrest. As an independent strategy to understand the mechanism of toxicity, we selected variant RAW 264.7 cells that resist PAO1. Upon exposure to <em>P. aeruginosa</em>, they are hyper-responsive with regard to classical inflammatory cytokine production and show transient downregulation of transcripts that are required for cell growth. They do not show obvious morphologic changes. Although they do not increase interferon transcripts, when exposed to PAO1 they dramatically upregulate a subset of the responses that are characteristic of exposure to g-interferon, including several guanylate-binding proteins. The present observations provide a novel foundation for learning how to equip cells with resistance to a complex challenge.</p> </div

Morphological changes of naïve and resistant RAW 264.7 cells.

<p>Both naïve and resistant cells were exposed to g-interferon (25 ng/ml), soluble LPS (2 mg/ml) and PAO1 (MOI 10). After 1 h they were returned to culture for 48 h. The large differences of responsiveness of between the naïve and resistant cells are indicated by arrows. The naïve cells upon exposure to g-interferon, LPS, and PAO1 have extended processes and appear vacuolated, whereas the appearance of the resistant cells upon treatment does not differ from that of untreated control cells.</p

NF-kB activation in naïve and resistant RAW 264.7 cells.

<p><b>A</b>) Naïve and resistant RAW 264.7 cells (ResRAW) were exposed to PAO1 (MOI 10) or to LPS (100 ng/ml) for 1 h. Note the translocation of the p65 subunit of NF-kB to the nucleus in both cell types. For the right-hand most images, preparations that received no primary antibody were viewed in the red channel. <b>B</b>) To quantitate the inhibitor of NF-kB (IKB), cells were exposed to PAO1 for 1 h, harvested and processed for Western blot analysis to detect IkB and b-actin. Note the major reduction in total IKB.</p

Toxicity of PAO1 toward RAW 264.7 cells.

<p><b>A</b>) RAW 264.7 cells were exposed to PAO1 in the presence of different kinase inhibitors: SP600125 (for JNK, 10 mg/ml), U0126 (for MEK, 10 mg/ml) and BMS345541 (for IKK, 10 mg/ml). MTT assays quantitated cell viability after 1 day. <b>B</b>) RAW 264.7 cells were treated with PAO1 (MOI 10, 1 h) and washed. Caspase-3 activity was measured by colorimetric assays after 1–24 h. Control cells not exposed to bacteria were collected at the same time points. Sample treated with staurosporine (STS, 1 µg/ml) with or without caspase-3 inhibitor (Inh) for 3 h were used as a positive control. <b>C</b>) RAW 264.7 cells were exposed to PAO1 (MOI 10) for 1 h. DNA was isolated from cells at the indicated time points and analyzed by agarose gel electrophoresis. Control cells were not exposed to PAO1. Cells incubated with staurosporine (S) for 3 h were used as a positive control. M denotes the DNA ladder. <b>D</b>) RAW 264.7 cells were exposed to PAO1 +/− prior incubation with caspase 3 inhibitor (Inh). The viability of the cells was measured by MTT assay after 2 days. Cells treated with staurosporine were used as a positive control. Data are indicative of three repeat experiments with similar results and error bars represent standard deviations. (***) designates p<0.0003.</p

Overeview of the responses of wildtype and mutant cells.

<p>As is detailed in the text, the present observations show that both cell types exhibit comparable initial changes, including NF-kB activation, cytokine secretion, and downregulation of transcripts that are essential for continued growth. Nevertheless, only the wildtype cells are ultimately killed. The mutant cells show distinct upregulation of transcripts that are associated with responses to g-interferon. These changes could be of central importance for survival.</p

Impact of <i>P. aeruginosa</i> on viability on RAW 264.7 cells.

<p><b>A</b>) Phase overview of cell morphology, uninfected cells (RAW) and resistant cells (ResRAW - see text) were exposed to <i>P. aeruginosa</i> (MOI 10) and returned to culture for 6 or 24 h. Note the major change in cell shape and size of the wt cells upon exposure to <i>P. aeruginosa</i>. The resistant cells appear unaffected over the same time period. <b>B</b>) Actin cytoskeleton, stained with rhodamine-phalloidin after fixation. Uninfected cells compared to resistant cells after exposure to <i>P. aeruginosa</i> (MOI 10) and reculture for 6 or 24 h. Note the enlargement of wt cells and appearance of a subset of cells with double nuclei (shown by an arrow). The nuclei were stained blue with DAPI. For the panels at the right, phalloidin staining was omitted and the images were viewed with the red filter. <b>C</b> and <b>D</b>: MTT assays to quantitate cell number. Uninfected cells compared to cells exposed to <i>P. aeruginosa</i> and then recultured over 4 days for both naïve RAW 264.7 (<b>C</b>) and resistant cells (<b>D</b>). In both cases, cells were exposed to <i>P. aeruginosa</i> for 1 h before washing and return to culture. Activity measurements (per culture) were normalized to values for cells cultured without PAO1 over four days.</p

Comparative Sensitivity of the Mutant and Wildtype Cells.

<p>Sensitivity was evaluated by exposing cells to the lectins or to LPS continuously for 2–3 days.</p

Overview of Transcript Changes.

<p>Overview of Transcript Changes.</p

MTT assay to determine the viability of RAW 264.7 cells.

<p>RAW 264.7 cells were (<b>A</b>) exposed to Type II and Type III mutants of PAO1 for 1 h, or (<b>B</b>) to PAO1 in the presence of rifampicin (Rfp, 40 µg/ml), or gentamicin (Gm, 200 µg/ml). In (<b>C</b>), cells were exposed to different mutant strains of PAO1 containing mutations in pili and flagella. In (<b>D</b>), cells were exposed to muramyldipeptide (MDP, 25 µg/ml) or CpG DNA (5 µg/ml). In all cases cell viability was estimated by measuring MTT activity after 24–48 h as mentioned in text. (***) designates p<0.0003. ns: not-significant.</p

Cytokine production in response to PAO1.

<p>IL6 (<b>A</b>), MIP2 (<b>B</b>) and TNFα (<b>C</b>) secretion by naïve (solid line) and resistant (dotted line) RAW 264.7 cells were quantitated by ELISA, as described in Methods. Cells were exposed to PAO1 at MOI 10 for 1 h and recultured. Supernatants were collected at 0, 1 and 6 h. Cytokine production of mean +/− SD of three wells for each condition.</p