ASPECTOS J URÍDICOS S OBRE V IRUS I NFORMÁTICOS

In this study we also utilised RAW 264.7 cells to further elucidate whether V antigen could modulate NF-κB and caspase-1 activation in response to LPS, and consequently the secretion of inflammatory cytokines.

Experiments were performed in RAW 264.7 cells order to test the hypothesis that V antigen is able to modulate NF-κB and caspase-1 activation in response to LPS. The presence of phospho-IκB in the cell lysate indicates the transcription of pro- inflammatory cytokines. Similarly, the presence of caspase-1 is an indicative of inflammasome activation, which also leads to a secretion in inflammatory cytokines. Western blotting was utilised in order to determine the presence of both phospho-IκBα and caspase-1 p-10.

3.3.3.1 Presence of phospho-IκBα and caspase-1 p10 in RAW 264.7 cells

in response to LPS

Initially, in order to determine whether LPS can lead to NF-κB and caspase-1 activation, RAW 264.7 cells were stimulated for 1, 2, 4 and 6 hours with 100ng/ml LPS. The supernatant was collected, at the respective time-points, frozen and stored for later cytokine assays. The cells were lysed and the samples were separated by size via SDS-PAGE (Section 2.9) and transferred onto a membrane (Section 2.10.1), which was probed for phospho-IκBα or caspase-1 p-10, with their respective primary antibody, followed by the appropriate secondary antibody conjugated to horse-radish peroxidase (HRP). Membranes were imaged via enhanced chemiluminescence

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(Section 2.11). It was shown that LPS could trigger NF-κB (Figure 3.19) and caspase-1 (Figure 3.20).

Figure 3.19: Western blot of phospho-IκBα from lysates of unstimulated RAW 264.7 cells and

those stimulated for 1, 2, 4 and 6 hours with 100ng/ml LPS. Lysates were separated by SDS- PAGE and transferred onto a nitrocellulose membrane. The membrane was probed with an antibody specific for phospho-IκBα followed by the appropriate secondary antibody conjugated to HRP. Enhanced chemiluminescence was used for detection. β-actin was used as a loading control.

Figure 3.20: Western blot of caspase-1 p10 from lysates of unstimulated RAW 264.7 cells and those stimulated for 1, 2, 4 and 6 hours with 100ng/ml LPS. Lysates were separated by SDS- PAGE and transferred onto a nitrocellulose membrane. The membrane was probed with an antibody specific for caspase-1 p10 followed by the appropriate secondary antibody conjugated to HRP. Enhanced chemiluminescence was used for detection. β-actin was used as a loading control.

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3.3.3.2. Presence of phospho-IκBα and caspase-1 p10 in RAW 264.7 cells

in response to LPS pre- and post-incubated with V antigen

Once it was established that LPS is able to trigger NF-κB signalling cascade and caspase-1 activation in RAW 264.7 cells, whether V antigen was able to modulate this response was investigated. In order to do so, RAW 264.7 cells were stimulated for 1 hour with 100ng/ml of LPS and either pre- or post-incubated for 1 hour or 1, 2, 4 and 6 hours, respectively, with 50µg/ml of V antigen. The cells were lysed and the samples were separated by size via SDS-PAGE (Section 2.9) and transferred onto a membrane (Section 2.10.1), which was probed for phospho-IκBα or caspase p-10, with their respective primary antibody, followed by the appropriate secondary antibody conjugated to horse-radish peroxidase (HRP). Membranes were imaged via enhanced chemiluminescence (Section 2.11).

The results obtained from pre- and post-incubating with V antigen show that there is a still some activation of NF-κB in response to LPS, however it appears to be slightly inhibited. Although post-incubating with V antigen initially seems to show some NF-κB activation, when it hits the 4 hour and 6 hour post-incubation mark it seems to be inhibited (Figure 3.21).

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Figure 3.21: Western blot of phospho-IκBα from lysates of unstimulated RAW 264.7 cells; stimulated for 2 and 4 hours with 100ng/ml LPS; stimulated for 1 hour with 50µg/ml of V antigen; pre-incubated for 1 hour with 50µg/ml of V antigen and subsequently stimulated for 1 hour with 100ng/ml LPS; and stimulated for 1 hour with 100ng/ml LPS and subsequently post-incubated for 1, 2, 4 and 6 hours with 50µg/ml of V antigen. Lysates were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was probed with an antibody specific for phospho-IκBα followed by the appropriate secondary antibody conjugated to HRP. Enhanced chemiluminescence was used for detection. β-actin was used as a loading control.

Concerning caspase-1 activation, the results show that pre-incubation with V antigen seems not to have an effect on caspase-1 activation. On the contrary and post- incubating with V antigen results on a reduction of caspase-1. This reduction seems to gradually occur through the four post-incubation time-points, most prominent at 4 hour post-incubation (Figure 3.22).

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Figure 3.22: Western blot of caspase-1 p10 from lysates of unstimulated RAW 264.7 cells; stimulated for 2 and 4 hours with 100ng/ml LPS; stimulated for 1 hour with 50µg/ml of V antigen; stimulated for 1 hour with 50µg/ml of V antigen; pre-incubated for 1 hour with 50µg/ml of V antigen and subsequently stimulated for 1 hour with 100ng/ml LPS; and stimulated for 1 hour with 100ng/ml LPS and subsequently post-incubated for 1, 2, 4 and 6 hours with 50µg/ml of V antigen. Lysates were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was probed with an antibody specific for caspase-1 p10 followed by the appropriate secondary antibody conjugated to HRP. Enhanced chemiluminescence was used for detection. β-actin was used as a loading control.

3.3.3.3. Cytokine secretion in RAW 264.7 cells in response to V antigen

Given that V antigen was found to modulate the expression of PRRs and the activation of both NF-κB and caspase-1 activation, the next step in this study was to examine the release of cytokines, the inflammatory mediators, caused by LPS and whether or not V antigen is able to modulate their secretion. Out of the inflammatory cytokines analysed (Interlukin-8 (IL-8), Interlukin-1β (IL-1β), Interlukin-6 (IL-6), Interlukin-10 (IL-10), Tumor Necrosis Factor (TNF) and Interlukin-12p70 (IL-12p70)), only the release of IL-1β, IL-6, IL-10 and TNF were significantly increased.

RAW 264.7 cells were stimulated for 1, 2, 4 and 6 hours with 100ng/ml of LPS as well as with 50µg/ml of V antigen for 1 hour. Additionally, RAW 264.7 cells were stimulated for 1 hour with 100ng/ml of LPS and either pre- or post-incubated for 1 hour or 1, 2, 4 and 6 hours, respectively, with 50µg/ml of V antigen. All stimulations were carried out in

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25cm2 flasks. The supernatants were collected post stimulations for cytokine analysis using the Human Inflammation BDTM Cytometric bead array kit (Section 2.8.2.2).

Figure 3.23: Secretion of TNF in RAW 264.7 cells in response to LPS, V antigen and pre- or post-incubation with V antigen. RAW 264.7 cells were stimulated with: 100ng/ml LPS for 1, 2, 4 and 6 hours; 50µg/ml of V antigen for 1 hour; pre-incubated with 50µg/ml V antigen for 1 hour and stimulated with 100ng/ml of LPS for 1 hour; or stimulated with 100ng/ml LPS for 1 hour and post-incubated for 1, 2, 4 and 6 hours with 50µg/ml V antigen. The cytokines were measured in the cell supernatant using a flow cytometric cytokine bead array system (Becton Dickinson). Unstimulated controls were performed. The data is statistically significant (p<0.05). The data represents the mean of three independent experiments.

Unsti mul ated 1h 2h 4h 6h g/m l  V Sol o 50 1h V + 1h LPS 1h L PS + 1h V 1h L PS + 2h V 1h L PS + 4h V 1h L PS + 6h V 0 1000 2000 3000 LPS Post-incubation Pre-inc V antigen

In document AMENAZAS LÓGICAS CAPÍTULO 7 (página 40-44)