RA protects normal TLR9-stimulated B cells against spontaneous and irradiation-

The role of MCL1

In line with previous reports (210;447), we showed in paper II that TLR9-mediated stimulation of B cells reduces both spontaneous-, and irradiation-induced apoptosis. The enhanced survival was accompanied by elevated levels of anti-apoptotic proteins like BCL2, BCL-XL and MCL1. Importantly, we found that RA potentiated the anti-apoptotic effects of TLR9-stimulation on both spontaneous, doxorubicin- and irradiation-induced cell death.

Notably, this enhanced RA-mediated survival involved a strong and specific increase in expression of the anti-apoptotic protein MCL1.

The finding that RA specifically augmented the expression of MCL1 was particularly intriguing, as MCL1 has been ascribed a prominent role in the survival of GC B cells and memory B cells, as well as for the longevity of plasma cells (211;448). With the identification of an RARE in the MCL1-promoter, our results suggested that the enhancing effect of RA on TLR9-mediated B cell survival could be explained by the ability of RA to directly induce the transcription of MCL1. Our results supported the previous in silico-based classification made by Balmer and Blomhoff, of MCL1 as a gene that appears to be a direct target of RA (306). It should however be noted, that we found a differential effect of RA on MCL1 expression in non-irradiated and irradiated cells. Thus, whereas RA alone had no effect on MCL1 expression in irradiated B cells, RA significantly increased the expression of MCL1 in non-irradiated cells. The inability of RA by itself to induce MCL1 expression in non-irradiated cells could be explained by the previous findings that MCL1 is degraded in response to irradiation (449;450). Interestingly, the differential effects of RA on MCL1 expression correlated with its ability to protect the B cells from cell death, i.e. the protective effect of RA alone against spontaneous cell death was lost in irradiated cells.

Although we demonstrated in paper II that siRNA against MCL1 reduced the protective effect of RA on cell death, our results could not exclude the possibility that also other mechanisms might contribute to the anti-apoptotic effects of RA. In general, the level of TLR9 expression dictates the responsiveness to CpG-ODN (451). We found that treatment of CD19⁺ B cells with CpG-ODN resulted in increased TLR9 expression, and that RA further enhanced this expression (unpublished data, Kristine Lillebø Holm). The RA-induced expression of TLR9 could be due to the enhanced IL-10 secretion imposed by RA in TLR9-stimulated B cells (paper I and (381;382)), as stimulation of naïve B cells with IL-10 has previously been shown to increase the expression of TLR9 (65). We cannot exclude the possibility that RA also promotes survival of TLR9-stimulated cells by acting as an antioxidant. Oxidative stress is a well-known factor in irradiation-mediated apoptosis (452;453), and RA may therefore inhibit apoptosis by its ability to act as an antioxidant (454;455).

In a study on murine T cells, TLR9-stimulation was found to protect the T cells against irradiation-induced apoptosis by increasing the activation of the DDR kinases ATM/ATR and CHK1/2 (456). Based on this study, we used wortmannin (inhibitor ATM/ATR) as well as debromohymenialdisine (DBH) and 7-hydroxystaurosporine (UCN-01) (inhibitors of CHK1/2) to explore the possible involvement of these kinases in the radioprotective effect of TLR9 in human B cells. In contrast to the result obtained in the study by Zheng and coworkers, we did not find that these kinase inhibitors affected the protective effect of CpG-ODN against spontaneous or irradiation-induced apoptosis (unpublished data, Kristine Lillebø Holm). The inhibitors tended to slightly reduce the radioprotective effects of RA, but the effects were not statistical significant. In support of these results, we did not find any increased activation of ATM or ATR in B cells stimulated with CpG-ODN and RA (paper III).

The impact of RA on DNA lesions and DDR in TLR9-stimulated B cells

In paper II we identified a vital role of MCL1 in the anti-apoptotic effects of RA on normal B cells. However, there were still two aspects that required investigation. First, we explored the possibility that the RA/TLR9-mediated survival of B cells involved reduced DNA damage and/or increased DNA repair. On the other hand, it was also important to investigate the alternative possibility i.e. that stimulating the B cells with RA and CpG-ODN would result in survival of cells with even more damaged DNA. The latter possibility could potentially

contribute to mutagenesis and cancer development. In paper III we addressed these aspects, by assessing the induction and repair of DNA strand breaks, as well as measuring the levels and activity of proteins involved in the DDR.

The DDR is a signaling network that detects and responds to DNA damage (219;457).

Upon DNA damage, kinases such as ATM and DNA-PKcs are activated and are able to phosphorylate and activate downstream targets such as p53 and H2AX (219;457). To evaluate the induction of DNA damage in response to stimulation and irradiation, we therefore measured the activation of these components. We did not find support for the first hypothesis, i.e. that RA alone or in the presence of CpG-ODN promoted cell survival by reducing the level of DNA damage in response to irradiation. When addressing the second possibility, i.e.

that the RA/TLR9-stimulated B cells would survive with increased DNA damage, we first obtained some disturbing results. Thus, stimulation of the cells with CpG-ODN alone or in the presence of RA resulted in enhanced phosphorylation and activation of the downstream DDR target p53 in irradiated cells, suggesting increased level of DNA damage. However, closer examination revealed that the effects on p53 simply was due to CpG-ODN enhancing the expression of p53 at the mRNA level in both irradiated and non-irradiated cells, with RA having no additional effect. The p53-target gene p21^Cip was not induced upon TLR9-stimulation in non-irradiated cells, supporting the notion that TLR9-induced expression of non-phosphorylated p53 was due enhanced transcription of the TP53 gene. The induced expression of p53 at the mRNA level was consistent with studies from the 80`s demonstrating mitogenic induction of TP53 transcription (458;459). In fact, these early studies were taken as support for the widely accepted misimpression that p53 was an oncogene and not a tumor suppressor. The activity of the DDR kinases upstream of p53, ATM and DNA-PKcs, were not affected by stimulation with CpG-ODN and RA, supporting the notion that these stimulants did not promote DNA damage in B cells.

Having given a plausible explanation for the TLR9-mediated induction of p53 independent of DNA damage, the finding that CpG-ODN increased the levels of phosphorylated H2AX (H2AX) gave us a new surprise. As an established target of the DDR kinases, the level of H2AX is considered to coincide with DNA lesions (225;420). It turned out, however, that the enhanced level of H2AX in response to TLR9-stimulation was due to increased levels of H2AX itself – both in irradiated and non-irradiated cells. This conclusion was supported by previous reports suggesting that γH2AX-levels in various cell types and

under certain conditions are related to the expression of H2AX (460). The mechanism whereby TLR9-stimulation of B cells enhances the expression of H2AX is unknown.

However, we believe that it may be the same mechanisms as the one behind the observed TLR9-mediated transcription of TP53. It is possible that stimulation of B cells via TLR9 results in changes in the chromatin structure, favoring certain transcriptional events. An important practical consequence of our results is that analyses of both γH2AX and phosphorylated p53 should be interpreted with caution when studying stimulated lymphocytes.

A final proof for the notion that TLR9-stimulation does not enhance the levels of DNA lesions came from measurement of DNA strand breaks by using the alkaline comet assay. As expected, we found that irradiation of B cells rapidly induced DNA strand breaks.

However, neither CpG-ODN alone or in the presence of RA affected the levels of lesions. The comet assay also gave us valuable information regarding the repair of damaged DNA, as did also the analyses of γH2AX levels. Both assays suggested that the repair of irradiation-induced DNA damage was efficient, and that neither of the stimulants affected the rate of repair. In general, the comet assay displayed more rapid repair than what was indicated by the decline in γH2AX levels. The differential repair kinetics measured by the two methods is probably due to a previously described delay in the phosphorylation of H2AX in response to DSBs (460). Although the number of DSBs is at its highest immediately after irradiation, the phosphorylation of H2AX shortly after irradiation is too low to reflect the true number of DSBs due to a rate-limiting factor upstream of H2AX phosphorylation.

In conclusion, we did not find support for RA/TLR9-mediated stimulation of B cells limiting the induction of – or inducing the repair of DNA damaging lesions. Neither did the results imply that non-irradiated or irradiated B cells survive with increased levels of DNA lesions when treated with CpG-ODN alone or with RA. Instead, the results strengthen the hypothesis proposed in paper II that B cells stimulated via TLR9 and with RA survive due to increased expression of anti-apoptotic BCL2-proteins. We suggest that RA/TLR9-mediated stimulation of B cells may provide the cells with p53 as a “fit for fight” protective barrier against potentially harmful consequences of enhanced B cell activation and proliferation. In the absence of such damage, p53 will remain at low levels and in an inactive state - ready for being rapidly activated by phosphorylation upon induction of DNA damage.

4.2.1.2 CpG-ODN and vitamin A in prevention and treatment of B-cell malignancies