Discussion
To determine the effect of CdA on murine microglia, we examined phenotypic and functional properties of naïve, LPS-, and IL-4-stimulated microglia treated with different concentrations of CdA for 24 hours. We found that CdA only affects stimulated and not naïve microglia. Furthermore, different concentrations of CdA influenced different aspects of microglia function. CdA concentrations putatively overlapping with CSF concentrations in humans (0.1-1 µM) significantly reduced the granularity, phagocytotic ability, and motility of LPS-stimulated microglia, while higher CdA concentration (10 µM) increased the mRNA expression of pro- and anti-inflammatory molecules, but not protein secretion. In addition, we found that activation of microglia increased the expression of DCK responsible for the activation of the prodrug.
CdA can cross the BBB and thereby potentially exerts an effect on CNS resident microglia (2, 3). The maximum plasma concentration of CdA after administration of a 10 mg tablet is in the range of 0.07-0.1 µM (23). As the CSF concentration is 25% of the plasma concentration (2, 3), the maximum CSF concentration attainable is approximately 0.019-0.025 µM. The active form of CdA is CdA triphosphate, which is the phosphorylated form of the drug (2, 3). The first step of this process is catalyzed by deoxycytidine kinase (DCK), the activity of which is 10 times lower in mice than humans (17, 18). The maximum CdA concentration detected in the CSF of patients thus may correspond to murine concentrations in the range of 0.1-1 µM. Therefore, we applied CdA concentrations of 0.01-10 µM in this study of murine microglia.
We first investigated whether the applied CdA concentrations were toxic to microglia. We found that the viability of microglia was not affected during the short culture conditions, similar to a previous study that applied the intravenous compound to rat microglia cultures (16). Therefore, we considered that using these concentrations for short time stimulation would not have a depleting effect on microglia.
LPS stimulation resulted in a morphological change of microglia characterized by an increase in SSC and thus a more granular morphology, as expected. Co-treatment with CdA 0.1 µM showed a tendency towards decreased granularity suggesting that CdA may have effect on the functional properties of microglia.
Therefore, we examined the phagocytotic ability and motility of microglia. We found that LPS stimulation significantly increased the phagocytotic ability of microglia, which correlated with the pro-inflammatory phenotype induced by LPS. Such increased phagocytotic ability of LPS-stimulated microglia was decreased by CdA in the range of 0.1-1 µM. Furthermore, the decrease in phagocytosis by CdA corresponds to the observed decreased granularity of microglia. CdA had no effect on naive microglia, which is consistent with findings of former studies performed on rat microglia (16), and murine dendritic cells (24).
Microglia motility is important for maintaining CNS homeostasis via interaction with other cells and scanning of the CNS environment (8, 11, 25, 26). We measured the random migration by (i) track displacement, i.e. the distance from start to end point, and (ii) total distance travelled that includes twists and turns and describes the patrolling of migrating cells. LPS stimulation significantly increased track displacement and showed a tendency towards increased total distance travelled. This is consistent with the acquisition of effector functions induced by LPS and a tendency towards increased patrolling of the surroundings. In contrast to random migration, LPS decreased chemotactic migration of rat microglia (27). We also found that CdA (0.1-1 µM) did not affect the motility of resting microglia.
Observing changes in morphology, phagocytotic ability and migration of LPS-stimulated microglia, we next examined how CdA alters gene expression and protein secretion of microglia stimulated with LPS and with anti-inflammatory IL-4. LPS increased the expression of pro- and anti-inflammatory markers consistent with a recently published study (27). Co-treatment with CdA in the high concentration (10 µM) increased the gene expression of both pro- and anti-inflammatory markers, such as IL-1β, TNF, iNOS, Arg1, and TNFR2 suggesting a potentiating effect on activated microglia. Interestingly, only the gene expression of anti-inflammatory TNFR2, but not the pro-inflammatory TNFR1 (28) was potentiated by CdA. The increased protein secretion of IL-1β, TNF, and IL-10 induced by LPS was not further increased by CdA (0.1-10 µM) consistent with a previous study on rat microglia (16). This suggests that CdA only affects gene expression but not protein secretion. We may speculate that accumulation of CdA in microglia may interfere with RNA processing besides interference with DNA replication and this may alter translational mechanisms.
We also found that IL-4 stimulation resulted in an increased gene expression only of Arg1 but it was not affected by CdA co-treatment. This could indicate that IL-4 is a less potent stimulus in the short-term, and microglia may not be sufficiently activated for the potentiating effect of CdA. In summary, CdA in the applied concentration did not have effect on cytokine secretion of activated or resting microglia in short-term primary cultures. The observed effect on gene expression was achieved by higher concentrations of CdA than may be expected in the human CSF during treatment.
Activation of microglia by LPS and IL-4 also increased the gene expression of DCK that phosphorylates CdA to its active form. This may suggest that activated microglia may increase its ability to respond to CdA. In lymphocytes, CdA interferes with DNA synthesis and repair resulting in depletion of both proliferating and resting cells (2, 3). The mechanism by which CdA affects microglia functions remains to be determined, and our data do not indicate microglia depletion in the short term. A previous study also suggested that an additional mechanism beside depletion may be responsible for the effects on microglia, such as reduced proliferation (16). In dendritic cells, the function of CdA is partially independent from its phosphorylation (24); but it may not be true in microglia, as the effect of CdA on proliferation was lost upon addition of a competitive substrate of DCK (16).
In summary, we found that CdA used in a concentration that may be reached in the human CSF during approved treatment of MS, affected activated but not naïve microglia; this may be related to the upregulated gene expression of DCK upon activation. Co-treatment with CdA reduced the phagocytotic ability and random migration of activated microglia, while viability was not affected among the applied conditions. Although gene expression of both pro- and anti-inflammatory molecules was potentiated by CdA, it was induced only by higher concentration than should be expected in the CSF, and protein secretion was not altered. CdA also potentiated the gene expression of anti-inflammatory TNFR2. These data suggest that beside depletion of peripheral lymphocytes, CdA may induce additional effects particularly in CNS resident cells that contribute to its beneficial effects in MS.