To examine the effect of cladribine (CdA) on microglia function in vitro.

The lymphocyte reducing CdA is an approved therapy for relapsing-remitting multiple sclerosis (MS). Since CdA can be detected in the CSF during treatment, we hypothesized that it modulates function of microglial cells that play pivotal role in maintaining chronic inflammation in MS.

CdA was added to mouse primary microglia in concentrations of 0.01-10 μM considering (i) the putative CSF concentration and (ii) the activity of deoxycytidine kinase (DCK), a key enzyme in the generation of the active drug. Microglia were either naïve, or activated with proinflammatory LPS or anti-inflammatory IL-4. Cytotoxicity was examined with MTT assay; phagocytosis and granularity were assessed by flow cytometry; random migration was investigated by the IncuCyte live-cell imaging system; production of pro- and anti-inflammatory molecules was evaluated by qPCR and Mesocale. The expression of DCK was quantified by qPCR.

CdA had effect only on activated microglia, and this was unrelated to its cytotoxic effect. Medium CdA concentrations (0.1–1 µM) reduced phagocytosis, random migration, and granularity. High concentration CdA (10 μM) increased expression of pro-inflammatory IL-1β, TNF, iNOS and anti-inflammatory Arg1 and TNFR2 in LPS-stimulated microglia. Expression of Arg1 was also increased by IL-4 stimulation. DCK expression was increased by LPS and by IL-4 in microglia, and this was unaffected by CdA.

Activation of microglia with LPS or IL-4 increased the expression of DCK that is responsible for the active form of CdA. Medium range CdA that corresponds to CSF levels in humans induced a less activated microglia phenotype. Higher concentration than attainable in human CSF increased the expression of both pro- and anti-inflammatory markers. These data indicate a potential alternative mechanism of cladribine with direct effect on CNS resident cells.