Cladribine (10 µM) alters expression but not secretion of pro- and anti-inflammatory molecules by activated microglia
Next, we investigated the effect of CdA on the relative mRNA expression and secretion of selected pro- and anti-inflammatory markers after stimulation with LPS or IL-4 for 24 hours. The expression of IL-1β, TNF, IL-10, TNF-receptors, iNOS, and Arg1 were examined by qPCR. The secretion of IL-1β, TNF, and IL-10 were investigated by Meso Scale Discovery.
LPS stimulation resulted in an increased mRNA expression of IL-1β, TNF, and IL-10 compared to control (Figure 4A ). Treatment with CdA 10 µM further increased the mRNA expression of IL-1β and TNF in LPS-stimulated cells (Figure 4A ). CdA (0.01-10 µM) did not affect the expression of these cytokines in naïve microglia (data not shown).
The protein secretion of IL-1β, TNF, and IL-10 was increased by LPS stimulation (Figure 4B ). In contrast, CdA (0.1-10 µM) did not affect the secretion of IL-1β, TNF, and IL-10 from LPS-stimulated or naïve microglia (Figure 4B ).
LPS stimulation increased the mRNA expression of both TNFR1 and TNFR2 compared to control. CdA 10 µM further increased the expression of TNFR2 but not TNFR1 (Figure 5 ). CdA (0.01-10 µM) did not affect the expression of TNF receptors in naïve microglia (data not shown).
LPS stimulation resulted in a significantly increased expression of iNOS and Arg1 mRNA compared to the untreated control, but these were reduced by DMSO co-treatment (Figure 6 ). Co-treatment with 10 µM CdA significantly increased the expression of iNOS and Arg1 in LPS-stimulated microglia compared to DMSO control (Figure 6 ). CdA did not affect the mRNA expression of iNOS or Arg1 in naïve microglia (data not shown).
IL-4 stimulation for 24 hours only increased Arg1 expression, whereas no changes were observed in mRNA expression of IL-1β, TNF, IL-10, TNFR1, and TNFR2. Furthermore, the expression of these genes was not affected by CdA co-treatment (Figure 7 and Supplementary Figure 1-2 ). IL-4 did not induce the expression of iNOS (data not shown). Similarly, no difference was observed in the secretion of IL-1β, TNF, and IL-10 from IL-4-stimulated and CdA co-treated microglia (Supplementary Figure 1) .