RESULTS 4. MICROGLIA PROLIFERATION AND CYTOKINE SECRETION

3.4 Microglia proliferation and cytokine secretion

As I could demonstrate that microglia are able to secrete multiple cytokines as once, I investi-gated how microglia subpopulations are composed in case of diverse functions, i.e. TNFα secre-tion and proliferasecre-tion.

Figure 3.10–Proliferation rate of challenged microglia isolated from WT and transgenic mice. A) Prolifera-tion rate of WT microglia stimulated with 0.01 ng/ml, 1 ng/ml or 100 ng/ml of LPS or Pam3CSK4or 25µg/ml, 50µg/ml and 100µg/ml of Fibronectin (FN); n= 14-20. B) Proliferation rate of myd88^−/−and trif^lps2cells stimulated with 100 ng/ml of LPS and Pam3CSK4or 100µg/ml of FN for 24 hours. n=30-48.The difference to WT medium levels was analyzed via 2-way ANOVA and Tukey´s multiple comparison with asterisks in-dicating p-values *≤0.05; **≤0.005; ***≤0.0005. C) Viability assessment via the WST-1 assay of Microglia treated with increasing concentrations of BrdU for 24 hours. n=6. Data was analyzed via multiple t-test.

Data are presented as mean±SEM.

3. RESULTS 3.4. MICROGLIA PROLIFERATION AND CYTOKINE SECRETION

Since previous studies reported very controversial results on microglia proliferation upon chal-lenges such as LPS stimulation (Gebicke-Haerter et al., 1989; Chen et al., 2012; Fukushima et al., 2015; George et al., 2015), Ifirst examined the effect of any challenge on microglia proliferation, before investigating the temporal orchestration of proliferation and cytokine secretion. There-fore, I stimulated microglia with increasing concentrations of LPS, Pam3CSK4(both 0.01 ng/ml; 1 ng/ml; 100 ng/ml), and Fibronectin (FN) (25µg; 50µg; 100µg) for up to 48 hours and performed subsequently an ELISA based BrdU cell proliferation assay.

Analysis revealed that challenged microglia proliferate significantly less compared to untreated controls, and that this reduction becomes more pronounced the longer the challenge lasts (see figure 3.10A, p.37). Microglia stimulated with LPS showed a significant reduction in the prolif-eration rate for all tested incubation times and concentrations, ranging to a minimum of 22.1%

compared to medium control. The FN-stimulated microglia showed afirst impairment of prolif-eration after 24 hours for all used concentrations. At 48 hours, the decreasing effect seems to be reversed for low levels of FN (25µg/ml) but stayed consistent for the other concentrations. Here, the minimum proliferation rate reached 51.6 % of the medium control. Microglia stimulated with Pam3CSK4 showedfirst reductions in the proliferation rate after 12 hours of incubation with 1 ng/ml and 100 ng/ml. After 24 hours, the proliferation rate was significantly reduced for all used concentrations with a minimum of 28.6 %.

After demonstrating this reduction in the proliferation rate, I wanted to identify the responsible signaling pathway. Thus, I performed additional proliferation assays in myd88^−/− and trif^lps2 KO cells. For this purpose, I stimulated WT and KO microglia with high concentrations of the previously used stimuli for 24 hours (see figure 3.10B, p.37). A comparison between WT and myd88^−/− cells showed that, in contrary to stimulated WT cells, stimulation of myd88^−/− cells with Pam3CSK4and Fibronectin did not reduce the proliferation rate significantly as it was ob-served in stimulated WT cells. Only LPS-stimulated KO cells showed a significant reduction com-pared to unstimulated WT cells. This reduction, however, was much weaker than in LPS-treated WT cells. In contrast to MyD88-/- cells, trif^lps2cells behaved similar to WT cells. The significant reduction after stimulation was demonstrated for all used stimuli.

3. RESULTS 3.4. MICROGLIA PROLIFERATION AND CYTOKINE SECRETION

Additionally, I performed an WST-1 viability experiment with increasing concentrations of BrdU to exclude toxic effects of BrdU, which could bias the obtained proliferation data (see figure 3.10C, p.37). Microglia incubated with increasing concentrations of BrdU for 24 hours showed no differences in their viability compared to untreated control cells.

After assessment of the general proliferative behavior of challenged microglia, I tested if mi-croglia proliferation and TNFαsecretion occur simultaneously in the same cell once they are stimulated.

A

TNFα TNFα

EdU EdU

unstimulated control LPS stimulation

Microglia

Figure 3.11–Determination of TNFα-producing and proliferating microglia subpopulation sizes via FACS analysis: Microglia plated at 300000 cells/well were stimulated with medium or LPS (1 ng/ml) containing 5 µM EdU for 8 hours. TNFαand EdU positive cells were analyzed via FACS. A) Representative FACS plots for unstimulated and stimulated microglia. B) Quantification of the individual subpopulations. C) Quantifica-tion of all TNFαand EdU positive cells. Data was analyzed via multiple t-test (*≤0.05; ***≤0.0005). Data are presented as mean±SEM. n=7-8

3. RESULTS 3.4. MICROGLIA PROLIFERATION AND CYTOKINE SECRETION

Hence, I performed combined FACS stainings for TNFαand EdU, a marker for proliferating cells.

To do so, I stimulated microglia with 1 ng/ml LPS mixed with 5µM EdU for 8 hours. The obtained results of this double staining behaved quite similar to the previously performed one for TNFα and RANTES (seefigure 3.11A, p.39). Unstimulated, TNFα-positive microglia are composed of a small population of TNFαsecreting microglia (10.4 %) and a small population that is positive for both, secretion and proliferation (6.4 %) (seefigure 3.11B, p.39). This composition was also ob-served for the proliferating microglia subpopulation which included a subpopulation of dividing microglia with a size of 6.8 %.

Cell viability

Figure 3.12–Effect of EdU on microglia viability: mi-croglia were treated with increasing concentrations of EdU for 24 hours. Viability was assessed via the WST-1 assay.Data was analyzed via multiple t-test.

Data are presented as mean±SEM. n=6 LPS stimulation increased the TNFα

pro-ducing subpopulation to 23.95 %, whereas the double positive microglia population was stable at a size of 8 %. The proliferat-ing microglia subpopulation was not sig-nificantly affected by LPS stimulation but was slightly reduced to 3.8 %. However, the total percentage of proliferating mi-croglia decreased significantly upon LPS stimulation from approximately 13.1 % to 11.8 % (seefigure 3.11C, p.39), which con-firms my data obtained from the BrdU

proliferation assay. The viability of microglia remained unchanged after a stimulation with in-creasing concentrations of EdU (seefigure 3.12).

In summary, the proliferation rates in LPS, Pam3CSK4 and FN challenged microglia were re-duced, primariliy driven by the Myd88 signaling pathway. In addition, microglia can multitask as some cells are able to perform proliferation and cytokine secretion simultaneously. A corrup-tion of these results by a toxic effect of the used cell division markers could be excluded.