Only small amounts of CD14 are internalized by microglia during the TLR4-triggered response CD14 was shown to be especially important for LPS-triggered responses that are dependent on TRIF

Here, CD14 regulates the internalization of the TLR4-LPS complex, thereby enabling for the second wave of signaling from endosomes. However, the CD14 internalization has been so far documented only in DCs and peripheral macrophages (Ling et al., 2014; Zanoni et al., 2011). It was thus interesting to investigate whether (i) the endocytosis of a TLR4-LPS complex could be shown also in microglia, (ii) whether it requires CD14 assistance and (iii) whether this happens to the same extent as in BMDM. Microglia, and as a control BMDM, were stimulated with S- as well as Re-LPS chemotypes. The rationale behind using both was that even though S-LPS was shown to have a direct link to TRIF-dependent signaling in peritoneal macrophages (Jiang et al., 2005) and thus its use would appear better for these studies, in microglia the situation differs. Microglia did not differentiate between S-LPS and Re-LPS for induced secretion of the respective cyto- and chemokines. Both used MyD88 and TRIF adaptor proteins, even in

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nearly the same way (Regen et al., 2011). After 20 min of incubation with Re- and S-LPS at two different concentrations, surface CD14 was measured by flow cytometry in order to detect a loss due to the internalization of the complex with LPS-TLR4. Figure 4.29A shows that CD14 levels on the surface of microglia did not significantly change upon stimulation with either of the LPS chemotypes. Less CD14 was detected on BMDM as stimulated with higher concentrations of both LPS variants. However, the decrease was not as dramatic as described in previous reports. In order to rule out that a failure in detecting a CD14 loss was due to inappropriate timing, microglia were stimulated with LPS for 10, 30 and 90 min. As recapitulated by the histograms in Figure 4.29B, LPS did not lead to any changes in CD14 expression on microglia at any time point after stimulation.

Figure 4.29: Flow cytometry analysis cannot show a significant loss of CD14 surface expression upon TLR stimulation.

(A) Microglia or BMDM were stimulated for 20 min with S-LPS or Re-LPS at the indicated concentrations.

Subsequently, cells were FcγRII/III-blocked, stained with PE-anti-CD14 and Pacific Blue-anti-CD11b Abs for acquisition by flow cytometry. For data analysis, only CD11b⁺ events were included. MFI was normalized to values obtained with unstimulated cells (set to 100%). Data are mean ± SEM with n=3-4. (B) Microglia were stimulated with Re-LPS (10 ng/ml) for the indicated time points. Loss of CD14 surface expression was assessed as in (A).

(C) Cells were stimulated with Pam₃CSK₄, Re-LPS (‘high’) (both 10 ng/ml), Re-LPS (‘low’, 0.1 ng/ml), poly(A:U) (50 μg/ml) or bovine pFN (100 µg/ml)) for the indicated time points. Loss of CD14 surface expression was assessed as in (A). Data is mean ± SEM with n=2 from 2 experiments. (Flow cytometry analysis of surface CD14 on BMDM was performed by Anne-Sophie Ernst during her Master Thesis period.)

The previous results showed that the microglial TRIF-dependent production of CCL5 in the response to pFN but also to other TLR agonists was reduced in a CD14-dependent manner (Figs. 4.9B and 4.16B). To see, whether it is the CD14 assistance that is needed for the full response by these agonists and whether this could differ as compared to a LPS stimulation, microglia were stimulated with pFN, with Pam3CSK4

(as a TLR2/6 agonist) and poly(A:U) (as a TLR3 agonist). However, as seen upon stimulation with LPS, CD14 surface levels were not changed upon stimulation with any of the ligands, no matter how long the cells were incubated (Fig. 4.29C).

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In a second approach, internalized CD14 was detected by confocal microscopy. For that, microglia were stimulated with LPS for 30 min or left untreated. After fixation, the cells were stained to detect intracellular CD14. Prior to stimulation, CD14 was located at a high density at the surface of microglia (white arrows) (Fig. 4.30A-D), but some pool of CD14 was also located inside the cells, i.e. in proximity to the nucleus (Fig. 4.30M). 30 minutes after LPS stimulation, the layer of membrane CD14 was rearranged (Fig. 4.30E-L, indicated by white star), and CD14 was found to be concentrated inside the cell (Fig. 4.30N). However, the amount of internalized CD14 was low and most of the CD14 remained at the cell surface. (A more detailed study involving confocal imaging is a part of the Master Thesis of Anne-Sophie Ernst, who performed all these evaluations.)

Due to the difficulty to track a loss of CD14 from the surface membrane by flow cytometry, it was not clear whether there were differences between microglia and BMDM and to which extent CD14 truly facilitated endocytosis. Apparently, microglia internalize only a small amount of the total surface-expressed CD14. This is probably sufficient to support a cellular incorporation of the LPS-TLR4-complex.

Indeed, the amount of TLR4 could be relatively small (unpublished observations), and as a consequence, not much reduction of the surface CD14 can be noticed. Yet our functional data on TRIF-dependent inductions (e.g. MHC I) clearly indicated that TLR4 did have an intracellular action.

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Figure 4.30: Microglia internalize only small amount of CD14 upon LPS stimulation.

(A) Microglia (wt) were stimulated for 30 min with S-LPS at indicated concentrations or left untreated.

Subsequently, cells were stained with fluorescently labeled antibody against CD14 (red), lectin (green) and the nucleus (blue). Images were obtained by scanning confocal laser microscopy and collected at 1 µm intervals to create a stack in the z axis (Z). (A-L) Representative images show (A-D) unstimulated microglia, (E-H) microglia stimulated with LPS 0.1 ng/ml, (I-L) microglia stimulated with LPS 10 ng/ml. (M) Higher magnification of (B), red channel only. (N) Higher magnification of (J), red channel only. Dotted line indicates accumulation of CD14 close to the nucleus. Arrow heads indicate evenly distribution of CD14 on cell surface, stars indicate accumulation of CD14 on the surface. (Imaging was performed by Anne-Sophie Ernst during her Master Thesis period.)

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