Identification of cell types recognizing RNase A-derived fragments

5.3.4. Identification of cell types recognizing RNase A-derived

immune activation of monocytes (4.1.9.3) in comparison to PBMC (4.1.9.2) cells. The purity of the monocytes was controlled by FACS analysis (4.7). As shown in Figure 15, the isolated monocytes showed a purity of about 90%, whereas PBMCs contained only 28%

monocytes. The monocytes were stimulated in the same way as described for PBMC cells. The supernatants were used for detection of IFN-α. Upon transfection of CpG 2216 and RNA 40, there was practically no IFN-α secretion detectable in human monocytes (we found only a weak secretion for RNA 40), whereas these ligands induced type-I interferon in human PBMC cells. Stimulation with fragments derived by RNase A treatment induced a strong type-I interferon response in human PBMC cells and monocytes (Figure 47).

Influence of the serum concentration for the immunostimulatory ability of RNase A-derived fragments

Our initial findings showed that fragments derived by partially RNase treatment of self-RNA induced an immune response in human PBMC cells when complexed to DOTAP.

We then wondered whether different serum concentrations might influence the immunostimulatory potential of RNase-derived fragments in human PBMC cells.

Therefore, we used fragments generated by RNase treatment, complexed them to

0,0 0,2 0,4 0,6 0,8 Medium

CpG ODN 2216 RNA 40 MDCK-RNA RNase A digest

IFN-α (ng/ml) DOTAP

Monos PBMCs

Figure 47: Analysis of the cell type of human PBMC cells responsible for recognizing RNase A-derived fragments. Fragments derived by RNase A treatment were complexed to DOTAP and used for stimulation of human PBMC cells and monocytes.

IFN-α production was measured 24 hours post stimulation by ELISA. CpG 2216 (1 µM) and RNA 40 served as positive controls (for ca. 90 % purity monocytes n = 2, for ca. 70 % purity monocytes n = 1; one representative experiment is shown).

DOTAP and tested their immunostimulatory potential in human PBMC cells exposed to different serum concentrations. As shown in Figure 48, the generated fragments complexed to DOTAP showed a serum-dependent IFN-α induction in human PBMC cells.

The fragments induced the highest IFN-α response in human PBMC cells when using 1-2% serum, whereas for higher serum concentrations like 10% serum the immune response was decreased. With regard to other immune cells, like murine immune cells or human HEK293 cells which were maintained in 10% serum, this result is very important concerning the immunostimulatory ability of RNase-derived fragments.

IFN-α (ng/ml) 0,0 0,5 1,0 4,0

Medium CpG 2216 A/PR/8/MDCK-RNA HEK-RNA 5 µg/ml HEK-RNA 10 µg/ml RNase A digest 5 µg/ml RNase A digest 10 µg/ml Medium CpG 2216 A/PR/8/MDCK-RNA HEK-RNA 5 µg/ml HEK-RNA 10 µg/ml RNase A digest 5 µg/ml RNase A digest 10 µg/ml Medium CpG 2216 A/PR/8/MDCK-RNA HEK-RNA 5 µg/ml HEK-RNA 10 µg/ml RNase A digest 5 µg/ml RNase A digest 10 µg/ml Medium CpG 2216 A/PR/8/MDCK-RNA HEK-RNA 5 µg/ml HEK-RNA 10 µg/ml RNase A digest 5 µg/ml RNase A digest 10 µg/ml Medium CpG 2216 A/PR/8/MDCK-RNA HEK-RNA 5 µg/ml HEK-RNA 10 µg/ml RNase A digest 5 µg/ml RNase A digest 10 µg/ml

FCS

IFN-α (ng/ml) 0,0 0,5 1,0 6,0 12,0

AB-Serum

0 % 1 % 2 % Serum concentration

4 % 10 %

RNA/DOTAP

Figure 48: Influence of the serum concentration for stimulation of PBMC cells. PBMC cells were stimulated at different serum concentrations from 0 % to 10 % with different ligands. IFN-α production was measured 24 hours post stimulation by ELISA (n = 2, one representative experiment is shown).

CpG 2216 (1µM) and RNA 40 served as positive controls.

Variable IFN-α responses for RNase A-derived fragments in murine immune cells

To investigate whether RIG-I or MDA-5 mediates the recognition of the fragments derived from RNase A treatment; we intended to use Flt3-derived dendritic cells lacking the downstream of RIG-I and MDA-5 working adaptor molecule IPS. The ability of the fragments to induce IFN-α in PBMC cells was additionally controlled (4.1.9.2).

First, we analyzed the immune activation of Flt3-derived dendritic cells (4.1.9.1) and measured IFN-α secretion by ELISA (4.9). Positive controls for IFN-α induction were the TLR ligands RNA 40 and CpG 2216. Upon transfection of RNase A-derived fragments complexed to DOTAP, there was always little-to-no IFN-α response detectable in Flt3-derived DCs. From n = 17 experiments performed in Flt3-Flt3-derived dendritic cells and bone-marrow-derived dendritic cells, we observed in n = 5 experiments that the RNase A-derived fragments were not recognized by TLR7 (data not shown). For all the other experiments, there was no IFN-α secretion detectable upon stimulation with RNase A- generated fragments in wild-type Flt3-derived dendritic cells. The reason for the variable data might be a change in the FCS lot number. Regarding the result we found in human PBMC cells (namely, that the IFN-α inducing ability of the RNase A-derived fragments was dependent on the serum concentration), we tested different serum concentrations for Flt3-derived DCs. PBMC cells were normally stimulated in medium containing 2% AB serum, whereas Flt3-derived dendritic cells were cultivated in medium containing 10%

FCS. For Flt3-derived dendritic cells, we found also a serum-dependent IFN-α induction for the fragments derived by RNase treatment (Figure 49). Whereas for serum concentrations below 5%, there was an IFN-α response detectable, for 10% serum concentration there was no IFN-α response. But this observation has to be reproduced.

RIG-I dependent recognition of RNase A-derived fragments in HEK immune cells

To investigate whether RNase-derived fragments are recognized in an IPS-dependent way, HEK293 cells which lack functional TLRs (with the possible exception of TLR3) were transfected with plasmids encoding for RIG-I and MDA-5 (4.12) (Figure 50).

Figure 49: Analysis of the influence of serum for the recognition of fragments derived from RNase A treated self-RNAs. Either mock- or RNase A (0.0075 U/ml)-treated self-RNAs were complexed to DOTAP and used for stimulation of Flt3-derived dendritic cells generated from a wild-type mouse at a final RNA concentration of 10 µg/ml. RNA 40 and CpG 2216 served as positive controls. IFN-α secretion was measured 24 hours post stimulation by ELISA (for FCS Titration n = 1, one representative experiment is shown).

IFN−α (U/ml)

0 1 2 3 4 200

Medium CpG 2216 RNA 40 no RNA MDCK-RNA 1 µg/ml MDCK-RNA 2 µg/ml MDCK-RNA 5 µg/ml MDCK-RNA 10 µg/ml RNase A digest 1 µg/ml RNase A digest 2 µg/ml RNase A digest 5 µg/ml RNase A digest 10 µg/ml

DOTAP

2 % FCS 5 % FCS 10 % FCS

Lipofectamine 2000

DOTAP

no RNA A/PR/8/MDCK-RNA A/PR/8/MDCK-RNA + CIP A/PR/8/MDCK-RNA + RNase III no RNA A/PR/8/MDCK-RNA A/PR/8/MDCK-RNA + CIP A/PR/8/MDCK-RNA + RNase III RIG-I ligand 5`-3P RNA RIG-I ligand 5`-3P RNA + CIP RIG-I ligand 5`-3P RNA + RNase III 500 ng/ml 1 µg/ml 2 µg/ml 5 µg/ml 500 ng/ml 1 µg/ml 2 µg/ml 5 µg/ml 500 ng/ml 1 µg/ml 2 µg/ml 5 µg/ml 500 ng/ml 1 µg/ml 2 µg/ml 5 µg/ml

+ RNase A + RNase A + RNase III

+ RNase A + CIP

mock

MDCK-RNA

relative Light Unit (RLU) mock

+ RNase A + RNase A + RNase III + RNase A + CIP

MDCK-RNA

DOTAP Lipofectamine 2000

0 800 1600 4000

Medium no RNA Poly IC A/PR/8/MDCK-RNA A/PR/8/MDCK-RNA + CIP A/PR/8/MDCK-RNA + RNase III no RNA Poly IC A/PR/8/MDCK-RNA A/PR/8/MDCK-RNA + CIP A/PR/8/MDCK-RNA + RNase III RIG-I ligand 5`-3P RNA RIG-I ligand 5`-3P RNA + CIP RIG-I ligand 5`-3P RNA + RNase III 500 ng/ml 1 µg/ml 2 µg/ml 5 µg/ml 500 ng/ml 1 µg/ml 2 µg/ml 5 µg/ml 500 ng/ml 1 µg/ml 2 µg/ml 5 µg/ml 500 ng/ml 1 µg/ml 2 µg/ml 5 µg/ml

B)

recognition of RNA fragments in non-immune cells. A) HEK293 cells were cotransfected with plasmids encoding the IFN-ß luciferase reporter construct, RIG-I and MDA-5 by electroporation. B) summarizes the interesting part for RIG-I-transfected cells. Transfected cells were treated with Poly I:C (10 µg/ml), RIG-I ligand 5`-3P RNA (0.2 µg/ml), uninfected and A/PR/8/MDCK-RNA (2 µg/ml) each complexed to DOTAP or Lipofectamine 2000. In addition, the transfected cells were stimulated with RNase A

(0.0075 U/ml)-derived fragments from self-RNA

complexed to Lipofectamine 2000. These fragments were also RNase III- and CIP-treated. IFN-ß activation was determined by Dual Luciferase Assay (for RNAs in a concentration range between 200 ng/ml to 20 µg/ml complexed to Lipofectamine 2000 n = 6,

one representative experiment is shown; for

RNAs in a concentration range between 200 ng/ml to 50 µg/ml complexed to DOTAP n = 7, no experiment is shown).

The normally MDA-5-specific ligand Poly I:C was found to show enhanced IFN-ß activation in RIG-I-transfected cells, but when complexed to Lipofectamine 2000 there was little detectable IFN-ß increase in MDA-5-transfected cells. The question arose as to whether our MDA-5 was functional. RIG-I expressing HEK293 cells gained responsiveness to the RIG-I-specific control RIG-I ligand 5`-3P RNA complexed to Lipofectamine 2000. The A/PR/8/MDCK-RNA complexed to DOTAP or Lipofectamine 2000 was also recognized in RIG-I-transfected HEK293 cells in a CIP-independent but RNase III-dependent way, as described in section 5.2. Upon stimulation with the fragments derived from RNase A treatment complexed to Lipofectamine 2000 at a final RNA concentration of 1 µg/ml, IFN-ß activation was enhanced. As a control we also performed stimulation with untreated self-RNA, but there was no immune induction. The observed IFN-ß activation upon stimulation with RNase A-derived fragments was only detectable by using the transfection reagent Lipofectamine 2000, but not with DOTAP (data not shown). Upon RNase III treatment of the fragments derived from RNase A treatment, the immune response was abrogated showing the importance of the ds character of the fragments for the RIG-I-dependent immune response. The 5`-triphosphate has no influence on the recognition of the fragments, because dephosphorylation did not reduce IFN-ß activation. Thus, the RNase A-derived fragments are recognized in different ways by immune cells. Whereas in human PBMC cells IFN-α secretion was observed upon transfection with RNase A-derived fragments complexed to DOTAP at a final RNA concentration in the range of 5-50 µg/ml (further concentrations have not been tested; data only shown until 10 µg/ml), in human HEK293 cells IFN-ß activation was enhanced when the fragments were complexed to Lipofectamine 2000 at a final RNA concentration of 1 µg/ml. In conclusion, we could say that, depending on the immune cell type, different concentrations of the RNA fragments and different transfection reagents are necessary for inducing an immune activation. Regarding the results obtained by using HEK293 cells overexpressing RIG-I, we observed that RIG-I is the responsible receptor for recognizing RNase A-derived fragments. This result could not be confirmed by using respective knockout mice. Reasons for this variable immune response to the RNase A-derived fragments on murine immune cells might be that FCS is a problem or that the absence of a 5`-triphosphate end of the fragments might be essential for murine immune signaling.