Buffy coats of healthy donors were acquired from the blood service (Institute of Transfusion Medicine University Hospital Leipzig; ethics license 272-12-13082012). Human peripheral blood mononuclear cells were obtained from buffy coats by Ficoll-Paque Plus density centrifugation. After washing with PBS containing 0.3 mM EDTA, monocytes were isolated by counter-flow elutriation using the JE-5.0 elutriation system (Beckman Coulter, Brea, CA, USA), as described previously (12).

Monocytes (5×10⁵/ml) with a purity of at least 90%, as assessed by flow cytometry using anti-CD14-APC Ab (M5E2, BioLegend, San Diego, CA, USA) were suspended in RPMI 1640 medium supplemented with 10% (v/v) FCS, 100 U/ml penicillin and 100 mg/ml streptomycin and differentiated with 500 IU/ml GM-CSF (Leukine, sargramostim) to GM-MΦ or 50 ng/ml M-CSF (R&D Systems, Minneapolis, MO, USA) to M-MΦ at 37°C and 5% CO₂ in teflon bags (Zell-Kontakt, Nörte-Hardenberg, Germany; fluorinated ethylene propylene foil, 50 µm, hydrophobic). After seven days, MΦ (5×10⁵/ml) were suspended in RPMI 1640 medium supplemented with 10% (v/v) FCS, 100 U/ml penicillin and 100 mg/ml streptomycin and incubated for 2 h in cell culture plates before starting experiments. Flow cytometry analysis of MΦ cell surface markers was performed as described previously (13), using direct dye-labelled antibodies anti-CD14-APC (M5E2, BioLegend), anti-CD40-PerCp (Elabscience Biotechnology, Houston, TX, USA), anti-CD80-PE (L307, BD Pharmingen, San Jose, CA, USA) and the unlabelled antibody anti-CD163 (GHI/61 BD Pharmingen) in combination with a FITC-labelled goat anti-mouse secondary antibody.

Virus infection was carried out as previously described (11). Stock virus preparations of the low-passaged (up to passage 10) clinical isolate RVi/Wuerzburg.DEU/47.11_12-00009 (Wb-12) were prepared and titered by standard plaque assay on Vero cells (green monkey kidney epithelial cell line, ATCC CCL-81). Vero cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific) with 10% (v/v) FCS. Human MΦ were infected with RV (approved by the ethics committee at the Medical Faculty of the University of Leipzig; ethics license 001/19-ek) at a multiplicity of infection (MOI) of 1.5. Cells that were not infected (mock) or treated with RV inactivated by exposure to UV-light (750,000 μJ/cm² (UV-Stratalinker 2000, Stratagene)) (UV-RV) served as controls. UV-RV was applied at the same MOI as RV. Before infection culture media were discarded and RV or the respective controls (mock and UV-RV) were added. After 2 h of incubation the media were removed, MΦ were washed with PBS and suspended in fresh culture media.

The immunofluorescence analysis of RV-infected MΦ was performed as described previously (14). Briefly, MΦ were cultivated on glass slides, washed at 24 hours post-infection (hpi) with PBS, and fixed with 2% (w/v) paraformaldehyde (PFA) in PBS for 15 min. After fixation, cells were washed with PBS and permeabilized with 0.3% Triton X-100 (v/v) in PBS at 37°C for 30 min followed by blocking with 0.3% Triton X-100 (v/v) and 5% normal goat serum (v/v) in PBS for 30 min at 37°C in a humidified chamber. Thereafter cells were incubated with primary mouse monoclonal antibody to rubella capsid protein (clone 2-36; 1:200 dilution; Meridian Life Science Inc, Saco, ME USA) for 60 min at 37°C followed by a three-time washing step with PBS. After incubation with the Cy3 conjugated donkey anti-mouse IgG secondary antibody (1:200 dilution; Dianova, Hamburg, Germany) for 45 min, the cells were washed again three times with PBS. To label F-actin, a 1:40 dilution of Alexa Fluor 488 phalloidin (Thermo Fisher Scientific) was added followed by DNA counterstaining during the mounting step with Fluoromount G (Invitrogen Life Technologies, Thermo Fisher Scientific, Schwerte, Germany) containing DAPI at a suitable working concentration. Stained cell samples were analyzed with a Leica TCS SP8 laser scanning confocal microscope and processed using Corel DRAW x7 with slight alterations to brightness and contrast.

Viral genome copies were determined after isolation of total cellular RNA by ReliaPrep™ RNA Miniprep System (Promega) followed by one-step Taq Man RT-qPCR as described previously (15).

TNF-α and IFNs were determined in culture supernatants of MΦ (5×10⁵/ml) using a human TNF-α ELISA (PeproTech) or a LEGENDPLEX human type 1/2/3 Interferon panel (5-plex) kit (BioLegend), respectively according to the manufacturer’s protocol.

Western blot analysis was carried out as described previously (12). After washing with PBS, MΦ (5 ×10⁵) on culture plates were suspended in 100 µl RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Nonidet P 40, 0.5% deoxycholate, 0.1% SDS; pH 7.5) supplemented with Complete EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and with phosphatase inhibitors (1 mM Na₃VO₄ and 50 mM NaF). After sonication on ice, samples were centrifuged for 5 min at 15,000 g and 4°C. In resulting supernatants protein concentrations were determined using a DC Protein Assay (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol. Cell lysates (25–30 µg) were boiled in 1× Laemmli sample buffer, run on a 12% SDS-polyacrylamide gel (Protean II, Bio-Rad GmbH) and transferred to polyvinylidene difluoride membranes (PVDF membrane, Amersham Biosciences, Munich, Germany).

After blocking with 5% milk powder, PVDF membranes were probed with the following antibodies: anti-phospho-Stat-1 rabbit Ab (Tyr701, D4A7, 1: 1,000); anti-phospho-NF-κB rabbit Ab (p65, Ser536, 93H1, 1: 1,000); anti-phospho-TBK1 rabbit Ab (Ser172, D52C2 XP^®, 1: 1,000); anti-phospho-ERK1/2 mouse Ab (Thr202/Tyr204, E10, 1: 2,000); anti-phospho-c-Jun amino-terminal kinase (JNK) rabbit Ab (Thr183/Tyr185, 81E11, 1: 1,000) (all from Cell Signaling, Danvers, MA, USA), anti-rubella (E1) mouse Ab (1: 500, Merck, Darmstadt, Germany) or anti-β-actin mouse Ab (clone AC 74, 1: 2,000, Sigma-Aldrich, St. Louis, USA). Primary Antibody were detected with the following POD-conjugated secondary antibodies: goat anti-rabbit IgG Ab (1: 20,000, Dianova) or goat anti-mouse IgG Ab (1: 8,000, Sigma-Aldrich, St. Louis, USA). Chemiluminescent detection on membranes by using ECL-A/ECL-B substrate (both from Sigma Aldrich) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) were analyzed on a CCD-camera Stella (raytest Isotopenmessgeräte GmbH, Straubenhardt, Germany). For Densitometric analysis, the AIDA Image Analyzer Software (Elysia-raytest GmbH, Straubenhardt, Germany) was used and sample values were expressed as density relative to β-actin. For stripping the membrane, blots were washed 5 min with distilled water followed by a 3 times incubation with 0.1 M glycine-HCl (pH 2.0) for 5 min. After an additional washing step, membranes were again blocked with 5% milk powder before re-probing with the next primary antibody.

Macrophages (3×10⁵) were washed with PBS and total RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. DNase I treatment and reverse transcription of equal amounts of RNA (at least 250 ng) to cDNA were performed as previously described (12).

For real-time PCR 1.5 µl of cDNA template were added to a reaction mixture containing 7.5 µl of the SYBR Green PCR mastermix (Bio-Rad, Hercules, CA, USA), 250 nM forward and reverse primers (see below) in a final volume of 15 µl. The following primers were used: GNB2L1 (fwd 5`-GAGTGTGGCCTTCTCCTCTG-3`; rev 5`-GCTTGCAGTTA GCCAGGTTC-3`) (16), IFN-β (fwd 5`-AACTTTGACATCCC TGAGGAGATTAAGCAG-3`; rev 5`-GACTATGGTCCAGGC ACAGTGACTGTACTC-3`) (17), IFN-λ1 (fwd 5`-GCAGGTTC AAATCTCTGTCACC-3`; rev 5`-AAGACAGGAGAGCTGC AACTC-3`) (18), IFNAR1 (fwd 5`-TCAGGTGTAGAAGAAA GGATTGAAA-3`; rev 5`-AGACACCAATTTTCCATGACGT A-3`) (19), IFNLR1 (fwd 5`-TGGGTGGAGTCCGAATACCT-3`; rev 5`-GAGTGATCTGGACTGGCTGG-3`).

IFNLR1 primers were designed using the BLAST and the Primer3 program. The PCR reactions were performed using the CFX connect real-time PCR detection system (Bio-Rad, Hercules, CA, USA) according to the following protocol: initial denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s (denaturation), at 60°C for 30 s (primer annealing) and at 72°C for 30 s (extension/synthesis). Product quantification was carried out at 72°C. Negative controls using water as template were run under the same conditions. Gene expression was calculated using the ΔΔCt method as previously described with GNB2L1 (Guanine nucleotide-binding protein subunit beta-2-like 1) as the reference gene (20).

The OCR and ECAR were measured using an XFp analyzer (Agilent Seahorse Technologies, Santa Clara, CA, USA). XFp Seahorse plates were seeded with MΦ (200 µl per well) at a density of 2x10⁵/ml. After 2 h cells were analyzed or subjected to virus infection. Thereafter culture medium was replaced by XF base medium (Agilent Seahorse Technologies) supplemented with 2 mM glutamine and 11 mM glucose. The cells were then incubated at 37°C in a CO₂-free incubator for 45 to 60 min. Hereafter basal OCR and ECAR were determined in the XFp analyzer, before LPS (100 ng/ml) was injected (measurement point 6). Further measurements were taken for another 1 h. Agilent Seahorse software Wave 2.3 was used for data analysis.

Statistical analysis was done using GraphPad PRISM or SigmaPlot^® software. Statistical significance was calculated with Student’s t-test or ANOVA test as indicated in the respective figure legend. Statistical significance is classified as follows: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. ANOVA test of percentage or fold change data was performed after log transformation.

Before infecting cells we verified the well documented phenotypical characteristics of GM- and M-MΦ (21) by showing that M-MΦ specifically expressed CD163 (M-MΦ 613.5 ± 663.6; GM-MΦ 23.5 ± 15.2) and CD14 (M-MΦ 5094.0 ± 773.7; GM-MΦ 1700.3 ± 933.1), while expression levels of CD80 (GM-MΦ 31.7 ± 8.4; M-MΦ 7.0 ± 7.2) and CD40 (GM-MΦ 167.7 ± 29.3; M-MΦ 65.7 ± 17.0) were constitutively higher on GM- than on M-MΦ. Based on recent findings, showing that after RV-infection neither the amount of extracellular virus particles nor the number of RV-positive cells varied between GM- and M-MΦ (11), we here examined the cellular morphology of both subsets after infection with RV by staining of F-actin. We found cluster formation ( Figure 1A ) which was slightly more prevalent in M-MΦ than in GM-MΦ.

Similar to RV, UV-inactivated RV (UV-RV) was detected in both cell types. However, the genome copies, which hardly varied between GM- and M-MΦ were 2 to 3 log steps lower than after RV infection indicating that UV-RV does not replicate within the cell and that the copies reflect the original incoming virus genome ( Figure 1B ).

The fact that UV-RV is taken up by human MΦ without initiation of RV replication and that UV-RV contains the virion components as well as factors released from Vero cells during virus stock preparation, make it a useful control when studying RV infection.

According to the time course of virion production performed over 24 h the number of infectious virus particles generated by GM- and M-MΦ was almost identical between 4 and 24 h post infection (hpi) ( Figure 1C ). At 6 hpi the number slightly decreased (indicating the eclipse phase) and at 24 hpi after a delay phase between 8 and 12 hpi virion production and release to the medium were increased.

The similarity of RV replication rates between GM-MΦ and M-MΦ was also reflected by expression levels of viral E1 protein as detected by western blot analysis at 24 hpi ( Figure 1D ). Thus, possible differences between GM- and M-MΦ in LPS-induced responses are unlikely to be due to differences in the course of infection.

Next, we determined the impact of RV infection on the inflammatory response of GM- and M-MΦ by analyzing the production of the cytokines TNF-α, IFN-α2 and IFN-β (type I), IFN-γ (type II), IFN-λ1 and λ2/3 (type III). IFN-γ and IFN-λ2/3 were not detectable under any assay conditions.

As seen in Figure 1E , RV induces TNF-α production in both subtypes, the amounts secreted by M-MΦ slightly exceeding those of GM-MΦ.

GM- and M-Mϕ also responded to the RV-infection with an increase in IFN-α2, IFN-β and IFN-λ1 concentrations ( Figure 1F ), whereas no IFN-γ was detectable (data not shown).

The observation that the increase of virion production ( Figure 1C ) also started 12 hpi and that a medium change carried out 12 hpi had no inhibitory effect on RV-induced IFN production (except IFN-β) ( Figure 1G ) points to a close relation between virion generation and IFN production. M-MΦ produced far more IFNs than GM-MΦ, the concentrations of IFN-λ1 being highest in both subtypes. UV-RV and mock controls did not produce any IFNs.

These differences in IFN-α2, -β and -λ1 production can hardly be explained by the kinetics of their release. Secretion of all three IFNs started at 12 hpi and high concentrations were reached at 24 hpi ( Figures 1H–J ).

As the expression levels of IFN-receptors play an important role for the production of IFNs in a positive feedback loop we determined the mRNA receptor levels of type III interferon-lambda receptor 1 (IFNLR1) and type I interferon-α/β receptor 1 (IFNAR1) before infection with RV as well as at 8 and 24 hpi. Before starting the experiments, we addressed the expression level of both types of receptors and showed that they hardly differed between uninfected GM- and M-MΦ (relative to M-MΦ expression = 1, GM-MΦ IFNAR = 0.95 ± 0.14, IFNLR1 = 1.24 ± 0.16). At 24 hpi IFNLR1 transcripts increased in GM- and M-MΦ about 5- and 3-fold respectively as compared to 0 h controls (before treatment) ( Figure 1K ) and at 8 hpi only a minor increase was observed in M-MΦ.

We also found IFNAR1 transcript to be moderately elevated in both subtypes at 24 hpi; the small rise at 8 hpi was restricted to mock- and UV-RV-infected controls ( Figure 1L ). However, as the RV-induced rise of both IFN receptor transcripts in GM- and M-MΦ also occurred in mock and UV-RV, a correlation between increased mRNA levels of the receptors and an elevated RV-induced IFN-response cannot be drawn.

These data show that RV infects GM- and M-MΦ to a similar extent and that both subtypes are competent to respond to RV mounting a cytokine response consisting of a release of a relative minor elevated TNF-α production and an increase in type I IFN-concentrations, the latter being more pronounced in M-MΦ than in GM-MΦ.

Having shown that RV induces TNF-α and IFN-production in both MΦ subtypes we next tested whether stimulation with LPS after RV infection affects the outcome of the cytokine response.

Therefore, cells were infected with RV and the respective controls (mock, UV-RV) for 24 h before fresh medium with or without LPS was added for another 6 h. As seen in Figure 2A , GM- and M-MΦ infected with RV only, produced small amounts of TNF-α (see Table 1 , Figure 2A ). The TNF-α levels increased after exposure to LPS, the rise being largely independent of the viral infection, albeit a small but insignificant increase was observed in M-MΦ after RV infection ( Figure 2A ).

Both subtypes secreted small amounts of IFNs (except for IFN-β in GM-MΦ) after RV infection in the absence of LPS ( Figure 2B , Table 1 ). However, addition of LPS to the infected cells resulted in an increase of IFN-β and IFN-λ1, but not of IFN-α2 levels. Neither UV-RV infected (data not shown) nor mock controls ( Table 1 ) produced any IFNs in response to LPS.

As IFN-β, known to act in an autocrine fashion (22), was produced by GM- and M-MΦ in response to RV-infection ( Figure 1F ), we tested whether it can mimic the effects of RV on LPS-induced cytokine production when added exogenously. We found that IFN-β (20 ng/ml) did not induce TNF-α production whereas after challenge with LPS the TNF-α levels increased, in M-MΦ similar to controls and in GM-MΦ even less ( Figure 2A ). Treatment with IFN-β also failed to induce IFN production ( Figure 2B ). Only after exposure to LPS IFN-β and IFN-λ1 levels increased, IFN-α2 levels were unaffected. The small amounts of IFN-β present in the absence of LPS likely stem from the added IFN-β ( Figure 2B ).

When diluting IFN-β concentrations starting with 20 ng/ml the LPS-induced enhancement of IFN-β and IFN-λ1 production decreased in a concentration dependent manner ( Figures 2C, D ). As little as 0.2 to 0.02 ng/ml were sufficient to amplify the IFN-β and -λ1 response. M-MΦ produced slightly more IFN-β and -λ1 than GM-Mϕ and the IFN-λ1 release always exceeded that of IFN-β.

As seen in Figures 2E, F exposure of GM- and M-MΦ to small concentrations (0.2 to 0.02 ng/ml) of IFN-β results in low phosphorylation levels of Stat1, sufficient for enhanced responsiveness to the secondary stimulus LPS ( Figures 2C, D ).

Taken together our data show that RV-infection hardly effects the LPS-induced TNF-α production and that the virus primes both subtypes to produce appropriate amounts of IFN-β and -λ1 in response to LPS. The same holds true at least for IFN-β and -λ1 production when RV is replaced by IFN-β.

Next, we tested whether the priming effect of RV on IFN-β and -λ1 production also occurred at the mRNA level. The mRNA expression of IFN-β ( Figure 3A ) and -λ1 ( Figure 3B ) started to increase at 8 hpi and 12 hpi in M- and GM-MΦ respectively and continued to rise up to 24 hpi. At all time points the values reached by M-MΦ exceeded those of GM-MΦ ( Figures 3A, B ). Negligible amounts of IFN-mRNA were produced after UV-RV infection. Extending the incubation time for another 6 h after medium change, in the presence of LPS resulted in a further increase of IFN-β ( Figure 3C ) and -λ1 ( Figure 3D ) mRNA in both MΦ subtypes.

The mRNA level of IFN-β in LPS challenged GM- and M-MΦ peaked after 2 h and reached basal values after 6 h. IFN-λ1 mRNA expression, however, peaked after 4 h in both subtypes and declined thereafter. In the absence of LPS the production of IFN-β and -λ1 mRNA remained at a rather constant level ( Figures 3C, D ).

Thus, IFN-β and -λ1 protein kinetics up to 24 hpi ( Figures 1I, J ) clearly reflect the corresponding mRNA expression levels ( Figures 3A, B ) and the IFN-β and -λ1 mRNA levels as well as the protein concentrations reached by M-MΦ 6 h after exposure to LPS both exceeded those of GM-MΦ ( Table 1 ).

Taken together these data demonstrate that RV as well as IFN-β regulate the IFN-production in response to a secondary challenge with LPS. Under assay conditions that result in a very low LPS-induced IFN-production the virus primes both macrophage subsets to react to LPS treatment with a rise in IFN concentrations. The same holds true when substituting RV by IFN-β. Notably low concentrations of IFN-β (pg/ml) are sufficient for enhanced LPS-induced responsiveness to this cytokine.

Having shown that exposure to LPS following infection with rubella alters the biological response of GM-MΦ and M-MΦ we tested whether distinct components of the virus and LPS-dependent signalling pathways are affected by the treatment. The role played by components in these pathways is outlined in a short and simplified way in Figure 4 .

As shown in Figures 5A, B we found the transcription factor NFκB to be phosphorylated in response to RV infection. NFκB plays an important role in the gene-regulatory network in immune responses including the induction of the genes that encode IFN-β and pro-inflammatory cytokines (32, 33). It associates with the transcription factors AP1 and IRF3 resulting in the direct binding and activation of the IFN-β promoter (34).

After RV-infection we detected only minor amounts of phosphorylated TBK1 ( Figure 5A ), a central kinase in the pathway leading to type I IFN-production. However, p-Stat1 expression was greatly enhanced in both MΦ subtypes, which was consistent with an increased production of IFNs following infection with RV.

Next, we tested the effect of LPS on the phosphorylation profile of NFκB, ERK1/2, JNK, TBK1 and Stat1 when applied 24 h after virus infection.

As shown in Figures 5A, C treatment of controls (mock, UV-RV) with LPS had no effect on Stat1 phosphorylation and led to an increased phosphorylation of NFκB, ERK1/2, JNK and TBK1, indicating that both TLR4 dependent pathways have been engaged.

Prior infection with RV resulted in an increased phosphorylation of NFκB (M-MΦ), while it caused a drastic downregulation of p-ERK1/2 in both subtypes. This effect was also slightly visible in the absence of LPS challenge.

In conclusion, an established RV infection leads to a decrease of the LPS-induced ERK1/2 phosphorylation in both MΦ subtypes.

Having shown that exposure of GM- and M-MΦ to RV prior to incubation with LPS results in an enhanced production of IFNs, we asked whether activation by the respective stimuli alone or applied successively is associated with metabolic changes.

To determine the metabolism of GM- and M-MΦ after RV-infection followed by exposure to LPS we measured extracellular acidification rate (ECAR) as an index of lactic acid production and oxygen consumption rate (OCR) as an index of oxidative phosphorylation.

OCR and ECAR were measured every 6 min at 16 measurement points. As seen in Figures 6A–D GM-MΦ exhibited elevated basal OCR and ECAR levels compared to M-MΦ indicating that the bioenergetics profile differs between the twoMΦ subsets. Challenge with LPS for 0.5 h and 1 h induced a profound increase in ECAR in GM-MΦ and to a lesser extent in M-MΦ, whereas OCR remained unaffected ( Figures 6B, D ).

These data as illustrated in the Energy Phenotype Profile of ECAR and OCR ( Figure 6E ) demonstrate that M-MΦ are in a more quiescent state than GM-MΦ. In response to LPS GM-MΦ undergo metabolic reprogramming towards glycolysis slightly more than M-MΦ.

As a next step, the bioenergetics profile that is reflected by the ratio of OCR and ECAR was determined in MΦ having been infected with RV and UV-RV for 24 h ( Figure 6F ).

The OCR/ECAR ratio serves as a useful parameter to compare metabolic states between different cell types (35). The lower the ratio, the higher the glycolytic activity.

The ratio analysis of GM- and M-MΦ 24 hpi ( Figure 6F ) confirmed their metabolic identity, which relies on glycolysis and oxidative phosphorylation, respectively.

Notably in M-MΦ, this ratio is lower after infection with RV than in the mock-control ( Figure 6F ) suggesting that during RV-infection glycolytic activity is increased in M-MΦ.

When determining the metabolic activity after LPS-challenge OCR values slightly increased in RV- and to a lesser extent in UV-RV- and mock-infected GM-MΦ whereas M-MΦ did not respond to LPS ( Figure 6G ).

Compared to basal OCR levels the rise at 0.5 h after treatment with LPS was only significant in RV-infected GM-MΦ and at 24 hpi ( Figure 6H ).

Independent of the preceding treatment both MΦ subtypes responded to LPS with a substantial increase in ECAR levels ( Figures 6I, J ). In relation to basal ECAR the values reached 0.5 h after LPS treatment were significantly lower in RV-infected MΦ than in mock and UV-RV treated controls ( Figure 6K ).

Taken together these data indicate that stimulation with LPS resulted in a slight increase in glycolytic activity of both GM- and M-MΦ. During RV-infection glycolytic activity was slightly increased in M-MΦ, while in both cell types RV-infection significantly dampened the LPS-induced increase in glycolysis. This latter metabolic reprogramming indicates that RV signalling followed by LPS signalling results in a specific biological response, which is associated with a diminished glycolysis.

As an overall conclusion, the here presented data are summarized in Figure 7 .