This study was done according to the ethical guidelines of the Academic Medical Center and human material was obtained in accordance with the AMC Medical Ethics Review Committee according to the Medical Research Involving Human Subjects Act. Buffy coats obtained after blood donation (Sanquin blood supply) are not subjected to informed consent, which is according to the Medical Research Involving Human Subjects Act and the AMC Medical Ethics Review Committee. All samples were handled anonymously. Ethical review and approval was not required for this study in accordance with the local legislation. Monocytes were isolated from buffy coats by density gradient centrifugation on Lymphoprep (Nycomed) and Percoll (Pharmacia). DCs or macrophages were generated by culturing monocytes for 6 days in IMDM (Lonza) containing 5% FBS (Biowest) and 86 μg/mL gentamicin (Gibco), supplemented with 20 ng/mL GM-CSF (Invitrogen) and 2 ng/mL IL-4 (Miltenyi Biotec) for DCs or 50 ng/mL recombinant human M-CSF (BioLegend) for macrophages. At day 2 or 3, half of the medium was replaced by new medium containing cytokines.

For silencing at day 3, cells were harvest by resuspending (DCs) or by using TrypLE Select (Invitrogen) (macrophages). Cells were microporated in the presence of 500 nM IRF5 si-RNA or control si-RNA (Dharmacon) and cultured for 3 more days in IMDM without gentamicin with supplemented cytokines.

DCs were harvested at day 6 by putting the cells on ice for 30 min and macrophages were harvested at day 6 by TrypLE Select. For cIgG stimulation, 96-well high-affinity Maxisorp plates (Nunc) were coated with 2 μg/mL IgG from pooled IgG (Nanogam; Sanquin Blood Supply) diluted in PBS overnight at 4°C, followed by blocking with PBS containing 10% FBS for 1 h at 37°C. Cells were stimulated (30,000–50,000 cells per well) with 10 μg/mL Pam3CSK4 (Invivogen). Co-stimulation experiments were performed by simultaneous exposure of the cells to cIgG and Pam3. Syk was inhibited with 1 μM R406 (Selleckchem), TBK1/IKKε was inhibited with 2 μM BX795 (Invivogen) and glycolysis was blocked using 10 mM 2-Deoxy-D-glucose (2DG; Sigma Aldrich). Cells were incubated with the inhibitor or the corresponding volume of DMSO (Sigma-Aldrich) or medium for 30 min at 37°C before stimulation.

For mRNA-level analysis, cells were lysed at the indicated time points, after which mRNA extraction was performed using RNeasy Mini Kit (Qiagen) and cDNA synthesis using RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas). Quantitative RT-PCR (StepOnePlus Real-Time PCR System; Thermo Fisher Scientific) was performed using Taqman Master Mix and the following Taqman primers (all from Thermo Fisher Scientific): GAPDH (4310884E), IRF5 (Hs00158114_m1), and TNF (Hs00174128_m1). mRNA levels were normalized to the geometric mean of the Ct-values of housekeeping gene GAPDH [2^{Ct(housekeeping)−Ct(target)}], and folds were calculated compared with an unstimulated control sample (t = 0 h).

For analysis of cytokine production, supernatants were harvested after overnight stimulation and stored at −20°C. Cytokine levels in supernatants were measured by ELISA, using antibody pairs for TNF (eBioscience), IL-Iβ, IL-6, and IL-23 (U-CyTech Biosciences).

For analysis of IRF5 translocation, DCs or macrophages were stimulated as indicated in Maxisorp plates. After 2 h stimulation, cells were washed with PBS, fixed with 3.7% formaldehyde (Sigma-Aldrich) for 15 min at room temperature, washed in PBS and stored in PBS containing 0.5% bovine serum albumin (BSA; PAA) and 0.1% sodium azide (Merck) at 4°C. Cells were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 5 min at room temperature and blocked for 30 min in PBS containing 0.5% BSA and 0.1% sodium azide. Cells were then stained with a rabbit-anti-human-IRF5 antibody (1:400) (Cell Signaling) or rabbit-anti-human NF-kB p65 antibody (1:100) (Cell Signaling) for 45 min at room temperature, washed with PBS and stained with a Cy3-labeled goat-anti-rabbit-IgG antibody (1:50) (Jackson ImmunoResearch). Cells were again washed with PBS and nuclei were stained using 1 μg/mL Hoechst (Immunochemistry Technologies) for 1 min at room temperature. Cells were imaged using a DM IRB inverted fluorescence microscope (Leica), combined with a DFC 300FX digital color camera (Leica).

For analysis of TBK1/IKKε phosphorylation, DCs or macrophages were stimulated as indicated in 48-well plates (Greiner Bio-One) for 30 min and fixed using Lyse/Fix buffer (BD Biosciences) for 10 min at room temperature. For analysis of IRF5, unstimulated DCs were also lysed and transferred in a 96-well plate following the same protocol as TBK1 phosphorylation. Cells were harvested by gentle scraping, transferred to a 96-well round-bottom plate (Greiner Bio-One), washed in PBS, and permeabilized using Perm III buffer (BD Biosciences) for at least 30 min at −20°C. Cells were then washed in PBS containing 0.5% BSA and 0.1% sodium azide and stained for 1 h at RT with a rabbit-anti-human-IRF5 antibody (1:200) (Cell Signaling) or a rabbit-anti-human-pTBK1 antibody (1:50) (Ser172; Cell Signaling), which also reacts to pIKKε, followed by a 30 min staining at room temperature with Alexafluor488-labeled goat-anti-rabbit-IgG antibody (1:400) (Molecular Probes). Fluorescence was determined by flow cytometry (Canto II, BD Biosciences).

Real-time analysis of the extracellular acidification rate (ECAR) and the oxygen consumption rate (OCR) of DCs were analyzed using an XF-96 Extracellular Flux Analyzer (Seahorse Bioscience). 30,000 DCs were plated per well. To trigger FcγR on DCs XF-96 cell culture plates were coated with 4 μg/ml IgG prior to seeding of the cells. DCs were plated in glucose-free medium after which glucose was added (10 mM) to the cells during the assay to be able to determine true glycolysis-driven ECAR. Thirty minutes after glucose addition cells were stimulated with 10 μg/mL Pam3CSK4 during the essay after which OCR and glycolysis-driven ECAR were determined 30 min post stimulation.

For analysis of IRF5 phosphorylation, DCs were stimulated as indicated in 6-well plates (1,250,000–2,000,000 cell per well) (Costar) for 30 min. Cells were gently scraped and collected in cold PBS. After washing, cells were lysed on ice for 10 min using RIPA lysis buffy (Cell signaling) supplemented with protease and phosphatase inhibitors (both from Roche). Lysates were briefly sonificiated for 10 s at 30% and centrifuged for 10 min at 14,000 × g. BCA assay was performed (Thermo Scientific) and samples were boiled with 4x Laemmli Sample Buffer (Bio-Rad) for 15 min at 95°C. Cell lysates were run on a 4–12% Bis-Tris protein gel (Invitrogen) using MES-running buffer (Invitrogen). Proteins were transferred to a PVDF membrane (GE healthcare) using transfer buffer (Invitrogen) and blocked with 2% milk (Bio-Rad) afterwards. Membrane was incubated in TBS Tween o/n at 4°C with indicated antibodies: Phospho-IRF5 (Ser437) polyclonal antibody (1:1000) (Thermo Scientific), IRF5 (1:1000) (E1N9G) rabbit mAb (Cell Signaling), or Actin antibody (I-19) (1:2000) (Santa Cruz). Afterwards membrane washed with TBS Tween and incubated for 1 h at room temperature with polyclonal swine anti-rabbit immunoglobulins HRP (1:3000) (Dako).

Co-localization quantification of the fluorescence microscopy data was done using Huygens Professional software (SVI, Hilversum, The Netherlands) calculating the Manders Coefficients. Western blots were analyzed using ImageJ. Data were analyzed for statistical significance using student's t-test with GraphPad Prism version 7 software (GraphPad Software).

FcγR-TLR cross-talk plays an important role in inducing inflammation during both bacterial infections and autoimmune diseases (1, 2, 6, 8). As illustrated in Figures 1A,B (representative donor and multiple donors, respectively), FcγR-TLR cross-talk synergistically amplifies the production of key pro-inflammatory cytokines TNF, IL-1β, and IL-23, while other cytokines such as IL-6 are not affected. Here, we set out to identify the molecular mechanisms underlying this response, using TNF production as a main read-out for FcγR-TLR cross-talk. FcγR-TLR cross-talk is known to amplify TNF production at the level of gene transcription (1, 8). Here, we hypothesized a role for IRF5, since this transcription factor is known to be involved in enhancing TNF transcription (10–14), is highly expressed in human myeloid APCs (15), and since IRF5 polymorphisms are a known risk factor for several autoimmune diseases (16–22).

To study the role of IRF5 in FcγR-TLR cross-talk, we made use of a small interfering (si)-RNA approach, which on average resulted in a 60 % reduction of IRF5 mRNA expression and a similar reduction in IRF5 protein in monocyte-derived DCs (moDCs) (Figures 1C,D). For stimulation of FcγRs and TLR2 we used plate-bound complexed IgG (cIgG) and Pam3CSK4 (Pam3), respectively. While individual stimulation with cIgG or Pam3 induced moderate amounts of TNF, combined stimulation strongly and synergistically amplified TNF production (Figure 1E). However, strikingly, silencing of IRF5 specifically reduced TNF protein production by FcγR-TLR cross-talk, without affecting cytokine production induced by the individual ligands (Figure 1E). In addition, we assessed whether IRF5 is also responsible for FcγR-TLR cross talk-induced gene transcription. Indeed, (partial) silencing of IRF5 reduced TNF mRNA production upon FcγR-TLR co-stimulation (Figure 1F for kinetics of representative donor, Figure 1G for multiple donors). In contrast, TNF mRNA induced by TLR stimulation alone was not affected by IRF5 silencing (Figure 1F), indicating that IRF5 specifically controls TNF transcription induced by FcγR-TLR cross-talk.

To determine whether IRF5 is only essential for FcγR-TLR cross-talk in moDCs, or whether it is also required for FcγR-TLR cross-talk in other cell types, we assessed the effect of IRF5 silencing on human macrophages, which are the main source of TNF in inflamed synovia of RA patients (23). Similar to moDCs, silencing of IRF5 in monocyte-derived macrophages (Figure 1C) specifically reduced TNF production induced by FcγR-TLR synergy, both on protein (Figure 1H) and mRNA (Figures 1I,J).

Combined, these data demonstrate that the synergistic induction of TNF by FcγR-TLR cross-talk in human moDCs and macrophages is dependent on IRF5.

The transcription factor IRF5 is constitutively expressed by myeloid APCs (15), but to regulate gene transcription IRF5 needs to be translocated to the nucleus (24). Therefore, we assessed IRF5 localization in human moDCs by fluorescence microscopy upon FcγR-TLR co-stimulation. IRF5 contains two nuclear localization signals (NLS) as well as a nuclear export signal (NES) and therefore continuously shuttles in and out of the nucleus (25–27). Indeed, in unstimulated moDCs IRF5 was present throughout the cell, both in the nucleus and the cytoplasm (Figure 2A). Similar to unstimulated cells, TLR2-stimulated moDCs also displayed an even distribution of IRF5 (Figure 2A). In contrast, stimulation with cIgG, either combined with TLR stimulation or not, resulted in near exclusive accumulation of IRF5 in the nucleus (Figure 2A; quantified in Figure 2B). As a control we also ascertained that (individual) TLR stimulation of moDCs results in nuclear translocation of NF-κB subunit p65 (Figures 2C,D), which is responsible for TLR-induced pro-inflammatory cytokine production. Very similar to moDCs, FcγR stimulation induced IRF5 nuclear translocation in human macrophages (Figures 2E,F), suggesting that nuclear translocation of IRF5 induced by FcγR stimulation is a general mechanism in myeloid APCs.

Since FcγR-TLR cross-talk is known to depend on signaling through the kinase Syk, we next assessed whether Syk is required for IRF5 nuclear translocation. As shown in Figure 2G, Syk inhibition by therapeutic small-molecule inhibitor R406 indeed suppressed IRF5 nuclear translocation both upon individual stimulation with cIgG and upon cIgG+Pam3 co-stimulation.

These data indicate that, in human moDCs and macrophages, stimulation with IgG immune complexes is responsible for nuclear translocation of IRF5.

While individual FcγR stimulation induced IRF5 translocation into the nucleus, it is not sufficient to induce TNF transcription (1, 6, 8). Importantly, in addition to nuclear translocation, IRF5 needs to be activated by phosphorylation in order to be transcriptionally active (14, 25, 26, 28, 29). Therefore, we determined IRF5 phosphorylation upon (co-)stimulation of human moDCs by Western blot. Interestingly, while Pam3 stimulation induced IRF5 phosphorylation, stimulation with cIgG did not (Figure 3A, quantified as pIRF5/IRF5 ratio for multiple donors in Figure 3B). These data indicate that while FcγR stimulation induces IRF5 nuclear translocation, TLR stimulation is required for IRF5 phosphorylation.

IRF5 phosphorylation can be induced by TBK1, a member of the Iκ kinase (IKK) family that shares larges structural and functional similarity to IKKε (25, 26, 30). Since TBK1/IKKε also needs to be phosphorylated in order to execute kinase activity (31), we assessed TBK1/IKKε phosphorylation by flow cytometry after (co-)stimulation. Similar to IRF5 phosphorylation, we found that stimulation with Pam3 induced TBK1/IKKε phosphorylation, while stimulation with cIgG did not (Figure 3C, quantified for multiple donors in Figure 3D).

To determine whether TBK1/IKKε is required for cytokine production by FcγR-TLR cross-talk, we inhibited TBK1/IKKε using small-molecule inhibitor BX795. Indeed, BX795 abrogated FcγR-TLR cross-talk-induced TNF production (Figure 3E).

Thus, while FcγR stimulation induces nuclear translocation of IRF5, TLR stimulation induces phosphorylation of TBK1/IKKε and IRF5, which combined results in nuclear translocation of phosphorylated IRF5 to modulate cytokine gene transcription.

Amplification of cytokine production can be orchestrated at both the transcriptional and translational level. Interestingly, upon FcγR co-stimulation of moDCs, the fold increase in expression of TNF mRNA was lower than that fold increase at the protein level (Figure 4A), suggesting that increased translation also contributes to the amplified cytokine response. In DCs, increased cytokine mRNA translation in response to TLR stimulation has been shown to be underpinned by a rapid increase in glycolytic rate, to serve as a carbon source for de novo fatty acid synthesis to support expansion of the endoplasmic reticulum required for increased cytokine gene translation (32, 33). This, together with the recent finding that IRF5 is able to increase the glycolysis in macrophages (34), led us to hypothesize that FcγR (co-) stimulation induces a similar metabolic reprogramming via IRF5 to support increased translation. To this end, we stimulated moDCs and analyzed them for changes in rates of extracellular acidification (ECAR), as a measure of lactate production (a proxy for the glycolytic rate), and the rate of oxygen consumption (OCR), as a measure of oxidative phosphorylation. Notably, stimulation with cIgG indeed increased the ECAR, which was even further enhanced upon co-stimulation with cIgG and Pam3 (Figure 4B). In contrast, the OCR was not affected by individual stimulation with cIgG or Pam3, and only moderately increased upon co-stimulation (Figure 4C).

Next, we set out to investigate whether the amplification of the glycolytic response by FcγR-TLR cross-talk was also dependent on IRF5. While silencing of IRF5 did not affect the ECAR induced by individual stimulation with cIgG or Pam3, IRF5 silencing did inhibit the increased ECAR induced upon co-stimulation (Figure 4D). These data indicate that FcγR-TLR cross-talk amplifies the glycolytic response via IRF5.

To assess whether the increased glycolysis by FcγR-TLR cross-talk indeed contributes to the induction of cytokine responses, we stimulated moDCs in the presence of 2-deoxyglucose (2DG), which blocks glycolysis by inhibiting hexokinase activity (35). In line with previous findings, 2DG suppressed cytokine production induced by individual TLR stimulation (Figure 4E). In addition, 2DG also strongly suppressed cytokine production upon co-stimulation with cIgG and Pam3 (Figure 4E). Interestingly, while 2DG strongly impaired FcγR-TLR cross-talk-induced TNF protein production, blocking of glycolysis had very little effect on FcγR-TLR cross-talk-induced TNF gene transcription (representative donor Figure 4F, multiple donors Figure 4G). These data indicate that the glycolytic changes induced by FcγR-TLR cross-talk, although essential for protein production, have little effect on cytokine gene transcription.

Taken together, these data identify that IRF5 activation by FcγR-TLR cross-talk does not only enhance cytokine gene transcription, but also boosts translation through glycolytic reprogramming that together account for the strongly increased pro-inflammatory profile of moDCs activated by FcγR-TLR cross-talk.

DB is also an employee of Union Chimique Belge. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.