Buffy coats (Oklahoma Blood Institute) and apheresis cones from Mayo Clinic were utilized as blood sources. PBMCs were collected from the blood by density gradient centrifugation utilizing Ficoll-Paque Plus (GE Healthcare). DCs were enriched from PBMCs with Pan-DC Enrichment Kit (Stemcell Technologies). pDCs (Lin-1^-HLA-DR⁺ CD123⁺CD11c^-) were sorted on a FACS Aria II (BD Biosciences). Purity after sorting was above 98%. pDCs were washed with complete RPMI (cRPMI) and resuspended in cRPMI/10% FBS (Gemini Bio-Products) containing 5 ng/mL IL-3 (R&D Systems). Complete RPMI consisted of RPMI1640 containing L-glutamine (ThermoFisher) supplemented with MEM non-essential amino acids, sodium pyruvate (Sigma Aldrich), and penicillin/streptomycin (ThermoFisher).

EGA (Cayman Chemical or Sigma Aldrich) and YM201636 (Cayman Chemical) were each dissolved in dimethyl sulfoxide (DMSO) (ThermoFisher), aliquoted, and frozen at -30°C. Fresh aliquots were utilized for each experiment. Cells were pre-incubated with 1μM YM201636, 20 μM EGA, or vehicle for 15-30 minutes at 37 °C. Approximately 5×10⁴ cells were stimulated with 5 μg/mL R837, 2 μg/mL R848 (Invivogen), 2 μg/mL ssRNA40 (Miltenyi Biotec), or 2 multiplicity of infection (MOI) live influenza A/PR8/34 (kindly provided by Dr. Adolfo Garcia-Sastre, Mount Sinai, NY). Complexes of ssRNA40 with 10 μg/mL 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP Transfection Reagent; Santa Cruz Biotechnology) were prepared in LAL water (Invivogen) at least 15 minutes prior to be used. After 2 hours, GolgiPlug (BD Biosciences) was added according to the manufacturer’s instructions. Additional 3 hours later, cells were washed with 2 mM EDTA/DPBS (ThermoFisher), stained for viability, fixed, permeabilized with Cytofix/CytoPerm (BD Biosciences), and stained for intracellular cytokine expression in PermWash solution (BD Biosciences). Cells were also stained for surface TNFα on pDCs.

Supplementary Table 1 lists the antibodies utilized for FACS analyses in this study. Cells were run on a FACS Celesta (BD Biosciences) or Cytek Aurora (Cytek Biosciences). Flow cytometry data were analyzed utilizing FlowJo v10 (FlowJo). Intracellular and surface cytokine staining gating was based on the examination of cytokine staining for unstimulated cells and isotype control antibody staining.

Bead-based Luminex kits assessing human IFNα and TNFα (ThermoFisher) were utilized for measuring cytokine concentrations in supernatants from overnight stimulated pDCs. Beads were assessed on a Luminex 200 machine and the Xponent 3.1 software (Luminex) was utilized to analyze the data.

FACS-sorted pDCs were extensively washed and rested for at least one hour in cRPMI containing IL-3 (5 ng/mL) at 37 °C. pDCs were then treated with indicated inhibitors at 37 °C for 30 minutes. Approximately 1×10⁵ - 1.4×10⁵ pDCs/well in 96-well plates were stimulated with 5 μg/mL R837 for the indicated time frame upon cell lysates were harvested in RIPA buffer (ThermoFisher) supplemented with HALT™ protease and phosphatase inhibitor cocktail (ThermoFisher). Cell lysates were reduced, ran on Novex 4-20% Tris-Glycine gels (Invitrogen), and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad), as previously described (24). Protein normalization was performed utilizing a No-Stain Protein labeling reagent (ThermoFisher) according to the manufacturer’s instructions. Antibodies utilized for immunoblotting are listed in Supplementary Table 2 . PageRuler prestained protein ladder (ThermoFisher, Catalog 26616) was used as a reference for protein size. Images were acquired on a ChemiDoc MP (Bio-Rad, CA) and analyzed in Image Lab V6.0 (Bio-Rad). Protein intensity was normalized either to total reference protein or total protein concentration. Relative intensity was obtained by comparison with vehicle-treated group at time zero.

3F compound has previously been characterized as a TLR7 agonist with similar efficacy to the imidazoquinoline, resiquimod (R848) (23). 3F and AF488 NHS (lumiprobe) were combined in equimolar concentrations in DMSO-D₆ (Cambridge Isotopes). TEA (Sigma Aldrich) was added, and the mixture was placed at 25°C for 16 hours. The reaction product was purified into precipitate form with washing of ethyl acetate to yield a red solid which was dissolved in DMSO. Reactions were monitored utilizing NMR and Thermo Scientific Dionex Ultimate 3000 HPLC equipped with an Eclipse Plus C18 3.5 mM, 3.0 x100 mm column using a 5:95 to 95:5 H₂O + 0.1% Formic Acid : Acetonitrile gradient.

HEK Blue hTLR7 cell line (Invivogen) and the parental cell line (Null-1k) were used to examine the ability of 3F-AF488 to activate cells via TLR7. Equivalent numbers of HEK Blue hTLR7 cells were stimulated with 10 μM 3F and 3F-AF488, and vehicle for 15 hours in HEK Blue detection media (Invivogen). NF-κB activity was measured by assessing the optical density at 620-655 nm using a SpectraMax 12 (Molecular Device) and analyzed with SoftMaxPro V5 software (Molecular Device).

Total RNA was extracted from cell lysates using protocol from the RNAqueous™ Micro Total RNA Isolation Kit (ThermoFisher). 200 ng of input total RNA was used to generate cDNAs using iScript Reverse Transcription Supermix for RT-PCR (Biorad) by following manufacturer’s protocol. The primer sequences used for human S18 were forward 5’-TGCCATCACTGCCATTAAGG-3’ and reverse 5’-TGCTTTCCTCAACACCACATG-3’, for human TNFα forward 5’- CTCTTCTGCCTGCTGCACTTTG-3’ and reverse 5’-ATGGGCTACAGGCTTGTCACTC-3’ and for human IFNα forward 5’-CCAGTTCCAGAAGGCTCCAG-3’ and reverse 5’-TCCTCCTGCATCACACAGGC-3’. SYBR green-based qPCRs with 2µl of cDNA were ran on a Roche LightCycler 480 instrument (Roche). The reactions were performed in 20 µl as follows: 50°C for 2 mins, 95°C for 2 mins, followed by 40 cycles of 95°C for 15 seconds, 52°C for 15 seconds and 72°C for 1mins. Results were analyzed using the 2-ΔΔ Ct method and normalized to the corresponding level of the housekeeping gene S18.

pDCs were enriched utilizing a human plasmacytoid DC enrichment kit (Stemcell Technologies). Enriched cells were assessed for purity utilizing the same surface markers for sorting. After three consecutive enrichments, pDC purity was above 92 - 97%. pDCs were pre-incubated with inhibitors for 1 hour at 37 °C before the addition of 20 μM 3F-AF488. Cells were affixed to coverslips after 1, 2, and 5 hours of stimulation and stained as previously described (25). Briefly, pDCs were attached to poly-D-lysine (35 μg/mL; ThermoFisher)-coated coverslips, fixed with ice-cold 4% paraformaldehyde (Electron Microscopy Sciences), permeabilized with 0.15% Triton™ X-100 Surfactant-Amps (ThermoFisher), and then washed extensively. Cells were blocked and stained in a saline blocking buffer containing 5% goat serum (Invitrogen), 1% glycerol (Sigma-Aldrich), and 0.1% bovine serum albumin (MP Biomedicals). After blocking for 1 hour, cells were incubated with primary and secondary antibodies in a blocking buffer with extensive washing between steps. Cells were stained with antibodies listed in Supplementary Table 3 . Cells were counterstained with Hoechst 33342 (ThermoFisher) according to the manufacturer’s instructions and washed. Coverslips were mounted on slides in Prolong Gold Antifade Mounting reagent (ThermoFisher). Slides were imaged on an LSM 800 Confocal Microscope (Zeiss) utilizing a 63x/1.4N.A objective. Identical acquisition settings were used for all experimental samples and images were scanned in frame mode to avoid any crosstalk between fluorophore signals.

Cell images were processed via Airyscan in the Zen software module (Zeiss). Images were further processed in the open-source software Fiji (26). Baseline intensity thresholds for analysis were defined by cellular autofluorescence and isotype antibody intensity staining for each experiment. Manders’ coefficients were determined utilizing an in-house customized plugin coded for thresholding of 3F signal and region of interest selection (27).

SLE patients ( Supplementary Table 4 ) were recruited at Mayo Clinic under a protocol approved by the institutional review board. Patients provided informed consent in accordance with the Declaration of Helsinki. PBMCs were isolated from the whole blood of SLE patients. PBMCs were washed in cRPMI and resuspended in cRPMI/10% FBS (Gemini Bio-Products). PBMCs were pre-incubated with 20 μM EGA or vehicle for 30 minutes at 37 °C. Approximately 8×10⁵ - 1×10⁶ PBMCs per well in 96-well plates were stimulated with 5 μg/mL R837 or 2 MOI live influenza virus A/PR8/34. After 2 hours incubation, GolgiPlug (BD Biosciences) was added according to the manufacturer’s instructions. After an additional 3 hours of incubation, PBMCs were stained for viability and surface markers, fixed and permeabilized with Cytofix/CytoPerm (BD Biosciences), and stained for intracellular cytokine expression. Cells were run on FACS Fortessa (BD Biosciences).

Graphs with error bars represent mean ± SD (or SEM). Data were analyzed in Prism 7.0 (GraphPad) utilizing one-way analysis of variance (ANOVA), and two-way ANOVA with Tukey, Sidak, or t-test. Significance was set at p<0.05.

We began by assessing how EGA, a late endosomal trafficking inhibitor, would affect pDC responses to R837, a TLR7-specific imidazoquinoline. As shown in Figure 1A , EGA treatment decreased the frequency of IFNα⁺ pDCs when pDCs were stimulated with R837. Summarized data generated with pDCs of 10 healthy donors are presented in Figure 1B . EGA treatment also reduced the amount of IFNα secreted by R837-stimulated pDCs ( Figure 1C ).

Interestingly, however, the effects of EGA treatment on TNFα expression by R837-stimulated pDCs were variable among healthy donors especially when the frequency of TNFα⁺ pDCs was assessed by intracellular TNFα staining that could detect both surface and intracellular TNFα ( Figures 1D, E ). EGA increased the frequency of TNFα⁺ pDCs from some donors whereas it decreased the frequency of TNFα⁺ pDCs from others. Regardless of the frequency of intracellular TNFα⁺ pDCs, we found that EGA consistently decreased the amount of TNFα secreted from R837-stimulated ( Figure 1F ). In addition, EGA also decreased mRNA transcripts of both IFNα ( Figure 1G ) and TNFα ( Figure 1H ). It was of note that pDCs tested in Figures 1F, H were those that showed increased intracellular TNFα expression ( Figure 1E ) in the presence of EGA.

To further understand the TNFα expression data, showing discrepancy between the frequency of intracellular TNFα⁺ pDCs ( Figures 1D, E ) and the amount of TNFα in culture supernatants ( Figure 1F ) as well as the expression level of TNFα transcripts ( Figure 1H ), we assessed the frequency of surface pro-TNFα⁺ pDCs ( Supplementary Figure 1 ). We found that the amount of TNFα secreted in the supernatants and TNF mRNA transcripts were consistent with the frequency of surface pro-TNFα⁺ pDCs, not intracellular TNFα⁺ pDCs ( Figures 1D, E ). It is known that newly made pro-TNFα is transported from the Golgi to the cell membrane where it can be cleaved by metalloproteinases to be released from cells (28, 29). However, it is also known that some of the pro-TNFα are endocytosed and traffic from early to late endosomes/lysosomes in which they can be degraded. As an endosomal trafficking inhibitor, EGA could inhibit such endocytosed pro-TNFα trafficking to late endosomes/lysosomes (30, 31), resulting in the accumulation of total TNFα intracellularly which could contribute to the increased frequency of intracellular TNFα⁺ pDCs. However, this needs to be further studied in the future. Similar to the R837-treated pDCs ( Supplementary Figure 1 ), EGA also decreased surface pro-TNFα expression by pDCs stimulated with CpG DNAs (not shown). Anti-TNFα antibody used in this study can detect both pro-TNFα and active form of TNFα. We also found that EGA did not decrease the frequency of intracellular TNFα⁺ pDCs ( Supplementary Figure 2A ), the frequency of surface TNFα⁺ pDCs ( Supplementary Figure 2B ), or the amount of TNFα secreted by pDCs when pDCs were stimulated with R848. R848 is a TLR7/8 agonist and a high concentration of R848 (e.g., 2µg/ml used in this study) could mainly activate pDCs via TLR8, not TLR7 (32), but it remains to be better understood in the future.

We next tested whether EGA could also alter IL-6 expression by R837-stimulated pDCs, as both TNFα and IL-6 expression is largely dependent on the activation of NF-κB pathway upon TLR7 stimulation. We measured pDC expression of IL-6 by both intracellular staining ( Supplementary Figure 3A ) and assessing the amount of IL-6 in culture supernatants ( Supplementary Figure 3B ). First of all, we noticed that R837-stimulated pDCs expressed much lower levels of IL-6 than TNFα. In addition, EGA did not significantly affect such low levels of IL-6 expression by R837-stimulated pDCs. Although additional experiments need to be performed to explain our results in Supplementary Figures 3A, B , both secreted and intracellular TNFα might also affect IL-6 expression by pDCs incubated overnight. Similar to the IL-6 expression, EGA did not decrease CD86 expression level on R837-stimulated pDCs ( Supplementary Figure 3C ).

We also tested whether EGA could also decrease both IFNα and TNFα secretion by ssRNA40-stimulated pDCs. As shown in Supplementary Figure 4A , ssRNA40 complexed with DOTAP (33) induced IFNα secretion by pDCs and EGA decreased IFNα secretion. However, the amount of TNFα secreted by ssRNA40-stimulated pDCs was minimal ( Supplementary Figure 4B ), as reported in a previous study (33), showing that TNFα expression by ssRNA40 stimulation was mainly observed in CD11c⁺ cells in PBMCs of healthy blood samples.

Therefore, we conclude that EGA can modulate cytokine expression by R837- treated human blood pDC by decreasing the expression of IFNα and TNFα as well as the amount of TNFα secreted by pDCs stimulated with R837. EGA was also capable of decreasing IFNα secretion by pDCs stimulated with ssRNA40.

Inhibition of PIKfyve activity can also result in the disruption of endo-lysosomal trafficking (34). It is also known that PIKfyve-mediated type 1 IFN expression by mouse macrophages can also induce ATF3 which negatively regulates type 1 IFN expression (35). Therefore, we tested whether the PIKfyve inhibitor, YM201636, would have similar effects as EGA on cytokine expression of R837-stimulated pDCs. Both EGA- and YM201636-treatments reduced IFNα expression in R837-stimulated pDCs; however, EGA was more effective than YM201636 ( Figures 2A, B ). As shown in Figure 2C , the amount of IFNα secreted by R837-stimulated pDCs was also decreased by EGA treatment.

In contrast to EGA, YM201636 did not significantly alter the frequency of intracellular TNFα⁺ pDCs ( Figures 2D, E ) or the amount of TNFα secreted in culture supernatants ( Figure 2F ) of R837-stimulated pDCs. Similar to Figures 1E, F , the effects of EGA treatment were variable among pDC donors when the frequency of intracellular TNFα⁺ pDCs was assessed. Unlike YM201636, EGA decreased the amount of TNFα secreted by pDCs when measured ( Figure 2F ) as in Figure 1F . Altered cytokine expression by EGA- and YM201636-treatments were not due to the effects on pDC viability ( Supplementary Figure 5 ).

We concluded that both EGA and YM201636 alters IFNα expression by pDCs stimulated via TLR7, but EGA was more effective than YM201636 at reducing IFNα expression by R837-stimulated pDCs. EGA was also more effective than YM201636 at decreasing the amount of TNFα secreted by R837-stimulated pDCs.

To investigate the mechanisms of action of EGA as well as differences with YM201636 at altering R837-stmulated pDCs, we assessed kinases and regulatory proteins that are known to be involved in cytokine expression by pDCs. These kinases included IKKα/β activation (36, 37) for type 1 IFN expression, IκBα (38) and p65 (39) for pro-inflammatory cytokine expression, p38 and STAT1 phosphorylation for interferon-stimulated gene factor 3 (ISGF3) formation (40, 41) which controls IFN-inducible gene expression, and IRF7 as a regulator of type 1 IFN expression (42). We also assessed whether ATF3 was enhanced with EGA- and YM201636-treatments as a possible mechanism for depressed type 1 IFN expression (35, 43). ATF3 can modulate IFN response as well as the expression of genes downstream of IFN signaling (35, 43). In addition, blocking the trafficking process through PIKfyve inhibition could rapidly induce the expression of the transcription repressor ATF3, resulting in the suppression of type I IFN expression by pDCs (35, 43).

Similar to the previously reported data generated with DCs, both mDCs and pDCs (36), and pDC cell line, Gen2.2 (37), R837-stimulation increased IKKα/β phosphorylation in pDCs. However, this increase was transient ( Figures 3A, B ). As shown in Figure 3C , IκBα levels were too low to examine the effects of EGA treatment by 1 hr. The effects of EGA on IκBα levels were seen at 2 hrs after activation. In contrast, IKKα/β activation and EGA-mediated suppression of IKKα/β activation was observed at 1 hr ( Figure 3B ). Interestingly, EGA treatment significantly reduced R837-induced IKKα/β phosphorylation while YM201636 treatment did not. This EGA-induced suppression of IKKα/β activation followed by reduced secretion of IFNα from R837-stimulated pDCs is supported by the data from previous studies (36, 37), showing that both IKKα and IKKβ contribute to type 1 IFN expression by pDCs. Notably, EGA-treatment also decreased IκBα accumulation after degradation ( Figures 3A, C ). Our results showed that EGA suppresses IKKα/β phosphorylation but prolongs IκBα degradation in R837-stimulated pDCs. This data could be surprising as degradation of IκBα could be the result of IKKα/β phosphorylation. However, previous studies have shown that IkBα degradation could also be independent of IKKα/β phosphorylation (44, 45). p65 ( Figures 3A, D ) and p38 ( Figures 3A, E ) phosphorylation also occurred at 1 hour in vehicle- and YM201636-treated pDCs with EGA-treatment suppressing phosphorylation of both p65 and p38 at 1 hour. These differences were not observed after 1 hour. STAT1 phosphorylation ( Figures 3A, F ) was enhanced 2 hours post-R837-stimulation with EGA-treatment inhibiting its phosphorylation. R837-stimulation of pDCs caused a decline in IRF7 levels at 2 and 4 hours; however, this decline occurred in all groups ( Figures 3A, G ). ATF3 expression was upregulated after IKKα/β phosphorylation and had similar kinetics to STAT1 phosphorylation in all treated groups ( Figure 3A ), suggesting autocrine effects of type 1 IFN expression (43) instead of an effect of EGA- or YM201636-treatments (35, 43). However, EGA-treatment inhibited ATF3 (35, 43) upregulation compared to vehicle ( Figure 3H ) further confirming EGA diminished the expression of type 1 IFN.

Taken these data together, we concluded that EGA could affect multiple kinases involved in R837-induced cytokine responses by pDCs. EGA-treatment suppressed the phosphorylation of IKKα/β, p38, and STAT1 which are involved in the expression of type 1 IFN. This was further confirmed through EGA-mediated reduction of ATF3 upregulation, which is an interferon-inducible gene (43). EGA-treatment suppressed p65 phosphorylation at early time point (1 hour). EGA also suppressed IκBα, supporting the decreased secretion of TNFα by R837-activated pDCs treated with EGA.

We next investigated whether EGA could alter the trafficking of TLR7 agonists in pDCs which could affect cytokine response by pDCs. We employed a previously characterized TLR7-specific substituted adenine, 3F (23, 46) that has a similar TLR7 activity to the imidazoquinoline (R848) and R837 (23, 46).

3F was conjugated with AF488 as depicted in Figure 4A . 3F-AF488 was purified by a high-pressure liquid chromatography (HPLC) ( Supplementary Figure 6 ). Purified 3F-AF488 was further characterized with a nuclear magnetic resonance (NMR) spectroscopy ( Supplementary Figure 7 ). There was no free TLR7 ligand.

Reporter cell assay using HEK-Blue TLR7 cells showed that both 3F and 3F-AF488 conjugates were comparable at activating HEK-Blue cells via the ligation of TLR7 ( Figure 4B ), as there was no activation of parental cell line (Null-1k) treated with 3F.

We next investigated whether EGA would alter trafficking of 3F-AF488 in pDCs by examining its colocalization with EEA1, VAMP3, LAMP2 and LAMP1. After analyzing the kinetics of 3F-AF488 uptakes ( Supplementary Figures 8A ), we performed experiments at 5 hours after 3F-AF488 treatment. We found that 3F-AF488 had reduced colocalization with LAMP1⁺ compartments in EGA-treated cells when compared to either vehicle- or YM201636-treatments ( Figures 4C–E ). EGA did not affect the colocalization of 3F-AF488 with other endo-lysosomal markers. YM201636-treatment did affect the colocalization of 3F-AF488 with VAMP3⁺ compartments compared to vehicle-treatment ( Figure 4E ) although this was not significant when compared to EGA-treatment ( Figure 4E ). EGA did not reduce the accessibility of 3F-AF488 into pDCs ( Supplementary Figures 8B, C ).

Therefore, we concluded that EGA-treatment could inhibit the trafficking of the small TLR7 agonists to LAMP1⁺ compartments. As previously described (17), TLR7 accumulation in late endosomes and lysosomes was required for type 1 IFN expression by pDCs stimulated with TLR7 agonists. The inhibition of 3F trafficking to LAMP1⁺ compartments by EGA ( Figure 4 ) support the EGA-mediated decreased IFNα expression by R837-stimulated pDCs ( Figures 1 , 2 ). Another study also reported that TLR7 localization was increased in late endosomes/lysosomes in pDCs of SLE patients (47). The enhanced IFNα production by TLR7-stimulated SLE pDCs was associated with increased retention of TLR7 in late endosomes/lysosomes in SLE pDCs (47). EGA treatment did not alter 3F colocalization with other endo/lysosomal markers, including EEA1, LAMP2, and VAMP3.

Previous studies (47, 48) reported that pDCs from SLE patients, when compared to pDCs of control subjects, have an enhanced cytokine response to TLR7 agonists. Such altered phenotype of SLE pDCs correlated with an increased colocalization of TLR7 within late endosomes (47). Therefore, we investigated how EGA affects TLR7-mediated cytokine responses by pDCs from the blood of SLE patients. Aberrant activation of TLR7 has also been associated with the interferon signatures in SLE (49–52) and with the pathogenesis of SLE (13, 47, 53).

With limited volume of blood samples from SLE patients, experiments in this section were performed with PBMCs. After in vitro cultures of PBMCs as indicated, pDCs were gated as in Supplementary Figure 9A . Figure 5A shows that EGA-treatment reduced IFNα expression by pDCs from both healthy and SLE patients in response to both R837 and PR8 influenza virus. Summarized data (right panel, Figure 5A ) also show that SLE pDCs were more potent than healthy pDCs at expressing IFNα in response to both R837 and PR8 influenza viruses, and this is in line with the previously published data (47, 48), showing that greater numbers of SLE pDCs expressed IFNα than pDCs from healthy controls in response to TLR7 ligands. Similar to the data generated with purified pDCs ( Figures 1 , 2 ), EGA treatment did not significantly reduce or increase the frequency of intracellular TNFα⁺ pDCs when they were stimulated with R837 (right panel, Figure 5B ). The effects of EGA were also variable among donors. However, EGA reduced the frequency of intracellular TNFα⁺ pDCs when they were stimulated with PR8 influenza viruses. As previously described (22), EGA inhibits influenza viral uncoating by blocking their trafficking to late endosomes and lysosomes.

We further analyzed cytokine expression by mDCs/monocyte populations (CD3^-CD19^-CD56^-HLA-DR⁺CD11c⁺CD123^- in Supplementary Figures 9A, B ). As shown in Figure 6A , the frequency of intracellular IFNα⁺ mDCs/monocytes were minimal (less than 1%) when they were stimulated with R837, and there was no significant effect of EGA treatment (right panel in Figure 6A ). However, PR8 influenza virus-induced IFNα expressions by mDCs/monocytes from both healthy individuals and SLE patients were decreased by EGA treatment.

Although the frequency of TNFα⁺ mDCs/monocytes (less than 15%, Figure 6B ) are less than the frequency of TNFα⁺ pDCs (25-70%, Figure 5B ), EGA could decrease the frequency of intracellular TNFα⁺ mDCs/monocytes stimulated with R837 and PR8 influenza virus. mDCs/monocytes from healthy subjects and SLE patients showed similar results. It was of note that the effects of EGA treatment on the frequency of intracellular TNFα⁺ pDCs were variable among donors ( Figures 1E , 2E ), but EGA could consistently decrease the frequency of intracellular TNFα⁺ mDCs/monocytes ( Figure 6B ), suggesting that cellular machineries for TLR7-mediated cytokine expression and/or secretion might not be the same in all cell types that will need to be further investigated in future studies. For example, in addition to the potential differences in the types and activation of membrane proteinases, cell type-specific metabolomic machineries are also known to differentially control inflammatory mediators, including TNFα, in human DC subsets, pDCs versus mDCs (54).

We therefore concluded, EGA can suppress IFNα expression by SLE pDCs treated with R837 and influenza virus. EGA can also effectively suppress PR8 influenza virus-induced TNFα expression. EGA is also capable of suppressing PR8 influenza virus-induced IFNα as well as R837-induced TNFα expression by mDCs/monocytes.