Isolation of primary human monocytes and differentiation of moDCs and macrophages

Human blood was collected from healthy donors (negative for human immunodeficiency virus, hepatitis B, and hepatitis C) under Protocol VD-057-0217 from the La Jolla Institute for Immunology and Protocol 181624 from UC San Diego. All recruited volunteers provided written informed consent. PBMCs were isolated by density gradient centrifugation on Histopaque 1077, and monocytes were purified by negative selection using a Human Pan Monocyte Isolation kit [Miltenyi Biotec, cat. #130-096-537] according to the manufacturer’s instructions.

For moDC differentiation, monocytes were resuspended in complete RPMI medium [RPMI 1640 with GlutaMAX [Thermo Fisher Scientific, cat. #61870036], 10% (v/v) fetal bovine serum [FBS; Gemini Bioproducts, cat. #A82G00K], 2.5% (v/v) HEPES buffer [Gibco, cat. #15630-080], and 1% (v/v) penicillin/streptomycin [Gibco, cat. #15140-122) supplemented with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) 100 ng/ml [Pepro Tech, cat. #300-03] and recombinant human IL-4 (rhIL-4) 100 ng/ml [Pepro Tech, cat. #200-04], plated at 1.5 × 10⁶ cells/ml in 6-well plates and incubated at 37 °C in a 5% CO₂ atmosphere for 7 days. The medium was changed after 3–4 days. Differentiation of CD14⁻ CD1a^high immature moDCs was confirmed on day 7 of culture by flow cytometry, as described below.

For macrophage differentiation, monocytes were resuspended in a complete macrophage medium (RPMI 1640 with l-glutamine [Corning, cat. #10-040-CV], 1% (v/v) penicillin/streptomycin [Gibco, cat. #15140-122], non-essential amino acids [Gibco, cat. #11140-050], and 10% (v/v) FBS [Biowest, cat. #S1620] supplemented with 100 ng/ml freshly-added recombinant human macrophage colony-stimulating factor (rhM-CSF) [PeproTech, cat. #300-25). Cells were plated at 8 × 10⁴ cells/well, 4 × 10⁵ cells/well, or 8 × 10⁵ cells/well in 96-, 24-, or 12-well plates, respectively, and allowed to differentiate for 7 days at 37 °C in a 5% CO₂ atmosphere. Medium was replaced every 3–4 days.

Virus propagation and focus-forming assay

ZIKV SD001 is an Asian lineage strain isolated in 2016 from a visitor to Venezuela returning to California, USA⁴⁰; ZIKV FSS13025 is an Asian lineage strain isolated from a patient in Cambodia in 2010 and was obtained from the World Reference Center for Emerging Viruses and Arboviruses; ZIKV PRVABC59 is an Asian lineage strain isolated from a patient in Puerto Rico in December 2015 and was obtained from BEI Resources. The African lineage ZIKV Dakar-1984 (Senegal, 1984) and DENV2 UIS353 (Colombia, 2004) strains were obtained from the World Reference Center for Emerging Viruses and Arboviruses. All viruses were propagated in C6/36 Aedes albopictus mosquito cells using standard conditions⁴¹.

Viral titers were measured using a baby hamster kidney (BHK)-21 cell-based focus-forming assay as previously described⁴¹. Briefly, BHK-21 cells were resuspended in complete MEMα [Thermo Fisher Scientific, cat. #12-561-072) containing 10% (v/v) FBS, 1% (v/v) HEPES buffer, and 1% (v/v) penicillin/streptomycin, plated at 2 × 10⁵ cells/400 μl/well in 24-well plates, and incubated overnight at 37 °C. Ten-fold serially diluted culture supernatants or viral samples were added to the cells and the plates were incubated with shaking every 15 min for 1 h at 37 °C. The supernatants were removed, and the cells were overlaid with 1 ml/well of 1% (w/v) carboxymethyl cellulose (Sigma, cat. #C5678) in a medium and incubated at 37 °C for 3 days. The cells were then fixed with 4% formalin, permeabilized with 1% Triton X-100, blocked with PBS/10% FBS, and incubated with clone 4G2 anti-pan-flaviviral envelope (E) protein [Absolute Antibody, cat. #Ab00230] at 1 μg/ml for 1 h at room temperature. The cells were washed and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody [Jackson ImmunoResearch, cat. #115-035-072] at room temperature for 2 h. The cells were then stained with KPL True Blue peroxidase substrate [SeraCare Life Science, cat. #5510-0050] and foci were counted manually. Viral titers were expressed as focus-forming units per ml of sample.

Cell infection and flow cytometry of moDCs and macrophages

On day 7 of culture, moDCs were infected with ZIKV SD001, ZIKV FSS13025, ZIKV Dakar-1984, or ZIKV PRVABC59 (all at MOI of 0.5), or with DENV2 UIS353 (MOI of 1) for 2 h at 37 °C, washed once, and resuspended in complete RPMI medium. The cells were then placed in 24-well plates at a density of 5 × 10⁵ cells/0.5 ml/well and incubated at 37 °C for the indicated times. Uninfected moDCs treated with vehicle (medium) or LPS [InvivoGen, cat. #tlrl-eblps] at 1 μg/ml served as controls. At 24 or 48 hpi, culture supernatants were collected and stored at − 80 °C for analysis. MoDCs were collected, washed once with PBS, and stained with eFluor 455UV viability dye [Invitrogen, cat. #65-0868-14]. The cells were surface-stained with combinations of anti-CD80-PE [clone 2D10, BioLegend), anti-CD86-PerCP-Cy5.5 [clone IT2.2, BioLegend], anti-CD40-APC-Cy7 [clone 5C3, BioLegend], anti-CD83-BV421 [clone HB15e, BioLegend], and anti-HLA-DR-PE/Cy7 [clone G46-6, BD Bioscience]. The cells were then fixed and permeabilized using Cytofix/Cytoperm [BD Bioscience, cat. #554714] and intracellularly stained with AF647-conjugated 4G2 at 5 μg/ml for 20 min at 4 °C. All antibodies were prepared in 200-fold dilution. Data were acquired on an LSRFortessa and analyzed using FlowJo software v10.3. Where indicated, IL-6 [BD Bioscience, cat. #555220] and TNF [BD Bioscience, cat. #555212] in the culture, supernatants were quantified using cytokine-specific ELISAs according to the manufacturer’s instructions.

On day 7 of culture, differentiated macrophages were infected with ZIKV SD001 or DENV2 UIS353 at a MOI of 1(both viruses) via ADE. Viruses were mixed with predetermined optimal concentrations of anti-flavivirus E protein antibody, 5 μg/ml for ZIKV or 0.156 μg/ml for DENV [human IgG1-4G2, Absolute Antibody, cat. # Ab00230-10.0], in complete macrophage medium (as above except 1% FBS) and incubated at 37 °C, 5% CO₂ for 1 h. The medium was then removed from macrophages in 96-, 24, or 12-well plates, and the virus/antibody mixture was added at 40, 250, or 500 µl/well, respectively. Control macrophages received mock infection, consisting of Conditioned medium (“mock” viral preparation; medium incubated 7 days on uninfected C6/36 cells and concentrated by the identical PEG purification procedure used for preparation of concentrated DENV and ZIKV stocks) incubated without antibodies in macrophage medium in place of virus/antibody mixture. The plates were incubated with rocking at 37 °C for 1 h, the virus/antibody supernatant was removed, and the macrophages were washed once in PBS before the addition of fresh complete macrophage medium. For intracellular cytokine staining, the cells were incubated for 18 h in medium alone and then for an additional 6 h in the presence of 3.0 μg/ml brefeldin A [ThermoFisher, cat. #00-4506-51]. At 24 hpi, macrophages were collected by scraping, stained with Zombie Violet Fixable Viability dye [BioLegend, cat. #423113], incubated with Human TruStain FcX Fc Receptor Blocking Solution [BioLegend, cat. #422302], and surface-stained with anti-CD80-PE [ThermoFisher Scientific, cat. #12-0809-42)]. The cells were then fixed with CytoFix [BD, cat. #554655], permeabilized with BD PermWash [cat. #554723, and stained intracellularly with 1 μg/ml 4G2-AF647 (as above) plus either 1.25 μg/ml anti-IL-6-FITC [BioLegend, cat. #501103] or 2.5 μg/ml anti-TNF-AF488 [BioLegend, cat. #502917]. Data were acquired on a Sony MA900 flow cytometer and analyzed using FlowJo software v10 (TreeStar).

qRT-PCR

At 24 hpi, total RNA was isolated from macrophages using a Direct-Zol RNA micro-prep kit [Zymo, cat. #R2062] and RNA was reverse transcribed using an iScript kit [Bio-Rad, cat. #1708891]. qPCR was performed on an Applied Biosystems QuantStudio 5 Real-time PCR system [ThermoFisher Scientific] using iTaq Universal SYBR Green Supermix [Bio-Rad cat. #1725121] with the following primers: ribosomal protein lateral stalk subunit P0 (RPLP0) forward 5′-GTGTTCGACAATGGCAGCAT-3′; RPLP0 reverse 5′-GACACCCTCCAGGGAGCGA-3′; CD80 forward 5’-ATCCTGGGCCATTACCTTAATC-3’; CD80 reverse 5’-CTCTCATTCCTCCTTCTCTCTCT-3’; IL-6 forward 5’-AGACAGCCACTCACCTCTTCAG-3’; IL-6 reverse 5’-TTCTGCCAGTGCCTCTTTGCTG-3’; TNF forward 5’-CTCTTCTGCCTGCTGCACTTTG-3’; TNF reverse 5’-ATGGGCTACAGGCTTGTCACTC-3’; and IL-1-beta forward 5’-CCACAGACCTTCCAGGAGAATG-3’;IL-1-beta reverse 5’-GTGCAGTTCAGTGATCGTACAGG-3’.

Western blot analysis

moDC were infected with ZIKV PRVABC59 or DENV-PL046 for 24 h and stained with Zombie Violet™ Fixable Viability stain [BioLegend, cat. #423113] or 7-AAD Viability Staining Solution [BioLegend, cat. #420403] for 20 min at 4 °C. Live cells were then sorted using a SONY SH800S Cell Sorter (≥ 10⁶ cells per condition) and subjected to subcellular fractionation using NE-PER Nuclear and Cytoplasmic Extraction Reagents [Thermo Scientific, cat. #78833] per the manufacturer’s instructions. A sample of the moDCs was fixed, permeabilized, and stained with 4G2-AF647 antibody as described above to determine the infection rate. Nuclear and cytoplasmic cell fractions containing Complete protease inhibitor [Sigma, cat. # 04693116001] were heated for 10 min at 70 °C in LDS sample buffer [Invitrogen, cat. #0007] and proteins were then separated on Bolt 4–12% Bis-Tris Plus gels and transferred to PVDF membranes. The membranes were blocked in a blocking buffer composed of 1x Tris-buffered saline [Fisher cat. #BP2471-1] plus 0.1% Tween 20 plus 5% non-fat milk [Research Products International, cat. #M17200-500.0], for 1 h at room temperature and then incubated at 4 °C overnight with primary antibodies: histone deacetylase (HDAC1) control nuclear protein [Cell signaling Technology, cat. #5356], glyceraldehyde 3-phosphate dehydrogenase (GAPDH) control cytoplasmic protein [Cell Signaling Technology, cat. #5174], or NF-κB p65 [Cell Signaling Technology, cat. #8242] all at 1:1000 dilution in blocking buffer. The membranes were washed again and incubated with HRP- or fluorophore-conjugated anti-rabbit or anti-mouse secondary antibodies [IRDye 800CW Goat anti-Mouse IgG Secondary Antibody, LI-COR, cat. # NC0824545; IRDye 680RD Goat anti-Rabbit IgG Secondary Antibody LI-COR, cat. # NC0885458; Goat anti-Rabbit IgG (H + L) Secondary Antibody, DyLight 800, Thermofisher, cat. # SA535571; Peroxidase AffiniPure goat anti-mouse IgG, F(ab′)₂fragment-specific, Jackson ImmunoResearch, cat. #115-035-072; Goat Ant-Rabbit, Polyclonal Immunoglobulins, HRP, Agilent, cat. #044801-2] at 1:10,000 for 1 h at room temperature. Protein detection was performed using a ChemiDoc MP Imaging System, and the data were analyzed with Image Lab 6.0.1 [both Bio-Rad].

Mixed leukocyte reaction

moDCs were infected with ZIKV SD001 (MOI of 0.5) or DENV2 UIS353 (MOI of 1) for 48 h and mixed with freshly prepared allogeneic PBMCs at a ratio of 1:10 (5 × 10⁴ moDCs, 5 × 10⁵ PBMCs) in 200 μl complete RPMI medium containing 10% human serum (Innovative Research) in 96-well round-bottom plates. PBMCs mixed with uninfected moDCs and anti-CD3/CD28 (1 μg/ml each)-stimulated PBMCs served as controls. After 1 or 3 days, MLR cultures were spun down, and culture supernatants were collected. IL-2 and IFNγ levels in the culture supernatants were quantified by ELISA (BD Bioscience). The cells were resuspended with fresh medium containing 5 μg/ml brefeldin A [BioLegend] for an additional 6 h at 37 °C and then stained with efluor 455UV viability dye [Invitrogen, cat.#65-0868-18)], anti-CD3 PerCP-Cy5.5 [clone HIT3a, BioLegend], anti-CD4 eF450 [clone OKT4, Thermo Fisher Scientific], anti-CD8 APC-Cy7 [clone HIT8a, BioLegend], anti-CD69 BV785 [clone FN50, BioLegend], followed by fixation and permeabilization with the Cytofix/Cytoperm kit [BD]. The cells were then stained intracellularly with a combination of anti-IFNγ PE or FITC [clone 4S.B3, BioLegend], anti-IL-2 BV711 [clone MQ1-17H12, BioLegend], and anti-granzyme B AF647 [clone QA16A02, BioLegend]. All antibodies were diluted 200-fold for staining. Data were acquired on an LSRFortessa and analyzed using FlowJo software.

Antigen-specific T cell stimulation

moDCs infected with ZIKV SD001 (MOI of 0.5) or DENV2 UIS353 (MOI of 1) for 48 h were prepared as described above. Autologous T cells were negatively selected [STEMCELL, cat. #19051] from fresh PBMCs (same individual as for moDC preparation) and co-cultured with the infected moDCs with 1 µg/mL of defined immunodominant peptide megapools [CD4 peptide pool: JPT, cat. #PM-CEFT-MHCII-3, including 14 peptide sequences derived from Clostridium tetani, Epstein-Barr virus, human cytomegalovirus, and Influenza A; CD8 peptide pool: Miltenyi Biotec, cat. #130-098-426, including 32 peptide sequences derived from Epstein-Barr virus, human cytomegalovirus, and Influenza A; EFX Ultra SuperStim Pool: JPT, cat. #PM-EFX-2, including 146 peptides from Clostridium tetani, Coxsackievirus B4, Haemophilus influenza, Helicobacter pylori, Human adenovirus 5, Human herpesvirus 1, Human herpesvirus 2, Human herpesvirus 3, Human herpesvirus 4, Human herpesvirus 6, Human papillomavirus, JC polyomavirus, Measles virus, Rubella virus, Toxoplasma gondii, and Vaccinia virus; or SARS-CoV-2 peptide pool: Miltenyi Biotec, cat. #130-129-712] at a ratio of 10⁶ autologous T cells to 10⁵ moDCs in 96-well round-bottomed plates. Unstimulated controls consisted of T cells cultured with an equal concentration of peptide vehicle (DMSO). After 24 h, cell culture supernatants were collected for IFNγ quantification by ELISA, and the cells were stained with efluor 455UV viability dye [Invitrogen, cat. #65-0868-18], anti-CD3 AF700 [clone OKT3, BioLegend, cat #317340], anti-CD4 BV605 [clone RPA-T4, BD Biosciences, cat. #562658], anti-CD8 BV650 [clone RPA-T8, BioLegend, cat. #301042], and anti-CD69 PE [clone FN50, BD Biosciences, cat. #555531]. Antibodies were stained at 200-fold dilution. Data were acquired on an LSRFortessa and analyzed with FlowJo software.

Cell sorting for RNA-seq and ChIP-seq

moDCs were collected from control or virus-containing cultures at the indicated times, stained with Zombie Violet™ Fixable Viability stain [BioLegend, cat. #423113] for 20 min at 4 °C, and washed. For RNA-seq, the cells were fixed and permeabilized with 4% paraformaldehyde and 0.1% saponin in the presence of RNasin Plus ribonuclease inhibitor (400 µ/ml; Promega, cat. #N2615), centrifuged, resuspended in wash buffer (0.2% BSA, 0.1% saponin, 400 µ/ml of RNasin Plus in PBS), and centrifuged. The supernatant was discarded, and the cells were resuspended in human Fc blocker buffer (1% BSA, 0.1% saponin, 1600 µ/ml RNasin Plus in PBS) for 5 min at 4 °C and incubated with 4G2-AF647 antibody for 30 min at 4 °C. The cells were centrifuged at 1000 × g for 3 min at 4 °C, resuspended in wash buffer, and re-centrifuged. Finally, the cells were resuspended in a sorting buffer (0.5% BSA and 1600 µ/ml RNasin Plus in PBS) and sorted into ZIKV +, DENV +, ZIKV, or DENV − populations using a FACSAria [BD Biosciences]. For ChIP-seq experiments, washed moDCs were cross-linked with 4 mM disuccinimidyl glutarate in PBS for 30 min at room temperature, followed by 1% formaldehyde for 15 min at room temperature and quenched with 0.125 M glycine. The moDCs were then prepared and sorted into virus + and virus − populations as described above for RNA-seq experiments, except the RNasin Plus inhibitor was replaced by 1x cOmplete protease inhibitors [Roche, cat. #4693132001]. After sorting, the cells were washed in a sorting buffer, aliquoted into tubes at 5 × 10⁵ cells/sample, pelleted, snap-frozen, and stored at − 80 °C.

NF-κB p65 ChIP-seq

Samples of 5 × 10⁵ cells were resuspended on ice in 500 µl RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% IGEPAL CA-630, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.5 mM DTT, 1x protease inhibitor cocktail). All subsequent steps were performed at 4 °C. Chromatin was sheared by sonication in epitubes using 14 cycles of 10 s at ~ 13 W output and 30 s pause with a Sonic Dismembrator 60 [Fisher Scientific]. Samples were recovered and spun at 21,000 × g for 5 min, and the pellet was discarded. An aliquot of 2% of the sample volume was reserved as DNA input control and stored at − 20 °C. To prepare antibody-coupled beads for ChIP, protein A Dynabeads [Invitrogen cat. #10002D], 20 μl per sample, were washed twice with 1 ml 0.5% BSA in TET (10 mM Tris-HCl, pH 8, 1 mM EDTA, 0.1% Tween 20), and resuspended in the same buffer. Anti-NFκB p65 antibody [C-20, Santa Cruz Biotechnology cat. #sc-372] was added to the beads, and the sample was rotated for 1 h at room temperature. The supernatant was then removed, and the Dynabeads were collected using a magnet, washed once with 0.1% BSA/TET, and resuspended in 10 µl RIPA buffer. For ChIP, 10 μl of prepared antibody-conjugated Dynabeads was added to each sample and rotated overnight at 4 °C. The beads were then washed three times with 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), three times with 10 mM Tris-HCl, pH 7.4, 250 mM LiCl, 1 mM EDTA, 1% Triton X-100, 0.7% sodium deoxycholate, and twice with TET/0.2% (as above except 0.2% Tween 20). Dynabeads were resuspended in 25 μl TT (10 mM Tris-HCl, pH 8, 0.05% Tween 20). Input samples were resuspended in 25 μl TT, and libraries were generated in parallel with ChIP samples. Library NEBNext End Prep and Adapter Ligation were performed using NEBNext Ultra II DNA Library Prep kit [New England BioLabs, cat. #E7645L] according to the manufacturer’s instructions with barcoded adapters [NextFlex, Bio Scientific. Cat #514123]. Libraries were incubated with proteinase K at 55 °C for 1 h and then at 65 °C overnight to reverse formaldehyde crosslinks. Libraries were PCR amplified for 14 cycles with NEBNext High Fidelity 2X PCR MasterMix [New England BioLabs, cat. #NEBM0541]. Libraries were size selected for 200–400 bp fragments by gel extraction from 10% TBE gels [Life Technologies] and were single-end sequenced on an Illumina NextSeq 500 (San Diego).

RNA Isolation and library preparation for RNA-seq

Total RNA was isolated from cell pellets using a RecoverAll Total Nucleic Acid Isolation Kit [Invitrogen, cat. #AM1975] starting at the protease digestion step. All steps were performed according to the manufacturer’s recommendations, except that cells were incubated for 3 h at 50 °C in digestion buffer supplemented with RNasin Plus (100-fold dilution). Thereafter, the RNA was treated with in-column DNase and eluted per the kit manufacturer’s instructions. RNA quality was determined using a BioAnalyzer system [Agilent] with the Eukaryote Total RNA Pico Chip. Samples with RNA integrity values > 8.0 were used for library preparation. RNA libraries were generated using the TruSeq Stranded Total RNA Library Prep Kit (Illumina) kit, and single-end sequenced on an Illumina Hi-Seq 4000, NextSeq 500, or NovaSeq 6000, all according to the manufacturer’s instructions.

NGS Data preprocessing

FASTQ files were mapped to the following reference genomes: UCSC build hg38 (human), GenBank KU955593.1 (ZIKV), or NC_001474.2 (DENV2). STAR with default parameters was used to map RNA-seq, and Bowtie2 with default parameters was used to map ChIP-seq data. HOMER was used to convert uniquely aligned reads into “tag directories” for further analysis.

Integrated NGS data analysis

RNA-seq reads aligned to a combined GRCh38/hg38 and either the ZIKV genome (KU955593.1) or DENV2 genome (NC_001474.2) were used to calculate the percentage of reads aligned to the ZIKV or DENV genome as follows: ([number of reads aligned to ZIKV or DENV genome / number of reads aligned to hg38 + ZIKV or hg38 + DENV genomes] × 100). RNA-seq reads aligned to the GRCh38/hg38 assembly were used to generate gene expression FPKM values using HOMER⁴². To measure gene expression, HOMER’s analyzeRepeats.pl utility was used to quantify reads in transcript exons defined by GENCODE. Differentially expressed genes and regularized logarithm (rlog) normalization values for each gene were calculated using DESeq2 while accounting for individual donors in the design matrix. NF-κB p65 ChIP-seq peaks were called from two biologic replicates of DENV- or ZIKV-infected samples with mock-infected conditions as the background (-b) and input DNA as input (-i) using HOMER’s getDifferentialPeaksReplicates.pl with the “-style factor” and default parameters. Differentially regulated peaks between conditions were calculated by first merging features from each condition (or assay) into the union of nonredundant features using mergePeaks. Then, differentially enriched peaks were compared between conditions using HOMER’s getDifferentialPeaks⁴³. Known motif enrichment and de novo motif discovery were performed using HOMER’s findMotifsGenome.pl utility with default parameters. NF-κB p65 peaks were analyzed from − 100 to + 100 base pairs relative to the center of the peaks, reflecting the locations where TFs and collaborating TF motifs are located. Normalized histograms of ChIP-seq peaks were generated using HOMER’s annotatePeaks.pl and reported relative to a total of 10⁷ uniquely aligned reads per experiment. Functional enrichment calculations were performed on differentially expressed genes (RNA-seq) and NF-κB p65 ChIP-seq peaks using Metascape⁴⁴. Fold change values were clustered using Cluster 3.0⁴⁵ and visualized using Java TreeView⁴⁶.

Statistical analysis

Data were analyzed for distribution using normality and lognormality tests (D’Agostino–Pearson or Shapiro–Wilk) and compared using either a paired parametric t test or repeated measures one-way analysis of variance (ANOVA) with multiple comparisons test (indicated in each figure legend) or a paired non-parametric, Mann–Whitney or Friedman’s test with Dunn’s multiple comparisons test. Geometric mean and geometric standard deviation (SD) are shown for data plotted on a log₁₀ scale, and mean and standard error of the mean (SEM) or SD are shown for data plotted on a linear scale. Exact P-values are indicated or are categorically defined as: throughout as follows: not significant (P  >  0.05), *P  <  0.05, **P  <  0.01, and ***P  <  0.001. All data were analyzed, and graphs were generated using Prism software v10 (GraphPad).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.