DOI: 10.34894/9q0fvo
title: Data and R Scripts for: "Transcriptome network analysis implicates CX3CR1-positive type 3 dendritic cells in non-infectious uveitis"
title: CD1c+ cDC2 RNA seq in non-infectious uveitis

description: This dataset contains the processed RNA sequencing data of purified CD1c-positive conventional type 2 dendritic cells (CD1c+ cDC2s), functional enrichment analysis, manual and automatic gating data of (i.e., flowSOM) flow cytometry, and multiplex cytokine analyses as outlined in &lt;i&gt;Hiddingh et al. 2022 "Transcriptome network analysis implicates CX3CR1-positive type 3 dendritic cells in non-infectious uveitis &lt;/i&gt; &lt;dl&gt;see preprint on &lt;a href="https://www.biorxiv.org/content/10.1101/2021.11.16.468816v4"&gt;&lt;i&gt;BioRxiv&lt;/i&gt;&lt;/a&gt;
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Data are from two cohorts (cohort I, n=36, and cohort II, n=42) of in total 51 patients with non-infectious uveitis (&lt;i&gt;HLA-B27-positive acute anterior uveitis&lt;/i&gt;, &lt;i&gt;idiopathic intermediate uveitis&lt;/i&gt;, &lt;i&gt;HLA-A29-positive Birdshot Uveitis (Birdshot chorioretinopathy)&lt;/i&gt;, and 27 sex/age-matched healthy controls without ocular inflammatory disease). &lt;br&gt;&lt;/br&gt;
All raw sequencing data are available at NCBI SRA under the accession number: &lt;dl&gt;

&lt;li&gt; &lt;a href="https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE195501"&gt;&lt;i&gt;GSE195501&lt;/i&gt;&lt;/a&gt; (FACS-sorted cohort I). &lt;/li&gt;
&lt;li&gt;&lt;a href="https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE194060"&gt;&lt;i&gt;GSE194060&lt;/i&gt;&lt;/a&gt; (MACS-sorted cohort II).&lt;/li&gt;
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This dataverseNL dataset contains additional raw, processed, and metadata (see &lt;a href="https://dataverse.nl/api/access/datafile/299114"&gt;readme&lt;/a&gt; file and reproducible R notebooks (R script and Image) used for the analysis in the manuscript: &lt;br&gt;&lt;/br&gt;
&lt;dl&gt;
&lt;dt&gt;R scripts (markdown + R image) with &lt;i&gt;step-by-step&lt;/i&gt; analyses &lt;/dt&gt; 
&lt;ul&gt;&lt;li&gt;&lt;dd&gt; Figure_1.rmd (see &lt;a href="https://dataverse.nl/api/access/datafile/320666"&gt;"Figure_1.html"&lt;/a&gt;)&lt;/dd&gt;&lt;/li&gt;
&lt;li&gt;&lt;dd&gt;Figure_2.rmd (see &lt;a href="https://dataverse.nl/api/access/datafile/320651"&gt;"Figure_2.html"&lt;/a&gt;)&lt;/dd&gt;&lt;/li&gt;
&lt;li&gt;&lt;dd&gt;Figure_3.rmd (see &lt;a href="https://dataverse.nl/api/access/datafile/320723"&gt;"Figure_3.html"&lt;/a&gt;)&lt;/dd&gt;
&lt;/li&gt;
&lt;li&gt;&lt;dd&gt;Figure_4.rmd (see &lt;a href="https://dataverse.nl/api/access/datafile/320726"&gt;"Figure_4.html"&lt;/a&gt;)&lt;/dd&gt;
&lt;/li&gt;
&lt;li&gt;&lt;dd&gt;Figure_5.rmd(see &lt;a href="https://dataverse.nl/api/access/datafile/320692"&gt;"Figure_5.html"&lt;/a&gt;)&lt;/dd&gt;&lt;/li&gt;
&lt;/dd&gt;&lt;/li&gt;

&lt;li&gt;&lt;dd&gt;Processed RNA seq data (including WGCNA) (see folder &lt;b&gt;Uveitis_mDC&lt;/b&gt; in files)&lt;/dd&gt;&lt;/li&gt;&lt;/ul&gt;

&lt;dt&gt;Experimental data&lt;/dt&gt;
&lt;ul&gt;&lt;li&gt;&lt;dd&gt;Manual gating data of MACS-sorted fractions cohort I (see &lt;a href="https://dataverse.nl/api/access/datafile/320648"&gt;here&lt;/a&gt;)&lt;/dd&gt;&lt;/li&gt;
&lt;li&gt;&lt;dd&gt;Manual gating data for CD1c+ cDC2 subsets in PBMCs (see &lt;a href="https://dataverse.nl/api/access/datafile/320646"&gt;here&lt;/a&gt;)&lt;/dd&gt;&lt;/li&gt;
&lt;li&gt;&lt;dd&gt;Manual gating CD14+ and CD14- CD1c+ cDC2 fractions from Buffy (see &lt;a href="https://dataverse.nl/api/access/datafile/320697"&gt;here&lt;/a&gt;)&lt;/dd&gt;&lt;/li&gt;
&lt;li&gt;&lt;dd&gt;qPCR data for &lt;i&gt;CX3CR1&lt;/i&gt;,&lt;i&gt;CCR5&lt;/i&gt;,&lt;i&gt;CCR2&lt;/i&gt;,&lt;i&gt;IRF8&lt;/i&gt;,&lt;i&gt;TLR7&lt;/i&gt;,&lt;i&gt;RUNX3&lt;/i&gt; and &lt;i&gt;CD36&lt;/i&gt; in sorted CD14+ and CD14- CD1c+ DCs (see &lt;a href="https://dataverse.nl/api/access/datafile/320682"&gt;here&lt;/a&gt;)&lt;/dd&gt;&lt;/li&gt;
&lt;li&gt;&lt;dd&gt;qPCR data (fold change compared to medium) for &lt;i&gt;RUNX3&lt;/i&gt; and &lt;i&gt;CD36&lt;/i&gt; in overnight stimulated cDC2 cultures (see &lt;a href="https://dataverse.nl/api/access/datafile/320652"&gt;here&lt;/a&gt;)&lt;/dd&gt;&lt;/li&gt;
&lt;li&gt;&lt;dd&gt;Cell phenotypes identified by flowSOM (7x7 grid) using the cDC2-subset flow cytometry panel (see &lt;a href="https://dataverse.nl/api/access/datafile/320674"&gt;here&lt;/a&gt;)&lt;/dd&gt;&lt;/li&gt;
&lt;li&gt;&lt;dd&gt;IL-23 ELISA concentration in supernatant of overnight LTA-stimulated cDC2 subset cultures (see &lt;a href="https://dataverse.nl/api/access/datafile/320658"&gt;here&lt;/a&gt;)&lt;/dd&gt;&lt;/li&gt;
&lt;li&gt;&lt;dd&gt;Luminex Multiplex Cytokine analysis of supernatant of overnight LTA-stimulated cDC2 subset cultures (see &lt;a href="https://dataverse.nl/api/access/datafile/320644"&gt;here&lt;/a&gt;)&lt;/dd&gt;&lt;/li&gt;&lt;/ul&gt;


&lt;dt&gt;Other transcriptomic data used in the R scripts (above)&lt;/dt&gt; 
&lt;ul&gt;&lt;li&gt;&lt;dd&gt; WT Untreated cDC2 versus cDC2 from &lt;i&gt;Runx3&lt;/i&gt;-11cKO mice&lt;a href="https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE48590"&gt;GSE48590&lt;/i&gt; &lt;/a&gt; generated by &lt;a href="https://pubmed.ncbi.nlm.nih.gov/24204843/"&gt;&lt;i&gt; Dicken et al., PLoS One
2013&lt;/i&gt; &lt;/a&gt;&lt;/li&gt;
&lt;li&gt;&lt;dd&gt;WT Untreated cDC2 versus cDC2 from &lt;i&gt;Notch2&lt;/i&gt;-11cKO mice&lt;a href="https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE119242"&gt;GSE119242&lt;/i&gt; &lt;/a&gt; generated by &lt;a href="https://pubmed.ncbi.nlm.nih.gov/30279176/"&gt;&lt;i&gt; Briseño et al., Proc Natl Acad Sci U S A 2018&lt;/i&gt; &lt;/a&gt;&lt;/li&gt;

&lt;li&gt;&lt;dd&gt;Sorted CD14+CD5-CD163+ and CD14-CD5-CD163+ cDC2s from SLE and Scleroderma patients &lt;a href="https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE136731"&gt;GSE136731&lt;/i&gt; &lt;/a&gt; generated by &lt;a href="https://www.cell.com/immunity/fulltext/S1074-7613(19)30334-6?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1074761319303346%3Fshowall%3Dtrue"&gt;&lt;i&gt; Dutertre et al., Immunity 2019&lt;/i&gt; &lt;/a&gt;&lt;/li&gt;

&lt;li&gt;&lt;dd&gt;Single-cell RNA-seq of aqueous humor from 4 HLA-B27-positive uveitis patients and control &lt;a href="https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE178833"&gt;GSE178833&lt;/i&gt; &lt;/a&gt; generated by &lt;a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8594918/"&gt;&lt;i&gt; Kasper et al., Elife 2021&lt;/i&gt; &lt;/a&gt;&lt;/li&gt;

&lt;li&gt;&lt;dd&gt;Inflammatory [inf-]cDC2s&lt;a href="https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE149619"&gt;GSE149619&lt;/i&gt; &lt;/a&gt; generated by &lt;a href="https://pubmed.ncbi.nlm.nih.gov/32392463/"&gt;&lt;i&gt; Bosteels et al., Immunity 2020&lt;/i&gt; &lt;/a&gt;&lt;/li&gt;

&lt;li&gt;&lt;dd&gt;RNA-seq data from cDC2s generated from murine bone marrow cells in co-culture with stromal OP-9 cell line transduced with or without expression of the Notch ligand Delta-like 1&lt;a href="https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE110577"&gt;GSE110577&lt;/i&gt; &lt;/a&gt; generated by &lt;a href="https://pubmed.ncbi.nlm.nih.gov/29925006/"&gt;&lt;i&gt; Kirkling et al., Cell Rep 2018 &lt;/i&gt; &lt;/a&gt;&lt;/li&gt;
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description: <b>Transcriptome network analysis implicates CX3CR1-positive type 3 dendritic cells in non-infectious uveitis</b>
<br>
<b>Background:</b>Type I interferons (IFNs) promote the expansion of subsets of CD1c+ conventional dendritic cells (CD1c+ DCs), but the molecular basis of CD1c+ DCs involvement in conditions not associated without elevated type I IFNs remains unclear.
<br>
<b>Methods: </b>We analyzed CD1c+ DCs from two cohorts of non-infectious uveitis patients and healthy donors using RNA-sequencing followed by high-dimensional flow cytometry to characterize the CD1c+ DC populations. <br>
<b>Results:</b> We report that the CD1c+ DCs pool from patients with non-infectious uveitis is skewed towards a gene module with the chemokine receptor CX3CR1 as the key hub gene. We confirmed these results in an independent case-control cohort and show that the disease-associated gene module is not mediated by type I IFNs. An analysis of peripheral blood using flow cytometry revealed that CX3CR1+ DC3s were diminished, whereas CX3CR1- DC3s were not. Stimulated CX3CR1+ DC3s secrete high levels of inflammatory cytokines, including TNF-alpha, and CX3CR1+ DC3-like cells can be detected in inflamed eyes of patients. <br><b>Conclusion:</b> These results show that CX3CR1+ DC3s are implicated in non-infectious uveitis and can secrete proinflammatory mediators implicated in its pathophysiology.