Using single-cell RNA-sequencing, we identify a new thymic CD14+Sirp+ population of monocyte-derived dendritic cells (CD14+moDC) that are enriched in the thymic medulla and effectively acquire mTEC-derived antigens in response to the above chemokines
Using single-cell RNA-sequencing, we identify a new thymic CD14+Sirp+ population of monocyte-derived dendritic cells (CD14+moDC) that are enriched in the thymic medulla and effectively acquire mTEC-derived antigens in response to the above chemokines. (3β,20E)-24-Norchola-5,20(22)-diene-3,23-diol e, 4c, 5e, f, 6c, e, h, 7aCd, f, h and 8b, c, e are provided as a Source Data file. The raw RNA sequencing data are deposited at the ArrayExpress database [https://www.ebi.ac.uk/arrayexpress/] under accession numbers E-MTAB-8024 (Fig.?2a, b), E-MTAB-8025 (Fig.?2d, e) and E-MTAB-8028 (Fig.?5aCc). Abstract The development of thymic regulatory T cells (Treg) is mediated by Aire-regulated self-antigen presentation on medullary thymic epithelial cells (mTECs) and dendritic cells (DCs), but the cooperation between these cells is still poorly understood. Here we show that signaling through Toll-like receptors (TLR) expressed on mTECs regulates the production of specific chemokines and other genes associated with post-Aire mTEC development. Using single-cell RNA-sequencing, we identify a new thymic CD14+Sirp+ population of monocyte-derived dendritic cells (CD14+moDC) that are enriched in the thymic medulla and effectively acquire mTEC-derived antigens in response to the above chemokines. Consistently, the cellularity of CD14+moDC is diminished in mice with MyD88-deficient TECs, in which the frequency and functionality Rabbit Polyclonal to PARP (Cleaved-Gly215) of thymic CD25+Foxp3+ Tregs are decreased, leading to aggravated mouse experimental colitis. Thus, our findings describe a TLR-dependent function of mTECs for the recruitment of CD14+moDC, the generation of Tregs, and thereby the establishment of central tolerance. and and mRNA expression is determined by qRT-PCR from FACS sorted mTECs and DCs. The expression is calculated relative to Casc3 and normalized to the highest value within each experiment=1 (mean??SEM, and cytokines, (ii) chemokines. These mediators act through receptors that are primarily expressed by myeloid cells and DCs32. Specifically, IL36R, the receptor for IL1F6, is expressed by DCs and T cells33 while Csf2r, the receptor for Csf2, is expressed mostly by monocytes, macrophages, and granulocytes34. The Ccr9, the receptor for Ccl25, is expressed by both thymocytes and pDCs driving their migration into the thymus14,35. Both Ccr5 (receptor for Ccl4) and Ccr3 (receptor for Ccl24) are expressed predominantly on granulocytes and DCs modulating their migration into inflamed tissues32,36. qRT-PCR analysis confirmed MyD88-regulated expression of selected genes in mTECshigh (Fig.?2c). Since the TLRs were postulated to sense both microbial and endogenous molecules21, we examined which of them could potentially act as a trigger. The analysis of mRNA expression of MyD88-dependent cytokines and chemokines (Fig.?2b, c) (3β,20E)-24-Norchola-5,20(22)-diene-3,23-diol in the mTEChigh population isolated from either Germ-free (GF) or specific-pathogen-free (SPF) mice was comparable (Supplementary Fig.?2b), indicating that these signals are likely of endogenous origin. Open in a separate window Fig. 2 TLR/MyD88 signaling (3β,20E)-24-Norchola-5,20(22)-diene-3,23-diol in mTECshigh drives the expression of cytokines and chemokines.a Principal component analysis of bulk RNA-sequencing data from mTECshigh (sorted as in Supplementary Fig.?1a) derived from MyD88fl/fl and MyD88TECs mice. Data represents the analysis of and which signal via various chemokine receptors, including Ccr1, 3, 5, 6 which are expressed mostly on myeloid cells32. Cytokines (and and chemokines after in vitro (Fig.?2f) as well as in vivo intrathymic TLR9 stimulation (Fig.?2g) was confirmed by qRT-PCR analysis. As shown in Supplementary Fig.?2c, repeated intraperitoneal (i.p.) injection of CpG ODN was insufficient for the upregulation of chemokines in mTECshigh. It is (3β,20E)-24-Norchola-5,20(22)-diene-3,23-diol of note that in vitro stimulation of TLR4 on mTECshigh by LPS also resulted in the upregulation of the previously noted chemokines, albeit at a lower level (Supplementary Fig.?2d). In addition to TLRs, MyD88 also conveys signals generated by IL-1 family cytokines, such as IL-1, IL-18 or IL-3338. Even though the receptors for these cytokines are expressed by mTECshigh (Supplementary Fig.?3a), only in vitro stimulation with IL-1 lead to the upregulation of cytokines and chemokines induced by TLR9 stimulation (Supplementary Fig.?3b). Besides chemokines and cytokines, TLR/MyD88 signaling in mTECshigh (Fig.?2b) also regulated the expression of molecules associated with cornified epithelial pathway39 (Supplementary Data?1C4). This specifically relates to genes that are associated with post-Aire mTECs40,41, (3β,20E)-24-Norchola-5,20(22)-diene-3,23-diol such as and (Supplementary Fig.?3c). Moreover, previously published data has shown the enhanced.