好了淳玩，這一篇回歸我們的主業(yè)厌蔽，分享單細(xì)胞空間聯(lián)合分析的運(yùn)用畦贸，參考文章在Non-lesional and Lesional Lupus Skin Share Inflammatory Phenotypes that Drive Activation of CD16+ Dendritic Cells告希，主要運(yùn)用單細(xì)胞空間表征損傷后的皮膚免疫激活狀態(tài)和分布桥温，看來(lái)單細(xì)胞空間技術(shù)運(yùn)用的地方引矩，無(wú)處不在~~~
ABSTRACT
Cutaneous lupus erythematosus（皮膚紅斑狼瘡） (CLE) is a disfiguring and poorly understood condition frequently associated with systemic lupus（系統(tǒng)性狼瘡）. Studies to date suggest that non-lesional keratinocytes（非損傷角質(zhì)形成細(xì)胞） play a role in disease predisposition（傾向）, but this has not been investigated in a comprehensive manner or in the context of other cell populations. To investigate CLE immunopathogenesis（免疫發(fā)病機(jī)制）, normal-appearing skin, lesional skin, and circulating immune cells from lupus patients were analyzed via integrated single-cell RNA-sequencing and spatial-seq. We demonstrate that normal-appearing skin of lupus patients represents a type I interferon-rich, ‘prelesional’ environment that skews gene transcription in all major skin cell types and dramatically distorts cell-cell communication（“病灶前”環(huán)境會(huì)扭曲所有主要皮膚細(xì)胞類型的基因轉(zhuǎn)錄并顯著扭曲細(xì)胞間通訊 ）. Further, we show that lupus-enriched CD16+ dendritic cells undergo robust interferon education in the skin, thereby gaining pro-inflammatory phenotypes. Together, our data provide a comprehensive characterization of lesional and non-lesional skin in lupus and identify a role for skin education of CD16+ dendritic cells in CLE pathogenesis.(總之，我們的數(shù)據(jù)提供了狼瘡病變和非病變皮膚的綜合特征，并確定了 CD16+ 樹突狀細(xì)胞在 CLE 發(fā)病機(jī)制中的skin education作用旺韭。)
INTRODUCTION
Cutaneous lupus erythematosus (CLE) is a disfiguring inflammatory skin disease(毀容炎癥性皮膚病) that affects 70% of patients with systemic lupus erythematosus (SLE,系統(tǒng)性紅斑狼瘡). While about 50% of patients respond to SLE-directed therapies, many patients suffer from refractory skin lesions(難治性皮膚病變), even when their systemic disease is controlled. Lack of knowledge regarding the inflammatory composition of CLE and the drivers that instigate disease has delayed effective therapy development.(缺乏關(guān)于 CLE 的炎癥成分和引發(fā)疾病的驅(qū)動(dòng)因素的知識(shí)延遲了有效治療的發(fā)展)氛谜。
Intriguingly, the etiology of skin lesions in CLE may be, at least partially, found in abnormalities in non-lesional, normal-appearing skin（正常的皮膚也可能金玉其外，敗絮其中）. Recent data support a role for increased epidermal type I interferon (IFN) production and dysfunction of Langerhans cells as important for priming inflammatory and apoptotic responses. However, the role of other cells in the skin, the skewed communication networks between them, and cellular mediators of this inflammatory predisposition have not been well-defined（多細(xì)胞的相互作用來(lái)認(rèn)識(shí)疾病）。
In this paper, we examined the cellular composition of paired lesional and non-lesional skin samples from SLE patients with active CLE lesions to comprehensively define the cellular makeup and to characterize the principal mediators of inflammatory changes that contribute to the disease. We further examined the peripheral blood of the same patients to investigate the cutaneous education of monocyte-derived dendritic cells, which were found to be prominent in lesional and non-lesional skin. Overall, we found an IFN-rich signature and a unique, pro-inflammatory cellular communication network between stromal and inflammatory cells in lesional and non-lesional skin that supports a critical role for the skin itself in priming inflammatory responses in SLE patients（看來(lái)疾病的發(fā)生都是結(jié)尾输吏，真正的過程隱藏在正常的皮膚之中）百新。
RESULTS
Single-cell RNA-sequencing (scRNA-seq) of lesional and non-lesional skin from patients with CLE identifies diverse skin and immune cell populations（首先是單細(xì)胞分析，印象里單細(xì)胞做皮膚組織的情況不多見）惭嚣。
To investigate the cellular composition and comprehensive transcriptional effects of CLE, we performed scRNA-seq on lesional and sun-protected non-lesional skin from 7 patients with active CLE, 6 of whom also carried a diagnosis of SLE. Samples were analyzed in parallel with skin from 14 healthy controls from diverse sites. The final dataset comprised 46,540 cells, with an average of 2,618 genes and 11,645 transcripts per cell. Visualization using Uniform Manifold Approximation and Projection (UMAP) revealed 26 distinct cell clusters that were annotated as 10 major cell types, each comprising cells from lesional, non-lesional, and healthy control skin biopsies. Conspicuous clustering by disease state was evident for many cell types, including keratinocytes (KCs), myeloid cells, and melanocytes. Cell composition analysis revealed an increase in the proportion of myeloid cells in both lesional and non-lesional skin relative to healthy control。（單細(xì)胞開始的標(biāo)準(zhǔn)分析）
KCs from both lesional and non-lesional skin of patients with CLE exhibit a pathologic type I IFN signature
KCs constituted the majority of cells sequenced (25,675 cells). Sub-clustering analysis of KCs identified 14 sub-clusters, including several (5, 6, 8, 13) dominated by KCs from lupus patients（細(xì)胞類型的比例變化）. Analyses of characteristic KC subtype markers identified 5 KC states: basal, spinous, supraspinous, follicular, and cycling（我們?cè)谧x每篇文章的時(shí)候悔政，細(xì)胞定義的marker多多搜集一下）. Lupus-dominated sub-clusters corresponded to subpopulations within basal and spinous KC states.
The relatively shallow depth of scRNA-seq precludes direct examination of transcript levels for many cytokines implicated in CLE – particularly IFNs（scRNA-seq 的相對(duì)較淺的深度排除了直接檢查 CLE 中涉及的許多細(xì)胞因子的轉(zhuǎn)錄水平 - 特別是干擾素 ）. Thus, to investigate whether cytokine responses were driving this KC sub-clustering by disease, we calculated KC module scores derived from genes induced in cultured KCs upon stimulation with the indicated cytokines and generated feature plots displaying module scores for each cytokine（計(jì)算了 KC 模塊評(píng)分晚吞，該評(píng)分源自在用指定細(xì)胞因子刺激培養(yǎng)的 KC 中誘導(dǎo)的基因，并生成顯示每種細(xì)胞因子模塊評(píng)分的特征圖 ）. Lupus-enriched sub-
clusters corresponded best with cells exhibiting high scores for type I IFN (IFN-α), type II IFN (IFN-γ), and to a lesser degree TNF. Our and others’ prior work has identified a critical role for type I IFN in SLE and CLE keratinocytes. Accordingly, cytokine module violin plots revealed that lupus-enriched basal (sub-cluster 8) and spinous (5 and 6) sub-clusters consisted almost entirely of KCs with high IFN-α module scores, whereas the separation was less striking for IFN-γ and TNF. Notably, cells scoring highest in these clusters originated from non-lesional biopsies（正常的皮膚也顯示了疾病的表達(dá)程序）. This suggests that even normal-appearing skin from patients with CLE exists in a ‘prelesional’ state, primed by heightened type I IFN signaling. Follicular KC sub-clusters (10 and 11) showed elevated IFN-α cytokine module scores in non-lesional and lesional samples relative to healthy control as well, suggesting the follicular epithelium also represents an abnormal, IFN-rich environment in CLE; however, scores were far lower for follicular than basal KCs, implicating the interfollicular epidermis more strongly than the follicular epithelium in type I IFN education of neighboring stromal and skin-infiltrating cells谋国。
For a broader understanding of the transcriptomic differences in lesional KCs of patients with CLE, we performed differential expression analysis（差異表達(dá)分析雖然大家都做槽地，但是方法千差萬(wàn)別，大家可以參考我的文章10X單細(xì)胞（10X空間轉(zhuǎn)錄組）22種基因差異分析方法匯總） between the non-lesional and lesional CLE vs. healthy basal KCs and identified type I IFN downstream genes (e.g., MX1, IFITM1, IFITM3, IFI6, ISG15, IFI27) among the top upregulated genes in the lesional cells. We then used Ingenuity Pathway Analysis (IPA芦瘾，富集分析) to identify the top cytokines predicted to serve as upstream regulators for the genes induced in lesional samples, identifying primarily IFNs as upstream regulators of CLE-enriched transcripts. Corroborating this, canonical pathway analysis distinguished IFN signaling as highly enriched in lesional samples（INF信號(hào)在病變樣本中高度富集）捌蚊。
scRNA-seq identifies a CLE-enriched fibroblast subtype exhibiting a strong IFN response signature and IL-17A influence restricted to lesional skin
We next analyzed fibroblasts（成纖維細(xì)胞） (FBs), the other major stromal cell constituent of the skin. Sub-clustering analysis of 8,622 FBs identified 10 sub-clusters. Only one sub-cluster (4) was dominated by FBs from lupus patients. Annotation of these sub-clusters based on published dermal FB marker genes revealed three subtypes as previously described (SFRP2+, COL11A1+, and SFRP4+ FBs) and a small cluster marked by expression of COL66A1 and RAMP1 (RAMP1+ FBs)（細(xì)胞定義的結(jié)果真的需要搜集一下）. Immunohistochemistry of these key markers confirmed that SFRP2+ FBs constituted the majority of FBs. The lupus-enriched sub-cluster lay within the SFRP2+ FBs and was analyzed as an independent subtype（一般分析的過程中出現(xiàn)了新的細(xì)胞類型，那大家偷著樂吧近弟，發(fā)的文章肯定不低）.Analysis of the top gene markers of each FB subtype indicated that these FBs were distinguished by high IFN-stimulated gene (ISG) expression, and thus we designated these IFN FBs. Consistent with this, feature and violin plots depicting FB cytokine module scores calculated using genes induced in cultured FBs stimulated by cytokines as above revealed that IFN FBs were most uniquely distinguished by IFN-α and IFN-γ cytokine signatures . As in KCs, IFN cytokine module scores were highest in non-lesional FBs, reinforcing that normal-appearing skin of lupus patients represents a prelesional, IFN-primed environment（IFN 細(xì)胞因子模塊評(píng)分在非病變 FB 中最高缅糟，這強(qiáng)化了狼瘡患者外觀正常的皮膚代表了病變前、IFN 引發(fā)的環(huán)境 ）. We compared the cytokine upstream regulators identified in the comparisons of non-lesional vs. healthy basal KCs and non-lesional IFN FBs vs. healthy SFRP2+ FBs. This revealed that type I IFNs, such as IFNA2, IFNL1, and IFNB1, served as the top upstream regulators in both cell types. Notably, high FB IFN module scores were primarily restricted to the single sub-cluster of IFN FBs, indicating that only a specific subset of FBs in skin of patients with CLE exhibits robust IFN education.This is an interesting contrast to KCs, where non-lesional and/or lesional KCs showed elevated IFN module scores for the majority of sub-clusters祷愉。
T cells infiltrating lesional and non-lesional skin of patients with CLE demonstrate IFN education across multiple subsets including regulatory T cells
Having analyzed the major stromal cell types of the skin, we moved on to examination of the immune cells（免疫細(xì)胞）. Sub-clustering and annotation based on established marker genes identified nine T cell subsets. Cells of one sub-cluster were distinguished by expression of ISGs and were therefore designated IFN T cells（看來(lái)作者早有預(yù)謀啊窗宦，和IFN較上勁了）. IFN T cells derived primarily from lupus samples, constituting 13% and 15% of T cells from non-lesional and lesional samples, respectively, but <1% of healthy control T cells.（不過確實(shí)差異明顯）
Regulatory T cells (Tregs), annotated based on FOXP3 expression（FOXP3是Tregs的marker）, were detected in similar proportions across healthy control, non-lesional, and lesional samples. Investigations of Treg abundance in peripheral blood of patients with SLE have yielded conflicting results, possibly due to differences in methodology or definition of Tregs, but there appears to be consensus that Treg function is altered in patients with SLE（對(duì) SLE 患者外周血中 Treg 豐度的研究產(chǎn)生了相互矛盾的結(jié)果，可能是由于方法學(xué)或 Treg 定義的不同二鳄，但似乎一致認(rèn)為 SLE 患者的 Treg 功能發(fā)生了改變）. To investigate whether the non-lesional skin environment might influence Treg function in lupus patients, we examined the top DEGs upregulated in non-lesional vs. healthy control Tregs.（又是差異分析） This identified numerous ISGs, suggestive of chronic IFN stimulation. Overproduction of type I IFNs by antigen-producing cells (APCs) in SLE has been proposed as a cause of Treg dysfunction in SLE, and we have previously demonstrated that lupus-prone NZM2328 mice treated with ultraviolet light exhibit type I IFN-dependent suppression of Treg function. Thus, the chronic IFN stimulation of Tregs that we observe in non-lesional skin may contribute to impaired Treg ability to maintain immune homeostasis and self-tolerance in lupus（在非病變皮膚中觀察到的 Treg 的慢性干擾素刺激可能導(dǎo)致 Treg 維持免疫穩(wěn)態(tài)和狼瘡自我耐受的能力受損 ）
One sub-cluster of T cells clustered closely with Tregs but did not express FOXP3. Rather, this sub-cluster was distinguished by expression of CXCL13, a B cell-attracting chemokine and SLE biomarker that appears to play a pathogenic role, as well as ICOS and PDCD1 (encoding PD-1), leading us to annotate these as T follicular helper (Tfh)-like cells（該亞群的特征在于 CXCL13（一種 B 細(xì)胞吸引趨化因子和 SLE 生物標(biāo)志物赴涵，似乎起致病作用）以及 ICOS 和 PDCD1（編碼 PD-1）的表達(dá)，導(dǎo)致我們將它們注釋為 T 濾泡輔助因子 (Tfh) 樣細(xì)胞）. Abundance of these cells varied by disease state at 1%, 4%, and 2% of healthy control, non-lesional, and lesional T cells, respectively（差異不大）. Closer inspection revealed that Tfh-like cells from healthy control and non-lesional samples differed in expression of ISGs including CXCL13 (expressed by 0% of healthy control vs. 76% of non-lesional Tfh-like cells). A similar population of Tfh-like cells was detected in scRNA-seq of kidney biopsies from patients with lupus nephritis, where they were theorized to promote B cell responses such as local antibody production and antigen-specific T cell activation by B cells. Their detection in our dataset suggests a similar process might be occurring in non-lesional skin of lupus patients as well.（在我們的數(shù)據(jù)集中的檢測(cè)表明订讼，類似的過程也可能發(fā)生在狼瘡患者的非病變皮膚中髓窜。）
Altogether, T cell imbalances and the presence of IFN T cells and other IFN-educated T cell subsets including Tregs in non-lesional samples indicate the presence of an abnormal and likely pathologic T cell infiltrate poised in the prelesional environment of normal-appearing skin of patients with CLE。
Major shifts in myeloid cell subsets are detected in lesional and non-lesional skin of CLE patients
We then evaluated myeloid cells, the other major immune cell type detected in our skin samples. Sub-clustering and annotation identified nine myeloid cell subsets with largely distinct marker genes: classical type 1 dendritic cell (cDC1; CLEC9A, IRF8), classical type 2 dendritic cell subset A (cDC2A; LAMP3 and CD1B), classical type 2 dendritic cell subset B (cDC2B; CLEC10A, IL1B), plasmacytoid dendritic cell (pDCs; GZMB, JCHAIN), CD16+ dendritic cell (CD16+ DC; FCGR3A,HES1), Langerhans cell (LC; CD207, CD1A), lipid-associated macrophage (LAM; APOE, APOC1), perivascular macrophage (PVM; CD163, SELENOP), and plasmacytoid dendritic cell-like cell (pDC-like; PPP1R14A, TRPM4)（細(xì)胞類型定義的marker還是要多多搜集一下）. Myeloid cell subsets showed far greater variability in representation among healthy control, non-lesional, and lesional samples than the above cell types欺殿。（變化幅度較大）
plasmacytoid dendritic cell (pDCs; GZMB, JCHAIN), CD16+ dendritic cell (CD16+ DC; FCGR3A, HES1), Langerhans cell (LC; CD207, CD1A), lipid-associated macrophage (LAM; APOE, APOC1), perivascular macrophage (PVM; CD163, SELENOP), and plasmacytoid dendritic cell-like cell (pDC-like; PPP1R14A, TRPM4) that was previously described by Villani et al（看來(lái)都是根據(jù)前人的marker定義的）. Myeloid cell subsets showed far greater variability in representation among healthy control, non-lesional, and lesional samples than the above cell types. In healthy control skin, cDC2Bs accounted for nearly half (47%) of the myeloid cells; this differed greatly from non-lesional lupus skin, where pDCs dominated (41%), although most were from a single patient. In keeping with prior reports, LCs were decreased in lesional and non-lesional lupus skin. Among the most striking differences between healthy control and lupus skin, however, was the overrepresentation of CD16+ DCs in both non-lesional and lesional CLE samples compared to healthy skin（細(xì)胞類型在三種樣本中變化很大）. While the exact identity of these cells remains somewhat in flux, CD16+ DCs are gaining recognition as a unique DC subset characterized by expression of FCGR3A/CD16a that can be detected as a transcriptomically distinct population（這個(gè)子集最為特殊）. This population is thought to overlap with CD16+ DCs previously described by MacDonald et al. as expressing high levels of CD86 and CD40 and possessing potent T cell stimulatory capabilities. CD16+ DCs also exhibit enhanced capacity relative to cDC2Bs for secretion of inflammatory cytokines upon toll-like receptor stimulation, a capacity that is further enhanced in CD16+ DCs isolated from peripheral blood of patients with SLE. Expansion and enhanced function of CD16+ DCs in lupus patients could therefore promote pathogenesis. Based on shared surface marker expression, this subset may also overlap with 6-Sulfo LacNAc-dendritic cells (slanDCs), a pro-inflammatory myeloid DC subset that has been linked to lupus immunopathogenesis and is increased in lesional skin of lupus patients（這個(gè)地方還是細(xì)胞類型比例變化明顯寄纵，容易研究，后續(xù)我們要看看整合分析作者是怎么做的）脖苏。
Ligand-receptor (L-R) analysis demonstrates lupus-enriched cell-cell interactions prominently involving CD16+ DCs（通訊分析）
Following identification of cellular populations, we then sought to understand how cell-cell communication differed in the skin of lupus patients. We thus performed L-R analyses among all major cell populations within healthy control, non-lesional, and lesional skin samples using CellPhoneDB（又是CellPhoneDB）. Each L-R pair was then assigned to the condition in which it showed the highest interaction score, and the number of interactions for each cell type pair was plotted. Few L-R interactions were strongest in healthy control skin, and the majority of these represented KC-KC crosstalk. Non-lesional skin, in contrast, showed many more interactions. FBs represented the main ligand-expressing cell type among non-lesional-enriched pairs, but myeloid and endothelial cells (ECs) were also highly interactive. Additionally, eccrine gland cells participated in a high number of interactions in non-lesional skin as expressers of both ligands and receptors, which is of interest given that perieccrine inflammation is a hallmark of CLE. In lesional skin, however, myeloid and ECs were most prominent among cell-cell interactors擂啥。
These analyses indicate a prominent role for myeloid cells in non-lesional and lesional skin（這些分析表明骨髓細(xì)胞在非損傷和損傷皮膚中的重要作用 ）. Given the functional heterogeneity within the myeloid cell population, we sought to define more precisely the myeloid and other cellular participants in these interactions. Thus, we divided KCs, FBs, T cells, and myeloid cells into their respective subsets and repeated analysis of L-R pairs（精細(xì)分析通訊）. Plotting the L-R interactions revealed an even denser network of candidate cellular interactors. Regarding stromal cells, ligands expressed by KC subsets primarily signaled to receptors on ECs, suggesting a mechanism by which KCs may influence tissue infiltration by immune cells. IFN FBs were among the most active of all cell subsets. However, CD16+ DCs represented the top interactors. Expressing both ligands and receptors, CD16+ DCs showed numerous enriched interactions involving IFN FBs, Tfh-like cells, and cDC2B cells, as well as within-CD16+ DC crosstalk. pDCs were comparatively inert in comparison, rarely participating in L-R pairs, consistent with recent literature supporting a more senescent phenotype of pDCs in SLE skin。
Integration of spatial-seq and scRNA-seq analyses provides architectural context shaping cell-cell interactions within lupus skin（輪到我空間上場(chǎng)了吧）
L-R analysis derives exclusively from differential gene enrichment and therefore lacks critical spatial context to substantiate putative interactions（L-R 分析完全來(lái)自差異基因富集帆阳，因此缺乏關(guān)鍵的空間背景來(lái)證實(shí)假定的相互作用 ）. To bolster our interaction analyses, we analyzed discoid lupus lesional skin sections using spatial sequencing on the 10x Genomics Visium platform. A section containing multiple hair follicle segments was selected for in depth analysis（選擇包含多個(gè)毛囊片段的部分進(jìn)行深度分析）. We detected 632 spatially defined spots with an average of 3,704 genes and 10,176 transcripts per spot. Abundant dermal deposition of extracellular glycosaminoglycans, termed mucin, is a frequent feature of cutaneous lupus and is evident in the section as collagen fiber splaying; accordingly, many dermal areas showed very low transcript detection and were excluded during quality control.
At a diameter of 55μm, each spot may account for multiple cells of intermixed types（空間轉(zhuǎn)錄組固有缺點(diǎn)）. This was corroborated by spatial heatmaps showing overlapping expression of representative marker genes corresponding to the major cell types（單細(xì)胞確定的細(xì)胞類型的marker在空間上是重疊表達(dá)的）. Accordingly, rather than assigning a single cell type to each spot, we generated a pie chart for each spot showing the representation of the transcriptomic signature of each major cell type（餅圖顯示各個(gè)spot細(xì)胞類型的比例）. This approach recapitulated the architecture visible on H&E staining, with the epidermis and follicle showing high KC signature detection and the dermis showing a mix of signatures corresponding primarily to FBs, ECs, and smooth muscle cells, which include both vascular smooth muscle and the cells of the arrector pili muscle attached to the hair follicle. The majority of spots with high immune cell signatures localize to the subepidermal and perifollicular regions, corresponding respectively to the characteristic interface dermatitis and periadnexal infiltrate of discoid lupus.（最為直觀的分析就是看看細(xì)胞類型的空間分布） Subepidermal spots showed a particularly prominent myeloid signature with a comparatively weak T cell signature, suggesting strong localization of myeloid cells to the interface, possibly as a direct effect of signaling initiated by KCs or FBs in the prelesional CLE environment（表明骨髓細(xì)胞在界面上的強(qiáng)烈定位哺壶，可能是在病灶前 CLE 環(huán)境中由 KCs 或 FBs 引發(fā)的信號(hào)傳導(dǎo)的直接影響）屋吨。
To further understand how the KC, FB, T cell, and myeloid cell heterogeneity observed in our scRNA-seq mapped onto the architecture of the lesional tissue, we generated spatial heatmaps showing prediction scores corresponding to the subsets defined above（為了進(jìn)一步了解在我們的 scRNA-seq 中觀察到的 KC、FB山宾、T 細(xì)胞和骨髓細(xì)胞異質(zhì)性如何映射到病變組織的結(jié)構(gòu)上至扰，我們生成了空間熱圖，顯示了對(duì)應(yīng)于上面定義的子集的預(yù)測(cè)分?jǐn)?shù)资锰。 ）. Spatial KC subset analysis demonstrated appropriate localization of the supraspinous, spinous, and basal KC signals to the superficial, mid, and basal epidermis, respectively. The follicular KC signal localized to the follicular epithelium and the cycling KC signal primarily to the deeper portion of the follicle, where the stem cells that give rise to the follicle are located. Consistent with the interfollicular epidermis representing the primary site of exaggerated IFN education in CLE, spatial FB subset analysis revealed prominent localization of the IFN FB signal to the superficial dermis and detection of other FB subsets in the perifollicular dermis. Spatial T cell subset analysis also showed subset-specific localization within the tissue section（主要關(guān)注的還是細(xì)胞類型的空間定位）敢课。
Myeloid cell subset spatial heatmaps complemented our immunostaining results. As expected, spots showing a strong LC gene signature were restricted to the epidermis and the follicular epithelium. Many spots in the perifollicular dermis scored highly for pDCs. Spots scoring highly for CD16+ DC clustered most densely in the superficial dermis, again suggesting these cells can be modulated in the IFN-rich environment generated by the basal KCs of the interfollicular epidermis。
To further dissect CD16+ DC cell-cell communication at the level of the individual cell, we performed imaging mass cytometry of lesional DLE and subacute CLE (SCLE) skin biopsies, defining CD16+ DCs as CD14+CD11c+CD16+ cells. This identified CD16+ DCs primarily concentrated in the superficial dermis directly under the dermo-epidermal junction. Enumeration of neighboring cells revealed that diverse immune cell types are detected within 4 μm distance of CD16+ DCs, with monocytes and macrophages being more common than lymphocytes（這里開始分析生態(tài)位绷杜，目標(biāo)細(xì)胞類型的生態(tài)位）. Among stromal cell types included in the analysis, epithelial cells (here, keratinocytes), occurred more commonly in proximity to CD16+ DCs than ECs, supporting interaction between CD16+ DCs and basal KCs. Statistical analysis demonstrated significant overrepresentation of innate inflammatory cells including pDCs and monocytes in proximity to CD16+ DCs in both DLE and SCLE（統(tǒng)計(jì)分析表明直秆，在 DLE 和 SCLE 中，先天性炎癥細(xì)胞（包括 pDC 和靠近 CD16+ DC 的單核細(xì)胞）顯著過度表達(dá) ）鞭盟。
Pseudotime analysis of paired circulating and skin-infiltrating myeloid cells suggests that CD16+ DCs arise from non-classical monocytes that undergo IFN education in lupus skin（擬時(shí)分析）
Overall, our data thus far supported close communication of CD16+ DCs with stromal cells in the skin（臨近細(xì)胞通訊）, so we next wanted to understand the origin and phenotype of CD16+ DCs that infiltrate the skin in lupus patients. Reasoning that these likely arise from circulating mononuclear cells similar to the DC4 subset, we examined peripheral blood mononuclear cells (PBMCs) by scRNA-seq from the same seven lupus patients above as well as from four healthy controls. PBMC and skin cell data were aggregated for clustering, and clusters containing myeloid cells were selected for further analysis based on expression of established markers. Myeloid cells from PBMCs and skin were then re-clustered together to assess for connections between circulating and skin-infiltrating subsets.
聚集的骨髓細(xì)胞的sub-cluster顯示跨越 PBMC 和皮膚的明顯轉(zhuǎn)變圾结。 注釋將bridging cells鑒定為 CD14+CD16++ 非經(jīng)典單核細(xì)胞 (ncMos)，它完全來(lái)自 PBMC齿诉，以及來(lái)自 PBMC 和皮膚的 CD16+ DC筝野。 使用 Monocle 對(duì) ncMos 和 CD16+ DC 進(jìn)行的偽時(shí)間分析沿單一軌跡排列細(xì)胞，反映了從循環(huán) ncMos 到皮膚浸潤(rùn) CD16+ DC 的轉(zhuǎn)變——基于相對(duì)豐度粤剧，這一過程似乎在狼瘡中比健康對(duì)照皮膚更頻繁地發(fā)生歇竟。 這種進(jìn)入皮膚的增加甚至可以解釋狼瘡患者中觀察到的 ncMos 和循環(huán) CD16+ DCs 的比例相對(duì)于數(shù)據(jù)集中的對(duì)照組的減少 。
為了了解伴隨這種轉(zhuǎn)變的轉(zhuǎn)錄變化抵恋，沿著偽時(shí)間進(jìn)行了差異表達(dá)分析焕议。 這確定了跨越從 ncMo 到 CD16+ DC 過渡的五種基因表達(dá)模式。 對(duì)前 80 個(gè)最重要的標(biāo)記基因進(jìn)行仔細(xì)檢查后發(fā)現(xiàn)弧关，這種轉(zhuǎn)變的主要標(biāo)志是編碼趨化因子的眾多基因的晚期上調(diào)号坡，這可以支持 CD16+ DC 的保留和其他炎癥細(xì)胞募集到 CLE 患者皮膚以及 ISG 中 ，與這些細(xì)胞遷移到富含 I 型干擾素的狼瘡皮膚環(huán)境一致梯醒。 從廣義上講，這些變化支持我們先前的發(fā)現(xiàn)腌紧，即角質(zhì)形成細(xì)胞衍生的 I 型干擾素可以促進(jìn) DC 激活茸习，使它們能夠刺激皮膚中的免疫反應(yīng) 。
接下來(lái)分析了這五種基因表達(dá)模式以進(jìn)行典型途徑富集壁肋，以深入了解在這種轉(zhuǎn)變過程中可能獲得和丟失的細(xì)胞功能号胚。在轉(zhuǎn)變?cè)缙诒磉_(dá)的基因模式中豐富的頂級(jí)經(jīng)典途徑（主要與外周相關(guān)）往往與白細(xì)胞販運(yùn)有關(guān)。這些包括 ephrin 受體信號(hào) (p=7.44x10-3)浸遗，在模式 A 中富含的頂部通路猫胁，以及肌動(dòng)蛋白細(xì)胞骨架信號(hào) (p=5.57x10-7) 和整合素信號(hào) (p=4.57x10-4)，頂部在模式 B 中富集的通路跛锌。在轉(zhuǎn)變后期表達(dá)的基因模式中富集的頂級(jí)通路（主要與皮膚相關(guān)）與細(xì)胞因子信號(hào)傳導(dǎo)更相關(guān)弃秆。富含模式 D 的頂部通路是高細(xì)胞因子血癥/高趨化因子血癥在流感發(fā)病機(jī)制中的作用 (p=6.79x10-6)，其中 IFN 信號(hào)傳導(dǎo) (p=2.80x10-4) 也排名靠前。富含模式 E 的頂部通路是 IL-6 (p=1.85x10-6)菠赚，這與支持繼發(fā)于 IFN 信號(hào)調(diào)節(jié)的狼瘡 KCs 增加 IL-6 產(chǎn)生作用的數(shù)據(jù)一致
為了分析影響從 ncMo 到皮膚浸潤(rùn) CD16+ DC 轉(zhuǎn)變的細(xì)胞因子脑豹，計(jì)算了每個(gè)細(xì)胞 IPA 中包含的所有細(xì)胞因子的upstream regulator scores，并確定了這些評(píng)分與偽時(shí)間的相關(guān)性衡查。一組 I 型 IFN（IFN-α1/13瘩欺、IFN-β 和 IFN-κ）和 IFN-γ 的分?jǐn)?shù)表明非常高的相關(guān)性（分別為 r = 0.774、0.880拌牲、0.666 和 0.873）俱饿，具有更明顯的相關(guān)性在偽時(shí)間后期上升，與代表這一轉(zhuǎn)變的重要終端步驟的穩(wěn)健的 IFN education相一致塌忽。許多上游upstream regulators在偽時(shí)間上表現(xiàn)出更漸進(jìn)的誘導(dǎo)和更高的相關(guān)性分?jǐn)?shù)拍埠，表明在過渡中發(fā)揮了更早的作用。這些包括 IL-1β (r= 0.928)砚婆、最相關(guān)的細(xì)胞因子和 TNF (r= 0.903)械拍；值得注意的是，這些在偽時(shí)間 DEG 分析中也成為最重要的基因装盯，顯示從 ncMo 到 CD16+ DC 過渡的晚期上調(diào)坷虑。這與我們之前的報(bào)告一致，即長(zhǎng)時(shí)間的 I 型 IFN 暴露會(huì)引發(fā)單核細(xì)胞的炎癥小體激活并增強(qiáng)它們的 IL-1β 產(chǎn)生埂奈。
介導(dǎo) CD16+ DC 滲入 CLE 患者正常外觀皮膚的相互作用尚不清楚迄损。 為了突出可以促進(jìn)這種積累的 L-R 對(duì)，我們生成了所有細(xì)胞因子相互作用對(duì)的 circos 圖账磺，其中 CD16+ DCs 表達(dá)受體或配體芹敌。 CD16+ DCs 表達(dá) 12 種細(xì)胞因子受體，涉及 L-R 對(duì)垮抗。 ECs 和平滑肌細(xì)胞氏捞，包括血管平滑肌細(xì)胞，表達(dá)了最多數(shù)量的相互作用配體冒版。 基質(zhì)細(xì)胞中次高的是 IFN FB液茎。 以類似的模式，CD16+ DCs 表達(dá)了 23 種參與 L-R 對(duì)的配體辞嗡，其中 ECs 和 IFN FBs 表達(dá)了最多數(shù)量的相互作用受體捆等。 總之，這些 L-R 數(shù)據(jù)表明與 EC 和 IFN FB 的增強(qiáng)相互作用使 CD16+ DC 能夠在 CLE 患者的皮膚中積聚续室，其中富含 IFN 的環(huán)境增強(qiáng)了它們的促炎特性和細(xì)胞間通訊的能力栋烤。
DISCUSSION
Collectively, our data describe the cellular composition and architecture of cutaneous lupus at unprecedented resolution. We demonstrate the pervasive effects of IFN, of which the epidermis is a critical source, on skin stromal and immune cells alike. Most intriguingly, these effects are pronounced in non-lesional samples, suggesting that normal-appearing skin of patients with CLE exists in an immunologically primed, ‘prelesional’ state. This state skews the transcriptional programs of many of the major cell types in the skin, with dramatic effects on the capacity for cell-cell communication. Indeed, even the minor cellular constituents of the skin not examined in detail here exhibited transcriptional shifts in non-lesional CLE skin that alter their potential to engage other stromal and immune cells。
This investigation highlighted CD16+ DCs, a myeloid cell subset increasingly implicated in lupus pathogenesis, as proficient intercellular communicators even in non-lesional skin of CLE patients, where they are highly abundant. Unsupervised clustering followed by pseudotime analysis of combined myeloid cells from skin and peripheral blood suggests that progenitor ncMos in circulation give rise CD16+ DCs. Clustering of DCs isolated from peripheral blood of healthy patients identified a subset of so-called DC4 cells characterized by expression of FCGR3A, encoding CD16a, with high transcriptional similarity to a monocyte subset with features of ncMos, inspiring some discussion that DC4 cells may in fact represent monocytes挺狰。 Here, however, utilization of single-cell technologies across blood and tissues has enabled identification of a CD16+ DC population enriched in the skin of patients with SLE and defined their probable precursors and the transcriptomic changes accompanying this transition that arm CD16+ DCs to instigate tissue inflammation. ScRNA-seq analysis of immune cells isolated from kidney biopsies of patients with lupus nephritis (LN) and healthy controls suggests that this paradigm may not be limited to the skin. Arazi et al. identified several myeloid subpopulations resembling DC4 cells (CM0, CM1, and CM4); these were highly enriched in LN biopsies and showed upregulation of IFN response scores relative to steady-state kidney macrophages and conventional DCs16, suggesting IFN education is a characteristic feature of tissue-infiltrating CD16+ DCs that facilitates their pathogenicity in lupus
皮膚中 CD16+ DC 的積累不僅代表了病灶而且代表了病灶前 CLE 環(huán)境的顯著特征明郭。在 CLE 患者中买窟，CD16+ DCs 從循環(huán)到非病變皮膚的退出可能通過 CD16+ DCs 和脈管系統(tǒng)之間強(qiáng)大的 L-R 相互作用得到增強(qiáng)。組織浸潤(rùn)后达址，CD16+ DCs 可能會(huì)被表皮下 FBs 產(chǎn)生的配體梯度引導(dǎo)積聚在淺表真皮中蔑祟，這些 FBs 已通過 KC 分泌的 IFN 的作用轉(zhuǎn)化為 IFN FB 表型。在遇到富含 IFN 的非病變 CLE 環(huán)境后沉唠，CD16+ DC 上調(diào)了一系列 ISG 編碼的細(xì)胞因子和趨化因子疆虚。這使它們能夠與包括緊鄰的先天免疫細(xì)胞在內(nèi)的多種細(xì)胞類型進(jìn)行廣泛的細(xì)胞間通訊，從而可能導(dǎo)致 CLE 病變的發(fā)生满葛。因此径簿，CLE 患者皮膚的病灶前環(huán)境代表了基質(zhì)細(xì)胞和免疫細(xì)胞之間的協(xié)作，其中 KC嘀韧、FB 和 CD16+ DC 做出了重要貢獻(xiàn)篇亭。針對(duì)非病變和病變 CLE 之間細(xì)胞間通訊差異的進(jìn)一步研究將提供對(duì)下游事件的進(jìn)一步了解，這些事件導(dǎo)致臨床明顯的炎癥和病變形成锄贷，并提供有針對(duì)性的治療策略译蒂，以改善患有這種破壞性疾病的患者的治療反應(yīng) 。
Method（關(guān)注一下重點(diǎn)的分析方法）
Cell clustering and cell type annotation
Spatial sequencing data analysis（單細(xì)胞空間聯(lián)合主要還是Seurat的方法）
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