好了，这一篇回归我们的主业，分享单细胞空间联合分析的运用，参考文章在Non-lesional and Lesional Lupus Skin Share Inflammatory Phenotypes that Drive Activation of CD16+ Dendritic Cells，主要运用单细胞空间表征损伤后的皮肤免疫激活状态和分布，看来单细胞空间技术运用的地方，无处不在~~~
ABSTRACT
Cutaneous lupus erythematosus（皮肤红斑狼疮） (CLE) is a disfiguring and poorly understood condition frequently associated with systemic lupus（系统性狼疮）. Studies to date suggest that non-lesional keratinocytes（非损伤角质形成细胞） play a role in disease predisposition（倾向）, but this has not been investigated in a comprehensive manner or in the context of other cell populations. To investigate CLE immunopathogenesis（免疫发病机制）, 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”环境会扭曲所有主要皮肤细胞类型的基因转录并显著扭曲细胞间通讯 ）. 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.(总之，我们的数据提供了狼疮病变和非病变皮肤的综合特征，并确定了 CD16+ 树突状细胞在 CLE 发病机制中的skin education作用。)
INTRODUCTION
Cutaneous lupus erythematosus (CLE) is a disfiguring inflammatory skin disease(毁容炎症性皮肤病) that affects 70% of patients with systemic lupus erythematosus (SLE,系统性红斑狼疮). While about 50% of patients respond to SLE-directed therapies, many patients suffer from refractory skin lesions(难治性皮肤病变), 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.(缺乏关于 CLE 的炎症成分和引发疾病的驱动因素的知识延迟了有效治疗的发展)。
Intriguingly, the etiology of skin lesions in CLE may be, at least partially, found in abnormalities in non-lesional, normal-appearing skin（正常的皮肤也可能金玉其外，败絮其中）. 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（多细胞的相互作用来认识疾病）。
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（看来疾病的发生都是结尾，真正的过程隐藏在正常的皮肤之中）。
RESULTS
Single-cell RNA-sequencing (scRNA-seq) of lesional and non-lesional skin from patients with CLE identifies diverse skin and immune cell populations（首先是单细胞分析，印象里单细胞做皮肤组织的情况不多见）。
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。（单细胞开始的标准分析）
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（细胞类型的比例变化）. Analyses of characteristic KC subtype markers identified 5 KC states: basal, spinous, supraspinous, follicular, and cycling（我们在读每篇文章的时候，细胞定义的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 的相对较浅的深度排除了直接检查 CLE 中涉及的许多细胞因子的转录水平 - 特别是干扰素 ）. 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（计算了 KC 模块评分，该评分源自在用指定细胞因子刺激培养的 KC 中诱导的基因，并生成显示每种细胞因子模块评分的特征图 ）. 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（正常的皮肤也显示了疾病的表达程序）. 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。
For a broader understanding of the transcriptomic differences in lesional KCs of patients with CLE, we performed differential expression analysis（差异表达分析虽然大家都做，但是方法千差万别，大家可以参考我的文章10X单细胞（10X空间转录组）22种基因差异分析方法汇总） 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，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信号在病变样本中高度富集）。
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（成纤维细胞） (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)（细胞定义的结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（一般分析的过程中出现了新的细胞类型，那大家偷着乐吧，发的文章肯定不低）.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 细胞因子模块评分在非病变 FB 中最高，这强化了狼疮患者外观正常的皮肤代表了病变前、IFN 引发的环境 ）. 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。
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（免疫细胞）. 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（看来作者早有预谋啊，和IFN较上劲了）. 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.（不过确实差异明显）
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（对 SLE 患者外周血中 Treg 丰度的研究产生了相互矛盾的结果，可能是由于方法学或 Treg 定义的不同，但似乎一致认为 SLE 患者的 Treg 功能发生了改变）. 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.（又是差异分析） 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（在非病变皮肤中观察到的 Treg 的慢性干扰素刺激可能导致 Treg 维持免疫稳态和狼疮自我耐受的能力受损 ）
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（该亚群的特征在于 CXCL13（一种 B 细胞吸引趋化因子和 SLE 生物标志物，似乎起致病作用）以及 ICOS 和 PDCD1（编码 PD-1）的表达，导致我们将它们注释为 T 滤泡辅助因子 (Tfh) 样细胞）. Abundance of these cells varied by disease state at 1%, 4%, and 2% of healthy control, non-lesional, and lesional T cells, respectively（差异不大）. 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.（在我们的数据集中的检测表明，类似的过程也可能发生在狼疮患者的非病变皮肤中。）
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)（细胞类型定义的marker还是要多多搜集一下）. Myeloid cell subsets showed far greater variability in representation among healthy control, non-lesional, and lesional samples than the above cell types。（变化幅度较大）
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（看来都是根据前人的marker定义的）. 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（细胞类型在三种样本中变化很大）. 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（这个子集最为特殊）. 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（这个地方还是细胞类型比例变化明显，容易研究，后续我们要看看整合分析作者是怎么做的）。
Ligand-receptor (L-R) analysis demonstrates lupus-enriched cell-cell interactions prominently involving CD16+ DCs（通讯分析）
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。
These analyses indicate a prominent role for myeloid cells in non-lesional and lesional skin（这些分析表明骨髓细胞在非损伤和损伤皮肤中的重要作用 ）. 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（精细分析通讯）. 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（轮到我空间上场了吧）
L-R analysis derives exclusively from differential gene enrichment and therefore lacks critical spatial context to substantiate putative interactions（L-R 分析完全来自差异基因富集，因此缺乏关键的空间背景来证实假定的相互作用 ）. 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（选择包含多个毛囊片段的部分进行深度分析）. 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（空间转录组固有缺点）. This was corroborated by spatial heatmaps showing overlapping expression of representative marker genes corresponding to the major cell types（单细胞确定的细胞类型的marker在空间上是重叠表达的）. 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（饼图显示各个spot细胞类型的比例）. 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.（最为直观的分析就是看看细胞类型的空间分布） 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（表明骨髓细胞在界面上的强烈定位，可能是在病灶前 CLE 环境中由 KCs 或 FBs 引发的信号传导的直接影响）。
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（为了进一步了解在我们的 scRNA-seq 中观察到的 KC、FB、T 细胞和骨髓细胞异质性如何映射到病变组织的结构上，我们生成了空间热图，显示了对应于上面定义的子集的预测分数。 ）. 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（主要关注的还是细胞类型的空间定位）。
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（这里开始分析生态位，目标细胞类型的生态位）. 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（统计分析表明，在 DLE 和 SCLE 中，先天性炎症细胞（包括 pDC 和靠近 CD16+ DC 的单核细胞）显著过度表达 ）。
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（拟时分析）
Overall, our data thus far supported close communication of CD16+ DCs with stromal cells in the skin（临近细胞通讯）, 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.
聚集的骨髓细胞的sub-cluster显示跨越 PBMC 和皮肤的明显转变。 注释将bridging cells鉴定为 CD14+CD16++ 非经典单核细胞 (ncMos)，它完全来自 PBMC，以及来自 PBMC 和皮肤的 CD16+ DC。 使用 Monocle 对 ncMos 和 CD16+ DC 进行的伪时间分析沿单一轨迹排列细胞，反映了从循环 ncMos 到皮肤浸润 CD16+ DC 的转变——基于相对丰度，这一过程似乎在狼疮中比健康对照皮肤更频繁地发生。 这种进入皮肤的增加甚至可以解释狼疮患者中观察到的 ncMos 和循环 CD16+ DCs 的比例相对于数据集中的对照组的减少 。
为了了解伴随这种转变的转录变化，沿着伪时间进行了差异表达分析。 这确定了跨越从 ncMo 到 CD16+ DC 过渡的五种基因表达模式。 对前 80 个最重要的标记基因进行仔细检查后发现，这种转变的主要标志是编码趋化因子的众多基因的晚期上调，这可以支持 CD16+ DC 的保留和其他炎症细胞募集到 CLE 患者皮肤以及 ISG 中 ，与这些细胞迁移到富含 I 型干扰素的狼疮皮肤环境一致。 从广义上讲，这些变化支持我们先前的发现，即角质形成细胞衍生的 I 型干扰素可以促进 DC 激活，使它们能够刺激皮肤中的免疫反应 。
接下来分析了这五种基因表达模式以进行典型途径富集，以深入了解在这种转变过程中可能获得和丢失的细胞功能。在转变早期表达的基因模式中丰富的顶级经典途径（主要与外周相关）往往与白细胞贩运有关。这些包括 ephrin 受体信号 (p=7.44x10-3)，在模式 A 中富含的顶部通路，以及肌动蛋白细胞骨架信号 (p=5.57x10-7) 和整合素信号 (p=4.57x10-4)，顶部在模式 B 中富集的通路。在转变后期表达的基因模式中富集的顶级通路（主要与皮肤相关）与细胞因子信号传导更相关。富含模式 D 的顶部通路是高细胞因子血症/高趋化因子血症在流感发病机制中的作用 (p=6.79x10-6)，其中 IFN 信号传导 (p=2.80x10-4) 也排名靠前。富含模式 E 的顶部通路是 IL-6 (p=1.85x10-6)，这与支持继发于 IFN 信号调节的狼疮 KCs 增加 IL-6 产生作用的数据一致
为了分析影响从 ncMo 到皮肤浸润 CD16+ DC 转变的细胞因子，计算了每个细胞 IPA 中包含的所有细胞因子的upstream regulator scores，并确定了这些评分与伪时间的相关性。一组 I 型 IFN（IFN-α1/13、IFN-β 和 IFN-κ）和 IFN-γ 的分数表明非常高的相关性（分别为 r = 0.774、0.880、0.666 和 0.873），具有更明显的相关性在伪时间后期上升，与代表这一转变的重要终端步骤的稳健的 IFN education相一致。许多上游upstream regulators在伪时间上表现出更渐进的诱导和更高的相关性分数，表明在过渡中发挥了更早的作用。这些包括 IL-1β (r= 0.928)、最相关的细胞因子和 TNF (r= 0.903)；值得注意的是，这些在伪时间 DEG 分析中也成为最重要的基因，显示从 ncMo 到 CD16+ DC 过渡的晚期上调。这与我们之前的报告一致，即长时间的 I 型 IFN 暴露会引发单核细胞的炎症小体激活并增强它们的 IL-1β 产生。
介导 CD16+ DC 渗入 CLE 患者正常外观皮肤的相互作用尚不清楚。 为了突出可以促进这种积累的 L-R 对，我们生成了所有细胞因子相互作用对的 circos 图，其中 CD16+ DCs 表达受体或配体。 CD16+ DCs 表达 12 种细胞因子受体，涉及 L-R 对。 ECs 和平滑肌细胞，包括血管平滑肌细胞，表达了最多数量的相互作用配体。 基质细胞中次高的是 IFN FB。 以类似的模式，CD16+ DCs 表达了 23 种参与 L-R 对的配体，其中 ECs 和 IFN FBs 表达了最多数量的相互作用受体。 总之，这些 L-R 数据表明与 EC 和 IFN FB 的增强相互作用使 CD16+ DC 能够在 CLE 患者的皮肤中积聚，其中富含 IFN 的环境增强了它们的促炎特性和细胞间通讯的能力。
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。 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
皮肤中 CD16+ DC 的积累不仅代表了病灶而且代表了病灶前 CLE 环境的显著特征。在 CLE 患者中，CD16+ DCs 从循环到非病变皮肤的退出可能通过 CD16+ DCs 和脉管系统之间强大的 L-R 相互作用得到增强。组织浸润后，CD16+ DCs 可能会被表皮下 FBs 产生的配体梯度引导积聚在浅表真皮中，这些 FBs 已通过 KC 分泌的 IFN 的作用转化为 IFN FB 表型。在遇到富含 IFN 的非病变 CLE 环境后，CD16+ DC 上调了一系列 ISG 编码的细胞因子和趋化因子。这使它们能够与包括紧邻的先天免疫细胞在内的多种细胞类型进行广泛的细胞间通讯，从而可能导致 CLE 病变的发生。因此，CLE 患者皮肤的病灶前环境代表了基质细胞和免疫细胞之间的协作，其中 KC、FB 和 CD16+ DC 做出了重要贡献。针对非病变和病变 CLE 之间细胞间通讯差异的进一步研究将提供对下游事件的进一步了解，这些事件导致临床明显的炎症和病变形成，并提供有针对性的治疗策略，以改善患有这种破坏性疾病的患者的治疗反应 。
Method（关注一下重点的分析方法）
Cell clustering and cell type annotation
Spatial sequencing data analysis（单细胞空间联合主要还是Seurat的方法）
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