這一篇我們繼續(xù)分析單細胞空間聯(lián)合分析的文章，參考文章在Immunoglobulin-producing AT2-like cells secrete IgA into the extracellular matrix in pulmonary fibrosis括堤，肺纖維化后免疫細胞反應(yīng)的變化路媚，其實越來越多的文章顯示互站，單細胞空間的研究琅轧，更多注重于組織與免疫之間的關(guān)系炸枣。

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Abstract

Pulmonary fibrosis（肺纖維化） is an interstitial lung disease（間質(zhì)性肺猜杉摺） that can be caused by various factors. Here, we first observed extensive IgA deposition（沉積） in the extracellular matrix (ECM) of the lungs of mice with pulmonary fibrosis induced by silica inhalation（二氧化硅吸入民镜，小鼠也是夠可憐的）. Consistent with this phenomenon, spatial transcriptomic sequencing of fresh mouse lung tissues from control mice and model mice showed that transcripts were highly expressed in the lesion area(空間轉(zhuǎn)錄組描述疾病后的免疫變化). Single-cell RNA sequencing (scRNA-seq) and reconstruction of B cell receptor (BCR) sequences revealed a new cluster of cells with a shared BCR and high expression of genes related to immunoglobulin IgA production（單細胞 + BCR測序）. Surprisingly, these clonal cells had more characteristics of AT2 (alveolar epithelial cell type 2) cells than B cells; thus, these cells were named AT2-like cells. Therefore, we propose that secretion of IgA into the ECM by AT2-like cells is an important process that occurs during lung fibrosis（也就是說這篇文章主要是發(fā)現(xiàn)了這一現(xiàn)象）。

Main

Pulmonary fibrosis is a chronic progressive disease. The extracellular matrix (ECM), as the framework supporting lung tissue, is mainly composed of 5 types of substances, namely, collagen, noncollagen, elastin, proteoglycan and aminoglycan（細胞外基質(zhì)（ECM）作為支撐肺組織的骨架险毁，主要由膠原蛋白制圈、非膠原蛋白、彈性蛋白畔况、蛋白聚糖和氨基聚糖5種物質(zhì)組成鲸鹦。）. These components are affected by lung cells and can in turn act on lung cells（這些成分受肺細胞影響，進而作用于肺細胞）. Lung epithelial injury and apoptosis（細胞凋亡） are thought to be the initiating factors of pulmonary fibrosis（內(nèi)皮細胞損傷和凋亡是引起肺纖維化最初的影響因子）. To maintain the normal alveolar structure, lung epithelial progenitor cells proliferate and differentiate to replenish（補充） alveolar epithelial cells. In fact, abnormal and excessive proliferation of progenitor cells fails to restore normal structure, and these cells secret various cytokines that affect fibroblasts（事實上跷跪，祖細胞的異常和過度增殖無法恢復(fù)正常結(jié)構(gòu)馋嗜，這些細胞會分泌影響成纖維細胞的各種細胞因子 ）. In recent years, it has been reported that dysregulated repair and regeneration of epithelial cells is a driving factor of lung fibrosis; however, the specific molecular mechanism by which the epithelium communicates with other cells is still unclear（又是細胞通訊）。

In this study, we started with lung ECM proteomics（蛋白質(zhì)組學） and found that IgA was the most highly deposited protein in the fibrotic lung ECM（這個地方倒是很值得研究）. To explore the source of IgA at the cellular and molecular levels, we applied spatial transcriptomics sequencing and single-cell sequencing to mouse lung tissue（單細胞空間聯(lián)合分析）. By integrating proteomics and sequencing data, we found that AT2-like cells obviously proliferated in lungs with fibrosis induced by SiO₂（這小鼠真的挺慘的）. Surprisingly, a cluster of AT2-like epithelial cells in fibrotic lungs shared the same BCR made up of , which was exactly consistent with the immunoglobulin IgA deposited in lung ECM from fibrotic lungs(令人驚訝的是吵瞻，纖維化肺中的一組 AT2 樣上皮細胞共享由 組成的相同 BCR葛菇，這與纖維化肺沉積在肺 ECM 中的免疫球蛋白 IgA 完全一致 ). It has been reported that IgA is distributed on the surface of the mucosal membrane of the respiratory tract(呼吸道粘膜) and promotes pulmonary fibrosis by activating fibroblasts(通過激活成纖維細胞促進肺纖維化). However, in our study, we found that a large amount of immunoglobulin IgA was deposited in fibrotic lung ECM. This is the first report in the world that epithelial cells, but not B cells, produce immunoglobulin during pulmonary fibrosis(上皮細胞產(chǎn)生免疫球蛋白，講道理橡羞，有點顛覆的意味). We believe that this phenomenon is closely related to the occurrence of pulmonary fibrosis. Blocking AT2-like cells secreting IgA may be a potential way to treat pulmonary fibrosis.（基因敲除眯停，這個臨床運用也很難）。

Results

A large amount of immunoglobulin IgA is deposited in the lung ECM of mice with SiO₂-induced fibrosis

Pneumoconiosis(塵肺) is a type of pulmonary fibrosis caused by inhalation of free silica（吸入游離二氧化硅）. Here, we established a lung fibrosis model through intratracheal instillation of a silica suspension (50 mg/ml, 100 μl) (SiO₂ group); the control group received normal saline (NS group). The CT and H&E staining results showed that acute inflammation(急性炎癥) was observed seven days after instillation, and collagen deposition(膠原沉積) was observed in the lungs 28 days after instillation, consistent with previous studies using this model. At 56 days after instillation, the levels of several indicators of pulmonaryfunction, including FEV75, IC and MMEF, were decreased. In addition to increased lung coefficients (lung weight/mouse body weight), strip- or sheet-like high density in CT and collagen deposition were observed by Sirius red staining in the SiO2-56d group compared to the NS-56d group. Therefore, we considered mice to have entered the fibrosis stage at 56 days after instillation of the SiO₂ suspension(我們認為小鼠在滴注 SiO₂ 懸浮液后 56 天進入纖維化階段).

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To explore the ECM components of fibrotic lungs, we generated a decellularized lung matrix(去細胞肺基質(zhì)) from NS-56d (n=3) and SiO₂-56d (n=3) samples and analysed the protein components through mass spectrometry(質(zhì)譜)-based proteomics. All 6 specimens showed similar results, and the 3 NS-56d samples and the 3 SiO₂-56d samples were most similar to each other. Ultimately, we identified 143 proteins with upregulated expression and 127 proteins with downregulated expression in the SiO₂-56d group compared to the NS-56d group.(差異分析無處不在)卿泽。

IgA was identified as the most highly upregulated protein. Moreover, of the top 20 proteins with upregulated expression, the surfactant proteins SFTPB, SFTPD and SFTPA are canonically thought to be secreted by mature AT2 cells（表面活性蛋白 SFTPB莺债、SFTPD 和 SFTPA 通常被認為是由成熟的 AT2 細胞分泌的）. Western blotting analysis confirmed that the IgA and SFTPB levels were substantially upregulated in the ECM of the SiO₂-56d samples. The deposition of IgA is accompanied by the deposition of surfactant protein, suggesting that IgA may be related to AT2（IgA的沉積伴隨著表面活性蛋白的沉積，提示IgA可能與AT2有關(guān)）. Gene Ontology (GO) analysis（富集分析） of upregulated proteins of SiO₂-56d samples identified lysosome, alveolar lamellar body, secretory IgA immunoglobulin complex, and immunoglobulin receptor binding as enriched terms. The chordal graph（弦圖） shows that the upregulated proteins are related to extracellular regions and membrane attack complexes（上調(diào)的蛋白質(zhì)與細胞外區(qū)域和膜攻擊復(fù)合物有關(guān)）. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed related pathways, such as rheumatoid arthritis, systemic lupus erythematosus, and intestinal immune network for IgA production. In summary, these results indicated that lung fibrosis was closely associated with immunoglobulin production and AT2 cell activity（這些結(jié)果表明肺纖維化與免疫球蛋白的產(chǎn)生和 AT2 細胞活性密切相關(guān)）

To further explore the spatiotemporal characteristics of transcription in our lung fibrosis model, we performed spatial transcriptomic sequencing of four samples（空間轉(zhuǎn)錄組） (NS-7d, SiO₂-7d, NS-56d, and SiO₂-56d). Before sequencing, all mice were scanned by computed tomography (CT) , and both the SiO₂-7d and SiO₂-56d mice exhibited hyperdense areas on their lungs, indicating that the models were successfully established. To obtain transcripts from the same structures and eliminate potential confounding by differences in anatomical position, we collected the left lung from each mouse and sliced it in the horizontal direction（為了獲得相同結(jié)構(gòu)的轉(zhuǎn)錄本并消除解剖位置差異造成的潛在混淆，我們收集了每只小鼠的左肺并在水平方向切片齐邦，相同的切片位置椎侠，這個空間轉(zhuǎn)錄組的設(shè)計還是很不錯的）. Then, we observed the tissue under a microscope. If the hilum of the lung and left main bronchus were observed, the tissue section was mounted onto the spatial transcriptomics arrays. In this way, we obtained four slices (one per experimental group) with approximately the same shape and anatomical location, with the major vessels and hilum of the lung clearly visible. H&E staining showed that the alveoli of the NS-7d and NS-56d samples had a normal structure, with no infiltration of inflammatory cells. For the SiO₂-7d sample, many inflammatory cells had accumulated, and the alveolar structures were completely destroyed. For the SiO₂-56d sample, infiltration of inflammatory cells was still detected but was weaker than that in the SiO₂-7d sample, and some alveoli were filled with neutrophils. This indicates that the SiO₂-7d and SiO₂-56d mouse models were successfully established（這小鼠真的挺慘的）

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After establishing the basic histology of the lung sections from each group, we performed spatial transcriptomic sequencing according to the experimental procedures recommended by 10x Genomics and obtained a total of 6778 spots (a single spot can hold cells within 60 μm in diameter)（10X的空間技術(shù)）. The numbers of genes and unique molecular identifiers (UMIs) detected in the tissue sections of model mice was obviously greater than that of control mice. Then, we performed graph-based clustering, and we captured 11 clusters of spots（空間spot聚類）. The NS-7d and NS-56d samples were highly consistent in terms of gene number and clusters of spots(平行樣本間的聚類結(jié)果很相近). The SiO₂-7d and SiO₂-56d samples were significantly different from their respective control samples in terms of spot clustering, and they were also significantly different from each other（SiO₂-7d 和 SiO₂-56d 樣品在斑點聚類方面與各自的對照樣品有顯著差異，彼此之間也有顯著差異 ）. These findings indicate that mRNA expression at the same anatomical location changed substantially from the inflammatory stage (7d) to the fibrosis stage (56d).（變化還挺大）

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• 注：a, UMAP graph of spots coloured by four samples. b, Clusters of spatial transcriptome spots on UMAP: 11 clusters in total, labelled in different colours. c. UMAP graph of individual sample, separated from (b). d, Clusters of spatial transcriptome spots shown in the tissue space

and , inflammatory factors, were highly expressed in SiO₂-7d lesions but showed decreased expression in SiO₂-56d lesions. In terms of the expression of specific genes, and were highly expressed in SiO₂-56d lesions, consistent with the observed high expression of the IgA protein. In addition, the expression of genes encoding immunoglobulin light chains, such as and , was highly expressed in SiO₂-56d lesions. Combined with proteomic findings for the lung ECM, these results indicated a strong relationship between immunoglobulin expression IgA and lung fibrosis.(這些結(jié)果表明免疫球蛋白表達 IgA 與肺纖維化之間存在密切關(guān)系措拇。)

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Single-cell sequencing detected highly amplified AT2-like cells in the SiO₂-56d sample(單細胞技術(shù))

To identify the cellular origin of IgA, we obtained whole lungs from four mice (NS-7d, SiO2-7d, NS-56d, and SiO2-56d, model time and groups were consistent with those used for spatial transcriptomic sequencing) and performed scRNA-seq（單細胞技術(shù)識別目標細胞的來源）. All mice were scanned by CT prior to tissue collection to confirm effective modelling. After scRNA-seq, the numbers of UMIs and genes were evaluated, and a total of 43,397 cells were obtained for data analysis. We normalized the transcriptomic data from the four groups and then applied graph-based clustering to the combined cells from all groups to analyse and compare the samples, which yielded 24 cell clusters（單細胞的常規(guī)分析）. We then annotated the cell type for each cluster according to canonical cell markers（依據(jù)傳統(tǒng)的細胞marker來定義細胞類型） from Cell Marker and assigned these 24 clusters to 15 types of cells, including endothelial cells, monocytes, macrophages, dendritic cells, B cells, T cells, Ccl3- Ccl4- neutrophils, Ccl3+ Ccl4+ neutrophils, fibroblasts, AT2-like cells (cluster 1, cluster 3, cluster 15), red blood cells, and AT1 cells. The marker genes of the different cell types are shown in the uniform manifold approximation and projection (UMAP) diagram. To explore the cell compositions of the different samples, we calculated the relative percentages of the main 15 cell types in the four samples（每種細胞類型在各個樣本中的占比）. The relative percentage of macrophages in the SiO₂-7d sample was obviously increased compared to NS-7d, representing up to 18% of the total number of cells, and the percentage remained higher than that in the NS-56d sample, even in the SiO₂-56 sample, indicating that macrophages were major participants in the inflammatory stage and were still present in the fibrotic stage. GO and KEGG analyses of differentially expressed genes (DEGs) of macrophages identified genes classically involved in the development of silicosis. Ccl3+ Ccl4+ Neutrophils, which are often involved in various inflammatory reactions induced by bacteria, viruses, and foreign objects, were activated upon stimulation with silica after 7 days and were detected until day 56.

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Because cells of clusters 1, 3, and 15 expressed Sftpc, Sftpb and Sftpa1, we identified the three clusters as AT2-like cells(目標細胞的識別). The proportion of AT2-like clusters 1 and 15 both showed an increase, but cluster 15 had far fewer cells than cluster 1. Interestingly, the proportion of AT2-like cluster 1 cells in the SiO₂-56d sample was notably increased, reaching 40%. GO and KEGG analyses of up-regulated genes (Fc>2, p<0.05) of AT2-like cluster 1 cells indicated that they were enriched in immune-related terms, such as systemic lupus erythematosus; immunoglobulin complex, circulating; humoural immune response; and immunoglobulin production . Thus, we hypothesized that AT2-like cluster 1 plays an important role during the fibrosis stage.(群1起到了關(guān)鍵作用)

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Immunoglobulin-producing AT2-like cluster 1 cells have more characteristics of AT2 cells than B cells

To gain further insight into the function of cluster 1, we focused on the DEGs in this cluster compared with all other cell clusters（這種找差異基因的方式就是我們普通的方式）. We found that , and were the top 3 DEGs in AT2-like cell cluster 1 . All three belong to the V gene group, with and encoding the mouse immunoglobulin light chain and encoding the heavy chain(這個地方的方法大家要多多注意). Furthermore, all three genes were expressed at high levels in the SiO₂-56d sample and were mainly expressed in cluster 1 cells. Moreover, was verified to be highly expressed in cluster 1 in this sample, consistent with the observed high levels of IgA deposition.

These genes code for immunoglobulin and are thought to be expressed by B cells. To ensure that cluster 1 was not B cells, we analysed the expression of several B cell marker gene distributions in all the clusters by Loupe Brower 4.0(使用loupe查看). B cell marker genes such as Cd79a, Cd79b and Cd19（B細胞常用的marker大家都應(yīng)該知道） were relatively highly expressed in the noted B cells. However, in the sections, no visible differences in the expression of these genes were observed between the SiO₂-7d and NS-56d or SiO₂-56d and NS-56d samples. This phenomenon indicated that B cells did not play a major role in the disease. In contrast, genes encoding AT2 markers such as Sftpc, Sftpa1 and Sftpb were relatively highly expressed in cluster 1 and showed significantly upregulated expression in the SiO₂-7d and SiO₂-56d sections. Scgb3a1 and Scgb3a2, markers of secretory epithelial or Clara cells (the progenitor cell of AT2), were both expressed in AT2-like cluster 1 cells. Spatially, they were expressed in the trachea(在空間上肺蔚，它們在氣管中表達). As these five genes (Sftpa1, Sftpb, Sftpc, Scgb3a1 and Scgb3a2) showed relatively higher expression in cluster 1, our findings suggested that cluster 1 cells originated from bronchial epithelial cells.(由于這五個基因（Sftpa1、Sftpb儡羔、Sftpc宣羊、Scgb3a1 和 Scgb3a2）在cluster 1 中表現(xiàn)出相對較高的表達，我們的研究結(jié)果表明cluster 1 細胞起源于支氣管上皮細胞,上皮細胞在行使B細胞該有的免疫功能)汰蜘。

Furthermore, of the five genes, Sftpa1, Sftpb and Sftpc were more likely to be activated in the SiO₂-56d sample than in the NS-56d sample Simultaneous examination of the expression of and cell markers of AT2 or Clara cells revealed that more than half of the cells coexpressed Sftpc (AT2 cell marker) or Scgb3a1 (Clara cell marker), but few coexpressed Cd79a or Cd19. This result strongly suggested that these cells were likely not derived from B cells. Based on the analysis results, we speculated that cluster 1 represents a specialized type of AT2 cell (alveolar epithelial cell type 2). In other words, silica inhalation could trigger the transformation of normal AT2 cells into AT2-like cells, which can transcribe BCR genes.(吸入二氧化硅可以觸發(fā)正常的 AT2 細胞轉(zhuǎn)化為 AT2 樣細胞仇冯，后者可以轉(zhuǎn)錄 BCR 基因)

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BCR clonal expansion in the SiO₂-56d sample

Because we observed the differential expression of the gene encoding the immunoglobulin light chain in the SiO₂-56d sample, we wanted to further explore the usage of the V(D)J and C genes of BCR in the four samples. We reconstructed BCR sequences from scRNA sequencing. Across the four groups, several V genes, including , , , and , were found at notably higher frequencies in the SiO₂-56d sample. Among these five genes, , , and were categorized as highly expressed DEGs in AT2-like cell cluster 1(再次證明了上皮細胞行使B細胞的功能). and were also mainly expressed in AT2-like cell cluster 1. Among IGH C genes, the expression of was substantially increased in the SiO₂-56d sample compared with the other three samples, which also indirectly led to a decline in the proportion of and usage. There was no obvious difference in the usage of other BCR genes across the four samples.

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On the basis of traditional immunology theory, we believe that clonal expansion is closely related to disease progression(基于傳統(tǒng)免疫學理論，我們認為克隆擴增與疾病進展密切相關(guān)). According to an analysis of the top 30 clonotypes from each sample, we found no identical clonal expansions among the four samples(有點尷尬啊). A heat map composed of different V-J gene pairing frequencies showed 9 kinds of V-J gene combinations in the SiO₂-56d sample in clonal populations that had expanded to over 100 cells. In the SiO₂-56d sample, clonotypes comprising over 100 cells constituted more than 75% of the total clonal cell population, and approximately 75% of the total clonal cells came from AT2-like cluster 1. Combined with the scRNA sequencing information, these results showed that BCR genes were expressed not only in B cells but also in AT2-like cluster 1 cells（結(jié)合scRNA測序信息族操，這些結(jié)果表明BCR基因不僅在B細胞中表達苛坚，而且在AT2樣cluster 1細胞中也有表達）. In fact, more BCR+ cells were identified as AT2-like cluster 1 cells. As with BCR, at least half of the clonal cells were identified as AT2-like cluster 1 cells through scRNA barcodes. These clones produce IgA with the progression of pulmonary fibrosis. To determine which cluster of cells the clones came from, we merged the barcodes of the top 3 clonal cell populations with barcodes from single-cell sequencing. Most of these clonal cells were identified as AT2-like cluster 1 cells. Compared with B cells, top 1 clonal cells highly expressed and 。

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The use of the V gene varies among different individuals. As expected, , and were barely expressed in the sections used for spatial transcriptomic sequencing(空間轉(zhuǎn)錄組檢測BCR基因的表達). We next focused on , a gene encoding the constant region of the immunoglobulin heavy chain that is crucial for determining the immunoglobulin isotype. As shown, this gene was coexpressed with . Moreover, the expression of in the SiO₂-56d sample was much higher than that in the SiO₂-7d sample, consistent with the expansion of IgA-related clonotypes in the SiO₂-56d sample. Therefore, the increased frequency of use is common in pathological conditions. The IgA-expressing AT2-like cell population might be important in pulmonary fibrosis.

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we concluded that the increased transcripts in the SiO₂-56d group were derived from AT2-like cluster 1 cells

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TCR is not amplified in the SiO₂-7d or SiO₂-56d sample compared with the NS-7d or NS-56d sample, respectively(TCR居然不起作用)

T cells are primarily responsible for cellular immunity, as opposed to humoral immunity(T 細胞主要負責細胞免疫泼舱，而不是體液免疫). To evaluate the role of T cell immunity in lung fibrosis, we rebuilt TCR sequences for the NS-7d, SiO₂-7d, NS-56d, and SiO₂-56d samples from scRNA-seq(單細胞TCR測序分析). There were no notable differences in V(D)J and C gene usage among the four samples. The amino acid sequences of the top 10 complementarity determining regions (CDR3) in the four samples were different, without a specific pattern or shared CDR sequences. The extent of clonal expansion in the four samples showed that more than 80% of the clonotypes of each sample were represented by only one cell, and the clonotype with the greatest expansion represented no more than 3% of all cells. Then, to explore the similarities and differences in clonotypes among the four samples, we analysed the top 30 clonotypes of each sample. Each sample had a unique preferred clonotype, and no clonotype was shared among the four samples（每個樣本都有一個獨特的首選克隆型，四個樣本之間沒有共享克隆型）

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然后枷莉，我們使用 Loupe（版本 4.1.0娇昙，10x Genomics）整合 scRNA 序列和 TCR 的Barcode。 簡而言之笤妙，攜帶 TCR 或配對克隆型的細胞在每個樣本中被識別為 T 細胞冒掌，并且四個樣本之間沒有顯著差異。 總之蹲盘，這些發(fā)現(xiàn)表明樣本之間沒有顯著差異股毫，表明 T 細胞沒有參與肺纖維化。

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Immunoglobulin IgA was increased in mouse lung epithelium under TGF-β treatment

GF-β 是一種已知的強纖維化因子召衔，常用于刺激細胞建立體外纖維化模型铃诬。 在這里，我們培養(yǎng)了具有AT2特征的小鼠肺上皮細胞系MLE-12苍凛，并用TGF-β處理了不同時間趣席。 然后，我們通過蛋白質(zhì)印跡評估 IgA毫深。 使用抗 IgA 抗體吩坝，我們檢測到 IgA，一條 75 kDa 的條帶哑蔫，恰好等于 IgA 輕鏈和重鏈的總分子量。 細胞用 TGF-β 處理 72 小時后，條帶變暗闸迷。 為了驗證 AT2 細胞可以將 IgA 分泌到 ECM 中嵌纲，我們將 MLE-12 細胞重新接種到從健康小鼠肺獲得的肺 ECM 上，然后用 TGF-β 處理腥沽。 蛋白質(zhì)印跡分析表明逮走，與純肺 ECM 相比，在 MLE12 細胞和 TGF-β 培養(yǎng)的 ECM 中檢測到更多的 IgA今阳。 這種現(xiàn)象表明肺上皮向肺 ECM 分泌 IgA师溅。

Combined analysis of scRNA-seq and spatial transcriptomics showed that more AT2-like cluster 1 cells were accumulated in the SiO₂-56d sample

為了進一步研究與纖維化病變相關(guān)的肺中cluster 1 細胞的位置，我們使用 Seurat V3.2 基于錨點特征整合我們的數(shù)據(jù)集盾舌，并預(yù)測包含在四個部分 (NS) 空間轉(zhuǎn)錄點中的主要細胞類型-7d墓臭、SiO₂-7d、NS-56d妖谴、SiO₂-56d)(單細胞空間聯(lián)合分析)窿锉。在四個部分中預(yù)測了通過單細胞測序鑒定的所有 15 種細胞類型。所有類型的細胞都分散在四組樣本中膝舅，沒有任何明顯的模式嗡载，這與大腦和腎臟的情況不同，其中特定的細胞cluster與特定的解剖位置相關(guān)仍稀。正如預(yù)期的那樣洼滚，SiO2-7d 樣品中的巨噬細胞比 NS-7d 樣品中的巨噬細胞更密集，這與 scRNA-seq 觀察到的巨噬細胞數(shù)量增加一致技潘。類似地判沟，在 SiO₂-56d 樣品的病變中發(fā)現(xiàn) AT2 樣cluster 1 細胞比在 NS-56d 樣品中的密度更高。值得注意的是崭篡，SiO₂-56d 樣品中的 B 細胞沒有特殊的分布模式挪哄。從空間角度來看，這一結(jié)果證實巨噬細胞參與了矽肺琉闪，尤其是在早期迹炼，BCR₊ AT2 樣細胞往往在纖維化階段起重要作用。

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Discussion

通常認為成纖維細胞在肺纖維化中起重要作用颠毙。阻斷成纖維細胞的藥物雖然可以減緩肺纖維化患者肺功能的下降，但不能降低患者的死亡率蛀蜜。因此煎楣，在確定肺纖維化的重要事件和分子機制為肺纖維化治療提供靶點方面還有很多工作要做。我們的研究發(fā)現(xiàn) AT2 樣細胞增殖并將 IgA 分泌到肺 ECM 中。這些細胞在細胞總數(shù)中所占的比例遠遠超過其他肺細胞户誓，這些細胞的激活極大地改變了肺 ECM 的組成。 IgA通常分布于消化道和呼吸道的黏膜表面帝美，由B細胞分泌。在我們的肺纖維化模型中庇忌，IgA 大量沉積在肺 ECM 中舰褪，這是迄今為止世界上首次報道。事實上墙基，肺纖維化疾病在臨床上與自身免疫性疾病如結(jié)節(jié)病和系統(tǒng)性硬化癥有關(guān)。研究表明刷喜，纖維化肺中的 IgA 可以激活成纖維細胞残制，促進其轉(zhuǎn)分化為肌成纖維細胞并分泌膠原蛋白。我們的發(fā)現(xiàn)為 IgA 和成纖維細胞之間的良好接觸提供了基礎(chǔ)掖疮。

肺泡祖細胞可以自我更新初茶、增殖和補充受損的肺泡上皮。 當它們異常增殖時浊闪，它們會導(dǎo)致肺泡結(jié)構(gòu)紊亂恼布。 已經(jīng)報道了三種肺祖細胞。 在我們的研究中搁宾，AT2 樣細胞cluster 1 更類似于 Sftpc⁺ Scgb3a1⁺ 肺祖細胞折汞，它們向 ECM 分泌 IgA。 雖然免疫球蛋白被認為來源于 B 細胞盖腿，但研究表明爽待，在肺癌中，上皮細胞也可以分泌免疫球蛋白 IgG翩腐。 這是首次發(fā)現(xiàn)上皮細胞在非癌癥疾病中產(chǎn)生免疫球蛋白.

有人建議特發(fā)性肺纖維化將在未來 10 年內(nèi)重新分類鸟款。 考慮到這一點，本研究的發(fā)現(xiàn)激勵我們研究其他類型的肺纖維化茂卦，并根據(jù) IgA 沉積的存在與否對它們進行分類何什。 靶向消融過度增殖的 AT2 樣細胞或阻斷 IgA 的產(chǎn)生可能成為治療肺纖維化的潛在靶點.

Method

Cell type annotation(marker要多搜集一下)

Cell types were determined by clustering and marker gene expression. Cluster

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BCR and TCR sequence reconstruction

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單細胞空間聯(lián)合的方法就是Seurat的方法。

生活很好等龙，有你更好