Immunoglobulin-producing AT2-like cells secrete IgA into the extracellular matrix in pulmonary fibrosis

Pulmonary fibrosis is an interstitial lung disease that can be caused by various factors. Here, we first observed extensive IgA deposition in the extracellular matrix (ECM) of the lungs of mice with pulmonary fibrosis induced by silica inhalation. Consistent with this phenomenon, spatial transcriptomic sequencing of fresh mouse lung tissues from control mice and model mice showed that Igha transcripts were highly expressed in the lesion area. 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. 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.

identified lysosome, alveolar lamellar body, secretory IgA immunoglobulin 126 complex, and immunoglobulin receptor binding as enriched terms (Fig. 1f). 127 The chordal graph shows that the upregulated proteins are related to 128 extracellular regions and membrane attack complexes (Extended Data Fig.   129 1e). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed 130 related pathways, such as rheumatoid arthritis, systemic lupus 131 erythematosus, and intestinal immune network for IgA production (Fig. 1g). 132 In summary, these results indicated that lung fibrosis was closely associated 133 with immunoglobulin production and AT2 cell activity. 134 To further explore the spatiotemporal characteristics of transcription in our lung 135 fibrosis model, we performed spatial transcriptomic sequencing of four 136 samples (NS-7d, SiO 2 -7d, NS-56d, and SiO 2 -56d). Before sequencing, all 137 mice were scanned by computed tomography (CT) (Fig. 1i, top), and both the 138 SiO 2 -7d and SiO 2 -56d mice exhibited hyperdense areas on their lungs, 139 indicating that the models were successfully established. To obtain transcripts 140 from the same structures and eliminate potential confounding by differences in 141 anatomical position, we collected the left lung from each mouse and sliced it in 142 the horizontal direction (Fig. 1h). Then, we observed the tissue under a 143 microscope. If the hilum of the lung and left main bronchus were observed, the 144 tissue section was mounted onto the spatial transcriptomics arrays. In this way, 145 we obtained four slices (one per experimental group) with approximately the 146 same shape and anatomical location, with the major vessels and hilum of the 147 lung clearly visible (Fig. 1i, bottom). H&E staining showed that the alveoli of the 148 NS-7d and NS-56d samples had a normal structure, with no infiltration of 149 inflammatory cells (Fig. 1i, bottom). For the SiO 2 -7d sample, many 150 inflammatory cells had accumulated, and the alveolar structures were 151 completely destroyed (Fig. 1i). For the SiO 2 -56d sample, infiltration of 152 inflammatory cells was still detected but was weaker than that in the SiO 2 -7d 153 sample, and some alveoli were filled with neutrophils (Fig. 1i, bottom). This  Fig. 3b-c). Then, we performed graph-based 163 clustering, and we captured 11 clusters of spots (Extended Data Fig. 4a-b). 164 The NS-7d and NS-56d samples were highly consistent in terms of gene 165 number and clusters of spots (Extended Data Fig. 4c). The SiO 2 -7d and 166 SiO 2 -56d samples were significantly different from their respective control 167 samples in terms of spot clustering, and they were also significantly different 168 from each other (Extended Data Fig. 4c). These findings indicate that mRNA 169 expression at the same anatomical location changed substantially from the 170 inflammatory stage (7d) to the fibrosis stage (56d).

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Ccl2 and Fn1, inflammatory factors, were highly expressed in SiO 2 -7d lesions 172 but showed decreased expression in SiO 2 -56d lesions (Fig. 1j). In terms of the 173 expression of specific genes (Kumar et al., 2020), Igha and Jchain were highly 174 expressed in SiO 2 -56d lesions (Fig. 1k), consistent with the observed high 175 expression of the IgA protein (Fig. 1g). In addition, the expression of genes  To identify the cellular origin of IgA, we obtained whole lungs from four mice 183 (NS-7d, SiO 2 -7d, NS-56d, and SiO 2 -56d, model time and groups were 184 consistent with those used for spatial transcriptomic sequencing) and 185 performed scRNA-seq. All mice were scanned by CT prior to tissue collection 186 to confirm effective modelling (Fig. 2b). After scRNA-seq, the numbers of UMIs 187 and genes were evaluated, and a total of 43,397 cells were obtained for data 188 analysis (Extended Data Fig. 5a-c). We normalized the transcriptomic data    To gain further insight into the function of cluster 1, we focused on the DEGs in 228 this cluster compared with all other cell clusters. We found that Igkv13-85, 229 Ighv3-6 and Igkv6-32 were the top 3 DEGs in AT2-like cell cluster 1 (Fig. 3a). 230 All three belong to the V gene group, with Igkv13-85 and Igkv6-32 encoding 231 the mouse immunoglobulin light chain and Ighv3-6 encoding the heavy 232 chain (Babbage et al., 2006). Furthermore, all three genes were expressed at 233 high levels in the SiO 2 -56d sample and were mainly expressed in cluster 1 234 cells (Fig. 3a). Moreover, Igha was verified to be highly expressed in cluster 1 in this sample (Fig. 3e), consistent with the observed high levels of IgA 236 deposition (Fig. 1g).

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These genes code for immunoglobulin and are thought to be expressed by B  Fig. 2c (Fig. 3b left). However, in the sections, 242 no visible differences in the expression of these genes were observed between 243 the SiO 2 -7d and NS-56d or SiO 2 -56d and NS-56d samples (Fig. 3b right). This 244 phenomenon indicated that B cells did not play a major role in the disease.  Fig. 3 d left). Spatially, they were expressed in the trachea (Fig. 3 d right). As  Furthermore, of the five genes, Sftpa1, Sftpb and Sftpc were more likely to be 256 activated in the SiO 2 -56d sample than in the NS-56d sample (Fig. 3c).

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Simultaneous examination of the expression of Igkv13-85 and cell markers of 258 AT2 or Clara cells revealed that more than half of the Igkv13-85 + cells 259 coexpressed Sftpc (AT2 cell marker) or Scgb3a1 (Clara cell marker), but few 260 coexpressed Cd79a or Cd19 (Fig. 3f). This result strongly suggested that 261 these Igkv13-85 + cells were likely not derived from B cells. Based on the 262 analysis results, we speculated that cluster 1 represents a specialized type of Igkv17-127, were found at notably higher frequencies in the SiO 2 -56d sample 273 ( Fig. 4a-b). Among these five genes, Igkv13-85, Ighv3-6, and Igkv6-32 were 274 categorized as highly expressed DEGs in AT2-like cell cluster 1 (Fig. 3a).

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Together, these findings indicate that there were no notable differences among 348 the samples, suggesting that T cells were not engaged in lung fibrosis.  The band became darker after the cells were treated with TGF-β for 72 hours.