scIVNL-seq resolves in vivo single-cell RNA dynamics of immune cells during Salmonella infection

Abstract

The immune response against pathogens involves multiple cell state transitions and complex gene expression changes. Here, we establish a single-cell in vivo new RNA labeling sequencing method (scIVNL-seq) and apply it to survey time-resolved RNA dynamics during immune response to acute enteric infection with Salmonella. We show that the detection of new RNA synthesis reflects more realistic information on cell activation and gene transcription than total RNA level. Interplay of RNA synthesis and degradation modulates the dynamics of total RNA. The bone marrow macrophages are first primed at a very early stage upon Salmonella infection. In contrast, the innate immune response of macrophages in intestine is limited. Notably, intestinal CD8⁺ T cells and plasma cells are rapidly and specifically activated at the early stage post infection. Intestinal late enterocytes quickly express MHC-I molecules and present Salmonella antigen to CD8⁺ T cells for their activation, serving as antigen presenting cells for the initiation of adaptive immunity. Our findings reveal the RNA control strategies and the dynamic activation rules of immune cells in response to Salmonella infection, challenging the doctrine boundary between innate immunity and adaptive immunity against bacterial infection.

Fig. 1. scIVNL-seq detects single-cell new RNA in vivo.

A Overview of scIVNL-seq. S⁴U was injected into mice via tail vein injection. BM cells were collected and re-suspended. Single-cell suspensions were loaded onto microfluidic device, followed by cell lysis and mRNA capture. S⁴U integrated in new RNA was converted into cytidine analogs and was further recognized as cytosine by reverse transcriptase. cDNAs were amplified and libraries were sequenced. New RNA was identified by T-to-C substitutions. The schematic diagrams were created with Adobe Photoshop (Version 22.0.0) (B) New RNA (red) imaging of femur sections, co-stained with CD45 (green), CD31 (cyan) antibodies and DAPI (blue). Scale bar, 100 μm. C UMAP showing cell type clusters (left), UMI counts of new RNA and NTRs (right) in bone marrow CD45⁺ immune cells. The uniform manifold approximation and projection (UMAP) method was used for dimension reduction. Unique molecular identifier (UMI) counts per cell are shown as ln(count+1). Ratios of new RNA to total RNA are shown as NTRs. HSPC, hematopoietic stem and progenitor cell; Mono, monocyte; Mφ, macrophage; Neu, neutrophil; Baso, basophil; NK, natural killer cell; DC, dendritic cell; Pre B, Pre-B cell; B, B cell; T, T cell. D Violin plot of NTR for each BM cell type. E Signature gene expression of new RNA (left) and total RNA (right) for each cell type. Genes in the heatmap were the same between new RNA and total RNA. Expressions were log-normalized. F Total RNA and new RNA expression (log-normalized) of Ptprc, Ccr2, Camp and Rpl13. G Scatter plot of transcription rate (α, normalized RNA counts per cells/h) and RNA half-life (t_1/2, h) of genes expressed in BM cells. H UMAP projection of BM cell types with new RNA. Cells with new RNA data were clustered by unsupervised classification. Cell types identified with total RNA data were mapped back on UMAP projection. Source data are provided as a Source Data file.

Fig. 2. New RNA is rapidly transcribed in BM macrophages post Salmonella infection.

A UMAP plot of CD45⁺ immune cells in BM and NTR at each time point post Salmonella infection. All cells from the indicated time points were combined to identify cell types (left). Ratio of new RNA to total RNA (NTR) was mapped onto UMAP plots (right). B New RNA imaging of femur sections (left) and mean fluorescence intensities (MFIs) of 5-EU labeled new RNA (right). New RNA was labeled with EU (red). Macrophages were stained with anti-F4/80 (green) antibody. Nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. n = 5 biologically independent replicates. C RNA velocity flow projected in PCA space. Macrophages (left) or monocytes (right) from different time points were combined. Dynamo was used to quantify time-resolved RNA velocity. Cells are color-coded by time points. Streamlines represent integration paths connecting local projections from observed state to inferred future state. D Heatmap showing new RNA expression of genes with significantly up-regulated new RNA (fold change > 1.5; FDR < 0.05) in the main BM cell types. Expression levels were scaled. Mφ macrophage; Mono monocyte; Neu neutrophil; NK natural killer cell; DC dendritic cell; B B cell; T T cell. E New RNA and total RNA expression (log-normalized) of indicated genes from sample at 6 h after infection. F Flow cytometry showing IL-1β and TNF-α expression of BM macrophages (CD11b⁺CD11c⁻F4/80⁺) at indicated post-infection time points. Proportions of IL-1β⁺TNF-α⁺ macrophages to BM cells are shown (left). n = 6 biologically independent replicates. Results are shown as mean ± SEM. p values were determined by one-way ANOVA. G Functional scores (inflammatory response and defense response) of new RNA for each cell type at 6 h post infection. In the box plot, the lower and upper hinges are defined as the first and third quartiles. The center represents the median, and the whiskers extend from the hinges to the largest or smallest values within 1.5× the interquartile range. Data in (B) and (F) are representative of at least three independent experiments. Source data are provided as a Source Data file.

Fig. 3. Different genes harbor various mRNA metabolic control strategies during immune response in macrophages.

A Numbers of genes that upregulated (log₂ (fold change) > 0.25, p value < 0.05, Wilcoxon Rank Sum test) in new RNA and total RNA in macrophages at the indicated time points after infection compared with 0 h. B Ridgeline plot of new and total RNA expression correlations in macrophages. Genes highly expressed at the indicated time points were selected for Spearman’s rank correlation analysis of new and total RNA. C Scatter diagrams showing NTR and fold change of new RNA in infected macrophages to new RNA in uninfected macrophages (top); total RNA expression and fold change of total RNA in infected macrophages to total RNA in uninfected macrophages (bottom). Red spots indicate upregulated genes (log₂(fold change) > 0.25); blue spots indicate downregulated genes (log₂(fold change) < −0.25); grey spots indicate no change. D Heatmap of new and total RNA expression pattern in macrophages at the indicated time points. Genes highly expressed at each time point were selected and ordered into 8 clusters by their total RNA expression patterns, and rank of genes was mapped to new RNA. Expressions were scaled. E Heatmap showing enrichment scores of selected GO pathways in new RNA and total RNA at each time points post infection. F Line graph showing new and total RNA expression (upper) and new RNA transcription rate (α, normalized RNA counts per cells/h) and RNA half-life (t_1/2, h) (lower) of Il1b, Ccl4 and Psmb9 at each time point in macrophages. G Dot plot showing average new RNA transcription rate (α) and RNA half-life (t_1/2) of genes in GO pathways in macrophages. Genes significantly upregulated at 6 h or 72 h compared to 0 h were selected for GO enrichment analysis. Average values of α and t_1/2 of these upregulated genes in the indicated GO were calculated and shown in the dot plot. Each dot shows an average value of α and t_1/2 in a GO. The size of each dot represents the number of genes in the GO term. Source data are provided as a Source Data file.

Fig. 4. Intestinal CD8⁺ T cells are rapidly activated early after Salmonella infection.

A UMAP plot showing cell type clusters of intestinal T cells, B cells, plasma cells and myeloid cells and their NTR at each time point post Salmonella infection. All cells from indicated time points were combined to identify cell types (left). NTR was overlaid on the UMAP plots (right). B Bubble plot showing log-normalized and scaled new RNA expression of genes. C New RNA (red) imaging of small intestine sections, co-stained with CD8a (green) and EpCAM antibodies (blue). Arrows denote new RNA signal positive CD8⁺ T cells. Scale bar, 20 μm. D GSEA analysis of new RNA in CD8⁺ T cells showing enrichment of indicated gene sets at 2 h post Salmonella infection (left) compared to 0 h (right). E Scatter plots showing fold change of new RNA (left) and total RNA (right) in CD8⁺ T cells between 2 h and 0 h. F UMAP plot showing new RNA expression of Gzma and Ccl5 at 0 h and 2 h. Cell type defining of this UMAP plot was the same as in (A). G Flow cytometry analysis of GzmA and GzmB expression in CD8⁺ T cells. H 3D imaging of GzmA⁺ T cells at different time points post infection. GzmA (red), CD8a (green) and EpCAM (cyan) antibodies were stained. GzmA positive granules per villus are provided (right). Scale bar, 100 μm. n = 5 biologically independent replicates, 30 villi of each mouse for statistics. Results are shown as mean ± SEM. p values were determined by one-way ANOVA. I 3D imaging of GzmA⁺ T cells. Imaris surface rendering model of CD8⁺ T cells was built to estimate the inside and outside states of GzmA⁺ granules. Outside GzmA⁺ granules are indicated with arrows. Scale bar, 10 μm. n = 9 biologically independent replicates, 30 villi of each mouse for statistics. Results are shown as mean ± SEM. p values were determined by two-way ANOVA. Data in (C), (G), (H) and (I) are representative of at least three independent experiments. Source data are provided as a Source Data file.

Fig. 5. IgA-producing plasma cells quickly expand after Salmonella infection.

A UMAP visualization of B cell and plasma cell development trajectory at each time point post infection. B Proportion of B cells and plasma cells at 0 and 72 h post Salmonella infection. C Flow cytometry analysis of IgA-producing plasma (IgA⁺B220⁻) and B cells (IgA⁻B220⁺). n = 4 biologically independent samples. Results are shown as mean ± SEM. p values were determined by two-way ANOVA. D Pie chart showing proportions of B cells and plasma cells expressing new RNA and total RNA of different immunoglobulin classes. Null, cells did not express Igha, Ighg, Ighm or Ighd. E Flow cytometry analysis of IgA and IgG coated fecal bacteria at each time point post Salmonella infection. n = 5 biologically independent replicates. Results are shown as mean ± SEM. p values were determined by two-way ANOVA. F Flow cytometry analysis of IgA-coated mcherry expressing Salmonella in faeces at each time point post Salmonella-mcherry infection. Samples were mixed for isotype control. n = 6 biologically independent replicates. Results are shown as mean ± SEM. p values were determined by one-way ANOVA. G Immunofluorescence imaging of IgA production in human ileum samples of Crohn’s disease patients and healthy people. IgA (green) and CD138 (red) antibodies were stained. Nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. This image represents 39 ileum samples of Crohn’s disease patients. Source data are provided as a Source Data file.

Fig. 6. Intestinal late enterocytes process and present Salmonella antigens to CD8⁺ T cells for their early activation.

A New RNA imaging of small intestines. Mice were infected with Salmonella and new RNA was labeled with EU (red). Scale bar, 100 μm. B UMAP plot of intestinal epithelial cells and NTR of each Salmonella infection time points. ISC, intestinal stem cell; TA, transit amplifying cell; EEC, enteroendocrine; E enterocyte. C Bubble plot showing log-normalized new RNA expression in IECs. D Intercellular communication network between IECs and immune cells at 2 h post Salmonella infection. Line width was proportional to the number of interactions. E Comparison of signaling pathways between 2 h and 0 h state based on the relative information flow. F Scatter plots showing outgoing and incoming interaction strength of MHC-I pathway at 2 h post infection. G Expression of Il15 in IECs. Late E was marked. H Flow cytometry showing MHC-I expression in CD45⁺ immune cells and Slc15a1⁺ late E at 0 h and 6 h post Salmonella infection. Mean fluorescence intensities (MFI) were calculated. n = 5 biologically independent replicates. Results are shown as mean ± SEM. p values were determined by two-way ANOVA. I Immunofluorescence imaging of MHC-I expression in enterocytes and CD8⁺ T cells at 6 h post Salmonella infection. Scale bar, 50 μm. J Late E or DCs from uninfected or Salmonella infected mice (6 h) were sorted and incubated with DQ-Ova. DQ-OVA antigen processing ability was detected via flow cytometry. n = 5 biologically independent replicates. Results are shown as mean ± SEM. p values were determined by one-way ANOVA. K Flow cytometry analysis showing presentation of SIINFEKL-H2Kb complex in late E and DCs at each time points post S.T-Ova infection. MFI was calculated. n = 6 biologically independent replicates. Results are shown as mean ± SEM. p values were determined by two-way ANOVA. L Late E induced CD8⁺ T cell proliferation in vitro. Flow cytometry analysis showing CFSE signals of naive CD8⁺ T cells from OT-I mice that were cultured alone or co-cultured with late E or DCs from uninfected and S.T-Ova infected mice. n = 5 biologically independent replicates. Results are shown as mean ± SEM. p values were determined by one-way ANOVA. Results are representative of at least three independent experiments. Source data are provided as a Source Data file.