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. 2021 May 28;10:e65762. doi: 10.7554/eLife.65762

Figure 3. Single cell transcriptome analysis reveals a sustained IFN signature in the neutrophil response against HSV-2.

Mice were infected as described in Figure 2. Vaginas were harvested at 5 d.p.i., and live cells were flow sorted and subjected to high-throughput single-cell RNA sequencing (scRNA-seq). (A) A t-Distributed Stochastic Neighbor Embedding (tSNE) visualization of 21,633 cells across all mice resolves 17 distinct clusters in the vaginal tissue. Clusters can be identified as myeloid cells (red border), epithelial cells (blue border), or lymphocytes (purple border). Neutrophils are encircled in black and contain three distinct clusters (0, 2, and 5). (B) Neutrophils are defined by high expression of S100a8 and Csf3r (G-SCFR). (C) tSNE plots of vaginal cell clusters from mock-inoculated or HSV-infected mice. Neutrophil populations are circled in black. (D) Distribution of expression for genes within the Hallmark IFNa Response gene set. (E) Production of IFNβ in vaginal washes collected at 4 and 5 d.p.i. as measured by ELISA (uninfected: n = 8, HSV-1: n = 9, HSV-2: n = 9). scRNA-seq in A-D was performed once. Data in E are pooled from two independent experiments. Statistical significance was measured by one-way ANOVA with Tukey's multiple comparisons test. *p<0.05, **p<0.01, ***p<0.005. Raw values for each biological replicate, specific p values, and complete lists of differentially expressed genes between clusters 0, 2, and 5 are provided in Figure 3—source data 1.

Figure 3—source data 1. Excel file with individual ELISA measurements, lists of differentially expressed genes between different neutrophil clusters, description of statistical tests and actual p values for Figure 3.
Figure 3—source data 2. Excel file with lists of differentially expressed genes between neutrophils between HSV-1 and HSV-2 infected mice, individual delta Cq values, ELISA measurements, description of statistical tests and actual p values for Figure 3—figure supplements 1–3.

Figure 3.

Figure 3—figure supplement 1. ISGs are differentially expressed in neutrophils during HSV-1 and HSV-2 infection.

Figure 3—figure supplement 1.

The t-Distributed Stochastic Neighbor Embedding (tSNE) plot shows clusters 5 and 2 within the neutrophil population. Tables show the top 20 differentially expressed genes in cluster 2 (left) and cluster 5 (right) at 5 d.p.i. between HSV-2 and HSV-1 infection. Interferon-stimulated genes (ISGs) listed in the Hallmark IFNα Response gene set are highlighted in gray. Complete lists of differentially expressed genes are provided in Figure 3—source data 2.
Figure 3—figure supplement 2. Validation of ISG expression in the vagina.

Figure 3—figure supplement 2.

Expression of CXCL10 (A, B), Gbp2 (C, D), or IL-15 (E, F) in the vagina of mock-inoculated mice or mice at 5 d.p.i. with HSV-1 or HSV-2. tSNE visualization of select ISG transcripts in live vaginal cells profiled by scRNA-seq (A, C, E). Expression of the same ISGs relative to Rpl13 was measured by quantitative RT-PCR (qRT-PCR) in whole vaginal tissue harvested from mock-inoculated mice (n = 3) or mice at 5 d.p.i. with HSV-1 (n = 9) or HSV-2 (n = 9) (B, D, F). Bars in B, D, and F show mean and SD. Data are pooled from two independent experiments. Statistical significance was measured by one-way ANOVA with Tukey's multiple comparisons test. ***p<0.005, ****p<0.001, ns = not significant. Raw values for each biological replicate and specific p values are provided in Figure 3—source data 2.
Figure 3—figure supplement 3. Type I IFN is produced early in the vagina and resolves by 1 week after acute HSV-1 or HSV-2 infection.

Figure 3—figure supplement 3.

C57BL/6J mice were infected as described in Figure 2. Type I IFN was measured by ELISA in vaginal washes collected from uninfected mice (n = 8), or mice infected with HSV-1 (n = 7–9) or HSV-2 (n = 9) at 2 (A) and 7 d.p.i. (B). (C) IFNβ was measured by ELISA from vaginal tissue homogenates collected at 7 d.p.i. Bars show mean with SD. All data are pooled from two independent experiments. Statistical significance was measured by one-way ANOVA with Tukey's multiple comparisons test. *p<0.05, ***p<0.005. Raw values for each biological replicate and specific p values are provided in Figure 3—source data 2.