Differential spleen immune signatures and germinal center responses during acute infection with Orientia tsutsugamushi Karp versus Gilliam strains

Figures

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  Figure 1. Disease progression kinetics in Ot Karp- or Gilliam-infected mice. C57BL/6 mice (5 per group) were intravenously infected with Karp or Gilliam strain (6.8 × 104 FFU) or PBS (mock controls) and monitored daily for (A) body weight, (B) survival rates, and (C) disease scores. Mice were euthanized at D0 (mock), D4, D8, and D12 for spleen and serum samples harvest and analyses. (D) Splenic bacterial burdens were measured by qPCR. Results are shown as mean ± SD from a single experiment and are representative of two independent experiments with similar trends. For statistical analysis, two-way ANOVA was used with a Šídák’s multiple comparisons test (A). Survival curves were analyzed by survival curve comparison and Log-rank (Mantel-Cox) test, log-rank test for trend, and Gehan-Breslow-Wilcoxon test (B). Disease score and splenic bacterial load data were analyzed by one-way ANOVA and Tukey’s multiple comparisons test (C, D). Asterisks or ns were representative of comparison between Karp- and Gilliam-infected mice at each timepoint, unless otherwise stated. ns, no significance; *p < 0.05; ***p < 0.001; ****p < 0.0001.

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  Figure 2. Differential serum IgM and IgG responses in Karp- vs. Gilliam-infected mice. Mice were infected, as described in Figure 1. Infected and mock serum samples (5 per group) were serially diluted at 1:3 ratios. Antibodies specific to TSA56 (both Karp and Gilliam recombinant proteins, 1:1 mixed) were measured via indirect ELISA assays for IgM and IgG responses. AUC analyses of ELISA antibody curves were performed on (A) IgM and (B) IgG data; AUC values shown in bar plots were calculated from OD450 measurements across the full dilution ranges. Data are shown as mean ± SEM from a single experiment and are representative of two independent experiments with similar trends. For statistical analysis, one-way ANOVA was used with a Tukey’s multiple comparisons test for comparison of Karp- and Gilliam-infected samples at each timepoint Asterisks were representative of comparison between Karp- and Gilliam-infected mice at each timepoint. *p < 0.05; **p < 0.01; ****p < 0.0001.

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  Figure 3. GC disorganization occurs in Ot Karp, but not Gilliam, infection. Mice were infected, as described in Figure 1. Spleen cryosections were prepared and co-stained for B cells (B220), T cells (CD3), and GC B cells (GL7). Images were captured by using confocal microscopy (Zeiss LSM 880). Shown are splenic white pulp overview (scale bar, 200 μm) and high-magnification images (scale bar, 100 μm) from (A) Karp and (B) Gilliam infection, respectively.

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  Figure 4. Gilliam infection induced more robust humoral immune responses as compared to Karp infection. Mouse spleens were harvested at given time points (5 per group), as described in Figure 1. Single-cell suspensions were prepared and stained for indicated cell surface markers. Gating strategies are shown for (A) T cell subsets and (B) B cell subsets, respectively. Flow cytometric results are presented as absolute numbers of various (C) T cell populations and (D) B cell populations. Results from mock groups are presented as dashed lines. Data are shown as mean ± SEM from a single experiment and are representative of two independent experiments with similar trends. For statistical analysis, one-way ANOVA was used with a Tukey’s multiple comparisons test for comparison of Karp- and Gilliam-infected samples at each timepoint. Asterisks were representative of comparison between Karp- and Gilliam-infected mice at each timepoint. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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  Figure 5. Abrogation of marginal zone occurs exclusively in Karp-infected mice. Mice were infected, as described in Figure 1. Frozen cryosections of spleen were co-stained for B cells (B220), MZ macrophages (SIGNR-1), and metallophilic macrophages (CD169). Images were captured by using confocal microscopy (Zeiss LSM 880). Shown are overviews (scale bar, 100 μm) and high-magnification images (scale bar, 50 μm) from (A) Karp and (B) Gilliam infection, respectively.

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  Figure 6. Transcriptomic analysis of splenic environment in Karp- vs. Gilliam-infected mice. Mice were infected, and spleen tissues were collected at indicated time-points, as described in Figure 1. RNAseq was performed (4 per group) and analyzed by Novogene. Differentially expressed gene comparisons (D4 Karp vs Mock, D4 Gilliam vs Mock, D8 Karp vs Mock, D8 Gilliam vs Mock, D12 Karp vs Mock, and D12 Gilliam vs Mock) were identified. (A) Select genes are plotted using log2-fold change and displayed. (B) Differentially expressed genes relating to inflammatory and innate immune cell pathways were identified by gene set enrichment analysis from D4 Karp vs Gilliam comparison. Pathways upregulated in Karp infection appear as positive NES values. (C) IPA analysis of pathways involved in acute phase response from Karp versus Gilliam comparisons at D4, D8, and D12. Results were filtered by a Benjamini-Hochberg corrected p-value. Pathways upregulated in Karp infection show positive z-scores, while pathways upregulated in Gilliam infection show negative z-scores. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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  Figure 7. Karp infection induces significant influx of myeloid cell subsets. Mock and infected mice were euthanized, and their spleens were harvested at given time points (5 per group), as described in Figure 1. Single-cell suspensions were prepared and stained for indicated cell surface markers. (A) Gating strategy for myeloid cell subsets is shown. Absolute cell numbers of (B) monocytes and inflammatory monocytes, (C) macrophages and M1 macrophages, and (D) neutrophils and activated neutrophils are displayed. The absolute cell numbers of the mock group are represented by the dashed grey line. Data are shown as mean ± SEM from a single experiment and are representative of two independent experiments with similar trends. For statistical analysis, one-way ANOVA was used with a Tukey’s multiple comparisons test for comparison of Karp- and Gilliam-infected samples at each timepoint. Asterisks were representative of comparison between Karp- and Gilliam-infected mice at each timepoint. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.