A humanized IFN-γ mouse model reveals skin eschar formation, enhanced susceptibility and scrub typhus pathogenesis

Figures

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  Fig 1. hIFNG/hIFNGR mice are susceptible to Ot infection. WT (n = 6), Ifngr1-/- (n = 4) and hIFNG/hIFNGR (n = 4) mice were intradermally infected with Ot (3 × 103 FFU) on the right flank. Mice were monitored daily for (A) body weight changes and (B) disease scores. (C) Skin eschar lesions at inoculation sites at 11 dpi are presented and (D) their sizes are measured. (E) Representative histological images of the skin eschar lesion are shown at 11 dpi. Scale bar = 100 µm. Data are presented as mean ± SD from two independent pooled experiments. Body weight changes were analyzed by two-way ANOVA with Tukey’s multiple comparisons between WT and hIFNG/hIFNGR mice at indicated time points. Disease scores (11 and 14 dpi) and skin eschar lesion sizes (11 dpi) were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test to compare differences among mouse strains at each time point. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Comparisons with no significant differences are not labeled.

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  Fig 2. Tissue histology of WT, Ifngr1-/- and hIFNG/hIFNGR mice following Ot infection. (A) Uninfected WT (n = 3), Ifngr1-/- (n = 4) and hIFNG/hIFNGR (n = 2) mice were i.d. injected with L929 cell culture control. (B) WT (n = 9), Ifngr1-/- (n = 4) and hIFNG/hIFNGR (n = 6) mice were infected as described in Fig 1. Representative histological images of the liver, lung and brain are shown at 14 dpi. The arrows indicate the focus of inflammatory infiltration in the tissues. Scale bar = 100 µm.

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  Fig 3. Serum cytokines and chemistry profiles in mice with Ot infection. Mice were infected as described in Fig 1. (A) Serum cytokine levels were analyzed at 14 dpi by a Bio-Plex assay. (B) Serum chemistry parameters were detected by using VetScan Comprehensive Diagnostic Profile Reagent Rotor. The parameters include alanine aminotransferase (ALT), albumin (ALB), alkaline phosphatase (ALP), amylase (AMY), total calcium (CA++), globulin (GLOB), glucose (GLU), potassium (K+), sodium (NA+), total protein (TP), blood urea nitrogen (BUN), and phosphorus (PHOS). Data are shown as mean ± SD from three pooled independent experiments and analyzed by one-way ANOVA with a Tukey’s multiple comparisons test. Comparisons were displayed among mouse strains under either uninfected or infected conditions. In addition, comparisons were displayed within each mouse strain between uninfected and infected conditions. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Comparisons with no significant differences are not labeled.

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  Fig 4. hIFNG/hIFNGR mice exhibit an increased bacterial burden and reduced expression of interferon stimulated genes (ISGs) following Ot infection. Mice were infected as described in Fig 1 and were euthanized at 14 dpi. Genomic DNA was isolated from the lung, spleen, brain, kidney and liver of (A) infected WT, hIFNG/hIFNGR and (B) Ifngr1-/- mice. Bacterial burden was measured by qPCR. (C) The transcript levels of ISGs in the brain were analyzed by qRT-PCR. Data is shown as mean ± SD from three pooled independent experiments. Unpaired t-test was performed for bacterial burdens. ISG expression data of three infected groups were analyzed by one-way ANOVA with a Šídák’s multiple comparisons test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Comparisons with no significant differences are not labeled.

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  Fig 5. Imbalanced T cell and NK cell responses in hIFNG/hIFNGR mice following infection. Mice were infected as described in Fig 1 and were euthanized at 14 dpi. Spleen cells were isolated and analyzed by flow cytometry. Single cells were gated first by FSC/SSC, followed by the exclusion of dead cells by live/dead staining. CD4+ and CD8+ T cells were gated on CD3+CD4+ and CD3+CD8+, respectively. CD44+CD62L- T cells were identified as activated populations on (A) CD4+ and (B) CD8+ T cells. (C-D) NK cells were gated on CD3-NK1.1+, and CD69 was used as an activation marker of NK cells. The percentages of cell populations were shown as mean ± SD on the flow cytometric images and the statistical analysis between groups were labeled. The absolute numbers of cell populations were also calculated and shown below the flow cytometric images. One-way ANOVA with a Šídák’s multiple comparisons test was used for data analysis. Comparisons were displayed among mouse strains under either uninfected or infected conditions. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Comparisons with no significant differences are not labeled.

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  Fig 6. Excessive neutrophils and reduced innate immune cell activation in hIFNG/hIFNGR mice following Ot infection. Mice were infected as described in Fig 1 and were euthanized at 14 dpi. Spleen cells were isolated and analyzed by flow cytometry. Single cells were gated first by FSC/SSC, followed by the exclusion of dead cells by live/dead staining. (A-B) Neutrophils and monocytes were identified by CD11b+Ly6G+ and CD11b+Ly6Chi, respectively. (C) CD11b+Ly6C- cells were further gated by CD11c and F4/80 markers. Macrophages and dendritic cells were characterized by CD11c-F4/80+ and CD11chiF4/80+, respectively. The mean fluorescent intensity (MFI) of MHCII on macrophages and dendritic cells were measured. The percentages of cell populations were shown as mean ± SD on the flow cytometric images and the statistical analysis between infected groups were labeled. The absolute numbers and MFI of cell populations were also calculated and were shown below the flow cytometric images. One-way ANOVA with a Šídák’s multiple comparisons was used for data analysis. Comparisons were displayed among mouse strains under either uninfected or infected conditions. *, p < 0.05; **, p < 0.01; ***, p < 0.001;…