1. Volume 22, Issue 3, Pages 354-365.e5 (September 2017)
Interferon-γ-Driven iNOS: A Molecular Pathway to Terminal Shock in Arenavirus Hemorrhagic Fever 
Melissa M. Remy, Mehmet Sahin, Lukas Flatz, Tommy Regen, Lifen Xu, Mario Kreutzfeldt, Benedict Fallet, Camille Doras, Toni Rieger, Lukas Bestmann, Uwe-Karsten Hanisch, Beat A. Kaufmann, Doron Merkler, Daniel D. Pinschewer 
Cell Host & Microbe 
Volume 22, Issue 3, Pages e5 (September 2017)
DOI: /j.chom
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2. Cell Host & Microbe 2017 22, 354-365. e5DOI: (10. 1016/j. chom. 2017
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3. Figure 1
LCMV-WE Infection of HHD Mice Enables the Investigation of AHF Pathogenesis under BSL-2 Conditions
(A–F) We infected HHD mice intravenously (i.v.) with LCMV-WE or LCMV-ARM and measured viremia (A) and serum AST (B) over time. n = 3–5. (C) Animals reaching humane endpoints were euthanized in accordance with Swiss law. n = 6 (ARM) and n = 43 (WE) from one and four experiments, respectively. At the peak of disease (day 8) pleural effusions were measured (D) and wet/dry ratios of skin flaps were determined (E). nd, not detectable. (F) Body temperature was monitored over time. Statistics were performed on day 8. in (D) to (F), n = 5–7.
(G) NP69-77-tetramer-binding CD8+ T cells in blood on day 7 after infection with LCMV-WE i.v. Uninfected mice are shown as technical controls. Symbols representing individual animals and the mean ± SEM of 6–23 mice per group from two experiments are plotted.
(H) HHD mice were depleted of CD8+ and CD4+ T cells or were given isotype control antibody prior to infection with LCMV-WE. Additional HHD control mice were left uninfected and undepleted. Viremia and AST were monitored. n = 3–5.
Data are shown as individual mice and/or mean ± SEM. Broken connection lines indicate that individual animals reached humane endpoints and were euthanized. In (A), (B), (G), and (H) one of two similar experiments is shown. ∗p < 0.05; by unpaired Student's t test on log-converted values (G) and by one-way ANOVA with Dunnett's post test (D–F); ns, not significant. See also Figure S1.
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4. Figure 2
LCMV-WE Infection Induces a Shock Syndrome due to Microvascular Leak
We infected HHD mice i.v. with LCMV-WE on day 0.
(A) Systolic blood pressure. n = 4 uninfected and 7 infected mice.
(B) Wet/dry ratios of skin flaps harvested at the peak of disease (d8-10). n = 4 uninfected and 7 infected mice.
(C) Serum albumin concentration. n = 6–14. day-8 values were statistically analyzed.
(D) Albumin concentration in serum and pleural fluid on day 8. The mean ± SEM of the serum/pleural fluid ratios of 9–11 mice is indicated.
(E) Echocardiography was performed on day 8 of infection (LCMV+) and in uninfected controls (LCMV−). A subgroup of HHD-WT mice with detectable pleural effusions at necropsy (effusions+) was separately analyzed. LVID-s, left ventricle internal diameter in systole. n = 6–11.
(F–H) Body temperature at peak of disease correlated inversely with pleural effusions (F), ventral edema (G), and reduction in systolic blood pressure (compared with day 0) (H). n = 32 mice from three experiments (F), n = 18 mice from two experiments (G); in (H) 11 mice were analyzed (days 6–10). Data are shown as individual mice and/or mean ± SEM.
Broken connection lines indicate individual animals reached humane endpoints and were euthanized. One representative experiment of two (B, D, and E) or three (A and C) independent experiments is shown. ∗p < 0.05, ∗∗p < 0.01 by one-way ANOVA (A: Dunnett's post test in comparison with day 0; B and E: Tukey's post test), unpaired Student's t test (C), or Pearson's correlation (F–H); ns, not significant. See also Figure S2.
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5. Figure 3
LCMV-WE Infection of HHD Mice Triggers a “Cytokine Storm” and Induces iNOS Expression
We infected HHD-WT (iNOS+/+) and HHD-iNOS−/− mice i.v. with LCMV-WE on day 0.
(A–C) Eight days later, liver (A), spleen (B), and lung (C) tissues were processed for histological analysis in comparison with uninfected HHD mice. Sections were either stained with H&E or were processed for immunohistochemical detection of monocyte/macrophages and T cells. Scale bars, 200 μm (scale bars in insets, 20 μm).
(D) Total IL-12p40 levels were determined in the serum of infected HHD-WT mice. n = 15.
(E) Inflammatory cytokines were quantified in the serum on day 0 and day 8 of infection. n = 10.
(F) Total nitrite (NO2−) and nitrate (NO3−) levels in serum (NOx) were measured in uninfected mice and in day-7 LCMV-infected mice. n = 5–7.
(G) Induction of iNOS mRNA expression upon LCMV-WE infection was determined in the indicated organs. For this, values of day-8 infected mice were expressed as a multiple of the values in uninfected controls. n = 4. Statistical analyses compared uninfected and infected mice within each organ.
(H and I) Liver tissue from day-8 LCMV-infected HHD-WT (iNOS+/+) and HHD-iNOS−/− mice was processed for immunohistochemical detection of iNOS (H) or immunologically co-stained for iNOS and IBA1 (I). Naive HHD-WT mice were included for reference. Scale bars, 100 μm (scale bars in insets, 20 μm).
Data are shown as dots for individual mice and/or mean ± SEM. One representative experiment of three independent experiments is shown in (D) to (G). ∗∗p < 0.01 by paired (E) or unpaired (F and G) Student's t test; ns, not significant. See also Figure S3.
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6. Figure 4
iNOS Is Essential for LCMV-WE-Induced Terminal Shock but Not for Generalized Inflammation
HHD-WT (iNOS+/+) and HHD-iNOS−/− mice were infected with LCMV-WE on day 0.
(A) Total nitrite (NO2−) and nitrate (NO3−) concentration (NOx) in serum on day 8. n = 8–20.
(B) Animals reaching humane endpoints were euthanized in accordance with Swiss law. n = 13 from 2 experiments.
(C) Body temperature was monitored. n = 8. Statistical analyses were performed individually for each time point.
(D) Pleural effusions were quantified on day 8. The numbers of animals exhibiting detectable pleural effusions/number of animals assessed and the resulting percentage of effusion-exhibiting animals are indicated. nd, not detectable. Symbols show 23 HHD-WT and 8 HHD-iNOS−/− mice from two experiments. The lack of pleural effusion in iNOS−/− mice was confirmed in three additional experiments.
(E) Skin edema at the peak of disease in HHD-WT mice.
(F–H) Viremia (F), serum AST (G), and serum IL-12p40 (H) concentrations were determined. n = 9–13.
(I) Inflammatory cytokines in the serum of uninfected and day-8 infected mice. n = 23–32 (HHD-WT) and n = 6–9 (HHD-iNOS−/−) from four independent experiments.
(J) NP tetramer-binding CD8+ T cell frequencies in blood on day 8. n = 5–9.
Data are shown as symbols for individual mice and/or mean ± SEM. Broken connection lines indicate that individual animals reached humane endpoints and were euthanized. Data representative of two (A, E, F–H, and J) or five (B–D) independent experiments are shown. ∗p < 0.05, ∗∗p < 0.01 by unpaired Student's t test (A and C), log-rank test (B), and one-way ANOVA with Tukey's post test (E, I, and J); ns, not significant. See also Figure S4.
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7. Figure 5
IFN-γ Blockade Prevents iNOS Induction and Terminal Disease in LCMV-Infected HHD Mice
We infected HHD mice with LCMV-WE on day 0, and on day 5 treated them with anti-IFN-γ-blocking or isotype control antibody. LCMV-WE-infected anti-CD4/CD8 double-depleted mice were included for comparison, alongside uninfected animals.
(A and B) iNOS mRNA (relative units, RU) in the liver. (A) n = 6–7; (B) n = 3–4.
(C) Total nitrite (NO2−) and nitrate (NO3−) concentrations (NOx) in serum of day-8 infected and uninfected mice. n = 16–17.
(D) Hepatic iNOS expression detected by immunohistochemistry. Scale bar, 100 μm.
(E) Animals reaching humane endpoints were euthanized in accordance with Swiss law. n = 10 from two independent experiments.
(F) Body temperature was monitored. n = 5–6 mice per group are shown.
(G–I) Pleural effusions (G), peritoneal effusions (H), and skin edema (I) were quantified at the peak of disease (day 8). n = 6–7. Uninfected mice were included as controls in (I).
(J) Schematic of the postulated mechanism and cascade of events leading to terminal shock in AHF.
Data are shown as individual mice and/or mean ± SEM. Broken connection lines indicate that individual animals reached humane endpoints and were euthanized. Data representative of two (A–C, E, and H) or three (F, G, and I) independent experiments are shown. ∗p < 0.05, ∗∗p < 0.01 by one-way ANOVA (Tukey’s post test, A–C and I), log-rank test (E), or unpaired Student's t test at individual time points (F–H); ns, not significant. See also Figure S5.
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