1. Volume 168, Issue 3, Pages 503-516.e12 (January 2017)
Pathogen-Mediated Inhibition of Anorexia Promotes Host Survival and Transmission 
Sheila Rao, Alexandria M. Palaferri Schieber, Carolyn P. O’Connor, Mathias Leblanc, Daniela Michel, Janelle S. Ayres 
Cell 
Volume 168, Issue 3, Pages e12 (January 2017)
DOI: /j.cell
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3. Figure 1 A Salmonella Effector Promotes Survival of its Host
(A) Survival of B6 mice orally infected with WT (n = 20) or ΔslrP (n = 20) ST. Data represent two independent experiments combined.
(B and C) Weight loss of B6 mice orally infected with WT or ΔslrP ST on days 1–3 (B) and day 6 (C) post-infection. n = 18/group. Data represent three independent experiments combined (B). n = 12 WT-infected and n = 17 ΔslrP-infected mice (C).
(D and E) MRI analysis of lean (D) and fat mass (E) of B6 mice orally infected with WT (n = 9) or ΔslrP (n = 10) ST for 72 hr. Measurements at 72 hr are normalized to those taken on day 0 for each mouse. Data represent two independent experiments combined.
∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05 by unpaired Student’s t test or Log rank analysis (survival). Error bars ± SEM.
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4. Figure 2
Inhibition of the Sickness-Induced Anorexia Response Controls Virulence
(A) RER at 72 hr post-infection of B6 mice orally infected with WT or ΔslrP ST. Black bar indicates dark/night cycle, and white bar indicates light/day cycle. n = 5/group.
(B) Food consumption of B6 mice orally infected with WT or ΔslrP ST. Quantification is expressed as a ratio of grams of food per animal at each time point to that of vehicle (PBS)-gavaged animals. 0–24 hr: n = 30 mice/group; 24–48 hr: n = 30 mice/group; 48–72 hr: n = 15 mice/group. Data represent three experiments combined.
(C) CFU in indicated tissues of B6 mice orally infected with WT or ΔslrP ST for 24 hr (top) and 48 hr (bottom). n = 10–16 mice/group. Data represent two independent experiments combined. Dotted line indicates limit of detection, and mice with no CFU detected are indicated by symbols below this line. ND, none detected.
(D) Animals from (C) were assigned a dissemination score of “1” (at least 1 CFU at the level of detection in indicated systemic organ) or “0” (no CFU detected in indicated systemic organ) 48 hr post-infection. n = 10/group.
(E) Food consumption of germ-free B6 mice orally infected with WT (n = 12) or ΔslrP (n = 14) ST for 24hr. Grams of food per animal normalized to uninfected consumption value of germ-free mice. Data combined from two independent experiments.
(F) Weight loss from days 1–3 of WT ST infection (n = 14), ΔslrP ST infection (n = 15), and WT ST infection during which food consumption was restricted (n = 15), as indicated in table. Right graph shows weight loss on day 3 post-infection from left graph. Data represent two independent experiments combined.
(G) Survival of mice from (F). ∗∗∗ p = between WT-infected and WT-infected food-restricted mice by Log rank analysis.
(H) Weight loss from days 1–3 of WT ST infection (n = 19), ΔslrP ST infection (n = 30), and ΔslrP ST infection during which mice were force-fed twice daily in addition to ad libitum feeding (n = 29). Right graph shows weight loss on day 3 post-infection from left graph. Data represent three independent experiments combined.
(I) Survival of mice from (H). ∗∗∗ p = between ΔslrP-infected mice and ΔslrP-infected force-fed mice by Log rank analysis.
∗∗∗∗ p < , ∗∗∗ p < 0.01, ∗∗ p < 0.05 by unpaired Student’s t test. Error bars indicate ± SEM.
See also Figure S1.
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5. Figure 3
Canonical Mediators of Sickness-Induced Anorexia Are Equivalent between WT and ΔslrP ST
(A) Activity of B6 mice orally infected with WT or ΔslrP ST. Black bar indicates dark/night cycle, and white bar indicates light/day cycle. n = 6 mice/group.
(B) B6 mice were infected i.p. with WT or ΔslrP ST, and food consumption was measured every 24 hr post-infection and normalized to consumption of uninfected animals. Grams of food per animal consumed within the first four days of infection shown. n = 6/group.
(C) H&E staining and histological scoring of spleen, liver, and ileum 24 hr after oral infection of B6 mice with WT or ΔslrP ST. ND indicates pathology above limit of detection not detected. NS, not significant. Scale bar, 200 μm. n = 5/group.
(D) Total cellularity of MLN and SI LP 48 hr after oral infection of B6 mice with WT or ΔslrP ST. n = 12–13/group. Data represent three independent experiments combined.
(E) Numbers of inflammatory cell populations 48 hr after oral infection of B6 mice with WT or ΔslrP ST. LP: T/B lymphocytes (P1, n = 8–9/group), neutrophils (P2, n = 13/group), phagocytic macrophages (P3, n = 8–9/group), inflammatory monocytes (P4, n = 8–9/group); MLN: migratory DCs (P5, n = 8/group). Data represent three independent experiments combined.
(F and G) ELISA measurements of TNFα (F) and IL-6 (G) in indicated tissues 48 hr after oral infection of B6 mice with WT or ΔslrP ST. n = 4–10/group. ND, not detected.
Vehicle indicates PBS. Error bars indicate ± SEM.
See also Figure S2.
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6. Figure 4
ST SlrP Prevents Anorexia and Regulates Virulence through the Inhibition of IL-1β
(A) Levels of mature IL-1β were determined by IL-1R reporter assay of whole-organ homogenates in liver, spleen, and serum of B6 mice 48 hr post-infection (left, n = 4–10/group) and in SI (right; n = 7 vehicle-gavaged, n = 24 WT-infected, and n = 24 ΔslrP-infected mice). Graph shown depicts reporter fluorescence (arbitrary units), indicative of active IL-1β. Data represent two experiments combined.
(B–D) Food consumption (B), weight loss (C), and survival (D) during ΔslrP infection of B6 (n = 6-20) and Il1β−/− (n = 7–15) mice. (B) Grams of food per animal was normalized to consumption of uninfected animals of appropriate genotype. Data represent two experiments combined.
(E and F) B6 mice were injected with PBS (n = 10–20 WT-infected mice; n = 10–20 ΔslrP-infected mice) or rIL-1β (0.05 μg/gram mouse, n = 15 WT-infected mice). Food consumption (E) and weight loss (F) were measured. (E) Data represent two to three experiments combined.
(G) Western blot analysis of CASPASE-1 cleavage (p20) in IECs (top) and LP leukocytes (bottom) isolated from infected mice. Leukocytes were combined from 5 WT-infected mice and from 5 ΔslrP-infected mice at 24 hr and 48 hr post-infection.
(H) Quantitative PCR (qPCR) analysis of Il1β expression in IECs and LP leukocytes 48 hr after oral infection of B6 mice with WT or ΔslrP ST. n = 5/group.
∗∗∗∗ p < , ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05 by unpaired Student’s t test or Log rank analysis for survival. NS indicates not significant by unpaired Student’s t test. ND indicates none detected above limit of detection. Error bars indicate ± SEM.
See also Figure S3.
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7. Figure 5
Salmonella Regulates the Anorexic Response and Virulence via the Gut-Brain Axis
(A and B) Il1β−/− mice orally infected with ΔslrP were fed ad libitum (n = 6) or given restricted amounts of food (n = 6). ΔslrP-infected B6 mice (n = 5) were fed ad libitum. Weight loss (A) and survival (B) were measured.
(C) Heatmap of differentially expressed genes in the hypothalamus of WT (n = 3) and ΔslrP (n = 3) infected B6 mice 48 hr post-infection.
(D) qPCR analysis of genes identified in (C) in the hypothalamus of B6 mice infected with WT (n = 4) or ΔslrP (n = 4) and Il1β−/− (n = 10) mice infected with ΔslrP ST for 48 hr.
(E) Feeding of ΔslrP-infected vagotomized or sham B6 mice. Values normalized to feeding amounts of uninfected mice. n = 9–10/group. Data represent two independent experiments combined.
(F) qPCR analysis of genes identified in (C) in the hypothalamus of ΔslrP-infected vagotomized or sham B6 mice at 48 hr post-infection. n = 4–5/group.
(G) Levels of mature IL-1β were determined by IL-1R reporter assay 48 hr post-infection in SI of ΔslrP-infected vagotomized or sham B6 mice. Graph shown depicts reporter fluorescence (arbitrary units), indicative of active IL-1β. n = 4–5/group.
∗∗∗∗ p < , ∗∗∗ <0.01, ∗∗ p < 0.05, ∗ p = Unpaired Student’s t test, one-sample t test, or Log rank analysis for survival. Error bars indicate ± SEM Il1β for (D) is from hypothalamus from 48 hr and 72 hr post infection.
See also Figures S4 and S5.
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8. Figure 6 Anorexia Creates Tradeoffs between Virulence and Transmission
(A) Animals were assigned a liver dissemination score of “1” or “0” at 72 hr post-infection. n = 10/group.
(B) Animals from (A) were assigned a score of “1” or “0” to determine fecal shedding index at 72 hr post-infection.
(C) CFU in liver of mice from (A) was plotted against fecal shedding index (B) for each individual mouse. “−” denotes mice with no fecal shedding, and “+” denotes mice with fecal burden.
(D) CFU detected in feces of mice from (A) was plotted against liver dissemination index (C) for each individual mouse. “−” denotes mice with no liver dissemination, and “+” denotes mice with liver burden.
(E) B6 mice were orally infected with WT or ΔslrP ST (WT-primary or ΔslrP-primary) and co-housed with an uninfected B6 mouse (WT-secondary or ΔslrP-secondary). Fecal samples were collected every 24 hr post-infection from primary and secondary hosts for CFU analysis. Mice were assigned a score of “1” or “0” n = 8–15 mice/group. Data combined from two experiments.
(F) Survival of mice from (E). 8–10 mice/group. ∗∗ represent statistical significance between both primary groups or both secondary groups.
(G) Model of how WT and ΔslrP ST regulate anorexia, virulence, and transmission.
∗∗∗∗ p < , ∗∗∗ p < 0.01, ∗∗ p < unpaired Student’s t test, one way ANOVA with tukey post-test, or Log rank analysis. Dotted line indicates limit of detection, and mice with no CFU detected in indicated tissue are indicated by symbols below this line.
See also Figure S6.
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9. Figure S1
SlrP Modulates Food Consumption during Infection, Related to Figure 2
(A and B) Rate of oxygen consumption (VO2) (A) and rate of CO2 production (VCO2) (B) were determined by CLAMS 72hr after oral infection of B6 mice with stated strain of S. Typhimurium. Black bar indicates dark/night cycle, and white bar indicates light/day cycle. n = 6/group.
(C) Food consumption of B6 mice orally infected with WT ST, ΔslrP ST with an empty control vector, or ΔslrP complemented with a vector expressing WT slrP under the native promoter. Grams of food per animal was normalized to consumption of uninfected animals. Data are two independent experiments combined, n = animals per group.
(D) CFU in the SI of B6 mice infected with WT ST, ΔslrP ST with an control empty vector, or ΔslrP ST complemented with WT slrP under the native promoter. n = 6-8 per group and representative of two independent experiments.
(E-F) Uninfected B6 mice were food restricted as described in methods, and weight (E) and survival (F) were monitored. n = 5 mice.
∗ indicates p < 0.05 and ∗∗ indicates p < by unpaired Student’s t test; error bars indicate ± SEM.
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10. Figure S2
SlrP Modulates Feeding Behavior Independent of Cell Infiltration, Pathology, TNFα, or IL-6, Related to Figure 3
(A) Histological scoring of spleen, liver, and ileum 72hr after oral infection of B6 mice with stated strain of S. Typhimurium. n = 5/group. “ND” indicates none detected – no pathology above limit of detection.
(B) Gating strategy for flow cytometric analysis of leukocyte populations in the small intestine LP: T/B lymphocytes (P1), neutrophils (P2), phagocytic macrophages (P3), inflammatory monocytes (P4).
(C) Gating strategy for flow cytometric analysis of migratory dendritic cells (P5) in the MLNs.
(D) Flow cytometric analysis of percentages of LP and MLN leukocytes 48hr after oral infection of B6 mice with stated strain of S. Typhimurium. Animal numbers shown.
(E) Total cellularity (left) and flow cytometric analysis of numbers (top) and percentages (bottom) of LP and MLN leukocytes 24hr after oral infection of B6 mice with stated strain of S. Typhimurium. n = 8-9 mice per group.
(F) Gating strategy for flow cytometric analysis of leukocyte populations in the spleen and liver: T/B lymphocytes (P1), neutrophils (P2), macrophages (P3), inflammatory monocytes (P4), and dendritic cells (P5).
(G) Total cellularity (left) and flow cytometric analysis of numbers (top) and percentages (bottom) of spleen and liver leukocytes 24hr after oral infection of B6 mice with stated strain of S. Typhimurium. n = 5 mice per group.
(H) Total cellularity (left) and flow cytometric analysis of numbers (top) and percentages (bottom) of spleen and liver leukocytes 48hr after oral infection of B6 mice with stated strain of S. Typhimurium. n = 5 mice per group.
(I) ELISA measurements of IL-6 in the indicated tissues 24hr after oral infection of B6 mice with WT or ΔslrP S. Typhimurium. n = 4-7 mice per group. ND denotes Not Detected. NS denotes Not Significant.
Error bars indicate ± SEM. All comparisons not significant by unpaired Student’s t test.
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11. Figure S3
SlrP Modulates Feeding Behavior by Regulating Inflammasome Activation and IL-1β Maturation, Related to Figure 4
(A) Levels of active/mature IL-1β determined by IL-1β bioassay of whole organ homogenates in liver, spleen and serum of B6 mice 24hr post-infection (n = 9-13/group). Data represent two independent experiments combined. A. U. indicates arbitrary units of reporter activation.
(B) Levels of active/mature IL-1β determined by IL-1β bioassay of the stomach and pancreas 24hr and 48hr after oral infection of B6 mice with WT and ΔslrP S. Typhimurium. n = 5 infected mice per group and n = 3 vehicle (PBS)-gavaged mice. A.U. indicates arbitrary units of reporter activity.
(C) Levels of active/mature IL-1β determined by IL-1β bioassay of the SI 24hr and 48hr after oral infection of B6 mice with WT ST, ΔslrP ST with an empty control vector, or ΔslrP ST complemented with a vector expressing WT slrP under the native promoter. Data are two independent experiments combined, n = animals per group. ∗ indicates p < 0.05 and ∗∗∗ indicates p < by unpaired t test. A.U. indicates arbitrary units of reporter activity.
(D-F) Food consumption (D), weight loss (E), and survival (F) of ΔslrP infected B6 (n = 9) and Il1β−/− (n = 10) mice. (C) Grams of food per animal was normalized to consumption of uninfected animals. Bedding from B6 and Il1β−/− cages was mixed to control for differences in microbiota as described in methods.
(G and H) B6 (n = 14) and Il1β−/− (n = 15) mice were orally infected with WT S. Typhimurium, and weight (G) and survival (H) were monitored. Data represent two experiments combined.
(I) Original western blot images from Figure 4.
(J) Cleaved Caspase-1 p20 from western blots was quantified as percent of full-length Caspase-1 using ImageJ densitometry analysis. n = 2/group for 24hr and n = 3/group for 48hr.
(K) Myeloid cells (CD45+CD3-CD19-) were sorted from LP preparations from naive B6, Casp1/11−/− or Il1β−/− mice and infected in vitro with MOI 1 of WT or ΔslrP S. Typhimurium. Mature IL-1β levels in supernatant were determined by IL-1β bioassay 6hr post-infection. Graph depicts two independent experiments for cells from B6 mice and represents two independent experiments for cells from genetic knockout mice.
(L) Intracellular CFU in sorted myeloid cells from the indicated genotypes infected for 6hr in vitro with WT or ΔslrP S. Typhimurium. n = 3-4 per group.
(M) western blot analysis of CASPASE-1 cleavage (p20) in in vitro infected myeloid cells from (J). Blot representative of two independent experiments. Due to space constraints, blot was cropped to remove membrane not relevant for the manuscript.
(N) Quantification of CASPASE-1 p20 from (l) normalized to β-actin using ImageJ densitometry analysis. Graph depicts quantification from two independent experiments for each genotype.
Error bars indicate ± SEM. ∗p < 0.05, unpaired Student’s t test; ∗∗∗p < by log rank analysis.
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12. Figure S4 IL-1β Causes Anorexia during Infection, Related to Figure 5
(A) Il1β−/− mice infected with ΔslrP were given food ad libitum (n = 6) or given restricted amounts of food (n = 6) as described in methods and Figure 2E. ΔslrP-infected B6 (n = 5) were fed ad libitum. Grams of food per animal was normalized to consumption of uninfected animals of appropriate genotype.
(B and C) Uninfected Il1β−/− mice were food restricted as described in methods and Figure 2E. Weight (B) and survival (C) were monitored. n = 6.
(D) qPCR validation of expression of genes from Figure 5C on RNA isolated from hypothalamus of B6 or Il1β−/− mice infected with WT or ΔslrP at 48hr. n = 5-10 mice/group.
Error bars indicate ± SEM. ∗p < 0.05 for B6 WT versus ΔslrP unpaired Student’s t test.
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13. Figure S5
The Vagus Nerve Is Required to Induce the Anorexic Response in the Absence of SlrP, Related to Figure 5
(A) Total amount of grams of food consumed per mouse in 24 hr under uninfected conditions of vagotomized (n = 5) and sham-vagotomized (n = 4) mice.
(B) qPCR validation of expression of genes from Figure 5C on RNA isolated from hypothalamus of sham or vagotomized B6 mice infected with ΔslrP for 48hr. n = 4-5 mice/group.
Error bars indicate ± SEM. ∗p < 0.05 unpaired Student’s t test.
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14. Figure S6
Food Consumption Creates Trade-offs between Virulence and Transmission, Related to Figure 6
(A) Splenic and liver burden of B6 mice infected IP with WT and ΔslrP S. Typhimurium for 48hr (left) and 96hr (right). n = 7-8 mice per group.
(B) Orally infected animals from the indicated groups were assigned a dissemination score of “1” (at least 1 CFU at the level of detection in spleen) or “0” (no CFU detected in spleen) at 72 hr post infection. Dissemination index refers to splenic burden 72hr post-infection. n = 10/group.
(C) CFU in spleen of mice from (B) was plotted against fecal shedding index for each individual mouse. “−” denotes mice with no fecal shedding, and “+” denotes mice with fecal burden at 72 hr postinfection. Dotted line indicates limit of detection, and mice with no CFU detected in the spleen are indicated by symbols below this line.
(D) CFU detected in feces of mice from (C) was plotted against spleen dissemination index for each individual mouse. “−” denotes mice with no spleen dissemination, and “+” denotes mice with spleen burden at 72 hr postinfection. Dotted line indicates limit of detection, and mice with no CFU detected in the feces are indicated by symbols below this line. This graph looks the same as shown in Figure 6D, because the animals used for analysis of fecal burden and spleen/liver dissemination are the same and the same animals with liver dissemination event had a spleen dissemination event yielding similar plots.
(E) Dissemination index for the PP and MLN 72hr post-infection of the indicated groups. n = 10/group.
(F) CFU in PP (left) and MLN (right) of mice from (E) was plotted against fecal shedding index for each individual mouse. “−” denotes mice with no fecal shedding, and “+” denotes mice with fecal burden. Dotted line indicates limit of detection, and mice with no CFU detected in the indicated tissue are indicated by symbols below this line.
(G) CFU detected in feces of mice from (F) was plotted against PP dissemination index (left) and MLN dissemination index (right) for each individual mouse. “−” denotes mice with no organ dissemination, and “+” denotes mice with organ burden. Dotted line indicates limit of detection, and mice with no CFU detected in the feces are indicated by symbols below this line.
(H) Dissemination index of indicated organs of ΔslrP-infected Il1β−/− or B6mice that were fed ad libitum or ΔslrP-infected Il1β−/− food-restricted. n = per group. Graph represents two to three experiments combined.
(I) CFU in indicated tissue of the mice from (H).
(J) qPCR analysis of Cldn2 and Cldn4 expression in intestinal epithelial cells 48hr after infection of B6 mice with indicated strain of S. Typhimurium. n = 5/group. Data are representative of two individual experiments.
Error bars indicate ± SEM. ∗∗∗∗p < ; ∗∗p < 0.01; ∗p < 0.05 by unpaired Student’s t test.
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