Immunostaining for S100A9 in the liver and spleen of mice during <i>S</i>. Typhimurium infection.

<p>Positive immunostaining for S100A9 in liver parenchyma (A) and the red pulp of the spleen (C) tissue was observed in uninfected control animals. Five days after infection with <i>S</i>. Typhimurium (10⁶) there was a marked increase of S100A9 in both liver (B) and spleen (D) corresponding with inflammatory cell influx. Magnification 10 × and 40 ×.</p

Patient characteristics.

<p>Baseline characteristics of patients with acute typhoid fever (admitted to the hospital, Bangladesh study) or during convalescence (treated in an outpatient setting, Indonesian study). Days of fever prior to admission or when sample was taken, median. Specific complications were: pneumonia (n = 3); encephalopathy (n = 2); gastrointestinal bleeding (n = 1); severe anemia requiring blood transfusion (n = 1); acute hepatitis (n = 1); hepatic abscess (n = 1); septic arthritis (n = 1); and renal failure, cardiopulmonary arrest and death (n = 1). N/A: not applicable.</p><p>Patient characteristics.</p

S100A8/A9 inhibits bacterial growth of <i>S</i>. Typhimurium and <i>S</i>. Typhi in vitro.

<p>Growth of <i>S</i>. Typhimurium (14028; A) and <i>S</i>. Typhi (Vivotif, Crucell; B) was assessed for a maximum of 24 hours in the presence of recombinant S100A8/A9 (50 μg/ml; grey bars) or control (white bars). Bacterial growth was dose dependently inhibited by S100A8/A9. Growth arrested, CFSE-labelled heat-inactivated <i>S</i>. Typhimurium bacteria were incubated with peripheral blood neutrophils (C) from wildtype (WT; white) and <i>S100A9</i>^<i>-/-</i> mice (grey) (n = 4 per mouse strain) for 0, 15 and 60 minutes respectively after which phagocytosis was quantified (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003663#sec005" target="_blank">Methods</a>). Data are expressed, as mean ± SD. **** P<0.0001, determined via non-parametric <i>t</i> tests comparing different dosages per time point.</p

<i>S100A9</i>^<i>-/-</i> mice are equally susceptible to <i>S</i>. Typhimurium infection when compared to wildtype mice in a <i>Salmonella</i> mouse model.

<p>Bacterial loads in wildtype (WT; white bars) mice compared with <i>S100A9</i>^<i>-/-</i> mice (grey bars; data represent 5–8 mice per group) from mesenterial lymph nodes (MLN; A), liver and spleen homogenates (B, C), and blood (D), 2 and 5 days post infection with oral 1x10⁵ or 1x10⁶ CFU of <i>S</i>. Typhimurium. Data are expressed as box-and-whisker diagrams (log scale) depicting the smallest observation, lower quartile, median, upper quartile and largest observation. Survival curve of WT and <i>S100A9</i>^<i>-/-</i> mice after oral inoculation with 1x10⁶ CFU of <i>S</i>. Typhimurium (E; n = 20 mice per group in each experiment).</p

S100A8/A9 complexes are abundantly present in plasma and feces from patients with typhoid fever and correlate with duration of fever.

<p>Increased levels of S100A8/A9 (ng/ml) complexes are measured in plasma of patients with confirmed typhoid fever caused by <i>S</i>. Typhi admitted to the hospital (A; n = 28 for admission) and compared to healthy controls (n = 38). However S100A8/A9 levels were not yet normalized at time of discharge (n = 15). In a community based control study during convalescence (3 weeks post infection) S100A8/A9 remains elevated (B; n = 60) compared to healthy controls (n = 60). Increased levels of S100A8/A9 (μg/g) measured in feces from hospitalized patients (C; n = 14) compared to healthy controls (n = 36). No differences were seen in levels of S100A8/A9 between hospitalized patients with complicated or uncomplicated typhoid fever as detailed in method section (D). Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation. Correlation curve log alanine aminotransferase (ALT) versus log plasma S100A8/A9 (E). Correlation curve days of fever prior to admission versus log plasma S100A8/A9 (F). For linear regression analysis, P value and Spearman rho are reported.</p

No effect of S100A8/A9 deficiency on organ injury during <i>S</i>. Typhimurium infection.

<p>Representative slides of liver (A, B) and spleen (D, E) hematoxylin and-eosin (HE) staining of wildtype (WT) and <i>S100A9</i>^<i>-/-</i> mice, 5 days post infection. Liver and spleen from both WT and <i>S100A9</i>^<i>-/-</i> mice displayed advanced inflammation with necrosis (#) and thrombosis (*). Magnification 10 ×. Total pathology score was determined at indicated time points in WT (white bars) and <i>S100A9</i>^<i>-/-</i> (grey bars) mice according to the scoring system described in the methods section (C, F). Aspartate aminotransferase (AST; G), alanine aminotransferase (ALT; H), and lactate dehydrogenase (LDH; I) were measured in plasma. Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation (5–8 mice per group at each time point).</p

Systemic cytokine profile during experimental <i>S</i>. Typhimurium infection in wildtype and <i>S100A9</i>^<i>-/-</i> mice.

<p>Cytokines were measured in plasma and liver tissue 2 and 5 days after oral infection with <i>S</i>. Typhimurium. Data are means ± SEM of 5–8 mice per group. *P <0.05 determined via non-parametric <i>t</i> test. Abbreviations: TNF-α: tumor necrosis factor-alpha; IFN-γ: interferon-gamma; MCP-1: monocyte chemo attractive protein-1; IL: interleukin.</p><p>Systemic cytokine profile during experimental <i>S</i>. Typhimurium infection in wildtype and <i>S100A9</i>^<i>-/-</i> mice.</p

S100A/A9 is up regulated in a murine <i>Salmonella</i> model.

<p>Levels of S100A8/A9 complexes measured in plasma (A) and liver homogenates (B) from wildtype (WT) mice orally challenged with 1x10⁵ or 1x10⁶ CFU <i>S</i>. Typhimurium after 0 (uninfected; UI; n = 3 per group), 2 or 5 days of infection (n = 5–8 per group). (C) S100A8/A9 complexes measured in murine whole blood 4 hours after stimulation with 1x10⁵ or 1x10⁷ heat inactivated <i>S</i>. Typhimurium (ATCC) diluted in RPMI to a final volume of 200 μl (n = 4). Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation. * P<0.05, determined using Kruskal-Wallis testing.</p