Targeting the "Rising DAMP" during a Francisella tularensis Infection

Abstract

Antibiotic efficacy is greatly enhanced the earlier it is administered following infection with a bacterial pathogen. However, in a clinical setting antibiotic treatment usually commences following the onset of symptoms, which in some cases (e.g., biothreat agents) may be too late. In a BALB/c murine intranasal model of infection for Francisella tularensis SCHU S4 infection, we demonstrate during a time course experiment that proinflammatory cytokines and the damage-associated molecular pattern HMGB1 were not significantly elevated above naive levels in tissue or sera until 72 h postinfection. HMGB1 was identified as a potential therapeutic target that could extend the window of opportunity for the treatment of tularemia with antibiotics. Antibodies to HMGB1 were administered in conjunction with a delayed/suboptimal levofloxacin treatment of F. tularensis We found in the intranasal model of infection that treatment with anti-HMGB1 antibody, compared to an isotype IgY control antibody, conferred a significant survival benefit and decreased bacterial loads in the spleen and liver but not the lung (primary loci of infection) 4 days into infection. We also observed an increase in the production of gamma interferon in all tested organs. These data demonstrate that treatment with anti-HMGB1 antibody is beneficial in enhancing the effectiveness of current antibiotics in treating tularemia. Strategies of this type, involving antibiotics in combination with immunomodulatory drugs, are likely to be essential for the development of a postexposure therapeutic for intracellular pathogens.

Fig 1

Bacterial colonization in organs following intranasal infection with F. tularensis strain SCHU S4. Bacterial numbers were enumerated on supplemented BCGA plates for three organs: lung (A), liver (B), and spleen (C). Each data point represents a single mouse, and lines indicate the median. Data shown are for two independent experiments. The calculated doses for experiments 1 and 2 were 99 and 89 CFU, respectively: 7 mice (experiment 1) and 5 mice (experiment 2) were culled at days 1 to 4 p.i., and 4 mice (experiment 1) and 1 mouse (experiment 2) were culled at day 5 p.i. (remaining survivors). An additional 5 mice were used to establish a baseline/naive concentration (day 0).

Fig 2

Organ cytokine profile following intranasal infection with F. tularensis strain SCHU S4. Levels of IL-6, MCP-1, IFN-γ, TNF-α, IL-10, and IL-12p70 were measured via Cytometric Bead Array (CBA) plate counts. Cytokine concentrations are displayed as pg/g per organ (lung, liver, and spleen). Each data point represents a single mouse, and the lines indicate the medians. Data shown are for two independent experiments. The calculated doses for experiments 1 and 2 were 99 and 89 CFU, respectively. Seven mice (experiment 1) and 5 mice (experiment 2) were culled at days 1 to 4 p.i. Four mice (experiment 1) and 1 mouse (experiment 2) were culled at day 5 p.i. (remaining survivors). An additional 5 mice were used to establish a baseline/naive concentration (day 0).

Fig 3

Serum HMGB1 release in mice following intranasal infection with F. tularensis strain SCHU S4. Levels of serum HMGB1 (ng/ml) were assessed via ELISA. Each data point represents a single mouse, and the lines indicate the medians. Data shown are for two independent experiments. The calculated doses for experiments 1 and 2 were 99 and 89 CFU, respectively. Seven mice (experiment 1) and 5 mice (experiment 2) were culled at days 1 to 4 p.i.; 4 mice (experiment 1) and 1 mouse (experiment 2) were culled at day 5 p.i. (remaining survivors). An additional 13 mice were used to establish a baseline/naive concentration (day 0).

Fig 4

Survival of mice following anti-HMGB1 Ab treatment in combination with levofloxacin. Male BALB/c mice were challenged with 87 CFU (experiment 1), 77 (experiment 2), or 103 CFU (experiment 3) F. tularensis SCHU S4 via the intranasal route. Groups were treated with either 600 μg of anti-HMGB1 Ab or 600 μg of an isotype control (IgY) at 48 and 72 h p.i. Levofloxacin (40 mg/kg) was administered via the intraperitoneal route at 96 h p.i. and continued once daily for 7 days for both groups. Each data point represents the percentage of survivors in each group at the corresponding time point. In the first experiment, 7 animals received anti-HMGB1 Ab and 17 animals received isotype control IgY; in the second and third experiments, 10 animals received anti-HMGB1 Ab and 10 animals received isotype control IgY. Data were analyzed by stratified log rank test.

Fig 5

Bacterial colonization in anti-HMGB1- and IgY Ab-treated mice. Male BALB/c mice were challenged with 87 CFU (experiment 1), 77 CFU (experiment 2), and 103 CFU (experiment 3) F. tularensis SCHU S4 via the intranasal route. Mice were culled at 96 h p.i. and bacteria enumerated on supplemented BCGA for the lung, liver, and spleen. Groups were treated with either 600 μg of anti-HMGB1 Ab or 600 μg of an isotype control Ab (IgY) at 48 and 72 h p.i. Each data point represents a mouse, and the lines represent the medians. In the first experiment, 4 animals received anti-HMGB1 Ab and 7 animals received isotype control IgY Ab; in the second and third experiments, 5 animals received anti-HMGB1 Ab and 5 animals received isotype control IgY Ab. Data were analyzed by univariate linear models of the log₁₀ transformed data.

Fig 6

Cytokine production in anti-HMGB1- and IgY Ab-treated mice. Male BALB/c mice were challenged with 87 CFU (experiment 1), 77 CFU (experiment 2), and 103 CFU (experiment 3) F. tularensis SCHU S4 via the intranasal route. Mice were culled at 96 h p.i. and IFN-γ and MCP-1 cytokines assayed for the lung, liver, and spleen. Groups were treated with either 600 μg of anti-HMGB1 Ab or 600 μg of an isotype control (IgY) Ab at 48 and 72 h p.i. Each data point represents a mouse, and the lines represent the medians. In the first experiment, 4 animals received anti-HMGB1 Ab and 7 animals received isotype control IgY Ab; in the second and third experiments, 5 animals received anti-HMGB1 Ab and 5 animals received isotype control IgY Ab. Data were analyzed by the univariate linear model of the log₁₀ transformed data. Significance markers have been included to show differences associated with the different treatments. Levels of IL-6, IL-10, TNF-α, and IL-12p70 were also determined but are not shown.