Mucosal immunotherapy for protection from pneumonic infection with Francisella tularensis

Abstract

Previous studies have demonstrated that systemically administered immunotherapy can protect mice from systemic challenge with the bacterial pathogen Francisella tularensis. However, for protection from inhalational challenge with this bacterium, we wondered if mucosally administered immunotherapy might be more effective. Therefore, we administered cationic liposome-DNA complexes (CLDC), which are potent activators of innate immunity, intranasally (i.n.) and assessed the effectiveness of protection from lethal inhalational challenge with F. tularensis. We found that pretreatment by i.n. administration of CLDC 24h prior to bacterial challenge elicited nearly complete protection of BALB/c mice from lethal challenge with F. tularensis LVS strain. We also observed that mucosal CLDC immunotherapy provided a statistically significant increase in survival time in mice challenged with the highly virulent F. tularensis Schu4 strain. Protection was associated with a significant reduction in bacterial burden in the lungs, liver, and spleen. Mucosal administration of CLDC elicited significantly increased expression of IL-12, IFN-gamma, TNF-alpha, IFN-beta and IFN-alpha genes in the lung as detected by real-time quantitative PCR. In vitro treatment of F. tularensis infected macrophages with CLDC-elicited cytokines also significantly suppressed intracellular replication of F. tularensis in infected macrophages. In vivo, depletion of NK cells prior to administration of CLDC completely abolished the protective effects of CLDC immunotherapy. CLDC-elicited protection was also dependent on induction of IFN-gamma production in vivo. We conclude therefore that activation of local pulmonary innate immune responses is capable of eliciting significant protection from inhalational exposure to a virulent bacterial pathogen.

Fig. 1

Mucosally administered CLDC immunotherapy generates rapid protection against F. tularensis pneumonia. BALB/c mice (n = 5 per group) were administered CLDC by the i.n. route, as described in Section 2, or were sham-treated with diluent (5% dextrose water) alone. Twenty-four hours later, mice were subjected to i.n. challenge with 10⁵ CFU F. tularensis LVS, as described in Section 2. In (a) survival times for each group of mice were plotted using a Kaplan–Meier curve. In (b), organ bacterial burdens were determined on day 6 after bacterial challenge, as noted in Section 2. Bacterial burden in all 3 organs assessed were significantly lower in CLDC-treated mice than diluent treated mice as assessed by Student’s two-tailed t test (***p < 0.001). Group means are plotted (±SD). Each experiment was repeated once with similar results.

Fig. 2

Effects of route and timing of CLDC delivery on protection. In (a) mice (n = 5 per group) were treated with established immunostimulatory doses of CLDC via four different routes, as described in Section 2. Treated mice were then challenged 24 h later i.n. with a lethal dose of F. tularensis LVS. Survival was evaluated by Kaplan–Meier survival curves, followed by log-rank analysis using the Bonferroni corrected threshold for multiple comparisons. Only mice treated with CLDC by the i.n. route had a statistically significant increase in survival (p = 0.0015; Bonferroni corrected threshold for significance: p < 0.0125), compared to control mice. In (b) the effects of timing of CLDC mucosal administration on protection from F. tularensis LVS were evaluated. Groups of mice (n = 5) were administered CLDC by the i.n. route at the times noted (relative to the time of challenge). Mice were subjected to lethal i.n. challenge with F. tularensis LVS. CLDC treatment 24 h prior to challenge provided complete protection (p = 0.0015; Bonferroni corrected threshold for significance: p < 0.0125) while treatment at 24 h after challenge provided partial protection, with a significant increase in survival time compared to untreated controls (p = 0.0088; Bonferroni corrected threshold for significance: p < 0.0125). CLDC treatment 72 h prior to challenge and 0 h after challenge did not significantly increase survival time (p = 0.0807, p = 0.0160, respectively; Bonferroni corrected threshold for significance: p < 0.0125). Each experiment was repeated once with similar results.

Fig. 3

Induction of cytokine production in the lungs by mucosal administration of CLDC. Cytokine gene expression in lung tissues of untreated control mice and mice (n = 4 per group) treated with i.n. administration of CLDC 24 h prior to sacrifice was assessed by quantitative real-time PCR, as described in Section 2. The fold increase in lung mRNA expression in CLDC-treated versus control mice was plotted for IFN-γ (a), IFN-α4 (b), IFN-β (c), IL-12b (d) and TNF-α (e). Statistical significance was assessed using Student’s two-tailed t test (**p < 0.01, *p < 0.05). Results are expressed as group means ± SD and are representative of at least two repeated experiments.

Fig. 4

IFN-γ is required for mucosal CLDC protection. Wild-type and IFN-γ^−/− mice (n = 5 per group) were treated with i.n. CLDC or diluent 24 h prior to F. tularensis LVS i.n. challenge. (a) All CLDC-treated wild-type mice survived, whereas there were no survivors among the CLDC-treated IFN-γ^−/− mice. In (b), organ bacterial burdens were assessed at day 6 after challenge. In wild-type mice, CLDC treatment resulted in a significant decrease in bacterial burden in all organs compared to sham-treated mice (***p < 0.001, one-way ANOVA with Tukey’s multiple comparison test). However, in IFN-γ^−/− mice (n = 5 per group), CLDC treatment failed to produce a significant change in bacterial burdens. Bars represent group means ± SD. Each experiment was repeated once with similar results.

Fig. 5

NK cells are necessary for mucosal CLDC protection from F. tularensis challenge. NK cells were depleted from wild-type BALB/c mice (n = 5 per group) by i.p. injection of anti-asialo-GM1 antibody 24 h prior to CLDC treatment and again 5 days later, as described in Section 2. Another group of mice was treated with irrelevant rabbit IgG instead of asialo-GM1 antibody. Mice were treated with CLDC or sham-treated with diluent and 24 h later challenged i.n. with F. tularensis LVS. In (a), non-depleted mice and mice pre-treated with control rabbit antibody and then treated with CLDC all survived F. tularensis challenge. However, NK cell depleted mice treated with CLDC failed to survive the F. tularensis challenge. In (b), organ bacterial burdens were determined 6 days after F. tularensis challenge as described in Section 2. CLDC treatment alone resulted in a significant (***p < 0.001) reduction in bacterial burden in all organs evaluated. Control antibody treatment prior to CLDC treatment did not alter the effect of CLDC treatment on bacterial burden. In contrast, NK cell depletion prior to CLDC treatment significantly increased bacterial dissemination to the spleen and liver (***p < 0.001, one-way ANOVA with Tukey’s multiple means comparison test), compared to mice treated with CLDC alone. Bars represent group means ± SD. Each experiment was repeated once with similar results.

Fig. 6

Type I interferons provide partial protection from F. tularensis LVS challenge but are not required for CLDC-induced protection. Wild-type 129 mice (n = 5 per group) and IFN-α/β receptor^−/− mice on the 129 background were challenged with 10⁵ CFU F. tularensis LVS, as noted in Section 2. In (a), mice lacking IFN-α/βR expression were all euthanized on day 9 while wild-type 129 mice were completely resistant to death from F. tularensis challenge. Kaplan-Meier survival curve differences assessed by log-rank analysis demonstrated a significant increase in survival time (p = 0.0027) in wild-type mice compared to IFN-α/βR^−/− mice. Similar results were found in one additional experiment. In (b), i.n. administration of CLDC to IFN-α/βR^−/− mice 24 h prior to challenge resulted in a significant increase in survival time (p = 0.0358) compared to sham-treated IFN-α/βR^−/− mice. These results are representative of two independent experiments.

Fig. 7

CLDC-induced cytokines inhibit F. tularensis LVS growth in infected macrophages in vitro. Mouse alveolar macrophage cells (AMJ cell line) in triplicate wells were incubated 16 h with supernatant from spleens of CLDC or sham-treated mice, as described in Section 2. The cells were then infected with F. tularensis LVS and the effects of pre-treatment on intracellular replication of F. tularensis in infected macrophages 48 h later were assessed. Pre-treatment of cells with CLDC supernatant resulted in a nearly two log decrease in intracellular growth of F. tularensis in infected macrophages. Similar results were obtained in three additional experiments. The effects of neutralizing IFN-γ or TNF-α activity in CLDC supernatant were assessed in the same in vitro infection assay. To neutralize IFN-γ and TNF-α activity, supernatants were pre-incubated for 30 min with specific neutralizing antibodies. Control irrelevant antibodies were also included for specificity. Addition of anti-IFN-γ antibody nearly eliminated the F. tularensis inhibitory activity present in CLDC supernatant, whereas addition of anti-TNF-α antibody did not result in a significant change. Statistical significance was assessed by one-way ANOVA with Tukey’s multiple means comparison test (***p < 0.001, **p < 0.01). Bars represent group means ± SD. These results are representative of two independent experiments with similar results.

Fig. 8

Mucosal CLDC provides protection against virulent F. tularensis Schu4 challenge. Mice (BALB/c, n = 5 per group) were pre-treated with i.n. CLDC (or sham treated with diluent) and then challenged i.n. 24 h later with 40 CFU F. tularensis Schu4. In (a), survival was assessed using Kaplan–Meier survival curves, followed by log-rank analysis. Mucosal administration of CLDC to mice resulted in a significantly greater survival time than in sham-treated mice (p = 0.0027). In (b), body weight was monitored twice daily in CLDC and sham-treated mice infected with F. tularensis Schu4 and the mean percent of starting weight (±SD) for each mouse for each day was plotted. The body weight data was analyzed by two-way ANOVA with repeated measures and Bonferroni post-tests. The percentage change in body weights was calculated for the time points until the first mice in each treatment group were euthanized (day 4.5 for diluent group, day 5.5 for CLDC group). Diluent treated mice had significantly greater weight loss on days 3.5 through 4.5 (***p < 0.001, *p < 0.05) compared with CLDC-treated mice. In (c), organ bacterial burdens were determined at day 3 post-challenge and compared using Student’s two-tailed t test. Bars represent group means ± SD. CLDC-treated mice had significantly lower bacterial burden than diluent treated mice for all organs examined (***p < 0.001, **p < 0.01, *p < 0.05). Each experiment was repeated once with similar results.