A TLR4 agonist liposome formulation effectively stimulates innate immunity and enhances protection from bacterial infection

Abstract

Stimulation of innate immunity can protect against infectious insult and could be used in combination with other therapies. Since antibiotic resistance is an increasing concern, strategies to reduce the dose or eliminate the need for these drugs are warranted. Lipo-CRX is a formulation in which the TLR4 agonist CRX-527 is incorporated into lipid membranes in liposomes. Lipo-CRX is less inflammatory than either CRX-527 or LPS, but retains unique capacity to enhance host defense responses. We compared lipo-CRX to other agonists in vitro using mammalian cells and in vivo in mice, and assessed indicators of innate immune responses and protection from bacterial infection. Lipo-CRX is similar to E. coli LPS in its capacity to activate bovine γδ T cells and to recruit neutrophils into mouse lungs, but with less reactivity in the LAL assay. However, lipo-CRX uniquely induced the production of systemic innate immune cytokines. In the mouse model of brucellosis, delivery of lipo-CRX to the lungs reduced the dissemination of B. abortus. While lipo-CRX or the antibiotic ampicillin alone did not alter B. abortus burdens in the lung, the combination had a synergistic beneficial effect. Our data suggest that stimulating the innate immune system with lipo-CRX, either alone or when combined with antibiotics, can enhance bacterial clearance in the mouse model of brucellosis.

Keywords: Adjuvant; Brucella; antibiotics; brucellosis; innate immunity.

Figure 1.

Lipo-CRX activates bovine γδ T cells in mixed cultures in vitro, despite lack of LAL reactivity. a. Cells were treated with agonists as shown at varying concentrations. After 24 h, IL-2R expression was measured by flow cytometry on bovine γδ T cells. This assay was performed in single wells precluding statistical analyses. This procedure was repeated on cells from 3 (1 ng/ml) or 4 (100 ng/ml) different bovine calves at two concentrations of agonist. b. 1 ng/ml and c. 100 ng/ml. Standard error bars are shown. d. To normalize the data between experiments shown in Figure 1C, we calculated % of maximum, with the CRX value being the maximum value, for control and lipo-CRX treatment for each calf experiment. Data were pooled, means and SEM calculated and significance tested by unpaired Student's t-test. e. LPS, CRX-527, lipo-CRX and vehicle only control (liposomes without CRX-527) were compared in an LAL assay in duplicate showing development of Endotoxin Units (EU) over time and EU values as calculated by comparison to a standard curve are shown for each. LPS was strongly reactive, CRX-527 less so, and lipo-CRX had no reactivity and appeared similar to vehicle only (ND-not detected).

Figure 2.

Lipo-CRX treatment contributes to protection of THP-1 macrophages from in vitro infection with two different intracellular bacterial pathogens. a. THP-1 macrophages were pretreated with agonists then infected with S. Typhimurium. Intracellular bacteria were measured in triplicate wells for each agonist. b. A similar titration experiment was performed in THP-1 macrophages using the S19 vaccine strain of B. abortus. These experiments were each repeated 3 times and representative data are shown. For A,*p < 0.05 as measured by one-way ANOVA. For B, *p < 0.05 between LPS positive and medium negative controls as measured by Student's t-test.

Figure 3.

Lipo-CRX delivered i.t. induces rapid immune stimulation in the lung. a. A titration of Lipo-CRX was applied to mouse lungs and compared to vehicle (delivered at the equivalent volume as the 10μg lipo-CRX dose). After 24 h, neutrophil recruitment to the lungs was measured by flow cytometry. b. A 1μg dose of each agonist was delivered into lungs of mice and neutrophils in lungs were measured by flow cytometry 24 h later. This figure displays combined data from three similar experiments. c-f. Cytokine levels were measured in BAL supernatant fluids and serum collected 24 h after treatment, using ELISA assays in two repeat experiments. *p < 0.1, **p < 0.01, ****p < 0.0001, as calculated by one-way ANOVA with Bonferroni's Multiple Comparison Test.

Figure 4.

Lipo-CRX reduces bacterial burden in virulent B. abortus-infected mice. Mice were treated either i.t. or i.p. with 1μg lipo-CRX, MPL (C-F only), or the same volume of vehicle only on days −1, 2 and 7 post- i.t. or i.p. infection with 10⁵ CFU B. abortus, or with lipo-CRX just on day −1. Fourteen days after infection mice were euthanized and bacteria in lungs and spleens were enumerated. a, b. Agonists were delivered intraperitoneally and mice were infected with B. abortus in the lung. NA-not assessed. c, d. Mice were both treated and infected by the intraperitoneal route. e, f. Mice were both treated and infected by the intratracheal route. Figures 4E and F are combined data from two different experiments, while A-D were single experiments with n = 5 mice per group. *p < 0.05 as calculated by Student's t test.

Figure 5.

Innate agonists synergize with a suboptimal dose of antibiotics to reduce B. abortus burdens in mouse spleens and lungs. a, b. Ampicillin was delivered to mice in the drinking water at the indicated doses starting at day 6 post infection with 10⁵ CFU B. abortus. c, d. A dose of 100 mg/kg of ampicillin was delivered starting at day 6 post infection combined with lipo-CRX, 1μg i.t. on the day before infection (−1 dpi) and compared to vehicle, lipo-CRX or ampicillin alone. e, f. A combination treatment was also delivered i.t. at therapeutic intervals, 6 and 10 days after infection combined with a lower dose of ampicillin (50 mg/kg) and compared to vehicle, lipo-CRX or ampicillin alone. In each experiment, mice were euthanized 14 days post infection and bacterial burdens in spleens and lungs were assessed. Figures 5E and F are combined data from two different experiments, while A-D were single experiments with n = 5 mice per group. *p < 0.0 5, **p < 0.01 as calculated by the Mann-Whitney test.