Innate immune protection against infectious diseases by pulmonary administration of a phospholipid-conjugated TLR7 ligand

Abstract

Pulmonary administration of Toll-like receptor (TLR) ligands protects hosts from inhaled pathogens. However, systemic side effects induced by TLR stimulation limit clinical development. Here, a small-molecule TLR7 ligand conjugated with phospholipid, 1V270 (also designated TMX201), was tested for innate immune activation and its ability to prevent pulmonary infection in mice. We hypothesized that phospholipid conjugation would increase internalization by immune cells and localize the compound in the lungs, thus avoiding side effects due to systemic cytokine release. Pulmonary 1V270 administration increased innate cytokines and chemokines in bronchial alveolar lavage fluids, but neither caused systemic induction of cytokines nor B cell proliferation in distant lymphoid organs. 1V270 activated pulmonary CD11c+ dendritic cells, which migrated to local lymph nodes. However, there was minimal cell infiltration into the pulmonary parenchyma. Prophylactic administration of 1V270 significantly protected mice from lethal infection with Bacillus anthracis, Venezuelan equine encephalitis virus and H1N1 influenza virus. The maximum tolerated dose of 1V270 by pulmonary administration was 75 times the effective therapeutic dose. Therefore, pulmonary 1V270 treatment can protect the host from different infectious agents by stimulating local innate immune responses while exhibiting an excellent safety profile.

Fig. 1

Pulmonary administration of 1V270 induces local innate immune activation. C57BL/6 mice (n = 5/group) were treated with 1 nmol 1V270 i.t. and BAL fluids were harvested 6, 24, 48, and 72 h after administration. IL-6 (a), MCP-1 (b), KC (c), and IP-10 (d) were measured in BAL fluids by Luminex beads assay. Data shown are means ± SEM. The data are representative of 2 independent experiments. * p < 0.05 vs. vehicle-treated mice by one-way ANOVA with Dunnett's post hoc testing. e C57BL/6 mice (n = 2-3/group) were intranasally inoculated with CFSE. Mice were then intrantracheally administered 1 nmol 1V270 or vehicle and mediastinal lymph nodes were collected 24 h after treatment. CFSE+CD11c+ cells were enumerated by flow cytometry. Data are means ± SD of 2 independent experiments. * p < 0.05 by Student's t test. f, g Mice (n = 4) were intranasally administered 1, 2, and 4 nmol 1V270 or vehicle control, and BAL and sera were collected 24 h after treatment. TNFα (f) and IL-6 (g) were determined in BAL fluids and sera by Luminex beads assay. * p < 0.05 vs. vehicle-treated mice by one-way ANOVA with Dunnett's post hoc testing.

Fig. 2

Pulmonary administration of 1V270 does not increase the B cell population. Mice were administered 1 nmol 1V270 i.t. Mediastinal (a), cervical (b), mesentric (c), and inguinal lymph nodes (d) were harvested 7 days after treatment. B cells were identified as a B220+ population by flow cytometry. NS = Not significant.

Fig. 3

Pulmonary 1V270 treatment induces minimal cell infiltration into lung parenchyma and has negligible adverse effects. a C57BL/6 mice (n = 5) were administered with 1 nmol 1V270 i.t. Lungs were harvested 6 and 24 h after treatment. Fixed sections were stained with HE (original magnification ×200). Scale bar = 200 μm. The sections are representative of 5 mice/group. b Mice were intratracheally treated with 1 nmol 1V270 or vehicle. c Wild-type or Tlr7−/− mice (n = 3-4/group) were administered 75 nmol 1V270 i.t. Body weights were monitored daily. * p < 0.05 vs. vehicle-treated mice by one-way ANOVA with Dunnett's post hoc testing. WT = Wild type.

Fig. 4

Pulmonary administration of 1V270 promotes neutrophil infiltration in BAL in a TLR7- and MyD88-dependent manner. C57BL/6 mice (n = 5/group) were administered 1 nmol 1V270 i.t. and BAL fluids were harvested 6, 24, 48, and 72 h later. Total cell numbers (a) were determined using a Guava cytometer. Numbers of neutrophils (b) and mononuclear cells (c) in BAL fluids were identified after Wright-Giemsa staining. d, e Mice (n = 5/group) were intratracheally treated with the indicated doses of 1V270 and BAL fluids were harvested 24 h after treatment. Numbers of total cells (d) and neutrophils (e) were determined as described above. f, g Wild-type (WT), Myd88−/− or Tlr7−/− mice (n = 5/group) were treated with 1 nmol 1V270 i.t. and BAL fluids were harvested 24 h after treatment. Numbers of total cells (f) and neutrophils (g) were determined as described above. Data shown are means ± SEM. The data are representative of 2 independent experiments. * p < 0.05 vs. vehicle-treated mice by one-way ANOVA with Dunnett's post hoc testing.

Fig. 5

Pulmonary treatment with 1V270 protects mice from bacterial and viral infections. a A/J mice (n = 16) were treated with 1V270 (1 nmol i.n.) or vehicle at 2-week intervals for three times and challenged with heat-activated live B. anthracis spores 4 weeks after the last immunization. Animal survival was monitored daily for up to 30 days. Kaplan-Meier survival curves and log-rank (Mantel-Cox) tests were performed to determine significance. The data were pooled from 2 independent experiments. b 1V270 (1 nmol/mouse i.n.) was administered to BALB/c mice (n = 20 in the placebo group; n = 10 in the drug group) once a day on days −3 and −1 relative to virus exposure. Mice were challenged subcutaneously with VEE virus (Trinidad donkey, NR-332) on day 0. c 1V270 (1 nmol/mouse) or placebo were administered intranasally to BALB/c mice (n = 20 in placebo group; n = 10 in other groups) once a day on days −3 and −1 prior to virus exposure. Mice were challenged intranasally with an influenza A/California/04/2009 (H1N1) virus on day 0. * p < 0.01, ** p < 0.0005, and *** p < 0.0001 vs. the vehicle-treated group by log-rank (Mantel-Cox) test.