Mucosal pre-exposure to Th17-inducing adjuvants exacerbates pathology after influenza infection

Abstract

Mucosal vaccines are thought to confer superior protection against mucosal infectious diseases. In addition, mucosal routes of vaccine delivery preferentially induce the generation of T helper 17 (Th17) cells, which produce the cytokine IL-17. Th17 cells are critical in mediating vaccine-induced immunity against several mucosal infectious diseases. However, IL-17 is also a potent proinflammatory cytokine, and we recently showed that IL-17 mediates immunopathology and lung injury after influenza infection in mice. In the present study, we tested the hypothesis that mucosal pre-exposure to Th17-inducing adjuvants can promote disease exacerbation upon subsequent infection with influenza virus. Mice mucosally pre-exposed to Th17-inducing adjuvants, such as type II heat-labile enterotoxin or cholera toxin, resulted in increased morbidity and exacerbated lung inflammation upon subsequent infection with influenza virus. Furthermore, the increased morbidity was accompanied by increased expression of inflammatory chemokines and increased accumulation of neutrophils. Importantly, blockade of the IL-17 pathway in mice pre-exposed to Th17-inducing adjuvants resulted in attenuation of the inflammatory phenotype seen in influenza-infected mice. Our findings indicate that, before mucosal Th17-inducing adjuvants can be used in vaccine strategies, the short- and long-term detrimental effects of such adjuvants on disease exacerbation and lung injury in response to infections, such as influenza, should be carefully studied.

Figure 1

Mucosal pre-exposure to LT-IIb adjuvant promotes increased morbidity and mortality after H1N1 influenza A/PR/8/34 infection. A: IL-17 Thy1.1 reporter mice were mucosally exposed to ESAT-6_1-20 peptide in combination with 1 μg LT-IIb adjuvant. On day 6 after exposure, the numbers of Thy1.1⁺ IL-17-producing αβ⁺ T cells and γδ⁺ T cells were determined by flow cytometry. B and C: C57BL/6 mice were mucosally exposed to 1 μg LT-IIb. On day 3 after exposure, mice were infected with 100 PFU of H1N1 influenza strain A/PR/8/34 in 50 μL of PBS. Mice were weighed every day, and percentage weight change was calculated (B) or survival was monitored over time (C). D: CXCL1, CXCL9, CXCL10, and CCL2 protein levels were determined from lung homogenates using a mouse Luminex assay. E: Relative lung viral loads were determined by RT-qPCR of viral M protein mRNA. Data are expressed as means ± SD. n = 4 or 5 mice. ^∗P ≤ 0.05, ^†P ≤ 0.005, and ^‡P ≤ 0.0005.

Figure 2

Mucosal pre-exposure to LT-IIb adjuvant promotes lung pathology after H1N1 influenza A/PR/8/34 infection. A: C57BL/6 mice were mucosally exposed to 1 μg LT-IIb or PBS and 3 days later were infected with 100 PFU of H1N1 influenza strain A/PR/8/34. B: Formalin-fixed, paraffin-embedded (FFPE) lung sections were stained with H&E, and the percentage of lung affected by inflammation was scored on a scale from 0% to 100%, based on the severity of inflammation. C and D: FFPE lung sections were assayed for CXCL9 (C) and CXCL10 (D) mRNA localization by in situ hybridization, using the corresponding murine chemokine mRNA probes. The arrows and arrowheads indicate expression of CXCL9 and CXCL10 mRNA, respectively. E: FFPE lung sections were analyzed by immunofluorescence using antibodies specific for Gr1 (red). The numbers of Gr1⁺ cells per ×20 field were counted. F: C57BL/6 mice were mucosally exposed to 1 μg LT-IIb or PBS and 3, 7, or 15 days later were infected with 100 PFU of H1N1 influenza strain A/PR/8/34. Weight loss was monitored in individual mice over time. G: FFPE lung sections were stained with H&E for assessment of inflammation. Data are expressed as means ± SD. n = 4 to 8 mice. ^∗P ≤ 0.05, ^†P ≤ 0.005, and ^‡P ≤ 0.0005. Original magnification: ×40 (B–D); ×100 (E, upper row); ×200 (E, lower row).

Figure 3

Mucosal pre-exposure to Th17-inducing adjuvants mediates increased pathology after novel pandemic H1N1 influenza infection. A: C57BL/6 mice were mucosally exposed to 1 μg LT-IIb, 1 μg CT, or PBS. On day 3 after exposure, mice were infected with 1 × 10⁶ PFU of novel H1N1 influenza strain A/California/7/2009. Percentage weight change was determined in individual mice over time. B–D: FFPE lung sections were stained with H&E (B) or were assayed for CXCL9 mRNA localization (arrows) by in situ hybridization using a murine CXCL9 mRNA probe (C), and Gr1-accumulating neutrophils were detected by immunohistochemistry (D). E: The numbers of Gr1⁺ cells per ×20 field were counted. F: The levels of myeloperoxidase activity (MPO) were determined in lung homogenates. Data are expressed as means ± SD. n = 4 or 5 mice. ^∗P ≤ 0.05, ^†P ≤ 0.005, and ^‡P ≤ 0.0005. Original magnification: ×40 (B and C); ×200 (D).

Figure 4

The IL-17 pathway mediates pathology in mice mucosally exposed to Th17-inducing adjuvants and then infected with influenza virus. A and B: C57BL/6 mice were mucosally exposed to 1 μg LT-IIb, 1 μg CT, or PBS. On day 3 after immunization, mice were infected with 5 × 10³ PFU of the highly pathogenic H5N1 influenza strain A/Vietnam/1203/2004. From day 0 of influenza infection, mice were treated with isotype control antibody or with 500 μg IL-17RA neutralizing antibody (αIL-17R). Percentage weight change was determined over time. Both LT- and CT-treated mice were compared with the same PBS-treated mice. C and D: FFPE lung sections were stained with H&E (C) or were analyzed by immunofluorescence using antibodies specific for Gr1 (red) (D). E: The numbers of Gr1⁺ cells per ×20 field were counted. F: C57BL/6 (B6) or IL-17RA knockout mice were mucosally exposed to 1 μg LT-IIb or PBS. On day 3 after immunization, mice were infected with 1 × 10⁶ PFU of novel H1N1 influenza strain A/California/7/2009, and weight loss was monitored in individual mice over time. G: FFPE lung sections were analyzed by immunofluorescence using antibodies specific for Gr1. The numbers of Gr1⁺ cells per ×20 field were counted. Data are expressed as means ± SD. n = 4 or 5 mice. ^∗P ≤ 0.05, ^†P ≤ 0.005, and ^‡P ≤ 0.0005. Original magnification, ×40 (C); ×100 (D, upper row); ×200 (D, lower row).