Activation of innate immune responses by Haemophilus influenzae lipooligosaccharide

Abstract

A Gram-negative pathogen Haemophilus influenzae has a truncated endotoxin known as lipooligosaccharide (LOS). Recent studies on H. influenzae LOS highlighted its structural and compositional implications for bacterial virulence; however, the role of LOS in the activation of innate and adaptive immunity is poorly understood. THP-1 monocytes were stimulated with either lipopolysaccharide (LPS) from Escherichia coli or LOS compounds derived from H. influenzae Eagan, Rd, and Rd lic1 lpsA strains. Cell surface expression of key antigen-presenting, costimulatory, and adhesion molecules, as well as gene expression of some cytokines and pattern recognition receptors, were studied. Eagan and Rd LOS had a lower capacity to induce the expression of ICAM-1, CD40, CD58, tumor necrosis factor alpha (TNF-α), and interleukin-1β (IL-1β) compared to LPS. In contrast, antigen-presenting (HLA-ABC or HLA-DR) and costimulatory (CD86) molecules and NOD2 were similarly upregulated in response to LOS and LPS. LOS from a mutant Rd strain (Rd lic1 lpsA) consistently induced higher expression of innate immune molecules than the wild-type LOS, suggesting the importance of phosphorylcholine and/or oligosaccharide extension in cellular responses to LOS. An LOS compound with a strong ability to upregulate antigen-presenting and costimulatory molecules combined with a low proinflammatory activity may be considered a vaccine candidate to immunize against H. influenzae.

FIG 1

Schematic representation of LOS from H. influenzae strains Eagan, Rd, and Rd lic1 lpsA as determined by MALDI. Represented in the LOS structure are 2-keto-3-deoxyoctulosonic acid (Kdo), heptose (Hep), glucose (Glc), galactose (Gal), N-acetylgalactosamine (GalNac), phosphate (P), phosphorylcholine (PCho), and phosphoethanolamine (PEtn).

FIG 2

Flow cytometry analysis of costimulatory and antigen-presenting molecule expression in response to LOS stimulation on THP-1 cells. The negative control was untreated medium alone, and the positive control was LPS stimulation at 1 μg/ml. THP-1 cells were stimulated with LOS compounds for 24 h at concentrations of 1, 5, 10, and 15 μg/ml, and cell surface expression of CD54 (A), CD40 (B), CD58 (C), CD86 (D) HLA-ABC (E), and HLA-DR (F) was measured. Data represent the mean fluorescence intensity (MFI) ± the standard error of the mean (SEM) (n = 3). Significant differences are indicated as follows: *, P < 0.05 compared to the unstimulated control; +, P < 0.05 between Rd lic1 lpsA and Rd wild-type compounds; ●, P < 0.05 between LPS and LOS compounds.

FIG 3

Relative gene expression of THP-1 cells in response to LOS stimulation. THP-1 cells were stimulated with LOS compounds for 4 h at concentrations of 0.1 and 1 μg/ml. RNA was extracted, and genetic expression was measured using real-time PCR and is presented as fold change relative to the unstimulated control. Expression of the genes coding for IL-1β (A), TNF-α (B), IL-10 (C), TLR4 (D), NOD1 (E), and NOD2 (F) was measured. Data represent means ± SEM (n = 3). Significant differences are indicated as follows: *, P < 0.05 compared to unstimulated control; +, P < 0.05 between Rd lic1 lpsA and Rd wild-type compounds; ●, P < 0.05 between LPS and LOS compounds.