Phosphocholine-Modified Lipooligosaccharides of Haemophilus influenzae Inhibit ATP-Induced IL-1β Release by Pulmonary Epithelial Cells

Abstract

Phosphocholine-modified bacterial cell wall components are virulence factors enabling immune evasion and permanent colonization of the mammalian host, by mechanisms that are poorly understood. Recently, we demonstrated that free phosphocholine (PC) and PC-modified lipooligosaccharides (PC-LOS) from Haemophilus influenzae, an opportunistic pathogen of the upper and lower airways, function as unconventional nicotinic agonists and efficiently inhibit the ATP-induced release of monocytic IL-1β. We hypothesize that H. influenzae PC-LOS exert similar effects on pulmonary epithelial cells and on the complex lung tissue. The human lung carcinoma-derived epithelial cell lines A549 and Calu-3 were primed with lipopolysaccharide from Escherichia coli followed by stimulation with ATP in the presence or absence of PC or PC-LOS or LOS devoid of PC. The involvement of nicotinic acetylcholine receptors was tested using specific antagonists. We demonstrate that PC and PC-LOS efficiently inhibit ATP-mediated IL-1β release by A549 and Calu-3 cells via nicotinic acetylcholine receptors containing subunits α7, α9, and/or α10. Primed precision-cut lung slices behaved similarly. We conclude that H. influenzae hijacked an endogenous anti-inflammatory cholinergic control mechanism of the lung to evade innate immune responses of the host. These findings may pave the way towards a host-centered antibiotic treatment of chronic airway infections with H. influenzae.

Keywords: A549; CHRNA10; CHRNA7; CHRNA9; Calu-3; immune evasion; inflammasome; lung; nicotinic acetylcholine receptor; phosphocholine-modification.

Figure 1

Nicotine (Nic) and phosphocholine (PC) inhibit the release of IL-1β by A549 cells. Human LPS-primed A549 cells were stimulated with 2′(3′)-O-(4-benzoylbenzoyl)adenosine-5′-triphosphate (BzATP, 100 µM) in the presence or absence of Nic (100 µM) (A) or PC (100 µM) (B) and the IL-1β released to the supernatant was measured 30 min later. The inhibitory effects of Nic and PC were sensitive to nicotinic antagonists mecamylamine (Mec; 100 µM), strychnine (Stry; 10 µM), α-bungarotoxin (α-Bun; 1 µM), [V11L, V16D]ArIB (500 nM), or RgIA4 (200 nM). Data are presented as individual data points, bars represent median, whiskers encompass the 25th to 75th percentile; n-numbers of independent experiments are indicated in the figure. Experimental groups were compared by Kruskal Wallis test followed by Mann Whitney rank sum test.

Figure 1

Nicotine (Nic) and phosphocholine (PC) inhibit the release of IL-1β by A549 cells. Human LPS-primed A549 cells were stimulated with 2′(3′)-O-(4-benzoylbenzoyl)adenosine-5′-triphosphate (BzATP, 100 µM) in the presence or absence of Nic (100 µM) (A) or PC (100 µM) (B) and the IL-1β released to the supernatant was measured 30 min later. The inhibitory effects of Nic and PC were sensitive to nicotinic antagonists mecamylamine (Mec; 100 µM), strychnine (Stry; 10 µM), α-bungarotoxin (α-Bun; 1 µM), [V11L, V16D]ArIB (500 nM), or RgIA4 (200 nM). Data are presented as individual data points, bars represent median, whiskers encompass the 25th to 75th percentile; n-numbers of independent experiments are indicated in the figure. Experimental groups were compared by Kruskal Wallis test followed by Mann Whitney rank sum test.

Figure 2

Nicotine (Nic) and phosphocholine (PC) inhibit the release of IL-1β by Calu-3 cells. Human LPS-primed Calu-3 cells were stimulated with BzATP (100 µM) in the presence or absence of Nic (100 µM) or PC (100 µM). [V11L, V16D]ArIB (500 nM) or RgIA4 (200 nM) was added together with BzATP and IL-1β released to the supernatant was measured after 30 min. In one of the experiments, IL-1β levels released in response to BzATP was low throughout. Data are presented as individual data points, bars represent median, whiskers encompass the 25th to 75th percentile, n = 4 per experiment. Experimental groups were compared by Kruskal Wallis test followed by Mann Whitney rank sum test.

Figure 3

Phosphocholine-modified lipooligosaccharides (PC-LOS) isolated from H. influenzae strains inhibit the release of IL-1β by A549 and Calu-3 cells. Human LPS-primed A549 (A,C) or Calu-3 (B) cells were stimulated with BzATP (100 µM) in the presence or absence of PC-LOS isolated from the H. influenzae strains RM118 (RMPC; 1 µg/mL) and NTHi 1233 (NTHiPC; 1 µg/mL) and IL-1β released to the supernatant was measured after 30 min. PC-free LOS from the corresponding lic1-mutant strains RM7004-lic1 (RM; 1 µg/mL) and NTHi1233-lic1 (NTHi; 1 µg/mL) lacking the PC-modification were included as a negative control. (C) The inhibitory effects of RMPC and NTHiPC were reversed by nicotinic antagonists mecamylamine (Mec; 100 µM)), strychnine (Stry; 10 µM), α-bungarotoxin (α-Bun; 1 µM), [V11L, V16D]ArIB (500 nM), or RgIA4 (200 nM). Data are presented as individual data points, bars represent median, whiskers encompass the 25th to 75th percentile, n = 4 per experiment. Experimental groups were compared by Kruskal Wallis test followed by Mann Whitney rank sum test.

Figure 4

Phosphocholine-modified lipooligosaccharides (PC-LOS) isolated from H. influenzae strains inhibit the release of IL-1β by wild-type mouse precision cut lung slices (PCLS, n = 6). (A) A fixed PCLS, lightly stained with Richardson’s stain depicts normal pulmonary morphology; (B,C) PCLS were primed with LPS (100 ng/mL), IFN-ϒ (20 ng/mL) and TNF-α (10 ng/mL) for 24 h followed by application of BzATP (150 µM) in the presence or absence of PC-LOS isolated from the H. influenzae strains RM118 (RMPC; 1 µg/mL) and NTHi 1233 (NTHiPC; 1 µg/mL); (B) Lactate dehydrogenase (LDH) was measured in the cell culture supernatant 30 min later and expressed as % of the total LDH content of the individual PCLS (n = 4). Due to a technical problem, LDH release was not measured in two out of six experiments; (C) IL-1β was measured in cell culture supernatant at the same time point as the LDH (n = 6). The IL-1β concentration in experiments where primed PCLS were stimulated with BzATP was set to 100% and all other values were calculated accordingly. (B,C) Data are presented as individual data points, bars represent median, whiskers encompass the 25th to 75th percentile. Experimental groups were compared by the Wilcoxon signed-rank test.