Nontypeable Haemophilus influenzae induces sustained lung oxidative stress and protease expression

Abstract

Nontypeable Haemophilus influenzae (NTHi) is a prevalent bacterium found in a variety of chronic respiratory diseases. The role of this bacterium in the pathogenesis of lung inflammation is not well defined. In this study we examined the effect of NTHi on two important lung inflammatory processes 1), oxidative stress and 2), protease expression. Bronchoalveolar macrophages were obtained from 121 human subjects, blood neutrophils from 15 subjects, and human-lung fibroblast and epithelial cell lines from 16 subjects. Cells were stimulated with NTHi to measure the effect on reactive oxygen species (ROS) production and extracellular trap formation. We also measured the production of the oxidant, 3-nitrotyrosine (3-NT) in the lungs of mice infected with this bacterium. NTHi induced widespread production of 3-NT in mouse lungs. This bacterium induced significantly increased ROS production in human fibroblasts, epithelial cells, macrophages and neutrophils; with the highest levels in the phagocytic cells. In human macrophages NTHi caused a sustained, extracellular production of ROS that increased over time. The production of ROS was associated with the formation of macrophage extracellular trap-like structures which co-expressed the protease metalloproteinase-12. The formation of the macrophage extracellular trap-like structures was markedly inhibited by the addition of DNase. In this study we have demonstrated that NTHi induces lung oxidative stress with macrophage extracellular trap formation and associated protease expression. DNase inhibited the formation of extracellular traps.

Fig 1. Nontypeable Haemophilus influenzae generates the production of oxidative stress mediators in lung tissue.

Panel A shows immunoreactivity for 3-nitrotyrosine (a toxic oxidative stress product) in lung tissue from uninfected (control) and NTHi-infected mice and Panel B shows intensity of 3-nitrotyrosine immunoreactivity which is significantly higher in NTHi group (control 1141 (1084–1227) NTHi 2538 (1877–2497)), (n = 6, p = 0·002) (A = airway). Panel C shows the ROS production in human cells by; lung fibroblasts (n = 8), lung epithelial cells (n = 8), lung macrophages (n = 44) and blood neutrophils (n = 10) as measured by flow cytometry of DHR cleavage-induced fluorescence. There is a progressive increase in ROS fluorescence between the cell types (fibroblasts 21 (13–31), epithelial cells 69 (26–76), macrophages 107 (71–201) and neutrophils 134 (70–242)) with the highest levels in the phagocytes (P < 0·001, ANOVA); with significant between-group differences between 1), the fibroblasts and i) epithelial cells ii), macrophages and iii), neutrophils (all p<0·05) and 2), the epithelial cells and i), macrophages and ii), neutrophils (all p<0·05).

Fig 2. Response by human bronchoalveolar macrophages to nontypeable Haemophilus influenzae.

Panel A shows adherent BAL macrophages with staining for NTHi and production of ROS fluorescence in macrophages infected with NTHi by confocal microscopy. Panel B shows the ROS production in control (uninfected) macrophages and NTHi-infected macrophages over a 17 hour time-period (n = 15 subjects for each time-point) measured by confocal microscopy. At each one-hour time-point ROS production is significantly higher in the NTHi group (p<0·01). Panel C shows the increase over 17 hours in ROS production (1 hour 13012±2214, 17 hours 15175±2247)), (n = 15, p = 0·009). Panel D shows upregulation of ROS as measured by the effect of three different strains of NTHi using chemiluminescence (control 5937 (1839–10855), NTHi-1 8193 (3569–15538), NTHi-2 8953 (5105–17111), NTHi-3 10093 (4136–20848)), (RLUs = reactive light units/second). The chemiluminescence predominantly measures extracellular ROS production. Panels E and F demonstrates an examples of the presence of high and low-producing ROS fluorescence populations in control (unstimulated) and NTHi-stimulated macrophages from a patient (using flow cytometry). The high-producing ROS populations have significantly increased surface expression of the M1 marker HLA-DR (p<0·001): Panel G shows the unstimulated (baseline) macrophage population (low peak 6 (1–45), high peak 98 (94–99)), (n = 17), whilst Panel H shows the NTHi-stimulated macrophage population (low peak 11 (7–40), high peak 95 (80–100)), (n = 15).

Fig 3. Production of phagocyte extracellular traps and proteases in response to nontypeable Haemophilus influenzae.

Panel A shows the % of neutrophils expressing neutrophil extracellular traps (NETs), (control 7 ±2, NTHi 40±6)), (n = 7, p = 0·002)) and Panel B shows the % of BAL macrophages expressing macrophage extracellular traps-like structures (METs), (control 0, NTHi 19±5), (n = 8, p = 0·008); after stimulation with NTHi compared to control. Panel C shows neutrophils producing NETs with co-expression of neutrophil elastase (arrow). Panel D shows macrophages producing a MET with co-expression of macrophage metalloproteinase-12 (arrow). Panel E shows that neutrophils (40±6, n = 7) have a higher expression of extracellular traps compared to macrophages (19±5, n = 8) (p = 0·013). Panel F; NTHi increases surface expression of MMP12 by BAL macrophages as measured by flow cytometry (control 804±78, NTHi 1075±142)), (n = 8, p = 0·005).

Fig 4. Production of extracellular traps is associated with ROS production and is inhibited by DNase.

Panels A shows that neutrophils expressing NETs have a four-fold increase in ROS fluorescence compared to NET negative (-ve) cells (NET-ve 16 706 (5879–42 332), NET+ve 64 146 (44 738–122921)), (n = 7, p = 0·008). Panel B shows that macrophages expressing MET-like structures have a two-fold increase in ROS fluorescence compared to MET negative (-ve) cells (MET-ve 45 644 (6257–118907), MET+ve 93 636 (28 113–392 973)), (n = 8, p = 0·023). Panels C and D demonstrate inhibition of NET (NTHi 44 (24–53), NTHi & DNase 3 (0–10)), (n = 6, p = 0·004) and MET-like structure (NTHi 19±5, NTHi & DNase 0.1±0.1)), (n = 8, p = 0·008) formation by the addition of DNase.