The effects of airway microbiome on corticosteroid responsiveness in asthma

Abstract

Rationale: The role of airway microbiome in corticosteroid response in asthma is unknown.

Objectives: To examine airway microbiome composition in patients with corticosteroid-resistant (CR) asthma and compare it with patients with corticosteroid-sensitive (CS) asthma and normal control subjects and explore whether bacteria in the airways of subjects with asthma may direct alterations in cellular responses to corticosteroids.

Methods: 16S rRNA gene sequencing was performed on bronchoalveolar lavage (BAL) samples of 39 subjects with asthma and 12 healthy control subjects. In subjects with asthma, corticosteroid responsiveness was characterized, BAL macrophages were stimulated with pathogenic versus commensal microorganisms, and analyzed by real-time polymerase chain reaction for the expression of corticosteroid-regulated genes and cellular p38 mitogen-activated protein kinase (MAPK) activation.

Measurements and main results: Of the 39 subjects with asthma, 29 were CR and 10 were CS. BAL microbiome from subjects with CR and CS asthma did not differ in richness, evenness, diversity, and community composition at the phylum level, but did differ at the genus level, with distinct genus expansions in 14 subjects with CR asthma. Preincubation of asthmatic airway macrophages with Haemophilus parainfluenzae, a uniquely expanded potential pathogen found only in CR asthma airways, resulted in p38 MAPK activation, increased IL-8 (P < 0.01), mitogen-activated kinase phosphatase 1 mRNA (P < 0.01) expression, and inhibition of corticosteroid responses (P < 0.05). This was not observed after exposure to commensal bacterium Prevotella melaninogenica. Inhibition of transforming growth factor-β-associated kinase-1 (TAK1), upstream activator of MAPK, but not p38 MAPK restored cellular sensitivity to corticosteroids.

Conclusions: A subset of subjects with CR asthma demonstrates airway expansion of specific gram-negative bacteria, which trigger TAK1/MAPK activation and induce corticosteroid resistance. TAK1 inhibition restored cellular sensitivity to corticosteroids.

Figure 1.

A schematic diagram of the analysis approaches for the bronchoalveolar lavage (BAL) 16S rRNA sequencing data to characterize airway microbiome of corticosteroid-resistant (CR) and corticosteroid-sensitive (CS) subjects with asthma.

Figure 2.

Bacterial load and composition of the airway microbiome based on 16S rRNA sequencing of the bacterial DNA isolated from bronchoalveolar lavage samples from normal control subjects, and corticosteroid-resistant (CR) and corticosteroid-sensitive (CS) subjects with asthma. (A) Bacterial load based on 16S RNA copy number in bronchoalveolar lavage samples from normal control subjects, and patients with CR and CS asthma. (B) The taxonomic composition of the airway microbiome in normal control subjects, and subjects with CR and CS asthma. A mean % of sequences for the major bacterial taxonomic groups is presented. The following phyla are shown: Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Fusobacteria. The remaining microorganisms that do not belong to these phyla are presented as “Other.” *P < 0.05 as compared to normal control.

Figure 3.

The taxonomic composition of the airway microbiome in corticosteroid-resistant (CR) and corticosteroid-sensitive (CS) subjects with asthma with (bact. exp.) and without (no bact. exp.) bacterial expansions. Microbial genera were considered as expanded in asthmatic airway microbiome if they represented more than 5% of the total 16S rRNA sequences and the % of sequences was increased at least twofold over control subjects for the genera present both in the airways of subjects with asthma and normal control subjects; for the genera found only in subjects with asthma they were considered as expanded if they represented more than 5% of the total 16S rRNA sequences. Twenty-four subjects with CR asthma and nine subjects with CS asthma were found to have bacterial expansions as compared with normal control subjects; five CR subjects with asthma and one CS subject with asthma did not have bacterial expansions. A mean % sequences of major bacterial phyla per group is shown. *P < 0.05, ***P < 0.001 as compared to normal control.

Figure 4.

Effects of bacteria from the airways of subjects with asthma on bronchoalveolar lavage (BAL) macrophages activation and response to corticosteroids in vitro. Incubation of asthmatic BAL macrophages with Haemophilus parainfluenzae (H. para) results in p38 mitogen-activated protein kinase activation in the cells as detected by Western blot (A), up-regulation of IL-8 (B) and mitogen-activated kinase phosphatase 1 (MKP-1) mRNA production (C), and reduced responsiveness to corticosteroids in vitro (D) as shown by real-time polymerase chain reaction. Cells cultured with airway commensal organism Prevotella melaninogenica (P. melan) do not activate p38, do not up-regulate IL-8 mRNA and MKP-1 mRNA expression, and remain sensitive to corticosteroid treatment. (B–D) For IL-8 mRNA and MKP-1 mRNA production the cells were cultured overnight in X-Vivo 15 medium, incubated with bacteria for 15 minutes followed by 3 hours of treatment with 10⁻⁶M dexamethasone (DEX) or medium, and analyzed by real-time polymerase chain reaction. 0.25 × 10⁶ cells was used per condition (bacterium to cell ratio 0.1:1 and 1:1). The amount of bacteria shown was calculated per 1 × 10⁶ cells/ml per condition. The responses of BAL macrophages from five subjects with asthma were examined. *P < 0.05, **P < 0.01 as compared with medium-treated cells.

Figure 5.

Influence of Toll-like receptor pathway inhibitors on cellular response to corticosteroids in the presence of bacteria. Pretreatment of asthmatic peripheral blood monocytes with transforming growth factor-β–associated kinase-1 (TAK1) but not p38 mitogen-activated protein kinase (MAPK) or p38 MAPK/ERK/JNK inhibitors results in significant inhibition of mitogen-activated kinase phosphatase 1 (MKP-1) mRNA (A) and IL-8 mRNA (B) induction by Haemophilus parainfluenzae (H. para) and restoration of cellular sensitivity to corticosteroids in vitro (C) as shown by real-time polymerase chain reaction. (D) A total of 2 μM TAK1 and 10 μM p38 inhibitors fully inhibited activation of downstream signaling targets in response H. para. Phosphorylation of ERK and hsp27 as a downstream read out targets for TAK1 and p38 MAPK activation, respectively, in response to 15 minutes of treatment of monocytes from subjects with asthma with H. para (bacterium to cell ratio 1:1) with and without corresponding inhibitor is shown by Western blot. For IL-8 mRNA and MKP-1 mRNA production the cells were cultured overnight in X-Vivo 15 medium, incubated with inhibitors for 1 hour, stimulated with bacteria for 15 minutes followed by 3 hours of treatment with 10⁻⁶ M dexamethasone (DEX) or medium, and analyzed by real-time polymerase chain reaction. Bacteria were added to 0.5 × 10⁶ cells per condition (bacterium to cell ratio 1:1). The responses of monocytes from four subjects with asthma were examined.

Figure 6.

Proposed schematic diagram of monocyte-macrophage activation and cellular response to corticosteroids in the presence of Haemophilus parainfluenzae and Prevotella melaninogenica. H. parainfluenzae short length acyl chains lipid A LPS interacts with Toll-like receptor (TLR) 4 and activates transforming growth factor-β–associated kinase-1 (TAK1) by MyD88 pathway, resulting in p38 mitogen-activated protein kinase phosphorylation and nuclear factor-κB (NF-κB) activation, which activate transcription of the proinflammatory cytokines like IL-8 (21, 26, 27, 53). On the contrary, P. melaninogenica long acyl chains lipid A LPS is a poor agonist for TLR4 (54). On interaction cytoplasmic glucocorticoid receptor (GR) with dexamethasone receptor translocates to the cell nuclei and activates mitogen-activated kinase phosphatase 1 (MKP-1) mRNA production. MKP-1 dephosphorylates activated p38 mitogen-activated protein kinase. GR interacts with NF-κB and inhibits IL-8 transcription. Monocyte-macrophage activation by H. parainfluenzae results in reduced cellular responses to corticosteroids. TAK1 activation by H. parainfluenzae inhibits GR-mediated MKP-1 production and suppresses GR inhibition of NF-κB–induced IL-8 production. The cells remain corticosteroid sensitive in the presence of P. melaninogenica.