Discussion
The novelty of our analysis is being able to pin down to the microbial
species level differences at the severity, inflammatory and molecular
phenotype level of severe asthma using sputum metagenomic sequencing of
a large cohort of patients. We focused on 3 pathogenic species, namelyHaemophilus influenzae, Moraxella catarrhalis andTropheryma whipplei because of their potential pathogenecity in
respiratory diseases. We found reduced bacterial α-diversity of
microbial species in the 2 severe asthma groups where there was higher
abundance of Haemophilus influenzae and Moraxella
catarrhalis in SAn compared to SAs/ex, MMA and HC, and ofHaemophilus influenzae and Tropheryma whipplei in SAs/ex
compared to MMA. In terms of inflammatory status, α-diversity was lowest
in the mixed granulocytic group followed by a slight reduction in the
neutrophilic group, with neutrophilic inflammation associated with
higher abundance of Haemophilus influenzae and Moraxella
catarrhalis , while eosinophilic inflammation was associated with high
abundance of Tropheryma whipplei . In terms of associated
molecular pathways, Haemophilus influenzae was most abundant in
TAC1 and TAC2, accompanied by an increase in Tropheryma whippleiin TAC1 and in Moraxella catarrhalis in TAC2. The abundance ofMoraxella catarrhalis in TAC1 and TAC3 was very low. The
increased abundance of Haemophilus influenzae in both TAC1, an
eosinophilic phenotype, and in TAC2, a neutrophilic phenotype, but not
in TAC3, a paucigranulocytic phenotype, is of interest. One possibility
is that this increased abundance may be due to the reduced phagocytosis
of Haemophilus influenzae by lung macrophages from patients with
severe asthma (27). Haemophilus influenzae can induce the release
of IL1α, IL1β, IL-6, IL-8, MCP-1 and TNFα from human tracheal epithelial
cells (28), through the toll-like receptor, TLR2, activation (29), that
could lead to neutrophil activation and inflammation.
In both SAn and SAs/ex, the species most positively correlated with
sputum neutrophils was Moraxella catarrhalis while in MMA,Haemophilus influenzae was inversely correlated. Moraxella
catarrhalis is a known airway pathogen that causes respiratory
infections linked to neutrophilic airway inflammation in severe or
poorly-controlled asthma (9, 12). Moraxella catarrhalis was also
most abundant in the TAC2 molecular phenotype characterized by
neutrophilic and inflammasome activation, supporting the possibility
that it may be associated with or be responsible for the induction of
neutrophilic inflammation in severe asthma. This link betweenMoraxella catarrhalis and neutrophilic inflammation is further
strengthened by the highest abundance of Moraxella catarrhalis in
TAC2 where it was positively correlated with sputum neutrophilia. This
potential activating role of Moraxella catarrhalis is supported
by its activation of TLR 2, 4 and 9 (30), induction of IL-6, IL-8 and
prostaglandin E2 release through NF-κB and activation in lung epithelial
cells (31, 32).
Although Tropheryma whipplei is known to cause Whipple’s disease
with gastrointestinal symptoms, it has also been implicated as a cause
of aspiration, ventilator-associated and community-acquired pneumonia
(33). Tropheryma whipplei in bronchoalveolar lavage fluid has
been isolated in asymptomatic immunosuppressed patients suffering from
Human Immunodeficiency virus, which was reduced by anti-retroviral
therapy (34). The increased abundance of Tropherymawhipplei in SAs/ex was positively associated with sputum
eosinophilia and also with pack years of smoking in the TAC1 and TAC2
phenotypes. A previous study has reported the presence ofTropheryma whipplei by PCR in sputum samples of severe asthma
patients who were mainly eosinophilic (12). We found thatTropheryma whipplei was most prominent in severe asthmatics who
were either current smokers or ex-smokers. These differences between NSA
and SAs/ex severe asthma groups in terms of the sputum metagenome is
demonstrated here for the first time. Previous studies using 16S
ribosomal RNA microarray comparing the lung microbiome of smokers with
non-smokers have reported either no differences (35, 36), or a higher
abundance of Veillonella and Megasphaera with a decrease
in Haemophilus (37). The pathogenic role of Tropheryma
whipplei, if any, in SAs/ex remains unclear.
Other bacterial species were also noted to be differentially-abundant,
being mainly reduced in the severe asthma groups of SAn and SAs/ex when
compared to MMA and/ or HC. These include species of Prevotella,
Veillonella, Neisseria and Rothia, of which we know little in
terms of their potential lung pathogenecity. SAn and SAS/ex were
different from MMA with a decreased abundance of Prevotella
intermedia and Rothia mucilaginosa consistent with the previous
report of decreased abundance of the genus Prevotella in asthma
compared to non-asthmatic individuals (7, 38). Most of
these species were negatively correlated with sputum neutrophilia. One
bacterial species, Haemophilus parainfluenzae, was reduced in
abundance in SAn and in TAC1 and TAC2, with negative correlation with
sputum neutrophilia in SAn and MMA, and in TAC1 and TAC3. Our data
indicate that Haemophilus parainfluenzae is decreased in current
smokers compared to ex-smokers; however, since this same species is
reduced also in the smoking/ex-smoking group on OCS compared to those
not on OCS, it may be related to OCS therapy. Haemophilus
parainfluenzae has been reported (39) to increase IL-8 expression with
activation of p38 MAPK and inhibit corticosteroid responses in alveolar
macrophages from corticosteroid-resistant asthma patients, thus may be
involved in induction of corticosteroid resistance.
Overall, the microbiome was stable when repeated at one year in severe
asthma with no shift in α or β-diversity. In an unbiased
microbiome-driven clustering to identify severe asthma phenotypes using
the same microbial data, we found 2 distinct robust phenotypes that
exhibited relative overtime stability (40). This stability does not
exclude the possibility of changes in the microbiome during acute
exacerbations of asthma as occurs in children where an increase in
gram-negative microbes have been detected in induced sputum (41). Out of
these 2 phenotypes (40), one had worse asthma outcomes, more sputum
neutrophilia and greater enrichment of the gammaproteobacteria,Haemophilus influenza e and Moraxella catarrhalis . Further
analysis of this phenotype has led to its association with
differentially-expressed genes, in particular TNFα and related
regulatory genes, the IL-1 family of interleukins, Toll-like receptors
and inflammasomes (42).
We highlight some technical issues in our analysis. First, the
sequencing technology of using paired-end 100 base-pairs at high
sampling depth provides less accurate taxonomy profiling compared to
reads of 150 base-pairs or longer at lower depth. However, we have used
MetaPhlAn2 because it provides a more sensible taxonomic composition for
respiratory microbiome experts compared to a protein-based method such
as Kaiju (43). Secondly, the use of a marker-based approach may lead to
exclusion of samples for downstream analysis due to their lack of marker
genes. This is likely due to sputum having high amounts of host DNA,
contributed by neutrophils and neutrophil extracellular traps resulting
in low level of microbial reads, with lack of marker genes for
estimating taxonomic composition, thus excluding some samples in the
downstream analysis. Finally, for differential analysis, DESeq2 was used
because it took into account the compositional nature of the metagenomic
data, with the addition of pseudocount to overcome lack of taxa shared
by all samples.
In conclusion, this sputum metagenomic study of severe asthma patients
during a stable state has revealed an important association of reduced
bacterial α-diversity at the species level to neutrophilic airway
inflammation and neutrophil and inflammasome activation whereHaemophilus influenzae and Moraxella catarrhalis were in
greater abundance. On the other hand, in eosinophilic inflammation, bothHaemophilus influenzae and Tropheryma whipplei were most
abundant with no Moraxella catarrhalis found. Tropheryma
whipplei was mostly linked to the smoking/ex-smoking severe asthma and
to eosinophilic inflammation. These bacterial species may play an
important role in influencing the severity and inflammatory phenotype of
severe asthma, particularly linked to neutrophilic inflammation, and
further studies are needed to confirm the importance of these bacteria.