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. 2024 Sep;14(9):e70007.
doi: 10.1002/ctm2.70007.

A severe asthma phenotype of excessive airway Haemophilus influenzae relative abundance associated with sputum neutrophilia

Ali Versi  1 , Adnan Azim  2 , Fransiskus Xaverius Ivan  3 , Mahmoud I Abdel-Aziz  4 , Stewart Bates  5 , John Riley  5 , Mohib Uddin  6 , Nazanin Zounemat Kermani  1 , Anke-H Maitland-Van Der Zee  4 , Sven-Eric Dahlen  7 , Ratko Djukanovic  2 , Sanjay H Chotirmall  3   8 , Peter Howarth  2 , Ian M Adcock  1 , Kian Fan Chung  1 ; U‐BIOPRED study group
Affiliations

A severe asthma phenotype of excessive airway Haemophilus influenzae relative abundance associated with sputum neutrophilia

Ali Versi et al. Clin Transl Med. 2024 Sep.

Abstract

Background: Severe asthma (SA) encompasses several clinical phenotypes with a heterogeneous airway microbiome. We determined the phenotypes associated with a low α-diversity microbiome.

Methods: Metagenomic sequencing was performed on sputum samples from SA participants. A threshold of 2 standard deviations below the mean of α-diversity of mild-moderate asthma and healthy control subjects was used to define those with an abnormal abundance threshold as relative dominant species (RDS).

Findings: Fifty-one out of 97 SA samples were classified as RDSs with Haemophilus influenzae RDS being most common (n = 16), followed by Actinobacillus unclassified (n = 10), Veillonella unclassified (n = 9), Haemophilus aegyptius (n = 9), Streptococcus pseudopneumoniae (n = 7), Propionibacterium acnes (n = 5), Moraxella catarrhalis (n = 5) and Tropheryma whipplei (n = 5). Haemophilus influenzae RDS had the highest duration of disease, more exacerbations in previous year and greatest number on daily oral corticosteroids. Hierarchical clustering of RDSs revealed a C2 cluster (n = 9) of highest relative abundance of exclusively Haemophilus influenzae RDSs with longer duration of disease and higher sputum neutrophil counts associated with enrichment pathways of MAPK, NF-κB, TNF, mTOR and necroptosis, compared to the only other cluster, C1, which consisted of 7 Haemophilus influenzae RDSs out of 42. Sputum transcriptomics of C2 cluster compared to C1 RDSs revealed higher expression of neutrophil extracellular trap pathway (NETosis), IL6-transignalling signature and neutrophil activation.

Conclusion: We describe a Haemophilus influenzae cluster of the highest relative abundance associated with neutrophilic inflammation and NETosis indicating a host response to the bacteria. This phenotype of severe asthma may respond to specific antibiotics.

Keywords: Haemophilus influenzae; Moraxella catarrhalis; Tropheryma whipplei; metagenome; neutrophils; severe asthma; α‐diversity.

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Conflict of interest statement

Mr Versi has nothing to declare. Dr Azim reports employment through AstraZeneca. Dr Chotirmall has received lecture fees from Chiesi Farmaceutici and AstraZeneca, serves on advisory boards for Boehringer‐Ingelheim, CSL Behring and Pneumagen Ltd. and is on Data and Safety Monitoring Boards (DSMB) for Inovio Pharmaceuticals all outside of the submitted work. Dr Maitland‐van der Zee has received grants from Health Holland and she is the PI of a P4O2 (Precision Medicine for more Oxygen) public private partnership sponsored by Health Holland involving many private partners that contribute in cash and/or in kind (Boehringer Ingelheim, Breathomix, Fluidda, Ortec Logiqcare, Philips, Quantib‐U, Smartfish, SODAQ, Thirona, TopMD and Novartis), received unrestricted research grants from GSK, Boehringer Ingelheim and Vertex, received consulting fees paid to her institution from Boehringer Ingelheim and AstraZeneca, and received honoraria for lectures paid to her institution from GlaxoSmithKline; outside the submitted work. Dr. Dahlén reports personal fees from AZ, Cayman Chemicals, GSK, Novartis, Regeneron, Sanofi, TEVA, outside the submitted work. Dr Chung has received honoraria for participating in Advisory Board meetings of Roche, Merck, Shionogi and Rickett‐Beckinson and has also been remunerated for speaking engagements for Novartis and AZ. Dr Riley worked for and had shares in GSK. Dr. Bates reports to be currently an employee of Johnson & Johnson and to have previously worked and holds stock in GSK. Dr Uddin is an employee and holds shares in AstraZeneca. Dr Djukanovic declares consulting fees from Synairgen, Sanofi and Galapagos, lecture fees from GSK, AZ and Airways Vista and he holds shares from Synairgen. Dr Howarth is an employee of GSK. Dr Montuschi, Dr Kermani, Dr Adcock, Dr Ivan and Dr Abdel‐Aziz have nothing to declare.

Figures

FIGURE 1
FIGURE 1
Flow chart showing the determination of relative dominant species (RDS) by α‐diversity threshold in the sputum samples and applied to samples of severe asthma patients and hierarchical clustering of RDS samples. MMA/HC: mild‐moderate asthma/Healthy controls; SA: severe asthma. The numbers studied in each cohort are shown.
FIGURE 2
FIGURE 2
Transcriptomic differentially expressed genes between RDS groups and MMA/HC cohort. Volcano plot depicting the sputum transcriptomic DEG between (A) Haemophilus influenzae RDS vs. MMA, (B) Moraxella catarrhalis RDS vs. MMA, Venn diagram depicts the overlap between upregulated genes (C) and downregulated genes (D) from the differentially expressed genes of the sputum transcriptomics between the Haemophilus influenzae, Moraxella catarrhalis and Tropheryma whipplei RDSs and MMA. MMA: mild‐moderate asthma.
FIGURE 3
FIGURE 3
Diversity of Aitchison distance clusters of RDS samples. α‐diversity using Shannon's index of each of the clusters C1 and C2 with non‐RDS and MMA/HC groups (A). β‐diversity of the clusters with non‐RDS and MMA/HC groups (B). Species‐level relative abundance of each of the 2 clusters compared to that of non‐RDS and MMA/HC groups (C).
FIGURE 4
FIGURE 4
Haemophilus influenzae component of C1 and C2 clusters of RDS samples. (A) Haemophilus influenzae abundance of C1 and C2 with non‐RDS. (B) Sputum eosinophils and sputum neutrophils (%) of C1 and C2 with non‐RDS. (C) Composition log fold‐change between each of the C1 (i) and C2 (ii) clusters versus non‐RDS group. RDS: relative dominant species of severe asthma groups; MMA/HC: mild‐moderate asthma and healthy controls.
FIGURE 5
FIGURE 5
Microbial pathway prevalence in C1 and C2 cluster samples. (A) Microbial pathway relative abundance of C1 and C2 clusters compared to that of non‐RDS. (B) β‐diversity of the relative abundant microbial pathways of the C1 and C2 clusters with non‐RDS.
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