Airway bacterial colonization is associated with bronchial remodeling in severe preschool wheezers

Severe recurrent preschool wheezers (SPW) are frequently characterized by the presence of pathogenic bacteria in their broncho-alveolar lavage (BAL).1, 2 Non-typable Haemophilus influenzae (NTHi), Moraxella catarrhalis and Streptococcus pneumoniae are most frequently detected and appear to play a role in the pathogenesis and the development of the disease.2, 3 Indeed, airway colonization with at least one of these bacteria has been associated with the inception and persistence of wheeze, as well as an increased risk of exacerbations (i.e., acute wheeze symptoms requiring ≥3 days of systemic steroids treatment1) and hospitalizations for wheeze in preschool age.2, 3 In terms of pathophysiology, the presence of these bacteria in the BAL has been associated with neutrophilic inflammation2 and could participate in the pathogenesis of bronchial remodeling (e.g., increased reticular basement membrane (RBM) thickness and bronchial smooth muscle (BSM) mass),4 which has been associated with poorer clinical outcomes from preschool to school age.1, 5, 6 To date, however, there is a lack of data on the presence of pathogenic bacteria in the BAL and their association with bronchial remodeling in SPW. We enrolled and pooled all SPW (n = 100, median age: 2.9 years) from the two previously described independent cohorts PTIT'ASTHME (NCT02806466) and RESPIRE (NCT04558671) with the same inclusion criteria.1 All SPWs underwent fiberoptic bronchoscopy with BAL. Bronchial biopsies were performed for research after obtaining informed consent (PTIT'ASTHME, approved by the French South–West region Ethics committee (no. 2010-A01134-35)) or as part of their routine care (RESPIRE), and data recorded prospectively in the patient's medical file were collected after having obtained the parents' non-opposition according to current French regulations, as previously described.1 A detailed description of methods has been previously published.1 Seven patients were excluded (BAL bacterial cultures not performed, n = 1; diagnosis of chronic bronchopathy (e.g., primary ciliary dyskinesia and non-cystic fibrosis bronchiectasis) confirmed between the bronchoscopy and December 2023 by either a new bronchoscopy or computed tomography scan, n = 6). All SPW had initially uncontrolled severe recurrent preschool wheeze despite high doses of inhaled corticosteroids with a second controller (i.e., long-acting beta2-agonists or leukotriene receptor antagonists) (Table 1). BAL fluid bacterial cultures identified NTHi, Moraxella catarrhalis and/or Streptococcus pneumoniae in 58/93 (62.4%) SPW (i.e., colonized SPW). In term of prevalence, NTHi was the most frequently identified bacteria in the BAL (45/93 (48.3%)) followed by Moraxella catarrhalis (25/93 (26.9%)) and Streptococcus pneumoniae (18/93 (19.4%)), which is consistent with previous findings.2 Colonized SPW were younger than non-colonized SPW (n = 35, i.e., SPW without NTHi, Moraxella catarrhalis or Streptococcus pneumoniae in the BAL) at the time of the bronchoscopy and at the first episode of wheezing and were hospitalized for wheeze more frequently in the first and the second year of life (Table 1). Moreover, the number of exacerbations in the year following bronchoscopy was higher in colonized SPW than in non-colonized SPW whereas the number of exacerbations in the year prior to bronchoscopy, antibiotic treatment after bronchoscopy, and treatment at inclusion and at 1-year follow-up were not significantly different between the two groups (Table 1). Regarding pathophysiological characteristics of the disease, there were no significant differences between the two groups in terms of atopy-related parameters (i.e., history of atopic dermatitis and food/aeroallergen sensitizations), blood test results (i.e., total IgE, eosinophils, and neutrophils) (Table 1). However, the proportion of neutrophils and the prevalence of respiratory viruses and other bacterial species were increased in the BAL of colonized SPW compared to non-colonized SPW (Table 1), which is consistent with a previously described steroid-refractory neutrophilic phenotype of preschool wheezers.2, 7 Regarding bronchial biopsies, colonized SPW showed increased normalized BSM mass compared to non-colonized SPW, while all other bronchial remodeling parameters and inflammatory cell densities were not significantly different between the two groups (Table 1). We then performed a multiple regression analysis including clinical and BAL parameters in the absence of bronchial remodeling parameters to predict BSM mass and exacerbations. Atopy (β = 5.48 [1.93; 9.03]) and the presence of NTHi in the BAL (β = 5.42 [1.86; 8.98]; p = .003) were the only two parameters independently associated with BSM mass (r2 = 0.231; p < .001, constant β = 17.59) in agreement with previous studies.6, 8 In addition, atopy (i.e., atopic dermatitis since birth and/or food/aeroallergen sensitization) (β = 1.20 [0.29; 2.12], p = .011) and NTHi in the BAL (β = 1.18 [0.26; 2.09], p = .012) were the only two parameters independently associated with the number of exacerbations in the year following bronchoscopy (r2 = 0.14; p = .001, constant β = 1.17). However, when the bronchial remodeling parameters are included in the regression analysis, the best model (r2 = 0.397; p < .001, constant β = −0.16), only identified the BSM mass (β = 0.13 [0.09; 0.18], p < .001) as the independent factor associated with the number of exacerbations in the year following the bronchoscopy. Based on these results, we then compared four groups of patients based on the presence of atopy and/or NTHi in the BAL: non-atopic without NTHi in the BAL (i.e., no atopy/NTHi−, n = 24), non-atopic with NTHi in the BAL (i.e., no atopy/NTHi+, n = 19), atopy without NTHi in the BAL (i.e., atopy/NTHi−, n = 24), atopy and NTHi in the BAL (i.e., atopy/NTHi+, n = 26) (Table 2). The group with no atopy/NTHi+ was characterized by a younger age at the time of the bronchoscopy than the other three groups, which is consistent with previous studies.9, 10 There were no differences between the atopy/NTHi− and atopy/NTHi+ groups in terms of atopic dermatitis history, food/aeroallergen sensitizations, and IgE levels and both groups had higher total blood IgE levels than the two non-atopic groups (Figure 1A). The two NTHi+ groups were more likely to have Moraxella catarrhalis in their BAL. Inflammatory cells in the blood, BAL and bronchial biopsies (data not shown) did not differ significantly between the four groups. The atopy/NTHi+ group was also characterized by an increased prevalence of hospitalizations in the first year of life compared to the two NTHi− groups but not to the group no atopy/NTHi+ (Figure 1B). In addition, the atopy/NTHi+ group had the highest number of exacerbations within a year prior (Figure 1C) and in the year after bronchoscopy (Figure 1D), and the lowest delay since the first exacerbation after bronchoscopy (Figure 1E), while treatment at the time of bronchoscopy and at 1-year follow-up did not differ significantly between the four groups (Table 2). There was no significant difference between the four groups in the percentage of neutrophils in the BALF (Figure 1F). In terms of bronchial remodeling, epithelial integrity, mucus gland area, submucosal fibrosis area, and RBM-BSM distance did not differ significantly between the four groups. In contrast, the atopy/NTHi+ group showed increased RBM thickness compared to the non-atopy/NTHi+ group (Figure 1G), which is consistent with previous studies.6 However, blood vessel density was lower (Figure 1H) and BSM mass was similar (Figure 1I) in the atopy/NTHi− group compared to the non-atopy/NTHi− group, in contrast to previous studies.6 However, in these studies, the presence of bacteria in the BAL was not assessed and considered. Indeed, the atopy/NTHi+ group showed an increased BSM mass compared to all other groups (Figure 1H). Several limitations must be pointed out: (i) We pooled patients from two different cohorts. However, the same inclusion criteria were used for both cohorts (i.e., patients with severe recurrent preschool wheezing requiring fiberoptic bronchoscopy). As previously reported, both cohorts were only different according to the age at bronchoscopy, prevalence of gastroesophageal reflux, and asthma treatment, higher in RESPIRE cohort than in PTIT'ASTHME cohort,1 limiting the impact of the initial inclusion cohort on our results. (ii) BAL samples were analyzed using bacterial cultures and not the next-generation microbiome sequencing method. However, the major bacterial species associated with childhood wheezing and asthma were captured, as previously described,9 and using methods accessible to all physicians. (iii) Several numbers of tests have been performed and the p-values are expressed without correction for multiple comparisons increasing the risk of type-I error whereas the small number of patients in each group according to atopy and NTHi status may have increased the risk of type-II error in class comparisons. Overall, bacterial colonization appears to be associated with BSM remodeling in SPW. While atopy appears to be the main driver in IgE level, both atopy and NTHi airway colonization are independently associated with the BSM mass and the number of exacerbations in the year following the bronchoscopy. In addition, when associated, atopy and NTHi airway colonization identified a group of SPW characterized by very early and severe disease, high-IgE levels, an increased risk of exacerbations despite high-dose-inhaled corticosteroids with a second controller, increased BSM mass compared to patients with atopy or NTHi airway colonization only, which is consistent with preclinical results suggesting that NTHi status would enhance remodeling in atopic mice.8 This increase in airway remodeling may result from a steroid-resistant Th17-associated inflammation induced by NTHi airway colonization instead of steroid-sensitive Type 2-associated eosinophilic inflammation as demonstrated in mice in vivo.8 Further studies are now needed to better characterize the inflammation in this SPW and to understand the association between NTHi, atopy, and bronchial remodeling in SPW. Fabien Beaufils: Conceptualization; writing – review and editing; formal analysis; writing – original draft; investigation; funding acquisition; methodology; validation; visualization. Laurence Delhaes: Conceptualization; writing – review and editing; formal analysis; writing – original draft; validation; supervision. Pauline Esteves: Writing – review and editing; investigation; validation. Marine Michelet: Writing – review and editing; validation; investigation. Cécile Bébéar: Writing – review and editing; validation. Hugues Begueret: Writing – review and editing; validation; investigation. Michael Fayon: Writing – original draft; writing – review and editing; funding acquisition; conceptualization; supervision; validation; formal analysis; investigation. Patrick Berger: Writing – original draft; writing – review and editing; validation; conceptualization; supervision; project administration; methodology; formal analysis. This study has received a research grant from the French Ministry of Health no. B101222-30; 2010-A01134-35, and the “Fondation Bordeaux Université (Fonds FGLMR/AVAD pour les Maladies Chroniques).” Clinical Trial nos. NCT02806466 and NCT04558671. FB has received a research grant from AstraZeneca and reports personal fees and non-financial support from Novartis, Chiesi, and AstraZeneca. LD and PE report no conflict of interest. MM reports personal fees and non-financial support from Astra Zeneca, GSK, Novartis, and Sanofi. CB reports no conflict of interest. HB reports no conflict of interest. MF has received a research and educational grant from Novartis and reports personal fees as a speaker from GlaxoSmithKline. In addition, MF has 1 co-patent (No. FR 19/14751; i.e., New in situ pulmonary treatment device in premature babies). PB is the medical coordinator of the French national cohort (i.e., COBRA), which received grants from AstraZeneca, GlaxoSmithKline, Sanofi and Chiesi. Moreover, PB reports grants and personal fees from Novartis, personal fees and non-financial support from Chiesi, grants, personal fees and non-financial support from Boehringer Ingelheim, personal fees and non-financial support from AstraZeneca, personal fees and non-financial support from Sanofi, outside the submitted work. In addition, PB has four patents (i) (No. WO/2016/120369A1; i.e., use of plerixafor for treating and/or preventing acute exacerbations of chronic obstructive pulmonary disease), delivered; (ii) (No. WO/2017/203064A1; i.e., MRI method for the geometrical characterization of pulmonary airways), delivered; (iii) (No. WO/2021/018439A1; i.e., method for generating a biomarker system), (delivered); and (iv) (No. WO/2021/224356; i.e., new compositions and methods of treating COVID-19 disease), licensed. The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer-review/10.1111/pai.14253.