Altered Stool Microbiota of Infants with Cystic Fibrosis Shows a Reduction in Genera Associated with Immune Programming from Birth.

Previous work from our group indicated an association between the gastrointestinal microbiota of infants with cystic fibrosis (CF) and airway disease in this population. Here we report that stool microbiota of infants with CF demonstrates an altered but largely unchanging within-individual bacterial diversity (alpha diversity) over the first year of life, in contrast to the infants without CF (control cohort), which showed the expected increase in alpha diversity over the first year. The beta diversity, or between-sample diversity, of these two cohorts was significantly different over the first year of life and was statistically significantly associated with airway exacerbations, confirming our earlier findings. Compared with control infants, infants with CF had reduced levels of Bacteroides, a bacterial genus associated with immune modulation, as early as 6 weeks of life, and this significant reduction of Bacteroides spp. in the cohort with CF persisted over the entire first year of life. Only two other genera were significantly different across the first year of life: Roseburia was significantly reduced and Veillonella was significantly increased. Other genera showed differences between the two cohorts but only at selected time points. In vitro studies demonstrated that exposure of the apical face of polarized intestinal cell lines to Bacteroides species supernatants significantly reduced production of interleukin 8 (IL-8), suggesting a mechanism whereby changes in the intestinal microbiota could impact inflammation in CF. This work further establishes an association between gastrointestinal microbiota, inflammation, and airway disease in infants with CF and presents a potential opportunity for therapeutic interventions beginning in early life.IMPORTANCE There is growing evidence for a link between gastrointestinal bacterial communities and airway disease progression in CF. We demonstrate that infants with CF ≤1 year of age show a distinct stool microbiota versus that of control infants of a comparable age. We detected associations between the gut microbiome and airway exacerbation events in the cohort of infants with CF, and in vitro studies provided one possible mechanism for this observation. These data clarify that current therapeutics do not establish in infants with CF a gastrointestinal microbiota like that in healthy infants, and we suggest that interventions that direct the gastrointestinal microbiota closer to a healthy state may provide systemic benefits to these patients during a critical window of immune programming that might have implications for lifelong health.

Use of a Multiplex Transcript Method for Analysis of Pseudomonas aeruginosa Gene Expression Profiles in the Cystic Fibrosis Lung.

The discovery of therapies that modulate Pseudomonas aeruginosa virulence or that can eradicate chronic P. aeruginosa lung infections associated with cystic fibrosis (CF) will be advanced by an improved understanding of P. aeruginosa behavior in vivo We demonstrate the use of multiplexed Nanostring technology to monitor relative abundances of P. aeruginosa transcripts across clinical isolates, in serial samples, and for the purposes of comparing microbial physiology in vitro and in vivo The expression of 75 transcripts encoded by genes implicated in CF lung disease was measured in a variety of P. aeruginosa strains as well as RNA serial sputum samples from four P. aeruginosa-colonized subjects with CF collected over 6 months. We present data on reproducibility, the results from different methods of normalization, and demonstrate high concordance between transcript relative abundance data obtained by Nanostring or transcriptome sequencing (RNA-Seq) analysis. Furthermore, we address considerations regarding sequence variation between strains during probe design. Analysis of P. aeruginosa grown in vitro identified transcripts that correlated with the different phenotypes commonly observed in CF clinical isolates. P. aeruginosa transcript profiles in RNA from CF sputum indicated alginate production in vivo, and transcripts involved in quorum-sensing regulation were less abundant in sputum than strains grown in the laboratory. P. aeruginosa gene expression patterns from sputum clustered closely together relative to patterns for laboratory-grown cultures; in contrast, laboratory-grown P. aeruginosa showed much greater transcriptional variation with only loose clustering of strains with different phenotypes. The clustering within and between subjects was surprising in light of differences in inhaled antibiotic and respiratory symptoms, suggesting that the pathways represented by these 75 transcripts are stable in chronic CF P. aeruginosa lung infections.

Low levels of insulin-like growth factor-1 contribute to alveolar macrophage dysfunction in cystic fibrosis.

Alveolar macrophages are major contributors to lung innate immunity. Although alveolar macrophages from cystic fibrosis (CF) transmembrane conductance regulator(-/-) mice have impaired function, no study has investigated primary alveolar macrophages in adults with CF. CF patients have low levels of insulin-like growth factor 1 (IGF-1), and our prior studies demonstrate a relationship between IGF-1 and macrophage function. We hypothesize that reduced IGF-1 in CF leads to impaired alveolar macrophage function and chronic infections. Serum and bronchoalveolar lavage (BAL) samples were obtained from eight CF subjects and eight healthy subjects. Macrophages were isolated from BAL fluid. We measured the ability of alveolar macrophages to kill Pseudomonas aeruginosa. Subsequently, macrophages were incubated with IGF-1 prior to inoculation with bacteria to determine the effect of IGF-1 on bacterial killing. We found a significant decrease in bacterial killing by CF alveolar macrophages compared with control subjects. CF subjects had lower serum and BAL IGF-1 levels compared with healthy control subjects. Exposure to IGF-1 enhanced alveolar macrophage macrophages in both groups. Finally, exposing healthy alveolar macrophages to CF BAL fluid decreased bacterial killing, and this was reversed by the addition of IGF-1, whereas IGF-1 blockade worsened bacterial killing. Our studies demonstrate that alveolar macrophage function is impaired in patients with CF. Reductions in IGF-1 levels in CF contribute to the impaired alveolar macrophage function. Exposure to IGF-1 ex vivo results in improved function of CF alveolar macrophages. Further studies are needed to determine whether alveolar macrophage function can be enhanced in vivo with IGF-1 treatment.

Characterization of SARS-CoV-2 Convalescent Patients' Serological Repertoire Reveals High Prevalence of Iso-RBD Antibodies.

While our understanding of SARS-CoV-2 pathogenesis and antibody responses following infection and vaccination has improved tremendously since the outbreak in 2019, the sequence identities and relative abundances of the individual constituent antibody molecules in circulation remain understudied. Using Ig-Seq, we proteomically profiled the serological repertoire specific to the whole ectodomain of SARS-CoV-2 prefusion-stabilized spike (S) as well as to the receptor binding domain (RBD) over a 6-month period in four subjects following SARS-CoV-2 infection before SARS-CoV-2 vaccines were available. In each individual, we identified between 59 and 167 unique IgG clonotypes in serum. To our surprise, we discovered that ∼50% of serum IgG specific for RBD did not recognize prefusion-stabilized S (referred to as iso-RBD antibodies), suggesting that a significant fraction of serum IgG targets epitopes on RBD inaccessible on the prefusion-stabilized conformation of S. On the other hand, the abundance of iso-RBD antibodies in nine individuals who received mRNA-based COVID-19 vaccines encoding prefusion-stabilized S was significantly lower (∼8%). We expressed a panel of 12 monoclonal antibodies (mAbs) that were abundantly present in serum from two SARS-CoV-2 infected individuals, and their binding specificities to prefusion-stabilized S and RBD were all in agreement with the binding specificities assigned based on the proteomics data, including 1 iso-RBD mAb which bound to RBD but not to prefusion-stabilized S. 2 of 12 mAbs demonstrated neutralizing activity, while other mAbs were non-neutralizing. 11 of 12 mAbs also bound to S (B.1.351), but only 1 maintained binding to S (B.1.1.529). This particular mAb binding to S (B.1.1.529) 1) represented an antibody lineage that comprised 43% of the individual's total S-reactive serum IgG binding titer 6 months post-infection, 2) bound to the S from a related human coronavirus, HKU1, and 3) had a high somatic hypermutation level (10.9%), suggesting that this antibody lineage likely had been elicited previously by pre-pandemic coronavirus and was re-activated following the SARS-CoV-2 infection. All 12 mAbs demonstrated their ability to engage in Fc-mediated effector function activities. Collectively, our study provides a quantitative overview of the serological repertoire following SARS-CoV-2 infection and the significant contribution of iso-RBD antibodies, demonstrating how vaccination strategies involving prefusion-stabilized S may have reduced the elicitation of iso-RBD serum antibodies which are unlikely to contribute to protection.

A pilot study of cystic fibrosis exacerbation response phenotypes reveals contrasting serum and sputum iron trends.

The cystic fibrosis (CF) community seeks to explain heterogeneous outcomes of pulmonary exacerbation (PEX) treatment. Serum and sputum inflammatory mediators may identify people with CF (PwCF) at risk for suboptimal responses. However, lack of an established association between response phenotypes and these mediators limits clinical application. In this pilot study, we prospectively characterized treatment response phenotypes by assessing health-related quality-of-life (HRQoL) during PEX. We also measured lung function and iron-related biochemical parameters in serum and sputum. We classified subjects as sustained symptom-responders (SRs) or non-sustained symptom-responders (NSRs) based on the absence or presence, respectively, of worsened symptom scores after initial improvement. We used linear mixed models (LMMs) to determine whether trends in lung function, hematologic, serum, and sputum indices of inflammation differed between response cohorts. In 20 PwCF, we identified 10 SRs and 10 NSRs with no significant differences in lung function at PEX onset and treatment durations. SRs had better model-predicted trends in lung function than NSRs during PEX. Non-linear trends in serum and sputum iron levels significantly differed between SRs and NSRs. In adults with cystic fibrosis, PEX treatment response phenotypes may be correlated with distinctive trends in serum and sputum iron concentrations.

Serum Iron Level Is Associated with Time to Antibiotics in Cystic Fibrosis.

BACKGROUND: Serum levels of hepcidin-25, a peptide hormone that reduces blood iron content, are elevated when patients with cystic fibrosis (CF) develop pulmonary exacerbation (PEx). Because hepcidin-25 is unavailable as a clinical laboratory test, we questioned whether a one-time serum iron level was associated with the subsequent number of days until PEx, as defined by the need to receive systemic antibiotics (ABX) for health deterioration. METHODS: Clinical, biochemical, and microbiological parameters were simultaneously checked in 54 adults with CF. Charts were reviewed to determine when they first experienced a PEx after these parameters were assessed. Time to ABX was compared in subgroups with and without specific attributes. Multivariate linear regression was used to identify parameters that significantly explained variation in time to ABX. RESULTS: In univariate analyses, time to ABX was significantly shorter in subjects with Aspergillus-positive sputum cultures and CF-related diabetes. Multivariate linear regression models demonstrated that shorter time to ABX was associated with younger age, lower serum iron level, and Aspergillus sputum culture positivity. CONCLUSIONS: Serum iron, age, and Aspergillus sputum culture positivity are factors associated with shorter time to subsequent PEx in CF adults.

Iron supplementation does not worsen respiratory health or alter the sputum microbiome in cystic fibrosis.

BACKGROUND: Iron supplementation for hypoferremic anemia could potentiate bacterial growth in the cystic fibrosis (CF) lung, but clinical trials testing this hypothesis are lacking. METHODS: Twenty-two adults with CF and hypoferremic anemia participated in a randomized, double-blind, placebo-controlled, crossover trial of ferrous sulfate 325mg daily for 6weeks. Iron-related hematologic parameters, anthropometric data, sputum iron, Akron Pulmonary Exacerbation Score (PES), and the sputum microbiome were serially assessed. Fixed-effect models were used to describe how ferrous sulfate affected these variables. RESULTS: Ferrous sulfate increased serum iron by 22.3% and transferrin saturation (TSAT) by 26.8% from baseline (p<0.05) but did not affect hemoglobin, sputum iron, Akron PES, and the sputum microbiome. CONCLUSIONS: Low-dose ferrous sulfate improved hypoferremia without correcting anemia after 6weeks. We did not observe significant effects on sputum iron, Akron PES, and the sputum microbiome. Although we did not identify untoward health effects of iron supplementation, a larger blinded randomized controlled trial would be needed to fully demonstrate safety.

Iron homeostasis during cystic fibrosis pulmonary exacerbation.

BACKGROUND: Hypoferremia is a marker of disease severity in cystic fibrosis (CF). The effect of systemic antibiotics on iron homeostasis during CF pulmonary exacerbation (CFPE) is unknown. Our central hypotheses were that, by the completion of treatment, serum iron would increase, serum concentrations of interleukin-6 (IL-6) and hepcidin-25, two mediators of hypoferremia, would decrease, and sputum iron would decrease. METHODS: Blood and sputum samples were collected from 12 subjects with moderate-to-severe CF (median percentage-predicted forced expiratory volume in 1 second (FEV(1) %) = 29%; median weight = 56 kg) within 24 hours of starting and completing a course of systemic antibiotics. RESULTS: After treatment, subjects showed median FEV(1) % and body weight improvements of 4.5% and 2.0 kg, respectively (p < 0.05). Median serum iron rose by 2.4 µmol/L (p < 0.05), but 75% of patients remained hypoferremic. Median serum IL-6 and hepcidin-25 levels fell by 12.1 pg/mL and 37.5 ng/mL, respectively (p < 0.05). Median serum erythropoietin (EPO) and hemoglobin levels were unaffected by treatment. We observed a trend toward lower sputum iron content after treatment. CONCLUSIONS: Hypoferremia is a salient characteristic of CFPE that improves with waning inflammation. Despite antibiotic treatment, many patients remain hypoferremic and anemic because of ineffective erythropoiesis.