Neonatal Gastrointestinal and Respiratory Microbiome in Cystic Fibrosis: Potential Interactions and Implications for Systemic Health

Abstract

Purpose

The gastrointestinal microbiome plays a critical role in nutrition and metabolic and immune functions in infants and young children and has implications for lifelong health. Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) mutations in CF result in viscous mucous production, frequent exposure to antibiotics, and atypical colonization patterns, resulting in an evolving dysbiosis of the gastrointestinal and respiratory microsystems; dysbiosis in CF results in systemic inflammation, chronic infection, and dysregulation of immune function. Dysbiosis in both the respiratory system and gut contributes to undernutrition, growth failure, and long-term respiratory and systemic morbidity in infants and children with CF. Understanding the role that the gut and respiratory microbiome plays in health or disease progression in CF will afford opportunities to better identify interventions to affect clinical changes.

Methods

Summary was done of the pertinent literature in CF and the study of the microbiome and probiotics.

Findings

New studies have identified bacteria in the respiratory tract in CF that are typically members of the intestinal microbiota, and enteral exposures to breast milk and probiotics are associated with prolonged periods of respiratory stability in CF.

Implications

Understanding the complex interactions between the CFTR mutations, microbial colonization, and mucosal and systemic immunity is of major importance to inform new treatment strategies (such as restoring a healthier microbiome with probiotics or dietary interventions) to improve nutritional status and immune competence and to decrease morbidity and mortality in CF.

Introduction

The composition and function of the human microbiome and its critical role in health in infants, young children, and adults is just beginning to be elucidated.¹ The gut microbiome, in particular, which co-evolved with humans, is critical for immune maturation in addition to drug and energy metabolism.¹ Clarifying the role of the microbiome and the metabolome, in particular in the neonatal period, may provide important understanding about immunity and lifetime disease risk, both in high-risk patient populations and in the healthy. Meaningful patterns in the microbiome/metabolome and their associations with disease risk and disease progression have been identified in disorders such as allergic disease, diabetes, obesity, autism, and respiratory disease.² Clarifying these patterns affords researchers possibilities for identifying targeted therapies to alter the microbiome to treat disease or to enhance health.³ The neonatal period is a critical window for immune programming that may affect health for a lifetime, and patterns in the microbiome may allow for prediction of disease risk, disease progression, or elimination of disease altogether.

The role of the gut microbiome in systemic health is a new focus in the care and evaluation of infants and young children with cystic fibrosis (CF). Mutations in CF transmembrane conductance regulator (CFTR) fundamentally affect the airway and intestinal microenvironment and result in altered colonization patterns of microorganisms in patients with CF even in the absence of antibiotic exposure.⁴ Intestinal dysfunction, secondary to structural and functional differences and frequent antibiotic exposure, results in an evolving dysbiosis of the gastrointestinal (GI) microsystem and macrosystem, or the microbe–microbe interactions and microbe–epithelium and immune crosstalk, and its holistic structural and functional role in systemic health.^5–8 This abnormal colonization likely contributes significantly to undernutrition, growth failure, and long-term morbidity from inflammation and infection in children and adults with CF.^5–8 Gut colonization and interactions with systemic immune training likely have implications for respiratory disease progression in CF.⁹ Understanding the complex interactions between CFTR mutations, microbial colonization, and mucosal and systemic immunity is of major importance to inform new treatment strategies to improve nutritional status and to decrease morbidity and mortality in CF. The role of microbes in systemic health and disease risk in CF has implications for healthy populations as well. The ability to investigate the microbiome of multiple organ systems with the use of high-throughput microbial sequencing methods provides more significant understanding of the landscape of multisystem microbial colonization and disease progression in CF.^10–12 Clarifying further the functional roles these microbes play with investigation of metabolomics has important implications for identifying interventions to alter disease progression in CF.¹³

The Exposome and the Developing Intestinal Microbiome in Healthy Children

The totality of human exposures, beginning in fetal life, and its interaction with the human genome, is called the exposome. The microbiome is established within the first 1 to 3 years of life and remains relatively stable throughout the life span.¹⁴ The developing microbiome in infancy is affected by mode of delivery, gestational age, infant diet, hospitalizations, and medications, particularly antibiotics; environmental toxicants and other exposures and their interaction with the developing microbiome are being investigated.¹⁵ The greatest intrapersonal and interpersonal variation in microbial communities occurs during infancy, potentially reflecting the differential development of the microbiome in relation to environmental factors, many of which can be altered.¹⁴ Human gene-environment interactions, and the interaction between “barrier organs,” such as the gut, respiratory tract, and the skin, are an important consideration in the study of the microbiome and its role in health. Environmental exposures and environmental stressors interact with us directly through these barrier organs, which house millions of microbes.¹⁶ Genetic predisposition to patterns of immune regulation or dysregulation direct homeostasis and inflammation of the gut, respiratory tract, and skin; CF is an important example of dysregulation of homeostasis of barrier organs. The microbes in the gut and respiratory tract play an important role in regulation of inflammation, and manipulation of the microbiome may be an important strategy to affect disease in CF.

The Intestinal Microbiome and Immunity

Seminal studies in germ-free animals found that absence of microbial colonization in neonatal life results in altered gut epithelialization, growth, and immune function.^17,18 The establishment of symbiotic bacteria can act as a central stimulus for maturation of the immune system and may alter risk of disease manifestation.^19,20 Both innate and adaptive immunity in humans have evolved to require microbial interactions during their development, including Toll-like receptors, class II major histocompatibility complexes, CD4 T cells, and the gut-associated lymphoid tissue.^21,22 Competent immune maintenance and homeostasis also require ongoing interaction with the gut microbiome.²³ It is theorized that fetal and neonatal exposures and events (such as exposure to toxins, antibiotics, and illness) during gut colonization and immune development are relevant to modifying disease risk for a lifetime.¹⁸ Targeted investigation of neonatal microbial colonization patterns with Bifidobacterium found associations between enhanced maturation of protective mucosal immunoglobulins, and early intense colonization with Bacteroides fragilis decreases immune responsiveness in infancy.²⁴ Interestingly, specific bacteria have been associated with early-onset allergy and atopy, particularly an increase in Clostridia and a decrease in Bifido-bacteria.²⁵ Murine models of type 1 diabetes have found that incidence of diabetes can be attenuated by modifying bacterial exposures²⁶; in a separate murine model oral administration of probiotics before 6 weeks of age modified risk of development of diabetes.²⁷ A study in human patients has identified that exposure to probiotics supplementation before 1 month of life in infants at high risk of developing type 1 diabetes is associated with a decreased risk of islet cell autoantibodies.²⁸

Observational studies in school-aged children have identified that lack of intestinal microbial diversity in the first weeks of life is related to risk of allergy and atopy.²⁹ Correspondingly, gut microbes influence the maturation of T helper cells (Th1) immune responses, CD4⁺ T-cell phenotype, Th1/Th2/Th17 development and activity, and regulatory T-cell function.³⁰ Th17 cells are found in abundance in early life in the lungs of patients with CF and are associated with Pseudomonas colonization and disease severity.³¹ It is possible that abnormal microbial patterns from birth, with repeated disturbances of the gut microflora, might cumulatively affect immune function over time in CF and might correlate with respiratory and intestinal disease progression.³²

Evidence for an Altered Gut Microbiome in CF

In the gut, CF affects digestion, nutrient absorption, and growth, secondary to CFTR gene mutations that deplete functional chloride secretion and subsequently transfer of water and other ions. The pH is altered, and thick, viscous secretions obstruct the gut lumen, altering the microenvironment of the intestines and causing inflammation and degradation of the pancreas, liver, gallbladder and intestine.⁸ New evidence suggests that the intestinal microenvironment predisposes young children with CF to intestinal and respiratory dysbiosis, or an altered and imbalanced microbiome, possibly from birth.³³ Dysbiosis is attributable to antibiotics, altered mucin clearance, pancreatic insufficiency, intestinal inflammation, small bowel bacterial overgrowth, and specifics of individual host genetics.^6,33 Evidence of chronic gut inflammation is seen in CF even in the absence of overt GI symptoms and is thought to be a driver of systemic inflammation in the disease.³⁴ Recent work in a murine model has clarified that CFTR knockout mice acquire an aberrant GI microbiota in the absence of antibiotic exposure.³² Murine models have identified that gut colonization is strongly affected by the loss of functional CFTR, resulting in overall increased bacterial load, with decreased richness, diversity, and reduced presence of symbionts, including Lactobacillus.^32,35 In addition, murine models have identified structural differences in the gut with CFTR mutations, including ileal crypt to villus axis distension, goblet cell hyperplasia, and muscularis externa thickness.³⁵ The gut mucosa is inflamed which results in enhanced adhesion and translocation of pathogenic gut microbes, leading to systemic inflammation and immune sequelae.³⁶ Broad-spectrum antibiotic treatment occurs frequently in the management of CF, resulting in disruption of the gut and respiratory flora that can also facilitate pathogenic behavior of microbes.³⁷

Multiple studies in young children and adults with CF have identified the importance of the gut microbiota in GI complications in CF.^7,8 A cross-sectional and longitudinal comparison of the microbiota of children with CF with their healthy siblings identified instability and decreased microbiome diversity with a decrease in Clostridia, Bifidobacterium, and Veillonella in patients with CF.³⁸ CFTR allelic variants correlate with patterns of GI dysbiosis, and patients with homozygous ΔF508 deletion have more significant alteration in gut microbiome patterns.³⁹ Schippa et al³⁹ identified specific bacterial species related to CF disease phenotype, and that more severe disease, in addition to ΔF508 homozygosity, correlated with a predominant pattern of Escherichia coli and Escherichia biform. These studies highlight that dysbiosis in CF may be related not simply to antibiotic exposure and impaired mucous clearance but point to CF as a systemic disease with a likely interrelationship between the intestinal and respiratory systems.

Potential Interaction Between the Gut and Respiratory Microbiome in CF

Respiratory dysbiosis in CF is the focus of most research and treatment strategies for the disease. Significant morbidity and ultimately mortality in CF often stems from chronic inflammation and infection of the respiratory tract.^4,40 New evidence exists for altered microbial communities in the respiratory tract, beginning in early life in CF compared with healthy controls, even in the absence of significant exposures.⁴¹ Several studies have identified bacteria in the respiratory tract in CF that are typically part of the intestinal microbiota and are theorized to contribute to the continuum of interactions between the host and microbial community in CF that relates to both the lung and gut microbiota.⁹ A longitudinal study of infants and young children with CF revealed distinct microbiota in the respiratory tract and gut with an overlapping core, with significant concordance between genera in both systems; >8 genera were highlighted as colonizing in the gut before the respiratory tract, identifying potential interactions.⁴⁰ In that same study, significant relations were discovered between patterns of respiratory colonization and diet, specifically breastfeeding, outlining relations between intestinal microbiome, immune development, and respiratory microbiome in CF. More recent work has identified the profound effects of breastfeeding on the CF gut and respiratory microbiome, with evidence for gut microbial diversity and specific bacterial communities associated with prolongation of respiratory health in patients exposed to breastmilk.³³ Correspondingly, studies of probiotics administration in children and young adults with CF linked alterations of the gut microflora with decreased pulmonary exacerbations,⁴² highlighting the interaction between the gut flora, immunity, and respiratory disease.

Is Therapeutic Alteration of the Gut Microbiome in CF a Viable Strategy?

Targeting alteration of the gut microbiome in CF is an innovative focus for altering disease expression. Breast milk exposure is reported to have positive benefits in CF, prolonging periods of respiratory sufficiency and health.³³ Breast milk provides the gold standard probiotic to infants, and it is likely that many of the benefits of breast milk exposure in CF are related to shifts in gut colonization and immune programming. Interventions that alter the gut microbiome in CF are likely to affect clinical benefits, secondary to the multiple antibiotic exposures, intestinal inflammation and increased permeability, abnormal microflora attributable to CFTR mutations, and because of altered innate immune mediators that are dysregulated in CF. Therapies to alter the gut microbiome in CF include targeted antibiotic therapies, prebiotic or symbiotic therapies, probiotic therapies, dietary interventions, and microbiome or fecal transplantations. Probiotics were studied most rigorously, and murine models of probiotic interventions found that Lactobacillus casei administration improves clearance of Pseudomonas aeruginosa from the lungs and treats bacterial and viral pneumonia.^43,44 Lactobacillus GG reduces P aeruginosa bacteremia in irradiated mice, and Lactobacillus plantarum was found to inhibit pathogenicity of P aeruginosa in murine models.⁴⁵ In humans, probiotics Lactobacillus and Bifidobacterium are the most examined probiotic therapies. In patients with CF, small pilot studies and randomized controlled trials of probiotic administration have been undertaken and have found promising, but limited, results. Lactobacillus was found to modulate allergic pulmonary inflammation and CF exacerbation in several studies.^46,47 Enteral probiotics in patients with CF are linked with alterations in the gut microflora which resulted in decreased frequency and severity of pulmonary exacerbations and reduced hospital admissions.^42,47,48,49 Probiotics are also associated with increased pulmonary function (forced expiratory volume timed) and increase in body weight.⁴⁸ Overall, alteration of the gut microbiome in CF may delay respiratory impairment and highlights the potential benefits of alteration of systemic inflammation and bolstering immune competence.

These small-scale studies are limited in their size and scope; undertaking large-scale randomized controlled trials of prebiotic or probiotic administration in patients with CF, ideally beginning in early life, would further expand our understanding of the potential risks and benefits of altering the microbiome and its relation to immune training and systemic health. Ideally, human microbiome studies would clearly define healthy microbiome acquisition patterns in young life to tailor probiotic interventions to alter the microbiome in high-risk populations. In addition, probiotic studies should outline changes in the structure of the microbiome and its metabolic functions, and investigation of clinical outcomes and immune modulation will further our understanding of the mechanisms behind the potential benefits of probiotics in CF.

Conclusion

Evidence is emerging that variable disease expression in infants and young children with CF who share identical CFTR mutations may be explained by differences in microbial acquisition, the interaction between the microbiome and the immune system, and gut–lung interactions through immunity in this systemic disease. This highlights the potential importance of future studies to better elucidate the relation between the gut microbiome and immunity in CF; the potential therapeutic benefits of altered antibiotic or probiotic interventions in CF; and potential dietary interventions, especially in infants and young children with CF, that might have short- and long-term health implications. It is likely that the neonatal period affords us with a critical window during which time alteration of the microbiome will result in changes in immune programming and prolongation of systemic health in CF.