The nasopharynx (NP) is a reservoir for microbes associated with acute respiratory infections (ARIs). Lung inflammation resulting from ARIs during infancy is linked to asthma development. We examined ...the NP microbiome during the critical first year of life in a prospective cohort of 234 children, capturing both the viral and bacterial communities and documenting all incidents of ARIs. Most infants were initially colonized with Staphylococcus or Corynebacterium before stable colonization with Alloiococcus or Moraxella. Transient incursions of Streptococcus, Moraxella, or Haemophilus marked virus-associated ARIs. Our data identify the NP microbiome as a determinant for infection spread to the lower airways, severity of accompanying inflammatory symptoms, and risk for future asthma development. Early asymptomatic colonization with Streptococcus was a strong asthma predictor, and antibiotic usage disrupted asymptomatic colonization patterns. In the absence of effective anti-viral therapies, targeting pathogenic bacteria within the NP microbiome could represent a prophylactic approach to asthma.
Display omitted
•The nasopharynx microbiome of infants has a simple structure dominated by six genera•Microbiome composition affects infection severity and pathogen spread to lower airways•Early asymptomatic colonization with Streptococcus increases risk of asthma•Antibiotic usage disrupts asymptomatic colonization patterns
Teo et al. characterize bacterial and viral communities within the infant nasopharynx during the first year of life, comparing between asymptomatic colonization and episodes of acute respiratory infections. Microbiome composition affects infection severity and spread to lower airways and risk for future asthma development.
Repeated cycles of infection-associated lower airway inflammation drive the pathogenesis of persistent wheezing disease in children. In this study, the occurrence of acute respiratory tract illnesses ...(ARIs) and the nasopharyngeal microbiome (NPM) were characterized in 244 infants through their first five years of life. Through this analysis, we demonstrate that >80% of infectious events involve viral pathogens, but are accompanied by a shift in the NPM toward dominance by a small range of pathogenic bacterial genera. Unexpectedly, this change frequently precedes the detection of viral pathogens and acute symptoms. Colonization of illness-associated bacteria coupled with early allergic sensitization is associated with persistent wheeze in school-aged children, which is the hallmark of the asthma phenotype. In contrast, these bacterial genera are associated with “transient wheeze” that resolves after age 3 years in non-sensitized children. Thus, to complement early allergic sensitization, monitoring NPM composition may enable early detection and intervention in high-risk children.
Display omitted
•Six genera dominate airway microbiota from birth to 2 years, but diversifies thereafter•Acute respiratory illness associates with pathogenic bacteria in the airway microbiota•Pathogenic airway bacteria may precede viral incursions and acute respiratory illness•Colonization with pathogens predicts chronic wheeze in allergic-sensitized children
Teo et al. characterize nasopharyngeal microbiota (NPM) in 244 children from birth to age 5 years during periods of illness and health. NPM colonization with illness-associated bacteria may promote acute respiratory illness independent of viral infection, and is associated with chronic or transient wheeze in allergic-sensitized or non-sensitized children, respectively.
Recurrent acute otitis media (rAOM, recurrent ear infection) is a common childhood disease caused by bacteria termed otopathogens, for which current treatments have limited effectiveness. Generic ...probiotic therapies have shown promise, but seem to lack specificity. We hypothesised that healthy children with no history of AOM carry protective commensal bacteria that could be translated into a specific probiotic therapy to break the cycle of re-infection. We characterised the nasopharyngeal microbiome of these children (controls) in comparison to children with rAOM (cases) to identify potentially protective bacteria. As some children with rAOM do not appear to carry any of the known otopathogens, we also hypothesised that characterisation of the middle ear microbiome could identify novel otopathogens, which may also guide the development of more effective therapies.
Middle ear fluids, middle ear rinses and ear canal swabs from the cases and nasopharyngeal swabs from both groups underwent 16S rRNA gene sequencing. The nasopharyngeal microbiomes of cases and controls were distinct. We observed a significantly higher abundance of Corynebacterium and Dolosigranulum in the nasopharynx of controls. Alloiococcus, Staphylococcus and Turicella were abundant in the middle ear and ear canal of cases, but were uncommon in the nasopharynx of both groups. Gemella and Neisseria were characteristic of the case nasopharynx, but were not prevalent in the middle ear.
Corynebacterium and Dolosigranulum are characteristic of a healthy nasopharyngeal microbiome. Alloiococcus, Staphylococcus and Turicella are possible novel otopathogens, though their rarity in the nasopharynx and prevalence in the ear canal means that their role as normal aural flora cannot be ruled out. Gemella and Neisseria are unlikely to be novel otopathogens as they do not appear to colonise the middle ear in children with rAOM.
Chronic immune-mediated diseases of adulthood often originate in early childhood. To investigate genetic associations between neonatal immunity and disease, we map expression quantitative trait loci ...(eQTLs) in resting myeloid cells and CD4
T cells from cord blood samples, as well as in response to lipopolysaccharide (LPS) or phytohemagglutinin (PHA) stimulation, respectively. Cis-eQTLs are largely specific to cell type or stimulation, and 31% and 52% of genes with cis-eQTLs have response eQTLs (reQTLs) in myeloid cells and T cells, respectively. We identified cis regulatory factors acting as mediators of trans effects. There is extensive colocalisation between condition-specific neonatal cis-eQTLs and variants associated with immune-mediated diseases, in particular CTSH had widespread colocalisation across diseases. Mendelian randomisation shows causal neonatal gene expression effects on disease risk for BTN3A2, HLA-C and others. Our study elucidates the genetics of gene expression in neonatal immune cells, and aetiological origins of autoimmune and allergic diseases.
Appropriate innate immune function is essential to limit pathogenesis and severity of severe lower respiratory infections (sLRI) during infancy, a leading cause of hospitalization and risk factor for ...subsequent asthma in this age group. Employing a systems biology approach to analysis of multi-omic profiles generated from a high-risk cohort (n=50), we found that the intensity of activation of an LPS-induced interferon gene network at birth was predictive of sLRI risk in infancy (AUC=0.724). Connectivity patterns within this network were stronger among susceptible individuals, and a systems biology approach identified IRF1 as a putative master regulator of this response. These findings were specific to the LPS-induced interferon response and were not observed following activation of viral nucleic acid sensing pathways. Comparison of responses at birth versus age 5 demonstrated that LPS-induced interferon responses but not responses triggered by viral nucleic acid sensing pathways may be subject to strong developmental regulation. These data suggest that the risk of sLRI in early life is in part already determined at birth, and additionally that the developmental status of LPS-induced interferon responses may be a key determinant of susceptibility. Our findings provide a rationale for the identification of at-risk infants for early intervention aimed at sLRI prevention and identifies targets which may be relevant for drug development.
Vitamin D may be essential for restricting the development and severity of allergic diseases and asthma, but a direct causal link between vitamin D deficiency and asthma has yet to be established. We ...have developed a 'low dose' model of allergic airway disease induced by intraperitoneal injection with ovalbumin (1 µg) and aluminium hydroxide (0.2 mg) in which characteristics of atopic asthma are recapitulated, including airway hyperresponsiveness, antigen-specific immunoglobulin type-E and lung inflammation. We assessed the effects of vitamin D deficiency throughout life (from conception until adulthood) on the severity of ovalbumin-induced allergic airway disease in vitamin D-replete and -deficient BALB/c mice using this model. Vitamin D had protective effects such that deficiency significantly enhanced eosinophil and neutrophil numbers in the bronchoalveolar lavage fluid of male but not female mice. Vitamin D also suppressed the proliferation and T helper cell type-2 cytokine-secreting capacity of airway-draining lymph node cells from both male and female mice. Supplementation of initially vitamin D-deficient mice with vitamin D for four weeks returned serum 25-hydroxyvitamin D to levels observed in initially vitamin D-replete mice, and also suppressed eosinophil and neutrophil numbers in the bronchoalveolar lavage fluid of male mice. Using generic 16 S rRNA primers, increased bacterial levels were detected in the lungs of initially vitamin D-deficient male mice, which were also reduced by vitamin D supplementation. These results indicate that vitamin D controls granulocyte levels in the bronchoalveolar lavage fluid in an allergen-sensitive manner, and may contribute towards the severity of asthma in a gender-specific fashion through regulation of respiratory bacteria.
Cyclophilin 40 (CyP40) is a tetratricopeptide repeat (TPR)-containing immunophilin and a modulator of steroid receptor function
through its binding to heat shock protein 90 (Hsp90). Critical to this ...binding are the carboxyl-terminal MEEVD motif of Hsp90
and the TPR domain of CyP40. Two different models of the CyP40-MEEVD peptide interaction were used as the basis for a comprehensive
mutational analysis of the Hsp90-interacting domain of CyP40. Using a carboxyl-terminal CyP40 construct as template, 24 amino
acids from the TPR and flanking acidic and basic domains were individually mutated by site-directed mutagenesis, and the mutants
were coexpressed in yeast with a carboxyl-terminal Hsp90β construct and qualitatively assessed for binding using a β-galactosidase
filter assay. For quantitative assessment, mutants were expressed as glutathione S -transferase fusion proteins and assayed for binding to carboxyl-terminal Hsp90β using conventional pulldown and enzyme-linked
immunosorbent assay microtiter plate assays. Collectively, the models predict that the following TPR residues help define
a binding groove for the MEEVD peptide: Lys-227, Asn-231, Phe-234, Ser-274, Asn-278, Lys-308, and Arg-312. Mutational analysis
identified five of these residues (Lys-227, Asn-231, Asn-278, Lys-308, and Arg-312) as essential for Hsp90 binding. The other
two residues (Phe-234 and Ser-274) and another three TPR domain residues not definitively associated with the binding groove
(Leu-284, Lys-285, and Asp-329) are required for efficient Hsp90 binding. These data confirm the critical importance of the
MEEVD binding groove in CyP40 for Hsp90 recognition and reveal that additional charged and hydrophobic residues within the
CyP40 TPR domain are required for Hsp90 binding.
Vitamin D has been suggested as a possible adjunctive treatment to ameliorate disease severity in human inflammatory bowel disease. In this study, the effects of diets containing high (D++, 10,000 ...IU/kg), moderate (D+, 2,280 IU/kg) or no vitamin D (D-) on the severity of dextran sodium sulphate (DSS) colitis in female C57Bl/6 mice were investigated. The group on high dose vitamin D (D++) developed the most severe colitis as measured by blinded endoscopic (p < 0.001) and histologic (p < 0.05) assessment, weight loss (p < 0.001), drop in serum albumin (p = 0.05) and increased expression of colonic TNF-α (p < 0.05). Microbiota analysis of faecal DNA showed that the microbial composition of D++ control mice was more similar to that of DSS mice. Serum 25(OH)D
levels reduced by 63% in the D++ group and 23% in the D+ group after 6 days of DSS treatment. Thus, high dose vitamin D supplementation is associated with a shift to a more inflammatory faecal microbiome and increased susceptibility to colitis, with a fall in circulating vitamin D occurring as a secondary event in response to the inflammatory process.
The C-terminal domain of Hsp90 displays independent chaperone activity, mediates dimerization, and contains the MEEVD motif essential for interaction with tetratricopeptide repeat-containing ...immunophilin cochaperones assembled in mature steroid receptor complexes. An α-helical region, upstream of the MEEVD peptide, helps form the dimerization interface and includes a hydrophobic microdomain that contributes to the Hsp90 interaction with the immunophilin cochaperones and corresponds to the binding site for novobiocin, a coumarin-related Hsp90 inhibitor. Mutation of selected residues within the hydrophobic microdomain significantly impacted the chaperone function of a recombinant C-terminal Hsp90 fragment and novobiocin inhibited wild-type chaperone activity. Prior incubation of the Hsp90 fragment with novobiocin led to a direct blockade of immunophilin cochaperone binding. However, the drug had little influence on the pre-formed Hsp90-immunophilin complex, suggesting that bound cochaperones mask the novobiocin-binding site. We observed a differential effect of the drug on Hsp90-immunophilin interaction, suggesting that the immunophilins make distinct contacts within the C-terminal domain to specifically modulate Hsp90 function. Novobiocin also precluded the interaction of full-length Hsp90 with the p50cdc37 cochaperone, which targets the N-terminal nucleotide-binding domain, and is prevalent in Hsp90 complexes with protein kinase substrates. Novobiocin therefore acts locally and allosterically to induce conformational changes within multiple regions of the Hsp90 protein. We provide evidence that coumermycin A1, a coumarin structurally related to novobiocin, interferes with dimerization of the Hsp90 C-terminal domain. Coumarin-based inhibitors then may antagonize Hsp90 function by inducing a conformation favoring separation of the C-terminal domains and release of substrate.
Virus-associated febrile lower respiratory tract infections (fLRIs) during infancy have been identified as risk factors for persistent wheeze development. We hypothesized that variations in innate ...immune defense capacity during this period, as exemplified by production of type 1 and 3 interferons (T1/3IFNs), might be an underlying determinant of risk.
We sought to investigate relationships between postnatal development of innate interferon response capacity and susceptibility to early infections and persistent wheeze.
We studied a subset of subjects from a birth cohort at high risk for asthma/allergy and determined the capacity of cord blood cells (n = 151) to produce any of a panel of 17 T1/3IFNs in response to the viral mimic polyinosinic-polycytidylic acid using a sensitive PCR assay. We investigated relationships between neonatal interferon responses and lower respiratory tract infection history during infancy, wheezing history to 5 age years, and ensuing maturation of innate immune capacity by age 4 years (n = 160) and 10 years (n = 125).
Although cohort subjects produced an average of 2.6 ± 0.3 of the 17 innate interferons tested at birth, 24% showed no T1/3IFN production. This nonproducer subgroup showed increased risk for infant fLRIs (odds ratio, 2.62; 95% CI, 1.14-6.06; P = .024) and persistent wheeze (odds ratio, 4.24; 95% CI, 1.60-11.24; P = .004) at age 5 years relative to those producing 1 or more T1/3IFNs, whereas risk for infant wheezy lower respiratory tract infections or “transient early wheeze” was unaffected. Moreover, infants who experienced fLRIs subsequently demonstrated accelerated development of T1/3IFN response capacity between 1 and 4 years of age.
T1/3IFN response capacity appears strongly developmentally constrained at birth. Infants in whom this negative regulation is strongest manifest increased risk for severe respiratory tract infections during infancy and subsequent persistent wheeze.
Display omitted
PDF
