Impaired interferon response in plasmacytoid dendritic cells from children with persistent wheeze.

BACKGROUND: Impaired interferon response and allergic sensitization may contribute to virus-induced wheeze and asthma development in young children. Plasmacytoid dendritic cells (pDCs) play a key role in antiviral immunity as critical producers of type I interferons. pDCs also express the high-affinity IgE receptor through which type I interferon production may be negatively regulated. Whether antiviral function of pDCs is associated with recurrent episodes of wheeze in young children is not well understood. OBJECTIVE: We sought to evaluate the phenotype and function of circulating pDCs in children with a longitudinally defined wheezing phenotype. METHODS: We performed multiparameter flow cytometry on PBMCs from 38 children presenting to the emergency department with an acute episode of respiratory wheeze and 19 controls. RNA sequencing on isolated pDCs from the same individuals was also performed. For each subject, their longitudinal exacerbation phenotype was determined using the Western Australia public hospital database. RESULTS: We observed a significant depletion of circulating pDCs in young children with a persistent phenotype of wheeze. The same individuals also displayed upregulation of the FcεRI on their pDCs. Based on transcriptomic analysis, pDCs from these individuals did not mount a robust systemic antiviral response as observed in children who displayed a nonrecurrent wheezing phenotype. CONCLUSIONS: Our data suggest that circulating pDC phenotype and function are altered in young children with a persistent longitudinal exacerbation phenotype. Expression of high-affinity IgE receptor is increased and their function as major interferon producers is impaired during acute exacerbations of wheeze.

Nasal Delivery of Haemophilus haemolyticus Is Safe, Reduces Influenza Severity, and Prevents Development of Otitis Media in Mice.

BACKGROUND: Despite vaccination, influenza and otitis media (OM) remain leading causes of illness. We previously found that the human respiratory commensal Haemophilus haemolyticus prevents bacterial infection in vitro and that the related murine commensal Muribacter muris delays OM development in mice. The observation that M muris pretreatment reduced lung influenza titer and inflammation suggests that these bacteria could be exploited for protection against influenza/OM. METHODS: Safety and efficacy of intranasal H haemolyticus at 5 × 107 colony-forming units (CFU) was tested in female BALB/cARC mice using an influenza model and influenza-driven nontypeable Haemophilus influenzae (NTHi) OM model. Weight, symptoms, viral/bacterial levels, and immune responses were measured. RESULTS: Intranasal delivery of H haemolyticus was safe and reduced severity of influenza, with quicker recovery, reduced inflammation, and lower lung influenza virus titers (up to 8-fold decrease vs placebo; P ≤ .01). Haemophilus haemolyticus reduced NTHi colonization density (day 5 median NTHi CFU/mL = 1.79 × 103 in treatment group vs 4.04 × 104 in placebo, P = .041; day 7 median NTHi CFU/mL = 28.18 vs 1.03 × 104; P = .028) and prevented OM (17% OM in treatment group, 83% in placebo group; P = .015). CONCLUSIONS: Haemophilus haemolyticus has potential as a live biotherapeutic for prevention or early treatment of influenza and influenza-driven NTHi OM. Additional studies will deem whether these findings translate to humans and other respiratory infections.

Mapping Lung Hematopoietic Progenitors: Developmental Kinetics and Response to Influenza A Infection.

The bone marrow is a specialized niche responsible for the maintenance of hematopoietic stem and progenitor cells during homeostasis and inflammation. Recent studies, however, have extended this essential role to the extramedullary and extravascular lung microenvironment. Here, we provide further evidence for a reservoir of hematopoietic stem and progenitor cells within the lung from Embryonic Day 18.5 until adulthood. These lung progenitors display distinct microenvironment-specific developmental kinetics compared with their bone marrow counterparts, exemplified by a rapid shift from a common myeloid to a megakaryocyte-erythrocyte progenitor-dominated niche with increasing age. In adult mice, influenza A viral infection results in a transient reduction in multipotent progenitors within the lungs, with a parallel increase in downstream granulocyte-monocyte progenitors and dendritic cell populations associated with acute viral infections. Our findings suggest that lung hematopoietic progenitors play a role in reestablishing immunological homeostasis in the respiratory mucosa, which may have significant clinical implications for maintaining pulmonary health after inflammatory perturbation.

LPS binding protein and activation signatures are upregulated during asthma exacerbations in children.

Asthma exacerbations in children are associated with respiratory viral infection and atopy, resulting in systemic immune activation and infiltration of immune cells into the airways. The gene networks driving the immune activation and subsequent migration of immune cells into the airways remains incompletely understood. Cellular and molecular profiling of PBMC was employed on paired samples obtained from atopic asthmatic children (n = 19) during acute virus-associated exacerbations and later during convalescence. Systems level analyses were employed to identify coexpression networks and infer the drivers of these networks, and validation was subsequently obtained via independent samples from asthmatic children. During exacerbations, PBMC exhibited significant changes in immune cell abundance and upregulation of complex interlinked networks of coexpressed genes. These were associated with priming of innate immunity, inflammatory and remodelling functions. We identified activation signatures downstream of bacterial LPS, glucocorticoids and TGFB1. We also confirmed that LPS binding protein was upregulated at the protein-level in plasma. Multiple gene networks known to be involved positively or negatively in asthma pathogenesis, are upregulated in circulating PBMC during acute exacerbations, supporting the hypothesis that systemic pre-programming of potentially pathogenic as well as protective functions of circulating immune cells preceeds migration into the airways. Enhanced sensitivity to LPS is likely to modulate the severity of acute asthma exacerbations through exposure to environmental LPS.

The maternal gut microbiome during pregnancy and offspring allergy and asthma.

Environmental exposures during pregnancy that alter both the maternal gut microbiome and the infant's risk of allergic disease and asthma include a traditional farm environment and consumption of unpasteurized cow's milk, antibiotic use, dietary fiber, and psychosocial stress. Multiple mechanisms acting in concert may underpin these associations and prime the infant to acquire immune competence and homeostasis following exposure to the extrauterine environment. Cellular and metabolic products of the maternal gut microbiome can promote the expression of microbial pattern recognition receptors, as well as thymic and bone marrow hematopoiesis relevant to regulatory immunity. At birth, transmission of maternally derived bacteria likely leverages this in utero programming to accelerate postnatal transition from a TH2- to TH1- and TH17-dominant immune phenotype and maturation of regulatory immune mechanisms, which in turn reduce the child's risk of allergic disease and asthma. Although our understanding of these phenomena is rapidly evolving, the field is relatively nascent, and we are yet to translate existing knowledge into interventions that substantially reduce disease risk in humans. Here, we review evidence that the maternal gut microbiome impacts the offspring's risk of allergic disease and asthma, discuss challenges and future directions for the field, and propose the hypothesis that maternal carriage of Prevotella copri during pregnancy decreases the offspring's risk of allergic disease via production of succinate, which in turn promotes bone marrow myelopoiesis of dendritic cell precursors in the fetus.

Metabolic dysfunction induced by a high-fat diet modulates hematopoietic stem and myeloid progenitor cells in brown adipose tissue of mice.

Brown adipose tissue (BAT) may be an important metabolic regulator of whole-body glucose. While important roles have been ascribed to macrophages in regulating metabolic functions in BAT, little is known of the roles of other immune cells subsets, particularly dendritic cells (DCs). Eating a high-fat diet may compromise the development of hematopoietic stem and progenitor cells (HSPCs)-which give rise to DCs-in bone marrow, with less known of its effects in BAT. We have previously demonstrated that ongoing exposure to low-dose ultraviolet radiation (UVR) significantly reduced the 'whitening' effect of eating a high-fat diet upon interscapular (i) BAT of mice. Here, we examined whether this observation may be linked to changes in the phenotype of HSPCs and myeloid-derived immune cells in iBAT and bone marrow of mice using 12-colour flow cytometry. Many HSPC subsets declined in both iBAT and bone marrow with increasing metabolic dysfunction. Conversely, with rising adiposity and metabolic dysfunction, conventional DCs (cDCs) increased in both of these tissues. When compared with a low-fat diet, consumption of a high-fat diet significantly reduced proportions of myeloid, common myeloid and megakaryocyte-erythrocyte progenitors in iBAT, and short-term hematopoietic stem cells in bone marrow. In mice fed the high-fat diet, exposure to low-dose UVR significantly reduced proportions of cDCs in iBAT, independently of nitric oxide release from irradiated skin [blocked using the scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO)], but did not significantly modify HSPC subsets in either tissue. Further studies are needed to determine whether changes in these cell populations contribute towards metabolic dysfunction .

Nasal Delivery of a Commensal Pasteurellaceae Species Inhibits Nontypeable Haemophilus influenzae Colonization and Delays Onset of Otitis Media in Mice.

Nasopharyngeal colonization with nontypeable Haemophilus influenzae (NTHi) is a prerequisite for developing NTHi-associated infections, including otitis media. Therapies that block NTHi colonization may prevent disease development. We previously demonstrated that Haemophilus haemolyticus, a closely related human commensal, can inhibit NTHi colonization and infection of human respiratory epithelium in vitro We have now assessed whether Muribacter muris (a rodent commensal from the same family) can prevent NTHi colonization and disease in vivo using a murine NTHi otitis media model. Otitis media was modeled in BALB/c mice using coinfection with 1 × 104.5 PFU of influenza A virus MEM H3N2, followed by intranasal challenge with 5 × 107 CFU of NTHi R2866 Specr Mice were pretreated or not with an intranasal inoculation of 5 × 107 CFU M. muris 24 h before coinfection. NTHi and M. muris viable counts and inflammatory mediators (gamma interferon [IFN-γ], interleukin-1ß [IL-1ß], IL-6, keratinocyte chemoattractant [KC], and IL-10) were measured in nasal washes and middle ear tissue homogenate. M. muris pretreatment decreased the median colonization density of NTHi from 6 × 105 CFU/ml to 9 × 103 CFU/ml (P = 0.0004). Only 1/12 M. muris-pretreated mice developed otitis media on day 5 compared to 8/15 mice with no pretreatment (8% versus 53%, P = 0.0192). Inflammation, clinical score, and weight loss were also lower in M. muris-pretreated mice. We have demonstrated that a single dose of a closely related commensal can delay onset of NTHi otitis media in vivo Human challenge studies investigating prevention of NTHi colonization are warranted to reduce the global burden of otitis media and other NTHi diseases.

Oestrogen amplifies pre-existing atopy-associated Th2 bias in an experimental asthma model.

BACKGROUND: The prevalence and severity of asthma, particularly the most common (atopic) form of the disease, increase amongst females but not males after puberty, and asthma activity also changes throughout the menstrual cycle and during pregnancy. The contribution of female sex hormones to asthma pathogenesis is incompletely understood. OBJECTIVE: To obtain insight into the role of oestrogen (E2) in experimental atopic asthma, and guide future research on sex-related variations in atopic asthma susceptibility/intensity in humans. METHODS: We utilized an experimental model comprising rat strains expressing dichotomous Th2-high vs Th2-low immunophenotypes exemplified by eosinophilia, mirroring differences between human atopics/non-atopics. We compared the efficiency of Th2-associated immunoinflammatory mechanisms, which differed markedly between the two strains, and between sexes in the Th2-high strain, and determined the effects of E2 administration on these differences. RESULTS: Unique to the Th2-high strain, eosinophil: neutrophil ratios in the airways at baseline and following sensitization/aeroallergen challenge were logfold higher in females relative to males, and this was reflected by higher baseline blood eosinophil numbers in females. Pretreatment of Th2-high males with E2 abrogated this sex difference by selectively boosting Th2-associated genes in the airways and eosinophilia, but was without corresponding effect in the Th2-low strain. In contrast, parallel E2 effects on myeloid and lymphoid cell populations were relatively modest. CONCLUSIONS AND CLINICAL RELEVANCE: E2 acts to amplify the eosinophilic component of pre-existing Th2-high immunophenotype, possibly acting at the level of the common eosinophil/neutrophil precursor in bone marrow to preferentially drive eosinophil differentiation. Constitutive granulocyte profiles in which the balance between eosinophils and neutrophils is skewed towards eosinophils have been identified in independent cohort studies as markers of asthma risk, and these findings suggest that more detailed studies on the role of E2 in this context, and in relation to asthma pathogenesis in post-pubertal females in particular, appear warranted.

In infants with sufficient vitamin D status at birth, vitamin D supplementation does not impact immune development.

BACKGROUND: Low vitamin D levels have been associated with allergic diseases. Vitamin D has potent immunomodulatory properties, but the mechanisms remain unclear. We have investigated the effect of oral vitamin D supplementation on circulating immune cell phenotypes in infants. METHOD: A double-blinded randomised controlled trial was conducted to investigate the effect of oral vitamin D supplementation (400 IU/d) on eczema and immune development. A subset of 78 infants was included in this analysis. Phenotypic analysis of immune cell subsets was performed using flow cytometry. RESULTS: Vitamin D supplementation resulted in median 25(OH)D levels of 80.5 vs 59.5 nmol/L in the placebo group at 3 months of age (P = .002) and 87.5 vs 77 nmol/L at 6 months of age (P = .08). We observed significant changes in immune cell composition from birth (cord blood) to 6 months of age. Vitamin D supplementation did not impact these changes, nor did immune cell composition correlate with plasma 25(OH)D levels. Through exploratory analysis, we identified possible associations with eczema development and increased abundance of naïve CD4- T cells at birth, as well as associations with basophils, iNKT and central memory CD4+ T cells, and altered expression patterns of IgE receptor (FcεR1) on monocytes and dendritic cells with eczema at 6 months. CONCLUSIONS: Vitamin D supplementation in infants who were vitamin D sufficient at birth did not affect developmental changes in immune cells during the first 6 months of life. However, immune cell profiles at birth and at 6 months of age were associated with early life eczema.

Personalized Transcriptomics Reveals Heterogeneous Immunophenotypes in Children with Viral Bronchiolitis.

Rationale: A subset of infants are hypersusceptible to severe/acute viral bronchiolitis (AVB), for reasons incompletely understood. Objectives: To characterize the cellular/molecular mechanisms underlying infant AVB in circulating cells/local airway tissues. Methods: Peripheral blood mononuclear cells and nasal scrapings were obtained from infants (<18 mo) and children (≥18 mo to 5 yr) during AVB and after convalescence. Immune response patterns were profiled by multiplex analysis of plasma cytokines, flow cytometry, and transcriptomics (RNA-Seq). Molecular profiling of group-level data used a combination of upstream regulator and coexpression network analysis, followed by individual subject-level data analysis using personalized N-of-1-pathways methodology. Measurements and Main Results: Group-level analyses demonstrated that infant peripheral blood mononuclear cell responses were dominated by monocyte-associated hyperupregulated type 1 IFN signaling/proinflammatory pathways (drivers: TNF [tumor necrosis factor], IL-6, TREM1 [triggering receptor expressed on myeloid cells 1], and IL-1B), versus a combination of inflammation (PTGER2 [prostaglandin E receptor 2] and IL-6) plus growth/repair/remodeling pathways (ERBB2 [erbb-b2 receptor tyrosine kinase 2], TGFB1 [transforming growth factor-ß1], AREG [amphiregulin], and HGF [hepatocyte growth factor]) coupled with T-helper cell type 2 and natural killer cell signaling in children. Age-related differences were not attributable to differential steroid usage or variations in underlying viral pathogens. Nasal mucosal responses were comparable qualitatively in infants/children, dominated by IFN types 1-3, but the magnitude of upregulation was higher in infants (range, 6- to 48-fold) than children (5- to 17-fold). N-of-1-pathways analysis confirmed differential upregulation of innate immunity in infants and natural killer cell networks in children, and additionally demonstrated covert AVB response subphenotypes that were independent of chronologic age. Conclusions: Dysregulated expression of IFN-dependent pathways after respiratory viral infections is a defining immunophenotypic feature of AVB-susceptible infants and a subset of children. Susceptible subjects seem to represent a discrete subgroup who cluster based on (slow) kinetics of postnatal maturation of innate immune competence.

Pregnancy Induces a Steady-State Shift in Alveolar Macrophage M1/M2 Phenotype That Is Associated With a Heightened Severity of Influenza Virus Infection: Mechanistic Insight Using Mouse Models.

BACKGROUND: Influenza virus infection during pregnancy is associated with enhanced disease severity. However, the underlying mechanisms are still not fully understood. We hypothesized that normal alveolar macrophage (AM) functions, which are central to maintaining lung immune homeostasis, are altered during pregnancy and that this dysregulation contributes to the increased inflammatory response to influenza virus infection. METHODS: Time-mated BALB/c mice were infected with a low dose of H1N1 influenza A virus at gestation day 9.5. Inflammatory cells in bronchoalveolar lavage (BAL) fluid were assessed by flow cytometry. RESULTS: Our findings confirm previous reports of increased severity of influenza virus infection in pregnant mice. The heightened inflammatory response detected in BAL fluid from infected pregnant mice was characterized by neutrophil-rich inflammation with concomitantly reduced numbers of AM, which were slower to return to baseline counts, compared with nonpregnant infected mice. The increased infection severity and inflammatory responses to influenza during pregnancy were associated with a pregnancy-induced shift in AM phenotype at homeostatic baseline, from the M1 (ie, classical activation) state toward the M2 (ie, alternative activation) state, as evidence by increased expression of CD301 and reduced levels of CCR7. CONCLUSION: These results show that pregnancy is associated with an alternatively activated phenotype of AM before infection, which may contribute to heightened disease severity.

Progressive increase of FcεRI expression across several PBMC subsets is associated with atopy and atopic asthma within school-aged children.

BACKGROUND: Antigen-specific IgE binds the Fcε receptor I (FcεRI) expressed on several types of immune cells, including dendritic cells (DCs). Activation of FcεRI on DCs in atopics has been shown to modulate immune responses that potentially contribute to asthma development. However, the extent to which DC subsets differ in FcεRI expression between atopic children with or without asthma is currently not clear. This study aimed to analyse the expression of FcεRI on peripheral blood mononuclear cells (PBMCs) from atopic children with and without asthma, and non-atopic/non-asthmatic age-matched healthy controls. METHODS: We performed multiparameter flow cytometry on PBMC from 391 children across three community cohorts and one clinical cohort based in Western Australia. RESULTS: We confirmed expression of FcεRI on basophils, monocytes, plasmacytoid and conventional DCs, with higher proportions of all cell populations expressing FcεRI in atopic compared to non-atopic children. Further, we observed that levels of FcεRI expression were elevated across plasmacytoid and conventional DC as well as basophils in atopic asthmatic compared to atopic non-asthmatic children also after adjusting for serum IgE levels. CONCLUSION: Our data suggest that the expression pattern of FcεRI on DC and basophils differentiates asthmatic from non-asthmatic atopic children. Given the significant immune modulatory effects observed as a consequence of FcεRI expression, this altered expression pattern is likely to contribute to asthma pathology in children.

Functional differences in airway dendritic cells determine susceptibility to IgE-sensitization.

Respiratory IgE-sensitization to innocuous antigens increases the risk for developing diseases such as allergic asthma. Dendritic cells (DC) residing in the airways orchestrate the immune response following antigen exposure and their ability to sample and present antigens to naïve T cells in airway draining lymph nodes contributes to allergen-specific IgE-sensitization. In order to characterize inhaled antigen capture and presentation by DC subtypes in vivo, we used an adjuvant-free respiratory sensitization model using two genetically distinct rat strains, one of which is naturally resistant and the other naturally susceptible to allergic sensitization. Upon multiple exposures to ovalbumin (OVA), the susceptible strain developed OVA-specific IgE and airway inflammation, whereas the resistant strain did not. Using fluorescently tagged OVA and flow cytometry, we demonstrated significant differences in antigen uptake efficiency and presentation associated with either IgE-sensitization or resistance to allergen exposures in respective strains. We further identified CD4+ conventional DC (cDC) as the subset involved in airway antigen sampling in both strains, however, CD4+ cDC in the susceptible strain were less efficient in OVA sampling and displayed increased MHC-II expression compared with the resistant strain. This was associated with generation of an exaggerated Th2 response and a deficiency of airway regulatory T cells in the susceptible strain. These data suggest that subsets of cDC are able to induce either sensitization or resistance to inhaled antigens as determined by genetic background, which may provide an underlying basis for genetically determined susceptibility to respiratory allergic sensitization and IgE production in susceptible individuals.

Immunological Processes Driving IgE Sensitisation and Disease Development in Males and Females.

IgE sensitisation has increased significantly over the last decades and is a crucial factor in the development of allergic diseases. IgE antibodies are produced by B cells through the process of antigen presentation by dendritic cells, subsequent differentiation of CD4⁺ Th2 cells, and class switching in B cells. However, many of the factors regulating these processes remain unclear. These processes affect males and females differently, resulting in a significantly higher prevalence of IgE sensitisation in males compared to females from an early age. Before the onset of puberty, this increased prevalence of IgE sensitisation is also associated with a higher prevalence of clinical symptoms in males; however, after puberty, females experience a surge in the incidence of allergic symptoms. This is particularly apparent in allergic asthma, but also in other allergic diseases such as food and contact allergies. This has been partly attributed to the pro- versus anti-allergic effects of female versus male sex hormones; however, it remains unclear how the expression of sex hormones translates IgE sensitisation into clinical symptoms. In this review, we describe the recent epidemiological findings on IgE sensitisation in male and females and discuss recent mechanistic studies casting further light on how the expression of sex hormones may influence the innate and adaptive immune system at mucosal surfaces and how sex hormones may be involved in translating IgE sensitisation into clinical manifestations.

Identification and Characterization of a Dendritic Cell Precursor in Parenchymal Lung Tissue.

The pulmonary parenchymal and mucosal microenvironments are constantly exposed to the external environment and thus require continuous surveillance to maintain steady-state immunological homeostasis. This is achieved by a mobile network of pulmonary dendritic cells (DC) and macrophages (mø) that constantly sample and process microenvironmental antigens into signals that can initiate or dampen inflammation, either locally or after onward migration to draining lymph nodes. The constant steady-state turnover of pulmonary DC and mø requires replenishment from bone marrow precursors; however, the nature of the pulmonary precursor cell (PC) remains unclear, although recent studies suggest that subsets of pulmonary DC may derive from circulating monocytic precursors. In the current study, we describe a population of cells in steady-state mouse lung tissue that has the surface phenotypic and ultrastructural characteristics of a common DC progenitor. Irradiation and reconstitution studies confirmed the bone marrow origins of this PC and showed that it had rapid depletion and reconstitution kinetics that were similar to those of DC, with a 50% repopulation by donor-derived cells by Days 7-9 after reconstitution. This was significantly faster than the rates observed for mø, which showed 50% repopulation by donor-derived cells beyond Days 16-21 after reconstitution. Purified PC gained antigen-presenting function and a cell surface phenotype similar to that of pulmonary DC after maturation in vitro, with light and electron microscopy confirming a myeloid DC morphology. To the best of our knowledge, this is the first study to describe a PC for DC in lung tissue; the findings have implications for the restoration of pulmonary immunological homeostasis after bone marrow transplant.

Altered immunity and dendritic cell activity in the periphery of mice after long-term engraftment with bone marrow from ultraviolet-irradiated mice.

Alterations to dendritic cell (DC) progenitors in the bone marrow (BM) may contribute to long-lasting systemic immunosuppression (>28 d) following exposure of the skin of mice to erythemal UV radiation (UVR). DCs differentiated in vitro from the BM of mice 3 d after UVR (8 kJ/m(2)) have a reduced capacity to initiate immunity (both skin and airways) when adoptively transferred into naive mice. Studies in IL-10(-/-) mice suggested that UV-induced IL-10 was not significantly involved. To investigate the immune capabilities of peripheral tissue DCs generated in vivo from the BM of UV-irradiated mice, chimeric mice were established. Sixteen weeks after reconstitution, contact hypersensitivity responses were significantly reduced in mice reconstituted with BM from UV-irradiated mice (UV-chimeric). When the dorsal skin of UV-chimeric mice was challenged with innate inflammatory agents, the hypertrophy induced in the draining lymph nodes was minimal and significantly less than that measured in control-chimeric mice challenged with the same inflammatory agent. When DCs were differentiated from the BM of UV-chimeric mice using FLT3 ligand or GM-CSF + IL-4, the cells maintained a reduced priming ability. The diminished responses in UV-chimeric mice were not due to different numerical or proportional reconstitution of BM or the hematopoietic cells in blood, lymph nodes, and skin. Erythemal UVR may imprint a long-lasting epigenetic effect on DC progenitors in the BM and alter the function of their terminally differentiated progeny.