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Background: Specific allergy sensitization pattern, using "component-resolved diagnosis" (CRD), is a central component of allergy and asthma in childhood. Besides this, allergic asthma has been characterized by a Th2-shifted endotype with elevation of classical Th2 cytokines. Recently, other endotypes with distinct mechanisms focusing on cytokine regulation evolved, yet those pathways are still not well understood., Objective: (a) To define reproducible immunological endotypes using cytokine expression in an asthma cohort and (b) to characterize their sensitization profile and clinical phenotype., Methods: Supernatants from PBMCs of 234 children (median age 10 years) of an asthma cohort were analysed for cytokine expressions. The children were split into a training (n = 49) and validation (n = 185) group. The training group was used to identify immunological endotypes by clustering cytokine expressions, which were then assessed regarding clinical characteristics and specific IgE of recombinant allergen components. Next, our findings were validated in the validation group., Results: We identified novel endotypes based on primarily unstimulated cytokine expression. One endotype showed an IFN-γ/Interleukin (IL)-17/IL-5 predominance, a different sensitization pattern (high in birch/apple; p < .01), and inferior lung function (p < .01). A second endotype grouped young children with food allergy and reduced lung function. Our findings were reproducible in the validation group., Conclusion and Clinical Relevance: We identified two novel clinical asthma endotypes via cytokine expression pattern with distinct sensitization patterns. These novel findings are critical for clinical guidance and open avenues for identifying underlying mechanisms and more patient-specific therapies., (© 2021 The Authors. Clinical & Experimental Allergy published by John Wiley & Sons Ltd.)

Levels of cytokines are used for in-depth characterization of patients with asthma; however, the variability over time might be a critical confounder. To analyze the course of serum cytokines in children, adolescents and adults with asthma and in healthy controls and to propose statistical methods to control for seasonal effects. Of 532 screened subjects, 514 (91·5%) were included in the All Age Asthma Cohort (ALLIANCE). The cohort included 279 children with either recurrent wheezing bronchitis (more than two episodes) or doctor-diagnosed asthma, 75 healthy controls, 150 adult asthmatics and 31 adult healthy controls. Blood samples were collected and 25 μl serum was used for analysis with the Bio-Plex Pr human cytokine 27-Plex assay. Mean age, body mass index and gender in the three groups of wheezers, asthmatic children and adult asthmatics were comparable to healthy controls. Wheezers (34·5%), asthmatic children (78·7%) and adult asthmatics (62·8%) were significantly more often sensitized compared to controls (4·5, 22 and 22·6%, respectively). Considering the entire cohort, interleukin (IL)-1ra, IL-4, IL-9, IL-17, macrophage inflammatory protein (MIP)-1- α and tumor necrosis factor (TNF)- α showed seasonal variability, whereas IL-1β, IL-7, IL-8, IL-13, eotaxin, granulocyte colony-stimulating factor (G-CSF), interferon gamma-induced protein (IP)-10, MIP-1 β and platelet-derived growth factor (PDGF)-BB did not. Significant differences between wheezers/asthmatics and healthy controls were observed for IL-17 and PDGF-BB, which remained stable after adjustment for the seasonality of IL-17. Seasonality has a significant impact on serum cytokine levels in patients with asthma. Because endotyping has achieved clinical importance to guide individualized patient-tailored therapy, it is important to account for seasonal effects., (© 2020 British Society for Immunology.)

Background: Respiratory tract infections and their symptoms are frequent during early childhood, but their risk factors, including the effect of early immune regulation, are less known. The aim of the study was to analyze whether stimulated cord blood cytokine production is associated with the frequency of respiratory tract infection symptoms or infections during the first year of life., Methods: The study population consisted of children of mothers from farm or non-farm rural environment from Austria, Finland, Germany, and Switzerland who participated in a prospective birth cohort study (PASTURE: Protection against Allergy-Study in Rural Environments) (N = 550). Cord blood samples were stimulated with the combination of phorbol ester and ionomycin (P/I) for 24 h, and the production of IL-5, IL-10, TNF-α, and IFN-γ was determined using ELISA. Information about infectious morbidity was collected using weekly diaries., Results: P/I-stimulated production of IL-5 (adjusted risk ratio (aRR) for ≤median production, 0.37; 95% confidence interval (CI), 0.25-0.55, aRR for >median production, 0.41; 95% CI, 0.27-0.61 vs. production median production, 0.39; 95% CI, 0.25-0.62 vs. production

Background: Farm exposure has been shown to protect from childhood asthma and allergic diseases, but underlying immunological mechanisms are not clear yet., Objective: To explore whether farming lifestyle determines cytokine profile of peripheral blood mononuclear cells (PBMCs) of 4.5-year-old children (n = 88) from the Finnish PASTURE birth cohort study., Methods: We analysed regulatory (IL-10, IL-2), T helper 1 (Th1)-associated (IL-12, IFN-γ), inflammatory (IL-1β, TNF, CXCL8) and Th2-associated (IL-13) cytokines in unstimulated PBMCs and after a short-term (5 h) stimulation with lipopolysaccharide (LPS). Specific farm exposures (stables, hay barn, farm milk) at age 4 years were assessed from questionnaires., Results: The unstimulated PBMCs of farm children produced more IL-10 (GMR 1.22, P = 0.032), IL-12 (GMR 1.24, P = 0.012) and IFN-γ (GMR 1.24, P = 0.024) than those of non-farm children. Also, specific farm exposures were associated with higher spontaneous production of cytokines. The number of specific farm exposures tended to be dose dependently associated with higher spontaneous production of IFN-γ (test for trends, P = 0.013) and lower LPS-induced production of TNF (test for trends, P = 0.025)., Conclusion and Clinical Relevance: Farming lifestyle seemed to be associated with increased spontaneous production of Th1 and regulatory cytokines. Decreased TNF responses to short-term LPS stimulation in farm-exposed children may imply tolerogenic immune mechanisms. These novel findings might contribute to the asthma and allergy protection in farm environment., (© 2015 John Wiley & Sons Ltd.)

Background: The transcription factor STAT6 is crucial for activation of the interleukin (IL)-4/IL-13 pathway and has been linked to regulatory T cells (Tregs). Associations of STAT6 polymorphisms with IgE levels were described; however, their impact on neonatal immune responses and early disease development is unknown., Methods: STAT6 polymorphisms were genotyped in cord blood mononuclear cells by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Gene expression was assessed by real-time polymerase chain reaction (PCR) and cytokines by Multiplex. At age 3 years, atopic diseases were assessed by questionnaires., Results: STAT6 rs324011 but not rs1059513 polymorphism was associated with significant or borderline significant decreased mRNA expression of Treg-associated genes (FOXP3, GITR, LAG3). Heterozygotes and minor allele homozygotes of rs324011 had low levels of tumor necrosis factor alpha (TNF-α) and increased interferon gamma (IFN-γ) (P ≤ 0.04), while heterozygotes and minor allele homozygotes of rs1059513 had increased TNF-α and Granulocyte-macrophage colony-stimulating factor (GM-CSF) (P ≤ 0.05). In minor allele homozygotes of rs324011, expression of Treg-associated genes was strongly inverse correlated with IFN-γ (unstimulated, r = -0.7, P = 0.111; LpA stimulation, r = -0.8, P = 0.011), but not in heterozygotes or major allele homozygotes. Heterozygotes and minor allele homozygotes of rs324011 presented a lower risk of atopic dermatitis and obstructive bronchitis until age 3 years., Conclusions: Two STAT6 polymorphisms were associated with altered immune responses already at birth. STAT6 rs324011 was associated with lower neonatal Treg and increased Th1 response. Those neonates had a lower risk of atopic dermatitis and obstructive bronchitis until 3 years. Our data suggest a role for STAT6 polymorphisms in early immune regulation and implications on early atopic disease development., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: For several immune-mediated diseases, immunological analysis will become more complex in the future with datasets in which cytokine and gene expression data play a major role. These data have certain characteristics that require sophisticated statistical analysis such as strategies for non-normal distribution and censoring. Additionally, complex and multiple immunological relationships need to be adjusted for potential confounding and interaction effects., Objective: We aimed to introduce and apply different methods for statistical analysis of non-normal censored cytokine and gene expression data. Furthermore, we assessed the performance and accuracy of a novel regression approach in order to allow adjusting for covariates and potential confounding., Methods: For non-normally distributed censored data traditional means such as the Kaplan-Meier method or the generalized Wilcoxon test are described. In order to adjust for covariates the novel approach named Tobit regression on ranks was introduced. Its performance and accuracy for analysis of non-normal censored cytokine/gene expression data was evaluated by a simulation study and a statistical experiment applying permutation and bootstrapping., Results: If adjustment for covariates is not necessary traditional statistical methods are adequate for non-normal censored data. Comparable with these and appropriate if additional adjustment is required, Tobit regression on ranks is a valid method. Its power, type-I error rate and accuracy were comparable to the classical Tobit regression., Conclusion: Non-normally distributed censored immunological data require appropriate statistical methods. Tobit regression on ranks meets these requirements and can be used for adjustment for covariates and potential confounding in large and complex immunological datasets.

Background: TBX21 (T cell specific T-box transcription factor) and HLX1 (H.20-like homeobox 1) are crucial transcription factors of T(H)1-cells, inducing their differentiation and suppressing T(H)2 commitment, particularly important for early life immune development. This study investigated the influence of TBX21 and HLX1 single nucleotide polymorphisms (SNPs), which have previously been shown to be associated with asthma, on T(H)1/T(H)2 lineage cytokines at birth., Methods and Findings: Cord blood mononuclear cells (CBMCs) of 200 neonates were genotyped for two TBX21 and three HLX1 SNPs. CBMCs were stimulated with innate (Lipid A, LpA; Peptidoglycan, Ppg), adaptive stimuli (house dust mite Dermatophagoides pteronyssinus 1, Derp1) or mitogen (phytohemagglutinin, PHA). Cytokines, T-cells and mRNA expression of T(H)1/T(H)2-related genes were assessed. Atopic diseases during the first 3 years of life were assessed by questionnaire answered by the parents. Carriers of TBX21 promoter SNP rs17250932 and HLX1 promoter SNP rs2738751 showed reduced or trendwise reduced (p≤0.07) IL-5, IL-13 and TNF-α secretion after LpA-stimulation. Carriers of HLX1 SNP rs2738751 had lower IL-13 levels following Ppg-stimulation (p = 0.08). Carriers of HLX1 exon 1 SNP rs12141189 showed increased IL-5 (LpA, p = 0.007; Ppg, p = 0.10), trendwise increased IL-13 (LpA), higher GM-CSF (LpA/Ppg, p≤0.05) and trendwise decreased IFN-γ secretion (Derp1+LpA-stimulation, p = 0.1). Homozygous carriers of HLX1 promoter SNP rs3806325 showed increased IL-13 and IL-6 (unstimulated, p≤0.03). In carriers of TBX21 intron 3 SNP rs11079788 no differences in cytokine secretion were observed. mRNA expression of T(H)1/T(H)2-related genes partly correlated with cytokines at protein level. TBX21 SNP rs11079788 carriers developed less symptoms of atopic dermatitis at 3 years of age (p = 0.03)., Conclusions: Polymorphisms in TBX21 and HLX1 influenced primarily IL-5 and IL-13 secretion after LpA-stimulation in cord blood suggesting that genetic variations in the transcription factors essential for the T(H)1-pathway may contribute to modified T(H)2-immune responses already early in life. Further follow-up of the cohort is required to study the polymorphisms' relevance for immune-mediated diseases such as childhood asthma.

Background: There is strong evidence that reduced exposures to microbial compounds triggering innate immune responses early in life are critical for the development of allergic illnesses. The underlying mechanisms remain unknown, but will include T-cell responses either along T helper type 1 (Th1)/Th2 pathways or via T regulatory and Th17 cells. Yet, little is known about innate immune responses and the function of T regulatory/Th17 cells at birth. The aim of this study was to investigate T-cell responses to innate (Lipid A/LpA, peptidoglycan/Ppg) and adaptive (phytohemagglutinin) stimuli at birth and to compare these findings with adult immune responses., Methods: Cord and peripheral blood mononuclear cells including T regulatory and Th17 cells from 25 neonates and 25 adults were examined for proliferation, cytokine secretion, surface, mRNA expression and functional suppression assays., Results: Proliferation and cytokine responses to innate stimuli were less mature at birth than in adulthood. T regulatory and Th17 cells were less expressed in cord than in adult blood (Ppg-induced Foxp3, P = 0.001, LpA-induced CD4(+) CD25(+) high, P = 0.02; Th17 : P < 0.0001). Mitogen-induced suppression of T-regulatory cells on T-effector cell function was less efficient in cord than in adult blood (P = 0.01). At both ages, Th17 cells were correlated with Th1/Th2 cells (P < 0.01), but not with interleukin-10 secretion following innate-stimulation., Conclusion: Innate immune responses are immature at birth. Furthermore, the function of T regulatory and Th17 cells is impaired. Th17 cells in association with Th1/Th2 cells may be involved in early immuno-modulation. Potent innate immune stimulation early in life can potentially contribute to protection from allergic diseases.

Objectives: Little is known about the relation between cytokine profile at birth and acute lower respiratory illnesses in the first year of life. The purpose of this work was to examine the relation between cytokine secretions by cord blood mononuclear cells and acute lower respiratory illness in a birth cohort of 297 children., Methods: Cord blood mononuclear cells were isolated, and secretion of interferon-gamma, interleukin-13, interleukin-10, and tumor necrosis factor-alpha at baseline and in response to allergens (Blatella germanica 2 and Dermatophagoides farinae 1) and mitogen (phytohemagglutinin) were quantified using enzyme-linked immunosorbent assay. Acute lower respiratory illness was defined as a parental report of a diagnosis of bronchiolitis, pneumonia, bronchitis, and/or croup by a health care professional in the first year of life. Differences in the levels of cord blood cytokines between children with and without acute lower respiratory illness were examined using 2-sample Wilcoxon tests. Logistic regression models were used to examine the relation between various categories of cord blood cytokines and acute lower respiratory illness., Results: Median levels of interferon-gamma secreted by cord blood mononuclear cells in response to Blatella germanica 2 and Dermatophagoides farinae 1 were higher among children without acute lower respiratory illness as compared with children with acute lower respiratory illness. After adjustment for other covariates, the odds of acute lower respiratory illness was reduced among children in the top category (at or more than the median of detectable values) of interferon-gamma level, significantly so in response to Blatella germanica 2., Conclusions: In a cohort of children from the general population, we found that upregulated interferon-gamma secretion at birth is associated with reduced risk of acute lower respiratory illness in the first year of life.

Abstract: Cross‐sectional studies have shown that exposure to indoor moisture damage and mold may be associated with subclinical inflammation. Our aim was to determine whether early age exposure to moisture damage or mold is prospectively associated with subclinical systemic inflammation or with immune responsiveness in later childhood. Home inspections were performed in children's homes in the first year of life. At age 6 years, subclinical systemic inflammation was measured by serum C‐reactive protein (CRP) and blood leukocytes and immune responsiveness by ex vivo production of interleukin 1‐beta (IL‐1β), IL‐6, and tumor necrosis factor alpha (TNF‐α) in whole blood cultures without stimulation or after 24 hours stimulation with phorbol 12‐myristate 13‐acetate and ionomycin (PI), lipopolysaccharide (LPS), or peptidoglycan (PPG) in 251‐270 children. Moisture damage in child's main living areas in infancy was not significantly associated with elevated levels of CRP or leukocytes at 6 years. In contrast, there was some suggestion for an effect on immune responsiveness, as moisture damage with visible mold was positively associated with LPS‐stimulated production of TNF‐α and minor moisture damage was inversely associated with PI‐stimulated IL‐1β. While early life exposure to mold damage may have some influence on later immune responsiveness, it does not seem to increase subclinical systemic inflammation in later life. [ABSTRACT FROM AUTHOR]

This study investigated the association between confirmed moisture damage in homes and systemic subclinical inflammation in children. Home inspections were performed in homes of 291 children at the age of 6 years. Subclinical inflammation at the age of 6 years was assessed by measuring the circulating levels of C-reactive protein ( CRP) and leukocytes in peripheral blood and fractional exhaled nitric oxide (Fe NO). Proinflammatory cytokines interleukin ( IL)-1 β and IL-6 and tumor necrosis factor ( TNF)- α were measured in unstimulated, and in phorbol 12-myristate 13-acetate and ionomycin ( PI), lipopolysaccharide ( LPS), or peptidoglycan ( PPG)-stimulated whole blood. Major moisture damage in the child's main living areas (living room, kitchen, or child's bedroom) and moisture damage with mold in the bathroom were associated with increased levels of CRP and stimulated production of several proinflammatory cytokines. There were no significant associations between moisture damage/visible mold and leukocyte or Fe NO values. The results suggest that moisture damage or mold in home may be associated with increased systemic subclinical inflammation and proinflammatory cytokine responsiveness. [ABSTRACT FROM AUTHOR]

Background: Interleukin (IL) 37 has been described as a negative regulator of innate immunity, as it reduces the activation and cytokine production of different innate immune cells. Recently, results from the CLARA childhood asthma cohort suggested an implication of IL-37 for human asthma pathogenesis. This study aimed to investigate the effects of IL-37 on allergic airway inflammation in a mouse model of experimental asthma. Methods: Peripheral blood mononuclear cells (PBMCs) of children were cultured for 48 h (anti-CD3/anti-CD28 stimulation or unstimulated), and IL-37 concentrations in supernatants were determined. Wild-type, IL-18Rα-deficient (-/-), and SIGIRR -/- C57BL/6 mice were sensitized to ovalbumin (OVA) and challenged with OVA aerosol to induce acute experimental asthma, and IL-37 was applied intranasally prior to each OVA challenge. Airway hyper-responsiveness (AHR), airway inflammation, cytokine levels in broncho-alveolar lavage fluid, and mucus production were determined. Results: IL-37 production of human PBMCs was significantly lower in allergic asthmatics vs healthy children. In wild-type mice, intranasal administration of IL-37 ablated allergic airway inflammation as well as cytokine production and subsequently diminished the hallmarks of experimental asthma including mucus hyperproduction and AHR. In contrast, local application of IL-37 produced none of these effects in mice lacking either IL18Rα or SIGIRR/IL-1R8. Conclusions: This study demonstrates that IL-37 is able to ablate a TH2 celldirected allergic inflammatory response and the hallmarks of experimental asthma in mice, suggesting that IL-37 may be critical for asthma pathogenesis. Furthermore, these data suggest a mode of action of IL-37 that involves IL18Rα as well as the orphan receptor SIGIRR/IL-1R8. [ABSTRACT FROM AUTHOR]

The collectin surfactant protein D (SP-D) confers protection against pulmonary infection and inflammation. Recent data suggest a role for SP-D in the modulation of allergic inflammation.The aim of this study is to characterize the immune responses of SP-D-deficient (SP-D−/−) mice in a kinetic model of allergic inflammation. We determined whether allergic parameters were enhanced in SP-D−/− micein vivo. Further, we examined whether functional immune responsesin vitrosuch as lymphocyte proliferation (LP) and cytokine production were modulated in the absence of SP-D.In vivo, wild-type (WT) and SP-D−/− mice were sensitized and challenged with the allergen ovalbumin (OVA) and assessed for allergic parameters (bronchoalveolar lavage (BAL) eosinophils, IL-13 production, pulmonary IFN-γ, IL-10 expression) at early time points (1 and 3 days of challenge) in comparison with late time points (7 days of challenge).In vitro, spleen cells from WT and SP-D−/− mice were stimulated with the mitogen concanavalin A (ConA) and lipid A (LpA) and analysed for LP, IL-13 and IFN-γ production. Toll-like receptor 4 (TLR4), ligand for LpA, was assessed by mRNA expression and immunohistochemistryin vivo.Following allergen exposurein vivo, SP-D−/− mice expressed higher BAL eosinophils and IL-13 concentrations and lower IFN-γ expression at early time points compared with WT mice. IL-10 expression was increased at early time points in SP-D−/− compared with WT mice. Allergen-induced TLR4 expression was increased in WT, but not in SP-D−/− mice. After stimulation with LpA and ConAin vitroLP was increased and IFN-γ concentration was decreased in SP-D−/− mice.SP-D may be critical for the modulation of early stages of allergic inflammationin vivo. [ABSTRACT FROM AUTHOR]