Natural protective immunity against grass pollen allergy is maintained by a diverse spectrum of response types
2017; Elsevier BV; Volume: 140; Issue: 6 Linguagem: Inglês
10.1016/j.jaci.2017.07.030
ISSN1097-6825
AutoresAlmedina Kurtaj, Christoph Hillebrand, Gerda Fichtinger, E. Hattinger, Melanie Lietzenmayer, Yoan Machado, Sandra Scheiblhofer, Angelika Stoecklinger, Theresa Thalhamer, Susanne Suessner, Martin Danzer, Sabine Keplinger, Johannes Weinberger, Susanne Schaller, Stephan Winkler, Christian Gabriel, Josef Thalhamer, Richard Weiss,
Tópico(s)Dermatology and Skin Diseases
ResumoAlthough allergen-specific TH2-type immune responses in allergic patients have been studied in great detail, the mechanisms underlying natural protection from allergy are far less understood. As highlighted in a recently published editorial ("It's a lot of work to be nonallergic"),1Sette A. Schulten V. It's a lot of work to be non-allergic.J Allergy Clin Immunol. 2017; 139: 769-770Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar a new concept is arising that nonallergic subjects mount "healthy" immune responses against allergens, including regulation of both epithelial barrier function2Ahuja S.K. Manoharan M.S. Harper N.L. Jimenez F. Hobson B.D. Martinez H. et al.Preservation of epithelial cell barrier function and muted inflammation in resistance to allergic rhinoconjunctivitis from house dust mite challenge.J Allergy Clin Immunol. 2017; 139: 844-854Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar and T-cell responses.3Akdis M. Verhagen J. Taylor A. Karamloo F. Karagiannidis C. Crameri R. et al.Immune responses in healthy and allergic individuals are characterized by a fine balance between allergen-specific T regulatory 1 and T helper 2 cells.J Exp Med. 2004; 199: 1567-1575Crossref PubMed Scopus (772) Google Scholar, 4Bellinghausen I. Klostermann B. Knop J. Saloga J. Human CD4+CD25+ T cells derived from the majority of atopic donors are able to suppress TH1 and TH2 cytokine production.J Allergy Clin Immunol. 2003; 111: 862-868Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 5Hinz D. Seumois G. Gholami A.M. Greenbaum J.A. Lane J. White B. et al.Lack of allergy to timothy grass pollen is not a passive phenomenon but associated with the allergen-specific modulation of immune reactivity.Clin Exp Allergy. 2016; 46: 705-719Crossref PubMed Scopus (9) Google Scholar, 6Skrindo I. Farkas L. Kvale E.O. Johansen F.E. Jahnsen F.L. Depletion of CD4+CD25+CD127lo regulatory T cells does not increase allergen-driven T cell activation.Clin Exp Allergy. 2008; 38: 1752-1759PubMed Google Scholar We have studied the latter in a large cohort of 228 nonallergic subjects and found that the diversity of naturally acquired immunity against the major grass pollen allergen Phl p 5 is much higher than hitherto postulated. Thus it becomes clear that the established viewpoint attributing protection from type I allergic diseases primarily to induction of IL-10–producing type 1 regulatory (TR1) cells has been too narrow. Furthermore, for the first time, we demonstrated that the environment has a significant effect on the prevalence of naturally acquired immune response types. In our study we compared healthy donors from 2 different environmental backgrounds: traditional farming with a high level of grass pollen and microbial exposure7Hanski I. von Hertzen L. Fyhrquist N. Koskinen K. Torppa K. Laatikainen T. et al.Environmental biodiversity, human microbiota, and allergy are interrelated.Proc Natl Acad Sci U S A. 2012; 109: 8334-8339Crossref PubMed Scopus (291) Google Scholar and urban life (see Table E1 in this article's Online Repository at www.jacionline.org for demographic characteristics and farm-related exposures). Although all subjects of the allergic control group were seropositive for Phl p 5–specific IgG1 and IgG4, we found 4 different groups of healthy subjects: donors producing only IgG1, only IgG4, both, or neither subclass. As shown in Fig 1, A, farmers displayed a significantly different IgG response pattern compared with townspeople because they were mainly IgG nonresponders (24%) or IgG1 single responders (73%). In general, IgG titers were lower in nonallergic subjects, especially with respect to IgG4, levels of which were significantly reduced in townspeople compared with allergic donors (Fig 1, B). This was also reflected by a significantly higher IgG1/IgG4 ratio in both nonallergic groups (Fig 1, C). Interestingly, we detected similar levels of Phl p 5–specific IgA within the 3 study groups (Fig 1, D). Our data are in agreement with previous findings8Pereira E.A. Silva D.A. Cunha-Junior J.P. Almeida K.C. Alves R. Sung S.J. et al.IgE, IgG1, and IgG4 antibody responses to Blomia tropicalis in atopic patients.Allergy. 2005; 60: 401-406Crossref PubMed Scopus (32) Google Scholar showing that antibody production against a ubiquitous antigen is not an exception but the rule, thus questioning the hypothesis of immunologic ignorance as the dominant mechanism to prevent allergy. To analyze T-cell polarization types, we expanded Phl p 5–specific memory T cells from PBMCs. T-cell receptor clonotype analysis confirmed that only a restricted pool of T cells was enriched, which showed specific reactivity against Phl p 5 but not an irrelevant antigen (see Figs E1 and E2 in this article's Online Repository at www.jacionline.org). Although expanded T cells from nonallergic farmers and townspeople display similar levels of proliferation (data not shown), they differed significantly in their patterns of secreted cytokines. Both nonallergic groups showed significantly higher numbers of IFN-γ–secreting cells compared with IL-10–secreting cells (Fig 2, A), which was confirmed by a higher expression of T-box transcription factor (T-bet) compared with forkhead box protein 3 (FoxP3; Fig 2, B). Surprisingly, townspeople had an enhanced potential for proinflammatory immune responses compared with farmers, as indicated by higher expression of T-bet and retinoic acid–related orphan receptor γt (RORγt), as well as higher prevalence for a TH1 polarization pattern (Fig 2, C). The high prevalence for inflammatory response types in both nonallergic populations was confirmed by means of multiplex analysis of a broader cytokine panel (Fig 2, D-F). Induction of IL-10–secreting TR1 cells in the course of allergen immunotherapy and detection of IL-10–secreting regulatory T (Treg) cells in nonatopic subjects3Akdis M. Verhagen J. Taylor A. Karamloo F. Karagiannidis C. Crameri R. et al.Immune responses in healthy and allergic individuals are characterized by a fine balance between allergen-specific T regulatory 1 and T helper 2 cells.J Exp Med. 2004; 199: 1567-1575Crossref PubMed Scopus (772) Google Scholar suggested "tolerance" as the general dominant mechanism underlying a healthy nonallergic status. However, in our current study IL-10 levels were significantly lower in nonallergic donors than in allergic subjects (Fig 2, G), ruling out a dominant role of TR1 cells in naturally acquired protection against Phl p 5. As expected, T cells of allergic subjects showed increased expression of TH2-related cytokines, but also several nonallergic donors mounted T-cell responses with substantial expression levels of IL-5 and IL-13 (Fig 2, E). However, these subjects apparently compensate and balance this partial TH2 polarization, with T cells expressing proinflammatory or regulatory cytokines. In contrast to allergic donors, farmers and townspeople secreted considerable amounts of proinflammatory cytokines together with TH2 cytokines (see Fig E3 in this article's Online Repository at www.jacionline.org). Taking a closer look at the cytokine profiles of nonallergic donors who produced increased levels of TH2 cytokines, we found the pattern of proinflammatory or regulatory cytokines to be highly diverse, ranging from almost exclusive use of IFN-γ and GM-CSF as compensatory cytokines to mixed responses comprised of up to 8 different cytokines (of 9 measured). A dominant TR1 response was rarely observed (3/19 donors) and always accompanied by proinflammatory cytokines (see Figs E4 and E5 in this article's Online Repository at www.jacionline.org). As shown in Fig E6 in this article's Online Repository at www.jacionline.org, the compensatory secretion of proinflammatory and regulatory cytokines was reflected by a statistically significant correlation with TH2 cytokines in both farmers and townspeople. No such correlation was observed in allergic donors. Previous studies have shown contradictory results regarding the contribution of TR1, CD4+CD25+ Treg, and IFN-γ–secreting cells to protection from allergy in healthy subjects.3Akdis M. Verhagen J. Taylor A. Karamloo F. Karagiannidis C. Crameri R. et al.Immune responses in healthy and allergic individuals are characterized by a fine balance between allergen-specific T regulatory 1 and T helper 2 cells.J Exp Med. 2004; 199: 1567-1575Crossref PubMed Scopus (772) Google Scholar, 4Bellinghausen I. Klostermann B. Knop J. Saloga J. Human CD4+CD25+ T cells derived from the majority of atopic donors are able to suppress TH1 and TH2 cytokine production.J Allergy Clin Immunol. 2003; 111: 862-868Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 5Hinz D. Seumois G. Gholami A.M. Greenbaum J.A. Lane J. White B. et al.Lack of allergy to timothy grass pollen is not a passive phenomenon but associated with the allergen-specific modulation of immune reactivity.Clin Exp Allergy. 2016; 46: 705-719Crossref PubMed Scopus (9) Google Scholar, 6Skrindo I. Farkas L. Kvale E.O. Johansen F.E. Jahnsen F.L. Depletion of CD4+CD25+CD127lo regulatory T cells does not increase allergen-driven T cell activation.Clin Exp Allergy. 2008; 38: 1752-1759PubMed Google Scholar Nevertheless, together with our data, it can be clearly ruled out that IL-10–secreting Treg cells represent the only players defining the non-allergic status. Notably, we identified a small percentage of nonallergic subjects who mount allergen-specific TH17/TH22 responses against Phl p 5 (Fig 2, F), which putatively contribute to regulation of immune responses. Although TH17 responses have been described mainly for their potential to exacerbate lung inflammation in allergic patients, recently, a high degree of instability and plasticity of TH17 cells toward regulatory functions has been described.9Gagliani N. Amezcua Vesely M.C. Iseppon A. Brockmann L. Xu H. Palm N.W. et al.Th17 cells transdifferentiate into regulatory T cells during resolution of inflammation.Nature. 2015; 523: 221-225Crossref PubMed Scopus (540) Google Scholar In summary, a picture emerges indicating that induction and maintenance of the nonallergic state is a dynamic and active immunologic process using multiple mechanisms in addition to Treg cell–based tolerance or TH1-based immune deviation. For the first time, the present study unequivocally demonstrates in a large cohort of healthy subjects that protection from type I allergic diseases is not exclusively mediated by IL-10–producing TR1 cells. Instead, a strikingly broad spectrum of TH response types counteracts allergic reactions. As discussed in more detail in the Methods and Discussion sections in this article's Online Repository at www.jacionline.org, our data are in principle agreement with the hygiene hypothesis10Romagnani S. Coming back to a missing immune deviation as the main explanatory mechanism for the hygiene hypothesis.J Allergy Clin Immunol. 2007; 119: 1511-1513Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar and point to a variable and complex immune deviation (predominantly of the proinflammatory phenotype) as a natural protective response against allergens. Moreover, in light of our results, it seems reasonable to rethink the numerous concepts that have been demonstrated to trigger antiallergic immune responses. Some of them might carry a potential to develop protective and therapeutic antiallergic immunity similar to that evolved in nature. We thank Dr Sven Mostböck for his contributions, suggestions, and critical discussion. Experiments involving human material were conducted in accordance with the guidelines of the World Medical Association's Declaration of Helsinki. Samples of human origin have been obtained from blood donations, and use of residual cells was approved by the Institutional Review Board of Upper Austria (EK Nr.E-6-10 2.1.5.). All participants provided informed written consent. After the blood samples had been collected, each participant was allocated a study number, demographic data were collected, and all data were anonymized in a database. The Red Cross Blood Transfusion Service Center Linz (Upper Austria, Austria) recruited a total of 228 nonallergic healthy adult subjects during the grass pollen season (May-June 2012) and screened for subjects who live within a farming or urban environment. Nonallergic healthy donors reported no history of any atopic diseases or allergic symptoms and had no detectable IgE titers against Phl p 5. Donors from a farming environment had to answer a questionnaire regarding their working and living conditions on the farm. As a control group, 11 allergic adult subjects with symptoms or a clinical history of allergic diseases and positive IgE titers against Phl p 5 were recruited during the grass pollen season (June 2015). The demographic and lifestyle characteristics are reported in Table E1. Human PBMCs were isolated from freshly drawn peripheral venous blood by means of density gradient centrifugation. Additionally, plasma samples from all donors were collected. PBMCs and plasma samples were stored in aliquots in liquid nitrogen until analysis. rPhl p 5 was expressed in Escherichia coli, as previously described.E1Vrtala S. Susani M. Sperr W.R. Valent P. Laffer S. Dolecek C. et al.Immunologic characterization of purified recombinant timothy grass pollen (Phleum pratense) allergens (Phl p 1, Phl p2, Phl p 5).J Allergy Clin Immunol. 1996; 97: 781-787Abstract Full Text PDF PubMed Scopus (100) Google Scholar Bacterial endotoxin was detected by using the Limulus Amebocyte Lysate assay. LPS levels were less than 1.5 pg/μg. Phl p 5–specific plasma IgA, IgE, IgG1, and IgG4 levels were determined by using an ELISA. High-binding half-area microtiter plates were coated with rPhl p 5 (1 μg/mL in Dulbecco-PBS) overnight at 4°C and then blocked with BSA at 3% in Dulbecco-PBS for 1 hour at room temperature. Samples and reference sera were diluted and incubated for 2 hours at room temperature. Subsequently, plates were incubated with horseradish peroxidase–conjugated secondary anti-human IgE (SouthernBiotech, Birmingham, Ala), anti-human IgA (Bio-Rad Laboratories, Hercules, Calif), anti-human IgG1 (Life Technologies, Grand Island, NY), or anti-human IgG4 (Life Technologies; all at 1:500) antibodies for 1 hour at room temperature. The assay was developed by adding Luminol (BM Chemiluminescence ELISA substrate, Roche Diagnostics, Mannheim, Germany). Plates were measured on a Tecan Infinite 200 Pro microplate reader (Tecan, Männedorf, Switzerland). All ELISA experiments were performed in triplicates. The reference curve was constructed from a pool of sera containing high amounts of IgA, IgE, IgG1, and IgG4 to Phl p 5, and units (corresponding to the specific titers of the reference serum pool) were calculated by using the reference line method, as described elsewhere.E2Reizenstein E. Hallander H.O. Blackwelder W.C. Kuhn I. Ljungman M. Mollby R. Comparison of five calculation modes for antibody ELISA procedures using pertussis serology as a model.J Immunol Methods. 1995; 183: 279-290Crossref PubMed Scopus (82) Google Scholar Subjects were divided into responders (IgG level greater than the cutoff) and nonresponders (IgG level less than the cutoff) based on the calculated cutoff:(2×[Meanofblanks+(2×SDofblanks)]). Frozen PBMCs were thawed and resuspended in X-VIVO 15 medium (Lonza, Basel, Switzerland) supplemented with 5% heat-inactivated male human AB serum (Sigma-Aldrich, St Louis, Mo), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (complete X-VIVO 15) at a concentration of 1 × 107 cells/mL in 6-well plates and stimulated with rPhl p 5 (30 μg/mL). Cells were kept at 37°C in 5% CO2, and 10 U/mL human rIL-2 (PeproTech, Rocky Hills, NJ) was added 6 days after culture setup. On day 12, cells were restimulated with rPhl p 5 (30 μg/mL) in the presence of autologous irradiated (60 Gy, 80 kV, and 26 mA) PBMCs (feeder cells) and rIL-2 (10 U/mL) for another 3 days. Finally, the nonadherent cells were harvested, washed, and cultured at a concentration of 1 × 107 cells/mL in 6-well plates in complete X-VIVO 15 and incubated for a resting phase of 13 days to enrich allergen-specific memory T cells. Production of IFN-γ, IL-10, and IL-4 from cultured cells in response to antigen stimulation was detected by using the ELISpot technique. Ninety-six-well polyvinylidene difluoride bottom plates (Millipore, Temecula, Calif) were activated for 10 minutes by using 70% ethanol, washed, and coated with 4 μg/mL anti-human IFN-γ (clone NIB42; eBioscience, San Diego, Calif), anti-human IL-10 (clone JES3-9D7; BioLegend, San Diego, Calif), or anti-human IL-4 (clone 8D4-8; BioLegend) overnight at 4°C. Then the plates were incubated for 1 hour at 37°C with blocking solution (X-VIVO 15 medium and 10% human serum) and washed afterward with Dulbecco-PBS. After the resting phase, cells were harvested, and 1 × 105 cells per well were cultured in the presence of 2 × 105 irradiated (60 Gy, 80 kV, and 26 mA) autologous PBMCs with or without 30 μg/mL rPhl p 5. After culture for 48 hours at 37°C in a 5% CO2 atmosphere, cells were removed by means of extensive washing, and cytokines were detected with a biotinylated anti-human IFN-γ (clone 4S.B3; BioLegend), anti-human IL-10 (clone JES3-12G8; eBioscience), or anti-human IL-4 (clone MP4-25D2; BioLegend), followed by horseradish peroxidase–conjugated streptavidin (BioLegend) and enzyme reaction with 3-amino-9-ethylcarbazole substrate. The reaction was stopped after 1 hour by washing with water, and plates were air-dried before spot counting by using ImageJ software (Wayne Rasband, National Institutes of Health, Bethesda, Md). Spot numbers in unstimulated wells (medium only) were subtracted from those obtained in stimulated wells (protein), and spot-forming units were calculated per 105 cells. According to their cytokine patterns, subjects were attributed to TH1 (IFN-γ > IL-10 and IL-4), TH2 (IL-4 > IFN-γ and IL-10), TR1 (IL-10 > IFN-γ and IL-4), and nonpolarized (<10 spots for all 3 cytokines) immune response types. Additionally, cytokines were measured in supernatants from identically prepared cocultures after 72 hours of incubation with a human cytokine 10-plex panel (Procarta Plex; eBioscience; IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IFN-γ, TNF-α, and GM-CSF) or a quantitative luminometric ELISA (Human ELISA Ready-SET-GO!; eBioscience; IL-2, IL-21, and IL-22), according to the manufacturer's protocol. The 5-parameter logarithmic curve fit (5PL) was used to create the standard curves using Procarta Plex Analyst (eBioscience). The lower Limit of quantitation was 3.9 pg/mL for IL-2, 11.7 pg/mL for IL-4, 9.4 pg/mL for IL-5, 9.9 pg/mL for IL-6, 7.2 pg/mL for IL-9, 1.9 pg/mL for IL-10, 11.4 pg/mL for IL-13, 8.3 pg/mL for IL-17A, 15.6 pg/mL for IL-21 and IL-22, 13.8 pg/mL for IFN-γ, 32.1 pg/mL for TNF-α, and 57.2 pg/mL for GM-CSF. Values from unstimulated cultures (medium only) were subtracted from stimulated cultures (protein), and concentrations are shown as picograms per milliliter. Supernatants for the assays were stored in aliquots at −70°C until analyzed. Samples were analyzed on a Luminex MAGPIX instrument or a Tecan Infinite 200 Pro microplate reader. After the resting phase, cells were harvested and washed with Dulbecco-PBS, and surface staining was performed by means of incubation for 30 minutes with various combinations of anti-human CD3-allophycocyanin (OKT3; eBioscience), anti-human CD4-BV510 (OKT4; BioLegend), anti-human CD62L–peridinin-chlorophyll-protein complex (PerCP)–eFluor 710 (DREG-56; eBioscience), anti-human CD45RO–fluorescein isothiocyanate (UCHL1; eBioscience), and viability Dye-eFluor 780 (eBioscience) at 4°C. Next, cells were washed in FACS Buffer (Dulbecco-PBS, 0.5% BSA, and 2 mmol/L EDTA), fixed, and permeabilized with FoxP3/Transcription Factor Staining Buffer Set (eBioscience). Intracellular staining was performed for 30 minutes with various combinations of anti-human T-bet–BV605 (4B10; BioLegend), anti-human GATA3-BV421 (16E10A23; BioLegend), anti-human FoxP3–PerCP-Cy5.5 (PCH101; eBioscience), anti-human RORγt-phycoerythrin (AFKJS-9; eBioscience), and anti-human Ki-67–phycoerythrin-Cyanine7 (20Raj1, eBioscience). Isotype controls were performed, and dead cells and doublets were excluded. Measurements were performed with a FACSCanto II flow cytometer or a FACSAria III cell sorter (BD Biosciences) and analyzed with FACSDiva software. Stimulation indices were calculated as the ratio of the percentage of Ki67+CD3+CD4+ cells under stimulating conditions (protein) to that under nonstimulating conditions (medium only). Clonally expanded T cells were evaluated regarding their T-cell receptor diversity by using next-generation sequencing. Cells before expansion, after expansion, and after the resting phase were stained with anti-human CD3-allophycocyanin (OKT3; eBioscience), anti-human CD4-BV510 (OKT4; BioLegend), and Viability Dye-eFluor 780 (eBioscience). Dead cells and doublets were excluded, and 106 CD3+CD4+ T cells were sorted with a FACSAria III cell sorter. Afterward, DNA was extracted by using a genomic DNA SV kit (Promega, Madison, Wis), concentration was determined by means of UV absorption at 280 nm with a NanoDrop photometer, and integrity was estimated by means of agarose gel electrophoresis. Genomic DNA was amplified, sequenced, and analyzed, as previously described.E3Weinberger J. Jimenez-Heredia R. Schaller S. Suessner S. Sunzenauer J. Reindl-Schwaighofer R. et al.Immune repertoire profiling reveals that clonally expanded B and T cells infiltrating diseased human kidneys can also be tracked in blood.PLoS One. 2015; 10: e0143125Crossref PubMed Scopus (10) Google Scholar Briefly, we used a 2-step multiplex PCR amplification of the rearranged T-cell receptor β chain based on Biomed-2 primer panels. Final PCR products were purified with Agencourt AMPureXP beads (Beckman Coulter, Brea, Calif) and subsequently sequenced with an Illumina MiSeq (Illumina, San Diego, Calif). Analysis of Illumina sequencing data was done by using several sequential steps, including filtering, trimming, sequence alignment, quantification of clonotypes, and diversity calculation.E4Schaller S. Weinberger J. Jimenez-Heredia R. Danzer M. Oberbauer R. Gabriel C. et al.ImmunExplorer (IMEX): a software framework for diversity and clonality analyses of immunoglobulins and T cell receptors on the basis of IMGT/HighV-QUEST preprocessed NGS data.BMC Bioinformatics. 2015; 16: 252Crossref PubMed Scopus (20) Google Scholar Statistical analysis was performed with GraphPad Prism version 6.01 software for Windows (GraphPad Software, La Jolla, Calif). Statistically significant differences in response-type distributions (Figs 1, A, and 2, C) were determined by using the χ2 test. Differences between IgG1 and IgG4 levels within a group were determined by using the paired Student t test. Comparisons between multiple groups for a specific subclass were done by using 1-way ANOVA, followed by the Tukey post hoc test (Fig 1, B). Statistical analysis of subclass distribution was performed by using the Kruskal-Wallis nonparametric ANOVA test with post hoc analysis using the Dunn multiple comparison test (Fig 1, C). Statistically significant differences between numbers of IFN-γ– and IL-10–secreting cells were determined by using the paired Student t test, and comparison of the numbers of IL-4–secreting cells between the individual groups was done by means of 1-way ANOVA, followed by the Tukey post hoc test (Fig 2, A). Comparison of transcription factor expression was done by using unpaired (farmers vs townspeople) or paired (comparison within 1 group) t test (Fig 2, B). Statistically significant differences in cytokine secretion were determined by using 1-way ANOVA, followed by the Tukey post hoc test (Fig 2, D-G). A P value of less than .05 was considered statistically significant. Correlation matrices were computed with R version 3.2.1.E5Team RDCR: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna2015Google Scholar The correlations of antigen-specific antibodies and cytokines (ELISpot and culture supernatants) were calculated for townspeople and farmers separately by using the pairwise.complete.obs option to handle missing values without discarding the whole sample.E6Correlation matrix: a quick start guide to analyze, format and visualize a correlation matrix using R software. Available at: http://www.sthda.com/english/wiki/correlation-matrix-a-quick-start-guide-to-analyze-format-and-visualize-a-correlation-matrix-using-r-software.Google Scholar The Spearman rho correlation coefficient was used, and significance was determined by computing the P value of correlations by using a CI of 95%. It is well accepted that exposure to a farming environment in early childhood prevents allergic diseases, and this effect is enhanced if the mother is exposed during pregnancy.E7Riedler J. Braun-Fahrlander C. Eder W. Schreuer M. Waser M. 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Laatikainen T. et al.Environmental biodiversity, human microbiota, and allergy are interrelated.Proc Natl Acad Sci U S A. 2012; 109: 8334-8339Crossref PubMed Scopus (678) Google Scholar Although townspeople are exposed seasonally to relatively low levels of pollen in the context of a low microbial load with limited diversity, the farmers in our study live in a microbially rich and diverse environment and are in contact with grass pollen allergens throughout the year. It has to be emphasized that the farming group in our study was comprised of selected subjects committed to a traditional type of farming on very small farms (closely resembling the farming style of the Amish people).E13Stein M.M. Hrusch C.L. Gozdz J. Igartua C. Pivniouk V. Murray S.E. et al.Innate immunity and asthma risk in Amish and Hutterite farm children.N Engl J Med. 2016; 375: 411-421Crossref PubMed Scopus (595) Google Scholar Because of their daily work, these farmers are perennially exposed to grass pollen and in daily close contact with farming animals and thus exposed to a vast microbial diversity originating from animal dander, dung, and soil. Despite higher perennial antigen exposure, farmers include a significantly higher proportion of IgG nonresponders and a reduced percentage of IgG4-reactive subjects compared with townspeople (Fig 1, A). This indicates that the farming environment triggers downregulation of IgG antibody responses. Nineteen percent of townspeople (but only 2% of farmers) mounted a nonallergic IgG4 antibody response, with 3% of them exclusively producing IgG4. IgG4 in the absence of IgE antibodies has been termed a "modified" TH2 immune response and has been found in nonallergic animal owners and beekeepers.E14Muller U. 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