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The prevalence of allergic diseases is on the rise, yet the environmental factors that contribute to this increase are still being elucidated. Laundry detergent (LD) that contains cytotoxic ingredients including microbial enzymes continuously comes into contact with the skin starting in infancy. An impaired skin barrier has been suggested as a route of allergic sensitization. We hypothesized that exposure of skin to LD damages the skin barrier resulting in systemic sensitization to allergens that enter through the impaired skin barrier. Mouse skin samples exposed in vitro to microbial proteases or LD exhibited physical damage, which was more pronounced in neonatal skin as compared to adult skin. Exposure of the skin to microbial proteases in vitro resulted in an increase in the levels of interleukin (IL)-33 and thymic stromal lymphopoietin (TSLP). BALB/c wild type mice epicutaneously exposed to LD and ovalbumin (OVA) showed an increase in levels of transepidermal water loss, serum OVA-specific immunoglobulin (Ig) G1 and IgE antibodies, and a local increase of Il33, Tslp, Il4 and Il13 compared with LD or OVA alone. Following intranasal challenge with OVA, mice epicutaneously exposed to LD showed an increase in allergen-induced esophageal eosinophilia compared with LD or OVA alone. Collectively, these results suggest that LD may be an important factor that impairs the skin barrier and leads to allergen sensitization in early life, and therefore may have a role in the increase in allergic disease., Competing Interests: M.E.R. is a consultant for Pulm One, Spoon Guru, ClostraBio, Serpin Pharm, Allakos, Celgene, Astra Zeneca, Arena Pharmaceuticals, GlaxoSmith Kline, Guidepoint and Suvretta Capital Management, and has an equity interest in the first five listed, and royalties from reslizumab (Teva Pharmaceuticals), PEESSv2 (Mapi Research Trust) and UpToDate. M.E.R is an inventor of patents owned by Cincinnati Children’s Hospital. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Background: Recent data associate eosinophilic esophagitis (EoE) with IgG4 rather than IgE, but its significance and function have not been determined. Our aims were to measure esophageal IgG4 levels and to determine functional correlations as assessed by histologic and transcriptome analyses., Methods: This case-control study included pediatric subjects with EoE (≥15 eosinophils/HPF) and non-EoE controls. Protein lysates were analyzed for IgA, IgM, and IgG1-IgG4 using the Luminex 100 system; IgE was quantified by ELISA. Esophageal biopsies were scored using the EoE histology scoring system. Transcripts were probed by the EoE diagnostic panel, designed to examine the expression of 96 esophageal transcripts., Results: Esophageal IgG subclasses, IgA, and IgM, but not IgE, were increased in subjects with EoE relative to controls. The greatest change between groups was seen in IgG4 (4.2 mg/g protein [interquartile range: 1.0-13.1 mg/g protein] vs 0.2 mg/g protein [0.1-0.9]; P < .0001). Tissue IgG4 levels correlated with esophageal eosinophil counts (P = .0006); histologic grade (P = .0011) and stage (P = .0112) scores; and IL4, IL10, IL13, but not TGFB1, expression and had strong associations with a subset of the EoE transcriptome. Esophageal IgG4 transcript expression was increased and correlated with IgG4 protein levels and IL10 expression., Conclusion: These findings extend prior studies on IgG4 in adult EoE to the pediatric population and provide deeper understanding of the potential significance and regulation of IgG4, demonstrating that IgG4 is a relevant feature of the disease; is closely related to esophageal eosinophil levels, type 2 immunity and T regulatory cytokines; and is likely produced locally., (© 2018 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Accumulating data have indicated a fundamental role of eosinophils in regulating adipose tissue homeostasis. Here, we performed whole-genome RNA sequencing of the small intestinal tract, which suggested the presence of impaired lipid metabolism in eosinophil-deficient ΔdblGATA mice. ΔdblGATA mice fed a high-fat diet (HFD) showed reduced body fat mass, impaired enlargement of adipocytes, decreased expression of adipogenic genes, and developed glucose intolerance. HFD induced accumulation of eosinophils in the perigonadal white adipose tissue. Concordantly, adipocyte-differentiated 3T3-L1 cells promoted the migration of eosinophils through the expression of CCL11 (eotaxin-1) and likely promoted their survival through the expression of interleukin (IL)-3, IL-5, and granulocyte-macrophage colony-stimulating factor. HFD-fed ΔdblGATA mice showed increased infiltration of macrophages, CD4 + T-cells, and B-cells, increased expression of interferon-γ, and decreased expression of IL-4 and IL-13 in white adipose tissue. Interferon-γ treatment significantly decreased lipid deposition in adipocyte-differentiated 3T3-L1 cells, while IL-4 treatment promoted lipid accumulation. Notably, HFD-fed ΔdblGATA mice showed increased lipid storage in the liver as compared with wild-type mice. We propose that obesity promotes the infiltration of eosinophils into adipose tissue that subsequently contribute to the metabolic homeostasis by promoting adipocyte maturation.

Loss of barrier integrity has an important role in eliciting type 2 immune responses, yet the molecular events that initiate and connect this with allergic inflammation remain unclear. We reveal an endogenous, homeostatic mechanism that controls barrier function and inflammatory responses in esophageal allergic inflammation. We show that a serine protease inhibitor, SPINK7 (serine peptidase inhibitor, kazal type 7), is part of the differentiation program of human esophageal epithelium and that SPINK7 depletion occurs in a human allergic, esophageal condition termed eosinophilic esophagitis. Experimental manipulation strategies reducing SPINK7 in an esophageal epithelial progenitor cell line and primary esophageal epithelial cells were sufficient to induce barrier dysfunction and transcriptional changes characterized by loss of cellular differentiation and altered gene expression known to stimulate allergic responses (for example, FLG and SPINK5 ). Epithelial silencing of SPINK7 promoted production of proinflammatory cytokines including thymic stromal lymphopoietin (TSLP). Loss of SPINK7 increased the activity of urokinase plasminogen-type activator (uPA), which in turn had the capacity to promote uPA receptor-dependent eosinophil activation. Treatment of epithelial cells with the broad-spectrum antiserine protease, α1 antitrypsin, reversed the pathologic features associated with SPINK7 silencing. The relevance of this pathway in vivo was supported by finding genetic epistasis between variants in TSLP and the uPA-encoding gene, PLAU We propose that the endogenous balance between SPINK7 and its target proteases is a key checkpoint in regulating mucosal differentiation, barrier function, and inflammatory responses and that protein replacement with antiproteases may be therapeutic for select allergic diseases., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Eosinophilic esophagitis (EoE) is a Th2 cytokine-associated disease characterized by eosinophil infiltration, epithelial cell hyperplasia, and tissue remodeling. Recent studies highlighted a major contribution for IL-13 in EoE pathogenesis. Paired Ig-like receptor B is a cell surface immune-inhibitory receptor that is expressed by eosinophils and postulated to regulate eosinophil development and migration. We report that Pirb is upregulated in the esophagus after inducible overexpression of IL-13 (CC10-Il13(Tg) mice) and is overexpressed by esophageal eosinophils. CC10-Il13(Tg)/Pirb(-/-) mice displayed increased esophageal eosinophilia and EoE pathology, including epithelial cell thickening, fibrosis, and angiogenesis, compared with CC10-Il13(Tg)/Pirb(+/+) mice. Transcriptome analysis of primary Pirb(+/+) and Pirb(-/-) esophageal eosinophils revealed increased expression of transcripts associated with promoting tissue remodeling in Pirb(-/-) eosinophils, including profibrotic genes, genes promoting epithelial-to-mesenchymal transition, and genes associated with epithelial growth. These data identify paired Ig-like receptor B as a molecular checkpoint in IL-13-induced eosinophil accumulation and activation, which may serve as a novel target for future therapy in EoE., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Eosinophils are multifunctional leukocytes that reside in the gastrointestinal (GI) lamina propria, where their basal function remains largely unexplored. In this study, by examining mice with a selective deficiency of systemic eosinophils (by lineage ablation) or GI eosinophils (eotaxin-1/2 double deficient or CC chemokine receptor 3 deficient), we show that eosinophils support immunoglobulin A (IgA) class switching, maintain intestinal mucus secretions, affect intestinal microbial composition, and promote the development of Peyer's patches. Eosinophil-deficient mice showed reduced expression of mediators of secretory IgA production, including intestinal interleukin 1β (IL-1β), inducible nitric oxide synthase, lymphotoxin (LT) α, and LT-β, and reduced levels of retinoic acid-related orphan receptor gamma t-positive (ROR-γt(+)) innate lymphoid cells (ILCs), while maintaining normal levels of APRIL (a proliferation-inducing ligand), BAFF (B cell-activating factor of the tumor necrosis factor family), and TGF-β (transforming growth factor β). GI eosinophils expressed a relatively high level of IL-1β, and IL-1β-deficient mice manifested the altered gene expression profiles observed in eosinophil-deficient mice and decreased levels of IgA(+) cells and ROR-γt(+) ILCs. On the basis of these collective data, we propose that eosinophils are required for homeostatic intestinal immune responses including IgA production and that their affect is mediated via IL-1β in the small intestine.

Eosinophilia and its cellular activation are hallmark features of asthma, as well as other allergic/Th2 disorders, yet there are few, if any, reliable surface markers of eosinophil activation. We have used a FACS-based genome-wide screening system to identify transcriptional alterations in murine lung eosinophils recruited and activated by pulmonary allergen exposure. Using a relatively stringent screen with false-positive correction, we identified 82 candidate genes that could serve as eosinophil activation markers and/or pathogenic effector markers in asthma. Carbonic anhydrase IV (Car4) was a top dysregulated gene with 36-fold induction in allergen-elicited pulmonary eosinophils, which was validated by quantitative PCR, immunohistochemistry, and flow cytometry. Eosinophil CAR4 expression was kinetically regulated by IL-5, but not IL-13. IL-5 was both necessary and sufficient for induction of eosinophil CAR4. Although CAR4-deficient mice did not have a defect in eosinophil recruitment to the lung, nor a change in eosinophil pH-buffering capacity, allergen-challenged chimeric mice that contained Car4(-/-) hematopoietic cells aberrantly expressed a series of genes enriched in biological processes involved in epithelial differentiation, keratinization, and anion exchange. In conclusion, we have determined that eosinophils express CAR4 following IL-5 or allergen exposure, and that CAR4 is involved in regulating the lung transcriptome associated with allergic airway inflammation; therefore, CAR4 has potential value for diagnosing and monitoring eosinophilic responses., (Copyright © 2014 by The American Association of Immunologists, Inc.)

The desmosomal cadherin desmoglein-1 (DSG1) is an essential intercellular adhesion molecule that is altered in various human cutaneous disorders; however, its regulation and function in allergic disease remains unexplored. Herein, we demonstrate a specific reduction in DSG1 in esophageal biopsies from patients with eosinophilic esophagitis (EoE), an emerging allergic disorder characterized by chronic inflammation within the esophageal mucosa. Further, we show that DSG1 gene silencing weakens esophageal epithelial integrity, and induces cell separation and impaired barrier function (IBF) despite high levels of desmoglein-3. Moreover, DSG1 deficiency induces transcriptional changes that partially overlap with the transcriptome of inflamed esophageal mucosa; notably, periostin (POSTN), a multipotent pro-inflammatory extracellular matrix molecule, is the top induced overlapping gene. We further demonstrate that IBF is a pathological feature in EoE, which can be partially induced through the downregulation of DSG1 by interleukin-13 (IL-13). Taken together, these data identify a functional role for DSG1 and its dysregulation by IL-13 in the pathophysiology of EoE and suggest that the loss of DSG1 may potentiate allergic inflammation through the induction of pro-inflammatory mediators such as POSTN.

Most in vivo studies of granulocytes draw conclusions about their trafficking based on examination of their steady-state tissue/blood levels, which result from a combination of tissue homing, survival, and egress, rather than direct examination of cellular trafficking. Herein, we developed a unique cell transfer system involving the adoptive transfer of a genetically labeled, bone-marrow-derived unique granulocyte population (eosinophils) into an elicited inflammatory site, the allergic lung. A dual polychromatic FACS-based biomarker-labeling system based on the IL4-eGFP transgene (4get) or Cd45.1 allele was used to track i.v. transferred eosinophils into the airway following allergen or T(H)2-associated stimuli in the lung in multiple mouse strains. The system was amenable to reverse tagging of recipients, thus allowing transfer of nonlabeled eosinophils and competitive tracking of multiple populations of eosinophils in vivo. The half-life of eosinophils in the blood was 3 h, and migration to the lung was dependent upon the dosage of transferred eosinophils, sensitive to pertussis toxin pretreatment, peaked at ∼24 h after adoptive transfer, and revealed a greater than 8-d eosinophil half-life in the lung. Eosinophil migration to the lung was dependent upon recipient IL-5 and IL-13 receptor α1 and donor eosinophil C-C chemokine receptor type 3 (CCR3) and interleukin 1 receptor-like 1 (ST2) in vivo. Taken together, this unique eosinophil transfer system provides an unprecedented opportunity to examine airway eosinophil migration without the need for extensive efforts to acquire donor source and time-consuming genetic crossing and has already been used to identify a long eosinophil half-life in the allergic lung and a definite role for ST2 in regulating eosinophil trafficking.

MiR-21 is one of the most up-regulated miRNAs in multiple allergic diseases associated with eosinophilia and has been shown to positively correlate with eosinophil levels. Herein, we show that miR-21 is up-regulated during IL-5-driven eosinophil differentiation from progenitor cells in vitro. Targeted ablation of miR-21 leads to reduced eosinophil progenitor cell growth. Furthermore, miR-21(-/-) eosinophil progenitor cells have increased apoptosis as indicated by increased levels of annexin V positivity compared to miR-21(+/+) eosinophil progenitor cells. Indeed, miR-21(-/-) mice have reduced blood eosinophil levels in vivo and reduced eosinophil colony forming unit capacity in the bone marrow. Using gene expression microarray analysis, we identified dysregulation of genes involved in cell proliferation (e,g, Ms4a3, Grb7), cell cycle and immune response as the most significant pathways affected by miR-21 in eosinophil progenitors. These results demonstrate that miR-21 can regulate the development of eosinophils by influencing eosinophil progenitor cell growth. Our findings have identified one of the first miRNAs with a role in regulating eosinophil development.

CD22 is currently recognized as a B cell-specific Siglec and has been exploited therapeutically with humanized anti-CD22 mAb having been used against B cell leukemia. In this study, tissue-specific eosinophil mRNA microarray analysis identified that CD22 transcript levels of murine gastrointestinal (GI) eosinophils are 10-fold higher than those of lung eosinophils. To confirm the mRNA data at the protein level, we developed a FACS-based protocol designed to phenotype live GI eosinophils isolated from the murine lamina propria. Indeed, we found that jejunum eosinophils expressed remarkably high levels of surface CD22, similar to levels found in B cells across multiple mouse strains. In contrast, CD22 was undetectable on eosinophils from the colon, blood, thymus, spleen, uterus, peritoneal cavity, and allergen-challenged lung. Eosinophils isolated from newborn mice did not express CD22 but subsequently upregulated CD22 expression to adult levels within the first 10 d after birth. The GI lamina propria from CD22 gene-targeted mice harbored more eosinophils than wild type control mice, whereas the GI eosinophil turnover rate was unaltered in the absence of CD22. Our findings identify a novel expression pattern and tissue eosinophilia-regulating function for the "B cell-specific" inhibitory molecule CD22 on GI eosinophils.

An altered balance between Th1 and Th2 cytokines is responsible for a variety of immunoinflammatory disorders such as asthma, yet the role of posttranscriptional mechanisms, such as those mediated by microRNAs (miRs), in adjusting the relative magnitude and balance of Th cytokine expression have been largely unexplored. In this study, we show that miR-21 has a central role in setting a balance between Th1 and Th2 responses to Ags. Targeted ablation of miR-21 in mice led to reduced lung eosinophilia after allergen challenge, with a broadly reprogrammed immunoactivation transcriptome and significantly increased levels of the Th1 cytokine IFN-γ. Biological network-based transcriptome analysis of OVA-challenged miR-21(-/-) mice identified an unexpected prominent dysregulation of IL-12/IFN-γ pathways as the most significantly affected in the lungs, with a key role for miR-21 in IFN-γ signaling and T cell polarization, consistent with a functional miR-21 binding site in IL-12p35. In support of these hypotheses, miR-21 deficiency led dendritic cells to produce more IL-12 after LPS stimulation and OVA-challenged CD4(+) T lymphocytes to produce increased IFN-γ and decreased IL-4. Further, loss of miR-21 significantly enhanced the Th1-associated delayed-type hypersensitivity cutaneous responses. Thus, our results define miR-21 as a major regulator of Th1 versus Th2 responses, defining a new mechanism for regulating polarized immunoinflammatory responses.

Genome-wide screening and positional cloning have linked neuropeptide S receptor 1 (NPSR1) with asthma and airway hyperresponsiveness. However, the mechanism by which NPSR1 regulates pulmonary responses remains elusive. Because neuropeptide S and its receptor NPSR1 are expressed in brain regions that regulate respiratory rhythm, and Npsr1-deficient mice have impaired stress and anxiety responses, we aimed to investigate whether neuropeptide S and NPSR1 regulate respiratory function through a central-mediated pathway. After neuropeptide S intracerebroventricular administration, respiratory responses of wildtype and Npsr1-deficient mice were monitored by whole-body or invasive plethysmography with or without serial methacholine inhalation. Airway inflammatory and hyperresponsiveness were assessed in allergen-challenged (ovalbumin or Aspergillus fumigatus) Npsr1-deficient mice. Analysis of breathing patterns by whole-body plethysmography revealed that intracerebroventricular neuropeptide S, as compared with the artificial cerebral spinal fluid control, increased respiratory frequency and decreased tidal volume in an NPSR1-dependent manner but did not affect enhanced pause. Following serial methacholine inhalation, intracerebroventricular neuropeptide S increased respiratory frequency in wildtype mice, but not in Npsr1-deficient mice, and had no effect on tidal volume. Intracerebroventricular neuropeptide S significantly reduced airway responsiveness to methacholine as measured by whole-body plethysmography. Npsr1 deletion had no impact on airway inflammation or hyperresponsiveness in ovalbumin- or A. fumigatus-induced experimental asthma. Our results demonstrate that neuropeptide S and NPSR1 regulate respiratory function through a central nervous system-mediated pathway., (Copyright © 2010 Elsevier Inc. All rights reserved.)

NPSR1 is a G protein coupled receptor expressed in multiple brain regions involved in modulation of stress. Central administration of NPS, the putative endogenous ligand of NPSR1, can induce hyperlocomotion, anxiolytic effects and activation of the HPA axis. The role of NPSR1 in the brain remains unsettled. Here we used NPSR1 gene-targeted mice to define the functional role of NPSR1 under basal conditions on locomotion, anxiety- and/or depression-like behavior, corticosterone levels, acoustic startle with prepulse inhibition, learning and memory, and under NPS-induced locomotor activation, anxiolysis, and corticosterone release. Male, but not female, NPSR1-deficient mice exhibited enhanced depression-like behavior in a forced swim test, reduced acoustic startle response, and minor changes in the Morris water maze. Neither male nor female NPSR1-deficient mice showed alterations of baseline locomotion, anxiety-like behavior, or corticosterone release after exposure to a forced swim test or methamphetamine challenge in an open-field. After intracerebroventricular (ICV) administration of NPS, NPSR1-deficient mice failed to show normal NPS-induced increases in locomotion, anxiolysis, or corticosterone release compared with WT NPS-treated mice. These findings demonstrate that NPSR1 is essential in mediating NPS effects on behavior., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Background: Eosinophilic oesophagitis (EO) is an emerging yet increasingly prevalent disorder characterised by a dense and selective eosinophilic infiltration of the oesophageal wall. While EO is considered an atopic disease primarily triggered by food antigens, disparities between standard allergen testing and clinical responses to exclusion diets suggest the participation of distinct antigen-specific immunoglobulin E (IgE) in the pathophysiology of EO., Aim: To find evidence for a local IgE response., Methods: Endoscopic biopsies of the distal oesophagus of atopic and non-atopic EO and control individuals (CTL) were processed for immunohistochemistry and immunofluorescence to assess the presence of B cells, mast cells, and IgE-bearing cells. Oesophageal RNA was analysed for the expression of genes involved in B cell activation, class switch recombination to IgE and IgE production, including germline transcripts (GLTs), activation-induced cytidine deaminase (AID), IgE heavy chain (Cepsilon) and mature IgE mRNA using polymerase chain reaction and microarray analysis., Results: Regardless of atopy, EO showed increased density of B cells (p<0.05) and of IgE-bounded mast cells compared to CTL. Both EO and CTL expressed muGLT, epsilonGLT, gamma4GLT, AID, Cepsilon and IgE mRNA. However, the frequency of expression of total GLTs (p = 0.002), epsilonGLT (p = 0.024), and Cepsilon (p = 0.0003) was significantly higher in EO than in CTL, independent of the atopic status., Conclusion: These results support the heretofore unproven occurrence of both local immunoglobulin class switching to IgE and IgE production in the oesophageal mucosa of EO patients. Sensitisation and activation of mast cells involving local IgE may therefore critically contribute to disease pathogenesis.

Background: Intestinal anaphylaxis (manifested by acute diarrhea) is dependent on IgE and mast cells., Objective: We aimed to define the respective roles of IL-4 and IL-13 and their receptors in disease pathogenesis., Methods: Wild-type mice and mice deficient in IL-4, IL-13, and IL-13 receptor (IL-13R) alpha1 (part of the type 2 IL-4 receptor [IL-4R]) were sensitized with ovalbumin (OVA)/aluminum potassium sulfate and subsequently given repeated intragastric OVA exposures. The IL-4R alpha chain was targeted with anti-IL-4R alpha mAb before or after intragastric OVA exposures., Results: IL4(-/-) (and IL4/IL13(-/-)) mice produced almost no IgE and were highly resistant to OVA-induced diarrhea, whereas allergic diarrhea was only partially impaired in IL13(-/-) and IL13Ralpha1(-/-) mice. IL13Ralpha1-deficient mice had decreased IgE levels, despite having normal baseline IL-4 levels. Intestinal mast cell accumulation and activation also depended mainly on IL-4 and, to a lesser extent, on IL-13. Prophylactic anti-IL-4R alpha mAb treatment, which blocks all IL-4 and IL-13 signaling, suppressed development of allergic diarrhea. However, treatment with anti-IL-4R alpha mAb for 7 days only partially suppressed IgE and did not prevent intestinal diarrhea., Conclusion: Endogenously produced IL-13 supplements the ability of IL-4 to induce allergic diarrhea by promoting oral allergen sensitization rather than the effector phase of intestinal anaphylaxis.

Periostin is an extracellular matrix protein that has been primarily studied in the context of the heart, where it has been shown to promote cardiac repair and remodeling. In this study, we focused on the role of periostin in an allergic eosinophilic inflammatory disease (eosinophilic esophagitis (EE)) known to involve extensive tissue remodeling. Periostin was indeed markedly overexpressed (35-fold) in the esophagus of EE patients, particularly in the papillae, compared with control individuals. Periostin expression was downstream from transforming growth factor-beta and interleukin-13, as these cytokines were elevated in EE esophageal samples and markedly induced periostin production by primary esophageal fibroblasts (107- and 295-fold, respectively, at 10 ng ml(-1)). A functional role for periostin in eliciting esophageal eosinophilia was demonstrated, as periostin-null mice had a specific defect in allergen-induced eosinophil recruitment to the lungs and esophagus (66 and 72% decrease, respectively). Mechanistic analyses revealed that periostin increased (5.8-fold) eosinophil adhesion to fibronectin. As such, these findings extend the involvement of periostin to esophagitis and uncover a novel role for periostin in directly regulating leukocyte (eosinophil) accumulation in T helper type 2-associated mucosal inflammation in both mice and humans.

Background: Surfactant protein (SP) D has been proposed to be protective in allergic airway responses., Objective: We aimed to determine the effect of SP-D deficiency on murine and human airway allergy., Methods: Immunologic responses of SP-D gene-deficient mice (Sftpd-/-) at baseline and after 4 intranasal Aspergillus fumigatus exposures were assessed. In addition, the significance of a single nucleotide polymorphism (Met(11)Thr) in the human SP-D gene (known to decrease SP-D function) was investigated., Results: Macrophage and neutrophil bronchoalveolar lavage fluid levels and large airway mucus production were increased in naive Sftpd-/- mice in association with increased lung CCL17 levels and CD4+ T cell numbers. T(H)2-associated antibody levels (IgG1 and IgE) were significantly lower in 4- to 5-week-old Sftpd-/- mice (P < .05). Accordingly, naive Sftpd-/- splenocytes released significantly less IL-4 and IL-13 on anti-CD3/CD28 stimulation (P < .01). After intranasal allergen exposures, a modest decrease in bronchoalveolar lavage fluid eosinophilia and IL-13 levels was observed in Sftpd-/- mice compared with values seen in wild-type mice in association with decreased airway resistance (P < .01). A single nucleotide polymorphism in the SFTPD gene, affecting SP-D levels and pathogen binding, was associated with decreased atopy in black subjects and potentially lower asthma susceptibility in white subjects., Conclusion: Sftpd-/- mice have an impaired systemic T(H)2 response at baseline and reduced inflammation and airway responses after allergen exposure. Translational studies revealed that a polymorphism in the SFTPD gene was associated with lower atopy and possibly asthma susceptibility. Taken together, these results support the hypothesis that SP-D-dependent innate immunity influences atopy and asthma.

Background: Eosinophilic esophagitis (EE) is an emerging worldwide disease that mimics gastroesophageal reflux disease. Early studies have established that esophageal eosinophilia occurs in association with T(H)2 allergic responses, and we recently identified an EE-specific esophageal transcriptome that included eotaxin-3., Objective: We sought to determine the mechanism by which this T(H)2 response leads to EE., Methods: Real-time PCR and microarray analysis were performed on RNA extracted from esophageal biopsy specimens and primary esophageal epithelial cell cultures stimulated with IL-13 (0-100 ng/mL). Transient transfections in esophageal cell lines were performed with plasmids containing the luciferase gene driven by eotaxin-3 promoter fragments and modified forms of signal transducer and activator of transcription 6., Results: The IL-13 mRNA level was markedly increased (16-fold) in esophageal biopsy specimens from patients with EE compared with those from healthy individuals. Furthermore, IL-13 treatment of primary esophageal epithelial cells was sufficient to induce a global-expression transcript profile that remarkably overlapped with the EE-specific esophageal transcriptome. In addition, esophageal epithelial cells markedly produce eotaxin-3 after IL-13 stimulation through a transcriptional mechanism dependent on signal transducer and activator of transcription 6. Lastly, increased IL-13 mRNA levels and the EE transcriptome were largely reversible with glucocorticoid treatment in vivo., Conclusions: Taken together, we propose that the pathogenesis of EE is mediated by an IL-13-stimulated keratinocyte-derived transcriptome that is largely reversible with corticosteroid treatment. Furthermore, we identify an in vivo IL-13-induced transcriptome that has potential utility for target assessment after anti-IL-13 therapeutics., Clinical Implications: IL-13-induced pathways and genes are fundamental processes in the cause and manifestations of EE; as such, therapeutic agents that interfere with IL-13 might be particularly useful for disease treatment.

A new class of organellar proteins, characterized by pentatricopeptide repeat (PPR) motifs, has been identified in plants. These proteins contain multiple 35-amino acid repeats that are proposed to form a super helix capable of binding a strand of RNA. All PPR proteins characterized to date appear to be involved in RNA processing pathways in organelles. Twenty-three PPR proteins have been identified in Trypanosoma brucei and database research indicates that most of these proteins are predicted to contain the traditional mitochondrial target sequence. Orthologues of each of the 23 proteins have also been identified in Leishmania major and Trypanosoma cruzi, indicating that these proteins represent a highly conserved class of proteins within the kinetoplastid family. Preliminary experiments using RNAi to specifically silence one identified PPR gene (TbPPRl- Tb927.2.3180), indicate that cells depleted of TbPPRl transcripts show a slow growth phenotype and altered mitochondrial maxicircle RNA profiles. This initial characterization suggests that PPR proteins will play important roles in the complex RNA processing required for mitochondrial gene expression in trypanosomes.

In Trypanosoma brucei, two classes of transcripts are produced from two distinct mitochondrial genome components. Guide RNAs (gRNAs) are usually minicircle encoded and exist as primary transcripts, while the maxicircle-encoded rRNAs and mRNAs are processed from a polycistronic precursor. The genes for the gRNAs gMURF2-II and gCYb(560) each have uncommon kinetoplast DNA (kDNA) locations that are not typically associated with transcription initiation events. We demonstrate that the conserved maxicircle gRNA gMURF2-II has an unusual location within the ND4 gene. This is the first report of a completely intragenic gene in kDNA. In addition, the gMURF2-II and ND4 transcripts are generated by distinctly different events; the ND4 mRNA is processed from a polycistronic precursor, while transcription of the gRNA initiates downstream of the 5' end of the ND4 gene. The gCYb(560) gene has an atypical minicircle location in that it is not flanked by the inverted repeat sequences that surround the majority of minicircle gRNA genes. Our data indicate that the mature gCYb(560) gRNA is also a primary transcript and that the 5'-end heterogeneity previously observed for this gRNA is a result of multiple transcription initiation sites and not of imprecise 5'-end processing. Together, these data indicate that gRNA genes represent individual transcription units, regardless of their genomic context, and suggest a complex mechanism for mitochondrial gene expression in T. brucei.